The document discusses electrosomes, a novel surface-display system combining redox enzymes for fuel oxidation to generate electricity, consisting of a hybrid anode and cathode. It details the preparation methods, advantages, disadvantages, and applications of electrosomes, which include drug targeting and cancer treatment. Key components are enzyme binding to scaffoldin and the characterization of biofuel cells, highlighting the potential for high power outputs using enzymatic cascades.

1. Preparation And Application Of
Electrosomes
HOD And Assistant Professor
Department of Pharmaceutics
Presented by:
M. Pharm 2nd Semester
Department of Pharmaceutics
MR.SHUBHAM SHARMA

2. Contents
 Introduction
 Objectives of Electrosomes
 Method of preparation
 Advantages Electrosomes
 Disadvantages Electrosomes
 Application
 Reference

3. Introduction

4.  The electrosomes, a novel surface-display system based on the specific
interaction between the cellulosomal scaffoldin protein and a cascade of
redox enzymes that allows multiple electron-release by fuel oxidation. The
electrosomes is composed of two compartment:(i) a hybrid anode, which
consists of dockerin-containing enzymes attached specifically to cohesin sites
in the scaffoldin to assemble an ethanol oxidation cascade, and (ii) a hybrid
cathode, which consists of a dockerin-containing oxygen-reducing enzyme
attached in multiple copies to the cohesin-bearing scaffoldin.
 The electrosomes was designed for use both in an anode and a cathode
compartment; in each compartment, the unique attributes of the cellulosome
scaffoldin give a different advantage.

5.  In the anode, the ethanol oxidation cascade consists of two enzymes, ADH and
formaldehyde dehydrogenase (FormDH), both containing a different dockerin
module of Acetivibrio cellulolyticus and of Clostridium thermocellum, C.
thermocellum (zADH-Ac and pFormDH-Ct), respectively, assembled on a
‘designer’-scaffoldin chimera displayed on the surface of S. cerevisiae.
 At the cathode, copper oxidase (CueO) was selected for surface-display. CueO is
a multi-copper oxidase enzyme expressed by E. coli that catalyzes the oxidation
of Cu(I) ions coupled to oxygen reduction to water.
 The different constructs used for assembly are depicted. We report the
characterization of the dockerin-containing enzymes and their electrochemical
activity using a diffusing redox mediator.

9. Objective of Electrosomes
 The molecular roots of our actions and the thoughts and feelings that drive
us to act are ion channels, proteins that form macromolecular pores in cell
membranes. These transmembrane proteins generate and propagate the
electrical signals that allow us to sense our surroundings, process
information, make decisions, and move.
 Ion channel proteins act as gates that span the lipid bilayer that surrounds
all cells where they open and close to allow the flow of ions down their
electrochemical gradients.
 The ion flux through a channel pore can be extremely high, ≈106 ions per
second. Because of their central role in the function of the excitable tissues
such as heart, brain, muscles, and nervous system, investigators have long
sought to understand ion channel properties from a molecular perspective.

10.  The pore-forming domains of most ion channels are multimeric assemblies
possessing cyclic symmetry in a general architecture known as barrel-stave. A
fixed number of subunits assemble around the axis of the ion conduction
pore.
 The very nature of these molecules—transmembrane proteins that are
difficult to obtain in the large quantities and high purity necessary for
structural investigation—has impeded attempts to obtain the most essential
information for understanding their functions, a three-dimensional
description of their molecular architectures at high resolution.

11. Method of Preparation
1. Strains and Constructs method.
2. Enzyme Binding to Scaffoldin.
3. Biofuel-Cell Assembly and Characterization.
4. Protein Expression.
5. Enzyme Activity Assays.
6. Construction of YSD of Chimeric Scaffoldins.
7. Cyclic Voltammetry (CV) and Chronoamperometry (CA).

