CC BY
17
УДК 544.013
A. Krivtsov, U. Olsson, B. Lindman, A. Bilalov
SEPARATION OF POLYAMINE-DNA COMPLEX FROM AQUEOUS LECITHIN
Key words: spermine, spermidine, DNA, phase separation.
In order to estimate the DNA isolation effect of polyamines, spermine (Spm) and spermidine (Spd), we have investigated the apparent equilibration time of the 3 phase samples where PolyamineDNA (1:1 Amine:Phosphate ratio) complexes, SpmDNA and SpdDNA, were mixed with lecithin at the excess of water. The apparent equilibration time is higher for the SpdDNA compared with the SpmDNA system was found. The SpmDNA complex can be separated from aqueous lecithin easier than the SpdDNA complex and this difference increases with lecithin content.
Ключевые слова: спермин, спермидин, ДНК, фазовое расслоение.
С целью оценки влияния полиаминов, спермина (Spm) и спермидина (Spd), на выделение ДНК мы исследовали кажущееся время установления равновесия в 3-х фазных образцах, приготовленных путем смешения комплексов полиамин-ДНК (соотношение аминогруппа:фосфатная группа равно 1:1), SpmDNA и SpdDNA, с лецитином при избытке воды. Установлено, что кажущееся время установления равновесия выше для системы SpdDNA, чем для системы SpmDNA. Комплекс SpmDNA может быть отделен от лецитина в воде легче, чем комплекс SpdDNA и это различие усиливается с увеличением содержания лецитина.
Introduction
Spermine (Spm) and spermidine (Spd) being a Luis bases, as well as a simple amines strongly interacting with protons, easily hydrolyze in water. Thus, partly positively charge forms on the each aminogroup. Spm having 4 aminogroups has the total charge equivale to +4. Molecule of Spd, in the difference from Spm molecule, consists only 3 aminogroups. As result of dissolution in water, cation of this polyamine has maximal charge equivale to +3.
Polyamines are consisted in all eukaryotic cells and in certain viruses. Spm and Spd induce DNA condencation and stabilize DNA packaging by neutralization of DNA due to non-specific electrostatic interaction and shielding of the DNA phosphates [1]. They stabilize DNA and RNA in living cells [2]. An influence of these polyamines on biological processes does not known in details and an investigation of the effect of polyamines on DNA-lipid interactions and self-assembly "in vitro" looks interesting.
Some properties of the spermine-DNA (SpmDNA) and spermidine-DNA (SpdDNA) complexes are described in [3]. The model of the complexes [3], taking into account an average distance between charged groups of polyamine and DNA, suggests that the polyamine molecules are attracted to the surface of DNA along the main grooves of the double helix. It is known, that starting from low concentrations polyam-ines practically fully substitute inorganic monovalent counterions from the surface of DNA [1, 3].
PolyamineDNA (1:1 Amine:Phosphate ratio) complexes, SpmDNA and SpdDNA are insoluble in pure water and form white fiber-like or film-like precipitation in aqueous media [1]. The precipitation has liquid crystalline structure with hexagonal or cholesteric symmetry where the double-helix DNA molecules are packed in hexagonal order on the distance 29-33 A between rods [1].
Solubility of SpmDNA or SpdDNA in lecithin is very low as well as solubility of lecithin in the polyaminDNA complexes.
In order to estimate effect of polyamines on association of DNA with cell membrane recently [4] we investigated ternary systems where 1:1 (in terms of charge) polyamine-DNA complex salt was first component. Lecithin and water were another two components. Phase diagrams of the SpmDNA/lecithin/water system and SpdDNA/lecithin/water system were obtained. We found [4] that lamellar phase formed by lecithin in water does not solubilise SpmDNA and SpdDNA.
In this work we have demonstrated an additional supporting for our previous findings. Here we have investigated also the macrophase separation rate of the polyamineDNA/lecithin aqueous dispersions.
Results and Discussion
Coexistence of pure hydrated SpmDNA (or SpdDNA) with vesicules of lecithin in water is supported also by analysis of 35 samples from the three-phase area (polyamineDNA+Lamellar aqueous lecithin+D2O). 2H NMR spectra of these 3-phases samples consist of intensive narrow singlet [4]. From visual observation of the samples (Fig. 1) one can be noticed that upper phase is viscous turbid and slightly yellow, lower phase is white viscous fiber-like and the middle phase is transparent isotropic liquid solution. The relative volume of the phases has strong dependence from polyamineDNA/lecithin ratio at constant water content.
