Method for studying transport of an agent across a bilayer membrane in bioanalytical sensor applications

Abstract

The present invention provides a method for studying transport of an agent across a membrane comprising the steps a) providing at least one surface with a bilayer structure tethered to the surface, said bilayer structure comprising a detection volume, b) contacting the bilayer with at least one agent to be analyzed, and c) detecting a change in refractive index in the detection volume resulting from transportation of the agent across the membrane. Further there is provided a device comprising a) at least one surface, b) at least one bilayer structure tethered to the surface, and c) at least one sensor capable of detecting a change in refractive index in a detection volume, wherein the bilayer structure encloses a first volume of the detection volume and wherein the volume not enclosed by the bilayer structure but within the detection volume is a second volume and wherein the ratio between the first volume and second volume is above about 0.001.

Claims

1. A method comprising: contacting a bilayer structure with at least one agent to be analyzed, the bilayer structure being tethered to at least one metal surface and enclosing a detection volume; detecting a change in refractive index only in the detection volume enclosed by the bilayer structure resulting from transportation of the at least one agent across a membrane of the bilayer structure; and determining a rate at which the at least one agent is transported across the bilayer structure using the detected change in the refractive index of the detection volume.

2. The method of claim 1 wherein the bilayer structure comprises at least one liposome.

3. The method of claim 2 wherein the at least one liposome is tethered to the at least one metal surface with a spacer molecule.

4. The method of claim 1 wherein at least one of a membrane protein or an ionophore is associated with the bilayer structure.

5. The method of claim 4 wherein the bilayer structure comprises at least one liposome.

6. The method of claim 5 wherein the at least one liposome is tethered to the at least one metal surface with a spacer molecule.

7. The method of claim 1 wherein the bilayer structure comprises at least two layers of liposomes.

8. The method of claim 1 wherein the change in refractive index is detected with a surface plasmon resonance sensor, an ellipsometry sensor, or an optical waveguide laser spectroscopy sensor.

9. A method comprising: contacting a bilayer structure with at least one agent to be analyzed, the bilayer structure being tethered to at least one metal surface and enclosing a detection volume; detecting a change in refractive index only in the detection volume enclosed by the bilayer structure resulting from transportation of the at least one agent across a membrane of the bilayer structure; and determining a permeation coefficient using the detected change in the refractive index of the detection volume.

10. The method of claim 9 wherein the bilayer structure comprises at least one liposome.

11. The method of claim 10 wherein the at least one liposome is tethered to the at least one metal surface with a spacer molecule.

12. The method of claim 9 wherein at least one of a membrane protein or an ionophore is associated with the bilayer structure.

13. The method of claim 12 wherein the bilayer structure comprises at least one liposome.

14. The method of claim 13 wherein the at least one liposome is tethered to the at least one metal surface with a spacer molecule.

15. The method of claim 9 wherein the bilayer structure comprises at least two layers of liposomes.

16. The method of claim 9 wherein the change in refractive index is detected with a surface plasmon resonance sensor, an ellipsometry sensor, or an optical waveguide laser spectroscopy sensor.

17. A method comprising: contacting a bilayer structure with at least one agent to be analyzed, the bilayer structure being tethered to at least one surface and enclosing a detection volume; detecting a change in refractive index only in the detection volume enclosed by the bilayer structure resulting from transportation of the at least one agent across a membrane of the bilayer structure, the change in refractive index being detected with a surface plasmon resonance sensor, an ellipsometry sensor, or an optical waveguide laser spectroscopy sensor; and determining a rate at which the at least one agent is transported across the bilayer structure using the detected change in the refractive index of the detection volume.

18. The method of claim 17 wherein the bilayer structure comprises at least one liposome.

19. The method of claim 18 wherein the at least one liposome is tethered to the at least one surface with a spacer molecule.

20. The method of claim 17 wherein at least one of a membrane protein or an ionophore is associated with the bilayer structure.

21. The method of claim 20 wherein the bilayer structure comprises at least one liposome.

22. The method of claim 21 wherein the at least one liposome is tethered to the at least one surface with a spacer molecule.

