Matija Rojnik

In vitro and in vivo characterization of temoporfin-loaded PEGylated PLGA nanoparticles for use in photodynamic therapy

AIMS In this study we evaluated temoporfin-loaded polyethylene glycol (PEG) Poly-(D,L-lactide-co-glycolide) (PLGA) nanoparticles (NPs) as a new formulation for potential use in cancer treatment. MATERIALS & METHODS NPs were characterized for their photophysical properties, temoporfin release, cellular uptake and intracellular localization, and dark and photocytotoxicities of temoporfin by using A549, MCF10A neoT and U937 cell lines. In vivo imaging was performed on athymic nude-Foxn1 mice. RESULTS Temoporfin was highly aggregated within the NPs and the release of temoporfin monomers was faster from PEGylated PLGA NPs than from non-PEGylated ones. PEGylation significantly reduced the cellular uptake of NPs by the differentiated promonocytic U937 cells, revealing the stealth properties of the delivery system. Dark cytotoxicity of temoporfin delivered by NPs was less than that of free temoporfin in standard solution (Foscan(®), Biolitec AG [Jena, Germany]), whereas phototoxicity was not reduced. Temoporfin delivered to mice by PEGylated PLGA NPs exhibits therapeutically favorable tissue distribution. CONCLUSION These encouraging results show promise in using PEGylated PLGA NPs for improving the delivery of photosensitizers for photodynamic therapy.

Procoagulant properties of bare and highly PEGylated vinyl-modified silica nanoparticles.

AIMS Undesired alterations of the blood clotting balance may follow the intravascular injection of nanotherapeutics/diagnostics. Here, we tested the procoagulant activity of synthetic amorphous silica (SAS) and organically modified silica (ORMOSIL) nanoparticles (NPs) and whether a high-density polyethylene glycol coating minimizes these effects. MATERIALS & METHODS Hageman factor- and tissue factor-dependent activation of human blood/plasma coagulation, and binding to human monocytes, endothelial cells and platelets were quantified in vitro using naked and PEGylated ORMOSIL-NPs. Their effects were compared with those of SAS-NPs, present in many industrial products, and of poly(lactic-co-glycolic acid)- and small unilamellar vesicles-NPs, already approved for use in humans. RESULTS Both SAS-NPs and ORMOSIL-NPS presented a significant procoagulant activity. However, highly PEGylated ORMOSIL-NPs were particularly averse to the interaction with the soluble factors and cellular elements that may lead to intravascular blood coagulation. CONCLUSION Stealth, highly PEGylated ORMOSIL-NPs with a poor procoagulant activity can be used as starting blocks to design hemocompatible nanomedical-devices.

Proinflammatory effects of bare and PEGylated ORMOSIL-, PLGA- and SUV-NPs on monocytes and PMNs and their modulation by f-MLP

AIMS We wanted to test the proinflammatory effects of vinyltriethoxysilane-based organically modified silica nanoparticles (ORMOSIL-NPs) in vitro on blood leukocytes. MATERIALS & METHODS Cell selectivity, cytokines/chemokines and O(2) (-) production were analyzed using nonpolyethylene glycol (PEG)ylated and PEGylated ORMOSIL-NPs, poly(lactic-co-glycolic acid) (PLGA)-NPs and small unilamellar vesicles (SUV)-NPs. RESULTS ORMOSIL-NPs mostly bound to monocytes while other NPs to all leukocyte types similarly. Cell capture of PEGylated-NPs decreased strongly (ORMOSIL), moderately (PLGA) and weakly (SUV). Bare ORMOSIL-NPs effectively stimulated the production of IL-1β/IL-6/TNF-α/IL-8 by monocytes and of IL-8 by polymorphonuclear leukocytes (PMNs). NP PEGylation inhibited such effects only partially. Formyl-methionine-leucine phenylalanine (f-MLP) further increased the release of cytokines/chemokines by monocytes/PMNs primed with bare and PEGylated ORMOSIL-NPs. PEGylated SUV-NPs, bare and PEGylated ORMOSIL- and PLGA-NPs sensitize PMNs and monocytes to secrete O(2) (-) upon f-MLP stimulation. CONCLUSION ORMOSIL-NPs are preferentially captured by circulating monocytes but stimulate both monocytes and PMNs per se or by sensitizing them to another agonist (f-MLP). PEG-coating confers stealth effects but does not completely eliminate leukocyte activation. Safe nanomedical applications require the evaluation of both intrinsic and cooperative proinflammatory potential of NPs.

Cathepsin H Mediates the Processing of Talin and Regulates Migration of Prostate Cancer Cells

Background: Cathepsin H (CtsH) is an aminopeptidase that is involved in tumor progression. Results: CtsH cleaves talin, and its inhibition reduces the migration of prostate cancer cells. Conclusion: CtsH affects cell migration by influencing the activity of integrins, a process that could be regulated by talin cleavage. Significance: Identification of novel CtsH proteolytic targets is important to understand and control tumor progression. The cytoskeletal protein talin, an actin- and β-integrin tail-binding protein, plays an important role in cell migration by promoting integrin activation and focal adhesion formation. Here, we show that talin is a substrate for cathepsin H (CtsH), a lysosomal cysteine protease with a strong aminopeptidase activity. Purified active CtsH sequentially cleaved a synthetic peptide representing the N terminus of the talin F0 head domain. The processing of talin by CtsH was determined also in the metastatic PC-3 prostate cancer cell line, which exhibits increased expression of CtsH. The attenuation of CtsH aminopeptidase activity by a specific inhibitor or siRNA-mediated silencing significantly reduced the migration of PC-3 cells on fibronectin and invasion through Matrigel. We found that in migrating PC-3 cells, CtsH was co-localized with talin in the focal adhesions. Furthermore, specific inhibition of CtsH increased the activation of αvβ3-integrin on PC-3 cells. We propose that CtsH-mediated processing of talin might promote cancer cell progression by affecting integrin activation and adhesion strength.

