Biogenic gold nanoparticles: As a potential candidate for brain tumor directed drug delivery

Abstract

Gold nanoparticles have tremendous application in the area of nanotechnology that raises new possibility in the treatment of brain tumor. These nanoparticles can be used for selectively gaining access to tumor due to their small size and modifiability. Gold nanoparticles are functionalized with various molecules such as anticancer drug, transferrin and monoclonal antibody to produce nanocarriers. These nanocarriers have ability to deliver the drug at targeted site. Transferrin crosses the blood-brain barrier because of the receptor-mediated endocytosis. The monoclonal antibody facilitates the release of anticancer drug at targeted sites. This approach of delivery saves the normal cells surrounding the tumor.

Keywords::

• brain tumor
• blood–brain barrier (BBB)
• targeted drug delivery
• transferrin
• gold nanoparticles

Introduction

Nanobiotechnology, defined as biological applications of nanoscale material and structure (usually ranging from 1 to 100 nm), is emerging area of nanoscience and nanotechnology (Yezhelyev et al. 2006, Saxena et al. 2010). Gold nanoparticles have tremendous application in sensor, medicine, catalysis and many emerging area of nanotechnology (Lim and Zhong 2006). Radiation and chemotherapy were used for the treatment of cancer but these involve harmful side effects such as lowered immune response to infection (Calvert et al. 1993, Leon et al. 2001, Shah and Schwartz 2001). Cancer tissue has to create its own blood supply if it has to grow bigger than few millimeters. This causes growth of tumor blood vessels in a process called tumor angiogenesis. Malignancies with the central nervous system pose unique chemotherapeutic problem because the blood–brain barrier (BBB) may limit penetration of antineoplastic drug into brain tumor (Siegal et al. 1995, Grossman 1991, Levin et al. 1980). The major factors governing the passage of chemotherapeutic substance across the BBB (like molecular weight, polarity and lipid solubility) differ from the factors that influence the amount of drug that diffuse passively from capillaries into brain tumor. Other factors like permeability of capillaries with respect to the specific drug, the luminal surface area of the capillaries available for exchange, blood flow into the tumor, the concentration of unbound drug in the plasma and the length of time of drug circulations through the capillaries are also involved in passage of chemotherapeutic materials (Siegal et al. 1995, Blasberg and Groothius 1981). The above problems can be minimized by targeted drug delivery by smart metal nanovectors. Some scientists are working on photo-thermal tumor ablation therapy (Gobin et al. 2007, Loo et al. 2005, O’Neal et al. 2004). Photo-thermal tumor therapy is a new method for destruction of tumor (Bernardi at al. 2008). Infrared radiation absorbing nanoparticles are called nanoshells and these are a new class of optical tunable nanoparticles composed of a dielectric core (silica) coated with an ultrathin metallic layer (gold) (Oldenburg et al. 1999). This type of nanoshell was used in photothermal therapy for the destruction of tumor.

In this article, we will describe the use of biologically synthesized gold nanoparticles for targeted drug delivery because this can reduce the toxicity and have selective drug delivery properties. Gold nanoparticles may have advantages over other metallic particles in terms of biocompatibility and non-toxicity (Dhar et al. 20008, Shukla et al. 2005) and can be readily conjugated to a large range of biomolecules, such as amino acids (Selvakannan et al. 2004), proteins/enzymes (Niemeyer and Ceyhan 2001), DNA (Alivisatos et al. 1996) and other molecular species without altering the biological activity of the conjugated species. The benefits of targeted drug delivery over conventional resection are numerous; most approaches are minimally or totally non-invasive, relatively simple to perform and have the potential of treating embedded tumors in vital regions where surgical resection is not feasible. Gold nanoparticles are functionalized according to their use. Several reviews concentrating on nanoparticles-biomacromolecules have been published. The nanoparticles featured a mixed monolayer composed of cationic ligands (tetra ethylene glycolylated) and fluorogenic ligands. The cationic nature facilitates the crossing of cell-membrane barrier and fluorophore probes possible drug release mechanism. The hydrophobic dye conjugated gold nanoparticles are non-fluorescent as the gold core quenches fluorescence via energy and/or electron transfer process (Akbuga and Durmaz 1994). The development of colloidal gold (cAu) nanoparticle vector that targets the delivery of tumor necrosis factor (TNF) to a solid tumor growing in mice. The optimal vector, designated PT-cAu-TNF, consists of molecules of thiol-derivatized PEG (PT) and recombinant human TNF that are directly bound onto the surface of the gold nanoparticles (Paciotti et al. 2004). The gold nanoparticles stabilized by heterobifunctional polyethylene glycol (PEG) were synthesized using about 15-nm spherical gold cores and covalently coupled to f19 monoclonal antibodies (Eck et al. 2008).

