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3. Introduction

The main aim of this research project is to investigate and characterise the hydrophobic interactions between propranolol with β-cyclodextrin, detergent micelles and a model lipid bilayer system using various aspects of fluorescence spectroscopy. The Stern-Volmer equation has been used to interpret the results in terms of the type of quenching occurring. The accessibility of propranolol to the quencher ions provides valuable information about the types of interactions taking place.

3.1 Drug

Propranolol is a non-selective β-adrenergic blocker, blocking the action of both epinephrine and norepinephrine on both β1 and β2 receptors (Bylund, 2007). These β antagonist properties are used to lower blood pressure in the treatment of hypertension. Propranolol has also been used to treat anxiety, angina pcctoris and convulsions (Glenn 2004).

Propranolol is an alkyl amine, it is aryl substituted, contains a naphthalene ring responsible for its fluorescence and an alkyl side chain which has a chiral centre (Glenn, 2005). Being a relatively hydrophobic, non-polar drug propranolol favours interactions with neutral and non-polar solvents, its fluorescence spectrum can be described as being solvent dependant (Hunt2006). The hydrophobic nature of propranolol causes it to form aggregates in water, this effect is called the hydrophobic effect. Propranolol is highly soluble in lipids, giving it a larger volume of distribution and making it able to penetrate the central nervous system, crossing the blood/brain barrier. Varied bioavailability is recorded and a reduction in drug concentration is observed during absorption in the liver and gut despite it being rapidly metabolised. The clinical form is propranolol hydrochloride which helps to prolong its biological activity. It has been noted that at high concentrations propranolol can act as a membrane stabilizing agent by blocking myocyte sodium channels increasing toxicity (Rodgers 1985). This property has been linked to specifically to (R)-(+) enantiomer (Hyunmyung 2003) and would be a excellent development for further study.

Propranolol is suggested to bind by polar alignment to cellular bilayers, the phospholipid component of biological membranes (Hallifax and Houston 2006). Propranolol has a characteristic fluorescence; it is thought that the naphthalene ring is responsible for this (Glenn, 2005). The naphthalene ring can be referred to as the fluorophore. This project will use fluorescence spectroscopy to image the uptake and distribution of propranolol in both detergent micelles and model bilayer systems. This will allow further analysis and quantification of the interactions of propranolol.

Propranolol is an optically active molecule, contains an asymmetric centre, and its enantiomers may interact differently, with the possibility of one being favoured. The enantiometric properties of (R)-(+) and (S)-(-) propranolol β-CD complexes have shown to require an interaction between the chiral centre and the secondary hydroxyls of the CD cavity (Glenn et al 2004). In the presence of chiral alcohol the fluorescence intensity of either enantiomer is dependent on the β-CD concentration, (S)-(-)-propranolol increasing and (R)-(+)-decreasing. The addition of a chiral alcohol suggests a change in propranolol's immediate environment may favour the interactions of a specific enantiomer. The enantioselectivity of SR-propranolol has been further investigated through the use of chiral activated membranes (Gumí et al 2004). A clear difference in the transport rate of the enantiomers was observed, further indicating that chirality strongly influences the chemical interactions of propranolol.

It is thought that for some drug interactions a slight change in the position of methyl group alters the binding affinity for each enantiomer, experimental conditions have not made it possible to confirm this yet. The crystal structure of propranolol has been determined using x-ray crystallography. It shows propranolol to be almost planar with the charged amine group 5.5 Å from the naphthalene ring. Neutron diffraction is used in conjunction with the crystal structure to give conformation, orientation and location of propranolol's position within lipid bilayers. The naphthalene ring was found to be in the hydrophobic region of the bilayer, approximately 10 Å from the surface. The charged amine is thought to be positioned within a phospholipid head group (Herbette et al 1986). This information suggests ionic interactions between the amine and the phospholipid region. The hydrophobic binding between the membrane and naphthalene ring is of particular interest with alteration of pH affecting the extent of association (Butler et al 2005).

