FIELD OF INVENTION

The invention is directed to a process to prepare an object coated with an active agent and to a coated object, especially a microarray of microneedles.

BACKGROUND OF INVENTION

A microarray of microneedles is a medical device which includes one or more microneedles capable of piercing the stratum corneum to facilitate the transdermal and/or dermal delivery of therapeutic agents through the skin.

Vaccination is a successful approach in the reduction of infectious diseases. When vaccinating toddlers and children, a major disadvantage is the injection used to induce protective immune responses. Injections cause serious stress, fear and concern in children and parents. Transcutaneous immunisation offers multiple advantages over current vaccination methods. First, the skin is an easily accessible rout of administration. Second, the skin is highly immune responsive, owing to the presence of large numbers of dendritic cells (DCs) and Langerhans cells (LCs) in the viable layers. These antigen-presenting cells take up antigens and are crucial for initiating an immune response. Finally, transcutaneous immunisation can be potentially pain free. From this point of view, microneedles are a very promising tool for transcutaneous immunisation.

There are two major types of microneedles, namely solid and hollow

microneedles. Here we focus on the solid (non-dissolvable) microneedles because of their ease of use. Such microneedles can be used in three different manners: coat and poke, poke and patch, scrape and patch. Our focus lies on the coat and poke method, because this method offers several advantages. First of all, there is much less antigen needed when microneedles are coated. Secondly, it will favour a high patient compliance because the vaccine is directly delivered after the microneedles are applied into the skin. Finally, a dry and stable formulation coated on the surface of the microneedles enables easy storage and transportation. Examples of coating microneedles with an active agent are described in the following publications. US2010280457 discloses a method for applying a high viscosity coating onto the surface of the microneedle. US2008/02941 16 discloses a microneedle which is coated with a composition comprising an active agent and a biologically active salt.

WO2010/042996 describes a method to coat a microneedle with a viscous solution wherein the silicon surface of the microneedle may be modified with 3- aminopropyl triethoxysilane (APTES) with the object to increase the hydrophilic properties of the surface.

US2009/0016935 discloses coating a microneedle with a formulation comprising a polyphosphazene polyelectrolyte and a biologically active agent. US2008/0051699 describes a method to coat microneedles wherein a film is used.

A disadvantage of coating the microneedles with a high viscosity coating as described in US2010280457 is that the coating method is complex and may results in a non-uniform distribution of the coating onto the surface of the microneedle.

The object of the present invention is to provide a process to coat a microarray with active agents in a more simple and uniform manner.

SUMMARY OF INVENTION

This object is achieved by the following process.

Process to coat an active agent to a modified surface of an object by electrostatic bonding, by

(a) contacting the object with a buffered aqueous coating solution comprising a negatively charged active agent, wherein the surface of the object is modified with organic molecules comprising ionisable groups which are weak bases and wherein said buffered solution has a pH below the pKa of the surface, or

(b) contacting the object with a buffered aqueous coating solution comprising a positively charged active agent, wherein the surface of the object is modified with organic molecules comprising ionisable groups which are weak acids and wherein said buffered solution has a pH above the pKa of the surface, or

(c) contacting the object with a buffered aqueous coating solution comprising a negatively charged active agent, wherein the surface of the object is modified with organic molecules comprising ionisable groups which are bases and ionisable groups which are acids and wherein said buffered solution has a pH below the isoelectric point of the surface or below the pKa of the surface, or

(d) contacting the object with a buffered aqueous coating solution comprising a positively charged active agent, wherein the surface of the object is modified with organic molecules comprising ionisable groups which are bases and ionisable groups which are acids and wherein said buffered solution has a pH above the isoelectric point of the surface or above the pKa of the surface.

Applicants found that with the above process it is possible to easily and uniformly coat the surface of an object, preferably microneedles with an active agent having the opposite charge compared to the surface of the object. The coating layer is very thin resulting in that for example the sharpness of the individual needle of a microneedle is retained. When the resultant microneedle is contacted with the skin the active agents are easily released through changes in the direct environment of the surface of the microneedle, such as pH and ionic strength, due to the physiological properties of the skin. Applicants further found that a high release of active agent is achievable. Without wishing to be bound to the following theory applicants believe that active compounds encapsulated in a viscous coating will have more barriers to be released from the surface that the active compounds bound to the surface as obtainable by the above process. By simply reducing the charge of the surface resulting from the physiological properties of the skin electrostatic bonding with the active agent will decrease and release of said agent results. A further advantage is that significantly less additives and carrier compounds are required when the process of the present invention is applied to coat with an active agent as when a viscous coating technology is used.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows a reaction scheme for the modification of silicon surfaces.

Figure 2: shows a synthesis of modified nanoparticles. Figure 3a shows a resulting S-shaped curves for the amine-modified surfaces with sulphate-modified nanoparticles.

Figure 3b shows a resulting S-shaped curves for the carboxyl-modified surfaces with CPTA-modified nanoparticles.

