Biodegradable and biocompatible spherical dendrimer nanoparticles with a gallic acid shell and a double-acting strong antioxidant activity as potential device to fight diseases from “oxidative stress”

Article abstract of DOI:10.1007/s13346-019-00681-8

Gallic acid (GA) is a natural polyphenol with remarkable antioxidant power present in several vegetables and fruits. A normal feeding regime leads to a daily intake of GA which is reasonably regarded as “natural” and “safe” for humans. It owns strong pote

Full text of DOI:10.1007/s13346-019-00681-8

Drug Delivery and Translational Research
https://doi.org/10.1007/s13346-019-00681-8
ORIGINAL ARTICLE
Biodegradable and biocompatible spherical dendrimer nanoparticles
with a gallic acid shell and a double-acting strong antioxidant
activity as potential device to fight diseases from Boxidative stress^
Silvana Alfei1 & Silvia Catena1 & Federica Turrini1
#
Controlled Release Society 2019
Abstract
Gallic acid (GA) is a natural polyphenol with remarkable antioxidant power present in several vegetables and fruits. A normal feeding
regime leads to a daily intake of GA which is reasonably regarded as Bnatural^ and Bsafe^ for humans. It owns strong potentials as
alternative to traditional drugs to treat several diseases triggered by oxidative stress (OS), but poor gastrointestinal absorbability,
pharmacokinetic drawbacks, and fast metabolism limit its clinical application. In this work, a fifth-generation polyester-based dendri-
mer was firstly prepared as a better absorbable carrier to protect and deliver GA. Then, by its peripheral esterification with GA units, a
GA-enriched delivering system (GAD) with remarkable antioxidant power and high potential against diseases from OS was achieved.
Scanning electron microscopy results and dynamic light scattering analysis revealed particles with an average size around 387 and 375
nm, respectively, and an extraordinarily spherical morphology. These properties, by determining a large particles surface area, typically
favour higher systemic residence time and bio-efficiency. Z-potential of − 25 mV suggests satisfactory stability in solution with
tendency to form megamers and low polydispersity index. GAD showed intrinsic antioxidant power, higher than GA by 4 times and
like prodrugs, and it can carry contemporary several bioactive GA units versus cells. In physiological condition, the action of pig liver
esterase (PLE), selected as a model of cells esterase, hydrolyses GAD to non-cytotoxic small molecules, thus setting free the bioactive
GA units, for further antioxidant effects. Cytotoxicity studies performed on two cell lines demonstrated a high cell viability.
.
.
.
.
Keywords Gallic acid Biocompatible dendrimer particles Gallic acid delivery device Radical scavenging activity Pig liver
.
esterase Spherical nanoparticles
Introduction
human disorders, including cardiovascular malfunction, cata-
racts, cancer, rheumatism, and many other auto-immune and
neurodegenerative diseases and ageing [1].
A number of synthetic drugs may provide protection against
the deleterious effects of OS, but these are also associated with
adverse side effects [1]. Phytochemicals present in unprocessed
or minimally processed plant foods, in addition to the basic
nutritional value, own extra health benefits and can be consid-
ered Bpharmaceutical-grade nutrients^.
In particular, polyphenols, such as hydroxyl benzoic acids
(HBAs), form an important class of naturally occurring bioac-
tive entities, having innumerable biological activities, all at-
tributable to their remarkable antioxidant power. HBAs pos-
sess the ability to scavenge reactive species such as superox-
ide radicals and hydroxyl radicals, to reduce lipid peroxyl
radicals and to inhibit lipid peroxidation, thus protecting cells
from OS via a number of pathways [1, 2]. Among HBAs,
gallic acid (3, 4, 5-trihydroxybenzoic acid) 1 (Fig. 1) is a
Oxidative stress (OS) is caused by the accumulation of free
oxygen and nitrogen radical species (ROS and RNS) in the
cells and is considered the key triggering factor for several
Silvia Catena and Federica Turrini contributed by determining the RSA%
by DPPH test.
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s13346-019-00681-8) contains supplementary
material, which is available to authorized users.
* Silvana Alfei
alfei@difar.unige.it
1
Dipartimento di Farmacia, Sezione di Chimica e Tecnologie
Farmaceutiche e Alimentari, Università di Genova, Viale Cembrano
4, I-16148 Genova, Italy

Drug Deliv. and Transl. Res.
In order to not waste the GA strong potentialities for
human’s wellness, researcher efforts are increasingly fo-
cused on implementing strategies to overcome GA phar-
macokinetics limitations [10, 14, 15].
Nanoparticles [16–19] are widely investigated to prepare
multifunctional platforms [20] and are extensively adopted in
several biomedical applications [21–29]. Among nanoparti-
cles, dendrimers are highly branched and symmetric macro-
molecules, characterised by a monodisperse tree-like struc-
ture, with both internal cavities for guest molecule entrapment
and many peripheral groups that allow further functionalisa-
tion. Differently from traditional polymers, they have an un-
usually low intrinsic viscosity that consents their easy trans-
port in the blood [30, 31] and are characterised by a very low
PDI < 1.1 or are even monodispersed [32]. These nonpareil
properties make dendrimers ideal carriers both for gene and
drug delivery (DD). In DD, they allow to control molecular
weight, hydrophilicity, solubility [33–35], bioavailability, and
pharmacokinetic behaviour of transported drugs.
