Nanoplatforms of Manganese Ferrite Nanoparticles Functionalized with Anti-Inflammatory Drugs

Article abstract of DOI:10.1002/ejic.201801539

Targeted drug delivery by magnetic nanoparticles (MNPs) is at the leading edge of the rapidly developed methods for the diagnosis and treatment of various diseases. Functionalization of nonsteroidal anti-inflammatory drugs (NSAIDs) onto MNP-based platforms constitutes a challenging endeavor, as it is based on the physicochemical characteristics of the final carrier/system. MnFe₂O₄ MNPs of relatively small size and enhanced magnetization with aminated (AmMNPs) and non-aminated (Non-AmMNPs) surface were prepared solvothermally in the presence of octadecylamine(ODA) through synthetic variations. Three nonsteroidal anti-inflammatory drugs (NSAIDs), acetylsalicylic acid (ASA), mefenamic acid (MEF) and Naproxen (NAP), of different pharmacochemical properties, were used for the loading of the nanoplatforms. In the case of AmMNPs, attachment of the NSAIDs was achieved by direct coupling of carboxylate donors with the NH₂ groups via the formation of an amide bond, whereas indirect coupling of the drugs through encapsulation was applied on Non-AmMNPs by taking advantage of the PEGylation approach. FT-IR and UV/Vis data confirmed the distinct drug release behavior, which is attributed to different cross-linking forces depending on the method of functionalization. The biological behavior and anti-inflammatory activity of the MNPs@NSAIDs was evaluated in vitro; AmMNPs@ASA inhibited lipoxygenase (LOX) and protected albumin denaturation with values comparable with those of NSAIDs.

Full text of DOI:10.1002/ejic.201801539

DOI: 10.1002/ejic.201801539
Full Paper
Nanoparticles for Drug Delivery
Nanoplatforms of Manganese Ferrite Nanoparticles
Functionalized with Anti-Inflammatory Drugs
Kleoniki Giannousi,[a] Emmanouil Koutroumpis,[a] Violetta Georgiadou,[a]
Vasilis Karagkounis,[a] and Catherine Dendrinou-Samara*[a]
Abstract: Targeted drug delivery by magnetic nanoparticles the loading of the nanoplatforms. In the case of AmMNPs, at-
(MNPs) is at the leading edge of the rapidly developed methods tachment of the NSAIDs was achieved by direct coupling of
for the diagnosis and treatment of various diseases. Functionali- carboxylate donors with the NH2 groups via the formation of
zation of nonsteroidal anti-inflammatory drugs (NSAIDs) onto an amide bond, whereas indirect coupling of the drugs through
MNP-based platforms constitutes a challenging endeavor, as it encapsulation was applied on Non-AmMNPs by taking advan-
is based on the physicochemical characteristics of the final tage of the PEGylation approach. FT-IR and UV/Vis data con-
carrier/system. MnFe2O4 MNPs of relatively small size and en- firmed the distinct drug release behavior, which is attributed
hanced magnetization with aminated (AmMNPs) and non-ami- to different cross-linking forces depending on the method of
nated (Non-AmMNPs) surface were prepared solvothermally in functionalization. The biological behavior and anti-inflamma-
the presence of octadecylamine(ODA) through synthetic varia- tory activity of the MNPs@NSAIDs was evaluated in vitro;
tions. Three nonsteroidal anti-inflammatory drugs (NSAIDs), AmMNPs@ASA inhibited lipoxygenase (LOX) and protected al-
acetylsalicylic acid (ASA), mefenamic acid (MEF) and Naproxen bumin denaturation with values comparable with those of
(NAP), of different pharmacochemical properties, were used for NSAIDs.