3.2 Fluorescence

Photoluminescence is a process in which a substance absorbs electromagnetic radiation (photons) and then re-radiates photons. It is an important tool in analytical chemistry, the most widely used form of this being fluorescence. All fluorescent molecules have an absorption and emission spectrum. In fluorescence the photon emitted is often of a longer wavelength length and therefore lower energy than the photon absorbed. The difference between the maxima of both the absorption and emission spectra is called Stokes shift, these are dependent on the molecule being studied. In order for a molecule to fluoresce it must be promoted it to an excited quantum state. The large difference between the ground state and the excited state requires electromagnetic radiation, normally in the form of a photon of ultraviolet radiation to be used. The molar absorptivity, , is defined by the Beer-Lambert Law:

The Beer-Lambert law relates the absorption of light to the properties of the material through which the light is travelling. The absorption process causes an electron to be promoted from the ground state to a higher energy orbital, S1 as shown in the Jablonski diagram . In the diagram the electronic states are depicted as S0, S1, S2 and S3, each containing a number of vibrational energy levels. The type of orbital classifies the transition type, n → π* and π → π*, the electron is transferred to an anti-bonding orbital.

Radiationless internal conversion causes the electron to rapidly relax to the lowest vibration energy level of the S1 singlet state. The electron will then make the transition to the ground state by the emission of a photon (fluorescence) lasting only a few picoseconds or less. Phosphorescence is a similar to fluorescence, but it achieved by a process called intersystem crossing. Instead of the electron making the transition to the ground state, S0, it undergoes interconversion and is transferred to a triplet state T1. The stability of the T1 excited state determines whether a material is fluorescent, unstable, or phosphorescent. Phosphorescence is therefore a much slower process than fluorescence, in the region of a few nanoseconds.

3.3 Fluorescence Quenching

By observing the fluorescence of propranolol in different environments and under different conditions we are able to understand the interactions taking place. There are several ways fluorescence intensity can be influenced. Inclusion in micelles or other molecular cavities can lead to an increase, and quenching can lead to a decrease.

There are various mechanisms by which quenching can occur. The excited molecule can lose energy through an interaction with a second molecule causing the fluorophore to become deactivated. This process is called dynamic (collisional) quenching where the fluorophore is returned to the ground state without the previously observed fluorescence. Dynamic quenching reduces the concentration of molecule in the excited state and therefore the fluorescence. Static quenching occurs when the quencher and fluorophore form a non-fluorescent ground state complex. The Stern-Volmer equation can be used to describe the decrease in fluorescence intensity related to quenching:

Where K is the Stern-Volmer quenching constant for either process (static, Ks, or dynamic, Kd), kq is the bimolecular quenching constant, is the unquenched lifetime and [Q] is the quencher concentration.

Fluorescence data is usually plotted as versus with the relationship is expected to be linearly dependant of quencher concentration. The gradient is equal to K with an intercept on the y-axis of 1. When referring to static quenching Ks it can be considered the association constant for the formation of the complex and can be used to explain for a decrease in fluorescence intensity which cannot be explained by dynamic quenching. The type of quenching that has occurred cannot be proven simply using a Stern-Volmer plot, as both types of quenching will give a linear Stern-Volmer plot.

The important characteristics of a fluorophore are fluorescence lifetime and quantum yield. The lifetime gives us the approximate time that a given fluorophore will spend in the excited state and therefore how long it has to interact with any molecules present. The quantum yield is a reference to the fluorescence intensity, it takes into account the number of emitted photons compared with the number absorbed. The fluorescence lifetime can be used to identify the type of quenching which has caused the decrease in fluorescence. For static quenching , and for dynamic quenching . It is possible that both types of quenching can take place at the same time; this is reflected in the Stern-Volmer plot when an upward curvature is observed as the extent of quenching is large. The modified second order Stern-Volmer equation for this is:

A quenching sphere of action can be used to analyse an upward curving Stern-Volmer plot. A series of calculated F0/F values are compared to those gained experimentally. Using the difference squared and Solver to manipulate the KSV and V from the SV plot, to achieve the best fit line observed on the plot. This takes into account both types of quenching, showing that static ground state complexes are not formed. It is in fact the proximity of the quencher to the fluorophore during excitation which is responsible for the static quenching component. The sphere of action is an interpretation of this quenching and a further modified Stern-Volmer equation describes this:

