CC BY
21
УДК 544.013
A. Krivtsov, U. Olsson, B. Lindman, A. Bilalov
COLLOIDAL STABILITY OF THE SURRFACTANT/LIPID/DNA PARTICLES
Key words: DNA; cationic surfactant, lecithin, colloidal stability.
The DNA incorporated 1 ¡um size vesicular multi-lamellar aggregates were obtained. Colloidal stability of the cationic surfactant-DNA/lecithin self-assemblies obtained by "solvent shifting" method increases with lecithin content was found.
Ключевые слова: ДНК,катионное ПАВ, лецитин, коллоидная устойчивость.
Получены мультиламеллярные везикулярные агрегаты размером порядка 1 мкм содержащие ДНК. Установлено, что коллоидная устойчивость самоассоциатов катионное ПАВ -ДНК/лецитин полученных методом "solvent shifting" увеличивается с увеличением содержания лецитина.
Introduction
Double helix DNA is highly charged rigid polymer. As result, a water solubility of DNA is countered by entropic gain from dissociated counterions, while a gain from conformational entropy of the polymer chain is very small. DNA associates strongly with multicharged cations, such as, e.g., multicharged metal ions, positively charged polyelectrolytes (polycations), linear multivalent polyamines (spermine, spermidine), and cationic surfactants and lipids, which self-assembly with formation of multicharged assembles (micelles). This work is focused on the last category of the counterions.
Interaction between counterion of DNA and phospholipid play a key-role in the DNA embedding in liquid crystalline structures formed by different phospholipids. For example, in the ternary system with dodecyltrimethylammonium (DTA) as counterion of DNA at the presence of lecithin in aqueous media three liquid crystalline (LC) phases were found: lamellar, reversed hexagonal and cubic [1].
Important transfection agents, lipoplexes, are based on a mixture of cationic amphiphiles (surfactants or lipids) and neutral zwitterionic lipids [2]. In a gene-delivery vectors discovery, a lipoplex particle size has a major impact on bioassays and formulation for in vivo dosing. Furthermore, the medical procedure of the lipoplex injection demands syringe-able form of last. A good goal for the size of a lipoplex discovery particles is 100 nm.
Here we have investigated the colloidal stability of the DNA-lipid microdispersions. Since DTADNA is much studied complex among others, we focused our attention on the study of colloidal stability of the modeling lipoplex particles forming in the DTADNA:lecithin:water system. In order to obtain microdispersions we used "solvent shifting" method.
Results and Discussion
The mechanism of particle formation at "solvent shifting" method was published in [3, 4]. The key significance for the particle size has the DTADNA content in the stock solution, which then must be rapidly mixed with the stabilizer solution. Ethanol was used as a good solvent for the DTADNA on the base of the
DTADNA/ethanol/water phase diagram obtained previously [5]. In ethanol, more than 70 wt % of the complex can be solubilized. We kept condition of the diluted polyelectrolyte solution. With this aim the "overlap concentration" of the DTADNA was estimated experimentally by using rheometry, z-potential measurements and DLS and was calculated theoretically using equations presented in the experimental section. Results of the experimental estimations of the "overlap concentration" are presented in Appendix. The overlap concentration- Si =1.4%wt.
To minimize the Ostwald ripening effect the DTADNA was dissolved in ethanol, and the resulting stock solution was then rapidly mixed with an aqueous stabilizer solution of the vesicular lecithin. Ostwald ripening is a process where the difference in (local) solubility with particle size leads to a transport of material from small to larger particles, with an accompanying increase in the mean particle size with time. The incorporation of a second component with low aqueous solubility leads to a difference in composition between large and small particles during the Ostwald ripening process. This difference may counterbalance the driving force for Ostwald ripening and eventually results termination of it. In our system, Ostwald ripening was inhibited by self assemble process resulting in formation of multilamellar vesicules by lecithin and DTA. DNA is incorporated between lamellas formed by surfactants.