Figure 4 shows a reaction scheme for the modification of silicon surfaces into phenol and pyridine modified surfaces

Figure 5 a shows a resulting S-shaped curve for the phenol-modified surfaces with CPTA-modified nanoparticles

Figure 5b shows a resulting S-shaped curve for the pyridine-modified surfaces with sulphate-modified nanoparticles.

DETAILED DESCRIPTION

The material of the object or microneedle as part of the microarray may be made from various materials such as non-dissolvable polymers, silicon or a metal, preferably silicon and metal. Suitable metals are stainless steel, iron and gold. An example of a suitable material of the microneedle is silicon. Examples of microarrays of microneedles are described in EP2303766, EP2289843,

CN101830428 and JP2008035874. Applicants have illustrated that a silicon surface may be modified according to the present invention to achieve the desired property. A skilled person will understand that such a modification may also be easily performed on other surfaces as described above.

In a first embodiment (a) a negatively charged active agent is coated on the surface. Preferably the pKa of the surface modified with weak bases is below 9.5. More preferably the surface is positively charged at pH<7 and becomes neutral at pH>7, wherein the pKa of the surface is between 4 and 7.4, more preferably between 6 and 7. Coating with a negatively charged active agent is performed by contacting this surface with an aqueous buffered coating solution having a pH below the pKa of the surface and comprising the negatively charged active agent.

In a second embodiment (b) a positively charged active agent is coated on the surface. Preferably the pKa of the surface modified with weak acids is above 4. More preferably the surface is negatively charged at pH>8 and becomes neutral at pH<8, wherein the pKa of the surface is preferably between 7.4 and 10, preferably between 8-9. Coating with a positively charged active agent is performed by contacting the surface with an aqueous buffered coating solution having a pH greater than the pKa of the surface and comprising the positively charged active agent.

To achieve the desired pKa property the surface is modified with organic molecules having ionisable groups. The ionisable groups are weak acids or weak bases. Depending on the charge of the active agent to be coated on the surface of the microneedle a positively charged or negatively charged ionisable group is chosen. In the context of the present invention organic molecules are molecules comprising carbon atoms. Thus the ionisable groups as present on the surface are linked to said surface via structures comprising carbon atoms.

Examples of ionisable groups which can bind via electrostatic bonding a negatively charged active agent are groups which are weak bases. In the context of the present invention a weak base is characterised in that it does not ionise fully in an aqueous solution. Examples of suitable ionisable groups which are weak bases comprise optionally substituted pyridinyl groups, imidazole groups and aromatic amine groups and glucosamine groups. Combinations of such groups are also possible.

Examples of ionisable groups which can bind via electrostatic bonding a positively charged active groups are groups which are weak acids. In the context of the present invention a weak acid is characterised and it does not fully ionise in an aqueous solution. Examples of suitable ionisable groups which are weak acids comprise carboxyl groups, hydroxyl groups and phosphate groups. Combinations of such groups are also possible. The carboxyl group or hydroxyl group itself may advantageously be substituted on an aryl group, preferably a phenyl group. The carboxyl group may be linked to the surface via an alkyl silane group, for example an alkyl silane group comprising between 4 and 30 carbon atoms. An example of a preferred weak acid group comprising a hydroxyl group is an optionally substituted hydroxyl phenyl group. In embodiments (c) and (d) the surface property may be expressed by its isoelectric point. Possible combinations are weak acid/weak base, weak

acid/strong base, strong acid/weak base, and weak acid/weak base. The weak acid and the weak base will provide a decrease or increase in charge of the surface when the pH is varied. The midpoint of the S-curve describing this increase or decrease is the pKa as measured as if the surface was only modified with said weak acid or weak base. At the isoelectric point the charge of the surface reverses. Such a combination results in an even more sharp transition between a situation wherein the active agent is bonded and a situation wherein the active agent is to be released. For example a surface modified with both weak base groups, such as a pyridine group, and acid groups, such as benzenesulfonate, may be used to bond a negatively charged active agent. Such a surface may have an isoelectric point between 4 and 10. The value of the isoelectric point may be influenced by varying the molar ratio between the acid and base groups on the surface. When a microarray of microneedles is used having such a surface, the charge of the surface may reverse from positive to negative when the needle is inserted in the skin, due to the physiological properties of the skin enabling an even quicker release of the negatively charged active agent into the skin as compared to a surface comprising only a weak acid or weak base as in (a) and (b). By choosing a weak acid or weak base having a certain pKa and a suitable base or acid respectively resulting in an isoelectric point one may make use of both the charge change and the charge reversal to assist in release of the active agent when the process is applied to a microneedle. Preferably in embodiments (c) and (d) the surface is modified with organic molecules comprising ionisable groups which are weak acids and with organic molecules comprising ionisable groups which are bases or wherein the surface is modified with organic molecules comprising ionisable groups which are weak bases and with organic molecules comprising ionisable groups which are acids. The pKa of the surface modified with such a weak base or such a weak acid, as measured as if the surface was only modified with said weak acid or weak base, is between 4 and 10, preferably between 4.5 and 9.5. In case the object is contacted with a buffered aqueous coating solution

comprising a negatively charged active agent it is preferred that the isoelectric point of the surface is between 4 and 7.4 and wherein said buffered solution has a pH below the isoelectric point of the surface.

