GlcNAc Conjugated Atorvastatin with Enhanced Water Solubility and Cellular Internalization

Article abstract of DOI:10.1021/acs.bioconjchem.7b00295

Targeting ligands facilitate cell specific drug delivery and improve pharmaceutical properties. Herein, we designed two ligand drug conjugates by conjugating GlcNAc (N-acetylglucosamine) with atorvastatin. These two conjugates, termed G-AT and G-K-AT, exhibited enhanced water solubility and cellular uptake. Moreover, both G-AT and G-K-AT were able to release atorvastatin and consequently achieve significant inhibition against 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase.

Full text of DOI:10.1021/acs.bioconjchem.7b00295

Subscriber access provided by AUBURN UNIV AUBURN
Article
GalNAc conjugated atorvastatin with enhanced
water solubility and cellular internalization
Xinfu Zhang, Xiaofang Chen, Weiyu Zhao, Chunxi Zeng,
Xiao Luo, Wenqing Li, Bin Li, Justin Jiang, and Yizhou Dong
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00295 • Publication Date (Web): 26 Jul 2017
Downloaded from http://pubs.acs.org on July 31, 2017
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Bioconjugate Chemistry is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 19
Bioconjugate Chemistry
1
2
3
4
5
6
7
8
9
GalNAc Conjugated Atorvastatin with Enhanced
Water Solubility and Cellular Internalization
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Xinfu Zhang a ‡, Xiaofang Chen a ‡, Weiyu Zhao a, Chunxi Zeng a, Xiao Luo a, Wenqing Li a, Bin
Li a, Justin Jiang a and Yizhou Dong a, b, c, d, e, f
*
*Eꢀmail: dong.525@osu.edu.
a Division of Pharmaceutics & Pharmaceutical Chemistry, College of Pharmacy, The Ohio State
University, Columbus, Ohio 43210, United States; b Department of Biomedical Engineering, The
Ohio State University, Columbus, Ohio 43210, United States; c The Center for Clinical and
Translational Science, The Ohio State University, Columbus, Ohio 43210, United States; d The
Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio 43210, United
States. e Dorothy M. Davis Heart & Lung Research Institute; f Department of Radiation
Oncology, The Ohio State University, Columbus, OH 43210, United States.
ACS Paragon Plus Environment
1

Bioconjugate Chemistry
Page 2 of 19
1
2
3
4
ABSTRACT
5
6
7
8
9
Targeting ligands facilitate cell specific drug delivery and improve pharmaceutical properties.
Herein, we designed two ligand drug conjugates by conjugating GalNAc (Nꢀ
Acetylgalactosamine, a hepatocyteꢀtargeting ligand) with atorvastatin. These two conjugates,
termed G-AT and G-K-AT, exhibited enhanced water solubility and cellular uptake. Moreover,
both G-AT and G-K-AT were able to release atorvastatin and consequently achieved significant
inhibition against 3ꢀhydroxyꢀ3ꢀmethylꢀglutarylꢀcoenzyme A (HMGꢀCoA) reductase.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Introduction:
Atorvastatin with a trade name Lipitor is one of the most prescribed drugs in the statin family for
the treatment of dyslipidemia and the prevention of cardiovascular diseases.1ꢀ3 Atorvastatin
functions in hepatocytes in the liver as a lipidꢀlowering agent through inhibiting 3ꢀhydroxyꢀ3ꢀ
methylꢀglutarylꢀcoenzyme A (HMGꢀCoA) reductase, a crucial enzyme in the early stage of
biosynthesis of cholesterol.1ꢀ3 However, the bioavailability of atorvastatin is relatively low
(approximate 12% with a 40mg oral administration),4 due to its low water solubility, dissolution
rate, and membrane permeability.5ꢀ6 Extensive efforts have been made to enhance its dissolution
rate by incorporating a calcium salt or additional ingredients in its pharmaceutical formulation
(tablets, capsules or powders).7ꢀ8 In order to further improve pharmaceutical profiles of
atorvastatin, a new strategy is needed such as incorporation of targeting ligands.
Small molecule ligands have been widely investigated for the delivery of drugs to targeted cells,
which improve drug properties such as chemical stability, pharmacokinetics, and cellular
uptake.9ꢀ16 A number of ligands including folate acid,13 glucosamine,12 biotin,14 and
ACS Paragon Plus Environment
2

