DOI:10.30919/es.180330

Received: 14 Mar 2018
Accepted: 30 Mar 2018
Published online: 30 Mar 2018

Surface engineering of microbial cells: Strategies and applications

Sabella Jelimo Kiprono1, 2, 3, # , Muhammad Wajid Ullah 1, 2, #  and Guang Yang 1, 2, *

1 Department of Biomedical Engineering, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, PR China

2 National Engineering Research Centre for Nano-Medicine, Huazhong University of Science and Technology, Wuhan 430074, China

3 Department of Medical Laboratory Sciences, Masinde Muliro University of Science and Technology, 190-50100, Kakamega, Kenya

* Corresponding Author(E-mail) : yang_sunny@yahoo.com


Abstract

Microbial cells (bacteria, fungi, and algae) and viruses are important part of life; which besides their harmful effects, perform several useful functions owing to their unique cell surface properties. The unique structures present on their surfaces serve as barriers between the cell and its environment and bestow them with unique functional properties. The current review describes strategies to decorate microbial cells by using different materials. It details various strategies such as layer by layer (LbL) decoration, mineralization, encapsulation, and genetic engineering among others to modify the surfaces of different microbial cells for potential applications such as environmental biotechnology, toxicology, medical microbiology, and nano-biotechnology, etc. Besides, it discusses the effects of various materials on cell viability, physiology, and functionality used for surface engineering.  This review provides fundamentals to the novice readers and insights to the seasoned researchers to pave way for their future research in the area.


Table of Content

The current review describes strategies to decorate microbial cells by using different materials.

 

 

 

Keywords

Microbial cell         Surface properties         Cell surface modification         Applications


1. Introduction

Biomineralization is a biological process used for the formation of protective structures around different microscopic single-cell organisms like diatoms algae and foraminifers. These microorganisms possess inorganic shells on their surfaces which are made of calcium carbonate and silica and potentially serve as a boundary between the cell and its environment. However, most of the microorganisms lack such structures that necessitates the introduction of artificial biomimetic shells on their surfaces.1 The process of introducing minerals or macromolecules helps in the modification of microbial cell surface thereby imparting specialized functions to them. This is achieved by using two main biological strategies: functional integration and biomimetic approach for modification of living cells.2 Entrapping  live cells inside a polymer layer at a micrometer scale where the polymer coating restricts the cell movement within the microsphere and offer protection against the varying microenvironment (pH, temperature, ionic strength, etc.) is a strategy that has been broadly applied in recent years.3,4 The pores, present in most cases in the encapsulating layer, allow the diffusion of nutrients, oxygen, wastes, and electrolytes to move across the barrier, thus, maintaining cell growth.5 Cells can also be coated with magnetic nanoparticles that allow effective spatial manipulation by application of magnetic fields. This property helps to improve control over size distribution, cell distribution and geometry within multicellular constructs, thus, giving way in tissue engineering which is a potential application in advanced regenerative medicine and many other fields.6 Polymer and nanoparticle coating of cells have been done on cells from different origin 1. The mostly studied eukaryotic organism is the yeast cell because of its cell wall characteristics that provide cell resistance.7 Bacterial cells have also been decorated with polymers and magnetic nanoparticles to obtain functionalized cells.810 Viruses on their surface lack the negative charge so they have been engineered with different minerals and nanoparticles.11

To date, a variety of strategies have been developed for the surface modification of microbial cells such as layer by layer (LbL) decoration,12 mineralization,13 encapsulation14 and genetic engineering15 among others. For example, the LbL strategy is used to achieve magnetized functionalized cells by depositing magnetic (Fe3O4) nanoparticles onto the cell surface using different polymers as mediators for the immobilization of colloidal nanoparticles. The simplicity and versatility of the LbL assembly technique paves the way for extensive applications due to the production of hybrid nanostructures with promising collective and improved functional properties.16 However, the different techniques used for surface modification of microbial cells differ in their degree of biocompatibility, sensitivity, types of materials, and effect on the viability of target microorganisms. Thus, there is an extensive need for developing more compatible strategies to deposit a variety of materials for the fabrication of broad-spectrum functionalized microbial cells.17 To date, materials of different nature such as natural and synthetic polymers, organic, inorganic, and magnetic nanoparticles, polyelectrolytes, gene and DNA, etc. have been used for the surface modification of microbial cells. The polymer and nanoparticles–based fabrication of microbial cells has been achieved for a variety of microbial cells owing to their potential applications in different fields such as biotechnology and biomedicine.1

Despite the greater potential of microbial cells to be surface-modified and availability of various materials used for their modification, the coating of living cells with certain types of nanoparticles, polymeric– and non-polymeric, and polyelectrolytes tend to have toxic effects towards their viability. Therefore, any microencapsulation strategy used for surface modification should ensure the viability of coated cells against any harmful effects of the materials used as well as environmental factors such as varying pH, ionic strength, temperature, metabolites, and osmotic stress, etc. Further, it should enhance the storage stability of the encapsulated cells. In line with cell viability, important considerations include the integrity of cell membrane, cell division, and intracellular enzymes of the functionalized cell.18 Recent interests of cell-surface modification by using various polymer nanofilms, hydrogels, minerals, and sol-gel shells, etc. have resulted in developments in several fields such as their applications in whole-cell biosensors19,20 toxicity microscreening devices17 and catalysts,21 tissue engineering,22 and bioanalytical chemistry.23

The use of inorganic micro-shells of different varieties for biomimetic encapsulation of microbial cells has been the recent area of research whose target is mainly yeast, human normal and cancerous cell lines, and bacteria, etc. for diverse applications.18 Biofabrication of microbes has provided an insight for wide range of applications such as micro devices, bio-nanomaterials and micro/nanorobots due to their different shapes and sizes.24 Therefore, this review is aimed to overview the current progress of surface engineering of a variety of microbial cells through different strategies for various applications. Emphasis has been laid on several microbes that can be potentially modified by using compatible materials. Further, various strategies employed to encapsulate different types of live microbial cells by creating an artificial shell around them have been described along with their potential merits and limitations. In addition, it addresses the effect of microbial encapsulation towards the viability of target cells to pave the way to future developments of the cell surface engineering strategies. Several important applications of surface modified microbial cells in different fields such as biomedical, pharmaceutics, environment, and industry, etc have been enumerated in detailed. Besides, this review provides a base for the development of new modification strategies, selection of appropriate materials and microbial cell types, and development of novel materials which can find potential applications in different fields.