We have mainly focused on the treatment of brain tumor by using biologically synthesized gold nanoparticles as carrier for drug delivery. These gold nanoparticles are functionalized to be attached with other molecules for making them prominent nanocarrier. This nanocarrier has ability to cross BBB and delivered the drug at targeted cells only.

Brain tumor

Cancer is characterized by a reduction or loss of cellular control and normal maturation mechanism. Its features include excessive cell growth, undifferentiated cells and tissues and the ability to grow into neighboring tissues (Lubbe et al. 2001). Brain benign tumor can be removed and they seldom grow back. The border or edge of benign tumor can be seen clearly. Benign tumor cells do not spread to other part of body. However, benign tumor can produce pressure on sensitive areas of the brain and cause serious health problems. Malignant tumor is generally more serious and often is life threatening. They grow rapidly and spread to other healthy parts of brain. The cell of malignant tumor can break and spread to other parts of brain, to the spinal cord, or even to other parts of the body. The spread of cancer is called metastasis. The symptoms of brain tumor depend on tumor size, type and location. The treatment of brain tumor depends on their features like size, position, etc. Craniotomy is the usual treatment for the most brain tumor (Figure 1).

Figure 1. CT scan image of brain tumor.

Gliomas, the most prominent primary tumors of the brain are classified on the basis of their origin, for example, Ependymomas originated from ependymal cells, Astrocytomas from astrocytes (glioblastoma multiforme is the most common astrocytoma), and Oligodendrogliomas from oligodendrocytes, moreover, mixed gliomas, such as oligoastrocytomas, contain cells from different types of glia (Arko et al. 2006).

Brain tumor biology

Microglia are a type of glial cells that are the resident macrophages of the brain and spinal cord, and thus act as the first and main form of active immune defense in the central nervous system (CNS). Microglia are constantly excavating the CNS for damaged neurons, plaques, and infectious agents (Gehrmann et al. 1995). The brain and spinal cord are considered “immune privileged” organs in that they are separated from the rest of the body by a series of endothelial cells known as the BBB, which prevents most infections from reaching the vulnerable nervous tissue. In the case where infectious agents are directly introduced to the brain or cross the BBB, microglial cells must react quickly to decrease inflammation and destroy the infectious agents before they damage the sensitive neural tissue. Due to the unavailability of antibodies from the rest of the body (few antibodies are small enough to cross the BBB), microglia must be able to recognize foreign bodies, swallow them, and act as antigen-presenting cells activating T-cells.

Angiogenesis is a complex process resulting in new blood vessel formation. It is also a fundamental step in the transition of tumors from a dormant state to a malignant one. It involves several coordinated mechanisms including degradation of the basement membrane by proteases, proliferation, and migration of endothelial cells (ECs), lumen formation, basement membrane resembling, recruitment of pericyte or vascular smooth muscle cells (VSMCs), vascular maturation, and finally blood flow (Carmeliet 2005, Jain 2003, Marchuk et al. 2003). The extracellular matrix (ECM), the network of proteins surrounding cells in the body, was long believed to function mainly as inert scaffolding for tissue. ECM is a key “signaling molecule” crucial for the normal functioning of cells. That is, the ECM is one of the environmental factors (along with hormones) that communicate with a cell nucleus, modifying nuclear structures and leading to selective gene expression (Chen et al. 2000). The angiogenic process is complex and involves endothelial cell proliferation and migration, degradation of the blood vessel basement membrane and associated extracellular matrix. Following endothelial cell proliferation and early tube formation, newly formed vessels differentiate into arterioles and venules, necessary to provide blood supply to tumors. Remodeling of the ECM is an integral component of the angiogenic process. A variety of mechanisms have been documented about how the ECM plays a pivotal role in regulating angiogenesis. Sooner or later during the development of cancer the primary tumor mass spawns pioneer cells that move out, invade adjacent tissues, and circulate to distant organs where they may form new colonies known as invasiveness of cancer.

