**4. Typical bioactive bead type cellulosic adsorbent for blood purification**

## **4.1. Cellulosic adsorbents for removing low density lipoprotein –cholesterol (LDL-C)**

Familial hypercholesterolemia is characterized by a high concentration of plasma cholesterol in the form of low-density lipoprotein-cholesterol (LDL-C). In order to decrease the LDL-C level in patients, drugs and surgical intervention were reported [35]. Sorbent-perfusion treatment is currently employed when the reduction of LDL-C level appears impossible to be achieved by drug administration [36-38]. Since the late 1970s, scientists have been engaged in developing different kinds of adsorbents to remove pathogenic substances [39-41].

Yaoting Yu and Shenqi Wang et al [42,43] have developed cellulosic adsorbent with amphiphilic ligands for the adsorption of (LDL-C) which was prepared by the following procedure: Cellulose beads were reacted with cholesterol N-(6-isocyanatohexyl) carbamate in the presence of pyridine in DMSO at 80℃ in order to introduce the hydrophilic moiety. It was then reacted with chlorosulfonic acid in dimethyl formamide to introduce the sulfonic group see **Figure 6** 

The effects of sulfonation and grafting time of cholesterol on the swelling property of adsorbent were studied. Results showed that sulfonation and grafting time of cholesterol Bioactive Bead Type Cellulosic Adsorbent for Blood Purification 203

**Figure 6.** Schematic structure of amphiphilic cellulose adsorbent

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


Note: n=6,

202 Cellulose – Medical, Pharmaceutical and Electronic Applications

L-lysine amount (mg/ml)

Source: Wang S Q et al, Reactive & Functional Polymers (2008), 68: 261-267 **Table 4.** Adsorption capacity of LDL –C by L-lysine ligand

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

different kinds of adsorbents to remove pathogenic substances [39-41].

Adsorption capacity(mg/g)

Average removal capacity of LDL (mg/ml)

CPS-lysine CPS-PEG1000-lysine

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

Adsorption percentage (%)

TC 0.664 8.769 0.402 5.31 LDL-C 1.493 33.559 4.158 93.29 TG 0.419 6.213 0.132 1.97 HDL-C 0.204 15.170 0.341 24.48

**4. Typical bioactive bead type cellulosic adsorbent for blood purification** 

Familial hypercholesterolemia is characterized by a high concentration of plasma cholesterol in the form of low-density lipoprotein-cholesterol (LDL-C). In order to decrease the LDL-C level in patients, drugs and surgical intervention were reported [35]. Sorbent-perfusion treatment is currently employed when the reduction of LDL-C level appears impossible to be achieved by drug administration [36-38]. Since the late 1970s, scientists have been engaged in developing

Yaoting Yu and Shenqi Wang et al [42,43] have developed cellulosic adsorbent with amphiphilic ligands for the adsorption of (LDL-C) which was prepared by the following procedure: Cellulose beads were reacted with cholesterol N-(6-isocyanatohexyl) carbamate in the presence of pyridine in DMSO at 80℃ in order to introduce the hydrophilic moiety. It was then reacted with chlorosulfonic acid in dimethyl formamide to introduce the sulfonic

The effects of sulfonation and grafting time of cholesterol on the swelling property of adsorbent were studied. Results showed that sulfonation and grafting time of cholesterol

**4.1. Cellulosic adsorbents for removing low density lipoprotein –cholesterol** 

— 121.6 0.130±0.013 0.001 1.018±0.098 1000 10.5 0.242±0.017 0.023 21.947±1.469 2000 9.8 0.263±0.013 0.027 25.556±1.178 4000 9.0 0.708±0.017 0.078 74.917±1.549 6000 8.6 0.903±0.003 0.105 100.000

Stoichiometric capacity (mg LDL/mg L-lysine)

Adsorption capacity(mg/g)

Efficiency of active site (%)\*

Adsorption percentage (%)

Mol .weight of PEG spacers (Da)

**(LDL-C)** 

group see **Figure 6** 

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

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


Bioactive Bead Type Cellulosic Adsorbent for Blood Purification 205

**Note:IU is abbreviation of international unit** 

to treated group with a significance of P>0.05.

