Abstract
Nanobiotechnology application, at the interface of nanocarrier and therapeutic enzyme, holds great promises in the nanomedicine. In this direction, gold nanocarriers contribute a plethora of nanobiotechnological applications due to their unique properties. The salient features of gold nanoparticle include high catalytic activity, unique optical properties, ease of surface functionalization, biocompatibility and long-period stability. The potential use of gold nanoparticle in conjunction with therapeutic enzymes can be further extended for curing many dreadful diseases.
We reviewed the suitability of gold nanocarrier-bound therapeutic enzyme delivery in biomedical modality, in particular to therapeutic application. The major health issues such as cancer, cardiovascular disease and brain disease are regulated with the intervention of gold nanoparticle-bound therapeutic enzyme delivery. Gold nanocarrier-bound therapeutic enzyme has increased the pharmacokinetic and pharmacodynamic correlation in drug delivery. Therapeutic fungal asparaginase covalently immobilized on the surface of gold nanoparticles demonstrated higher cytotoxicity effect against lung cancer and ovarian cell lines. It is further demonstrated that the gold nanoparticle-bound asparaginase has increased its bioavailability up to 85% more against lung cancer. The serratiopeptidase-bound gold nanoparticle has considerably increased anti-inflammatory response. The present chapter is concluded with recent literature discussion that gold nanoparticle-bound therapeutic enzyme has broadened the scope of traditional therapeutics to effective therapeutic enzyme delivery.
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Keywords
- Nanogold
- Biogenic methods
- Therapeutic enzyme
- Enzyme as a drug
- Bioconjugation
- Stability
- Applications
- Cell lines
- Drug delivery
- Anti-inflammatory
- Cancer
3.1 Introduction
Nanomaterials, in particular gold nanoparticles, have gained attention due to the simplicity in its mode of action, ease of surface modifications, a plethora of applications such as data storage, environment, especially in medical biotechnology as nanocarrier for enzyme immobilizations and for drug delivery (Chamundeeswari et al. 2018; Golchin et al. 2018; Kaphle et al. 2018; Dykman and Khlebtsov. 2017; Verma 2017a, b, c, d; Gupta et al. 2016; Shankar et al. 2015; Kumar et al. 2014a, b; Sharma et al. 2014a, b; Verma et al. 2013a, b, c, d). Drug delivery is a fascinating field of scientific research in nanobiotechnology. Drug delivery is defined as the process for the release of biologically active medicament at a definite speed and at a destined location (Xin et al. 2017). Functionalized gold nanocarriers present huge probabilities for multiple, locus-specific drug delivery to the disease locus as their diminutive size can effectively penetrate across obstacles through small capillaries into individual cells. Specifically, gold nanoparticles have revealed great capacity to be used as drug delivery platforms (Pelaz et al. 2017). Gold nanoparticles have tremendous potential to deliver multiple drug molecules, recombinant proteins, vaccines and nucleotides into their targets effectively. Targeted/localized drug delivery is possibly achieved through active or passive approaches. Active targeting is based on conjugating the therapeutic agent or carrier system to a cell- or tissue-specific ligand, whereas passive targeting is based on a therapeutic agent that passively reaches out to a localized organ for efficient biomedical application such as target tumours by incorporation in the macromolecule or nanoparticle (Daraee et al. 2016).
Gold nanoparticles have always been considered as potential target for localized drug delivery applications in the field of biomedicine (Baskar et al. 2018). Nanocarriers have unique physicochemical characteristics such as definite size, surface area to mass ratio, chemical stability with high reactivity and functionalized structure with admirable biocompatibilities (Kong et al. 2017). Today, nanocarriers can serve as drug depots exhibiting prolonged-release kinetics and long persistence at the target site. Nanotechnology-based biomedicines have improved the pharmacokinetic and pharmacodynamic potential of different drug molecules which are capable of targeted/localized drug delivery applications such as early detection of cancer lesions, determination of molecular signatures of the tumour by non-invasive imaging and, most importantly, molecular-targeted cancer therapy and cardiovascular and neurodegenerative disease treatments (Pietro et al. 2017). Biocompatibility of gold nanoparticles, with ease of their biological and chemical nature, mimics the function of some enzymes including superoxide dismutase, esterase, peroxidase and glucose oxidase for various therapeutic applications such as tissue regeneration (Golchin et al. 2018). Localized delivery of drug-coated nanoparticles and emergence of such nanotherapeutics/diagnostics based on therapeutic enzymes provides the way for deeper understanding of human longevity and human ills that include genetic disorders, cancer and cardiovascular disease (Peer et al. 2007).