In case the object is contacted with a buffered aqueous coating solution

comprising a positively charged active agent it is preferred that the isoelectric point of the surface is between 7.4 and 10 and wherein said buffered solution has a pH above the isoelectric point of the surface.

Preferably the object is a microarray of microneedles. "Microneedle" refers to a microscopic needle-like structures capable of piercing the stratum corneum to facilitate the transdermal delivery of an active agent. "Microarray of microneedles" refers to a medical device comprising a plurality of microneedles capable of piercing the stratum corneum to facilitate the transdermal delivery of therapeutic agents or the sampling of fluids through the skin.

The microarray of microneedles as obtained by the process according to the present invention suitably has a surface of microneedles which is chemically modified in order to coat and release an active agent on microneedles via electrostatic interactions and in a pH dependent manner. The surface of

microneedles is chemically modified with an organic compound which has a permanent charge resulting in either a permanent positive or negative surface charge at a certain pH. Coating of the active agent is accomplished by generating a surface charge which is opposite to the charge of the active agent. Release of the active agent, when contacting the skin, is accomplished through changes in ionic strength between active agent and the surface because of the physiological properties of the skin which has a pH of 7.4.

When a microarray of microneedles having the above obtained coated surface is used on the skin, the surfaces will loose their charge due to a pH shift in the skin and thereby breaking electrostatic interactions and releasing the active agent from the surface and into the skin. The invention is also directed to an object having a surface to which an active agent is bonded by electrostatic bonding, wherein

(i) the surface of the object has a pKa of between 4 and 7.4 and wherein the surface is modified with organic molecules comprising ionisable groups which are weak bases and wherein the active agent is a negatively charged active agent, or

(ii) the surface of the object has a pKa of between 7.4 and 10 and wherein the surface is modified with organic molecules comprising ionisable groups which are weak acids and wherein the active agent is a positively charged active agent, or

(iii) .the surface of the object is modified with organic molecules comprising ionisable groups which are bases and ionisable groups which are acids and wherein the active agent is a negatively charged active agent, or

(iv) the surface of the object is modified with organic molecules comprising ionisable groups which are bases and ionisable groups which are acids and wherein the active agent is a positively charged active agent.

Preferably the object is obtained by the process described above. In view of the above the invention may also be directed to an object having a surface which is chemically modified with organic molecules comprising ionisable groups to which an active agent is bonded by means of electrostatic bonding wherein the pKa or its isoelectric point of the modified surface is so chosen that it looses its charge due to the physiological properties of the skin thereby releasing the active agent.

The object may be any object, such as an immunoassay-based diagnostic device or spheres for use in ion-exchange separation. Ion-exchange separation may be performed by using a column, like for example an ion exchange column, coated with the surface as described above. By choosing the pKa and/or the isoelectric point of the surface it is possible to separate and isolate positively or negatively charged proteins from other proteins having a different charge. The isolated proteins thus retain onto the inner surface of the column and may be removed as the surface is contacted with an aqueous solution having a well chosen pH at which the surface loses or reverses its charge. The pKa and/or the isoelectric point may be between 2 and 12 and preferably between 4 and 10. For example, an ion exchange column with a surface having an isoelectric point of 7.5 may be used for cation exchange chromatography at a pH greater than 7.5, for example at a pH of 8 and for anion exchange chromatography at a pH smaller than 7.5, for example at a pH of 7.

Therefore the invention is also directed to the use of such an object having such a surface to isolate charged compounds from a solution. The object having such a surface is suitably the object described except that no active agent is present. More especially the invention is directed to the use of an object having a surface which is modified with organic molecules comprising ionisable groups which are bases and ionisable groups which are acids to bond negatively charged

compounds as present in a solution having a pH smaller than the isoelectric point of the surface and elute these charged compounds from the surface by contacting the thus obtained surface rich in negatively charged compounds with an eluting solution having a pH greater than the isoelectric point of the surface

or

to bond positively charged compounds as present in a solution having a pH above the isoelectric point of the surface and elute these charged compounds from the surface by contacting the surface rich in positively charged compounds with an eluting solution having a pH smaller than the isoelectric point of the surface.

Preferably the object is a single microneedle or a microarray of microneedles as described in this specification. The ionisable groups are preferably the weak acid or weak bases as described above. The coating of said agent onto said modified surface may be as described in this specification. The modified surface may thus have a pKa of between 4 and 7.4 or between 7.4 and 10 or have an isoelectric point between 4 and 10. Suitably the surface is a non-dissolvable polymer or preferably silicon or a metal as described above.

