Poly(amidoamine)-BSA conjugates synthesised by Michael addition reaction retained enzymatic activity

Polymer-protein conjugates are key to overcome some of the therapeutic protein limitations, including inefficient intracellular delivery. Poly(amidoamine)s are bioresponsive polyelectrolytes, which can form complexes with proteins and promote their delivery into the cytosol of cells. To investigate if conjugation would affect the activity of the protein, two poly(amidoamine)-BSA conjugates were synthesised using a “grafted to” method and Michael addition reaction. Following purification, the conjugates were characterised by electrophoresis, size exclusion chromatography (Mn(C1) = 140.7 kDa ; Mn(C2) = 218.6 kDa) and light scattering (Dh(C1) = 37.5 nm ; Dh(C2) = 75.1 nm). As a result of the conjugation with the cationic polymer, the conjugates had a positive zeta potential (?(C1) = +15.4 mV; ?(C2) = +20.2 mV). TNBS assays demonstrated that 16% to 25% of the protein amine groups were modified and HPLC analysis indicated that the amount of protein in the conjugate was 0.76 mg of BSA/mg of PAA (C1) and 0.43 mg of BSA /mg of PAA (C2). Enzymatic assays indicated the conjugates displayed an esterase activity similar (C1) or reduced ~ 35% (C2) compare to BSA. Altogether the results demonstrated that the conjugation of poly(amidoamine)s to a model protein can lead to the formation of bioconjugates that retain the enzymatic activity of the native protein. Such conjugates could have some application in protein delivery and enzyme engineering for biocatalysis and biosensors

Synthesis and applications of protein/peptide-polymer conjugates

Recent advances in synthetic methodologies have brought us closer than ever to the precision conferred by nature. For example, the control possible in reversible deactivation radical polymerization enables us to design and synthesize macromolecules with unprecedented control over not only the polymer chain ends, but also the side chain functionality. Furthermore, this functionality can be exploited to afford chemical modification of peptides and proteins, with ever-improving site-specificity, yielding a range of well-defined protein/peptide-hybrid materials. Such materials benefit from the amalgamation of the properties of proteins/peptides with those of the synthetic (macro)molecules in question. Here, the latest developments in the synthesis of functional polymers and their use for preparation of well-defined protein/peptide-polymer conjugates will be discussed, with particular attention focused on modulating the stability, efficacy and/or administration of therapeutic peptides

A new procedure for the synthesis of polyethylene glycol-protein adducts; Effects on function, receptor recognition, and clearance of superoxide dismutase, lactoferrin, and [alpha]2-macroglobulin

A new, simplified technique for the synthesis of polyethylene glycol (PEG) derivatives of proteins utilizing 1,1'-carbonyldiimidazole for PEG activation, is described. PEG derivatives of superoxide dismutase, [alpha]2-macroglobulin, [alpha]2-macroglobulin-trypsin, and lactoferrin were prepared and studied. Superoxide dismutase coupled to PEG preserved 95% of its original activity while its plasma half-life increased from 3.5 min to 9 or more hours depending on the PEG derivative studied. PEG-derivatized [alpha]2-macroglobulin showed decreased protease binding activity but PEG derivatives of preformed [alpha]2-macroglobulin-trypsin demonstrated no loss of activity. The plasma clearance of PEG-[alpha]2-macroglobulin-trypsin was prolonged significantly compared to native [alpha]2-macroglobulin-trypsin, particularly when a high-molecular-weight PEG was coupled to the protease inhibitor complex. The plasma clearance half-life of lactoferrin was increased 5-to 20-fold by this modification. Trinitrobenzenesulfonic acid titration studies demonstrated that [epsilon]-amino groups of lysine residues are modified by the coupling of carbonyldiimidazole-activated PEG to proteins.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/25222/1/0000663.pd

Restricting detergent protease action to surface of protein fibres by chemical modification

Due to their excellent properties, such as thermostability, activity over a broad range of pH and efficient stain removal, proteases from Bacillus sp. are commonly used in the textile industry including industrial processes and laundry and represent one of the most important groups of enzymes. However, due to the action of proteases, severe damage on natural protein fibres such as silk and wool result after washing with detergents containing proteases. To include the benefits of proteases in a wool fibre friendly detergent formulation, the soluble polymer polyethylene glycol (PEG) was covalently attached to a protease from Bacillus licheniformis. In contrast to activation of PEG with cyanuric chloride (50%) activation with 1,1′-carbonyldiimidazole (CDI) lead to activity recovery above 90%. With these modified enzymes, hydrolytic attack on wool fibres could be successfully prevented up to 95% compared to the native enzymes. Colour difference (ΔE) measured in the three dimensional colour space showed good stain removal properties for the modified enzymes. Furthermore, half-life of the modified enzymes in buffers and commercial detergents solutions was nearly twice as high as those of the non-modified enzymes with values of up to 63 min. Out of the different modified proteases especially the B. licheniformis protease with the 2.0-kDa polymer attached both retained stain removal properties and did not hydrolyse/damage wool fibres

PEG−peptide conjugates

The remarkable diversity of the self-assembly behavior of PEG−peptides is reviewed, including self-assemblies formed by PEG−peptides with β-sheet and α-helical (coiled-coil) peptide sequences. The modes of self-assembly in solution and in the solid state are discussed. Additionally, applications in bionanotechnology and synthetic materials science are summarized