Page 3 of 19
Bioconjugate Chemistry
1
2
pamidronate11, 15 were widely used to target specific cell populations, which showed potential for
therapeutic applications. NꢀAcetylgalactosamine (GalNAc), a galactose derivative, was reported
as a high affinity ligand for the hepatocyteꢀspecific asialoglycoprotein receptor (ASGPR).17ꢀ19
GalNAc modified oligonucleotides showed efficient permeability into hepatocytes in both
preclinical and clinical studies.17ꢀ19 A most recent study demonstrated the high delivery
efficiency of GalNAc derivatives towards hepatocytes via ASGPR. 20 Thus, GalNAc may be a
promising ligand for delivery of atorvastatin to hepatocytes.
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
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Hence, we designed two atorvastatin derivatives, G-AT and G-K-AT (Scheme 1), by
conjugating atorvastatin and GalNAc through a ‘click reaction’.21 Both conjugates showed
increased water solubility and cell permeability. The conjugates themselves showed no inhibiting
activity on HMGꢀCoA, while restored the activity after releasing atorvastatin. G-AT can be
hydrolyzed by esterase in biological pH (pH~7.40) and G-K-AT can be hydrolyzed in a more
acidic environment (pH~4.5, lysosomal pH value22). Importantly, cellular uptake of G-AT and
G-K-AT was dramatically increased compared to atorvastatin.
ACS Paragon Plus Environment
3

Bioconjugate Chemistry
Page 4 of 19
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
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 1. Synthetic routes of G-AT and G-K-AT.
Design and synthesis
First, we synthesized G-AT and G-K-AT following the synthetic routes as shown in scheme 1.
Two hydroxyl groups were protected with a ketal to provide compound a. Then, a propargyl
group was installed onto compound a and yielded compound b. After deꢀprotection of compound
b in diluted hydrochloric acid, intermediate c was obtained. GalNAc group was then introduced
through a ‘click reaction’ to afford G-AT. A similar ‘click reaction’ between compound b and
GalNAc was performed to produce G-K-AT. In order to visualize the cellular uptake of GalNAc,
Cy7, a near infrared fluorophore, was conjugated to GalNAc using the same synthetic method,
providing G-Cy7 (Supporting information, Scheme S1). Structures of these conjugates were
confirmed by 1H NMR and mass spectrometry (Supporting information).
Then, we measured the solubility of atorvastatin, G-AT and G-K-AT in water through a UVꢀ
spectra assay. Their solubility was calculated using a standard curve: 0.18±0.0025 mmole/L,
ACS Paragon Plus Environment
4

Page 5 of 19
Bioconjugate Chemistry
1
2
3
4
5
6
7
8
9
0.78±0 mmole/L and 1.58±0.0028mmole/L for atorvastatin (in a calcium salt form), G-AT, and
G-K-AT, respectively (Table 1 and Supporting information, Table S1). Water solubility of G-
AT and G-K-AT was approximately 4ꢀ and 9ꢀfold higher than atorvastatin, respectively. In
addition, atorvastatin showed enhanced water solubility at a lower pH environment, while G-AT
and G-K-AT displayed higher water solubility at pH 7.4 in comparison with pH 4.5 (Table S1).
To investigate whether G-AT and G-K-AT formed micelles, we conducted dynamic light
scattering (DLS) studies using a NanoZS Zetasizer. As shown in table S2, both G-AT and G-K-
AT produced relatively homogenous micelles. Particle size was 144±4.5 nm and 95.8±1.1 nm,
respectively. Regarding atorvastatin, no detectable particles were observed. The formation of
micelles may lead to enhanced water solubility of G-AT and G-K-AT.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 1. Water solubility of atorvastatin, G-AT and G-K-AT.
Water solubility
(mmole/L)
0.18±0.0025
Atorvastatin
G-AT
0.78±0
1.58±0.0029
G-K-AT
We next studied hydrolysis of G-AT and G-K-AT to release atorvastatin. When incubated with
esterase in PBS (pH 7.40) at 37 oC, G-AT was quickly transformed into atorvastatin through a
twoꢀstep process (Scheme S2). According to the mass spectra analysis, GalNAc was first
cleaved. The remaining moiety formed an intermolecular ester, and then released atorvastatin
(Figure S1). On the other hand, G-K-AT first underwent an incubation in an acidic PBS (pH 4.5)
to eliminate the ketal group; and then was hydrolyzed with esterase to afford atorvastatin (Figure
S3). No hydrolysis products of G-K-AT were detected through mass spectra analysis after
ACS Paragon Plus Environment
5