Preferably the surface of the object has a pKa of between 4 and 7.4 and wherein the surface is modified with organic molecules comprising ionisable groups which are weak bases according to (i). Preferably the weak base comprises an optionally substituted pyridinyl group, an imidazole group and an aromatic amine group and/or a glucosamine group.

Preferably the surface of the object has a pKa of between 7.4 and 10 and wherein the surface is modified with organic molecules comprising ionisable groups which are weak acids according to (ii). Preferably the weak acid comprises carboxyl groups, hydroxyl groups and/or phosphate groups.

Preferably in embodiments (iii) or (iv) the surface is modified with organic molecules comprising ionisable groups which are weak acids and with organic molecules comprising ionisable groups which are bases or wherein the surface is modified with organic molecules comprising ionisable groups which are weak bases and with organic molecules comprising ionisable groups which are acids and wherein the pKa of the surface modified with such a weak base or such a weak acid, as measured as if the surface was only modified with said weak acid or weak base, is between 4 and 10, preferably between 4.5 and 9.5. More preferably the isoelectric point of the surface is between 4 and 7.4 and wherein the active agent is a negatively charged active agent. More preferably the isoelectric point of the surface is between 7.4 and 10 and wherein the active agent is a positively charged active agent.

The invention is also directed to an object having a surface which is chemically modified with organic molecules comprising ionisable groups to which an active agent is bonded by means of electrostatic bonding and wherein the pKa or the isoelectric point of the modified surface is so chosen that the surface loses its charge due to the physiological properties of the skin. Preferably the surface is a modified silicon surface and wherein the ionisable groups are weak acid or weak bases. More preferably the surface has a pKa of between 4 and 7.4 and wherein the surface is modified with organic molecules comprising ionisable groups which are weak bases or wherein the surface has a pKa of between 7.4 and 10 and wherein the surface is modified with organic molecules comprising ionisable groups which are weak acids. The invention is also directed to a microarray as described above or an object, preferably having a modified silicon or metal surface, wherein an active agent is bonded to its surface by electrostatic bonding. Possible object may be silica nanoparticles for use as part of a drug delivery system. Active agent according to the present invention refers to one or more pharmacologically active or

pharmaceutically effective molecules, compounds, materials or substances producing one or more local or systemic effects in mammals, including humans. The active agent has a charge enabling electrostatic bonding to the surface of the microarray or object. Examples of active agents include, without limitation, small molecules, polypeptides, proteins, oligonucleotides, nucleic acids,

polysaccharides, drugs, adjuvants, vaccines or other immunologically active agents or an agent capable of triggering the production of an immunologically active agent. The active agent may also be a micro- or nanoparticle having a charge enabling electrostatic bonding to the surface of the microarray or object. Examples of nanoparticles are lipid particles, such as liposomes, which can be prepared with either a positive or a negative charge (J Drug Deliv.

201 1 ;201 1 :326497), or polymeric particles such as poly lactic acid particles, which have a negative charge (PLoS One. 2012;7(7):e41230. Epub 2012 Jul 23), or trimethylated chitosan particles, which have a positive charge (Eur J Pharm Sci. 2012 Mar 12;45(4):475-81 ).

The microarray is especially suited to administer vaccines. Examples of vaccines are conventional and/or commercially available vaccines including but not limited to flu vaccines, Lyme disease vaccines, rabies vaccines, measles vaccines, mumps vaccines, chicken pox vaccines, smallpox vaccines, hepatitis vaccines, pertussis vaccines, rubella vaccines, diphtheria vaccines, encephalitis vaccines, yellow fever vaccines, polio vaccines, cancer vaccines, herpes vaccines, pneumococcal vaccines, meningitis vaccines, whooping cough vaccines, tetanus vaccines, typhoid fever vaccines, cholera vaccines, and tuberculosis vaccines. The term "vaccine" thus includes, without limitation, antigens in the forms of proteins, polysaccharides, oligosaccharides, or weakened or killed viruses, viral vectors, recombinant protein antigens, plasmid DNA vaccines, as well as antigen- loaded carriers such as, but not limited to, virosomes, virus-like particles, liposomes, iscoms, polymeric nanoparticles and microparticles, surfactant vesicles.

Active agent may also be a dye compound. The microarray of microneedles may thus advantageously be used to color the skin, for example when applying a tattoo onto the skin.

Modification of surfaces with ionisable groups is known and for example described in Kusnezow et al., Proteomics 2003, 3, 254-264 and Kim et al., Langmuir 2010, 26(4), 2599-2608. Zhao et al., Electroanalysis 1999, 1 1 , No.15, 1 108-1 1 1 1 . (1999) describes the modification of a gold metal surface with carboxyl groups.

To obtain a silicon surface modified with molecules having ionisable groups starting from silicon may be performed by the following process steps:

(i) modification of the surface generating Si-OH groups, (ii) reacting said surface with an am ino-alkyl tri alkoxy silane and

(iii) reacting the surface thus obtained with a molecule having a weak base or weak acid group, which molecule can react with the terminal amine group as present on said surface. The invention is also directed to silicon surfaces modified with optionally substituted pyridinyl groups and silicon surfaces modified with optionally substituted hydroxyl phenyl groups.

