Lipid-coated microbubbles (LCMs), dispersed lipid-mesophase nanoparticles (LMNs), and large rodlike micelles probably all self-assemble simultaneously from the lipid mixture comprised within the Filmix® nanoemulsion, which contains cholesterol, cholesterol (ester) derivatives, and saturated glycerides. The LCMs’ structural characteristics, especially regarding (saturated) acyl chain length in addition to content of cholesterol compounds, help drive and govern a continual and reversible (molecular and/or supramolecular) lipid interchange with the nanoparticle subpopulations (i.e., mixed micelles and dispersed LMNs) in the stable nanoemulsion. Accordingly, the LCMs, mixed micelle, and dispersed LMNs (including dispersed cubic nanostructures) each represent separate bulk phases, that is, different colloidal species, in the colloidally stable Filmix® nanoemulsion.
Recent multidisciplinary analyses on newer particle-sizing instruments uncovered evidence that lipid-coated microbubbles (LCMs) actually represent a “microbubble/particle” population, the vast majority of which are submicron in size. Based upon the above (and other related) physicochemical factors, these predominant submicron-sized (so-called) LCMs likely largely represent liquid-crystalline lipid particles (i.e., dispersed “lipid-mesophase nanoparticles” or dispersed LMNs). Yet the same targeted drug-delivery attributes can logically be expected also from such a subpopulation of liquid-crystalline nanoparticles in the Filmix® nanoemulsion, since both categories of stable colloidal species (i.e., LCMs and dispersed LMNs) are formed simultaneously using the same patented mixture of powdered solid lipid surfactants.
Dilute gas-in-liquid emulsions, existing in natural waters, represent self-assembled (i.e., “self-organized”) coated microbubbles which are of great concern to workers in many fields of fundamental and engineering sciences. A detailed knowledge of the predominant physicochemical/biochemical mechanism by which such gas microbubbles, 0.5-100 µm in diameter, are stabilized in aqueous media is of practical importance to numerous and varied fields. Such fields are: hydrodynamic and acoustic cavitation, hydraulic and ocean engineering, waste-water treatment, commercial oil recovery, chemical oceanography, meteorology, marine biology, food technology, and various medical applications including echocardiology, decompression sickness and, more recently, cancer diagnosis and treatment.
Lipid polymorphism is complex, but important and useful for intravenous drug delivery. Various structural/physicochemical considerations lead to the resultant judgment that the dispersed (inverse) cubic phase probably represents the preferred (lipid polymorph or liquid-crystalline nanoparticle) structure of “dispersed lipid-mesophase nanoparticle (LMN)” in the “lipid-coated microbubble/nanoparticle-derived” nanoemulsions. This conclusion helps explain the observed efficacy of such nanoemulsion formulations in solubilizing, encapsulating, and delivering selected lipophilic drugs, by receptor-mediated endocytosis, to certain disease sites in animals. As concerns polymorphic mechanism, the expected physicochemical tendency of the dispersed LMN to adopt nonlamellar mesostructural topology is a function of head-group hydration, acyl chain length, and cholesterol content.
Lipid-coated microbubbles (LCMs) can be labeled with lipophilic fluorescent dye, and such dye-labeled LCMs are readily internalized by tumor cells both in vitro and in vivo. LCMs can also function as a targeted drug-delivery vehicle, for lipophilic drugs (such as the anticancer drug paclitaxel), specifically to tumors. For example, both in vitro and in vivo data (reviewed herein) indicate that paclitaxel can be lastingly incorporated into LCMs. In vivo treatment in rats, using two different tumor models, indicated that intravenously injected paclitaxel–LCM can be delivered to the tumor site, and can exert both a measurable biological effect and an antitumor activity.
Self-assembled (colloidal mesophase) lipid nanoemulsions, particularly those predominantly containing dispersed cubic-phase lipid nanoparticles, continue to receive growing research attention. The main reason for this attention is the fact that these nonlamellar lipid nanostructures, such as cubic liquid-crystalline phases, have wide potential as delivery systems for numerous drugs, cosmetics, and food applications. In a few cases, the self-assembled “lipid particle” structure itself (upon injection into the bloodstream) demonstrates the added advantage of successfully functioning as an “active” targeting ligand—which is directed via (simple adsorption of) plasma lipoproteins toward the appropriate receptors on the target-cell surface.
