A. GNTbm Nano-Gold

One nanometer equals 10-9 meters; nano-drugs refer to particles with diameters less than 100 nm. Mammalian cells are approximately 10,000 nm in size, E. coli is approximately 1000–1500 nm wide and 2,000–6,000 nm long, a virus is approximately 40–100 nm in size, a monoclonal antibody is approximately 10 nm, a lipid bilayer is approximately 3–5 nm, glucose is <1 nm, and an atom is approximately 0.1 nm. With a diameter of less than 100 nm, nano-drugs can enter the inflammatory region or tumor tissues through the enhanced permeability and retention effect (EPR).


Manufacturing process of GNTbm Nano-Gold

The manufacturing processes of nano-gold have many kinds, but they primarily comprise chemical and physical methods. GNTbm currently uses the following manufacturing processes.

(a) Chemical synthesis

The chemical method to manufacture GNTbm Nano-Gold uses HAuCl4 as the raw material, which is heated to boiling, mixed with an appropriate amount of reducing agent (Na3C6H5O7 or NaBH4), and heated again to boiling. The reaction occurs quickly and the Au3+ in HAuCl4 is reduced to Au0 gold atoms by the reducing agent. The continuous increase of Au0 gold atoms leads to increased concentration of Au0, concurrently, the synchronous nucleation and growth quickly promote further nucleation and the stacking of Au0 gold atoms to generate small nano-gold particles. Over time, with continuous stirring, the particle diameter of the nano-gold gradually increases, which is accompanied by a continuously color change. After immediate addition of reducing agent the pale yellow HAuCl4 solution transform to a colorless transparent solution. At this point, Au3+ has been reduced to Au0 gold atoms by the reducing agent, the color gradually becomes black, which indicates rapid nucleation and stacking of Au0 gold atoms; the size of nano-gold particles is <1 nm. When the color changes to black purple, the size of nano-gold particles is approximately 3 nm and when the color changes to orange, the particle diameter is approximately 5 nm. Eventually, the growth of the particles stops and they remain at a constant size; when this occurs, the color of the solution is burgundy red and the diameter of the nano-gold particles is approximately 20 nm (Figure 2). The concentration of the reducing agent determines the size of the nano-gold particles and the stirring speed determines the appearance and uniformity of the particles.


Figure 2. Chemical synthesis of gold nanoparticles using HAuCl4 and reducing agent.


(b)Physical synthesis

GNTbm uses the GNT nano-gold produced by a physical method obtained from GNT Inc. by technology transfer to develop nano-drugs. The physical method of GNT Inc. uses 99.99% pure gold in a high temperature vacuum vaporization environment, in which physical vapor deposition (PVD) technology is used to generate nano-gold (please refer to the GNT Inc. official website).

GNTbm now has both chemical and physical methods to produce nano-gold. The nano-gold particles produced by the two methods are both produced by nucleation and growth of Au0 gold atoms, and the physical and chemical properties of each method are yet to be further studied. However, in terms of purity, nano-gold produced by the physical method is higher than that produced by chemical method, whereas for the particle diameter control and uniformity, the chemical method is slightly superior to the physical method. Currently, the two manufacturing methods can produce a few liters of nano-gold each time, with a concentration of 100–200 ppm. In the future, the manufacture of nano-gold will be outsourced to CMO, which will allow mass production of nano-gold drugs.


Analysis of GNTbm Nano-Gold --- Chemical method

Figure 3. Gold nanoparticles with an average diameter of 5 nm, with or without PEG coating, are analyzed by using transmission electron microscopy and spectrophotometry

Figure 4 Gold nanoparticles with an average diameter of 11.8 nm are analyzed by using transmission electron microscopy and spectrophotometry


Figure 5. Gold nanoparticles with an average diameter of 15 nm are analyzed by using transmission electron microscopy and spectrophotometry


Figure 6. Gold nanoparticles with an average diameter of 25.2 mm are analyzed by using transmission electron microscopy and spectrophotometry

Figure 7. Gold nanoparticles of an average diameter of 31.3 nm are analyzed by using transmission electron microscopy and spectrophotometry

B. Proprietary controlled-release short-chain linkers

Linkers are the bridges between the nano-gold and the drugs. The design of the linker is very important. One end of the linker contains a specific functional group that binds to different chemotherapy agents or small molecule drugs and the specific functional group on the other end can form covalent bonds with the nano-gold surface. GNTbm has designed its own proprietary linkers to establish a nano-gold drug delivery platform.

