**11.2. Enclosing drugs within the MB shell**

Instead of co-injecting drugs and MBs, the bubbles might be modified to contain drugs or DNA [97, 106-110]. It is virtually possible to load the drugs within the interior of the MBs, which is very convenient since the drug is completely protected and is not released before applying the ultrasound [23]. However, since the MBs are relatively small,the space for drug loading is quite limited. This approach was applied for the first time in 1998 already by the team of Unger, who used lipid-shelled MB with an addition of paclitaxel containing oil phase. Nonetheless, Unger reported the methodology seems to be unsuitable for clinical setting for three reasons; firstly, as the monolayer of the lipid-shelled MB is very thin, only small amount of drug can be loaded; secondly, this strategy is limited only to hydrophobic drugs; last but not least, due to the very high mechanical indexes of ultrasound waves necessary for drug release, which resulted only in 30% of cell death, the in vivo use of such ultrasound setting appears unfeasible [110].

Recently, Kooiman et al. used oil-filled polymer microcapsule model for ultrasound-mediated delivery of lipophilic drugs; microcapsules with a shell of fluorinated end-capped poly(L-lactic acid) contained hexadecane oil as a drug-carrier reservoir. At higher diagnostic pressures, the microcapsules cracked and released the drug [111]. Despite offering a great potential in clinical use, there are limitations as this approach can be applied only for lipophilic drugs. In another paper, Tinkov *et al.* suggested the application of doxorubicin (DOX)-loaded phospholipid MBs for targeted tumour therapy. DOX as a potent chemotherapy was loaded into the DPPC/DPPG/ PEG2000-DPPE MBs and was tested in a pancreas carcinoma model of the rat. Although an agglomeration and capillary adhesion with embolisation/occlusion of the pulmonary arteries/ capillaries after intravenous infusion of MBs with any new formulation might appear, in this case no rat died or showed any signs of pulmonary vascular occlusion [112]. Therefore the strategy seems suitable for more extensive preclinical therapeutic studies.

#### **11.3. Nucleic acids and MBs**

Concerning gene delivery, [113] incorporated plasmid DNA inside the gas core of polymer MBs, so that the plasmid was even protected from the host nucleases; at the same time high plasmid load per bubble was achieved [113]. In a recent study, Sun et al. compared the transfection efficiency of commercial Definity MBs to modified cationic MBs with longer lipid chain; as a result, cationic bubbles were binding significantly more DNA, so that multi-fold enhancement was reached in gene delivery [114]. The results were consistent with the findings of Panje and Nomikou, *et al.* and the notion that gene delivery might be augmented with a higher local DNA concentration [115, 116]. In another study, Florinas *et al.* designed a nonviral siRNA gene carrier using a combination of an arginine-grafted bioreducible polymer (ABP), MBs and ultrasound, for targeting vascular endothelial growth factor (VEGF) in human ovarian cell line; as a result, MBs in combination with lipoplexes showed significantly higher loading capacity compared to naked siRNA; furthermore, only siRNA complexes with ABP and MBs showed significant knock down in human ovarian cell line compared to naked siRNA when incubated for a short time after sonication treatment [116]. The potential of MBs as gene carriers is still on its rise, offering a wide range of options by functionalization of the bubbles.

The drugs that have sufficient affinity for the lipid monolayer might be, in principle, incorpo‐ rated directly in lipid-shelled MBs [106]. However, due to the fact that the lipid monolayer is rather thin, the amount of drug available for incorporation is very low. Furthermore, the thin shell might not be able to prevent the leakage of drugs from the bubble during their circulation. Therefore other strategies were developed, such as preparation of MBs with a thick lipid shell containing triglyceride oil phase and drug dissolved in it [117]. Nevertheless, this approach is restricted only to hydrophobic fat-soluble drugs, e.g. paclitaxel. A thick polymer shell represents another variety for drug incorporation suitable for water-soluble materials such as DNA and proteins.