The present article is focussed on the applications of gold nanoparticle-mediated therapeutic enzyme delivery. Various physicochemical and biological methods of gold nanoparticle synthesis, biotechnology of therapeutic enzyme production, strategies of robust nanocarrier-enzyme bioconjugate development and biomedical applications of the gold nanocarrier-bound therapeutic enzyme are critically discussed.
3.2 Synthesis of Gold Nanoparticles
Various methods such as the Turkevich method, Brust-Schiffrin method, seeding growth method and biological method have been employed for the synthesis of gold nanoparticles (Herizchi et al. 2016; Rawat et al. 2016; Abdulghani and Hussain 2014; Singh et al. 2013; Siti et al. 2013; Bisker et al. 2012; Chithrani et al. 2010; Akbarzadeh et al. 2009; Mohanpuria et al. 2008; Brust et al. 1994; Turkevich et al. 1951).
Various chemical and physical methods of gold nanoparticle synthesis are most commonly used. However, these chemical methods involve the use of expensive and hazardous chemicals under extreme reaction conditions (Ahmed et al. 2015a; Ahmed et al. 2015b; Krishnaswamy et al. 2014; Kumar et al. 2011a, b). In addition, these nanoparticles may have harmful effects in biomedical applications (Noruzi et al. 2011; Shankar et al. 2004a, b). To overcome these problems, green synthesis of nanoparticles is an emerging field of research in the current era (Kulkarni and Muddapur 2014; Mittal et al. 2013). Hence, there is a growing need to develop eco-friendly and cost-effective procedures for the synthesis of nanoparticles. The inherent, clean, nontoxic and environment-friendly ability of microorganisms and plant systems to synthesize the gold nanoparticles is particularly important in the advancement of nanobiotechnology (Mohanpuria et al. 2008).
Recently, plants are commonly employed for the synthesis of gold nanoparticle (Table 3.1A). The biosynthesis of gold nanoparticles using plants and plant extracts is a very important aspect due to lack of pathogenicity and their diversity (Chandran et al. 2014). Green synthesis of nanoparticles uses extracts of various plants such as Aloe vera (Chandran et al. 2006), Pogostemon benghalensis ( Paul et al. 2015), Salix alba (Ul et al. 2015), Solanum nigrum (Muthuvel et al. 2014), Terminalia arjuna (MohanKumar et al. 2013), Piper pedicellatum (Sujitha and Kannan 2013), Terminalia chebula (Tamuly et al. 2013a, b), Citrus reticulata and Citrus sinensis (Mittal et al. 2013), Mangifera indica (Philip et al. 2011), Murraya koenigii (Das et al. 2011), Zingiber officinale (Kumar et al. 2011a, b), Cymbopogon citratus (Parida et al. 2011; Smithaa et al. 2009), Coriandrum sativum (Narayanan and Sakthivel 2008), Azadirachta indica (Shankar et al. 2004a, b) and Medicago sativa (Gardea-Torresdey et al. 2002). Plant extracts may act as both reducing agent and stabilizing agent in the synthesis of nanoparticles. In view of its simplicity, the use of plant extract for reducing metal salts to nanoparticles has attracted considerable attention (Mittal et al. 2013). Large-scale biosynthesis of nanoparticles is a main factor in green syntheses in which suitability of the reagents plays an important role (Chandran et al. 2014). Gold nanoparticles are rapidly synthesized using aqueous leaf extracts of Acalypha indica and Azadirachta indica as novel sources of bio-reductants (Krishnaraj et al. 2014). Biosynthesis of gold nanoparticles using leaf extracts of Zingiber officinale, which acted as a reducing and capping agent, was also reported (Singh et al. 2011). The use of plants and plant extracts for the preparation of gold nanoparticles is more advantageous. It does not require elaborate processes such as intracellular synthesis and multiple purification steps.
