WO2006114014A2 - Electrochemical patterning and dissolution of polyelectrolyte multilayers - Google Patents

Abstract

Described is an electric potential driven drug delivery system, which drug delivery system is suitable for the time dependent dispensing of at least one active ingredient. Said drug delivery system comprises at least one active ingredient, a substrate with at least a semi-conductive part, wherein at least said semi-conductive part is covered by at least one bilayer of oppositely charged monolayers. Said at least one active ingredient may form one of said monolayers, or said at least one active may be incorporated within one of the monolayers, or said active ingredient may form a layer that is placed between said two oppositely charged monolayers. For use, said multilayers covered substrate is connected to a regulating means that is suitable to change the potential of said substrate such that one or more of said monolayers - and therewith the active ingredient therein - are released.

Description

Electrochemical patterning and dissolution of polyelectrolyte multilayers

Cross References to Related Applications

This application claims the priority of European patent application no. 05 009 306.1, filed April 28, 2005, the disclosure of which is incorporated herein by reference in its entirety.

Technical Field

The present invention relates to a system for the administration of drugs and/or biologically active materials by release from the surface of an electrode, in particular a system utilizing a conductive substrate where a functionalized multilayer film is built, wherein the drug release is controlled by the application of an electric potential .

Background Art

The deposition of polyelectrolyte multilayers^1"3 through the layer-by-layer method (LBL) has stimulated a large number of studies aiming at the understanding of the physicochemical mechanisms^4"8 responsible for self-assembly and the development of numerous applica- tions.^9"11 The build-up of polyelectrolyte multilayers occurs through the alternate dipping of the substrate in poly-anion and poly-cation solutions, and is a consequence of a charge overcompensation of the substrate during the adsorption of the previous polyelectrolyte layer.¹²'¹³ Simple to construct, polyelectrolyte multilayers can easily be functionalized with biologically active molecules. This can be performed with biomolecules them- selves as materials to build the film^14"16 or by embedding them into the polyele.ctrolyte films^17"20. This technique allows, in particular, the preparation of supramolecular nanoarchitectures exhibiting specific properties in terms of cell activation control⁹'^21"23.

A precise control over the release of drugs is highly desirable in order to optimize drug therapy. A number of different drug delivery vehicles such as liposomes, microcapsules, hydrogels which respond to stimuli, e.g., temperature^24"26, pH^27"29, light³⁰, electric fields³¹'³², ultrasound³³, etc., are currently being investigated in an attempt to optimize drug therapy. In the field of polye- lectrolyte multilayers, a study from Hammond's group³⁴ reports a system, made by hydrolytically degradable thin films which allows controlled release of the film components under physiological conditions . A more recent study of the same group deals with the controllable dissolution of Prussian blue nanoparticles/linear poly (ethylene imine) multilayers (PB/LPEI)³⁵. In another field, Kwon et al . ³⁶ studied the release of heparin from poly (allylamine) /heparin complexes. Upon the application of an electric current, a rapid dissolving process occurred which is explained by the locally increased pH near the cathode (resulting from OH^" ions production) . This increase of pH induces the disruption of electrostatic bonds between the two oppositely charged polyelec- trolytes .

There are few studies on the influence of an electric field on the buildup process of polyelectrolyte multilayers. Layer-by-layer assembly of a strong polyelectrolyte, poly (diallyldimethylammonium chloride) (PDADMAC) , alternated with CdTe nanocrystals ^37"39 or enzymes ⁴⁰'⁴¹ under an electric field has been investigated by Gao and co-workers. They switched the polarity of the substrate (the working electrode) at each new adsorption to obtain a favorable deposition, with opposite signs between material and surface. Moreover by alternating fa- vorable and unfavorable depositions on two side by side indium-tin-oxide (ITO) -electrodes, they obtained a pattern of two different types of CdTe nanocrystals³⁷ or of enzymes⁴⁰'⁴¹, the latter keeping their activities. Hitherto, two different mechanisms have been suggested to explain the observed exponential growth. The first mechanism is based on the charge mismatch between the polycation and the polyanion, which increases the surface roughness and consequently increases the surface area for deposition of material.^49"51 The second mechanism suggests the diffusion into and out of the entire film of, at least, one of the polyelectrolytes which will result in a thickness-dependent growth of the multilayer.^52"

54 In spite of the above discussed state of the art, none of the hitherto studied models for controlled dissolution/drug release proved to be satisfactory for a long-term controlled drug delivery system.

Disclosure of the Invention

Hence, it is a general object of the invention to provide a drug delivery system for controlled dissolution/drug release satisfactory for a long-term controlled drug delivery.