Bioconjugate Chemistry
Page 6 of 19
1
2
3
4
5
6
incubated in PBS (pH 7.40, Figure S2). These results suggest that G-K-AT is more stable than
G-AT.
7
8
9
In order to test whether atorvastatin released from G-AT and G-K-AT is functional, we
measured their activity using a HMGꢀCoA Reductase Assay Kit. Neither G-AT nor G-K-AT
showed any inhibiting activity, while their hydrolysis products showed significant inhibition of
HMGꢀCoA reductase (Figure 1). After hydrolyzed by esterase, G-AT showed comparable
activity to atorvastatin. Since G-K-AT possesses an additional ketal protection, it underwent a
twoꢀstep hydrolysis after deprotecting the ketal group in an acidic environment. G-K-AT
displayed less activity than atorvastatin due to a slower release of atorvastatin.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 1. Inhibition of HMGꢀCoA reductase activity by atorvastatin, G-AT, G-K-AT and their
hydrolysis products. (Triplicate; **, P < 0.01; ***, P < 0.001; N.S., P > 0.05; t test, doubleꢀ
tailed)
To further study cellular uptake of GalNAc derivatives, G-Cy7 was synthesized as mentioned
above. After incubation of Hep3B cells, (a human hepatoma cell line) with G-Cy7, fluorescence
intensity was quantified through fluorescenceꢀactivated cell sorting (FACS) analysis. As shown
in figure 2A, the G-Cy7 group (intensity= 1727) showed much higher fluorescence intensity than
ACS Paragon Plus Environment
6

Page 7 of 19
Bioconjugate Chemistry
1
2
3
4
5
6
7
8
9
free Cy7 group (intensity= 349). When endocytosis was inhibited by decreasing the incubation
temperature to 4 °C,23 G-Cy7 treated cells displayed a significant lower fluorescence intensity
(intensity= 310; p< 0.001). These results indicate that endocytosis is the main pathway for
cellular uptake of G-Cy7.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
We then examined cellular uptake of G-AT and G-K-AT. Equal amount of atorvastatin, G-AT
and G-K-AT were used to treat Hep3B cells, respectively. After incubation, culture medium was
removed and the amount of drugs in cells was quantified using the same UVꢀspectra analysis as
mentioned above. The total amount of G-AT and G-K-AT were calculated to be 176% and
207% of atorvastatin group, respectively, significantly higher than atorvastatin (Figure 2B and
Supporting information, Table S3). In addition, both G-AT and G-K-AT showed release of free
atorvastatin in cells using mass spectra analysis (Supporting Information, Figure S4 and Figure
S5).
Figure 2. (A) Fluorescence intensity of Hep3B cell treated with Cy7 or G-Cy7. (B) Cellular
uptake of atorvastatin, G-AT or G-K-AT. The data were normalized to the amount of
atorvastatin. (Triplicate; *, P < 0.05; **, P < 0.01; ***, P < 0.001; t test, doubleꢀtailed)
ACS Paragon Plus Environment
7

Bioconjugate Chemistry
Page 8 of 19
1
2
3
4
5
6
7
8
9
Lastly, we examined biological function of G-AT and G-K-AT in cells using a method reported
previously.25 According to its mechanism of action, after internalized by hepatocytes,
atorvastatin inhibits HMGꢀCoA, and consequently induces increased expression of lowꢀdensity
lipoprotein (LDL) receptors.25 In the study, we measured the amount of LDL receptors on
Hep3B cells treated with atorvastatin, G-AT and G-K-AT using a flow cytometer. As shown in
figure 3, LDL receptors were significantly increased in the groups of atorvastatin, G-AT and G-
K-AT compared to the untreated group. These results indicate G-AT and G-K-AT are able to
release atorvastatin and then increase LDL receptors accordingly.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 3. Effects of atorvastatin, G-AT and G-K-AT on LDL receptors expression in Hep3B
cell line. The amount of LDL receptors were quantified by an immunofluorescence assay.
(Triplicate; *, P < 0.05; ***, P < 0.001; t test, doubleꢀtailed)
Conclusion
In conclusion, we designed and synthesized two GalNAcꢀconjugated atorvastatin, G-AT and G-
K-AT. Both G-AT and G-K-AT demonstrated enhanced water solubility and cellular uptake
than free atorvastatin. G-AT and G-K-AT were able to be hydrolyzed at different conditions due
to different chemical stability, which enables us to tune its pharmaceutical profiles in future
ACS Paragon Plus Environment
8