(Source: Wang L Y et al, Chemical Research in Chinese Universities(2004),20: 795-800)

RA patients were graded into 4 grades according to the functional ability of the sick joint. 11cases having grade II joint sickness, 20 cases having grade III and 1 case having gradeIV. Conventional method of treatment was performed on 30 cases. The control group compared

After perfusion the levels of rheumatoid factor was reduced and platelet count showed no significant changes in 35 RA patients (P<0.05) during treatment. After one week joint pain, swelling, tenderness, morning stiffness disappeared in 90 percent patients; 80 percent abnormal indexes recovered to normal value. After two to three weeks, joint function of 82 percent patients were improved and reached grade II or I. Two to three months later, X-ray examinations showed 80 percent bone matrix destruction was restored. Results of follow-up (0.67-2.5 years) proved that effective rate (97.14%, 81.25%) and total remission rate (82.86%, 59.38%) all had a significant improvement and recovery when compared to routine therapy. In conclusion, clinical results show that sorbent-perfusion by cellulosic adsorbents is an effective approach for the therapy of rheumatoid arthritis. The treatment is safe, without hemolysis or pyrogenic side reactions, the adsorbent is easy to sterilize and cost effective.

Bacterial endotoxins (ET), frequently named as lipopolysaccharides (LPS), are components of the outer cell wall of gram-negative bacteria and supposed to be a key factor in the pathogenesis of endotoxemia and septic shock [48,49] . Sorbent-perfusion is one of the best methods to remove endotoxin. Hui Fang and Yaoting Yu et al [10] synthesized a new type adsorbent for the removal of bacterial endotoxins by immobilizing lysine covalently onto cellulosic beads. Results showed that the adsorbent has good biocompatibility, see **Table 9**. In order to evaluate the adsorbent's properties, rabbit models were constructed by the following method [10]: Thirteen New Zealand white rabbits (weight 2.0-2.5 Kg) were

**Figure 7.** Adsorption capacity versus amount of DNA immobilized

**4.3. Cellulosic adsorbents for removal of endotoxin** 

Note: n=6

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

**Table 7.** Removal of LDL-C subfraction by amphiphilic adsorbent

The authors also developed the adsorbent with lysine and phosphate groups as ligand for the treatment of hyperlipidemia [34, 45]. Comparing to the amphiphilic adsorbent, the adsorbent with lysine as ligand has a lower adsorption capacity for LDL-C, total cholesterol and higher HDL-C. On the other hand, the phosphalated cellulosic adsorbent has a higher adsorption capacity for LDL-C and lower for HDL-C, see **Table 8**.


Source: Wang T et al, Chinese Journal of Biomedical Engineering ( 2008), 27: 132-136

**Table 8.** Effect of amount of coupled phosphate on adsorption capacity

Haofeng Yu et al [46] synthesized PAA-grafted cellulosic adsorbent for the removal of LDL-C from human plasma. In-vitro studies showed that this adsorbent could remove total cholesterol (TC), LDL-C, and triglyceride (TG) at levels of 5.55, 4.46, and 2.48 mg /m1 respectively. Unfortunately, it removed 30% HDL-C in the plasma.

## **4.2. Cellulosic adsorbents for removal of rheumatoid factors**

Rheumatoid Arthritis (RA) is a rather wide-spread immune disease. Spector reported that there is about 1% of the world's population is infected by this disease. Lianyong Wang and Yaoting Yu et al [25] covalently linked ss-DNA to cellulosic beads for the removal of rheumatoid factors. In vitro and in vivo studies showed that the adsorbent had a high adsorption capacity for IgMRF, IgGRF and IgARF, see **Figure 7**. Furthermore, the adsorbent attained good blood compatible properties.

Sorbent-perfusion using the above mentioned adsorbent was conducted on 35 RA patients clinically. Clinical protocol was designed as follows: 46 patients were hospitalized from Dec. 1998 until Nov.2000 and diagnosed as RA sickness. The extent of joint pain, swelling, morning stiffness, nodules under the skin, titer of rheumatoid arthritis factor (RF), value of immune-globulin and X-ray diagnosis of sick ankle etc. were all performed. The patients were divided into several groups from the above 46 patients. 35 of them matched the American Rheumatoid Arthritis Standard of 1987[47].

