Evaluation of Nanoparticles Covalently Bound with BODIPY for Their Photodynamic Therapy Applicability.

Photodynamic therapy (PDT) relies on the combined action of a photosensitizer (PS), light at an appropriate wavelength, and oxygen, to produce reactive oxygen species (ROS) that lead to cell death. However, this therapeutic modality presents some limitations, such as the poor water solubility of PSs and their limited selectivity. To overcome these problems, research has exploited nanoparticles (NPs). This project aimed to synthesize a PS, belonging to the BODIPY family, covalently link it to two NPs that differ in their lipophilic character, and then evaluate their photodynamic activity on SKOV3 and MCF7 tumor cell lines. Physicochemical analyses demonstrated that both NPs are suitable for PDT, as they are resistant to photobleaching and have good singlet oxygen (1O2) production. In vitro biological analyses showed that BODIPY has greater photodynamic activity in the free form than its NP-bounded counterpart, probably due to greater cellular uptake. To evaluate the main mechanisms involved in PDT-induced cell death, flow cytometric analyses were performed and showed that free BODIPY mainly induced necrosis, while once bound to NP, it seemed to prefer apoptosis. A scratch wound healing test indicated that all compounds partially inhibited cellular migration of SKOV3 cells.

Chitosan nanoparticles for enhancing drugs and cosmetic components penetration through the skin.

Chitosan nanoparticles (CT NPs) have attractive biomedical applications due to their unique properties. This present research aimed at development of chitosan nanoparticles to be used as skin delivery systems for cosmetic components and drugs and to track their penetration behaviour through pig skin. CT NPs were prepared by ionic gelation technique using sodium tripolyphosphate (TPP) and Acacia as crosslinkers. The particle sizes of NPs appeared to be dependent on the molecular weight of chitosan and concentration of both chitosan and crosslinkers. CT NPs were positively charged as demonstrated by their Zeta potential values. The formation of the nanoparticles was confirmed by FTIR and DSC. Both SEM and TEM micrographs showed that both CT-Acacia and CT:TPP NPs were smooth, spherical in shape and are distributed uniformly with a size range of 200nm to 300 nm. The CT:TPP NPs retained an average of 98% of the added water over a 48-hour period. CT-Acacia NPs showed high moisture absorption but lower moisture retention capacity, which indicates their competency to entrap polar actives in cosmetics and release the encapsulated actives in low polarity skin conditions. The cytotoxicity studies using MTT assay showed that CT NPs made using TPP or Acacia crosslinkers were similarly non-toxic to the human dermal fibroblast cells. Cellular uptake study of NPs observed using live-cell imaging microscopy, proving the great cellular internalisation of CT:TPP NPs and CT-Acacia NPs. Confocal laser scanning microscopy revealed that CT NPs of particle size 530nm containing fluorescein sodium salt as a marker were able to penetrate through the pig skin and gather in the dermis layer. These results show that CT NPs have the ability to deliver the actives and cosmetic components through the skin and to be used as cosmetics and dermal drug delivery system.

Expanded molecular diversity generation during directed evolution by trinucleotide exchange (TriNEx).

Trinucleotide exchange (TriNEx) is a method for generating novel molecular diversity during directed evolution by random substitution of one contiguous trinucleotide sequence for another. Single trinucleotide sequences were deleted at random positions in a target gene using the engineered transposon MuDel that were subsequently replaced with a randomized trinucleotide sequence donated by the DNA cassette termed SubSeq(NNN). The bla gene encoding TEM-1 beta-lactamase was used as a model to demonstrate the effectiveness of TriNEx. Sequence analysis revealed that the mutations were distributed throughout bla, with variants containing single, double and triple nucleotide changes. Many of the resulting amino acid substitutions had significant effects on the in vivo activity of TEM-1, including up to a 64-fold increased activity toward ceftazidime and up to an 8-fold increased resistance to the inhibitor clavulanate. Many of the observed amino acid substitutions were only accessible by exchanging at least two nucleotides per codon, including charge-switch (R164D) and aromatic substitution (W165Y) mutations. TriNEx can therefore generate a diverse range of protein variants with altered properties by combining the power of site-directed saturation mutagenesis with the capacity of whole-gene mutagenesis to randomly introduce mutations throughout a gene.

Investigating protein structural plasticity by surveying the consequence of an amino acid deletion from TEM-1 beta-lactamase.