Thus, we injected 100 microliters of the 1.4 % wt. DTADNA ethanolic solution into the 900 microliters of aqueous lecithin and measured the average size of particles starting from the injection time using DLS. Lecithin content was varied between 1 and 6 % wt. The DTA/lecithin mol/mol ratio was varied between 1.5 and 0.2. Dependences of the average diameter of particles vs. time from the beginning of injection, the kinetic curves, are shown on fig. 1.
Three periods can be distinguished dependently from character of the kinetic curve behavior. The initial period, where the size of the particles has lowest values and does not change or grows very slowly; the aggregation period, where the value of the average diameter grows sharply during 2 hours from 1 up to 11-14 micrometers; the last period, where particles reach the maximal size and the average diameter then does not
change or changes very slowly. Given that increasing of the initial period is observed when the lecithin content is increased, it is reasonable to assume that the aggregative stability of the colloidal DTADNA increases with decreasing of the DTA/lecithin molar ratio. At the DTA/lecithin molar ratio higher than 1, the initial period is very short (certain seconds), while at the excess of lecithin this period can be prolonged till hours and days. The initial period becoming higher than 1 day starting from the 1/6 DTADNA/lecithin weight ratio.
DTADNA:ethanol:water systems at high water contents where lamellar phase coexists with isotropic solution in accordance with the phase diagrams of the both systems.
Fig. 1 - The average particle size (dynamic light scattering data) as function from the time after injection of DTADNA ethanolic solution into the vesicular aqueous solution of lecithin. The DTADNA/lecithin wt. ratio is shown in the window
Three periods can be distinguished dependently from character of the kinetic curve behavior. The initial period, where the size of the particles has lowest values and does not change or grows very slowly; the aggregation period, where the value of the average diameter grows sharply during 2 hours from 1 up to 11-14 micrometers; the last period, where particles reach the maximal size and the average diameter then does not change or changes very slowly. Given that increasing of the initial period is observed when the lecithin content is increased, it is reasonable to assume that the aggregative stability of the colloidal DTADNA increases with decreasing of the DTA/lecithin molar ratio. At the DTA/lecithin molar ratio higher than 1, the initial period is very short (certain seconds), while at the excess of lecithin this period can be prolonged till hours and days. The initial period becoming higher than 1 day starting from the 1/6 DTADNA/lecithin weight ratio.
In order to know structure of the colloidal particles we separated phases by centrifuging and analyzed a dispersive phase of the particles by small angle X-ray scattering (SAXS). In fig. 2 we present a typical SAXS pattern for this paste-like phase. Two Bragg peaks can be identified in the diffraction pattern (fig. 2).
The relative positions of the Bragg peaks, at 1 : 2, indicate the existence of a lamellar phase. Position of the first Bragg peak correlates with a peak position obtained for similar paste-like phase from a two-phase sample (water + lamellar phase) of the DTADNA/lecithin/water system (fig. 3). This would be expected for the DTADNA:lecithin:ethanol:water system that can be viewed as reflection of the phase behavior in the DTADNA:lecithin:water and
Fig. 2 - SAXS diffraction patterns for the dispersed phase from the DTADNA/lecithin 1/6 wt./wt. dispertion obtained by "solvent shifting" method
Fig. 3 - SAXS diffraction patterns for sample with composition 4:48:48 DTADNA:lecithin:water %:% wt. from the two-phase area (water + lamellar LC phase) of the DTADNA/lecithin/water phase diagram [1]
Thus, we obtained colloidal DNA embedded into the multilamellar aqueous DTA/lecithin aggregates with 1-micrometer average size, which is stable at least during 1 day that is a good condition for lipoplex formulation. Schematic illustration of the transformation of the DTADNA structures found in the system during "solvent shifting" procedure is shown on representation of the phase diagram obtained in previous studies (fig. 4).
