Preparation and in vitro release profiling of PLGA microspheres containing BSA as a model protein

Conventional drug formulations are incapable of adequate delivery of proteins and peptides for therapeutic purposes. As these molecules have very short biological half-life, multiple dosing is required to achieve the desirable therapeutic effects. Microspheres are able to encapsulate proteins and peptide in the polymeric matrix while protecting them from enzymatic degradation. In this study Bovine Serum Albumin (BSA) matrix type microspheres were fabricated using Polylactide-co-glycolide (PLGA) by double emulsion solvent evaporation method. The effects of variables such as homogenizer speed, molecular weight of polymer and the effect of pH of the water phases, were investigated against factors such as drug loading, encapsulation efficiency, morphology, size, drug distribution and release profile of the microspheres. Results, suggested that an increase in homogenization speed leads to a decrease in microsphere size. The increase in homogenization speed also caused a significant effect on the release profile only when higher molecular weight of polymer had been used.. The pH change of the internal aqueous phase led to modification of surface morphology of spheres to a porous structure that significantly increased the total amount of released protein. Integrity of protein structure was intact as shown by SDS-PAGE. According to the results, it can be concluded that we achieved a reproducible method regarding controlled protein delivery for different sizes of particles.

Methods for Monitoring Macroautophagy in Pancreatic Cancer Cells.

Macroautophagy is a catabolic process through which redundant, aged, or damaged cellular structures are first enclosed within double-membrane vesicles (called autophagosomes), and thereafter degraded within lysosomes. Macroautophagy provides a primary route for the turnover of macromolecules, membranes and organelles, and as such plays a major role in cell homeostasis. As part of the stress response, autophagy is crucial to determine the cell fate in response to extracellular or intracellular injuries. Autophagy is involved in cancerogenesis and in cancer progression. Here we illustrate the essential methods for monitoring autophagy in pancreatic cancer cells.

Ex-Vivo Signal Transduction Studies in Chronic Lymphocytic Leukemia.

Microenvironmental signaling is pivotal to chronic lymphocytic leukemia (CLL) pathology; therefore understanding how to investigate this pathway by both protein and chemical methods is crucial if we are to investigate and correlate biological changes with therapeutic responses in patients. Herein, we describe the use of western blotting also referred to as immunoblotting as a method that can semiquantitatively evaluate changes in protein expression following receptor engagement; this includes B cell receptor (BCR) signaling following stimulation with anti-IgM (Blunt et al. Clin Cancer Res 23(9):2313-2324, 2017). It is important to note that immunoblotting should always be combined with other quantitative methods such as flow cytometry to confirm activation of these signaling pathways (Aguilar-Hernandez et al. Blood 127(24):3015-3025, 2016).

Protein Extraction from Gels: A Brief Review.

Protein gel electrophoresis is an important procedure carried out in protein studies. Elution and recovery of proteins separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) are often necessary for further downstream analyses. The process involves localizing the protein of interest on the gel following SDS-PAGE, eluting the protein from the gel, removing SDS from the eluted sample, and finally renaturing the protein (e.g., enzymes) for subsequent analyses. Investigators have extracted proteins from gels by a variety of techniques. These include dissolution of the gel matrix, passive diffusion, and electrophoretic elution. Proteins eluted from gels have been used successfully in a variety of downstream applications, including protein chemistry, proteolytic cleavage, determination of amino acid composition, polypeptide identification by trypsin digestion and matrix-assisted laser desorption ionization-time of flight mass spectroscopy, as antigens for antibody production, identifying a polypeptide corresponding to an enzyme activity, and other purposes. Protein yields ranging from nanogram levels to 100 µg have been obtained. Here, we review some of the methods that have been used to elute proteins from gels.

Professor Volodymyr Oleksiiovych Shlyakhovenko (on the 80th birth anniversary).

In September 2018, Professor Volodymyr Oleksiiovych Shlyakhovenko, well-known Ukrainian scientist in the field of cancer biochemistry, celebrated his 80th anniversary.

Controlling Dispersion during Single-Cell Polyacrylamide-Gel Electrophoresis in Open Microfluidic Devices.

New tools for measuring protein expression in individual cells complement single-cell genomics and transcriptomics. To characterize a population of individual mammalian cells, hundreds to thousands of microwells are arrayed on a polyacrylamide-gel-coated glass microscope slide. In this "open" fluidic device format, we explore the feasibility of mitigating diffusional losses during lysis and polyacrylamide-gel electrophoresis (PAGE) through spatial control of the pore-size of the gel layer. To reduce in-plane diffusion-driven dilution of each single-cell lysate during in-microwell chemical lysis, we photopattern and characterize microwells with small-pore-size sidewalls ringing the microwell except at the injection region. To reduce out-of-plane-diffusion-driven-dilution-caused signal loss during both lysis and single-cell PAGE, we scrutinize a selectively permeable agarose lid layer. To reduce injection dispersion, we photopattern and study a stacking-gel feature at the head of each <1 mm separation axis. Lastly, we explore a semienclosed device design that reduces the cross-sectional area of the chip, thus reducing Joule-heating-induced dispersion during single-cell PAGE. As a result, we observed a 3-fold increase in separation resolution during a 30 s separation and a >2-fold enhancement of the signal-to-noise ratio. We present well-integrated strategies for enhancing overall single-cell-PAGE performance.

