WO2012007623A2

WO2012007623A2 - Oligonucleotide-activated controlled-release system

Info

Publication number: WO2012007623A2
Application number: PCT/ES2011/070507
Authority: WO
Inventors: Estela Climent Terol; María Dolores MARCOS MARTÍNEZ; Ramón MARTÍNEZ MAÑEZ; Félix SANCENON GALARZA; Juan Soto Camino
Original assignee: Universidad Politécnica De Valencia
Priority date: 2010-07-12
Filing date: 2011-07-08
Publication date: 2012-01-19
Also published as: ES2377298B1; WO2012007623A3; ES2377298A1

Abstract

The invention describes a novel system for the controlled release preferably of active substances or detection substances in response to the presence of oligonucleotides as specific stimulus. Furthermore, a preparation method for systems containing the above-mentioned controlled-release system has been developed, together with a method for the determination of specific nucleic acid sequences in aqueous solutions.

Description

CONTROLLED RELEASE SYSTEM ACTIVATED BY

OLIGONUCLEOTIDOS DESCRIPTION

OBJECT OF THE INVENTION

The present describes a new system for controlled release, preferably of active substances, in response to the presence of oligonucleotides as a specific stimulus.

In addition, a preparation of an application of this system has been developed, its use for the detection of oligonucleotides and biological species, among others, and a methodology for its use.

BACKGROUND OF THE INVENTION

In recent decades the development of coordination chemistry and supramolecular chemistry has led to the birth of new concepts related to the design of devices on a nanometric scale that allow chemical substances to be released in a controlled manner. The control of the release of certain substances is very important, since when it is intended to release anticancer substances, such as Taxol or Doxorubicin, it is necessary that there is a release of this substance only in localized areas, in this case with tumor cells. The response to events that occur in biological systems, so that they are self-regulated, has been explored in the development of controlled release materials in response to the glucose / insulin ratio or changes in pH, or antibodies. However, so far, the use of DNA or RNA hybridization with its complements has not been explored as a stimulus for controlled release processes.

Certain current release systems take place by simple dispersion, while the mechanism of other systems, such as biodegradation of polymeric supports, requires organic solvents during loading process, which can affect the structure of the encapsulated substance.

Among the use of other types of supports, such as microcapsules (M. Hamidi, et al., Adv. Drug Delivery Rev. 2008, 17, 1638;), micelles (CW Pouton, et al., Adv. Drug Delivery Rev. 2008 , 17, 625;), vesicles (CJF Rijckenet al., Controlled Relay 2007, 120, 131;), or liposomes (TL Andresen, et al., Prog. Lipid Res. 2005, 44, 69), the use of supports Inorganic mesoporous has been little used in comparison. These supports can be synthesized in a wide variety of morphologies with a defined pore size, and have a high specific surface area. Among the different oxides that can be used as support, porous silicas of the type MCM-41, HMS, MSU-n, MSU-V, FSM-16, KSW-2, SBA-n (n = 1, stand out for their use 2, 3, 8, 11-16) UVM-7, UVM-8, M-UVM-7 or M-UVM-8. These are characterized by having a particle size between 1-100 and m and a specific surface area of 200-1100 m ² / g.

The chemical modification of this type of materials allows to develop more sophisticated controlled release systems, since molecular valves can be designed that contain chemical substances inside that can be transported to a specific site of the organism and be released in a controlled way by external stimuli (Barefoot AB, et al., Angew. Chem Int. Ed 2006, 45, 5924-48; and Ariga K, et al., Chem Rev 2007, 251, 2562-91;) or caused at the cellular level. Recently some functionalized materials with molecular doors have been described by the use of organized nanoscopic solid structures (mesoporous of the MCM-41 type) in which functional organic molecules have been anchored on their outer surface. In this way, functionalized materials with molecular doors have been described whose opening / closing cycles are controlled by the presence of certain anions, changes in the pH of the medium, temperature, redox reactions and irradiation with light (Casasús, R. Et al., J. Am. Chem. Soc, 2008, 130, 1903-1917; Angelos, S. Et al., Angew. Chem. Int. Ed. 2008, 47, 2222-2226; Fu, Q. Et al., Adv Mater. 2003, 15, 1262; Trewyn, BG Et al., Acc. Chem. Res, 2007, 40, 846-853; Radu, DR et al. , J. Am. Chem. Soc. 2004, 126, 13216-13217; Slowing, II et al., Adv Funct. Mater., 2007, 17, 1225-1236; Liu, N. et al., Nano Lett., 2004, 4, 551-554; Vivero-Escoto, JL et al., J. Am. Chem. Soc, 2009, 131, 3462-3463 and Aznar, E. Et al., J. Am. Chem, Soc, 2009 , 131, 6833-6843). Additionally, there are very few hybrid materials with molecular gates that show selective release in the presence of bio-molecules, but in no case has a similar system been described in which the stimulus is the presence of an oligonucleotide (DNA or RNA). Thus, very recently, three examples of siliceous materials have been described whose charge is released in the presence of an enzyme that is capable of hydrolyzing certain bonds in the organic molecules that act as a gate (Patel, K. et al., J. Am. Chem. Soc, 2008, 130, 2382-2383; Schlossbauer, A. et al., Angew. Chem. Int. Ed., 2009, 48, 3092-3095 and Bernardo, A. et al., Angew. Chem. Int Ed. 2009, 48, 5884-5887), and an example in which antibodies are used that interact with a hapten anchored to the surface of the material and that allow the release of bioactive species in the presence of the corresponding antigen, because the The affinity of the antibody for the antigen is greater than for the hapten anchored to the surface of the designed hybrid material (Climent, E. et al., J. Am. Chem. Soc, 2009, 131, 14075-14080).