1. Introduction
mentioned drawbacks, since they result to lower doses of active
agent, due to preservation of the drugs by simultaneously
avoiding their destruction.[8,9]
Nonsteroidal anti-inflammatory drugs (NSAIDs) are a wide
group of cyclooxygenase (COX) inhibitors that work by blocking
the production of prostaglandins (PGs), albeit with side effects,
such as stomach irritation, ulcers and renal toxicity.[1] Recent
epidemiological studies correlate the prolonged usage of
NSAIDs with slowing the progression of Alzheimer's Disease
(AD).[2] However, the potential of NSAIDs for the treatment of
AD are correlated with a number of factors, e.g. the physico-
chemical characteristics, brain penetration properties and dos-
age of the drugs. Conventional analgesic NSAIDs include acetyl-
salicylic acid (ASA),[3] mefenamic acid (MEF)[4,5] and naproxen
(NAP).[6,7] NSAIDs are widely administered against a wide range
of inflammatory diseases to treat moderate pain including fever,
headache, dental pain, postoperative and postpartum pain, dys-
menorrhea, osteoarthritis, rheumatoid arthritis.[3–7] However,
NSAIDs are accompanied with limitations such as lack of speci-
ficity toward desired tissues, high distribution volume, lack of
selectivity, poor aqueous solubility and dissolution rate. Con-
trolled drug release systems could overcome the above-
Magnetic nanoparticles (MNPs) based drug delivery plat-
forms have been proposed as suitable vehicles for overcoming
pharmacokinetic limitations associated with conventional drug
formulations.[10] Considering also the heterogeneity of en-
hanced permeability and retention (EPR) effect, magnetic tar-
geting boosts the accumulation of MNPs in the diseased
area.[11] However, biological barriers not only affect the move-
ment of MNPs, but also alter their magnetic and surface proper-
ties and/or induce a negative host response through biochemi-
cal signaling, resulting in an early uptake by cells before the
MNPs manage to reach the target tissue.[12] Nonetheless, appro-
priate functionalization of the MNPs could result in the design
of multifunctional drug carriers, finding applications as contrast
agents in magnetic resonance imaging and/or in magnetic
hyperthermia.[13,14] Even though polymeric nanoparticles and
MNPs have been studied in the area of theranostics in the sense
that diagnosis coexists with targeted drug and/or gene delivery
in case of tumors,[3–8,15] the use of MNPs as NSAIDs carriers has
been scarcely reported.[16]
Recently, manganese ferrite (MnFe2O4) nanoparticles due to
their unique properties have been the subject of extensive re-
search, concerning multiple functionalities of catalysis, adsorb-
ent, water treatment, imaging, hyperthermia and triggered
drug release.[17] MnFe2O4 MNPs show greater biocompatibility,
have high magnetization, large relaxivity and a strong T2 phase
contrast for magnetic resonance imaging compared to Fe3O4,
[a] Laboratory of Inorganic Chemistry, Department of Chemistry,
Aristotle University of Thessaloniki,
Thessaloniki, 54124 Greece
E-mail: samkat@chem.auth.gr
http://users.auth.gr/samkat
Supporting information and ORCID(s) from the author(s) for this article are
available on the WWW under https://doi.org/10.1002/ejic.201801539.
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γ-Fe2O3, CoFe2O4, and NiFe2O4 MNPs.[18] Interestingly, it has tion of albumin denaturation, with NSAIDs as the standard ref-
been reported that suitable surface modification onto the na- erence drugs.
noparticles not only can prevent aggregation and clustering,
but also results to prolonged circulation in the blood vessels,
2. Results and Discussion
2.1. Synthetic Aspects, Characterization, and
Functionalization of MNPs
since NPs are being less recognized by the reticulo-endothelial
system (RES).[12]
The prerequisites for using MNPs in bio-applications are mul-
We have shown before that based on the synthetic conditions,
tifaceted and include specific physicochemical characteristics
the linear configuration of ODA molecules can lead to un-
(size, shape, structure and surface chemistry known as 4S's) and
shielded NH2 groups on the surface of CoFe2O4 MNPs.[22] In the
magnetic properties, which lead to desirable biodistribution,
present study we tested if this efficacy is present in case of
image contrast, target selectivity and sustained drug release.
MnFe2O4 MNPs in addition to the prerequisites of size, crystalli-
The successful preparation of primary nanoparticles with de-
nity, magnetization. In case of implementing Fe(acac)3 and
sired characteristics is nowadays the preliminary step for mov-
Mn(acac)2 (2:1) as precursors and solely ODA in triple role of
ing forward to the next generation of secondary nanomaterials,
surfactant, reducing agent and solvent, free NH2 groups on the
which can be designed via appropriate synthetic and/or post-
surface of MnFe2O4 MNPs were isolated (aminated=AmMNPs).
synthetic functionalization of the primary MNPs. An ultimate
On the other hand, the decomposition of the same ratio of
rational way that such systems can be formed is missing, due
precursors in the presence of ODA as a surfactant in an aprotic
to the fact that each drug possesses its unique physicochemical
solvent (diphenyl ether) resulted to non-aminated MnFe2O4
characteristics. In so, direct and indirect methods of functionali-
MNPs (Non-AmMNPs). Additionally, the trivalent instead of the
zation have emerged. As far as direct conjugation concerned,
divalent manganese acetylacetonate precursor has been used
coupling with reactive functional groups such as a primary or
for the synthesis of MnFe2O4 MNPs as higher magnetization
a secondary amino, carboxyl, hydroxyl, alkyl halogen, or azide
values were expected. The similar decomposition/dissociation
are in practice.[19] On the other hand, indirect drug loading has
also been studied through intermediate biocompatible coatings
e.g. Polyethylene glycol (PEG) polymers, Polyethyleneimine (PEI)
molecules, dextranes, liposomes, chitosan etc. that offer excel-
lent chemical stability, precise control over composition and
structure and hence high efficiency in biomedical applications
but suffers from the final size;[12,20] when the size increases,
more NPs are entrapped within the liver and spleen.[10,11]
rate of MnIII with that one of FeIII precursor can lead to varia-
tions of manganese ions in tetrahedral (Td) and octahedral (Oh)
sites in the spinel structure[23] and eventually to higher magne-
tization values. However, the isolated MnFe2O4 MNPs with the
divalent MnII precursor showed higher magnetization values (64
emu/g) instead to those of MnIII precursor (10 emu/g) (Figure
S1), and under this evidence we did not proceed further with
this sample. Further studies are needed to unveil and manipu-
Herein, we propose a simple synthetic variation which allows late the physicochemical properties of MNPs prepared with
the preparation of octadecylamine (ODA) coated manganese MnIII. In case of Non-AmMNPs prepared with the divalent pre-
ferrite magnetic nanoparticles (MnFe2O4 MNPs) either with cursor the magnetization properties (Ms = 44 emu g–1) allowed
aminated (AmMNPs) or non-aminated (Non-AmMNPs) groups us to continue our studies (Figure S7d). TEM images of
aminated and non-aminated MNPs are illustrated in Figure 1.
