Research

Research and Applications

"Colloids and Surfaces, one particle or two particles at a time"

Overview:  SPSE is an interdisciplinary research center concerned with experimental and theoretical research within the broad field of colloid and surface chemistry.  Centered around a technique called micropipet manipulation it can form, study, and characterize materials as microparticles in both the inanimate realm of metals, ceramics, plastics and composites (traditonal materials) and the biological realm of lipids, sugars, amino acids, nucleotides, salts and water. With three states of matter (gas, liquid and solid) and therefore five interfaces (G-L, G-S, L-L, L-S, S-S), there are many combinations and compositonal variations.  What the center generates though is a fundamental understanding of the way in which these materials and their interfaces behave, including, forming microparticles of gas and liquid in a second liquid solvent, and measuring, for example, the rate of dissolution of the gas or liquid as a dissolving microsphere.  If the liquid contains a solute (such as a salt in water or a polymer in organic solvent) and the initial solvent dissolves, then we can observe crystallization or the formation of amorphous (glassy) materials as solids.  Interfacial interactions and adsorption of surfactants are seen as changes in response of a curved interface to pressure applied in the micropipet, and also from the formation of a skin of solidified material.  Interactions between particles are characterized by briging two microdroplets or particles together to observe spreading, coalescence or stable adhesion as shown at the top of the page for a so called Droplet Interface Bilayer (DIB) made from gyceryl mono-oleate between two water microdroplets in a squalene-decane mixture.  Basically it is surface and colloid science one particle or two particles at a time. 

The mission now is to bring all of these experimental techniques, understanding and characterized phenomena to specific applications for a range of specific materials, and material compositions.  Applications include physics and chemistry of soft materials; nucelation and growth of precipitates, crystals and amorphius materials;  development and behavior of pharmaceutics as advanced drug delivery systems; mixtures of materials in complex multiphase-multicompoent emulsions in, for example, the food industry or pharmacy; and the manipulation of individual and pairs of cells, their interactions with each other and other surfaces as well as their mechanical and physical properties.

Current Projects

1. The Dissolution of Liquid Microdroplets into a Second Solvent (fundamental studies into dissolution mechanisms, diffusion coefficients and solubilities, with applications in chemical, pharmaceutical, and food industries) 
2. The Precipitation  of Various Solutes (polymers, salts, drugs and oils) from Dissolving Solution Microdroplets (fundamental studies in phase behavior, again with applications in chemical, pharmaceutical, and food industries)
3. Microglassification of Proteins and Peptides (preservation and formulation of protein therapeutics, including antibodies)
4. Equilibrium and Dynamic Interfacial Tensions (development and testing of new lung surfactants; reactions and phase separations at interfaces)
5. Endogenous-Inspired Anti-Cancer Treatments: Formulation of Hydrophobic Drugs (Reverse engineering nature’s designs for new approaches to anti-cancer drug delivery, especially for metastatic cancer)
          A. Phase Behavior and Mixing (Non-Mixing) of Triolein-Cholesteryl Oleate-Cholesterol-Lecithin Emulsions
         B. Fabrication and testing of a Pure-Drug, Ligand-Targeted Nanoparticle for Metastatic Cancer
6. Chemical and Biological Reactions at Micro Droplet Interfaces (Project with FLinT)  (observing and characterizing interfacial chemistry at the surface of an aqueous microdroplet and an oil phase, or vice versa) 
7. Droplet Interface Bilayers (Using the bilayer formed between two water droplets in oil to study physical, chemical and transport properties of lipid bilayer membranes

1.The Dissolution of Liquid Microdroplets into a Second Solvent. 
Following recent work that characterized the dissolution of water into organic oils (Su et al, 2010), fundamental aspects of this project provide a direct measure of the diffusion coefficient of molecules of the microdroplet into the surrounding solvent.  Thus, the droplet could be an oil dissolving in an aqueous phase, or water dissolving in an organic liquid.   Either way, knowing the solubility of the dissolving material provides a direct measure of the diffusion coefficients for these molecules when analyzed using a solubility diffusion model.  Interesting applications arise when the surrounding medium contains other solutes or nanoparticles that can act as a sink for the dissolving material, such as liposomes where the rate of dissolution of an organic drug is now influenced by partitioning into bilayers. So, here we would be interested in the partitioning of an organic molecule not just into water but also into a membrane sink.  This kind of experiment can have a direct bearing on understanding stability of a drug or drug delivery system in the blood stream of a patient.  It can also lay the foundation for understanding the stability of one component of a multi-component system during processing in pharmaceuticals, or food/nutrition formulations.