Macrophages play important role in tumor growth. Macrophages are versatile, plastic cells which respond to micro-environmental signals with distinct functional programs (Mantivani et al. 2004). Macrophages have a pleiotropic biological role which includes antigen presentation, target cell cytotoxicity, removal of debris and tissue remodeling, regulation of inflammation, induction of immunity, thrombosis and various forms of endocytosis (Auger and Ross 1992). Macrophages are prominent in tumor tissues and called tumor associated macrophages (TAMs) which are reprogramed by cancer cells. In fact, TAMs actually release a number of promitogenic factors that stimulate the tumor cell proliferation (Lewis and Murdoch 2005). Because of their high concentration in tumor tissues, TAMs provide an ideal target for anti-cancer therapies, but selectively delivering drugs to kill TAMs with minimal side-effects is a major hurdle. Nanotechnology is a promising area for the treatment of tumor/cancer more especially by targeted drug delivery to minimize the side effect.

Gold nanoparticles

The bulk gold cannot be used in biomedical application because of their large size; it cannot cross the membrane barrier. But gold has good properties for medical application because of their chemical and physical characteristics. Nanoparticles often behave much differently than bulk samples of the same materials, resulting in unique electrical, optical, chemical, biological, and mechanical properties (Crabtree et al. 2003, Krolikowska et al. 2003, Zhao and Stevens 1998).

The gold nanoparticles were synthesized by reduction of chloroauric acid (H[AuCl₄]). The formation of gold nanoparticles can be observed by color change of liquid, usually either an intense red (for particles less than 100 nm), or a dirty yellowish color (for large particles). Generally, metal nanoparticles are synthesized and stabilized by using chemical methods such as chemical reduction (Krishna and Goia 2009, Tripathi et al. 2010), electrochemical techniques (Rodriguez-Sanchez et al. 2000), photochemical reactions in reverse micellers (Taleb et al. 1997) and now days via green chemistry route (Ara et al. 2009). Use of plant in synthesis of nanoparticles is quite novel leading to truly green chemistry which provide advancement over chemical and physical method as it is cost effective and environment friendly and in this method there is no need to use high pressure, energy, temperature, and toxic chemical. Now a days we can synthesize the gold nanoparticles by bacteria, fungi, and plant extract (Lengke and Southam 2006, Xin et al. 2013, Gérrard et al. 2013, Raliya and Tarafdar 2013, Zhang et al. 2012, Ahmad et al. 2003, Daniela 2013). Gold nanoparticles can be capable of delivering large molecules, without restricting themselves as nanocarriers of only small molecular drug. The tunable size and functionality of gold nanoparticles make them a useful scaffold for efficient recognition and delivery of biomolecules at targeted site. They have shown success in delivery of peptides, proteins, or nucleic acid like DNA or RNA (Ghosh et al. 2008).

Biosynthesis of gold nanoparticles

The emergence of nanotechnology and its applications in agriculture, medical, cancer therapy, diagnostics, drug delivery, and tissue engineering have opened up potential benefits to mankind. Many unknown biological processes are now being studied to understand the formation of new nanomaterials and their antibacterial activity (Tripathi et al. 2010). Biogenic gold nanoparticles were functionalized to use as biosensing enhancement (Torres-Chavolla et al. 2010). Chemical methods have been used for the synthesis of nanostructured materials (Jana et al. 2001, Lin et al. 2004, El-Sayed 2001). Chemical methods have some demerits like being toxic in nature, costly, non-environmental friendly, etc. Here we describe the various biological reducing as well as capping agents for the synthesis of gold nanoparticles. Many microorganisms, both unicellular and multicellular have been reported to produce inorganic materials either intracellularly or extracellularly. Moreover the preparation of nanoparticles of desired quality with low cost is difficult by conventional methods and offer potential benefits. Biological methods of nanostructured material synthesis are nontoxic and environment-friendly and thus some well known examples cited in literature are as follows.

Bacteria

Some bacteria like Methylosinus trichosporium 3011 have been reported to synthesize gold nanoparticles (Xin et al. 2013). Rhodopseudomonas capsulata has been screened and found to successfully produce gold nanoparticles of different sizes and shapes (Gérrard et al. 2013).

Fungi

Use of various fungi also has been made to obtain large quantities of metal nanoparticles. Fusarium oxysporum has been used to synthesize gold and silver nanoparticles (Ahmad et al. 2003, Mukherjee et al. 2002).

