Self-assembled polypeptide nanoparticles for intracellular irinotecan delivery.

In this research poly(l-lysine)-b-poly(l-leucine) (PLys-b-PLeu) polymersomes were developed. It was shown that the size of nanoparticles depended on pH of self-assembly process and varied from 180 to 650nm. The biodegradation of PLys-b-PLeu nanoparticles was evaluated using in vitro polypeptide hydrolysis in two model enzymatic systems, as well as in human blood plasma. The experiments on the visualization of cellular uptake of rhodamine 6g-loaded and fluorescein-labeled nanoparticles were carried out and the possibility of their penetration into the cells was approved. The cytotoxicity of polymersomes obtained was tested using three cell lines, namely, HEK, NIH-3T3 and A549. It was shown that tested nanoparticles did not demonstrate any cytotoxicity in the concentrations up to 2mg/mL. The encapsulation of specific to colorectal cancer anti-tumor drug irinotecan into developed nanocontainers was performed by means of pH gradient method. The dispersion of drug-loaded polymersomes in PBS was stable at 4°C for a long time (at least 1month) without considerable drug leakage. The kinetics of drug release was thoroughly studied using two model enzymatic systems, human blood serum and PBS solution. The approximation of irinotecan release profiles with different mathematical drug release models was carried out and allowed identification of the release mechanism, as well as the morphological peculiarities of developed particles. The dependence of encapsulation efficiency, as well as maximal loading capacity, on initial drug concentration was studied. The maximal drug loading was found as 320±55µg/mg of polymersomes. In vitro anti-tumoral activity of irinotecan-loaded polymersomes on a colon cancer cell line (Caco-2) was measured and compared to that for free drug.

The immunization with peptide 189-205, a derivative of serotonin receptor subtypes 1B, changes the sensetivity of adenylyl cyclase to hormones in the rat brain.

The aim of this work was to study the effect of multiple (during 12 months) immunization of rats with BSA-conjugated peptide 189-205 corresponding to the second extracellular loop of rat HT1BR on ACSS activity in the brain of immunized animals (group HT1BR) and its regulation by hormones.

[Peptide 612-627 of thyrotropin receptor and its modified derivatives as the regulators of adenylyl cyclase in the rat thyroid gland].

The regulation of the specific activity of the thyroid gland is carried by thyroid-stimulating hormone (TSH) through TSH receptor (TSHR). This receptor is coupled to different types of G-proteins, including the G(s)-proteins, through which TSH stimulates the enzyme adenylyl cyclase (AC). As the application of TSH in medicine is limited, the development of selective regulators of TSHR with agonistic and antagonistic activity is carried out. One of the approaches to their creation is to develop the peptides corresponding to functionally important regions of TSHR which are located in its intracellular loops (ICL) and are involved in the binding and activation of G-proteins. We have synthesized peptide corresponding to the C-terminal region 612-627 of the third ICL of TSHR and its derivatives modified by palmitic acid residue (at the N- or the C-terminus) or by polylysine dendrimer (at the N-terminus), and studied their effect on the basal and TSH-stimulated AC activity in the membrane fraction isolated from the rat thyroid. The most active was peptide 612-627-K(Pal)A modified by palmitate at the C-terminus, where in TSHR the hydrophobic transmembrane region is located. At the micromolar concentrations the peptide increased AC activity and reduced the AC stimulating effect of TSH. The action of the 612-627-K(Pal)A has been directed onto TSHR homologous to it, as indicated by the following facts: 1) the inhibition of G(s)-protein, the downstream component of AC system, by treating the membranes with cholera toxin led to the blocking of peptide AC effect, 2) this effect was not detected in the tissues where no TSHR, 3) the peptide did not significantly affect the AC stimulating effects of hormones acting via other receptors. The unmodified peptide and the peptide with N-terminal dendrimer are far behind the 612-627-K(Pal)A in their ability to activate AC in the thyroid, while the peptide modified by palmitate at the N-terminus was inactive. At the same time, the peptide modified by dendrimer was comparable to the 612-627-K(Pal)A in the ability to inhibit the AC effect of TSH, but, although to a lesser extent that it decreased the AC effects of other hormones, demonstrating the low receptor specificity. Thus, these data point to the high efficiency of peptide 612-627-K(Pal)A, as a regulator of TSHR, and the prospects of creating the drugs based on it to control the thyroid functions in pathology.

