Anticancer activity ofwater-soluble olsalazine-pamam-dendrimer-salicylic acid-conjugates

Article abstract of DOI:10.3390/biom9080360

Improving the activity and selectivity profile of anticancer agents will require designing drug carrier systems that employ soluble macromolecules. Olsalazine-PAMAM-dendrimer-salicylic acid-conjugates with dendritic arms of different lengths have shown good stability regarding the chemical link between drug and spacer. In this study, the drug release was followed in vitro by ultraviolet (UV) studies. Evaluation of the cytotoxicity of the olsalazine-PAMAM-dendrimer-salicylic acid-conjugates employing a sulforhodamine B (SRB) assay in PC-3 (human prostatic adenocarcinoma) and MCF-7 (human mammary adenocarcinoma) cell lines demonstrated that conjugate 9 was more active as an antiproliferative agent than cisplatin, and no cytotoxicity towards the African green monkey kidney fibroblast (COS-7) cell line was observed in any of the conjugates synthesized in the present work.

Full text of DOI:10.3390/biom9080360

biomolecules
Article
Anticancer Activity of Water-Soluble
Olsalazine-PAMAM-Dendrimer-Salicylic
Acid-Conjugates
Sandra Cortez-Maya 1, Luis Daniel Pedro-Hernández 1 , Elena Martínez-Klimova 2,
Teresa Ramírez-Ápan 1 and Marcos Martínez-García 1,
*
1
Instituto de Química, Universidad Nacional Autónoma de México, Ciudad Universitaria, Circuito Exterior,
Coyoacán, C.P. 04510, México D.F., México
Facultad de Química, Universidad Nacional Autónoma de México, Ciudad Universitaria, Circuito Interior,
Coyoacán, C.P. 04510, México D.F., México
2
*
Correspondence: margar@unam.mx
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 10 June 2019; Accepted: 5 August 2019; Published: 13 August 2019
Abstract: Improving the activity and selectivity profile of anticancer agents will require designing
drug carrier systems that employ soluble macromolecules. Olsalazine-PAMAM-dendrimer-salicylic
acid-conjugates with dendritic arms of different lengths have shown good stability regarding the
chemical link between drug and spacer. In this study, the drug release was followed in vitro by
ultraviolet (UV) studies. Evaluation of the cytotoxicity of the olsalazine-PAMAM-dendrimer-salicylic
acid-conjugates employing a sulforhodamine B (SRB) assay in PC-3 (human prostatic adenocarcinoma)
and MCF-7 (human mammary adenocarcinoma) cell lines demonstrated that conjugate 9 was more
active as an antiproliferative agent than cisplatin, and no cytotoxicity towards the African green
monkey kidney fibroblast (COS-7) cell line was observed in any of the conjugates synthesized in the
present work.
Keywords: olsalazine; salicylic acid; dendrimers; anticanceractivity
1. Introduction
Olsalazine (3,3’-azobis salicylic acid) is an anti-inflammatory drug used in the treatment of
inflammatory bowel disease (IBD), ulcerative colitis (UC) and other gastrointestinal problems [
Olsalazine consists of two 5-aminosalicylic acid (5-ASA) molecules joined by an azo bridge [
Olsalazine has been shown to inhibit the development of colorectal cancer in patients [
has also been proposed as a broad-spectrum anticancer agent [ ]. Colonic bacteria were found to
split the azo bond of this compound through the liberation of the two molecules of 5-ASA [ ].
1
,2].
2].
2,3], and it
4
5–9
Nanocarriers based on generation 5-poly(amidoamine) (PAMAM) dendrimers with salicylic acid
covalently attached to their surface were used as chemical exchange saturation transfer (CEST) imaging
agents, making them an effective non-toxic ligand that could be useful in the design of new materials
such as dendrimers [10]. Dendrimers are well-defined, globular, highly branched macromolecules
with a large number of functional groups at their surface. Moreover, their size, molecular weight
and surface properties can be easily controlled. Dendrimers have attracted much attention in drug
delivery due to their unique structural properties and good water-solubility [11–14]. Thus, 5-amino
salicylic acid has been conjugated to poly(ethylene glycol) dendrimers and polyamidoamine (PAMAM)
dendrimers to reduce their toxicity and improve their activity [11–21].
This article reports the rational design of olsalazine as a nucleus of PAMAM-dendrimer-salicylic
acid-conjugates to reach human cancer cells and the evaluation of drug release from these
Biomolecules 2019, 9, 360; doi:10.3390/biom9080360
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macromolecular products. In this study, the olsalazine-PAMAM-dendrimers of second generation (G2)
with 8 amino terminal groups and salicylic acid in the periphery were synthesized (Figure 1). The
release of the drug from the conjugates in vitro was also determined in comparison with free olsalazine.
Figure 1. Synthesis of olsalazine-PAMAM-dendrimer-salicylic acid-conjugates of the first (8) and
second (9) generation.
2. Materials and Methods
All reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). Salicylic acid
(SA), 5-amino-2-hydroxybenzoic acid (98%), hydrochloric acid (36.5–38%), sodium nitrite (98%),
hexamethylenediamine (98%), ethylendiamine titanium(IV) isopropoxide (97%), methyl bromoacetate,
methyl acrylate, Triton X-100 solution and phosphate buffered saline pH 7.4 were used without
additional purification. Ethanol and methanol were used without additional drying. Tetrahydrofuran
(THF) was dried with Na. N,N-Dimethylformamide (DMF) and dimethylsulfoxide (DMSO) (99.8%)
were used without any purification.
2.1. Instrumentation
1H and 13C NMR spectra were recorded on a Varian Unity-300 MHz with tetramethylsilane (TMS)
as an internal reference. Infrared (IR) spectra were measured on a Nicolet FT-SSX spectrophotometer
(Bruker, Billerica, MA, USA). Elemental analysis was determined by Galbraith Laboratories, INC
Knoxville (Sycamore, MO, USA). FAB+ mass spectra were taken on a JEOL JMS AX505 HA instrument
(JEOL manufacturers, Tokyo, Japan). Electrospray mass spectra were taken on a Bruker Daltonic,
Esquire 6000 (Bruker, Billerica, MA, USA). Matrix-assisted laser desorption/ionization-time of flight
(MALDI-TOF) mass spectra were taken on a Bruker Omni FLEX using 9-nitroanthracene (9NA) as a
matrix. The ultraviolet–visible (UV-Vis) absorption spectra were obtained at room temperature with a
Shimadzu 2401 PC spectrophotometer (Shimadzu, Kyoto, Japan).

