Nanostructured polymer and lipid carriers for sunscreen. Biological effects and skin permeation.

The interest in developing new sunscreens is increasing due to the harmful effects of UV radiation on the skin, such as erythema, accelerated skin ageing (photoageing) and the induction of skin cancer. However, many molecular sunscreens penetrate into the skin causing photoallergies, phototoxic reactions and skin irritation. Thus, the aim of this work was the preparation and characterization of polymeric and solid lipid nanoparticles to act carriers of benzophenone-3 (BZ3), aiming to improve the safety of sunscreen products by increasing the sun protection factor (SPF), decreasing BZ3 skin penetration and decreasing BZ3 concentration in sunscreen formulation. BZ3 was encapsulated in poly(epsilon-caprolactone) (PCL) nanoparticles by the nanoprecipitation method and in solid lipid nanoparticles (SLN) by the hot high pressure homogenization method. The particles were stable for 40 days. The BZ3 encapsulated in PCL nanoparticles was released faster than BZ3 encapsulated in SLN. The sun protection factor increased when BZ3 was encapsulated in both nanostructures. However, BZ3 encapsulated in PCL nanoparticles decreased its skin permeation more than SLN-BZ3. Furthermore, BZ3 encapsulated in SLN did not exhibit cytotoxic or phototoxic effects in human keratinocytes (HaCaT cells) and BABL/c 3T3 fibroblasts, whereas PCL nanoparticles with BZ3 showed phototoxic potential in HaCaT cells. Nevertheless, BZ3 free and encapsulated in PCL nanoparticles or in SLN did not show allergic reactions in mice. Our results suggest that these nanostructures are interesting carriers for sunscreen.

Cytotoxicity of materials used in perforation repair tested using the V79 fibroblast cell line and the granulocyte-macrophage progenitor cells.

AIM: To compare the cytotoxicity of materials used to repair perforations using permanent V79 fibroblasts and murine granulocyte-macrophage progenitor cells (CFU-GM). METHODOLOGY: Set specimens from amalgam, glass-ionomer, SuperEBA, N-Rickert, MTA and gutta-percha were eluted with culture medium for 72 h and their cytotoxicities were assessed by incubating the extracts with V79 and bone marrow-derived progenitors for 24 h and 7 days, respectively. Cytotoxicity on V79 cells was judged using the total nucleic acid content (NAC), neutral red uptake (NRU) and reduction of the tetrazolium salt (MTT). The number of bone marrow CFU-GM colonies determined in clonal cultures stimulated with recombinant murine granulocyte-macrophage colony-stimulating factor was used to assess cytotoxicity to progenitor cells. Statistical analyses were conducted using the one-way analysis of variance and Tukey's test where appropriate. RESULTS: All materials were cytotoxic in both cell systems; however, CFU-GM was more sensitive to the extracts than V79 cells. A similar rank order of toxicity was observed in V79 cells using the NAC and the MTT assays: glass-ionomer > N-Rickert congruent with SuperEBA > gutta-percha > amalgam congruent with MTA (P < 0.05). In contrast, the NRU test exhibited a lower sensitivity to MTA, gutta-percha and amalgam extracts. In the clonal culture assay, the toxicity was less pronounced in the presence of gutta-percha, SuperEBA and MTA. Similar cellular responses were found by placing the set specimens directly in the clonal culture dishes. CONCLUSIONS: The sensitivity of toxicity depended on the choice of the endpoint and the cell-culture system. Nevertheless, MTA was ranked as the least cytotoxic cement in both cell systems.

Violacein cytotoxicity on human blood lymphocytes and effect on phosphatases.