[N-palmitoylated peptide 232-245 of rat type 4 melanocortin receptor possessing agonistic activity].

Melanocortin receptors of the type 4 (M4R) play a key role in the regulation of feeding behavior, neuroendocrine functions, and energy metabolism. The alterations in their functional activity induce obesity, metabolic syndrome, depression, and mental disorders, which makes the search of selective regulators of M4R to be one of the actual problems of molecular endocrinology. Promising for the development of such regulators is to design peptides corresponding to functionally important regions of M4R. The purpose of this study was to study the influence of synthesized N-palmitoylated peptide Palm-Thr-Gly-Thr-Ile-Arg-Gln-Gly-Ala-Asn-(Nle)-Lys-Gly-Ala-Ile232-245-amide (Palm-232-245) structurally corresponding to the C-terminal half of the third intracellular loop (ICL-3) of rat M4R on functional activity of adenylyl cyclase signaling system (ACSS) in the fractions of synaptosomal membranes isolated from the brains of male rats. It has been shown that, at a concentration of 10(-7) M and higher, Palm-232-245 stimulates the basal activity of adenylyl cyclase (AC) in the synaptosomal membranes and increases the basal level of GTP binding with the EC50 values of 71 and 267 nM, respectively. Under the combined action of low concentrations of the peptide (10(-7)-10(-6) M) and M4R agonists, α-melanocyte-stimulating hormone (α-MSH) and THIQ (10(-7) M), we observed an additivi stimulatory effect on AC, which disappeared when the peptide concentration was increased to 10(-4)-10(-3) M. In the synaptosomal membranes preincubated with 10(-5) M peptide, the maximum stimulatory effect of M4R agonists on AC activity was lower than that in controls, and EC50 values for this effect, on the contrary, increased. In the case of combined action of the peptide and hormones (γ-MSH, serotonin, PACAP-38) that activate AC via the other receptors, the additivity of their stimulating effects on the ACSS persisted throughout the range of peptide concentrations. The effect of the peptide was not observed in myocardial and testicular membranes no in which there is M4R homologous to the peptide. Thus, N-palmitoylated peptide Palm-232-245 specifically activates the ACSS in the rat brain by acting as intracellular M4R agonist. This may be used to create drugs regulating brain melanocortin system and physiological processes that depend on it.

[The secondary structure of peptides derived from the third intracellular loop of the serpentine type receptors and its interrelation with their biological activity].

We and other authors have shown that synthetic peptides corresponding to regions of the third intracellular loop (ICL-3) of receptors of the serpentine type are capable of activating G-protein signaling cascades and trigger them in the absence of hormone. To create on the basis of these peptides the selective regulators of hormonal signaling systems the relationship between their biological activity and secondary structure are studied. It is assumed that most suitable is a helical conformation, which allows the peptide effectively interact with signaling proteins. The aim of this study was to test the biological activity and secondary structure of synthesized by us linear peptides and their dimeric and palmitoylated analogs, corresponding to C-terminal region of the ICL-3 of luteinizing hormone receptor (LHR) and 5-hydroxytryptamine receptor of the type 6 (5-HT6R). It is shown that LHR-peptides at the micromolar concentrations stimulate the basal activity of adenylyl cyclase (AC) and the GTP-binding of G-proteins in the plasma membranes of rat testes, while 5-HT6R-peptides activate AC and G-proteins in the synaptosomal membranes of rat brain. The action of peptides is tissue-specific and observed in the tissues where there are homologous receptors. The most effective were palmitoylated peptides. LHR-peptide reduced the AC stimulatory effect of human chorionic gonadotropin, while 5-HT6R-peptides the effect of 5-HT6R-agonist, EMD-386088, and the action of the peptides was not found in the case of non-homologous receptors. Using circular dichroism spectroscopy it is shown that in neutral (pH 7) and acidic (pH 2) medium all the peptides are exist predominantly in the antiparallel beta-sheet (37-42%) and disordered conformations (33-35%). In alkaline medium (pH 10) in the case palmitoylated peptides the increase of the contribution of the helical conformation to 12-27% was observed. In the presence of trifluoroethanol (10-80%), a helix-forming solvent, the contribution of helical conformation for the majority of peptides was slightly increased (for palmitoylated analogs to 14%), however, in this case the antiparallel beta-sheet and disordered conformation prevailed. The conclusion was made that the lack of clearly expressed ability to form helices in peptides derived the ICLs of receptors did not significantly affect their activity. This is consistent with proposed mechanism of peptides action, whereby peptide interacts with the complementary regions of homologous receptor that does not require the helix formation.

