Patent Application
20210356396
The invention relates to a second-harmonic generating, water-suspensable nanoparticle, a method for preparing an aqueous solution of second-harmonic generating nanoparticles, a method for second-harmonic generation imaging and the use of such second-harmonic generating nanoparticles for imaging applications.
In optical imaging there are many techniques to generate and acquire an optical signal. One of these imaging techniques is the so-called second-harmonic generation imaging. Second-harmonic generation imaging is based on second-harmonic generation (SHG).
SHG is a nonlinear optical scattering-process, in which due to a non-linear susceptibility term of the scattering material, two photons with the same frequency result in a single, new photon with twice the energy, and therefore twice the frequency of the two initial photons.
This effect is used in imaging applications like SHG imaging. In these applications the SHG probes respond with an SHG signal under intense illumination. As the SHG probe signal has a narrow signal profile and twice the wavelength of the excitation light, the SHG signal from the SHG probes can be detected with minimal background.
Furthermore, due to the scattering nature of SHG, SHG imaging does not suffer bleaching or fluorescence intermittency of the probe, as the SHG probe is not excited to higher energy levels (like in fluorescence imaging) from where bleaching and intermittency of the probe occurs.
SHG probes are known in the state-of-the-art, which are made of inorganic material that is not biodegradable, and therefore limiting the prospect of having them in clinical use or under clinical development (U.S. Pat. Nos. 8,945,471, 9,221,519).
Efforts have been made to prepare biocompatible SHG probes using peptides, however an inherent problem during preparation is that oligopeptides self-assemble to SHG structures only in organic solvents [1]-[3] that aggregate in aqueous solutions, rendering them useless for most biological or medical applications.
Therefore, the problem underlying the present invention is to provide a water-suspensable, biocompatible, biodegradable SHG probe and a method for preparing an aqueous suspension comprising said SHG probes.
This problem is solved by a second-harmonic generating nanoparticle having the features of claim 1, a method having the features of claims 9 and 14 as well as a use for such a nanoparticle in imaging applications having the features of claim 15. Preferred embodiments are stated in the sub claims.
According to claim 1, a biodegradable, biocompatible, water-suspensable nanoparticle, for generating a second- or third-harmonic light signal upon illumination with light, comprises
Such a nanoparticle advantageously solves the problem according to the invention, as it is water-soluble and biocompatible.
The water-suspensability of the nanoparticles is particularly achieved by the shell layer that comprises a biodegradable polymer, wherein the shell layer provides water-suspensability to the water-insoluble or not-well soluble, structured plurality of oligopeptides.
Another advantage of the nanoparticle according to the invention is that the nanoparticle does not tend to aggregate with other nanoparticles of the same kind in an aqueous solution, wherein the pure oligopeptides suspended in an aqueous solution might aggregate over time.
Self-assembling oligopeptides can form large scale assemblies that can generate SHG signal, as the orientation of the assembly lacks inversion symmetry. The SHG signal intensity is proportional to the number of peptides employed for the assembly, which is related to the size of the assembly.
Consequently, smaller nanoparticles particularly generate a smaller SHG signal. This invention provides a biodegradable, biocompatible, water-suspensable nanoparticle, particularly in the diameter range of 50 nm to 200 nm, that surprisingly does generate a sufficiently high SHG signal for example for use in microscopy applications.
Large-scale assemblies, as for example disclosed in [4] (particle sizes ˜1 μm to 10 μm), typically need to satisfy phase-matching conditions and their signal propagates predominantly in the forward direction, which limits the imaging capabilities of large assemblies. Moreover, these large scale assemblies exhibit a particularly broad size range is due to the dynamic nature of the assembly process, which can assemble and disassemble in response to environmental changes.
Given the preference of oligopeptides to form aggregates, the nanoparticle according to the invention solves this problem by providing a coating that ensures that the oligopeptide assemblies do not aggregate and growth is restricted. This way, a precise control of the size range and distribution of the nanoparticles is achieved.
Furthermore, suitable oligopeptides for the nanoparticle that generate an SHG signal upon illumination particularly comprise or consist of peptides that self-assemble into beta sheets and π-π stackings through crystallization.
In contrast, the example peptide LIVAGK disclosed in [4] relies on decreasing amino acid sizes within the peptide sequences, which do not self-assemble or generate SHG signal after encapsulation.