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2.2. Anticancer Screening
Human glioblastoma, U-251, PC-3 (human prostatic adenocarcinoma), K-562 (human chronic
myelogenous leukemia cells), HCT-15 (human colorectal adenocarcinoma), MCF-7 (human mammary
adenocarcinoma) and SKLU-1 (human lung adenocarcinoma) were supplied by the National Cancer
Institute (USA). The African green monkey kidney fibroblast (COS-7) cell line was acquired from Thermo
Fisher Scientific (Waltham, MA USA). Cytotoxicity assays were determined using the protein-binding
dye sulforhodamine B (SRB) in microculture to measure cell growth, as described previously [19,20].
Olsalazine-PAMAM-dendrimer-salicylic acid-conjugates were prepared in fresh culture medium with
2% DMSO immediately before use, then added to the culture medium. Control cells were treated with
2% DMSO. Mean values of half maximal inhibitory concentration (IC50) for each tested drug were
calculated from at least three experiments.
2.3. In Vitro Cellular Uptake of the Conjugates of Salicylic Acid
At 35 ◦C in a Synergy HT Microplate Reader (Bio-Tek Instruments, Winossky, VT, USA), the cellular
uptake of olsalazine-PAMAM-dendrimer-salicylic acid-conjugates was quantitatively determined in a
pH 7.4 buffer solution. The human mammary adenocarcinoma cancer cells, MFC-7 were seeded in a
96-well black plate at 10,000 cells/well and cultured. After 24 h, MFC-7 cancer cells were incubated with
either olsalazine-PAMAM-dendrimer-salicylic acid-conjugates or free olsalazine 1 at a concentration
of either 10 or 25
were washed four times with 50
µ
M for 16 h at 35 ◦C. After incubation, the suspension was removed and the cells
µL cold phosphate buffered saline solution (PBS, pH 7.4). All the
resulting solutions were kept at 37 ◦C for 24 h prior to measurements. The fluorescence intensity of
each sample well was measured by a microplate reader calibrated with standard solutions of olsalazine
(0.1–50 µM/mL) under similar conditions. The excitation and emission wavelengths were 300 and
400 nm; and 340 and 500 nm, respectively. All release measurements were carried out in triplicate and
the average values were plotted.
2.4. Hydrolytic Release of the Drug from Olsalazine-PAMAM-Dendrimer-Salicylic Acid-Conjugates
◦
◦
The hydrolytic release of the drugs was measured at 37 C using 25 mL PBS, at 37 C and
pH 7.4. The olsalazine-PAMAM-dendrimer-salicylic acid-conjugates were dried in vacuum at room
temperature. Figure 2 shows the time course of UV spectral change of the conjugates incubated in
phosphate buffer. The maximum UV absorption of the Schiff base in PBS was at 300 nm at pH 7.4.
The dendrimers and conjugates were dispersed in buffer solution and the hydrolysis was made. The
mixture solution was stirred by a magnetic stirrer continuously at 600 rev/min, and a sample solution
(3 mL) was withdrawn at selected intervals of time and replaced with the same volumes of media. The
quantity of the released drug was analyzed by means of UV spectroscopy and determined from the
calibration curve using a 1-cm quartz cell.