Where V is the volume of the sphere and N Avogadro's number. The volume of the sphere gives us the area around the fluorophore where if a quencher molecule were present quenching would occur. When the volume of the sphere is known the radius of the sphere can also then be calculated giving an estimate of the distance between quencher and fluorophore required for quenching to occur. This is calculated from . The probability of immediate quenching in this given volume is unity, only fluorophores with no adjacent quenchers are observed. The above equation is derived after calculation of the number of fluorophores without a quencher ion in their sphere of action. The Poisson distribution gives the probability of finding a quencher within a molecules sphere of action. The existence of the sphere reduces the amount of fluorescence that will be observed:

The mean number of quenchers in the given volume, sphere, is λ which is equal to . The probability that no quenchers are within the sphere can be given derived from:

Fluorescence should only occur from fluorophores with no adjacent quencher molecules. As the concentration of quencher is increased it will increase the probability that the quencher ion is in close proximity to the first solvent shell of the fluorophore at excitation and there for the chance of quenching.

If no lifetime measurements are available the next step in analysis is to calculate the apparent quenching constant, Kapp = , for each quencher concentration. This can then be plotted against [Q], to give an intercept will the value KD + KS and a slope which is equal to KDKS. By solving the quadratic equation both KD and KS for the quenching process can be found. The larger value is normally assumed to be the dynamic component.

To further evaluate the quenching processes the rates of emissive and non-radiative decay ( rates, which are responsible for the molecules return to the ground state, can be studied. This data will not be measured in the current set of experiments but the relationships will be shown. The affect on the quantum yield leads to a depopulation of the excited state, their relationship is given by:

Fluorescence lifetimes are also affected by emissive and non-radiative decay rates:

The fluorescence emission spectrum of propranolol is dependent on the environment of the fluorophore, including the solvent. The position of the fluorophore can alter the effectiveness of the quencher and therefore the quenching constant. The naphthalene ring in propranolol is responsible for its fluorescence and so by restricting access to it by inclusion in a micelle or β-cyclodextrin cavity. This will lower the value of K compared to that in free solution.

3.4 Cyclodextrin complexes

The cyclodextrin's (CDs) are a class of cyclic oligomers of α-D-glucose. The central cavity of CD is hydrophobic while the outer region of the oligosaccharide is hydrophilic due to the presence of primary and secondary hydroxyls. One of the most important properties of CD's is their ability to form complexes with organic compounds in solution. In terms of drugs this inclusion can lead to a reduction in free drug in the body and possible side effects. The cavity size and bonding interactions will therefore determine both bioavailability and therapeutic effects of the drug. The cavity size of α-CD was found to be too small to accommodate propranolol. The cavity of γ-CD, was able to form a complex with the drug but the binding interactions were not as stable as those of the β-CD complex. The thermodynamics of inclusion of propranolol in various cyclodextrin cavities have shown to favour β-CD (Castronnuovo and Niccoli 2006).

Hydrophobic Interactions of Propranolol Rebekah Sayers

The naphthalene ring comprises the fluorophore in propranolol and its partial or full inclusion in the hydrophobic CD cavity has shown an increase in fluorescence intensity due to the shielding of the excited singlet state (Glenn et al 2004). In the presence of quencher ions complexation of a naphthalene containing molecule with β-CD has shown decreasing efficiency with increasing β-CD concentration (Encinaset al 1992). This would affect the Stern-Volmer plot with a downward curved line and results would have to be attained from the linear section of the plot. Dynamic quenching would be observed between bound fluorophore and both free or CD associated quencher ions with static quenching disregarded.

3.5 Detergent micelles, liposomes and biological membranes.

Lipids are molecules which have both hydrophobic and hydrophilic domains, they are called amphiphiles. Detergents are composed of amphiphiles that allow hydrophobic molecules to be solubilised in water by forming micelles and bilayers.