Both samples, AmMNPs (Figure 1a) and Non-AmMNPs (Fig-
ure 1b), display nearly spherical shape with average sizes at
11 0.6 and 8.4 0.3 nm respectively. The size differences are
attributed on the applied synthetic conditions. It has to be
noted that the difference in Ms values between the AmMNPs
(64 emu/g) and Non-AmMNPs (44 emu/g) is attributed to the
size difference. The Ms value decreases as the particle sizes de-
crease.
on their surface. During synthesis, when octadecylamine (ODA)
has been used in a triple role (coating, solvent, reducing agent)
AmMNPs have been isolated, while in the presence of a non-
polar solvent (diphenyl ether, DPE) Non-AmMNPs have been
produced. The resulted MNPs were functionalized through post-
synthetic procedures with three NSAIDs of different molecular
weight and structure with a common feature of carboxylate
donor; ASA, MEF and NAP. By taking into account that different
inflammation-related diseases require different drug administra-
tion routes, we aimed at unique release profiles. The functionali-
zation of the MNPs with the drugs aimed at confining the side-
effects and giving them a specific delivery profile. For example,
in the long-term treatment of rheumatoid arthritis, osteo-
arthritis and ankylosing spondylitis, the transdermal topical
route is preferable with initial burst drug release followed by a
second slow release.[21] For that purpose, two procedures were
employed for the transformation of the synthesized MnFe2O4
MNPs as carriers: (i) an amide reaction through the carboxylate
donor of the drugs with the AmMNPs (direct coupling) and
(ii) loading of the drug on the PEG matrix of the Non-AmMNPs
MNPs (indirect coupling). The nanoplatforms were examined as
Figure 1. TEM images of AmMNPs (a) and Non-AmMNPs (b).
Aminated MnFe2O4 MNPs
anti-inflammatory agents by evaluating in vitro the inhibition The powder X-ray diffraction diagram of the sample showed all
of the enzymatic activity of lipoxygenase (LOX) and the protec- the characteristic peaks of pure manganese ferrite, while the
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average crystalline size of the sample was calculated by fitting tion of the vibration of carboxylic acid at ≈ 1730 cm–1 of the
the diffraction data with a pseudo-Voigt function (Jade6 Soft- NSAIDs is recorded. Simultaneously, FT-IR spectrum of AmMNPs
ware) and was found 12 nm (Figure S2a). The presence of the display peak at ≈ 1660 cm–1 attributed to hydrogen bonding
organic coating bearing free amines was certified by FT-IR spec- between the surfactant molecules, while in case of
troscopy (Figure S2b), while by thermogravimetric analysis (Fig- MNPs@NSAIDs peaks at ≈ 1630 (C=O), ≈ 1550–1535 cm–1
ure S2c) the total weight loss of the organic coating was meas- (N–H) correspond to amide I and at ≈ 1450 cm–1 amide II
ured at 30 % w/w. By taking into account that decomposition (C–N) vibrations. The spectrum of MNPs@ASA demonstrates
occurs in two steps and the linearity of ODA molecules, a con- also peak at ≈ 1700 cm–1 characteristic of carbonyl ester vibra-
figuration of a double layer on the top of the NPs is proposed, tion. UV/Vis spectra followed the ninhydrin colorimetric analysis
with the amines of the inner layer facing the metal ions and further confirmed that –NH2 groups take part in the amide
those of the outer layer remaining exposed for the formation bond after the reaction with NSAIDs, since the absence of Ruhe-
of the amide bonds with the drugs. The presence of –NH2 mann's purple complex is observed (Figure 2b). By combining
group on the MNPs surface was further confirmed by ninhydrin the above data, it can be deduced that the drugs were mostly
colorimetric assay through the absence or appearance of Ruhe- covalently linked with the MNPs. However, the absorption of
mann's purple complex at λmax ≈ 600 nm. The free –NH2 groups small quantity of the drugs onto MNPs cannot be excluded.
were found 0.0035 mg mL–1. The saturation magnetization (Ms)
Estimation of Drug Loading by Defining the Optimum pH
emu g–1 and 260 Oe, respectively (Figure S2d).[24,25] Detailed The efficacy of the nanoplatforms is pH-sensitive and thus a
and the coercive field (Hc) value of the MNPs was found 64
characterization of the AmMNPs is given in the SI.