Algae

Various Algae have been used to obtain metal nanoparticles. Such as Sargassum wightii has been used to synthesize gold nanoparticles (Singaravelu et al. 2007). Chlorella vulgaris have been reported to synthesize single-crystalline gold nanoplates (Xie et al. 2006).

Plants

Various Plant extracts have also been used to synthesize the metal nanoparticles. Gold and silver nanoparticles are synthesized by Neem (Azadirachta indica) extract (Shankar et al. 2004). Alfalfa plants extract was used for the synthesis of gold nanoparticles (Gardea-Torresdey et al. 2002). Lemongrass extract was used to synthesize Au core-Ag shell nanoparticles (Rai et al. 2007) and lemongrass extract was reported for the synthesis of triangular gold nano-prisms (Shankar et al. 2004).

The formation of nanoparticles was characterized by using UV-Vis spectroscopy, dynamic light scattering (DLS) and Fourier transform infrared spectroscopy (FTIR). The morphology of silver nanoparticles was confirmed by transmission electron microscopy (TEM).

Functionalization of gold nanoparticles

Nanotechnology is the development and improvement in the use of fluorescent marker for the diagnostic and screening purpose. Drug delivery systems (DDS) provide useful adjuncts for therapeutics, including drugs, nucleic acid, and proteins (Langer and Tirrell 2004). DDS can also be used for target specific tissues and cell types through appropriate functionalization. A diverse range of materials have been used as carrier, including linear polymer (Sershen and West 2002), dendrimers (Paleos et al. 2004), and nanomaterials like nanoparticles (Luo and Saltzman 2000). Mixed monolayer-protected clusters with gold cores provide desirable attitudes for use as carriers in DDS:

• (1) Gold core is essentially inert and non-toxic (Connor et al. 2005, Joshi et al. 2006).

• (2) Monodispersed nanoparticles can be formed with sizes 1.5–10 nm (Verma and Roltella 2005).

• (3) The monolayer can be tailored with a range of ligands, providing effective cellular uptake, controlled payload release and targeting a specific cell.

Biologically synthesized gold nanoparticles can be used as a nanocarrier for anticancer drug delivery in brain tumor. Gold nanoparticles will be functionalized with various materials to provide free group for the attachment of desirable materials. The functionalized gold nanoparticles will allow the desired molecule to bind on it. The anticancer drug and specific antibody (monoclonal antibody) are attached on the surface of functionalized gold nanoparticles. The monoclonal antibody facilitates the release of anticancer drug at specific site. The hetero-bifunctional PEG ligands contain a dithiol group for stable anchoring onto the gold surface and a terminal carboxy group for coupling of antibodies to the outside of the PEG shell (Eck et al. 2008). Ortho-pyridyl-disulfide-n-hydroxysuccinimide polyethylene glycol polymer (OPSS-PEG-NHS) was used to tether antibodies onto the surface of gold nanoparticles (Loo et al. 2004). Gold nanoparticles are surface functionalized by hetero-bifunctional poly(ethylene glycol) having a thiol group on one terminus and a reactive functional group on the other to use as a flexible spacer. Coumarin, a model fluorescent dye, was conjugated to one end of the PEG spacer and gold nanoparticles were modified with coumarin-PEG-thiol (Shenoy et al. 2006). Gold nanoparticles were functionalized by thiol-derivatized PEG for attachment of Tumor Necrosis Factor (TNF). TNF conjugated gold nanoparticles were used in treatment of tumor in mice (Paciotti et al. 2004). The targets of drug can be visualized by use of fluorescent nanomaterials having exceptional photostability, allowing the emission of fluorescent light over a long time without a rapid decline in emission (i.e., photobleaching). Gold nanoparticles were functionalized to produce negative charge on the surface of nanoparticles so that anticancer drug, monoclonal antibodies, and transferrin will be attached on it (Figure 2). The above bioconjugated nanocarrier molecules facilitate to cross the BBB by receptor-mediated endocytosis. This type of approach will deliver the drug at targeted site and protect the normal cells surrounding the tumor.

Figure 2. Diagrammatic representation of functionalization of biologically synthesized gold nanoparticles for the attachment of monoclonal antibody and anticancer drug.

