Prospective Bacterial Minicells for Drug Delivery Systems

Abstract

Drug delivery system (DDS) is an important challenge in medicine over the conventional drug delivery system in case of therapeutic efficacy. In recent years, due to the shortcomings of conventional chemotherapy such as poor bioavailability, low treatment index, and unclear side effects, the focus of drug development and research has shifted to new nanocarriers of chemotherapeutic drugs. By using biodegradable materials, nanocarriers generally have the advantages of good biocompatibility, low side effects, specific target, controlled release profile, and improved efficacy. There are many kinds of DDS such as lyposome, vesicle, peptide, gene, microchip, polysaccharide and so on being studied nowadays. Each DDS has the advantages and disadvantage. However, the materials made them are expensive and the preparation techniques sometimes are complicated. Moreover, those DDS are rarely shown the ability in drug delivery to target. In the study, nano sized bacterial minicells were showed to clarify the importance of this material in drug delivery and target therapy.

1. Introduction

The drug is delivered via entering the body, traveling through the bloodstream to go to the target and then eliminating out of the body [1, 2]. DDS is oriented to limit unwanted effects in human pharmacology. DDS must be biodegradable in the body, suggesting that DDS should be researched and exploited from various materials to obtain high bioavailability toward the target from which the possibility of drug resistance and side effects will be reduced, in addition to carrying drugs. Lyposomes, peptides, vesicles, polysaccharides, aptamers, viruses, and DNA are much being researched. However, each DDS can bring drugs and especially toward the target is valuable. Furthermore, targeted DDS is developed to achieve a higher level of treatment due to the disease target specificity but reduce side effects. Nanoparticles can also accumulate in the healthy organ where they can cause side effects [3, 4]. Nanoparticles must be potential to the target of the body, control drug release, and dramatically increase the bioavailability of active compounds. Various materials have been used to prepare nanoparticles for drug delivery systems. Liposome, polymer and minicell conjugated drugs were taken the preclinical trials. The first biodegradable nanoparticles are developed for drug delivery with the properties of lipid membranes such as Doxil [5]. According to the US National Cancer Institute, nanoparticles are currently authorized for use in the global market, while the cost of these products is ten times higher than conventional treatments [6]. At present, the use of nano-sized particles derived from bacteria which are called minicells to encapsulate a wide range of different chemotherapeutic drugs is a new technology that specifically targets to receptors on disease cell surface via dual-specific antibodies coated on the minicells. Minicell loaders have been shown the apoptosis effects on tumor cells both in vitro and in vivo with high specificity when delivering drug [7]. Although minicell is still a new concept in DDS, the term of minicell is appeared in a number of patents around the world as well as through clinical trials. Minicells are small cells produced by bacteria that could play many roles in the development of agents to treat cancer [7, 8]. With a bacterial structure with different functional groups and holes in the cell wall, minicells can bind with many drugs including chemicals and antibodies to reach the most favorable destination for developing a DDS. This study aims to point out the minicells derived nanoparticles for DDS to suggest a new opportunity for prospective nanoparticle development for pharmaceutical science.