2.  Surface modification strategies 

2.1 Layer by layer technique  

Layer by layer (LbL) is the most commonly used technique for encapsulation of microbial cells and is illustrated in Fig. 1. It involves multilayer coatings formation by exposing the cells to polyelectrolytes by alternating the charges existing of an acidic and basic component. The living cells are mainly used as functional elements of polyelectrolyte such as attachment of multilayers to the surface of the cells and the incorporation of polyelectrolyte into multilayers.12 This strategy involves formation of thin films and has received immense consideration owing to its wide choice of materials that can be used for coating particulate substrates and due to its ability to modulate nanometer control over film thickness. This fabrication technique has led to rise of functional and responsive thin films which have found potential applications in a various fields such as but not limited to bioelectronics, energy storage and conversion, drug delivery, and catalysis, etc..16

The LbL technique is a low-cost, simple, and possesses wall properties, such as texture or thickness and permeability. These properties can be controlled to a nanometer scale during the layer by modulating the ionic strength, pH or counteracting ions.25 Briefly, the first layer deposited onto the cell is composed of a polycation (cells mainly possess the negative charge in water), the second layer deposited is comprised of a polyanion. This layer is repeated until the required bilayers are obtained. Washing is done after every layer has been deposited so as to remove the traces of polymers used and finally centrifugation is carried out.18 Several studies have reported about the application of LbL strategy to encapsulate microbial cells. For instance, Fakhrullin and Minullina demonstrated the LbL to encapsulate yeast cells into artificial inorganic shells of calcium carbonate (CaCO3). Capsules of CaCO3 were formed because of precipitation of Ca2+ and CO32- ions onto the cell surfaces in aqueous solutions for several minutes. The resulting two component hybrid structures of cells and inorganic shells are formed, referred to as “core shell particles,” where the inorganic layer is 1–2 µm thick.26

Fig. 1 Illustration of (A) layer by layer strategy and (B) single layer step of polycation stabilized with nanoparticles on the surface bacterial cell.

2.2 Genetic engineering

As stated earlier, viral engineering methods like genetic recombination, PEGylation, and covalent modulations have become disadvantageous owing to their irreversibility that can easily affect several processes like viral production, infection, and the transduction processes.27,28 Fabrication strategies such as genetic engineering are more advantageous compared to the previously reported strategies. Genetic engineering involves the transformation of coat proteins by inserting amino acids which act as ligation handles for introducing peptide-based affinity tags, bio-conjugation, and to insert peptides as epitopes or targeting ligands in order to provoke the immune response.29 The changes lead to the insertion or exchange of individual amino acids to introduce side chains that allow functionalization, terminal extensions (adding sequences to C-terminus or N-terminus of each coat protein), or insertion of sequences that form surface loops30,31 or to alter the overall physicochemical properties of VNP.32 Examples of modifications include the introduction of targeting sequences that allow VNP to target-specific receptors, introduction of immunodetection tags/purification, and introduction of epitope sequences for functioning of VNP as a vaccine.33,34 The genetic material is located in the single-stranded or double-stranded fragments or in the interior of the capsid as circular. Enveloped viruses consists of a bi-lipid layer on the exterior which provides targeting specificity to the virus.35 The addition of unnatural amino acids as unique handles for subsequent chemical reactions is also possible using similar recombinant expression techniques.36

2.3 Encapsulation

Virus coat proteins self-assemble around the nucleic acids under physiological conditions, and this property, shared by the viral nanoparticles (VNPs), can be exploited to reassemble and disassemble them into more desirable structures around other cargo molecules.14 At present, two different strategies are used to trigger the cargo encapsulation; (a) unique binding interactions that occur during self-assembly, and (b) electrostatic interactions and surface charge. For efficient encapsulation process of the foreign cargo, self-assembly of viral coat proteins around a negatively charged nucleic acid is warrant.14 In viral encapsulation, the size of the cargo is the main key factor due to different sizes and its radius of curvature, which could lead to the morphological and physical characteristics of the capsid to be altered.37

2.4  Biomineralization

The deposition process of minerals around and in the cells and tissues of living organisms to accumulate and assemble is known as biomineralization. In viral nanoparticles (VNPs), this process involves the capability of virus coat proteins to nucleate mineralization or assemble around a mineral core.13 The biotemplate, that is a VNP, is exposed to other inorganic precursors or metallic, resulting in the nucleation of material on the internal or external surface due to the capsid amino acids interactions.13

A study by Pouget and Grelet described a novel mineralization process of a filamentous virus by stabilizing the virus surface with polyethylene glycol (PEG) covalently, followed by mineralization on the surface by use of silica and with titanium dioxide (TiO2) to achieve high quantity of the mineralized rods. The results showed aggregation of 1-2 nm nanoparticles on the virus surface forming an incomplete non-homogeneous mineral layer. However, the mean thickness of coated mineral layer was constant on the whole length surface of the virus 11. These three startegies have been summarized in Fig. 2.