Original contribution
Preparation, characterization and in vivo observation of phospholipid-based gas-filled microbubbles containing hirudin

https://doi.org/10.1016/j.ultrasmedbio.2005.05.007Get rights and content

Abstract

The objective of this work was to prepare echogenic phospholipid-based gas-filled microbubbles (PGM) and investigate their physical characteristics, echogenicity and loading ability of hirudin under various NaCl concentrations. PGM were prepared by a sonication-lyophilization method. Hirudin was used as a model drug to evaluate the drug encapsulation efficiency of the PGM. PGM loaded with hirudin were prepared by dissolving lyophilized powder with hirudin solution. The morphology, particle size and microbubble concentration of PGM were measured. The hirudin encapsulation efficiency as a function of NaCl concentration was determined. The mean particle size and microbubble concentration of PGM were unchanged by the presence of hirudin for at least 60 min after preparation. Hirudin encapsulation quantity was proportional to the hirudin concentration until saturation occurred at high concentration, and the encapsulation efficiency had an inverse relationship. Hirudin encapsulation efficiency was affected by NaCl concentration. When NaCl concentration was increased from 10 mg mL−1 to 20 mg mL−1 in PGM solution, hirudin encapsulation efficiency decreased from 35.8 to 26.7%, and microbubble concentration decreased from 2.7 × 108 to 1.7 × 108 microbubbles per mL. The PGM were shown easily to be visible in in vivo rabbit liver. There was no difference in echogenicity between the loaded and unloaded bubbles. PGM prepared by the sonication-lyophilization method exhibited satisfactory physical characteristics and loading ability and are suitable for use in imaging and ultrasound-triggered delivery. (E-mail: [email protected])

Introduction

Ultrasound (US) has been used in drug and gene delivery since the 1980s (Fechheimer et al 1987, Liang et al 2004). The gene delivery efficiency can be enhanced further if microbubbles are used in combination with US (Klibanov 1999, Lu et al 2003, Unger et al 2004). Peptide and protein drugs have strong physiological activity. However, most of these drugs cannot be used in their raw state, but have to be carried by suitable delivery systems, such as liposomes, micro- or nanoparticles, because of their instability and poor targeting ability in blood. Among these systems, the use of microbubbles as vehicles for peptide and protein has attracted considerable interest in recent years (Lindner and Kaul 2001).

Many different compounds, such as acrylates, palmitic acid, phospholipids, albumin and polymers, have been used as microbubble shells or surface-modifying agents. Among these, phospholipids are easily obtained chemically inert compounds that have no specific interactions leading to toxic reactions or side effects.

Phospholipid-based gas-filled microbubbles (PGM) are biocompatible and biodegradable echogenic agents of low toxicity. Many studies have highlighted their clinical application as contrast-enhancing agents. Moreover, because of their bioadhesive properties, these PGM also have potential application in targeted gene/drug delivery systems (Klibanov 1999).

The echogenic PGM delivery system enjoys many advantages in targeted drug and gene therapy. Like other particulate systems, PGM can be loaded with therapeutic agents. Peptide and protein or genes can be incorporated into the shell of PGM or attached to its surface. The PGM-carrying drugs can be tracked using US scanning. Active substances can be selectively released within designated organs by ultrasonic disruption of the echogenic PGM as they transit or accumulate in the intended organ. Furthermore, the US-triggered delivery system could help to protect surrounding healthy tissue from significant levels of toxic drugs. The normal, nonultrasonic disruption of the microbubbles would eventually occur throughout the body, but with low levels of toxic material. Contrast-enhanced US images taken at various time points may prove to be useful in monitoring therapeutic progress and, thus, may verify efficacy claims for the marketing approval of a drug.

