Flexible Spacer Arm Research Articles

Oligoglycine and fluoropolymer functionalized enzyme-responsive gene delivery surface for rapid in situ endothelialization of vascular grafts

The treatment success of small-caliber artificial vascular grafts is seriously limited by high thrombogenesis and low endothelialization. Gene transfection can effectively enhance the proliferation and migration of endothelial cells (ECs) and accelerating the formation of endothelium layer. How to efficiently and temporarily immobilize the gene complexes on the material surface, how to intelligently release them and effectively transfect ECs are still the challenges for in situ endothelialization. Herein, a matrix-metalloproteinase (MMP)-responsive gene delivery system is constructed for in situ smart release of genes from the graft surface. The fluorinated cationic polymer (F-ssPEI) is served as high-efficient and low-cytotoxic gene vector. The gene complexes are temporarily immobilized via a MMP-cleavable peptide (MCP) linker which contains a MMP-susceptible sequence as the principal part and oligoglycine sequence (Gm) with different units (m = 0, 4 and 8) as the flexible spacer arm. These gene delivery surfaces achieve the temporary immobilization, smart release and high internalization of gene complexes. The MCP(G8) group shows the highest transfection efficacy and the optimal HUVEC proliferation/migration/function. The enhanced endothelialization and contractile smooth muscle cell regeneration of grafts are achieved in vivo. The MMP-responsive gene delivery system provides an effective platform to promote regeneration of vascular graft.

Preparation of inert magnetic nano-particles for the directed immobilization of antibodies

Various activated supports (cyanogen bromide, glutaraldehyde, epoxy-chelates, primary amino) were evaluated for the immobilization of IgG anti-horseradish peroxidase. Cyanogen bromide and glutaraldehyde supports greatly reduced the recognition capacity of the antigen, probably due to the incorrect orientation of the antibody on the support. Hetero-functional epoxy-chelate and immobilization by the sugar chain on primary amino groups had little effect on high recognition of the antigen (near to the theoretically expected value). However, the immobilization by the sugar chain resulted in a higher adsorption rate of horseradish peroxidase, possibly due to a favourable orientation on a flexible spacer arm). Antibodies immobilized on aminated surfaces showed two major drawbacks. Firstly, the biological activity of the immobilized antibody sharply decreased over several days when stored at low ionic strength, although this effect could be partially reversed by incubation at high ionic strength. Secondly, a high level of non-specific proteins adsorption on the support surface was observed. Both problems could be successfully resolved by controlling the coating of the support with aldehyde-aspartic-dextran. We propose that the loss of biological activity was related to the ionic adsorption of the immobilized antibody on the support surface, leading to a blocking of the recognition areas. This optimized protocol was applied to the immobilization of IgG anti-horseradish peroxidase from rabbit on magnetic nano-particles. A 10 μg preparation of nanoparticles was able to capture more than 75% of the 0.1 microgram of recombinant horseradish peroxidase present in 10 L of crude protein extract (1 g/L) from Escherichia coli.

Attachment of Water-Soluble Proteins to the Surface of (Magnetizable) Phospholipid Colloids via NeutrAvidin-Derivatized Phospholipids

The present work describes the incorporation of a functionalized phospholipid derivative into the phospholipid bilayer of both classical small unilamellar vesicles and recently developed magnetoliposomes, resulting in unique biocolloid structures onto which peripheral water-soluble enzymes can be immobilized on the surfaces. In the first part of this work, a synthesis protocol is outlined for a universal membrane anchor for water-soluble proteins. Dioleoylphosphatidylethanolamine-N-dodecanyl was used as the starting lipid molecule. After activation of the terminal –COOH group, α,ω-diamino-poly(ethylene glycol), used as a hydrophilic, flexible spacer arm, was coupled covalently. Subsequently, NeutrAvidin was bound, after blocking the free –NH2 groups with citraconic anhydride. In the second part, the resulting lipid–NeutrAvidin derivative was incorporated into small unilamellar vesicles comprised of dimyristoylphosphatidylglycerol. FPLC with Superdex 200 as the column matrix clearly showed that biotinylated alkaline phosphatase, which served as a representative model of water-soluble proteins, was attached to the vesicles. Furthermore, magnetoliposomes, constructed of the same type of phospholipid molecules, were presented as interesting colloids to assess the degree of enzyme immobilization in a rapid and elegant manner. Potential applications that can emerge from this study are briefly discussed.

