2.4.1. Targeting

Localized BBB opening can be achieved using physical stimuli (such as FUS or magnetic field), while functionalized agents with a ligand can target overexpressed receptors associated with the treated pathology.

Fan et al. used magnetic targeting as a physical targeting option: particles can be magnetized and become physically sensitive to external magnetic fields. This approach was validated in vivo using the SPIO-DOX-microbubble complex described earlier (schematically represented in Figure 4A). It increased SPIO deposition in the rat brain by 2.8 fold [109]. As well, Wu et al. made lipid-shelled bubbles conjugated with cationic polyethylenimine-coated superparamagnetic iron oxide particles (PSPIO) to open the BBB on mice. The SPIO, here again, was used for magnetic targeting and enhanced the BBB opening by 2.8-fold compared with unconjugated bubbles [125].

Molecular targeting of the circulating microbubbles would allow a BBB opening in the desired area without affecting healthy tissues. Typical ligands used for molecular targeting are antibodies or peptides. Table 4 summarizes the different molecular targeting and drug/gene-complexed bubble options for BBB opening and therapeutic purposes found in the recent literature.

Table 4. Molecular targeting and drug/gene-complexed bubble for BBB opening and therapeutic purpose.

TGFβ1: transforming growth factor; GDNFp: a glial cell line-derived neurotrophic factor plasmid DNA; BDNFp: brain-derived neurotrophic factor plasmid DNA; NGR: Asn-Gly-Arg peptide; shBirc5: short hairpin RNA-Birc5 gene; VEGFR2: vascular endothelial growth factor receptor 2; pLUC: plasmid DNA encoding luciferase gene; pHSV-TK/GCV: plasmid DNA encoding herpes simplex virus type 1 thymidine kinase/ganciclovir gene; SPIO-DOX: superparamagnetic iron oxide-doxorubicin complex; pDC315/Nrf2: plasmid DNA encoding DC315-nuclear factor E2-related factor 2; pPrestin: plasmid DNA encoding Prestin protein; PSPIO-GDNFp: superparamagnetic iron oxide coated with cationic polyethylenimine conjugated with plasmid DNA encoding glial cell line-derived neurotrophic factor; NA: Not applicable).

Chen et al. developed des-octanoyl ghrelin-conjugated microbubbles loaded with TGFβ1 inhibitor to disrupt BBB on glioma-bearing mice. Des-octanoyl ghrelin is a ligand that can bind with BBB. Authors have observed higher BBB disruption with the des-octanoyl ghrelin-conjugated microbubbles than the unconjugated ones (negative contrast intensity of superparamagnetic iron oxide nanoparticles on the T2-weighted MRI images was 0.81-fold higher for the conjugated microbubbles) [88].

Specific biomarkers of the blood-tumor barrier (BTB) could be used to target microbubbles toward these tumors. Indeed, gliomas and brain metastases are tumors known to compromise the integrity of the BBB, resulting in a vasculature known as the BTB, which is highly heterogeneous and characterized by numerous distinct features, including non-uniform permeability and active efflux of molecules [153]. Vascular endothelial growth factor receptor 2 (VEGFR2) is one of the selected targets as it is a specific endothelial molecular marker of angiogenesis, which is exceptionally high in tumor growth and, thus, overexpressed in BTB [154]. Moreover, the inhibition of VEGFR2 with antibodies results in prolonged survival in cancer patients [154]. Functionalized bubbles/droplets with VEGFR2-targeted ligand were formulated for BTB targeting. Chang et al. have used anti-VEGFR2 antibody-conjugated cationic microbubbles to target VEGFR2 in the rat BTB. The microbubble targeting efficiency was evaluated in vitro on C6 glioma cells and was 99.4 ± 0.3%, while it was 6.4 ± 1.2% for unconjugated microbubbles [104]. Most of the targets studied, such as VEGFR2, are directed to endothelial cells. However, it is also possible to target receptors directly expressed on the malignant cells, i.e., folate receptors. Hence, Fan et al. used this receptor as a target for BBB opening and gene delivery in the C6 glioma rat model. They have demonstrated the targeting ability on C6 glioma cells of folate-conjugated DNA-loaded cationic microbubbles in vitro: the folate increased the targeting ability of the complex by 7.6 fold [107]. Besides, Zhao et al. used Asn-Gly-Arg (NGR), a peptide motif that can be used to target CD13 receptors. CD13 is overexpressed in glioma cells and neovascular endothelial cells. In vitro, NGR-linked-shBirc5-loaded liposome complex linked to lipid-shelled microbubble demonstrated a better shBirc5 gene transfection on C6 glioma cells for the targeted microbubble compared to the untargeted ones (36.25% of transfection efficiency vs. 21.26%), thereby demonstrating the targeting profits [110].