Now, in order to implement these and still further objects of the invention, which will become more readily apparent as the description proceeds, the elec- trie potential driven drug delivery system is manifested by the features that it is suitable for the time dependent -dispensing of at least one active ingredient, said drug delivery system comprising at least one active ingredient, a substrate with at least a semi-conductive part, at least said semi-conductive part being covered by at least one bilayer of oppositely charged monolayers, at least one of said monolayers comprising said at least one active ingredient, or said at least one active ingredient being placed between said two oppositely charged monolayers, said substrate being connected to a regulating means, and said regulating means being suitable to change the potential of said substrate.

In the scope of the present invention, it has been found that polyelectrolyte multilayers can be dissolved in a controlled manner and can be used for the production of an electric potential driven drug delivery system, said drug delivery system being suitable for the time dependent dispensing of at least one active ingredient. Said drug delivery system comprises at least one active ingredient, a substrate with at least a semi- conductive part, at least said semi-conductive part being covered by at least one bilayer of oppositely charged monolayers, at least one of said monolayers comprising said at least one active ingredient, or said at least one active ingredient being placed between said two oppositely charged monolayers, said substrate being connected to a regulating means, and said regulating means being suitable to change the potential of said substrate.

In the context of this invention, the terms drug, effective substance and active ingredient are used synonymous . These terms cover pharmaceutically active in- gredients, nutrients, trace elements, vitamins, substances necessary for cell selection in in vitro cell culture, etc.

In a preferred embodiment, the drug delivery system of the present invention has a substrate that as a whole is a semi-conductor.

The. inventive system uses the electrochemical behavior at the surface of an electrode, said electrode being used as a substrate, on which biocompatible and preferably biodegradable and resorbable multilayer films are applied. The formation and stability of the polyelectrolyte multilayers was found to depend on the applied potential and the ionic strength of the buffer. The ap- plication of potentials above a system dependent threshold leads to controllable dissolution of the polyelectro- lyte multilayer film following a single exponential kinetics. The rate of this process can be controlled by an on-off profile of the potential leading to a controlled drug release in the bulk. The electro-dissolution of the polyelectrolyte multilayers has been found to be a local phenomenom which leads to the formation of nanoporous films . Dependent on the doses to be administered by one and the same device, a plurality of bilayers can be provided, whereby for the delivery of one dose not a whole layer must be dissolved. Dependent on the thickness of the layer and due to the possibility to stop dissolu- tion or to at least slow it markedly down, partial dissolution is possible.

In an alternative embodiment, the substrate may be such that several semi-conductive parts of it are separated by insulating parts and that each semi- conductive part is individually addressable.

The number of bilayers forming the multilayer structure is dependent on the above mentioned criteria. In general, however, the number will be higher than 1, often between 2 and 100. If an active ingredient is sufficiently charged, such active ingredient can itself be one of the monolayers. Such molecules are e.g. those with highly acidic groups (see e.g. heparin, hyaluronic acid etc.) or basic groups (see e.g. polycationic peptides such as poly (L-lysin) , proteins such as lysozyme etc.). Such compounds are well suited to be applied on a substrate alternating with an oppositely charged molecular layer. The use of an active ingredient alone as one of the monolayers has the advantage of higher possible concentration and less possible side-effects due to reduced number of total ingredients, however the embedment of drugs, parti- cles or drug carriers into the polyelectrolyte multilayer is also convenient.

Preferably all ingredients of the monolayers are at least biologically degradable, preferably biologi- cally resorbable.

In view of its biological features, its easy producibility and its amino groups that upon physiological pH are positively charged, PLL is a suitable positively charged layer for being used with a negatively charged active ingredient.

If the drug has no or no sufficient net charge, it may be incorporated into a suitably charged layer. Possibly said layer may be chosen such that it can interact with the drug, e.g. by Hydrogen bridges, Van- der-Waals interactions etc.

If the active ingredient comprises differently charged substituents , it may possibly be incorporated in one or the other of the oppositely charged monolayers, or - if the charge separation is sufficiently distant, as e.g. in the case of proteins, it may form an intermediate layer between two oppositely charged monolayers.