2. Well-known drug delivery systems

2.1 Advantages of well-known DDSs

There are many drugs that are effective for treating diseases, but side-effects are still problematic when crossing the barriers in the body, for example eye barrier, colon, and brain membrane due to the complex structure of barriers of eye, colon, and brain. DDS can be microelectromechanical (MEM)-based device, polymer matrix or gene delivery system. DDS can be developed using pH, enzyme, time, pressure-controlled delivery method [9]. In some cases of colon treatment, a hole in the membrane is covered by an enteric-coated polymer to prevent drug release in the upper gastrointestinal tract. Brain-targeted drug delivery has received increasing attention over the past decade. Many strategies have been developed to improve brain-targeted drug delivery by fabricating particle-based drug delivery systems. Despite impressive progress, drug delivery efficiency remains unsatisfactory. Recent advances in the field of microfabrication have made possible the development of controlled release systems for drug delivery. Drug delivery has achieved tremendous development over the past two decades, but regulating drug delivery to the brain is going on a daunting task. By using such highly specialized transport mechanisms, it is possible to efficiently mediate the penetration of the nanodrug delivery system into the brain. Surface functionalization of DDS and selection of suitable materials for their membranes play an important role in drug release kinetics. The release of a particular drug from DDS can be regulated by external or internal stimuli. The pH-responsive DDS can only release the drug in the area it targets. Several pH-responsive DDSs have been successfully engineered to deliver drugs in tumor tissues because tumor tissues have a different pH than healthy tissues [10]. The reduced environment of the cytoplasmic fluid of the cell relative to the body fluid becomes a stimulus for redox-sensitive DDS to release active substances only in the cytosol but not in the body fluid [11]. Magnetic NPs loaded with therapeutic agents can be guided, using an externally applied magnetic field, to a specific organ and stimulated to release the drug only at that location [12]. Nanotechnology was first defined in 1974 by Norio Taniguchi of Tokyo University of Science, as the technique and technology for understanding and controlling matter at the length scale of about 1 to 100 nanometers [1]. Many nanomaterial preparations have significant potential applications for therapeutic drug delivery systems. Several nano-sized preparations such as liposomes, polymeric micelles and polymer drug conjugates have been developed in vitro, and several preclinical studies are underway [13, 14]. Properties including the size, surface charge, shape and density of surface-related targeting ligands could allow nanoparticles to evade renal clearance to reach cellular targets. Indicated cells in sufficient quantities, undergo active cellular uptake, and induce biological responses with minimal non-specific interactions [15]. The raw materials of nanoparticles are not only of biological origin such as lactic acid, dextran, phospholipids, lipids, carbon and chitosan but also of chemical origin such as polymers, silica and metals [16, 17, 18]. The nanodrugs currently approved for the treatment of cancer are mainly liposomal nanoparticles. They have their structural, physicochemical properties giving hope to patients. Additionally, the gene therapy field approved viral vector-based drugs of various designs and purposes such as cancer therapies. Currently, the three main vector strategies are based on adenoviruses, adeno-associated viruses, and lentiviruses that occured in clinical and preclinical processes for the past decades. However, there are still many challenges for drug targeting in cancer therapy [19].

2.2 Challenges of well-known DDSs

Undoubtedly, the controlled DDS presents an important challenge in medicine when compared with the conventional DDS in the case of therapeutic efficacy. Accordingly, there has been a search for drug delivery systems that enhanced activity for more drugs with fewer complications. Until now, there are very few reports on the effects of nanoparticles on the body. The long-term effects on our health are difficult to predict and understand. Nanocarriers can increase the stability of many anticancer drugs by integrating them into their structures, for example, the stability of doxorubicin has also been increased through its incorporation into liposomes [20]. Previous reports showed a beneficial effect on the long-term stability of DNA when the molecule was complexed into polymeric micelles [21, 22]. Furthermore, the nanoparticles are antibacterial agents but can also kill the good bacteria in the gut. In addition, DDSs using nanoparticles are more expensive to manufacture than traditional materials because of the many complex steps [18]. Consequently, it is difficult to scale up the production of nanoparticles leading to the limited production in the market and also causing to many risks due to their unknown side effects. Drug delivery using nanoparticles is increasingly popular and faces many challenges in pharmaceutical engineering. There are many reports on nanotoxicology, the potential negative effects of interactions between nanomaterials and biological systems. For example, naked quantum dots exhibit cytotoxicity by generating reactive oxygen species, leading to nuclear, mitochondrial, and plasma membrane damage [17]. For silica nanoparticles, concentrations above 0.1 mg/ml were found to be toxic, as demonstrated by decreased cell proliferation and availability [17]. The production of carbon nanotubes also induced the formation of reactive oxygen species, mitochondria dysfunction, lipid peroxidation, and changes in cell morphology. In addition, it has been reported that most cationic NPs can induce hemolysis and coagulation, while neutral and anionic NPs did not show toxicity [23, 24]. For hydrophobic drugs, their main limitation is their low solubility at the absorption site and poor biodegradability. The case of many common hydrophobic drugs is well illustrated by paclitaxel that have low oral bioavailability in conventional formulations but the activity increase when formulated in DDS like liposome [25, 26, 27]. However, DDS which can carry different kinds of drugs to the disease target are challenged. Therefore, finding out the potential DDS is essential. In this chapter, bacterial minicells that have nano size are focused to show its ability in drug delivery.