Given the importance of protein phosphorylation in the context of cellular functions, abnormal protein phosphatase activity has been implicated in several diseases, including cancer. These critical roles of protein phosphatases qualify them as potential targets for the development of medicinal compounds that possess distinct modes of action such as violacein. In this work, studies with this natural indolic pigment at a concentration of 10.0 micromol L(-1) demonstrated a 20% activation of total protein phosphatase extracted from human lymphocytes. Although no alteration was observed on protein tyrosine phosphatase (CD45), 30% of inhibition was achieved in cytoplasmatic protein phosphatase activity after incubation with 10.0 micromol L(-1) violacein. Additionally, 5.0 micromol L(-1) of violacein inhibited by 50% the serum tartrate-resistant acid phosphatase activity. Violacein presented toxic effect on lymphocytes with IC50 values of 3 and 10 micromol L(-1) for protein content and protein phosphatase activity, respectively. These findings suggest an important role for protein phosphatases in the mechanisms controlling proliferation and cell death.

Natural killer cell activity, lymphocyte proliferation, and cytokine profile in tumor-bearing mice treated with MAPA, a magnesium aggregated polymer from Aspergillus oryzae.

The present study examined the effects of MAPA, an antitumor aggregated polymer of protein magnesium ammonium phospholinoleate-palmitoleate anhydride, isolated from Aspergillus oryzae, on concanavalin A (Con A)-induced spleen cell proliferation, cytokine production and on natural killer (NK) cell activity in Ehrlich ascites tumor-bearing mice. The Ehrlich ascites tumor (EAT) growth led to diminished mitogen-induced expansion of spleen cell populations and total NK activity. This was accompanied by striking spleen enlargement, with a marked increase in total cell counts. Moreover, a substantial enhancement in IL-10 levels, paralleled by a significant decrease in IL-2 was observed, while production of IL-4 and interferon-gamma (IFN-gamma) was not altered. Treatment of mice with 5 mg/kg MAPA for 7 days promoted spleen cell proliferation, IL-2 production and NK cell activity regardless of tumor outgrowth. In addition, MAPA treatment markedly enhanced IFN-gamma levels and reduced IL-10 production relative to EAT mice. A 35% reduction in splenomegaly with normal number of nucleated cells was also found. Altogether, our results suggest that MAPA directly and/or indirectly modulates immune cell activity, and probably disengages tumor-induced suppression of these responses. Clearly, MAPA has an impact and may delay tumor outgrowth through immunotherapeutic mechanisms.

Evaluation of Caesalpinia ferrea extract on bone marrow hematopoiesis in the murine models of listeriosis and Ehrlich ascites tumor.

The capacity of hematopoietic tissues to produce and mobilize phagocytes to the site of infection and tumor growth is of central importance to mediate the early immunological response. In this perspective, studies from our laboratory have defined Listeria monocytogenes infection and the Ehrlich ascites tumor (EAT) as useful models to investigate the effects of natural compounds on the growth and differentiation of granulocyte-macrophage progenitor cells (CFU-GM). As expected, a significant reduction in the number of bone marrow CFU-GM was observed in the initial stages of infection with a sublethal dose of Listeria. Similarly, the bone marrow CFU-GM decreased sharply 4 days after the EAT transplantation. Treatment of infected and tumor-bearing mice with 500 and 1,000 mg/kg of Caesalpinia ferrea aqueous extract, given 3 times orally, significantly stimulated myelopoiesis, whereas no effects were observed with the 250 mg/kg dose. Similar results were obtained in normal mice. The administration of the two higher doses of the extract also protected 15-20% of mice from a lethal dose of Listeria and significantly prolonged survival of EAT-bearing mice. In summary, these results demonstrate that C. ferrea extract acts as a positive regulator of myelopoiesis, and suggest that the therapeutic effect of C. ferrea may be partially mediated by this action.

Adjuvant effect of Pluchea quitoc extract on the resistance of tumor-bearing mice by modulation of the host hematopoietic response.

