A novel high-capacity immunoadsorbent with PAMAM dendritic spacer arms by click chemistry

Article abstract of DOI:10.1039/C8NJ02142B

Polyamidoamine (PAMAM) dendrimers, bearing multiple peripheral end groups that can be used as clickable modules, make it possible to bind a large number of small-molecule ligands via click chemistry to prepare high-capacity immunoadsorbents. Thus, an immunoadsorbent with PAMAM dendritic spacer arms possessing pseudo-biospecific affinity for IgG from human plasma, Sep-PAMAM-AA, was designed and prepared by click chemistry using sepharose gel as a support and the amino acids His, Phe and Trp as ligands; two sepharose-based control samples, Sep-triazole-His and Sep-PA, with linear spacer arms were prepared using l-histidine and protein A as ligands, respectively. The ligand density and IgG adsorption performance of Sep-PAMAM-AA from human plasma were measured and evaluated. The influences of the structure and generation number of the PAMAM spacer arms on the performances of the products were also investigated. The results indicate that the immunoadsorbent with PAMAM G3 as a spacer arm and His as a ligand, Sep-G3-His, is the best among the prepared immunoadsorbents. Its ligand density reaches 1.58 mmol g^?1 sepharose gel, almost 5-fold higher than that of Sep-triazole-His; its IgG adsorption capacity is 28.43 mg g^?1, which is higher than those of Sep-triazole-His and Sep-PA. Moreover, Sep-G3-His exhibits a relatively low level of non-specific adsorption, which indicates that the immunoadsorbents with PAMAM as a spacer arm and His as a ligand are expected to have great application prospects in the field of blood purification.

Full text of DOI:10.1039/C8NJ02142B

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A novel high-capacity immunoadsorbent with PAMAM dendritic
spacer arms by click chemistry
Xiaoyan Hu, a Guangji Li,*a and Yinlei Lin*b
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
Polyamidoamine (PAMAM) dendrimers, bearing multiple peripheral end groups that can be used as clickable modules,
make it possible to bind a large number of small-molecule ligands via click chemistry to prepare high-capacity
immunoadsorbents. Thus, an immunoadsorbent with PAMAM dendritic spacer arms possessing pseudo-biospecific affinity
for IgG from human plasma, Sep-PAMAM-AA, was designed and prepared by click chemistry using sepharose gel as a
support and the amino acids His, Phe and Trp as ligands; two sepharose-based control samples, Sep-triazole-His and Sep-
PA, with linear spacer arms were prepared using L-histidine and protein A as ligands, respectively. The ligand density and
IgG adsorption performance of Sep-PAMAM-AA from human plasma were measured and evaluated. The influences of the
structure and generation number of the PAMAM spacer arms on the performances of the products were also investigated.
The results indicate that the immunoadsorbent using PAMAM G3 as a spacer arm and His as a ligand, Sep-G3-His, is the
best among the prepared immunoadsorbents. Its ligand density reaches 1.58 mmol/g sepharose gel, almost 5-fold higher
than that of Sep-triazole-His; its IgG adsorption capacity is 28.43 mg/g, which is higher than those of Sep-triazole-His and
www.rsc.org/
Sep-PA. Moreover, Sep-G3-His has
immunoadsorbents using PAMAM as a spacer arm and His as a ligand are expected to have great application prospects in
the field of blood purification.
a relatively low level of non-specific adsorption, which indicates that the
rotational flexibility, which can endow it with sufficient
mobility to allow it come in proximity with the target
molecules.6-9 In addition, the chemical structure of the spacer
arm is another important factor that affects the performance
of the immunoadsorbent.10, 11 Commonly used spacer arms,
bridge chains between the reactive group on the support
surface and the ligand, are linear molecular chains of
compounds.3, 4, 10 Generally speaking, one spacer arm with a
linear molecular structure can only conjugate one ligand
molecule. The performance of the corresponding
immunoadsorbent may be unsatisfactory, especially when the
ligand is a small molecule with only one active site for
adsorption. For this reason, it is difficult to enhance the ligand
density of an immunoadsorbent using a linear molecular chain
as a spacer arm. Nevertheless, employing dendrimers12-14 as
spacer arms can overcome this disadvantage. As the most
widely studied dendrimer, polyamidoamine (PAMAM), which
can be synthesized by the repetition of a given set of reactions
Introduction
In the conventional preparation of immunoadsorbents, if the
ligand is coupled directly to the support, steric hindrance
between the support surface and the target molecule will
occur.1 Therefore, the ligand that is critical for selectively
absorbing the target molecule should be sufficiently distant
from the solid support, which can be achieved by binding the
ligand at the end of a long chain or “spacer arm”.2 The
employment of a spacer arm may set the ligand away from the
solid matrix to make it more accessible to the target
molecules.