comparative study in the pH region 2 to 8 was undertaken in
order to define the appropriate pH values for the calculations
of drug loading and release. Solubility and permeability are the
Direct Coupling Functionalization
The direct coupling of NSAIDs was achieved via the formation two important characteristics that define the pharmacokinetic
of a covalent amide bond between the carboxyl groups of the profile of the drugs.[27] The balance between the dissolution
drugs and the NH2 group of ODA (AmMNPs).[26] FT-IR spectra rate and the absorption through lipid membranes in combina-
illustrates the presence of the drug along with the organic coat- tion with the targeted area of application determine the per-
ing of the MNPs. Figure 2a clearly shows the absence of the formance of the functionalized MNPs.[28] For example, the pH
free NH2 peak at ≈ 3300 cm–1 presented in the as-prepared of gastric acid is 1.5 to 3.5, whereas the pH gradually increases
MNPs. Moreover, by comparing the spectra of the functional- in the small intestine from pH 6 to approximately 7.4 in the
ized MNPs with those of neat NSAIDs (Figure S3), the attenua- terminal ileum. In so, ASA at pH > 5 exists in its ionic form
Figure 2. (a) FT-IR spectra of AmMNPs directly coupled with ASA, MEF and NAP in comparison with as prepared MNPs. (b) Ninhydrin colorimetric analyses
showing the absence of free NH2 groups after the formation of amide bond.
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(R-COO–) with high aqueous solubility, but lower absorption indicating that steric stabilization contributes to the colloidal
through lipid membranes, whereas at pH < 2 a neutral form (R- stability. In case of AmMNPs@MEF the PZC was recorded at pH
COOH) is formed which lowers the aqueous solubility by simul- ≈ 2.5, while at pH > 4 negative zeta potential values were re-
taneously enhancing the absorption.[28] The mixture of the two corded (from –18 to –22 mV). In all cases, hydrodynamic sizes
forms existing at the pH region of 2–5 seems to be the key of indicate that aggregation took place probably via Van der Waals
balanced dissolution and absorption rate.[28] As far as mefen- interactions, while the zeta potential values were less than
amic acid concerned the dissolution rate increases with pH
30 mV, which is generally considered as the threshold value
reaching its maximum at pH = 9.[29] In case of NAP, the solubility for electrostatic stabilization.[33]
is low at the pH region 2.2 to 5.8 due to unionization of drug,
Non-Aminated MnFe2O4 MNPs and PEGylation of the MNPs
while an intermediate dissolution rate is recorded at pH = 6.4.
At pH = 7.4 the solubility of the drug further improves, but
the permeability gets extremely low.[27] In order to define the
optimum pH for each drug, the stability of ODA in variable pH
was also investigated. Based on previous report, ODA molecules
are almost neutral charged at pH > 5.7 and thus the layer is
stable, while in the pH 3–4 the amine headgroups get proto-
nated and the ODA molecules either dissolve into the solution
subphase or are positioned loosely at the interface. By lowering
the pH further (< 3), ODA molecules form a layer but with de-
fects.[30] UV measurements were conducted in order to estimate
the drug loading in different pH based on the respective refer-
ence curves (Figures S4–S6). After all, by combining all the re-
marks, the optimum pH values for ASA, MEF and NAP studies
were set as 2.2, 7.4 and 6.4, while the loaded drug was esti-
mated ≈ 13.5 % w/w, 39.7 % and 27 % w/w, respectively.
The hydrodynamic sizes in terms of Z-average (d.nm) with
the relative polydispersity indexes (pdI) of the functionalized
by direct coupling MNPs, AmMNPs@ASA, AmMNPs@MEF and
AmMNPs@NAP, were measured in PBS and found 166.3 nm
(0.341), 220 nm (0.277) and 247.4 nm (0.205), respectively. The
primary AmMNPs were found with diameter of 137.8 nm
(0.160), implying that the functionalization process did not alter
significantly the physicochemical properties of the MNPs. The
pH-dependent zeta potential was measured for the functional-
ized MNPs and is shown in Figure 3. Zeta potential measure-
ments allow us to follow the charge of the nanoparticles in
different environments and to determine the point of zero
charge (PZC) which is the pH at which there is no charge on the
nanoparticles' surface.[31,32] AmMNPs@ASA and AmMNPs@NAP
displayed positive zeta potential (> +15 mV) at low pH (< 3),
with PZC occurring at pH ≈ 6 and 5, respectively. Though at the
pH range 3.5–7 the values were varied from +13 to –4 mV,
the particles were stable and no precipitation was observed,
The powder X-ray diffraction diagram of the sample showed all
the characteristic peaks of pure manganese ferrite, while the
average crystalline size of the sample was calculated by fitting
the diffraction data with a pseudo-Voigt function (Jade6 Soft-
ware) and was found 9 nm very close to the size provided by
TEM (Figure S7a). The encapsulation of Non-AmMNPs into PEG
matrix was initially carried out without any drug. In the FT-IR
spectra (Figure S7b) the peaks of Non-AmMNPs, clearly shown
at ≈ 489 and 600 cm–1, are corresponding to the manganese
and ferrite oxides, respectively. By comparing the spectra of
Am-MNPs and Non-AmMNPs, the disappearance of the NH2
group peak at ≈ 3300 cm–1 is recorded only in the last case,
indicating the absence of free amines onto the MNPs. Thermo-
gravimetric analysis data of Non-AmMNPs demonstrate that
ODA, despite the fact that is absorbed at higher percentage
(weight loss 50 %), is getting decomposed at one step (Figure
S7c), whereas in case of Am-MNPs two layers of the surfactant
were observed. Although AmMNPs were found of enhanced Ms
value, still Non-AmMNPs display appropriate magnetic proper-
ties (Figure S7d). The detailed characterization is given in the
SI.