Without intending to be bound by any theory, the controllable release of drugs from polyelectrolyte multilayers is assumed to be due to the following interpretation:

Polyelectrolyte multilayers can be considered as "layered complexes" with the same interactions as in soluble complexes⁵'^62"65, but exhibiting a higher segment density⁶⁶. Due to their high hydration and their swelling properties, they can be viewed as dense hydrogels⁶⁷ where the cross-linking density is controlled by the charge distribution along the chain⁶⁰'⁶⁸. In multilayers, the internal layers are electrically neutral⁵'⁶². The macro- scopic neutrality is due on one hand to intrinsic charge compensation, i.e. layered complex formed by an exact 1:1 stoichiometry of polycation and polyanion and on the other hand to extrinsic compensation by counter-ions present in the multilayers. When e.g. 1.8V is applied as a potential on the substrate (the anode) , the electrolysis of water takes place which produces H⁺ ions. The H⁺ ions render the surface charge positive which attracts chloride ions near the substrate. At the same time, sodium ions move toward the cathode. This production of H⁺ ions leads to an acidic pH near the anode (pH=2), equilibrated in deionized water or PBS³⁶'⁶⁹. This acidic pH could induce the neutralization of anionic groups, in case of e.g. the drug heparin, of carboxylic and sulfate and sulfamide groups. This neutralization can weaken the interactions between the cationic and the anionic layers and dissociate the multilayer as it has been suggested by Kwon et al.³⁶. However instead of having a fast dissolution of the film which would be due to a decrease of the pH near the substrate, an exponential decrease of the film mass as a function of time is obtained.

By applying a water electrolysis inducing electric potential, a local electro-dissolution of the multilayer is induced leading to nanoporous films. The diffusion of an anion, in particular chloride, toward the anode (the substrate) increases locally the ionic strength on the top of the film. This ionic diffusion is well known to cause the shrinking and the swelling of cross-linked hydrogels⁷⁰'⁷¹. This increase in ions concentrations is assumed to weaken the electrostatic interaction between the anionic and the cationic layers and to allow to remove the polyelectrolytes by the flow of the buffer. A higher ionic strength, obtained by a high concentration of salt in the buffer or by applying a potential during the buildup process of the films, induces a faster dissolution of the film. Furthermore, a salt concentration dependency of the dissolution process was found. The formation of nanoporous films during the dissolution process is assumed. Very surprisingly, the dis- solution process is also obtained for strong polyelectro- lyte multilayers which do not present a pH sensitivity. In view of the above demonstrated behaviour and dependencies of the deposited films, it is preferred that each monolayer is formed one by one on the substrate. This allows choosing the appropriate deposition parameters for the desired dissolution/decomposition characteristics .

In view of its biologically interesting fea- tures, poly (L-lysine) (PLL) is of great interest. For this molecule forming one type of the layers, it was found that when a suitable electric potential is applied on a semi-conductive substrate, e.g. 1.8V under a constant flow rate of buffer, the adsorbed mass of polyelec- trolyte multilayers evolves as an exponential decay function and decreases about 90% after 3h . A local electro- dissolution of multilayers composed of PLL and a negative charge comprising layer is obtained leading to nanoporous films. The electromigration of electrolyte species toward the substrate induces probably the disintegration of complexes between PLL and the negative charge comprising layer, composing the multilayers, into water-soluble polymers . It was found that the more permeable the multilayers are, the faster they are dissolved. Moreover, a controlled release of effective substance present in the bulk is obtained by an on-off profile of the potential The advantage of such electro-dissolution of multilayers is that it can be applied for all kind of materials or polyelectrolytes, strong or weak, biodegradable or not, in order to obtain a controlled drug and/or protein release .

Since the release of active ingredient can be regulated by applying a specific potential to the substrate, the drug delivery system of the present invention in general comprises a regulating means. Said means may be coupled to a sensor providing data that are indicative for the need of an active substance or that indicate that sufficient active substance has been delivered, i.e. that delivery can be stopped.

One of the main applications of the multilayer covered substrate of the present invention is in the need of controlled and dosed supply of specific substances to e.g. in vitro cell cultures. It has, however, also several applications in medicine.

The drug delivery system as a whole may be so small that it can be implanted, or the regulating means may be designed for extracorporal application in view of easier maintenance and coupled to an implantable multilayer carrying substrate and optionally a sensor. A multilayer substrate of implantable size is also termed "microchip drug delivery substrate". The extracorporal regulating means also allows for manual regulation, e.g. if complex data are required for the determination of the amount of effective substance needed.

Passive or active microchip devices, utiliz- ing patterned semi-conductive substrates, have numerous in vitro and in vivo applications. The microchip can be used in vitro to deliver small, controlled amounts of chemical reagents to solutions, cell cultures or reaction mixtures at precisely controlled times and rates. Ana- lytical chemistry and medical diagnostics are examples of fields where the microchip delivery device can be used. In vivo, the microchip can be implanted into a patient, either by surgical techniques or by injection. The microchips provide delivery of drugs to animals or to persons who are unable to remember or to be ambulatory enough to take medication. The microchips further provide the possibility for delivery of many different drugs at varying rates and at varying times of delivery, whereby one chip may carry different selectively addressable areas each area comprising one specific drug, or several different bilayers or multilayers containing different drugs, or whereby one regulating means is connected to more than one individually addressable microchip.

