Shell modification of microbubbles

Abstract

The present invention relates, inter alia, to a method for producing a microbubble, comprising (i) providing a polymerizable monomer and a non-polymerizable compound; and (ii) polymerizing the monomer in an aqueous solvent in the presence of the non-polymerizable compound to form a polymeric shell encapsulating an inner lumen; wherein the polymerizable monomer is selected from the group consisting of alkyl cyanoacrylates, alkyl acrylates, lactide, glycolide, and combinations thereof; and the non-polymerizable compound is capable of integrating into the polymeric shell as the polymerization proceeds so as to form a plurality of non-polymerized nanocavities.

Description

[0001] Munich, 17 July 2025

[0002] Our ref.: RM 5253-02WO SOE / TWS

[0003] Applicant: Rheinisch-Westfalische Technische Hochschule Aachen, abgekiirzt RWTH

[0004] Aachen, Korperschaft des offentlichen Rechts, 52062 Aachen, DE official file number: Subsequent application

[0005] Rheinisch-Westfalische Technische Hochschule Aachen, abgekiirzt RWTH Aachen, Korperschaft des offentlichen Rechts, 52062 Aachen, DE

[0006] Shell modification of microbubbles

[0007] Technical field

[0008] The invention generally lies in the field of microbubbles (MB) and their use as ultrasound (US) contrast enhancer and US-mediated drug delivery system. More specifically, the present invention relates to a method for the production of a MB through combined use of a polymerizable monomer and a non-polymerizable compound, resulting in enhanced performance as US contrast enhancers and US-mediated drug delivery systems.

[0009] Background to the invention

[0010] MB are widely used as contrast agents for US imaging and US-enhanced drug delivery. Polymeric MB are highly suitable for these applications because of their particular acoustic responsiveness, high drug loading capability, and ease of surface functionalization. MB are gas-filled vesicles (bubbles) of 1 to 5 pm in size whose shell is stabilized by lipids, proteins or polymers. Contrast enhancement is caused by a compressible gas core that enables the bubble to reflect the applied US waves. Loaded with drugs, the MB can also act as a US- mediated drug delivery system that can be disrupted by US pulses in vivo to release the drug locally. While many studies have focused on using polymeric MB for diagnostic and therapeutic purposes, relatively little attention has thus far been paid to improving their inherent imaging and drug delivery features.

[0011] The primary problem of the present invention was therefore to improve drug loading capacity and / or in vitro and / or in vivo acoustic responses of MB, to provide a method for producing such improved MB and to provide accordingly improved MB produced by said method.

[0012] Description of the invention

[0013] In a first aspect of the invention, the problem is solved by a method for producing a microbubble (MB), comprising:

[0014] (i) providing a polymerizable monomer and a non-polymerizable compound; and

[0015] (ii) polymerizing the monomer in an aqueous solvent in the presence of the non- polymerizable compound to form a polymeric shell encapsulating an inner lumen; wherein: the polymerizable monomer is selected from the group consisting of alkyl cyanoacrylates, alkyl acrylates, lactid, glycolid, and combinations thereof; and the non-polymerizable compound is capable of integrating into the polymeric shell as the polymerization proceeds so as to form a plurality of non-polymerized nanocavities.

[0016] The invention is based on the inventors’ innovation that during the formation of the shell by polymerization, the non-polymerizable compound integrates into the forming polymeric shell as the polymerization proceeds, thereby forming a plurality of non-polymerized nanocavities in the shell. This allows to produce an MB having an advantageous property profile, including (i) a drug loading capacity that is at least 50% higher, (ii) a signal intensity in non-linear contrast (NLC) mode ultrasound imaging that is at least 50% higher, (iii) a signal intensity in single and pulse inversion modes that is at least 100% higher, and (iv) a destruction rate upon ultrasound exposure that is at least 5% higher, as compared to an MB produced by a corresponding standard method lacking the non-polymerizable compound.

[0017] Further aspects, embodiments and advantages of the invention become apparent from the following description and the experimental part together with the drawings and the claims. In the context of the present invention, the term “inner lumen” denotes a non-solid core, space or cavity inside the MB surrounded by the shell that is not solid, but contains a liquid or a gas. It is preferably large relative to the thickness of the shell. For instance, the diameter of the inner lumen at the narrowest point may be greater than the thickness of the shell at its thickest point.

[0018] An aqueous solvent means a solvent that primarily comprises water, e.g., more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, or even more than 95% water, or that consists of water. Further optional constituents include detergents, preferably non-ionic detergents, in particular Triton X-100, which may be included in an amount of 0.1 % to 6.0%, preferably 0.2% to 4.0%, more preferably 0.5% to 2.0%. Percentages are by weight, relative to the total weight of the aqueous solvent.

[0019] With respect to the non-polymerized compound, any compound can be used that is capable to integrate into the polymeric shell as the polymerization proceeds. On the basis of the inventors’ experiments, it is assumed that the non-polymerizable compound is compatible with the polymerizable monomer in this regard if they do not excessively deviate from each other in their physico-chemical property profile defined by miscibility, solubility, hydrophobicity and size. This means, the non-polymerizable compound and the polymerizable monomer should fulfil at least a few of the following properties: (i) be fully miscible with each other, (ii) have a similar solubility in the aqueous solvent, (iii) have a similar hydrophobicity, and (iv) have a similar size.

