Solubility of nicotinic acid in polyamidoamine dendrimer solutions

Article abstract of DOI:10.1016/j.ejmech.2005.08.001

In the present study we investigated the effect of ethylenediamine (EDA) core polyamidoamine (PAMAM) dendrimers on the aqueous solubility of nicotinic acid. The aqueous solubility of nicotinic acid was measured in the presence of dendrimers at room temperature in distilled water. The effect of variables, such as pH condition, concentration, surface functional group and generation (molecule size) of dendrimer, has been investigated. Results showed that the solubility of nicotinic acid in the dendrimer solutions was proportional to dendrimer concentration, both amine and ester-terminated dendrimers caused the higher increase in nicotinic acid solubility at higher pH conditions. The order in which the dendrimers increased the solubility at a constant pH condition was G4 > G3 > G2 > G1. In addition, at each pH, the solubility of nicotinic acid was greater in the presence of amine-terminated dendrimers compared to the amine ester-terminated dendrimers possessing the same number of surface functional groups. Under suitable conditions PAMAM dendrimers can be highly effective used to enhance the solubility of nicotinic acid.

Full text of DOI:10.1016/j.ejmech.2005.08.001

European Journal of Medicinal Chemistry 40 (2005) 1384–1389
www.elsevier.com/locate/ejmech
Preliminary communication
Solubility of nicotinic acid in polyamidoamine dendrimer solutions
a,
b
ChengYiyun *, Xu Tongwen
a
Laboratory of Structural Biology, Department of Polymer Science and Engineering, School of Life Science,
University of Science and Technology of China (USTC), Hefei, Anhui 230027, China
Department of Chemistry, University of Science and Technology of China, Hefei, Anhui 230026, China
b
Received 13 July 2005; accepted 2 August 2005
Available online 13 October 2005
Abstract
In the present study we investigated the effect of ethylenediamine (EDA) core polyamidoamine (PAMAM) dendrimers on the aqueous
solubility of nicotinic acid. The aqueous solubility of nicotinic acid was measured in the presence of dendrimers at room temperature in
distilled water. The effect of variables, such as pH condition, concentration, surface functional group and generation (molecule size) of
dendrimer, has been investigated. Results showed that the solubility of nicotinic acid in the dendrimer solutions was proportional to dendrimer
concentration, both amine and ester-terminated dendrimers caused the higher increase in nicotinic acid solubility at higher pH conditions. The
order in which the dendrimers increased the solubility at a constant pH condition was G4 > G3 > G2 > G1. In addition, at each pH, the
solubility of nicotinic acid was greater in the presence of amine-terminated dendrimers compared to the amine ester-terminated dendrimers
possessing the same number of surface functional groups. Under suitable conditions PAMAM dendrimers can be highly effective used to
enhance the solubility of nicotinic acid.
©
2005 Elsevier SAS. All rights reserved.
Keywords: Polyamidoamine dendrimers; PAMAM; Solubility enhancer; Nicotinic acid
1
. Introduction
solubility and reactivity. These specific properties make den-
drimers suitable for drug delivery systems [5–7]. Drugs or
other molecules can either be attached to dendrimers end
groups or encapsulated in the macromolecule interior [8]. The
high density of amino groups and special structure in PAMAM
dendrimers may be expected to have potential applications in
enhancing the solubility of the low aqueous solubility drugs
and as delivery systems for bioactive materials [9]. This study
uses PAMAM dendrimers (G1-G4) to investigate the poten-
tial of PAMAM dendrimers to increase the solubility of drugs
as exemplified by nicotinic acid.
Nicotinic acid, 3-carboxylic pyridine, the deficiency of
which leads to Pellagra, is an essential dietary vitamin. It is
required for cell respiration, helps in the release of energy
and metabolism of carbohydrates, fats and proteins, proper
circulation and healthy skin, functioning of the nervous sys-
tem and normal secretion of bile and stomach fluids. It is also
used in the synthesis of sex hormones, treating schizophrenia
and other mental illnesses, and a memory-enhancer [10]. The
physiologically active form of nicotinic acid is nicotinamide
adenine dinucleotide (NAD) or its phosphate (NADP). Both
NAD and NADP function as coenzymes for a wide variety of
Dendrimers, artificial polymers topologically based on the
structure of a tree, are synthetic, highly branched, spherical,
mono-disperse macromolecules. They combine typical char-
acteristics of small organic molecules and polymers that
results in special physical and chemical properties [1–4].
Polyamidoamine (PAMAM) with an ellipsoidal or spheroi-
dal shape is one of the most-studied starburst macromol-
ecules. Due to specific synthesis PAMAM dendrimers have
some interesting properties, which distinguish them from clas-
sical linear polymers, e.g. PAMAM has a much higher amino
group density comparing with conventional macromol-
ecules, a third generation PAMAM prepared from ammonia
–
4
core has 1.24 × 10 amine moieties per unit volume (cubic
–6
Angstrom units) in contrast to the 1.58 × 10 amine moi-
eties per unit volume of a conventional star polymer [4];Also,
PAMAM Dendrimers possess empty internal cavities and
many functional end groups which are responsible for high
*
Corresponding author. Tel./fax: +86 55 1360 3913.
E-mail address: yycheng@mail.ustc.edu.cn (C. Yiyun).
0223-5234/$ - see front matter © 2005 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2005.08.001