The transformation of the DTADNA structures found in the studied system during "solvent shifting" procedure is shown by arrows. The DTADNA was dissolved in ethanol, and the resulting stock solution was then rapidly mixed with an aqueous stabilizer solution of the aqueous lecithin (going from the left bottom to the left top and then to the right). As result, after injection of the ethanolic DTADNA into the vesicular aqueous lecithin solution, the DNA was embedded within the water layers in the multi-lamellar structure of DTA-lecithin vesicular dispersions.
Fig. 4 - Principle schematic drawing of possible structural transitions in the
DTADNA/lecithin/ethanol/water system
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 [6, 7]. Sodium bromide (Riedel-deHaen extra pure quality) was used as received. The cationic surfactant,
dodecyltrimethylammonium bromide (DTAB), was used as received and obtained from Tokyo Kasei Kogyo Co., Ltd. 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/1 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. Ethanol (Aa purity 99,7 %) was obtained from Solveco and used as received. The water used was from a Milli-Q filtration system (Millipore).
Preparation of the complex salt DTADNA
DNA solutions were prepared by weighing the desired amount and dissolving it in 10 mM NaBr. The pH of all solutions was 7±0.2. DNA-surfactant aggregates were prepared by mixing equal molar amounts of negative charges of DNA and positive charges of DTA (200 mL of 5 mM solutions) under stirring. Under these conditions, the counterion release should be maximal and nearly complete. The precipitate was equilibrated in solution for 48 h. It was then separated from the aqueous phase by filtration and washed extensively with Millipore water. The macromolecular complex salt
(DTADNA) was dried for 5 days in a DW6-85 freeze dryer.
Preparation of the DTADNA-lecithin microdispersions
To disperse DTADNA in aqueous media we designed fast and easy method based on "solvent shifting" technique. To obtain a micro-dispersion, the DTADNA was firstly dissolved in ethanol, and the resulting solution was then rapidly mixed with an aqueous stabilizer solution. To choose optimal concentration of the DTADNA in ethanolic solution for "solvent shifting" procedure the DTADNA overlap concentration was estimated in advance by using dynamic light scattering (DLS), z-potential measuring and viscosimetry methods, described below. The overlap concentration (OC) for DTADNA in ethanol was around 1 % wt. Theoretical layouts for calculation of OC are following.
OC is the quantity of polymer per volume unit at which polymer occupy all volume but no overlap occurs yet. This condition described by following equation:
v , =yv~-
sol. ag.1
where Vsol. is the volume unit of solution; Vag. is the hy-drodynamic volume of averaged polymer macromole-cule, calculated by simple geometric equation:
- 4 —3
Vag. = 3 x xx Rh ;
Exact hydrodynamic radius of DTADNA can be calculated using bead model. In this model DNA macromolecule assumed as chain of number of equal sized spheres of linear shape. This theory calculates hydrodynamic radii using following equation:
Rh =-=i-x (^X
M2^) - 0,45 d
where Lext. is the extended length of DTADNA; d* is the diameter of hydrated DTADNA.
Average size of DTADNA inherited after DNA, which is about 700 base-pairs. Each base-pair have size of 3,4 A [8]. Consequently, average length of DTADNA is about 240 nm. Diameter of complex salt is about 5,2 nm, which is the sum of two length of DTA molecule (which is about 1,6 nm) and diameter of DNA rod (which is around 2 nm).
Averaged hydrodynamic radius equals 29,75 nm. Calculations of overlap concentration of DTADNA were performed using following equation:
overlap ^^'^erADh'A^^n] DTADNA
■ t1
where M j ^ is the molar weight of monomer (two bases + two attached DTA cations) of DTADNA and equals 1116 g/mol; nm is the average number of monomers, about 700; NA is Avogadro number; p is the ethanol density, at room temperature is 0,789 g/ml; Rh is the hydrodynamic radius.
Resulting theoretical overlap concentration of DTADNA is about 1,45 % wt. Thus, theoretical and experimental results are in a good agreement; therefore we can conclude that optimal concentration of
DTADNA in ethanol solution for "solvent shifting" procedure equals or below 1 % wt.