It is known that the length and chemical structure of spacer
arms have direct effects on the adsorption performance of an
immunoadsorbent.3-5 An increase of the length of spacer arms,
to a certain extent, will correspond to an increase of its
using divergent strategies, possesses a highly branched,
regular and well-defined architecture.15-18 For this reason, an
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advantage of using PAMAM as a spacer arm is that its multiple
The immunoglobulin-G (IgG) adsorption performance of the
peripheral end groups can covalently bind more ligands than prepared immunoadsorbents from human blood was
the corresponding linear spacer arm with only one end group. evaluated to estimate their application prospects in treating
Thus, the immunoadsorbent containing PAMAM spacer arms is various autoimmune diseases by means of blood purification,
expected to show increased adsorption capacity for the target and the influences of the structure and generation number of
molecules.
the PAMAM spacer arms on the performance of the products
On the other hand, the Cu(I)-catalysed Huisgen 1,3-dipolar were also investigated.
cycloaddition reaction19-22 between an azide and an alkyne
O
NH2
O
NH
AA
N
H
N
H
Amino acid methyl ester hydrochlorides
N
O
H
N
N
O
H
N
O
N
H
HCl H2
N
OCH3
NH2
NH2
N
H
NH AA
NH
generating 1,2,3-triazole or click chemistry, developed by
Sharpless,22-24 appears to offer a simple, reliable, and fruitful
method of preparing immunoadsorbents.25-28 Because of the
high yields and lack of by-products provided by the click
chemistry for coupling ligands and supports,29 it can be readily
adopted for the preparation of immunoadsorbents.
O
O
O
N
R
N
O
H
N
H
N
AA
N
H
N
H
N
O
H
N
N
H
N
O
NH2
n
NH AA
O
n
O
PAMAM dendrimers
Gm (m=n, n=0,1,2,3)
PAMAM-AA
Gm-AA (R =AA=His, Phe, Trp)
O
NH AA
N
H
Sep-N3
N
O
H
N
N
H
NH
AA
O
N3
O
OH
N
O
N
NH AA
H
N
O
N
H
N
N
OH
N
O
H
N
NH AA
n
O
N
In this study, a novel immunoadsorbent with PAMAM
dendritic spacer arms was prepared by click chemistry, and its
preparation principle is shown in Figure 1. Sepharose (Sep) was
used as the support for the immunoadsorbents, and the
R=(
Sep
Trp)
=
His,
Phe, HN
Immunoadsorbent Sep-PAMAM-AA
Sep-Gm-AA
N
H
Figure 1. Schematic of the preparation principle of immunoadsorbents
using PAMAM as spacer arms.
pseudo-biospecific30-33 amino acid ligands with molecular Results and discussion
recognition and selective adsorption for the target molecules
Structure characterization of PAMAM-AA
were bonded to the amine-terminated groups of the PAMAM
spacer arms. Finally, the PAMAM-ligand complexes with
terminal alkynes were coupled with the azide groups of the
functionalized support through click chemistry. For purposes
of comparison, protein A immunoadsorbent with linear spacer
arms, Sep-PA, prepared using the same sepharose as a support
and protein A as a ligand, was used as a control in this work.
With respect to the preparation process, the synthesis of
the novel immunoadsorbent with PAMAM dendritic spacer
arms designed in this work is completely different from that of
Sep-PA in the key technical aspects involved. Protein ligands
are difficult to immobilize on the support matrix in the proper
orientation34. Moreover, protein ligands possess some
drawbacks that need be taken seriously, such as potential
immunogenicity, safety concerns caused by protein ligands
detached from the support matrix, sensitivity to harsh
sanitization procedures, and high cost. Accordingly, compared
with Sep-PA, the novel immunoadsorbent using amino acids as
ligands will possess high stability and low cost and be easy to
store and handle, thereby having advantages over Sep-PA in
cost and use security.