Furthermore the peptides disclosed in [4] require a specific peptide concentration and pH value in order to self-assemble. Thus, the assembly process of peptides in [4] is vulnerable to environmental changes. Upon dilution or PH-change these assemblies disassemble rapidly. The instability of the peptide assemblies taught in [4] render them impractical for any tracking experiment within cells and tissues, where imaging probes typically encounter lower pH values for an extended period of time.
In contrast, the nanoparticles according to the invention remain stable under such environmental changes.
Given that the peptide assemblies in [4] are not coated with any polymer, any attempt to functionalize and target this assembly to a cell or protein of interest would necessitate adding functional groups on the peptides, which would disrupt the assembly process.
The shell layer of the nanoparticle allows the nanoparticle i) to be targeted to sites of interest with great precision and ii) to protect the assembled peptides from dissociation or disassembly. Hence, the nanoparticles according to the invention are particularly applicable as imaging agents in various biomedical applications.
[4] specifies a restricted sequence order in order to generate the SHG-generating peptides. Given that the SHG signal was measured in a hydrogel, such restriction is necessary. In contrast, the oligopeptides suitable for the nanoparticles according to the invention, do not suffer from such restrictions and their self-assembly particularly depends on their crystal structure as opposed to their shape.
This difference particularly shows that the SHG generating mechanism in [4] is different to the SHG generation of the nanoparticles according to the invention as will be explained in the following.
The peptides disclosed in [4] require certain shapes and sizes as well as sequences to generate an SHG signal. This indicates that their crystallisation plays little role in SHG generation.
SHG signal generation of a nanoparticle according to the invention particularly relies on SHG generated by crystal unit cells.
The shell layer might not necessarily be understood as a complete and tight coating of the oligopeptides, but it might comprise pores and openings, through which particularly an organic solvent can evaporate. The shell layer or parts of the shell layer particularly extend to the inner part of the nanoparticle, wherein other parts of the layer might extend to the environment of the nanoparticle, e.g. into an aqueous solution. The exact structure of the shell layer is of no great importance, as long as it renders the nanoparticle water-suspensable, biodegradable and non-aggregating.
It is the structured plurality of oligopeptides in the nanoparticle that gives rise to the SHG signal upon illumination with light. The capability of the structured plurality of oligopeptides of generating a second-harmonic signal in turn roots in the lack of the inversion symmetry of the structured plurality of oligopeptides. Thus, without a structured plurality of oligopeptides or with a centrosymmetric structure of oligopeptides no appreciable SHG signal can be generated.
A nanoparticle according to the invention has a diameter ranging particularly between 10 nm and 5 μm, particularly between 50 nm and 1 μm, more particularly between 50 nm and 500 nm.
Furthermore, a nanoparticle according to the invention is particularly capable of generating a third-harmonic light signal upon illumination with light, wherein the third-harmonic light comprises three-times the energy than the illumination light.
A biocompatible nanoparticle in the context of the present invention is particularly non-toxic to biological tissue, cells or a living body, as long as it does not comprise additional and specific epitopes or substances that are designed for triggering for example cell death or for altering cellular signalling pathways.
A biodegradable nanoparticle according to the invention refers the property of the nanoparticle of being degradable particularly by specific enzymes, bacteria, fungi or cells.
Illustrative biodegradable materials suitable for use in the practice of the invention include naturally derived polymers, such as acacia, gelatin, dextrans, albumins, alginates/starch, and the like; or synthetic polymers, whether hydrophilic or hydrophobic.
As used herein, the terms “biodegradable” and “biocompatible” therefore denote any synthetic or naturally-derived material that is known as being suitable for uses in the body of a living being, i.e., is particularly biologically inert and physiologically acceptable, non-toxic, and, in the context of the present invention, is biodegradable in the environment of use, i.e., can be resorbed by the body or degraded by specific enzymes or bacteria.
An oligopeptide consists of at least two amino acids and comprises particularly less than 100 amino acids that are chemically linked. Particularly oligopeptides consisting of two, three, four, five, six or seven amino acids are suitable for the invention, as long as they are capable of forming a structured plurality that generates an SHG signal when comprised by the nanoparticle and when illuminated with light.
