Lysozyme Conjugates Research Articles

Evaluation of the antibacterial activity of selenium nanoparticles and those conjugated with lysozyme against Bacillus cereus spiked in pasteurized skim milk

Selenium nanoparticles (SeNPs) and SeNPs conjugated with lysozyme (SeNPs-lysozyme) were synthesized via sodium selenite reduction with ascorbic acid and polysorbate-80 stabilization. These were physiochemically characterized to impart antibacterial properties. X-ray diffraction analysis (XRD) revealed that both formulations had hexagonal crystalline structures. Fourier transform infrared (FT-IR) confirmed the successful conjugation of lysozyme to SeNPs through the emergence of amine peaks at 1542 cm−1. Dynamic light scattering (DLS) showed lysozyme conjugation increased nanoparticle size from 76.6 to 92.4 nm and charge from − 12.6 to + 17.6 mV. Transmission electron microscopy (TEM) images confirmed that spherical morphologies and size increased from 73–79 nm for SeNPs to 84–98 nm for SeNPs-lysozyme after lysozyme capping. Both formulations exhibited identical minimum inhibitory concentrations (MIC) of 125 μg/mL. However, lysozyme conjugation reduced SeNPs cytotoxicity by 2.4-fold based on IC50 values from an MTT (Thiazolyl Blue Tetrazolium Bromide) assay. When tested in phosphate-buffered saline (PBS), SeNPs and SeNPs-lysozyme fully inhibited Bacillus cereus (B. cereus) proliferation up to 6 log10 CFU/mL inoculums over 24 h of incubation. However, at (7 log10 CFU/mL) inoculum, only partial inhibition occurred, with (2.5 ± 0.3 log10 CFU/mL) growth still detected for SeNPs and (2.2 ± 0.2 log10 CFU/mL) for SeNPs-lysozyme. At all inoculum levels (5, 6, and 7) log10 CFU/mL, B. cereus grew a lot in milk (more than 8 log10 CFU/mL). This was probably because SeNPs or SeNPs-lysozyme tend to aggregate around milk components, making them less effective.

Conjugation of Lysozyme and Epigallocatechin Gallate for Improving Antibacterial and Antioxidant Properties.

One of the main interests in the food industry is the preservation of food from spoilage by microorganisms or lipid oxidation. A novel alternative is the development of additives of natural origin with dual activity. In the present study, a chemically modified lysozyme (Lys) with epigallocatechin gallate (EGCG) was developed to obtain a conjugate (Lys-EGCG) with antibacterial/antioxidant activity to improve its properties and increase its application potential. The modification reaction was carried out using a free radical grafting method for the Lys modification reaction, using ascorbic acid and hydrogen peroxide as radical initiators in an aqueous medium. The synthesis of Lys-EGCG conjugate was confirmed by spectroscopic (FT-IR, 1H-RMN, and XPS) and calorimetry differential scanning (DSC) analyses. The EGCG binding to the Lys biomolecule was quantified by the Folin-Ciocalteu method; the antibacterial activity was evaluated by minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MCB) against Staphylococcus aureus and Pseudomonas fluorescens; the antioxidant activity was evaluated by ABTS, DPPH, and FRAP. The spectroscopic results showed that the Lys-EGCG conjugate was successfully obtained, and the DSC analysis revealed a 20°C increase (P < 0.05) in the denaturation temperature of Lys due to EGCG modification. The EGCG concentration in Lys-EGCG was 97.97 ± 4.7µmol of EGCG/g of sample. The antibacterial and antioxidant activity of the Lys-EGCG conjugate was higher (P < 0.05) than pure EGCG and Lys. The chemical modification of Lys with EGCG allows for the bioconjugate with a dual function (antibacterial/antioxidant), broadening the range of Lys and EGCG applications to different areas such as food, cosmetic, and pharmaceutical industries.