To examine whether the amino acid ligand was functionalized
with terminal alkynes, the structure of the produced PAMAM-
AA, such as PAMAM G3-His, was analysed by 1H NMR
spectrum in D2O, as shown in Figure 2. From the 1H NMR
spectra, besides the original characteristic peaks of PAMAM
G3 (a
∼ j), there appeared new peaks of histidine units at 3.97
ppm (k), 3.39 ppm (l), 7.81 ppm (m), and 7.00 ppm (n) in
PAMAM G3-His, and the peak of methyl in His-OMe∙2HCl
disappeared here. All of these demonstrated that His ligands
were successfully bonded to the PAMAM spacer arms.
1
According to the H NMR spectra of PAMAM G3-His in Figure
2., the area of each peak m, n, k and l is 6, 6, 6 and 12 times
larger than the area of peak a, respectively; and the number of
protons in the corresponding positions, m, n, k, l and a, in the
molecular structure of PAMAM G3 is 1, 1, 1, 2 and 1,
respectively. From the above data, it was then calculated that
6 histidine units were conjugated to the terminal amines of
PAMAM G3. This number of conjugated units means that 75%
of the terminal amines reacted with His as a ligand.
2 | J. Name., 2012, 00, 1-3
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6
6
6
3
6
6
75
75
2475.96
2536.14
2770.38
3890.87
4362.47
4596.71
G3-His
G3-Phe
G3-Trp
G4-His
8
75
19
75
G4-Phe
G4-Trp
16
37.5
Structure characterization of Sep-PAMAM-AA
Figure 2. The 1H NMR spectra of PAMAM G3 and PAMAM G3-His.
The FTIR spectra of Sep-PAMAM-His, Sep-PAMAM-Phe and
Sep-PAMAM-Trp are presented in Figure 3. Compared to the
FTIR spectrum of Sep-N3, the peak at 2100 cm-1, attributed to
azide groups, disappeared almost completely in the FTIR
spectra of Sep-PAMAM-AA. This implies that the azide groups
of Sep-N3 have been consumed completely in the course of the
click reaction of Sep-N3 and PAMAM-AA, which clearly
exhibited that the coupling efficiency of the support and the
ligand with spacer arms was nearly 100%. For this reason, the
ligand density could be evaluated according to the number of
amino acid units conjugated to PAMAM. As the azide content
in Sep-N3 was calculated to be 751.4 μmol/g gel, according to
the elemental analysis data, the ligand density of Sep-PAMAM-
AA, DLigand, was then calculated and listed in Table 2. Compared
to the immunoadsorbent Sep-triazole-His with linear spacer
arms, the immunoadsorbents using PAMAM dendrimers as the
spacer arms exhibited greatly enhanced ligand density,
especially when the spacer arm was G2 or G3. Among them,
the ligand density of Sep-G3-His reaches 1.58 mmol/g
sepharose gel, almost 5-fold as high as that of Sep-triazole-His.
In addition to the above method, the ligand density may be
calculated from the data on the N content of Sep-N3 and Sep-
PAMAM-AA, which can be determined by elemental analysis,
as well as the N content of PAMAM, which can be calculated
from its molecular structure. Unfortunately, this way is
complicated and has low accuracy in practice, so that it is
rarely adopted.
The number of amino acid units conjugated with other
generations was calculated by the same method as PAMAM
G3-His, which was shown in Figure 2. According to the number
of –NH2 in different generations of PAMAM dendrimers,
PAMAM G1, G2, G3 and G4 can theoretically combine 2, 4, 8
and 16 amino acid units, respectively. From the results listed in
Table 1, 100% of the terminal amines at the outer sphere in
PAMAM G1 and PAMAM G2 reacted with amino acid His, while
only 75% and 37.5% of the terminal amines for PAMAM G3
and PAMAM G4 reacted with His, respectively, although both
PAMAM G3 and G4 can conjugate 6 His ligands. This implies
that as the generation number increases, the number of
terminal groups increases exponentially, until steric crowding
at the surface occurs. Thus, the number of ligands conjugated
to PAMAM is lower than the theoretical value. Obviously,
when more ligands are conjugated to PAMAM, the prepared
immunoadsorbent will possess more active sites and higher
adsorption capacity for the target molecules, which is the
advantage of choosing PAMAM dendrimers as the spacer
arms. Compared with PAMAM G4, PAMAM G3 can conjugate
as many ligands as PAMAM G4, but its preparation is less
complicated than that of G4. Therefore, PAMAM G3 is the
optimal spacer arm. Consequently, the molecular masses of
PAMAM-AA (MGm-AA) were obtained, as listed in Table 1.
