Molecular Assemblies of Biomimetic Systems and Nanostructures

Junbai Li's Group Research Interests and Plan

  Molecular assemblies of biomimetic systems: Membrane hydrolysis. Complex assembly of lipids, proteins,
   enzymes and biomolecular motors: F0F1, Kinesin, Actin. Biogenic microcapsules. Self assembly and in vitro
   characterizations of biological molecules such as DNA, peptide and single cells as well as their mixtures with
   surfactant and polymers.
  Bio-interfaces: Molecular patterns, surface modification, molecular recognition of enzymes,
   chemical recognition at cell surfaces, transport through cell membranes.
  Design and synthesis of bioinspired molecules and materials for drug release and gene delivery
  Nanostructures: Design, synthesis, characterization and functionalization of nanoparticles, nanopatterns,
   nanotubes, nanocrystals.

Molecular Assembly of Biomimetic Systems

- Membrane hydrolysis catalyzed by enzyme

        Several newly developed microscopic and spectroscopic techniques enable to provide quantitative and qualitative information with the molecular orientation and reorganization process of lipid monolayers at the air/water interface. Combination of these techniques is used to in situ trace the hydrolysis process of spreading lipid monolayer catalyzed by different phospholipases, such as Phospholipase A2, C and D. With information of morphology, structure and component analysis of assembled lipid monolayer after or during the hydrolysis, it can provide a better understanding of hydrolysis mechanism of membrane catalyzed by enzymes.

                  
                        Enzymatic Reaction with the Interface of a Membrane Consists of a
                                      Molecular Recognition Process and the Cleavage Reaction

- ATPase assembled into microcapsules for ATP biosynthesis

        Biomolecular motor, ATPase assembled in lipid-modified polyelectrolyte microcapsules is able to perform the process of ATP biosynthesis and provides a novel routine to fabricate bionanodevices. This assembled complex can not only help us to understand the biological function of ATPase molecules but also such an artificial designed system containing ATPase provides a well-defined container for the storage of energy currency, ATP. When vital activities need energy, ATP will be released across the wall of the capsules as power supply.

                         
                                         CFoF1-ATPase in Lipid-coated Capsules

- Capsules as “Cargo” driven by linear biomotor along microtubules

        Modification of capsules with specific motor proteins enables to move along microtubules. It is particular interesting to take use of capsules as “cargo” to delivery drugs, biological molecules or catalysis in the living systems. This not only simulates the transport of vesicles, but also be useful for studying the process of drug release and designing novel bionanodevices. Biocompatible micro- and nano-capsules can be constructed by using layer-by-layer assembly technique and the template method. Through assembling lipid bilayers containing some recognition units on the surfaces of various capsules, it anticipates that the movement of the capsules along microtubules will be observed directly by fluorescence microscopy.
                              
                                    Microcapsule as “Cargo”                         In vitro record of assembled capsules running along the microtubes

- Biocompatible microcapsules

        Coating of phospholipid bilayers on the surface of multilayer polyelectrolyte microcapsules may improve the biocompatibility and permeation of the polymer shells. Leaving such lipid-modified capsules in an enzyme, for instance, phospholipase A2 solution, one can expect to change the permeability of the lipid coated microcapsules due to the hydrolysis reaction occurred in the lipid bilayer. Thus a capsule with adjustable permeability can be obtained. With this treatment, the system is possibly considered as a model for the controlled drug release.

- Biogenic capsules

        Polyelectrolyte capsules were eventually made by coating size controlled templates with alternating polyelectrolyte adsorption and followed by removal of the template cores. However, if core dimensions approach the persistence length and charge density for sufficient coating, this is especially demanding for biocompatible materials. Therefore, as an important extension of shell materials, the preparation of biogenic capsules with properties as good as hitherto for synthetic polymers is a big challenge. Proteins or the mixture with lipids, alginate with chitosan as efficient encapsulation materials can meet perfectly the demand.

                            
         Preparation of Hollow DMPA/HSA Multilayer Capsules                 Sustained Release Encapsulated Ibuprofen from
                                                                                                                                                 HSA/DMPA Microcapsules

Design and Synthesis of Bioinspired Materials for Drug and Gene Delivery       

- Assembled peptide vesicles for gene delivery and release in cells

        The synthesized multivalent cationic peptide-lipid (MCPL) is considered as DNA delivery to mammalian cells. The MCPL can form dispersed liposomes. The binding of MCPL liposomes with DNA can be detected by a standard ethidum bromide (EtBr)-DNA fluorescence quenching exclusion assay. As such assembled DNA/EtBr/MCPL solution is incubated with a trypsin. The fluorescence intensity increasing after trypsin hydrolyzed the headgroup of MCPL suggests the DNA release from the complexes. The enhanced transfecting efficiency of MCPL with DNA is highly expected. We intend to design and synthesized several different types of cationic peptides for this purpose.