The biological method for the synthesis of nanoparticles by using microbes like bacteria, fungi, actinomycetes, yeast and algae is providing a wide range of resources for the synthesis of nanoparticles (Table 3.1B). Use of diverse microorganisms such as Bacillus marisflavi (Nilofar and Shivangi 2016), Bacillus subtillus (Reddy et al. 2010), Bacillus licheniformis (Kalishwaralal et al. 2009), Pseudomonas veronii (Baker and Satish 2015), Galaxaura elongata (Abdel-Raouf et al. 2017), Chlorella vulgaris (Xie et al. 2007), Trichoderma asperellum and Trichoderma reesei (Vahabi et al. 2011; Mukherjee et al. 2008), Fusarium oxysporum (Das et al. 2012), endophytic fungus Verticillium sp. (Bharde et al. 2006) and Rhizopus oryzae (Mukherjee et al. 2002) was employed for the synthesis of gold nanoparticles. It is a relatively new area of research with considerable prospects that can be used either extracellularly or intracellularly due to their innate potential. Mukherjee et al. (2002) also demonstrated that fungi secrete a significantly higher amount of proteins than bacteria; this would amplify the productivity of nanoparticle synthesis. Further, it is environmentally acceptable, economic, time saving and easily scaled up. Due to this ability to adapt to extreme conditions, these fungi can be used as a potential resource for biosynthesis of nanoparticles.
It can be inferred from the above-stated various methods of gold nanoparticle synthesis that the biological route provides an attractive possibility for the scale-up of gold nanoparticle production.
3.3 Biotechnology of Therapeutic Microbial Enzymes
Enzymes are the excellent biocatalysts that catalyse complex chemical reactions under appropriate physiological conditions. Enzymes possess a unique chiral-selective property, a prerequisite step for enantiomerically pure pharmaceutical drug production (Mane and Tale 2015; Bankar et al. 2009; Underkofler et al. 1957). Use of enzymes as drug target exhibits advantages over conventional drugs due to their unique target specificity and multiple substrate conversion (SKumar and Abdulhameed 2017). Therapeutic enzymes are obtained from bacteria, fungi and yeast (Table 3.2). Microbial enzyme production offers cost-effective technology that has a potential profitable market (Mane and Tale 2015; Gurung et al. 2013; Teal and Wymer 1991). Nowadays therapeutic enzymes are used for treating a diverse spectrum of life-threatening diseases such as cancer and gastrointestinal disorders and enzyme replacement therapy. Thus, therapeutic enzymes served as oncolytics, thrombolytics or anticoagulants and anti-inflammatory agents (Mane and Tale 2015; Gurung 2013; Gurung et al. 2013; Vellard 2003; Ozcan et al. 2002; Gonzalez and Isaacs 1999).
3.3.1 Different Types of Therapeutic Enzymes
Specificity of therapeutic enzymes makes them the most desirable therapeutic agents for the treatment of various diseases. Digestive and metabolic enzymes can be used either alone or in combination with other therapies for treating a variety of diseases safely (Mane and Tale 2015; Kaur and Sekhon 2012; Sabu 2003; Vellard 2003; Cooney and Rosenbluth 1975).