Applicants found that with the above process silicon surfaces can be modified with ionisable groups enabling electrostatic bonding with an active agent under controlled pH conditions and wherein the surfaces are able to lose their charge at the physiological properties of the skin thereby releasing the active agent. The silicon surface used may be part of any object and especially a single microneedle or a microarray of microneedles.

The amino-alkyl tri alkoxy silane is preferably a compound according to the general formula:

(NH ₂ -(CH ₂ ) _m Si(0(CH)2n ₊ l )3 Wherein m and n may be varied and wherein m may for example be 3-10 and n may for example be from 1 to 4. A suitable amino-alkyl tri alkoxy silane is

(3-aminopropyl)triethoxysilane (APTES). When preparing a silicon surface modified with hydroxyl phenyl groups it is preferred to react in step (ii) the surface is reacted with hydroxy-benzaldehyde. When preparing a surface modified with such ionisable pyridinyl groups it is preferred to react in step (iii) the surface with a pyridine aldehyde. The above process wherein an APTES-modified surface is used and further modified is suitably used to screen different weak acids and weak bases for their surface pKa value. This because the amine groups of the APTES-modified surface are easily derivatised into a group of choice. A skilled person will understand that when a suitable weak acid or weak base has been identified more simple processes can be identified to make a surface comprising the weak acid or weak base of choice. For example a pyridine-modified surface may also be prepared by reacting a suitable pyridyl alkoxy silane with the general formula (NC5H4-

(CH2) _m Si(0(CH)2n+l )3, such as 2-(4-pyridylethyl)triethoxysilane, with the surface comprising Si-OH groups as obtained in step (i) described above and wherein m and n may be as above. This alternative is preferred because less reaction steps are required.

In a preferred embodiment the active agent or different active agents are bonded to the surface in a multitude of layers. Such a multilayer can be prepared starting from a chemically modified surface as described above. The addition of further layers may be performed by well known techniques as for example described in DeMuth, P., Xingfang, S. et al., Adv.Mater.2010, 22, 4851 -4856 and in Saurer, E., et al., Biomacromolecules 2010, 1 1 , 3136-3143. This multilayer can be composed of one or more layers of a positively charged active agent and a negatively charged polymer or a negatively charged active agent and a positively charged polymer. Examples of cationic polymers are chitosan, polyethylenimine, and polybrene. Examples of anionic polymers are dextran sulphate, hyaluronic acid, and polyaspartic acid, In order to attach the first layer, the surface should first be modified with ionisable groups as described above. Subsequently a compound with the opposite charge, which may either be the active agent or the polymer, is coupled to this surface via electrostatic interactions and thereby forming a new layer. Subsequently, a new layer can be formed by coupling a compound having the opposite charge, which may be the active compound or the polymer. Because this process can be done multiple times, the dose of the active compound can be influenced in a controlled manner. Another benefit of this process is that different types of active agents can be present in one layered system. Examples are combination of adjuvants and the pharmaceutically active agent or different vaccines against different diseases. This enables for example that in one single use vaccination against multiple diseases can be achieved. Furthermore, in this lamellar system different adjuvant can be incorporated to potentiate the immune reaction. The invention is also directed to a microarray of microneedles according to the present invention wherein an active agent is bonded to the surface of the microneedle by electrostatic bonding for use in administration of the active agent through the skin of a patient. The invention is also directed to a method to administer an active agent via the skin of a patient using a microarray of microneedles according to the present invention wherein an active agent is bonded to the surface of the microneedle by electrostatic bonding. The invention is also directed to an object as described above not comprising an active agent and suited to bond an active agent. This microarray of microneedles may be loaded with an active agent. The non-loaded microarray of microneedles may be manufactured at one location, while loading takes place at another location or by another company. For this reason the invention extends also to this inventive intermediate product.

The pKa of the surface is measured according to the below described method based on fluorescence, which method is also illustrated in the Examples. This method is developed by applicant because it can provide a pKa value over a large pH range in a relatively easy manner as compared to the known contact angle titration technique. The technique is especially suited for chemically modified silicon surfaces as described above. If in this description mention is made of a pKa of a surface wherein the surface also has an isoelectric point property then the pKa is meant which would be measured as if the surface of the object would have been modified with said weak acid or weak base only.

The invention is thus also directed to this new method of determining the pKa of a chemically modified surface by first loading the surface with positively or negatively charged fluorescently labelled polystyrene nanoparticles and measuring the relative fluorescence at different pH values between pH of 2 and pH of 12 resulting in a S-curve in the domain of relative fluorescence and pH, wherein the pKa is the pH value at the midpoint of the resulting S-shaped curve. To achieve a desired accuracy of the measurement it is advised to repeat the above procedure at least 3 times, wherein the pKa is the average value of said measurements.