Electrostatic interactions of the lipids and plasmid DNA (possessing an overall negative charge) are made possible by the positively charged groups of some synthetic lipids within the shell [109]. DNA can be loaded on the charged MB shell by simply mixing a plasmid with the lipid bubbles before use. After insonation, the surface-coupled DNA remains intact and at the same time is protected against enzymatic degradation [118, 119]. Moreover, the transfection efficiency is increased by the adherence of DNA to the MBs in comparison with co-adminis‐ tration of MBs and non-adsorbed DNA. At the same time, unbound plasmids should not be able to penetrate into non-targeted cells and tissues and should be degraded, resulting in high specificity with a low transfection in non-insonated regions [19]. Nevertheless, in comparison with the payload of DNA inside the gas core of the bubbles, the loading capacity is restricted to their surface area, which means that high numbers of MBs must be injected. The develop‐ ment of multi-layered assemblies by sequential adsorption of DNA on the positive MB template offers another alternative; because DNA can be directly incorporated as an anionic layer within these assemblies, layer-by-layer methods allow precise control over the loading (or dose) of DNA by controlling film thickness or the number of layers deposited during the fabrication. Besides that, these methods are entirely aqueous and, unlike conventional methods for the encapsulation of DNA in thin polymer films, do not require the use of organic solvents which residua could be presented in these materials post-assembly. [120].

In conclusion, an important advantage of ultrasound-mediated MB-based transfection is the ability to attain DNA delivery and expression only in the insonated areas and not in nontargeted regions. Even if the transfection efficacy is not very high but the encoded protein is produced in the target tissue in an amount sufficient to achieve functional biological response, therapeutic intervention should meet its success [121]. Considering the improvement of transfection efficacy via implementation of viral particles using positively charged assemblies, a significant transfection was observed [122]. However, one should be always careful since undesired immune responses after systemic administration might appear.

### **11.4. Incorporation of drug loaded LNPs with MBs**

transfection efficiency of commercial Definity MBs to modified cationic MBs with longer lipid chain; as a result, cationic bubbles were binding significantly more DNA, so that multi-fold enhancement was reached in gene delivery [114]. The results were consistent with the findings of Panje and Nomikou, *et al.* and the notion that gene delivery might be augmented with a higher local DNA concentration [115, 116]. In another study, Florinas *et al.* designed a nonviral siRNA gene carrier using a combination of an arginine-grafted bioreducible polymer (ABP), MBs and ultrasound, for targeting vascular endothelial growth factor (VEGF) in human ovarian cell line; as a result, MBs in combination with lipoplexes showed significantly higher loading capacity compared to naked siRNA; furthermore, only siRNA complexes with ABP and MBs showed significant knock down in human ovarian cell line compared to naked siRNA when incubated for a short time after sonication treatment [116]. The potential of MBs as gene carriers is still on its rise, offering a wide range of options by functionalization of the bubbles.

The drugs that have sufficient affinity for the lipid monolayer might be, in principle, incorpo‐ rated directly in lipid-shelled MBs [106]. However, due to the fact that the lipid monolayer is rather thin, the amount of drug available for incorporation is very low. Furthermore, the thin shell might not be able to prevent the leakage of drugs from the bubble during their circulation. Therefore other strategies were developed, such as preparation of MBs with a thick lipid shell containing triglyceride oil phase and drug dissolved in it [117]. Nevertheless, this approach is restricted only to hydrophobic fat-soluble drugs, e.g. paclitaxel. A thick polymer shell represents another variety for drug incorporation suitable for water-soluble materials such as

Electrostatic interactions of the lipids and plasmid DNA (possessing an overall negative charge) are made possible by the positively charged groups of some synthetic lipids within the shell [109]. DNA can be loaded on the charged MB shell by simply mixing a plasmid with the lipid bubbles before use. After insonation, the surface-coupled DNA remains intact and at the same time is protected against enzymatic degradation [118, 119]. Moreover, the transfection efficiency is increased by the adherence of DNA to the MBs in comparison with co-adminis‐ tration of MBs and non-adsorbed DNA. At the same time, unbound plasmids should not be able to penetrate into non-targeted cells and tissues and should be degraded, resulting in high specificity with a low transfection in non-insonated regions [19]. Nevertheless, in comparison with the payload of DNA inside the gas core of the bubbles, the loading capacity is restricted to their surface area, which means that high numbers of MBs must be injected. The develop‐ ment of multi-layered assemblies by sequential adsorption of DNA on the positive MB template offers another alternative; because DNA can be directly incorporated as an anionic layer within these assemblies, layer-by-layer methods allow precise control over the loading (or dose) of DNA by controlling film thickness or the number of layers deposited during the fabrication. Besides that, these methods are entirely aqueous and, unlike conventional methods for the encapsulation of DNA in thin polymer films, do not require the use of organic solvents

