J.S. D'Arrigo
© 2011, Elsevier
TABLE OF CONTENTS
Part I: NATURAL COATED MICROBUBBLES IN THE BIOSPHERE
Chapter 1. OCCURRENCE OF DILUTE GAS-IN-LIQUID EMULSIONS IN NATURAL WATERS
1.1 Practical Importance of Stable Microbubbles
1.1.1 Hydrodynamic cavitation, hydraulic and ocean engineering
1.1.2 Acoustic cavitation
1.1.3 Waste-water treatment: Microflotation
1.1.4 Marine biology, chemical oceanography
1.1.5 Meteorology
1.2 Background Observations
1.2.1 Problems with the crevice model for bubble nuclei
1.2.2 Reduction of gaseous diffusion across the air/water interface by selected surfactant monolayers
1.3 Demonstration of Film-Stabilized Microbubbles in Fresh Water
1.3.1 Acoustical measurements
1.3.2 Light-scattering measurements
1.3.3 Gas-diffusion experiments
1.4 Demonstration of Film-Stabilized Microbubbles in Sea Water
1.4.1 Acoustical measurements
1.4.2 Light-scattering measurements
1.4.3 Photographic identification
Chapter 2. EARLY WORK WITH AQUEOUS CARBOHYDRATE GELS
2.1 Development of the Agarose Gel Method for Monitoring Bubble Formation
2.2 Results from Dilute Electrolyte Additions and pH Changes in Agarose Gels
2.3 Results from Concentrated Electrolyte Additions and 1% Phenol in Agarose Gels
2.4 Detailed Comparison with Published Data in the Physicochemical Literature for Salting Out of Identified Nonionic Surfactants
2.5 Concluding Remarks
Chapter 3. COMPARISON OF AQUEOUS SOIL EXTRACTS WITH CARBOHYDRATE GELS
3.1 Functional Microbubble Residues in Soil and Agarose Powder
3.2 Adaptation of (Filtered) Aqueous Soil Extracts for Use with the Agarose Gel Method
3.3 Ninhydrin Effect on Bubble Formation in Commercial Agarose and Aqueous Soil Extracts
3.4 Photochemical Experiments Using Methylene Blue
3.5 2-Hydroxy-5-Nitrobenzyl Bromide Experiments
3.6 Conclusions
Chapter 4. CHARACTERISTIC GLYCOPEPTIDE FRACTION OF NATURAL MICROBUBBLE SURFACTANT
4.1 Analytical Methods
4.1.1 Isolation of microbubble glycopeptide surfactant from commercial agarose and forest soil
4.1.2 Decompression tests with agarose gels
4.1.3 Amino acid analyses of the isolated glycopeptide surfactant
4.1.4 Sodium dodecyl sulfate/polyacrylamide-gel electrophoresis
4.1.5 Carbohydrate analyses of partially purified glycopeptide surfactant
4.1.6 Sephadex column chromatography of dansylated glycopeptide surfactant
4.1.7 Edman degradation analyses
4.2 Biochemical Results
4.2.1 Protein extraction and bubble production in agarose gels
4.2.2 Amino acid composition of microbubble glycopeptide surfactant
4.2.3 Molecular weight determinations by gel electrophoresis
4.2.4 HPLC determination of carbohydrate content
4.2.5 Gel-filtration column chromatography: Determination of average molecular weight and the NH2-terminus
4.3 Review of Natural-Product Literature and Possible Animal Sources of the Glycopeptide Fraction of Microbubble Surfactant
4.4 Concluding Remarks
Part II: PHYSICOCHEMICAL PROPERTIES OF NATURAL MICROBUBBLE SURFACTANT
Chapter 5. ECOLOGICAL CHEMISTRY OF MICROBUBBLE SURFACTANT
5.1 Analytical Methods
5.1.1 Preparation of aqueous soil extract
5.1.2 Elemental, infrared, and x-ray diffraction measurements
5.1.3 Pyrolysis mass spectrometry
5.1.4 Isolation of microbubble surfactant
5.1.5 Gel-filtration column chromatography, amino acid analysis and carbohydrate determination
5.2 Experimental Results
5.2.1 Abundant mineral content and characteristic IR absorption bands
5.2.2 Comparison of pyrolysis mass spectra for aqueous soil extract, fulvic acid, and water-soluble humic acid
5.2.3 Further purification of the microbubble surfactant mixture by gel-filtration column chromatography
5.2.4 Amino acid composition of the main glycopeptide subfraction from microbubble surfactant
5.3 Biochemical/Geochemical Considerations
5.3.1 Interaction of forest soil organic matter with abundant mineral content
5.3.2 Dispersal of microbubble surfactants in natural waters
5.3.3 Bonding within the microbubble surfactant complex