Fig. 1 Structure of Gallic acid (GA) 1
low molecular weight, tri-phenolic acid, extensively distribut-
ed in many different families of plants, and contained in com-
mon foodstuffs.
It can be found both in the free state and as a part of more
complex molecules, i.e. as aglycone, bound to its food matrix,
as ester derivatives or in polymeric forms known as
gallotannins (GTs). After food consumption or its oral admin-
istration as pure compound, GA is slowly absorbed in gastro-
intestinal tract (GIT) and, after fast metabolism, it is excreted
in the urine as 4-O-methylgallic acid. Its ester derivatives,
such as catechin gallate esters and GTs, are firstly hydrolysed
to GA and then metabolised to methylated derivatives [3]. GA
is an efficient apoptosis inducing agent that, thanks to a fine
amalgam between its antioxidant and pro-oxidant power, fits a
large range of applications both clinical, and industrial. GA is
able to exert strong antioxidant activity also in emulsion or
lipid systems [4, 5] and is used in processed foods, cosmetics,
and food packing materials to prevent rancidity induced by
lipid peroxidation and food spoilage. GA is also a potent
inhibitor of iron and NADPH-dependent microsomal lipid
peroxidation but is ineffective against xenobiotic-induced
lipids oxidation [6]. It showed neuroprotective actions in
different animal models of neurodegeneration, neurotoxici-
ty, and OS as well as anti-mutagenic and anti-carcinogenic
activity [7–9].
Unfortunately, the therapeutic application of GA is
limited by its pharmacokinetic drawbacks and oral poor
bioavailability [10, 11]. Other phenols, such as ferulic
acid or p-coumaric acid, are adsorbed by the monocar-
boxylic acid transporter (MCT), whereas GA is not. The
serum pharmacokinetic profile obtained from studies in
rats showed that GA is slowly absorbed, with a tmax for
intact GA of 60 min and a max concentration (Cmax) of
0.71 μmol/L [12], value irrelevant to provide a thera-
peutically valid antioxidant effect.
Dendrimers offer the possibility to anchor active directing
agents, allowing cell-targeted therapies [36–38]. Dendrimers
are also capable of establishing strong interactions with sev-
eral drugs, thus facilitating drug loading and limiting systemic
toxicity by minimising the initial massive drug release in
parenteral administration [33–35].
Well-known poly(amidoamine) dendrimers (PAMAMs)
have been and are unceasingly under study both for drug
and gene delivery applications [39, 40]. Unfortunately, due
to their amine-terminated, not biodegradable scaffold and to
the excessive positive charge, spread across the whole surface,
PAMAMs frequently interact, non-specifically with negative
charges of biological membranes. As direct consequence, sev-
eral problems occur, such as cytotoxicity, haemolytic toxicity,
and fast clearance from the blood circulation, as well as high
level of uptake in the reticuloendothelial system. For these
reasons PAMAMs, if not properly modified, are not advisable
for clinical applications [41]. Uncharged dendrimer matrices,
decorated with protonable amino acid residues [42–45], rep-
resent a better and less toxic alternative [46–49]. Within this
category, polyester dendrimer scaffolds are the most attractive,
because of their good biodegradability [32, 50, 51].
Against this background, the first scope of this work was to
develop a successful strategy, resorting to dendrimer nanopar-
ticles, for ameliorating GA Lipophilic Hydrophilic Balance
(LHB) and gastrointestinal absorbability, for limiting its phar-
macokinetic drawbacks and for opposing its fast metabolism
and pro-oxidant counterpart. In this regard, a fifth-generation
polyester-based dendrimer, built on the di-functional core 1,
3-propandiol (4) was prepared. The so obtained dendrimer 4,
thanks to its sixty-four OH peripheral groups (4) exploitable
for binding GA, was used as a permeability enhancer carrier
[52] for promoting a more efficient GA administration.
Therefore, 4 was peripherally esterified with bioactive GA
Furthermore, GA undergoes a very fast metabolism
that translates in an insignificant drug-life. Its major
metabolites, i.e. 4-O-methylgallic acid and pyrogallol,
are in turn promptly metabolised to other molecules that, both
possess less antioxidant activity and are speedily excreted in
the urine [13].

Drug Deliv. and Transl. Res.
units, achieving a biodegradable GA-dendrimer prodrug
(GAD) 9, endowed with high molecular weight (MW), un-
common particles size of about 380 nm, particles morphology
amazingly spherical and consequently large surface area, that
typically increase the drug delivery systems (DDSs) systemic
retention time, favouring higher bio-efficiency [52]. The sec-
ond scope of the present study was, to perform significant
Bpreliminary tests^, to assess whether GAD could have any
potentiality to be subsequently taken into consideration for
more in-depth and specific evaluations concerning its actual
activity against disorders triggered by OS. In this regard, GAD
was firstly investigated to evaluate its antioxidant power, a key
and mandatory property to be able to hope for possible thera-
peutic properties against diseases caused by OS. Then, addi-
tional investigations, to evaluate its behaviour in an in vitro
simulation of the physiological conditions inside the cell,
where esterase hydrolytic attacks may occur, were also per-
formed, with exciting results. Finally, cytotoxicity studies on
two opportunely selected cell lines were performed to exclude
any damage to the cells by exposure to GAD.
carried out using a Varian 3400 gas chromatograph con-
nected to a Finnigan MAT SSQ710A mass spectrometer.