Methods for Assessing Apoptosis and Anoikis in Normal Intestine/Colon and Colorectal Cancer.

Caspase-dependent apoptosis, including its distinct cell death subroutine known as anoikis, perform essential roles during organogenesis, as well as in the maintenance and repair of tissues. To this effect, the continuous renewal of the human intestinal/colon epithelium is characterized by the exfoliation by anoikis of differentiated cells, whereas immature/undifferentiated cells may occasionally undergo apoptosis in order to evacuate daughter cells that are damaged or defective. Dysregulated epithelial apoptosis is a significant component of inflammatory bowel diseases. Conversely, the acquisition of a resistance to apoptosis represents one of the hallmarks of cancer initiation and progression, including for colorectal cancer (CRC). Furthermore, the emergence of anoikis resistance constitutes a critical step in cancer progression (including CRC), as well as a limiting one that enables invasion and metastasis.Considering the implications of apoptosis/anoikis dysregulation in gut physiopathology, it therefore becomes incumbent to understand the functional determinants that underlie such dysregulation-all the while having to monitor, assess, or evidence apoptosis and/or anoikis. In this chapter, methodologies that are typically used to assess caspase-dependent apoptosis and anoikis in intestinal/colonic normal and CRC cells, whether in vivo, ex vivo, or in cellulo, are provided.

Proteomic Profiling for Colorectal Cancer Biomarker Discovery.

Nowadays, the ideal biomarker for colorectal cancer (CRC) has not been found. Two-dimensional electrophoresis (2-DE) and mass spectrometry (MS) are suitable techniques for searching new biomarkers. In this chapter, we describe methodology for biomarker discovery based on a proteomic approach. In addition, special attention is given to the sample preparation, including protein extraction, fractionation, and cleanup, as we consider this a critical step. Comparing the proteomic profile of tumor and mucosa, we identified the nucleoside diphosphate kinase A (NDKA) protein as a candidate biomarker for CRC. Finally, we validated NDKA with an ELISA kit using serum samples from individuals of a screening cohort. Our results suggest that serum NDKA is a potential biomarker for screening of CRC and premalignant advanced adenomas (AA).

Dissecting the Recombination Mediator Activity of BRCA2 Using Biochemical Methods.

Homologous recombination (HR) is an essential pathway to restart stalled replication forks, repair spontaneous DNA double-strand breaks, and generate genetic diversity. Together with genetic studies in model organisms, the development of purification protocols and biochemical assays has allowed investigators to begin to understand how the complex machinery of HR functions. At the core of the HR process is the recombination enzyme RecA in bacteria or RAD51 and DMC1 in eukaryotes. The main steps of HR can be reconstituted in vitro and involve: (1) The formation of a ssDNA-RAD51 complex into a helical structure termed the nucleoprotein filament after one DNA strand has been resected at the site of the break. (2) The homologous DNA pairing with an intact copy of the damaged chromatid to form a joint molecule also called displacement loop (D-loop). (3) The exchange of DNA strands and de novo DNA synthesis to restore the damaged/lost DNA. (4) The resolution of joint molecules by nucleolytic cleavage. The human tumor suppressor BRCA2 is a mediator of HR as it actively facilitates the DNA transactions of the recombination proteins RAD51 and DMC1 in a variety of ways: It stabilizes ssDNA-RAD51/DMC1 nucleoprotein filaments. It limits the assembly of RAD51 on dsDNA. It facilitates the replacement of replication protein A by RAD51. The result of these activities is a net increase of DNA strand exchange products as observed in vitro. Here, we describe some of the biochemical assays used to dissect the mediator activities of BRCA2.

Methods to Study DNA End Resection II: Biochemical Reconstitution Assays.