5.3.4 Probable biological source of the glycopeptide fraction of microbubble surfactant
Chapter 6. SURFACE PROPERTIES OF MICROBUBBLE-SURFACTANT MONOLAYERS
6.1 Modified Langmuir Trough Method
6.1.1 Surface pressure measurements with a cylindrical rod
6.1.2 Advantages of method when testing complex biochemical mixtures
6.1.3 Langmuir trough apparatus and solutions
6.2 Surface Pressure-Area (Π-A) Curves
6.2.1 Initial compression-expansion cycle
6.2.2 Effect of salt concentration, pH, and selected nonelectrolytes
6.2.3 ΠA-Π plots
6.3 Selective Desorption from Compressed Monolayers
6.4 Bonding within Compressed Microbubble-Surfactant Monolayers
6.5 Glycopeptide:Acyl Lipid Area Ratio and Association of Complexes within Monolayers
6.6 Conclusions
Chapter 7. STRUCTURE OF PREDOMINANT SURFACTANT COMPONENTS STABILIZING NATURAL MICROBUBBLES
7.1 1H-NMR Spectroscopy of Isolated Microbubble Surfactant
7.2 Langmuir-Trough Measurements and Collection of Monolayers
7.3 1H-NMR Spectroscopy of Compressed Monolayer Material
7.4 Chemical Similarities between Microbubble-Surfactant Monolayers and Lipid Surface Films at the Air/Sea Interface
Chapter 8. STABLE MICROBUBBLES IN PHYSIOLOGICAL FLUIDS: COMPETING HYPOTHESES
8.1 Comparison of Different Decompression Schedules: Correlation between Bubble Production in Agarose Gels and Incidence of Decompression Sickness
8.1.1 Background observations
8.1.2 Methods
8.1.3 Experimental results
8.1.4 Water depth at first stop, and total decompression time
8.2 Comparison of Cavitation Thresholds for Agarose Gels and Vertebrate Tissues
8.3 Contradictory Findings
8.4 Homogeneous Nucleation Hypothesis
8.5 Clinical Use of Injected Gas Microbubbles: Echocardiography; Potential for Cancer Detection
Part III: PHYSICOCHEMICAL PROPERTIES OF ARTIFICIAL COATED MICROBUBBLES AND NANOPARTICLES
Chapter 9. CONCENTRATED GAS-IN-LIQUID EMULSIONS IN ARTIFICIAL MEDIA. I. DEMONSTRATION BY LASER-LIGHT SCATTERING
9.1 Physiological Hints for the Production of Artificial Microbubbles
9.2 Laser-Based Flow Cytometry and Forward-Angle Light Scattering
9.3 Synthetic Microbubble Counts versus the Control
9.4 Microbubble Flotation with Time
9.5 Microbubble Persistence with Time
Chapter 10. CONCENTRATED GAS-IN-LIQUID EMULSIONS IN ARTIFICIAL MEDIA. II. CHARACTERIZATION BY PHOTON CORRELATION SPECTROSCOPY
10.1 Brownian Motion and Autocorrelation Analysis of Scattered Light Intensity
10.2 Background Observations on Micellar Growth
10.3 Solubilization of Gases in Micelles
10.4 Size Distribution of Synthetic Microbubbles: Formation, Coalescence, Fission, and Disappearance
10.4.1 Bimodal size distribution of the microbubble-surfactant particle population
10.4.2 Combined evidence that the larger-diameter Filmix particles (subpopulation) are surfactant-stabilized gas microbubbles
10.4.3 Apparent reversible and/or cyclical behavior: Microbubble formation and coalescence versus microbubble fission and disappearance
Chapter 11. CONCENTRATED GAS-IN-LIQUID EMULSIONS IN ARTIFICIAL MEDIA. III. REVIEW OF MOLECULAR MECHANISMS INVOLVED IN MICROBUBBLE STABILIZATION
11.1 Microbubble Longevity and Interaggregate Interactions
11.2 Molecular Packing within the Microbubble's Surfactant Monolayer
11.3 Repulsive Head-Group Interactions and Monolayer Curvature
11.4 Microbubble Fission, Collapse, and Re-emergence
Part IV: LIPID-COATED MICROBUBBLES AND RELATED LIPID NANOPARTICLES IN BIOMEDICAL STUDIES ON ANIMALS
Chapter 12. TARGETED IMAGING OF TUMORS, AND TARGETED CAVITATION THERAPY, WITH LIPID-COATED MICROBUBBLES (L.C.M.)
12.1 Description of the LCM Agent (Filmix®)
12.2 Targeted Ultrasonic Imaging of Tumors with LCM as a Contrast Agent
12.3 Tumor Detection versus Tumor Therapy with LCM
12.4 Use of LCM as a Targeted, Susceptibility-Based, MRI Contrast Agent for Tumors
12.5 LCM-Facilitated Ultrasonic Therapy of Tumors
Chapter 13. TARGETED DRUG-DELIVERY THERAPY OF TUMORS USING L.C.M.