Hydrophobic Interactions of Propranolol Rebekah Sayers

Biological membranes are composed of amphiphilic phospholipids that are made up of lipids arranged so that their polar heads face outwards and non-polar regions form the hydrophobic core. The hydrophobic interactions between lipid molecules are thermodynamically favourable as they reduce the amount of hydrophobic molecule exposed to the water, increasing entropy. This prevents any external water mixing with the internal aqueous environment of a cell. This is the reason why the formation of lipid bilayers and micelles is observed. Lipid bilayers are the basic structural element of biological membranes. Membranes are therefore impermeable to charged or polar molecules, favouring non-polar alternatives. When using quencher ions in biological membranes the barrier effect of the bilayer has been shown to allow the permeation of both iodide and acrylamide at comparable rates (Moroetal1993). Non-polar fluorophores appear to be embedded in the bilayer and in this situation quenching is observed for iodide but not acrylamide.

Sodium dodecyl sulphate (SDS) is anionic surfactant, it has a polar head and a non-polar tail (NaOSO3C12H25) making it amphiphilic. When dissolved in water the surface tension of SDS decreases and its ability to solubilise hydrocarbons is increased. When these changes take place the concentration is called the critical micelle concentration (CMC). Above the CMC the monomers of SDS will form SDS micelles. Micelles have an interior made up of hydrocarbons with a polar exterior composed of OSO3-. The hydrocarbon core has the ability to accommodate hydrophobic molecules, a property which can be used in the study of propranolol. The importance of the anionic head group of SDS in binding has been observed compared to the micelles from neutral and cationic solutions, where the interactions were not as strong. The quenching of fluorophores held within SDS micelles is complex due to the bimodal size distribution at high ionic strength. When using iodide as a quencher, it is important to remember that it is an anionic quencher and access to a SDS micelle will require a large amount of energy (Cramb and Beck 2000). It is therefore possible that quenching is a result of the I3- ions which are formed in solution or following the partition of I2 into SDS micelles (Lakowicz).

The binding between drug and membrane is important for drug delivery into cell membranes. Fluorescence analysis of drug/glycosaminoglycans complex formation identifies an increase in fluorescence intensity (Santos et al2007). In order to understand the interactions between propranolol and liposomal membranes in the body, a model liposomal system can be prepared. Some research into the use of spectrophotometry to determine drug partition coefficients has been done using water/dimyristoyl-L-α-phosphatidylglycerol (DMPG) (Rodriguesetal2001) and water/dimyristoyl-L-α-phosphatidylcholine (DMPC) (Rodrigues et al 2000) liposomes. A quantative measure of a drugs lipophilicity can be made by working out its distribution between aqueous and water immiscible phases. The equilibrium constant for this relationship is the partition coefficient (Kp) and can be calculated using spectrophotometric data gathered over a range of pH2 to pH12 at 37. A pH dependant dissociation of propranolol is seen above pH4.5. Below pH4.5 an acid induced change influences the drug partitioning (Pauletti and Wunderli-Allenspach 1994).

4. Experimental

4.1 Apparatus

Fluorescence measurements were recorded using a FluoroMax 3 fluorescence spectrometer equipped with a thermostatic cell housing to allow manipulation of temperature. The slit widths for emission and excitation were kept at 5 nm for all experiments. An excitation wavelength of 290 nm was used and the emission spectra recorded between 295-450 nm. The emission maxima were determined experimentally and recorded at approximately 354 nm. The solutions were measured in a 3 cm3 acrylic cuvette.

4.2 Materials

Propranolol hydrochloride (RS), Β-cyclodextrin and SDS were obtained from Sigma-Aldrich Corporation. NaI and Acrylamide were obtained from???. PBS was used to prepare all the solutions. The lipids were kept at -200C and used without further treatment. 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) was obtained from ???. All other chemicals were reagent grade and were used as received, any solutions were made up with PBS.

4.3 Fluorescence and fluorescence quenching of propranolol using NaI and acrylamide ions

A stock solution of propranolol (2 mM) in phosphate buffered saline (PBS) was prepared. Using suitable dilutions an absorbance (100 μM, 200-600 nm) and emission (10 μM, 295-450 nm) spectra were recorded at room temperature in order to determine the wavelengths at which the absorbance and emission are at a maximum. Excitation was found to be at approximately 290 nm and the emission maximum at approximately 354 nm.

