**3. Mechanism study of molecular recognition between the ligand and the pathogenic toxic molecule.**

## **3.1. Molecular recognition**

To understand the interaction mechanism of pathogenic toxins with different ligands is essential, since it not only provides fundamental insight to biomaterial science, but also can lead to the discovery of more efficient ligands for the removal of pathogenic toxins in human blood. Chemical modification of proteins has been frequently used in the studies of structure-function relationships of proteins, especially in the determination of the active sites in biologically active proteins [23,24]. In the present study, we selectively modified the arginine, tryptophan, lysine residues and carboxyl terminus on the protein for the molecular recognition studies.

Lianyong Wang et al [25] investigated the interaction between ss- DNA and IgGRF by selectively modification of the arginine, tryptophan, lysine residues and carboxyl terminus on IgGRF, which was purified from patients' serum. It is well known that the density of negative charge is high on the surface of ss-DNA molecule, due to the large amount of phosphate groups. After the ss-DNA was covalently attached to the cellulose carrier, the immunoadsorbent is negatively charged, so it has a high adsorption capacity for the positively charged *N*-bromosuccinimide (NBS) modified IgGRF. The same situation occurred when *N*-Ethyl-*N'*-[3-(dimethylamino)propyl]carbodiimide( EDC) modified IgGRF because of its decrease in negatively charged density. The low adsorption capacity for 1,2 cyclohexanedion(CHD) and pyridoxal 5-phosphate (PP) modified IgGRF may be attributed to the reduction of positively charged density after modification. From all the experimental results, it is assumed that there is an ionic bond formed between the modified IgGRF and the ss-DNA immobilized immunoadsorbent.

Shenqi Wang and Yaoting Yu et al [24] studied the mechanism of recognition and interactions of low density lipoprotein cholesterol(LDL-C) with different charged ligands on the adsorbents. Tryptophan, lysine residues and carboxyl terminus on LDL were chemically modified by PP,EDC and NBS respectively. Due to the effectiveness of L-lysine in the removal of LDL-C, it was selected to study the interaction of ligand with the modified LDL. Experimental results show that positive charge on the surface of LDL interacted with the negatively charged carboxyl groups of L-lysine by electrostatic force, thus resulting in the adsorption of LDL by the absorbent. We also found that increasing the positive charge on the surface of LDL could enhance the adsorption capacity of the adsorbent. On the contrary, increasing the negative charge could decrease the adsorption ability. Thus, different adsorbents containing sulfonic groups, phosphoric groups, L-lysine and carboxyl groups as the ligand were synthesized for investigating the effect of electric charge on their adsorption capacity. Results show that the adsorption capacity increases with the increase of the electro-negativity of the ligand on the adsorbent. See **Table1**


Source: Wang S Q et al, Reactive & Functional Polymers (2008), 68: 261-267

198 Cellulose – Medical, Pharmaceutical and Electronic Applications

**pathogenic toxic molecule.** 

**3.1. Molecular recognition** 

recognition studies.

**Figure 3.** Amount of epoxy groups on cellulose versus concentration of NaOH

IgGRF and the ss-DNA immobilized immunoadsorbent.

**3. Mechanism study of molecular recognition between the ligand and the** 

To understand the interaction mechanism of pathogenic toxins with different ligands is essential, since it not only provides fundamental insight to biomaterial science, but also can lead to the discovery of more efficient ligands for the removal of pathogenic toxins in human blood. Chemical modification of proteins has been frequently used in the studies of structure-function relationships of proteins, especially in the determination of the active sites in biologically active proteins [23,24]. In the present study, we selectively modified the arginine, tryptophan, lysine residues and carboxyl terminus on the protein for the molecular