While the deletion of an amino acid is a common mutation observed in nature, it is generally thought to be disruptive to protein structure. Using a directed evolution approach, we find that the enzyme TEM-1 beta-lactamase was broadly tolerant to the deletion mutations sampled. Circa 73% of the variants analysed retained activity towards ampicillin, with deletion mutations observed in helices and strands as well as regions important for structure and function. Several deletion variants had enhanced activity towards ceftazidime compared to the wild-type TEM-1 demonstrating that removal of an amino acid can have a beneficial outcome.

Bulgecin A: a novel inhibitor of binuclear metallo-beta-lactamases.

Bulgecin A, a sulphonated N-acetyl-D-glucosamine unit linked to a 4-hydroxy-5-hydroxymethylproline ring by a beta-glycosidic linkage, is a novel type of inhibitor for binuclear metallo-beta-lactamases. Using steady-state kinetic analysis with nitrocefin as the beta-lactam substrate, bulgecin A competitively inhibited the metallo-beta-lactamase BceII from Bacillus cereus in its two-zinc form, but failed to inhibit when the enzyme was in the single-zinc form. The competitive inhibition was restored by restoring the second zinc ion. The single-zinc metallo-beta-lactamase from Aeromonas veronii bv. sobria, ImiS, was not inhibited by bulgecin A. The tetrameric L1 metallo-beta-lactamase from Stenotrophomonas maltophilia was subject to partial non-competitive inhibition, which is consistent with a kinetic model in which the enzyme bound to inhibitor retains catalytic activity. Docking experiments support the conclusion that bulgecin A co-ordinates to the zinc II site in metallo-beta-lactamases via the terminal sulphonate group on the sugar moiety.

Genetic linkage of the penicillinase gene, amp, and blrAB, encoding the regulator of beta-lactamase expression in Aeromonas spp.

Aeromonas hydrophila T429125, a human clinical isolate, possesses three coordinately inducible beta-lactamases encoded by ampH (class D beta-lactamase), cepH (class C beta-lactamase) and imiH (class B beta-lactamase). We report that upstream of ampH there are two genes, blrA and blrB, encoding a putative two-component regulatory system. PCR studies revealed the same blrAB-amp gene arrangement in all Aeromonas spp. isolates tested; namely, Aeromonas veronii bv. sobria, Aeromonas jandaei, Aeromonas mediae, Aeromonas salmonicida and Aeromonas trota. A dominant mutation in the predicted BlrB kinase domain results in beta-lactamase overexpression in A. hydrophila T429125, but in other beta-lactamase-overexpressing mutants blrAB remains intact. Relative to the parent strain, A. hydrophila T429125, beta-lactamase- overexpressing mutants show a clear hierarchy of increased beta-lactamase expression: ImiH > CepH > AmpH. The same hierarchy is seen following beta-lactam challenge of A. hydrophila T429125, and correlates with the number of blr-tag sequences (TTCAC) found upstream of each beta-lactamase gene: ampH (one), cepH (two) and imiH (three).

Biochemical characterization of the acquired metallo-beta-lactamase SPM-1 from Pseudomonas aeruginosa.

SPM-1 is a new metallo-beta-lactamase recently identified in Pseudomonas aeruginosa strain 48-1997A, isolated in Sao Paulo, Brazil. Kinetic analysis demonstrated that SPM-1 has a broad hydrolytic profile across a wide range of beta-lactam antibiotics. Considerable variation was observed within the penicillin, cephalosporin, and carbapenem subfamilies; however, on the whole, SPM-1 appears to preferentially hydrolyze cephalosporins. The highest k(cat/)K(m) ratios (in micromolar per second) overall were observed for this subgroup. The hydrolytic profile of SPM-1 bears the most similarity to that of the metallo-beta-lactamase IMP-1, yet for the most part, SPM-1 has k(cat)/K(m) values higher than those of IMP-1. Zinc chelator studies established that progressive inhibition of SPM-1 by EDTA, dipicolinic acid, and 1-10-o-phenanthroline demonstrated a biexponential pattern in which none of the chelators completely inhibited SPM-1. A homology model of SPM-1 was developed on the basis of the IMP-1 crystal structure, which showed the protein folding and active-site structure characteristic of metallo-beta-lactamases and which provides an explanation for the kinetic profiles observed.

