Surface modification of micro-bubbles and nanoparticles based on poly(alkyl cyanoacrylate)

Abstract

The invention relates to, inter alia, a method for functionalising a cyanoacrylate-based material with the steps of: providing a cyanoacrylate-based material with ester groups; providing a ligand with an amino group; bringing the cyanoacrylate-based material into contact with the ligand under conditions that promote an aminolysis of the amino group of the ligand and the ester groups of the cyanoacrylate-based material; and optionally obtaining the cyanoacrylate-based material functionalised with the ligand.

Description

[0001] Surface modification of microbubbles and nanoparticles based on poly(alkyl cyanoacrylate) Technical field The invention lies in the field of the functionalization of cyanoacrylate-based material, in particular of butyl cyanoacrylate-based material. More specifically, the present invention relates to a method for coupling a compound to cyanoacrylate-based material by aminolysis and its application for the production or functionalization of ultrasound contrast enhancers and US-mediated drug delivery systems. Background of the invention Ultrasound (US) is one of the most frequently used diagnostic methods. It is a non-invasive, comparatively cost-effective, imaging technique with a broad range of applications. In combination with microbubbles (MBs), US allows functional and molecular imaging of (patho-)physiological phenomena.MBs are gas-filled bubbles measuring 1 to 5 µm in size, whose shell is stabilized by lipids, proteins, or polymers. Contrast enhancement is caused by a compressible gas core, which allows the bubble to reflect the applied US waves. Loaded with drugs, the MBs can also function as a US-mediated drug delivery system. They can be disrupted in vivo by US pulses to release the drug locally. In particular, poly(butylcyanoacrylate) (PBCA)-MBs, an MB variant with a rather hard shell, are considered a suitable candidate for diagnostics and therapy. PBCA is a biodegradable polymer and is approved by the FDA as a surgical superglue for wound closure. The shell of PBCA-MBs is typically composed of relatively small polymer chains with an average molecular weight (MW) of 4 kDa, with more than 99% of the chains being below 40 kDa.The diameter of PBCA-MBs is typically about 2 µm, and the thickness of the shell can vary between 50 and 300 nm. This relatively thick shell enables stable encapsulation of the gas and prevents its diffusion from the MBs. Coupling functional compounds to the shell of the MBs can expand the field of application of US-MBs. Accordingly, since the first PBCA-MBs were developed by emulsion polymerization, attempts have been made to couple antibodies and peptides using multistep syntheses to produce targeted PBCA-MBs for molecular US imaging. A common method for functionalizing MBs is biotin-streptavidin conjugation. Palmowski et al. (Invest Radiol. 2008 Mar;43(3):162-9) prepared streptavidin-coated PBCA-MBs and investigated their physicochemical properties.Subsequently, several studies demonstrated that these MBs can be functionalized by conjugation with monoclonal antibodies that recognize receptor proteins involved in various diseases. A disadvantage of biotin-streptavidin conjugation is that the resulting MBs have limited clinical application due to streptavidin's potential immunogenicity in humans. Furthermore, biotin-streptavidin conjugation involves a non-covalent bond, which poses the risk of the functional linkage detaching from the MBs. The lower binding force also complicates the binding of larger substances, such as nanoparticles and micelles. Furthermore, biotin-streptavidin conjugation is a relatively slow process, which can lead to a low yield of intact MBs due to the relatively short lifetime and instability of the MBs.The relatively high cost and complexity of the synthesis also hinder widespread application. Another modification strategy is based on 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), which is capable of activating carboxyl groups of PBCA-MBs in aqueous solution for the coupling of amines to form amide bonds. The challenges of this modification strategy are control and reproducibility of the reaction. The technique requires hydrolysis to access carboxyl groups, which enables further coupling with EDC. The degree of hydrolysis is difficult to control. Typically, a pH value of 10 to 11 is required. The excess of hydroxyl groups enhances the hydrophilic properties of PBCA