NL2007382C2

NL2007382C2 - Method to coat an active agent to a surface.

Info

Publication number: NL2007382C2
Application number: NL2007382A
Authority: NL
Inventors: Koen Maaden; Johanna Aaltje Bouwstra; Wim Jiskoot; Niels Tas
Original assignee: Univ Leiden; Univ Twente
Priority date: 2011-09-09
Filing date: 2011-09-09
Publication date: 2013-03-12
Also published as: WO2013036115A1

Abstract

The invention is directed to a process to prepare an object coated with an active agent by contacting an object, wherein the surface of the object has a pKa of between 4 and 7.4, with a buffered aqueous coating solution comprising a negatively charged active agent and wherein said buffered solution has a pH below the pKa, or by contacting an object, wherein the surface of the object has a pKa of between 7.4 and 10, with a buffered aqueous coating solution comprising a positively charged active agent and wherein said buffered solution has a pH above the pKa of the surface. The surface of the object may also be modified with organic molecules comprising ionisable groups which are bases and ionisable groups which are acids.

Description

METHOD TO COAT AN ACTIVE AGENT TO A SURFACE FIELD OF INVENTION

The invention is directed to a microarray of microneedles and to a method to coat an 5 active agent to a surface. The invention is also directed to a method to coat an active agent to a microarray of microneedles.

BACKGROUND OF INVENTION

A microarray of microneedles is a medical device which includes one or more 10 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 15 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-20 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 25 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 30 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 35 publications. US2010280457 discloses a method for applying a high viscosity coating onto the surface of the microneedle. US2008/0294116 discloses a microneedle which is coated with a composition comprising an active agent and a biologically active salt.

2 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.

5 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 microarray on which active agents can 10 more easily be coated in a uniform manner and wherein the sharpness of the needle to be coated is retained as much as possible after coating.

SUMMARY OF INVENTION

This object is achieved by the following microarray. Microarray of microneedles wherein 15 the surface of the microneedle has a pKa of between 4 and 10 and wherein the surface is modified with organic molecules comprising ionisable groups which are weak bases or weak acids.

Applicants found that a microarray having the claimed surface it is possible to easily and 20 uniformly coat the surface of the microneedles with an active agent having the opposite charge compared to the surface of the microneedle. The coating layer is very thin resulting in that the sharpness of the individual needle 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 25 strength, due to the physiological properties of the skin.

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.

30 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 35 pyridine modified surfaces

Figure 5 a shows a resulting S-shaped curve for the phenol-modified surfaces with CPTA-modified nanoparticles 3

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

DETAILED DESCRIPTION

5 “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 10 delivery of therapeutic agents or the sampling of fluids through the skin.

The microarray of microneedles 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 15 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 because of the physiological 20 properties of the skin.

In a first embodiment a negatively charged active agent is coated on the surface. The surface is suitably positively charged at pH<7 and becomes neutral at pH>7, wherein the pKa of the surface is preferably between 4 and 7.4. Coating with a negatively charged 25 active agent is performed by contacting this surface with an aqueous buffered solution having a pH below the pKa of the surface and comprising the active agent.

In a second embodiment a positively charged active agent is coated on the surface. The surface is suitably negatively charged at pH>8 and becomes neutral at pH<8, wherein the 30 pKa of the surface is preferably between 7.4 and 10. Coating with a positively charged active agent is performed by contacting the surface with an aqueous buffered solution having a pH greater than the pKa of the surface and comprising the active agent. The surfaces of both embodiments will loose their charge due to the physiological properties of the skin and thereby breaking electrostatic interactions and releasing the active agent 35 from the surface and into the skin.

4

The material of the 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. A suitable material of the microneedle is silicon. Examples of microarrays of microneedles are described in 5 EP2303766, EP2289843, CN101830428 and JP2008035874. Applicants found that the silicon surface may be modified according to the present invention to achieve the desired pKa property. To achieve the desired pKa property he surface of the silicon 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 10 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.

15 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 are optionally substituted pyridinyl groups, imidazole groups and aromatic amine groups and 20 glucosamine groups.

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.