23. The method of claim 17 wherein the bilayer structure comprises at least two layers of liposomes.

24. A method comprising: contacting a bilayer structure with at least one agent to be analyzed, the bilayer structure being tethered to at least one surface and enclosing a detection volume; detecting a change in refractive index only in the detection volume enclosed by the bilayer structure resulting from transportation of the at least one agent across a membrane of the bilayer structure, the change in refractive index being detected with a surface plasmon resonance sensor, an ellipsometry sensor, or an optical waveguide laser spectroscopy sensor; and determining a permeation coefficient using the detected change in the refractive index of the detection volume.

25. The method of claim 24 wherein the bilayer structure comprises at least one liposome.

26. The method of claim 25 wherein the at least one liposome is tethered to the at least one surface with a spacer molecule.

27. The method of claim 24 wherein at least one of a membrane protein or an ionophore is associated with the bilayer structure.

28. The method of claim 27 wherein the bilayer structure comprises at least one liposome.

29. The method of claim 28 wherein the at least one liposome is tethered to the at least one surface with a spacer molecule.

30. The method of claim 24 wherein the bilayer structure comprises at least two layers of liposomes.

Description

DETAILED AND EXEMPLIFYING DESCRIPTION

Brief Description of the Drawings

(1) FIG. 1 shows the successive addition of Neutravidin/Biotin DNA (N/B), 0.1 M sucrose (S1), 2 mg/ml cholesterol DNA tagged POPC-liposomes (POPC), 0.1 M sucrose (S2), 40 μM melittin, 0.1 M sucrose (S3), 0.1 M sucrose (S4), 0.1 M sucrose (S5) to a PEG/PEG-biotin functionalized Biacore® sensor.

(2) FIG. 2 shows the response (RU) from the five sucrose additions (S1-S5). The insert shows a partial enlargement of FIG. 2.

(3) FIG. 3 shows the uptake of sucrose into the liposomes as a function of time, which is obtained by subtracting the change in RU upon addition of sucrose to closed liposomes (S2) from the change in RU upon addition of S3-S5.

(4) FIGS. 4a and 4b show liposome permeability for glycerol, urea and hydroxyurea.

(5) FIGS. 5a and 5b show the release and transport rate for glycerol over different temperatures.

(6) FIGS. 6a to 6c show the behaviour of cholesterol comprising liposomes when subjected to cyclodexterin.

(7) FIG. 7 illustrates membrane transport of iodide and chloride ions.

Example 1, Study of Melittin and its Effect on Membrane Transport

(8) Material and Methods

(9) 1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phophocholine (POPC) was purchased from Avanti Polar Lipids®. PEG-polymers (3 kDa), HS-PEG-NHCO—CH.sub.2CH.sub.2—OH (HS-PEG) HS-PEG-NHCO—CH.sub.2CH.sub.2-Biotin (HS-PEG-Biotin) were purchased from Rapp Polymere® GmbH. The Sensor chips were purchased from GE Healthcare®. The membrane protein analysis kit came from LayerLab®. HEPES-2 is 10 mM HEPES, 150 mM NaCl, pH 7.4.

(10) Preparation of Liposomes

(11) Liposomes were made by first evaporating the solvent, chloroform, from the POPC lipids using a flow of nitrogen gas. The dried lipids were then kept under the flow of nitrogen gas for at least one hour after which HEPES-2 was added to yield a 4 mg/ml concentration of POPC. The dissolved POPC lipids were vortexed for at least 30 min to yield multi-laminar liposomes. Uni-laminar liposomes were produced using a mini extruder and polycarbonate membranes with pore size of 50 nm from Avanti Polar Lipids® with a 50 nm pore size. The radiuses of the liposomes had a Gaussian size distribution with a mean radius of 36 nm. The liposomes were stored under nitrogen gas at 4° C. until use (within 2 days).

(12) Functionalization of the Biosensor Surface

(13) A mixture (300 μl) of HS-PEG (200 μg/ml) and HS-PEG-Biotin (20 μg/ml), dissolved in HEPES-2, were injected over the Biacore® Au sensor chip at a flow rate of 5 μl/min. Then a mixture (150 μl) of NeutrAvidin (40 μg/ml) and Biotin-DNA (0.7 μM) was injected and bound to HS-PEG-Biotin at the surface.