and reduces the hydrophobic interactions in the MBs, resulting in structural disruption and the formation of short PBCA chains.The experiment showed that the number of MBs was reduced by approximately 48% after hydrolysis and EDC coupling. The primary objective of the present invention was therefore to provide a process for modifying or functionalizing cyanoacrylate-based MBs, which can be used in particular as US contrast enhancers or US-mediated drug delivery systems. Furthermore, the process should be as efficient, easily controllable, inexpensive, and / or simple as possible. Furthermore, it was desired that the MBs produced exhibit a high functionalization density and be immunologically safe for humans.According to the invention, this object is at least partially achieved in that a method for functionalizing a cyanoacrylate-based material comprises the following steps: a) providing a cyanoacrylate-based material having ester groups; b) providing a ligand having an amino group; c) contacting the cyanoacrylate-based material and the ligand under conditions that favor aminolysis of the amino group of the ligand and the ester groups of the cyanoacrylate-based material; and optionally: d) obtaining the cyanoacrylate-based material functionalized with the ligand.The present invention is based on the discovery that aminolysis enables the covalent coupling of a ligand with an amino group in a particularly efficient, reproducible, simple, and cost-effective manner, and is capable of producing a functionalized material that exhibits a number of advantageous properties beyond functionalization. Thus, compared to biotin-streptavidin conjugation chemistry, the coupling efficiency is many times higher. Furthermore, the reaction is better controllable, allowing consistent coupling efficiency to be achieved. The coupling is gentler; the yield of intact material is higher. The process is simple and cost-effective. The material produced in this way has good acoustic properties (for additional information, see the examples section). A further advantage is that the material produced according to the invention is fundamentally immunologically safe.As already indicated above, there are concerns regarding the immunogenicity of streptavidin present on the known MBs, as it is of non-human origin and has a high molecular weight. The method according to the invention thus represents a safe, or at least less harmful, form of coupling in vivo. Due to the low immunogenicity, a longer in vivo circulation time is also to be expected. The present method is not based on the use of amphiphilic shell material as known, for example, from DE 19882362 T5. This type of microparticle has a two-layer shell. The outer layer exposed to the blood and tissue consists of an amphiphilic, biologically compatible material such as polyethylene glycol, polyethylene oxide, polypropylene glycol, and combinations or derivatives thereof.The inner layer consists of a biodegradable polymer, which is intended to impart the desired mechanical and acoustic properties or drug-transport properties to the shell. An emulsification process is suitable for the production of the microparticles. Due to the amphiphilicity of the material forming the outer layer, stable oil / water emulsions can be produced. The shell can be stabilized by crosslinking using glutaraldehyde or carbodiimide. As already explained, the cyanoacrylate-based material according to the invention is not an amphiphilic material. For example, the monomer n-butyl cyanoacrylate (PubChem ID: 23087) has a logP value of 2.4 and is therefore hydrophobic. The PBCA polymer based on this monomer can therefore only be more hydrophobic and not hydrophilic.Thus, the material according to the invention is not produced by conventional emulsification and / or emulsion polymerization. The described advantages can be particularly well exploited when the process according to the invention is used to produce functionalized poly(alkyl cyanoacrylate) (PACA)-MBs that are to be used as functionalized US contrast enhancers or as functionalized US-mediated drug delivery systems. The ligand to be coupled is preferably a compound that functionalizes the produced PACA-MBs such that they accumulate in specific areas of the body, thus enabling or at least supporting targeted diagnostics or targeted therapy. Further aspects, embodiments, and advantages of the invention emerge from the detailed description and the experimental section, including the figures and the claims.