Progressive tumor growth is regularly accompanied by changes in the cellular constituents of the immune system. Evidence suggests that soluble factors generated during tumor growth can affect the amount of granulocyte-macrophage progenitors. In vitro colony growth of progenitor cells may be an early indicator of the cellular changes associated with tumor growth. Pluchea quitoc has been previously found to modulate the hematopoietic response during bacterial infection. This study was designed to investigate the effects of P. quitoc on the growth and differentiation of bone marrow granulocyte-macrophage progenitor cells (CFU-GM) in Ehrlich ascites tumor-bearing mice. In contrast to the myelosuppression developed in the tumor-bearing animals, treatment with P. quitoc ethanolic extract (250, 500 or 1000 mg/kg) for 3 consecutive days after tumor challenge reversibly stimulated myelopoiesis, restoring the number of CFU-GM to normal. This same dose-schedule also increased colony formation in normal mice as compared to controls. In addi tion, P. quitoc significantly enhanced survival of tumor-bearing mice. These results suggest an immunoregulatory role for P. quitoc in counteracting the tumor-induced myelopoietic suppression as well as usefulness as adjuvant treatment of cancer.

Stimulation of myelopoiesis in Listeria monocytogenes-infected mice by an aggregated polymer isolated from Aspergillus oryzae.

In this work, we investigated the effects of the proteic aggregated polymer of magnesium ammonium phospholinoleate-palmitoleate anhydride (MAPA) isolated from Aspergillus oryzae on the growth and differentiation of bone marrow granulocyte-macrophage progenitor cells (CFU-GM) in Listeriamonocytogenes-infected mice. A significant reduction in the CFU-GM number was observed in the initial phase of infection with a sublethal dose of Listeria. Treatment of mice with 0.5, 2.0 and 5.0 mg/kg MAPA for 7 days prior to infection significantly stimulated myelopoiesis in a dose-dependent manner. Moreover, treatment with 0.5 and 5.0 mg/kg MAPA resulted in 30% and 40% cures of mice lethally infected with Listeria, respectively. MAPA added directly to the culture dishes hardly affected colony formation by bone marrow cells, suggesting an indirect effect ofthis compound on myelopoiesis in vivo. In summary, the data show that MAPA can modulate the CFU-GM generation and antibacterial resistance in listeriosis. As the ability of hematopoietic tissues to produce phagocytes is of particular significance to mediate resistance to Listeria, the promotion of bone marrow CFU-GM by MAPA may contribute to a rapid restoration of phagocyte numbers in infected sites, thus mitigating the course of infection.

Effects of the green algae Chlorella vulgaris on the response of the host hematopoietic system to intraperitoneal ehrlich ascites tumor transplantation in mice.

Chlorella vulgaris extract (CVE) was examined for its effects on the Ehrlich ascites tumor-induced suppression in the numbers of bone marrow and spleen granulocyte-macrophage progenitor cells (CFU-GM) in mice. No effects on bone marrow and spleen CFU-GM, as compared to controls, were observed in normal mice given 50, 100 and 200 mg/kg CVE orally for 5 days. In tumor-bearing mice, myelosuppression concomitant with increased number of spleen CFU-GM were observed. The number of CFU-GM in the bone marrow was restored to control levels after the administration of CVE (50, 100 and 200 mg/kg) to tumor-bearing mice, and a slight reduction in spleen colony formation was observed in these animals. In addition, CVE significantly prolonged the survival of mice inoculated with the Ehrlich ascites tumor. These results suggest a protective antitumor effect of CVE which might be attributable, at least in part, to the stimulation of the production and, possibly, maturation of granulocytes and macrophages.

Stimulatory action of Pluchea quitoc extract on the hematopoietic response during murine listeriosis.

The importance of both granulocytes and macrophages in the response to Listeria monocytogenes infection make this infection a suitable choice to investigate the effects of Pluchea quitoc on hematopoiesis. A significant depletion of bone marrow granulocyte-macrophage progenitor cells (CFU-GM) was observed at 48 and 72 h after intraperitoneal infection of mice with 1 x 10(4) L. monocytogenes. However, the treatment of infected animals with P. quitoc ethanolic extract (250, 500 or 1000 mg/kg) given orally for 3 consecutive days prior to infection produced a stimulatory effect on myelopoiesis, restoring the number of CFU-GM to normal. This same dose-schedule also increased colony formation in normal mice as compared to controls. In addition, P. quitoc significantly enhanced survival of infected mice. Thus, it is probable that the ability of P. quitoc to induce a higher reserve of granulocyte-macrophage precursors in the bone marrow is of major significance in determining early resistance to infection.