Table 1 The analysis of PAMAM-AA
Efficiency of
conjugation
(%)
Number of
–NH2 in Gm
Number of
His in Gm-AA
MGm-AA
(g/mol)
Sample
2
2
2
4
4
4
100
100
100
100
100
100
557.65
577.71
655.79
1288.51
1328.63
1484.79
G1-His
G1-Phe
G1-Trp
G2-His
G2-Phe
G2-Trp
2
4
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which was poor in ligand density. Furthermore, the IgG
adsorption capacity of the immunoadsorbents with His ligand
was higher than those with Phe and Trp ligands when the
spacer arms were the same. This indicates that His is better
than Phe and Trp to act as a pseudo-biospecific ligand.
Moreover, the immunoadsorbents with PAMAM G2 or
PAMAM G3 spacer arms were relatively preferable in
adsorption capacity, which was consistent with the ligand
density.
Table 3 The adsorption capacity of Sep-PAMAM-AA
Sample
Sep-PA
V (ml)
6.002
m (g)
3.635
A280
Q (mg /g)
22.97
18.782
Sep-triazole-His
Sep-G1-His
Sep-G2-His
Sep-G3-His
Sep-G4-His
Sep-G1-Phe
Sep-G2-Phe
Sep-G3-Phe
Sep-G4-Phe
Sep-G 1-Trp
Sep-G2-Trp
Sep-G3-Trp
Sep-G4-Trp
6.035
6.004
6.110
6.015
6.006
6.013
6.005
6.997
6.010
6.008
6.022
6.001
6.005
0.685
0.662
0.661
0.652
0.690
0.690
0.692
0.620
0.689
0.683
0.680
0.656
0.680
2.527
3.153
3.656
4.252
3.306
3.085
3.655
3.302
3.831
2.590
3.147
3.523
3.414
16.49
20.72
24.49
28.43
20.85
19.48
22.98
27.00
24.22
16.51
20.20
23.35
21.85
Figure 3. The FTIR spectra of Sep-PAMAM-AA. (a) Sep-PAMAM-His; (b) Sep-
PAMAM-Phe; (c) Sep-PAMAM-Trp.
Table 2 The ligand density of Sep-PAMAM-AA
Based on the above results, Sep-G3-His exhibits the highest
adsorption capacity for IgG from human plasma. Thus, the
adsorption performance of Sep-G3-His was studied intensively.
In the static adsorption experiment using Sep-G3-His as an
adsorbent, the IgG concentration in the human plasma before
and after adsorption was measured via the standardized
immunoturbidimetric assay. The obtained results and analysis
are shown in Table 4. From the difference in IgG concentration
between the initial plasma and the plasma after the
adsorption test, the adsorption ratio or the yield of the static
adsorption process can be calculated to be 44%, while the
amount of adsorbed IgG by Sep-G3-His is calculated to be
28.99 mg/g. This value is considered to be the accurate and
true IgG adsorption capacity of Sep-G3-His, and the IgG
adsorption capacity of Sep-G3-His given in Table 3 (28.43
mg/g) is actually the amount of IgG eluted from the adsorbent
D
Ligand (mmol/g)
Spacer arm
Sep-Gm-His
1.06
Sep-Gm-Phe
1.05
Sep-Gm-Trp
1.01
G1
G2
G3
G4
1.53
1.50
1.42
1.58
1.55
1.46
0.57
1.05
1.01
Adsorption capacity of IgG from human plasma
The IgG adsorption capacity of Sep-PAMAM-AA from human
plasma was determined and compared with Sep-PA and Sep-
triazole-His, as shown in Table 3. It is observed that
immunoadsorbents with PAMAM as the spacer arms had a
higher IgG adsorption capacity than those with linear spacer
arms, and they were comparable to Sep-PA. Strictly speaking,
the immunoadsorbents with His as the ligand showed
remarkable IgG adsorption capacity, except for Sep-G4-His,
4 | J. Name., 2012, 00, 1-3
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from Sep-G3-Phe; Lane 6: eluate from Sep-G2-Trp; Lane 7: eluate from Sep-
G3-Trp.