Brief Description of the Drawings

The invention will be better understood and objects other than those set forth above will become apparent when consideration is given to the following detailed description thereof. Such description makes refer- ence to the annexed drawings, wherein:

Figure 1 is a schematic representation of an EC-OWLS setup comprising a three electrode configuration within a waveguide spectrometer wherein the indium-tin- oxide (ITO) -coated waveguide was the working electrode, a silver wire inserted into the cell served as the reference electrode and a platinum wire in the export tubing served as the counter electrode.

Figure 2 shows the evolution of the adsorbed mass, r (ng/cm²) , of PLL/HEP multilayers measured after deposition of each layer using the in-situ EC-OWLS in HEPES buffer (1OmM, pH 7.4). The symbols (O) refer to the layer formation in 0.15M NaCl and applying an external potential of OV, (Δ) in 0.15M NaCl built at 1.5V, and (D) in IM NaCl built at OV. Figure 3 shows the evolution of the adsorbed mass r (ng/cm²) , obtained in-situ by EC-OWLS, as a function of time during the alternate deposition of PLL and HEP layers at an electric potential of OV in HEPES buffer (1OmM, pH 7.4) at 0.15M NaCl. Black and gray arrows cor- respond to the injection of PLL and HEP respectively.

Lines represent the application of an electric potential of 1.8V and OV.

Figure 4 shows the evolution of the normalized mass of (PLL/HEP) ₉ multilayers, measured in-situ by EC-OWLS, as a function of time during the application of 1.8V potential. The multilayers were built in HEPES buffer (1OmM, pH 7.4) (O) in 0.15M NaCl at OV, (Δ) in 0.15M NaCl at 1.5V, and (D) in IM NaCl at OV.

Figure 5 shows atomic force microscopy (AFM) height mode images (3μmx3μm) and profilometric sections in liquid of (PLL/HEP)₉ multilayers, built at 0 V in HEPES buffer (1OmM, pH 7.4) and 0.15M NaCl, wherein (a) and (b) visualize multilayers before the application of 1.8 V with the maximum Z range at 20nm, (c) and (d) after the application of 1.8 V potential during Ih, with the maximum Z range at lOOnm and (e) and (f) after the application of 1.8V during 3h, with the maximum Z range at 50nm.

Figure 6 shows the evolution of the mass of (PLL/HEP)₉ multilayer built at OV (dark curve), obtained by in-situ EC-OWLS, as a function of time during the on/off application of 1.8V/0V. Lines represent the application of electric potential of 1.8V and OV. The dashed curve was obtained by putting one after the other the three exponential decay curves due to the application of 1.8V.

Modes for Carrying out the Invention

In one embodiment of the present invention the in-situ buildup process and the dissolving process of multilayers upon the application of a constant electric potential of 1.8 V was investigated for the system poly (L-lysine) /Heparin (PLL/HEP; formulas see below).

sine)

n

This system was selected because both substances are biodegradable and biocompatible polyelectro- lytes. PLL is a polypeptide with a pK_a of 10.5⁴², due to its amino groups. HEP is a polysaccharide widely used as an effective anti-coagulant for the prevention and treatment of coagulation disorders. The sulphate monoesters and the sulphamido groups of HEP are both highly acidic, having pK_a values ranging from 0.5 to 1.5⁴³ and the car- boxylate groups have a pK_a between 2.8 and 3.1⁴³'⁴⁴. The multilayers were built on an indium tin oxide (ITO) coated substrate, which served as the working electrode in a three electrode setup. Electrochemical Optical Waveguide Lightmode Spectroscopy (EC-OWLS) (see Figure 1) , introduced and developed recently^45"48, was used to follow in-situ the buildup and the dissolution of the multilayers under an applied potential. Atomic Force Mi- croscopy (AFM) was used to study the topography of the multilayers films after the dissolution process .

As already mentioned above, this system uses the electrochemical behavior at the surface of an elec- trode, said electrode being used as a substrate, on which biocompatible and preferably biodegradable and resorbable multilayer films are applied.