Blood clots in the circulation, heart, brain and pulmonary emboli are a common cause of death. Thrombolysis is the treatment to break up abnormal blood clots that are restricting blood flow. Increasing evidence from in vitro, animal and initial patient studies (Cohen et al. 2003) indicates that application of US as an adjunct to thrombolytic therapy offers unique potential to improve effectiveness and decrease bleeding complications. When US and thrombolytics are combined, the improved lysis is thought to be caused by better clot penetration by the drug (Siddiqi et al. 1995). Microbubbles oscillating during US exposure may increase penetration of fibrinolytic agents into the thrombus and, thus, accelerate fibrinolysis. Acoustic cavitation play a major role in increasing the bioavailability of fibrinolytic agents at the surface of the thrombus (Rosenschein et al. 1994). Microbubbles could lower acoustic cavitation thresholds by as much as two thirds (Holland and Apfel 1990). This phenomenon opens new possibilities for applying microbubbles for therapeutic ends to induce cavitation at a lower US energy level.

Contrast in vascular imaging with US is also important because an acute thrombus is typically difficult to detect. Filling the vein with an echogenic microbubble medium allows for clear visualization of clots and even the tiny tortuous open channels that form as a clot begins to recanalize (Schutt et al. 2003).

The cargo space available in the membrane of the microbubble is usually relatively small, which means that only potent drugs can be considered. Thicker shells can be used, but at the expense of US scattering efficacy (Schutt et al. 2003). Hirudin is a peptide with a molecular weight of 7 kDa. As a direct thrombin inhibitor, it is potent but has a short half-life of 0.84 h in man (Markwardt et al. 1984). In clinical use, effective and safe antithrombotic therapy in interventional cardiology requires the right balance between prevention of clotting and creation of bleeding. Targeted delivery could minimize bleeding. One of the criteria for a successful PGM containing peptides or proteins is that it must have the stability to survive ambient conditions during storage long enough for it to be used clinically (Basude et al. 2000). The objectives of the present work were to prepare stabilized PGM, to determine its value as a peptide and protein delivery system and to demonstrate its in vivo imaging echogenicity.

Hirudin was used as a model drug to evaluate the drug encapsulation efficiency of PGM and their echogenicity. Echogenic PGM may provide a targeted delivery system with imaging properties offering the monitoring of clotting and bleeding. It has been reported that the loading of peptides with lipid membranes depends both on electrostatic attraction at the head group level and the apolar part of the membrane (Jones 1995). The electrostatic attraction of the membrane was affected by the ion concentration in solution. The effect of the ion concentration on the characteristics of gas-filled liposomes and their interactions with hirudin was determined using different NaCl concentrations in the preparation of PGM.

Section snippets

Preparation of PGM containing hirudin

The preparation of PGM was performed by the sonication-lyophilization method (Igartua et al. 1997). As shown in Fig. 1, the required components (8 mg hydrogenated phosphatidylcholine (HPC) (HPC >99%, Doosan Corporation Biotech BU, Kyonggi Do, Korea), 50 mg polyethylene glycol 1500 (Qingming Chemical Plant, Zhejiang Province, China) and 100 mg poloxamer 188 (Shenyang Chemical Plant, Liaoning Province, China)) were mixed in a test tube placed in a 65 °C water bath. Then, 5 mL normal butanol

Preparation of PGM

PGM lyophilized product was provided as lyophilized powder, which was an aggregation of hollow and porous particles (Fig. 2a). The lyophilized powder, after the addition of water, dissolved to leave a single-layer phospholipid membrane that enclosed ambient gases to create a dispersion of PGM (Fig. 2b). PGM solution showed a majority of spherical vesicles and no aggregation or fusion was observed.

Physical characteristics of PGM

PGM loaded with hirudin were prepared by dissolving lyophilized powder with hirudin solution. The

Discussion

In this work, PGM loaded with hirudin were prepared and their physical characteristics and hirudin encapsulation efficiencies were investigated. The influence of NaCl concentration on coating efficiency of PGM was studied. From the present study, incorporation of hirudin in PGM did not have significant effect on mean particle size and microbubble concentration. When hirudin dissolved in water solution, it was integrated on the microbubble shell by the embedding and electrostatic attraction of

References (25)

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