                  
          DNA Release from Cationic Lipopeptide Vesicles                    Gene Transfection Efficiency and Cytotoxicity
                                                                                                                                          Results after 48h in HeLa Cells

- Encapsulated Photosensitive Drugs by Biodegradable Microcapsules to Kill Cancer Cells

       Water-insoluble drugs, photodynamic HB can be accumulated in hollow microcapsules via nonspecific binding. The surface charge of the microcapsules show the key factor for cellular uptake, and the outer layer of the microcapsules with positively charge is effective in transporting HB cargos into the cells. The biocompatible microcapsules which loaded with HB showed high cytotoxicity after exposure to visible light, indicating that microcapsules are efficient anti-cancer agent cargo. The uniqueness of microcapsules as molecular transporters is beginning to emerge and needs to be fully developed. Microcapsules could become a new class of molecular transporters for in vitro and in vivo delivery applications.

                  
        Images of (ALG/CHI)4 capsules filled with hypocrellin B taken up by                   Photocytotoxic effects on MCF-7 cells
                               MCF-7 cells cultured for 2 days

                      
                  Hypocrellin B (HB) is accumulated in water-solubilized natural polyelectrolyte microcapsules
                  for drug delivery. HB loaded capsules are subsequently taken up by cancer cells, HB will be
                           released with a cytotoxicity to kill cancer cells after irradiation

- Conversion of dipeptide for gene delivery through cell membrane

       Positively charged dipeptides can self-assemble into the structure of vesicles spontaneously under a certain condition. Such a conversion could readily bring gene into cells through membrane. The self-assembly behavior of dipeptide nanostructure can be exploited as a new class of molecular transporter for the delivery of a wide range of foreign substances such as drugs and proteins. We are interesting in investigating the conversion process quantitatively and building up models. Several relevant systems will be developed.
       
                              Intercellular Uptake of Vesicles from Reassembly of Dipeptide
                                                          Nanotubes with Green Labeled Ss-DNA

- Self-assembled organogel of diphenylpeptide

       Low molecular weight organogelators are a distinct class of soft material and low molecular weight gelator compounds can self-assemble into the organogelators with 3D networks through nonconvalent forces. Subsequently, they can encapsulate a large volume of organic solvents under suitable conditions and lead to the formation of gel-like phases. We found the formation of a novel self-assembled organogel using a small molecular building block, diphenylalanine peptide. Formation of this newly discovered gel is driven by the hydrogen bond of peptide main chains and the π-π interactions between aromatic residues of the peptide. Such an organogel may find its applications in structure-directing agents for synthesis of nanoporous materials, templates for assembling nanoparticles or nanotubes, electro-optical display materials, drug delivery and others.

Nanostructure

                                                                                                                                                  gel
                  
        Self-assembly mechanism of the dipeptide: Aromatic groups of FF peptides stack through π-π interactions, the resulting
        molecular stacks further assemble to nanofibrils. Such nanofibrils intertwist each other to form the peptide organogel

- Protein supported lipid patterns for the targeted recognition

        Supported lipid micro- nanopatterns is one of the most popular biomembrane models which can be applied to fundamental studies of cell membrane science and the engineering of integrated lipid membrane microdevices. Our aim is to fabricate stable lipid bilayer patterns to create possibility in corporating specific components like channels or receptors for specific recognition, which allows transferring materials (like drugs) to a solid surface for the medical application.
                                      
              Human Serum Albumin Supported Lipid Patterns for the Targeted Recognition of Microspheres
                                               Coated by Membrane based on ss-DNA Hybridization

- E-Coli recognition via glycolipid pattern

        Biomembrane surfaces contain a number of glycolipids and glycoproteins acting as intermediates for biological communications and recognition processes. Bacterial adhesion on biomembrane surface will eventually cause infection. Therefore, the investigation of such adhesions accompanying recognition will help us to understand the mechanism of interaction to establish a rapid detection method to E.coli. Glycolipid arrays play a key role in a rapid detection and classifying method for pathogens based on their adhesion profile. We try to build up a simple design to fabricate glycolipid microarrays, which is capable of detecting E.coli bacteria. Such patterns are expected to extend the technique of bacteria detection with a higher resolution.

         
- Biointerfacing molecular pattern

        Selective electron irradiation on self-assembled thiol-aromatic biphenyl monolayer (collaborated with Prof. M. Grunze's group) will cause the reduction of the terminal nitro groups into amino groups, which allows introducing initiators for the further polymerization. The resulting polymer structures will exhibit a good contrast between the functionalized and unfunctionalized regions in term of their chemical and physical properties. Such a combination is anticipated to result in a pattern formation with a superior control of amplification and chemical functionalities. Lipid coating on the patterned surface will create a biointerface, which is used as supporters for controlled adsorption and growth of cells. The detailed investigation of adhesion forces and growing behavior of living cells needs to develop a fine well-defined pattern structure at a nanoscale.