In one specific embodiment, multilayers of poly (L-lysine) and heparin, were built on an indium tin oxide (ITO) semi-conductor substrate. The buildup and the dissolution process of the multilayers was followed by electrochemical optical waveguide lightmode spectroscopy (EC-OWLS) . The formation and stability of the polyelec- trolyte multilayers was found to depend on the applied potential and the ionic -strength of the buffer. The application of potentials above a threshold of 1.8V induced dissolution of the polyelectrolyte multilayer film following a single exponential kinetics. The rate of this process could be controlled by an on-off profile of the potential leading to a controlled release of heparin in the bulk. AFM investigations showed that the electro- dissolution of the polyelectrolyte multilayers is a local phenomenom which leads to the formation of nanoporous films . The multilayer system PLL/HEP was investigated at 1.8V under a constant flow rate of buffer. It was found that the adsorbed mass of polyelectrolyte multilayers evolves as an exponential decay function and decreases about 90% after 3h. A local electro-dissolution of PLL/HEP multilayers was obtained leading to nanoporous films. The electromigration of electrolyte species toward the substrate is assumed to induce the disintegration of PLL/HEP complexes, composing the multilayers, into water- soluble polymers. It seems that the more permeable the multilayers are, the faster they are dissolved. Moreover, a controlled release of heparin in the bulk was obtained by an on-off profile of the potential The advantage of such electro-dissolution of multilayers is that it can be applied for all kind of materials or polyelectrolytes, strong or weak, biodegradable or not, in order to obtain a controlled drug and/or protein release. In addition to the build-up of multilayers of oppositely charged molecules, it is also possible to build up layers of proteins utilizing their charge separation to anchor them within a suitably charged layer or to connect two appropriately charged layers, thereby gen- erating a "sandwich" structure of the type negatively charged layer-protein-positively charged layer.

It is also possible to incorporate neutral molecules (also molecules without charge separation) within a charged layer, e.g. if the layer molecules and the molecules to be incorporated have some interaction/affinity. Also in case of incorporation, the incorporated molecules are set free upon dissolution of the incorporating layer.

Materials Preparation and Analytical Methods :

Polyelectrolyte Solutions . All the experiments were performed at pH 7.4. The buffer medium used was prepared at pH 7.4 from 1OmM 4- (2-hydroxyethyl) pi- perazine-1-ethanesulfonic acid (HEPES) , purchased from Fluka and supplemented at 0.15 M or IM of sodium chloride. All aqueous solutions were prepared using ultrapure water filtered through Milli-Q Gradient AlO filters, pur- chased from Millipore AG, Switzerland. Poly (L-lysine) hy- drobromide (PLL, P-7890, MW=15700) was purchased from Sigma and heparin (HEP, 51543) was purchased from Fluka Biochemika. Poly ( 4-styrene sulfonate) sodium (PSS, 243051, MW=70000), poly (allylamine hydrochloride) (PAH, 283215, MW=15000), poly (diallyldimethylammonium) chloride (PDADMAC, 409030, MW= 500000) and poly (acrylic acid sodium salt) (PAA, 416037, MW=15000) were purchased from Aldrich. PLL, PDADMAC, PAH, HEP, PSS and PAA polyelectro- lytes were used at a lmg/mL concentration in HEPES buffer solution .

Electrochemical Optical Waveguide Lightmode

Spectroscopy. The in-situ optical waveguide lightmode spectroscopy (OWLS, Bios-I, Artificial Instruments AG, Zurich, Switzerland) is an optical technique that allows access to the optical film thickness and its optical mass. This technique was modified to permit an electrochemical control of the waveguide chip, treated by an indium tin oxide (ITO) coating to be conductive. This technique has been described in more details elsewhere⁴⁸. The flow cell contained three-electrode electrochemical test- ing configuration, with the ITO-coated waveguide serving as a working electrode (Figure 1) . A silver (Ag) wire served as a quasi-reference electrode and a platinum (Pt) wire served as a counter electrode. Although the exact reference voltage of the Ag wire with respect to the nor- mal hydrogen electrode (NHE) was not known, it is acknowledged that a silver wire adopts a reasonably steady potential (0.78V) that is reproducible to within 10 to 20 mV,. when immersed in a halide-containing supporting electrolyte, as used in the present system⁷⁴. The thickness and the optical mass were determined from the mode equation using a four-layer model.⁷⁵'⁷⁶ The adsorbed mass was then calculated according to Feijter's formula.⁷⁷'⁷⁸ To simplify the calculations, a refractive index increment of 0.182 cm³/g was used for adsorbed mass density calcu- lations of all the polyelectrolytes used.

Example 1 :

Buildup of polyelectrolyte multilayers . The buildup of the polyelectrolyte multilayers was performed in stop-flow mode using a flow-through cell with a volume of 15 μL, which was maintained at room temperature (24°C). After the stabilization of the baseline at the chosen potential (0 V or 1.5 V) in contact with the HEPES buffer solution, 500μL of polyelectrolyte were injected and maintained in contact at rest. After 5 min of contact, the polyelectrolyte solution was replaced by ImL of HEPES buffer solution and left at rest during 5 min. This was done alternatively with the polycation and the polyanion solution. These polycation/polyanion adsorption steps were repeated until the multilayer film was built to the desired number of layers.