Ethyl Acetate Microdroplet (30 micron diameter) dissolving in water

Related manuscript: 

  • J. T. Su, P. B. Duncan, A.Jutilla, and D. Needham, (2010).  The Effects of Hydrogen Bonding on the Diffusion of Water in n-Alkanes and n-Alcohols Measured with a Novel Single Microdroplet Method, J. Chem Phys 132, (4),  044506http://www.ncbi.nlm.nih.gov/pubmed/20113048

2. The Precipitation  of Various Solutes (polymers, salts, drugs) from Dissolving Solution Microdroplets
Just as in Project 1, in this project we can dissolve a solvent into a second phase solvent, but this time the microdroplet is a solution. Now the solute, which could be for example, a polymer, a salt or a drug, will be left behind if it is relatively insoluble in the dissolving medium.   In this experiment, the whole solvent loss and so process of passing along the phase diagram is observed in real time, including any initial liquid-liquid phase separation, crystallization, glassification or how a single phase microparticle forms.  There is an infinite number of single solute or mixed solute solutions that can be studied (see Projects 3 for proteins and peptides, and 4 for cholesteryl ester and lipids), it all depends on the application.  In any event this project teaches the fundamentals of phase behavior, mixing, de-mixing and phase separation.  The example shown below is of a solution of polystyrene (PS) in ethyl acetate (EtAc), formed as a microdroplet in an aqueous phase.  As the ethyl acetate leaves, a new polystyrene-rich phase forms as internal nano and then microdroplets inside the large dissolving microparticle.  These PS-rich droplets coalesce as more and more EtAc dissolves out, eventually leaving the polystyrene microdroplet on the end of the pipet.   So bring a system you are interested in, or an application that might need a new solution using micro or nano particle formation, and let’s work out a project.

 

Microdroplet solution of polystyrene in ethyl acetate, formed as a microdroplet in an aqueous phase; EtAc leaves as PS-ric phase eventualy forms PS microsphere.

Related manuscripts: 

  • Chiming Yang, David Plackett, David Needham, Helen M. Burt (2009) PLGA and PHBV microsphere formulations and solid-state characterization: Local delivery of fusidic acid for the treatment and prevention of orthopaedic infections, Pharmaceutical Research, Vol. 26, No. 7
  • Samuel E. Gilchrist, Deborah L. Rickard, Kevin Letchford, David Needham & Helen M. Burt (2012). The phase separation behavior of fusidic acid and rifampicin in PLGA microspheres, published on line,  Molecular Pharmaceutics. DOI: 10.1021/mp300099f • Publication Date (Web): 07 Apr 2012.

3. Microglassification of Proteins and Peptides
A third project is along the lines of experiments we have already done on a protein like lysozyme (Rickard et al 2010), where we formed microglassified beads of protein by removing the water solvent into a second dissolving solvent like octanol.  An accurate measurement of the diameter of the protein-solution microdroplet as the water is removed into the surrounding alcohol not only gives the concentration of protein (which can reach ~1100mg/ml), but also the amount of water that remains bound to each protein molecule.  A model for protein packing then gives a measure of the water of hydration and even the decay constant for this water of hydration of 1.7Å, i.e., after the removal of bulk water solution and before strong protein-protein steric forces are encountered.  We have used the same technique to characterize the micro-glassification of Albumin (Alb) and Hemoglobin (Hb), and even seen that Hb can still exchange oxygen in its microglassified state.  The technique is now ready to be used to characterize a range of proteins, peptides and other macromolecules including new biologic therapeutics. Applications are again in new pharmaceutical formulations where micro and nano microglassified beads are more suitable for polymer encapsulation, and where the more sensitive and delicate new biologic therapeutics require a gentler processing environment.