(14) Tethering of Liposomes

(15) POPC liposomes (2 mg/ml, in HEPES-2) containing cholesterol-DNA tags (3 DNA tags per liposome) were injected at a flow rate of 5 μl/min. The end of the DNA-part of the cholesterol-DNA tag is single-stranded and complementary in sequence to the Biotin-DNA at the surface enabling tethering of the liposomes to the sensor surface.

(16) Melittin Binding to the Liposomes

(17) The reversible binding of melittin was measured in the Biacore® instrument directly after the tethering of POPC liposomes was finished. Melittin solutions at a concentration of 0.5-40 μM in HEPES-2 were injected in sequence. During injection the melittin molecules bind to the liposomes and after the injection is terminated, running buffer (HEPES-2) flows over the surface and melittin dissociates. After the dissociation was complete a new melittin solution of different concentration was injected.

(18) Rupture of Liposomes by Melittin

(19) Rupture of liposomes by melittin was measured in the Biacore® instrument directly after the tethering of POPC liposomes. Melittin dissolved in HEPES-2 at a concentration of 360 μM (1 mg/ml) was injected and the decrease in response (RU) as the liposomes rupture was monitored.

(20) Melittin Mediated Uptake of Sucrose

(21) After tethering of NeutrAvidin/Biotin-DNA the following injections were made in sequential order; 0.1 M sucrose, 2 mg/ml cholesterol-DNA tagged POPC-liposomes, 0.1 M sucrose, 40 μM melittin, 0.1 M sucrose, 0.1 M sucrose, 0.1 M sucrose. All samples were dissolved in HEPES-2.

(22) Results

(23) Tethering of Liposomes

(24) There was binding of liposomes to the biosensor surface. The Carboxylic groups at the surface were activated for amide binding using EDC/NHS. Thereafter PLL-PEG/PLL-PEG-Biotin (5:1), NeutrAvidin/Biotin-DNA (1:1) and cholesterol DNA tagged POPC-liposomes were added.

(25) The bilayer structure enclosed a first volume of the detection volume and the volume not enclosed by the bilayer structure but within the detection volume is a second volume. The ratio between the first and second volumes was in this particular case about 0.1.

(26) Melittin Binding and Pore Formation

(27) Melittin solutions of various concentrations (0.5-40 μM) were injected over the bound liposomes in the Biacore® instrument at 5 μl/min. The change in response (RU) as a function of time (t) was fitted to RU=RU.sub.0+RU.sub.1*(1−exp(−t/τ.sub.1))+RU.sub.2*(1−exp(−t/τ.sub.2)). However, the change in RU during the injection of 0.5 μM melittin could be described using only one exponential (i.e. RU.sub.2=0). It is known that high concentrations of melittin can rupture cells and liposomes. Unspecific binding (i.e. binding to the biosensor surface without liposomes) of melittin was small, less than 5% of the binding of melittin to the liposome membrane surface (data not shown). The observed rate constant, k.sub.1, (k.sub.1=1/τ.sub.1) and the response (RU.sub.1), were studied as a function of the concentration of melittin in the bulk solution [Mel].sub.B. The microscopic on-rate (k.sub.+1) and off-rate (k.sub.−1) of melittin with the liposome membrane surface were obtained from a fit of the data to k.sub.1=[Mel].sub.B*k.sub.+1+k.sub.−1, which gave k.sub.−1=0.032 s.sup.−1 and k.sub.+1=3684 M.sup.−1 s.sup.−1. The amplitude, RU.sub.1, was also determined by the microscopic rate constants, RU.sub.1=RU.sub.max*(k.sub.+1/(k.sub.+1+k.sub.−1). There was a good fit of RU.sub.1 as a function of [Mel].sub.B when using k.sub.−1=0.032 s.sup.−1 and k.sub.+1=3684 M.sup.−1 s.sup.−1 (which are obtained from the fit of k.sub.1 as a function of [Mel].sub.B).