DNA end resection initiates the largely accurate repair of DNA double-strand breaks (DSBs) by homologous recombination. Specifically, recombination requires the formation of 3' overhangs at DSB sites, which is carried out by nucleases that specifically degrade 5'-terminated DNA. In most cases, DNA end resection is a two-step process, comprising of initial short-range followed by more processive long-range resection. In this chapter, we describe selected assays that reconstitute both the short- and long-range pathways. First, we define methods to study the exonuclease and endonuclease activities of the MRE11-RAD50-NBS1 (MRN) complex in conjunction with phosphorylated cofactor CtIP. This reaction is particularly important to initiate processing of DNA breaks and to recruit components belonging to the subsequent long-range pathway. Next, we describe assays that reconstitute the concerted reactions of Bloom (BLM) or Werner (WRN) helicases that function together with the DNA2 nuclease-helicase, and which are as a complex capable to resect DNA of kilobases in length. The reconstituted reactions allow us to understand how the resection pathways function at the molecular level. The assays will be invaluable to define regulatory mechanisms and to identify inhibitory compounds, which may be valuable in cancer therapy.

Analysis of Enzymatic Activity of Matrix Metalloproteinase (MMP) by Collagen Zymography in Melanoma.

Protein zymography is the most commonly used technique to study the enzymatic activity of matrix metalloproteinases (MMPs) and their inhibitors. MMPs are proteolytic enzymes that promote extracellular matrix degradation. MMPs are frequently mutated in malignant melanomas as well as other cancers and are linked to increasing incidence of tumor metastasis. Substrate zymography characterizes MMP activity by their ability to degrade preferred substrates. Here we describe the collagen zymography technique to measure the active or latent form of MMPs using MMP-8 as an example, which is a frequently mutated MMP family member in malignant melanomas. The same technique can be used with the modification of substrate to detect metalloproteinase activity of other MMPs. Both wild-type and mutated forms of MMPs can be analyzed using a single gel using this method.

Multiplex Cathepsin Zymography to Detect Amounts of Active Cathepsins K, L, S, and V.

Cysteine cathepsins are powerful proteases that can degrade other proteins, among which are the extracellular matrix proteins collagen and elastin. Multiplex cathepsin zymography is an assay that can quantify the amount of active cathepsins in a cell or tissue preparation. This method works for measuring the amounts of active cathepsins K, L, S, and V in a cell or tissue preparation without requiring the use of antibodies for specific identification which tremendously reduces cost. This chapter will demonstrate the utility and interpretation of this method with mammalian cells and tissue to quantify amounts of active cathepsins K, L, S, and V without complicating signals of the procathepsin. Multiplex cathepsin zymography has many advantages: (1) it separates cathepsins K, L, S, and V by electrophoretic migration distance, (2) allows visual confirmation of cathepsin identity, (3) does not detect procathepsins, and (4) can be quantified with densitometry.

Kinetic Rate Determination via Electrophoresis along a Varying Cross-Section Microchannel.

High throughput, efficient, and readily adoptable analytical tools for the validation and selection of reliable antibody reagents would impact the life sciences, clinical chemistry, and clinical medicine. To directly quantify antibody-antigen association and dissociation rate constants, kon and koff, in a single experiment, we introduce a microfluidic free-standing kinetic polyacrylamide gel electrophoresis (fsKPAGE) assay. Here, an antibody is immobilized in zones along the length of a single freestanding polyacrylamide gel lane of varying cross-sectional width. Fluorescently labeled antigen is electrophoresed through each immobilized antibody zone, with local cross-sectional area determining the local electric field strength and, thus, the local interaction time between immobilized antibody and electromigrating antigen. Upon crossing, the interaction yields immobilized immunocomplex. The kon is quantified by assessing the amount of immunocomplex formed at each interaction time. To quantify koff, immobilized zones of fluorescently labeled immunocomplex are subjected to a buffer dilution and monitored over time. We determine kon and koff for prostate-specific antigen (PSA) and make a comparison to gold-standard values. The fsKPAGE assay determines kon and koff in a single experiment of less than 20 min, using 45 ng of often limited antibody material and standard laboratory equipment. We see the fsKPAGE assay as forming the basis for rapid, quantitative antibody-screening tools.

Hydrogel Pore-Size Modulation for Enhanced Single-Cell Western Blotting.

Pore-gradient microgel arrays enable thousands of parallel high-resolution single-cell protein electrophoresis separations for targets accross a wide molecular mass (25-289 kDa), yet within 1 mm separation distances. Dual crosslinked hydrogels facilitate gel-pore expansion after electrophoresis for efficient and uniform immunoprobing. The photopatterned, light-activated, and acid-expandable hydrogel underpins single-cell protein analysis, here for oncoprotein-related signaling in human breast biopsy.

Bifunctional glass membrane designed to interface SDS-PAGE separations of proteins with the detection of peptides by mass spectrometry.

We describe the construction and characterization of a novel membrane designed to allow proteins separated by gel electrophoresis (SDS-PAGE) to be detected as peptides by mass spectrometry in an efficient and comprehensive manner. The key attribute of the membrane is a bifunctional design that allows for the digestion of protein(s) and retention of the resulting peptides with minimal lateral diffusion. Silane chemistries are used to differentially treat the opposing surfaces of a glass filter paper to enable this unique capability.