Page 9 of 19
Bioconjugate Chemistry
1
2
3
4
5
6
7
8
9
applications. The hydrolysis products of G-AT and G-K-AT dramatically inhibited the activity
of HMGꢀCoA reductase. Moreover, both G-AT and G-K-AT increased LDL receptors in Hep3B
cells. Reflecting the data above, these results prove the concept of our design and merit further
development of cell specific delivery of atorvastatin for therapeutic applications.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Experimental Section
Materials.
Esterase from porcine liver, HMGꢀCoA Reductase Assay Kit and other chemicals were
purchased from SigmaꢀAldrich or AlfaꢀAesar. Eagle’s Minimum Essential Medium (EMEM)
was purchased from Corning Incorporated (NY, USA). All chemicals were used without further
purification. Compounds a, b and c were synthesized according to the methods reported
previously.24
Synthesis of G-AT: Compound c (20 mg, 0.034 mmole) and 2ꢀAzidoethyl 2ꢀAcetamidoꢀ2ꢀdeoxyꢀ
βꢀDꢀglucopyranoside (20 mg, 0.069 mmole) were dissolved in 4 mL ethanol and stirred at RT
under Ar. CuSO4 (10 mg, 0.063 mmole) in 0.5 mL water and sodium ascorbate (20 mg, 0.10
mmole) in 0.5 mL water were added to the reacting mixture. The resulting mixture was stirred
for another 12h. After washing with water and CH2Cl2, the organic phase was collected and
evaporated. The crude product was purified by column chromatography using a Combiflash Rf
system with CH2Cl2/MeOH, (85/15 by volume) to give the product in a white solid (18 mg, yield
59.8%). 1H NMR (400 MHz, CD3OD) δ = 8.01 (1H, s), 7.32ꢀ7.24 (6H, m), 7.15ꢀ7.05 (7H, m),
5.28ꢀ5.20 (2H, m), 4.60ꢀ4.58 (2H, d, J = 8), 4.42ꢀ4.40 (1H, m), 4.26ꢀ4.24 (1H, m), 4.06ꢀ4.05
(2H, m), 3.93ꢀ3.85 (3H, m), 3.72ꢀ3.66 (3H, m), 3.47ꢀ3.45 (2H, m), 3.37 (7H, s), 3.33 (4H, s),
ACS Paragon Plus Environment
9

Bioconjugate Chemistry
Page 10 of 19
1
2
2.54ꢀ2.41 (2H, m), 1.94 (3H, s), 1.70 (2H, s), 1.51ꢀ1.49 (6H, d, J = 4). MS (m/z): [M + H]+ calcd.
3
4
5
6
for C46H56FN6O11, 886.3991; found, 887.3953.
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Synthesis of G-K-AT: Compound b (20 mg, 0.031 mmole) and 2ꢀAzidoethyl 2ꢀAcetamidoꢀ2ꢀ
deoxyꢀβꢀDꢀglucopyranoside (20 mg, 0.069 mmole) were dissolved in 4 mL ethanol and stirred at
RT under Ar. CuSO4 (10 mg, 0.063 mmole) in 0.5 mL water and sodium ascorbate (20 mg, 0.10
mmole) in 0.5 mL water were added to the reacting mixture. The resulting mixture was stirred
for another 12h. After washing with water and CH2Cl2, the organic phase was collected and
evaporated. The crude product was purified by column chromatography using a Combiflash Rf
system with CH2Cl2/MeOH, (90/10 by volume) to give the product in a white solid (19 mg, yield
65.0%). 1H NMR (400 MHz, CD3Cl) δ = 7.78 (1H, s), 7.20ꢀ7.17 (8H, m), 7.09ꢀ7.07 (2H, d, J =
8), 7.02ꢀ6.98 (3H, t, J = 8), 6.91 (1H, s), 5.20 (2H, m), 4.53ꢀ4.44 (6H, m), 4.22ꢀ4.20 (2H, m),
4.10ꢀ4.05 (1H, m), 3.95 (1H, s), 3.84ꢀ3.82 (3H, m), 3.68 (2H, s), 3.59ꢀ3.56 (2H, m), 3.32 (1H, s),
2.52ꢀ2.46 (1H, m), 2.38ꢀ2.28 (1H, m), 2.07 (3H, s), 1.96 (3H, s), 1.67ꢀ1.64 (2H, m), 1.54ꢀ1.53
(6H, d, J = 4), 1.34 (6H, s). MS (m/z): [M + H]+ calcd. for C49H60FN6O11, 927.4304; found,
927.4240.
Synthesis of G-Cy7: PropargylꢀCy7 (20 mg, 0.027 mmole) and 2ꢀAzidoethyl 2ꢀAcetamidoꢀ2ꢀ
deoxyꢀβꢀDꢀglucopyranoside (15 mg, 0.052 mmole) were dissolved in 3 mL ethanol and stirred at
RT under Ar. CuSO4 (5 mg, 0.032 mmole) in 0.3 mL water and sodium ascorbate (10 mg, 0.05
mmole) in 0.3 mL water were added to the reacting mixture. The resulting mixture was stirred
for another 12h. After washing with water and CH2Cl2, the organic phase was collected and
ACS Paragon Plus Environment
10