[0002] Brief Description of the Figures Figure 1 shows an HPLC chromatogram at 220 nm wavelength of cRGD-modified PBCA-MBs. Unmodified PBCA-MBs showed no absorption peak due to the lack of a corresponding chromogenic group. cRGD-modified PBCA-MBs had a different retention time compared to free cRGD, indicating the formation of cRGD-modified PBCA-MBs. Figure 2 shows the particle size distribution of PBCA-MBs modified according to the invention at pH 8 ("aminolysis") compared to unmodified PBCA-MBs ("control") and compared to PBCA-MBs modified via EDC at pH 10 ("hydrolysis"). Figure 3 shows a comparison of the targeting efficiency of cRGD-modified PBCA-MBs (Fig. 3C, RGD-MBs) to TNF-alpha activated human umbilical vein endothelial cells (HUVEC) compared to cRAD-modified PBCA-MBs (Fig. 3B, RAD-MBs) and unmodified PBCA-MBs (Fig.3A, control) using in vitro flow chamber tests (100x, scale bar 50 µm). The MBs were loaded with Rhodamine B. Hoechst and WGA 488 were used to stain the cell nuclei and the cell membrane, respectively. The arrows indicate the red staining. Figure 4 shows the echogenicity of cRGD-modified PBCA-MBs (Fig. 4A bottom, Fig. 4C) compared to the control (Fig. 4A top, Fig. 4B) before (Fig. 4A, left) and after (Fig. 4A, right) rupture. The time of rupture is indicated in Fig. 4B and Fig. 4C. No contrast enhancement was measurable in US imaging after rupture (black image, cf. Fig. 4A right), indicating that the contrast signal measured before rupture originated from the (still intact) MBs. Figure 5 illustrates an example of a possibility for the production of modified MBs using the example of the modification of PBCA-MBs with an antibody as a ligand.Detailed Description In a first aspect, the invention relates to a method for functionalizing a cyanoacrylate-based material (also referred to herein for short as "material"). The method according to the invention comprises the following steps: a) providing a cyanoacrylate-based material with ester groups; b) providing a ligand with terminal amino groups; c) contacting the cyanoacrylate-based material and the ligand under conditions that favor aminolysis between the amino groups and the ester groups; and optionally: d) obtaining the cyanoacrylate-based material functionalized with the ligand (also referred to herein for short as "functionalized material"). The term "cyanoacrylate" or "alkyl cyanoacrylate" refers to an alkyl ester of cyanoacrylic acid. A polymeric shell composed of alkyl cyanoacrylates accordingly comprises polyalkyl cyanoacrylates (also abbreviated herein as "PACA").Polyalkyl cyanoacrylates refer to polymers composed of one or more alkyl cyanoacrylates that are essentially free of free acid and alcohol groups. The term “cyanoacrylate-based material” refers to a material that contains cyanoacrylate in unpolymerized, partially polymerized, or polymerized form. Optionally, the material can contain further components, in particular further polymers that are not based on cyanoacrylates. In a preferred embodiment of the invention, the cyanoacrylate-based material is at least partially present as PACA, more preferably as PACA with an alkyl group that has 2 to 10 C atoms, preferably 3 to 9 C atoms, more preferably 4 to 8 C atoms. A particularly preferred PACA within the scope of the present invention is PBCA and is known, for example, from EP 3223864. Chemically speaking, PBCA is referred to as poly(n-butyl cyanoacrylate).If the material is unpolymerized in step c), the modification takes place at the ester groups of the cyanoacrylate monomers. After the modification, polymerization can optionally take place, so that modified material based on PACA, such as PACA-MBs, in particular PBCA-MBs, can also be obtained in this way. Preferably, the material is already polymerized in step c), and the (accessible) ester groups of the polymer are modified (see also Fig. 5). In a preferred embodiment of the invention, the amino group is a primary amino group (-NH2) that is directly bonded to the ligand R (H2N-R) or bonded to the ligand R via oxygen (H2N-OR). Aminolysis according to the following reaction equation is particularly preferred (the split-off 1-butanol is not shown). , where -R represents the ligand or -XR', where X represents oxygen and R' represents the ligand. Regardless of whether the term "ligand" is used in the singular or plural, it collectively refers to a group of identical molecules to be coupled. The same applies to the following term "functional compound". However, the term "a ligand" does not exclude the cyanoacrylate-based material from being functionalized with more than one ligand, e.g., having a first molecular group and a second molecular group that is different from the first molecular group. Preferred methods within the scope of the present invention use a functional compound as the ligand. A functional