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Figure 2. Proton nuclear magnetic resonance, 1H NMR, spectra of the olsalazine-PAMAM-conjugates
of salicylic acid (8) and (9) in D2O.
3. Synthesis of Olsalazine-PAMAM-Dendrimers
Olsalazine was obtained from salicylic acid and 5-amino-2-hydroxybenzoic acid (5-ASA) with
sodium nitrite in HCl at 0 ◦C. The olsalazine derivative 2 was obtained using hexamethylenediamine
in presence of molecular sieves of 4 Å and titanium (IV) isopropoxide in THF at 70 ◦C (Scheme 1).
Scheme 1. Synthesis of: (a) olsalazine 1 (NaNO3, HCl, 0 ◦C); and (b) olsalazine derivative 2
(hexamethylenediamine, MS 4 Å, Ti(OiPr)4, THF, 70 ◦C).
The olsalazine-dendrimers intermediates 3 and 5 were obtained using methyl acrylate in a mixture
of methanol, toluene 5:1 and reflux (Scheme 2). The olsalazine-PAMAM-dendrimers 4 and 6 with the
NH2-terminated groups were obtained with ethylenediamine in a mixture of methanol, toluene 5:1
and reflux (Scheme 2).

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Scheme 2. Synthesis of olsalazine-PAMAM-dendrimers of the first (4) and second (6) generation.
The compounds 4 and 6 were characterized by 1H NMR spectroscopy. In the spectrum, the broad
signals due to the internal -CH2 of the hexylenediamine groups were observed from δH 1.37 to 1.63,
and the signals due to the CH2- groups were observed at the dendritic arms from δH 1.94 to 3.31. One
triplet due to the -CH2 at 3.62 was also observed (see NMR spectra for all the compounds can be found
in Supplementary Materials; Figures S25 and S32).
3.1. Synthesis of Olsalazine-PAMAM-Dendrimer-Salicylic Acid-Conjugates
After that, the methyl salicylate was obtained from methyl salicylic acid in methanol and sulfuric
acid at the reflux. The olsalazine-PAMAM-dendrimer-salicylic acid-conjugates 8 and 9 with methyl
salicylate were obtained in a mixture of methanol/toluene at the reflux (Figure 1).
The conjugates were soluble in water and characterized by proton nuclear magnetic resonance
(1H NMR) spectroscopy (Figure 2). The following signals were observed: from δH 1.03 to 1.86, three
broad signals were observed due to the internal CH2- groups at the hexyl moiety; from δH 2.54 to
3.83, the signals for the CH2 groups assigned to the dendritic branches and the terminal CH2 groups
at the hexyl fragment were observed; and from δH 6.5 to 7.5 the protons for the aromatic fragments
were observed.
3.2. Hydrolytic Release of Olsalazine-PAMAM-Dendrimer-Salicylic Acid-Conjugates 8 and 9
The stability of the conjugates was studied in conditions of gastric fluid pH 1.2 and intestinal
fluid pH 7.4 for 2 and 12 h, respectively, where a negligible amount of 5-ASA was hydrolyzed. No
degradation of conjugates at pH 1.2 and 7.4 showed that the conjugates were stable and remained
intact at the gastric pH (see Supplementary Materials Figure S57).
At pH 7.4, the olsalazine-PAMAM-dendrimer-salicylic acid-conjugates 8 and 9 showed a relatively
slow release in the first minutes, followed by a sustained release after 500 min. Salicylic acid was
released from the corresponding first generation 8 and the second generation 9. Figure 3 shows the time
course of UV spectral change from the conjugates. The amount of drug released from the conjugates
was calculated from their UV absorption at 300 nm at pH 7.4. The amount of drug released was