Lianyong Wang et al [25] investigated the interaction between ss- DNA and IgGRF by selectively modification of the arginine, tryptophan, lysine residues and carboxyl terminus on IgGRF, which was purified from patients' serum. It is well known that the density of negative charge is high on the surface of ss-DNA molecule, due to the large amount of phosphate groups. After the ss-DNA was covalently attached to the cellulose carrier, the immunoadsorbent is negatively charged, so it has a high adsorption capacity for the positively charged *N*-bromosuccinimide (NBS) modified IgGRF. The same situation occurred when *N*-Ethyl-*N'*-[3-(dimethylamino)propyl]carbodiimide( EDC) modified IgGRF because of its decrease in negatively charged density. The low adsorption capacity for 1,2 cyclohexanedion(CHD) and pyridoxal 5-phosphate (PP) modified IgGRF may be attributed to the reduction of positively charged density after modification. From all the experimental results, it is assumed that there is an ionic bond formed between the modified **Table 1.** Adsorption capacity and percentage of total cholesterol(TC), LDL-C by cellulosic beads having different terminus groups

(From Wang S Q et al, Reactive & Functional Polymers (2008), 68: 261-267, adapted)

**Figure 4.** The relationship of absorption capacity versus electronegativity of ligands immobilized on the adsorbent

Experimental results show that the adsorption capacity (mg/ ml or percentage) for TC and LDL-C decreased with decreasing of electro-negativity of ligands on the adsorbents (-SO32->-PO43->-COO- ; -PO43->PP-PO43->DNA-PO43-), which demonstrate that the electro-negativity of ligand on adsorbent plays an important role in adsorbing TC and LDL-C. This relationship of the adsorption capacity to its electro-negativity is shown in Figure. 4

Bioactive Bead Type Cellulosic Adsorbent for Blood Purification 201

TC LDL-C

Removal capacity (%)

Stoichiometric capacity (mg LDL-C/mg L-lysine)

Removal amount (mg/ml)

Efficiency of active site (%)\*

Removal amount (mg/ml)

1000 11.45±0.35 0.351±0.011 12.01±0.79 0.242±0.017 2000 14.90±0.69 0.458±0.020 13.04±0.71 0.263±0.013 4000 28.94±0.33 0.889±0.011 35.13±0.69 0.708±0.017 6000 33.48±0.33 1.028±0.011 44.76±0.36 0.903±0.003

**Table 2.** Adsorption capacity and adsorption percentage of TC, and LDL-C by cellulosic beads with

Average removal amount of LDL- C (mg/ml)

1000 10.5 0.242 0.023 22 2000 9.8 0.263 0.027 26 4000 9.0 0.708 0.079 75 6000 8.6 0.903 0.105 100

In this study, carboxyl modified PEG spacer was synthesized and linked covalently to cellulose beads. L-lysine ligand was coupled to the spacer and its selective affinity for lowdensity lipoprotein-cholesterol (LDL-C) was determined. It was found that the adsorption capacity and the efficiency of the ligand for adsorption of LDL-C were increased when PEG spacer was used. Experimental results showed that by increasing the molecular weight of PEG spacers from 1000Da to 6000Da, the average adsorption capacity of LDL-C was enhanced from 0.242mg/ml to 0.903mg/ml. According to the analytical data of cellulose adsorbents, the amount of L-lysine ligand could be calculated. Although the amount of Llysine linked to the adsorbent with PEG spacers (10.5, 9.8, 9.0, 8.6 mg per ml cellulose adsorbent respectively) was lower than those without PEG spacers (121.6mg per ml cellulose adsorbent), see **Table 2**, the average adsorption capacity for LDL-C per ml cellulose adsorbent increased from 0.130 mg/ml to 0.903 mg/ml. After the introduction of PEG spacers, (see **Table 3)** and consequently the adsorption capacity for LDL-C per unit ligand increased significantly from 0.001 mg/mg L-lysine to 0.105 mg/mg L-lysine, see **Table 4**, the adsorption capacity of LDL-C per unit L-lysine ligand (0.027mg LDL-C/mg L-lysine) was much higher than that without PEG spacer (0.001mg LDL-C/mg L-lysine). This result indicated that in the presence of PEG spacer, the adsorption efficiency of L-lysine ligands was enhanced significantly, see **Table 5**. It is postulated that appropriate increasing the amount of the L-lysine ligands and the use of PEG spacers can enhance the adsorption

capacity for LDL-C.