compound refers to a compound that imparts a desired function to the material modified with it.The functional compound is preferably selected from the group consisting of target ligands, diagnostics, therapeutics, macromolecules, and nanomedicine constructs. Target ligands are compounds that have an affinity for a predetermined target site, such as a specific tissue type, and thus effect or at least support the transport of the correspondingly functionalized material to a desired target site. Preferred target ligands are peptides for integrin-mediated cell adhesion or non-peptide analogues thereof, or antibodies directed against specific target structures. RGD peptides are particularly preferred. The term "RGD peptide" refers to a peptide with an amino acid sequence consisting of the three amino acids arginine, glycine, and aspartic acid, abbreviated to RGD. The RGD sequence occurs naturally in the extracellular matrix (EM). This allows cells to bind to the EM with the help of integrins.This property can be utilized within the scope of the present invention by presenting RGD peptides (or non-peptide analogues) on the surface of the cyanoacrylate-based material. The aim is to achieve accumulation of the functionalized material in integrin-rich tissues, such as cytokine-activated blood vessels (e.g., damaged arteries), tumors and their blood vessels, and inflamed intestinal segments. Another preferred target ligand is E-selectin, a cell adhesion molecule expressed by endothelial cells activated by cytokines such as TNFα or IL-1. Antibodies that bind integrins, selectins, and / or cell adhesion molecules are further examples of preferred target ligands.Although it is preferred that – in the case of use as a drug carrier described below – an active ingredient is passively incorporated into the polymer shell, the coupling process can also be used to conjugate active ingredients. A preferred ligand with therapeutic effect here is doxorubicin, an anthracycline used in chemotherapy (cytostatic agent). Macromolecules to be coupled include, in particular, proteins and other polymers such as RNA or DNA, especially mRNA and plasmid DNA, which impart desired (surface) properties to the cyanoacrylate-based material. For example, polymers such as polyethylene glycol (PEG) can be coupled to increase the in vivo circulation time. Modification of the surface with a hydrophilic compound such as PEG can (also) minimize the interaction of active substance present on the surface with the blood vessel cells.This can, for example, prevent the active ingredient from exerting its (possibly undesired) effect en route to its target site. The term "nanomedicine constructs" refers to nanoparticles with an average particle diameter of 1 nm to 999 nm that exhibit therapeutically or diagnostically relevant properties, such as polymer micelles and liposomes. The most preferred ligands are the aforementioned peptides for integrin-mediated cell adhesion or non-peptide analogues thereof, in particular RGD peptides such as RGDfK. RGD peptides are preferably coupled to PBCA-MBs to allow them to bind specifically and support the diagnosis of inflammatory bowel disease, arteriosclerosis, arterial wall injury, cancer, or other diseases associated with vascular inflammation.Before using the functionalized material, in particular the functionalized MBs, characterization can optionally be carried out. Exemplary characterization methods are described in the examples section. One specific test method is the determination of the targeting efficiency of RGD-functionalized material compared to a RAD-functionalized material (negative control). In an alternative embodiment, the ligand is a linker, which is first bound to the cyanoacrylate-based material (in step c). A functional compound (as defined above) can then be bound via the linker. For this purpose, the linker has, in addition to the amino group, preferably a primary amino group, one or more further groups that can be coupled to the desired functional compound.Alternatively, the ligand to be coupled in step c) can comprise a functional compound and a linker that are already bonded together. Particularly suitable linkers are PEG or alkanes with a preferably terminal coupling group opposite the amino group. Preferred coupling groups are selected from the group consisting of azides, alkynes, and maleimides. The structure of specific linkers can be described as follows: X-R1-Y, where R1 is -(CH2). n - with n = 1, 2, 3 or more, or -(CH2-CH2-O) n - where n = 1, 2, 3 or more, X is -NH2 or -O-NH2, and Y is azide, alkyne, or maleimide. After coupling the linker as a ligand to the cyanoacrylate-based material during aminolysis (step c)), the functional compound R2 can be attached to the linker, for example, by one of the following catalyzed click reactions: Huisgen dipolar cycloaddition:

[0003] Michael Addition: In a preferred embodiment, the cyanoacrylate-based material is already polymerized in step c), preferably in the form of particles, capsules, or bubbles whose average diameter is preferably in the nanometer (1 nm to 999 nm) and / or micrometer range (1 µm to 999 µm). More preferably, the particles, capsules, or bubbles have an average diameter of 500 nm to 8 µm. Deviating from the meaning of the terms nanometer and micrometer range, bubbles with an average diameter of 0.1 µm to 100 µm are referred to herein as MBs. A particularly preferred embodiment relates to the use of the functionalized material as a US contrast enhancer. For this purpose, the cyanoacrylate-based material is provided in the form of bubbles, in particular MBs, and subjected to aminolysis.The bubbles have a polymer shell and a gas core enclosed by the polymer shell, wherein the gas core preferably contains air, oxygen, nitrogen oxide and / or perfluorocarbon. In principle, it is also possible, and encompassed by the present invention, for the gas core to arise from a liquid core only as a result of ultrasound excitation. The wall thickness is preferably in the range from 10 to 400 nm, in particular 50 to 300 nm. The average molecular weight is preferably 1 kDa to 20 kDa, in particular 2 kDa to 10 kDa, with preferably more than 90%, in particular more than 95%, of the chains being below 50 kDa, in particular below 40 kDa. Alternatively, it is possible to first functionalize the cyanoacrylate monomers and then form bubbles. The production of bubbles from cyanoacrylate monomers is well known to those skilled in the art.A further, likewise preferred embodiment relates to the use as a US-mediated drug delivery system. For this purpose, the gas-filled bubbles further comprise an active ingredient, which can be embedded, for example, in the matrix or the lumen and / or bound to the surface of the polymer shell. For example, the active ingredient can be bound to PACA via physicochemical interactions. The release of the active ingredient from the drug delivery system can be controlled by ultrasound treatment. The intensity and wavelength of the ultrasound source, which is usually arranged outside the body, are selected so that the radiation penetrates the tissue to the desired release site. US pulses cause the bubbles to burst, releasing the active ingredient.Drug delivery systems that release an embedded drug when exposed to ultrasound are known to those skilled in the art under the English term "ultrasound-mediated drug delivery." Instead of coupling another compound, such as a targeting ligand, to the ester group, it is fundamentally possible for the drug to act as the ligand. Targeted release of the drug from the drug delivery system can be controlled by selecting the body region to be treated with ultrasound. However, it is preferred that the surface of the drug delivery system be functionalized so that it binds specifically to certain in vivo structures, as described above.Step c) contacting the cyanoacrylate-based material and the ligand under conditions that favor aminolysis of the amino groups of the ligand and the ester groups of the cyanoacrylate-based material is preferably carried out in an aqueous, particularly buffered solution, preferably at a pH of 7.25 to 8.75, more preferably 7.5 to 8.5, most preferably 7.75 to 8.25. Alternatively or additionally, step c) can be carried out in the presence of a catalyst, in particular a lithium catalyst such as lithium methoxide. Furthermore, it is preferred to carry out the aminolysis in the presence of a surfactant in order to prevent or at least reduce aggregation of the particles, capsules, or bubbles. Triton X-100 is mentioned here merely as an example, which can be added to the reaction mixture, for example, in a final concentration of 0.02%.A further aspect of the present invention relates to a cyanoacrylate-based material functionalized with a ligand, produced by the process according to the invention. The features and embodiments described in connection with the process according to the invention form corresponding features and embodiments of the functionalized material produced thereby. This applies accordingly to the further aspects of the invention described here. A US contrast enhancer and a US-mediated drug delivery system comprising the aforementioned functionalized cyanoacrylate-based material represent further aspects of the present invention. For this purpose, the functionalized material is in the form of gas-filled bubbles, in particular MBs. As described above, the bubbles can be imparted with desired properties by the process according to the invention.In the case of the US contrast enhancer according to the invention and the US-mediated drug delivery system according to the invention, functionalization with target ligands is particularly envisaged in order to achieve enrichment at the desired target site. Examples that equally represent further aspects of the invention include the use of the contrast agent or drug delivery system according to the invention in the context of diseases associated with vascular inflammation, in particular inflammatory bowel disease, arteriosclerosis, arterial wall injury, or cancer. Thus, the US contrast enhancer according to the invention is particularly suitable for their detection in the context of a diagnosis, and the US-mediated drug delivery system according to the invention is particularly suitable for their treatment.In particular, it is intended that the contrast agent is functionalized with one or more ligands that specifically bind to one or more tissue markers that are indicative of the presence of the disease, in order to be able to determine the expression of the tissue marker(s) in a tissue using US imaging. For use as a drug delivery system, it is particularly intended that the drug delivery system is functionalized with one or more ligands that specifically bind to one or more tissue markers that are indicative of the presence of the disease, in order to specifically enrich the drug delivery system in a tissue in which the expression of the tissue marker(s) is high.Accordingly, the use of the contrast enhancer according to the invention for detecting, diagnosing, or supporting a diagnosis of a disease associated with vascular inflammation, in particular an inflammatory bowel disease, arteriosclerosis, arterial wall injury, or cancer, as well as a corresponding diagnostic method, represent further aspects of the invention. For this purpose, the ultrasound contrast enhancer comprises vesicles functionalized with a peptide for integrin-mediated cell adhesion, having a polymer shell and a gas core enclosed by the polymer shell, produced according to the method according to the invention. Further aspects relate to the drug delivery system according to the invention for use in the treatment of a disease associated with vascular inflammation, in particular an inflammatory bowel disease, arteriosclerosis, arterial wall injury, or cancer, as well as a corresponding treatment method.It is envisaged that the ultrasound-mediated drug delivery system comprises vesicles functionalized with a peptide for integrin-mediated cell adhesion, having a polymer shell and a gas core enclosed by the polymer shell, produced according to the method of the invention, as well as an active ingredient. A preferred active ingredient is the above-mentioned doxorubicin. A further aspect of the invention relates to a composition comprising the US contrast enhancer according to the invention or the US-mediated drug delivery system according to the invention and a dispersant in which the US contrast enhancer or the US-mediated drug delivery system is present, or a lyophilisate thereof. Suitable dispersants include sodium chloride, PBS (phosphate buffered saline), HBSS (Hanks' balanced salt solution), and other physiological buffers and solutions.The present invention will now be further described with reference to the following examples and the accompanying drawings.