3. Minicells in drug delivery

3.1 Minicells generated by microorganisms

Minicells are the small cells in nano size that can be call nanoparticles. They are made from the membranes of mutant bacteria during cell division cycle. The term “minicells” refers to cells that contain RNA and proteins but only small amounts of chromosomal DNA, if any at all. The genetic and biochemical characterization of the minicells of Escherichia coli revealed that most were defective in the multiprotein system known as the Min system, which mediates the alignment correct arrangement of the middle cells of the cell division septum [28, 29]. In Escherichia coli, the three proteins which are MinC, MinD, and MinE, synergistically mediate the niche of the cell division machinery by inhibiting its growth at sites location other than the middle cell [30]. In Escherichia coli, fts is temperature-sensitive filament involving mutants and partition (par) mutants [31]. There are FtsZ, FtsA, FtsK, FtsQ , FtsL, FtsB, FtsW, FtsI, and FtsZ. FtsZ, a tubulin homolog which are the essential components of septal ring, called a Z-ring at potential division positions, where more division proteins are recruited to form the complete division [31]. In Escherichia coli, Min proteins rapidly migrate from one pole of the cell to the opposite pole, forming a bipolar inhibitory gradient that prevents assembly of the Z ring near the poles but allows FtsZ to stay at the poles near the middle of the cell. Min system is not dependent on nucleoid occlusion and forms anucleate cells which are chromosome segregation mutants. Otherwise, when Min system lacks in cells, nucleoid occlusion is not enough to direct Z-ring assembly. Consequently, the division occurs at any one of three potential division site, a chromosome less-minicell and a multinucleate filament are produced [32]. The minicell mutants in Escherichia coli were mapped to the min locus which encodes MinC, MinD, and MinE [31]. In Bacillus subtilis, the Min system includes MinC, MinD, DivIVA and DivIVB which are different from Escherichia coli. Bacillus subtilis does not have MinE. DivIVA localizes from poles to midcell of division sites. DivIVA has some functions beyond that of involving MinCD location. Together with DivIVA, DivIVB also participate in minicell formation [33]. Both Gram-positive and Gram-negative bacteria can produce minicells. Minicells were also produced in Lactobacillus acidophilus [34] or by gene expressing the cell division inhibitor [35]. Gram-negative cell wall consists of lipopolysaccharide, O-specific side chains, outer membrane, peptidoglycan, periplasmic space, and porins. Gram-positive cell wall is composed peptidoglycans, teichoic acid, and periplasmic space. A peptidoglycan network can cover with a variety of substances. Teichoic acid is a linear polymer of polyols in the form of glycerol or various monosaccharides linked by phosphodiester bridges [36]. The polysaccharides associated with different drugs, antibodies and so on. Minicells with a uniform diameter around 400 nm were produced from both Gram-positive and Gram-negative bacteria. Based on bacterial properties, minicells can play a potential role in drug delivery (Figure 1).

Figure 1.

Gram staining and observing Lactobacillus acidophilus. (A): Minicells started producing from rod cells in supplemented MRS medium in 36 hours. (B): Minicells produced from rod cells in supplemented MRS medium in 48 hours. (C): Purified Minicells.