Demands of therapeutic enzymes are growing rapidly due to massive biomedical applications. At present, the most prominent medical uses of microbial enzymes are the removal of dead skin and burns by proteolytic enzymes and clot busting by fibrinolytic enzymes (Singh et al. 2016). For example, a good agent for thrombosis therapy is nattokinase, a potent fibrinolytic enzyme (Sumi et al. 1987). Enzymes, namely, L-asparaginase, L-glutaminase, L-tyrosinase and galactosidase, are used as antitumour agents, and streptokinase and urokinase act as anticoagulants. Acid protease, dextranase and rhodanase may be used to treat alimentary dyspepsia, tooth decay and cyanide poisoning, respectively (Okafor, 2007). Microbial lipases and polyphenol oxidases are involved in the synthesis of diltiazem intermediate (2R,3S)-3-(4-methoxyphenyl)methyl glycidate and 3,4-dihydroxylphenyl alanine (DOPA, for treatment of Parkinson’s disease), respectively (Faber 1997). Tyrosinase, an important oxidase enzyme, is involved in melanogenesis and in the production of L-DOPA. Dopamine, a potent drug to control the myocardium neurogenic injury and for the treatment of Parkinson’s disease, is produced using L-DOPA as a precursor (Zaidi et al. 2014; Ikram-ul-Haq and Qadeer 2002). Chitosanase catalyses hydrolysis of chitosan to biologically active chitosan oligosaccharides, which are used as antimicrobial and antioxidant, in lowering blood cholesterol and high blood pressure, controlling arthritis, protecting against infections and improving antitumour properties (Thadathil and Velappan 2014; Zhang et al. 2012; Ming et al. 2006; Kim and Rajapakse 2005).
3.3.2 Therapeutic Enzyme Production
In the pharmaceutical industry, bioprocessing of enzymes for use as drugs is an important aspect that is now being capitalized at every research and development centre across the globe (Cassileth 1998). Microbial therapeutic enzymes offer economic feasibility. That is why the use of microbial enzymes is increasing day by day (Gurung et al. 2013). Various methods involving fermentation technology are available for the production of microbial enzymes (Sabu et al. 2000). These include solid-state fermentation and submerged fermentation. On commercial scale, these methods are utilized for mass production of therapeutic enzymes than liquid cultures in huge bioreactors (Lozano et al. 2012). These important enzymes can be produced by different methods of fermentation. On an industrial scale, liquid cultures in huge bioreactors are preferred for producing therapeutic enzymes in bulk. Other processes like solid-state fermentations and submerged fermentations are also widely used for the production of therapeutic enzymes (Sabu 2003). Large-scale productions of microbial therapeutic enzymes using various production techniques and downstream processing have been reported (Sabu et al. 2005; Sabu 2003).
Gold nanoparticle was employed for some enzyme deliveries such as superoxide dismutase, esterase, peroxidase and glucose oxidase for various therapeutic applications (Golchin et al. 2018). It is very pertinent that only a few therapeutic enzymes have been explored for gold nanoparticle-mediated drug delivery so far. Thus, it can be inferred that many therapeutic enzymes have to be employed for nanocarrier-mediated drug delivery.
3.4 Methods for Developing Robust Gold Nanocarrier for Therapeutic Enzyme Delivery
Therapeutic enzymes are susceptible to denaturation under harsh environmental conditions (Abraham et al. 2014; Puri et al. 2013; Verma and Kanwar 2010, 2012; Verma et al. 2009, 2011, 2012). In order to make a robust and biocatalytic stable enzyme, enzymes need protection and cost-effective recyclability by immobilizing the suitable inert carrier (Verma et al. 2016). Nanomaterials possess many physicochemical advantages over their bulk materials. Immobilization of enzymes on the nanoparticles holds a great promise to improve their functionality and biocatalytic potentials. Nano-immobilization methods are generally categorized into four types, namely, (1) electrostatic adsorption, (2) conjugation of the ligand on the nanoparticle surface, (3) conjugation to a small cofactor molecule that the protein can recognize and bind to and (4) direct conjugation to the gold nanoparticle surface (Fig. 3.1; Verma et al. 2016; Verma and Barrow 2015; Puri et al. 2013; Yeh et al. 2012; Ackerson et al. 2010; Aubin and Hamad 2008). Each of these techniques has its pros and cons (Table 3.3; Kanwar and Verma 2010; Kanwar et al. 2008; Kanwar et al. 2007a,b; Kanwar et al. 2006; Kanwar et al. 2005). Thus, sometimes a combination of these nano-immobilization techniques is employed in order to get robust gold nanoparticle conjugates.