**Note:IU is abbreviation of international unit**  (Source: Wang L Y et al, Chemical Research in Chinese Universities(2004),20: 795-800)

**Figure 7.** Adsorption capacity versus amount of DNA immobilized

204 Cellulose – Medical, Pharmaceutical and Electronic Applications

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

**Table 7.** Removal of LDL-C subfraction by amphiphilic adsorbent

adsorption capacity for LDL-C and lower for HDL-C, see **Table 8**.

Source: Wang T et al, Chinese Journal of Biomedical Engineering ( 2008), 27: 132-136 **Table 8.** Effect of amount of coupled phosphate on adsorption capacity

respectively. Unfortunately, it removed 30% HDL-C in the plasma.

**4.2. Cellulosic adsorbents for removal of rheumatoid factors** 

attained good blood compatible properties.

American Rheumatoid Arthritis Standard of 1987[47].

Note: n=6

Adsorption capacity (mg/ml)

Before perfusion After perfusion Change=(A-B)

TC 0.893 0.735 0.939 1.586 LDL 1.235 1.515 1.707 2.721 HDL 0.134 0.039 0.058 0.063

mmol/l % mmol/l % LDL-1 0.51±0.061 13.95±2.1\* 0.12±0.046 15.1±2.3 8.24±2.6 LDL-2 1.65±0.019 45.4±5.3\* 0.38±0.043 53.4±5.8\* 17.36±3.4 LDL-3 1.45±0.052 40.1±4.7\* 0.23±0.024 31.6±3.6\* -21.20±3.9

The authors also developed the adsorbent with lysine and phosphate groups as ligand for the treatment of hyperlipidemia [34, 45]. Comparing to the amphiphilic adsorbent, the adsorbent with lysine as ligand has a lower adsorption capacity for LDL-C, total cholesterol and higher HDL-C. On the other hand, the phosphalated cellulosic adsorbent has a higher

Sample 1 2 3 4 Amount of coupled phosphate(μmol/ml) 0 68.4 94.3 128.6

Haofeng Yu et al [46] synthesized PAA-grafted cellulosic adsorbent for the removal of LDL-C from human plasma. In-vitro studies showed that this adsorbent could remove total cholesterol (TC), LDL-C, and triglyceride (TG) at levels of 5.55, 4.46, and 2.48 mg /m1

Rheumatoid Arthritis (RA) is a rather wide-spread immune disease. Spector reported that there is about 1% of the world's population is infected by this disease. Lianyong Wang and Yaoting Yu et al [25] covalently linked ss-DNA to cellulosic beads for the removal of rheumatoid factors. In vitro and in vivo studies showed that the adsorbent had a high adsorption capacity for IgMRF, IgGRF and IgARF, see **Figure 7**. Furthermore, the adsorbent

Sorbent-perfusion using the above mentioned adsorbent was conducted on 35 RA patients clinically. Clinical protocol was designed as follows: 46 patients were hospitalized from Dec. 1998 until Nov.2000 and diagnosed as RA sickness. The extent of joint pain, swelling, morning stiffness, nodules under the skin, titer of rheumatoid arthritis factor (RF), value of immune-globulin and X-ray diagnosis of sick ankle etc. were all performed. The patients were divided into several groups from the above 46 patients. 35 of them matched the RA patients were graded into 4 grades according to the functional ability of the sick joint. 11cases having grade II joint sickness, 20 cases having grade III and 1 case having gradeIV. Conventional method of treatment was performed on 30 cases. The control group compared to treated group with a significance of P>0.05.

After perfusion the levels of rheumatoid factor was reduced and platelet count showed no significant changes in 35 RA patients (P<0.05) during treatment. After one week joint pain, swelling, tenderness, morning stiffness disappeared in 90 percent patients; 80 percent abnormal indexes recovered to normal value. After two to three weeks, joint function of 82 percent patients were improved and reached grade II or I. Two to three months later, X-ray examinations showed 80 percent bone matrix destruction was restored. Results of follow-up (0.67-2.5 years) proved that effective rate (97.14%, 81.25%) and total remission rate (82.86%, 59.38%) all had a significant improvement and recovery when compared to routine therapy.