The build-up of the multilayers in dependence of ionic strength and potential applied to the substrate surface is shown in Figure 2. Said Figure supports exponential growth of a (PLL/HEP)₉ multilayer, measured by EC-OWLS in HEPES buffer at 0.15 M NaCl and at an electric potential of 0 V (O), in 0.15 M NaCl built at 1.5 V (Δ) , and in 1 M NaCl built at 0 V (D) .

Example 2 : Dissolution of PLL/HEP multilayers .

After the buildup of the multilayers as described in Example 1, the electric potential of the substrate was increased to 1.8 V and a constant flow rate (10 mL/h) of HEPES buffer solution was maintained. This process was applied during 3h.

Figure 3 shows the evolution of the adsorbed mass as a function of time, measured by EC-OWLS, during the buildup process of the (PLL/HEP) ₉ multilayer in 0.15 M NaCl and at an electric potential of 0 V and its disso- lution at 1.8V. During the buildup of the film, the mass increases when the film is brought into contact with a new polyelectrolyte solution (see also Example 1 above and Figure 2) . When the buildup of the film was finished, the increase of the voltage from 0 V to 1.8 V induced a rapid decrease of the mass following an exponential decay function. Table 1 summarizes the parameters obtained when the evolution of mass at 1.8V is fitted by an exponential decay function. Knowing that the optical properties of the ITO coated waveguides do not change over days upon application of a few volts external potential⁴⁵, the remaining mass of the film can be calculated. This calcula- tion is obtained using the baseline of the buffer obtained at 0 V at the beginning of the experiment (see Figure 3). After applying 1.8 V for 3 h, only 10% of the film remained on the substrate.

Example 3 :

Influence of the ionic strength and the applied voltage on the build up of PLL/HEP multilayers :

In an attempt to tailor the dissolution ki- netics and to understand the effect of the local ionic strength and pH in the presence of an external potential on the formation and stability of the polyelectrolyte multilayers, the influence of the ionic strength on the (PLL/HEP) system was studied. The buildup and dissolution process of multilayer films built at 1.5V/0.15M NaCl, at 0V/1M NaCl and at 0V/0.15M NaCl was investigated. Figure 2 represents the evolution of adsorbed mass for these three cases. By increasing the salt concentration and maintaining the electric potential at OV, the amount of adsorbed PLL and HEP decreased. This result is similar to the study of Dubas and Schlenoff⁵⁵ on a poly (diallylmethylammonium) /poly (acrylic acid) (PDADMAC/PAA) system. They have shown that the thickness of these polyelectrolyte films has a maximum when using 0.15M salt concentration. On the other hand, the buildup of (PLL/HEP) film at 1.5V, keeping the salt concentration at 0.15M NaCl, led to less adsorbed mass of PLL and HEP. This suggests that the applied external potential have an equivalent effect to an increase in the salt concentra- tion of the buffer. Example 4 :

Influence of the ionic strength and the applied voltage on the dissolution of PLL/HEP multilayers :

The dissolution processes of the films of Ex- ample 4 were compared in Figure 4. The dissolution of the film built at 0 V/l M NaCl was similar to the one built at 1.5 V/0.15 M. Indeed, the fitted time constants of the exponential decay functions were very close (τ=55 min for 1.5 V/0.15 M NaCl; and τ=52 min for 0 V/l M NaCl:) (Table 1) .

By keeping the salt concentration of the buffer constant, it was found that the buildup of multilayers at 1.5 V induced a higher ionic strength near the substrate compared to the buildup at 0 V. This finding is assumed to be due to the positive charge of the ITO coating at this voltage. Consequently, a higher ionic strength inside the film, obtained by a high concentration of salt in buffer, or by applying 1.5 V during the buildup, induces a faster dissolution of the film. The exponential decay parameters of each fitted non-normalized dissolution curves of Figure 4 are shown in Table 1. Said curves are based on a three- parameter exponential fit and the percentage of PLL/HEP multilayers remaining. This percentage was obtained by a ratio of the total mass of multilayer after application of 1.8 V (during 3h) and before the application of this potential. The masses were calculated with the baseline recorded at the potential of the buildup.

Table 1

5 Example 5 :

Dissolution Process of Different Multilayers Systems .