which residua could be presented in these materials post-assembly. [120].

In conclusion, an important advantage of ultrasound-mediated MB-based transfection is the ability to attain DNA delivery and expression only in the insonated areas and not in nontargeted regions. Even if the transfection efficacy is not very high but the encoded protein is

DNA and proteins.

98 Advances in Bioengineering

An intensive research has been devoted to the development of nanocarrier-MB hybrids, wherein drug-containing LNPs are physically attached to the surface of MB [21]. These hybrids therefore possess a high drug loading capacity of the nanocarriers and the systemic targeting capability and vascular permeabilizing effect of bubbles. Lum et al. were the first to attach loaded LNPs to MBs via avidin-biotin interaction; they reported on biotinylated lipid MBs to which avidinylated polystyrene beads were attached [117]. The biotinylated MBs were prepared by inclusion of DSPE-PEG-biotin in the lipid shell. Since up to 105 LNPs were bound to each of the bubbles, the drug loading capacity is reasonably enhanced [106]. LNPs seem highly advantageous as (1) they have been long proved as versatile drug delivery systems; (2) a plethora of knowledge is available on LNPs for drug delivery; (3) some LNPs are perfectly and already used in clinical practice. They might also enclose both amphiphilic and hydro‐ phobic drugs in their lipid bilayer, as well as hydrophilic drugs in an aqueous core. Never‐ theless, owing to the fact that biotin-avidin linkage might develop an undesired immune response, this approach is not suitable for clinical use. Therefore Geers et al. prepared doxor‐ ubicin (DOX)-LNPs loaded MBs containing pDNA or siRNA that in combination with ultrasound boosted both DOX cytotoxicity and pDNA and siRNA delivery to the cells in vitro [108, 119, 123, 124]. In their last work, Geers et al. developed a self-assembly of functionalized phospholipids, DOX-loaded LNPs and perfluorobutan gas [125]. Compared to the other methods discussed above to load MBs with drugs, this strategy seems the most advantageous from the loading capacity point of view; though it was estimated that approximately 600 to 1300 LNPs might be bound per singe MB, the amount of loaded DOX is still rather limited. On the other hand, Klibanov et al. prepared liposome-MB pendants incorporating calcein, a hydrophilic self-quenching fluorescent dye, or thrombin, a crucial enzyme of the thrombosis cascade, and they achieved a significant payload volume – up to several cubic micrometres of internal liposome aqueous core space per bubble-LNP [126].

A different approach was introduced by Fan et al. who developed a novel DOX-loaded MB containing super-paramagnetic iron oxide (SPIO) nanoparticles for dual modality imaging and improved image-guided drug delivery [127]. The authors showed that the delivery of SPIO to brain tumours was improved using magnetic targeting in combination with focused ultra‐ sound. Unfortunately, the improvement of DOX delivery using magnetic targeting was not the object of assessment. Hernot et al. prepared nanobody-targeted MBs using metabolic and site-specific biotinylation of nanobodies [128]. The researchers developed a system of carmelidderived single-domain antibody-fragments, i.e. molecular tracers, coupled to targeted MBs. Although they demonstrated the functionality of these modified MBs both in vitro and vivo, since the biotinylated nanobodies were linked to the surface of biotinylated MBs via strepta‐ vidin linker, these are not suitable for clinical application due to the undesired immune response.