C. Yiyun, X. Tongwen / European Journal of Medicinal Chemistry 40 (2005) 1384–1389
1385
methyl acrylate, methanol (HPLC grade) were obtained from
Shanghai Chemical Co. (Shanghai, China). For both solubil-
ity studies, water used to prepare solutions was two-distilled
water.
2
.2. Synthesis of PAMAM dendrimers
PAMAM dendrimers were synthesized by the following
method (Fig. 2) [2,4]. Ethylenediamine (10.0 g, 0.166 mol)
was dissolved in 100 ml methanol in a 1-l round-bottomed
flask. Methyl acrylate (94.6 g, 0.751 mol) was added at 40 °C
and the system stirred for 24 h under nitrogen. Excess methyl
acrylate was removed under vacuum at room temperature. A
Michael addition between the amine and the acrylate yielded
a product bearing four terminal methyl ester groups, defined
as the G0.5 PAMAM. Subsequently, ethylenediamine (120 g,
2.00 mol) was dissolved in methanol and added to the
G0.5 PAMAM and, after stirring for 48 h under nitrogen and
removing excess reactants by vacuum distillation, a product
bearing four terminal amino groups were obtained, defined
as the G1 PAMAM. By repeating the above cycle, higher gen-
eration PAMAM dendrimers (up to G4) were synthesized.
Since the dendrimers are highly hygroscopic, they were stored
as 10% (w/w) solutions in methanol. The characteristic data
of different generations of PAMAM are listed in Table 1.
Fig. 1. Schematically molecular structures of G3 PAMAM dendrimer (A)
and nicotinic acid (B).
proteins that catalyze oxidation–reduction reactions essen-
tial for tissue respiration. However, it is not freely soluble in
water at room temperature, so we choose this important and
common drug for an investigation.
The aim of the present work was (1) to study the potential
of PAMAM dendrimers as a solubility enhancer of the men-
tioned drug nicotinic acid; (2) to study the effect of pH, con-
centration, surface functional group and generation on inter-
actions between PAMAM dendrimers and nicotinic acid
(Fig. 1).
2. Experiments
2
.3. Solubility testing experiments
2.1. Materials
The solubility of nicotinic acid in PAMAM dendrimer solu-
Nicotinic acid was purchased from Weifang Sunwin
Chemicals Co., Ltd (Shangdong, China). Ethylenediamine,
tions in the range 0–10 mg/ml was determined at pH 3, 4 and
6 in phosphate buffers (0.05 M NaH PO ). The method used
2
4
Fig. 2. Synthesis strategy for PAMAM dendrimers.
Table 1
The characteristic data of PAMAM dendrimers
Generation
Molecular formula
Molecular weight
Number of terminal
Number
Radius from SAXS (Å)
amino/ester groups
of total amino groups
G1
G2
G3
G4
G5
C22H48O N
516
4
10
–
4
10
C62H128O12N26
1428
3252
6900
14196
8
26
–
C142H288O28N58
C302H608O60N122
C622H1248O124N250
16
32
64
58
15.8
17.1
24.1
122
250

