Covalent cell surface recruitment of chemotherapeutic polymers enhances selectivity and activity

Synthetic macromolecular chemotherapeutics inspired by host defence peptides can disrupt cell membranes and are emerging as agents for the treatment of cancer and infections. However, their off-target effects remain a major unmet challenge. Here we introduce a covalent recruitment strategy, whereby metabolic oligosaccharide engineering is used to label targeted cells with azido glycans, to subsequently capture chemotherapeutic polymers by a bio-orthogonal click reaction. This results in up to 10-fold reduction in EC₅₀ and widening of the therapeutic window. Cell death is induced by not only membrane leakage, but also by apoptosis due to the conjugated chemotherapeutic being internalised by glycan recycling. Covalent recruitment also lead to increased penetration and significant cell death in a 3-D tumour model in just 3 hours, whereas doxorubicin required 24 hours. This conceptual approach of ‘engineering cells to capture polymers’ rather than ‘engineering polymers to target cells’ will bring new opportunities in non-traditional macromolecular therapeutics.

Chemotherapeutic polymers are targeted to cells by introduction of unnatural glycans to their glycocalyx, enhancing their cytotoxic effect.     

Covalent immobilization of carbohydrates on sol-gel-coated microplates

Carbohydrate microarrays have attracted increasing attention in recent years because of their ability to monitor biologically important protein-carbohydrate interactions in a high-throughput manner. Here we have developed an effective approach to immobilizing intact carbohydrates directly on polystyrene microtiter plates coated with amine-functionalized sol-gel monolayers. Lectin binding was monitored by fluorescence spectroscopy using these covalent arrays of carbohydrates that contained six mono- and di-saccharides on the microplates. In addition, binding affinities of lectin to carbohydrates were also quantitatively analyzed by determining IC(50) values of lectin-specific antibody with these arrays. Our results indicate that microplate-based carbohydrate arrays can be efficiently fabricated by covalent immobilization of intact carbohydrates on sol-gel-coated microplates. The microplate-based carbohydrate arrays can be applied for screening of protein-carbohydrate interactions in a high-throughput manner.     

Covalent and selective immobilization of fusion proteins

A general method for the covalent immobilization of fusion proteins is presented. The approach is based on the unusual mechanism of the human O6-alkylguanine-DNA alkyltransferase, which irreversibly transfers the alkyl group from its substrate, alkylated or benzylated guanine, to a reactive cysteine residue. By attaching the benzyl group to a surface, hAGT fusion proteins immobilize themselves in a specific and covalent manner. The specificity of the reaction of hAGT with its substrate even allows the specific immobilization of hAGT fusion proteins directly out of cell extracts, making the approach an attractive alternative to currently used immobilization procedures.     

Covalent immobilization of heparin on a collagen film

Four procedures for the covalent immobilization of heparin (Hp) on a collagen film (CF) have been investigated. In three of them (methods (I–A, B, C), the CF was first treated with epichlorohydrin and ammonia and the Hp was added with the aid of CMBC (method I–A), by reductive amination in the presence of NaCH CN (method I–B), and with the aid of CMEC after succinylation (method I–C). In the fourth procedure (method II), the CF was activated by treatment with alkali and the Hp was added with the aid of CMEC. It was shown that the maximum amount of Hp was immobilized by method II.     

Covalent immobilization of heparin on a collagen film

Four procedures for the covalent immobilization of heparin (Hp) on a collagen film (CF) have been investigated. In three of them (methods (I–A, B, C), the CF was first treated with epichlorohydrin and ammonia and the Hp was added with the aid of CMBC (method I–A), by reductive amination in the presence of NaCH CN (method I–B), and with the aid of CMEC after succinylation (method I–C). In the fourth procedure (method II), the CF was activated by treatment with alkali and the Hp was added with the aid of CMEC. It was shown that the maximum amount of Hp was immobilized by method II.M. V. Lomonosov Moscow State University. Translated from Khimiya Prirodnykh Soedinenii, No. 5, pp. 700–704, September–October, 1987.     