[0020] Preferably, the non-polymerizable compound and the polymerizable monomer have a hydrophobicity difference expressed in terms of A (delta) log P of not more than 3, preferably not more than 2.5, more preferably not more than 1 .5, most preferably not more than 1 ; and / or a molecular weight difference of not more than 50%. It is further preferred that the non-polymerizable compound and the polymerizable monomer are not or poorly soluble in the aqueous solvent (at the conditions during polymerization).

[0021] Moreover, the non-polymerizable compound should further be at least partially removable from the shell so that MB can be produced in which the nanocavities are not, or at least not completely, occupied by the non-polymerizable compound.

[0022] In a preferred embodiment of the invention, the non-polymerizable compound is a mimetic of the polymerizable monomer, but lacking the polymerizable monomer’s reactive group facilitating polymerization. In other words, the non-polymerizable compound differs from the polymerizable monomer only in the absence of the polymerizable group.

[0023] A preferred polymerizable monomer is an alkyl cyanoacrylate. The term "alkyl cyanoacrylate" refers to an alkyl ester of cyanoacrylic acid. A polymeric shell composed of alkyl cyanoacrylate accordingly comprises polyalkyl cyanoacrylate (also abbreviated herein as "PACA"). Polyalkyl cyanoacrylate refers to a polymer consisting of one or more alkyl cyanoacrylates that are essentially free of free acid and alcohol groups.

[0024] A preferred non-polymerizable compound is an alkyl cyanoacetate. As compared to the alkyl cyanoacrylate, the alkyl cyanoacetate lacks the reactive group required for polymerization.

[0025] In a preferred embodiment of the invention, the alkyl cyanoacrylate and / or the alkyl cyanoacetate have an alkyl group with 1 to 10 C atoms, preferably 2 to 7 C atoms, more preferably 3 to 5 C atoms, such as methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n- heptyl, n-octyl, n-nonyl or n-decyl, as well as their non-linear isomers, such as sec-, iso-, tert-, and neo-. It is further preferred that the length of the alkyl group of the alkyl cyanoacrylate differs by not more than 2 C atoms as compared to the alkyl group of the alkyl cyanoacetate. Most preferable, the alkyl groups of the alkyl cyanoacrylate and the alkyl cyanoacetate are identical.

[0026] A particular preferred polymerizable monomer is butyl cyanoacrylate (BCA). Using BCA as polymerizable monomer results in a MB variant with a rather hard shell made from poly(butyl cyanoacrylate) (PBCA). PBCA is a biodegradable polymer and is approved by the FDA as a surgical superglue for wound closure. The shell of PBCA MB preferably consists of relatively small polymer chains with an average molecular weight (MW) of 2 to 15 kDa, preferably 5 to 10 kDa, with preferably more than 90% of the chains below 20 kDa. The diameter of PBCA MB may range from 0.5 pm to 10.0 pm, preferable 1.0 to 5.0 pm, and the thickness of the shell can vary between 20 and 500 nm, preferably 50 to 300 nm. This relatively thick shell enables stable encapsulation of the gas and prevents its diffusion out of the MB. PBCA is also (chemically exactly) referred to as poly(n-butyl cyanoacrylate), and is disclosed, among others, in EP 3 223 864.

[0027] A particular preferred non-polymerizable compound is butyl cyanoacetate (BCC). BCC is a mimetic of BCA, but lacking the reactive group of BCA. It integrates during polymerization with high efficiency into the shell, thereby forming a plurality of nanocavities. Generally, the molar ratio of the polymerizable monomer to the non-polymerizable compound present in (ii) may range from 99:1 to 70:30, preferably 90:10 to 70:30, more preferably 80:20 to 70:30. On the basis of the mode of action, one of skill in the art will appreciate that the actual molar ratio depends primarily on the desired number of nanocavities (determining the porosity of the MB shell) as well as the combination of polymerizable monomer and non-polymerizable compound used, and can be determined by one skilled in the art using routine experiments.

[0028] Preferred methods of the invention further comprise the step of (iii) washing the microbubble to remove the non-polymerizable compound. The removal aims at making the nanocavities and / or the inner lumen substantially free of non-polymerizable compound using a washing agent. A preferred washing agent is an aqueous solvent that primarily comprises water, e.g., more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, or even more than 95% water, or that consists of water. Further optional constituents include detergents, preferably non-ionic detergents, in particular Triton X-100, which may be included in an amount of 0.0001 to 5.0%, preferably 0.001 to 0.50%, more preferably 0.005% to 0.10%, most preferably 0.01 % to 0.05%. Percentages are by weight, relative to the total weight of the aqueous solvent.

[0029] After washing, the MB may be stored in a storage solution. As a preferred storage solution an aqueous solvent can be used that primarily comprises water, e.g., more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, or even more than 95% water, or that consists of water. Further optional constituents include detergents, preferably nonionic detergents, in particular Triton X-100, which may be included in an amount of 0.0001 to 5.0%, preferably 0.001 to 0.50%, more preferably 0.005% to 0.10%, most preferably 0.01 % to 0.05%. Percentages are by weight, relative to the total weight of the aqueous solvent. In certain embodiments, the MB is stored directly in the washing agent.

[0030] Another aspect of the invention is a MB, preferably produced by the method disclosed herein. The MB has an inner lumen surrounded by a polymeric shell. The polymeric shell is formed from a polymer selected from the group consisting of (i) homopolymers of poly(alkyl cyanoacrylates), poly(alkyl acrylates), polylactide (PLA), polyglycolide (PGA), and (ii) copolymers comprising two or more monomers of the homopolymers such as poly(lactide-co-glycolides) (PLGA). Moreover, the MB has a higher porosity as compared to a standard MB. For instance, as compared to a standard MB the shell of the MB of the invention may be characterized by having a porosity that is at least 50% greater. The term “standard microbubble / MB” denotes a MB produced by a method that corresponds to the method used for producing the MB of the invention, but lacks polymerization in the presence of a non-polymerizable compound.

