Soft Hybrid Nanostructures Composed of Phospholipid Liposomes Decorated with Oligonucleotides

C H A P T E R

T H I R T E E N

Soft Hybrid Nanostructures Composed of Phospholipid Liposomes Decorated with Oligonucleotides Martina Banchelli, Francesca Baldelli Bombelli, Debora Berti, and Piero Baglioni Contents 250 251 252 253 253 254 256

1. 2. 3. 4.

Introduction Materials Liposome Preparation and Determination of Lipid Content Incorporation of Oligonucleotides 4.1. Choice of the lipid anchor 4.2. Choice of the grafting density 5. Characterization of the Soft Hybrid Nanostructure 5.1. Purification of the liposome–oligonucleotide systems (size exclusion chromatography) 5.2. Dynamic light scattering measurements 6. Applications of Oligo-Decorated Liposomes 6.1. Hybridization with complementary oligonucleotides in solution 6.2. Hybridization with self-assembled DNA nanostructures 6.3. Different preparation procedures 6.4. Kinetics aspects 7. Challenges and Perspectives Acknowledgments References

256 259 262 262 265 266 273 275 276 276

Abstract This chapter reports on the design, preparation, and characterization of liposomes decorated with synthetic lipid–oligonucleotide conjugates. Several key parameters should be considered for a successful preparation of these

Department of Chemistry and CSGI, University of Florence, Sesto Fiorentino, Florence, Italy Methods in Enzymology, Volume 464 ISSN 0076-6879, DOI: 10.1016/S0076-6879(09)64013-1

#

2009 Elsevier Inc. All rights reserved.

249

250

Martina Banchelli et al.

functional nanostructures that can be employed further as building blocks in DNA-directed assembly of nano-objects. These parameters are reviewed explicitly in this report and their contributions are discussed.

1. Introduction The insertion of synthetic lipid–oligonucleotide conjugates into fluid amphiphilic surfaces, such as lipid vesicles, is currently gaining increasing interest for DNA-directed self-assembly into nanometer-scaled arrays of functional objects. The structural and functional properties of such nanomaterials are defined through the choice of the sequence of the oligonucleotide decoration and encoded by base pairing. These hybrid soft nanomaterials merge the unique features offered by DNA structural fidelity and specificity, to the characteristics of lipid self-assembly, in terms of ease of preparation, responsiveness and hierarchical aggregation in functional arrays of nano units (Chan et al., 2008). In the design of DNA/membrane hybrids, several points yet remain to be addressed, as the guidelines for the choice of the anchoring unit, which is the lipophilic portion of the oligonucleotide conjugate. For instance, the hydrophobic portion can be directly attached to the oligonucleotide or can be separated from the functional part by a hydrophilic spacer acting as a flexible joint that guarantees conformational freedom to the oligonucleotide. In terms of its length, the choice of the oligonucleotide portion, linked to the spacer–assembler portions through its 50 or 30 end, is mainly guided by the applications purposes and is responsible for the coupling specificity and efficiency. Lipid–oligonucleotide molecules are currently being used for two different purposes: (i) to direct their assembly to soft surfaces (e.g., SLB, supported lipid bilayer or other vesicles) (Pfeiffer and Ho¨o¨k, 2004; Stengel et al., 2007) regulated by several different mechanisms as reversible clustering (Beales and Kyle Vanderlick, 2007) or vesicle fusion (Chan et al., 2008), depending on the above-mentioned structural parameters of the synthetic lipid conjugate (Chan et al., 2009) and (ii) liposomes or SLB as scaffolds of DNA nanostructures to build soft DNA/lipid hybrids (Baldelli Bombelli et al., 2009). Our group has recently studied the incorporation of a cholesteryltetraethylene glycol (TEG) functionalized oligonucleotide in phospholipid vesicles (Banchelli et al., 2008). Its hybridization with a complementary strand has also been investigated in detail. The results have been interpreted in terms of the average distance between noncovalent grafting sites onto the membrane. Both the oligonucleotide conformation and the hybridization kinetics are strongly dependent on macromolecular crowding at the liposomal surface. These investigations have been extended to the construction

Liposomes Decorated with Oligonucleotides

251

of a lipid membrane/pseudohexagonal DNA hybrid, anchored to the membrane, thanks to a cholesteryl-TEG functionalization of one of the single strands. The effects of grafting density, lipid/DNA ratio, liposome number density, and preparation procedure on the final structure and yield of the resulting hybrid nanomaterial, are demonstrated. A particularly interesting result is the fact that above a critical grafting density, connected to the lipophilic oligonucleotide excluded area, coupling kinetics are slowed down with respect to strand pairing in solution. Conversely, when the average interoligonucleotide density on the liposomal surface is low, coupling is faster than in bulk medium. In this chapter, we will describe the preparation and structural characterization of oligonucleotide-decorated liposomes with particular emphasis on the experimental procedure and on the control parameters that will ultimately lead to the preparation of well-characterized systems, intended as building blocks for further assembly into nanostructured arrays. As an applicative example, we will report on their use as scaffolding hosts for DNA nanostructures that we have investigated in our laboratory, again stressing how the experimental preparation protocol affects the final yield of the hybrid nanostructures.

2. Materials POPC (1-palmitoyl, 2-oleoyl-sn-glycero-3-phosphocholine) is purchased from Avanti Polar Lipids Inc. (Alabama). Its purity is checked by TLC, to ensure the absence of oxidation or lysis products, and the lipid is used as received if it is intact. All other chemicals (TRIS base, NaCl) are purchased from Fluka (Milan, Italy) at the highest purity commercially available. All the modified and unmodified oligonucleotides presented in this chapter are synthesized with an Applied Biosystems 394 automated DNA/RNA synthesizer at the School of Chemistry in Southampton in the group of professor Tom Brown. Chemical modifications are introduced using the appropriate phosphoramidite monomers and incorporating them during oligonucleotide assembly. Purification of oligonucleotides is carried out by reversed phase HPLC (Banchelli et al., 2008). Cholesterol-derivative oligonucleotides (18-mers) are synthesized by modifying one end of the oligonucleotide sequence (50 -end or 30 -end). An oligoethylene glycol spacer is attached between the oligonucleotide sequence and the cholesteryl group. In this way, the hydrophilic group forms a bridge between the lipophilic group and the phosphate backbone of the DNA chain and enables the oligonucleotide to interact with lipid membranes and, at the same time, to remain in a hydrophilic environment at a fixed distance from the lipid surface.

252

Martina Banchelli et al.