Molecular characterization of SPM-1, a novel metallo-beta-lactamase isolated in Latin America: report from the SENTRY antimicrobial surveillance programme.

The gene encoding the metallo-beta-lactamase SPM-1 was cloned from a genomic library of Pseudomonas aeruginosa strain 48-1997 A. The insert carrying spm-1 possessed a GC content of 47%, indicating that it is of non-Pseudomonas origin. Upstream of spm-1 there is a small open reading frame (ORF), which is homologous to the LysR family of proteins (69% identity to the LysR protein from Salmonella enterica serovar Typhimurium). Downstream of spm-1 there is the start of an ORF, the product of which shows close homology with the GroEL-type proteins from Xanthomonas campestris. No transmissible element could be identified upstream or downstream of spm-1. The spm-1 gene is carried on a plasmid that can transform both Escherichia coli and P. aeruginosa to ceftazidime resistance. SPM-1 contains the classic metallo-beta-lactamase zinc-binding motif HXHXD and shows the highest identity (35.5%) to IMP-1. SPM-1 is a distinctly different metallo-beta-lactamase from VIM and IMP and, accordingly, represents a new subfamily of mobile metallo-beta-lactamases. The predicted molecular weight of the protein was 27 515 Da, significantly higher than that of IMP (25 041 Da) or VIM (25 322 Da). SPM-1 possesses a unique loop of 23 residues that accounts for the higher molecular mass.

Sequence and genome context analysis of a new molecular class D beta-lactamase gene from Legionella pneumophila.

Legionella pneumophila Philadelphia-1 (ATCC 33152) produces a serine active site beta-lactamase. The chromosomal gene that encodes this enzyme, loxA, has been cloned by PCR using information from the L. pneumophila Philadelphia-1 genome sequencing project. LoxA is a class 2d penicillinase, and its sequence puts it into the molecular class D beta-lactamase family, although phylogenetic analysis shows that LoxA forms a distinct branch in the OXA family along with the LoxA homologue, OXA-29, from Legionella gormanii ATCC 33297(T). Upstream of loxA on the L. pneumophila Philadelphia-1 chromosome is a two-gene locus similar to that found linked to the beta-lactamase genes of Gram-positive bacteria. The unit consists of loxI, encoding a homologue of the Gram-positive beta-lactamase expression regulator, and pbpX, encoding a putative penicillin-binding transpeptidase. Despite the presence of beta-lactamase regulator homologues, we could find no evidence of LoxA induction upon challenge of L. pneumophila Philadelphia-1 with beta-lactams.

Characterization of monomeric L1 metallo-beta -lactamase and the role of the N-terminal extension in negative cooperativity and antibiotic hydrolysis.

The L1 metallo-beta-lactamase from Stenotrophomonas maltophilia is unique among this class of enzymes because it is tetrameric. Previous work predicted that the two regions of important intersubunit interaction were the residue Met-140 and the N-terminal extensions of each subunit. The N-terminal extension was also implicated in beta-lactam binding. Mutation of methionine 140 to aspartic acid results in a monomeric L1 beta-lactamase with a greatly altered substrate specificity profile. A 20-amino acid N-terminal deletion mutant enzyme (N-Del) could be isolated in a tetrameric form but demonstrated greatly reduced rates of beta-lactam hydrolysis and different substrate profiles compared with that of the parent enzyme. Specific site-directed mutations of individual N terminus residues were made (Y11S, W17S, and a double mutant L5A/L8A). All N-terminal mutant enzymes were tetramers and all showed higher K(m) values for ampicillin and nitrocefin, hydrolyzed ceftazidime poorly, and hydrolyzed imipenem more efficiently than ampicillin in contrast to wild-type L1. Nitrocefin turnover was significantly increased, probably because of an increased rate of breakdown of the intermediate species due to a lack of stabilizing forces. K(m) values for monomeric L1 were greatly increased for all antibiotics tested. A model of a highly mobile N-terminal extension in the monomeric enzyme is proposed to explain these findings. Tetrameric L1 shows negative cooperativity, which is not present in either the monomer or N-terminal deletion enzymes, suggesting that the cooperative effect is mediated via N-terminal intersubunit interactions. These data indicate that while the N terminus of L1 is not essential for beta-lactam hydrolysis, it is clearly important to its activity and substrate specificity.