[0004] Examples Using the example of the functionalization of PBCA-MBs with cRGD (Cyclo(Arg-Gly-Asp-D-Phe-Lys)), the known methods described in the introduction (hereinafter also referred to as streptavidin or EDC methods) were compared with the method according to the invention based on the aminolysis described below. The results are as follows: 1. Efficiency of modification According to previous studies, quantification of the number of coupled streptavidin molecules on the surface of PBCA-MBs by FACS revealed an average of 95 streptavidin proteins / MB (corresponding to 15.7 x 10 -23 mol / MB). This means that no more than 95 binding sites are available for functionalization. In comparison, the method according to the invention achieved a coupling efficiency of 3.84 x 10 -16mol / MB (according to the quantitative colorimetric peptide assay by Pierce) was achieved using the example of the coupling of cRGD to PBCA-MBs. Furthermore, a coupling efficiency of 10.53 ± 1.5% was achieved (see Fig. 1). This means that a good 10% of cRGD was coupled to the PBCA-MBs. 2. Controllability The previous biotin-streptavidin conjugation chemistry for modifying PBCA-MBs has the disadvantage that the coupling efficiency is difficult to control. Even under the same coupling conditions, the average efficiency varies depending on the synthesis method and streptavidin coating protocol (e.g., degree of MB hydrolysis prior to streptavidin coating). The method according to the invention is easily controllable. In particular, a consistent coupling efficiency can be achieved, allowing the production of material with consistent properties (data not shown). 3.Yield: Compared to the EDC process, the yield of the inventive method is higher. As already mentioned, the known technique requires hydrolysis to access carboxyl groups, which enables further coupling with EDC. The degree of hydrolysis is difficult to control. Typically, a pH value of 10 to 11 is required. The excess of hydroxyl groups can trigger rapid depolymerization of the PCA-MBs, disrupting the structure and generating short PBCA chains. Experiments showed that the number of MBs was reduced by approximately 48% after hydrolysis and EDC coupling. In contrast, the inventive method can largely prevent the degradation of PCA-MBs. For example, when coupling cRGD from PBCA-MBs, a loss of only approximately 10% of the MBs was observed (see Fig. 2).Costs and Complexity As already described in the introduction, the streptavidin method is relatively expensive, complicated, and time-consuming (multi-step synthesis, long incubation times). The method according to the invention, on the other hand, is inexpensive and can be carried out in a simple one-pot step. 5. Properties of the functionalized PBCA-MBs Using in vitro flow chamber tests, the targeting efficiency of cRGD-modified PBCA-MBs (cf. Fig. 3C) on TNF-alpha activated human umbilical vein endothelial cells (HUVEC) was compared with cRAD-modified PBCA-MBs (cf. Fig. 3B) and unmodified PBCA-MBs (cf. Fig. 3A). Human umbilical vein endothelial cells (HUVEC) were activated with TNF-alpha for 4 hours before the activated HUVECs were placed in flow chamber 10. 7MBs. cRAD-modified PBCA-MBs, prepared under the same conditions, served as a negative control for cRGD-modified PBCA-MBs. The MBs were stained with Rhodamine B. Hoechst and WGA 488 were used to stain the cell nuclei and the cell membrane, respectively. It was found that the cRGD-modified PBCA-MBs exhibited a significantly higher affinity for activated HUVECs. In particular, a targeting efficiency of 0.22 ± 0.09 per HUVEC was achieved (see Fig. 3D). Echogenicity was assessed using the Vevo 3100 ultrasound device. For this purpose, 3 × 10 5MBs were suspended in 4.5 ml of 2% gelatin solution, and the mixture was embedded in 10% gelatin solution. US imaging was performed in nonlinear contrast mode at a center frequency of 18 MHz, initially at a power of 4% (mechanical index 0.2), followed by a power of 100% (mechanical index 0.9) to disrupt the MBs in the gelatin phantom. Quantitative analysis shows that cRGD-modified PBCA-MBs and unmodified PBCA-MBs exhibited similar contrast signals, each with a high and stable enhancement of the monodisperse contrast signal, without being significantly altered by the modification (see Fig. 4). The acoustic behavior of the MBs modified according to the invention is comparable to the echogenicity achieved using the streptavidin method.