To evaluate the influence of the pH and the ionic strength sensitivity of the film on the dissolution 0 process, a system composed of strong polyelectrolytes and a system composed of weak polyelectrolytes were studied. The system composed of strong polyelectrolytes was poly (diallyldimethylammonium) /poly (styrene sulfonate) (PDADMAC/PSS) , which is only sensitive to the ionic 5 strength⁵'⁵⁶'⁵⁷ and not to the pH and the system composed of weak polyelectrolytes was poly (allylamine) /poly (acrylic acid) (PAH/PAA) , which is sensitive to pH and ionic strength⁸'⁹. Multilayers of both systems were built at 0 V with 0.15 M NaCl and the same conditions of dissolution 0 process were applied (data not shown) . Whereas a regular multilayers formation was observed for the PDADMAC/PSS system at 0 V, the PAH/PAA system presented a cycling behavior of the adsorbed mass after three bilayers (data not shown) . Indeed, the mass increased at the injection 5 of PAH but decreased at the injection of PAA. Such an effect might be due to a partial film dissolution similar to the one observed by Kovacevic et al . ⁵⁸'⁵⁹ at low ionic strength for different systems. Although partial dissolution at each injection of PAA might be induced by the loose interactions between PAH and PAA in the film, this is not suitable to explain the behavior at 0 V. Comparison of the dissolu- 5 tion process (in the same conditions) of (PLL/HEP) multilayers with (PDADMAC/PSS) and (PAH/PAA) ones, showed a higher constant of time for the strong polyelectrolytes system and a lower one for the weak polyelectrolytes system. As expected, the time constant for (PLL/HEP) was be-

10 tween the one of (PDADMAC/PSS) and the one of (PAH/PAA)

(see Tables 1 and 2) . Moreover, the remaining mass of the PDADMAC/PSS films was about 41% after 3h compared to less than 10% for PLL/HEP or PAH/PAA (see Tables 1 and 2) .

Besides of the remaining mass, Table 2 shows

15 the fit of the dissolution curves obtained at 1.8 V for PDADMAC/PSS and PAH/PAA multilayers built in HEPES buffer in 0.15 M NaCl at 0 V. Exponential decay parameters of each fitted non-normalized dissolution curves based on a three-parameter exponential fit and percentage of multi-

20 layers remaining were gathered. This percentage was obtained by a ratio of the total mass of multilayer after application of 1.8V (during 3h) and before the application of this potential. The mass were calculated with the baseline recorded at the potential of the buildup.

25

Table 2:

30 * data from Table 1 PDADMAC/PSS multilayers are known to be less permeable to ions than multilayers composed by weak poly- electrolytes, as PAH/PAA.⁶⁰'⁶¹ The results show that the more permeable the multilayers are, the faster they are dissolved. Moreover, PDADMAC/PSS multilayers are more resistant to high ionic strength compared to PDADMAC/PAA multilayers . Indeed, the first system was found to be stable up to 3.5 M and the second one was found to be rapidly removed at salt concentration of 0.6 M, suggesting that the slow process of dissolution of PDADMAC/PSS multilayers is due to the low ionic strength used, since PDADMAC/PSS multilayers need a higher ionic strength to be dissolved than systems composed of at least one weak polyelectrolyte .

Example 6 :

Topography of the multilayers , studied by means of atomic force microscopy.

To learn more about the process of dissolution atomic force microscopy (AFM) measurements were performed. AFM images allow access to the topography of the multilayers films before and after the application of 1.8V. Multilayers samples, for being studied by AFM, were built on ITO coated wafers (IMT Neuchatel, Switzerland) having an average roughness at lnm using the EC-OWLS setup and following the same procedure for the buildup of multilayers as described in Example 1. AFM images were obtained in contact mode in buffer with a Nanoscope Ilia from Digital Instruments (Santa Barbara, CA) . Cantilevers with a spring constant of 0.58 N/m and with silicon nitride tips were used. Always several scans were performed over a given surface area. These scans had to produce re- producible images to ascertain that there was no sample damage induced by the tip. Height mode images were scanned at a fixed scan rate (2 Hz) with a resolution of 512x512 pixels. The AFM height mode^' images (3μmx3μm) together with the corresponding profilometric sections in liquid of (PLL/HEP)₉ multilayers are shown in Figure 5.

Figure 5a represent a typical in-situ AFM im- age obtained for (PLL/HEP)₉ multilayers built at 0 V in HEPES buffer (1OmM, pH 7.4 ) at 0.15M NaCl. A corresponding typical profilometric section is given in Figure 5b and shows a maximum Z range at about 20nm. The outer structure of the film appears to be constituted of regu- larly distributed small hills with characteristic sizes ranging from lOOnm to 150nm. A few isolated large hills appear also with characteristic sizes ranging from 250nm to 350nm. The thickness of the film, measured by AFM after scratching of the film, was found to be about lOOnm. The comparison between the film thickness and the pro- filometric section (Figure 5b) revealed that the adsorbent surface was fully coated with polyelectrolytes . Moreover, it can be seen that the outer surface is not structurally flat but rough with a RMS value of the film around 8 nm.