25 Examples of suitable ionisable groups which are weak acids are carboxyl groups, hydroxyl groups and phosphate groups. The carboxyl group or hydroxyl group itself may advantageously be substituted on an aryl group, preferably a phenyl group. A preferred weak acid group is an optionally substituted hydroxyl phenyl group.

30 The invention is also directed to an object having a surface of silicon wherein the surface has a pKa of between 4 and 10 and wherein an active agent is bonded to said surface by electrostatic bonding and wherein the surface of the silicon is modified with organic molecules comprising ionisable groups which are weak bases or weak acids. The preferred embodiments are as described above.

35

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 5 an active agent is bonded by means of electrostatic bonding wherein the pKa 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.

5 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 10. Suitably the surface is a non-dissolvable polymer, a metal or preferably silicon as 10 described above.

The invention is also directed to a microarray as described above or an object having a modified silicon surface wherein an active agent is bonded to its surface by electrostatic bonding. Active agent according to the present invention refers to one or more 15 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, 20 adjuvants, vaccines or other immunologically active agents or an agent capable of triggering the production of an immunologically active agent.

The microarray is especially suited to administer vaccines. Examples of vaccines are conventional and/or commercially available vaccines including but not limited to flu 25 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 30 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.

35 6

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.

5 Modification of surfaces with ionisable groups is known and for example described in Kusnezow et al., Proteomics 2003, 3, 254-264; Kim et al., Langmuir 2010, 26(4), 2599-2608 and Zhao et al., Electroanalysis 1999, 11, No. 15, 1108-1111. (1999).

To obtain a silicon surface modified with molecules having ionisable groups starting from silicon may be performed by the following process steps: 10 (i) modification of the surface generating Si-OH groups, (ii) reacting said surface with an amino-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 15 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 20 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 25 formula: (NH2-(CH2)mSi(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 30 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 35 surface modified with such ionisable pyridinyl groups it is preferred to react in step (iii) the surface with a pyridine aldehyde.

7

The invention is also directed to a process to prepare a microarray as described above or an object having a modified surface wherein an active agent is bonded to the surface by electrostatic bonding and wherein the active agent is a negatively charged active agent. The surface is positively charged at pH<7 and becomes neutral at pH>7, wherein the pKa 5 of the surface is preferably between 4 and 7.4. This process of coating with a negatively charged active agent is performed by contacting this surface with an aqueous buffered solution having a pH below the pKa of the surface and comprising the negatively charged active agent. The active agent may be dissolved or dispersed in said aqueous buffered solution.

10

The invention is also directed to a process to prepare a microarray as described above or an object having a modified surface wherein an active agent is bonded to the surface by electrostatic bonding and wherein the active agent is a positively charged active agent. The surface is suitably negatively charged at pH>8 and becomes neutral at pH<8, wherein 15 the pKa of the surface is preferably between 7.4 and 10. This process of coating with a positively charged active agent is performed by contacting the surface with an aqueous buffered solution having a pH greater than the pKa of the surface and comprising the positively charged active agent. The active agent may be dissolved or dispersed in said aqueous buffered solution.

20

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. This multilayer can be composed of one or more layers of a positively charged active agent and a negatively charged polymer or a 25 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 30 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 35 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 8 achieved. Furthermore, in this lamellar system different adjuvant can be incorporated to potentiate the immune reaction.

Preferably a surface modified with both weak base groups and strong acid groups is used 5 to bond a negatively charged active agent. The surface is preferably modified with organic molecules having ionisable groups which are weak bases and with organic molecules having strong acid groups and wherein the surface has an isoelectric point of between 5 and 9. This 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. When a 10 microarray of microneedles is used having such a surface the charge of the surface will change from positive to negative due to the physiological properties of the skin enabling an even quicker release of the negatively charged active agent into the skin. Examples of suitable strong acid groups are sulphonate groups.

15 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.

20 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 pKa of the surface is measured according to the below described method based on 25 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.

30 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 35 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.

9

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-5 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.

10

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.

15 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.

20 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.