3. Liposome Preparation and Determination of Lipid Content Phospholipid vesicles are generally not a thermodynamically stable amphiphilic phase, and do not form spontaneously; they are metastable structures with a shelf life of several months. The physical properties of lipid vesicles very much depend on how and under which conditions they are prepared. Thus, the mean size, the lamellarity, and the physical stability of the vesicles not only depend on the chemical structure of the lipid used, but particularly on the method of vesicle preparation as well. Physical instabilities of lipid vesicle systems involve vesicle aggregation and fusion. For the study here presented, POPC has been chosen as the lipid for vesicle preparation. POPC is a major component of biological membranes and it has a lamellar gel-to-liquid crystalline phase transition temperature Tm of 2.5  C. At 25  C the POPC bilayer is fluid, hence it is characterized by high lateral and rotational lipid diffusion rather similar to a liquid (Cevc, 1993). This lipid fluid state provides in biological membranes the optimal environment to host membrane proteins. Small unilamellar POPC vesicles with a mean hydrodynamic diameter of about 60–70 nm are prepared for the incorporation of the cholesteryl– oligonucleotides. The vesicles are prepared by the FAT-VET50 method. First, the lipids are dissolved in a chloroform:methanol (5:1) mixture and the organic solvent is completely removed by rotatory evaporation and high vacuum drying in a round bottom flask. To the formed thin, dry lipid film is added a 50 mM of TRIS and 100 mM of NaCl at pH 7.5 aqueous solution. Vigorous shaking with the help of a Vortex mixer leads to the dispersion of the lipid multilayers (MLV) in the aqueous solution, which results in the formation of a heterogenous population of vesicles. Upon repetitively freezing the MLV suspension in liquid nitrogen (at 195  C) and thawing at 45  C (far above the Tm of POPC), the vesicles’ aqueous interior and the external bulk aqueous phase equilibrate and possibly a fragmentation of MLV into smaller vesicles is achieved. Freezing and thawing (FAT) cycles are repeated six times. The MLV suspension is then passed under a stream of N2 at moderate pressure repetitively (10 times) through track-etch polycarbonate filters which contain almost cylindrical pores of a defined size. The whole extrusion process leads to a mechanical transformation of the large vesicles into smaller ones. The filtration is started with filters containing larger pores (mean diameter of 100 nm), followed by a filtration through smaller pores (50 nm). The corresponding vesicle preparation is abbreviated as VET50, where ‘‘50’’ indicates the mean pore diameter used for the final extrusion. By this procedure we achieved mainly unilamellar vesicles with a mean hydrodynamic diameter of 65–70 nm and a homogenous size distribution, assessed by dynamic light scattering (DLS)

Liposomes Decorated with Oligonucleotides

253

experiments. The total membrane area and the number of lipid molecule in the outer leaflet are determined from the hydrodynamic radius considering an area per POPC of 0.72 nm2 and a membrane thickness of 3.7 nm, for the bilayer in the liquid crystalline state, respectively. Unilamellarity and low polidispersity of the liposomes are required for accurate knowledge of the grafting density (see Section 6.3.2) to interprete results from DLS. Extrusion might lead to a decrease of the lipid content caused by adsorption on the extruder walls or onto the filter. It is thus essential to determine the lipid concentration in solution after vesicles are prepared. The importance of knowing the total surface will become clear in next paragraphs: if the radius of the vesicles is precisely known, the polydispersity low (<0.07 from the second cumulant of the DLS fitting function), the total external lipid surface is determined and the oligonucleotide/lipid ratio determines the grafting density. The POPC concentration in the vesicles is determined by the Steward– Marshall method (Stewart, 1980), using an appropriate calibration curve obtained with known amounts of POPC. This colorimetric method is based on the formation of a complex between phospholipid and ammonium ferrothiocyanate that is soluble in chloroform. An aliquot of 7 ml of liposomes is mixed with 2 ml 0.1 M ammonium thiocyanate (NH4SCN) in chloroform. After shaking with vortex for 15 s, the sample is centrifuged for 5 min at 1000 rpm. The red lower layer (chloroform) is removed with a Pasteur pipette and the absorbance is read in a Lambda 900 UV–vis spectrophotometer at 485 nm. The phospholipid concentration is determined by comparison to the calibration curve.

4. Incorporation of Oligonucleotides 4.1. Choice of the lipid anchor The chemical nature and the position of the lipid anchor play a fundamental role in the attachment of the lipophilic oligonucleotides to the liposomal membrane. The anchorage determines the incorporation yield and the stability of the oligonucleotides in the membrane. The distribution of the oligonucleotides on the liposomal surface and the conformation of the attached oligonucleotides are dependent on the anchorage mode also. The main problem related to the insertion of lipophilic oligonucleotides into existing membranes is associated with their hydrophobic nature (i.e., amphiphilic properties) and their supramolecular self-organization in aqueous solution. The lipid moiety in the lipophilic oligonucleotide should be chosen to enhance partition in the liposomal bilayer rather than promote the formation of stable micelle-like aggregates in solution by self-aggregation process. Cholesterol was shown to be a good anchoring group for the insertion of lipophilic DNA into membranes: cholesterol conjugates have been shown to have a lower

254

Martina Banchelli et al.

tendency to self-aggregation with respect to alkyl chain conjugates (Gosse et al., 2004) and cholesterol-conjugated oligonucleotides can be directly incorporated into lipid bilayers through direct spontaneous insertion of the cholesteryl moiety into the bilayer. In fact, cholesterol itself embeds within the hydrophobic interior of the bilayer, forming a mobile anchor and also stabilizing the lipid bilayer. Two competing aspects have to be taken into account: the affinity for the membrane and the self-aggregation of the lipophilic oligonucleotides in solution. Of course these two points are interconnected, because, while the cholesteryl–oligonucleotide conjugate inserts into the membrane, it is subtracted from other aggregation equilibria. We have determined the aggregation threshold for cholesteryl– oligonucleotide conjugates, CAC (critical aggregation concentration), that can be conveniently monitored by light scattering or fluorescent probe techniques (Kalyanasundaram and Thomas, 1977); surface tension is not the method of choice, because the surface tension decrease is scarce and the adsorption at the interface slow. Two different anchoring strategies are pursued: a single-cholesterol anchoring group and multiple-cholesterol functions (Banchelli et al., 2009a,b) (Scheme 13.1). In this latter case, the number of cholesterol tagged is in the range 3–4, and the lipophilic units are spaced by three nucleotides along an oligonucleotide chain (12 thymidine bases). This is achieved through a novel synthetic strategy for ssDNA carrying lipophilic modified nucleotides in different positions within the oligonucleotide chain. In this way, the hydrophobic anchors can be separated by a number of nucleotides thus reducing the stability of micellar self-organization. It has been shown that membrane binding of oligonucleotides with one single-cholesterol anchor may be weak when the hydrophobic moiety is directly attached to the ssDNA (Pfeiffer and Ho¨o¨k, 2004). For this reason, we tested a new cholesterol-conjugated ssDNA where an oligoethylene glycol is introduced into the chain to act as a spacer between the cholesterol and the oligonucleotide portion. The introduction of the oligoethylene linker would improve the hybridization of the membraneanchored ssDNA with a complementary strand and the stability of the duplex at the membrane surface. Flexible spacer chains are commonly utilized to enhance the hybridization of terminally anchored oligonucleotide probes of DNA microarrays, since they create distance between the probes and the impenetrable surface, thus approaching the hybridization conditions of free chains in solution.

4.2. Choice of the grafting density Cholesteryl–oligonucleotide conjugates are added to the pre-prepared liposomal suspension (POPC 1.3 mM) from the solution at different concentrations (in the range 0.3–57 mM).

255

Liposomes Decorated with Oligonucleotides

ss-DNA 6.3 nm

TEG chol 1.4 nm 1.6 nm

6.3 nm ss-DNA

1.8 nm HEG

4.8 nm chol-ss-DNA

Scheme 13.1 Molecular diagram of single-cholesterol (ON-Chol) and multiplecholesterol oligonucleotide (ON-multichol).

In order to perform studies on the grafted liposomes it is useful to introduce the parameter hN i, defined as the average number of cholesteryl-ssDNA per vesicle. The distribution of guest molecules among colloidal hosts has been the subject of several studies that point to a Poisson distribution (Zana, 1987). Since hN i is quite high, the probability of oligonucleotide occupancy P(N ) can be well approximated by a Gaussian distribution function centered at hN i. Assuming a negligible translocation of the cholesteryl–oligonucleotide during the experimental time window, we can consider that the oligonucleotide derivative is entirely distributed in the outer vesicular leaflet. Therefore, it is meaningful to consider the stoichiometry with respect to POPC in the outer leaflet. The knowledge of liposomal size and the narrow size distribution allow a fairly accurate estimate of hN i. These hN i values, reported in the second column of Table 13.1, can be converted into an ‘‘average distance’’ (G 1/2 ) between anchoring sites onto vesicular surface. It should be stressed here that anchoring is due to intermolecular interactions of hydrophobic nature between the cholesterol of the ON-Chol and the phospholipid bilayer. The estimation of hNi is obtained by calculating the mean aggregation number (NAGG) of the POPC liposomes (35 nm radius, from DLS measurements), then the molar concentration of the liposomes, and finally the average number of cholesteryl–oligonucleotides per liposome, described in details as follows:

256

Martina Banchelli et al.

