Evaluation of the potential of doxorubicin loaded microbubbles as a theranostic modality using a murine tumor model

In this study, a novel phospholipid-based microbubble formulation containing doxorubicin and perfluoropropane gas (DLMB) was developed. The DLMBs were prepared by mechanical agitation of a phospholipid dispersion in the presence of perfluoropropane (PFP) gas. An anionic phospholipid, distearoyl phosphatidylglycerol (DSPG) was selected to load doxorubicin in the microbubbles by means of electrostatic interaction. The particle size, zeta potential, echogenicity and stability of the DLMBs were measured. Drug loading was ?92%. The potential of the DLMBs for use as a theranostic modality was evaluated in tumor bearing mice. Gas chromatography analysis of PFP showed significant enhancement of PFP retention when doxorubicin was used at concentrations of 10-82% equivalent to DSPG. The inhibitory effects on the proliferation of B16BL6 melanoma murine cells in vitro were enhanced using a combination of ultrasound (US) irradiation and DLMBs. Moreover, in vivo DLMBs in combination with (US) irradiation significantly inhibited the growth of B16BL6 melanoma tumor in mice. Additionally, US echo imaging showed high contrast enhancement of the DLMBs in the tumor vasculature. These results suggest that DLMBs could serve as US triggered carriers of doxorubicin as well as tumor imaging agents in cancer therapy

Ultrasound induced cancer immunotherapy.

Recently, the use of ultrasound (US) has been shown to have potential in cancer immunotherapy. High intensity focused US destruction of tumors may lead to immunity forming in situ in the body by immune cells being exposed to the tumor debris and immune stimulatory substances that are present in the tumor remains. Another way of achieving anti-cancer immune responses is by using US in combination with microbubbles and nanobubbles to deliver genes and antigens into cells. US leads to bubble destruction and the forces released to direct delivery of the substances into the cytoplasm of the cells thus circumventing the natural barriers. In this way tumor antigens and antigen-encoding genes can be delivered to immune cells and immune response stimulating genes can be delivered to cancer cells thus enhancing immune responses. Combination of bubbles with cell-targeting ligands and US provides an even more sophisticated delivery system whereby the therapy is not only site specific but also cell specific. In this review we describe how US has been used to achieve immunity and discuss the potential and possible obstacles in future development

PEG and lipids in solid dispersions and liquid crystals - structure and phase behaviour

In this thesis, two different kinds of nano-structured systems have been studied; solid dispersion systems of polyethylene glycol (PEG) and liquid crystalline systems containing the polar lipids monoolein (MO) and phytantriol (PT). Solid dispersions of PEG were studied with addition of different substances, lipids or parabens. The dispersions have been examined in the solid state, using differential scanning calorimetry and X-ray methods with the focus of the behaviour of the PEG carrier material. It was found that incorporation of MO increased with decreasing PEG molecular weight and that MO addition also affected the lamellae thickness of PEG. Different lipids affected the structure of PEG differently. To find which properties of the lipid that governed the behaviour of the solid dispersions twelve different lipids in PEG 4000 were examined. All lipids increased the fraction of the PEG that was in its folded conformation where small and hydrophilic lipids increased the folding the most. The unfolding of the folded form to unfolded was slower with lipids present in the PEG. Large and hydrophobic lipids like triolein and tristearin stabilized the folded form most effectively. The relation between solubility in a liquid PEG (PEG 400) and the structure of PEG 4000 solid dispersions was examined using a homologous series of parabens (methyl- to butyl- paraben). A high solubility in liquid PEG correlated to a high solubility in the solid dispersions and a larger increase of the lamellae thickness. The thickening of the lamellae was shown to be due to a swelling of the amorphous domains of the structure.The studies of the liquid crystalline phases of MO and PT revealed large similarities. The two-component lipid-water systems are almost identical with the difference that the PT phases swell much less. In three component solvent-lipid-water systems (solvents: propylene glycol (PG), polyethylene glycol 400 (PEG 400) and 2-methyl-2,4-pentanediol (MPD)) the same differences could be seen between PT and MO where PT phases swelled less than MO. One other major difference was the formation of L3 phase. With MO L3 phase was found in all three solvent systems whereas it with PT was only found in the system with MPD, the most hydrophobic solvent. The differences between the PT and MO systems were explained by the fact that PT is more hydrophobic and forms less flexible bilayers

PEG and lipids in solid dispersions and liquid crystals - structure and phase behaviour

In this thesis, two different kinds of nano-structured systems have been studied; solid dispersion systems of polyethylene glycol (PEG) and liquid crystalline systems containing the polar lipids monoolein (MO) and phytantriol (PT). Solid dispersions of PEG were studied with addition of different substances, lipids or parabens. The dispersions have been examined in the solid state, using differential scanning calorimetry and X-ray methods with the focus of the behaviour of the PEG carrier material. It was found that incorporation of MO increased with decreasing PEG molecular weight and that MO addition also affected the lamellae thickness of PEG. Different lipids affected the structure of PEG differently. To find which properties of the lipid that governed the behaviour of the solid dispersions twelve different lipids in PEG 4000 were examined. All lipids increased the fraction of the PEG that was in its folded conformation where small and hydrophilic lipids increased the folding the most. The unfolding of the folded form to unfolded was slower with lipids present in the PEG. Large and hydrophobic lipids like triolein and tristearin stabilized the folded form most effectively. The relation between solubility in a liquid PEG (PEG 400) and the structure of PEG 4000 solid dispersions was examined using a homologous series of parabens (methyl- to butyl- paraben). A high solubility in liquid PEG correlated to a high solubility in the solid dispersions and a larger increase of the lamellae thickness. The thickening of the lamellae was shown to be due to a swelling of the amorphous domains of the structure.The studies of the liquid crystalline phases of MO and PT revealed large similarities. The two-component lipid-water systems are almost identical with the difference that the PT phases swell much less. In three component solvent-lipid-water systems (solvents: propylene glycol (PG), polyethylene glycol 400 (PEG 400) and 2-methyl-2,4-pentanediol (MPD)) the same differences could be seen between PT and MO where PT phases swelled less than MO. One other major difference was the formation of L3 phase. With MO L3 phase was found in all three solvent systems whereas it with PT was only found in the system with MPD, the most hydrophobic solvent. The differences between the PT and MO systems were explained by the fact that PT is more hydrophobic and forms less flexible bilayers