3.2 Minicell loading with different drugs

Interestingly, minicells can be packed with 1–10 million drug molecules [17]. Minicells traverse tumor cells by receptor-mediated endocytosis. In vivo experiments, doxorubicin-loaded minicells inhibited tumor growth in mice such as breast, ovary, leukemia or lung [37]. Importantly, a relatively small amount of drug is needed to induce tumor regression, 100 times the higher dose required with liposome-encapsulated doxorubicin. The antitumor efficacy of minicells was further evaluated in dogs with end-stage T-cell non-Hodgkin lymphoma; treatment resulted in marked tumor regression and tumor lysis [38]. Importantly, multiple studies have shown that minicells have no adverse side effects despite repeated dosing and do not increase proinflammatory cytokines. Other animal experiments confirmed no adverse reactions [39]. Using microcells derived from bacteria for the first time used in humans with clear results is safe, well tolerated. Solomon reported that minicells encapsulated the chemotherapy drug paclitaxel and coated with antibodies targeting tumors that express an epidermal growth factor receptor (EGFR) protein found on the surface of many cancer cells. The study was then conducted in phase I for treating the small groups of patients. Phase II trials of minicells were continued in one group of patients with glioblastoma (a type of brain tumor) using doxorubicin-loaded minicells [39, 40]. In vitro studies, the small cells produced by Lactobacillus are nano-sized which are useful in drug delivery. Lactobacillus minicells can be loaded and reloaded with hydrophilic and hydrophobic drugs such as paclitaxel, cephalosporins without phagocytosis that suggested to one of valuable DDS [41]. There are many applications of minicells reported such as therapeutic research such as drug discovery, delivery of nucleic acids and other bioactive compounds to cells. MacDiarmid and colleagues described the use of chemotherapeutic-encapsulated bacterial small cells guided by antibodies to deliver their payloads to target cells by a recognized surface lipopolysaccharide dual-specific antibody (epidermal growth factor receptor (EGFR), HER2/neu (ERBB2), CD33 or CD3 [38].

By molecular docking application, different kinds of proteins of bacteria are expected to interact with a variety of ligands such as drugs. Molecular docking is a method to predict the drug molecule which can be bind to the proteins of the disease target based on a computation aid. In this study, protein-ligand concatenation was performed to see the ability of drugs in interacting with the target protein [42]. The 3D crystal structures of proteins from bacteria was obtained from the Protein Data Bank (PDB). Moreover, all the 3D structures of ligands like drugs were downloaded in Pubchem and ChemSpider [43]. Then, Autodock Vina were used to screen protein and ligand interaction, providing the binding affinity of each profile between the ligand and protein interactions. The Genetic Algorithm was calculated based on 10 runs for 10 different configurations of drugs to show the binding ability to the target protein [43]. Validation is based on Root Mean Standard Deviation (RMSD) value and the binding energy value (Kcal/mol) [44]. Interactions with amino acid residues in the receptor’s active binding pocket were analyzed using BIOVIA Discovery Studio binding [45, 46]. The interactions between some proteins of Escherichia coli, Salmonella enterica subsp. enterica serovar Typhimurium, Bacillus subtilis with hydrophilic and hydrophobic drugs used as ligands were shown in Table 1. Maltose binding protein of Escherichia coli (ID: 3OSR), outer membrane protein A (OmpA) from Salmonella enterica subsp. enterica serovar Typhimurium (ID: 5VES), the outer membrane protein A and C (OmpA and C) of Escherichia coli (ID: 3NB3), Escherichia coli MinD (ID: 3Q9L), Bacillus subtilis N-terminal domain of MinC (ID 2M4I) could bind to ligands with negative energy, showing the interaction. Depending on the protein and ligand, the binding ability was different.