       
                         Thermosensitive Polymer Nanopatterns through Atom Transfer Radical Polymerization

- 3D polymer gradient structures created by Chemical lithography and one-step polymerization

        Advances in nanoscience and nanotechnology require a simple and rapid technique to fabricate complex topographic nanostructures of functional organic thin films with different height and/or varying height on solid surfaces. Potential applications are envisioned in optical and mechanical nanodevices, and in microfluidic systems. Chemical Lithography with ATRP can fabricate complex gradient structures of stimulus-responsive polymer brushes with controlled size, shape, position and thickness on a gold substrate. 3D micro- and nanostructures were grown by grafting the polymer brush from an initiator-coated surface. The dependence of the PNIPAM brush height on the density of the initiator (controlled by the e-beam doses in the chemical lithography process) allows the preparation of spatially defined polymer patterns with varying heights. Their swelling behavior in water and the temperature dependence of swelling follows what is known for PNIPAM hydrogels, and also their Young′s modulus in the swollen state agrees with the bulk value.

           
         The preparation of different-height polymer brush patterns by Chemical lithography and surface-initiated ATRP.

- Template synthesized polymer nanotubes

        Most synthesized nanotubes exhibits a good perspective applied in the biological or medical field, for instance for bioseparations or materials transport. At this stage, precisely controlling of the inner diameter and biocompatibility of the synthesized nanotubes are highly required. Template synthesized polymer nanotubes have the obvious features of good flexibility and mechanical stability. With the combination of self-assembly or layer-by-layer assembly techniques one can modify the inner pores in different way through electrostatic absorption, covalent bond, hydrogen bond or chemical reaction to obtain micro-nanosize tubes. The achieved nanotubes may contain the features of biocompatibility, luminescence, biodegradability or thermosensitivity, which expects the potential applications in polar cells, drug delivery, biocatalysis or tissue engineering.

	(Chitosan/Alginate) Nanotubes Uptaken in Living Cells
Highly Flexible (PAH/PSS) n Nanotubes
Thermosensitive PNIPAM-co-MBAA Nanotubes

- Decoration of gold nanoparticles

        The nanocomposites of gold nanoparticle (AuNPs) with various macromolecules display much potential applications in the fields of biology and nanotechnology. Surface-initiated atom-transfer radical polymerization on the AuNPs surface provides a perfect core-shell nanostructure and will alter the property of nanoparticles and response to interface and environment. Such nanosized hybrids are considered for delivery of biomolecules, catalysts or drugs.

         
                    Thermosensitive complex Nanoparticles                        pH-sensitive Polymer and Au Nanoparticles Composite

- Binary thermo-sensitive copolymer

        Smart nanocomposite of gold nanoparticles and block copolymer of poly(N-isopropylacrylamide) and poly(methoxy-oligo(ethylene glycol) methacrylate) was fabricated by consecutive surface-initiated ATRP. These Au@copolymer nanocomposite displays core-shell nanostructures and has two thermosensitive points at about 33 and 55°C, respectively corresponding to the conformational transition of inner homopolymer and peripheral oligo(ethylene glycol) moieties. Interestingly, the slightly cross-linked Au@copolymer nanostructures can successfully encapsulate silver nanoparticles in-situ to obtain Au@Ag nanocomposites which display a thermally modulated catalytic activity.

                 
                              25°C                                   40°C                              65°C

- Semiconductive nanocrystals

        We created a simple route to construct hierarchical hollow microspheres and microcubes of MnO2 nanosheets through self-assembly with intermediate crystal templating process The discrete spherical and cubic hollow MnO2 nanostructures can be prepared with controlled morphologies by changing the morphologies of MnCO3 precursors, which can be simply obtained by adding the (NH4)2SO4 solution in the reaction system, and the thicknesses of the shells of hierarchical hollow nanostructures can be adjusted readily by the relative quantities of KMnO4 reacted followed by selective removal of MnCO3 crystal template with HCl. When used as adsorbent in waste-water treatment.

                 
                                                   CdS Nanocrystal                                                          Absorption of Congo red with time by new MnO2
                                                                                                                                          microspherical hollow hierarchical nanostructures
                                                                                                                                                       at different time intervals
                 
        Process of MnO2 hierarchical hollow nanostructures formation after MnCO3 was added in the KMnO4 solution
                at different time intervals and after the removal of the core with hollow shell and microcubic structure.