In conclusion, clinical results show that sorbent-perfusion by cellulosic adsorbents is an effective approach for the therapy of rheumatoid arthritis. The treatment is safe, without hemolysis or pyrogenic side reactions, the adsorbent is easy to sterilize and cost effective.

#### **4.3. Cellulosic adsorbents for removal of endotoxin**

Bacterial endotoxins (ET), frequently named as lipopolysaccharides (LPS), are components of the outer cell wall of gram-negative bacteria and supposed to be a key factor in the pathogenesis of endotoxemia and septic shock [48,49] . Sorbent-perfusion is one of the best methods to remove endotoxin. Hui Fang and Yaoting Yu et al [10] synthesized a new type adsorbent for the removal of bacterial endotoxins by immobilizing lysine covalently onto cellulosic beads. Results showed that the adsorbent has good biocompatibility, see **Table 9**. In order to evaluate the adsorbent's properties, rabbit models were constructed by the following method [10]: Thirteen New Zealand white rabbits (weight 2.0-2.5 Kg) were injected intravenously with 0.20 mg LPS (*E.coli O55: B5, sigma*) to induce endotoxemia after being anaesthetized. The rabbits were classified into two groups, one was the treated group (n=8) perfused through adsorbents while the other was the control group (n=5) without undergoing perfusion. Sorbent-perfusion was conducted 1.5 hours after LPS administration and conventional equipments for perfusion were used. Blood was drawn from the artery and returned to the vein by peristaltic pump (Pharmacia-LKB). The perfusion was carried out at a rate of 5 ml/min for 2 hours and the adsorbent showed a strong ET-binding capacity. After perfusion, the blood ET level was decreased from 5.56±0.54 EU/ml to 0.41±0.26 EU/ml, see **Figure 8**. Liver function and renal function tests as well as SOD, malondialdehyde (MDA) assays were conducted. Results all showed that the septic symptoms were ameliorated with the removal of large amounts of ET in the blood which obviously prevented further damage to the organs, see **Table 10, Figure 9** and **10**.

Bioactive Bead Type Cellulosic Adsorbent for Blood Purification 207

Parameter *Groups 0 min 60 min 120 min* 

Note: the NU is a unit that is used to indicate the activity of SOD, (From Fang H and Yu Y T et al, Biomaterials (2004),

**Figure 9.** Improved activities of serum superoxide dismutase (SOD) versus ET removal by perfusion

Perfusion 26.7±2.4 24.5±1.8 23.2±2.3 Control 26.1±3.7 22.0±2.2 24.1±4.6

Perfusion 0.04±0.01 0.05±0.02 0.05±0.01 Control 0.05±0.01 0.08±0.03 0.09±0.02

Perfusion 15.3±4.0 12.1±3.2 14.0±4.2 Control 17.9±5.7 27.6±6.9 35.7±8.0

Perfusion 34.4±9.5 25.9±9.1 24.8±8.5 Control 35.3±9.9 34.5±6.2 41.4±1.8

Perfusion 113.1±8.5 116.9±19.2 118.1±25.7 Control 109.4±10.3 153.0±17.3 195.5±31.2

Perfusion 5.2±0.6 4.9±1.3 4.6±0.6 Control 5.6±1.1 7.3±0.8 7.8±0.7

Perfusion 61.8±4.8 59.3±7.5 70.9±2.0 Control 62.7±4.2 80.4±3.7 94.3±4.7

*SA (g/L)*

*TBL (mg/ml)*

> *AST (U/L)*

> *ALT (U/L)*

*AKP (U/L)*

*BUN (mmol/L)* 

*CRK (umol/L)* 

Source: Fang H and Yu Y T ,et al, Biomaterials (2004), 25: 5433-5440

**Table 10.** Liver and renal function tests

Note: n=3

25: 5433-5440)

Note: EU is the abbreviation of Endotoxin unit, which is used to indicate the content of endotoxin (From Fang H and Yu Y T,et al, Biomaterials(2004), 25: 5433-5440)


**Figure 8.** Removal of ET from rabbit's blood by perfusion using Lys immobilized cellulosic beads

R\* index of sample=R index of extract – R index of sample tested=0.5/0- 0.5/0=0/0, PII\*\* is the skin stimulation index