Method for Orienting DNA Molecules on Mica Surfaces in One Direction for Atomic Force Microscopy Imaging

An efficient method was developed to stretch DNA molecules on an atomically flat surface for AFM imaging. This method involves anchoring DNA molecules from their 5′ ends to amino silanized mica surfaces. N-Succinimidyl6-[3′-(2-pyridyldithio) propionamido]hexa-noate (LC-SPDP), a heterobifunctional cross-linker with a flexible spacer arm was used for this purpose. Immobilization was carried out by introducing a thiol group to the 5′ end of DNA by PCR. Thiolated molecules were then reacted with the cross linker to conjugate with its 2-pyridyl disulphide group via sulfhydryl exchange. The resulting complex was deposited on amino silanized mica where NHS-ester moiety of the cross linker reacted with the primary amino group on the surface. Samples were washed by a current of water and dried by an air jet in one direction parallel to the surface. DNA molecules were fully stretched in one direction on imaging them by AFM.

Modulation of lipase properties in macro-aqueous systems by controlled enzyme immobilization: enantioselective hydrolysis of a chiral ester by immobilized Pseudomonas lipase.

Lipase from Pseudomonas fluorescens (PFL) has been immobilized by using different immobilization protocols. The catalytic behavior of the different PFL derivatives in the hydrolytic resolution of fully soluble (R,S) 2-hydroxy 4-phenyl butanoic acid ethyl ester (HPBE) in aqueous medium was analyzed. The soluble enzyme showed a significant but low enantioselectivity, hydrolyzing the S isomer more rapidly than the R-isomer (E = 7). The enzyme, immobilized via a limited attachment to a long and flexible spacer arm, showed almost identical activity and specificity to the soluble enzyme. However, other derivatives, e.g. PFL adsorbed on supports covered by hydrophobic moieties (octyl, decaoctyl), exhibited significant hyperactivation on immobilization (approximately 7-fold). Simultaneously, the enantioselectivity of the PFL-immobilized enzyme was significantly improved (from E = 7 to E = 80). By using such derivatives, almost pure R ester isomer (e.e. > 99%) has been obtained after 55% hydrolysis of the racemic mixture of a solution of 10% (w/v) (R,S) HPBE. The derivatives could be used for 10 cycles without any significant decrease in the activity of the biocatalyst.

Method for stretching DNA molecules on mica surface in one direction for AFM imaging.

An efficient method was developed to stretch DNA molecules on an atomically flat surface for AFM imaging. This method involves anchoring DNA molecules from their 5' ends to amino silanized mica surfaces. N-Succinimidyl6-[3'-(2-pyridyldithio) propionamido]hexanoate (LC-SPDP), a heterobifunctional cross-linker with a flexible spacer arm was used for this purpose. The immobilization process was carried out by introducing a thiol group to the 5' end of DNA by PCR. Thiolated molecules were then reacted with the cross linker to conjugate with its 2-pyridyl disulphide group via sulfhydryl exchange. The resulting complex was deposited on amino silanized mica where NHS-ester moiety of the cross linker reacted with the primary amino group on the surface. Samples were washed by a current of water and dried by an air jet in one direction parallel to the surface. DNA molecules were shown to be fully stretched in one direction on imaging them by AFM.

Highly potent and small neuropeptide Y agonist obtained by linking NPY 1–4 via spacer to α-helical NPY 25–36

Analogues of neuropeptide Y (NPY) containing small N- and C-terminal segments linked via flexible spacer arms were found to exhibit receptor binding affinity constants almost as high as NPY as well as post- and presynaptic NPY-agonistic activities. One of the most active analogues contains N-terminal NPY segment 1–4 linked via ε-aminocaproic acid (Aca) to the C-terminal partially α-helical peptide amide segment 25–36. NPY 1–4-Aca-25–36 is the first highly potent NPY agonist, which is of considerably reduced size in comparison to the native hormone. The analogues are accessible by solid-phase synthesis using Fmoc strategy.

Diagnosis of antibody-mediated drug allergy. Pyrazolinone and pyrazolidinedione cross-reactivity relationships.

Based on the 1-phenyl-2,3-dimethyl-3-pyrazolin-5-one series and on the 1,2-diphenyl-pyrazolidine-3,5-dione series of drugs, haptenic reagents and conjugates were synthesized and evaluated by passive cutaneous anaphylaxis in guinea pigs, and by ELISA tests using rabbit antisera against the haptens. No cross-reactivity between pyrazolinone and pyrazolidinedione haptenic reagents was found in any of the test systems. But also within each series, the animal antibodies showed rather strict specificities upon interaction with related haptens or drugs. These results are somewhat unexpected, because the haptens were used in connection with long and flexible spacer arms. Furthermore, they are not typical for certain other drug allergies. The strict specificity reduces the diagnostic potential of the haptenic reagents when used in serological tests or in skin testing.