Loading on the PEG Matrix
Encapsulation of NSAIDs in colloidal nanodispersions represents
a promising strategy with the significant potential for enhance-
ment of bioavailability. PEGylation is a commonly used proce-
dure for providing “stealth” characteristics, since enable the
MNPs to deceit RES.[34] The amphiphilic properties of PEG poly-
mers (molecular mass lower than 100 kDa) allow for entrap-
ment of the MNPs through a biphasic system (H2O/CHCl3) with-
out the need of any other linker. Under that prism, PEG8000
created a polymeric matrix around the MNPs where the drugs
were loaded/adsorbed in a second step through Van der Waals
interactions and hydrogen bonding. Drug loading on PEGylated
Non-AmMNPs was utilized as an alternative functionalization
method. PEG8000 was used for the loading of ASA, MEF and
NAP on the MNPs in order to study the release rate compared
to the AmMNPs directly coupled with the drugs. Although there
is a permanent interest to enhance the aqueous solubility and
dissolution rate of NSAIDs in order to achieve faster onset of
action and to minimize the absorption variability, the simulta-
neous encapsulation of MNPs and NSAIDs is scarcely re-
ported.[35]
The presence of the drugs onto the PEG chains of the
Non-AmMNPs was evidenced by FT-IR spectroscopy. The char-
acteristic vibrations of methylene groups of PEG chains
at
≈
2934 cm–1, multiple CH2–O–CH2 vibration modes
Figure 3. Zeta potential as a function of pH for AmMNPs@ASA, AmMNPs@MEF
and AmMNPs@NAP.
at ≈ 1107 cm–1, and carboxylic group at ≈ 1640–1700 cm–1 of
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the drugs, respectively, are presented in Figure 4. The peaks 20.8 %. On the other hand, the percentages of drug release
corresponding to ODA have been shaded by those of the bulk- from the PEG matrix were significantly higher up to 24 h (91 %
ier PEG8000, while in case of ASA, MEF and NAP the bending NAP and 62 % ASA release), due to the weaker Van der Waals
vibration of –C–H group is depicted at ≈ 1380 cm–1. Moreover, interactions between the adsorbed NSAIDs and the PEG chains.
in case of MEF the peak at ≈ 700 cm–1 corresponds well with
Table 1. Percentage of cumulative release (Q%) at 3 h and 24 h and release
exponent (n) according to Ritger–Peppas model.
bending vibration of –NH. The relative amounts of ASA and
NAP were estimated ≈ 18.8 % and 24.2 % w/w at pH 2.2 and
n3h
Q3h
%
n24h
Q24h%
6.4 respectively, of the total mass of the samples by UV meas-
urements (Figure S4, Figure S6). However, in case of MEF, count-
able amount of ODA molecules was released together with the
drug at pH 7.4, implying a loose conformation of the surfactant
layer on the surface of the MNPs and in that case quantification
was not allowed.
AmMNPs@ASA
AmMNPs@MEF
AmMNPs@NAP
Non-AmMNPs@PEG-NAP
Non-AmMNPs@PEG-ASA
0.63
0.66
0.45
> 1
32
17
27
66
38
0.2
0.09
0.1
0.14
0.18
42
20.8
34.6
91
0.28
62
In all cases of amide bond formation, under the assumption
that there is not observed any swelling phenomena and the
nanoparticles remain spherical, the respective limits of the dif-
fusion index n were implemented to determine the mechanistic
aspects of the drug release. The estimated n3h value indicated
a non-Fickian diffusion (0.43 < n <1) and suggested that the
release profile is a combination of both diffusion and erosion
by means of the amide bond hydrolysis.[38] The second step
shows a very small diffusion exponent that can be ascribed to
a long-term release (sustained) process mainly due to the stabil-
ity of the amide bond between ODA and the NSAIDs located
in the inner organic layer (Figure 5a).[39] In the case of PEGy-
lated MNPs the limits of the diffusion index n differ, since the
polymer swelling phenomenon is observed. In case of ASA the
diffusion index n remains low through time, which is indicative
of limited swelling and Fickian diffusion in one stage opposite
to NAP, where two stages were recorded. In the latter case the
diffusion exponent had a high value (n3h > 1) for the first 5 h
that indicates a case II transport mechanism. The non-Fickian
diffusion is associated with stresses and state-transition of the
hydrophilic PEG8000 chains that swell in physiological solution.
The lower value (n24h = 0.14) for the rest 19 h corresponds to
Fickian diffusion and a sustained release from the intermediate
layers of the polymer is indicated. We assume that the chemical
structure and the smaller size of the acetylsalicylic acid as well
as the stronger Van der Waals interactions and hydrogen bond-
ing between the molecule and the polymer could justify the
above remark (Figure 5b).