Lamellar phase of lecithin is typical model of the neutral phospholipid bilayers. Obviously, neutralization and compactization are not sufficient conditions for the DNA transport across neutral lipidic membranes. The hydrocarbon chain of the amphiphilic counterion can play a double role. Firstly, as mentioned in the introduction, it is responsible for a multiply charged counterion formation (micellization) that provides a strong association between counter-ion and DNA (the stability of the complex). Secondly, due to incorporation into the hydrocarbon lipidic bilayer it plays the role of an anchor that holds the counterion together with the DNA inside the neutral phospholipid membrane (Fig. 2).
Fig. 1 - Photos of the samples from the three-phase area (polyamineDNA+Lamellar aqueous leci-thin+D2O) at constant water content (80 % wt. of water) and different lecithin/SpmDNA or leci-thin/SpdDNA ratios (the Lecithin/SpDNA ratio)
[6] In the studied systems, polyamine is strongly associated with phosphatic groups of DNA. As result, the association constant for the hypothesized reaction between spermine and lecithin is very low. By another words, the neutral polyamine-DNA complex does not interact with lecithin.
It must be said that, since spermine can interact with more than one negative acidic group forming higher order complexes with DNA (for example, 2:1 cation-ic counterion-to-DNA ratio in terms of positive and negative charges), at the high excess of polyamines the positively recharged polyamine-DNA associate binds to zwitterionic phospholipids [3] (electrostatic mechanism of the embedding into the lipidic membrane).
Competition between polyamines and Ca2+ has also been suggested as a mechanism to modulate DNA solubility in negatively charged lipidic membranes formed by mixtures of acidic and zwitter-ionic phospho-lipids. [7]
Mixing on
MB vibrator i
Tor 1 min.
I
• ■III
50min lii i,sh 2h 1
Fig. 3 - Visual observation of the macrophase separation in the sample with composition 1.5:0.5:98 SpmDNA:lecithin:D2O %:% wt. The apparent equilibration time (the lightning time) is around 2 hours
Fig. 2 - Neutral SpDNA complexes (SpmDNA or SpdDNA) do not penetrate into the lamellar phase formed by neutral zwitter-ionic phospholipid (lecithin) in water, while neutral DTADNA complex can be easily solubilized by this lamellar phase due to strong hydrophobic interaction between amphiphilic counterion of DNA and phospholipid
Interaction between counterion of DNA and phospholipid play a key-role in the DNA embedding in liquid crystalline structures formed by different phos-pholipids. Binding of polyamine to lipidic bilayer appears to be a charge interaction. Positively charged pol-yamine strongly interacts with acidic phospholipid. The binding strength depends on the acidic group type, being stronger, for example, for phosphatidate than for phosphatidylserine and phosphatidylinositol; whereas no significant binding of polycations like spermine to zwitterionic lipids, e.g., phosphatidylcholine (lecithin), has been observed [5].
Positively charged polyamine (spermine or spermidine, etc.) binds most likely to negatively charged phosphate groups in a possible competition with the internal electrostatic interaction between phosphate and a sterically hindered choline group of lecithin.
lecithin / SpDNA, % wt. / % wt.
Fig. 4 -The apparent equilibration time (the lightening time) vs. lecithin / polyamine-DNA complex (SpDNA) wt. ratio in the SpmDNA:lecithin:water (the lower curve) and SpdDNA:lecithin:water (the upper curve) systems at 80 % wt. of water
Though the neutral complex polyamine-DNA is virtually insoluble in lecithin, we noticed that the macro-phase separation time depends not only from water content, but also from lecithin content in the
three-phase samples of the studied SpmDNA:lecithin:water and SpdDNA:lecithin:water ternary systems (Fig. 3, 4).
From Fig. 4 one can be noticed that the apparent equilibration time ("the lightening time") is higher for the SpdDNA compared with the SpmDNA system and this difference increases with the leci-thin/polyamine-DNA (lecithin/SpDNA) ratio. Practically, the SpmDNA complex can be separated from aqueous lecithin twice easier than the SpdDNA complex. It may be important for isolation of genetic material of the cell.
Unfortunately, these systems have not high practical interests from a gene therapy point of view, since in both of the systems the lecithin phase is free from DNA as noticed above. From the DNA-delivery point of view, since the amphiphilic DNA counterion with hydrocarbon chain has much higher permissibility with cell membrane than Spm or Spd, it is more reasonable to obtain cationic surfactant-DNA complex/lecithin microdispersions.
