Exploring the Potential of Lapatinib, Fulvestrant, and Paclitaxel Conjugated with Glycidylated PAMAM G4 Dendrimers for Cancer and Parasite Treatment.

Fulvestrant (F), lapatinib (L), and paclitaxel (P) are hydrophobic, anticancer drugs used in the treatment of estrogen receptor (ER) and epidermal growth factor receptor (EGFR)-positive breast cancer. In this study, glycidylated PAMAM G4 dendrimers, substituted with F, L, and/or P and targeting tumor cells, were synthesized and characterized, and their antitumor activity against glioma U-118 MG and non-small cell lung cancer A549 cells was tested comparatively with human non-tumorogenic keratinocytes (HaCaT). All cell lines were ER+ and EGFR+. In addition, the described drugs were tested in the context of antinematode therapy on C. elegans. The results show that the water-soluble conjugates of G4P, G4F, G4L, and G4PFL actively entered the tested cells via endocytosis due to the positive zeta potential (between 13.57-40.29 mV) and the nanoparticle diameter of 99-138 nm. The conjugates of G4P and G4PFL at nanomolar concentrations were the most active, and the least active conjugate was G4F. The tested conjugates inhibited the proliferation of HaCaT and A549 cells; in glioma cells, cytotoxicity was associated mainly with cell damage (mitochondria and membrane transport). The toxicity of the conjugates was proportional to the number of drug residues attached, with the exception of G4L; its action was two- and eight-fold stronger against glioma and keratinocytes, respectively, than the equivalent of lapatinib alone. Unfortunately, non-cancer HaCaT cells were the most sensitive to the tested constructs, which forced a change in the approach to the use of ER and EGFR receptors as a goal in cancer therapy. In vivo studies on C. elegans have shown that all compounds, most notably G4PFL, may be potentially useful in anthelmintic therapy.

Lapatinib- and fulvestrant-PAMAM dendrimer conjugates promote apoptosis in chemotherapy-induced senescent breast cancer cells with different receptor status.

Lapatinib (L) and fulvestrant (F) are used in targeted anticancer therapies, in particular, against phenotypically different breast cancer cells. L, a dual inhibitor of EGFR and HER2 tyrosine kinases, is active against HER2-positive breast cancer cells, while F, a selective estrogen receptor degrader (SERD), is active against ER-positive breast cancer cells. However, the action of L and F can be limited due to their relatively low water solubility and bioavailability. In the present study, poly(amidoamine) (PAMAM) dendrimer G3 was functionalized with L or F or L and F to compare their effects with free L or F against breast cancer cells with different receptor status (ER-positive MCF-7, triple negative MDA-MB-231 and HER2-positive SK-BR-3 cells). L-PAMAM and F-PAMAM conjugates potentiated cytostatic and cytotoxic action of L and F that was accompanied by elevated levels of autophagy. TRDMT1, RNA methyltransferase, was also involved in this response as judged by TRDMT1 nuclear translocation and nano-drug resistance of TRDMT1 gene knockout cells. Nano-drugs also promoted elimination of doxorubicin-induced senescent breast cancer cells by apoptosis-mediated senolysis regardless of receptor status. In conclusion, we propose a novel anticancer approach based on L-PAMAM and F-PAMAM nanoplatforms being effective, at least, against breast cancer cells with different phenotypic features.

Distribution and Morphological Characteristics of Oligodendrocytes in Selected Areas of the Brain of Male and Female Red Kangaroos (Macropus rufus).

This study was carried out on six adult red kangaroos of both sexes. To determine the location of the oligodendrocytes (OLGs) of the hippocampus (Hip) and corpus callosum (CC), the method of impregnation of the neuroglia with silver salts was applied. The iron distribution in the OLGs was determined by the histochemical method. The Nissl method was used to determine the location of the brain structure and to analyze the number of OLGs. In the Hip, these cells are located one beside another, mainly in blood vessels and neurons; in the neocortex (NC), they are located in layers I-VI; and in the CC, they are arranged in characteristic rows and accompany both nerve fibers and blood vessels. The analysis of the results obtained by the chosen methods in the Hip, NC, and CC in males and females did not show statistically significant differences in the distribution and location of the red kangaroo OLGs. The involvement of these cells is a physiological process that proceeds in a similar manner throughout the life of individuals and actively influences the metabolism of neurons and myelin.

Synthesis of Biotinylated PAMAM G3 Dendrimers Substituted with R-Glycidol and Celecoxib/Simvastatin as Repurposed Drugs and Evaluation of Their Increased Additive Cytotoxicity for Cancer Cell Lines.

Recent achievement in anticancer therapy considers the application of repurposed drugs in optimal combinations with the use of specific carriers for their targeted delivery. As a result, new optimized medications with reduced side effects can be obtained. In this study, two known anticancer drugs, celecoxib and/or simvastatin, were conjugated covalently with PAMAM G3 dendrimer and tested in vitro against human squamous carcinoma (SCC-15-15) and glioblastoma (U-118 MG) cells, as well as normal human fibroblasts (BJ). The obtained conjugates were also substituted with biotin and R-glycidol to increase their affinity for cancer cells and were characterized with NMR spectroscopy and dynamic light scattering technique. Conjugates furnished with two celecoxib and four simvastatin residues revealed the very high effectiveness and dramatically decreased the SCC-15 and U-118 MG cell viability at very low concentrations with IC50 equal to about 3 µM. Its action was 20-50-fold stronger than that of either drug alone or as a mixture. Combined conjugate revealed also additive action since it was 2-8-fold more effective than conjugates with either single drug. The combined conjugate revealed rather low specificity since it was also highly cytotoxic for BJ cells. Despite this, it may be concluded that biotinylated and R-glycidylated PAMAM G3 dendrimers substituted with both celecoxib and simvastatin can be considered as a new perspective anticancer agent, effective in therapy of malignant, incurable glioblastomas.