Among the existing patented systems, in the patents described by J. Zink, F. Stoddart et al. (WO 2009/094580 and WO 2009/097439) use nanodevices to produce a controlled release using oxidation-reduction reactions or pH changes as stimuli for the opening / closing mechanism while that in other examples, such as those described by V. Lin et al. in US2006 / 154069 or US2009 / 0252811 use CdS nanoparticles or different polymers to coat the synthesized material. Thus, there are not too many specific stimuli described to produce new controlled release systems, being a field that due to its potential applications is of great interest.

DESCRIPTION OF THE INVENTION

The invention is within the field of controlled release system design in response to specific external stimuli. Because the use of nucleic acids has not been described as a specific stimulus in the controlled release of bioactive substances, the present invention combines the use of active substance containers with nucleic acids to develop a new controlled release system.

In this invention, the system is based on the surface modification of the material containing the active substance, which by supramolecular interaction blocks the exit of the pores of the material by an oligonucleotide, inhibiting its release. The interface between the support and the oligonucleotide occurs through a coordinating group attached to the container material by covalent bonding and by supramolecular interaction to the oligonucleotide. The system is activated by contacting it with a solution containing the complementary nucleic acid, preferably under tested conditions, so that hybridization occurs between both nucleotide chains and in this way the pore will open and an exit of the substance will occur. active inside the solid nanodevice.

Depending on the content of the support, its release can be applied for the detection of an oligonucleotide hybridization that covers the pores of the support or can be applied to the controlled release of a substance biologically active when there is hybridization of the oligonucleotide.

Another object of the present invention is the process for the preparation of systems containing the controlled release system described in the preceding paragraphs.

Furthermore, it is an object of the present invention to develop a method for the determination of specific nucleic acid sequences in aqueous solutions.

The different particular ways of carrying out this invention defined by dependent claims 2 to 14, 16 to 18, 20 to 23 and 25 to 27 are considered included in this description by reference.

DESCRIPTION OF THE DRAWINGS

The present specification is complemented with drawings, illustrative of a preferred and never limiting example of the invention.

Figure 1 shows a preferred embodiment of the invention, indicating a set of a nanodevice, in this case of the type MCM-41, loaded with fluorescein and modified superficially by reaction with aminopropyltrietaxysilane, which in turn is bound to an oligonucleotide by supramolecular interactions .

Figure 2 represents the reaction of the oligonucleotide attached to the nanodevice in the presence of its completely complementary oligonucleotide, leading to a more specific hybridization and freeing the pores of the nanodevice, releasing the fluorescein contained therein.

Figure 3 compares the reaction of the oligonucleotide bound to the nanodevice with several oligonucleotides, measured in the variation of the fluorescence of the solution measured at 516 nm (X _exc 419 nm) according to the amount of fluorescein released after hybridization.

DETAILED EXHIBITION OF THE INVENTION The first object of the present invention is a new opening system activated by the presence of specific oligonucleotides and which can be applied in new detection formats or in controlled release processes, depending on the content of the support.

