Ultrasound-Sensitive Liposomes for Triggered Macromolecular Drug Delivery: Formulation and In Vitro Characterization

Abstract

Mistletoe lectin-1 (ML1) is a nature-derived macromolecular cytotoxin that potently induces apoptosis in target cells. Non-specific cytotoxicity to normal cells is one of the major risks in its clinical application, and we therefore propose to encapsulate ML1 in a nanocarrier that can specifically release its cargo intratumorally, thus improving the efficacy to toxicity ratio of the cytotoxin. We investigated the encapsulation of ML1 in ultrasound-sensitive liposomes (USL) and studied its release by high-intensity focused ultrasound (HAccessedIFU). USL were prepared by entrapment of perfluorocarbon nanodroplets in pegylated liposomes. The liposomes were prepared with different DPPC/cholesterol/DSPE-PEG2000 lipid molar ratios (60/20/20 for USL20; 60/30/10 for USL10; 65/30/5 for USL5) before combination with perfluorocarbon (PFC) nanoemulsions (composed of DPPC and perfluoropentane). When triggered with HIFU (peak negative pressure, 2–24 MPa; frequency, 1.3 MHz), PFC nanodroplets can undergo phase transition from liquid to gas thus rupturing the lipid bilayer of usl. Small unilamellar liposomes were obtained with appropriate polydispersity and stability. ML1 and the model protein horseradish peroxidase (HRP) were co-encapsulated with the PFC nanodroplets in USL, with 3% and 7% encapsulation efficiency for USL20 and USL10/USL5, respectively. Acoustic characterization experiments indicated that release is induced by cavitation. HIFU-triggered release of HRP from USL was investigated for optimization of liposomal composition and resulted in 80% triggered release for USL with USL10 (60/30/10) lipid composition. ML1 release from the final USL10 composition was also 80%. Given its high stability, suitable release, and ultrasound sensitivity, USL10 encapsulating ML1 was further used to study released ML1 bioactivity against murine CT26 colon carcinoma cells. Confocal live-cell imaging demonstrated its functional activity regarding the interaction with the target cells. We furthermore demonstrated the cytotoxicity of the released ML1 (I.E., After USL were treated with HIFU). The potent cytotoxicity (IC50 400 ng/ml; free ML1 IC50 345 ng/ml) was compared to non-triggered USL loaded with ML1. Our study shows that USL in combination with HIFU hold promise as trigger-sensitive nanomedicines for local delivery of macromolecular cytotoxins.

Recent Advancements in Stimuli Responsive Drug Delivery Platforms for Active and Passive Cancer Targeting

“…The presence of unsaturated acyl chains phospholipids and PEG-Lipopolymers improves the susceptibility of vesicle bilayers to low frequency ultrasound. The ultrasound-responsive liposomes were also prepared by different lipids in combination with perfluorocarbon DPPC/cholesterol/DSPE-PEG 2000 [153]. Some phosphatidylcholinebased lipids as well as other phospholipids used in ultrasound-triggered drug release from liposomes have been discussed here.…”

“…Frenkel and coworkers demonstrated that high-frequency intensity-focused ultrasound is an advanced non-invasive technique for targeted treatment by focusing a high energy focal point as well as high-frequency ultrasonic beams within the body [95]. Matos et al, 2019 [153], prepared ultrasound-responsive liposomes using various lipid ratios of DPPC/cholesterol/DSPE-PEG 2000 and studied its release by high-intensity focused ultrasound (HIFU). Ultrasound-responsive liposomes were fabricated by mixing nanodroplets of perfluorocarbon (PFC) with pegylated liposomes.…”

The Phospholipid Research Center: Current Research in Phospholipids and Their Use in Drug Delivery

“…Heat [ 54 ] induced a phase transition in the lysolipid-containing lipid membrane leading to bilayer destabilization and pore formation, and the ML-I could leave the cargo [ 55 ]. Secondly, ultrasound [ 56 ] also induced a disruption (cavitation) of the lipid bilayer, releasing the contents [ 57 ]. Since the formulation in ultrasound-sensitive liposomes (USL) showed the triggered release of over 90% compared to 10% release from temperature-sensitive liposomes (TSL), USL are therefore a promising approach, despite the complex purification procedure and the initial losses of the encapsulated cargo.…”

Stability of Engineered Micro or Nanobubbles for Biomedical Applications

“…For example, when applying sonothrombolysis, a technology that dissolves blood clots in blood vessels by causing cavitation using ultrasound, the use of MNB can lower the ultrasonic energy for cavitation. This reduces the burden on the patient when dissolving blood clots [ 17 ] and internal drugs can be released using cavitation [ 18 ]. Additionally, the combination of ultrasound (US) and bubble can increase drug permeability to BBB, with delivering antisense to BBB using US and bubble as an example [ 19 ].…”