A Theoretical Consideration of the Loading Capacity of Nano-Gold

Johnson K. Gao and Catherine W. Gao August 7, 2006 Nano technology has been recognized as the future direction in biomedicine and manufacturing industry (1). One of benefits of nano particle is its small size with relatively large surface area. That property of a nano-meter gold permits us to make drug carriers which can penetrate into cells (or tissues) easier and load more drug molecules. In order to seek a novel drug carrier, we have recently modified the procedure (2) and prepared a peculiar carrier called ngSAB, which denotes the nano-meter gold particles assembled in sub-cellular size agarose bead(s). We had successfully loaded such a carrier with a kind of polypeptide “α-endo” (an abbreviation of an antagonist to endothelial cell growth factor). It was found that ngSAB-α-endo were internalized by cultured cells as evidently shown with light and electron microscopy (3) Since “α-endo” may “wash” away unwanted color spots in skin and to increase its beauty, ngSAB-α-endo might become a potential candidate for cosmetic product manufacturing in the future. One nano meter equals to 1/1,000,000,000 of a meter. Suppose that we have a cubic meter of certain material, such that a huge block of gold. The total surface area of one cubic meter gold is (1 m x 1 m) x 6 = 6 m2, or, six square meters. In the current article, we intend to make an effort to explain how large area it could be created as one cubic meter of gold (or any solid maters) is divided into as many as tiny cubic nano particles of gold. First let us divide one cubic meter of gold with three cuts, and each cut is exactly perpendicular to one of its three dimensions, and the cutting plane is just passing through the middle of three different dimensions, and without loss of any materials of the gold. Thus, we can get 8 blocks of ½ m x ½ m x ½ m, i.e. 8 pieces of 1/8 m 3. The total volume of 8 small blocks still equals to one cubic meter. However, after such a 3halfway-cut division the total surface area of those eight pieces of 1/8 cubic meter increases to ½ m x ½ m x 6 x 8 = 12 m2, i.e. 12 square meters. So, when we shall run another similar 3-halfwaycut division, which reduce the length of each dimension of every newly produced block to ¼ m. Thus, each original 1/8 m3 block of gold can produce eight pieces of 1/64 m3 of gold blocks. The total surface area equals to ¼ m x ¼ m x 8 x 8 x 6 = 24 m2. Now, we can conclude that by performing every 3-halfway-cut division it will gain a surface increment of two folds without reducing the original volume of the block(s). Table 1 is the result of all 30 consecutive 3-halfway cuts. Those figures were obtained by calculation after each 3-halfway-cut division. The equation used for calculation of these figures is given as following: Sn = Qn x Un ---------- (Equation 1) In that equation, Sn denotes the sum of surface areas of all cubic blocks obtained after n times of 3-halfway-cut divisions. Since, after n times of 3-halfway-cut division, the length of each dimension (or we may roughly say the diameter) of the resulting cubes Ln = 1/ 2n (m). (When the particle size is getting smaller and smaller, the difference between diameter of a sphere (Dn) and

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dimension of a cube could be neglected). So, the unit surface area of each final block at n times of 3-halfway-cut division, Un = 6 x 1/22n (m2). Hince the quantity of total blocks created at the end of the last 3-halfway-cut division Qn = 23n, Sn = Qn x Un = 23n x 6 x 1/22n (m2) = 23n x 6 x 2-2n (m2), or, Sn = 6 x 2n (m2) ---------- (The practical calculation of Equation 1) As one can see from that table clearly, when n = 30, the diameter of gold particle is 0.9 nm. The total surface area of all collected nano-meter gold particles is larger than 6,442 million square meters. In such a circumstance, for a city with a population of 10 million people, everyone in that city can share a surface of 644 square meter of gold. That seems incredible. And that is the very reason why the nano gold shall have so great loading power for a drug carrier. Table 1 has another function. It can be used to predict how much is the minimum amount of protein needed to cover all the surface of fixed amount of nanogold, if we know the average size of a given nanogold and the volume of the pellet of that a nanogold. To measure the volume of nanogold pellet, one simple method can be tried. After a colloidal gold solution was prepared, it normally needs to spin down the colloidal particle to the bottom of centrifuge tubes at a high speed, such that 100,000 g (gravity forth) with a super ultra centrifuge at low temperature. Then, suck away the supernatant. Now, add certain amount of water (A) over the pellet of colloidal gold, and sonicate the tube for few minutes, and re-calculate the suspended colloidal gold volume (B). Subtract A from B and get the difference (C). C is the approximate volume of that pellet. We can use Table 1 to estimate how large is the total surface area of all those nanogold particles in that pellet. For example, the nanogold we made has an average size of 20 nm. And the volume of pellet of nanogold is 100 micro litre (102 μl). Searching from the Table 1, it can be found, when n = 26, D26 = 19.9 nm, which is very close to 20 nm gold in experiment. In that case, one cubic meter gold can produce a total surface area of S26 = 0.4 x 109 m2. Because one cubic meter = 1003 cm3 = 106 ml = 109 μl, we can calculate out the surface area of 100 μl 20 nm gold. The value X in question can be obtained as following. 109 μl : 102 μl = 0.4 x 109 m2 : X X = 0.4 x 109 m2 x 102 μl x 10-9 μl-1 = 0.4 x 102 m2, or, 40 m2. Colloidal gold is a negatively charged lyophobic sol. It is sensitive to eletrolytes and will be flocculated by salts. Colloidal gold can be stabilized by protein by adsorbing protein molecule on to gold particle surface through electronic-chemical interaction. (4) When the diameter of a protein (or polypeptide) molecule is smaller than the diameter of nanogold particle, it is protein (or polypeptide) coating the nanogold. When the diameter of a protein molecule is larger than the diameter of nanogold particle, then, there shall be more than one nanogold particle attaching to each protein molecule. However, in either the case, the minimum amount of protein required in

