Preparation and characterization of dextran nanobubbles for oxygen delivery

https://doi.org/10.1016/j.ijpharm.2009.07.010Get rights and content

Abstract

Dextran nanobubbles were prepared with a dextran shell and a perfluoropentan core in which oxygen was stored. To increase the stability polyvinylpirrolidone was also added to the formulation as stabilizing agent. Rhodamine B was used as fluorescent marker to obtain fluorescent nanobubbles. The nanobubble formulations showed sizes of about 500 nm, a negative surface charge and a good capacity of loading oxygen, no hemolytic activity or toxic effect on cell lines. The fluorescent labelled nanobubbles could be internalized in Vero cells. Oxygen-filled nanobubbles were able to release oxygen in different hypoxic solutions at different time after their preparation in in vitro experiments. The oxygen release kinetics could be enhanced after nanobubble insonation with ultrasound at 2.5 MHz. The oxygen-filled nanobubble formulations might be proposed for therapeutic applications in various diseases.

Introduction

Oxygen is one of the key elements in cell metabolism, and its concentration in tissues plays an important role in regulating biochemical reactions (Wagner, 2008, Soleymanlou et al., 2005).

Reduced oxygen supply from the arterial blood to cells substantially increases the risk of inflammation, infection and scarring, reduces the efficacy of medical treatments and may ultimately lead to tissue necrosis. Many medical conditions, such as diabetes, burns, bedsores and wounds are related to insufficient supply of oxygen to the tissues; oxygen deficiency, together with acidosis, is also the main hallmark of cancerous solid tumors and a major factor limiting the effectiveness of radiotherapy (Asimov et al., 2007). These are therefore possible fields of application of oxygen-filled nanobubbles besides other oxygenation approches (Al-Waili et al., 2005, Overgaard, 1994). Another potential field for nanobubbles application is that of anaerobic infections. Anaerobic bacteria grow where oxygen is completely, or almost completely, lacking and they can cause infections when a normal barrier, such as the skin, gums, or intestinal wall, is damaged due to surgery, injury, or disease. The targeted release of oxygen by means of delivery system may be a useful adjuvant to antibiotic therapy of anaerobic infections.

The delivery of oxygen has been examined in various studies (Van Liew and Burkard, 1996, Burkard and Van Liew, 1994) Hemoglobin-loaded particles (Hb-particles) encapsulated within a biodegradable polymer have been prepared for use as oxygen carriers (Zhao et al., 2007). The Hb-particles exhibited oxygen-carrying capacity similar to that of native hemoglobin and showed prolonged circulation time after in vivo administration in mice. Dextrose albumin microbubbles for oxygen and nitrogen delivery have been formulated (Porter et al., 1998) for use as contrast agents. Oxygen-enriched lipid-coated perfluorocarbon microbubbles have been prepared for oxygen delivery; these oxygen-enriched microbubbles were tested in a model of anemic rats (Unger et al., 2004, Unger et al., 2002) and maintained the rat's survival at very low hematocrit levels.

Oxygen-filled chitosan microbubbles showed to act as an efficient oxygen delivery system (Cavalli et al., 2009). The in vitro oxygen release from the chitosan microbubbles in hypoxic media was previously evaluated before and after ultrasound (US) sonication, and the results supported the hypothesis that US enhances gas delivery. Preliminary investigations on the biological effect of such oxygen release on the cellular activity were also undertaken, and an effect on the expression of Hypoxia Inducible Factor-1α (HIF-1) from hypoxic human choriocarcinoma cells after oxygen-filled microbubble delivery was demonstrated (Bisazza et al., 2008).

The aim of the present study was to develop a new bubble formulation in the nanometer size range for use as an innovative oxygen-delivery system.

New formulations were developed, consisting of nanobubbles with a dextran coating and bubble stabilization was improved, both by adding a poorly soluble gas (perfluoropentan) to the core and (Lundgren et al., 2006) by adding polyvinylpyrrolidone (PVP) to the shell. The resulting nanobubbles were characterized by their sizes, chemical properties, oxygen release efficiency, biocompatibility and cellular uptake, in view of their possible applications in treating chronic wounds and anaerobic infections.

Section snippets

Materials

Ethanol 96° was from Carlo Erba (Milan, I). Epikuron® 200 (soya phosphatidylcholine 95%) was a kind gift from Degussa (Hamburg, D). Palmitic acid, perfluoropentan, dextran sulphate sodium salt (Mw = 100,000), polyvinylpyrrolidone (PVP) (Mw = 24,000) were from Fluka (Buchs, CH). Rhodamine B and diethylamino-ethyl-dextran hydrocloride (DEAE) (Mw = 500,000) were from Sigma Aldrich (St. Louis, USA). Ultra-pure water was obtained using a system 1-800 Milli-Q (Millipore, F). Dulbecco's modified Eagle's

Results

The average diameter, polydispersity index, zeta potential values of oxygen-filled nanobubbles, plus the pH values of their aqueous suspensions, are reported in Table 2.

The sizes of the three nanobubble formulations were below 1 μm, with a rather uniform size distribution. The presence of the fluorescent marker did not affect the diameter of nanobubbles while the presence of PVP formed smaller nanobubbles with a lower polydispersity index. PVP acts as stabilizer agent on the nanobubble surface

Discussion

In this study oxygen-filled nanobubbles were prepared using perfluoropentan as core and dextran sulphate, a polysaccharide polymer, as shell. The biocompatibility of dextran-based formulations have been extensively studied (Bos et al., 2005, De Groot et al., 2001). Recently dextran-based hydrogels have been studied as matrices in tissue engineering; no sign of inflammation was detected in in vivo experiments (Möller et al., 2007).

The two types of nanobubble formulations as well as the

Acknowledgements

The authors would like to thank PRIN 2006 Italian Ministry of University and Ricerca Sanitaria Finalizzata 2006 and 2008 Projects of Regione Piemonte for their support.

References (25)

Cited by (107)

  • Antimicrobial oxygen-loaded nanobubbles as promising tools to promote wound healing in hypoxic human keratinocytes

    2022, Toxicology Reports
    Citation Excerpt :

    Both OLNB and OLND delivery systems exploit the Laplace’s law for spherical surfaces, therefore, the smaller bubble’s radius, the higher differential pressure of the gas, and the faster diffusion of the gas [33]. Oxygen release from OLNBs or OLNDs either occurs spontaneously, through passive diffusion, or it can be induced upon ultrasound administration, destabilizing its shell and allowing instant release of the gas from the inner core [34]. Additionally, OLNB and OLND peculiar architecture allows for further functionalization of the carrier with additional molecules (drugs, dyes, antibodies, etc.) [30,32,35,36].

View all citing articles on Scopus
View full text