Covalent VEGF protein immobilization on resorbable polymeric surfaces

Vascular endothelial growth factor type protein (VEGF), a potent angiogenic effector molecule, was successfully covalently immobilized onto the surfaces of the resorbable polymers poly(L‐lactic acid) (PLLA) and poly(ε‐caprolactone) (PCL) through a three‐step strategy. The surfaces were first covalently grafted with poly(acrylic acid) using non‐destructive and solvent free vapor‐phase grafting. A diamine spacer was coupled to the carboxylic acid pendant groups on the graft chains using EDC/NHS chemistry and VEGF was finally covalently attached to the amine linkers. The chemistry and topography of the modified substrates were quantitatively and qualitatively verified with XPS, ATR‐FTIR, UV–VIS, SEM, and ELISA. Copyright © 2010 John Wiley & Sons, Ltd.     

Covalent immobilization of liposomes on plasma functionalized metallic surfaces

A method was developed to functionalize biomedical metals with liposomes. The novelty of the method includes the plasma-functionalization of the metal surface with proper chemical groups to be used as anchor sites for the covalent immobilization of the liposomes. Stainless steel (SS-316) disks were processed in radiofrequency glow discharges fed with vapors of acrylic acid to coat them with thin adherent films characterized by surface carboxylic groups, where liposomes were covalently bound through the formation of amide bonds. For this, liposomes decorated with polyethylene glycol molecules bearing terminal amine-groups were prepared. After ensuring that the liposomes remain intact, under the conditions applying for immobilization; different attachment conditions were evaluated (incubation time, concentration of liposome dispersion) for optimization of the technique. Immobilization of calcein-entrapping liposomes was evaluated by monitoring the percent of calcein attached on the surfaces. Best results were obtained when liposome dispersions with 5mg/ml (liposomal lipid) concentration were incubated on each disk for 24h at 37°C. The method is proposed for developing drug-eluting biomedical materials or devices by using liposomes that have appropriate membrane compositions and are loaded with drugs or other bioactive agents.     

Covalent growth factor immobilization strategies for tissue repair and regeneration

Growth factors play a critical role in regulating processes involved in cellular differentiation and tissue regeneration, and are therefore considered essential elements in many tissue engineering strategies. The covalent immobilization of growth factors to biomaterial matrices addresses many of the challenges associated with delivering freely-diffusible growth factors and has thus emerged as a promising method of achieving localized and sustained growth factor delivery. This Feature Article discusses methods that have been used to immobilize growth factors to substrates, followed by an overview of several tissue repair and regeneration applications in which immobilized growth factors have been used.     

Covalent immobilization of proteases and nucleases to poly(methylmethacrylate)

The increased popularity of microfabricated devices formed from plastics such as poly(methylmethacrylate) (PMMA) will benefit from approaches adding (bio)chemical functionality to such surfaces. Here, various proteases and nucleases have been covalently immobilized to PMMA surfaces and shown to retain their enzymatic activity as monitored by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Immobilized enzymes yield structural information at a level equivalent to or exceeding that obtained from conventional homogeneous solution-based approaches. Such an approach could be used to expand the functionality of polymer-based microfabricated devices for biological mass spectrometry.     

Covalent immobilization of protein onto a functionalized hydrogenated diamond-like carbon substrate

Hydrogenated diamond-like carbon (HDLC) has an atomically smooth surface that can be deposited on high-surface area substrata and functionalized with reactive chemical groups, providing an ideal substrate for protein immobilization. A synthetic sequence is described involving deposition and hydrogenation of DLC followed by chemical functionalization. These functional groups are reacted with amines on proteins causing covalent immobilization on contact. Raman measurements confirm the presence of these surface functional groups, and Fourier transform infrared spectroscopy (FTIR) confirms covalent protein immobilization. Atomic force microscopy (AFM) of immobilized proteins is reproducible because proteins do not move as a result of interactions with the AFM probe-tip, thus providing an advantage over mica substrata typically used in AFM studies of protein. HDLC offers many of the same technical advantages as oxidized graphene but also allows for coating large surface areas of biomaterials relevant to the fabrication of medical/biosensor devices.     