The next step was to determine the effect of quenching by sodium iodide and acrylamide ions on propranolol. This was carried out by measuring the fluorescence spectrum of propranolol (10 μM) in PBS whilst titrated with a solution of the given quencher ions (2 M, 20-100 μl additions). NaI solutions were freshly prepared before each set of experiments to avoid I3- formation. All solutions were made up accurately in volumetric flasks using PBS. Special care was taken whilst handling acrylamide. It was weighed and diluted in the fume cupboard as well as taking all the usually safety precautions. The buffered propranolol (10 μM, 2.5ml) was measured into the cuvette and the quencher additions (20 μl) added through a parafilm lid using a micro-syringe. After each addition the cuvette was inverted to allow the quencher ions to diffuse into the buffered propranolol solution. The spectra were recorded at room temperature on the FluoroMax 3 spectrometer from 295-450 nm after exciting the sample at 290 nm. The Stern-Volmer plot equation was then used and adapted using the sphere of action to show the types of quenching occurring.

4.4 Effect on the fluorescence of propranolol on inclusion in SDS detergent micelle cavities using NaI and acrylamide quencher ions

A titration using sodium dodecyl sulphate, SDS (100 mM, 10-120 μl additions), was performed with propranolol (10 μM, 2.5 ml). The buffered propranolol (10μM, 2.5 ml) was measured into the cuvette and the SDS additions (20 μl) added through a parafilm lid using a micro-syringe. After each addition the cuvette was inverted to allow the buffered propranolol solution to diffuse into any newly formed micelles. The fluorescence spectra were recorded for each addition at room temperature on the FluoroMax 3 spectrometer from 295-450 nm after excitation at 290 nm.

After performing the SDS titration further investigation was done to assess the position of propranolol in the SDS micelles by measuring the effect of quencher ions on the fluorescence. A cuvette was filled with propranolol (10 μM, 2.5 ml) and SDS (100 mM, 125 μl) and a titration was performed for each of the quencher ions, NaI and acrylamide (2 M, 10-120 μl). The SDS concentration in the cuvette was in excess of the CMC, approximately 5 times greater, this was to ensure that there were enough micelles to include all the propranolol in solution. The additions were added to the cuvette through a parafilm lid using a micro-syringe and the cuvette inverted after each addition to allow the quencher ions to diffuse. The fluorescence spectrum was recorded for each addition at room temperature on the FluoroMax 3 spectrometer from 295-450 nm after excitation at 290 nm. The Stern-Volmer equation can then be used and adapted to fit the sphere of action model to show the types of quenching occurring and the bonding relationship between propranolol and the SDS micelle.

4.5 Effect on fluorescence of propranolol on inclusion in β-CD cavities using NaI and acrylamide quencher ions.

In this set of experiments the inclusion of propranolol (10 µM) in β-cyclodextrin solutions of varying concentration (3-12 mM) was observed. Due to the high molecular weight of β-CD a stock solution (15 mM) was prepared, this was made to the highest concentration possible due to the insolubility of β-CD. The solution had to be warmed to approximately 25 0C to enable the β-CD to fully dissolve. A series of dilutions were then made in volumetric flasks from the stock (3 mM, 6 mM, 9 mM, and 12 mM) using PBS, each contained an equal amount of propranolol (50 µM, 2 ml). The propranolol solution used was of a higher concentration than in previous experiments. This allowed it be diluted with the β-CD and the concentration in the cuvette to remain as in all previous experiments (10 µM). Each of the propranolol/β-CD solutions (2.5 ml) was measured into a cuvette and an emission spectra was ran for each concentration at room temperature on the FluoroMax 3 spectrometer, from 295-450 nm after excitation at 290 nm. The position of the propranolol in the β-CD complexes was assess by measuring the effects of quenching on fluorescence using sodium iodide (2M) and acrylamide (2 M) ions. The quencher ions (2 M) were titrated against each dilution by addition (10 x 10 µl) to the cuvette using a micro-syringe. Once again after each addition the cuvette was inverted to allow the quencher ions to diffuse through the solution. The Stern-Volmer equation can then be used and adapted to show the types of quenching occurring and the bonding relationship between propranolol and the β-cyclodextrin cavity discussed.