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constant regardless of the generation number. This result could be related to the relative stability of the
conjugates at the pH 7.4.
Figure 3. Ultraviolet (UV) spectra showing drug release from conjugates 8 and 9.
Drug release from both olsalazine-PAMAM-dendrimer-salicylic acid-conjugates 8 and 9 was
calculated from their UV absorption at 300 nm, as depicted in Figure 4. At physiological pH 7.4,
conjugate 8 showed a less controlled release pattern compared to conjugate 9.

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Figure 4. Release of drug from compound 8 (dotted line), and from compound 9 (continuous line) as a
function of time and pH (pH 7.4, 37 ◦C).
3.3. Cytotoxicity of Olsalazine-PAMAM-Dendrimer-Salicylic Acid-Conjugates
The cytotoxic activity of the synthesized olsalazine-PAMAM-dendrimers 4 and 6 and the
olsalazine-PAMAM-dendrimer-salicylic acid-conjugates 8 and 9 were chosen for evaluation of their
biological activity against cancer cell lines. We screened in vitro against six human cancer cell
lines: U251 (human glioblastoma), PC-3 (human prostatic adenocarcinoma), K-562 (human chronic
myelogenous leukemia cells), HCT-15 (human colorectal adenocarcinoma), MCF-7 (human mammary
adenocarcinoma) and SKLU-1 (human lung adenocarcinoma). As a control, we also tested against the
COS-7 African green monkey kidney fibroblast cell line. Free olsalazine and cisplatin were used to
compare the antiproliferative activity of the dendrimers. The dendrimers 4 and 6, and the conjugates 8
and 9 were not active against the cell lines U251 and SKLU-1. The compounds 4–9 showed almost the
same activity against cell lines PC-3, K562, HCT-15 and MFC-7 at 25 and 10 µM. The concentration of
the conjugates 8 and 9 was diluted four and eight times, respectively, to have the anticancer activity of
one molecule of salicylic acid to compare with the free salicylic acid, olsalazine or cisplatin. Table 1
shows the normalized percentage of inhibition of the growth that allows comparing the activity of
the same amount of salicylic acid in its free state versus when it is contained in the conjugates 8 and
9. Cisplatin was tested at a concentration of 10
acid-conjugate 8 at 25 M, the growth inhibitions were 50.1, 55.0 and 56.4% for the PC-3, K-565 and
MCF-7 cell lines, respectively. This behavior was also observed when the concentration was diminished
to 10 M. Conjugate 8 showed higher activity than cisplatin against cell lines PC-3 and MFC-7. In
the case of the conjugate 9, the antiproliferative activity was lower than with conjugate . The free
µM. For the olsalazine-PAMAM-dendrimer-salicylic
µ
µ
8
olsalazine and the free salicylic acid showed very low antiproliferative activity. None of the studied
samples showed any activity against the control COS-7 cell line in comparison with free cisplatin
(42.4%).