Myelopoietic response in tumour-bearing mice by an aggregated polymer isolated from Aspergillus oryzae.

The effects of magnesium ammonium phospholinoleate-palmitoleate anhydride (MAPA), a proteic aggregated polymer isolated from Aspergillus oryzae, on the growth and differentiation of granulocyte-macrophage progenitor cells (colony-forming unit-granulocyte-macrophage [CFU-GM]) in normal and Ehrlich ascites tumour-bearing mice were studied. Myelosuppression concomitant with increased numbers of spleen CFU-GM was observed in tumour-bearing mice. Treatment of these animals with MAPA (0.5-10 mg/kg) stimulated marrow myelopoiesis in a dose-dependent manner and reduced spleen colony formation. No changes were observed in total and differential marrow cell counts. The dose of 5.0 mg/kg MAPA, given prior or after tumour inoculation, was the optimal biologically active dose in tumour-bearing mice and this dose schedule also stimulated myelopoiesis in normal mice. MAPA significantly enhanced survival and concurrently reduced tumour growth in the peritoneal cavity. We propose that the modulatory effect of MAPA on the myelopoietic response may be related to its antitumour activity as a possible mechanism for regulation of granulocyte-macrophage production and expression of functional activities.

Sensitized photooxygenation and peroxidase-catalyzed inactivation of xanthine oxidase--evidence of cysteine damage by singlet oxygen.

Xanthine oxidase (XO) has been investigated for its decreased activity in several cancerous tissues and constitutive generation of reactive oxygen species (ROS) in vivo seems to contribute significantly to its inactivation. Singlet oxygen (1O2) production has been suggested to be relevant when considering folic acid metabolism by cancer cells. Thus, the susceptibility of XO to inactivation by 1O2 generated either by the bioenergized systems folic acid/peroxidase/GSH/Mn2+/O2 and malonaldehyde/peroxidase/Mn2+/O2 or by methylene blue (MB) or eosin-sensitized photooxygenation was studied. Our results showed that other ROS were also responsible for XO inactivation when MB was used. In contrast, eosin produced almost exclusively 1O2. Kinetic studies of XO oxidation in the malonaldehyde/peroxidase system showed that histidine (His) is a competitive inhibitor with respect to XO. A similar result was observed in the eosin-photosensitized process, suggesting the involvement of 1O2 in both processes. In addition, an efficient quenching of XO oxidation by guanosine in the folic acid/peroxidase system was observed. Amino acid analysis revealed that cysteine (Cys) is more affected than other XO amino acids also prone to oxidation such as tyrosine (Tyr), methionine (Met) and His. These results indicate that 1O2 may cause oxidative damage to the Cys residues of XO, with loss of enzyme activity. Alteration of the flavin prosthetic site is hypothesized.

Chemical and photochemical generated carbon-centered radical intermediate and its reaction with desoxyribonucleic acid.

The possible action of carbon-centered radicals in promoting damage to DNA is explored by generation of the 2-phenylethyl radical. The radical is generated during oxidation of phenelzine (2-phenylethyl hydrazine) by ferricyanide, as well as optically from phenylpropionic acid. Covalent binding of the 2-phenylethyl radical to DNA is suggested by studies with the plasmid pBR 322 DNA. Other sensitive techniques used to study DNA damage were the interaction with formaldehyde at 60 degrees C and the fluorescence of DNA-Tb(III) and DNA-DAPI complexes. Theoretical MNDO calculations indicated a preferential attack at position 8 of the guanine residues. This study shows that the 2-phenylethyl radical is able to induce primary effects on nucleic acid structure, leading to alkylated products, especially at purine rings.