Targeted Microbubbles for Immunotherapy Applications: History
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Contributor:
J. Angel Navarro-Becerra
,
Mark A. Borden

Microbubbles are 1–10 μm diameter gas-filled acoustically-active particles, typically stabilized by a phospholipid monolayer shell. In cancer immunotherapy, the microbubble shell can be engineered through the bioconjugation of molecular ligands to facilitate the delivery and uptake of drugs, genes, or cells. This approach allows for precise control of immune stimulation and improves the delivery and pharmacokinetics of immunomodulatory agents at the target site. It has emerged as an attractive strategy for treating various cancer immunotherapy modalities, including monoclonal antibodies, immune checkpoint inhibitors, adoptive cell transfer, cytokine therapy, and vaccines.

  • targeted microbubbles
  • ultrasound imaging probes
  • ultrasound-targeted delivery

1. Ultrasound Targeted Microbubbles

Ultrasound Targeted Microbubble (tMB) formulations have been engineered as ultrasound-responsive carriers to promote and enhance the local delivery and uptake of a wide variety of drugs, genes, and cells for immunotherapy applications. tMBs play a crucial role in facilitating the targeted delivery of immune cells, cytokines, antigens, and antibodies, thereby promoting the activation and infiltration of immune cells at various levels, including single cells, tissues, organs, and physiological systems [1][2][3][4]. In the context of cancer treatment, for instance, the aim is to induce modulation and modification of the tumor microenvironment, leading to enhanced adaptive immune-cell activation and subsequent destruction of the primary tumor and its metastases.