By comparing the release profile of NAP among the two
ways of functionalization, direct coupling and PEGylation, differ-
ent release profiles are revealed. In the first case, the hydrolysis
of the amide bond of the outer layer is followed by hydrolysis
occurring in the inner coating. On the other hand, when NAP
is loaded on PEG matrix, the release is happening via diffusion
from the outer polymer layer due to polymer swelling, while
later NAP molecules that are more protected are released by
interlayer diffusion among the stacking of PEG chains. Similar
behavior was recorded when ASA and MEF molecules were di-
rectly coupled onto the aminated MNPs. However, when ASA is
adsorbed on PEG matrix, the diffusion remains gradual but the
release records higher cumulative percentages.
Figure 4. FT-IR spectra of PEGylated Non-AmMNPs loaded with ASA, MEF and
NAP.
The hydrodynamic sizes of the PEGylated Non-AmMNPs
loaded with the drugs were measured in PBS. The relative diam-
eters were found 178.7 nm (pdI:0.277) and 194.6 (pdI:0.189) for
Non-AmMNPs@PEG-NAP and Non-AmMNPs@PEG-ASA, respec-
tively, indicating that those systems slightly differ from the anal-
ogous direct functionalized MNPs. This is attributed to the rela-
tively low molecular weight of PEG and/or to the low amount
of the polymer. The zeta potential values varied from +20 to
+25 mV, while the dispersions were stable in the pH range 2–
9 and no precipitation occurred. In case of PEGylation, steric
stabilization dominates making electrostatic repulsions insignif-
icant.[36]
Drug Release
The drug release studies of the functionalized samples were
conducted in diverse pH according to the stability and disper-
sion of the drugs in conjugation with minimization of the disso-
lution of ODA molecules. The release profile corresponds well
with a two-step procedure. The Ritger–Peppas model was ap-
plied to estimate the drug release rate in each case. The estima-
tion of cumulative release in percentage (Q) after 3 h and 24 h
is presented in Table 1. In case of direct coupling functionaliza-
tion, the covalent attachment of the drugs onto MNPs resulted
in lower Q24h% drug release, since the amide bond is more
stable.[37] The cumulative NAP and ASA release in percentage
It is worth to mention that direct coupling proved to be a
(Q) after 24 h was estimated 34.6 %, and 42 %, respectively, simple and efficient method for incorporating ASA, MEF and
while in case of MEF the respective percentage hardly reaches NAP and achieve a sustained therapeutic release rate. Moreover,
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Figure 5. (a) Comparative cumulative drug release in percentage (Q %) for direct functionalization in a 24 h time period, (b) Comparative cumulative drug
release in percentage (Q %) for PEGylated MNPs-NSAIDs in a 24 h time period. Adjusted R-squared values are also included.
it does not require preliminary modifications that would cause not seem to be affected by the way of functionalization. MNPs
the loss of NSAIDs activity and any external source to trigger coupled/loaded with ASA recorded the most remarkable func-
the drug release.[40]
tion (respective IC50 values; 6.7, 7.6 μg/mL) and for this reason
was selected to be studied further.
Albumin Denaturation Experiments
2.2. Anti-Inflammatory Activity of MNPs and
Functionalized MNPs
Protein denaturation is a process during which the secondary
and tertiary structures of proteins are destroyed resulting in loss
of their biological function caused by a spectrum of reasons
including heat, electrolytes, or alcohols which generate well-
studied alterations in the solubility of albumins and globu-
lins.[43] Inflammation and protein denaturation are closely re-
lated, and inhibition of the protein collapse is achieved by
NSAIDs. Acetylsalicylic acid has shown a dose-dependent ability
to inhibit the induced protein denaturation.[44] Zinc peroxide,
silver and iron nanoparticles have also been tested as anti-
inflammatory agents via inhibition of denaturation and have
been found of concentration dependent anti-inflammatory ac-
tivity.[45,46,47] Herein, we aimed at examining both the anti-
inflammatory capacity of MnFe2O4 MNPs and the collective
properties of MNPs coupled and/or loaded with ASA. Protein
denaturation was triggered by heating and acidifying of bovine
serum albumin (BSA), which results in antigen expression and
is related with type-III hypersensitivity reaction, which in turn is
associated to various diseases including rheumatoid arthritis,
systemic lupus erythematosus, serum sickness, and glomerulo-
nephritis.[48] The assay implemented is based on albumin de-
naturation optical density measurements and results derived
from 5 different concentrations (5, 10, 25, 50, and 100 μg/mL).
Results are summarized in Table 3 where ASA values are also
included as reference.