Effects of conjugation of egg lysozyme to theaflavins on their structure and function

Proteins modified by polyphenols have more advantages. In this study, we investigated the effect of covalent conjugation of egg white lysozyme (LYZ) with theaflavin (TF) on its structure and related functions. The LYZ-TF covalent conjugation was prepared by an alkaline method (pH 9.0) with a branching rate of about 80%. The results such as SDS-PAGE and UV spectroscopy proved the formation of the covalent conjugation. The results of fluorescence and infrared spectroscopy showed that the conformation of the LYZ-TF coupling was altered, with an increase in α-helix content by more than 10% and an increase in surface hydrophobicity compared to LYZ. The results of relevant functional characterization experiments showed the thermal stability and antioxidant properties of LYZ modified by TF, but its enzymatic activity and in vitro antimicrobial properties were inhibited. Notably, the amount of TF addition also affected the structure and function of the conjugates. These results provide some theoretical guidance for the development of novel food functional materials with enhanced properties.

Enzyme-Polymer Conjugates with Photocleavable Linkers for Control Over Protein Activity.

Reversible conjugation of polymers to proteins is important for a variety of applications, for example to control protein activity. Light is often employed as an external trigger to allow for spatio and temporal control over release of a payload. In this report, we demonstrate preparation of photocleavable poly(polyethylene glycol) acrylate)-lysozyme (pPEGA-Lys) conjugates via ortho-nitrobenzyl linkages. The conjugates were made by both grafting-to and grafting-from in order to compare and contrast the two synthetic approaches. First, a lysine-reactive ortho-nitrobenzyl atom transfer radical polymerization (ATRP) initiator was synthesized. For the grafting-to strategy, the initiator was employed in the ATRP of PEGA, and the subsequent polymer was conjugated to the lysine residues of lysozyme. For the grafting-from strategy, lysozyme was modified first with the photocleavable initiator, and the purified macroinitiator was then subjected to polymerization conditions to synthesize the protein-polymer conjugate. The polymer was cleaved from the protein via UV light, and activity before and after polymer removal was evaluated, showing 83% recovery. This work provides evidence that reversing conjugation is successful for activity modulation for ortho-nitrobenzyl linked protein-polymer conjugates.

Impedance nanobiosensor based on enzyme-conjugated biosynthesized gold nanoparticles for the detection of Gram-positive bacteria.

In this report, gold nanoparticles (GNPS) were synthesized using cell-free extracts of seven different isolates, namely, Pseudomonas aerogenosa CEBP2, Pseudomonas sp. CEBP1, Pseudomonas pseudoalcaligenes CEB1G, Acinetobactor baumani CEBS1, Cuprividus sp. CEB3, Micrococcus luteus CUB12, and Pandoraea sp. CUB2S. The spectroscopic (UV-vis, FTIR, DLS, XRD, EDS) and microscopic (FESEM, TEM) results confirm the reduction of Au3+ to Au0 in the presence of biomolecules having reducing as well as self-stabilizing activity. In this green synthesis approach, the average particle size of biosynthesized GNPS might vary (4-60 nm) depending on the bacterial species, pH of the media, incubation time, and temperature. In this study, GSH-modified BSGNPs (Au-GSH) have shown antimicrobial activity with better stability against Gram-positive bacteria. After conjugation of lysozyme with Au-GSH (lyso@Au-GSH), the zone of inhibition was enhanced from 12 to 23 mm (Au-GSH). The TEM study shows the spherical GNP (16.65 ± 2.84) turns into a flower-shaped GNP (22.22 ± 3.12) after conjugation with lysozyme due to the formation of the protein corona. Furthermore, the nanobioconjugate (lyso@Au-GSH) was immobilized with Nafion on a glassy carbon electrode to fabricate a label-free impedance biosensor that is highly sensitive to monitor changes in the transducer surface due to biomolecular interactions. The uniquely designed biosensor could selectively detect Gram-positive bacteria in the linear range of 3.0 × 101-3 × 1010 cfu mL-1 with RE <5%. The proposed simplest biosensor exhibited good reproducibility (RSD = 3.1%) and excellent correlation (R2 = 0.999) with the standard plate count method, making it suitable for monitoring Gram-positive bacterial contamination in biofluids, food, and environmental samples.