Molecular weight of PEG spacers (Da)

Mol. weight of PEG spacers (Da)

Removal capacity (%)

Source: Wang S Q et al, Reactive & Functional Polymers (2008), 68: 261-267

Source: Wang S Q et al, Reactive & Functional Polymers (2008), 68: 261-267

**Table 3.** Adsorption capacity of LDL-C per mg L-lysine

different molecular weight of PEG as a spacer

L-Lysine amount (mg/ml)

## **3.2. Spacer effect**

Spacers have a significant effect on the adsorption property of the resin adsorbents. It can reduce the steric hindrance between the ligand and the large toxic molecules, resulting in an increase of adsorption capacity of the adsorbent. Different spacers have an obvious effect on the adsorption properties of adsorbents. The density of ligands on the carrier and the effect of steric hindrance are both important factors in specific adsorption. When the target substance is a small molecule, there may be no steric hindrance, see **Figure 5 a,** so the enhancement of the density of ligands can improve the adsorption capacity. But when the target substance is a large molecule, due to the presence of steric hindrance [26-33], a high density of ligands linked may display a low adsorption capacity of target protein, see **Figure 5b**. In theory, a flexible spacers can reduce the steric hindrance, see **Figure 5c**. In order to study flexible spacers play the role in reducing steric hindrance between the target protein and immobilized ligands, Xinji Guo et al [34]designed and prepared cellulosic adsorbents with L-lysine acid as ligands and PEG having different molecule weights as spacers.

**Figure 5.** Schematic diagram of interaction between the ligand and target toxins

Note: a, Interaction between small target molecules and the immobilized ligands; b, Large target molecules having a steric hindrance to the immobilized ligands; c, Flexible spacer can reduce the steric hindrance,

In this study, carboxyl modified PEG spacer was synthesized and linked covalently to cellulose beads. L-lysine ligand was coupled to the spacer and its selective affinity for lowdensity lipoprotein-cholesterol (LDL-C) was determined. It was found that the adsorption capacity and the efficiency of the ligand for adsorption of LDL-C were increased when PEG spacer was used. Experimental results showed that by increasing the molecular weight of PEG spacers from 1000Da to 6000Da, the average adsorption capacity of LDL-C was enhanced from 0.242mg/ml to 0.903mg/ml. According to the analytical data of cellulose adsorbents, the amount of L-lysine ligand could be calculated. Although the amount of Llysine linked to the adsorbent with PEG spacers (10.5, 9.8, 9.0, 8.6 mg per ml cellulose adsorbent respectively) was lower than those without PEG spacers (121.6mg per ml cellulose adsorbent), see **Table 2**, the average adsorption capacity for LDL-C per ml cellulose adsorbent increased from 0.130 mg/ml to 0.903 mg/ml. After the introduction of PEG spacers, (see **Table 3)** and consequently the adsorption capacity for LDL-C per unit ligand increased significantly from 0.001 mg/mg L-lysine to 0.105 mg/mg L-lysine, see **Table 4**, the adsorption capacity of LDL-C per unit L-lysine ligand (0.027mg LDL-C/mg L-lysine) was much higher than that without PEG spacer (0.001mg LDL-C/mg L-lysine). This result indicated that in the presence of PEG spacer, the adsorption efficiency of L-lysine ligands was enhanced significantly, see **Table 5**. It is postulated that appropriate increasing the amount of the L-lysine ligands and the use of PEG spacers can enhance the adsorption capacity for LDL-C.