Table 13.1 Composition of the POPC liposome/cholesteryl–oligonucleotide samples Cholesteryl– oligonucleotide (mM)

POPCext leaflet/oligos

hN i

˚) G 1/2 (A

0.3 2.0 4.1 8.3 16.6 17.5 57.0

2050:1 307:1 150:1 75:1 40:1 37:1 11:1

9 60 125 250 500 525 1700

410 158 110 78 55 53 30

NAGG ¼ Total liposome surface / POPC molecular surface 2 2 ¼ (4pRext þ 4pRint ) / 0.7 nm2 Rext ¼ 35 nm, Rint ¼ 31 nm (thickness of POPC bilayer  4 nm) NAGG  39,200 [Liposome] ¼ [POPC]/NAGG hNi ¼ [cholesteryl–oligonucleotides]/[liposome] The hNi values can be then converted into an ‘‘average distance’’ (G 1/2) between anchoring sites onto vesicular surface by dividing the vesicle surface in hNi parts, and calculating their sides. The grafting density parameters reported in Table 13.1 are relative to the systems that we have investigated. The G 1/2 values must then be compared with the size of the hydrophilic portion of the lipophilic oligonucleotide. The relative values of these two structural parameters determine the conformation of the oligonucleotides and therefore their binding efficiency and kinetics.

5. Characterization of the Soft Hybrid Nanostructure 5.1. Purification of the liposome–oligonucleotide systems (size exclusion chromatography) As already mentioned, self-aggregation of lipid–oligonucleotide conjugates might compete or slow down the insertion in membranes; therefore, for a given G 1/2, a quantitative determination of oligonucleotide in solution is necessary. A quantitative determination of the ON-Chol incorporation in the lipid bilayer is assessed by gel filtration on Sephadex G-50. The technique of gel exclusion chromatography can be used to separate macromolecules by their size through columns of beads of gels that have small pores, so that smaller molecules are more retained within the pores of

Liposomes Decorated with Oligonucleotides

Figure 13.1

257

Representation of a typical size gel exclusion experiment.

the support medium, and hence elute more slowly than larger molecules (Fig. 13.1). The medium chosen for the ON-Chol/vesicles gel filtration is dextran Sephadex, which is one of the most commonly used for nucleic acids purification. In this experiment, the smaller molecule is represented by the ON-Chol monomer, whereas the larger particles consist of the oligonucleotidedecorated vesicles. The purpose of the experiment is to separate the free oligonucleotides from those anchored to the vesicles, therefore determining the percentage of oligonucleotides inserted into the vesicle bilayer. The ON-Chol-decorated vesicles are separated from unincorporated ON-Chol by a mini-column centrifugation method (Fry et al., 1978), which requires only a small amount of sample (aliquots of 200 ml by using 1 ml mini-columns) and has the advantage that the vesicles can be recovered with practically no dilution. This method is satisfactory for solutes of molecular weight less than 7000 Da; therefore, it is appropriate for separating ON-Chol (Mw ¼ 6193) from the larger POPC vesicles ( 30,000 kDa). Possible ON-Chol small oligomers may be excluded as well from the dextran pores. The Sephadex powder (10 g) is hydrated with 120 ml of TBS and stored for 24 h at 4  C before use. As an example, we report the results for minicolumn gel filtration performed on different samples simultaneously: POPC vesicles (1.3 mM ) and ON-Chol (4.1 mM ), POPC vesicles as a blank, 4.1 mM ON-Chol solution as a control. Fractions of 0.2 ml are collected and subjected to DLS and UV analysis to quantify ON-Chol concentration relatively to vesicles. UV absorption is only due to the oligonucleotides; however, scattering from the vesicles can provide a nonnegligible contribution to the extinction cross section. DLS measurements confirm that the

258

Martina Banchelli et al.

vesicles are recovered in the first fraction eluted with virtually no dilution: in fact the same value of the intensity of scattered light I(sample)/I(toluene) (where I(toluene) is the scattering of toluene measured in the same experimental conditions as the sample) is obtained from the vesicle dispersion before elution and the first eluted fraction. The light scattering contribution of the vesicles to the UV absorption spectra is evaluated by polynomial fitting of the absorption curve between 600 and 350 nm, where no absorption from the DNA bases is expected, according to the relationship (Barrow and Lentz, 1980):   I ln ¼ Clg ð13:1Þ I0 where l is the wavelength, and the exponent g (4 for Rayleigh scatterers) and C are parameters that can be adjusted in the fitting procedures. The light scattering curve has been extrapolated to 220 nm and subtracted from the absorption spectrum. UV analysis of the fractions collected in the gel separation experiments leads to the results shown in Fig. 13.2, where the absorption spectra of the vesicles/ON-Chol system are corrected for the light scattering contribution due to the vesicles. The percentage absorbance of the ON-Chol in the eluted fractions with respect to the solution before the filtration is calculated and the results are reported in Fig. 13.3. The first fraction eluted from the mixed vesicle/ONChol system contains the major amount of ON-Chol (95.8%), almost absent in the subsequent fractions. The 4.2% loss can be due to some free monomers entrapped inside the gel pores that are partially recovered by repeating the elution. The control experiment performed with free ON-Chol in solution shows that 26.5% of the oligonucleotide is inside the first eluted fraction, consistent with the occurrence of some aggregates which do not obviously form in the presence of vesicles. The presence of vesicles therefore significantly alters the relative abundance of ON-Chol monomers/oligomers. However, the relatively low cutoff of the pore size of the gel is sufficient to exclude dimers so that the first fractions of the ON-Chol gel filtration might contain a certain amount of these dimeric structures. This is not expected instead for the vesicle/ON-Chol system, since the self-aggregation process is not favored when the ON-Chol can insert into a preformed lipid membrane via hydrophobic intermolecular interactions between the cholesterol of the ON-Chol and the phospholipid bilayer. The incorporation of the cholesteryl–oligonucleotide into POPC vesicle bilayer is demonstrated to be highly efficient, and for further calculations it will be considered as complete (i.e., 100%).

259

Liposomes Decorated with Oligonucleotides

A 0.8

Abs

0.6

0.4

0.2

0.0 220

240

260 280 Wavelength (nm)

300

320

220

240

260 280 Wavelength (nm)

300

320

B 0.8

Abs

0.6

0.4

0.2

0.0

Figure 13.2 UV absorption spectra of the different fractions from the gel separation experiment: system before the separation (black), first fraction (red), second fraction (green), third fraction (blue); (A) POPC vesicles (1.3 mM) and ON-Chol (4.1 mM) and (B) free ON-Chol as the control.

5.2. Dynamic light scattering measurements DLS experiments are performed on the decorated vesicles at different lipid/ cholesteryl–oligonucleotide ratios. To test hybridization on the vesicles, the complementary sequence is also added afterwards in a 1:1 ratio with respect to the cholesteryl-ssDNA. This technique was used previously as a powerful and sensitive probe of hybridization on DNA-functionalized colloidal particles, for instance gold nanoparticles. The experimental DLS autocorrelation functions, g(t), for the decorated vesicles are analyzed by the method of cumulants (Koppel, 1972)

260

Martina Banchelli et al.