Methods
Small Angle X-ray scattering (SAXS).
SAXS measurements were performed on a Kratky compact small-angle system equipped with a position sensitive detector (OED 50M from M Braun, Graz, Austria) containing 1024 channels with 53.0 mm width. Cu Ka radiation of wavelength 1.542 A was provided by a Seifert ID300 X-ray generator operating at 50 kV and 40 mA. A 10 mm thick nickel filter was used to remove the Kb radiation, and a 1.55 mm tungsten filter was used to protect the detector from the primary beam. The sample-to-detector distance was 277 mm. The camera was kept under vacuum during data collection in order to minimize the background scattering. The temperature was kept constant at 25 °C (±0.1 °C) with a Peltier element.
Microscopy.
Zeiss Axioplan Universal microscope equipped with differential interference contrast and a High Resolution Microscopy Camera AxioCam MRm Rev. 3 FireWire, Illuminator HBO 100 as well as with a 100W mercury short-arc lamp and a system of filters to allow the fluorescence microscopy observations. The microscope is further equipped with a high-sensitivity SIT video camera and an image processor, AxioVision 4 together with the Macintosh-based image.
Colloidal stability estimation
Main subjects of our colloidal stability studies were diluted colloidal systems comprising particles of micrometer scale obtained by "solvent shifting" method. Most of colloids were transparent and we used light scattering method to characterize particle size.
Dynamic light scattering (DLS) and z-potential measurement.
Experiments were carried out on a «Zetasizer Nano ZS» from «Malvern Instruments Ltd», Worshestershire, UK. The instruments measures DLS and SLS at a set angle of 173° using the NIBS technol-
°gy.
3D Dynamic light scattering DLS.
To avoid influence of multiple scattering in case of comparably turbid sample 3D DLS was applied. The instrument is equipped with a HeNe laser light source with a wavelength of 632.8 nm and a maximum power of 35 mW. The sample is filled into cylindrical glass cells of a diameter of 3.5 mm cells and placed in the temperature controlled index-matching bath. 3D DLS measurements were performed at 20 °C. The scattered light is detected at angle of 1350 by two efficient Avalanche Photo Diodes and processed by a Flex correlator in a 3D cross-correlation configuration. In aqueous samples we have access to scattering vectors between 0.0034 and 0.025 nm-1.
The overlap concentration of the DTADNA in ethanol was estimated experimentally by two independent methods, z-potential measuring and rheometry.
The zeta-potential (or electrophoretic mobility) measurements using M3-PALS technology are performed at a set angle of 17° on a «Zetasizer Nano ZS». The laser used is a 4 mW He-Ne laser (632.8 nm) and the detection unit comprises an avalanche photodiode.
The samples were filled into 10 mm width polypropylene cells. The temperature range of the instrument is 290 °C. DLS measurements were performed at 20 °C.
Rheometry.
The rheological studies of the DTADNA ethanolic solutions were performed on Advanced Rheometric Expansion System (ARES) true strain-controlled instrument, where the application of strain and the measurement of stress are separated. Solutions were studied in coquette geometry (32 mm bob and 34 mm cup). The instrument is implemented with the transducer 1 K FRT (torque range 0.004 - 20.0 g-cm and normal force range 2.0 - 2000.0 gmf). Rheological measurements were performed at 20 °C with a temperature stability at thermal equilibrium of ±0.1 °C, which is controlled with a Peltier system.
Appendix
DTA DNA/ethanol binary system was studied using dynamic light scattering (DLS), z-potential measuring and viscosimetry methods.
DLS data interpretation was carried out with taking into account a high polydispersity of DNA mac-romolecules. We were not interested in exact size of particles, we studied tendency of size changing with concentration. On Fig. A1 correlation functions of solutions with different DTADNA concentration are plotted. Stepwise changing of shape of curves testifies gain of average relaxation time with concentration. However this effect was not observed for the DTADNA concentration range below 1 weight percent. All samples from mentioned composition range were characterized by constant relaxation time.
o,o- V\ / ___1 _;_i,___2/
1.........I-1 1 1 '""I-1 < 1 '""I- 1 1 '""I-' 1 1 ""'I-1 1 ' '""I-1-,-r'
1E-6 1E-5 1E-4 1E-3 0,01 0,1 1
Time (sec.)