4.6 Determination of drug-liposome binding

Liposomes were prepared using the ethanol injection method (Pons 1992). DPPC (7.3 mg, 50 mM) was dissolved in ethanol (0.2 ml) and then injected into PBS (5ml, 500C) whilst on a mixer. The solution was then allowed to settle. The first set of results was recorded at room temperature, below the phase transition temperature of the liposomes. Spectra were recorded for propranolol in PBS (10 µM, 2.5 ml), DPPC in PBS (1 mM, 2.5 ml) and for the addition of propranolol (2 mM, 12.5 µl) to the DPPC liposome (1 mM, 2.5 ml). The experiment was repeated at 500C, above the phase transition temperature of the liposomes.

5. Results and discussion

5.1 Fluorescence and fluorescence quenching of propranolol using NaI and acrylamide ions

The absorption spectrum of propranolol gave the maximum absorption wavelength to be 290 nm. This wavelength was used to excite the sample when recording the emission spectra. The emission spectrum was recorded over 295-500 nm after initially exciting the sample at 290 nm. A maximum emission was recorded at approximately 354 nm and it was at this wavelength that fluorescence intensity was to be recorded for all further experiments.

Propranolol fluorescence was analysed further with the use of NaI and acrylamide quencher ions. Stern-Volmer dynamics were used to describe the observed decrease in fluorescence intensity in relation to increased quencher concentration. A Stern Volmer plot of the quencher concentration against fluorescence intensity without quencher/fluorescence intensity with quencher (F0/F) can be derived from the data .

Hydrophobic Interactions of Propranolol Rebekah Sayers

The SV plots show florescence to be proportional to the concentration of quencher ions in a linearly dependant relationship. Where dynamic quenching has occurred, the fluorophore was deactivated by the loss of energy through interaction with a second molecule, the quencher ion, during the lifetime of the excited state. This linear dependence is not conclusive of dynamic quenching as static quenching is also linearly dependant with quencher concentration. A slight upward curvature towards the y-axis is observed in both instances and the quenching process can be described as second order, two different types of fluorescence quenching were taking place, dynamic and static. Static quenching is thought to occur in this situation not by complex formation, but due to the closeness of the quencher ion to the first solvent shell of the fluorophore at the moment of excitation. The probability of this type of quenching occurring can therefore be considered to be unity. The data was then fitted to a sphere of action quenching model to interpret the different types of quenching occurring, using the modified Stern-Volmer equation: F0/F=(1+Kd[Q])exp(V[Q]) . The sphere dictates the area in which if a quencher molecule were present quenching would occur.

The value for KSV is given by the slope of the plot and is indicative of the sensitivity of the fluorophore to the quencher. For the NaI quenching there is little change to the experimental values and values obtained from the sphere of action, KSV = 67.43 to 62.89 and V=0.99 to 1.02. For the acrylamide quenching there is a significant change to the experimental values, Ksv = 64.78 to 37.34 and V=0.978 to 7.453. This indicates that iodide quenching was largely a dynamic process with only a small static quenching component observed and that acrylamide is a combined quencher, having both dynamic and static components. From the values for the volume of sphere can be used to estimate the radius and therefore the distance between the quencher and fluorophore required for quenching to occur. The iodide molecule is approximately 7.4 Å and acrylamide approximately 14.4 Å from the fluorophore. These distances are much greater distance than the average C-C bond (1.54 Å) confirming that static quenching is due to the proximatey of the quencher not through the formation of a ground state complex.

The results can be analysed further by calculating the apparent quenching constant, Kapp for each quencher concentration, given by: . These values were then plotted against [Q], the plot giving an intercept of KD + KS and the slope equal to KDKS.

The values are calculated by solving a quadratic equation, assuming that the larger of the two values is the dynamic component and the other corresponding to the static component.





[Q] (M)






































Table 1. Table of calculated Kapp values for each [Q]. Additional table of calculated and experimental quenching constants.

The static quenching component can also be called the association constant as it also expresses the extent of association between quencher and fluorophore.