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C. Yiyun, X. Tongwen / European Journal of Medicinal Chemistry 40 (2005) 1384–1389
for sample preparation was similar for each system, i.e. excess
nicotinic was added to 5 ml vials containing 4 ml of each test
solution. The vials were then incubated in a shaking water
bath at 37 °C for 24 h. Then the solutions were centrifuged at
nificantly with PAMAM concentrations. In the presence of
G3 PAMAM dendrimer at a fixed pH condition, the solubil-
ity of nicotinic acid in the dendrimer solutions increased in
an approximately linear manner with an increase in den-
drimer concentration. This was presumably due to the increase
in the number of surface amines and internal cavities that are
available to interact with nicotinic acid molecules. Molecu-
lar simulations of structures of the PAMAM dendrimers
showed that lower generation dendrimers (G < 4 or 4.5) pos-
sess an open structure and an ellipsoidal shape; where as later
generations (G > 4 or 4.5) are characterized by closed struc-
ture with a density packed surface and a spherical shape [11].
Due to this specific and interesting property of PAMAM den-
drimers, the cavities in PAMAM dendrimers can keep small
guest molecules inside and make dendrimers suitable for
enhancing the solubility of drug molecules such as nicotinic
acid in aqueous solutions. Also, there are tertiary amines in
these internal cavities, which could interact with the atoms of
the nicotinic acid molecules by hydrogen bond formation.
Further, PAMAM dendrimers have primary amines on the
surface, which could interact electrostatically with the car-
boxyl group in the nicotinic acid molecules. Therefore,
G3 PAMAM dendrimers possess open and internal cavities
and many functional terminal groups that are responsible for
high solubility and reactivity. These specific properties make
dendrimers suitable for drug delivery systems [5].
5000 rpm for a minute and the absorbance of the nicotinic
acid test solutions at the characteristic wavelength 262 nm
were tested using the Varian Cary VIII spectrophotometer.
Three repeats were conducted for each sample.
3. Results and discussion
3.1. Structural characterization of the PAMAM dendrimers
Hydrodynamic diameters of ester-terminated PAMAM
dendrimers (G 0.5, G 1.5, G2.5, and G3.5) in ethyl acetate
solution were determined by dynamic light scattering method
(
DYNAPRO-99, USA). For sample preparation, 1–2 wt%
PAMAM/ethyl acetate solution was repeatedly filtrated
through a 0.1 um nylon filter. Purity of the amine-terminated
PAMAM dendrimers were characterized via FT-IR
1
(
MAGNA-IR 750, Nicolet Instrument Co., U.S.A), H and
1
3
C NMR (DMX-500, German), Mass spectral analysis (BIFI
EXTM 3, German) and Element analysis (VARIO EL 3,
Elementar Instrument Co., German), and the results agreed
with that reported in the literature [2,4]. Since the dendrim-
ers are highly hygroscopic, they were stored as 10% (w/w)
solutions in methanol.
3.3. Effect of pH condition on solubility of nicotinic acid
3
.2. Effect of PAMAM concentration on solubility
To ascertain the most effective pH condition on solubility
of nicotinic acid using PAMAM dendrimers, samples of nico-
tinic acid solution were produced at a range of pH values, the
concentration of G3 PAMAM dendrimer being constant. The
results are shown in Fig. 3 and, as can be seen, the process
was pH-dependent. The solubility of nicotinic acid in
PAMAM dendrimer solutions was highest at pH 6, less at pH
4, and least at pH 3. Studies have shown that lower genera-
tion dendrimers are very weak pH-dependent conforma-
tional changes [12].Also rheological studies of PAMAM den-
drimers showed that the lower generation dendrimers showed
excellent fit with the view that with higher generation a quali-
tative change in molecular conformations may occur where
as lower generations are less sensitive to conformational
changes [13]. All these mean that the lower generation den-
drimers are more accessible for drug inclusion. As the
PAMAM dendrimers used in this study was lower generation
ones (G1–G4), we can presume that the conformation of
PAMAM dendrimers did not change at various pH condi-
tions.As discussed in 3.2, the solubility enhancement of nico-
tinic acid is due to an electrostatically interaction between
the surface amine groups of dendrimer molecule and the car-
boxyl group of nicotinic acid and to hydrogen bond forma-
tions between tertiary amines in internal cavities of dendrim-
ers and the atoms of nicotinic acid (Fig. 4). At lower pH
conditions, there is a lower increase of solubility of nicotinic
acid in dendrimer solution compared to that at higher pH con-
of nicotinic acid
The effect PAMAM dendrimer concentration on solubil-
ity of nicotinic acid was carried out using G3 PAMAM den-
drimer of molecular weight 3252 Da and 16 amine groups in
the outer shell, and the results were shown in Fig. 3. It was
observed that the solubility of nicotinic acid increased sig-
Fig. 3
. Solubility of nicotinic acid in the presence of increasing concentra-
tion of G3 PAMAM dendrimer.