Among different nanoparticles (fullerenes, single-walled and multiwalled carbon nanotubes, magnetic nanoparticles, modified silicon nanowires, dendrimers and quantum dots), only gold nanoparticles can be marked as the most used and widespread for biomedical applications (Zhang et al. 2015). Besides the common properties typical for nanomaterials, the main specific characteristics of gold nanostructures are its stability for a long period of time, easy surface functionalization, biocompatibility, unique optical properties and high catalytic activity providing the successful use of gold nanoparticles (Yu et al. 2016). Gold nanoparticles can be attached to those functional groups which have positive charge because of negative charge on their surface. Likewise, the presence of six free electrons in the conduction band of gold nanoparticles makes them potential candidates to bind with reactive functional groups like thiols and amines. Silica, aluminium oxide and titanium oxides facilitate the attachment of different functional groups on the surface of gold nanoparticles (Sharma et al. 2015; Sharma et al. 2010; Kim et al. 2010; Sun et al. 2008; Tkachenko et al. 2004). Thus, gold nanoparticles can be easily tagged with various proteins and biomolecules that are rich in amino acids (Giljohann et al. 2010; Eustis and El-Sayed 2005). Therapeutic and diagnostic efficiency can strongly be influenced by changing the surface characteristics of nanoparticles such as size, shape and surface charge which in turn change cellular uptake and functional surface area (Jazayeri et al. 2016).
The conjugation of different functionalized groups to nanoparticles is prerequisite for improving stability, functionality and biocompatibility (Delong et al. 2010). It has also been reported that it is possible to control the interactions of gold nanoparticles with cell membranes in order to improve their cellular uptake while minimizing their toxicity by rigid change of the surface charge densities (Lin et al. 2010). Physical and chemical interactions are used for attaching functional groups (DNA, RNA, enzymes, peptides, bovine serum albumin, polyethylene glycol and proteins) to gold nanoparticles’ surface (Cho et al. 2012; Lee et al. 2008). Non-covalent interaction between functional groups and gold nanoparticles depends on three phenomena: (a) ionic attraction between the negatively charged gold and the positively charged functional group, (b) hydrophobic attraction between the functional group and the gold surface and (c) dative binding between the gold conducting electrons and functional group. Covalent interactions between functional groups and nanoparticle surface are achieved in a number of ways like (i) through chemisorption via thiol derivatives, (ii) through the use of bifunctional linkers and (iii) through the use of adapter molecules like streptavidin and biotin (Delong et al. 2010). Other functional groups like citrate, tannic acid and polyvinylpyrrolidone can be capped to gold nanoparticles (Marcelo et al. 2015; Senoudi et al. 2014; Mirza and Shamshad 2011).
Gold nanoparticles are useful for important biomedical applications including targeted drug delivery, cellular imaging and biosensing (Hwang et al. 2012; Hong et al. 2012; Giljohann et al. 2010; Huang and El-Sayed 2010). In a recent study, therapeutic fungal asparaginase was covalently immobilized on the surface of gold nanoparticles or nanoporous gold nanoparticles (Baskar et al. 2018). Immobilized gold nanoparticle was further targeted for drug delivery with respect to cancer treatment. It has been demonstrated that the synthesized gold nanobiocomposite of asparaginase can be used as an effective anticancer drug with increased bioavailability against lung cancer.
Gold nanoparticles proved robust nanocarriers for neurotrophin peptides (Patrizia et al. 2017). The immobilization of neurotrophin peptide was achieved by direct physisorption and lipid bilayer-mediated adsorption methods. The nano-bioconjugates were characterized by UV-vis spectroscopy, X-ray photoelectron spectroscopy, dynamic light scattering, zeta-potential analyses and atomic force microscopy. Both peptide- and lipid-dependent features were identified to have a modulation in the peptide coverage of nanoparticles as well as in the cellular uptake of nerve growth factors and brain-derived neurotrophic factors. Robust hybrid gold peptide nanointerface demonstrated a promising approach to neurotrophin for crossing blood-brain barriers. Gold nanocarrier provided new multipotential therapeutic nanoplatform for the treatment of central nervous system disorders.
Gupta et al. (2016) reported a new generation of surface ligands based on a combination of short oligo(ethylene glycol) chains and zwitterions capable of providing non-fouling characteristics while maintaining colloidal stability and functionalization capabilities. Moreover, conjugation of gold nanoparticles with avidin helped in the development of a universal toolkit for further functionalization of nanomaterials.