#### **11.5. Infusion of MBs**

Besides imaging applications, MBs showed to be a suitable tool for therapeutic use, e.g. targeted drug delivery, gene therapy, thrombolysis, and vascular permeability enhancement [129]. Certain US-contrast-imaging or therapeutic strategies, such as destruction-reperfusion or MB-enhanced drug delivery, require a continuous injection of the MBs over several minutes. Results of previous *in vitro*, animal, and human studies have shown that continuous infusion of a US contrast agent can markedly prolong vascular Doppler US enhancement. [130-132]. Most quantitative imaging strategies rely on the ability to administer a consistent dose of contrast agent. The spatial distribution of MBs inside a syringe varies over time owing to the buoyancy of the MBs. These changes are rapid and result in significant inhomogeneity in the concentration and size distribution of the MBs during continuous infusion. The population of MBs that is pumped from a horizontal syringe outlet differs from the initial population as the MBs float to the top of the syringe [133]. This effect is often overlooked and underestimated by experimenters. We believe that this is the main reason for contradictory results published by several authors that compare bolus versus continuous infusion methods of MB application [131]. A superior signal contrast of liver metastasis has been achieved by a continuous application of MBs to patients. US contrast agents for hepatic imaging are traditionally injected as a bolus.

For i.v. applications, precise concentration and size distribution of MBs is required. Continuous application of MBs might present a problem, owing to their strong tendency to float and accumulate directly below the surface. The concentration and size distribution of specific MBs injected into a patient's blood vessel can be quite different from the ones in the syringe.Re‐ cently, we have described the device called Infucon for continuous infusion of MBs which prevents flotation of MBs and their accumulation below the surface. The device has been tested on New Zealand White rabbits using both commercial Sono-Vue diagnostic MBs and PEGy‐ lated DPPC MBs prepared in our laboratory [48, 134].

The Infucon device consists of several parts: a programmable syringe pump (e.g. LAMBDA VIT-FIT Brno, Czech Republic) with a syringe filled with SF6, an Infucon stirred vessel positioned on magnetic stirrer (e.g. Heidolph MR 3000 D, Germany) and fixed in the position by a holder, and the cannula for application of MBs into the rabbit via ear vein. The schemes of the device together with its photographs are presented in Figure. 14. The role of the syringe pump is to force the gas (e.g. SF6) into the Infucon stirred vessel and to inject the MBs into bloodstream via cannula. The same gas which is used for filling MBs is used in the syringe to ensure stability of the MBs during the infusion. The main function of the turbine-like stirrer (which contains two teflon-coated ferrits) is to maintain a uniform dispersion of the MBs in the solution and to prevent their accumulation to the surface owing to their flotation. The stirrer is hung up on the collar of the carrying-off capillary outlet. The direction of rotation is selected to maintain down-stream movement of the fluid and to strip MBs from the surface. The device itself is designed to be hermetically insulated using an O-ring seal. The cell is made of inert materials (Teflon®, polypropylene, glass, and Viton®). All parts of the Infucon are compatible with chemical or heat sterilization. The filter installed in the Infucon head prevents possible air-born particles from entering the cell. The vial (conical bottom) is filled up with a solution of MBs (up to 1.5 mL) via an inlet capillary. The flow rate (usually in the range of 100 - 200 μL/ min) and the time of pump action are programmable parameters. The silicon piston of the syringe is properly lubricated to ensure very low friction and smooth movement preventing sudden jumps of the piston resulting in pulsation. A turbine-like stirrer fits the conical bottom of the vial, therefore very low dead volume of residual MB solution is obtained. The turbinelike stirrer freely hangs on the fringe of the Teflon outlet capillary. The capillary is tightly embedded into the Teflon body in order to be prevented from bending. There is also an inlet aperture for incoming gas in this Teflon body. The gas is delivered from the syringe through the inlet capillary linked tightly to the head part of the Infucon (Figure 15, 16). The stirred Infucon cell represents solution of the problem of the continuous infusion of MBs. This device is independent on mechanical stirring of the infusion syringe. Unlike the system based on the rotation of the entire syringe, Infucon cell provides a genuinely homogeneous MB dispersion. The MBs are prevented from flotation by stirring the suspension in a separate cell. The infusion is driven by inert gas filled in a syringe placed in the infusion pump. The inert gas used both for filling the MBs and driving the MB infusion is supposed to have stabilizing effect on the MBs during the process of infusion.