Understanding polymer-lipid solid dispersions-The properties of incorporated lipids govern the crystallisation behaviour of PEG

A deeper insight into the crystallisation process of semi-crystalline polymers during formation of solid dispersions is crucial to improve control of product qualities in drug formulation. In this study we used PEG 4000 with 12 different lipids as a model system to study the effect that incorporated components may have on the crystallisation of the polymer. The lipids were melted with PEG 4000 and the crystallisation of the polymer studied with differential scanning calorimetry (DSC) and small angle X-ray diffraction (SAXD). PEG 4000 can crystallise into lamellar structures with either folded or fully extended polymer chains. All lipids increased the fraction of the folded form and lowered the crystallisation temperatures. Some lipids were incorporated to a high extent into the amorphous domains of the PEG lamellae and thereby swelling the structure, which also resulted in a high degree of chain folding. Partial least squares (PLS) modelling indicated that small hydrophilic lipids increased the folding of PEG and that large non-polar lipids retarded the unfolding during secondary crystallisation. This work shows that there is a large difference in the behaviour of PEG depending on lipid added. Differences are explained in terms of molecular properties for the lipids, demonstrated by the use of PLS modelling to describe the behaviour of PEG solid dispersions. (C) 2009 Elsevier B.V. All rights reserved

Effect of salt and surfactant concentration on the structure of polyacrylate gel/sutrfactant complexes

Small-angle X-ray scattering was used to elucidate the structure of crosslinked polyacrylate gel/ dodecyltrimethylammonium bromide complexes equilibrated in solutions of varying concentrations of surfactant and sodium bromide (NaBr). Samples were swollen with no ordering (micelle free), or they were collapsed with either several distinct peaks (cubic Pm3n) or one broad correlation peak (disordered micellar). The main factor determining the structure of the collapsed complexes was found to be the NaBr concentration, with the cubic structure existing up to similar to 150 mM NaBr and above which only the disordered micellar structure was found. Increasing the salt concentration decreases the polyion mediated attractive forces holding the micelles together causing swelling of the gel. At sufficiently high salt concentration the micelle-micelle distance in the gel becomes too large for the cubic structure to be retained, and it melts into a disordered micellar structure. As most samples were above the critical micelle concentration, the bulk of the surfactant was in the form of miscelles in the solution and the surfactant concentration thereby had only a minor influence on the structure. However, in the region around 150 mM Nal3r, increasing the surfactant concentration, at constant NaBr concentration, was found to change the structure from disordered micellar to ordered cubic and back to disordered again

Effect of salt and surfactant concentration on the structure of polyacrylate gel/sutrfactant complexes

Small-angle X-ray scattering was used to elucidate the structure of crosslinked polyacrylate gel/ dodecyltrimethylammonium bromide complexes equilibrated in solutions of varying concentrations of surfactant and sodium bromide (NaBr). Samples were swollen with no ordering (micelle free), or they were collapsed with either several distinct peaks (cubic Pm3n) or one broad correlation peak (disordered micellar). The main factor determining the structure of the collapsed complexes was found to be the NaBr concentration, with the cubic structure existing up to similar to 150 mM NaBr and above which only the disordered micellar structure was found. Increasing the salt concentration decreases the polyion mediated attractive forces holding the micelles together causing swelling of the gel. At sufficiently high salt concentration the micelle-micelle distance in the gel becomes too large for the cubic structure to be retained, and it melts into a disordered micellar structure. As most samples were above the critical micelle concentration, the bulk of the surfactant was in the form of miscelles in the solution and the surfactant concentration thereby had only a minor influence on the structure. However, in the region around 150 mM Nal3r, increasing the surfactant concentration, at constant NaBr concentration, was found to change the structure from disordered micellar to ordered cubic and back to disordered again

Studies on an ester-modified cationic amphiphile in aqueous systems: Behavior of binary solutions and ternary mixtures with conventional surfactants

The aqueous behavior of an ester-modified cationic amphiphile with the molecular structure CH3CH2O(C=O)(CH2)(6)(C=O)O(CH2)sN+(CH3)(3)Br-, in the following referred to as A, has been investigated. Systems with A as the only solute, as well as different aqueous mixtures with conventional cationic surfactants, primarily dodecyltrimethylammonium bromide (DTAB), were included in the study. Isotropic solution samples were characterized using H-1 NMR, C-13 NMR, NMR diffusometry, and conductivity measurements, whereas liquid crystalline samples were investigated by optical polarization microscopy and small-angle X-ray diffraction. The results are compared to the behavior of the binary system of DTAB and water. A does not exhibit a typical surfactant behavior. When it is present as the only solute in a binary aqueous system, it forms neither conventional micelles nor liquid crystalline phases. However; there is clear evidence that it assembles with lower cooperativity into loose clusters at concentrations above 25-30 mM. When A is mixed with DTAB in solution, the two amphiphiles form mixed assemblies, the structure of which varies with the total amphiphile concentration. In concentrated mixtures with alkyltrimethylammonium surfactants, A can participate in hexagonal liquid crystalline phases even when it constitutes a significant fraction of the total amphiphile content