Photomechanical transdermal delivery of insulin in vivo.

BACKGROUND AND OBJECTIVE: Previous studies have shown that photomechanical waves transiently permeabilize the stratum corneum in vivo. The aim of the present work was to investigate the potential of photomechanical waves for systemic drug delivery. STUDY DESIGN/MATERIALS AND METHODS: Photomechanical waves were generated by ablation of a polystyrene target by a Q-switched ruby laser. Systemic insulin delivery in a streptozotocin-diabetic rat model was monitored by measuring the blood glucose level. RESULTS: After photomechanical insulin delivery, the blood glucose decreased 80 +/- 3% and remained below 200 mg/dl for more than 3 hours. Whereas in control experiments (for which insulin was applied without photomechanical waves), there was no dramatic change in the blood glucose (standard deviation of measurements over 4 hours was 7%). CONCLUSION: The application of the photomechanical waves allowed approximately 6-kDa protein molecules (insulin) to pass through the stratum corneum and into the systemic circulation.

Photomechanical drug delivery into bacterial biofilms.

PURPOSE: To investigate whether photomechanical waves generated by lasers can increase the permeability of a biofilm of the oral pathogen Actinomyces viscosus. METHODS: Biofilms of Actinomyces viscosus were formed on bovine enamel surfaces. The photomechanical wave was generated by ablation of a target with a Q-switched ruby laser and launched into the biofilm in the presence of 50 microg/ml methylene blue. The penetration depth of methylene blue was measured by confocal scanning laser microscopy. Also, the exposed biofilms were irradiated with light at 666 nm. After illumination, adherent bacteria were scraped and spread over the surfaces of blood agar plates. Survival fractions were calculated by counting bacterial colonies. RESULTS: Confocal scanning laser microscopy revealed that a single photomechanical wave was sufficient to induce a 75% increase in the penetration depth of methylene blue into the biofilm. This significantly increased the concentration of methylene blue in the biofilm enabling its photodestruction. CONCLUSIONS: Photomechanical waves provide a potentially powerful tool for drug delivery that might be utilized for treatment of microbial infections.

Determination of nonligand amino acids critical to [4Fe-4S]2+/+ assembly in ferredoxin maquettes.

The prototype ferredoxin maquette, FdM, is a 16-amino acid peptide which efficiently incorporates a single [4Fe-4S]2+/+ cluster with spectroscopic and electrochemical properties that are typical of natural bacterial ferredoxins. Using this synthetic protein scaffold, we have investigated the role of the nonliganding amino acids in the assembly of the iron-sulfur cluster. In a stepwise fashion, we truncated FdM to a seven-amino acid peptide, FdM-7, which incorporates a cluster spectroscopically identical to FdM but in lower yield, 29% relative to FdM. FdM-7 consists solely of the. CIACGAC. consensus ferredoxin core motif observed in natural protein sequences. Initially, all of the nonliganding amino acids were substituted for either glycine, FdM-7-PolyGly (.CGGCGGC.), or alanine, FdM-7-PolyAla (.CAACAAC.), on the basis of analysis of natural ferredoxin sequences. Both FdM-7-PolyGly and FdM-7-PolyAla incorporated little [4Fe-4S]2+/+ cluster, 6 and 7%, respectively. A systematic study of the incorporation of a single isoleucine into each of the four nonliganding positions indicated that placement either in the second or in the sixth core motif positions,.CIGCGGC. or.CGGCGIC., restored the iron-sulfur cluster binding capacity of the peptides to the level of FdM-7. Incorporation of an isoleucine into the fifth position,.CGGCIGC., which in natural ferredoxins is predominantly occupied by a glycine, resulted in a loss of [4Fe-4S] affinity. The substitution of leucine, tryptophan, and arginine into the second core motif position illustrated the stabilization of the [4Fe-4S] cluster by bulky hydrophobic amino acids. Furthermore, the incorporation of a single isoleucine into the second core motif position in a 16-amino acid ferredoxin maquette resulted in a 5-fold increase in the level of [4Fe-4S] cluster binding relative to that of the glycine variant. The protein design rules derived from this study are fully consistent with those derived from natural ferredoxin sequence analysis, suggesting they are applicable to both the de novo design and structure-based redesign of natural proteins.

Cell loading with laser-generated stress waves: the role of the stress gradient.

PURPOSE: To determine the dependence of the permeabilzation of the plasma membrane on the characteristics of laser-generated stress waves. METHODS: Laser pulses can generate stress waves by ablation. Depending on the laser wavelength, fluence, and target material, stress waves of different characteristics (rise time, peak stress) can be generated. Human red blood cells were subjected to stress waves and the permeability changes were measured by uptake of extracellular dye molecules. RESULTS: A fast rise time (high stress gradient) of the stress wave was required for the permeabilization of the plasma membrane. While the membrane was permeable, the cells could rapidly uptake molecules from the surrounding medium by diffusion. CONCLUSIONS: Stress waves provide a potentially powerful tool for drug delivery.

Ferredoxin and ferredoxin-heme maquettes.

A 16-amino acid residue peptide derived from a consensus motif of natural ferredoxins incorporates a tetranuclear iron sulfur cluster under physiological conditions. Successful assembly of the [4Fe-4S]2+/1+ cluster within a monomeric peptide was demonstrated using size exclusion chromatography, UV-visible, visible CD, and cryogenic EPR spectroscopies. The robustness of [4Fe-4S]2+/1+ formation was tested using peptides with either the ligating cysteine exchanged for alanine or with the intervening amino acids replaced by glycine. The small size of the peptide allows for modular incorporation into more complex protein structures. In one larger structure, we describe a tetra-alpha-helix bundle that self-assembles both iron-sulfur clusters and hemes, thereby demonstrating feasibility for the general synthesis of maquettes containing multiple, juxtaposed redox cofactors. This is a motif common to the catalytic sites of native oxidoreductases.

Design and synthesis of multi-haem proteins.

A water-soluble, 62-residue, di-alpha-helical peptide has been synthesized which accommodates two bis-histidyl haem groups. The peptide assembles into a four-helix dimer with 2-fold symmetry and four parallel haems that closely resemble native haems in their spectral and electrochemical properties, including haem-haem redox interaction. This protein is an essential intermediate in the synthesis of molecular 'maquettes', a novel class of simplified versions of the metalloproteins involved in redox catalysis and in energy conversion in respiratory and photosynthetic electron transfer.