The positively charged fluorescently labelled polystyrene nanoparticles are preferably quaternary ammonium-modified nanoparticles because these particles are stable over a broad pH range, especially at the higher pH values. A preferred quaternary ammonium-modified nanoparticles is obtained according to the scheme shown in Figure 2 wherein the reaction product of (3- carboxypropyl)trimethylammonium chloride (CPTA) and N-hydroxysuccinimide (NHS) is reacted with amine-modified polystyrene fluorescent orange

nanoparticles (Par-NH2). The amine-modified polystyrene fluorescent orange nanoparticles may be obtained from Sigma Aldrich.

The negatively charged fluorescently labelled polystyrene nanoparticles are preferably sulphate-modified nanoparticles for example as obtained from Sigma Aldrich as Latex beads, sulphate-modified, fluorescent orange/ yellow-green/ blue/ or red.

The isolecetric point of a surface may be determined by making use of elements of the above method wherein the surface is loaded with positively charged

fluorescently labelled polystyrene nanoparticles and wherein the relative fluorescence at different pH values between pH of 2 and pH of 12 is measured. In addition the surface is coated with negatively charged fluorescently labelled polystyrene nanoparticles and the relative fluorescence at different pH values between pH of 2 and pH of 12 is measured. The isolecetric point is the pH value at which the two curves intersect.

Applicants have thus provided a method to easily develop a microarray for administration of an active compound wherein first molecules (with an expected pKa) are selected to modify the surface of the microarray with. Then the newly developed method to determine the surface pKa is used to check whether this modification results in the desired surface properties regarding coating with and release of the specific active agent.

Example 1

In Example 1 a silicon surface was modified wherein first SiOH groups are generated, subsequently these groups were modified with (3- aminopropyl)triethoxysilane (APTES) yielding an amine-modified surface and subsequent reacted with succinic anhydride SA yielding a carboxyl-modified surface as illustrated in Figure 1 .

Silicon wafers <1 10> (dsp of 0.7 mm thickness) were cut in pieces of either 1 by 1 cm or 1 by 2.5 cm. Cleaning of the silicon surfaces was performed by treating them once with acetone and twice with methanol and subsequently drying in a vacuum oven at 50°C for 30 minutes. Then, the silicon slides were incubated for 60 minutes at 80°C in a freshly prepared piranha mixture (a mixture of 30% H2O2 and 70% H2SO4). Finally, the silicon slides were washed twice in MQ water and five times in methanol and then dried in a vacuum oven at 50°C for 30 minutes. An amine-modified surface was obtained by incubating the silicon slides for 24 h at room temperature on a shaking device in a 2% APTES solution in toluene.

Subsequently, the amine-modified silicon slides were washed once with toluene and three times with methanol. Then, the amine-modified silicon slides were cured under argon for 30 minutes at 120°C, and were incubated in MQ water (as produced by a Millipore water purification system) for 2 h at 40°C to remove unreacted ethoxy groups. Finally, the slides were flushed once with methanol and dried in a vacuum oven at 50°C for 30 minutes. A carboxyl-modified surface was obtained by incubating the amine-modified silicon slides in a 20 mg/ml_ solution of succinic anhydride in 1 ,4-dioxane for 30 minutes at 80°C. Then, the silicon slides were washed three times with methanol and dried in a vacuum oven for 30 minutes at 50°C.

All modified silicon slides were stored under argon until use.

The amine modified surface (APTES modification) as obtained above was confirmed by means of using a fluorescent dye which specifically binds to primary amines, and subsequent analysing the surface by fluorescence microscopy. The results confirmed the existence of the amine modified surface.

The carboxyl modified surface as obtained above was confirmed by means of using a fluorescent dye which specifically binds to primary amines, and

subsequent analysing the surface by fluorescence microscopy. The results confirmed the existence of the carboxyl modified surface.

Example 2

Example 2 describes the preparation of fluorescent quaternary ammonium- modified nanoparticles according to the scheme shown in Figure 2 for use in the method to determine the pKa of the silicon surface as prepared in Example 1 . In Figure 2 the nanoparticle is shown as the circle named Par.