Page 11 of 19
Bioconjugate Chemistry
1
2
3
4
5
6
7
8
9
evaporated. The crude product was purified by column chromatography using a Combiflash Rf
system with CH2Cl2/MeOH, (85/15 by volume) to give the product in a blue solid (14 mg, yield
49.0%). 1H NMR (400 MHz, CD3OD) δ = 8.01 (1H, s), 7.80 (2H, s), 7.39ꢀ7.37 (2H, d, J = 8),
7.33ꢀ7.31 (2H, m), 7.09 (3H, s), 5.81 (1H, s), 5.20 (2H, s), 4.58ꢀ4.55 (2H, m), 4.42ꢀ4.39 (1H, m),
4.25ꢀ4.20 (1H, m), 3.92ꢀ3.88 (2H, m), 3.80ꢀ3.76 (2H, t, J = 8), 3.70ꢀ3.66 (2H, m), 3.46ꢀ3.41 (5H,
m), 3.34ꢀ3.33 (5H, m), 3.32 (2H, m), 2.57 (3H, s), 2.43ꢀ2.39 (2H, t, J = 8), 1.93 (3H, s), 1.88ꢀ
1.81 (4H, m), 1.74ꢀ1.72 (1H, m), 1.51ꢀ1.43 (3H, m), 1.31 (2H, s), 0.94ꢀ0.87 (1H, m). MS (m/z):
M+ calcd. for C51H68N7O8, 906.5124; found, 906.5132.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Quantification of water solubility:
Standard curves of absorption intensity at 290 nm against concentration for atorvastatin and two
conjugates were plotted (Supporting information, Table S1). By measuring the absorption
intensity at 290 nm of saturated solution of three compounds in water (diluted with methanol
before spectra test), water solubility of each conjugate was determined.
Hydrolysis assay:
G-AT was dissolved in DMSO to obtain a 3.5 mmol/L stock solution. 15 µL of stock solution
was then added into 485 µL of PBS (pH 7.40) with esterase (0.5 mg/mL) in a 1.5 mL sealed tube
and shaken at 37 oC for fixed time as 0, 3, and 24h. The hydrolysis reaction was terminated by
adding 0.5 mL of dichloromethane and shaken strongly. The organic phase was used for mass
spectrometry analysis. G-K-AT was dissolved in DMSO to obtain a 3.5 mmol/L stock solution.
ACS Paragon Plus Environment
11