Experimental
Materials
Herring testes Na-deoxyribonucleic acid sodium salt (Sigma) was used as received. This DNA is highly polydisperse with an average molar mass of 700 bp, determined by electrophoresis. The concentration of DNA was determined by UV methods. The A260/A280 ratio of DNA solutions was determined to be 1.8 suggesting that DNA was free of proteins [8, 9]. Sodium bromide (Riedel-deHaen extra pure quality) was used as received. Soybean lecithin (1,2-diacyl-sn-3-phosphatidylcholine), with the trade name Epikuron 200, was obtained from Lucas Meyer (Hamburg, Germany). Its density is 1.02 g/mL and the main fatty acid component is the C-18 acid with two double bonds. The high content of unsaturated fatty acid chains (>78%) gives a chain melting point well below 0 °C. Epikuron 200 contains about 2.5% water, with the molecular weight of lecithin being 773. Lecithin contains P-carotene as an added anti-oxidant. b-Carotene is the lipophilic pigment responsible for the orange-yellow color of lecithin. Spermine and spermidine (Sigma) were used as received. The water used was from a Milli-Q filtration system (Millipore). D2O was obtained from Dr Glaser AG, Basel. Its density is 1.11 g/mL.
Preparation of the complex salts SpmDNA and
SpdDNA
DNA-polyamine aggregates were prepared by mixing equal molar amounts of negative charges of DNA and positive charges of spermine or spermidine under stirring. The fiber-like or film-like white precipi-
tate was equilibrated in solution at +4 °C for 72 h. It was then separated from the aqueous phase by centrifu-gation and washed with Millipore water. The centrifug-ing and washing procedure was continued repeatedly. The macromolecular complex salt (SpmDNA or SpdDNA) was dried for 5 days in a DW6-85 freeze dryer.
Sample preparation
Appropriate amounts of the complex salt (SpmDNA or SpdDNA), lecithin, and water (for the NMR experiments, heavy water was used) were loaded in 8 mm (i.d.) glass tubes, which were flame-sealed immediately. Firstly the components were mixed with a Vortex vibrator; the mixing was continued in a centrifuge over a few days at 4000 rpm and 40 °C, where the tubes were repeatedly turned end over end. The samples were finally left to equilibrate in a temperature-controlled room at 25 ± 0.5 °C for two to six months.
References
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2. C.W. Tabor, H. Tabor, Spermidine synthase of Escherich-ia coli: localization of the speE gene. Polyamines, Annu. Rev. Biochem., 1984. 53, 749 - 790.
3. T. Dewa, T. Asai, N. Oku, and M. Nango, Polyamine-Lipid Conjugates as Effective Gene Carriers: Chemical Structure, Morphology, and Gene Transfer Activity, Non-Viral Gene Therapy, 2011, Prof. Xubo Yuan (Ed.), ISBN: 978-953-307-538-9, InTech, Available from: http://www.intechopen.com/books/non-viral-gene thera-py/polyamine-lipid-conjugates-as-effective-genecarriers-chemical-structure-morphology-and-gene-transfer.
4. A. Krivtsov, A. Bilalov, U. Olsson, B. Lindman, Affect of the multi-charged counterion nature on solubility of DNA in the lipid membrane, Kazan State Technological University Bulletin, 2013, 16, 203 - 208.
5. B. Tadolini, G. Hakim, Interaction of polyamines with phospholipids: spermine and Ca2+. Competition for phosphatidylserine containing liposomes, Progress in pol-yamine research. Advances in experimental medicine and biology, 1988, 250, 481-490.
6. F. Momo, A. Wisniewska, R. Stevanato, EPR study of spermine interaction with multilamellar phosphatidylcholine liposomes, Biochimica et Biophysica Acta, 1995, 1240, 89-94.
7. S. K. Ballas, N.A. Mohandas, L. J. Marton, S. B. Shohet, Stabilization of erythrocyte membranes by polyamines, Proc. Nati. Acad. Sci. USA 1983, 80, 1942-1946.
8. A. Bilalov, U. Olsson, B. Lindman, A Cubic DNA-Lipid Complex, Soft Matter, 2009, 5, 3827-3830.
9. C. Leal, A. Bilalov, B. Lindman, The Effect of Postadded Ethylene Glycol Surfactants on DNA-Cationic Surfactant/Water Mesophases, J. Phys. Chem. B, 2009, 113, 9909-9914.
© A. Krivtsov - PhD, technical manager, TECHLUB Ltd., alexey_krivtsov@mail.ru; U. Olsson - Professor, Physical Chemistry, Lund University (Sweden), Ulf.Olsson@fkem1.lu.se; B. Lindman - Professor, Physical Chemistry, Lund University (Sweden), Bjorn.Lindman@fkem1.lu.se; A. Bilalov- Professor, Physical and Colloid Chemistry, Kazan National Research Technological University, azus2004@bk.ru.
© A. Кривцов - к.х.н, технический менеджер, ООО «ТЕХЛЮБ»; У. Oлссон - профессор, кафедра физической химии, университет города Лунда (Швеция); Б. Линдман - профессор, кафедра физической химии, университет города Лунда (Швеция); A. Билалов - профессор, кафедра физической и коллоидной химии, Казанский национальный исследовательский технологический университет, azus2004@bk.ru.