Chromatographic separation was performed with a RTX-
5MS (Restek Corporation) capillary column (30 m ×
0.25 mm i.d., 0.25-μm film thickness). Scanning electron mi-
croscopy (SEM) images were obtained with a Leo Stereoscan
440 instrument (LEO Electron Microscopy Ltd). Thin layer
chromatography (TLC) system employed aluminium-backed
silica gel plates (Merck DC-Alufolien Kieselgel 60 F254) and
detection of spots was made by UV light. Elemental analyses
were determined by an EA1110 Elemental Analyser
(Fison Instruments). Organic solutions were dried over
anhydrous magnesium sulphate and were evaporated using a
rotatory evaporator operating at reduced pressure of about
10–20 mmHg.
Synthesis of compounds 3-9
Compounds 3-9 were prepared according to the synthetic
paths described in Schemes 1 and 2. Experimental details
were reported in Table S1 in Electronic Supplementary
Material (ESM) (Section S1). Synthetic and purification pro-
cedures are available in Sections S1-S3 in ESM. An additional
table (Table 1), reporting the yield (%) of all the intermediates,
except for not isolated compound 8, and of final product GA-
dendrimer (9), is available in the BResults and discussion^
section.
Materials and methods
Materials and Instruments
All the reagents including pig liver esterase (carboxylic-ester
hydrolase) and solvents were purchased from Merck (e.g.
Sigma-Aldrich). Reagents were used without further purifica-
tions, while solvents were dried and purified by distillation
according to standard procedures. Petroleum ether refers to
the fraction with boiling point 40–60 °C. Dendrons D4BnA,
D4BnOH, D5BnA, and 2 were prepared as previously report-
ed [53–55]. Melting points were determined on Mettler
Toledo MP50 Melting Point System and are uncorrected.
FT-IR spectra were recorded as films or KBr pellets on a
Physicochemical characterisation of compounds 3-9
Spectroscopic and Elemental analysis data
FT-IR, NMR, and Elemental analysis data are available
in ESM (Section S4). Copies of FT-IR and NMR spec-
tra of compounds 3-8 and of GA-dendrimer (9)
(Figure S7.1-S7.21) are available in Section S7 of ESM.
1
Perkin Elmer System 2000 spectrophotometer. H and 13C
NMR spectra were acquired on a Bruker Avance DPX 300
Spectrometer at 300 and 75.5 MHz, respectively, and
assigned through DEPT-135 and decoupling experiments.
Coupling constant values were given in Hertz. Fully
decoupled 13C NMR spectra were reported. Chemical shifts
were reported in δ (parts per million) units relative to the
internal standard tetramethylsilane (δ = 0.00 ppm) and the
splitting patterns were described as follows: s (singlet), d (dou-
blet), t (triplet), q (quartet), m (multiplet) and br (broad signal).
Centrifugations were performed on an ALC 4236-V1D
Centrifuge at 3400–3500 rpm. Freeze-drying was performed
on an EDWARDS Super Modulyo Freeze Dryer, Ice capacity
8 kg, 8 kg/24 h, refrigeration down to − 55 °C with 24-place
Drum Manifold. Dynamic light scattering (DLS) was per-
formed on a Malvern Zetasizer Nano ZS instrument
(Southborough, MA). Gas chromatography analysis was
Morphology, size, and Z-potential of GAD (9) particles
The morphology and size of GAD particles were firstly inves-
tigated by scanning electron microscopy (SEM). In addition,
the hydrodynamic size (diameter) and Z-potential (mV) of
GA-dendrimer (9) particles were also determined by dynamic
light scattering (DLS) analysis for further confirmation of
SEM results.
Scanning electron microscopy The sample was fixed on alu-
minium pin stubs and sputter-coated with a gold layer (30 mA
for 1 min) and was examined at an accelerating voltage of
20 kV. The micrographs were recorded digitally using
DISS 5 digital image acquisition system (Point Electronic
GmbH, Halle, Germany) and the average size of particles
was provided by the instrument.

Drug Deliv. and Transl. Res.
Scheme 1 Synthetic procedure
for preparing GA-dendrimer 9
Dynamic light scattering The hydrodynamic size (diameter) of
GAD was measured in batch mode at the physiological tem-
perature of 37 °C in a low volume quartz cuvette (path length
10 mm) using a back scattering detector (173°, 633-nm laser
wavelength). The dendrimer sample was prepared at a con-
centration of 1.0 mg/mL in in PBS and filtered through a
0.02-μm filter. A minimum of 12 measurements were per-
formed. Z average diameter (Z-AVE), derived from a
cumulants analysis of the measured correlation curve, was
reported as the intensity-weighted average (Int-Peak) hydro-
dynamic radius. The Z-potential was measured at 37 °C in
PBS as medium and an applied voltage of 100 V was used.
The GAD sample was loaded into pre-rinsed folded capillary
cells and twelve measurements were performed.
determined by performing the DPPH• (2, 2-diphenyl-1-
pycrylhydrazyl) assay. For comparison, commercially avail-
able samples of GA, ascorbic acid (AA) and Trolox, indicated
as standards, were similarly essayed.