[Peptides derived from the third cytoplasmatic loop of serotonin 1B receptor subtype selectively inhibit serotonin signal transduction via a homologous receptor].

The third intracellular loops of hormonal receptors play the main role in the interaction of majority of the serpentine type receptors with heterotrimeric G-proteins. In recent years, it was shown that synthetic peptides corresponding to membrane-proximal regions of these loops could be selectively influenced with hormonal signal transduction via the receptors homologous to them and trigger signalling cascade in absence of the hormone. For the first time, we succeeded in synthesizing the peptides derived from C-terminal region of the third intracellular loop of the IB-subtype serotonin receptor and studied their influence on serotonin-sensitive adenylyl cyclase system in the rat brain. The peptides 300-316 and 306-316 (the numbers correspond to amino acid positions in the rat IB-subtype serotonin receptor) at micromolar concentrations in absence of hormone-stimulated GTP-binding of Gi,-proteins coupled with the IB-subtype serotonin receptors and inhibited forskolin-stimulated adenylyl cyclase activity. Using selective agonists and antagonists of serotonin receptors it was shown that the peptides 300-316 and 306--316 inhibited serotonin signal transduction via homologous to them receptor and weakly influenced other types of serotonin receptors. The peptide 300-316 is more active compared with its shorter analogue 306-316 in the selectivity and efficiency of action on adenylyl cyclase signalling system regulated via the IB-subtype serotonin receptors. These findings indicate that the regions 300-316 of the IB-subtype serotonin receptor are involved in interaction with Grproteins and consist of the main molecular determinants responsible for serotonin signal transduction to adenylyl cyclase.

[Hyperbranched polylysines modified with histidine and arginine: the optimization of their DNA compacting and endosomolytic properties].

The DNA compacting and transfection properties of hyperbranched polylysines whose N-terminal amino groups were modified with histidine and arginine were studied. The histidine-modified hyperbranched polylysines were shown to provide higher efficacy of binding and transfection in comparison with unmodified or hyperbranched arginine-containing polylysines. This fact was explained by the intrinsic endosomolytic activity of the histidine-modified polymers. The dependence between the quantity of the amino acids that modified the terminal lysine residues in the hyperbranched polylysines, the efficacy of their DNA binding, and the transfection activity of the DNA complexes with the corresponding carriers was found. The possibility to increase the transfection activity of the DNA complexes with the hyperbranched polylysines by glycerin or the JTS-1 amphipathic nonapeptide was studied. At the same time, their simultaneous use was found to result in a transfection decrease.

[Lysine dendrimers as vectors for delivering genetic constructs to eukaryotic cells].