Muthurasu and Ganesh (2016) prepared glucose oxidase-stabilized gold nanoparticles by changing the pH and showed feasibility of employing such nanocarrier as an ideal sensor for dual-mode sensing of glucose. Gold nanoparticles were able to detect glucose at a low concentration with high sensitivity, good stability and reproducibility suggesting promising applications in the field of nanobiosensors.
Malda et al. (2010) developed a conjugate of gold nanoparticle and therapeutically important superoxide dismutase at specific physiochemical reaction condition. Binding of enzyme-nanoparticle was confirmed by gel electrophoreses. Superoxide dismutase is a metalloenzyme that catalysed the dismutation of superoxide radicals into hydrogen peroxide and oxygen. Reactive oxygen species, such as superoxide radicals, are the root cause to pathogenesis of several diseases, such as familial amyotrophic lateral sclerosis, Parkinson’s disease, Alzheimer’s disease, Down syndrome and several neurological disorders (Halliwell and Gutteridge 2012; Pissuwan et al. 2007). Gold nanoparticle-superoxide dismutase enzyme conjugates proved its therapeutic potential in the prevention of oxidative damage from superoxide radicals (He et al. 2013; Zhao et al. 2012).
Synthesis of gold nanoparticles using the therapeutic enzyme serratiopeptidase was done at 25 °C and physiological pH 7 (Venkatpurwar and Pokharkar 2010). The formation of serratiopeptidase-reduced gold nanoparticles was confirmed by UV-visible spectroscopy, transmission electron microscopy, X-ray diffraction and Fourier transform infrared spectroscopy. This study successfully demonstrated that physiological condition is an important process parameter for the controlled synthesis of highly stable gold nanoparticles with respect to retention of biocatalyst activity. Researchers further confirmed use of gold nanoparticle as a carrier for serratiopeptidase led to an improved anti-inflammatory response (Venkatpurwar and Pokharkar 2010).
It is inferred from the above-stated studies that the binding of gold nanocarrier either non-covalently or covalently to therapeutically important enzyme depends on the immobilization reaction conditions and enzyme stability. This is a very critical step to immobilize fragile enzyme on the non-functionalized surface of gold nanoparticle. Robust gold nanocarrier immobilized enzyme successfully demonstrated various biomedical applications such as neurological and inflammatory issues.
3.5 Potential Applications of Gold Nanocarriers in Enzyme-Mediated Drug Delivery
Gold nanoparticle-based targeted drug deliveries have considerable applications to overcome the limitations in traditional therapeutics (Daraee et al. 2016). For example, antineoplastics, antiviral drugs and various other types of drugs are manifestly stuck due to their inability to cross the blood-brain barrier. Nanoparticle application to deliver drugs across this barrier is enormously promising. Researchers have reported that nanoparticles can cross several biological barriers for sustained delivery of therapeutic agents for difficult-to-treat diseases like brain tumours (Nazir et al. 2014; Hainfeld et al. 2013).
The potential of nanomedicine with respect to targeted drug delivery has improved with the ease of nanoformulation technique and widened the scope of delivering a range of drugs. Nanomedicine has developed novel diagnostic and screening techniques that have extended the scope of molecular diagnostics. They have been used in vivo to protect the drug entity in the systemic circulation, restrict access of the drug to the chosen sites and deliver the drug at a controlled and sustained rate to the site of action, minimizing undesirable side effects of the drug and allowing for more efficient use of the drug (Bosio et al. 2016).
Today, therapeutic enzymes are considered as one of the most promising applications in the pharmaceutical field. It has been reported by various researchers that enzymatic biocatalyst properties improved considerably by enzyme immobilization on nanomaterials, thereby increasing its stability and reusability and most importantly enhancing their targeting/localization to specific cell and tissues (Golchin et al. 2018; Xin et al. 2017). Gold nanoparticle-based therapeutic biocatalyst provides new tools for the diagnosis and treatment of old and newly emerging pathologies and presents distinctive modality for therapeutic delivery (Table 3.4; Golchin et al. 2018). Thus, gold nanoparticle-based therapeutic enzymes represent a highly promising alternative for treating a variety of pathologies by localized drug delivery approach.