Chiral Recognition of Homochiral Poly (amidoamine) Dendrimers Substituted with R- and S-Glycidol by Keratinocyte (HaCaT) and Squamous Carcinoma (SCC-15) Cells In Vitro.

The generation 2 and 3 poly(amidoamine) dendrimers (PAMAM G2 and G3) were converted into N-(2,3-dihydroxy)propyl derivatives by the addition of enantiomerically pure S- and R-glycidol. The homochiral dendrimers bind to HaCaT and SCC-15 cell membranes with an R/S glycidol enantioselectivity ratio of 1.5:1, as was quantitatively determined by fluorescence microscopy and visualized by confocal microscopy. Fully substituted G2 and G3 dendrimers were equipped with 32 and 64 N-(2,3-dihydroxy)propyl residues and showed effectively radial symmetry for homochiral derivatives in 13C NMR spectrum in contrary to analogs obtained by reaction with rac-glycidol. The sub-stoichiometric derivatives of G2 and G3 were also obtained in order to characterize them spectroscopically. The homochiral dendrimers were labeled with two different fluorescent labels, fluorescein, and rhodamine B, using their isothiocyanates to react with G2 and G3 followed by the addition of S- and R-glycidol. Obtained fluorescent derivatives were deficiently filled with N-(2,3-dihydroxy)propyl substituents due to steric hindrance imposed by the attached label. Nevertheless, these derivatives were used to determine their ability to bind to the cell membrane of human keratinocytes (HaCaT) and squamous carcinoma cells (SCC-15). Confocal microscopy images obtained from cells treated with variously labeled conjugates and fluorescence analysis with fluorescence reader allowed us to conclude that R-glycidol derivatives were bound and entered the cells preferentially, with higher accumulation in cancer cells. The G3 polyamidoamine (PAMAM)-based dendrimers were taken up more efficiently than G2 derivatives. Moreover, S- and R-glycidol furnished dendrimers were highly biocompatible with no toxicity up to 300 µM concentrations, in contrast to the amine-terminated PAMAM analogs.

Atrial natriuretic factor binding sites in experimental congestive heart failure.

A quantitative in vitro autoradiographic study was performed on the aorta, renal glomeruli, and adrenal cortex of cardiomyopathic hamsters in various stages of heart failure and correlated, in some instances, with in vivo autoradiography. The results indicate virtually no correlation between the degree of congestive heart failure and the density of 125I-labeled atrial natriuretic factor [(Ser99, Tyr126)ANF] binding sites (Bmax) in the tissues examined. Whereas the Bmax was increased in the thoracic aorta in moderate and severe heart failure, there were no significant changes in the zona glomerulosa. The renal glomeruli Bmax was lower in mild and moderate heart failure compared with control and severe heart failure. The proportion of ANF B- and C-receptors was also evaluated in sections of the aorta, adrenal, and kidney of control and cardiomyopathic hamsters with severe heart failure. (Arg102, Cys121)ANF [des-(Gln113, Ser114, Gly115, Leu116, Gly117) NH2] (C-ANF) at 10(-6) M displaced approximately 505 of (Ser99, Tyr126)125I-ANF bound in the aorta and renal glomeruli and approximately 20% in the adrenal zona glomerulosa in both series of animals. These results suggest that ANF may exert a buffering effect on the vasoconstriction of heart failure and to a certain extent may inhibit aldosterone secretion. The impairment of renal sodium excretion does not appear to be related to glomerular ANF binding sites at any stage of the disease.

Processing of the atrial natriuretic factor propeptide by atrial cardiocytes as revealed by immunocryoultramicrotomy.

Antibodies were raised against three fragments of the rat ANF molecule: C-terminal atrial natriuretic factor (ANF)-(101-126), N-terminal ANF-(11-37), and the putative cleavage site of the ANF propeptide, ANF-(94-103). These antibodies were purified by affinity chromatography and revealed a major band (17K) corresponding to the propeptide by Western blot analysis. Antibodies against ANF-(94-103) were used in a RIA. All of the peptides that possess this region (98-99) were able to displace iodinated pro-ANF from the antibody. However, peptides that have only one part of the fragment, such as ANF-(99-126) or ANF-(1-98), failed to displace pro-ANF; human ANF-(79-98) (100 pmol) demonstrated about 0.01% cross-reactivity. Immunohistochemical studies revealed that all atrial cardiocytes are reactive with the three antibodies. Immunocryoultramicrotomy revealed that the propeptide travels, uncleaved, from the Golgi complex to immature granules and mature secretory granules. These results indicate that cleavage of the ANF propeptide does not occur at these sites.