Determination of 1, 1-diphenyl-2-picryl hydrazyl radical
scavenging activity of GA-dendrimer (9), GA, AA, and Trolox
The DPPH• (2, 2-diphenyl-1-pycrylhydrazyl) assay [56] was
performed, to determine the radical scavenging activity (RSA)
of GA-dendrimer (9), GA, AA, and Trolox. Briefly, aliquots
(0.250 mL) of the water solutions of compounds under study
were transferred into a 10-mL volumetric flask and a daily
prepared DPPH mother solution (approximately 10−4 M in
methanol) was added. Depending on the compounds solubil-
ity, the samples solutions were prepared in the range of con-
centration of 340–6760 μg/mL (GAD), 35–6760 μg/mL
(AA), 25–6760 μg/mL (Trolox), and 17–6760 μg/mL (GA).
The reaction flask was kept in the dark for 30 min, and the
GAD antioxidant activity in vitro investigation
In order to evaluate the antioxidant power of GA-dendrimer
(9), its radical scavenging activity (RSA %) has been
Scheme 2 Synthetic three steps
procedure for preparing acid
chloride derivative of GA 7, via
compounds 5 and 6

Drug Deliv. and Transl. Res.
Table 1 Yield (%) of each
reaction step performed, MW and
physical state of compounds 3-9
Entry
Product
Yield
(%)
MW
Physical state
(g; mmol)
(g; mmol; %)
2
3
98.0
8557.28a
Glassy
2.64; 0.600
2.34; 0.27
Off-white solid
3
4
99.0
7275.24a
626.37
Fluffy white hygroscopic solid
2.34; 0.270
1
1.94; 0.267
5
1.35b; 2.15b
6
91.9b
99.0
Viscous resin
398.7; 2.34
5
512.90
White crystals
1.20; 1.91
0.94; 1.89
6
7
100.0
95.5c
97.6
531.35
Pale yellow waxy solid
Not isolated
0.93; 1.81
4
0.96; 1.81
8
0.82c; 0.02c
9
Not isolated
17,010.02a
159.3; 0.022
8
Brownish glassy hygroscopic solid
1.00; 0.0256
0.43; 0.025
a Estimated by 1 H NMR spectra and confirmed by Elemental analysis
b Weight, mmol, and yield have been calculated considering the impurities
c Not purified product
residual absorbance (A) was measured by an Agilent 8453
UV-VIS spectrophotometer at 515 nm, at 25 °C. For each
sample, A was measured at different sample concentrations
within the ranges reported above. Blanks (solution without
samples) were measured before each sample and subtracted.
The results were expressed as a percentage (%) of the DPPH
free radical scavenging activity, calculated with the following
Eq. (1):
(50 mg). The mixture was stirred at physiological tem-
perature (37 °C) for 24 h. The suspension was then
filtered, through Celite plug, and the filtrate was extract-
ed with ethyl acetate. The organic layer was dried over
MgSO4, filtered, and evaporated, to leave a crude prod-
uct named 10, as a brownish residue (0.0878 g). Firstly,
10 was qualitatively investigated (Section S6 in ESM),
in order to define its composition and to identify its
components. After having established by qualitative
analysis on crude 10 that the main detectable compo-
nent was GA (Section S6 in ESM), a recrystallisation
from ethanol was performed, obtaining white crystals, to
which the name 10c was assigned (0.0632 g, 0.3715
mmol). 10c was analysed by NMR spectroscopy and
elemental analysis, that confirmed the structure of GA
and a very good level of purity. Detailed characterisation data
and copies of 1H and 13C NMR of 10c are available in ESM
(Section S6).
½AðblankÞ−Aðsample or standardÞ ꢀ 100ꢁ
Scavenging activityð%Þ ¼
AðblankÞ
ð1Þ
where A (blank) is the absorbance of DPPH radicals without
sample or standard and A (sample or standard) is the absor-
bance of DPPH radicals with sample or standard, i.e. commer-
cially available compounds. All the analytical determinations
were developed in three replicated, and standard deviation
( SD) was calculated in order to assess the repeatability of the
method.
Enzymatic hydrolysis kinetic quantification by gas
chromatography-mass spectrometry
Evaluation of biodegradability of GAD by performing
an in vitro simulation of hydrolysis by cell esterase
Procedure A sample of GA-dendrimer (9) was subjected to
enzymatic hydrolysis by performing the same procedure de-
scribed above, taking aliquots of hydrolysate (1 mL) at
different time points.
Procedure for enzymatic hydrolysis of GA-dendrimer (9)
by pig liver esterase
Each aliquot was extracted with ethyl acetate and rapidly
dried over MgSO4. The solvent was separated and removed
and the residue was incubated with 250 μL of 1:1 N, O-
bis(trimethylsilyl)trifluoroacetamide (BTSFA)/anhydrous
pyridine at 70 °C.
GA-dendrimer 9 (0.100 g, 0.0058 mmol) was dissolved in
acetone (10 mL), then PBS phosphate buffer (pH = 7.4, 20
mL) was added. The solution was treated with pig liver ester-
ase (PLE EC 3.1.1.1) lyophilised powder, ≥ 15 units/mg solid

Drug Deliv. and Transl. Res.
Instrumental conditions Helium flow 39 ml/min; injector tem-
perature 260 °C; transfer line temperature 280 °C; energy of
electron 70 eV; oven temperature 90 °C for 1 min, from 90 to
240 °C at ramp rate of 20 °C/min, 240 °C for 10 min, from 240
to 280 °C at ramp rate of 20 °C/min, 280 °C for 1 min; split
less injection mode was used to introduce 1 μL of sample. All
experiments were done in duplicate repeating the analysis if
the error between duplicate samples was greater than 5%.
Statistical analyses
Data are expressed as means SD. Statistical significance of
differences was determined by one-way analysis of variances
(ANOVA). P < 0.05 was considered statistically significant.