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Table 1. Cytotoxic activity of the compounds 4, 6, 8 and 9 at 25 µM and at 10 µM*.
Percentage of growth inhibition
Compound
U-251
PC-3
K-562
HCT-15
MCF-7
SKLU-1
COS-7
NC
NC*
20.3
16.8*
39.6
37.3*
29.0
14.9*
28.1
18.2*
NC
NC*
NC
NC*
4
NC
NC*
14.8
13.5*
38.2
23.1*
13.4
17.8*
29.1
15.8*
NC
NC*
NC
NC*
6
8
9
NC
NC*
50.1
45.6*
55.0
54.6*
28.9
19.9*
56.4
49.5*
NC
NC*
NC
NC*
NC
NC*
11.3
10.4*
19.9
13.3*
13.1
14.9*
15.7
16.7*
NC
NC*
NC
NC*
Olsalazine
Salicylic Acid
Cisplatin
NC
NC
11.9
4.9
8.1
6.6
6.3
6.8
3.1
0.5
NC
NC
NC
NC
87.5*
42.7*
79.2*
32.4*
32.4*
81.4*
42.4*
NC: non-cytotoxic.
In general, the PC-3 (prostate grade IV adenocarcinoma), K-562 (chronic myelogenous leukemia)
and MCF-7 (human mammary adenocarcinoma) cancer cell lines appeared to be more sensitive to
growth inhibition by the olsalazine-PAMAM-dendrimer-salicylic acid-conjugates than the U-251,
HCT-15 and SKLU-1 cancer cell lines. The compound 9 was the best inhibitor of this series against
the PC-3, K-562 and MCF-7 cell lines with an IC50 of 17.5
±
2.1 and 11.6
±
3.6, respectively, and the
activity was very close to that of cisplatin. Moreover, these compounds were tested against the COS-7
African green monkey kidney cell line (Table 2) in order to determine their selectivity for cancer cells
compared to normal cells. All the compounds showed better selectivity for the cancer cells than for the
COS-7 cell line.
Table 2. The effect of olsalazine-PAMAM-dendrimers 4 and 6 and olsalazine-PAMAM-dendrimer-
salicylic acid-conjugates 8 and 9 on the viability of different tumor cell lines and the COS-7 cell line
expressed as IC50 in µM.
IC50 (µM)
Compound
PC-3
K-562
HCT-15
33.3 ± 4.8
24.7 ± 2.8
34.9 ± 4.3
24.1 ± 2.8
12.3 ± 0.8
MCF-7
COS-7
NC
4
32.3 ± 5.2
31.0 ± 6.3
26.9 ± 2.4
18.5 ± 1.8
17.5 ± 1.4
29.2 ± 3.1
22.0 ± 2.7
38.3 ± 6.3
17.5 ± 2.1
1.2 ± 0.08
40.6 ± 2.5
31.6 ± 3.6
20.3 ± 8.6
11.6 ± 3.6
17.4 ± 0.8
6
NC
8
9
NC
NC
Cisplatin
15.2 ± 0.9
Half maximal inhibitory concentration (IC50) values are presented as the mean
separate experiments. NC: non-cytotoxic.
±
SD (standard deviation) from five
4. Conclusions
In summary, we report the development of novel water-soluble olsalazine-PAMAM-dendrimer-
salicylic acid-conjugates that inhibit the proliferation of the PC-3, K-562, HCT-15 and MCF-7 cell
lines in vitro. The drug release was followed by UV spectroscopy at pH 7.4. These conjugates were
designed and synthesized for maximal MCF-7 inhibition and exhibited high comparable selectivity
against human mammary adenocarcinoma cancer cells. The olsalazine-PAMAM-dendrimer-salicylic
acid-conjugate
9 revealed an IC50 value that was lower than that of cisplatin for the MCF-7 cell line. In
addition, the use of olsalazine-PAMAM-dendrimer-salicylic acid-conjugates could be an effective way
to diminish the damage towards normal cells.

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Supplementary Materials: The following are available online at http://www.mdpi.com/2218-273X/9/8/360/s1
Figures S1–S57: full characterization of all the conjugates (1H, 13C, FTIR, and mass spectrometry).
Author Contributions: Methodology, S.C.-M., L.D.P.-H. and E.M.-K.; formal analysis, L.D.P.-H. and E.M.-K.;
investigation, S.C.-M., L.D.P.-H., E.M.-K., T.R.-Á. and M.M.-G.; writing—original draft preparation, E.M.-K. and
M.M.-G.; writing—review and editing, M.M.-G.; supervision, S.C.-M., L.D.P.-H., E.M.-K. and M.M.-G.
Funding: This research was funded by DGAPA, grant number IN101117 and EMK was supported by a postdoctoral
fellowship from CONACyT.
Acknowledgments: We would also like to thank Rios O. H., Velasco L., Huerta S. E., Patiño M. M. R. and Peña
Gonzalez M. A. for their technical assistance.
Conflicts of Interest: The authors declare no conflict of interest.
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2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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