Bioconjugate Chemistry
Page 12 of 19
1
2
3
4
5
6
7
8
9
15 µL of stock solution was then added into 235 µL of PBS (pH 4.50) and incubated at 37 oC for
24h. pH of the solution was adjusted to 7.40 by adding 1 mmole/L NaOH aqueous. Then, 250
µL PBS (pH 7.40) with esterase (1.0 mg/mL) was added. The mixture was shaken at 37 oC for
24h. The hydrolysis was terminated by adding 0.5 mL of dichloromethane and shaken strongly.
The organic phase was used for mass spectrometry analysis.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Quantification of HMG-CoA Reductase activity:
Activity of HMGꢀCoA Reductase was measured through a HMGꢀCoA Reductase Assay Kit
following the standard procedure. After adding the regents in requested order, the absorption at
340 nm was immediately collected every 20 seconds for up to 10 minutes. The activity of HMGꢀ
CoA Reductase was calculated according to the following equation:
∆ꢉꢊꢋꢌ
∆ꢉꢊꢋꢌ
ꢈ
−
ꢖ × ꢗꢘ
ꢅꢂꢁꢍꢎꢏꢐꢑꢒ ꢅꢂꢁꢓꢑꢎꢔꢕ
ꢀꢁꢂꢃꢄ(ꢅꢆꢇ) =
12.44 × ꢘ × 0.6 × ꢙꢇ
Where:
12.44 = εmM ꢀ the extinction coefficient for NADPH at 340 nm is 6.22 mM–1cm–1. 12.44
represents the 2 NADPH consumed in the reaction.
TV = Total volume of the reaction in ml (0.2 ml is used in this assay)
V = Volume of enzyme used in the assay (12×10ꢀ3 ml)
0.6 = Enzyme concentration in mgꢀprotein (mgP)/ml
LP = Light path in cm (0.55 cm is used for this assay)
ACS Paragon Plus Environment
12

Page 13 of 19
Bioconjugate Chemistry
1
2
3
4
5
6
7
Cell uptake of Cy7 and G-Cy7:
8
9
Human hepatocellular carcinoma Hep3B cell line (ATCC) was maintained in Eagle’s Minimum
Essential Medium (EMEM) supplemented with 10% heat inactivated FBS at 37 °C with an air
atmosphere of 5% CO2. Hep3B cells were seeded in 6ꢀwell clear bottom plates (15 x 104
cells/ml, 2ml/well). After 24h incubation, 200 µL stock solution of Cy7 and G-Cy7 (5 mM in
DMSO) was added with DMSO as a control group, which make the final concentration of each
dye to be 5 µM. 2h after treatment at 4 oC or 37 oC, cells were collected and the fluorescence
signal from Cy7 or G-Cy7 was quantified with BD LSR II flow cytometry analyzer (BD
Biosciences, San Jose, CA).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Cellular uptake of atorvastatin, G-AT and G-K-AT:
Hep3B cells were seeded in 6ꢀwell clear bottom plates (2 x 104 cells/500 µl). After 24h
incubation, 10 µL stock solution of atorvastatin, G-AT and G-K-AT (21 mM in DMSO) were
added with DMSO as a control group. 2h after treatment, cells were collected and extracted
using a mixture of 0.5 mL CH2Cl2 and 0.5 mL methanol. After filtration, the solution was used
for mass spectra and UVꢀspectra analysis. The amount of drugs were determined through
standard curves (Supporting information, Table S3).
Immunofluorescent staining analysis of LDL receptors:
ACS Paragon Plus Environment
13

Bioconjugate Chemistry
Page 14 of 19
1
2
Hep3B cells were seeded in 6ꢀwell clear bottom plates (1 x 105 cells/ml, 2mL/well). After 24h
3
4
5
6
7
incubation, 1 µL stock solution of atorvastatin, G-AT and G-K-AT (10 mM in DMSO) were
8
9
added (final concentration is 5 µM). After 48h incubation, cells were collected and washed with
PBS twice. Resuspend the cells to approximately 1 x 106 cells/ml in ice cold PBS, 1% sodium
azide in 1.5 mL tube. Cells of three test groups and one control group were incubated with antiꢀ
LDL receptor antibody (primary monoclonal antibody, clone 2H7.1, EMD Millipore
Corporation) for 30 min at room temperature. Wash the cells 2ꢀtimes with cold PBS, cells were
resuspended in 1 mL ice cold PBS, 1% sodium azide. They were incubated with Alexa 647ꢀ
labeled secondary antibody (Goat AntiꢀMouse IgG H&L) for 25 min at room temperature in
dark. Wash the cells 2ꢀtimes with cold PBS. The cells were resuspended in 0.3 mL cold PBS and
the fluorescence was quantified immediately with BD LSR II flow cytometry analyzer (BD
Biosciences, San Jose, CA).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ASSOCIATED CONTENT
Supporting Information. The Supporting Information is available free of charge on the ACS
1
Publications website. This material includes synthesis, mass spectra and H NMR spectra of the
compounds.
AUTHOR INFORMATION
Corresponding Author
* Eꢀmail: dong.525@osu.edu.
ORCID
ACS Paragon Plus Environment
14