Profilin 1 as a target for cathepsin X activity in tumor cells.

Cathepsin X has been reported to be a tumor promotion factor in various types of cancer; however, the molecular mechanisms linking its activity with malignant processes are not understood. Here we present profilin 1, a known tumor suppressor, as a target for cathepsin X carboxypeptidase activity in prostate cancer PC-3 cells. Profilin 1 co-localizes strongly with cathepsin X intracellularly in the perinuclear area as well as at the plasma membrane. Selective cleavage of C-terminal amino acids was demonstrated on a synthetic octapeptide representing the profilin C-terminal region, and on recombinant profilin 1. Further, intact profilin 1 binds its poly-L-proline ligand clathrin significantly better than it does the truncated one, as shown using cathepsin X specific inhibitor AMS-36 and immunoprecipitation of the profilin 1/clathrin complex. Moreover, the polymerization of actin, which depends also on the binding of poly-L-proline ligands to profilin 1, was promoted by AMS-36 treatment of cells and by siRNA cathepsin X silencing. Our results demonstrate that increased adhesion, migration and invasiveness of tumor cells depend on the inactivation of the tumor suppressive function of profilin 1 by cathepsin X. The latter is thus designated as a target for development of new antitumor strategies.

Targeting EGFR-overexpressed A431 cells with EGF-labeled silica-coated magnetic nanoparticles

Human epidermal growth-factor receptor (EGFR) has emerged as an attractive target for cancer therapy. In this study, amino- or carboxyl-functionalized silica-coated maghemite nanoparticles were conjugated with epidermal growth-factor (EGF) using five different binding modes: carbodiimide chemistry, two types of homo-bifunctional cross-linking reagents, and electrostatic interactions between the nanoparticles and the EGF. The nanoparticles and their aqueous suspensions were characterized by transmission electron microscopy, zeta-potential measurements and dynamic light scattering. The binding efficiency of the EGF to the nanoparticles was measured by flow cytometry using a specific anti-EGF antibody. The ability of EGF bioconjugates to target the EGF receptors was tested using EGFR over-expressing A431 cells in comparison to EGFR negative HeLa cells. Our results showed that the bioconjugates where the EGF was bonded by carbodiimide chemistry are the most effective for the specific targeting of EGFR-expressing cells in vitro.

Cathepsin H indirectly regulates morphogenetic protein-4 (BMP-4) in various human cell lines

Cathepsin H indirectly regulates morphogenetic protein-4 (BMP-4) in various human cell lines Background. Cathepsin H is a cysteine protease considered to play a major role in tumor progression, however, its precise function in tumorigenesis is unclear. Cathepsin H was recently proposed to be involved in processing of bone morphogenetic protein 4 (BMP-4) in mice. In order to clarify whether cathepsin H also regulates BMP-4 in humans, its impact on BMP-4 expression, processing and degradation was investigated in prostate cancer (PC-3), osteosarcoma (HOS) and pro-monocytic (U937) human cell lines. Materials and methods. BMP-4 expression was founded to be regulated by cathepsin H using PCR array technology and confirmed by real time PCR. Immunoassays including Western blot and confocal microscopy were used to evaluate the influence of cathepsin H on BMP-4 processing. Results. In contrast to HOS, the expression of BMP-4 mRNA in U937 and PC3 cells was significantly decreased by cathepsin H. The different regulation of BMP-4 synthesis could be associated with the absence of the mature 28 kDa cathepsin H form in HOS cells, where only the intermediate 30 kDa form was observed. No co-localization of BMP-4 and cathepsin H was observed in human cell lines and the multistep processing of BMP-4 was not altered in the presence of specific cathepsin H inhibitor. Isolated cathepsin H does not cleave mature recombinant BMP-4, neither with its amino- nor its endopeptidase activity. Conclusions. Our results exclude direct proteolytic processing of BMP-4 by cathepsin H, however, they provide support for its involvement in the regulation of BMP-4 expression.

The influence of differential processing of procathepsin H on its aminopeptidase activity, secretion and subcellular localization in human cell lines

Cathepsin H is a unique member of the cysteine cathepsins that acts primarily as an aminopeptidase. Like other cysteine cathepsins, it is synthesized as an inactive precursor and activated by proteolytic removal of its propeptide. Here we demonstrate that, in human cells, the processing of the propeptide is an autocatalytic, multistep process proceeding from an inactive 41kDa pro-form, through a 30kDa intermediate form, to the 28kDa mature form. Tyr87P and Gly90P were identified as the two major endopeptidase cleavage sites, converting the 30kDa form into the mature 28kDa form. The level of processing differs significantly in different human cell lines. In monocyte-derived macrophages U937 and prostate cancer cells PC-3, the 28kDa form is predominant, whereas in osteoblasts HOS the processing from the 30kDa form to the 28kDa form is significantly lower. The aminopeptidase activity of the enzyme and its subcellular localization are independent of the product, however the 30kDa form was not secreted in HOS cells. The activity of the resulting cathepsin H in U937 cells was significantly lower than that in HOS cells, presumably due to the high levels of endogenous cysteine protease inhibitor cystatin F present specifically in this cell line. These results provide an insight into the dependence of human cathepsin H processing and regulation on cell type.