Photoinduced effects of m-tetrahydroxyphenylchlorin loaded lipid nanoemulsions on multicellular tumor spheroids.

BACKGROUND: Photosensitizers are used in photodynamic therapy (PDT) to destruct tumor cells, however, their limited solubility and specificity hampers routine use, which may be overcome by encapsulation. Several promising novel nanoparticulate drug carriers including liposomes, polymeric nanoparticles, metallic nanoparticles and lipid nanocomposites have been developed. However, many of them contain components that would not meet safety standards of regulatory bodies and due to difficulties of the manufacturing processes, reproducibility and scale up procedures these drugs may eventually not reach the clinics. Recently, we have designed a novel lipid nanostructured carrier, namely Lipidots, consisting of nontoxic and FDA approved ingredients as promising vehicle for the approved photosensitizer m-tetrahydroxyphenylchlorin (mTHPC). RESULTS: In this study we tested Lipidots of two different sizes (50 and 120 nm) and assessed their photodynamic potential in 3-dimensional multicellular cancer spheroids. Microscopically, the intracellular accumulation kinetics of mTHPC were retarded after encapsulation. However, after activation mTHPC entrapped into 50 nm particles destroyed cancer spheroids as efficiently as the free drug. Cell death and gene expression studies provide evidence that encapsulation may lead to different cell killing modes in PDT. CONCLUSIONS: Since ATP viability assays showed that the carriers were nontoxic and that encapsulation reduced dark toxicity of mTHPC we conclude that our 50 nm photosensitizer carriers may be beneficial for clinical PDT applications.

Serotonin reverts age-related capillarization and failure of regeneration in the liver through a VEGF-dependent pathway.

The function of the liver is well-preserved during the aging process, although some evidence suggests that liver regeneration might be impaired with advanced age. We observed a decreased ability of the liver to restore normal volume after partial hepatectomy in elderly mice, and we identified a pathway that rescued regeneration and was triggered by serotonin. 2,5-dimethoxy-4-iodoamphetamine (DOI), a serotonin receptor agonist, reversed the age-related pseudocapillarization of old liver and improved hepatosinusoidal blood flow. After hepatectomy, the open fenestrae were associated with a restored attachment of platelets to endothelium and the initiation of a normal regenerative response, including the up-regulation of essential growth mediators and serotonin receptors. In turn, hepatocyte proliferation recovered along with regain of liver volume and animal survival. DOI operates through the release of VEGF, and its effects could be blocked with anti-VEGF antibodies both in vitro and in vivo. These results suggest that pseudocapillarization in the aged acts as a barrier to liver regeneration. DOI breaks this restraint through an endothelium-dependent mechanism driven by VEGF. This pathway highlights a target for reversing the age-associated decline in the capacity of the liver to regenerate.

Chitosan-thioglycolic acid as a versatile antimicrobial agent.

As functionalized chitosans hold great potential for the development of effective and broad-spectrum antibiotics, representative chitosan derivatives were tested for antimicrobial activity in neutral media: trimethyl chitosan (TMC), carboxy-methyl chitosan (CMC), and chitosan-thioglycolic acid (TGA; medium molecular weight: MMW-TGA; low molecular weight: LMW-TGA). Colony forming assays indicated that LMW-TGA displayed superior antimicrobial activity over the other derivatives tested: a 30 min incubation killed 100% Streptococcus sobrinus (Gram-positive bacteria) and reduced colony counts by 99.99% in Neisseria subflava (Gram-negative bacteria) and 99.97% in Candida albicans (fungi). To elucidate LMW-TGA effects at the cellular level, microscopic studies were performed. Use of fluorescein isothiocyanate (FITC)-labeled chitosan derivates in confocal microscopy showed that LMW-TGA attaches to microbial cell walls, while transmission electron microscopy indicated that this derivative severely affects cell wall integrity and intracellular ultrastructure in all species tested. We therefore propose LMW-TGA as a promising and effective broad-band antimicrobial compound.

Overcoming Endocytosis Deficiency by Cubosome Nanocarriers.

The use of lipid-based nanoparticles for the delivery of biomacromolecules has attracted considerable attention due to the current interest in protein-based therapeutics. Cubosomes protect the incorporated therapeutics, which are susceptible to degradation by enzymes, thereby improving their bioavailability, and concomitantly enhance cellular uptake. The cubosome nanoparticles presented herein were loaded with bovine serum albumin (BSA) and characterized by small-angle X-ray scattering and dynamic light scattering techniques, while the BSA encapsulation and its release were evaluated in vitro. The ability of this formulation to increase the cellular uptake of albumin by 2-fold was tested on various types of renal tubular cells and confirmed by in vivo renal uptake experiments in mice. The obtained results show that cubosomes are able to deliver BSA inside the cell through distinct uptake and intracellular routing. These data were substantiated, with evidence of a high cubosome-mediated uptake of BSA in Clcn5 knockout mice characterized by defective receptor-mediated endocytosis. The use of cubosomes as a delivery system thus represents a promising approach to overcome the low endocytic uptake in diseased epithelial cells and to treat dysfunctions of the kidney proximal tubule.

The role of phospholipase D in modulating the MTOR signaling pathway in polycystic kidney disease.

The mammalian target of rapamycin (mTOR) signaling pathway is aberrantly activated in polycystic kidney disease (PKD). Emerging evidence suggests that phospholipase D (PLD) and its product phosphatidic acid (PA) regulate mTOR activity. In this study, we assessed in vitro the regulatory function of PLD and PA on the mTOR signaling pathway in PKD. We found that the basal level of PLD activity was elevated in PKD cells. Targeting PLD by small molecule inhibitors reduced cell proliferation and blocked mTOR signaling, whereas exogenous PA stimulated mTOR signaling and abolished the inhibitory effect of PLD on PKD cell proliferation. We also show that blocking PLD activity enhanced the sensitivity of PKD cells to rapamycin and that combining PLD inhibitors and rapamycin synergistically inhibited PKD cell proliferation. Furthermore, we demonstrate that targeting mTOR did not induce autophagy, whereas targeting PLD induced autophagosome formation. Taken together, our findings suggest that deregulated mTOR pathway activation is mediated partly by increased PLD signaling in PKD cells. Targeting PLD isoforms with pharmacological inhibitors may represent a new therapeutic strategy in PKD.

Peptidomimetic antibiotics target outer-membrane biogenesis in Pseudomonas aeruginosa.

Antibiotics with new mechanisms of action are urgently required to combat the growing health threat posed by resistant pathogenic microorganisms. We synthesized a family of peptidomimetic antibiotics based on the antimicrobial peptide protegrin I. Several rounds of optimization gave a lead compound that was active in the nanomolar range against Gram-negative Pseudomonas spp., but was largely inactive against other Gram-negative and Gram-positive bacteria. Biochemical and genetic studies showed that the peptidomimetics had a non-membrane-lytic mechanism of action and identified a homolog of the beta-barrel protein LptD (Imp/OstA), which functions in outer-membrane biogenesis, as a cellular target. The peptidomimetic showed potent antimicrobial activity in a mouse septicemia infection model. Drug-resistant strains of Pseudomonas are a serious health problem, so this family of antibiotics may have important therapeutic applications.