[0031] In particular embodiments, the porosity is at least 4.50 m2 / g, preferably at least 5.00 m2 / g, at least 5.50 m2 / g, at least 6.00 m2 / g, at least 6.5 m2 / g, or at least 7.00 m2 / g, as determined by the Brunauer, Emmett and Teller method. Further, the shell may have a thickness ranging from 200 to 600 nm, preferably 300 to 500 nm, as determined by STED (Stimulated Emission Depletion) microscopy; and / or a thickness ranging from 20 to 200 nm, preferably 50 to 150 nm, as determined by cryoSEM (Scanning electron cryomicroscopy). The absolute values may, however, depend on the actually used combination of polymerizable monomer and non-polymerizable compound, as well as their concentrations.

[0032] As mentioned above, the method of the invention allows the production of an MB having an advantageous property profile. In accordance with this, the MB of the invention is preferably characterized by having (i) a drug loading capacity that is at least 50% higher as compared to a standard microbubble, as determined using Coumarin 6 as drug model loaded into the MB post-synthesis; and / or (ii) a signal intensity in non-linear contrast (NLC) mode ultrasound imaging that is at least 50% higher as compared to a standard microbubble; and / or (iii) a signal intensity in single and pulse inversion modes that is at least 100% higher as compared to a standard microbubble; and / or (iv) a destruction rate upon ultrasound exposure that is at least 5% higher as compared to a standard microbubble.

[0033] The explanations and embodiments for the polymerizable monomer and the non- polymerizable compound given above in relation to the production method of the invention apply correspondingly with respect to the MB of the invention. Accordingly, in one embodiment, the polymeric shell is composed of polyalkyl cyanoacrylate (PACA). PACA refers to a polymer consisting of one or more alkyl cyanoacrylates that are essentially free of free acid and alcohol groups. Optionally, the shell may contain other components, in particular other polymers. Preferably the PACA has an alkyl group having 1 to 10 C atoms, preferably 2 to 7 C atoms, more preferably 3 to 5 C atoms, such as methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl or n-decyl, as well as their non-linear isomers, such as sec-, iso-, tert-, and neo-. A particularly preferred PACA in the context of the present invention is PBCA. In some embodiments, the MB comprises a non-polymerizable compound (as defined herein). This product may be regarded as an intermediate product in the production of an MB that is essentially free of non-polymerizable compound, following the method described herein.

[0034] The MB may further comprise a ligand that imparts a desired function to the MB. The ligand may be selected from the group consisting of targeting ligands, diagnostics, therapeutics, macromolecules and nanomedicine constructs. The MB may further comprise a linker between the matrix of the MB and the ligand.

[0035] A further aspect of the invention relates to an ultrasound imaging contrast agent comprising a plurality of MB as defined herein. For this purpose, the shell and / or the nanocavities enclose a gas core as inner lumen. The gas core preferably contains air, oxygen, nitrogen oxide and / or perfluorocarbons. In principle, it is also possible, and encompassed by the present invention, that the gas core is only formed from a liquid core as a result of excitation with ultrasound. 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.

[0036] An ultrasound-assisted drug delivery vehicle comprising a plurality of MB as defined herein concerns another aspect of the invention. For this purpose, the MB include a drug that can be embedded in the shell matrix, in particular in the nanocavities, the inner lumen and / or bound to the surface of the polymer shell. Ultrasonic treatment can be used to control the release of the drug from the drug delivery system. The intensity and wavelength of the ultrasound source, which is usually located outside the body, is selected so that the radiation passes through the tissue to the desired release site. US pulses are used to burst the MB to release the drug.

[0037] Another aspect of the invention relates to a composition comprising the US contrast enhancer or US-mediated drug delivery system of the invention and a dispersant in which the US contrast enhancer or US-mediated drug delivery system is present, or a lyophilizate thereof. Suitable dispersants include sodium chloride, PBS (Phosphate Buffered Saline), HBSS (Hanks' Balanced Salt Solution) and other physiological buffers and solutions.

[0038] The present invention will now be described further with reference to the following examples and the accompanying drawings. Brief description of the drawing

[0039] Fig. 1 : Morphological characteristics of butyl cyanoacetate-enhanced polymeric MB. (a) Concentration, (b) mean diameter and (c) diameter distribution profile of synthesized polymeric MB. (d) Representative STED and cryoSEM micrographs of MB. (e) Shell thickness values of MB measured by STED and cryoSEM. 100 / 0, 90 / 10, 80 / 20 and 70 / 30 refer to the specific BCA / BCC ratios used in the synthesis of each sample. Values represent mean ± standard deviation of three different batches of polymeric MB, measured in triplicates. (*) and (**) indicate groups that are significantly different with p < 0.05 and p

[0040] < 0.01 , respectively; (ns) indicates groups that are not significantly different with p > 0.05 (one-way ANOVA with post hoc Tukey HSD test).