It is noted that not all structured pluralities of oligopeptides are capable of generating a second-harmonic signal, even though theoretically they should do so. Thus, each different oligopeptide has to be tested for the SHG property separately. Furthermore, it is noted that even if the oligopeptides assemble in a second-harmonic generating structure, it is observed that after preparation of the nanoparticle according to the invention, said nanoparticle might not generate the SHG signal anymore. Also here, a thorough testing of various oligopeptides is necessary.
The term “structured” refers to the fact, that the plurality of oligopeptides is arranged at least area by area in an assembly that exhibits a certain regularity or repeated pattern and that the plurality of structured oligopeptides is particularly not arranged in a random coil configuration. Nonetheless it is possible that a fraction of enclosed oligopeptides in the nanoparticle is not adopting a structured configuration and does not produce an SHG signal upon illumination. This fraction is then simply not considered to be part of the structured plurality of oligopeptides.
Furthermore, the structured oligopeptides are particularly not arranged in a lattice formed exclusively by chemical bonds, such as covalent bonds.
As stated above, the structured plurality of peptides is particularly lacking inversion symmetry, such that the structured plurality of oligopeptides is capable of second-harmonic generation.
The structured plurality of oligopeptides generates a second-harmonic signal upon illumination with light, wherein the excitation light comprises preferably wavelengths in the range of 400 nm to 2000 mm.
The SHG strength or SHG susceptibility of the nanoparticle particularly depends on the number and assembly orientation of the structured oligopeptides comprised by the nanoparticle. The structured plurality of oligopeptides particularly comprises more than 100 oligopeptides.
According to another embodiment of the invention, the plurality of oligopeptides comprises or consists of self-assembling oligopeptides, wherein the plurality of structured oligopeptides is arranged in a self-assembled structure.
This embodiment is particularly advantageous as the preparation of nanoparticles is greatly simplified by using self-assembling oligopeptides.
The term “self-assembling” in the context of the invention refers to the property of the oligopeptides that the oligopeptides assemble in predefined structures or assemblies, wherein structure of the self-assembly particularly depends on the specific amino acid sequence of the oligopeptides and/or the specific mixture or mixing proportion of different oligopeptides as well as the solvent properties. The self-assembling process of the oligopeptides is particularly triggered by the oligopeptide concentration.
An example of a self-assembling oligopeptide is the tripeptide Phe-Phe-Phe. Said tripeptide, triphenylalanine, is capable of self-assembling into nanorods.
According to another embodiment of the invention, the plurality of oligopeptides is crystalized in crystal unit cells, wherein the SHG signal is generated from the crystal unit cells upon illumination of the nanoparticle. According to another embodiment of the invention the plurality of oligopeptides comprises or consists of at least one of:
According to another embodiment of the invention the plurality of oligopeptides comprises at least one structured and shape-persistent region exhibiting a high degree of internal order. Said shape-persistent region is capable of second-harmonic generation.
The degree of internal order of the structured plurality has to be so high that an SHG signal is generated upon illumination of the nanoparticle. Thus, ideally all enclosed oligopeptides are part of a particularly self-assembled structure, wherein also assemblies are included in the meaning of the invention, where the plurality of structured oligopeptides is assembled in a plurality of structures, wherein at least one of these structures generates a second-harmonic signal upon illumination. Thus, particularly also nanoparticles comprising a plurality of structured pluralities of oligopeptides, e.g. a plurality of nanorods or any other second-harmonic generating structure are to be summarized under the claimed invention.
These particularly identical structures might have different orientations within the nanoparticle, which is particularly advantageous with regard to its SHG susceptibility. A high degree of internal order refers particularly to the fact that the majority of oligopeptides are part of a structured plurality of oligopeptides and particularly only less than half of the enclosed oligopeptides are in an un-ordered state, such as a random coil configuration.
A high degree of internal order also refers to the fact that particularly more than 50% of the structured plurality of oligopeptides are ordered in a non-centrosymmetrical structure. All centrosymmetrically structured pluralities, such as for example a random coil configuration, lead to a cancellation of the SIHG signal due to destructive interference.