This system is based on blocking the pores of the support by an oligonucleotide, thus containing the active substance within the support. For the activation of the release of the substance occurs in the presence of a second activating oligonucleotide and complementary to this first oligonucleotide, fixed to the support. Since hybridization of the second oligonucleotide with the first oligonucleotide is a stronger interaction than that existing with the active substance's container material, hybridization unlocks the exit of the pores by releasing the substance within the support.

The interaction of the first oligonucleotide that has to be fixed to the support, occurs with it through supramolecular interactions, preferably electrostatic with the material containing the substance to be released.

We obtain through this system an innovative approach to the development of controlled release systems, which is also highly selective since it is based on the hybridization of complementary strands of DNA or RNA.

In a particular embodiment, the system is composed of:

- a porous support capable of containing the active substance or indicator;

an interface layer between the porous support and the oligonucleotide that ensures its fixation on the surface and

- the complementary oligonucleotide of the one to be recognized, which is adsorbed to the surface by supramolecular interactions (such as electrostatic interactions) blocking the release of the active substance.

The support may consist of metals, semiconductors, organic polymers, carbons or oxides, preferably of different inorganic species, such as titanium, zirconium, silicon, magnesium or boron. Among the high surface specific silicas that can be used as support, there is the type MCM-41, HMS, MSU-n, MSU-V, FSM-16, KSW-2, SBA-n (n = 1, 2, 3 , 8, 11-16) UVM-7, UVM-8, M-UVM-7 or M-UVM-8, among others. Preferably the MCM-41 support has been used.

The interface layer is covalently bound to the support and is capable of giving supramolecular, preferably electrostatic, interactions with the oligonucleotide.

The choice of support must be made taking into account its compatibility with the active substance to be released and the possibility of surface modification with the interface layer. The interaction between this layer and the oligonucleotide must be strong enough to prevent separation of the blocking oligonucleotide in the absence of the activating oligonucleotide and 100% complementary, but weak enough compared to hybridization between the oligonucleotides so that in the presence of the activator Liberation occurs. Thus, it is essential to select the species, topology and surface concentration in the composition of the interface layer.

In relation to the blocking oligonucleotide, this may be a DNA or RNA chain formed between 10 and 50 nucleotides. In a particular embodiment, said nucleotides can be covalently bound to other inorganic particles, such as cadmium sulfide nanoparticles or magnetic nanoparticles.

The systems thus developed can be part of polymers or scaffolds for, among other applications, tissue regeneration or cell differentiation, depending of the biological active substance present in the support and which is released after hybridization of the oligonucleotides. If the support comprises an indicator, dye, fluorescent substance, or the like, its release can be used for the detection of the oligonucleotide, both for monitoring and quantification.

In a preferred embodiment, the support is of the mesoporous silicon oxide type and the interface layer consists of a layer of compounds with positive charge at the test pH of the system, such as amines or guanidinium salts, and the oligonucleotide is formed by DNA strands. or RNA between 15 and 30 nucleotides.

The second aspect of the invention refers to the process for the preparation of controlled release systems. Said procedure comprises the following steps:

a) Suspend a porous material capable of forming supramolecular interactions with oligonucleotides in an aqueous solution containing the oligonucleotide;

b) Stir between 10 minutes and 3 hours preferably 30 minutes, the suspension simultaneously maintaining the constant temperature between 15 and 50 ° C, preferably at 37 ° C;

c) Remove the solution, preferably by centrifugation for 5 minutes at 5,000 rpm and d) Wash with aqueous solution.

Although different media may be used as an aqueous solution, buffered media at physiological pH, especially pH 7.5 with a 37.5 mM MgCl ₂ mixture, 20 mM Tris-HCl, are preferred.

Third, the invention comprises the use of the controlled release systems set forth above for the determination of oligonucleotide chains - in the event that the materials are loaded with indicators, dyes, fluorescent or similar substances - or for controlled release in response to the presence of oligonucleotides in solution for the release of other active substances (drugs, biocides, etc.) in cellular media, or biological systems.

Finally, the method for the detection of oligonucleotides comprising the following steps is included in the invention:

a) Suspend the material with controlled release properties in aqueous solution;

b) Mix the solution to be tested containing the oligonucleotide with the solution prepared in step a) Y

c) Measure the macroscopic signal produced in step b) and optionally quantify said signal by interpolation in a calibration curve. The signal produced will depend on the indicator present in the support and released by the hybridization of the oligonucleotides. For example, absorbance, fluorescence, etc. could be measured.

The following examples illustrate the invention.