Note: The biocompatibility and toxicology tests of the adsorbent were conducted by the Testing and Evaluation Research Centre of Biomedical Materials in Tianjin, China according to the Criteria of GB/T16886.5-1997 in correlation to ISO 10993.4:2002, Source: Fang H and Yu Y T et al, Biomaterials (2004), 25: 5433-5440

**Table 9.** Biocompatibility and toxicology properties of the adsorbent

Bioactive Bead Type Cellulosic Adsorbent for Blood Purification 207


Note: n=3

206 Cellulose – Medical, Pharmaceutical and Electronic Applications

Yu Y T,et al, Biomaterials(2004), 25: 5433-5440)

**Parameters tested** Results

Hemolysis 1.1% (standard <5% ),(n=3)

injected intravenously with 0.20 mg LPS (*E.coli O55: B5, sigma*) to induce endotoxemia after being anaesthetized. The rabbits were classified into two groups, one was the treated group (n=8) perfused through adsorbents while the other was the control group (n=5) without undergoing perfusion. Sorbent-perfusion was conducted 1.5 hours after LPS administration and conventional equipments for perfusion were used. Blood was drawn from the artery and returned to the vein by peristaltic pump (Pharmacia-LKB). The perfusion was carried out at a rate of 5 ml/min for 2 hours and the adsorbent showed a strong ET-binding capacity. After perfusion, the blood ET level was decreased from 5.56±0.54 EU/ml to 0.41±0.26 EU/ml, see **Figure 8**. Liver function and renal function tests as well as SOD, malondialdehyde (MDA) assays were conducted. Results all showed that the septic symptoms were ameliorated with the removal of large amounts of ET in the blood which obviously

Note: EU is the abbreviation of Endotoxin unit, which is used to indicate the content of endotoxin (From Fang H and

**Figure 8.** Removal of ET from rabbit's blood by perfusion using Lys immobilized cellulosic beads

Platelet adhesion 18.76% (n=3) (10% is excellent; 10-30% is good)

Note: The biocompatibility and toxicology tests of the adsorbent were conducted by the Testing and Evaluation Research Centre of Biomedical Materials in Tianjin, China according to the Criteria of GB/T16886.5-1997 in correlation

Allergic reaction Grade 1, not higher than negative control (n=10), none allergic

21.50.61 (g) 24.42.04 (g) (n=5)

Whole body toxicology Wt. of rat before test Wt. of rat after test

Cytotoxicology R\* index of sample=0/0 (n=6), none toxic Skin stimulation PII\*\*=0.0-0.4, (N=15), very mild toxic R\* index of sample=R index of extract – R index of sample tested=0.5/0- 0.5/0=0/0, PII\*\* is the skin stimulation index

to ISO 10993.4:2002, Source: Fang H and Yu Y T et al, Biomaterials (2004), 25: 5433-5440

**Table 9.** Biocompatibility and toxicology properties of the adsorbent

prevented further damage to the organs, see **Table 10, Figure 9** and **10**.

Source: Fang H and Yu Y T ,et al, Biomaterials (2004), 25: 5433-5440

**Table 10.** Liver and renal function tests

Note: the NU is a unit that is used to indicate the activity of SOD, (From Fang H and Yu Y T et al, Biomaterials (2004), 25: 5433-5440)

**Figure 9.** Improved activities of serum superoxide dismutase (SOD) versus ET removal by perfusion

Bioactive Bead Type Cellulosic Adsorbent for Blood Purification 209

The rabbits underwent extracorporeal whole blood perfusion for 2 h. Results showed no significant damages on blood cells and changes in the concentration of electrolytes. Whole blood sorbent-adsorption improved clinical manifestation and neuromuscular function of

No. of rabbits Pre WBIA 5th day after WBIA 1 + 0 - + 2 + 0 3 + 0 4 + 0 5 + 0 6 + 0 7 ++ + 8 + 0