These results were compared to the (PLL/HEP)g multilayers after the dissolution process when applied, during Ih (Figure 5c) and during 3h (Figure 5e) . After Ih of dissolution process (Figure 5c), the topography of the film, with dispersed granules, did not really change but the maximum height amplitude (maximum Z range) was about lOOnm. In addition to hills, the. presence of holes can also be noticed at the surface of the film, with the characteristic size ranging from 3nm to IOnm (Figure 5c) and a depth of 20-30nm (Figure 5d) . The thickness of the film, determined by AFM after scratching, was about lOOnm. The profilometric section shows that holes did not cross over the entire film. Moreover, the roughness of the film was increased, the RMS value was about 14nm. After the dissolution process of 3h, the surface of the (PLL/HEP)g film seemed to be composed by large holes with characteristic sizes ranging from 300nm to 500nm (Figure 5e) and a depth of IOnm (Figure 5f) . The holes became larger when the time of dissolution was longer. Here, the RMS value of the film was 7nm. Additionally to the holes, the presence of isolated larges cracks can be observed (Figure 5e) with a depth of 30nm and maximum Z range about 50nm (Figure 5f) . After 3h of dissolution process, the thickness of the film, measured by AFM, decreased from lOOnm to 45nm.

Example 7 :

Controlled heparin release. In order to investigate the possible application of this process, an on/off dissolution of PLL/HEP multilayers was performed by applying a cycle of 1.8V and OV. Figure 6 shows the evolution of multilayers mass as a function of time, measured by EC-OWLS, during this on/off dissolution. It was found that the dissolution was stopped as soon as the voltage was decreased from 1.8V to OV. When the 1.8V po- tential was switched off, the increase of the mass was due to ions accumulation near the substrate. The exponential decay relative to the dissolution process was dif- ferent at each 1.8V step and became slower with ongoing dissolution/decay. The dashed curve in Figure 6 repre- ■ sents the exponential decay function obtained by putting one after the other the three dissolution curves . In total the dissolution process took 80 min, 4.5% of the film remained on the substrate and the constant of time of this curve was about 40 min. In comparison to this re- suit, the application of constant dissolution during 3h resulted in a constant of time of 89 min and 10% of the film remained.

While there are shown and described presently preferred embodiments of the invention, it is to be distinctly understood that the invention is not limited thereto but may be otherwise variously embodied and prac^¬ ticed within the scope of the following claims.

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Claims

Claims

1. An electric potential driven drug delivery system, said drug delivery system being suitable for the time dependent dispensing of at least one active ingredient, said drug delivery system comprising at least one active ingredient, a substrate with at least a semi- conductive part, at least said semi-conductive part being covered by at least one bilayer of oppositely charged monolayers, at least one of said monolayers comprising said at least one active ingredient, or said at least one active ingredient being placed between said two oppositely charged monolayers, said substrate being connected to a regulating means, and said regulating means being suitable to change the potential of said substrate.

2. The drug delivery system of claim 1, wherein the substrate as a 'whole is semi-conductive.

3. The drug delivery system of anyone of the preceding claims, wherein at least pne of the monolayers is composed of an active ingredient.

4. The drug delivery system of anyone of the preceding claims, wherein at least one active ingredient is incorporated into a suitably charged mono layer.

5. The drug delivery system of anyone of the preceding claims, wherein at least one active ingredient forms an intermediate layer between said two oppositely charged monolayers .

6. The drug delivery system of anyone of the preceding claims, wherein at least part, preferably all of the monolayers are biologically degradable, preferably bioresorbable.

7. The drug delivery system of anyone of the preceding claims, wherein the positively charged monolayers are poly (L-lysine) .

8. The drug delivery system of anyone of the preceding claims, wherein the effective substance has a net negative charge.

9. The drug delivery system of anyone of the preceding claims, wherein the regulating means is coupled to a sensor providing active ingredient delivery dependent data .

10. The drug delivery system of anyone of the preceding claims, wherein the substrate is covered by at least 1, preferably 2 to 10, more preferably 20 to 100 identical or different bilayers.

11. The drug delivery system of anyone of the preceding claims that is suitable for drug delivery in in vitro cell culture.

12. The drug delivery system of anyone of claims 1 to 10, wherein the whole system is suitable for implantation .

13. The drug delivery system of anyone of claims 1 to 10, wherein the multilayer covered substrate is suitable for implantation and the regulating means is not suitable for implantation.

14. A method for producing an electric poten- tial driven drug delivery system of anyone of the preceding claims, wherein the multilayer coated substrate is sequentially coated with each of the oppositely charged monolayers and optionally a layer of active ingredients between one monolayer and the oppositely charged mono- layer.

15. Use of a drug delivery system of anyone of claims 1 to 11 for the directed supplementation of cell culture medium with one or more necessary active ingredients .