[0041] Fig. 2: Butyl cyanoacetate-enhanced MB exhibit improved drug loading capabilities, (a) Number of coumarin 6 molecules per MB, and (b) percentage of model drug release by the different MB samples upon ultrasound irradiation, (c) Fluorescence intensity profile of different MB loaded with coumarin 6 as a drug model measured by flowcytometry. Values represent mean ± standard deviation of three different batches of polymeric MB, measured in triplicates. (**) and (**) indicate groups that are significantly different with p < 0.01 and p

[0042] < 0.005, respectively; (ns) indicates groups that are not significantly different with p > 0.05 (one-way ANOVA with post hoc Tukey HSD test).

[0043] Fig. 3: Butyl cyanoacetate-enhanced polymeric MB display improved acoustic properties (a) Representative NLC and B mode sonograms of polymeric MB at 4% power, (b) Quantified mean signal intensities of polymeric MB at 4% in NLC mode, (c) Power density spectra of polymeric MB in pulse inversion imaging mode. Destruction rate of MB after exposure to (d) 10% and (e) 15% power for 5 s; 100 / 0, 90 / 10, 80 / 20 and 70 / 30 refer to the specific BCA / BCC ratio used in the synthesis of each sample. Values represent mean ± standard deviation of three different batches of polymeric MB, measured in triplicates; (*) and (***) indicate groups significantly different with p < 0.05 and p < 0.005, respectively (one-way ANOVA with post hoc Tukey HSD test).

[0044] Fig. 4: Butyl cyanoacetate-enhanced MB display superior in vivo US imaging performance, (a) Representative US sonograms of mouse liver and kidney in B mode and NLC mode after injection of the different MB. (b) Representative NLC signal intensity over time curves of the different MB in the liver, (c) Average NLC signal intensity acquired in the liver of the mice at different time points after injection, (d) Representative NLC signal intensity graph acquired in the liver of the mice 5 min after injection and upon bursting with 100% US power, (e) Representative NLC signal intensity over time curves of the different MB in the kidneys, (f) Average NLC signal intensity acquired in the kidneys of the mice at different time points after injection, (g) Representative NLC signal intensity graph acquired in the kidneys of the mice 5 min after injection and upon bursting with 100% US power; measurements are done at 10% US power; 100 / 0, 90 / 10, 80 / 20 and 70 / 30 refer to the specific BCA / BCC ratio used in the synthesis of each sample. Each group contained four animals (n = 4). Values represent mean ± standard deviation. (*) and (**) indicate groups significantly different with p < 0.05 and p < 0.01 , respectively (one-way ANOVA with post hoc Tukey HSD test).

[0045] Fig. 5: Butyl cyanoacetate-enhanced MB are biocompatible and do not induce acute toxicity, (a) Red blood cells (RBC) count, (b) white blood cells (WBC) count, (c) platelet (PLT) count, and (d) hemoglobin (HGB) concentration of blood samples at different time points of the experiment, (e) Weight of the different organs, and (f) representative H&E- stained micrographs of mouse organs two days after MB administration. 100 / 0, 90 / 10, 80 / 20 and 70 / 30 refer to the specific BCA / BCC ratio used in the synthesis of each sample. Each group contained four animals (n = 4). Values represent mean ± standard deviation. (*) indicates groups that are significantly different with p < 0.05 (one-way ANOVA with post hoc Tukey HSD test).

[0046] Fig. 6: Morphological characteristics of porous shelled polymeric MB. (a) Concentration, (b) mean diameter, and (c) representative cryoSEM micrographs of MB. Numbers in parentheses indicate the molar ratio of butylcyanoacrylate monomer to non-polymerizable compound. Scale bar is 1 micrometer. Values represent mean ± standard deviation of three different batches of polymeric MB, measured in triplicate. (*), (**), (***) and (****) indicate groups that are significantly different with p < 0.05, p < 0.01 , p < 0.001 , and p < 0.0001 , respectively; (ns) indicates groups that are not significantly different with p > 0.05 (one-way ANOVA with post hoc Tukey HSD test).

[0047] Fig. 7: 1 H-NMR spectra of polymeric MB with peak assignment confirming the nonpolymerizable compound is washed away during purification steps.

[0048] Fig. 8 : Porous-shelled MB show up to a 10-fold increase in drug loading capacity, (a) Number of coumarin 6 molecules per MB, and (b) percentage of drug release by different MB samples upon ultrasound irradiation, (c) Representative STED micrographs of MB. The scale bar is 1 micrometer. Values represent mean ± standard deviation from three different batches of polymeric MB, measured in triplicates. (*), (**), (***) and (****) indicate groups that are significantly different with p < 0.05, p < 0.01 , p < 0.001 , and p < 0.0001 , respectively; (ns) indicates groups that are not significantly different with p > 0.05 (one-way ANOVA with post hoc Tukey HSD test).

[0049] Fig. 9 : Porous-shelled polymeric MB demonstrate up to 14-fold improved acoustic properties, (a) Quantified mean signal intensities of polymeric MB at 4% in NLC mode, (b) Destruction rate of MB after exposure to 10% power for 5 s; (c) Representative NLC and B mode sonograms of polymeric MB at 4% power. (*), (**), (***) and (****) indicate groups that are significantly different with p < 0.05, p < 0.01 , p < 0.001 , and p < 0.0001 , respectively; (ns) indicates groups not significantly different with p > 0.05 (one-way ANOVA with post hoc Tukey HSD test).