The neuromuscular transmission function was evaluated by the stimulation of the deep peroneal nerve. The mean decrement of potentials evoked from the anterior tibial muscle, at three stimulation frequencies in the therapeutic rabbit group were determined. At 3Hz, the potential decreased from 21.87% to 17.87%, at 5Hz, decreased from 22.25% to 18.75% and at 10 Hz, decreased from 24.37% to 23.25%. Table 12 shows the changes of the electrophysiological features of EAMG rabbits after WBIA which was on the 5th (D5) and the 8th (D8) day after passive transfer. The same RNS was performed in the control rabbit group,

Frequency Therapeutic group (%) Control group (%)

D5 D8 D5 D8

3Hz 21.875±3.226 17.875±1.642 21.375±2.615 21.125±2.416

5Hz 22.250±2.815 18.750±1.388 21.875±2.232 22.000±1.772

10Hz 24.375±1.685 23.250±1.388 23.875±1.126 24.125±2.696

The quantity of neuromuscular junction can reflect the neurotransmission function. On the 3rd day after WBIA (D8), the quantity of neuromuscular junction per unit area (25mm2) of the therapeutic rabbit group was determined. **Figure 11** shows the quantity of neuromuscular junction per unit area increased from 9.8253.401 to 10.902.879(P<0.05) after

**Table 12.** Comparison of the decrement of RNS between the therapeutic and the control group

the EAMG rabbits, see **Table 11** 

Note: 0 stands for the rabbits recovered normally after therapy Source: Yang L et al, Artif Cell Blood Sub(2004), 32: 519-528

but no decrement was found, see **Table 12**

Source: Yang L et al, Artif Cell Blood Sub(2004), 32: 519-528

WBIA, which was higher than that of the control group (P<0.01)

Grading of clinical manifestation pre and 5th **Table 11.** day after WBIA

(From Fang H and Yu Y T et al, Biomaterials (2004), 25: 5433-5440)

**Figure 10.** Decrease in malondialdehyde (MDA) concentrations versus ET removal by perfusion

## **4.4. Cellulosic adsorbents for removal of anti-DNA antibody in treatment of systematic lupus erythematosus**

The abnormally high levels of anti-DNA antibodies and immune complex in the sera of systemic lupus erythematosus (SLE) patients can be removed by sorbent- perfusion. Yaoting Yu and Deling Kong [50] synthesized cellulose adsorbents with DNA as ligand for the removal of anti-DNA antibodies. The activation of cellulosic beads were conducted according to reference [50]: In brief, 2 ml of activated cellulose beads was added to 4.0ml buffer solution containing 4.0mg DNA in a flask and stirred at 25℃ for 20 h on a shaker. Then the immobilized DNA beads were washed consecutively with buffer solution and water, until no leakage of DNA in the rinse water was detected at 260nm by UV spectrometer. The immunoadsorbent thus obtained was stored at 4℃. In vitro adsorption tests showed that the DNA immuno-adsorbent could remove 40%-70% of anti-DNA antibody from plasma [29]. The maximum decrease of anti-DNA level was 80% after 60 min in a dynamic experiment. This high adsorption capacity shows a high potential for clinical application.

### **4.5. Cellulosic adsorbent for the treatment of myasthenia gravis**

Myasthenia gravis (MG) is an autoimmune disorder characterized by a disturbance in neuromuscular transmission that results in muscle weakness. Yaoting Yu and Li Yang et al [51, 52] synthesized immobilized tryptophan cellulosic adsorbent and evaluated its adsorption capacity for binding acetylcholine receptor in the plasma of MG patients. Experimental autoimmune myasthenia gravis (EAMG) rabbits were induced by Ta183-200 peptide according to the following method: Briefly, Female rabbits weighing approximately 2 kg were injected intradermally at multiple sites with 500μg of T*a*183-200, which was emulsified with an equal volume of Freund's complete adjuvant. A booster injection of 500 μg T*a*183-200 with Freund's incomplete adjuvant was administered after 4 weeks.

The rabbits underwent extracorporeal whole blood perfusion for 2 h. Results showed no significant damages on blood cells and changes in the concentration of electrolytes. Whole blood sorbent-adsorption improved clinical manifestation and neuromuscular function of the EAMG rabbits, see **Table 11** 


Note: 0 stands for the rabbits recovered normally after therapy

Source: Yang L et al, Artif Cell Blood Sub(2004), 32: 519-528

208 Cellulose – Medical, Pharmaceutical and Electronic Applications

(From Fang H and Yu Y T et al, Biomaterials (2004), 25: 5433-5440)

**systematic lupus erythematosus** 

application.