Sep-G3-His used in the static adsorption experiment, which
indirectly reflects the IgG adsorption capacity of Sep-G3-His.
Accordingly, the efficiency of the IgG desorption is calculated
to be 98%.
Theoretically speaking, the adsorption selectivity of an
immunoadsorbent mainly depends on its ligand. The above
qualitative analysis obtained by SDS-PAGE shows that among
three small-molecule ligands His, Phe and Trp, His gives the
best performance. The immunoadsorbent using His as the
ligand, Sep-G3-His, indeed exhibits high adsorption selectivity
for IgG, which is consistent with the result obtained by MALDI-
TOF MS in our previous work.35 On the other hand, the non-
specific adsorption of Sep-G3-His and Sep-PA as a control was
evaluated by quantitative detection to further study the
adsorption selectivity of the prepared immunoadsorbents
from another aspect. The total protein and albumin
concentration in the human plasma before and after the static
adsorption experiment were measured via the standardized
biuret and coagulation methods, respectively; the obtained
results and analysis are shown in Table 5. Based on the data in
Table 5, Sep-G3-His exhibits a slightly higher adsorption ratio
for the total protein and albumin compared to Sep-PA, which
are 9% and 6%, respectively. Therefore, the non-specific
adsorption of Sep-G3-His is close to that of Sep-PA. That
indicates that the dendrimers of PAMAM as spacer arms do
not cause high non-specific adsorption, thus indirectly
confirming the adsorption selectivity of Sep-G3-His for IgG
from human plasma. Moreover, the blood purification mode of
both the prepared adsorbents with PAMAM dendritic spacer
arms is plasma adsorption. Therefore, the negative effects
from the PAMAM dendritic spacer arms of the adsorbent are
negligible, and the adsorbent cannot damage blood cells.
Table 4 IgG concentration in the human plasma before and after adsorption
using Sep-G3-His as an immunoadsorbent.
IgG concentration IgG adsorption Adsorption
Sample
(mg/ml)
capacity (mg/g)
ratio (%)
Plasma before
adsorption
7.11
———
———
Plasma
adsorption
after
3.96
28.99
44
Adsorption selectivity of IgG from human plasma
As the immunoadsorbents with G2 or G3 spacer arms are
preferable in terms of ligand density and adsorption capacity,
their adsorption selectivity was further evaluated by analysing
the purity of the adsorbed molecules in the eluate from the
adsorption experiment through SDS–PAGE. The result is
presented in Figure 4. As observed in Figure 4, the band
corresponding to the molecule (150 kDa) of IgG can be found
in the eluates from all of the immunoadsorbents. Moreover,
the light chain (25 kDa) and the heavy chain (50 kDa) of IgG
can be observed in the eluates from both Sep-G2-His and Sep-
G3-His (Lane 2 and Lane 3). In addition to IgG, traces of several
other proteins are faintly visible in the eluate from the latter,
while there are some impurities in the eluates from the
immunoadsorbents with Phe or Trp as the ligand, which means
that Phe and Trp are inferior to His in acting as a pseudo-
biospecific ligand. Furthermore, Sep-G3-His, with G3 spacer
arms and His as the ligand, is capable of highly selective
adsorption for IgG from human plasma, and its non-specific
adsorption is almost negligible.
Table 5 Total protein and albumin concentration in the human plasma
before and after adsorption using Sep-G3-His and Sep-PA as the
immunoadsorbent, respectively.
Total
Albumin
concentr
ation
Albumin
adsorptio
Total protein
concentratio
n (mg/ml)
protein
adsorption
ratio (%)
Sample
n
(%)
ratio
(mg/ml)
Plasma before
adsorption
57.4
52.2
———
36.1
———
Plasma
after
adsorption by
Sep-G3-His
9
34.0
6
Plasma
after
adsorption by 53.3
Sep-PA
7
34.5
4
Figure 4. SDS-PAGE analysis of fractions eluted from Sep-G2-AA and Sep-
G3-AA. Lane 1: protein markers; Lane 2: eluate from Sep-G2-His; Lane 3:
eluate from Sep-G3-His; Lane 4: eluate from Sep-G2-Phe; Lane 5: eluate
Adsorption stability over repeated use
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As Sep-G3-His possesses the best IgG adsorption declines slowly from the initial value of 28.43 mg/g to 24.97
performance, the adsorption-desorption cycle was repeated mg/g during 10 cycles of usage, which means that Sep-G3-His
10 times using Sep-G3-His to investigate its reusability. The can be repeatedly used without a noticeable loss in its IgG
data on the IgG adsorption capacity with the times of reuse are adsorption capacity to a certain extent.
listed in Table 6. It is observed that the IgG adsorption capacity
Table 6 Adsorption stability over repeated use of Sep-G3-His.