13.1 Internalization of LCM by Tumor Cells In Vivo and In Vitro
13.1.1 LCM reach tumors within minutes after i.v. injection: Light- and fluorescence-microscopy data
13.1.2 LCM preferentially interact with tumor cells in vivo: Data from confocal laser microscopy
13.1.3 LCM are found inside tumor cells in vivo: Serial optical sections
13.1.4 LCM are endocytosed by tumor cells in culture: Kinetics of uptake and temperature dependence
13.1.5 LCM are found in acidic compartments in tumor cells in culture: Confocal microscopy using dual-channel recording
13.1.6 Concluding remarks
13.2 Evaluation of LCM as a Delivery Agent of Paclitaxel (Taxol®) for Tumor Therapy
13.2.1 Experimental methods
13.2.2 Pharmacological results
Chapter 14. PROPOSED MECHANISM OF SELECTIVE L.C.M. UPTAKE BY TUMOR CELLS: ROLE OF LIPOPROTEIN RECEPTOR-MEDIATED ENDOCYTIC PATHWAYS
14.1 Low-Density Lipoprotein (LDL) Receptors, on Tumor Cells, and LCM
14.2 Multiligand Lipoprotein Receptors
14.2.1 LDL receptor-related protein (LRP), on tumor cells, and LCM
14.2.2 Scavenger receptors on tumor cells as well as "activated" macrophages: LCM binding, and its relation to certain disease sites
Chapter 15. ENDOCYTOTIC EVENTS VERSUS PARTICLE SIZE: MULTIDISCIPLINARY ANALYSES DEMONSTRATE L.C.M. SIZES ARE MOSTLY SUBMICRON
15.1 Chylomicron Remnant-Like Particle Sizes
15.2 Comparison with LCM Sizes: Proportion of LCM Population Between 0.1-0.2 µm
Part V: SELF-ASSEMBLING MIXED-LIPID MICROBUBBLES AND NANOPARTICLES FOR CLINICAL APPLICATIONS
Chapter 16. L.C.M. AND NANOPARTICLE SUBPOPULATIONS FOR DRUG DELIVERY
16.1 Stable Nanoemulsions
16.2 Mixed-Lipid "Microbubble versus Nanoparticle" Interrelationships in Filmix®
16.2.1 Nanoparticles based on solid lipids: Background literature
16.2.2 "Dispersed LMN" (or colloidal liquid crystals) and targeted drug delivery
16.2.3 Self-assembly and interplay of LCM, dispersed LMN, and mixed micelles: Correlations with bile colloids
Chapter 17. COMPOSITION OF L.C.M. GOVERNING INTERPLAY WITH NANOPARTICLE SUBPOPULATION
17.1 Patented LCM Components
17.2 LCM Structural Characteristics Affecting Molecular Interchange with Dispersed LMN and Mixed Micelles
17.3 Film-Shedding Transitions and/or Collapse in Lipid Monolayers Coating Microbubbles
17.3.1 Dissolution of microbubbles in degassed media
17.3.2 Dissolution of microbubbles via ultrasound
17.4 Concluding Remarks
Chapter 18. TARGETED NANOPARTICLE SUBPOPULATION: COMPARISON WITH SELF-NANOEMULSIFYING DRUG-DELIVERY SYSTEMS IN PHARMACEUTICAL RESEARCH
18.1 Small Energy Input for Production: Self-Nanoemulsification
18.2 Medium-Chain, and Long-Chain, Glycerides
18.3 Nonpolar Core of Nanoemulsion Particles
Chapter 19. CLINICAL DEVELOPMENT OF A "L.C.M./NANOPARTICLE-DERIVED" FORMULATION: A NANOEMULSION BASED UPON "DISPERSED L.M.N."
19.1 Details of Chemical Composition of the Clinical-Grade "Non-Gas Containing, Lipid Nanoparticles" (or Clinical-Grade "Dispersed LMN")
19.2 Particle Size Distribution of Clinical-Grade Dispersed LMN Prepared with, or without, Incorporated Drug and/or Additives
19.3 Targeting Properties of Clinical-Grade Dispersed LMN: Drug Delivery to Tumor Cells
19.4 Targeted Drug Delivery by Clinical-Grade Dispersed LMN: Effects of Different Additives
Chapter 20. SELECTED PARENTERAL LIPID NANOEMULSIONS UNDER CLINICAL STUDY: COMPARISON CONCERNING PASSIVE ACCUMULATION IN TUMORS, ACTIVE TARGETING
OF TUMORS, AND VALIDATION STATUS
20.1 Tocol Nanoemulsions for Solubilizing, and Drug Delivery, of Paclitaxel: Passive Accumulation in Tumors
20.2 Cholesterol-Rich/Phospholipid Nanoemulsions Containing Derivatized Paclitaxel: Active Uptake into Tumors via LDL Receptors
20.3 Stable (Non-Phospholipid, Non-Protein) Lipid Nanoemulsions for Targeting Tumors: Active Uptake of (Unmodified) Paclitaxel via Endocytosis
Chapter 21. SUPPLEMENTARY OPERATIONAL BENEFITS CONCERNING "L.C.M./NANOPARTICLE-DERIVED" FORMULATIONS: RELATION TO LIPID-NANOEMULSION STRUCTURE
21.1 Prolonged Circulation After Intravenous Injection
21.2 Filmix® Chemical Composition Supports Long-Term Stability of Liquid-Crystalline Lipid Nanoparticles (or Dispersed LMN)
21.3 Rapid (Nondestructive) Determination of Particle Size Distribution of the Parenteral Nanoemulsion by Standard Light-Scattering Methods
Part VI: "L.C.M./NANOPARTICLE-DERIVED" NANOEMULSIONS: BIOLOGICAL LIPID POLYMORPHISM, AND RECEPTOR-MEDIATED ENDOCYTOSIS, USED FOR CLINICAL STUDY
Chapter 22. BIOLOGICAL LIPID POLYMORPHS: PREFERRED CUBIC PHASE OF "DISPERSED L.M.N."