Denaturation of albumin in the presence of PBS is considered
as 0 % anti-inflammatory activity, while in all cases the ob-
served action is concentration dependent. AmMNPs and Non-
AmMNPs protected the protein unfolding but in lower extent
compared to the reference drug. However, in case of functional-
ized MNPs (AmMNPs@ASA and Non-AmMNPs@PEG-ASA) a
much stronger anti-inflammatory capacity comparable with
that of free ASA is recorded. The higher % protective ability
against protein denaturation is recorded when ASA is directly
coupled with the MNPs (82.5 %), which implies enhanced anti-
inflammatory response. Still, Non-AmMNPs@PEG-ASA display a
high protection value (79.7 %) towards albumin denaturation.
Evaluation of Lipoxygenase Activity
NSAIDs are primarily COX inhibitors and exert their activity by
reducing the production of PGs induced in the inflammatory
process. In recent years, it has been clarified that PG synthesis
is only one part of the arachidonic acid pathway.[41] Eicosanoids
are oxygenated metabolites of arachidonic acid and they are
implicated in a diversity of diseases. Lipoxygenase (LOX) cata-
lyzes the first two steps in the metabolism of arachidonic acid
to leukotrienes (LTs) which are important in the pathogenesis of
neutrophil-mediated inflammatory diseases.[42] In view of this
concept dual inhibition of the COX and LOX pathways could
produce a wider spectrum of anti-inflammatory effects and thus
the ability of nanoplatforms to inhibit soybean lipoxygenase
(LOX) was tested.
The results presented in Table 2 demonstrate that solely the
MNPs (either aminated or not) did not interfere with the ability
of LOX to bind with its substrate, in the tested concentrations.
However, the functionalized MNPs inhibited the LOX activity
with IC50 values comparable with the neat drugs (controls),
while the anti-inflammatory activity of the nanoplatforms does
Table 2. Anti-inflammatory activity of AmMNPs and Non-AmMNPs before and
after functionalization with NSAIDs evaluated by the half-minimal inhibitory
concentration (IC50) (μg/mL). Data represented are mean SD of three identi-
cal experiments made in three replicates.
LOX Inhibition
IC50 (μg/mL)
AmMNPs
> 50
> 50
Non-AmMNPs
AmMNPs@ASA
AmMNPs@MEF
AmMNPs@NAP
Non-AmMNPs@PEG-ASA
Non-AmMNPs@PEG-NAP
ASA
6.7 0.89
12 0.94
8.4 0.76
7.6 0.92
9.8 0.78
5.2 0.45
5.5 0.87
NAP
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Table 3. Anti-inflammatory activity of various concentrations [0 (control), 5, 10, 25, 50, and 100 μg/mL] of AmMNPs and Non-AmMNPs before and after
functionalization with ASA, as well as ASA (reference drug), measured as inhibitory percentage (%) of albumin denaturation. Data represented are mean SD
of three identical experiments made in three replicates.
Concentration
Denaturation protection/Anti-inflammatory activity [%]
(μg/mL)
AmMNPs
Non-AmMNPs
AmMNPs@ASA
Non-AmMNPs@PEG-ASA
ASA
0 (control)
0
0
0
0
0
5
7.9 0.84
10.9 0.88
23.7 0.99
34.9 0.49
57.2 0.64
6.7 0.79
9.2 0.97
19.6 0.66
30.6 0.78
48.4 0.39
12.4 0.76
21.5 0.75
48.8 0.53
63.2 0.74
82.5 0.59
10.9 0.65
16.1 0.65
43.6 0.69
51.4 0.94
79.7 0.86
11.2 0.77
25.3 0.54
47.6 0.78
65.7 0.43
88.9 0.58
10
25
50
100
(+)-2-(6-methoxy-2-naphthyl)propionic acid (Naproxen) and Mefen-
amic acid (MEF) was from Aldrich Chemistry. Deionized water was
used for the fabrication of MnFe2O4 MNPs and Milli pure water
(< 3.0 MR, Millipore, MilliQ Gradient) was used for the modification
of the NPs surface properties. Nanofilters Minisart RC 15 (Single use
syringe filter Non-sterile RC-membrane, Pore Size: 0.20 μm) were
from Sartorius Stedim Biotech GmbH. Pur-A-Lyzer Midi 1000 Dialysis
Kit (for Research & Development use only) by Sigma-Aldrich. Bovine
serum albumin was purchased from Sigma (1 % aqueous solution,
Sigma-Aldrich, Germany).
3. Conclusions
Diagnostic and therapeutic agents in complicated diseases,
which are correlated with chronic inflammation (e.g. cancer, Alz-
heimer's Disease), are one of the most promising aspects of
nanotechnology to be implemented in human health. The de-
sign and drug loading of nanoplatforms is challenging, as
unique release profiles can be exploited in different diseases.
The physicochemical properties of the magnetic nanocarriers
as well as those of drugs, have to be combined properly for the
desired outcome. ODA as a primary surfactant proved to be a
beneficial agent for synthesizing MnFe2O4 MNPs of high crystal-
linity, with superparamagnetic behavior and based on the syn-
thetic conditions with aminated or non-aminated surface,
which further can be tuned either by direct or indirect function-
alization processes, respectively. Three different NSAIDs (carb-
oxylate donors) were successfully coupled with the aminated
MNPs via formation of amide bond, as well as encapsulated
with Non-AmMNPs in a PEG matrix. In case of the covalent at-
tachment of ASA, MEF and NAP with the MNPs sustained drug
release was observed, whereas more rapid release profiles were
recorded when NSAIDs loaded onto PEGylated MNPs. The bal-
ance between the dissolution rate of the drugs and the absorp-
tion through lipid membranes is affected by the pH of the tar-
geted area and eventually the drug loading and release was
found pH-sensitive. The in vitro studies indicate that functional-
ized MNPs could be administered to a variety of inflammatory
conditions, depending on the release profile, giving the per-
spective to propose these nanocarriers as theranostics.