12. Strains and Constructs method
 The genes encoding dockerins of Acetivibrio cellulolyticus and Clostridium
thermocellum were cloned and ligated to the C-terminus of Zymomonas mobilis
alcohol dehydrogenase and to Pseudomonasputida formaldehyde dehydrogenase
by standard methods.
 The dockerin module of C.thermocellum was also ligated to the C-terminus of
CueO (CueO-Ct) of E.coli.
 All the dockerin-containing enzymes encoding genes have been cloned into the
pET15b vector for expression in E. coli, yielding the pET15b-zADH-Ac, pET15b-
pFormDH-Ct, and pET15b-CueO-Ct vectors.
 For controls, the genes encoding the native enzymes without an appended
dockerin module were also cloned in the same vector, yielding plasmids pET15b-
zADH, pET15b-pFormDH, and pET15b-CueO.
 All the chemicals used in this study are detailed in the SI section.

13. Enzyme Binding to Scaffoldin
 2.0 mL of yeast cells displaying scaffoldin, for which absorbance at a wavelength of
600 nm was 1.0 , were incubated with bacterial lysates containing the expressed
enzymes at room temperature for 1 h. 1.0 mL of the bacterial lysates were used for
the binding, which was performed in a final volume of 15 ml.
 As a binding buffer, 50 mM Tris buffer at pH 8.0 with 1 mM CaCl2 was used. Upon
binding, the yeast cells were precipitated, and binding was repeated using fresh
lysate.
 After the second binding cycle, the yeast cells were washed four times in the buffer
to remove non-specifically bound enzymes.
 For the CueO-Ct binding, the yeast cells were suspended in 0.1m acetate buffer pH
5.0 containing 1 mm CaCl2 after the last wash.
 Following binding, the yeast cells were resuspended in 2.0 ml of buffer.

14. Biofuel-Cell Assembly and
Characterization
 Air was continuously purged to the fuel-cells. A potentiostatically controlled anode
set to −0.2 V versus Ag/AgCl was used.
 In all experiments, the cells were left to stabilize overnight, following fuel cell
assembly, before characterization was performed.
 The characterization of fuel cell performance was done by measuring the voltage
of the cells under variable external loads.
 A background current cell was used as a negative control for all fuel cell
experiments and did not contain any yeast. Graphite rods of 5 mm diameter
served as both anodes and cathodes.
 The counter electrode that served for the potentiostatically controlled electrode
was of a larger surface area, as described for the CV and CA measurements.

15. Advantages
 It perpetuates the endurance of active drug molecule in the systemic
circulation.
 Deferment the elimination reactions of promptly metabolize drugs and
contributes to controlled release.
 Incorporates both hydrophilic and lipophilic drugs.
 Intensifies the stability of medicament.
 Cost of therapy is minimized by reducing the dose per unit formulation
 Elevate bioavailability especially in water disfavouring drugs.
 Selective uptake by tissues due to direct drug delivery.

16. Disadvantages
 The production cost of electrosomes are generally high since these come
under the class of nanotherapeutics.
 The constituent phospholipids present in lipid vesicular structures may
undergo oxidation or hydrolysis.

17. Application
 They use enzymatic reactions to catalyze the conversion of chemical energy to
electricity in a fuel cell.
 The use of enzymatic cascades in enzymatic fuel cell anodes resulted in very
high power outputs, as the electron density achieved was much higher when
the fuel was fully oxidized.
 Its used as a carrier in drug targeting.
 Used in the treatment of cancer.
 Used in studying immune response.
 Ear targeting
 Muscle targeting

18. Reference
1. SHEFRIN S, SREELAXMI C. S, VISHNU VIJAYAN, SREEJA C.
NAIR.ENZYMOSOMES: A RISING EFFECTUAL TOOL FOR TARGETED DRUG
DELIVERY SYSTEM.Int J App Pharm. 2017;9(6);1-9
2. Szczupak A, Aizik D, Moraïs S, Vazana Y, Barak Y, Bayer A E, Alfonta L. The
Electrosome: A Surface-Displayed Enzymatic Cascade in a Biofuel Cell’s
Anode and a High-Density Surface-Displayed Biocathodic Enzyme.Nano-
material.2017
3. www.science direct.com