[0050] Examples

[0051] 1 . Synthesis of polymeric MB and morphological characterization

[0052] Polymeric MB were synthesized following an anionic polymerization protocol, where BCA and BCC were simultaneously added dropwise to a 1 % (w / v) Triton X-100 surfactant solution under high-speed stirring. Based on our previous experience with PBCA MB, the pH of the solution was set at 2.5, since the polymerization reaction does not occur at a lower pH (because of the low hydroxide concentration), and at a higher pH, the reaction occurs too fast, producing either polydisperse MB or polymer flakes. The total content of BCA and BCC added to the solution was kept constant (30 mM), while their relative molar ratios were varied (i.e. BCA / BCC ratios of 100 / 0, 90 / 10, 80 / 20, and 70 / 30). After the synthesis, the polymeric MB were washed and stored in a 0.02% (w / v) T riton X-100 solution to avoid coalescence and aggregation.

[0053] The presence of BCC affected the final concentration of synthesized MB (Figure 1 a), as increasing the BCC content from 0 to 30% reduced the concentration of MB by 59 ± 6%. We hypothesize that due to the inability of BCC molecules to polymerize, their presence may disrupt the polymerization of BCA and, consequently, the final concentration of synthesized MB. This agrees with the observation that it was not possible to synthesize polymeric MB with a BCC content higher than 30%. Notably, the synthesis quenching above 30% BCC was not caused by an insufficient amount of BCA in solution since, in the absence of BCC, we could decrease the BCA concentration by 30% and still produce polymeric MB. The addition of BCC, however, did not have statistically significant effects on MB diameter distribution and average (Figures 1 b and 1 c). Next, the plain and fluorescent dye (coumarin 6) loaded polymeric MB were imaged by scanning electron cryomicroscopy (cryoSEM) and stimulated emission depletion (STED) microscopy, respectively, which demonstrated that the addition of BCC did not affect the MB morphology and shell thickness (Figures 1d and 1 e). Taken together, these results highlight that adding BCC (up to 30%) into the synthesis solution allowed polymeric MB to retain their morphology, although the MB concentration decreased with BCC content.

[0054] 2. BCC-enhanced MB and standard PBCA MB exhibit similar shell composition

[0055] In a previous study, we identified that by changing the MB synthesis conditions, we could modify the polymer chains in the shell and affect the MB acoustic and drug loading characteristics. Hence, we evaluated the extent to which the growth of the PBCA chains was affected by the addition of BCC during the synthesis with gel permeation chromatography (GPC). All MB were made of polymer chains with weight average molar mass (Mw) values below 40 kDa, which is the size cutoff for kidney clearance.

[0056] Next, we used1H-nuclear magnetic resonance (NMR) spectroscopy to characterize the shell compositions. The 1 H-NMR spectra of all the samples demonstrated that the shell materials were made of PBCA polymer with no traces of BCC or BCA. Thus, while the presence of BCC during the synthesis affected the polymerization reaction, the chemical was removed during the washing steps. Hence, improving the characteristics of PBCA MB with a chemical that can be washed away would benefit the translatability of the resulting microformulation, as the final formulation would not include any additional chemical. Taken together, the GPC and1H-NMR results demonstrate that BCC affects the polymerization reaction, however, the chemical is washed away and not retained in the final MB formulation.

[0057] 3. BCC improves drug loading capabilities of polymeric MB

[0058] Because shell characteristics determine MB drug delivery performance, we studied the drug loading and release capacities of the different polymeric MB. Coumarin 6 was selected as a drug model because of its strong fluorescence emission and hydrophobicity (logP value of 4.9), which is similar to clinical pharmaceuticals, such as tucatinib and neratinib. Moreover, coumarin is used as a prescribed drug for thrombosis and embolism therapy, and is being explored as a treatment for lymphedema. Coumarin 6 was loaded inside the MB shell post-synthesis, following a previously established protocol that exploits the hydrophobic interactions between the drug and the polymeric chains.

[0059] As shown in Figure 2a, the number of coumarin 6 molecules loaded in each sample increased with BCC content. For example, while standard PBCA MB encapsulated 1 .31 x 106drug molecules per MB, the 70 / 30 polymeric MB contained 2.2-fold more drug molecules per MB. Drug release rates upon destructive pulse irradiation of all samples, however, were in the same range, between 50 and 60% (Figure 2b). Hence, the MB grown in the presence of BCC could carry higher amounts of drug molecules than standard PBCA MB did, while releasing their payload with the same efficiency.

[0060] To better visualize the loading capacity of the polymeric MB, the fluorescence intensity of coumarin-loaded MB of each sample was quantified by flow cytometry (Figure 2c). While the profiles of 100 / 0 and 90 / 10 samples are roughly similar, there is a clear shift toward higher intensities in 80 / 20 and 70 / 30 samples. This observation indicates that these two samples are loaded with larger amounts of coumarin 6 dye per MB.

[0061] Considering the similar morphology among the samples, the increase in drug loading capacity observed in the MB synthesized with BCC was likely caused by differences in the physiochemical characteristics of the shell. We hypothesized that BCC molecules were entrapped between the PBCA polymer chains during the synthesis, and then removed from the shell during the washing process due to their high-water solubility, providing more space between the chains to entrap the coumarin 6 molecules.