Example 1

Obtaining support with the specific absorbed oligonucleotide (SI)

In a specific embodiment of the invention, 5 mg of an MCM-41 type carrier loaded with fluorescein and superficially modified with aminopropyltriethoxysilane (amine concentration determined by thermogravimetric analysis 1.98 mmol / g Si0 ₂ ) were weighed and suspended in 5 mL of a solution buffered to pH 7.5 (37.5 mM MgCl ₂ , 20 mM Tris-HCl) and containing oligonucleotide 0-1 (5'- AATGCTAGCTAAT CAATCGGG-3 ') at a concentration of 20 nmol / g solid and allowed to stir at 37 ° C for half an hour. The suspension was then centrifuged for 5 minutes at 5000 rpm and the solution was separated, washing the solid with 5 mL of the buffered solution. This suspension was separated again by centrifugation, thus obtaining the SI set.

Figure 1 shows a diagram of this SI set obtained, where 1.1 is the first 0-1 oligonucleotide, indicated in detail in the lower part of the figure; 1.2 is the aminopropylsilane chain generated by the reaction of the aminopropyltriethoxysilane with the surface of the support, indicated in detail at the bottom right; 1.3 is the MCM-41 support; 1.4 is the fluorescein contained in the pores of the support, indicated in detail in the upper right; and 1.5 indicates the SI set.

For characterization and quantification of the SI set, a 0-1 '(5'-AATGCTAGCTAATCAATCGGG-Cy5-3') oligonucleotide was used, labeled with Cy5 at the 3 'end, attached to the support by the mechanism described above. The quantification was carried out by calculating the fluorescence of the solution after the solid was anchored, having a content of 17 nanomoles / g solid.

Example 2

Behavior of the SI set in the presence of the complementary oligonucleotide and oligonucleotides

Similar .

In a specific embodiment of the invention, 5 mg of the SI assembly described above was suspended in 5 mL of the buffer solution at pH 7.5 (37.5 mM MgCl ₂ , 20 mM Tris-HCl). 100 yL of this suspension was pipetted and 300 yL of the buffered solution containing oligonucleotide 0-2 (5'-CCCGATTGATTAGCTAGCATT-3 ') in 15 nmol / g solid concentration was added, which is complementary 100% with oligonucleotide 0-1 attached to the support. Figure 2 illustrates the operation of the 0-1 oligonucleotide hybridization attached to the support in the presence of the 100% complementary 0-2 oligonucleotide. When the two strands of those comprising the oligonucleotides hybridize, the pore of the support is uncovered and the substance contained therein is released. The same numerical indications have been used for the same components included in Figure 1 and 2.1 indicating oligonucleotide 0-2, 100% complementary with oligonucleotide 0-1, has been added; 2.2 is the strand formed by oligonucleotides 0-1 and 0-2 after hybridization, uncovering the pore of support 1.3, leaving the aminopropyltriethoxysilane 1.2 anchored to the support and releasing the fluorescein 1.4 contained in the pores of support 1.3.

This experience was repeated in the presence of oligonucleotide 0-3 (5 '- CCCGATTGATTCTCTAGCATT-3'), with two different bases with respect to oligonucleotide 0-2 in the central position, or in the presence of oligonucleotide 0-4 (5 '-CCCGATTGATTGGCTAGCATT-3 ', with only a different base with respect to oligonucleotide 0-2 in the central position) at the same concentration, or in the absence of any nucleotide.

The fluorescence intensity was measured for a total of 180 minutes. The results are illustrated in Figure 3, where a) represents the fluorescence measured by adding oligonucleotide 0-2; b) represents the fluorescence measured by adding oligonucleotide 0-3; c) represents the fluorescence measured by adding oligonucleotide 0-4 and d) represents the fluorescence measured in the absence of any nucleotide, but by adding the rest of the components of the mixture. A slight release of the contents of the pores is observed even when no nucleotide is present, so there may be a small and insignificant degradation of the 0-1 oligonucleotide binding to the support. The liberation of Pore content is then much less when adding oligonucleotide 0-4 with respect to release with the presence of oligonucleotide 0-3. The most effective response is observed by adding oligonucleotide 0-2, indicating the specificity of oligonucleotide 0-1 to hybridize with its complete complementary partner and at a significantly lower level with oligonucleotides that differ on one or two bases.

All these embodiments are not limiting and present examples of the possibilities of the invention.

Claims

1. - A controlled release system characterized in that the activation of the release is produced by the hybridization of an oligonucleotide attached to and covering pores of a support, activating the release of a substance contained in the pores of the support.