Synthesis and Self-Assembly of Hyperbranched Multiarm Copolymer Lysozyme Conjugates Based on Light-Induced Metal-Free Atrp

In recent years, the coupling of structurally and functionally controllable polymers with biologically active protein materials to obtain polymer-protein conjugates with excellent overall properties and good biocompatibility has been important research in the field of polymers. In this study, the hyperbranched polymer hP(DEGMA-co-OEGMA) was first prepared by combining self-condensation vinyl polymerization (SCVP) with photo-induced metal-free atom transfer radical polymerization (ATRP), with 2-(2-bromo-2-methylpropanoyloxy) ethyl methacrylate (BMA) as inimer, and Di (ethylene glycol) methyl ether methacrylate (DEGMA) and (oligoethylene glycol) methacrylate (OEGMA, Mn = 300) as the copolymer monomer. Then, hP(DEGMA-co-OEGMA) was used as a macroinitiator to continue the polymerization of a segment of pyridyl disulfide ethyl methacrylate (DSMA) monomer to obtain the hyperbranched multiarm copolymers hP(DEGMA-co-OEGMA)-star-PDSMA. Finally, the lysozyme with sulfhydryl groups was affixed to the hyperbranched multiarm copolymers by the exchange reaction between sulfhydryl groups and disulfide bonds to obtain the copolymer protein conjugates hP(DEGMA-co-OEGMA)-star-PLZ. Three hyperbranched multiarm copolymers with relatively close molecular weights but different degrees of branching were prepared, and all three conjugates could self-assemble to form nanoscale vesicle assemblies with narrow dispersion. The biological activity and secondary structure of lysozyme on the assemblies remained essentially unchanged.

Adsorption of conjugates of lysozyme and fluorescein isothiocyanate in hydrophobic interaction chromatography

Bioconjugates, such as antibody-drug conjugates or fluorescent-labeled proteins, are highly interesting for various applications in medicine and biology. In their production, not only the synthesis is challenging but also the downstream processing, for which hydrophobic interaction chromatography (HIC) is often used. However, in-depth studies of the adsorption of bioconjugates in HIC are still rare. Therefore, in the present work, three different conjugates of lysozyme and fluorescein isothiocyanate (FITC) were synthesized and isolated, and their adsorption on the hydrophobic resin Toyopearl PPG-600 M was systematically studied in batch experiments. The influence of sodium chloride and ammonium sulfate with ionic strengths up to 2000 mM on the adsorption isotherms was investigated at pH 7.0 and 25 °C, and the results were compared to those for pure lysozyme. The conjugation leads to an increase of the adsorption in all studied cases. All studied conjugates contain only a single FITC and differ only in the position of the conjugation on the lysozyme. Despite this, strong differences in the adsorption behavior were observed. Moreover, a mathematical model was developed, which enables the prediction of the adsorption isotherms in the studied systems for varying ionic strengths.

N-Terminal Lysozyme Conjugation to a Cationic Polymer Enhances Antimicrobial Activity and Overcomes Antimicrobial Resistance.

Microbial resistance to antibiotics is one of the greatest global healthcare challenges. There is an urgent need to develop effective strategies to overcome antimicrobial resistance. We, herein, report photoinduced in situ growth of a cationic polymer from the N-terminus of lysozyme. The attachment of the cationic polymer improves the proteolytic and thermal stability of lysozyme. Notably, the conjugate can efficiently overcome lysozyme resistance in Gram-positive bacteria and antibiotics-resistance in Gram-negative bacteria, which may be ascribed to the synergistic interactions of lysozyme and the cationic polymer with the bacteria to disrupt their cell membranes. In a rat periodontitis model, the lysozyme-polymer conjugate not only greatly outperforms lysozyme in therapeutic efficacy but also is superior to minocycline hydrochloride, which is the gold standard for periodontitis therapy. These findings may provide an efficient strategy to dramatically enhance the antimicrobial activities of lysozyme and pave a way to overcome antimicrobial resistance.

Impact of Polymer Bioconjugation on Protein Stability and Activity Investigated with Discrete Conjugates: Alternatives to PEGylation.