[0062] 4. Higher porosity of BCC-enhanced MB assessed by nitrogen physisorption experiments

[0063] Because the simulations indicated that BCC induced the formation of nanocavities in the shell of the MB that improved their drug loading performance, we evaluated the shell porosity through nitrogen physisorption cycles. We tested the 100 / 0 and 70 / 30 formulations, as these two samples are the ones that displayed more different behaviors. The adsorption-resorption experiments were performed with shell fragments of the same size to avoid the oscillation and the bursting of the MB during the experiments. The adsorption-resorption curves displayed type H4 hysteresis loop according to IUPAC classification, which combined with the pronounced uptake at low p / p° indicated the presence of microporosities. A surface area of porous material of 4.27 m2 / g and 9.74 m2 / g was calculated for the 100 / 0 and 70 / 30 samples, respectively, via the Brunauer, Emmett and Teller method, which is commonly used to assess porosity of materials. Hence, the 70 / 30 sample displayed 2.2-fold higher pore capacity than the conventional PBCA MB. These results were consistent with the 2.2-fold enhanced drug loading capacity of the 70 / 30 samples compared to their 100 / 0 counterparts. The differential pore volume profiles were determined with the Barrett, Joyner and Halenda equation, which also indicated a higher degree of porosity of the 70 / 30 sample in comparison to the 100 / 0. In addition, the pore volume profiles also confirmed relatively large number of pores that were smaller than 2 nm, which was in agreement with the simulation data. Taken together, the physisorption experiments clearly proved that the presence of BCC during the synthesis of the MB increased the porosity of the final formulation.

[0064] 5. BCC-enhanced MB show higher acoustic responses

[0065] The shell features strongly dictate the acoustic responses of polymeric MB. Hence, we characterized the acoustic performance of the synthesized MB in non-linear contrast mode (NLC; specific to MB non-linear responses) and brightness mode (B; associated with the general acoustic impedance of the sample) at 4% power in a preclinical setup with a central transducer frequency of 18 MHz, which is commonly used in in vivo imaging and corroborated that all the samples were highly responsive in both modes (Figure 3a).

[0066] The MB synthesized with 20 and 30% BCC showed 1 .9-fold higher signal intensities than conventional PBCA MB did in the MB-specific NLC mode (Figure 3b). The acoustic responses of MB strongly depend on shell characteristics, such as thickness, diameter, and shell mechanical properties. Because MB diameter and shell thickness were not statistically different in all samples, the presence of BCC during the synthesis likely reduced the shell stiffness, which led to easier MB oscillation upon exposure to US and, therefore, higher contrast signal. We hypothesize that the higher shell elasticity of the MB synthesized with BCC may have been caused by their more porous shell structure, as identified by the nitrogen physisorption experiments.

[0067] To better understand the acoustic performance of the different samples, we characterized the backscattering spectra of the MB in both single and double pulse (pulse inversion signal) modes when excited with a transducer with a center frequency of 17.5 MHz. In the pulse inversion mode (Figure 3c), the peak intensities attributed to the ultraharmonics (27 ~ 28 MHz) and second harmonics (33 ~ 35 MHz) increased with BCC content during the synthesis, and were consistent with the NLC sonograms previously obtained. Hence, the ultraharmonics intensities in pulse inversion mode were calculated to be 1 .96, 4.84, 9.00, and 8.10 pW / Hz for the 100 / 0, 90 / 10, 80 / 20, and 70 / 30 samples, respectively. Similar trends were observed in the second harmonic intensities, which were 4.17, 9.29, 18.70, and 19.55 pW / Hz for the 100 / 0, 90 / 10, 80 / 20, and 70 / 30 samples, respectively. The same trends were observed in the single pulse mode, where the 70 / 30 sample displayed 4.7-fold higher intensity in the fundamental peak and up to 4.6-fold higher intensity in the (sub-) harmonic peaks than the 100 / 0 formulation.

[0068] Next, we determined the percentage of MB destruction after relatively higher power US exposures. MB synthesized with larger amounts of BCC showed greater destruction after US exposures of 10 and 15% power (Figures 3d and 3e). It is worth noting that more elastic MB tend to display greater NLC-mode signal intensities but also higher stability to high- power US irradiations. However, in our case, MB synthesized with higher BCC / BCA ratios displayed greater NLC contrast but lower stability to high-power US exposures. Although the exact reason for this observation is not fully understood, we hypothesize that the higher shell porosity of the MB synthesized with high BCC / BCA ratios facilitated their destruction by highly energetic irradiations. This sensitivity to US is beneficial for drug delivery applications, as it facilitates the release of MB cargo without the need to apply high mechanical indexes.

[0069] In summary, the polymeric MB synthesized with larger amounts of BCC displayed stronger signal intensities in NLC mode (up to 1.9-fold) and in single and pulse inversion signal modes (up to 4.7- and 5.3-fold, respectively), and higher destruction rates upon (relatively) higher power US exposures than conventional PBCA MB did. Those characteristics make polymeric MB synthesized with BCC better candidates for US-based imaging and drug delivery applications.

[0070] 6. BCC-enhanced MB exhibit superior US imaging performance in vivo

[0071] Based on the excellent in vitro results of the MB formulations, we explored their performance as US contrast agents in vivo. To that end, 16 Balb / cAnNRj mice (4 per group) were intravenously injected with 50 pL of MB with a concentration of 2 x 109MB / mL. After administration, the circulation and distribution of the MB in the liver and kidneys were imaged with a preclinical US device for 5 min.

[0072] Representative B-mode and NLC-mode sonograms of mice kidneys and livers after injection are shown in Figure 4a, which clearly proved that the MB synthesized with BCC were brighter than standard PBCA MB. This was further evidenced by Figure 4b, a representative graph illustrating the NLC signal intensity of the different MB over time in the liver of the mice. A rapid increase in signal intensity within the first 5 min after injection was observed for all samples, indicating accumulation of the MB in the organ. This was followed by a progressive decrease in intensity, which was more pronounced in the 80 / 20 and 70 / 30 samples. This decrease in intensity was likely caused by the gradual bursting of the MB due to exposure to US waves. In addition, the reduction in signal could also be affected by shadowing effects, since the 80 / 20 and 70 / 30 MB were significantly brighter than the other two samples. Furthermore, a portion of MB are also known to burst in the lungs, as they get stuck in the microcapillaries.

