Mikhail A. Voronin

Supramolecular Systems Based on Novel Mono- and Dicationic Pyrimidinic Amphiphiles and Oligonucleotides: A Self-Organization and Complexation Study

Novel mono- and dicationic pyrimidinic surfactants are synthesized and their aggregation behavior is studied by methods of tensiometry and nuclear magnetic resonance (NMR) self-diffusion. To estimate their potentiality as gene delivery agents, the complexation with oligonucleotides (ONus) is explored by dynamic light scattering (DLS) and zeta-potential titration methods and ethidium bromide exclusion experiments. Bola-type pyrimidinic amphiphile (BPM) demonstrates rather a weak affinity to ONus. Although it induces mixed associations with ONus, only slight charge compensation changes occur at a large excess of bola, with no recharging reached. Similarly, the ethydium bromide exclusion study reveals a slow increase in the binding capacity toward an ONu with an increment in BPM concentration. The monocationic pyrimidinic surfactant (MPM) and its gemini analogue (GPM-1) are ranked as intermediates in both their aggregative activity and complexing properties toward ONus. They both form mixed associates with ONus well below the critical micelle concentrations (cmcs) of 2 and 15 mM respectively. However, GPM-1 has a much lower isoelectric point at the molar ratio surfactant/ONu r~1 compared to r~3 for MPM. This probably indicates a larger electrostatic contribution to the ONu complexation in the case of GPM-1. The most hydrophobic pyrimidinic surfactant (GPM-2), bearing three alkyl tails, demonstrates enhanced aggregative activity and binding capacity toward ONus as compared to former pyrimidinic surfactants. Due to effective aggregative (low cmc of 0.04 mM) plus binding properties (fraction of bound ONu β=0.76 at r=2.5), GPM-2 may be ranked as a promising agent for wider biological applications.

Novel bolaamphiphilic pyrimidinophane as building block for design of nanosized supramolecular systems with concentration-dependent structural behavior.

A new macrocyclic bolaamphiphile with thiocytosine fragments in the molecule (B1) has been synthesized and advanced as perspective platform for the design of soft supramolecular systems. Strong concentration-dependent structural behavior is observed in the water-DMF (20% vol) solution of B1 as revealed by methods of tensiometry, conductometry, dynamic light scattering, and atomic force microscopy. Two breakpoints are observed in the surface tension isotherms. The first one, around 0.002 M, is identified as a critical micelle concentration (cmc), whereas the second critical concentration of 0.01 M is a turning point between the two models of the association involved. Large aggregates of ca. 200 nm are mostly formed beyond the cmc, whereas small micelle-like aggregates exist above 0.01 M. The growth of aggregates between these critical points occurs, resulting in a gel-like behavior. An unusual decrease in the solution pH with concentration takes place, which is assumed to originate from the steric hindrance around the B1 head groups. Because of controllable structural behavior, B1 is assumed to be a candidate for the development of biomimetic catalysts, nanocontainers, drug and gene carriers, etc.

Novel biomimetic systems based on amphiphilic compounds with a diterpenoid fragment: Role of counterions in self-assembly

Novel biomimetic systems are designed based on cationic surfactants composed of an isosteviol moiety and different counterions, namely bromide (S1) and tosylate (S2). The counterion structure is shown to play a crucial role in the surfactant association. A number of methods used provide evidence that only one type of aggregate, i.e., micelles are observed in the S2 systems, while a concentration-dependent association occurs in the case of S1. The DLS and fluorescence anisotropy measurements reveal that the micelle-vesicle-micelle transitions probably occur with the S1 system. The occurrence of small aggregates near the critical micelle concentration with radii of 2.5 nm is supported by NMR self-diffusion data. The Orange OT solubilization results strongly support the idea of a second threshold in the S1 system around 0.025 mM and provide evidence that hydrophobic domains occur in the aggregates. The latter property and the capacity to integrate with the lipid bilayer make it possible to suggest the newly synthesized surfactants as effective nanocontainers for hydrophobic guests.