Times of reuse
1
2
3
4
5
6
7
8
9
10
IgG Adsorption Capacity (mg/g)
28.43
27.55
27.81
26.81
27.09
26.13
25.71
25.22
25.46
24.97
Experimental
Synthesis of Amino Acid Methyl Ester Hydrochlorides
Reagents
Amino acid methyl ester hydrochlorides were synthesized according
to the procedures described in Figure 5. The carboxyl group of
amino acids should be converted into highly reactive methyl ester
groups, thereby generating amino acid methyl ester hydrochlorides.
TMSCl (50 mmol) was added to a solution of amino acid (10 mmol)
in methanol (30 ml) under stirring, and the reaction mixture was
refluxed until the solution turn clear. The volatiles were removed
under reduced pressure on a rotary evaporator, yielding the desired
product. The structure of amino acid methyl ester hydrochlorides
was confirmed by FTIR and 1H NMR analyses, as depicted in the ESI.
Sepharose 6FF (Sep) was purchased from Pharmacia (Sweden).
Azidated sepharose (Sep-N3) was prepared in our laboratory.
Human plasma was obtained from the Guangzhou blood station
donated by volunteers, related experiments were performed in
compliance with the relevant laws and institutional guidelines.
Human immunoglobulin G (IgG, 98%) was purchased from Sigma
(USA). L-Histidine (His), L-Phenylalanine (Phe), L-Tryptophan (Trp),
propargylamine, methyl acrylate (MA, 98%), 1,2-ethylenediamine
(EDA, 98%) and trimethyl chlorosilane (TMSCl) were purchased
from Jingchun (Shanghai) and were used as received. Methanol
(MeOH), triethylamine (TEA), sodium ascorbate, copper sulphate
pentahydrate and ethylene diamine tetraacetic acid (EDTA) were all
of analytical purity and were used without further purification.
Throughout this study only double-distilled water was used.
Cl
O
H2N
COOH
MeOH
CH3
CH3
H2N
C
HCl
Si
OCH3
H3C
R
R
Amino acid
TMSCl
Amino acid methyl ester hydrochlorides
N
R=(
Phe, HN
Trp)
His,
N
H
Figure 5. The route to preparing amino acid methyl ester hydrochlorides.
Synthesis of PAMAM Dendrimers
Synthesis of Amino Acid Modified PAMAM (PAMAM-AA)
PAMAM dendrimers were synthesized by the divergent approach
using propargylamine as the core.36-38 This methodology involves
typical stepwise and iterative two-step reaction sequences,
consisting of Michael addition of primary amines with MA and
amidation of methyl ester groups with a large excess of EDA to
produce amine terminations. The completion of this two-step
process gives a full generation (G = 1); if only one step is finished,
then a half generation (G = 0.5) is obtained. Repeating this two-step
reaction sequence together with purification at each step leads to
the second (G = 2), third (G = 3) and fourth generations (G = 4),
depending on the numbers of repetition. The specific steps of
preparing generation 1 ∼ 4 PAMAM dendrimers (G1 ∼ G4) are
described in the ESI. The structures of the synthesized PAMAM G1 ∼
Amino acids, His, Phe and Trp, were chosen as small molecular
ligands. The prepared PAMAM dendrimers (G1 ∼ G4) bearing
symmetrical end groups were end-capped with amino acids,39 thus
creating complexes of spacer arm and ligand, PAMAM-AA. TEA was
added to an aqueous solution of amino acid methyl ester
hydrochloride. After mixing to uniformity, an aqueous solution of
PAMAM was added. The resulting solution was stirred at 60 ˚C for
48 h. (Ashford et al., 2002) After dialyzing and freeze drying, the
product PAMAM-AA was obtained. Here, the molar ratio of the
amine end groups of the PAMAM to TEA was 1:6 and to AA-
OMe∙HCl was 1:2.5.