[0073] Similarly, the mean NLC signal intensities of the samples at different time points after injection (0, 2 and 5 min) showed that the 70 / 30 MB displayed between 4.5- and 5.9-fold higher NLC signal than the standard 100 / 0 PBCA MB (Figure 4c). Next, we explored the destruction of the MB in vivo by high (100%) US power. Immediately afterthe US irradiation, a sharp dip in the signal intensity (up to 84%) was observed in the liver (Figure 4d), which confirmed the destruction of a considerable number of MB accumulated in the organ. However, in the following seconds, the signal intensity started to increase again, suggesting that intact MB from the bloodstream began to replenish the liver. Nevertheless, because the initial bursting destroyed a large number of MB, the final signal was weaker than the signal before the destructive US pulse.

[0074] Similar MB behavior was observed in the kidneys, where the signal rapidly increased after intravenous administration, followed by a progressive and slow reduction (Figure 4e). The NLC signal reduction was more prominent (and rapid) in the kidneys than in the liver, which could be caused by the higher accumulation of MB in the latter through internalization of MB in Kupffer cells, compensating for the destruction of the MB by the imaging US pulses. Notably, the mean NLC signal intensities of the 80 / 20 and 70 / 30 samples were 7.7- and 10.4-fold higher than that of the standard 100 / 0 MB (Figure 4f). The intensity differences between the samples produced with larger BCC content and the conventional PBCA MB were more pronounced in the kidneys than in the liver, since the greater accumulation of MB in the liver likely resulted in stronger shadowing effects by the brighter samples. In addition, the perfusion rate and blood flow are higher in the kidney in comparison to the liver which leads to a relatively higher signal. Upon irradiation with a destructive US pulse, the NLC signal abruptly decreased, suggesting that most MB in the vasculature of the kidneys were destroyed (Figure 4g). Shortly after, however, the signal intensity rapidly increased, indicating that intact MB from the bloodstream started to replenish the kidney vasculature. Taken together, these findings demonstrate that the 70 / 30 and 80 / 20 MB, which were synthesized with larger amounts of BCC, displayed superior imaging capabilities in vivo compared to the other tested MB.

[0075] 7. In vivo biocompatibility and safety of BCC-enhanced MB

[0076] As mentioned earlier, PBCA is approved by the FDA as a surgical glue, and the safety of PBCA MB has already been confirmed through multiple in vitro and in vivo studies. Furthermore, our1H-NMR data demonstrated that no BCC remained in the final MB formulation, hence, the shells of all our samples contained the same materials than the shells of PBCA MB did. Nevertheless, to ensure the safety of these newly developed MB, we evaluated their in vivo biocompatibility and safety through blood analysis, physical examination and histopathology.

[0077] Blood samples were collected from the animals 7 days before, right after, and 2 days after the imaging procedure, and blood count was performed, including the quantification of red blood cells (RBC), white blood cells (WBC), platelets (PLT), hemoglobin (HGB), hematocrit (HCT), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and mean corpuscular volume (MCV). Although some statistically significant variations were observed (Figures 5a to 5d), such as WBC in the group administered with 100 / 0 MB, all the values were within normal biological ranges.

[0078] Two days after the administration of the MB (and after performing the in vivo imaging), the animals were euthanized, and the relevant organs collected for further histopathological analysis. After euthanasia, the organs were visually examined, showing no signs of distress or damage. The weights of the organs were also consistent throughout the groups (Figure 5e) and within healthy biological ranges.

[0079] Lastly, histopathological analysis of the organs was carried out. The organs were fixed in 4% v / v formalin and embedded in paraffin after dehydration. Tissue slices were cut from the paraffin blocks and deparaffinized with xylene and ethanol. The resulting slices were then stained with Hematoxylin and Eosin (H&E) to identify potential acute toxicological reactions in the different host tissues. Figure 5f shows representative H&E micrographs of each sample which did not display any pathological features. Collectively, all these results confirm that the different MB did not cause any acute toxicity or severe adverse effects.

[0080] 8. Systematic evaluation of non-polymerizable additives for modulating structure and function of porous-shelled PBCA MB

[0081] To identify useful modulators, i.e. non-polymerizable compounds, for tuning the shell properties of PBCA MB, a panel of non-polymerizable compounds with varying side chains - and accordingly varying physicochemical characteristics - was evaluated. These compounds are listed in the table below along with theirsequence, name, molecular weight, and hydrophobicity (log P).

[0082]

[0083] This chemical diversity allowed us to systematically investigate how the respective compound influences porosity and functional properties of the resulting polymeric MB shells. The influence of different non-polymerizable compounds on the morphology of PBCA MBs was assessed through quantitative and imaging-based analysis. As shown in

[0084] Fig. 6, the MB concentration (Fig. 6a), mean diameter (Fig. 6b), and cryo-scanning electron microscopy (cryoSEM) micrographs (Fig. 6c) were compared for MB prepared with varying molar ratios of PBCA monomer to non-polymerizable compound. Each sample was analyzed in triplicate across three independent batches. The data reveal that modulator incorporation significantly alters MB yield and size, while the cryoSEM images demonstrate the emergence of porosity within the shell. Statistical significance was determined using one-way ANOVA followed by Tukey HSD post hoc testing.