Number	Proteins	Ligands	Binding energy (kcal/mol)
1	Maltose-bound maltose sensor engineered by insertion of circularly permuted green fluorescent protein into Escherichia coli O157:H7 maltose binding protein at position 311 (ID: 3OSR)	Vancomycin	−23.4
		Vincristine	−20.7
		Paclitaxel	−19.8
		Stigmasterol	−9.9
		Quercetin	−8.6
		Cephalexin	−7.7
		Ciprofloxacin	−7.7
		Erythromycin	−7.6
		Penicillin	−7.6
		Hydroxychloroquine	−7.4
2	The 2.4A crystal structure of OmpA domain of OmpA from Salmonella enterica subsp. enterica serovar Typhimurium str. 14028S (ID: 5VES)	Vancomycin	−19.1
		Vincristine	−17
		Vinblastine	−16.8
		Paclitaxel	−15.9
		Quercetin	−8
		Stigmasterol	−7.3
		Penicillin	−7
		Ciprofloxacin	−7
		Erythromycin	−6.9
		Cephalexin	−6.8
		Hydroxychloroquine	−5.9
3	The host outer membrane proteins OmpA and OmpC are packed at specific sites in the Shigella phage Sf6 virion as structural components (ID: 3NB3)	Vancomycin	−21.6
		Vincristine	−17.3
		Vinblastine	−16.8
		Paclitaxel	−16.8
		Stigmasterol	−7.9
		Quercetin	−7.8
		Hydroxychloroquine	−5.7
		Erythromycin	−8.2
		Penicillin	−7
		Cephalexin	−7.3
		Ciprofloxacin	−7
4	The structure of the dimeric Escherichia coli MinD-ATP complex (ID: 3Q9L)	Paclitaxel	−19.7
		Vancomycin	−17.8
		Vinblastine	−14.7
		Vincristine	−14.6
		Erythromycin	−11.5
		Stigmasterol	−7.0
		Ciprofloxacin	−6.3
		Cephalexin	−6.2
		Quercetin	−6.1
		Penicillin	−5.7
		Hydroxychloroquine	−5.0
5	Solution structure of Bacillus subtilis MinC N-terminal domain (ID: 2M4I)	Vancomycin	−17.5
		Vincristine	−16.9
		Vinblastine	−15.9
		Paclitaxel	−13.1
		Erythromycin	−13.0
		Quercetin	−7.4
		Stigmasterol	−6.5
		Ciprofloxacin	−5.7
		Cephalexin	−5.3
		Penicillin	−5
		Hydroxychloroquine	−4.8

Table 1.

Interaction of some bacterial proteins with ligands using molecular docking.

As seeing in Table 1, beside outer membrane proteins could bind to drugs, cell division protein inhibitors such as MinC and MinD could interact to different drugs. Figure 2 showed the representative studies of molecular docking for screening the interaction of MinD cell division inhibitor of Escherichia coli could bind to anticancer drug such as paclitaxel via hydrogen bond, Pi lone pair, Pi-sigma, Pi-anion, Pi-alkyl while vincrisrine interaction was by means of hydrogen bond, Pi-cation, Pi-alkyl, amide-Pi stacked (Figure 2). MinD protein could also bind to hydroxychloroquine, an antimalarial drug at the active sites which were Thr 17, Thr 18, Pro 212, Val 217, Glu213, and Arg 212 via the determined bonds (Figure 2). Besides, antibiotics such as erythromycin belonging to macrolide as well as cephalexin and penicillin belonging to beta-lactam group or alkaloids and steroids represented such as quercetin and stigmasterol could also bind to MinD that was illustrated in Figure 2. By these interactions, drugs/ligands can be released again from minicells when going to the target sites. More studies will be developed for getting the ideal DDS. Therefore, with the structure of bacterial minicells, there are many kinds of drugs which can be bound to be delivered. As a result, bacterial minicells could be the potential drug delivery system for target treatment.

Figure 2.

Visualization of drugs with the highest binding energy interacts to MinD cell division inhibitor of Escherichia coli. (A): Erythromycin; (B): Penicillin; (C): Cephalexin; (D): CiproFloxacin; (E): Paclitaxel; F: Hydroxychloroquine; (G): Vincristine; (H): Quercetin; (I): Stigmasterol. (conventional hydrogen bond: Green color; pi-sigma bond: Purple color; pi-alkyl: Pink color; pi-cation: Orange colors).

4. Conclusion

Nowadays, DDS is developed for targeting to therapy. With the small size like nanoparticle, the bioavailability of drugs increases. However, nanotechnology is still challenged. Some materials are not convenient for DDS development. Moreover, DDS which carries drug to the target to reduce side effect is very important for human health care. Bacterial minicells are cheap and can be produced in large scale. With the complexity of cell wall structure, minicells can bind with hydrophobic and hydrophilic drugs. Especially, minicells could bind to monoclonal antibodies which are specific to the target cells. Exploiting minicells give a good picture for cancer therapy, pathogen treatment and vaccine development.

Acknowledgments

The study was developed by private fund.

Conflict of interest

The authors declare no conflict of interest.