Asparaginase obtained from Aspergillus terreus is a potent drug for the treatment of cancer and has antineoplastic or cytotoxic chemotherapy effect (Baskar and Renganathan 2012). Malignant cells lack asparagine synthase and employ the free circulating asparagine for its growth. Asparaginase converts the free circulating asparagine into aspartic acid and ammonia thereby lacking the asparaginase and leading to the death of tumour cells. So, research have been carried out across globe to target better asparaginase delivery system by immobilizing asparaginase on to gold nanoparticles followed by procedure of asparaginase gold nano-bioconjugate as potential drug candidate for curbing cancer, by testing against lung cancer cell line and ovarian cancer cell line.
Researchers studied gold nanoparticle-mediated delivery of fungal asparaginase against cancer cells (Baskar et al. 2018). The fungal asparaginase immobilized on gold nanoparticles showed efficient drug delivery in cancer treatment. Fourier transform infrared spectroscopy and nuclear magnetic resonance analysis of the synthesized asparaginase gold nano-bioconjugate showed that primary amines, secondary amines and allylic carbon are the main functional groups concerned with binding of asparaginase onto gold nanoparticles. Increment in the specific enzyme activity of asparaginase was recorded from crude (252.05 U/mg) to gold nano-bioconjugate (364 U/mg). Protein concentration was also increased from 0.018 mg/ml in crude asparaginase to 0.332 mg/ml in gold nano-bioconjugate. Nano-bioconjugate cytotoxicity effect was also observed to be higher against lung cancer cell line A549 than ovarian cancer cell line A2780. Finally, authors demonstrated that synthesized gold asparaginase nano-bioconjugate can be used as an effective anticancer drug and for targeted drug delivery with its increased bioavailability against lung cancer cell line (A549), given that toxicity is 84.51% (Baskar et al. 2018).
Serratiopeptidase, a proteolytic endopeptidase bioenzyme, is recognized as one of the most important therapeutic enzymes having anti-inflammatory activity (Salamone and Wodzinski 1997). Traditionally, therapeutic enzyme delivery is limited due to their poor uptake and vulnerability to degradation inside the gastrointestinal tract. For efficient drug delivery, nanoparticles such as gold nanoparticle complex have immense potential in the therapeutic perspective of biomedicine formulation. With this, the prerequisite is the nanocarrier which plays an important role in the bioavailability of the pharmaceutically active compound, efficiently improving absorption across the gastrointestinal mucosa (Dykman and Khlebtsov 2017).
Venkatpurwar and Pokharkar (2010) have reported the synthesis of gold nanoparticle using a therapeutic enzyme serratiopeptidase at physiological conditions which retained enzyme activity, and serratiopeptidase-capped gold nanoparticle complex led to improved therapeutic benefit. Characterization of synthesized gold nanoparticles has been reported using UV-visible spectroscopy, transmission electron microscopy, X-ray diffraction and Fourier transform infrared spectroscopy. Synthesized nanoparticle stability was assessed at ambient temperature up to 6 months. The retention of enzymatic activity was confirmed by in vitro enzymatic activity and in vivo anti-inflammatory activity of synthesized serratiopeptidase-capped gold nanoparticle complex. The tri-functional role of serratiopeptidase was reported, such as reduction, stabilization and therapeutic activity, finally demonstrating the gold nanoparticles as a nanocarrier for the immobilization and efficient and improved delivery of a therapeutic enzyme for an oral administration with improved therapeutic benefit (Venkatpurwar and Pokharkar 2010).
Tao et al. (2015) studied the bifunctionalized mesoporous silica-supported gold nanoparticles that showed intrinsic oxidase and peroxidase catalytic activities for antibacterial applications for their targeted delivery. Gold nanoparticles have exhibited both oxidase and peroxidase mimicking activities imparting end reactions as reactive oxygen species (ROS). Antibacterial properties proved against both Gram-negative and Gram-positive bacteria.
