Peptide-mediated cell penetration and targeted delivery of gold nanoparticles into lysosomes.

There is considerable interest in the sub-cellular targeting and delivery of biomolecules, therapeutic and imaging agents, and nanoparticles and nanoparticle conjugates into organelles for therapeutic and imaging purposes. To date, a number of studies have used sorting peptides for targeted delivery of cargo into different cell organelles but not into lysosomes. In this study, the delivery of 13-nm gold nanoparticles across the cell membrane followed by targeted localisation into the lysosomes of a mammalian cell line was examined using novel combinations of cell-penetrating peptides and lysosomal sorting peptides conjugated to the nanoparticles. Using a combination of fluorescence spectroscopy, fluorescence microscopy and transmission electron microscopy techniques, we show that these nanoconjugates were efficiently and selectively delivered into the lysosomes with minimal cytotoxic effects. This novel targeted delivery system may underpin the development of a new strategy for the treatment of lysosomal storage diseases by exploiting the large surface area of nanoparticles to deliver drugs or replacement enzymes directly to the lysosomes.

Analysis of Fasciola cathepsin L5 by S2 subsite substitutions and determination of the P1-P4 specificity reveals an unusual preference.

Fasciola parasites (liver flukes) express numerous cathepsin L proteases that are believed to be involved in important functions related to host invasion and parasite survival. These proteases are evolutionarily divided into clades that are proposed to reflect their substrate specificity, most noticeably through the S(2) subsite. Single amino acid substitutions to residues lining this site, including amino acid residue 69 (aa69; mature cathepsin L5 numbering) can have profound influences on subsite architecture and influence enzyme specificity. Variations at aa69 among known Fasciola cathepsin L proteases include leucine, tyrosine, tryptophan, phenylalanine and glycine. Other amino acids (cysteine, serine) might have been expected at this site due to codon usage as cathepsin L isoenzymes evolved, but C69 and S69 have not been observed. The introduction of L69C and L69S substitutions into FhCatL5 resulted in low overall activity indicating their expression provides no functional advantage, thus explaining the absence of such variants in Fasciola. An FhCatL5 L69F variant showed an increase in the ability to cleave substrates with P(2) proline, indicating F69 variants expressed by the fluke would likely have this ability. An FhCatL2 Y69L variant showed a decreased acceptance of P(2) proline, further highlighting the importance of Y69 for FhCatL2 P(2) proline acceptance. Finally, the P(1)-P(4) specificity of Fasciola cathepsin L5 was determined and, unexpectedly, aspartic acid was shown to be well accepted at P(2,) which is unique amongst Fasciola cathepsins examined to date.

Acoustic wave immunosensing of a meningococcal antigen using gold nanoparticle-enhanced mass sensitivity.

Bacterial meningitis is an infection of the thin membranes covering the brain and spinal cord by a number of microorganisms including Neisseria meningitidis, which can lead to permanent neurological damage in the event of late diagnosis. Given the quick onset and severity of the disease, there is a clear need for a rapid, sensitive and specific diagnostic technique. Here, we describe the development and evaluation of an acoustic wave sensor, the quartz crystal microbalance (QCM), as a rapid immunosensor employing antibodies against the cell surface outer membrane protein 85 (OMP85) of N. meningitidis as an immobilized selective layer. These antibodies were directionally orientated as receptors by thin film deposition of structured polyvinylidene fluoride and Protein A. The sensitivity of this QCM immunosensor was further increased by conjugation of the OMP85 antigen to 50 nm gold nanoparticles providing reproducible detection of the target down to 300 ng/mL. Subsequent treatment of the QCM surface with an acidic glycine solution regenerated the immunosensor allowing each crystal to be used several times.

Adult and juvenile Fasciola cathepsin L proteases: different enzymes for different roles.

Cathepsin proteases are promising vaccine or drug targets for prophylaxis or therapy against Fasciola parasites which express cathepsin L and B proteases during their development. These proteases are believed to be involved in important functions for the parasite, including excystment, migration, feeding and host immune evasion. Several cathepsin L transcripts, including FhCatL5, have been isolated from adult Fasciola, while certain cathepsin L proteases, including FgCatL1G, have only been identified in the juvenile forms of the parasite. In this study, Fasciola hepatica cathepsin FhCatL5 and F. gigantica FgCatL1G were expressed in yeast and their biochemical properties characterised and compared. The pH profiles of activity and stability of the two recombinant cathepsins was shown to differ, differences that are likely to be functionally important and reflect the environments into which the cathepsins are expressed in vivo. Biochemical analysis indicates that FgCatL1G can cleave substrates with proline residues at P(2), a characteristic previously described for the adult cathepsin FhCatL2. FgCatL1G and FhCatL5 show differences in their host substrate digestion patterns, with different substrates cleaved at varying efficiencies. Functional analysis of a recombinant FhCatL5 L69W variant indicates that the residue at position 69 is important for the S(2) subsite architecture and can influence substrate specificity.

The detection of influenza A and B viruses in clinical specimens using a quartz crystal microbalance.

Current methods for the accurate diagnosis of influenza based on culture of the virus or PCR are highly sensitive and specific but require specialised laboratory facilities and highly trained personnel and, in the case of viral culture, can take up to 14 days to obtain a definitive result. In this study, a quartz crystal microbalance-based immunosensor (QCM) has been developed and its potential evaluated for the rapid and sensitive detection of both influenza A and B viruses in laboratory-cultured preparations and clinical samples. The effective limit for detection by QCM for stock preparations of both A/PR/8/34 and B/Lee/40 viruses was 1 x 10(4) pfu/mL, associated with observed frequency shifts of 30 (+/-5) and 37 (+/-6.5) Hz, respectively. Conjugation of 13 nm gold nanoparticles to the detecting antibody improved the mass sensitivity of the immunosensor, resulting in a 10-fold increase in sensitivity and a detection limit of 1 x 10(3) pfu/mL for both preparations, with resulting frequency shifts of 102 (+/-11) and 115 (+/-5) Hz, respectively. Detection of virus in nasal washes with this technique was achieved by overnight passage in MDCK cultures prior to analysis. A comparison of results obtained from 67 clinical samples using existing RT-PCR, shell vial, cell culture and ELISA methods showed that QCM techniques were comparable in sensitivity and specificity to cell culture methods.