22.1 Biological Importance of Lipid Polymorphism: Focus on Cubic Phases
22.2 Inverse Micellar Cubic Phase: Special Relevance to "Dispersed LMN"
22.3 Physicochemical Tendency of the Dispersed LMN to Adopt "Non-Lamellar" Mesostructural Topology: Roles of Head-Group Hydration, Acyl Chain Length, and Cholesterol Content
Chapter 23. NON-LAMELLAR PHASE(S) FACILITATING MEMBRANE FUSION: ENDOCYTOSIS OF DISPERSED L.M.N.
23.1 Inverse Cubic Phase(s) Induce or Facilitate Membrane Fusion: The Stalk Mechanism
23.2 Inverse Bicontinuous Cubic Phases in Phosphoglyceride-Cholesterol Mixtures: Cholesterol as an Inducer of Biomembrane Fusion and Endocytosis
23.3 Endocytosis of Dispersed LMN: Competing Endocytic Pathways
Chapter 24. RECEPTOR-MEDIATED ENDOCYTOSIS OF (MIXED-LIPID) DISPERSED L.M.N.
24.1 Characteristics of Scavenger Receptors (versus LDL Receptor and LRP)
24.2 Biophysical Properties of a "class B" Scavenger Receptor: SR-BI
24.2.1 SR-BI versus CD36
24.2.2 SR-BI: Endocytosis and "selective" uptake?
24.2.3 SR-BI, membrane domains, and cholesterol
24.3 Endocytosis Mediated by SR-BI: Comparison of Human Tumor Cell Lines
Chapter 25. FURTHER CHEMOTHERAPY WITH LIPID NANOEMULSIONS: TARGETING CERTAIN HYPERPROLIFERATIVE DISEASES, AS WELL AS NEOPLASIAS, VIA "LIPOPROTEIN RECEPTOR"-MEDIATED ENDOCYTOSIS
25.1 Scavenger Receptors and Proliferative Processes: The Role of SR-BI on "Activated" Forms of Astrocytes, Vascular Smooth Muscle Cells, and Macrophages (besides Tumors and Hepatitis)
25.1.1 SR-BI at CNS-injury sites
25.1.2 SR-BI at vascular smooth muscle cells and macrophages
25.2 Mimicking of Targeting Behaviors of "Reconstituted Lipoprotein" Vehicle(s)
25.3 Expanded Clinical Study of Lipid Nanoemulsion for Targeted Drug Delivery of Paclitaxel (as well as Etoposide) to Human Tumors
25.4 Further Correlations between Overexpression of SR-BI and Much Increased Internalization of Cholesterol Compounds: More Support for Lipoprotein Receptor-Mediated Drug Delivery
25.4.1 Targeted drug delivery of paclitaxel, via lipid nanoemulsion, to atherosclerotic lesions
25.4.2 Added correlations between SR-BI overexpression, greater internalization of cholesterol compounds, and targeted drug delivery of antineoplastic drugs
25.5 Selected Patents with Relation to "Active-Targeting" Lipid Nanoemulsions: Comparison of Key Lipid Components, Field of Use, and Commercialization Path
Chapter 26. RELATED CLINICAL TRIALS AND HUMAN EPIDEMIOLOGICAL STUDIES
26.1 Added Background on "Actively" Targeted Drug Delivery and Clinical Trials
26.2 SR-BI/CLA-1 Located in Plaque and Platelets versus Liver: Roles in Atherogenesis and Implications for Treatment
26.3 Continued Investigational Use of (Protein-Free) Parenteral Lipid Nanoemulsions toward Targeted Chemotherapy of Cardiovascular Disease in Human Subjects
26.4 Associated Epidemiological Studies Implicating SR-BI/CLA-1 as Target for Chemotherapy of Atherosclerosis in Humans
Chapter 27. ASPECTS OF FUTURE R&D REGARDING TARGETED LIPID NANOEMULSIONS
27.1 SR-BI/CLA-1, Lipid Mediators in Membrane Microdomains, and Cellular Signaling: Implications for Future Targeted Treatment of Hyperproliferative Diseases in Humans
27.2 Added Clarifications Regarding the General Preparation Methods for Lipid Nanoemulsions (and Associated Liquid Crystals)
27.3 Latest Developments in the (Research and Patent) Literature: Further Evidence of Lipid Polymorphism Linked to Certain (Filmix®-like) Lipid Mixtures, Resulting in Targeted Nanoemulsion Drug-Delivery Vehicles
REFERENCES
SUBJECT INDEX
Chapter ABSTRACTS and KEYWORDS
Re: 2011 Book (Stable Nanoemulsions: Self-Assembly in Nature and Nanomedicine)
by J.S. D'Arrigo
CHAPTER 1:
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.
[Keywords: coated microbubbles, cavitation, gas-in-liquid emulsions, bubble nuclei, air/water interface]
CHAPTER 2:
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.