100% 90% 80% 70% 60%

Fraction 3 Fraction 2 Fraction 1

50% 40% 30% 20% 10% 0% Liposome + ON-Chol

ON-Chol

Figure 13.3 Relative values of % UV absorbance at 260 nm for the filtered fractions with respect to the absorption values before filtration.

1 1 lnð gðtÞÞ ¼ K  Gt þ m2 t2  m3 t 3 þ L 2 3

ð13:2Þ

where K is an experimental constant and mI are cumulants, adjustable constants fitting the curve. From the decay rate, G, we obtained the related z-average diffusion coefficient D ¼ G/q2 which is related to the hydrodynamic radius RH by the Stokes–Einstein equation RH ¼ kBT/(6pD). The polydispersity index (PDI) of the micellar aggregates is estimated from the m2 =m21 ratio. DLS experiments on decorated vesicles have been performed with different cholesterol-modified oligonucleotides. When the oligonucleotide portion, together with the hydrophilic spacer, protrudes outward from the membrane, the insertion should cause an increase of hydrodynamic radius of the vesicles, due to the added hydrodynamic thickness. This can be monitored using DLS to follow the time evolution of intensity autocorrelation functions. The logarithmic scale allows appreciating the slope variation that follows ON-Chol addition to the external milieu. The contribution to the scattering intensity due to possible cholesteryl– oligonucleotide aggregates must be completely negligible. In fact, if no self-aggregation of the lipophilic DNA occurs, the cholesteryl–oligonucleotide distribution onto the liposomal surface is controlled by the adsorption only, and the insertion properties of the cholesteryl function into the POPC bilayer. Therefore, it is extremely important to know the aggregation state of the lipophilic oligonucleotide in solution at various concentration because self-assembly is competitive toward the

261

Liposomes Decorated with Oligonucleotides

incorporation into the lipidic membrane and aggregation regimes should be avoided. For this reason, DLS measurements on the binary system (ON-Chol in buffer at the concentrations used with the liposomes) are required as a control experiment. The intensity autocorrelation functions are recorded at different equilibration times after dilution of the vesicular suspension with the solution containing the cholesteryl–oligonucleotides. The slope variation in the experimental curves (i.e., the relaxation rate of the concentration fluctuations, assuming a monomodal decay) can be directly correlated with a size variation, given the monodispersity of the liposomal suspension. The observed decrease of the decay rate corresponds to an increase in hydrodynamic radius with time until equilibrium is reached within 6 h. The observed radius increase corresponds to an increase of the hydrodynamic thickness around the vesicle, due to the hydrophilic portion of the guest molecules. Therefore, we have investigated the equilibrium thickness increase as a function of the added cholesteryl–oligonucleotide. The contribution of the oligonucleotide does not depend on the initial size of the vesicles, whose radius of curvature (33–35 nm) is higher than the fully extended length of the ON-Chol (9 nm). The increase of hydrodynamic thickness, termed H0, is reported as a function of ON-Chol concentration in Table 13.2. From the analysis of the dependence of the hydrodynamic layer thickness on surface coverage, we found that a conformational transition of the oligonucleotidic chain takes place (from a random coil- to a brush-state) as the surface coverage is increased and the average distance between anchoring sites on the vesicular surface is decreased (Banchelli et al., 2008).

Table 13.2 Hydrodynamic thickness H0, on the liposomes as a function of ON-Chol concentration Cholesteryl–oligonucleotide (mM)

H0 (nm)

0.3 2.0 4.1 8.3 16.6 17.5 57.0

0.45 1.25 1.55 2.15 4.20 5.25 5.50

262

Martina Banchelli et al.

6. Applications of Oligo-Decorated Liposomes 6.1. Hybridization with complementary oligonucleotides in solution As already mentioned, the grafting density on vesicles has important effects on hybridization kinetics. The coupling rates of ON-decorated liposomes with complementary strands in solution can be studied by means of UV stopped-flow experiments. The purpose of the experiment is the measure the hybridization kinetics of ON-Chol anchored to the vesicles with the free complementary strand in solution. The vesicular system is studied in parallel with the system of two oligonucleotides in solution and the effect of oligonucleotide surface density on the hybridization rate is also analyzed. UV absorbance spectroscopy is used to investigate duplex formation in a series of vesicle/ds-oligonucleotide hybrids at different POPC/oligonucleotide ratio, by varying the concentration of POPC and keeping the concentration of ds-oligonucleotide constant at 2 mM. Hybridization between ON-Chol incorporated in vesicles and complementary strand free in solution is observed by monitoring the decrease in UV absorbance at 260 nm upon rapid mixing of equal molar concentrations of the two oligonucleotide solutions in TBS saline. These solutions are mixed within approximately 103 s into a 1 cm path-length cuvette in a stopped-flow apparatus and hybridization is monitored at fixed temperature, that is, 25  C. The decrease in UV absorbance, caused by the hypochromism that occurs during duplex formation, is monitored until it has become invariant over time. Kinetic data are collected continuously and the time course of the absorbance (l ¼ 260 nm) is recorded in a range between 0.5 and 20 s from time zero, that is, when the injected solution volume reaches the instrumental beam height in the cuvette and a reasonable absorbance value can be obtained; typically, 400 pairs of data are taken in each experiment and data sets from three experiments performed under identical conditions are averaged (Fig. 13.4). Duplex formation of oligonucleotides is generally accepted as a process involving two main events (Wetmur and Davidson, 1968), initial formation of a nucleation complex followed by the sealing of the duplex. It is also generally accepted that the intermediate, initial association complex is unstable, with a marked tendency to either dissociate or seal, depending on the temperature. For short DNAs of up to several hundred base pairs, nucleation is rate-limiting at low concentrations and each duplex zips to completion almost instantly (>1000 bp s 1). The nucleation process is dependent on the concentration as well as on the complexity of the single strands at low DNA concentration nucleation is faster and for short heterogeneous oligonucleotides nucleation sites are fully extended by rapid zipping-up. For longer strands, a complex secondary structure of the

263

Liposomes Decorated with Oligonucleotides

70 ⫻ 10−3 60

A0-Ai

50 40 30 20 10 0 5

10 Time (s)

15

20

Figure 13.4 Kinetics of oligonucleotide hybridization onto POPC vesicles (35 nm hydrodynamic radius) at three different POPC/ON-Chol molar ratio: 82 (triangles), 164 (squares), and 657 (circles). Fitting curves are calculated through Eq. (5).

oligonucleotide, possibly containing intrastrand hairpin loops generated by various intramolecular base pairs, may profoundly decrease the hybridization kinetics with the complementary sequence. In fact, unimolecular intrastrand processes can be 100 times faster than the corresponding bimolecular pairing processes. Therefore, the existence of such hairpins within the oligonucleotide strand retards the rate of association, so that propagation of the duplex becomes the rate-limiting process (Gao et al., 2006). The rate constants of oligonucleotide hybridization are obtained by second-order fits to the absorbance data, for both solutions and vesicular suspensions. At time t, the concentration of ssDNA, Ct, is calculated using the following equation: Ct ¼

At  A1 C0 A0  A1

ð13:3Þ

where At is the absorbance at time t, A0 is the absorbance of the ssDNA at t ¼ 0, A1 is the absorbance of the dsDNA at equilibrium, and C0 is the initial concentration of ssDNA. C0 in our experiments is the bulk concentration, both in the case of the oligonucleotides in solution and on the vesicles. Hybridization of equal molar oligonucleotide single strands can be described by second-order reaction kinetics: 1 1  ¼ kon t Ct C0

ð13:4Þ

where kon is the association rate constant. Combining Eqs. (13.3) and (13.4), we find:

264

Martina Banchelli et al.