2. - A controlled release system according to claim 1 characterized in that the oligonucleotide attached to and covering pores of a support is attached to the surface of the support by supramolecular interactions.

3. - A controlled release system according to claim 2 characterized in that the union between the oligonucleotide and the surface of the support is due to electrostatic forces.

4. - A controlled release system according to any of claims 1 to 3 characterized in that the union between the oligonucleotide and the support is strong enough to bind an oligonucleotide to the surface of the support, blocking the pores of the support and weak enough so that it can hybridize the oligonucleotide attached to the support with the complementary oligonucleotide.

5. - A controlled release system according to any one of claims 1 to 4 characterized in that the system comprises a porous support capable of containing an active substance or an indicator, an oligonucleotide blocking the pores of the support and an interface layer between the porous support and the oligonucleotide that ensures fixation between these elements.

6. - A controlled release system according to claim 5, characterized in that the porous support is composed of metals, semiconductors, organic polymers, carbons or oxides of species such as the silicon, aluminum, titanium, zirconium, magnesium or boron, among others.

7. - A controlled release system according to claim 6, characterized in that the porous support is a silica with a high specific surface area.

8. - A controlled release system according to claim 7, characterized in that the porous support is a silica type MCM-41, HMS, MSU-n MSU-V, FSM-16, KSW-2, SBA-n (n = 1, 2, 3, 8, 11-16) UVM-7, UVM-8, M-UVM-7 or M-UVM-8, among others.

9. - A controlled release system according to any one of claims 1 to 8, characterized in that the oligonucleotide blocking the pores of the support is a DNA or RNA chain formed between 10 and 50 nucleotides.

10. - A controlled release system according to any one of claims 1 to 9 characterized in that the oligonucleotide blocking the pores of the support is complementary to an oligonucleotide to be detected.

11. A controlled release system according to any one of claims 1 to 10 characterized in that the support-blocking oligonucleotide of the support can be covalently bound to other inorganic particles, such as cadmium sulfide nanoparticles or magnetic nanoparticles.

12. A controlled release system according to claim 5, characterized in that the interface layer is composed of positively charged compounds covalently bonded to the surface of the porous support.

13. A controlled release system according to claim 12, characterized in that the positively charged compounds included in the interface layer are amines, ammonium salts or guanidinium salts.

14. - A controlled release system according to any of claims 5 to 13, characterized in that it further comprises a polymer or scaffold.

15. - Method of preparing a controlled release system according to claims 1 to 14, characterized in that it comprises:

b) Stir between 10 minutes and 3 hours, the suspension simultaneously maintaining the constant temperature between 15 and 50 ° C;

c) Remove the solution and

d) Wash with aqueous solution.

16. Method according to claim 15, characterized in that the solution is stirred for 30 minutes and the suspension is maintained at a constant temperature of 37 ° C in step b) and the solution is removed by centrifugation for 5 minutes at 5000 rpm in step c).

17. - Method according to any of claims 15 or 16 characterized in that the aqueous solution is buffered.

18. - Method according to claim 17, characterized in that the buffered aqueous solution has a pH of 7.5 and comprises a mixture of 37.5 mM MgCl ₂ and 20 mM Tris-HCl.

19. - Use of a controlled release system described in any of claims 1 to 14 for the detection of oligonucleotides, biological species or residues of biological species, among others.

20. - Use of a controlled release system described in any of claims 1 to 14 for the release of active substances in solution.

21. - Use of a controlled release system according to claim 20, wherein the aqueous solution is a cellular or biological medium.

22. - Use of a controlled release system according to any of claims 20 or 21, wherein the substance to be released has activity.

23. - Use of a controlled release system according to claim 22 wherein the substance to be released is a drug, a biocide, an aroma, or the like.

24. Method of using the controlled release system according to any of claims 19 to 23, characterized in that it comprises: a) Suspend the material with controlled release properties according to claims 1 to 14 in aqueous solution;

b) B) Mix the solution to be tested containing the oligonucleotide with the solution prepared in step a) and

c) Measure the macroscopic signal produced in step b)

25. Method according to claim 24, characterized in that the macroscopic signal produced in step b) is also quantified by interpolation in a calibration curve.

26.- Method according to any of claims 24 or 25, characterized in that the aqueous solution is buffered.

27. The method according to claim 17 to 26, characterized in that the buffered aqueous solution has a pH of 7.5 and comprises a mixture of 37.5 mM MgCl ₂ and

20 mM Tris-HCl.