(A) Assembled device, (B) Head of the Infucon. (Adapted from [144])

OC – outlet capillary for the infusion of microbubles (connection to the cannula)

IC – inlet capillary for inert gas from a syringe inserted into the syringe pump

O – O-ring

**11.5. Infusion of MBs**

100 Advances in Bioengineering

as a bolus.

Besides imaging applications, MBs showed to be a suitable tool for therapeutic use, e.g. targeted drug delivery, gene therapy, thrombolysis, and vascular permeability enhancement [129]. Certain US-contrast-imaging or therapeutic strategies, such as destruction-reperfusion or MB-enhanced drug delivery, require a continuous injection of the MBs over several minutes. Results of previous *in vitro*, animal, and human studies have shown that continuous infusion of a US contrast agent can markedly prolong vascular Doppler US enhancement. [130-132]. Most quantitative imaging strategies rely on the ability to administer a consistent dose of contrast agent. The spatial distribution of MBs inside a syringe varies over time owing to the buoyancy of the MBs. These changes are rapid and result in significant inhomogeneity in the concentration and size distribution of the MBs during continuous infusion. The population of MBs that is pumped from a horizontal syringe outlet differs from the initial population as the MBs float to the top of the syringe [133]. This effect is often overlooked and underestimated by experimenters. We believe that this is the main reason for contradictory results published by several authors that compare bolus versus continuous infusion methods of MB application [131]. A superior signal contrast of liver metastasis has been achieved by a continuous application of MBs to patients. US contrast agents for hepatic imaging are traditionally injected

For i.v. applications, precise concentration and size distribution of MBs is required. Continuous application of MBs might present a problem, owing to their strong tendency to float and accumulate directly below the surface. The concentration and size distribution of specific MBs injected into a patient's blood vessel can be quite different from the ones in the syringe.Re‐ cently, we have described the device called Infucon for continuous infusion of MBs which prevents flotation of MBs and their accumulation below the surface. The device has been tested on New Zealand White rabbits using both commercial Sono-Vue diagnostic MBs and PEGy‐

The Infucon device consists of several parts: a programmable syringe pump (e.g. LAMBDA VIT-FIT Brno, Czech Republic) with a syringe filled with SF6, an Infucon stirred vessel positioned on magnetic stirrer (e.g. Heidolph MR 3000 D, Germany) and fixed in the position by a holder, and the cannula for application of MBs into the rabbit via ear vein. The schemes of the device together with its photographs are presented in Figure. 14. The role of the syringe pump is to force the gas (e.g. SF6) into the Infucon stirred vessel and to inject the MBs into bloodstream via cannula. The same gas which is used for filling MBs is used in the syringe to ensure stability of the MBs during the infusion. The main function of the turbine-like stirrer (which contains two teflon-coated ferrits) is to maintain a uniform dispersion of the MBs in the solution and to prevent their accumulation to the surface owing to their flotation. The stirrer is hung up on the collar of the carrying-off capillary outlet. The direction of rotation is selected to maintain down-stream movement of the fluid and to strip MBs from the surface. The device itself is designed to be hermetically insulated using an O-ring seal. The cell is made of inert materials (Teflon®, polypropylene, glass, and Viton®). All parts of the Infucon are compatible with chemical or heat sterilization. The filter installed in the Infucon head prevents possible air-born particles from entering the cell. The vial (conical bottom) is filled up with a solution

lated DPPC MBs prepared in our laboratory [48, 134].

MS – turbine-like magnetic stirrer with blades

C – fringe of the outlet capillary (OC)

**Figure 14.** Schematic drawing of the stirred continuous injector Infucon and the photograph of the Infucon device.

I – inlet for gas

Blue line: gass pushing homogenaous dispersion of MBs Red line: homogenaous dispesion of MBs driving from Infucon cell into rabbit ear vein via canula

**Figure 15.** Schematic and real view of the device used for i.v. infusion of MBs into the rabbit.