Formation of a Microglassified Lysozyme bead (~15 microns diameter) by removing water of solution into decanol

Related manuscripts:

  • Deborah L Rickard, Brent Duncan and David Needham, (2010). Hydration Potential of Lysozyme: Protein Dehydration Using a Single Microparticle Technique, Biophysical Journal, Volume 98, Issue 6, 17 March 2010, Pages 1075-1084

4. Equilibrium and Dynamic Interfacial Tensions
Well-defined interfaces of air-water, air-oil, and water-oil, can readily be formed and manipulated inside a tapered micropipet.   Following methods developed in two papers in 2001, (Lee et al 2001), both equilibrium and dynamic interfacial tensions (T) can be measured just from a knowledge of the pressure (P) in the pipet required to move the interface to a position in the pipet characterized by a radius of curvature (R).   The simple Laplace equation of P = 2T/R then gives T from a plot of P vs 1/R from the slope (of 2T).  In addition to characaterizing clean interfaces or air with liquids or between immiscible liquids, this technique provides measures of the changes in interfacial tensions when, for example, surface active agents, proteins, or drugs adsorb at air-water or oil-water interfaces.  Applications here include measuring the interfacial tensions for new synthetic lung surfactants, and perhaps even using these pre-formed lipid-peptide monolayers as simple models for drug interactions with lung epithelia.  Additionally, this technique provides fast and reliable measurements of interfacial tensions for emulsions or gas microbubbles that are exposed to surfactants, proteins, peptides, drugs, or polymers, bringing new insights into formulation processing in pharmaceutical and food industries.

Air-water interfacial tension measurement in tapered micropipet (radius of curvature ~25 microns).

Related manuscripts:

  • S. Lee, D. H. Kim, and D. Needham.  (2001) Static and Dynamic Surface Tension Measurement of Microscopic Interfaces Using a Micropipet Technique: 1.  A New Method for Determination of Surface Tension, Langmuir 200117, 5537-5543.
  • S. Lee, D. H. Kim, and D. Needham.  (2001) Static and Dynamic Surface Tension Measurement of Microscopic Interfaces Using a Micropipet Technique: 2.  Dynamics of Phospholipid Monolyer Formation and Equilibrium Spreading Pressures at the Air-Water Interface, Langmuir 200117, 5544-5550. 

5. Endogenous-Inspired Anti-Cancer Treatments: Formulation of Hydrophobic Drugs

A. Phase Behavior and Mixing (Non-Mixing) of Triolein-Cholesteryl Oleate-Cholesterol-Lecithin  Emulsions
By making single droplets composed of mixtures of Triolein-Cholesteryl Oleate-Cholesterol-Lecithin we can determine the mutual solubilities of each lipid in another.  In the experiment, one or more suitable solvents are chosen; these may be for example chloroform, dichloromethane, or ethyl acetate.  Each component is dissolved in the solvent and single microdroplets are created at the end of the micropipet in an aqueous phase such as dilute (5mM sodium dodecyl sulfate).  We then observe what happens when the solvent dissolves out of the droplet into the water phase leaving the lipid solute behind as a solid or liquid phase microparticle.  For example, if cholesteryl oleate (CO) is mutually soluble in triolein (TO), then a single phase droplet is formed; if not then the liquid TO will separate from the solid phase CO.  Similarly for cholesterol and its binary and even tertiary mixtures.  The overall goal is to provide composition-structure-property relationships that characterize these mixtures, and to compare and contrast them with what is known about the natural LDL.   These data then feed directly into project 6 which is to generate an artificial version that can contain pure drug as the core and perhaps lipid and cholesterol as the encapsulating monolayer, and is tested for its ability to target LDL receptors and be taken up by, and kill cancer cells.