Page 15 of 19
Bioconjugate Chemistry
1
2
3
4
Yizhou Dong: 0000ꢀ0001ꢀ5786ꢀ0659
5
6
7
Author Contributions
8
‡X.Z. and X.C. contributed equally.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
Y.D. accknowledges the support from the Maximizing Investigators' Research Award
1R35GM119679 from the National Institute of General Medical Sciences as well as the startꢀup
fund from the College of Pharmacy at The Ohio State University.
ABBREVIATIONS
DLS: Dynamic light scattering; GalNAc: NꢀAcetylgalactosamine.
REFERENCES
(1)
(2)
(3)
(4)
Buhaescu, I., and Izzedine, H. (2007) Mevalonate pathway: A review of clinical and
therapeutical implications. Clinical Biochemistry 40, 575ꢀ584.
Endo, A. (1992) The discovery and development of HMGꢀCoA reductase inhibitors.
Journal of Lipid Research 33, 1569ꢀ1582.
Istvan, E. S. (2002) Structural mechanism for statin inhibition of 3ꢀhydroxyꢀ3ꢀ
methylglutaryl coenzyme A reductase. American Heart Journal 144, S27ꢀS32.
Lennernäs, H., and Fager, G. (1997) Pharmacodynamics and Pharmacokinetics of the
HMGꢀCoA Reductase Inhibitors. Clinical Pharmacokinetics 32, 403ꢀ425.
ACS Paragon Plus Environment
15

Bioconjugate Chemistry
Page 16 of 19
1
2
3
4
5
6
7
8
9
(5)
(6)
Juha P., Shai L., Gerardo H., Chirayth M. S., Arthur L., Basil M. R., Heaman A. T.
(1990) Tenꢀyear mortality from cardiovascular disease in relation to cholesterol level
among men with and without preexisting cardiovascular disease. The new England
journal of medicine 322, 1700ꢀ1707.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Yu R. Z., Gunawan R., Post N., Zanardi T., Hall S., Burkey J., Kim T.ꢀW., Graham M. J.,
Prakash T. P., Seth P. P., et al. (2016) Disposition and Pharmacokinetics of a GalNAc3ꢀ
Conjugated Antisense Oligonucleotide Targeting Human Lipoprotein (a) in Monkeys
Nucleic Acid Therapeutics 26, 272ꢀ280.
(7)
(8)
F., S. R. (1989) Compressed Tablets by Sirect Compression Granulation Pharmaceutical
Dosage Form., Vol. 1, USA.
Panghal, D., Nagpal, M., Thakur, G. S., and Arora, S. (2014) Dissolution Improvement of
Atorvastatin Calcium using Modified Locust Bean Gum by the Solid Dispersion
Technique. Scientia Pharmaceutica 82, 177ꢀ191.
(9)
Fan, W., Wu, Y., Li, X.ꢀK., Yao, N., Li, X., Yu, Y.ꢀG., and Hai, L. (2011) Design,
synthesis and biological evaluation of brainꢀspecific glucosyl thiamine disulfide prodrugs
of naproxen. European Journal of Medicinal Chemistry 46, 3651ꢀ3661.
(10) Grubmüller, H., Heymann, B., and Tavan, P. (1996) Ligand Binding: Molecular
Mechanics Calculation of the StreptavidinꢀBiotin Rupture Force. Science 271, 997ꢀ999.
(11) Bhushan, K. R., Tanaka, E., and Frangioni, J. V. (2007) Synthesis of Conjugatable
Bisphosphonates for Molecular Imaging of Large Animals. Angewandte Chemie
International Edition 46, 7969ꢀ7971.
(12) Chen, X., Bi, Y., Wang, T., Li, P., Yan, X., Hou, S., Bammert, C. E., Ju, J., Gibson, K.
M., Pavan, W. J., and Bi, L. (2015) Lysosomal Targeting with Stable and Sensitive
ACS Paragon Plus Environment
16