C. Yiyun, X. Tongwen / European Journal of Medicinal Chemistry 40 (2005) 1384–1389
1387
Fig. 4
. Two potential strategies for interactions between PAMAM dendri-
mers and nicotinic acid molecules.
ditions. This is because the nicotinic acid, being weakly acid,
is not fully ionized at low pH conditions and hence cannot
freely interact electrostatically with the surface amine groups
of dendrimer molecule. Further, Earlier studies have shown
that the cavity environment inside the PAMAM dendrimer is
more hydrophobicity than that of the water phase outside [14].
Therefore, in PAMAM dendrimer solutions, nicotinic acid
molecules could be solubilized in the cavities of the PAMAM
dendrimer at the site of low polarity [14]. However, at low
pH conditions, protonation of the tertiary amines in the full
generation dendrimers enhances the polar level of environ-
ment inside the dendrimer, which causes no significant
increase in the solubility of nicotinic acid as observed in the
experiment (Fig. 3). As a result, the solubility of nicotinic
acid in PAMAM dendrimer solutions was lowest at pH 3.
Fig. 5
.
Solubility of nicotinic acid in the presence of increasing concentra-
tion of G2.5 and G3 PAMAM dendrimer at pH 4.
3
.5. Effect of generation of PAMAM on solubility
of nicotinic acid
The effect of various generations of PAMAM dendrimers
(G1–G4) on the process was investigated at pH 3, 4 and 6,
respectively. The results are shown in Figs. 6–9, from which
it is clear that the solubility of nicotinic acid was affected by
the generation of PAMAM dendrimer. The solubility of nico-
tinic acid in higher generation PAMAM solution was in fact
higher that those in lower ones. The solubility of hydropho-
bic compounds in dendrimer solutions likely depends on the
dendrimer generation (size) [15]. Since the number of pri-
mary and tertiary amines in the dendrimer increases with gen-
eration size, at a given pH condition, higher generation den-
drimer has a tendency to entrap more hydrophobic compound
inside than lower ones. Also, the solubility of nicotinic acid
in PAMAM solutions depends on the surface area and pri-
mary amino groups of PAMAM particles, which cause the
higher generation PAMAM particles to have a higher ability
3.4. Effect of surface functional group of PAMAM on
solubility of nicotinic acid
The effect of surface functional group of PAMAM den-
drimers (amine-terminated and ester-terminated) on the pro-
cess was investigated at pH 4. The G3 PAMAM dendrimer,
used in the present paper, possesses 16 amino groups on a
surface whereas PAMAM G2.5 dendrimers possess 16 car-
boxylate groups on the surface. The results are shown in Fig. 5.
In contrast to the amine-terminated full generation dendrim-
ers, in the presence of ester-terminated G2.5 PAMAM den-
drimers, the aqueous solubility of nicotinic acid also increased
linearly with dendrimer concentration at pH 4. It is clear that
the solubility of nicotinic acid was affected by the surface
functional group of PAMAM dendrimer. The solubility of
nicotinic acid in amine-terminated dendrimer solution was
obviously higher that those in ester-terminated ones. This is
because that the solubility of nicotinic acid in amine-
terminated PAMAM solutions depends on the primary amines
on the surface and cavities inside the PAMAM particles, while
the solubility in ester-terminated PAMAM solutions depends
on cavities inside the PAMAM particles. Thus the amine-
terminated PAMAM dendrimers have a higher ability to
enhance the solubility of nicotinic than ester-terminated
PAMAM dendrimers.
Fig. 6
. Solubility of nicotinic acid in the presence of increasing concentra-
tion of G1 PAMAM dendrimer.