Related manuscripts:

  • Kurt W. Miller and Donald M. Small. Triolein-Cholesteryl Oleate-Cholesterol-Lecithin  Emulsions:  Structural  Models of  Triglyceride-Rich Lipoproteins , Biochemistry 1983, 22, 443-451

B. Make and Test a Pure Drug, Ligand-Targeted,  Mimetic of a Low Density Lipoprotein Particle
The overall scientific purpose of this project is to first explore the body’s endogenous processes for how we make, transport, and our cells take up the natural fat nanoparticle Low Density Lipoprotein (LDL). It is then to utilize what we learn in order to design, create, and test new formulations for anti-cancer drug delivery that follow these endogenous processes and pathways.  The reason this is important is that, in order to support their uncontrolled rapid growth and spread throughout the body, cancers are known to take up LDL by increasing their number of LDL Receptors (LDLR).  In fact, the cells that are found to take up most LDL are known to be the most metastatic.  A woman with LDLR+ beast cancer has a worse prognosis for survival than if she was LDLR-.  Our approach is to reverse engineer the LDL by first understanding how it is made in the hepatocyte, transported through the blood stream, and taken in and processed by cancer cells. It is then to develop similar bioinspired methods for fabrication, administration, and delivery of the new molecularly-targeted drugs to cancer cells with a pure-drug peptide-targeted nanoparticle --“put the drug in the cancer’s food”.  The project will be carried out in tandem with project 5A, and use the data from microparticle studies to understand the phase and mixing relationships between the natural LDL components, (cholesteryl ester, triglyceride, cholesterol and phospholipids). It would then take this data and explore drug-solvent-surfactant combinations, along with top-down (homogenization), bottom up (micelle swelling) and even nature’s own processes for making LDLs as new methods to develop the pure drug nanoparticles.  As with Project 5A, this is classic material science but now with cancer biology included; and so a strong background and interest in materials and cell and molecular biology will be required for this project.

6. Chemical and Biological Reactions at Micro droplet Interfaces (Project with The Center for Fundamental Living Technology (FLinT)

In a new project with the The Center for Fundamental Living Technology (FLinT), we are interested in observing and characterizing Chemical and Biological Reactions at Micro-droplet Interfaces.  An example of one reaction is that between a microdroplet of concentrated sodium hydroxide and olive oil.  When a single microdropet of 3.5M NaOH is formed in a liquid phase of olive oil (mainly triglyceride), the triglyceride is hydrolysed to the free fatty acid, which, because of the high sodium content, forms solid crystals of sodium oleate at the droplet-oil interface.  This so called, "Bütschli Dynamic Droplet System" is seen in real time in this video from a preliminary experiment is the formation and build-up of this "crusty" crystal coating.  The idea now is to more fully characterize this particular reaction and ten explore others including enzyme conversions of surface active materials

Sodium Oleate crystalization at the Interface between NaOH solution microdroplet (20pL) and excess Olive Oil.

Related manuscript:
Armstrong R, Hanczyc MM. 2012.  Bütschli Dynamic Droplet System. Artificial Life, accepted.
Links:  http://www.nature.com/news/2011/110223/full/news.2011.118.html

7. Droplet Interface Bilayers

In 2000 we did an experiment that formed a bilyer between two water droplets manipulated in a hydrocarbon oil phase.  Subsequently, a collaboration with Hagan Bayley's group in Oxford developed this system to include hemolysin protein channel and a new field was born.  The Bayley group and indeed other researchers, most notably Donald Leo at UVA, have taken this simple concept and expanded its potential with many differnt coppositions and multiple drioplet arrangements.  We can offer this as a system for graduate study, for collaboration and for developing new applications.

Forming the Droplet Interface Bilayer from glycerylmonooleate monolayers on the water droplets as they are brought into contact using the micropipets

Related manuscripts:
M.  Holden, D. Needham, H Bayley, (2007). Functional Bio-Networks from Nanoliter Water Droplets J. Am Chem Soc., vol. 129, pp. 8650-8655
Hagan Bayley,  Brid Cronin,  Andrew Heron,  Matthew A. Holden,  William Hwang,  Ruhma Syeda,  James Thompson, and  Mark Wallace.  Droplet interface bilayers. Mol Biosyst. 2008 4(12): 1191–1208