Covalent attachment of synthetic polymers to proteins, known as protein-polymer conjugation, is currently one of the main approaches for improving the physicochemical properties of these biomolecules. The most commonly employed polymer is polyethylene glycol (PEG), as evidenced by extensive research and clinical track records for its use in biopharmaceuticals. However, the occurrence of allergic reactions or hypersensitivity and the discovery of PEG antibodies, on the one hand, and the rise of controlled polymerization techniques and novel monomers, on the other hand, have been driving the search for alternative polymers for bioconjugation. The present study describes the synthesis, purification, and properties of conjugates of lysozyme with poly( N-acryloylmorpholine) (PNAM) and poly(oligoethylene glycol methyl ether methacrylate) (POEGMA). Particularly, conjugate species with distinct conjugation degrees are investigated for their residual activity, aggregation behavior, and solubility, by using a high-throughput screening approach. Our study showcases the importance of evaluating conjugates obtained by nonsite-specific modification through isolated species with discrete degrees of conjugation rather than on the batch level. Monovalent conjugates with relatively low molar mass polymers displayed equal or even higher activity than the native protein, while all conjugates showed an improved protein solubility. To achieve a comparable effect on solubility as with PEG, PNAM and POEGMA of higher molar masses were required.

Conjugates of α-lactalbumin, β-lactoglobulin, and lysozyme with polysaccharides: Characterization and techno-functional properties

Conjugates of protein (α-lactalbumin, β-lactoglobulin, and lysozyme) with polysaccharides (guar, locust, pectin, and carboxymethilcellulose) were prepared via Maillard reaction by the dry-heating method. The conjugates were characterizated by using the browning index, extent of reaction, grafting degree, sodium dodecyl sulfate – polyacrilamide gel electrophoresis, fluorescence, and circular dichroism. The emulsifying properties and foaming ability of the formed conjugates were also evaluated. Conjugates with pectin and Lz-CMC system showed an increase in the browning index with the increase of the heating time. Circular dichroism and fluorescence data pointed out to conformational changes of proteins during glycation. The lysozyme (lz) conjugates presented the highest degree of glycation (89.1%). The α-Lactalbumin (α-la) - polysaccharides (PS) conjugates showed foam stability higher than the mixture (α-la + PS), the pure α-la, and the conjugates of β-lactoglobulin (β-lg) and lysozyme (lz) for all studied time (30, 60, and 120 min). The α-la-carboxymethylcellulose (CMC) conjugate presented the highest value of foaming stability (85.71). The pure β-lg shows greater foam stability and volume than β-lg-PS conjugates and mixture (β-lg + PS). The lz conjugates do not exihibit foam stability, except for the lz-CMC conjugate that showed stability up to 60 min. Furthermore, emulsion stability of the systems was affected by sonication time. Conjugates of α-la have greatly potencial applications as novel foaming agents in food industry.

Effect of guar gum conjugation on functional, antioxidant and antimicrobial activity of egg white lysozyme

This study was undertaken to analyze the effect of conjugation of egg-white lysozyme with guar gum. Lysozyme is an antimicrobial polypeptide that can be used for food preservation. Its antibacterial activity is limited to gram positive bacteria. Conjugation with polysaccharides like guar gum may broaden its activity against gram negatives. Conjugate was developed through Maillard reaction. Assays carried out included sugar estimation, SDS-PAGE, GPC, color, FT-IR, DSC, thermal stability, solubility, emulsifying, foaming and antioxidant activity. In addition, antimicrobial activity of the conjugate was determined against two gram positive (Staphyllococcus aureus and Enterococcus) and two gram negative pathogens (E. coli and Salmonella). Results showed higher functional properties of lysozyme-guar gum conjugate. The antioxidant properties increased from 2.02–35.80% (Inhibition of DPPH) and 1.65–4.93AAE/g (reducing power) upon guar gum conjugation. Conjugate significantly inhibited gram negative bacteria and the antibacterial activity also increased significantly against gram positive pathogens.

Recovery of PEGylated and native lysozyme using an in situ aqueous two‐phase system directly from the PEGylation reaction