[Keywords: agarose gels, bubble formation, nonionic surfactants, salting-out sequences, decompression tests]
CHAPTER 3:
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".
[Keywords: soil extracts, hydrophobic amino acids, surface-active proteins, humic substances, microbubble residues]
CHAPTER 4:
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.
[Keywords: glycopeptide surfactant, natural microbubble surfactant, surfactant chromatography, surfactant electrophoresis, surfactant degradation]
CHAPTER 5:
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. Lastly, we provide additional data on the
biochemical heterogeneity of the microbubble surfactant mixture and identify the probable natural source of its characteristic glycopeptide fraction.
[Keywords: microbubble surfactant geochemistry, pyrolysis mass spectra, microbubble surfactant dispersal, glycopeptide-lipopolysaccharide complex, infrared spectrum]
CHAPTER 6:
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.
[Keywords: compressed monolayers, Langmuir trough, air/water interface, surface pressure, hydrogen bonding]
CHAPTER 7:
The monolayer-film material, contained in compressed microbubble-surfactant monolayers, was collected for structural determinations using
1H-nuclear magnetic resonance (NMR) spectroscopy. The resulting spectrum was then compared to the 1H-NMR spectrum which
was obtained beforehand from the partially purified, microbubble surfactant mixture prior to monolayer formation and compression. Comparison of the
above two spectra clearly demonstrated that the characteristic family of peaks originating from carbohydrate material is markedly reduced, and also
indicated some unsaturated lipids are ejected, following monolayer formation and compression. It is concluded that the most likely structural
candidates for the predominant acyl lipids stabilizing microbubbles are saturated glycerides.
[Keywords: acyl lipids, saturated glycerides, NMR spectroscopy, compressed monolayers, Langmuir trough]
CHAPTER 8:
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; on the other hand, injected artificial surfactant-stabilized microbubbles are likely to have
useful clinical applications.
[Keywords: decompression schedules, decompression sickness, caisson disease, hyperbaric exposure, ultrasound contrast agents]
CHAPTER 9:
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 x 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.
[Keywords: gas-in-water emulsions, flow cytometry, light scattering, synthetic microbubbles, Filmix surfactant mixture]
CHAPTER 10:
Particle size distributions, derived from photon correlatin 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.
[Keywords: photon correlation spectroscopy (PCS), micellar growth, Filmix surfactant mixture, artificial microbubbles, gas-in-water emulsions]
CHAPTER 11:
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
results in higher curvature, and greater monolayer (gas) permeability; the result is loss of gas and finally ultramicro-bubble collapse into a
rodlike micelle, which completes the cyclical process.
[Keywords: coated microbubbles, molecular packing, lipid exchange, head-group repulsion, monolayer curvature]
CHAPTER 12:
Filmix surfactant-stabilized microbubbles, also referred to as concentrated gas-in-liquid emulsions or "lipid-coated microbubbles" (LCM), have been
modeled primarily from natural microbubble surfactant. Accordingly, Filmix coated microbubbles or LCM 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.
[Keywords: targeted imaging, contrast agent, Filmix surfactant mixture, targeted cavitation therapy, LCM (lipid-coated microbubbles)]
CHAPTER 13:
LCM can be labeled with lipophilic fluorescent dye, and such dye-labeled LCM are readily internalized by tumor cells both in vitro and in vivo. LCM
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 LCM. 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.
[Keywords: targeted drug delivery, LCM (lipid-coated microbubbles), paclitaxel, confocal laser microscopy, endocytosis]
CHAPTER 14:
As concerns the actual endocytic pathways likely to be involved in LCM uptake by tumors, a few reasonable candidates emerge upon reviewing parts of
an extensive research literature describing significantly enhanced, receptor-mediated endocytosis in many different cancerous cells and solid
tumors. Basically, the LCM lipid formulation actively targets drug (e.g., paclitaxel) to tumor cells that commonly overexpress certain surface
receptors, i.e., "lipoprotein receptors". In addition, there are several other (noncancerous) lesion/injury sites involving hyperproliferative
diseases (e.g., neuro-injury [gliosis] sites, and atherosclerosis) which include overexpression of cell-surface lipoprotein receptors; therefore,
these sites are also suitable for targeted chemotherapy via LCM.
[Keywords: endocytic pathways, receptor-mediated endocytosis, lipoprotein receptors, LCM (lipid-coated microbubbles), active uptake]
CHAPTER 15:
Since the lipid composition of LCM is similar to that of chylomicron remnant particles, the active uptake of the chylomicron remnant particles by
their associated "lipoprotein receptor"-mediated endocytic pathways probably reflects a similar active-uptake pattern for LCM. Accordingly, much
of the LCM size distribution should be in the same diameter range as observed with chylomicron remnants. Consistent with this expectation, it was
found that the Filmix® agent contained close to 1010 LCM/ml = 0.1 µm, when measured using optical particle counters. A large majority
(~ 90%) of the LCM (i.e., Filmix® "coated-microbubbles/particles") were smaller than 0.2 µm in diameter.