At ¼ A0 þ ðA1  A0 Þ

C0 kon t 1 þ C0 kon t

ð13:5Þ

To obtain quantitative kinetic information about the hybridization onto vesicles as a function of oligonucleotide grafting density, the rate constant kon is obtained by fitting the raw experimental data to Eq. (13.5). Results from stopped-flow experiments for six samples measured at different POPC/ON molar ratios and for the two oligonucleotides in solution are listed in Table 13.3. In all cases, the expression describing a second-order reaction with equal concentrations of the reactants resulted in the best fit. A clear trend in the rate of formation of the nucleation complex emerges: as [POPC]/[ON] ratio is increased and as oligonucleotide grafting density is decreased, the association rate increases. Therefore, hybridization kinetics is modulated by grafting site density. This behavior can be explained rather simply on the basis of an increased electrostatic repulsion at high surface density of oligonucleotides that may hinder the association with the complementary strand. However, another important aspect could affect the hybridization kinetics at high surface coverage as well, that is, the conformation of the single-stranded oligonucleotides at the surface. As previously shown in detail in Paragraph 3, the oligonucleotides at the vesicular surface undergo a conformational transition from mushroom to brush state when the average distance between grafting sites, G 1/2, becomes comparable to the Flory radius, RF, of the oligonucleotidic portion, which, for TEG-ss-18-mer in our experiments, varies from 40 to ˚ . At higher G 1/2 values, the oligonucleotide is in a random coil 55 A Table 13.3 Association hybridization rate constants for ON-Chol/complementary ON (1:1) POPC vesicles at different POPC/ON-Chol ratio in TBS solution (Tris 50 mM and NaCl 100 mM) Conc. POPC (mg/ml)

[POPC]/ [ON]

Number of ON per vesicle

Average distance between grafting ˚) sites (A

– 0.125 0.25 0.5 1 2 4

– 82.2 164.4 328.9 657.8 1315.6 2631.2

– 440 220 110 55 27 15

– 28 40 57 80 114 160

kon (105 M 1 s 1)

2.94  0.05 1.13  0.02 2.04  0.05 2.68  0.05 3.02  0.1 3.23  0.1 5.10  0.8

The kon values are from the stopped-flow experiments using equal concentrations, 2 mM, of the oligonucleotides.

Liposomes Decorated with Oligonucleotides

265

conformation and chains at the surface do not overlap, whereas, at G 1/2 ˚ , the oligonucleotides start to overlap and form a values lower than 55 A brush the vesicular surface. The dense packing of the oligonucleotides inside the brush may affect the hybridization rate with complementary strand more than the mushroom state, where oligonucleotide chains develop as separate coils and duplex formation might occur faster. If compared to the oligonucleotide kinetic rate in solution, we can see that beyond a [POPC]/[ON] ratio of 330, that is consistent of about 110 duplexes per vesicle, the constant rate of the oligonucleotide hybridization at the vesicular surface is lower, while above this threshold concentration the kinetic rate of hybridization on the vesicle becomes higher than the value obtained in solution. It has been shown that the associative kinetics of surface DNA hybridization on planar gold surface are suppressed by a factor of 20- to 40-fold compared to solution-phase hybridization and a 5- to 10-fold suppression in hybridization rates of 22 mers is also observed on microparticles by means of FRET measurements (Henry et al., 1999). In the present case, the oligonucleotide can hybridize with the complementary strand faster on the vesicle rather than in solution, as long as grafting density is not too high. The advantage of having nanoparticles rather than solid planar supports in the hybridization kinetics relies on the diffusion of those particles that could help the association reaction. Also, noncovalent anchorage of oligonucleotides to vesicles, rather than covalent linkage to solid supports or gold nanoparticles, allows more flexibility and mobility of the grafted molecules onto the surface. Oligonucleotides can continuously rearrange and change their distribution on the vesicle, through the spontaneous motion of the cholesterol along the lipid bilayer, probably improving the association kinetics with a complementary strand in solution as well. In addition, the conformation of the anchored oligonucleotide is, as previously discussed, an important aspect in the hybridization kinetics: the threshold [POPC]/[ON] ratio at which hybridization on the vesicles becomes faster than in solution corresponds to a calculated average grafting site distance of ˚ , which is comparable to the Flory radius of the ON-TEG portion. 50 A This might be interpreted as if the main factor controlling the kinetic properties is the conformation at the surface. This is a very promising result for the efficient fabrication of nanosystems for DNA recognition and self-assembly.

6.2. Hybridization with self-assembled DNA nanostructures The realization of addressable DNA architectures requires the design of complex motifs with sticky ends, which assemble forming extended DNA scaffolds. These subunits can be spatially organized onto functionalized lipid surfaces to possibly achieve addressable platforms with sub-nm precision. The successful realization of the DNA nanohybrid structure resides in the accomplishment of an optimized preparation protocol, where grafting

266

Martina Banchelli et al.

density, lipid/DNA ratio, liposome number density, and preparation procedure on the final structure are taken into account, as detailed in the previous paragraphs. Two main approaches can be exploited to anchor DNA nanostructures onto oligonucleotide-decorated liposome surfaces: the first method consists of the direct immobilization of the preformed DNA nanostructures (previously obtained in solution) by hybridization of complementary sticky ends set on the nano-object and on the lipid surface (single-step strategy), while the other procedure involves the stepwise addition of each strand in a predefined order to form the desired nanostructure directly on the lipid surface (stepwise strategy) (Baldelli Bombelli et al., 2009). Here, we describe these approaches for the anchoring of two different DNA nano-objects: a closed pseudohexagon and an open linear nanostructure. The choice of two different structures allows the investigator to better address all the critical parameters to be considered in the formation of such hybrid systems. Both nanostructures can be formed in solution by hybridization of six defined linear oligonucleotides in which five are identical for the two structures and the sixth is partially modified to obtain close (B0 A0 ) and open structures (B0 X), respectively (Scheme 13.2). The strand labeled as 2 in Scheme 13.2 is the sticky end complementary to the ON-Chol incorporated into the lipid membrane (see Paragraph 3).

6.3. Different preparation procedures 6.3.1. Single-step strategy The first step is the formation of the DNA nanostructures in the same buffer solution used for the liposomes preparation. The hybridization process conditions have to be optimized as a function of the length, the geometry, and the sequence of the Oligo-Decorated Nucleotide (ODN) strands used as building blocks. Generally, buffer solution with ionic strength in the range of 0.1– 0.5 M, to assure stability at room temperature (high melting temperature), and DNA concentration of 0.3–5 mM, to minimize the formation of undesired polymerization products, are used. The concentration of each ODN has to be precisely set using nearest neighboring approximation (NNA) values for the extinction coefficients and their absorbance at 260 nm. The nanostructures are formed in 50 mM Tris buffer (pH ¼ 7.5) with 100 mM NaCl (Tm  56  C) by mixing equimolar amounts of the DNA sequences to have a 3 mM final concentration of each strand in solution, which means a 3 mM concentration of the nanoconstructs. The samples are annealed by heating to 90  C and then cooled to 5  C with a constant temperature gradient over 6 h. The protocol is optimized controlling the samples by gel electrophoresis, DLS, and atomic force microscopy (AFM) experiments (Baldelli Bombelli et al., 2008; Banchelli et al., 2008; Tumpane et al., 2007).