Results and discussion
General considerations
In vitro GAD cytotoxicity evaluation by cell viability
(%) determination
As far as the planning of 4 is concerned, the choice of a high
MW polyester-based dendrimer architecture and of the specif-
ic fifth generation has been pivoted on several considerations,
dictated by the first aim of the present work, i.e. to improve
GA stability, LHB, and gastrointestinal absorbability, to slow
down its fast metabolism and to decrease its pro-oxidant
counterpart.
Dendrimer architecture can both enhance oral drugs ab-
sorption and increase their blood residence time through three
modality.
Cell culture
Epithelial (fibrosarcoma) B14 cells (Cricetulus griseus, ATCC,
CCL-14.1, Sigma-Aldrich Inc.) were grown in high-glucose
Dulbecco’s modified Eagle medium with 10 % (v/v) fetal bo-
vine serum (FBS) and 4 mM glutamine; liver BRL 3A cells
(Rattus norvegicus, ATCC, CRL-1442, Sigma-Aldrich Inc.)
with fibroblast-like morphology, were grown in Ham’s F12
medium with 10% (v/v) FBS and 2 mM glutamine. All media
were supplemented with 0.1% (w/v) penicillin and 0.1% (w/v)
streptomycin. The cells were maintained in culture flasks in a
37 °C humidified atmosphere of 5% CO2/95 % air (incubator)
and passaged every 2–3 days. Cells were harvested and used in
experiments after obtaining 80–90% confluence. The number
of viable cells was determined by trypan blue exclusion with a
haemo-cytometer. Then cells were suspended in media at a
concentration of 1.0 × 10−5 cells per mL and plated in flat-
bottom 96-well plates. Plates with cells were incubated for
24 h at 37 °C in a humidified atmosphere of 5% CO2 to allow
adherence of the cells before the administration of dendrimer.
(i). Acting as Bexcipients^ or permeability enhancers,
dendrimers alter the barrier function of the intestinal ep-
ithelium and thereby enhance the permeability of a co-
administered drug [52].
(ii). The dendrimer–drug complex may itself be better
transported across the intestinal epithelium.
(iii). Big constructs with large surface and high molecular
weight are typically retained in the circulation for lon-
ger periods and provide enhanced opportunity for act-
ing to drugs [52].
The polyester-based scaffold, differently from well-work-
ing, but not biodegradable strongly cationic commercial
PAMAMs, is designed to hydrolyse under physiological con-
ditions thus resulting not toxic to cells [32, 50, 51].
Preliminary calculations were made, to determine the lipo-
philic hydrophilic balance (LHB) [57] of the GA-dendrimers
obtainable in function of the generation of dendrimer carrier
adopted. The fifth-generation architecture of 4 would have
allowed achieving a GA-enriched dendrimer with a little low-
er LHB value than GA and with a slightly more hydrophobic
character thus favouring its absorbability in GIT. On the con-
trary, the not too much low LHB value would permit GAD
water solubility.
3-(4, 5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay
Cytotoxicity of GAD 9 was assayed with MTT. After 24-h
incubation when cells attached on 96-well plates, they were
treated with GAD at a concentration from 0.5 to 20 μmol/L
(dimethylsulphoxide, DMSO). After 24-h incubation, a 20-μL
solution of MTT in phosphate-buffered saline (5 mg/mL) was
added to each well. Four hours later the medium was removed
and the formazan precipitate was dissolved in DMSO for ab-
sorbance measurement at 580 nm and reference at 700 nm.
The cell viability was related to the control wells, containing
untreated cells with fresh cell culture medium and was calcu-
lated according to the following Eq. (2):
The several peripheral OH functions of 4 could be
exploited for further esterification reactions and to bind a con-
siderable number of bioactive GA units. In addition, by the
esterification of 4 with GA, two goals would be rationally
achieved at once.
Absorption test
Absorption control
Cell viabilityð%Þ ¼
ꢀ 100
ð2Þ
The results were presented as the mean of three measure-
ments ( SD).
The synthesis of a biodegradable dendrimer in possession
of intrinsic antioxidant activity thanks to the numerous

Drug Deliv. and Transl. Res.
peripheral units of GA and the 192 phenol groups, potential-
ities as semi-natural alternative device to treat diseases caused
by the OS.
The preparation of a GA’s prodrug, i.e. a GA’s reservoir in
possession of the nonpareil characteristics of dendrimers
known as excellent materials for drug delivery and biomedical
applications [21–29].
GA-enriched dendrimer 9 could have allowed the simulta-
neous transport of many GA moieties towards cells and, once
inside the cells, thanks to its polyester-based structure, it
would been degraded by the hydrolytic action of the cellular
esterase, releasing GA for further antioxidant action.
HMPA) as building block [58]. Firstly, the fourth-generation
dendron homologous of 2 both benzyl and acetonide protected
to the carboxylic group and to the periphery respectively and
named D4BnA (Fig. S1 in ESM) was prepared [53]. Then, its
sixteen peripheral hydroxyls were set free by acidic treatment,
to obtain the compound named D4BnOH (Fig. S1 in ESM).
D4BnOH was increased by one generation, obtaining the fifth-
generation dendron D5BnA (Fig. S1 in ESM), that, after cata-
lytic hydrogenation to set free the carboxylic group, provided 2
as off-white foamy solid [54].