(3-carboxypropyl)trimethylammonium chloride (CPTA), (100 mg, 0.55 mmol) and N-hydroxysuccinimide (NHS) (108 mg, 0.94 mmol, 1 .7 eq.) were dissolved in 12.5 ml_ acetonitrile with molecular sieves 4A under magnetic stirring for 15 minutes at room temperature. Then, the solution was cooled on ice under magnetic stirring for 15 minutes. Subsequently, Ν,Ν'-dicyclohexylcarbodiimide (DCC) (216 mg, 1 .05 mmol, 1 .9 eq.) dissolved in 2.15 ml_ acetonitrile was added to the reaction mixture, and the solution was stirred for 16 h at room temperature. Subsequently, the stirrer was turned off and the solution was put on ice for 15 minutes to precipitate the dicyclohexylurea, and the resulting suspension was then filtered on a fritted funnel. The precipitate was washed with 5 ml_ acetonitrile, and was then discarded (the reaction product is in the acetonitrile). Subsequently, the acetonitrile with the reaction product was vacuum evaporated, leaving a brownish residue. Then, 6.25 ml_ tetrahydrofuran (THF) was added to this residue to dissolve non reacted NHS and DCC, and the mixture was put on ice for 2 h. Then, the product was collected on a glass filter and was washed with 5 ml_ THF. Finally, the product was dried in a vacuum oven for 16 h at 30°C, weighed (46 mg, 0.17 mmol, 31 % yield), and was stored at -80°C until use. For the confirmation of the desired reaction product 1 H and 13C NMR was used and the following peak shifts were identified: 1 H NMR D ₂ 0 300 MHz δ 3.45 (2H, m, N(CH ₃ ) ₃ CH ₂ ), 3.15 (9H, s, N(CH ₃ ) ₃ ), 2.95 (4H, s, CO(CH ₂ )2CO), 2.90 (2H, t, NHS-C0 ₂ CH ₂ CH ₂ ), 2.30 (2H, m, CH ₂ CH ₂ CH ₂ ). 13C NMR D ₂ 0 75 MHz δ 173.3 (CO of NHS), 169.3 (CO ₂ -NHS), 64.8

(CH ₂ CH ₂ N(CH ₃ ) ₃ ), 53.0 (N(CH ₃ ) ₃ ), 27.3 (CO ₂ CH ₂ ), 25.5 (CH ₂ of NHS), 17.9 (CH ₂ CH ₂ CH ₂ ).

100 μΙ_ of an amine-modified (5%) polystyrene fluorescent orange nanoparticles of 100 nm as obtained from Sigma Aldrich (L9904-1 ml_, latex beads, amine-modified polystyrene, fluorescent orange) were diluted with 900 μΙ_ solution 1 , composed of 1 :4 THF: 100 mM (4-(2-hydroxyethyl)-1 -piperazineethanesulfonic acid (HEPES) buffer (pH 6.0) under magnetic stirring. Then, 10 mg CPTA-NHS as obtained above dissolved in 100 μΙ_ solution 1 was added and the mixture was left to react for 1 h at room temperature. To confirm that the CPTA was coupled and thereby stable nanoparticles had been formed, the particle size and size distribution was determined by dynamic light scattering (DLS) and the zeta potential by laser

Doppler electrophoresis on a Zetasizer Nano (Malvern Instruments) at pH values between 2-12. Subsequently, the CPTA-modified polystyrene nanoparticles were dialysed against a 5 mM HEPES buffer (pH 6.0) for 6 h at room temperature, and were stored in a dark vial at 4°C until use.

Example 3

For the pKa determination of the modified silicon surfaces (APTES modification) of example 1 negatively charged nanoparticles (Sigma beads: L1528-1 ml_, latex beads, sulfate-modified polystyrene, fluorescent orange) were used for the titration of the positive (amine) surface, and positively charged nanoparticles were used for the titration of the negative (carboxyl) surface.

To determine the surface pKa a 1 mM EDTA buffer with 1 μΙ_ of 2.5% fluorescent nanoparticles per ml_ was prepared. Then, the pH was adjusted between 2 and 12 either with 0.01 M NaOH or 0.01 M HCI to make 20 nanoparticle suspensions, each with a different pH. For each pH value two aliquots of 0.75 ml_ were transferred to 1 .5 ml_ cuvettes, one with and one without the modified silicon slide. The amine-modified silicon slide (weak base) was incubated with the negatively charged nanoparticles for 4 h, and the carboxyl-modified silicon slide (weak acid) was incubated with the positively charged nanoparticles for 3 h. Incubations were done at room temperature and by using a shaking device. Then, two times 200 μΙ_ from each sample cuvette were transferred to a black 96-well plate and the fluorescence was measured at an excitation wavelength of 520 nm and en emission wavelength of 540 nm with a Tecan Infinite® M1000 plate reader.

Subsequently, the relative fluorescence was calculated for the different pH values. Relative fluorescence values (compared to the 100% fluorescence value for the nanoparticle sample from the cuvette without silicon slide) were plotted against the pH. Finally, according to the Henderson-Hasselbalch equation, the S-shaped curve was fitted in Origin version 8.1 . From this fitted curve the surface pKa was calculated as the pH value at the midpoint of the S-shaped curve. The resulting S- shaped curves are presented in Figures 3a for the amine-modified surfaces with sulphate-modified nanoparticles and in Figure 3b for the carboxyl-modified surfaces with CPTA-modified nanoparticles.