A water-soluble extract from a forest soil, rich in microbubble surfactants, has been geochemically characterized using elemental, infrared, and X-ray diffraction measurements, pyrolysis mass spectrometry, carbohydrate determinations, and amino acid analyses. Moreover, the low solubility in water, (soil-derived) pyrolysis mass spectra, and iodine-stained thin-layer chromatography of the microbubble surfactant mixture indicated that lipids, previously unidentified, probably also represent a major component of the surfactant mixture. Last, we provide additional data on the biochemical heterogeneity of the microbubble surfactant mixture and identify the probable natural source of its characteristic glycopeptide fraction.
The extreme longevity of (Filmix) surfactant-stabilized microbubbles is, in part, related to their continuous interaction with the mixed-micelle population in the colloidal system. Various factors (e.g., uncharged head groups, cholesterol condensing effect, and long hydrocarbon chains) favoring larger microbubbles (and rodlike micelles) are, in fact, eventually fully opposed by repulsive head-group interactions. As the hydrated head groups are forced closer together, microbubble growth slows. Thereafter, microbubble collisions and consequent fission into ultramicrobubbles result in higher curvature and greater monolayer (gas) permeability the result is loss of gas, and finally ultramicrobubble collapses into a rodlike micelle, which completes the cyclical process.
Overlap of lipid composition exists among high-density lipoprotein (HDL), low-density lipoprotein (LDL), modified LDL, lipid-coated microbubble (LCM), and dispersed lipid-mesophase nanoparticle (LMN). This overlap suggests that LCM and/or dispersed LMN themselves can act as ligands for multiligand lipoprotein receptors. In particular, the multiligand scavenger receptor (i.e., SR-BI) is known to bind both HDL and LDL particles. Accordingly, SR-BI represents the most likely candidate involved in enhanced endocytosis of “LCM/nanoparticle-derived” lipid nanoemulsions (i.e., mostly dispersed LMN) into tumor cells. Moreover, the overlap of lipid composition between HDL, LDL, modified LDL, LCM, and LMN can partially mimic the known heterogeneity (i.e., subpopulations or subspecies) of LDL and HDL particles.
The naturally occurring, largely hydrophobic surfactants which surround and stabilize long-lived gas microbubbles include proteinaceous compounds that contain, and whose surface activity depend upon, aromatic amino acid residues. The particular finding of protein/peptide-stabilized gas microbubbles in filtered aqueous extracts of forest soil may provide an explanation for the widespread occurrence of these long-lived microbubbles in nature. Specifically, humic substances, which are known to reversibly bind proteinaceous material (that can include surfactant-stabilized microbubbles) thereby forming complexes resistant to decomposition, are among the most widely distributed natural products on the Earth’s surface, occurring in soils, lakes, rivers, and in the sea.
Various investigators have proposed three different drug-delivery routes for targeted chemotherapy of atherosclerosis (i.e., via macrophages or platelets or the liver); all three routes entail receptor-mediated processes in which SR-BI plays a key role. This situation is suitable to certain “actively targeted” lipid nanoemulsions for which SR-BI (or CLA-1 in humans) emerged as the most likely candidate receptor, involved in ligand–receptor binding of such nanoemulsions, at target cells. Moreover, several of these closely related, “chylomicron-like” nanoemulsions mimic the metabolic fate of native chylomicrons and, hence, have been evaluated as targeted chemotherapeutic agents for potentially treating atherosclerosis in humans.
The chemical literature provides much (theoretical and experimental) support for a supramolecular or biophysical connection between inverse cubic phases, or isotropic phases, and the inducing or facilitating membrane fusion. These physicochemical factors (along with lipid composition, and cholesterol functioning as an added promoter of biomembrane fusion) together probably serve to facilitate cellular uptake or endocytosis of “lipid-coated microbubble (LCM)/nanoparticle-derived” nanoemulsions (representing mostly dispersed lipid-mesophase nanoparticle (LMN)). The next, but more problematic, question requiring analysis concerns the identification of which “lipoprotein receptor”-mediated endocytic pathway is the most likely candidate involved in this active uptake process, of dispersed LMN, into tumor cells.