Molecular targeting was always associated with drug/gene loaded into the bubble in the browsed literature. Indeed, molecular targeting may improve drug delivery: Fan et al. have observed better gene transfection efficiency in vivo for their folate-conjugated microbubbles than those without conjugation (luciferase gene expression 4.7 fold higher after 24 h) [107].

2.4.2. Drug and Gene Delivery

Drug delivery through the BBB with FUS can be reached by (i) co-injection of microbubbles and free drugs or drug carriers or (ii) encapsulating or covalently linking the therapeutics to the agent shell [32,129]. Loading drugs into microbubbles' shells enables better spatial control to deliver the drug to the treated site. The drug's encapsulation would also decrease the side effects induced by the circulation of a high drug dose in the vasculature [30,32].

Non-viral gene delivery by plasmid DNA complexation with bubbles was studied in numerous studies [89,104,105,107,110,111,112]. Thereby, glial cell line-derived neurotrophic factor (GDNF) [89,111,125], brain-derived neurotrophic factor (BDNF) [89] and nuclear factor E2-related factor 2 (Nrf2) [112] have been studied for Parkinson’s disease treatment. Gene therapy with neurotrophic factors is a promising approach to improving current Parkinson’s disease therapy. It has been found to reduce progressive neuronal loss and play a crucial role in the development, survival, and maintenance of the central and peripheral nervous system [89]. Nrf2 is a nuclear factor that activates the antioxidant response element pathway and protects the brain by regulating redox status. Nrf2 might be useful in Parkinson’s disease therapy as reactive oxygen species play an important role in disease development [112]. Thus, these factors have been encoded in plasmid DNA and complexed with microbubble for BBB disruption and drug delivery on Parkinson’s disease rodent model [89,111,112,125]. Neurotrophic factor delivery provided a neuroprotective effect by showing evidence of improvement of behavioral deficits [89,111,125], while Nrf2 gene transfection enabled the reduction of reactive oxygen species levels [112].

Interestingly, Wu et al. have used pPrestin-microbubble to disrupt the BBB, modify, and activate neurons within mice brain for spatiotemporal neuromodulation [105]. Prestin is a transmembrane protein that exists in the mammalian auditory system and functions as an electromechanical transducer. The cellular transfection rate with pPrestin-microbubble was 1.5-fold higher than with commercial transfection agents (LT-1) [105].

Chang et al. used anti-tumor suicide gene therapy for glioblastoma therapy on Sprague-Dawley rats using cationic VEGFR2-targeted microbubbles, complexed with luciferase gene and herpes simplex virus type 1 thymidine kinase/ganciclovir (gene suicide system) encoding plasmid DNA (pLUC and pHSV-TK/GCV). Anti-tumor suicide gene therapy involves tumor-targeted transfection of a suicide gene that encodes an enzyme for converting non-toxic prodrugs into toxic products to kill tumor cells. Both VEGFR2-targeting and pHSV-TK contributed to improving the anti-tumor efficiency: 25 days after tumor implantation, the tumor volume was 9.7 ± 5.2 mm³ for the pHSV-TK/GCV-loaded VEGFR2-targeted microbubbles-treated group, 40.1 ± 4.3 mm³ for the untargeted pHSV-TK/GCV-loaded microbubbles-treated group, and approximately 68 ± 8 mm³ for the untreated group [104].