25 Silicon wafers <110> (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 30 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 35 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 10 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, 5 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 as obtained above was confirmed by means of using a 10 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 15 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 20 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-25 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, N,N’-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 30 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 productwas vacuum evaporated, leaving 35 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 11 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 1H and 13C NMR was used and the following peak shifts were identified: 1H NMR D20 300 MHz 5 3.45 (2H, m, N(CH3)3CH2), 3.15 (9H, s, N(CH3)3), 2.95 (4H, s, 5 CO(CH2)2CO), 2.90 (2H, t, NHS-C02CH2CH2), 2.30 (2H, m, CH2CH2CH2). 13C NMR D20 75 MHz 5 173.3 (CO of NHS), 169.3 (C02-NHS), 64.8 (CH2CH2N(CH3)3), 53.0 (N(CH3)3), 27.3 (C02CH2), 25.5 (CH2 of NHS), 17.9 (CH2CH2CH2).

100 pL of an amine-modified (5%) polystyrene fluorescent orange nanoparticles of 100 10 nm as obtained from Sigma Aldrich (L9904-1mL, latex beads, amine-modified polystyrene, fluorescent orange) were diluted with 900 pL 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 pL solution 1 was added and the mixture was left to react for 1 h at room temperature. To 15 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 20 temperature, and were stored in a dark vial at 4°C until use.

Example 3

For the pKa determination of the modified silicon surfaces of example 1 negatively charged nanoparticles (Sigma beads: L1528-1 ml_, latex beads, sulfate-modified 25 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 pL of 2.5% fluorescent nanoparticles per ml. was prepared. Then, the pH was adjusted between 2 and 12 either 30 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 35 nanoparticles for 3 h. Incubations were done at room temperature and by using a shaking device. Then, two times 200 pL from each sample cuvette were transferred to a black 96- 12 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 5 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 10 Figure 3b for the carboxyl-modified surfaces with CPTA-modified nanoparticles.

Figure 3a shows a representative surface pKa determination of amine-modified silicon slides with sulphate-modified polystyrene fluorescent nanoparticles. Fitting an S-shaped curve according to the Henderson-Hasselbalch equation revealed two surface pKa’s for 15 the amine-modified silicon slides. The midpoint of the first S-shaped curve revealed a surface pKa of 6.55±0.73 (n=3, meaniSD) and a second surface pKa of 9.94±0.19 (n=3, meaniSD).

In figure 3b a representative picture is shown of a surface pKa determination of the 20 carboxyl-modified silicon surface with CPTA-modified polystyrene fluorescent nanoparticles. The surface pKa of the carboxyl-modified silicon surfaces was found to be 4.37±0.59 (n=3, meaniSD). 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 25 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.

30 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 35 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 13 phenol- and pyridine-modified surfaces via an imide bond. Subsequently, to stabilise the phenol and pyridine-modified surfaces, the reactants were removed and the imide 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 5 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 10 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 15 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 20 surface had a pKa of 8.71 ±0.60.

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 6.86±0.20.

25

Example 5

In example 5 an active compound was bound to pyridine-modified silicon surfaces with a surface of 5 cm2. First pyridine-modified surfaces were generated as described in example 4. As an active substance fluorescently labelled ovalbumin was used.

30 The pyridine-modified silicon surfaces were coated with 100 pg 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 35 pyridine-modified silicon surfaces at pH 5.5 was about 95%, meaning that the pyridine- modified silicon surface contained 19 pg/cm2, and that only 5% of the active compound 14 was lost during the coating process. Furthermore, fluorescence microscopy revealed that the pyridine-modified silicon surfaces were homogeneously coated with ovalbumin.

Claims

A microarray of micronaaides, wherein the surface of the micronaaides is in possession of a pKa that is between 4 and 10, and wherein the surface is modified with the aid of organic molecules containing ionizable groups that are weak bases or weak acids .

A microarray according to claim 1, wherein the material of the micronaaides is silicon. 10

The microarray of claim 1 or claim 2, wherein the ionizable group is a weak base and is an optionally substituted pyridinyl group.

4. Microarray according to claim 1 or claim 2, wherein the ionizable group is a weak acid and is an optionally substituted hydroxylphenyl group.