Figure 3a shows a representative surface pKa determination of amine-modified silicon slides (APTES modification) with sulphate-modified polystyrene fluorescent nanoparticles. Fitting an S-shaped curve according to the Henderson-Hasselbalch equation revealed two surface pKa's for the amine-modified silicon slides. The midpoint of the first S-shaped curve revealed a surface pKa of 6.5 (n=3) and a second surface pKa of 9.9 (n=3). If two pKa values may be determined it is defined that the main pKa of the surface as in the present invention is the midpoint of the S-shaped curve wherein the difference in relative fluorescence is the greatest. In Figure 3a it is clear that the difference in relative fluorescence is the greatest for the second pKa value 9.9. If such a surface would have been coated with a negatively charged active agent at a lower pH value only a small reduction of bonding strength would occur when the pH of the environment of the silicon surface reaches the physiological value of 7.4. This would result in that only a small percentage of the active agent would have been released as is illustrated in Comparative Example 6.

In figure 3b a representative picture is shown of a surface pKa determination of the carboxyl-modified silicon surface with CPTA-modified polystyrene fluorescent nanoparticles. The surface pKa of the carboxyl-modified silicon surfaces was found to be 4.4 (n=3). This is in compliance with pKa values found in the literature for -COOH terminated surfaces where pKa values of -COOH terminated surfaces between 4.4 and 6.5 have been reported. However, also pKa values of up to 10.3 have been reported for aliphatic -COOH terminated surfaces, which is dependent on the chain length (-CH2-) between the -COOH group and the surface

(substrate): longer chain lengths lead to higher surface pKa values. Overall, the surface pKa of our carboxyl-modified surface (short chain length) is within the expected and reported range.

If such a surface would have been coated with a positively charged active agent at a higher pH value almost no reduction of bonding strength would occur when the pH of the environment of the silicon surface reaches the physiological value of 7.4. Example 4

In Example 4 a silicon surface was modified with amine groups with APTES as described in example 1 , subsequently these groups were modified with either 4- hydroxybenzaldehyde yielding a phenol-modified surface or with 4- pyridinecarboxaldehyde yielding a pyridine-modified surface as illustrated in Figure 4. In order to generate phenol and pyridine-modified surfaces, the amine-modified surfaces from example 1 were first incubated for 16 h at room temperature in a 100 mM solution of either 4-hydroxybenzaldehyde or 4-pyridinecarboxaldehyde in isopropanol, yielding phenol- and pyridine-modified surfaces via an imine bond. Subsequently, to stabilise the phenol and pyridine-modified surfaces, the reactants were removed and the imine bond was converted into a primary amine bond through reductive amination in a 50 mM NaBH3CN solution in isopropanol for 2 h at room temperature. Finally, the modified silicon slides were washed twice with isopropanol and five times with methanol, and were then dried in a vacuum oven for 30 minutes at 50°C. The modified silicon slides were stored under argon until use. The phenol-modified surface as obtained above was confirmed by means of using a fluorescent dye which specifically binds to primary amines, and subsequently analyse the surface by fluorescence microscopy. The results confirmed the existence of the phenol-modified surface.

The pyridine-modified surface as obtained above was confirmed by means of using a fluorescent dye which specifically binds to primary amines, and

subsequently analyse the surface by fluorescence microscopy. The results confirmed the existence of the pyridine-modified surface.

The pKa of the phenol-modified surface as obtained above was determined as described by example 3, and is shown in figure 5a. The results showed that the phenol-modified surface had a pKa of 8.7.

The pKa of the pyridine-modified surface as obtained above was determined as described by example 3, and is shown in figure 5b. The results showed that the pyridine-modified surface had a pKa of 6.9.

Example 5

In example 5 an active compound was bound to pyridine-modified silicon surfaces with a surface of 5 cm^. First pyridine-modified surfaces were generated as described in example 4. As an active substance fluorescently labelled ovalbumin was used.

The pyridine-modified silicon surfaces were coated with 100 g fluorescent ovalbumin in 1.5 mL 1 mM EDTA at pH 5.5 for 1 h. The protein coated pyridine- modified silicon surfaces were photographed by fluorescence microscopy (GFP filter set, 100x magnification, exposure time of 5 s).

Fluorescence intensity measurements revealed that the coating efficiency of ovalbumin to pyridine-modified silicon surfaces at pH 5.5 was about 95%, meaning that the pyridine-modified silicon surface contained 19 g/cm^, and that only 5% of the active compound was lost during the coating process. Furthermore,

fluorescence microscopy revealed that the pyridine-modified silicon surfaces were homogeneously coated with ovalbumin. Comparative Example 6

An APTES modified microneedle as obtained by the above procedure of Example 1 was coated with ovalbumin. Of the total amount of ovalbumin used in the coating process 95 wt% has been found to actually bind to the surface as coated ovalbumin.

It was found that only 4.7% (n=3) of the ovalbumin protein as coated on the APTES surface was released into in vitro human skin after a single application of 60 seconds, as determined by radioactivity.

Example 7

A pyridine-modified microneedle as obtained by the procedure of Example 4 was coated with ovalbumin. Of the total amount of ovalbumin used in the coating process 95 wt% has been found to actually bind to the surface as coated ovalbumin.

It was found that 71 % (n=3) of the ovalbumin protein as coated on the surface was released into in vitro human skin after a single application of 60 seconds, as determined by radioactivity.