(28) A comparison of the response (RU) from binding of liposomes to that from binding of melittin, gives the number of melittin molecules that binds to each liposome. It is known that the α-helical melittin molecule binds to the membrane in a parallel orientation and the area of the liposome membrane that a melittin molecule occupies is approximately 210 Å.sup.2. The surface area of the liposome outer membrane is approximately 1.63*10^6 Å.sup.2 (liposome radius≈360 Å). Hence the concentration of melittin at the liposome outer membrane ([Mel].sub.O.M.) and the fraction (F.sub.O.M.) of the liposome surface that the melittin molecules cover can be calculated for each [Mel].sub.B.

(29) At concentrations of 2, 4, 8 and 40 μM of melittin in the bulk solution, association of melittin with the liposome were biphasic. It is known that pore formation is induced at a threshold concentration of melittin at the membrane surface. If pores are formed, melittin molecules in the bulk solution outside the liposomes should be able to cross the liposome membrane and bind to the inner membrane surface of the liposomes. It is thus assumed that the slower kinetic phase represent melittin binding to the inner membrane. The observed rate constant, k.sub.2, was found to be linear dependent on [Mel].sub.B which shows that transfer a melittin through the pore is not the rate limiting step (not dependent on [Mel].sub.B). A linear fit gave that k.sub.+2=56 M.sup.−1 s.sup.−1 and that k.sub.−2=0.0046 s.sup.−1. The lower value of the apparent diffusion coefficient k.sub.+2 (56 M.sup.−1 s.sup.−1) compared to k.sub.+1 (3684 M.sup.−1 s.sup.−1) can be explained by the lower probability of a melittin molecule finding the pores of the liposome than the liposome surface as a whole. The number of pores/liposome is Gaussian distributed and at low [Mel].sub.O.M. an increase in [Mel].sub.B increases the number of liposomes having one pore. Hence, at low [Mel].sub.B the response (RU.sub.2) will not only depend on [Mel].sub.B but also on the fraction of liposomes that do not have any pores. At high [Mel].sub.B, where all liposomes have at least one pore, the surface coverage of melittin at the inner membrane should equal that at the outer membrane, and RU.sub.2 should approach RU.sub.2max=RU.sub.1*(Area.sub.In/Area.sub.Out). Hence the above results show that the slower kinetic phase describes binding of melittin to the inner membrane. Further support comes from an observation that RU, after the injection of 4, 8 and 40 μM is stopped, does not return to the initial value (pre-injection), which is explained by the melittin molecules being trapped inside the liposomes as the pores disappear due to melittin dissociation from the membrane surface.

(30) Melittin Pore Mediated Sucrose Uptake

(31) A simple method for real-time and label-free uptake measurements of analytes across membranes at a biosensor surface would be of great value when investigating for example membrane transporters. Here we wanted to test if it was possible to measure the uptake of an analyte, sucrose, through the melittin pore by simply detecting the change in refractive index of the solution inside the liposome caused by the analyte as it is taken up.

(32) FIG. 1 shows the successive addition of Neutravidin/Biotin DNA (N/B), 0.1 M sucrose (S1), 2 mg/ml cholesterol DNA tagged POPC-liposomes (POPC), 0.1 M sucrose (S2), 40 μM melittin, 0.1 M sucrose (S3), 0.1 M sucrose (S4), 0.1 M sucrose (S5) to a PEG/PEG-biotin functionalized surface.

(33) FIG. 2 shows the response (RU) from the five sucrose additions (S1-S5). Adding sucrose changes the refractive index of the solution in the detection volume, i.e. the volume corresponding to the evanescent field above the sensor surface, which is manifested as a shift in RU. The smaller increase in RU upon adding S2 compared to adding S1 corresponds to the reduced accessible volume of the evanescent field caused by the surface-bound liposomes. Addition of melittin produces pores in the liposomes membrane and hence makes the interior volume of the liposomes accessible to the added sucrose molecules. As the melittin addition is stopped, melittin dissociates from the membrane surface and the number of liposomes having melittin-pores decreases with time. FIG. 2 shows the change in RU upon addition of sucrose as the number of pores/liposome is reduced (S3-S5).