Name

Sequence

FA-2

5’-ACGAGCCTTTGACGCTTGGA-TT-TAGTGCGTAACATAGGCTAC-TTCTGAAATTATGATAAAGA-3’

E’F’

5’-ATTTACCTGGAAGCAGCCAC-TT-TCCAAGCGTCAAAGGCTCGT-3’

ED

5’-GTGGCTGCTTCCAGGTAAAT-TT-CACTATGTAACTGGTCTCTTA-3’

D’C’

5’-TAGAGACCAGTTACATAGTG-TT-TGACCTCAGTCGCAAGGCTG-3’

CB

5’-CAGCCTTGCGACTGAGGTCA-TT-TCGGGTCAACGAATGGCTGC-3’

B’A’

5’-GCAGCCATTCGTTGACCCGA-TT-GTAGCCTATGTTACGCACTA-3’

B’X

5’-GCAGCCATTCGTTGACCCGA-TT-CCCCCCCCCTTTTTTTTTTT-3’

Pseudo-hexagonal structure D

E D⬘

C

E⬘

C⬘

F⬘ B⬘

B

F

A⬘ 5⬘

A

2 3⬘ Open structure

2

A

F⬘

E⬘

D⬘

C⬘

B⬘

F

E

D

C

B

Scheme 13.2 DNA nanostructure composing strands. Adapted from Baldelli Bombelli et al. (2008).

X

268

Martina Banchelli et al.

The binding experiment is designed on the basis of spatial considerations calculating the steric hindrance of the two nanostructures through geometrical estimates, and thus determining the maximum number of occupancy, Nmax, on the lipid surface, which corresponds to hNi mentioned earlier. The calculated Nmax are very approximate values and more likely overestimated since both orientation and conformation of the nanostructures and electrostatic repulsion contributions have not been considered in this calculation. Nonetheless, Nmax can be a first indicative parameter for the choice of the right grafting density range to use for preparing DNA/lipid hybrid complexes. As previously described, monodisperse liposome solutions of about 35 nm radius are used for which the resulting Nmax are about 90 and 1000 for the close and open DNA nanostructures, respectively. On this basis, preformed hexagons and open nanostructures are added to ON-Choldecorated vesicles in a stoichiometric fashion with respect to the anchoring sites, varying the occupancy number from 10 to 130. Therefore, the investigated surface density is safely chosen within the vesicle hosting capacity and should allow the buildup of isolated nanoconstructs. The samples, prepared either by adding the nanostructures to the decorated liposomes equilibrated overnight at room temperature, or mixing together all the components, are measured by DLS 1 and 24 h after the preparation (Fig. 13.5A). The two preparation methods provide different results in the shortest time. Indeed, after 1 h, samples obtained by simultaneously mixing give larger hydrodynamic radii and higher PDI than the corresponding ones prepared in two steps. After a few days, depending on the grafting densities, different samples reach the same values of RH and PDI (data not shown), indicating that the incorporation of the ON-Chol into the lipophilic environment is the slowest step of the process. Hence, all reported experiments are performed on samples prepared with the two steps method. Our first observation is that the attainment of an ‘‘equilibrium’’ state is time-variable, depending on the nanostructure grafting density, as shown in Fig. 13.5A. Moreover, the hydrodynamic radius of both hybrid nanostructures increases with respect to the naked liposomes and keeps growing with increasing the occupancy number (see Fig. 13.5B), while the polidispersity is almost invariant. This increase is related to the different orientation of the nanostructure as a function of the grafting density in term of steric and electrostatic repulsions between neighboring objects; these become stronger at higher packing density determining a larger hydrodynamic shell. Increasing hNi, while pseudohexagon-decorated vesicles reach a sort of saturation for N > 80, those with open nanostructures keep growing, indicating that the more flexible and less bulky open nanostructures can be more densely packed on the lipid surface. In the inset of Fig. 13.5B, we also report the hydrodynamic radius of lipid/DNA hybrids for N ¼ 30 at different

269

Liposomes Decorated with Oligonucleotides

A

44

RH (nm)

42

40

1 day

38

36

3

4

5

6 7 8 9

2

3

4

5

6 7 8 9

10

100 Time (min)

B

60

Pseudo-hexagon Open

RH (nm)

55

50 48

45

46 44

40

42 0.4

0.8

1.2

1.6

cPOPC [M]

35 0

20

40

60

80 N

100

120

140

Figure 13.5 (A) Time dependence of the hydrodynamic radius of the lipid/DNA hexagon hybrid for two grafting densities: (●) N ¼ 9, [POPC] ¼ 0.785 mM, [ODN] ¼ 0.18 mM; (□) N ¼ 30, [POPC] ¼ 0.235 mM, [ODN] ¼ 0.18 mM. The empty circle and the filled square represent the final equilibrium values reached after 1 day. (B) Hydrodynamic radii of lipid/DNA hybrids as a function of the occupancy number, [POPC] ¼ 1.33 mM. The inset shows the behavior of the hydrodynamic radius of the hybrid as a function of liposome density for N ¼ 30. (□) Pseudohexagons, (▲) open nanostructures.

liposome densities, the results show a slight increase in the absolute value, while PDI is of the same order of magnitude. This means that liposome density is not critical for the successful formation of the complexes.

270

Martina Banchelli et al.

The samples are monitored with time, and they are stable for weeks at room temperature, demonstrating the effectiveness of this method. It is important to underline that the achievement of a thermodynamically stable system requires variable equilibration times depending on the surface grafting density. 6.3.2. Stepwise strategy Ring and open nanostructures are also built on the liposome surface by a stepwise strategy, which consists of the sequential addition of equimolar amounts of each strand to ON-Chol-decorated liposomes, in stoichiometric fashion with respect to the anchoring sites, and then recruitment by the partially built hybrid. Two stepwise procedures differing by the mixing order of the oligonucleotides, illustrated in Fig. 13.7, are adopted for the construction of the more complex close nano-object to distinguish a possible kinetic control of each step due to the different shapes of the partially built nanoconstructs. Each strategy consists of eight steps where step 1 corresponds to POPC liposomes and step 2 to the insertion of ON-Chol into the lipid membrane. The spontaneous insertion of ON-Chol and the following coupling events between complementary oligonucleotides are revealed as an increase of the radius of the liposome, due to the added hydrodynamic thickness, by DLS (Fig. 13.7). The study is performed on eight different samples for each strategy representing the composing different steps (in the asymmetric strategy for close and open nanostructures the first seven steps coincide) prepared under the same conditions: the same ODN strand is added to different step samples at the same time, and the interval between two sequential additions is set at 1 h, and the final volume is adjusted with PBS buffer. ON-Chol is added previously to the liposomes, and eventually the samples are equilibrated overnight before the sequential addition of the other strands. First, samples are prepared at different grafting densities keeping constant the lipid concentration (1.33 mM) as a function of the added oligonucleotide concentration. For the open construct, the stepwise strategy gives comparable results, in terms of the hydrodynamic radius, to those obtained with the single-step procedure in all the grafting density ranges investigated. For the construction of closed nanostructures at this lipid concentration, instead, we need to distinguish between low (N < 15) and high grafting densities: pure isolated hybrid structures have only been obtained in the former condition. In fact, for low grafting densities, both symmetric and asymmetric strategies have given comparable RH values in the final step, in agreement with those obtained with the single-step, procedure, as well as a RH trend in the intermediate construction steps consistent with the expected partially built nanoconstructs (see Fig. 13.6A). However, the symmetric strategy ends up being more effective than the asymmetric one

271

Liposomes Decorated with Oligonucleotides

A = 9 (step-by-step) = 9 (single step) = 30 (step-by-step) = 30 (single step)

44

RH (nm)

42

40

38

36

34 1

2

3

4

5

6

7

8

Step B 44

42 RH (nm)

1 day

40

38 3

4

5 6 7 8 9

2

10

3

4

5

6 7 8 9

100 Time (min)

Figure 13.6 (A) Effect of the liposome number density on the formation of lipid/DNA nanohybrids for two different POPC/DNA ratios obtained with the symmetric stepwise procedure. The radii of the nanoconstructs obtained by addition of preformed hexagons are reposted with filled symbols for comparison. Readapted from Baldelli Bombelli et al. (2009). (B) Time dependence of the hydrodynamic radius of the lipid/ DNA hexagon hybrid for two grafting densities: (●) N ¼ 9, [POPC] ¼ 0.785 mM, [ODN] ¼ 0.18 mM; (□) N ¼ 30, [POPC] ¼ 0.235 mM, [ODN] ¼ 0.18 mM.

in terms of polydispersity and grafting density range. For occupancy numbers >15, although the intermediate steps are characterized by hydrodynamic radii in agreement with the expected assemblies, the addition of the

272

Martina Banchelli et al.

