Formulation, Delivery and Stability of Bone Morphogenetic Proteins for Effective Bone Regeneration

Drug Delivery Technolog

Label-Free, Flow-Imaging Methods for Determination of Cell Concentration and Viability

To investigate the potential of two flow imaging microscopy (FIM) techniques (Micro-Flow Imaging (MFI) and FlowCAM) to determine total cell concentration and cell viability. B-lineage acute lymphoblastic leukemia (B-ALL) cells of 2 different donors were exposed to ambient conditions. Samples were taken at different days and measured with MFI, FlowCAM, hemocytometry and automated cell counting. Dead and live cells from a fresh B-ALL cell suspension were fractionated by flow cytometry in order to derive software filters based on morphological parameters of separate cell populations with MFI and FlowCAM. The filter sets were used to assess cell viability in the measured samples. All techniques gave fairly similar cell concentration values over the whole incubation period. MFI showed to be superior with respect to precision, whereas FlowCAM provided particle images with a higher resolution. Moreover, both FIM methods were able to provide similar results for cell viability as the conventional methods (hemocytometry and automated cell counting). FIM-based methods may be advantageous over conventional cell methods for determining total cell concentration and cell viability, as FIM measures much larger sample volumes, does not require labeling, is less laborious and provides images of individual cells. PURPOSE METHODS RESULTS CONCLUSIONDrug Delivery Technolog

Towards the development of a supercritical carbon dioxide spray process to coat solid protein particles

The aim of this study was to develop a supercritical carbon dioxide (scCO2) spray process to coat solid protein particles with a hydrophilic polymer. The final purpose is to manufacture drug particles exhibiting controlled release behaviour in patients. Lysozyme microparticles (about 20 μm) were suspended in a vessel into which a dextran sulphate (DS) solution was dispersed by scCO2 via a nozzle. Upon interaction with the droplets, DS was deposited onto or mixed with suspended lysozyme particles. Particles of about 100 μm were obtained. The zeta-potential analysis and elemental analysis indicated that the top layer of the particles consisted of both lysozyme and DS. Some of the produced particulate materials showed retarded lysozyme release when exposed to water or phosphate buffered saline, holding promise for future production of controlled drug delivery systems for therapeutic proteins.Drug Delivery TechnologyBiopharmaceutic

Fc gamma receptor binding profile of anti-citrullinated protein antibodies in immune complexes suggests a role for FcγRI in the pathogenesis of synovial inflammation

OBJECTIVES: Anti-citrullinated protein antibodies (ACPA) are highly specific for rheumatoid arthritis (RA). Here, we studied binding of ACPA-IgG immune complexes (IC) to individual Fc gamma receptors (FcγR) to identify potential effector mechanisms by which ACPA could contribute to RA pathogenesis. METHODS: ACPA-IgG1 and control IgG1(IgG1 depleted of ACPA-IgG1) were isolated from plasma and synovial fluid (SF) of RA patients by affinity chromatography using CCP2 peptides. Subsequently, IC were generated using fluorescently labelled F(ab’)2 fragments against the F(ab’)2 region of IgG, or by using citrullinated fibrinogen. IC were incubated with FcγR-transfected CHO cell lines or neutrophils from healthy donors. FcγR binding of IC was analysed by flow cytometry in the presence or absence of specific blocking antibodies. RESULTS: ACPA-IgG1 IC predominantly bound to FcγRI and FcγRIIIA on FcγR-transfected CHO cell lines, while much lower binding was observed to FcγRIIA and FcγRIIB. ACPA-IgG1 IC showed reduced binding to FcγRIIIA compared to control IgG1 IC, in line with enhanced ACPA-IgG1 Fc core-fucosylation. Neutrophils activated in vitro to induce de novo expression of FcγRI showed binding of ACPA-IgG IC, and blocking studies revealed that almost 30% of ACPA-IgG IC binding to activated neutrophils was mediated by FcγRI. CONCLUSIONS: Our studies show that ACPA-IgG1 IC bind predominately to activating FcγRI and FcγRIIIA, and highlight FcγRI expressed by activated neutrophils as relevant receptor for these IC. As neutrophils isolated from SF exhibit an activated state and express FcγRI in the synovial compartment, this IC-binding could contribute to driving disease pathogenesis in RA.Drug Delivery Technolog