Drug targeting

Nanocapsules mean sandy nanoparticles that consist of shell and a space, in which desired substances may be placed. Drug filled nanocapsules can be covered with antibodies or cell-surface receptors that bind to the cancer or various cells and release their biological compound on the contact with that tissue. Nanoparticulates in drug delivery systems can exploit a characteristic of solid tumors such as the enhanced permeability and retention (EPR) effect in which tumor tissues show peculiar characteristic properties such as hyper vasculature, defective vascular architecture and a deficient lymphatic drainage which lead macromolecules and particulates to be accumulated preferentially and to be retained for a longer time in tumor (Natalie et al. 2007); this type of transportation according to the size of nanoparticles is known as passive targeting (Figure 3). But, nanoparticles can be modified to target inherent characteristics of cancer cells such as rapid proliferation and particular antigen presentation (Moghimi et al. 2001) and this type of targeting is known as active targeting (Figure 4). The nanocapsules are attached to bioadhesive materials which is performed either by incorporating a bioadhesive material in the pH-sensitive microsphere matrix, or by using a bioadhesive material in the nanocapsules matrix in conjunction with a bioadhesive material in the microsphere matrix. Thus, nanocapsules were designed to release the drug over an extended period of time by dissolving/swelling the microsphere at a pH that is typically found in or near cancerous tissue.

Figure 3. Representation of passive targeting of drug. Nanoparticles accumulated in tumor because blood brain barrier selectively disrupted at the site of malignant lesion.

Figure 4. Representation of active targeting of drug. Use of antibody-nanoparticles conjugates can target tumor cells only and deliver the drug at specific site (tumor cell).

Mechanism of drug delivery across BBB

The BBB is a homeostatic defense mechanism of the brain that restricts the passage of solutes. The brain cells are tightly packed so that drugs have to move through the cells themselves and astrocytes (star shaped glial cells in brain) provide biochemical support of endothelial cells which form the BBB (Perdan et al. 2009). This approach makes powerful barrier and very few drug can cross it. BBB has receptors for several substrates like insulin and transferrin, etc. These receptors regulate the entry of molecules inside the brain. Receptor mediated drug delivery is a prominent method to deliver the drug across the BBB.

Nanotechnology manipulates the complex biological systems with high selectivity and timing than the conventional pharmacological approaches such as BBB (Modi et al. 2009). Some previous reporters have used transferrin for receptor mediated drug delivery to cross the BBB (Yang et al. 2005, Li and Qian 2002, Qian et al. 2002). The intratumoral drug distribution is an important issue with conventional method because of physiological properties of tumor like their cellular packing and limited diffusion of drug. The distribution of drug can be enhanced by active targeting. In active targeting the drug will release inside the cell by the action of monoclonal antibody. Biologically synthesized gold nanoparticles are functionalized to upload the drug, transferrin, and monoclonal antibody (Figure 2). This nanocarrier will be able to transfer the anticancer drug to targeted site only because antibody has the higher affinity with the tumor cells. BBB cannot allow any molecule to enter into the brain, but the above suggested nanocarrier is conjugated with transferrin which is attached to transferrin receptor of BBB (Figure 5) and cross the barrier by endocytosis (Yang et al. 2005).

Figure 5. Diagrammatic representation of receptor-mediated endocytosis to cross the BBB and deliver the drug at targeted site only. (A) Representation of BBB, (B) Intravenously injection of nanocarrier, (C) Transferrin attached to BBB receptor. (D, E & F) Receptor mediated endocytosis of nanocarrier and reached into brain by exocytosis, (G) Finally, nanocarrier attached with tumor cells only through monoclonal antibody.

Conclusions

In this review article we have described the targeted drug delivery by biologically synthesized gold nanoparticles for the treatment of brain tumor. We have witnessed the use of biologically synthesized gold nanoparticles in developing a new generation of more effective brain tumor therapies capable of overcoming the many biological, biophysical, and biomedical barriers that the body stages against conventional brain tumor therapies. Biologically synthesized gold nanoparticles are functionalized to attach with anticancer drug, transferrin, and monoclonal antibody. Transferrin has ability to cross the blood–brain barrier (BBB) by receptor-mediated endocytosis. The use of nanoparticles conjugated to monoclonal antibodies allows the possibilities of simultaneously detecting multiple molecular targets in brain tumor, on which treatment decisions can be made. This approach of targeted drug delivery is a promising method to deliver drug at targeted site and save the normal cells surrounding the tumor.

Acknowledgements

Authors are thankful to Dr R. P. Singh (Advisor) and Dr L. M. Bhardwaj (HOI), Amity Institute of Nanotechnology, Amity University, Noida, India for their encouragement and giving precious advice continuously for the above review article.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.