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occupying all the surface of nanogold particles, or, the sigma of attachment area (not all the free surface) of protein should equal to the total surface provided by all nanogold particles. If there is a protein having a diameter of 8 nm for each molecule, and we want to use that protein to coat 100 μl of 20 nm gold, we can calculate out the sigma of attachment area as follows. Let us suppose that the protein molecule is globular in shape, and its attachment area to nanogold (that including its attachment points and its exclusive area that prohibits other molecules to bind geometrically to the same area on nanogold particle) is approximately equal to the surface area of a cycle. Because surface area = πr2, the area of attachment/exclusion of a protein molecule of 8 nm in diameter shall equal to 3.14 x 42 nm2 = 50.24 nm2. Since X = 40 m2, and 1 m2 = 109 x 109 nm2. So, X = 40 x 1018 nm2. The number of molecules needed to cover all those nanogold particles should be 40 x 1018 nm2 divided by 50.24 nm2 /per molecule. That is 7.96 x 1017 molecules, or, nearly 8 x 1017. According to Avogadro constant NA = 6.02 x 1023 /mol, Or, NA = 6.02 x 1017 /micro mol, And let us set Y as the amount of how many micro mol of protein needed that can fully load 100 μl of 4 nm gold , we can calculate out Y = 1.33 micro mol (μMol). Similar data of the number of micro mol of protein should be useful before consideration of drug labeling for a given amount of specific size of nanogold. Table 2, 3, and 4 are estimations of exclusion surface areas of different size of protein for every 100 μl of 19.9, 7.5, and 3.7 nanogold respectively. The last column of each table is the micro mol of protein needed for the saturation of 100 μl of those nanogold particles with variety of protein used. These data may be quite meaningful to figure out how to economically use of valuable protein, since the labeling of colloidal gold is always empirically to use protein in far excess range. That is an unwanted waste of valuable things. Those tables provide information that helps a reasonable consideration in drug loading to the colloidal gold and it saves money.

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Table 1. The relationship between numbers (n) of 3-halfway-cut division of a cubic meter of solid mass and the dimension (m), or, approximate diameter (expressed in different unit) of each single final block, and, the total surface area (m2) of all those cutting blocks obtained and added together. n 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Ln = 1/2n (m) 1 1/2 1/4 1/8 1/16 1/32 1/64 1/128 1/256 1/512 1/1,024 1/2,048 1/4,096 1/8,192 1/16,384 1/32,768 1/65,536 1/131,072 1/262,144 1/524,288 1/1,048,576 1/2,097,152 1/4,194,304 1/8,388,608 1/16,777,216 1/33,554,432 1/67,108,864 1/134,217,728 1/268,435,456 1/536,870,912 1/1,073,741,824 Approximate diameter Dn (vary in unit) 1.0 m 50.0 cm 25.0 cm 12.5 cm 6.3 cm 3.1 cm 1.6 cm 0.8 cm 3.9 mm 2.0 mm 1.0 mm 0.5 mm 0.2 mm 0.1 mm 61.0 μm 30.5 μm 15.3 μm 7.6 μm 3.8 μm 1.9 μm 1.0 μm 0.5 μm 0.2 μm 0.1 μm 59.6 nm 29.8 nm 19.9 nm 7.5 nm 3.7 nm 1.9 nm 0.9 nm Sn = 2n x 6 (m2) 6 12 24 48 96 192 348 768 1,536 3,072 6,144 12,288 24,576 49,152 98,304 196,608 393,216 786,432 1,572,864 3,145,728 6,291,456 12,582,912 25,165,824 50,311,648 100,663,296 201,326,592 402,653,184 805,306,368 1,610,612,736 3,221,225,472 6,442,450,944