Fig. A1 - Corrected correlation functions for samples from DTADNA/ethanol system with different DTADNA concentration (% wt.)
The relaxation time is in inverse ratio to diffusion coefficient, which is bound with hydrodynamic radius of the DTA-DNA aggregates according to Stokes-Einstein equation. Therefore, in accordance with obtained correlogramms (Fig. A1), hydrodynamic radii of aggregates decreases with DTADNA concentration till 1% wt. and below 1% wt. it remains constant.
This border between two states of a polymer solution we refer to overlap concentration of
DTADNA. Obviously, above concentration of 1 % wt. macromolecules overlap and form aggregates. Below estimated overlap concentration macromolecules of DTADNA exist in diluted regime.
Apart of DLS, z-potential of the DTADNA/ethanol aggregates was measured at different concentrations. At Fig. A2 z-potential as function from the DTA-DNA concentration is shown.
ос,о о -
0123-15678 Concentration of DTA-DNA (wt.%)
Fig. A2 - Z-potential of DTADNA/ethanol system vs. DTA-DNA concentration
Z-potential of the particles is negative in over the studied concentrations. Above 1 % wt. z-potential dicreases monotonically with decreasing of the DTADNA content, while below 1 % wt. z-potential riches minimal constant value (maximal negative charge). Obviously, a big part of amphiphilic cations are bound with DNA only slightly and these cations may be easily cut of the macroanion by weak electric field. This behavior is typical for simple counterion condensation of polyelectrolytes with non-specific interactions.
Also rheology of DTADNA complex ethanol solution was studied. Viscosity increases with concentration (Fig. A3). On this graph dependence curve for low concentrations (below 7 wt. %) can be distinguished into two regions: diluted region below 1 wt. % behaves as Neutonian liquid and concentrated region where dependence has complicated shape.
Fig. A3 - Viscosity of the DTADNA ethanolic solutions vs. concentration of the DTADNA complex
References
1. A. Bilalov, U. Olsson, B. Lindmann, DNA-lipid self-assembly: phase behavior and phase structures of a DNA-surfactant complex mixed with lecithin and water, Soft Matter, 2011, 7, 730-742.
2. G. Montalvo, A. Khan, Self-Assmbly of Mixed Ionic and Zwitterionic Amphiphiles: Associative and Dissociative Interactions between Lamellar Phases, Langmuir, 2002, 18, 8330-8339.
3. L. Lindfors, P. Skantze, U. Skantze, M. Rasmusson, A. Zackrisson, and U. Olsson, Amorphous Drug Nanosuspensions. 1. Inhibition of Ostwald Ripening, Langmuir, 2006, 22, 906-910.
4. L. Lindfors, S. Forsse'n, P. Skantze, U. Skantze, A. Zackrisson, and U. Olsson, Amorphous Drug Nanosuspensions. 2. Experimental Determination of Bulk Monomer Concentrations, Langmuir, 2006, 22, 911-916.
5. C. Leal, A. Bilalov, and B. Lindman, Phase Behavior of a DNA-Based Surfactant Mixed with Water and n-Alcohols, J. Phys. Chem. B, 2006, 110, 17221-17229.
6. A. Bilalov, U. Olsson, B. Lindman, A Cubic DNA-Lipid Complex, Soft Matter, 2009, 5, 3827-3830.
7. 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.
8. A. R. Thierry, V. Norris, F. Molina, and M. Schmutz, Lipoplex nanostructures reveal a general self-organization of nucleic acids, Biochim. Biophys. Acta, 2009, 1790, 385-394.
© 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. Олссон - 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.