The term “shape-persistent” refers to the property of the nanoparticles, that the particularly self-assembled structures do not spontaneously change their shape or organization, but generally maintain their shape until the nanoparticle is degraded. According to another embodiment of the invention the plurality of structured oligopeptides is structured by means of non-covalent interactions, particularly by hydrophobic interactions and/or hydrogen bonds. Structures formed by non-covalent interactions are particularly useful for preparing the nanoparticle according to the invention, as no further chemical reactants are needed in order to obtain such particularly self-assembling structures. Furthermore these structures tend be biodegradable.
According to another embodiment of the invention, the biodegradable polymer is one of:
These polymers are particularly suitable for forming a biodegradable and biocompatible shell layer of the nanoparticle.
According to another embodiment of the invention, the nanoparticle is digestable by an enzyme, a proteinase, particularly by proteinase K (CAS-Nr: 39450-01-6), particularly after removing any surfactant. Therefore the nanoparticles according to the invention are particularly biodigestable. This digestion might occur at a slower rate than a digestion of oligopeptides alone, i.e. without a polymer shell.
The property of being biodigestable is particularly important and advantageous when the nanoparticle is to be used in biological imaging or medical applications.
According to another embodiment of the invention the nanoparticle particularly the polymer comprises a fluorescent compound, wherein said fluorescent compound is particularly a covalently linked fluorescent dye. A nanoparticle comprising a fluorescent compound enables the co-localization of the fluorescence signal and the SHG signal stemming from the same nanoparticle and thus allows the monitoring of the preparation process of the nanoparticle.
The problem according to the invention is also solved by a method for preparing an aqueous suspension of biodegradable, water-suspended nanoparticles, wherein the nanoparticles generate a second-harmonic signal upon illumination with light, comprising the steps:
According to the definition of the UIPAC, a miniemulsion is an emulsion in which the particles or micelles of the dispersed phase have diameters in the range from approximately 50 nm to 1 μm. In the case of the present invention, the organic phase is the dispersed phase, the continuous phase corresponds to the aqueous phase and also an emulsion with micelle sizes up to 5 μm is referred to as a miniemulsion.
The oligopeptides assemble in the structured plurality of oligopeptides during preparation of the nanoparticle. The assembling is particularly starting when the micelles have formed in the miniemulsion, as the local concentration of the oligopeptides in the micelles is then high enough to trigger the assembling process. The assembling is particularly also happening during evaporation of the organic solvent from the miniemulsion, as the concentration during evaporation is increasing even more.
Given the preference of oligopeptides to form aggregates, the method according to the invention ensures that the oligopeptides do not aggregate and growth is restricted, once the oligopeptides are coated with the shell layer. This way, a precise control of the size and the size distribution of the nanoparticles according to the invention is achieved.
According to another embodiment of the invention the organic phase contains mixed stabilizers, for example an ionic surfactant, such as sodium dodecyl sulfate (n-dodecyl sulfate sodium) and a short aliphatic chain alcohol (referred to as co-surfactant) for colloidal stability, or a water-insoluble compound, such as a hydrocarbon (referred to as a co-stabilizer) limiting diffusion degradation. By adding a mixed surfactant, a co-surfactant and/or co-stabilizer the mini-emulsion according to the invention can be stabilized for several days.
The miniemulsion can be prepared by applying shear-forces to the mixed organic and aqueous phase.
According to another embodiment of the invention the miniemulsion is prepared by applying shear-forces to the organic phase and to the aqueous phase, particularly after mixing the two phases coarsely, particularly by shaking, wherein the shear-forces are applied by sonication.
According to another embodiment of the invention, the organic solvent is removed by evaporating the organic solvent from the miniemulsion, wherein evaporation is particularly achieved by heating and/or by reducing the surrounding atmospheric pressure.
The organic solvent can be removed to great extent from the miniemulsion by evaporation, such the miniemulsion transforms in an aqueous suspension comprising the aqueous, continuous phase and the water-suspended, biodegradable nanoparticles. Thus, the miniemulsion is not an emulsion anymore after the organic solvent is removed, but it is converted in an aqueous suspension of nanoparticles.
According to another embodiment of the invention, the organic solvent is chloroform, the oligopeptide is or comprises the tripeptide Phe-Phe-Phe and the polymers comprise or consist of Poly-Lactic Acid or the monomers comprise or consist of lactat and the aqueous solution comprises sodium dodecyl sulfate (n-dodecyl sulfate sodium) as surfactant.