AbstractBACKGROUNDPurification of PEGylated proteins from reactions is still a challenge due to the formation of isomers. In this work, the recovery of PEGylated lysozyme was studied in a novel integrative approach called in situ aqueous two‐phase systems (ATPS). The excess of PEG in the lysozyme PEGylation reaction (LPR) was used as part of the phase forming chemicals with different polymer (UCON, ficoll, dextran) and salt (sodium phosphate, potassium sulphate, sodium sulphate, ammonium sulphate, sodium carbonate) solutions.RESULTSThe best option for the in situ ATPS formation was the addition of a 4 mol L−1 ammonium sulphate in 20 mmol L−1 Tris–HCl (pH 7.0) solution to the PEGylation reaction. PEGylated conjugates of lysozyme exhibited a preferential partition to the top (PEG‐rich) phase while native protein was partitioned to the bottom (salt‐rich) phase. Di‐PEGylated lysozyme partitioned entirely to the top phase (recovery of 100%), while the mono‐PEGylated protein presented a recovery yield of 56% in the top phase. On its part, 97.7% of the native lysozyme was concentrated in the bottom phase.CONCLUSIONThe proof‐of‐concept presented in this work allows the recovery of PEGylated proteins directly from a PEGylation reaction. It is the first time that PEGylated proteins have been fractionated using a PEG–ammonium sulphate system taking advantage of the unreacted PEG left in a PEGylation reaction. Based on reaction engineering, in situ ATPS are a potential mechanism for PEGylation control, a good alternative for ‘packed‐bed’ or on‐column PEGylation processes and a pioneer strategy in the development of phase transfer catalysis in PEGylation. © 2017 Society of Chemical Industry

Comparative studies on interactions ofl-ascorbic acid, α-tocopherol, procyanidin B3, β-carotene, and astaxanthin with lysozyme using fluorescence spectroscopy and molecular modeling methods

l-Ascorbic acid, α-tocopherol, procyanidin B3, β-carotene, and astaxanthin are five classic dietary antioxidants. In this study, the interaction between them and lysozyme was investigated by fluorescence spectroscopy and molecular modeling methods. The quenching mechanisms of lysozyme by them are all static quenching at lower concentrations of antioxidants, but at higher concentrations, predominantly by the “sphere of action” mechanisms. The binding constants of lysozyme-antioxidants systems are in the following order as: astaxanthin>β-carotene > procyanidin B3 > l-ascorbic acid>α-tocopherol. Thermodynamic analysis reveals that the binding process of α-tocopherol to lysozyme is synergistically driven by enthalpy and entropy. For the other four antioxidants-lysozyme systems, the binding processes are all entropy process. Synchronous fluorescence spectroscopy shows that the five antioxidants may induce microenvironmental changes of lysozyme. Molecular docking results reveal that the five antioxidants bind into the enzyme active site and lysozyme activity is inhibited, in accordance with the results of lysozyme activity experiment. Practical applications Antioxidants are used worldwide as food additives to protect foodstuffs against deterioration caused by oxidation, such as fat rancidity and color changes. Dietary antioxidant is considered to be a safe natural product. However, it may act as an antinutritional factor, in terms of the inhibition of proteases, when ingested in excess. Lysozyme with high natural abundance is an enzyme known for its unique ability to damage bacterial cell walls, thereby providing protection against bacterial infections. When the diverse endogenous and exogenous ligands enter into the human body, ligand–lysozyme conjugation can be observed. Therefore, lysozyme is selected to investigate the binding characteristics of five classic dietary antioxidants, which is critical in order to understand their possible delivery, consequent availability, and relevant health risks.

Conjugation of Estrone Glucuronide with Human Lysozyme

Human milk lysozyme was conjugated with estrone glucuronide to give a monoacylated conjugate, two disubstituted isoforms, and one trisubstituted isoform in 99.4% yield. The conjugates were pure and highly inhibited (>98%) by the anti-estrone glucuronide antibody. The clearing curves were biphasic for all four conjugates but a 3 min initial rate assay was established and used to measure a normal menstrual cycle profile of estrone glucuronide excretion rates. The marked differences between the hen egg white and human milk lysozyme conjugates show that near identical tertiary structures do not necessarily imply similar physical, chemical, biochemical, and kinetic behavior.

Regenerated Cellulose Fiber and Film Immobilized with Lysozyme

The present work reports an initial engineering approach for fabricating lysozyme-bound regenerated cellulose fiber and film. Glycine-esterified cotton was dissolved in an ionic liquid solvent 1–Butyl–3–methylimidazolium Chloride (BMIMCl) in which lysozyme was activated and covalently attached to cotton cellulose through an enzymatic conjugation between its carboxyl groups and glycine cellulose’s amino groups. The resulting solution was extruded for fiber/film formation in a water bath. After performing a bicinchoninic acid (BCA) protein assay, quantity of attached lysozyme to cellulose fiber/film was evaluated. The study exhibited that a synthesis of lysozyme conjugation on cellulose in BMIMCl could be completed in a control manor, resulting in a cellulose solution suitable for fiber/film production. It was also found that lysozyme could be successfully immobilized onto the cellulose fiber and film regenerated from solution spinning with a reasonable amount ranging from 197.6 to 343.7 μg/mL.mg.