The differences observed in quenching using iodide suggests that it is largely a dynamic quencher. Whereas, with acrylamide the large differences in quenching constants implies that combined dynamic and static quenching has occurred. These differences can be accounted for by considering the individual quencher ions. Iodide is a heavy charged atom, whereas acrylamide is a neutral small atom. Both quenchers were able to successfully quench propranolol fluorescence. The quencher concentration increased the probability of observing both types of quenching. The differences between the two quenchers became more noticeable in the experiments where the access to the fluorophore was limited.

Hydrophobic Interactions of Propranolol Rebekah Sayers

5.2 Effect on fluorescence of propranolol on inclusion in SDS detergent micelles using NaI and acrylamide quencher molecules.

The results from the SDS titration with propranolol were plotted to show the relative fluorescence intensity versus the emission wavelength as the concentration of SDS was increased.

At low SDS concentrations, pre-micellization, the typical emission spectrum of propranolol is observed. This changes once the critical micelle concentration (CMC) is reached after 30 µl of 100 mM SDS has been added which translates to approximately 1 mmol dm-3. The micellization process is said to be promoted in the presence of a buffer containing a high salt content such as PBS which was used in this experiment. The charge transfer band is no longer visible in the spectrum due to the strong association between the naphthalene ring of propranolol and the micelle. It is the hydrophobic nature of propranolol which causes it to partition into the non-polar SDS micelle, shielding it from any solvent or quencher ions present in solution. The results above the CMC indicate that the bonding is strong; the spectrum indicating the inclusion of the naphthalene ring responsible for propranolol's fluorescence. Similar studies with neutral or cationic micelles were not as successful (Sarpal 1994), this indicates that the anionic head group in SDS contributes to the binding process.

In the following experiments the amount of SDS in solution was in excess of the CMC and it can be presumed that all the propranolol molecules have been accommodated within the available SDS micelles. Therefore in order to successfully quench the fluorescence of propranolol the quencher ion must also be able access the detergent micelle. The quenching of propranolol when held within a SDS micelle is complex due to the bimodial size distribution at high ionic strength (Cramb 2000). Iodide is a charged species and is easily polarised, therefore the environment of the fluorophore in this instance will affect its ability to quench. As an anionic quencher, iodide requires a large amount of energy to gain access to the SDS micelle.

The emission spectra do not change with increasing iodide concentration, the shape is representative of propranolol fluorescence in SDS above the CMC . From the results it is clear that iodide ions had no effect on the fluorescence, their inability to quench was probably due to the inaccessibility to the SDS micelle due to coulombic repulsion. It has been suggested that in some systems involving SDS micelles the anionic I- can be oxidised to form I3- which may be able to quench from outside the micelle through resonance energy transfer mechanisms. This could also lead to the partitioning of I2 into the micelle (Cramb 2000). It may have been possible to evaluate this further if the experiment had been conducted using higher concentrations of NaI.

Acrylamide is a neutral molecule and was able to diffuse into the micelle interior allowing it to quench the fluorescence through both dynamic and static quenching. The emission spectra are typical of propranolol in SDS above the CMC. A direct relationship between concentration and fluorescence intensity was observed . The results are represented as a Stern-Volmer plot showing the same upward curvature to the y-axis as in the previous acrylamide quenching .

The deviation from the Stern-Volmer dynamics can be accounted for by fitting the data to a sphere of action quenching model, which allows interpretation of the different types of quenching occurring. In this situation acrylamide has performed both dynamic and static quenching on the propranolol despite being contained in the SDS micelles. As the concentration of acrylamide ions increases the possibility of quenching increases and a decrease in fluorescence intensity was observed.

Comparison of the KSV values for acrylamide quenching of propranolol in PBS and in the presence of SDS shows a decrease in quenching of propranolol on inclusion in SDS micelles. The SDS micelle has provided a degree of protection from acrylamide molecules. The variation in quenching ability can also be assessed by calculating the individual quenching components. The results can be analysed further by calculating the apparent quenching constant, Kapp for each quencher concentration, given by: . These values were then plotted against [Q], the plot giving an intercept of KD + KS and the slope equal to KDKS .

The Kapp values for propranolol and SDS titrated with acrylamide against [acrylamide] at room temperature.