[Keywords: lipoprotein receptors, chylomicron remnants, size distribution, Filmix® agent, LCM (lipid-coated microbubbles)]
CHAPTER 16:
Recent multidisciplinary analyses on newer particle-sizing instruments uncovered evidence that LCM 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) LCM likely largely represent liquid-crystalline lipid particles (i.e., dispersed "lipid-mesophase nanoparticles" or
dispersed LMN). 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., LCM and dispersed LMN) are formed simultaneously
using the same patented mixture of powdered solid lipid surfactants.
[Keywords: lipid nanoemulsion, LCM (lipid-coated microbubbles), LMN (lipid-mesophase nanoparticles), liquid-crystalline nanostructures, colloidal systems]
CHAPTER 17:
LCM, dispersed LMN, and large rodlike micelles probably all self-assemble simultaneously from the lipid mixture comprised within the Filmix®
nanoemulsion, which contains saturated glycerides, cholesterol, and cholesterol (ester) derivatives. The LCM's 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 LMN) in the stable
nanoemulsion. Accordingly, the LCM, mixed micelle, and dispersed LMN (including dispersed cubic nanostructures) each represent separate bulk phases,
i.e., different colloidal species, in the colloidally stable Filmix® nanoemulsion.
[Keywords: lipid self-assembly, molecular interchange, colloidal liquid crystals, Filmix® nanoemulsion, liquid-crystalline nanostructures]
CHAPTER 18:
The Filmix® nanoemulsion (including its self-assembling mixed-lipid nanoparticle subpopulations) is easily produced using a relatively mild
dispersing technique. The small energy input needed for production of the Filmix® nanoemulsion (at room temperature) resembles the generally
mild conditions under which SNEDDS (self-nanoemulsifying drug-delivery system(s)) are formed. Moreover, medium-chain and long-chain glycerides,
which are employed for producing various SNEDDS reported in the chemical literature, are also major components of the Filmix® nanoemulsion
formulation. Such colloidal systems offer advantages over unstable dispersions (such as coarse lipid emulsions and suspensions) since they are
produced by low-energy methods and have a long shelf-life.
[Keywords: self-nanoemulsification, lipid nanoemulsions, medium-chain glycerides, long-chain glycerides, colloidal liquid crystals]
CHAPTER 19:
A commercial-scale automated method, which includes a modified Filmix® chemical formulation, to produce a clinical-grade drug-delivery agent has
been developed in the pharmaceutical industry. This lipid nanoemulsion product, as specified in recent patents, bears many similarities to
"dispersed LMN" (described in the preceding chapters and herein). The above manufacturing approach provides the resultant lipid nanoparticles
with a structure that facilitates high internalization levels when applied to targeted tissues and cells, i.e., tumor cells. "Internalization"
(as used in the above patents) means "active" uptake into the tumor cell, which is also precisely the same targeting behavior described earlier
for dispersed LMN.
[Keywords: nanoemulsions, tumor targeting, lipid nanoparticles, lipid formulation, paclitaxel]
CHAPTER 20:
A renewed interest in targetable lipid-based drug-delivery systems concerns solubilizing lipophilic drugs. One example is a tocol nanoemulsion to
deliver paclitaxel, without any toxic solvents; this paclitaxel formulation can passively accumulate in tumors (EPR effect). Also under clinical
study is a parenteral cholesterol-rich/phospholipid nanoemulsion (termed "LDE"), which undergoes active uptake into cancer cells that overexpress
lipoprotein receptors. However, LDE nanoemulsion requires derivatization of the drug to stabilize the LDE-paclitaxel complex. Another lipid
nanoemulsion that actively targets tumors, after intravenous injection, is the "LCM/nanoparticle-derived" drug-delivery agent. It is produced by
a commercial-scale method involving no chemical derivatization, of paclitaxel, at all.
[Keywords: active targeting, EPR effect, parenteral nanoemulsions, paclitaxel, receptor-mediated endocytosis]
CHAPTER 21:
Further operational benefits of "LCM/nanoparticle-derived" nanoemulsions include all of the factors outlined below. Various investigators have
reported that small-particle-size lipid emulsions, about 0.1 µm (or 100 nm) in diameter, displayed the following desirable characteristics regarding
drug delivery: reduced hepatic uptake, and a much slower plasma clearance. In addition, the Filmix®-like lipid formulation characteristically
yields a persistent, particulate, liquid-crystalline matrix in excess water (such as blood plasma) which is very suitable for intravenous drug
delivery. Finally, standard light-scattering methods can be readily employed to rapidly determine particle size distribution of "LCM/nanoparticle-
derived" nanoemulsions (for quality-assurance testing purposes).
[Keywords: liquid-crystalline nanoparticles, intravenous nanoemulsion, lipid formulation, particle size distribution, light-scattering measurements]
CHAPTER 22:
Lipid polymorphism is complex, but important and useful for intravenous drug delivery. Various structural/physicochemical considerations lead to
the resultant judgement that the dispersed (inverse) cubic phase probably represents the preferred (lipid polymorph or liquid-crystalline
nanoparticle) structure of "dispersed LMN" in the "LCM/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.
[Keywords: nonlamellar lipid mesophases, cubic phases, inverse lipid phases, lipid polymorphism, dispersed LMN]
CHAPTER 23:
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
"LCM/nanoparticle-derived" nanoemulsions (representing mostly dispersed 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.