Covalent immobilization of antibody fragments on well-defined polymer brushes via site-directed method

Well-defined polymer brushes and block copolymer brushes consisting of 2-methacryloyloxyethyl phosphorylcholine (MPC) and glycidyl methacrylate (GMA) were prepared by surface-initiated atom transfer radical polymerization (ATRP). The polymer brushes were used for the immobilization of antibody fragments in a defined orientation. Pyridyl disulfide moieties were introduced to the polymer brushes via a reaction of epoxy groups in GMA units. Fab’ fragments were then immobilized onto these surfaces via a thiol-disulfide interchange reaction and the reactivity of antibodies with antigens was investigated. Antigen/antibody binding on the polymer brushes was more preferable than that on epoxysilane films as a control surface. Furthermore, the activity of the antibodies immobilized on the block copolymer brushes having biocompatible PMPC was greater than that on other surfaces that did not have PMPC in their structures.     

Covalent immobilization of tyrosinase on magnetic multi-walled carbon nanotubes for inhibitor screening

The inhibition of tyrosinase is considered to be a common therapeutic strategy for some hyperpigmentation disorders. Screening of tyrosinase inhibitors is of great significance to the treatment of pigmentation diseases. In this study, tyrosinase was covalently immobilized on magnetic multi-walled carbon nanotubes for the first time, and the immobilized tyrosinase was applied for ligand fishing of tyrosinase inhibitors from complex medicinal plants. The immobilized tyrosinase was characterized by transmission electron microscopy, atomic force microscopy, Fourier-transform infrared spectroscopy, vibrating sample magnetometry, and thermo-gravimetric analyzer, which indicated that tyrosinase was immobilized onto magnetic multi-walled carbon nanotubes. The immobilized tyrosinase showed better thermal stability and reusability than the free one. The ligand was fished out from Radix Paeoniae Alba and identified as 1,2,3,4,6-pentagalloylglucose by ultra-performance liquid chromatography-quadrupole time-of-flight high-resolution mass spectrometry. 1,2,3,4,6-pentagalloylglucose was found to be a tyrosinase inhibitor with similar half maximal inhibitory concentration values of 57.13 ± 0.91 μM compared to kojic acid (41.96 ± 0.78 μM). This work not only established a new method for screening tyrosinase inhibitors but also holds considerable potential for exploring the new medicinal value of medicinal plants.     

Covalent immobilization of horseradish peroxidase via click chemistry and its direct electrochemistry

A simple and versatile approach for covalent immobilization of redox protein on solid surface via self-assembled technique and click chemistry is reported. The alkynyl-terminated monolayers are obtained by self-assembled technique, then, azido-horseradish peroxidase (azido-HRP) was covalent immobilized onto the formed monolayers by click reaction. The modified process is characterized by reflection absorption infrared spectroscopy (RAIR), surface-enhanced Raman scattering spectroscopy (SERS) and electrochemical methods. All the experimental results suggest that HRP is immobilized onto the electrode surface successfully without denaturation. Furthermore, the immobilized HRP shows electrocatalytic reduction for H₂O₂, and the linear range is from 5.0 to 700 μM. The heterogeneous electron transfer rate constant k_s is 1.11 s⁻¹ and the apparent Michaelis-Menten constant is calculated to be 0.196 mM.     

Covalent immobilization of oligonucleotides on p-aminophenyl-modified carbon screen-printed electrodes for viral DNA sensing

DNA-sensing platforms were prepared by covalently attaching oligonucleotide capture probes onto p-aminophenyl-functionalized carbon surfaces and applied to the determination of an amplified herpes virus DNA sequence in an electrochemical hybridization assay.     