(34) FIG. 3 shows the uptake of sucrose into the liposomes as a function of time, which is obtained by subtracting the change in RU upon addition of sucrose to closed liposomes (S2) from the change in RU upon addition of S3-S5. The amplitude of the uptake decrease from S3 to S5, which is explained by a decreasing number of liposomes having pores. The rate also decreases from S3 to S5, which is explained by decreasing number of pores per liposome. From the graph in FIG. 3 the decay time for uptake of sucrose can be estimated and τ.sub.3≈20 sec. Given that the concentration of sucrose inside the liposome after the uptake is equal to [sucrose].sub.B=0.1 M, then ˜7400 sucrose molecules cross the membrane in ˜20 sec. Table 2 show that RU.sub.2/RU.sub.2max at [Mel]B=40 μM is 0.85 which correspond to the number of liposomes having more than one pore/liposome. The amplitude of the response is in good agreement with the value that can be expected from filling the liposomes with 0.1 M of sucrose, which have a molecular weight (Mw) of 0.34 kDa. The expected RU of the 7400 sucrose molecules (0.1 M) inside the liposome can be calculated from the obtained response of immobilizing the liposome itself (RU=6790). The Mw of the liposome is 35800 kDa, which gives 0.19 RU/kDa. The Mw of 7400 sucrose molecules is 7400*0.342 kDa=2531 kDa, which gives that RU=2531*0.19=480. Since only ˜85% of the liposomes have pores the expected value is 480/0.85≈560 RU, which is in good agreement with the measured response of 530 RU from the S3 injection.

Example 2

SPR-Based Methods for Screening of Multiple Permeation Events by Investigating the Permeability of the Non-Electrolytes Glycerol, Urea and Hydroxyurea

(35) The efficiency of the SPR-based method and its compatibility with screening of multiple permeation events was evaluated by investigating the permeability of the non-electrolytes glycerol, urea and hydroxyurea, which are all biologically relevant molecules. For example, the efficiency by which glycerol is transported across the membranes of the adipocytes, where fat molecules are stored, has been suggested to be an important factor in the development of obesity and type II diabetes [17]. Urea, on the other hand, is a waste product in the metabolic process and is transported out from liver cells and released from the body via urine, while hydroxyurea is a drug that has been widely used in cancer chemotherapy [18] as well as in treatment of HIV-virus infections [19]. The applicability of the method is demonstrated for in situ alteration and simultaneous monitoring of liposome permeability by monitoring the increase in glycerol permeation upon a gradual cyclodextrin-induced reduction in the liposomal cholesterol content. Also shown is the compatibility of the method to probe transport of ions.

(36) At first 70 nm liposomes were provided in accordance with Example 1. FIG. 4a shows binding of 70 nm liposomes followed by repeated injection pulses of glycerol, urea, hydroxyurea at different concentrations (see inset). FIG. 4b shows a magnification of one of the rinsing steps, upon which the solute (glycerol) is released from the liposome interior. Also shown in FIG. 4b (dashed curve) is an identical rinsing step without immobilized liposomes, illustrating that the time constant of the fluidic exchange is faster than 100 ms, which is sufficient to temporally resolve these transport measurements. The rate of glycerol release was examined in a temperature interval from 8 to 22° C. The results are demonstrated in FIG. 5a. In agreement with expectations, the rate of transport across the lipid membrane increases with increasing temperature, and Arrhenius plots display a linear dependence between ln(1/) and 1/T, yielding activation energies in excellent agreement with literature data for the three solutes (glycerol, urea and hydroxyurea) investigated, see FIG. 5b.

(37) The results shown in FIGS. 4 and 5 demonstrate that the method can be used to accurately determine permeation coefficients of lipid membranes by following the release of solutes, rather than the uptake. Note that all these data were obtained from a single experiment using the same set of immobilized liposomes. The reproducibility between different experiments was better than 2%.

(38) 70 nm liposomes containing 40% cholesterol were provided in accordance with Example 1. Binding of the liposomes followed by repeated injection cyclodextrin, which removes cholesterol from the lipid bilyaer of the liposomes is shown in FIG. 6a. The SPR response is proportional to the interfacial refractive index, which makes it possible to quantify the removal of cholesterol. After seven injections of cyclodextrin, the cholesterol content was reduced to 14%.