Genetic differentiation of cercariae infrapopulations of the avian schistosome Trichobilharzia szidati based on RAPD markers and mitochondrial cox1 gene

Avian schistosome Trichobilharzia szidati is a member of the largest genus within the family Schistosomatidae (Trematoda). Population genetic structure of Trichobilharzia spp. schistosomes, causative agents of cercarial dermatitis in humans, has not been studied yet. The knowledge of the genetic structure of trichobilharzian populations is essential for understanding the host–parasite coevolutionary dynamics and epidemiology strategies. Here we examined genetic diversity in three geographically isolated local populations of T. szidati cercariae inhabiting Russia based on nuclear (randomly amplified polymorphic DNA, RAPD) and mt (cox1) markers. We analyzed T. szidati cercariae shed from seven naturally infected snails of Lymnaea stagnalis. Using three random primers, we demonstrated genetic variation among populations, thus posing genetic structure across geographic sites. Moreover, T. szidati cercariae have been genetically structured among hosts (infrapopulations). Molecular variance analysis was performed to test the significance of genetic differentiation within and between local populations. Of total parasitic diversity, 18.8% was partitioned between populations, whereas the higher contribution (48.9%) corresponds to the differences among individual cercariae within infrapopulations. In contrast to RAPD markers, a 1,125-bp fragment of cox1 mt gene failed to provide any significant within-species structure. The lack of geographic structuring was detected using unique haplotypes which were determined in the current work for Moscow and Western Siberian local populations as well as obtained previously for European isolates (Czech Republic and Germany). All T. szidati/Trichobilharzia ocellata haplotypes were found to be mixed across their geographical origin.

Tunable biomimetic systems based on a novel amphiphilic pyrimidinophane and a helper nonionic surfactant

Tunable nanosystems based on a novel water insoluble pyrimidinic amphiphile are designed. pH dependent aggregates composed of protonated pyrimidinophane 1 are formed at pH<4, which undergo reversible transition to precipitate at neutral and basic conditions. The approach assuming the application of a helper nonionic surfactant Triton-X-100 (TX-100) is used in this work. Different models of a self-assembly were found depending on the molar ratio of components and solution pH. In the equimolar 1-TX-100 solution, mixed assemblies contributed by aggregated molecules of both TX-100 and cationic form of 1 are formed in acidic conditions. Upon alkalization, deprotonated pyrimidinophane molecules shift toward the micellar core. The assemblies undergo reversible precipitation after 4-5h, while the excess of TX-100 leads to the formation of highly stable mixed aggregates. The acidification-alkalization cycles followed by the aggregation/precipitation and the re-charging of aggregates can be multiply repeated. Surprisingly, stable mixed aggregates are also formed under the excess of pyrimidinophane in both the acidic and alkaline conditions, but at a certain component ratio. They are characterized by the highest micellization degree among all the systems studied. The low concentration threshold of these assemblies in alkali solution is probably due to their nonionic character.

Cationic gemini surfactants as new agents for plasmid DNA delivery into cells

197 Gene therapy has a great potential for treating many human diseases that are currently considered incurable. Viral vectors based on adenoviruses or rett roviruses are very effective in gene delivery. Neverthe less, in view of the problems associated with immunoo genicity and biosafety of viral vectors, there remains a need to develop nonviral vectors. As an alternative to viral vectors, cationic polymers [1–3] and liposomes [4], which bind to DNA through electrostatic interacc tions and form nanosized complexes, have been invess tigated [5, 6]. Cationic agents protect DNA from degg radation by nucleases and serve as mediators in the penetration into the cell and subsequent release from endosomes, which increases the efficiency of transfecc tion. The use of cationic surfactants as nonviral vectors was described in several papers [6, 7]. It is shown that they are effective agents that provide the compaction of DNA and recharging of the complex. Despite the obvious advantages (simplicity of synthesis and forr mulation, low concentration, availability, and high complexing ability), a significant drawback of synn thetic agents based on cationic surfactants is the low transfection efficiency. In view of this, the search for new cationic vector systems is a relevant problem. In this study, we investigated alkylammonium gemm ine surfactants (AGSs) with the formula , where m = 4–12, R = СН 3 , С 2 Н 4 ОН, R 1 = nC n Н 2n + 1 , where n = 10, 12, 14, and 16. Aqueous solutions of these compounds ensure efficient delivery of DNA into cells. Gemine surfactants (alkanedyllα,ω bis(methyldialkylammonium bromides)) were pree pared under laboratory conditions as described in [8]. The structure of the obtained compounds was conn firmed by elemental analysis as well as by IR and NMR spectroscopy data. Cell line 293T (human embryonic kidney epithee lium cells), which was used as a producer of pseudoo lentiviruses and as a model target cell line, was mainn tained by the standard procedure. We used the peGFPPN1 plasmid (Clontech, United States), which is 5100 bp in length and expresses the green fluoress cent protein, and the pCIINEO plasmiddbased vector (Promega, United States), which is 5000 bp in length and does not contain the green fluorescent protein gene. HEK293T cells were replated 40 thousand cells per well one day before transfection. To transfect HEK293T cells, 50–100 thousand cells per well of a 966well plate were used. One hour before transfection, …

Amphiphilic macrocycles bearing biofragment: Molecular design as factor controlling self-assembly