5. Microarray according to any one of claims 1-4, wherein the pKa is measured by loading the surface with positively or negatively charged, fluorescently labeled polystyrene nanoparticles, and by measuring the relative fluorescence at different pH values that are between 2 and 12, resulting in an S-shaped curve in the domain of relative fluorescence and pH, the pKa being the pH value corresponding to the central point of the resulting S-shaped curve.

6. Microarray according to any of claims 1-3 or 5, wherein the surface of the micro needle is modified with organic molecules that contain ionizable groups that are weak bases, and with organic molecules that have strong acid groups, and wherein the surface has an iso- has an electrical point that is between 5 and 9.

The microarray of any one of claims 1-6, wherein an active agent is bonded to the surface of the micronaaids by an electrostatic bond.

A method for obtaining a silicon surface modified with the aid of organic molecules containing ionizable groups, starting from silicon and by carrying out the following steps: (i). modifying the surface by forming Si-OH groups, reacting the surface with an aminoalkyl-tri-alkoxy silane, and (iii). reacting the thus obtained surface with a molecule with a weak base or with a weak acid group, wherein the molecule can react with the terminal amino group present on the surface.

The method of claim 8, wherein the aminoalkyl is tri-alkoxy silane (3-aminopropyl) tri-ethoxy silane (APTES).

10. A method according to claim 8 or claim 9, wherein in step (ii) the surface is reacted with hydroxybenzaldehyde to obtain a silicon surface which has been modified with hydroxylphenyl groups.

The method of claim 8 or claim 9, wherein the surface is reacted with pyridine aldehyde in step (iii) to obtain a surface modified with ionizable pyridinyl groups. 20

12. Silicon surface modified with optionally substituted pyridinyl groups.

A silicon surface modified with optionally substituted hydroxylphenyl groups. 25

Use of a microarray according to claim 7, to administer an active agent through the skin of a patient by bringing the microaray of the microarray into contact with the skin.

An article that has a silicon surface, the surface of which has a pKa that is between 4 and 10, and wherein an active agent is bonded to the surface by an electrostatic bond, and wherein the surface of the silicon is modified with the aid of organic molecules containing ionizable groups that are weak bases or weak acids.

The article of claim 15, wherein the ionizable group is a weak base and an optionally substituted pyridinyl group.

The article of claim 15, wherein the ionizable group is a weak acid and is an optionally substituted hydroxylphenyl group.

The article of claim 15 or claim 16, wherein the surface of the silicon is modified with the help of organic molecules that contain ionizable groups that are weak bases, and with organic molecules that have strong acid groups, and wherein the surface has an isoelectric point has that is between 5 and 9. 15

19. Object having a surface that is chemically modified using organic molecules containing ionizable groups to which an active agent is bound by an electrostatic bond, and wherein the pKa of the modified surface is selected so that the surface is load loses under the influence of the physiological properties of the skin.

The article of claim 19, wherein the surface is a modified silicon surface, and wherein the ionizable groups are weak acids or weak bases. 25

The article of claim 20, wherein the surface is in the possession of a pKa that is between 4 and 10.

22. A method for producing a microarray according to claim 7, or an article according to any one of claims 15, 16, or 18-21, with a modified silicon surface with a pKa that is between 4 and 7.4 by the microarray or contacting the article with a buffered aqueous solution containing a negatively charged active agent, and wherein the buffered solution has a pH lower than the pKa of the surface.

23. Method of producing a microarray according to claim 7, or an article according to any one of claims 15, 17, 19-21, with a modified silicon surface with a pKa that is between 7.4 and 10, through the microarray or contacting the article with a buffered aqueous solution containing the positively charged active agent, and wherein the buffered solution has a pH higher than the pKa of the surface. 10

24. Method for determining the pKa of a chemically modified surface, by first loading the surface with positively or negatively charged, fluorescently labeled polystyrene nanoparticles, and by measuring the relative fluorescence at different pH values between 2 and 12, resulting in an S-shaped curve in the domain of relative fluorescence and pH, the pKa being the pH value corresponding to the central point of the resulting S-shaped curve.