Biodistribution, clearance, and long‐term fate of clinically relevant nanomaterials

Realization of the immense potential of nanomaterials for biomedical applications will require a thorough understanding of how they interact with cells, tissues, and organs. There is evidence that, depending on their physicochemical properties and subsequent interactions, nanomaterials are indeed taken up by cells. However, the subsequent release and/or intracellular degradation of the materials, transfer to other cells, and/or translocation across tissue barriers are still poorly understood. The involvement of these cellular clearance mechanisms strongly influences the long-term fate of used nanomaterials, especially if one also considers repeated exposure. Several nanomaterials, such as liposomes and iron oxide, gold, or silica nanoparticles, are already approved by the American Food and Drug Administration for clinical trials; however, there is still a huge gap of knowledge concerning their fate in the body. Herein, clinically relevant nanomaterials, their possible modes of exposure, as well as the biological barriers they must overcome to be effective are reviewed. Furthermore, the biodistribution and kinetics of nanomaterials and their modes of clearance are discussed, knowledge of the long-term fates of a selection of nanomaterials is summarized, and the critical points that must be considered for future research are addressed

Biocatalysis by transglutaminases: a review of biotechnological applications

The biocatalytic activity of transglutaminases (TGs) leads to the synthesis of new covalent isopeptide bonds (crosslinks) between peptide-bound glutamine and lysine residues, but also the transamidation of primary amines to glutamine residues, which ultimately can result into protein polymerisation. Operating with a cysteine/histidine/aspartic acid (Cys/His/Asp) catalytic triad, TGs induce the post-translational modification of proteins at both physiological and pathological conditions (e.g., accumulation of matrices in tissue fibrosis). Because of the disparate biotechnological applications, this large family of protein-remodelling enzymes have stimulated an escalation of interest. In the past 50 years, both mammalian and microbial TGs polymerising activity has been exploited in the food industry for the improvement of aliments’ quality, texture, and nutritive value, other than to enhance the food appearance and increased marketability. At the same time, the ability of TGs to crosslink extracellular matrix proteins, like collagen, as well as synthetic biopolymers, has led to multiple applications in biomedicine, such as the production of biocompatible scaffolds and hydrogels for tissue engineering and drug delivery, or DNA-protein bio-conjugation and antibody functionalisation. Here, we summarise the most recent advances in the field, focusing on the utilisation of TGs-mediated protein multimerisation in biotechnological and bioengineering applications

Drying and deposition of poly(ethylene oxide) droplets determined by P\'eclet number

We report results of a detailed experimental investigation into the drying of sessile droplets of aqueous poly(ethylene oxide) (PEO) polymer solutions under various experimental conditions. Samples are prepared with a range of initial concentrations c_0 and are filtered to remove traces of undissolved PEO clusters. In typical experiments, droplets with initial volumes between 5\muL and 50\muL are left to evaporate while temperature and relative humidity are monitored. Droplets either form a disk-like solid "puddle" or a tall conical "pillar". The droplet mass is monitored using a microbalance and the droplet profile is recorded regularly using a digital camera. Subsequent processing of the data allows values of droplet volume V, surface area A, base radius R, contact angle {\theta} and height h to be determined throughout drying. From this data we identify four stages during pillar formation: pinned drying; pseudo-dewetting; bootstrap building; solid contraction and propose physical models to explain key aspects of each stage and to predict the transition from each stage to the next. The experimental parameters of relative humidity, temperature, pressure, droplet volume and initial contact angle are all systematically varied and observed to influence the drying process and consequently whether the droplet forms a pillar or a puddle. We combine these parameters into a dimensionless P\'eclet number Pe, which compares the relative effects of evaporation and diffusion, and show that the drying behaviour is only dependent on c_0 and Pe.Comment: 21 pages, 10 figure

PEG-Albumin Plasma Expansion Increases Expression of MCP-1 Evidencing Increased Circulatory Wall Shear Stress: An Experimental Study

Treatment of blood loss with plasma expanders lowers blood viscosity, increasing cardiac output. However, increased flow velocity by conventional plasma expanders does not compensate for decreased viscosity in maintaining vessel wall shear stress (WSS), decreasing endothelial nitric oxide (NO) production. A new type of plasma expander using polyethylene glycol conjugate albumin (PEG-Alb) causes supra-perfusion when used in extreme hemodilution and is effective in treating hemorrhagic shock, although it is minimally viscogenic. An acute 40% hemodilution/exchange-transfusion protocol was used to compare 4% PEG-Alb to Ringer’s lactate, Dextran 70 kDa and 6% Hetastarch (670 kDa) in unanesthetized CD-1 mice. Serum cytokine analysis showed that PEG-Alb elevates monocyte chemotactic protein-1 (MCP-1), a member of a small inducible gene family, as well as expression of MIP-1α, and MIP-2. MCP-1 is specific to increased WSS. Given the direct link between increased WSS and production of NO, the beneficial resuscitation effects due to PEG-Alb plasma expansion appear to be due to increased WSS through increased perfusion and blood flow rather than blood viscosity