[Keywords: inverse cubic phases, membrane fusion, fusion promoters, receptor-mediated endocytosis, dispersed LMN]
CHAPTER 24:
Overlap of lipid composition exists among HDL, LDL, modified LDL, LCM, and dispersed 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.
[Keywords: scavenger receptors, multiligand lipoprotein receptors, dispersed LMN, receptor-mediated endocytosis, SR-BI ("class B, type I"
scavenger receptor)]
CHAPTER 25:
Besides tumors, some other (noncancerous) lesion/injury sites involving hyperproliferative disease processes (such as atherosclerosis, as well as
traumatic neuro-injury sites) often display an overexpression of cell-surface lipoprotein receptors (i.e., SR-BI and others). Accordingly,
targeted drug-delivery therapy of that given hyperproliferative disease or injury site, via "LCM/nanoparticle-derived" nanoemulsions (such as Filmix®
nanoemulsion), becomes quite practicable. A rather similar therapeutic capability, therefore, would also be expected for a few other types of
parenteral nanoemulsions known to actively target lipoprotein receptors. Finally, the related intellectual property, key lipid components, field of
use, and/or commercialization paths of these separate technologies are also compared herein.
[Keywords: hyperproliferative diseases, lipoprotein receptors, active targeting, parenteral nanoemulsions, intellectual property]
CHAPTER 26:
Various investigators have proposed three different drug-delivery routes for targeted chemotherapy of atherosclerosis (that is, 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 (i.e., 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 chylimicrons and, hence, have been evaluated as targeted chemotherapeutic agents for potentially treating atherosclerosis
in humans.
[Keywords: atherosclerosis, targeted chemotherapy, parenteral nanoemulsions, clinical trials, cardiovascular disease]
CHAPTER 27:
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.
[Keywords: colloidal mesophase, nonlamellar lipid nanoparticles, liquid-crystalline phases, cubic lipid phases, targeted drug delivery]
PREFACE
Re: 2011 Book (Stable Nanoemulsions: Self-Assembly in Nature and Nanomedicine)
by J.S. D'Arrigo
With the growth of complex system science and the expansion of nanotechnology, there is increased need to distinguish between two related mechanisms, "self-organization" and "self-assembly", occurring in physical and biological systems. Basically, as pointed out in a recent issue of the journal Complexity, self-organization is a nonequilibrium process; in contrast, self-assembly leads toward equilibrium. Nevertheless, self-organization and self-assembly are regularly used interchangeably, as both explain how collective order is developed from dynamic small-scale interactions [J.D. Halley and D.A. Winkler (2008) "Consistent concepts of self-organization and self-assembly", Complexity 14: 10-17]. Hence in this book, all use of the term "self-assembly" (which some chemists classify as either static or dynamic [Science 295:2418-2421 (2002)] ) is here only intended within a "dynamic" sense; specifically, "dynamic" self-assembly corresponds to what biologists understand as self-organization [Complexity 14:10-17 (2008)].
Stable gas nanoemulsions, existing in natural waters, represent self-assembled coated microbubbles (also known as "gas in-liquid emulsions"). Similarly, in certain artificial media (namely, lipid dispersions modeled from natural microbubbles), stable nanoemulsions are also able to self-assemble (self-organize) readily. [Consequently, the first (1986) and expanded second (2003) editions of a related earlier book were entitled Stable Gas-in-Liquid Emulsions (with the subtitle Production in Natural Waters and Artificial Media). Yet, this much-expanded current book, that is, 12 chapters longer than the 2003 monograph, is more inclusive in its scope and accordingly entitled Stable Nanoemulsions.] In this specific case, the nanoemulsions comprise both "lipid-coated microbubbles (LCM)" (i.e., the gas-emulsion subpopulation) and "related lipid nanoparticles" (i.e., a particle-like subpopulation including mostly colloidal liquid crystals). Various measurements and other published findings indicate that the LCM's structural characteristics help drive and govern a continual and reversible (molecular and/or supramolecular) lipid interchange, with the nanoparticle subpopulation, in these self-assembling lipid nanoemulsions.
The term "LCM" is utilized, in this multidisciplinary book, to accurately trace the chronological development (and functional conversion) of the "LCM/nanoparticle-derived" colloidal system: 1) from its (modeling after natural microbubble surfactant and) early biomedical application as an imaging agent (in Chapters 1-12, which focus mainly on the less numerous micron-scale colloidal species) into 2) the later adaptation of exactly the same mixed-lipid (e.g., "Filmix®") colloidal system (in Chapters 13-27, which focus more upon the vastly more numerous nanoscale colloidal species) for nanomedical application as a ("LCM/nanoparticle-derived") drug-delivery vehicle. In addition, as explained in the chapters, newer models of several selected particle-size-analysis instruments have revealed that approximately 90% of these "LCM/nanoparticle-derived" colloidal species are actually smaller than 200 nm in diameter, while over 99% of the same mixed-lipid colloidal species (detectable via optical-particle-counter data) are documented to be smaller than 300 nm in diameter.