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C. Yiyun, X. Tongwen / European Journal of Medicinal Chemistry 40 (2005) 1384–1389
to absorb and interact with the nicotinic acid molecule. In
this way, we could explain why higher generation dendrim-
ers could enhance the solubility of nicotinic acid more effi-
ciently than lower ones.
4
. Conclusion
Different generation (G1–G4) PAMAM dendrimers have
the potential to significantly enhance the solubility of nico-
tinic acid. The drug solubility depends on the concentration
of the dendrimer, the pH value of the solution, and surface
functional group and the generation of the dendrimer. Solu-
bility of nicotinic acid in the dendrimer solutions increase in
an approximately linear manner with an increase in den-
drimer concentration; Both amine and ester-terminated den-
drimers cause the higher increase in nicotinic acid solubility
at higher pH conditions; The order in which the dendrimers
increased the solubility at a constant pH condition is
G4 > G3 > G2> G1; In addition, at each pH, the solubility of
nicotinic acid is greater in the presence of amine-terminated
dendrimers compared to the amine ester-terminated dendrim-
ers possessing the same number of surface functional groups.
Both observations are evidence of the solubility enhance-
ment of nicotinic acid is due to an electrostatically interac-
tion between the surface amine groups of dendrimer mol-
ecule and the carboxyl group of nicotinic acid and to hydrogen
bond formations between tertiary amines in internal cavities
of dendrimers and the atoms of nicotinic acid.
Fig. 7
. Solubility of nicotinic acid in the presence of increasing concentra-
tion of G2 PAMAM dendrimer.
Acknowledgements
Financial supports from Innovation Foundation of Gradu-
ate Student in University of Science and Technology of China
(KD2004035) were highly appreciated.
Fig. 8
. Solubility of nicotinic acid in the presence of increasing concentra-
tion of G4 PAMAM dendrimer.
References
[
[
[
[
1] D.A. Tomalia, H. Baker, J. Dewald, A new class of polymers:
starburst-dendritic macromolecules, Polym. J. 17 (1985) 117.
2] D.A. Tomalia, H. Baker, J.R. Dewald, Dendritic molecules: synthesis
of starburst dendrimer, Macromolecules 19 (1986) 2466.
3] D.A. Tomalia, J.R. Dewald, (1986) Dense star polymers having two-
dimensional molecular diameter. U.S. Patent 4587329.
4] D.A. Tomalia, A.M. Naylor, W.A. Goddard, Starburst dendrimers:
molecular-level control of size, shape, surface chemistry, topology,
and flexibility from atoms to macroscopic matter, Angew. Chem. Int.
Ed. Engl. 29 (1990) 138.
[
[
5] E. Roseita, D.A. Tomalia, Poly (amidoamine) (PAMAM) dendrimers:
from biomimicry to drug delivery and biomedical applications, Drug
Delivery Today 6 (2001) 427–436.
6] O. Schiavon, G. Pasut, S. Moro, PEG–Ara–C conjugates for con-
trolled release, Eur. J. Med. Chem. 39 (2004) 123–133.
Fig. 9
. Solubility of nicotinic acid in the presence of increasing concentra-
tion of G1, G2, G3 and G4 PAMAM dendrimer at pH 4.
[7] M.F. Brana, G. Dominguez, B. Saez, Synthesis and anti-tumor activity
of new dendritic polyamines–(imide–DNA–intercalator) conjugates:
potent Lck inhibitors, Eur. J. Med. Chem. 37 (2002) 541–551.

C. Yiyun, X. Tongwen / European Journal of Medicinal Chemistry 40 (2005) 1384–1389
1389
[
[
[
8] P. Furuta, J.M. Frechet, Controlling solubility and modulating periph-
eral function in dendrimer encapsulated dyes, J. Am. Chem. Soc. 125
[12] W. Chen, D.A. Tomalia, L.J. Thomas, Unusual pH-dependent polarity
changes in PAMAM dendrimers: Evidence for pH-responsive confor-
mational changes, Macromolecules 33 (2000) 9169–9172.
(
2003) 13173–13181.
9] G.R. Newkome, C.N. Moorefield, G.R. Baker, Alkane cascade poly-
mers processing micellar topology: micellanoic acid derivatives,
Angew. Chem. Int. Ed. Engl. 30 (1991) 1178–1180.
10] C.T. Supuran, A. Scozzafava, L. Menabuoni, Carbonic anhydrase
inhibitors—part 70. Synthesis and ocular pharmacology of a new
class of water-soluble, topically effective intraocular pressure lower-
ing agents derived from nicotinic acid and aromatic/heterocyclic sul-
fonamides, Eur. J. Med. Chem. 34 (1999) 799–808.
[
13] S. Uppuluri, F.A. Morrison, P.R. Dvornic, Rheology of dendrimers. 2.
Bulk polyamidoamine dendrimers under steady shear, creep, and
dynamic oscillatory shear, Macromolecules 33 (2000) 2551–2560.
[
14] G. Postolis, A. Milliaris, D. Tsiourvas, Polyamidoamine dendrimers
as pH-sensitive controlled release systems, Chem. Eur. J. 5 (1999)
1
440–1445.
[15] K. Chie, K. Kenji, M. Kazuo, Synthesis of polyamidoamine dendrim-
ers having poly (ethylene glycol) grafts and their ability to encapsulate
anticancer drugs, Bioconjugate Chem. 17 (2000) 910–917.
[
11] A.M. Naylor, I.I.I. Goddard, D.A. Tomalia, Starburst dendrimers 5:
molecular shape control, J. Am. Chem. Soc. 111 (1989) 2339–2341.