The principles and advantages of controlled-release linkers

When nano-gold is used as a carrier to deliver drugs, it will be readily engulfed by tumor cells and the nano-gold drugs are delivered to lysosomes, which have a low pH. As chemotherapy drugs are bonded by a pH-sensitive functional group, the low pH environment causes the bond to break, leading to a fast release of the chemotherapy drug. Therefore, the design is classed as a controlled-release linker. Through the GNTbm nano-gold drug delivery system, a large amount of chemotherapy drugs can be delivered to cancer cells and effectively eliminate these cells to treat the cancer. As the diameter of the nano-gold drug complex is less than 100 nm, it will be taken up in the tumor site by the unique EPR effect, which reduces the toxicity and adverse effects of chemotherapy drugs; therefore, high concentrations of chemotherapy drugs can be delivered selectively to tumor areas and kill tumor cells. GNTbm has designed and developed a series of linkers, including short-chain linkers that can bind small molecule drugs. Short chain linkers have a molecular weight of less than 1,000 Da.

C. Proprietary long-chain linkers for controlled release

The delivery of protein drugs or monoclonal antibodies is an important part of nano-gold drug delivery. GNTbm has developed proprietary long-chain linkers to connect functional groups at a specific location on monoclonal antibody, which avoids impacts on the structure and antigen binding sites as much as possible and maintains the activity of monoclonal antibodies. In the proprietary GNTbm long-chain linkers, one end contains a unique functional group that can covalently bind with the surface of GNT nano-gold and the other end contains a functional group that can efficiently react with the glycosyl group of the monoclonal antibody for the delivery of protein drugs or monoclonal antibodies. The long chain linkers have a molecular weight of approximately 2,000–10,000 Da.

D. Unique drug absorption mechanism of nanoparticle drugs

All nanoparticle drugs have a unique drug absorption mechanism which is known as the Enhanced Permeability and Retention (EPR) effect. The size of small molecule drugs is less than 1 nm, the size of monoclonal antibodies with a molecular weight of 150 kDa is about approximately 10 nm, and the size of nanoparticle drugs (including liposomal drugs) is less than 100 nm. In most inflammatory regions or tumors, the arrangement of vascular endothelial cells is not very tight; often, as pores of approximately 200–400 nm are generated, most small molecule drugs, antibodies, protein drugs, and a variety of nanoparticle drugs can readily enter into these pores and will remain inside for a long time when circulated through these regions. Over time, the concentration will gradually increase, which enhances the efficacy. This phenomenon is called EPR effect. Most small molecule drugs molecules are too small, and their fast-in and fast-out behavior makes the EPR effect insignificant. Although the molecular weight of monoclonal antibodies is large enough and can enter the pore, it is difficult for them to accumulate and penetrate deeper into tumor tissues. Thus, the ERP effect is relatively insignificant, and the efficacy is compromised. Nano-gold is a very special carrier, which can be readily engulfed by cancer cells to allow an easier delivery of drugs into cancer cells and thereby achieve a profound therapeutic effect.


Polyethylene glycol (PEG), which coats the surface of the gold nanoparticles by bonding, has several functions. First, as PEG coating adequately protects the nano-gold from aggregation, the subsequent drug loading uses a substitution reaction to bind the linker and the drug to the remaining nano-gold surface. Second, PEG can increase the stability of nano-drugs and elongate their half-life in the blood. PEG coating leads to the formation of a hydrophilic membrane on the surface of nano-gold particles to prevent the clearance of nano-gold by the monocyte phagocytic system. In addition, PEG is approved by the FDA for use in drug development. The length, physical properties, and presence or absence of bifunctional groups can significantly affect whether the nano-drugs will induce immunological reaction, as well as the half-life of nano drugs, which both exert a significant influence on the efficacy. GNTbm will develop its own unique functional PEG for the Vaucarrin® technology platform.

Figure 8. Chemical structure of PEG