Intradermal vaccination with hollow microneedles: A comparative study of various protein antigen and adjuvant encapsulated nanoparticles

Drug Delivery TechnologySupramolecular & Biomaterials Chemistr

Measurement of the average mass of proteins adsorbed to a nanoparticle by using a suspended microchannel resonator

Drug Delivery TechnologyAnalytical BioScience

Potential Issues With the Handling of Biologicals in a Hospital

Drug Delivery Technolog

An Albumin-Free Formulation for Escherichia coli-Derived Interferon Beta-1b with Decreased Immunogenicity in Immune Tolerant Mice.

Human serum albumin (HSA)-free formulation of Escherichia coli-derived human interferon beta (IFNβ-1b) with a high percentage of monomeric protein and low immunogenicity is developed and characterized in the current study. UV spectroscopy, fluorescence spectroscopy, dynamic light scattering, sodium dodecyl sulfate polyacrylamide gel electrophoresis, Western blotting, Micro-Flow Imaging, resonant mass measurement, size exclusion, and reversed-phase high performance liquid chromatographies were applied to assess the effect of excipients on the stability of IFNβ-1b to establish a HSA-free formulation. The antiviral activity of IFNβ-1b was evaluated using human lung carcinoma cell line. Immune tolerant mice to hIFNβ were used to assess the immunogenicity of the HSA-free formulated IFNβ-1b in comparison to Betaferon drug product and Avonex drug substance as standards through IgG titering of plasma. HSA-free formulated IFNβ-1b, including 200 mM L-arginine, 200 mM trehalose, and 0.1% n-dodecyl β-D-maltoside in 10 mM sodium acetate buffer, pH 7.4, showed the highest biological activity. The stability of IFNβ-1b in the HSA-free formulation was monitored for 3 weeks at 4°C and 37°C with relative humidity of 10% and 75%, respectively. Protein aggregation and immunogenicity in transgenic mice were decreased in the HSA-free formulated IFNβ-1b compared to Betaferon. The stability, biological activity, and immunogenicity of the HSA-free formulation and Betaferon were evaluated. Incubation of formulations at 4°C and 37°C for 3 weeks showed that the HSA-free formulated IFNβ-1b was more stable and less immunogenic in transgenic FVB/N mice. Low immunogenicity and the absence of HSA, which reduces the potential risk of viral infection (eg, HIV and HCV), are promising for clinical studies.Drug Delivery Technolog

Facilitating the mineralization of oligo(poly(ethylene glycol) fumarate) hydrogel by incorporation of hydroxyapatite nanoparticles.

Exploring strategies to induce the mineralization of hydrogels is an important step toward the development of hydrogel-based materials for bone regeneration. In the current study, the effect of incorporating hydroxyapatite (HA) nanoparticles on the mineralization capacity of an inert poly(ethylene glycol) (PEG)-based hydrogel was investigated. HA nanoparticles were either directly loaded into oligo(poly(ethylene glycol) fumarate) (OPF) hydrogel or loaded into commonly used gelatin microsphere porogens that were subsequently integrated in the OPF matrix. Mineralization of composites after immersion of the samples in simulated body fluid up to 28 days was assessed. In contrast to the blank OPF hydrogel, the HA-containing constructs strongly mineralized such that the average rate of calcium uptake by the material was enhanced by orders of magnitude. The mineral formed was observed to be apatitic and needle shaped. The presented method allows modification of inert PEG-based hydrogels into bioactive biomaterials for applications in bone regeneration