Claims

Claims 1. A method for functionalizing a cyanoacrylate-based material comprising the following steps: a) providing a cyanoacrylate-based material with ester groups; b) providing a ligand with an amino group; c) contacting the cyanoacrylate-based material with the ligand under conditions that promote aminolysis of the amino group of the ligand and the ester groups of the cyanoacrylate-based material; and optionally: d) obtaining the cyanoacrylate-based material functionalized with the ligand. 2.A method according to claim 1, wherein the provided cyanoacrylate-based material comprises one or more polymers and preferably: one of the one or more polymers is poly(alkyl cyanoacrylate), in particular poly(n-butyl cyanoacrylate), and / or the cyanoacrylate-based material is provided in the form of particles, capsules, or vesicles, preferably in the form of echogenic vesicles with a polymer shell and a gas core enclosed by the polymer shell.

3. A method according to claim 1 or 2, wherein the amino group is a primary amino group.

4. A method according to any one of the preceding claims, wherein: the ligand is a functional compound selected from the group consisting of target ligands, diagnostics, therapeutics, macromolecules, and nanomedical constructs, wherein the functional compound is preferably a peptide for. the integrin-mediated cell adhesion, preferably an RGD, in particular an RGDfK peptide, or a RAD peptide, in particular a RADfK peptide; or the ligand is a linking element through which the functional compound is bound or can be bound in a further step or is bound in a further step.

5. Method according to any one of claims 2 to 4, wherein: the coupling rate is on the order of 10 -21 up to 10 -156. A method according to any one of the preceding claims, wherein: step c) is carried out at a pH of 7.25 to 8.75, preferably 7.5 to 8.5, more preferably 7.75 to 8.25, and / or step c) is carried out in the presence of a catalyst, preferably lithium methoxide.

7. A ligand-functionalized cyanoacrylate-based material produced according to any one of the preceding claims.

8. An ultrasonic contrast enhancer or ultrasonic drug delivery system comprising the ligand-functionalized cyanoacrylate-based material according to claim 7.

9. An ultrasound contrast enhancer for use in a method for diagnosing a disease associated with vasculitis, in particular inflammatory bowel disease, arteriosclerosis, arterial wall injury, or cancer, wherein the ultrasound contrast enhancer comprises echogenic vesicles functionalized with a peptide for integrin-mediated cell adhesion, having a polymer shell and a gas core enclosed by the polymer shell, produced according to the method of any one of claims 1 to 6. 10.An ultrasound-mediated drug delivery system for use in a method for treating a disease associated with vascular inflammation, in particular inflammatory bowel disease, arteriosclerosis, arterial wall injury or cancer, wherein the ultrasound-mediated drug delivery system comprises vesicles functionalized with a peptide for integrin-mediated cell adhesion, having a polymer shell and a gas core enclosed by the polymer shell, produced according to the method of any one of claims 1 to 6, and an active ingredient.

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