Asymmetrical lysine dendrimers are promising as vectors for delivering gene expression constructs into mammalian cells. The condensing, protective, and transfection properties were studied for pentaspherical lysine dendrimer D5 and its analog D5C10, modified with capric acid residues at the outer sphere; in addition, the transfection activity was assayed for complexes DNA-dendrimer-endosomolytic peptide JTS-1. Fatty acid residues incorporated in lysine dendrimers proved to improve their ability to bind DNA, to protect DNA from nuclease degradation, and to ensure its transfer into the nucleus. Peptide JTS-1 introduced in DNA-dendrimer complexes significantly increased their transfection activity. The potentiating effect of JTS-1 was especially high with the DNA-D5C10 complex. An excess of JTS-1 changed the structure of the complexes and reduced their transfection activity. It was assumed that dendrimers D5 and D5C10 are promising vectors for delivering DNA to eukaryotic cells and provide a basis for constructing more refined nonvirus module carriers.

[Dendrimers based lysine and their "starburst" polymeric derivatives: prospects of use in compacting DNA and in vitro delivery of genetic constructs]. / Dendrimery na osnove lizina i ikh "zvezdoobraznye" polimernye proizvodnye: vozmozhnost' ispol'zovaniia dlia kompaktizatsii DNK i dostavke ékspressiruiushchikh geneticheskikh konstruktsii in vitro.

We attempted to find some compounds for the effective delivery of gene constructs into cells and obtained two trispherical dendrimers on the basis of lysine, (Lys)8-(alpha, epsilon-Lys)4-(alpha, epsilon-Lys)2-(alpha, epsilon-Lys)-Ala-NH2 (D1) and (Lys)8-(alpha, epsilon-Lys)4-(alpha, epsilon-Lys)2-(alpha, epsilon-Lys)-Ala-[Lys(Plm)]2-Ala-NH2 (D2), as well as the starburst polymeric derivatives of D1, (pVIm)8-D1 and (pLys)n-D1, containing poly(N-vinylimidazole) and polylysine chains bound at a single point to the dendrimer amino groups. The conditions of dendrimer-plasmid DNA complex formation were studied. The intracellular localization of these complexes and the expression of gene constructs delivered with their help were analyzed in transfection experiments on the HeLa cell cultures of human epithelial carcinoma and on C2C12 mouse myoblasts. It was found that the chemical structure of dendrimer D1 and its derivatives significantly affected the structure and properties of complex. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 1; see also http://www.maik.ru.

[The periodontal status of schoolchildren in different age groups]. / Issledovanie sostoianiia parodonta u shkol'nikov raznykh vozrastnykh grupp.

Examinations of the periodontium, carried out in 350 schoolchildren, aged 12 to 16, in the town of Odessa, included registration of the CPITN, PMA, Green-Vermillion's, and CDL indexes. The findings evidence that adolescents aged 12 to 15 have but few periodontal segments with bleeding gingiva, their PMA indexes are reduced, and hygienic status of the oral cavity is getting better, but by the age of 16 accumulation of dental deposit results in inflammation enhancement. Though girls, whatever their age, take better care of the oral cavity, its status is not much better in them than in boys.

[Clinico-instrumental studies of the digestive system during diet therapy using culinary products prepared in ultra-high frequency ovens]. / Kliniko-instrumental'nye issledovaniia pishchevaritel'nogo apparata pri ispol'zovanii v lechebnom pitanii kulinarnykh izdelii, prigotovlennykh v SVCh-pechakh.

A total of 148 patients aged 18-66 years were examined. Of these, 86 subjects received an experimental diet, while 62 were placed on the common dietetics. Both single and 10-day nutrition with products manufactured in ultra-high ovens (UHF products) did not produce any adverse effect on the mean estimates of acid-forming function of the stomach and pH in the basal phase of gastric secretion or on the pattern of gastric mucus formation in patients. Histological study of the biopsy specimens of the gastric mucosa in patients who received UHF products did not reveal any specific time course of changes in its morphology and function. The blood flow in the gastric mucosa varied within an insignificant range (10-15 cal/min). Fifty per cent of patients who received UHF products without any additional treatment methods showed moderate positive shifts in the clinical and physiological parameters under study. Based on the data obtained the authors draw the conclusion about the lack of an unfavourable effect of UHF products, thinking it advisable to use such products, particularly in a complex of therapeutic measures.