Superoxide dismutase is an important metalloenzyme and antioxidant defence against free radicals. It catalyses the dismutation of superoxide radicals into hydrogen peroxide and oxygen. Also, catalase is classified under a therapeutic enzymatic group supporting the cell from oxidative damage by reactive oxygen species (Golchin et al. 2018). Reactive oxygen species, such as superoxide radicals, have received great attention due to their involvement in the pathogenesis of various diseases, such as Alzheimer’s disease, Down syndrome, cataract, familial amyotrophic lateral sclerosis, Parkinson’s disease, cardiac myocytes and several neurological disorders. Superoxide dismutase enzymes have vast physiological importance and therapeutic benefit in the prevention of the oxidative damage from superoxide radicals (He et al. 2013; Zhao et al. 2012). Malda et al. (2010) have synthesized gold nanoparticle-iron-bound enzyme that demonstrated vast efficacy of gold colloid nanoparticle-bound superoxide dismutase protein.
Maji et al. (2015) have developed the new nanostructured hybrid as a mimetic enzyme for in vitro detection and therapeutic treatment of cancer cells. For targeted drug delivery application in the emerging field of nanobiotechnology, an artificial therapeutic enzyme conjugate was prepared by the immobilization of gold nanoparticles on mesoporous silica-coated nanosized reduced graphene oxide conjugated with folic acid, a cancer cell-targeting ligand. In vitro experiments with bioconjugate hybrid using human cervical cancer cells led to an enhanced cytotoxicity to Henrietta Lacks (HeLa) cells. In the case of normal cells (human embryonic kidney HEK 293 cells), the treatment with the hybrid and H2O2 showed no obvious damage, proving selective killing effect of the hybrid to cancer cells. Hybrid therapeutic enzyme bioconjugate with peroxidase activity has dual applications: firstly, detection (selective quantitation and colorimetric) of cancer cells and, secondly, cancer therapy by activating oxidative stress. Both detection and therapeutic processes are selective to cancer cells, indicating high specificity and robustness of the hybrid (gold nanoparticle) conjugate proved as a promising candidate for clinical cancer diagnostics and treatment and their targeted drug delivery approach (Nasrabadi et al. 2016).
It can be inferred from few of the above-discussed studies of nanocarrier-bound therapeutic enzyme delivery that nanocarrier-based approach such as gold nanoparticle-immobilized enzymes represents an important modality within therapeutic and diagnostic biomedical applications including cancer, cardiovascular diseases and brain diseases.
3.6 Conclusion
Gold nanoparticles offer an excellent platform for biomedical applications due to their unique physical and chemical properties. Amongst the various physicochemical and biological methods of gold nanoparticle syntheses, the biological route has become most fascinating due to total avoidance of toxic chemical and ambient reaction conditions and more biocompatibity of the gold nanoparticles, since delivery of enzyme as drug along with the antimicrobial property of gold nanocarrier adds additional double effects on various health ailments. Very few therapeutic microbial enzymes are used till date, and more research on gold nanocarrier-bound therapeutic important enzyme is the need of the hour.
The selection of the most appropriate methods for robust gold nanocarrier design needs a thorough understanding of non-covalent and covalent interactions at the interface of different types of therapeutic enzymes and different functionalized gold nanoparticles. It is inferred that gold nanocarrier-bound limited therapeutic enzyme has shown promising results in the treatment of central nervous system disorders. To sum up, gold nanocarrier-mediated delivery of therapeutic enzymes holds a great potential for biomedical applications.
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The authors would like to thank the director of the Indian Institute of Information Technology Una for providing the necessary facility to carry out nanobiotechnology work.
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Verma, M.L., Kumar, P., Sharma, S., Dhiman, K., Sharma, D., Verma, A. (2020). Gold Nanoparticle-Mediated Delivery of Therapeutic Enzymes for Biomedical Applications. In: Daima, H., PN, N., Ranjan, S., Dasgupta, N., Lichtfouse, E. (eds) Nanoscience in Medicine Vol. 1. Environmental Chemistry for a Sustainable World, vol 39. Springer, Cham. https://doi.org/10.1007/978-3-030-29207-2_3
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