Another technique of gene delivery therapy is the RNA interference technique, which consists of protein expression inhibition. The RNA interference technique includes small interfering RNA (siRNA) and short hairpin RNA (shRNA). Zhao et al. used this technique on an orthotopic C6 glioma rat model with NGR-conjugated shBirc5-loaded liposome attached to microbubbles. Birc5 is a protein in the family of apoptosis inhibitors. The Birc5 gene is only expressed in malignant tumors and not in normal tissue. Thus, a plasmid containing shRNA for the Birc5 gene could enter the cell, decrease Birc5 gene transcription in a targeted manner, promote tumor cell apoptosis, and reduce angiogenesis without affecting normal cells. The triple function agent (tumor cell targeting, delivering gene, and BBB opening) in conjunction with FUS exhibited a significant therapeutic effect, higher than the control group: median survival times were 38 and 21 days, respectively [110].

Besides, Chen et al. have proposed another cancer treatment possibility combined with molecular targeting: des-octanoyl ghrelin-conjugated microbubbles were loaded with TGFβ1 inhibitor (LY364947). Transforming growth factor TGFβ plays an essential role in the functional regulation of tumor interstitium; it also controls the permeability of the BBB and reduces the permeability of the brain’s endothelial cell. Therefore, inhibition of TGFβ in cancer cells is expected to improve the therapeutic effects of chemotherapy. Thus, the deposition of doxorubicin in mice brain tissues was higher for the mice treated with TGFβ1 inhibitor-loaded conjugated bubbles (45 ± 5 µg/g of tissue) than for the group treated with unloaded conjugated bubbles (35 ± 5 µg/g of tissue). The median survival time was also increased (44 days for loaded microbubbles, 38 days for unloaded microbubbles) [88].

As mentioned earlier, anti-cancer drug-loaded bubbles have also been explored for cancer treatment (Figure 6) [109]. Interestingly, Fan et al. [106] proposed an alternative to boron neutron capture therapy consisting of radiotherapy based on boron agent delivery to the brain [151]. For a more efficient tumor-targeted boron delivery, the authors fabricated boron-containing nanoparticles-loaded microbubbles for the treatment of the glioma-bearing mice model. The complex successfully disrupted the BTB and delivered boron into the brain tumor (76.6 ± 3.6% of boron uptake in tumor 4 min after drug delivery) [106].

Several studies came up with other innovative drug delivery. Briefly, Zhao et al. used phosphatidylserine nanoparticles-microbubbles complexes to monitor inflammatory reaction [113]. The complex successfully and safely opened the BBB and activated the microglia/macrophage in the rat brain. In addition, Liu et al. used sulfur nanoparticles-quercetin complex embedded in the microbubble shell for Alzheimer’s treatment [86]. Those microbubbles successfully opened the BBB on mice and allowed for a rapid accumulation of nanoparticles-quercetin complex in the brain, leading to improved Alzheimer’s disease outcome (significant increase of success on Morris water maze experiment after treatment). Finally, Ha et al. proposed a drug delivery system by binding microbubble with ultrasound-sensitizing dye-incorporating nanoparticles. They successfully delivered those nanoparticles to the U87MG (human glioblastoma cell line) located in the mouse brain (fluorescence intensity 1.5 times higher than the control group) [90].

Among the multifunctional bubbles for drug delivery strategies found in the recent literature, most of them have to be destroyed to release the active principle embedded in their shell. However, bubble destruction results in a significant risk of brain damage (hemorrhage), which is not acceptable. Although safe BBB disruption with no evidence of acute or chronic inflammation was pointed out after bubble destruction by some studies [89,105], DNA delivery has been reported without destroying the DNA-loaded bubble [104]. Indeed, DNA loaded in bubbles might penetrate cells through endocytosis when the bubble undergoes stable cavitation [104].