Two novel macrocyclic 6-methyluracilic amphiphiles (uracilophanes) with four (UP1) and two (UP2) uracil moieties and ammonium groups have been synthesized. Tetracationic multi-uracilophane is composed of two macrocyclic units bridged each other with an external methylene spacer, while in the cryptand-like dicationic uracilophane pyrimidinic moieties are connected with an internal methylene spacer. This internal spacer provided a conformational rigidity to the macrocycle. The self-assembly of the uracilophanes is studied and compared with a reference dicationic uracilophane (UP3) with no spacer fragment. Compounds UP1 and UP3 are capable of aggregating, which is characterized by the analogous critical micelle concentration of 1mM, although the former has four decyl tails versus two decyl tails in UP3 molecule. NMR self-diffusion, fluorimetry and DLS techniques revealed that bimodal size distribution occurs in the UP1 solution, with small (≤2nm) and large (ca. 30-50 nm) aggregates contributed. Unexpectedly, the cryptand-like uracilophane UP2 with the same hydrophobicity as UP3 does not form aggregates. The balance of the geometry and energetic factors was analyzed and compared with those contributing to the aggregation of the reference compound UP3. It was established that it is the geometry that controls the packing of the cryptand-like uracilophanes upon aggregation, while hydrophobic effect plays a minor role. In contrast, both factors control the aggregation of oligomeric macrocycle, with energetic factor prevailing. These findings are of importance for (i) the understanding the diverse structural behavior of bioamphiphiles that have very similar chemical structure, but different conformations; and (ii) the design of amphiphiles with controlled model of self-assembly. Supramolecular systems studied can be recommended for biotechnological applications.

Regulation of the rate of hydrolysis of phosphorus acid esters in organized systems based on amphiphilic pyrimidinophanes

The aggregation and catalytic activity of supramolecular systems based on new macrocyclic dimeric surfactants (pyrimidinophanes) was studied both in the absence and in the presence of polyethyleneimine (PEI). It was found that the critical micelle concentrations measured by tensiometry were independent of the structure of surfactants. The morphology of aggregates was responsible for various characters of the catalytic effects of pyrimidinophanes in the hydrolysis of phosphonic acid esters. A less hydrophobic pyrimidinophane exhibited a typical effect of cationic surfactants to accelerate the reaction both in the absence and in the presence of PEI. Unlike cationic micelles, a heminal-type pyrimidinophane exhibited an anomalous behavior: it had no effect on the rates of hydrolysis of the substrates and did not inhibit the hydrolysis. Upon the addition of lanthanum ions, the catalytic activity of dimeric surfactants increased. The overall catalytic effect due to the action of supramolecular systems based on pyrimidinophanes, PEI, and lanthanum ions can increase the rates of hydrolysis of the substrates by three orders of magnitude, as compared with that of alkaline hydrolysis.

Counterion effect in SDS–calix[4]resorcinarene micelles

Here we report the clouding phenomenon, conductivity, (1)H NMR spectroscopy and dynamic light scattering data of mixed sodium dodecylsulfate-calix[4]resorcinarene micellar solutions in combination with tetrabutyl-, tetraethyl-, tetramethylammonium bromides and choline chloride. The NMR data reveal tight (the binding constant is about 10(4)) guest-host binding of calix[4]resorcinarene in alkaline conditions with tetraethyl-, tetramethylammonium and choline cations. This binding occurs at rather small concentrations of the external quaternary salts (0-0.0005 M) and leads to dramatic increase of the cloud point temperature. The obtained data are discussed in the correlation with the micellar size changes, caused by the ion exchange due to the variation of quaternary salt concentrations.

Monolayers and Langmuir–Blodgett films of amphiphilic tetramethylsulphonatocalix[4]resorcinarene and their interactions with polyethyleneimine and caeruloplasmin

The paper deals with the surface films of amphiphilic tetramethylsulphonatocalixresorcinarene (R = C11H23) 1 insoluble in water but forms rigid stable non-collapsing films at the water–air interface. Compression isotherms were used to investigate the interactions of the macrocycle films with two polymers fed to the aqueous subphases: synthetic – polyethyleneimine (PEI) and natural – an oxidase enzyme caeruloplasmin (CP). The interactions of the surface films of 1 with these substrates are predominantly dictated by the nature of the macromolecules and not by macrocycle interactions with their individual fragments. CP having retained its globular structure was extracted in the layer of 1 and became the dominating component of the film. The synthetic macromolecule of PEI, six times lower in its weight than CP, did not affect the morphology of the interfacial film, involved in the interactions only with its hydrophilic part directed inside the water.