**Figure 10.** Decrease in malondialdehyde (MDA) concentrations versus ET removal by perfusion

**4.4. Cellulosic adsorbents for removal of anti-DNA antibody in treatment of** 

**4.5. Cellulosic adsorbent for the treatment of myasthenia gravis** 

The abnormally high levels of anti-DNA antibodies and immune complex in the sera of systemic lupus erythematosus (SLE) patients can be removed by sorbent- perfusion. Yaoting Yu and Deling Kong [50] synthesized cellulose adsorbents with DNA as ligand for the removal of anti-DNA antibodies. The activation of cellulosic beads were conducted according to reference [50]: In brief, 2 ml of activated cellulose beads was added to 4.0ml buffer solution containing 4.0mg DNA in a flask and stirred at 25℃ for 20 h on a shaker. Then the immobilized DNA beads were washed consecutively with buffer solution and water, until no leakage of DNA in the rinse water was detected at 260nm by UV spectrometer. The immunoadsorbent thus obtained was stored at 4℃. In vitro adsorption tests showed that the DNA immuno-adsorbent could remove 40%-70% of anti-DNA antibody from plasma [29]. The maximum decrease of anti-DNA level was 80% after 60 min in a dynamic experiment. This high adsorption capacity shows a high potential for clinical

Myasthenia gravis (MG) is an autoimmune disorder characterized by a disturbance in neuromuscular transmission that results in muscle weakness. Yaoting Yu and Li Yang et al [51, 52] synthesized immobilized tryptophan cellulosic adsorbent and evaluated its adsorption capacity for binding acetylcholine receptor in the plasma of MG patients. Experimental autoimmune myasthenia gravis (EAMG) rabbits were induced by Ta183-200 peptide according to the following method: Briefly, Female rabbits weighing approximately 2 kg were injected intradermally at multiple sites with 500μg of T*a*183-200, which was emulsified with an equal volume of Freund's complete adjuvant. A booster injection of 500

μg T*a*183-200 with Freund's incomplete adjuvant was administered after 4 weeks.

Grading of clinical manifestation pre and 5th **Table 11.** day after WBIA

The neuromuscular transmission function was evaluated by the stimulation of the deep peroneal nerve. The mean decrement of potentials evoked from the anterior tibial muscle, at three stimulation frequencies in the therapeutic rabbit group were determined. At 3Hz, the potential decreased from 21.87% to 17.87%, at 5Hz, decreased from 22.25% to 18.75% and at 10 Hz, decreased from 24.37% to 23.25%. Table 12 shows the changes of the electrophysiological features of EAMG rabbits after WBIA which was on the 5th (D5) and the 8th (D8) day after passive transfer. The same RNS was performed in the control rabbit group, but no decrement was found, see **Table 12**


Source: Yang L et al, Artif Cell Blood Sub(2004), 32: 519-528

**Table 12.** Comparison of the decrement of RNS between the therapeutic and the control group

The quantity of neuromuscular junction can reflect the neurotransmission function. On the 3rd day after WBIA (D8), the quantity of neuromuscular junction per unit area (25mm2) of the therapeutic rabbit group was determined. **Figure 11** shows the quantity of neuromuscular junction per unit area increased from 9.8253.401 to 10.902.879(P<0.05) after WBIA, which was higher than that of the control group (P<0.01)

Bioactive Bead Type Cellulosic Adsorbent for Blood Purification 211

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NOTE: a and b stand for the results before and after WBIA (From Li X H et al, Artif. Cell Blood Sub (2010), 38:186-191)

**Figure 11.** Neuromuscular junctions of the therapeutic rabbit group before and after WBIA on the 3rd day(200)

In conclusion, extracorporeal whole blood sorbent-adsorption is an effective and safe approach in treating the passive experimental autoimmune myasthenia gravis by improving clinical manifestation, neuromuscular transmission function, enhancing the quantity of neuromuscular junction and antibody titer.