38

37

RH (nm)

36

35

Asymmetric FA−2 F⬘E⬘ DE D⬘C⬘ BC B⬘A⬘ *B⬘X(open)

34

Symmetric FA−2 B⬘A⬘ BC F⬘E⬘ D⬘C⬘ DE

33

32 2

4

6

8

Step

Figure 13.7 Trend of the hydrodynamic radii for the step-by-step construction of DNA pseudohexagons and open nanostructures on liposomes for N  10. Symmetric (filled circles) and asymmetric strategies (filled squares) used for close nanostructures are showed. The open nanostructures are formed by asymmetric strategy and the first seven steps coincide with those of the asymmetric strategy for the close nanostructure (empty square). The tables on the left indicate the addition order of the oligonucleotides in the two strategies (see Scheme 13.1). Adapted from Baldelli Bombelli et al. (2009).

last oligonucleotide strand induces the formation of large aggregates with a consequent significant increase in PDI. Size distribution analysis of the autocorrelation functions for these samples does not give well-defined populations, but we rather obtain a single broad population probably composed of isolated hybrid structures, together with dimers and trimers. The fact that we do not observe the same aggregation process for the addition of the last strand in the formation of the open nanostructure, suggests that the closure of the ring is the driving mechanism for the formation of aggregates. On the basis of this hypothesis, a determining factor in the aggregation process could be a too short average distance between liposomes in solution, causing the association of neighboring hybrid structures during the ring closure. This parameter can be optimized to make the symmetric strategy effective for higher occupancy numbers also. Hence, a different series of samples is prepared according to the symmetric strategy keeping constant

Liposomes Decorated with Oligonucleotides

273

DNA concentration (0.18 mM) and varying lipid concentration between 0.235 and 0.785 mM that corresponds to occupancy numbers of 30 and 9, respectively. Aggregation is not detected upon addition of the last ODN strand for these samples and the resulting hybrid nanostructures hydrodynamic sizes and PDI are comparable to those obtained for corresponding samples prepared with the single-step procedure (Fig. 13.6A). Moreover, the hybridization process is monitored with time, as reported in Fig. 13.6B. We do not observe a significant difference, as for the singlestep procedure, in the achievement of the ‘‘equilibrium’’ state between two grafting densities and after 10 min the hydrodynamic radius is invariant with time for both samples. It is important to stress that the formation of DNA nanoassemblies in solution requires a lengthy annealing process (heating at 90  C and then cooling down to 20  C with a constant temperature gradient within 6 h), while the step-by-step procedure on liposomes is performed at room temperature. The success of the stepwise strategy highlights the important role of the immobilization of ODN on the lipid surface, working as a sort of catalyzer for the hybridization reaction. We conclude that the stepwise strategy is a sucessful method for ‘‘in situ’’ construction of more complex DNA nanostructures on the liposome surface at room temperature, but, to avoid aggregation, special care is needed regarding the liposome density in solution.

6.4. Kinetics aspects DLS is shown to be an extremely powerful tool for studying the formation of soft hybrid nanostructures composed of phospholipid liposomes decorated with oligonucleotides nanostructures. DLS analysis allows the comparison of different preparation methods to infer a final optimized protocol for the preparation of monodisperse, isolated DNA/lipid nanohybrid structures. Several parameters have to be considered in the different preparation procedures and the optimal conditions may not be the same for diverse strategies. In fact, one of the main differences between single-step and stepwise procedures seems to be the kinetics of the attainment of a stable complex. Moreover, in the stepwise strategy the determining parameter is liposome crowding, that can promote aggregation during the closure step of the ring-like structure, while in the single-step protocol a longer time scale is needed to reach the equilibrium state of the system at higher grafting density. To study the kinetics of hybridization of the determining step of the formation of these hybrids, the time course of the 260 nm absorbance after mixing the solutions containing the coupling sides is followed via stoppedflow (see Section 4.1). The time-course of the absorbance is recorded between 0.04 and 17 min (longest time measurable with this equipment).

274

Martina Banchelli et al.

A 0.400

A

0.398

0.396

0.394

0 B

5

10 t (min)

15

0.400 0.398 0.396

A

0.394 0.392 0.390 0.388 0.386 0.384 0

200

400 600 t (min)

800

1000

Figure 13.8 (A) Kinetics of ON-Chol/pseudohexagon-2 hybridization onto POPC vesicles (single-step process) at two different occupancy numbers: 9 (empty circles) and 30 (filled squares). The absorbance has been normalized to 0.4 for a better comparison. (B) Kinetics of hybridization process that occurs at the step 8 of the symmetric stepwise procedure presented in Fig. 13.5 (DE addition).

In particular, for the stepwise procedure we monitor the absorbance decrease upon addition of the DE strand to the step 7 solution obtained according to symmetric strategy (Fig. 13.8A), while for the single-step procedure a preformed DNA pseudohexagon solution is added to oligoloaded vesicles (Fig. 13.8B). The final ODN concentration is kept constant in all experiments to 0.18 mM, while the lipid concentration is set to 0.235 and 0.785 mM which corresponds to N ¼ 30 and 9, respectively. The data are reported in Fig. 13.8A and B and, since the total absorption is strongly affected by vesicle scattering, the curves have been normalized to

Liposomes Decorated with Oligonucleotides

275

achieve a better comparison between different samples. Nonetheless, a quantitative estimation of the hybridization cannot be precisely determined. In the single-step procedure, hybridization kinetics curves cannot be analyzed assuming a single process with an uniform association rate constant, but are rather composed of an initial faster dynamics followed by a slower process. Presumably, the first process is driven by the diffusion of the nanostructures approaching the lipid surface, as suggested by the superimposition of the kinetics curves in Fig. 13.8A for the first minutes of the process. The higher steric and repulsive barrier of the more crowded interface comes into play later, and determines the association rate of the second process. Of course, there are several processes involved in the formation of the DNA/lipid hybrid, and a more accurate analysis is needed to be able to model it. Unfortunately, the hybridization process is not completed by 17 min, which is the longest experimental time achievable with this experimental setup. Nevertheless, 17 min seems to be a good time scale to study the kinetics of the ring closure mechanism in the symmetric stepwise preparation upon addition of the DE strand. In this case, the hybridization is mainly composed of a single association process characterized by a faster rate constant and completed by the investigated time scale. These data are in good agreement with that observed by DLS, confirming that different kinetics of binding are involved in the determining step of the two preparation procedures. This enhances the necessity to choose the right conditions depending on the chosen methodology to succeed in the preparation of DNA/lipid hybrid structures.