Both 3 and 4 were obtained in high yield and were
characterised by FT-IR, NMR, and elemental analysis, which
confirmed both the structure and the good level of purity.
Since GA 1 is a poly-functional substance and represents
an AB2 type monomer itself, before esterification of 4, some
precautions have been taken, in order to avoid undesired and
uncontrolled GA polymerisation.
Firstly, GA 1 was protected both to the three hydroxyls and
the carboxyl group, with tert-butyldimethylsilyl chloride
(TBDMSCl) obtaining 5 [59]. The derivative 5 was then trans-
formed in 6 by selective removal of the TBDMS protecting
group from carboxyl [59] and finally 6 was transformed in the
chloride acid derivative 7 (Scheme 2).
An attempt to obtain the derivative 6 in a single step [60]
provided a mixture of 6 and 5 that would have required chro-
matographic separation, so that the above described, longest
but most convenient procedure, was preferred.
The conversion of 6 in 7 was realised implementing the
common procedure that uses SOCl2 with small amounts of
DMF as catalyst. Thanks to the in situ formation of a reactive
chloroiminium intermediate (Vilsmeier–Haack reagent),
which is ideal as a halogenating reagent [61–64], the complete
substitution of OH with chlorine atom was achieved in less
than 2 h. The structure of all the compounds was confirmed by
FT-IR, NMR, and elemental analysis. NMR spectra of 5
showed the presence of two impurities, not previously
highlighted [59]. These pollutants, identified as 1, 3-di-tert--
butyl-1, 1, 3, 3-tetramethyl-disiloxane, (5.6%) and 1, 2-di-
tert-butyl-1, 1, 2, 2-tetramethyldisilane (6.0%) were easily
removed in the next step during crystallisation of com-
pound 6. Elemental analysis was not influenced by the
presence in traces of these impurities. The last two reaction
steps (Scheme 1) allowed achieving the GA-decorated dendri-
mer 9. The intermediate TBDMS-GA-dendrimer 8 was
analysed by FT-IR (Fig. S7.18 in ESM), and all the spectrum
signals were consistent with the desired structure and gave
proof of the successful coupling between 4 and 7. Then, since
an investigation by TLC of crude 8 showed only trivial traces
of unreacted 7, it was subjected to TBDMS removal reaction,
without further purification, postponing it to the next final
step. As reaction system, acidic conditions by anhydrous hy-
drochloric acid generated in situ from acetyl chloride and al-
cohols was selected. The reported procedure [65], that avoids
Preparation of GA-enriched dendrimer (9)
On the base of the common practice of making use of nano-
particles to ameliorate solubility, GIT absorbability and me-
tabolism profile of chemical bioactive entities with pharmaco-
kinetics drawbacks, the fifth-generation polyester-based den-
drimer 4 was prepared, and then, it was peripherally decorated
with sixty-four GA units achieving the GA-enriched
dendrimer 9 (Scheme 1).
Dendrimer 4 was synthetised by esterification of the diol
core, i.e. 1, 3-propandiol, with the fifth generation dendron 2
(Fig. 2), owing a free carboxyl group, to achieve acetonide
protected dendrimer 3.
Subsequently, intermediate dendrimer 3 was exposed to
acidic condition by treatment with Dowex resins to give 4
(Scheme 1). Table S1 (Section S1 in ESM) reports essential
experimental data of all reaction performed in the present
work. Table 1 summarises the percentage yield of each reac-
tion step performed, together with the MW and physical state
of compounds 3-9.
Dendron 2 was in turn prepared via convergent, double ex-
ponential, and divergent growth approaches, using the AB2
type monomer 2, 2'-bis(hydroxymethyl)propionic acid (bis-
Fig. 2 Structure of the fifth generation dendron 2

Drug Deliv. and Transl. Res.
Table 2 GAD particle hydrodynamic size (DLS) and Z-potential at
37 °C ( SD), average particles size by SEM analysis and cell
viability values ( SD) from cytotoxicity essay at GAD concentration of
340 μg/mL
water, would be respectful of the polyester architecture of 8
and, even if slightly modified, has been already extensively
exploited by the authors in previous works [58, 66, 67] to
remove BOC protecting group from similarly structured
polyester-based dendrimers. Differently from the reported
procedure [65], the reaction was developed using a strong
excess of acetyl chloride [58, 66, 67]. The complete
deprotection was achieved within 5 h at r.t. After purifying
procedures developed to remove the residual unbound GA,
GA-dendrimer (9) was obtained as brownish glassy hygro-
scopic solid that, in the FeCl3 test for the identification of
phenols, gave a green colouring (Fig. S3.1 in ESM).
Otherwise, from free GA, that in such test gives dark blue or
black colouring (Fig. S6.1, ESM), green colouring is charac-
teristic of high molecular weight GA esters as Gallotannins
[68], thus indicating the absence of unassociated GA and the
good outcome of purification procedure. Structure of GA-
dendrimer (9) was further confirmed by FT-IR, Elemental
analysis and NMR. In particular, as a consequence of esterifi-
cation, changes in the 1H NMR spectrum of 4 were observed
around 4.00 ppm, but the GA presence was definitively
asserted by a very intense peak concerning the CH = proton
atoms of the GA aromatic ring at 7.30 ppm.