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Table 2. Estimation of approximate micro mol of protein with different sizes of molecule that is needed to saturate per 100 μl of 20 nm gold*. Dp (nm) 5 10 20 40 80 19.63 78.50 314.00 1,256.00 5,024.00 Sp (nm2) Sg (nm2) 402.65 x 10 402.65 x 10 402.65 x 10 402.65 x 10 402.65 x 10
17 17 17 17 17

Mp (Mp = Sg /Sp) 20.51 x 1017 5.13 x 10 1.28 x 10 0.32 x 10 0.08 x 10
17 17 17 17

Micro mol of protein needed 3.41 0.85 0.21 0.05 0.01

* In this table, Dp represents the diameter of one molecule of protein or polypeptide. Sp represents the area of attachment/exclusion per molecule, which equals to πr2, or, π(0.5D)2. Sg represents the total binding area offered by 100 μl of 19.9 nm gold. Mp represents number of protein molecules needed to cover the total binding area offered by 100 μl of 19.9 nm gold, so that Mp = Sg /Sp.

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Table 3. Estimation of approximate micro mol of protein with different sizes of molecule that is needed to saturate per 100 μl of 7.5 nm gold*.* Dp (nm) 5 10 20 40 80 19.63 78.50 314.00 1,256.00 5,024.00 Sp (nm2) Sg (nm2) 805.30x 10
17 17 17 17 17

Mp (Mp = Sg /Sp) 41.02 x 1017 10.26 x 10 2.57 x 10 0.64 x 10 0.16 x 10
17 17 17 17

Micro mol of protein needed 6.82 1.70 0.43 0.11 0.03

805.30 x 10

805.30 x 10 805.30 x 10 805.30 x 10

** In this table, Dp represents the diameter of one molecule of protein or polypeptide. Sp represents the area of attachment/exclusion per molecule, which equals to πr2, or, π(0.5D)2. Sg represents the total binding area offered by 100 μl of 7.5 nm gold. Mp represents number of protein molecules needed to cover the total binding area offered by 100 μl of 7.5 nm gold, so that Mp = Sg /Sp. Table 4. Estimation of approximate micro mol of protein with different sizes of molecule that is needed to saturate per 100 μl of 3.7 nm gold.*** Dp (nm) 5 10 20 40 80 19.63 78.50 314.00 1,256.00 5,024.00 Sp (nm2) Sg (nm2) 1,610.61 x 10 1,610.61 x 10 1,610.61 x 10 1,610.61 x 10 1,610.61 x 10
17 17 17 17 17

Mp (Mp = Sg /Sp) 80.05 x 1017 20.52 x 10 5.13 x 10 1.28 x 10 0.32 x 10
17 17 17 17

Micro mol of protein needed 13.63 3.14 0.85 0.21 0.05

*** In this table, Dp represents the diameter of one molecule of protein or polypeptide. Sp represents the area of attachment/exclusion per molecule, which equals to πr2, or, π(0.5D)2. Sg represents the total binding area offered by 100 μl of 3.7 nm gold. Mp represents number of protein molecules needed to cover the total binding area offered by 100 μl of 3.7 nm gold, so that Mp = Sg /Sp.

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Reference: 1. Isha Nanotechnologie. What is nano Technology? http://www.ishananotech.com/nanotech.htm. 2. Gao, K. Colloidal gold-labeled agarose-gelatin microspherule method. In Hayat, M. A. edited “Colloidal Gold. Principles, Methods and Applications. Vol. 1. Academic Press Inc., 1989. Pp.391-415. 3. Gao, J. K. and Gao, C. W. Internalization of ngSAB-endo, and ngSAB-coll by cultured cells observed with light and electron microscopy. (In preparation) 4. Slot, J. W., and Geuze, H. J. (1985) A new method of preparing gold probes for multiplelabeling cytochemistry. Euro.J.Cell Biol. 38,87.

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