7. Challenges and Perspectives DNA coupling to hard nanoparticles (e.g., AuNp) to direct their hierarchical self-assembly is a relatively mature research field (Alivisatos et al., 1996; Mirkin et al., 1996; Nykypanchuk et al., 2008). Conversely, the literature on DNA coupling to soft nanoparticles (i.e., liposomes) is still at an early stage, even if interesting and innovative applications of this procedure are now being reported in the literature (Beales and Kyle Vanderlick, 2007; Jakobsen et al., 2008; Pfeiffer and Ho¨o¨k, 2004; Stengel et al., 2007). Coupling DNA to soft nanoparticles requires a noncovalent approach, that is, a lipid modification of the oligonucleotide, and its insertion into the membrane is ruled by thermodynamics, and can therefore be altered in response to experimental conditions (temperature, concentration, salinity, and so on). Hence, the preparation and characterization of DNAdecorated liposomes is probably more challenging, but should nevertheless be pursued in view of the many envisaged uses.

276

Martina Banchelli et al.

Several key factors should be taken into account: the choice of the lipidanchoring unit, the presence and nature of a spacer toward the membrane proximal end, and, of course, the length and the composition of the oligonucleotide portion, which is more directly dictated by the purpose of the application. It is difficult to rationalize these different contributions, in view of the still limited reports in the literature. The role of the lipid composition of the membrane has not investigated in detail, but we can easily predict that the affinity of the lipid-ON conjugate will be highly dependent on this parameter. It should be mentioned, however, that whatever the long-term envisaged application, the reliable knowledge of structural parameters, such as the size of the hybrid aggregates, oligonucleotide conformation, and grafting density, are vital for the use of these objects as functional building blocks for nanostructure arrays.

ACKNOWLEDGMENTS Financial support from CSGI, MIUR-PRIN, CNR-FUSINT, and the European Commission’s Sixth Framework Program (Project Reference AMNA, Contract No. 013575) are acknowledged. Dr. Alessio Innocenti is acknowledged for help in Stopped Flow Experiment. Dr. Gabriella Caminati is acknowledged for fruitful discussions.

REFERENCES Alivisatos, A. P., Johnsson, K. P., Peng, X. G., Wilson, T. E., Loweth, C. J., Bruchez, M. P., and Schultz, P. G. (1996). Organization of ‘nanocrystal molecules’ using DNA. Nature 382, 609–611. Baldelli Bombelli, F., Gambinossi, F., Lagi, M., Berti, D., Caminati, G., Brown, T., Sciortino, F., Norde´n, B., and Baglioni, P. (2008). DNA closed nanostructures: A structural and Monte Carlo simulation study. J. Phys. Chem. B 112, 15283–15294. Baldelli Bombelli, F., Betti, F., Gambinossi, F., Caminati, G., Brown, T., Baglioni, P., and Berti, D. (2009). Closed nanostructures assembled by step-by-step ss-DNA coupling assisted by phospholipid membranes. Soft Matter 5, 1639–1645. Banchelli, M., Betti, F., Berti, D., Caminati, G., Baldelli Bombelli, F., Brown, T., Wilhelmsson, L. M., Norde´n, B., and Baglioni, P. (2008). Phospholipid membranes decorated by cholesterol-based oligonucleotides as soft hybrid nanostructures P. J. Phys. Chem. B 112, 10942–10952. Banchelli, M., Gambinossi, F., Berti, D., Caminati, G., Brown, T., and Baglioni, P. (2009a). Anchoring amphiphilic DNA to phospholipid membranes. Part II: Modulation of grafting density and conformation in vesicles. J. Phys. Chem. B submitted. Banchelli, M., Gambinossi, F., Berti, D., Caminati, G., Brown, T., and Baglioni, P. (2009b). Anchoring amphiphilic DNA to phospholipid membranes. Part I: Modulation of grafting density and orientation in supported lipid bilayers. J. Phys. Chem. B manuscript in preparation. Barrow, D. A., and Lentz, B. R. (1980). Large vesicle contamination in small, unilamellar vesicles. Biochim. Biophys. Acta 597, 92–99.

Liposomes Decorated with Oligonucleotides

277

Beales, P. A., and Kyle Vanderlick, T. J. (2007). Specific binding of different vesicle populations by the hybridization of membrane-anchored DNA. J. Phys. Chem. A 111, 12372–12380. Cevc, G. (1993). Phospholipids Handbook. Marcel Dekker Inc., New York. Chan, Y.-H. M., Van Lengerich, B., and Boxer, S. G. (2008). Lipid-anchored DNA mediates vesicle fusion as observed by lipid and content mixing. Biointerphases 3, FA17–FA21. Chan, Y.-H. M., Van Lengerich, B., and Boxer, S. G. (2009). Effects of linker sequences on vesicle fusion mediated by lipid-anchored DNA oligonucleotides. Proc. Natl. Acad. Sci. 106, 979–984. Fry, D. W., White, C., and Goldman, D. J. (1978). Rapid separation of low molecular weight solutes from liposomes without dilution. Anal. Biochem. 90, 809–815. Gao, Y., Wolf, L. K., and Georgiadis, R. M. (2006). Secondary structure effects on DNA hybridization kinetics: A solution versus surface comparison. Nucleic Acids Res. 34, 3370–3377. Gosse, C., Boutorine, A., Aujard, I., Chami, M., Kononov, A., Cogne-Laage, E., Allemand, J.-F., Li, J., and Jullien, L. (2004). Micelles of lipid-oligonucleotide conjugates: Implication for membrane anchoring and base pairing. J. Phys. Chem. B 108, 6485–6497. Henry, M. R., Stevens, P. W., Sun, J., and Kelso, D. M. (1999). Real-time measurements of DNA hybridization on microparticles with fluorescence resonance energy transfer. Anal. Biochem. 276, 204–214. Jakobsen, U., Simonsen, A. C., and Vogel, S. (2008). DNA-controlled assembly of soft nanoparticles. J. Am. Chem. Soc. 130, 10462–10463. Kalyanasundaram, K., and Thomas, J. K. (1977). Environmental effects on vibronic band intensities in pyrene monomer fluorescence and their application in studies of micelar systems. J. Am. Chem. Soc. 99, 2039–2044. Koppel, D. E. (1972). Analysis of macromolecular polydispersity in intensity correlation spectroscopy: The method of cumulants. J. Chem. Phys. 57, 4814–4820. Mirkin, C. A., Letsinger, R. L., Mucic, R. C., and Storhoff, J. J. (1996). A DNA-based method for rationally assembling nanoparticles into macroscopic materials. Nature 382, 607–609. Nykypanchuk, D., Maye, M. M., Van der Lelie, D., and Gang, O. (2008). DNA-guided crystallization of colloidal nanoparticles. Nature 451, 549–552. Pfeiffer, I., and Ho¨o¨k, F. (2004). Bivalent cholesterol-based coupling of oligonucleotides to lipid membrane assemblies. J. Am. Chem. Soc. 126, 10224–10225. Stengel, G., Zahn, R., and Hook, F. (2007). DNA-induced programmable fusion of phospholipid vesicles. J. Am. Chem. Soc. 129, 9584–9585. Stewart, J. C. M. (1980). Colorimetric determination of phospholipids with ammonium ferrothiocyanate. Anal. Biochem. 104, 10–14. Tumpane, J., Sandin, P., Kumar, R., Powers, V. E. C., Lundberg, E. P., Gale, N., Baglioni, P., Lehn, J.-M., Albinsson, B., Lincoln, P., Wilhelmsson, L. M., Brown, T., et al. (2007). Addressable high-information-density DNA nanostructures. Chem. Phys. Lett. 440, 125–129. Wetmur, J. G., and Davidson, N. (1968). Kinetics of renaturation of DNA. J. Mol. Biol. 31, 349–370. Zana, R. (1987). Surfactant Solutions: New Methods of Investigation. Marcel Dekker, New York.