Z-AVE size Average particles Z-potential Cell viability
(nm)a
size (nm) by
(mV)a
(%)b
SEM analysis
375 7.9
387 11
− 25 0.34 B14
BRL
91.4 3.5 85.3 1.1
a N (degree of freedom) = 12
b N (degree of freedom) = 3
several poly hydroxylated GA units that can establish many
hydrogen bonds between the dendrimer molecules, giving rise
to dendrimers aggregates. Dendrimer particles, in SEM image,
appear amazingly spherical and consequently gifted with a
very large surface area that typically increases the DDSs sys-
temic retention time and their bio-efficiency [52]. For further
confirmation of SEM results, the particle hydrodynamic size
(diameter) of GAD was measured using DLS analysis and the
result matched the SEM ones with a very small error of 12 nm.
It was reported as the intensity-weighted average (Z-AVE) [N
(degree of freedom) = 12] in Table 2. In addition, by DLS
technique, Z-potential (mV) of GA-dendrimer (9) particles
was also measured, for having an idea of physical stability
of GAD in aqueous solution, for investigating the possible
tendency of the GAD particles to form aggregates
(megamers) and therefore obtaining a further validation of
the assumption referred to above, and for inquiring possible
toxicity. The result was reported in Table 2.
Morphology, size, and Z-potential of GAD particles
SEM images of the GA-enriched dendrimer (9) are shown in
Fig. 3 while in Table 2, the average particles size provided by
the instrument has been reported.
As all we know, particles size of about 380 nm is uncom-
mon for dendrimers of similar generation [69], but is typical
for the so called megamers [70]. Megamers are dendrimer
multi-molecular assemblies that, besides occurring thanks to
cross-linking agents introduced during the synthesis, can form
also because of the natural clustering assemblies of dendrimer
molecules into supramolecular assemblies. In the present case,
such an assembly process was rationally favoured by the
Zeta potential is the potential difference between the dis-
persion medium and the stationary layer of fluid attached to
particles. It is a measure of the electrical charge of particles
that are suspended in liquid and is crucial also for
nanoparticles-cell interactions. Low Z-potential favours the
particles binding to low serum proteins and a longer
Fig. 3 SEM images of GA-
enriched dendrimer particles

Drug Deliv. and Transl. Res.
circulation in blood. Values < 5 mV may lead to agglomera-
tion of particles conferring physical instability in solution. On
the contrary, Z-potential > 30 mV, either positive or negative,
leads to monodispersity and good physical stability in solution
[71, 72]. Finally, high positive Z-potential values are correlat-
ed to high cytotoxicity while negative values allow high cell
viability [73]. In these regards, the GA-dendrimer (9) nano-
particles here prepared, possess a Z-potential of − 25 mV SD
and therefore can be considered sufficiently stable in water
solution, with a tendency to form megamer aggregates as sup-
posed by the average particles size by SEM and DLS, and
would be endowed with low cytotoxicity.
In order to investigate the effect of heat on the antioxidant
property of GA-dendrimer (9) and GA, samples of GAD and
GA were dissolved in ethanol at the highest possible concen-
tration (6.67 mg/mL), heated until the solution was browned
and evaluated for RSA % again.
In the case of GAD, RSA (%) turned out to be decreased of
almost 60% if compared with RSA (%) determined on a not
heated sample of 9 at same concentration (6.67 mg/mL),
whereas in the case of GA, RSA (%) resulted decreased up
to 98%. According to these results, GAD can be considered
far more stable than GA to thermal induced oxidative
degradation.
In vitro simulation of physiological cell esterase
hydrolytic action
Radical scavenging activity of GA-dendrimer (9)
In order to evaluate the antioxidant power of GA-dendrimer (9)
the radical scavenging activity (RSA %) was determined by the
DPPH• assay [56]. In parallel, for comparison purposes, sam-
ples of commercially available GA, AA, and Trolox have been
tested in the same conditions. The RSA (%) was evaluated at
different concentrations of GA-dendrimer (9), GA, AA, and
Trolox and reported in graphs as functions of concentrations.
Concentrations were expressed in μg/mL, μmol/mL, and in
molarity (mM) (Fig. S5.1 in ESM). The results were then
expressed as IC50 that is the efficient concentration of sample
that inhibits 50% of the DPPH radicals [74].
Table S1. in ESM shows the determined IC50 expressed in
μg/mL, μmoL/mL, and millimolarity again, together with an
IC50 value of α-tocopherol (Vitamin E) reported in literature.
Since the molecular weight of GAD is considerably higher
than that of GA, AA, Trolox, and α-tocopherol, it is more
consistent to compare molar concentrations rather than the
concentrations in weight. According to these results, as early
supposed, GA-dendrimer (9) proved to possess an intrinsic
antioxidant activity even higher than that of Trolox, AA,
GA, and vitamin E. GAD proved to reach the IC50 action at
molar concentrations of 24, 15, 4, and 3 times more diluted
respectively (Fig. 4).
As it is extensively reported concerning polyester-based
dendrimers [32, 50, 51], once inside the cell, GA-dendrimer
(9) that belong to that category, it is supposed to undergo an
enzymatic attack by cell esterase, that should degrade it, to
small monomeric units, releasing GA. In order to have an
experimental confirmation of this assumption, as last experi-
ence of this study, an in vitro reproduction of these events, was
conceived and realised. Supposing an administration via i. v.
and a high accumulation of GAD in liver, as reported by
Palidda et al. [50] for high MW polyester-based analogous
dendrimers, PLE was selected as model esterase. GAD was
treated at physiological temperature and pH with the powdery
form of PLE, which belongs to mammalian liver microsomal
carboxylesterases. The solid residue obtained, named 10, was
then qualitatively investigated to evaluate its composition and
its components identity. Firstly, investigations by FeCl3 essay
and TLC analysis were performed. The FeCl3 essay on a crude
10 ethanol solution gave a blue colouring, identical to that
given by a GA solution (Section S6, Fig. S6.1 in ESM), while
TLC elution profile, showed as unique significant spot, a stain
with the same Rf. of GA (Section S6, Fig. S6.2 in ESM).