Covalent immobilization of invertase on chemically activated poly(2-hydroxyethyl methacrylate) microbeads

Properties of invertase immobilized on poly(2-hydroxyethyl methacrylate) microbeads activated by epichlorohydrin or cyanuric chloride were studied. After 20 repeated uses for 3 days, the activity of the immobilized enzyme was 92–93%. Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 10, pp. 1793–1797, October, 2006.     

Covalent anchoring of a racemization catalyst to CALB-beads: towards dual immobilization of DKR catalysts

The preparation of a heterogeneous bifunctional catalytic system, combining the catalytic properties of an organometallic catalyst (racemization) with those of an enzyme (enantioselective acylation) is described. A novel ruthenium phosphonate inhibitor was synthesized and covalently anchored to a lipase immobilized on a solid support (CALB, Novozym® 435). The immobilized bifunctional catalytic system showed activity in both racemization of (S)-1-phenylethanol and selective acylation of 1-phenylethanol.     

Covalent immobilization of glucose oxidase onto new modified acrylonitrile copolymer/silica gel hybrid supports

New polymer/silica gel hybrid supports were prepared by coating high surface area of silica gel with modified acrylonitrile copolymer. The concentrations of the modifying agent (NaOH) and the modified polymer were varied. GOD was covalently immobilized on these hybrid supports and the relative activity and the amount of bound protein were determined. The highest relative activity and sufficient amount of bound protein of the immobilized GOD were achieved in 10% NaOH and 2% solution of modified acrylonitrile copolymer. The influence of glutaraldehyde concentration and the storage time on enzyme efficiency were examined. Glutaraldehyde concentration of 0.5% is optimal for the immobilized GOD. It was shown that the covalently bound enzyme (using 0.5% glutaraldehyde) had higher relative activity than the activity of the adsorbed enzyme. Covalently immobilized GOD with 0.5% glutaraldehyde was more stable for four months in comparison with the one immobilized on pure silica gel, hybrid support with 10% glutaraldehyde and the free enzyme. The effect of the pore size on the enzyme efficiency was studied on four types of silica gel with different pore size. Silica with large pores (CPC-Silica carrier, 375 A) presented higher relative activity than those with smaller pore size (Silica gel with 4, 40 and 100 A). The amount of bound protein was also reduced with decreasing the pore size. The effect of particle size was studied and it was found out that the smaller the particle size was, the greater the activity and the amount of immobilized enzyme were. The obtained results proved that these new polymer/silica gel hybrid supports were suitable for GOD immobilization.     

Covalent immobilization of Candida antarctica lipase B on nanopolystyrene and its application to microwave-assisted esterification

Nanopolystyrene was used as a solid support for the covalent immobilization of Candida antarctica lipase B (CalB) using the photoreactive reagent 1-fluoro-2-nitro-4-azido benzene (FNAB) as a coupling reagent. The obtained derivative was then used as a biocatalyst in a microwave assisted esterification experiment. Factors such as contact time, pH, and enzyme concentration were investigated during immobilization. The hydrolytic activity, thermal, and operational stability of immobilized-CalB were determined. The maximum immobilized yield (218 μg/mg support) obtained at pH 6.8 exhibited optimum hydrolytic activity (4.42 × 10³ mU p-nitrophenol/min). The thermal stability of CalB improved significantly when it was immobilized at pH 10, however, the immobilized yield was very low (93.6 μg/mg support). The immobilized-CalB prepared at pH 6.8 and pH 10 retained 50% of its initial activity after incubation periods of 14 and 16 h, respectively, at 60 ℃. The operational stability was investigated for the microwave assisted esterification of oleic acid with methanol. Immobilized-CalB retained 50% of its initial activity after 15 batch cycles in the microwave-assisted esterification. The esterification time was notably reduced under microwave irradiation. The combined use of a biocatalyst and microwave heating is thus an alternative total green synthesis process.