Page 17 of 19
Bioconjugate Chemistry
1
2
3
4
5
6
Fluorescent Probes (Superior LysoProbes): Applications for Lysosome Labeling and
Tracking during Apoptosis. Scientific Reports 5, 9004.
7
8
9
(13) Low, P. S., Henne, W. A., and Doorneweerd, D. D. (2008) Discovery and Development
of FolicꢀAcidꢀBased Receptor Targeting for Imaging and Therapy of Cancer and
Inflammatory Diseases. Accounts of Chemical Research 41, 120ꢀ129.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(14) Weber, P. C., Ohlendorf, D. H., Wendoloski, J. J., and Salemme, F. R. (1989) Structural
Origins of HighꢀAffinity Biotin Binding to Streptavidin. Science 243, 85ꢀ88.
(15) Yin, Q., Tang, L., Cai, K., Tong, R., Sternberg, R., Yang, X., Dobrucki, L. W., Borst, L.
B., Kamstock, D., Song, Z., et al. (2016) Pamidronate functionalized nanoconjugates for
targeted therapy of focal skeletal malignant osteolysis. Proceedings of the National
Academy of Sciences 113, E4601ꢀE4609.
(16) Srinivasarao, M., Galliford, C. V., and Low, P. S. (2015) Principles in the design of
ligandꢀtargeted cancer therapeutics and imaging agents. Nat Rev Drug Discov 14, 203ꢀ
219.
(17) Akinc, A., Querbes, W., De, S., Qin, J., FrankꢀKamenetsky, M., Jayaprakash, K. N.,
Jayaraman, M., Rajeev, K. G., Cantley, W. L., Dorkin, J. R., et al. (2010) Targeted
Delivery of RNAi Therapeutics With Endogenous and Exogenous LigandꢀBased
Mechanisms. Molecular Therapy 18, 1357ꢀ1364.
(18) Kanasty, R., Dorkin, J. R., Vegas, A., and Anderson, D. (2013) Delivery materials for
siRNA therapeutics. Nat Mater 12, 967ꢀ977.
(19) Prakash, T. P., Graham, M. J., Yu, J., Carty, R., Low, A., Chappell, A., Schmidt, K.,
Zhao, C., Aghajan, M., Murray, H. F., et al. (2014) Targeted delivery of antisense
ACS Paragon Plus Environment
17

Bioconjugate Chemistry
Page 18 of 19
1
2
3
4
5
6
oligonucleotides to hepatocytes using triantennary Nꢀacetyl galactosamine improves
potency 10ꢀfold in mice. Nucleic Acids Research 42, 8796ꢀ8807.
7
8
9
(20) Sanhueza, C. A., Baksh, M. M., Thuma, B., Roy, M. D., Dutta, S., Préville, C., Chrunyk,
B. A., Beaumont, K., Dullea, R., Ammirati, M., et al. (2017) Efficient Liver Targeting by
Polyvalent Display of a Compact Ligand for the Asialoglycoprotein Receptor. Journal of
the American Chemical Society 139, 3528ꢀ3536.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(21) Kolb, H. C., Finn, M. G., and Sharpless, K. B. (2001) Click Chemistry: Diverse Chemical
Function from a Few Good Reactions. Angewandte Chemie International Edition 40,
2004ꢀ2021.
(22) Mindell, J. A. (2012) Lysosomal Acidification Mechanisms. Annual Review of
Physiology 74, 69ꢀ86.
(23) Zhang, X., Wang, C., Jin, L., Han, Z., and Xiao, Y. (2014) Photostable Bipolar
Fluorescent Probe for Video Tracking Plasma Membranes Related Cellular Processes.
ACS Applied Materials & Interfaces 6, 12372ꢀ12379.
(24) Mizoi, K., Takahashi, M., Haba, M., and Hosokawa, M. (2016) Synthesis and evaluation
of atorvastatin esters as prodrugs metabolically activated by human carboxylesterases.
Bioorganic & Medicinal Chemistry Letters 26, 921ꢀ923.
(25) Moon, J. H., Kang, S. B., Park, J. S., Lee, B. W., Kang, E. S., Ahn, C. W., Lee, H. C.,
Cha, B. S. (2010) Upꢀregulation of hepatic lowꢀdensity lipoprotein receptorꢀrelated
protein 1:
a
possible novel mechanism of antiatherogenic activity of
hydroxymethylglutarylꢀcoenzyme A reductase inhibitor Atorvastatin and hepatic LRP1.
Metabolism 60, 930ꢀ940.
ACS Paragon Plus Environment
18

Page 19 of 19
Bioconjugate Chemistry
1
2
3
4
TOC
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
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment
19