According to these qualitative investigation, GAwas the main
component of crude 10. To complete the investigation, a
recrystallisation of crude 10 was tried in the condition reported
in literature for GA (hot ethanol). Off-white crystals (10c)
were obtained whose m. p., NMR and elemental analysis con-
firmed the GA structure (Section S6 and Fig. S6.3 in ESM).
The weight of 10c and the mole number derived (64.05 mmol)
were in accordance with the number (64) of the estimated (1H
NMR) GA units peripherally present per dendrimer mole with
an error of less than + 0.08 %. Once identified that the main
product UV-detectable of hydrolysis product was GA 1, the
enzymatic hydrolysis was repeated, to investigate the hydro-
lysis profile and to quantify the esterase hydrolysis kinetic.
Another sample of GA-dendrimer (9) was treated with PLE
again, and the GA content in hydrolysate was determined at
different time points by GC/MS, after derivatisation of GA by
Fig. 4 Comparison between radical scavenging activity expressed as IC50
(mM) of 9, GA, Vitamins A and E and Trolox

Drug Deliv. and Transl. Res.
By merging the nonpareil properties of synthetic
dendrimers and the irreproducible multi-target health activity
of natural compounds, a GA-enriched biodegradable and non-
cytotoxic dendrimer (9), endowed with remarkable antioxi-
dant power and nanoparticles amazingly spherical of about
380 nm, due to hydrogen bond induced aggregation to supra-
molecular megamers, was achieved. Aggregation of GAD
nanoparticles is rationally favoured by Z-potential value (−
25 mV) which, however, allows a sufficient stability in aque-
ous solution. GAD nanoparticles are gifted with very large
surface area that typically increases the systemic retention
time of DDSs and their bio-efficiency. GAD cytotoxicity in-
vestigations on two cell lines proved high viability (%) of cells
exposed to GAD, its high degree of biocompatibility and an
advantageous action of the carrier dendrimer scaffold in re-
ducing the proven cytotoxicity of GA versus the cells tested.
Thanks to these features, GA-dendrimer (9) is advisable
for further more in deep investigations to evaluate its
specific pharmacological activities and its eligibility as
innovative therapeutic, against diseases triggered by OS.
In this regard, as crucial property, GA-dendrimer (9)
radical scavenging activity was investigated. GAD
proved to be an antioxidant more powerful than free
GA than other known antioxidants. Furthermore, as a
GA delivering system, it is able to carry sixty-four
GA units per mole at once, and when subjected to the
attack by PLE, selected as an esterase model in physi-
ological condition, it degrades, setting free the GA moi-
eties transported, for further antioxidant activity. It can
also be rationally thought that the GAD strong antioxi-
dant activity may be exploited to reduce the auto-
oxidative degradation of fatty acids, lipids, and foods
or to prepare nanocomposites for the creation of active
food packaging with a large positive economic impact.
Fig. 5 Esterase hydrolysis profile [wGA, weight of GA; wGAD, weight of
GA-dendrimer (9) treated with PLE (100 mg)]
using BTSFA. Figure 5 shows the results that highlighted a
plateau of hydrolysis and therefore the GA maximum concen-
tration within three hours.
In vitro cytotoxicity investigation by cell viability (%)
evaluation
To determine the actual biomedical applications of GAD, its
in vitro cytotoxicity profile, was investigated against two
model cell lines, i.e. epithelial B14 and liver BRL cells.
These particular cell lines where selected because they are
particularly sensitive to tannic, ellagic, and gallic acids [75];
therefore, a good cell viability would have been meant, both as
the low cytotoxicity of GAD and as the positive action of
dendrimer architecture in reducing toxicity of GAversus these
specific cell lines. MTT assay, a method which is based on
monitoring of NAD(P)H-dependent cellular oxidoreductase
enzyme activity and is therefore related to inhibition
of metabolic processes in mitochondria, was performed.
GAD concentration used in MTT assay was in the range
0.5–20 μmol/L according to literature data [76]. Regarding
GAD, this range means a concentration of 8.5–340 μg/mL.
The results referring to the fixed max concentration essayed
only marginally affected cell growth, since viability values
were in the range of > 90 % for B14 cells and > 82 % for
BRL cells (Table 2).
Acknowledgements The authors are very thankful to Mr. Gagliardo
Osvaldo for Elemental Analysis and Prof. Deirdre Kantz for language
help.
As the first author, I affectionately dedicate this work to my dear
parents and dear friend Rocco.
Funding information This work has been supported by University of
Genoa (Progetti di Ateneo).
Compliance with ethical standards
Conclusions
Conflict of interest The authors declare that they have no conflict of
interest.
The present work reports innovative and outstanding results
that meet the needs of one of the most important research
areas. Such current studies aims at optimising the antioxidant
properties of natural polyphenols and at improving their draw-
backs through the use nanosized carriers, for preparing inno-
vative DDSs with potentialities as alternative more efficient
therapeutics with reduced side effects.
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