Polar electrophoresis: shape of two-dimensional maps is as important as size.

The performance of two-dimensional electrophoresis in conventional gels in Cartesian coordinates (2-DE) vs. polar coordinates (2-PE) is here evaluated. Although 2-DE is performed in much longer Immobiline gels in the first dimension (17 cm) vs. barely 7-cm in 2-PE, an equivalent resolving power is found. Moreover, due to the possibility of running up to seven Immobiline strips in the radial gel format, the reproducibility of spot position is seen to be higher, this resulting in a 20% higher matching efficiency. As an extra bonus, strings of "isobaric" spots (i.e. polypeptides of identical mass with different pI values) are more resolved in the radial gel format, especially in the 10 to 30 kDa region, where the gel area fans out leaving extra space for spot resolution. In conclusion, this novel gel format in the second dimension of 2D gels is seen as an important improvement of this technique, still one of the most popular in proteome analysis.

Polyacrylamide gel photopatterning enables automated protein immunoblotting in a two-dimensional microdevice.

We demonstrate a two-dimensional microfluidic architecture that integrates polyacrylamide gel electrophoresis (PAGE) with immunoblotting in a fully automated format. This assay is designed to overcome performance limitations of conventional slab-gel immunoblotting, including multiple manual interventions, low-throughput transfer and blotting, and substantial consumption of reagents and sample. To unify PAGE with blotting in one device, this microfluidic approach makes use of high-resolution regional photopatterning of multiple polyacrylamide gel elements, and automated electrophoretic transport. A complete native immunoblot of free prostate specific antigen from human seminal fluid is demonstrated in less than 5 min. Further, the characterization of post-PAGE electrophoretic transfer showed high efficiency and minimal sample dispersion.

Quantitative computerized western blotting.

Western blotting allows analysis of antibody reactivity against multiple antigens separated according to their molecular weights. The distinction between immune dominant and recessive antigens is often difficult and carried out by qualitative or empirical means. Quantitative computerized western blotting (QCWB) addresses this difficulty by analyzing reactivity to specific antigens and providing a statistically measurable value for each band. This allows differentiation between immunodominant and immunorecessive determinants. QCWB is appropriate for either single time point analysis or longitudinal studies where multiple time points are evaluated and the reactivities against individual bands compared. This technique can be used to study humoral responses to complex antigenic mixtures such as allergens and infectious agents, or to identify serologic markers for early diagnosis of cancer, autoimmune, or infectious diseases, or to monitor patient's clinical status.

Peptide enrichment and protein fractionation using selective electrophoresis.

In recent times, the analysis of the peptidome has become increasingly valuable to gain a better understanding of the critical roles native peptides play in biological processes. Here, we show a technique using a novel electrophoretic device named MF10, for the fractionation of proteins and peptides based on size and also pH in low volume liquid phase under an electric field. A 1 microM, 7-protein and peptide standard mix ranging from 1 to 25 kDa has been used to show peptide migration into a fraction contained by 1-5 kDa membranes. Simultaneous fractionation of the higher mass protein standards to the correct fraction also occurred. To assess the MF10's ability to fractionate more complex samples, human plasma was used to enrich for the peptidome below 5 kDa in the presence of the proteome. Peptide enrichment was achieved while simultaneously fractionating higher mass proteins to three other mass restricted fractions. The utility of this approach is demonstrated with the identification (with at least 2 ppm mass accuracy) of 76 unique peptides, equating to 22 proteins enriched to the 1-5 kDa fraction of the MF10.

Development and application of a miniaturized gel electrophoresis device for protein analysis.

The present article describes a miniaturized polyacrylamide slab gel electrophoresis-chip (PASGE-Chip) that can rapidly separate a set of predefined samples as well as cell lysate samples for clinical diagnosis. The chip consists of a polymethyl methacrylate (PMMA) upper unit (25 x 30 x 10 mm, width x length x depth) with integrated buffer chambers, running electrodes and loading wells and a bottom unit comprising a silicon dioxide-coated silicon plate with embossed gel chamber (11 x 15 x 0.37 mm). This miniaturized device was designed to be fast, easy to use and cheap to produce. The polyacrylamide slab gel electrophoresis can be performed in less than 10 min with low voltage. The gel-to-gel repeatability is around 3.8%. The limit of detection is approx. 10 ng as determined by Coomassie staining of selected standard proteins, and corresponds to a 10-fold increase in sensitivity as compared with a common size PAGE analysis device (e.g. 10 x 7 cm). The device was successfully applied to peptide mass fingerprint analysis, protein sequencing and ultra-sensitive immunodetection, and the performance was compared to a commonly used regular PAGE device.