In this book, much experimental data is reviewed in detail and updated, along with the relevant current literature, which collectively demonstrate that this type of stable lipid nanoemulsion (upon intravenous injection) is capable of "active targeting" to tumors, and to certain lesion sites, via the process of receptor-mediated endocytosis. Hence, this "LCM/nanoparticle-derived" lipid formulation has been used successfully, in animals, as a drug-delivery agent that actively targets antineoplastic drug (e.g., paclitaxel) against tumor cells that commonly overexpress certain surface receptors, which fall within the category known as "lipoprotein receptors". Moreover, this "LCM/nanoparticle-derived" lipid nanoemulsion contains no phospholipids, no proteins nor peptides, no carbohydrates, -- and no chemical modification of the drug (paclitaxel) is required. Hence, this category of parenteral lipid nanoemulsion avoids various past problems reported for earlier versions of (actively) targeted drug-delivery agents utilizing such "lipoprotein receptor"-mediated endocytic pathway(s). ( Consequently, a human clinical trial is now in preparation, by a pharmaceutical company, for targeted drug delivery of paclitaxel to tumors in patients using a "LCM/nanoparticle-derived" drug-delivery agent.) In addition, as detailed in later chapters of the book, there are several noncancerous lesion/injury sites involving certain proliferative processes (e.g., atherosclerosis) which include overexpression of cell-surface "lipoprotein receptors". Therefore, the scope of potential clinical trials, which are applicable to the pharmaceutical category referred to as "LCM/nanoparticle-derived" lipid nanoemulsions, can now include the targeted chemotherapy of hyperproliferative diseases, for example, atherosclerosis and CNS-injury sites. In these last few chapters, several sections detail how one particular lipid-nanoemulsion agent (Filmix®) in this pharmaceutical (LCM-related) category, as well as a few other closely related protein-free parenteral lipid nanoemulsions, accordingly exhibit much (literature-supported) potential for providing "actively targeted" chemotherapy of atherosclerotic lesions in human subjects. (Such targeted chemotherapy is also in harmony with goals of the current U.S. National Nanotechnology Initiative, which include nanomedical approaches to drug delivery that focus on developing nanoscale particles (or macromolecules) to improve drug bioavailability, that is, often using targeted nanoparticles for delivering drugs with cell precision and less side effects.)
The book has been organized into six parts. Parts I and II (Chapters 1-8) describe coated microbubbles in the biosphere, as well as various biochemical, geochemical, surface, and structural properties of natural microbubble surfactant. Next, artificial lipid-coated microbubbles (LCM) and related lipid nanoparticles are described in Part III (Chapters 9-11), while their utilization in biomedical studies with animals is examined in detail in Part IV (Chapters 12-15).
Parts V and VI consist of completely new chapters (i.e., Chapters 16-27) that contribute to a strong nanomedicine focus. These 12 chapters further analyze and characterize this type of self-assembling mixed-lipid nanoemulsion, regarding LCM and especially its predominant mixed-lipid nanoparticle subpopulation. In addition, recent clinical studies with related parenteral (lipid) nanoemulsions are described; this limited clinical review provides added understanding of the development path leading to the human clinical trials -- evaluating these parenteral lipid nanoemulsions as new, (actively) targeted, drug delivery agents. Finally, throughout Parts V and VI, extensive cross-references to the earlier chapters are provided in the text. Furthermore, over 500 new literature references have been added by Parts V and VI, many of which are very recent.
The underlying chemical and biomedical principles covered in each chapter are presented in sufficient detail for this book to be useful to all interested readers worldwide with a working knowledge of chemistry, physics, and biology. Accordingly, the level of readership is intended to include graduate students, researchers, and professional people from widely varying fields. Furthermore, due to the many current and potential applications of stable lipid nanoemulsions, the appropriate readership of this book is likely to be found in industry, universities, government laboratories, and clinical facilities alike.
Thanks are due to the following colleagues for their collaboration on some of the original investigations described in this book and/or their generous
help with various experimental measurements: Elisa Barbarese, William Barker, J. Howard Bradbury, Kai-Fei Chang, Stephanie A. Ching, Michael A. Davis,
John F. Dunne, Donald C. Grant, Richard J. Guillory, Brendon C. Hammer, Shih-Yieh Ho, Toyoko Imae, Jacob N. Israelachvili, Inam U. Kureshi, Kathleen M.
Nellis, Barry W. Ninham, Noboru Oishi, Richard M. Pashley, Neil S. Reimer, P. Scott Rice, Cesareo Saiz-Jimenez, Richard H. Simon, Kent Smith,
Candra Smith-Slatas, Charles S. Springer, Ourai Sutiwatananiti, and Linda Vaught. Finally other acknowledgments, in addition to those appearing in
the chapters, include permission for: using quoted material appearing on p. 15, Copyright © 1981 by the AAAS; p. 26, Copyright © 1972 by
the ASME; pp. 9, 12, 18, and 98 Copyright © 1975, 1978, 1978, and 1974, respectively, by the Pergamon Press, Ltd.; p. 271, Copyright © 1973
by Springer-Verlag; pp. 271-272, Copyright © 1993 by the American Chemical Society; and the reprinting of Figure 12.1 on p. 216, Copyright ©
1991 by Sage Publications, Inc.
Joseph S. D'Arrigo