[Q] (M)








































Table 2. Tables to show the calculated Kapp values and quenching constants for acrylamide in propranolol in SDS solution.

The values obtained for the volume of the sphere can be used to estimate the radius of the sphere giving an approximate distance between the fluorophore and quencher required for quenching. A distance of less than an average bond length could suggest that static quenching may be a result of complex formation rather than being within the sphere of action. Using the V value form the SV plot and the SV equation derived for the sphere of quenching the volume can be adapted to be given as 1.663 x 10-27 m3 molecule-1 or 1.663 x 103 Å3. This can then be substituted into to gives the radius, 7.35 Å. This can be compared to the typical C-C bond length of 1.54 Å, indicating that as suggested static quenching has not occurred as a result of complex formation but due to the proximity of the fluorophore and quencher molecule.

The results were evaluated further, first by calculation of the aggregation number. A plot of vs. [Acrylamide] gave a slope equal to Nagg, 17.386 . This gives the number of molecules present each micelle at the CMC.

This was then used with the CMC value obtained earlier to calculate the concentration of micelles in solution. , 2.3x10-4 M. The number of quencher molecules per micelle, <Q>, was then calculated for each quencher concentration by dividing [Q] by [M]. This is plotted to show the directly proportional trend .

5.3 Effect on fluorescence of propranolol on inclusion in β-CD cavities using NaI and acrylamide quencher ions.

The following results analyse the effect of complexation and β-CD concentration on the fluorescence of propranolol. Results show an increase in fluorescence intensity of propranolol on inclusion of β-CD without significant change in the spectra. Many studies on this complexation have been carried out, confirming the propranolol interaction to be through the fluorophore containing naphthyl group, with significant contribution from hydrophobic interactions (Castronuovo2006). The changes in fluorescence intensity on incorporation of propranolol in β-CD were too small to evaluate binding constants, a double reciprocal plot producing a poor linear fit. This suggests weak interactions between propranolol and the β-CD cavity possibly due to hindered inclusion of the naphthalene group caused by the alkyl ammonium side chain (Glenn 2004). The small change in fluorescence with increasing β-CD concentration is a good indication that even at the lower β-CD concentration most of the drug is complexed. It can be assumed that the stoichiometry of the reaction is 1:1, only a single propranolol molecule may be accommodated in each β-CD cavity in solution. This provides shielding from any non-radiative deactivation processes and leaves very few propranolol molecules free in solution, leading to a decrease in any self quenching that may have been observed previously.

Further assessment of the interactions between propranolol and β-CD were made by performing quenching with NaI and acrylamide ions. Quenching was observed for both quenching ions with a clear trend in fluorescence intensity, fluorescence appearing to decrease with increasing β-CD concentration .

The β-CD cavity has provided some protection, shielding the excited singlet state of propranolol from quenching as well as any non-radiative deactivation processes. The resulting Stern-Volmer plots show that quenching has occurred, diffusing quencher ions have been able to access the fluorophore .

There is little change to the quenching constants after applying the sphere of action model. The differences in the quenching constants with increasing [B-CD] for both plots can be compared in the tables below (Table).












































Table 3. NaI quenching constants for SV and SA plots.












































Table 4. Acrylamide quenching constants for SV and SA plots.

Further analysis of results!! Radius of sphere, distance of quencher position in B-CD cavity?

The Kapp values were calculated but the results were inconclusive as the difference between B-CD concentrations was so small that the independent quenching components could not be calculated. The changes observed in fluorescent intensity are weak as a function of [B-CD] which indicates that the propranolol is not tightly bound to the B-CD (Glenn 2005).

Hydrophobic Interactions of Propranolol Rebekah Sayers

5.4 Determination of drug-liposome binding

Hydrophobic Interactions of Propranolol Rebekah Sayers


6. Conclusions and further work


Possible errors in results?

Results aided if fluorescence lifetime measurements were available?

Further area of interest, membrane stabilization, membrane protection from oxidation?

7. Acknowledgements

I would like to acknowledge the help and advice given to me throughout this research project from ProfR.H.Bisby.

Hydrophobic Interactions of Propranolol Rebekah Sayers

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