Reprint of "Nanobody — Shell functionalized thermosensitive core-crosslinked polymeric micelles for active drug targeting"

The aim of this study was to develop poly(ethylene glycol)-b-poly[N-(2-hydroxypropyl) methacrylamide-lactate] (mPEG-b-p(HPMAm-Lacn)) core-crosslinked thermosensitive biodegradable polymeric micelles suitable for active tumor targeting, by coupling the anti-EGFR (epidermal growth factor receptor) EGa1 nanobody to their surface. To this end, PEG was functionalized with N-succinimidyl 3-(2-pyridyldithio)-propionate (SPDP) to yield a PDP–PEG-b-p(HPMAm-Lacn) block copolymer. Micelles composed of 80% mPEG-b-p(HPMAm-Lacn) and 20% PDP–PEG-b-p(HPMAm-Lacn) were prepared and lysozyme (as a model protein) was modified with N-succinimidyl-S-acetylthioacetate, deprotected with hydroxylamine hydrochloride and subsequently coupled to the micellar surface. The micellar conjugates were characterized using SDS-PAGE and gel permeation chromatography (GPC). Using the knowledge obtained with lysozyme conjugation, the EGa1 nanobody was coupled to mPEG/PDP–PEG micelles and the conjugation was successful as demonstrated by western blot and dot blot analysis. Rhodamine labeled EGa1-micelles showed substantially higher binding as well as uptake by EGFR over-expressing cancer cells (A431 and UM-SCC-14C) than untargeted rhodamine labeled micelles. Interestingly, no binding of the nanobody micelles was observed to EGFR negative cells (3T3) as well as to14C cells in the presence of an excess of free nanobody. This demonstrates that the binding of the nanobody micelles is indeed by interaction with the EGF receptor. In conclusion, EGa1 decorated (mPEG/PDP–PEG)-b-(pHPMAm-Lacn) polymeric micelles are highly promising systems for active drug targeting.

Pluronic–lysozyme conjugates as anti-adhesive and antibacterial bifunctional polymers for surface coating

This paper describes the preparation and characterization of polymer–protein conjugates composed of a synthetic triblock copolymer with a central polypropylene oxide (PPO) block and two terminal polyethylene oxide (PEO) segments, Pluronic F-127, and the antibacterial enzyme lysozyme attached to the telechelic groups of the PEO chains. Covalent conjugation of lysozyme proceeded via reductive amination of aldehyde functionalized PEO blocks (CHO-Pluronic) and the amine groups of the lysine residues in the protein. SDS-PAGE gel electrophoresis together with MALDI-TOF mass spectrometry analysis revealed formation of conjugates of one or two lysozyme molecules per Pluronic polymer chain. The conjugated lysozyme showed antibacterial activity towards Bacillus subtilis. Analysis with a quartz crystal microbalance with dissipation revealed that Pluronic–lysozyme conjugates adsorb in a brush conformation on a hydrophobic gold-coated quartz surface. X-ray photoelectron spectroscopy indicated surface coverage of 32% by lysozyme when adsorbed from a mixture of unconjugated Pluronic and Pluronic–lysozyme conjugate (ratio 99:1) and of 47% after adsorption of 100% Pluronic–lysozyme conjugates. Thus, bifunctional brushes were created, possessing both anti-adhesive activity due to the polymer brush, combined with the antibacterial activity of lysozyme. The coating having a lower degree of lysozyme coverage proved to be more bactericidal.