Preparation of azidated sepharose (Sep-N3)
PAMAM G4 were characterized by FTIR, H and 13C NMR analyses,
1
Azidated sepharose, Sep-N3, can be prepared via two reaction steps
as depicted in our previous work.35 Firstly, sepharose was activated
as depicted in the ESI.
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through its hydroxy with epichlorohydrin to prepare epoxidized The IgG adsorption capacity of Sep-PAMAM-AA was measured by
sepharose; then, a nucleophilic displacement reaction was carried static adsorption experiments in a batch system. The human plasma
out in which an azide ion attacks the epoxide, resulting in the was thawed for 1 h at 37 ˚C prior to use and the prepared
opening of the ring to obtain azidated sepharose. The detailed immunoadsorbent Sep-PAMAM-AA was equilibrated with 0.02 M
experimental data can be observed in the ESI.
PBS buffer at pH 7.0 before being added to the human plasma. The
static adsorption experiments were conducted at 25 ˚C and at a
stirring rate of 100 rpm for 2 h. Then, the Sep-PAMAM-AA was
washed by 0.02 M PBS buffer at pH 7.0 to remove those proteins or
impurities not fully adsorbed by the adsorbent,40 followed by
suction-drying. Afterwards, the adsorbed IgG by the Sep-PAMAM-
AA was eluted with 0.02 M citric acid buffer at pH 2.5. IgG
concentration was determined by measuring the absorbance of the
eluate at 280 nm according to the Beer–Lambert law:
Preparation of immunoadsorbent Sep-PAMAM-AA by click
chemistry
Immunoadsorbents Sep-PAMAM-AA were prepared by coupling the
complexes PAMAM-AA to the “clickable” support Sep-N3 via click
chemistry. The azidated sepharose (3 g, in 5 ml water), CuSO4.5H2O
(50 mg, 0.2 mmol, in 2.5 ml water) and sodium ascorbate (79 mg,
0.4 mmol, in 2.5 ml water) were added to the solution of PAMAM-
AA (10 mmol, in 10 ml water). The mixture was stirred at room
temperature for 24 h and then washed sequentially with H2O, 0.1 M
EDTA and H2O to purify the prepared immunoadsorbents Sep-
PAMAM-His, Sep-PAMAM-Phe and Sep-PAMAM-Trp. The ligand
density of the immunoadsorbent was evaluated according to the
number of amino acid units conjugated to PAMAM, which was
calculated based on the 1H NMR data.
A
280 = εcl
(1)
Where A280 is the absorbance of the eluate at 280 nm; ε is the
molar absorptivity of IgG, which is 1.35 ml/mg∙cm according to the
standard curve in Fig. S3, ESI; c is the IgG concentration of the
eluate (mg/ml); and the path length l is 1.0 cm in the UV cell. The
amount of adsorbed IgG was then calculated from the following
equation:
For comparison purposes, protein A immunoadsorbent with
linear spacer arms using the same sepharose as a support and
Q = cV/m
(2)
Where Q is the amount of IgG adsorbed onto a unit mass of
immunoadsorbent (mg/g); c is the IgG concentration of the eluate
(mg/ml); V is the volume of the eluate (ml), and m is the mass of the
immunoadsorbent used in the experiment (g).
protein A as a ligand, Sep-PA, was also prepared in our laboratory35
.
To evaluate the effectiveness of PAMAM spacer arm, the
immunoadsorbent with linear spacer arms, Sep-triazole-His, was
prepared as depicted in our previous study.35
To facilitate comparative analysis, the IgG concentration in the
human plasma before and after static adsorption experiment were
Instrumental analyses
further
measured
using
a
standardized
commercial
1H NMR spectra were recorded on a 300 MHz NMR spectrometer
(Mercury-Plus 300, Varian, USA). The peaks of different shapes in
the 1H NMR spectra were expressed by the following abbreviations,
respectively: s, singlet; d, doublet; t, triplet; m, multiplet. FTIR
spectra were performed on an FTIR spectrophotometer (Nexus Por
Euro, Nicolet, USA). The absorbance of the eluate at 280 nm was
determined on an ultraviolet-visible spectrophotometer (UV-2450,
Shimadzu, Japan). The determination of different compositions in
human plasma such as IgG, total protein and albumin were carried
out on an automatic biochemistry analyser (P800, Roche,
Switzerland) by immunoturbidimetric assay, biuret method and
coagulation method, respectively.
immunoturbidimetric assay by KingMed Diagnostics in Guangzhou,
China.