[0085] To confirm that the non-polymerizable compounds do not remain entrapped within the MB shell after synthesis and purification, we performed1H-NMR spectroscopy on purified MB samples. Fig. 7 displays representative1H-NMR spectra with peak assignments. The absence of characteristic peaks corresponding to the modulator compounds after washing indicates their complete removal, validating that these small molecules function solely as transient modulators during shell formation without becoming covalently integrated into the polymer matrix.

[0086] Next, the functional impact of shell porosity on drug loading and release was evaluated. As illustrated in Fig. 8, porous-shelled MB loaded with the model hydrophobic compound coumarin 6 showed significantly increased encapsulation capacity, with up to a 10-fold enhancement compared to non-porous controls (Fig. 8a). Upon ultrasound exposure, these MBs also released a higher proportion of their cargo (Fig. 8b). Stimulated emission depletion (STED) microscopy confirmed the nanoscale distribution of the loaded dye within the porous shell (Fig. 8c).

[0087] To assess the acoustic behavior of porous-shelled MB, ultrasound signal intensities and stability were measured under insonation. Fig. 9a shows that MB incorporating porositymodulating non-polymerizable compounds yielded up to a 14-fold increase in nonlinear contrast (NLC) signal intensity. These MB also exhibited increased susceptibility to destruction at 10% ultrasound power (Fig. 9b), suggesting enhanced acoustic responsiveness. Representative NLC and B-mode images are shown in Fig. 9c to illustrate signal enhancement. These findings underscore the potential of porous MB for ultrasound imaging and triggered drug release applications.

Claims

Claims1 . A method for producing a microbubble, comprising:(i) providing a polymerizable monomer and a non-polymerizable compound; and(ii) polymerizing the monomer in an aqueous solvent in the presence of the non- polymerizable compound to form a polymeric shell encapsulating an inner lumen; wherein: the polymerizable monomer is selected from the group consisting of alkyl cyanoacrylates, alkyl acrylates, lactide, glycolide, and combinations thereof; and the non-polymerizable compound is capable of integrating into the polymeric shell as the polymerization proceeds so as to form a plurality of non-polymerized nanocavities.

2. The method of claim 1 , wherein the non-polymerizable compound and the polymerizable monomer have: a hydrophobicity difference expressed in terms of Alog P of not more than 3, preferably not more than 2.5, more preferably not more than 1 .5, most preferably not more than 1 ; and / or a molecular weight difference of not more than 50%.

3. The method of claim 1 or 2, wherein the non-polymerizable compound is a mimetic of the polymerizable monomer, but lacking the polymerizable monomer’s reactive group facilitating polymerization.

4. The method of any of claims 1 to 3, wherein: the polymerizable monomer is an alkyl cyanoacrylate, and / or the non-polymerizable compound is an alkyl cyanoacetate; preferably, the polymerizable monomer is butyl cyanoacrylate and / or the non- polymerizable compound is butyl cyanoacetate.

5. The method of any of claims 1 to 4, wherein the molar ratio of the polymerizable monomer to the non-polymerizable compound present in (ii) ranges from 99:1 to 70:30, preferably 90:10 to 70:30, more preferably 80:0 to 70:30.

6. The method of any of claims 1 to 5, further comprising:(iii) washing the microbubble to remove the non-polymerizable compound.

7. A microbubble comprising an inner lumen and a polymeric shell, preferably produced by the method of any of claims 1 to 6, wherein: the polymeric shell is formed from a polymer selected from the group consisting of: homopolymers of poly(alkyl cyanoacrylates), poly(alkyl acrylates), polylactide (PLA), polyglycolide (PGA), and copolymers comprising two or more monomers of the homopolymers such as poly(lactide-co-glycolides) (PLGA); and preferably: the polymeric shell has a porosity of at least 4.50 m2 / g, preferably at least 5.00 m2 / g, at least 5.50 m2 / g, at least 6.00 m2 / g, at least 6.50 m2 / g, or at least 7.00 m2 / g.

8. The microbubble of claim 7, wherein the shell is characterized by having: a porosity that is at least 50% larger greater as compared to a corresponding standard microbubble; and / or a thickness ranging from 200 to 600 nm, preferably 300 to 500 nm, as determined by STED; and / or a thickness ranging from 20 to 200 nm, preferably 50 to 150 nm, as determined by cryoSEM, wherein the standard microbubble is produced by a method corresponding to the method of any of claims 1 to 6 but lacking polymerization in the presence of a non- polymerizable compound.

9. The microbubble of any of claims 7 to 8, wherein the microbubble is characterized by having:a drug loading capacity that is at least 50% higher as compared to a corresponding standard microbubble, as determined using Coumarin 6 as drug model loaded into the microbubble post-synthesis; and / or a signal intensity in non-linear contrast (NLC) mode ultrasound imaging that is at least 50% higher as compared to a corresponding standard microbubble; and / or a signal intensity in single and pulse inversion modes that is at least 100% higher as compared to a corresponding standard microbubble; and / or a destruction rate upon ultrasound exposure that is at least 5% higher as compared to a corresponding standard microbubble, wherein the standard microbubble is as defined in claim 8.

10. The microbubble of any of claims 7 to 9, wherein the polymer is poly(butyl cyanoacrylate).11 . Ultrasound imaging contrast agent comprising a plurality of microbubbles as defined in any of claims 7 to 10.

12. Ultrasound-assisted drug delivery vehicle comprising a plurality of microbubbles as defined in any of claims 7 to 10.

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