Source: Wang S Q et al, Reactive & Functional Polymers (2008), 68: 261-267

200 Cellulose – Medical, Pharmaceutical and Electronic Applications

(-SO32->-PO43->-COO-

**3.2. Spacer effect** 

Experimental results show that the adsorption capacity (mg/ ml or percentage) for TC and LDL-C decreased with decreasing of electro-negativity of ligands on the adsorbents

electro-negativity of ligand on adsorbent plays an important role in adsorbing TC and LDL-C. This relationship of the adsorption capacity to its electro-negativity is shown in Figure. 4

Spacers have a significant effect on the adsorption property of the resin adsorbents. It can reduce the steric hindrance between the ligand and the large toxic molecules, resulting in an increase of adsorption capacity of the adsorbent. Different spacers have an obvious effect on the adsorption properties of adsorbents. The density of ligands on the carrier and the effect of steric hindrance are both important factors in specific adsorption. When the target substance is a small molecule, there may be no steric hindrance, see **Figure 5 a,** so the enhancement of the density of ligands can improve the adsorption capacity. But when the target substance is a large molecule, due to the presence of steric hindrance [26-33], a high density of ligands linked may display a low adsorption capacity of target protein, see **Figure 5b**. In theory, a flexible spacers can reduce the steric hindrance, see **Figure 5c**. In order to study flexible spacers play the role in reducing steric hindrance between the target protein and immobilized ligands, Xinji Guo et al [34]designed and prepared cellulosic adsorbents

with L-lysine acid as ligands and PEG having different molecule weights as spacers.

**Figure 5.** Schematic diagram of interaction between the ligand and target toxins

reduce the steric hindrance,

Note: a, Interaction between small target molecules and the immobilized ligands; b, Large target molecules having a steric hindrance to the immobilized ligands; c, Flexible spacer can

; -PO43->PP-PO43->DNA-PO43-), which demonstrate that the

**Table 2.** Adsorption capacity and adsorption percentage of TC, and LDL-C by cellulosic beads with different molecular weight of PEG as a spacer


Source: Wang S Q et al, Reactive & Functional Polymers (2008), 68: 261-267

**Table 3.** Adsorption capacity of LDL-C per mg L-lysine


Bioactive Bead Type Cellulosic Adsorbent for Blood Purification 203

SO3H

SO3H

SO3H

SO3H

HO SO3H

Cellulose bead

:Cholesterol

SO3H

was 3 and 5 h, respectively. The amphiphilic adsorbent had a high adsorption capacity for LDL-C without significantly adsorbing high-density lipoprotein. Rabbit model was constructed according to the following method [44]. In brief, Japanese white male rabbits were purchased from local experimental animal institute and housed in a standard facility. After feeding with standard chow and water *ad libitum* for one week, the healthy rabbits were divided into control group (group 1, n=6) and hyperlipidemia group. Rabbits in the control group consumed standard chow from 120-150g/d and water *ad libitum.* In the hyperlipidemia model group, the rabbits were fed with standard chow supplemented with 0.5-1% cholesterol, 15% egg yolk and 5% animal oil. After 8 weeks, the rabbits in the hyperlipidemia group were further divided into two groups, that was group No.2 (n=6), (without any treatment) and group No.3 (n=6), (treated by sorbent-perfusion.). Experimental results showed that the LDL-C levels decreased significantly after 2 h perfusion indicating the adsorbent could effectively remove LDL-C, see **Table 6.** Furthermore, sorbent-perfusion also reduced all the subfractions of LDL-C, therefore decreased the risk for the development

3S

SO3H

(From Wang S Q et al, Artif Cells Blood Sub (2002), 30: 285-292, adapted) **Figure 6.** Schematic structure of amphiphilic cellulose adsorbent

of atherosclerosis and myocardial infarction, see **Table 7**.

Source: Wang S Q et al, Biomaterials (2003), 24: 2189-2194

**Table 6.** Removal of lipoproteins by amphiphilic adsorbent

Note: n=6,

Parameter Before (mmol/l) After (mmol/l) Reduction (%) TC 8.54±1.01 3.33±0.63\*, 61.20±2.81 TG 1.845±0.191 1.05±0.153\*, 43.09±2.43 LDL-C 3.619±0.354 0.724±0.07\*, 78.56±0.147 HDL-C 0.216±0.06 0.205±0.057 5.09±0.042

SO3H

Source: Wang S Q et al, Reactive & Functional Polymers (2008), 68: 261-267



Source: Wang S Q et al, Reactive & Functional Polymers (2008), 68: 261-267

**Table 5.** Adsorption capacity of TC and LDL by CPS beads with and without PEG as spacer