Adsorption selectivity of IgG from human plasma
Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–
PAGE) was used to analyse the adsorption selectivity of Sep-
PAMAM-AA for IgG from human plasma. The concentration of
separation gel was 15% Tris–HCl, whereas stacking gel was 5% Tris–
HCl. Samples were mixed in a 1:1 ratio with loading buffer and
incubated for 5 min at 100 ˚C. The loading sample and molecular
weight marker was 10 μL, stained with Coomassie Brilliant Blue R-
250.
To further verify the adsorption selectivity of the
immunoadsorbents Sep-PAMAM-AA for IgG, the total protein
IgG adsorption capacity from human plasma
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and albumin concentration in the human plasma before and
after static adsorption experiment were measured using
standardized commercial biuret and coagulation methods,
respectively, by KingMed Diagnostics in Guangzhou, China.
Conflicts of interest
There are no conflicts to declare
Acknowledgements
The acknowledgements come at the end of an article after the
conclusions and before the notes and references.
Repeated use study
To evaluate the reusability of Sep-PAMAM-AA, a series of 10
adsorption cycles of IgG from human plasma were performed
as described above. Desorption testing was achieved with 0.02
M citric acid buffer at pH 2.5, and then re-equilibrated with
0.02 M PBS buffer at pH 7.0. IgG binding capacity was
measured for each cycle.
Notes and references
1
C. R. Lowe and P. D. G. Dean, Affinity Chromatography,
Wiley-Interscience: London, 1974.
2
G. T. Hermanson, A. K. Mallia and P. K. Smith, Immobilized
Affinity Ligand Techniques, Academic Press: California 1992,
137–158.
3
4
5
6
7
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Conclusions
In this study, immunoadsorbents with PAMAM dendritic
spacer arms possessing pseudo-biospecific affinity for IgG from
human plasma, Sep-PAMAM-AA, were designed and prepared
by click chemistry using sepharose gel as a support and using
amino acids, His, Phe and Trp, as ligands. The as-prepared
immunoadsorbents using PAMAM G3 as a spacer arm and His
as a ligand, Sep-G3-His, possessed the best IgG adsorption
performance among the prepared immunoadsorbents. Its
ligand density reaches 1.58 mmol/g sepharose gel, almost 5
times higher than that of Sep-triazole-His; its IgG adsorption
capacity is 28.43 mg/g, higher than those of Sep-triazole-His
and Sep-PA. Moreover, Sep-G3-His shows a relatively low level
of non-specific adsorption, which is of significance for the
immunoadsorbent to be applied to remove autoantibodies
from human plasma to treat various autoimmune diseases.
Therefore, the immunoadsorbents using PAMAM as a spacer
arm and His as a ligand are expected to have great application
prospects in the field of blood purification.
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New Journal of Chemistry
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DOI: 10.1039/C8NJ02142B
A novel high-capacity immunoadsorbent
with PAMAM dendritic spacer arms by click
chemistry
O
NH2
O
NH AA
N
H
N
H
Amino acid methyl ester hydrochlorides
O
O
N
H
O
O
N
O
H
N
N
N
H
HCl H2
N
Xiaoyan Hu, Guangji Li, Yinlei Lin
OCH3
NH2
NH2
N
H
NH
AA
O
O
O
O
N
R
N
H
N
NH AA
H
N
N
N
H
H
N
H
N
H
A
novel immunoadsorbent with polyamidoamine
N
N
n
NH2
NH
AA
O
n
O
PAMAM dendrimers
Gm (m=n, n=0,1,2,3)
PAMAM-AA
dendritic spacer arms was prepared. Click chemistry
can improve the reaction selectivity between the ligands
and support matrix under mild reaction conditions. The
designed and prepared immunoadsorbent exhibits
excellent adsorption for IgG. The IgG adsorption
capacity of Sep-G3-His is superior to those of
Sep-triazole-His and protein A immunoadsorbent.
Gm-AA
(R =AA=His, Phe, Trp)
O
NH
AA
N
H
H
N
Sep-N3
N
O
O
N3
N
H
NH AA
NH
O
O
OH
N
N
H
N
AA
O
N
N
N
H
H
N
OH
N
O
NH AA
n
O
N
R=(
Sep
=
Phe, HN
Trp)
His,
Immunoadsorbent Sep-PAMAM-AA
Sep-Gm-AA
N
H