(39) Each injection of cyclodextrin (FIG. 6a) was followed by an injection pulse of glycerol, which enabled the time constant of glycerol transport versus cholesterol content to be quantified, ranging from around 7 s at 40% cholesterol to 1 s at 14% cholesterol, see FIG. 6b. Note that a difference in transport rate was detectable for as little as 1% change in cholesterol content.

(40) FIG. 6c shows the variations in permeability (estimated from the rate of solute transfer and the vesicle area) and the amplitude of the response (which is proportional to the internal volume). In agreement with expectations, the permeability decreases linearly versus cholesterol content, while the internalized volume increases non-linearly. The latter observation reflects a structural change of the membrane at a cholesterol content of around 25%.

(41) With the measurements shown in FIGS. 3a to c, it is demonstrated that the method enables solute transfer to be quantified in terms of permeability, which can simultaneously be correlated with structural changes of the membrane properties. Information of this type is of high relevance in studies on how, for example, drugs influence the permeability of cell-membranes. Both passive and membrane-protein mediated transfer of uncharged solutes is of high biological and pharmaceutical relevance. However, for a method like this to be generically applicable for any type of membrane transport reaction, also ion translocation should be possible to measure, which is today typically measured using patch-clamp based assays. FIG. 7 illustrates membrane transport of iodide (˜10 s) and chloride (˜200 s) ions, in excellent agreement with literature data. Note that gramicidin was incorporated into the liposomes in order to ensure co-transport of counter ions (K+), thus prohibiting the establishment of an electrostatic transmembrane potential that would otherwise restrict the unhindered transport of anions.

(42) The results shown in FIG. 7 demonstrate the applicability of the method to time resolve membrane translocation reactions also of charged solutes. The natural extension of this work is studies of molecule and ion-channel controlled transport, of high relevance in the drug-screening process.

(43) As illustrated for glycerol, urea and hydroxyurea, the method according to the invention enables screening of multiple permeation events in a single measurement, and allows for in situ alterations in permeability to be quantified, which is exemplified by successive removal of cholesterol from the lipid bilayer. In comparison with alternative methods to probe non-electrolyte transfer, which rely on indirect measurements osmotically-induced size changes of suspended liposomes, the method improves the sensitivity and reduces the required amount of sample by orders of magnitude.

(44) In the set of results shown in FIGS. 4 to 7, it is demonstrated that solute transport across the lipid bilayer membrane of liposomes immobilized in an electromagnetic evanescent field can be determined by following the changes in refractive index of the liposome-internalized volume upon solute release, rather than uptake (FIG. 4). It is also demonstrated that the membrane properties can be varied in situ, and be correlated to changes in permeability (FIG. 5). In FIG. 6, we demonstrate that not only uncharged solutes, but also the transport of ions can be measured by following changes in the refractive index of the liposome-internalized volume upon solute release (or uptake).

(45) In summary, the present invention admits the possibility of using this sensing principle for direct measurements of molecular transport of non-electrolyte solutes across membranes with a time resolution of 100 ms. The method is unique in the sense that it enables direct, rather than indirect (c.f. the DLS and fluorescence quenching methods), measurements of solute transfer across lipid bilayers, which was so far possible only for charged molecules using patch-clamp [14] or chip-based alternatives [15]. In comparison with the indirect methods, it also offers all other advantages of surface-based methods, such as rapid sequential (or parallel) screening of multiple solutes as well as in situ perturbation of liposome permeability by addition of effector molecules. There is also no limitation with respect to the size of the analyzed liposomes, while the DLS-based method is applicable on relatively large liposomes only, since liposomes with a diameter smaller than around 80 nm are essentially non-compressible (<1%) [16].

(46) Although the invention has been described with regard to its preferred embodiments which comprise the best mode presently known to the inventors it should be understood that various changes and modifications as would be obvious to one having the ordinary skill in this art may be made without departing from the scope of the invention as set forth in the claims appended hereto.

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