2. Monoclonal Antibody Immunotherapy

Monoclonal antibody immunotherapy aims to induce cell death by targeting specific antigens, sequences, or epitopes expressed at the disease target site [5]. Therapeutic monoclonal antibodies (mAbs) can be administered unconjugated or conjugated with chemotherapeutic drugs and radioisotopes to specifically target tumors and minimize the toxicity effects of conventional chemotherapy [6]. Although mAb immunotherapy has potential therapeutic benefits, poor penetration and heterogenous distribution can impact the therapeutic effectiveness [5][6]. High concentrations of mAbs are often required, leading to adverse side effects due to the rapid metabolism and clearance rate through the kidneys [7]. The use of mAb-tMBs combined with ultrasound (US) (mAb-tMBs+US) has been proposed as an image-guided delivery method to enhance targeting, improve local penetration, and potentiate the therapeutic effect of mAbs in different cancer therapies (Table 1). Liao et al. [8] demonstrated improved glioma treatment by administrating EGFR-tMB + US, resulting in increased tumor vessel permeability and enhanced tumor-suppressing effects. Kang et al. [9] found that combining anti-DLL4-tMBs + US with DAPT for gastric tumor therapy was more effective than DAPT alone, showing synergistic antitumor proapoptotic effects. These effects were attributed to the regulation of apoptosis-related proteins Bcl-2 and BAX, as well as the tumor suppressor protein P53. Recently, Sun et al. [10] developed a tMB construct that delivers pyropheophorbide sensitizer and therapeutic trastuzumab mAbs for targeted combination of sonodynamic and antibody therapies in gastric cancer. This therapy enhanced antibody accumulation at the tumor site, increased tumor cell apoptosis and tumor growth inhibition by suppressing AKT phosphorylation.
Table 1. Targeted microbubbles used for cancer immunotherapy.
Immunotherapy
Modality		 Therapeutic
Molecule		 Conjugation Chemistry MicrobubbleComposition Ultrasound
Parameters		 Animal/Disease Model Reference
Monoclonal
antibody		 EGFR mAb Avidin-biotin Targestar™-SA
Shell: Phospholipid
Gas: C4F10
Diameter: 2.5 μm
Dose: 3.6 × 109 MBs/kg or 29.7 µL/kg MVD		 f = 400 kHz
PRF = 1 Hz
Power = 5 W
Time = 3–4 min		 Mouse/Glioma
tumor		 [8]
  DLL4 mAb Avidin-biotin Targestar™-SA
Shell: Phospholipids
Gas: C4F10
Diameter: 2 μm
Dose: 5.3 × 108 MBs/kg or 2.2 µL/kg MVD		 f = 1 MHz
DC= 50%
I = 2 W/cm2
Time = 1.5 min		 Mouse/Gastric
cancer		 [9]
  Trastuzumab mAb NHS Shell: DPSC, DSPE-PEG-2000-NHS, Cholesterol and pyropheophorbide
Gas: SF6
Diameter: 1.654 ± 1.07 μm
Dose: unknown		 f = 1 MHz
DC= 50%
I = 2 W/cm2
Time = 5 min		 Mouse/Gastric
cancer		 [10]
Immune checkpoint inhibitors PDL-1 mAb NHS Shell: DSPC,
DSPE-PEG-2000-NHS.
Gas: SF6
Diameter: 1.06 ± 0.31 μm
Dose: 6.9 × 1010 MBs/kg or 43.3 µL/kg MVD		 f = 1.1 MHz
DC = 5%
PRF = 100 Hz
Time = 0.5 min		 Mouse/Colon cancer [11]
  PDL-1 mAb and Cisplatin Avidin-biotin
Unbounded		 Shell: DPSC, DSPE-PEG-2000, -Biotin
Gas: C3F8
Diameter: 1.01 ± 0.14 μm
Dose: 1 × 108 MBs/kg or 0.06 µL/kg MVD		 f = 1 MHz
DC = 50%
I = 1W/cm2
PRF = 1 kHz
Time = 1.5 min		 Mouse/Cervical
cancer		 [12]
  PDL-1 mAb and miR-34a Avidin-biotin
Electrostatic		 Shell: DSPC, DSPE-PEG-2000, DSPE-PEG-2000-Biotin, PEI-600
Gas: C3F8
Diameter: 0.940 ± 0.080 μm
Dose: 4 × 109 MBs/kg or 1.7 µL/kg MVD		 f = 18 MHz
DC = 50%
I = 1 W/cm2
Time = 1.5 min		 Mouse/Cervical
cancer		 [13]
Vaccine CD11b mAb and
CGAMP		 Maleimide Shell: DSPC, DSPE-PEG-2000,
DSPE-PEG-5000-Maleimide
Gas: C4F10
Diameter: 2.6 μm
Dose: 1.4 × 109 MBs/kg or 13.1 µL/kg MVD		 f = 1 MHz
DC = 50%
I = 4 W/cm2
Time = 2 min		 Mouse/Breast cancer [14]
  HSP70-MAGEA1 Electrostatic Shell: Span 60 and Tween 80
Gas: SF6
Diameter: 6 μm
Dose: 1.3 × 109 MBs/kg or 144.7 µL/kg MVD		 MI = 0.75 Mouse/Melanoma tumor [15]
  Dendritic cell plasma membrane fragments Hydrophobic Shell: DPPC, DPPA, DSPE-PEG5000
Gas: C3F8
Diameter: 1.21  ±  1.0  μm
Dose: 5 × 108 MBs/kg or 0.5 µL/kg MVD		 f = 18 MHz
MI = 0.75
Time = 5 min		 Mouse/Breast cancer [16]
DSPC = 1,2-distearoyl-sn-glycero-3-phosphocholine; DPPA: 1,2-dipalmitoyl-sn-glycero-3-phosphate, DPPC: (1,2-dipalmitoyl-sn-glycero-3-phosphocholine); DSPE: 1,2-Distearoyl-sn-glycero-3-phosphorylethanolamine; PEI: Branched Polyethylenimine; DSPE-PEG-2000: (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxypolyethylene glycol)-2000]; DSPE-PEG-5000: (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxypolyethylene glycol)-5000]; C3F8 = perfluoropropane; SF6 = sulfur hexafluoride, C4F10 = perfluorobutane; MVD = Microbubble Volume Dose; f = frequency; DC = duty cycle; I = intensity; PRF = pulse repetition frequency; MI = mechanical index.