Nanoparticles made from this composition are robust and reliable in preparation.
According to another embodiment of the invention the monomers are polymerized in a polymerization step.
This step might be carried out simultaneously or subsequently to the removal of the organic solvent. The polymerization step is providing increased stability for nanoparticles that otherwise would consist only of monomers.
The problem according to the invention is further solved by a method for second-harmonic generation imaging of a sample comprising a nanoparticle according to the invention, comprising the steps of:
The sample is particularly obtained from a person or an animal. The at least one nanoparticle is particularly provided to the sample after the sample is obtained from the person or animal.
The problem according to the invention is further solved by a use of the nanoparticle according to the invention, in second-harmonic generation imaging of biological samples, cells, tissue preparations, tissue or living organisms, particularly applying the method for second-harmonic imaging.
Also here, the biological samples, cells tissue preparations, tissue or living organisms are particularly obtained from a person or an animal prior to the provision of nanoparticle to the sample and particularly prior to imaging.
Further features and advantages of the invention shall be described by means of a detailed description of embodiments with reference to the Figures, wherein it is shown in
In step A) a continuous phase, here the aqueous phase 12 comprising water and a surfactant 11 is provided. The continuous phase 12 rests on top of a dispersed phase, here the organic phase 10, which comprises chloroform, a preformed polymer 3, in this case PLLA, and oligopeptides 4, in this case triphenylalanines 41, capable of self-assembling into a second-harmonic generating structure, particularly to a nanotube or a nanorod, that generates a second-harmonic signal upon illumination with light. In this example the polymer 3 is linked to a fluorescent dye, in the present case to Alexa 488, such that co-localization of the fluorescence signal and the second-harmonic signal becomes possible. This is particularly advantageous to monitor a successful preparation of second-harmonic generating nanoparticles 1—a co-localized signal indicates that there are nanoparticles 1 suspended in the solution comprising polymer 3 and oligopeptides 4 assembled in a second-harmonic generating conformation.
In step B) the continuous phase 12 and the dispersed phase 10 are mixed together and sonicated 100 for several minutes such that micelles 13 comprising a plurality 40 of structured oligopeptides 4, the polymer 3 and the organic solvent are formed. Such emulsion is referred to as a miniemulsion 14.
The oligopeptides 4 are located on the inside of the micelles 13, where the organic solvent is encapsulated by the polymer 3. The polymer 3 thus forms a shell 2 for the oligopeptides 4. The micelles 13 are dispersed in the continuous phase 12.
The miniemulsion 14 obtained by the sonication 100 step B) is then heated (step C), such that the organic solvent evaporates 101 from the micelles 13. Once evaporated, the micelles 13 are now solid nanoparticles 1 suspended in the continuous phase 12. The nanoparticles 1 comprise the polymer shell 2 with a fluorescent dye, wherein the polymer shell 2 encapsulates the self-assembled oligopeptides 4. Upon illumination the nanoparticle 1 will generate a second-harmonic signal from the illumination light due to the self-assembled structured plurality 40 of oligopeptides 4.
One important property of these nanoparticles 1 is that besides being capable of generating a second-harmonic signal, said nanoparticles 1 also have the property that they do not aggregate over time. This is particularly useful for targeting and imaging applications that require inert probes for aqueous solutions.
As the polymer shell 2 is biodegradable the nanoparticles 1 themselves are biodegradable.
As can be seen, the overall SHG signal increases, which indicates the formation of more and larger self-assembled nanostructures that are capable of second-harmonic generation. It also indicates that the cluster size is increasing with time, which points to a progressing aggregation of self-assembled structures. Therefore the polymer shell is particularly advantageous in order to inhibit a progressing aggregation that would take place otherwise. The aggregation-inhibiting effect of the polymer shell 2 is shown in
The proteinase K digests the self-assembled structure into single phenylalanines.
In
In the following a brief, non-limiting example of the preparation of a suspension of nanoparticles according to the invention is given:
While the actual steps remain particularly the same, the concentrations of oligopeptide, polymer and surfactant might be different when using a different oligopeptide.
| Number | Date | Country | Kind |
|---|---|---|---|
| 16191538.4 | Sep 2016 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2017/074709 | 9/28/2017 | WO | 00 |