Nanobody — Shell functionalized thermosensitive core-crosslinked polymeric micelles for active drug targeting

The aim of this study was to develop poly(ethylene glycol) -b-poly[ N-(2-hydroxypropyl) methacrylamide-lactate] (mPEG- b-p(HPMAm-Lac n)) core-crosslinked thermosensitive biodegradable polymeric micelles suitable for active tumor targeting, by coupling the anti-EGFR (epidermal growth factor receptor) EGa1 nanobody to their surface. To this end, PEG was functionalized with N-succinimidyl 3-(2-pyridyldithio)-propionate (SPDP) to yield a PDP–PEG- b-p(HPMAm-Lac n) block copolymer. Micelles composed of 80% mPEG -b-p(HPMAm-Lac n) and 20% PDP–PEG- b-p(HPMAm-Lac n) were prepared and lysozyme (as a model protein) was modified with N-succinimidyl-S-acetylthioacetate, deprotected with hydroxylamine hydrochloride and subsequently coupled to the micellar surface. The micellar conjugates were characterized using SDS-PAGE and gel permeation chromatography (GPC). Using the knowledge obtained with lysozyme conjugation, the EGa1 nanobody was coupled to mPEG/PDP–PEG micelles and the conjugation was successful as demonstrated by western blot and dot blot analysis. Rhodamine labeled EGa1-micelles showed substantially higher binding as well as uptake by EGFR over-expressing cancer cells (A431 and UM-SCC-14C) than untargeted rhodamine labeled micelles. Interestingly, no binding of the nanobody micelles was observed to EGFR negative cells (3T3) as well as to14C cells in the presence of an excess of free nanobody. This demonstrates that the binding of the nanobody micelles is indeed by interaction with the EGF receptor. In conclusion, EGa1 decorated (mPEG/PDP–PEG) -b-(pHPMAm-Lac n) polymeric micelles are highly promising systems for active drug targeting.

Synthesis and bioactivity of poly(HPMA)–lysozyme conjugates: the use of novel thiazolidine-2-thione coupling chemistry

A novel thiazolidine-2-thione functionalized chain transfer agent (CTA) was synthesized and used as a reversible addition-fragmentation chain transfer (RAFT) polymerization agent to prepare well-defined poly-N-(2-hydroxypropyl) methacrylamide (PHPMA). The polymer chains had pre-designed molecular weights, narrow polydispersities and were chain-end functionalized. On incubation with protein (lysozyme) under different pH conditions, PHPMA was conjugated to the protein surface via covalent amide bonding. The bioactivity of the lysozyme-PHPMA conjugates was assessed using Micrococcus lysodeikticus (Ml) cells as substrates. The number of polymer chains attached to the protein could be controlled by both the pH of the conjugation reaction and the molecular weights of the polymers, thereby influencing significantly the bioactivity of the protein-polymer conjugates.

Complex Coacervate Core Micelles with a Lysozyme-Modified Corona

This paper describes the preparation, characterization, and enzymatic activity of complex coacervate core micelles (C3Ms) composed of poly(acrylic acid) (PAA) and poly(N-methyl-2-vinyl pyridinium iodide)-b-poly(ethylene oxide) (PQ2VP-PEO) to which the antibacterial enzyme lysozyme is end-attached. C3Ms were prepared by polyelectrolyte complex formation between PAA and mixtures containing different ratios of aldehyde and hydroxyl end-functionalized PQ2VP-PEO. This resulted in the formation of C3Ms containing 0-40% (w/w) of the aldehyde end-functionalized PQ2VP-PEO block copolymer (PQ2VP-PEO-CHO). Chemical conjugation of lysozyme was achieved via reductive amination of the aldehyde groups, which are exposed at the surface of the C3M, with the amine groups present in the side chains of the lysine residues of the protein. Dynamic and static light scattering indicated that the conjugation of lysozyme to C3Ms prepared using 10 and 20% (w/w) PQ2VP-PEO-CHO resulted in the formation of unimicellar particles. Multimicellar aggregates, in contrast, were obtained when lysozyme was conjugated to C3Ms prepared using 30 or 40% (w/w) PQ2VP-PEO-CHO. The enzymatic activity of the unimicellar lysozyme-C3M conjugates toward the hydrolysis of the bacterial substrate Micrococcus lysodeikticus was comparable to that of free lysozyme. For the multimicellar particles, in contrast, significantly reduced enzymatic rates of hydrolysis, altered circular dichroism, and red-shifted tryptophan fluorescence spectra were measured. These results are attributed to the occlusion of lysozyme in the interior of the multimicellar conjugates.