3. Immune Checkpoint Inhibitor Therapy

Immune checkpoint inhibitors (ICI) are variants of mAbs that activate T cells by blocking immune checkpoint receptors [5]. Approved ICIs for therapy include programmed cell death protein-1 (PD-1), programmed death ligand-1 (PD-L1), and cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) [5][17]. However, systemic ICI therapy is limited by severe side effects associated with dosage, low treatment response, and overactivation of the immune response [18]. To address these limitations, a controlled delivery strategy called PD-L1 mAb–tMBs + US has been proposed (Table 1). Kim et al. [11] reported that PD-L1 mAb–tMBs can improve therapeutic efficacy, increase the therapeutic index, reduce toxicity, and prevent immune responses and fatalities associated with systemic administration of PD-L1 mAb in colon cancer treatment. Ma et al. [12] and Liu et al. [13] both demonstrated that combining PD-L1 mAb–tMBs with chemotherapeutic drugs or loading PD-L1 mAb–tMBs with genes exhibited strong synergistic effects in inhibiting cervical tumor growth, improving survival rates, and reducing side effects compared to using either the drug/gene or PD-L1 mAb–tMBs alone. The combination treatment showed better immunological activity, as indicated by increased infiltration of CD8+ T cells and cytokine expression, ultimately resulting in an effective antitumor immune-killing effect.

4. Adoptive Cell Immunotherapy

Adoptive cell-mediated immunotherapy (ACT) involves the intravenous transfer of resident T cells or genetically modified T cells that specifically target tumor antigens and mediate anti-tumor functions. There are three types of ACT: tumor-infiltrating lymphocytes (TIL), T cell receptor (TCR) gene therapy, and chimeric antigen receptor-modified T cells (CAR-T) [5][19]. However, ACT is limited by the lack of in vivo persistence of transferred cells, toxicities associated with lymphodepletion, immune response, and cytokine release [19]. Preliminary studies utilizing MBs targeted with specific antibodies and retroviruses (CD3, CD8, CD45RA, CD62L, CD3/CD28, and CD-19CAR) have demonstrated that tMBs have the potential to stimulate [20][21], activate, transduce, and precisely sort specific phenotypes of CAR-T cells [20]. These simulations result in increased in vivo persistence, reduced toxicity, and improved antitumor response of adoptively transferred CAR-T cells compared to CAR-T cells obtained through conventional methods [21].

5. Cytokine Immunotherapy

Cytokine-mediated immunotherapy involves the systemic administration of cytokines to enhance the immune response [5]. Commonly used cytokines for immunotherapy in clinical and research settings include Interferon-alpha (IFN-α), Interleukins (ILs), and granulocyte-macrophage colony-stimulating factor (GM-CSF) [5][22][23]. However, this therapy has several limitations, including low efficacy, high levels of toxicity, and immune response activation [24]. IL-27-tMBs have shown promising results by enhancing cytokine bioactivity, inhibiting prostate tumor growth, and efficiently improving the recruitment of natural killer cells (NKT) and CD8+ cells to the tumor site compared to untargeted delivery [25]. Additionally, IL-16–tMBs [26] have been introduced for evaluating myocardial ischemia–reperfusion, detecting atherosclerosis, and detecting ovarian tumors, respectively.

6. Vaccine Immunotherapy

Vaccine-mediated immunotherapy involves administering specific antigens or protein fragments to stimulate an immune response [5]. Different types of vaccines are used, including peptide-based vaccines [27], DNA-based vaccines [28], and cell-based vaccines such as NK cells, dendritic cells (DC), and CAR-T cells [29]. However, the effectiveness of vaccine therapy is limited by tissue-specific antigens, low humoral responses, and heterogenous immune responses [5][30]. Gene- and protein-loaded MBs targeted with tumor-specific antigens offer promising image-guided vaccine immunotherapy for breast and melanoma tumors, as demonstrated by Li et al. [14] and Gao et al. [15]. This approach can increase delivery efficiency, prolong survival rates, activate systemic antitumor immunity, inhibit and delay tumor growth [14][15], reduce systemic toxicity, and inhibit cancer metastasis by bridging the innate and adaptive immune responses [14]. Recently, Jungio et al. [16] introduced the first cell-free vaccine in the form of tMBs with activated DC plasma membranes to enhance breast cancer tumor targeting, reduce tumor growth, and increase survival rates. Studies have also shown tumor growth inhibition and/or antigen-specific protection through DCs activated by mRNA-loaded MBs [31] or tMBs conjugated with NK cells to promote the controlled delivery at the target site [32].

This entry is adapted from the peer-reviewed paper 10.3390/pharmaceutics15061625

References

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