Experimental work in this laboratory was aimed at the systematic development of an efficient method for isolating the proteinaceous surfactants, which help stabilize natural microbubbles, from both commercial agarose powder and forest soil samples collected locally. The microbubble surfactant mixture was shown to contain low-molecular-weight glycopeptides of similar structure, which were invariably contaminated with a much greater quantity of oligosaccharide material. It appears likely that the micro-bubble-surfactant mixture's glycopeptide fraction is essentially a partial degradation product of larger (precursor) glycoproteins, which are probably of biological origin and are widely distributed in the environment.
Quantitative examination of the surface properties of monomolecular films of the isolated microbubble surfactant complex (or glycopeptide–lipid–oligosaccharide complex), at an air/water interface, was carried out using a Langmuir trough apparatus. Stabilized microbubbles are apparently formed from shrinkage of surfactant-coated macroscopic bubbles, and various data indicate that most of the carbohydrate material is selectively desorbed from the microbubble surfactant monolayer during this initial compression phase. The result is a stable, tightly packed, insoluble monolayer containing glycopeptide–acyl lipid complexes, which have been shown to be held intact primarily by hydrogen bonding. The glycopeptide:acyl lipid area ratio within this stable monolayer is approximately 1:60.
Agarose gels make possible well-controlled, surface-chemical studies on microbubble stabilization and related bubble growth. The relative effectiveness of different added ions, in decreasing bubble formation within these aqueous gels, showed many similarities with published data in the physicochemical literature for salting out of identified nonionic surfactants. In particular, the cation sequences obtained, which indicated strong salting out in all cases, rendered it quite unlikely that ether linkages contribute to the hydrophilicity of the nonionic surfactants stabilizing gas microbubbles. It was concluded that the nonionic or hydrophobic surfactants stabilizing long-lived gas microbubbles are probably mostly, if not all, of natural origin.
Experiments revealed that aqueous suspensions of saturated monoglycerides (with acyl chain lengths over 10 carbons) combined with cholesterol and cholesterol derivatives readily formed concentrated gas-in-water emulsions when shaken vigorously (in an air atmosphere). Using laser-based flow cytometry and forward-angle light scattering (detection limit = 0.3 µm), the calculated concentration of synthetic microbubbles in the filtered sample is approximately 7 × 105 microbubbles/ml. Further measurements also indicated that very slow dissolution of the newly formed, surfactant-coated microbubbles does continue for a period. Moreover, this gradual rate of dissolution of the artificial microbubbles can apparently be increased somewhat by circulating the liquid.
Filmix surfactant-stabilized microbubbles, also referred to as concentrated gas-in-liquid emulsions or lipid-coated microbubbles (LCMs), have been modeled primarily from natural microbubble surfactant. Accordingly, Filmix-coated microbubbles or LCMs contain specifically nonionic lipids exclusively throughout their microbubble coating. Interestingly, this nonionic-lipid monolayer coating causes this specific synthetic-microbubble agent to display marked tumor-targeting abilities well suited for both diagnostic and therapeutic applications. For example, in several published animal studies, targeted imaging of tumors (via ultrasonography or MRI) has been successfully carried out using LCM as a contrast agent. Similarly, targeted therapeutic applications were initiated with an investigation of LCM-facilitated ultrasonic therapy of tumors.
A documented correlation between bubble production in agarose gels and the incidence of decompression sickness, in humans decompressed after hyperbaric exposure, has been observed—which indirectly suggests that surfactant-stabilized gas microbubbles exist in physiological fluids. Specifically, the depth at which slow decompression commences is a major factor, along with total decompression time, in determining the extent of bubble formation in both situations. Despite these findings, more recent studies using various animal models indicate that stable microbubbles might not occur naturally in physiological fluids however, injected artificial surfactant-stabilized microbubbles are likely to have useful clinical applications.
Particle size distributions, derived from photon correlation spectroscopy (PCS) of microbubble surfactant solutions, are presented, which suggest the formation of gas microbubbles from large micellar structures as well as the reverse process of collapse of gas microbubbles into such micellar structures. Detailed inspection of the PCS data (combined with a review of relevant chemical literature) reveals that this reversible process is actually part of a cycle, namely microbubble formation and coalescence followed by microbubble fission and disappearance. This cyclical microbubble process is promoted by prior mechanical agitation of, and hence entrapment of macroscopic gas bubbles in, these saturated surfactant solutions.