Characterization Techniques: Powder X-ray diffraction (XRD) was
performed using a 2-cycle Rigaku Ultima+ diffractometer (40 kV,
30 mA, Cu-Kα radiation) with Bragg–Brentano geometry (detection
limit 2 % approximately). Fourier transform infrared spectroscopy
(280–4000 cm–1) was recorded using a Nicolet FTIR 6700 spectrom-
eter with samples prepared as KBr pellets. Thermogravimetric analy-
sis (TGA) was performed using a SETA-RAM SetSys-1200 instrument
at a heating rate of 10 °C min–1 under N2 atmosphere. Magnetic
measurements were acquired by a vibrating sample magnetometer
(1.2H/CF/HT Oxford Instruments VSM). The isolation of the organic
coating was achieved after the dissolution of the samples in CHCl3
and several cycles of sonication and centrifugation, until the super-
natant was limpid. The supernatant from each washing cycle was
removed and placed into glass vials. Then it was filtered with the
use of nanofilters to retain the dispersed NPs. UV/Visible measure-
ments were carried out with a double beam UV/Visible spectropho-
tometer U-2001 Hitachi. The dynamic light scattering (DLS) and zeta
potential measurements were performed with a Malvern Zetasizer
instrument. Conventional TEM images were obtained with a JEOL
100 CX microscope (TEM), operating at an acceleration voltage of
100 kV. For TEM observations we have used suspensions of the
nanoparticles deposited onto carbon-coated copper TEM grids.
Preparation of MnFe2O4 MNPs with Amine Groups on the Sur-
face (AmMNPs): MnFe2O4 MNPs with aminated surface (AmMNPs)
were prepared in an autoclave by the decomposition of acetylacet-
onate iron(III) and manganese(II) at a 2:1 ratio, Fe(acac)3 1.8 mmol/
Mn(acac)2 0.9 mmol in the presence solely of ODA 12.9 mmol. The
temperature of the oven was elevated with a steady rate (4 °C/min)
to 200 °C and was kept stable for 24 h. After the 24 h reaction the
autoclaves were left to cool down to room temperature with a rate
of 5 °C/min and MnFe2O4 MNPs were isolated after repeated wash-
ing cycles with EtOH and centrifugation (5000 rpm).
4. Experimental Section
Materials: All the reagents were used without any further purifica-
tion. Iron(III) acetylacetonate [Fe(acac)3] and octadecylamine
(> 90.0 %) were purchased by Fluka, while manganese(II) acetylacet-
onate [Mn(acac)2, ≥ 99.9 %] was supplied by Sigma-Aldrich. Ethanol
(100 %, 1 % MEK) was from Bruggermann GmbH and chloroform
(analytical reagent) from Chem. Lab NV. Diphenyl ether (DPE) and
ninhydrin (GR for analysis ACS, Reag. Ph Eur) were purchased by
Merck. Phosphate buffer pH 6.4 and 7.4 was prepared with NaH2PO4
(Merck), Na2HPO4 (Merck) and NaCl (Merck), while phosphate buff-
ered saline (PBS, pH = 7.2) was obtained by E.U Gibco BRL. 1-(3-
Direct Coupling of Acetylsalicylic Acid, Mefenamic Acid, and
Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, (EDC, Naproxen on AmMNPs: The direct coupling of NAP onto the
> 98.0 %, coupling agent for peptides) was purchased from TCI
(Tokyo Chemical Industry) and PEG8000 from Alfa Aesar GmbH &
AmMNPs surface coating was achieved via a modified procedure,[49]
where 20 mg of AmMNPs were mixed with EDC (47.9 mg), NHS
CoKG. Triethylamine (Et3N, ≥ 99.5 %), N-hydroxysuccinimide (NHS, (≈ 3 mg), ASA (45 mg), MEF (60 mg), NAP (57.5 mg) and Et3N
98.0 %) and acetylsalicylic acid (ASA) were from Sigma-Aldrich. (S)-
(0.1 mL) in CHCl3 (5 mL). The mixture was shaken vigorously for
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24 h and the AmMNPs-NSAIDs were isolated after repeated washing
cycles with EtOH and centrifugation.
Ninhydrin Colorimetric Assay: Aliquots of 0.1–0.6 mL of ODA
(0.25 mg mL–1) in DMF were pipetted into a series of tubes.[53]
0.7 mL of ninhydrin solution in MeOH 0.06
M
(10.7 mg mL–1) were
Preparation of Non-Aminated MnFe2O4 MNPs (Non-AmMNPs):
Non-aminated MnFe2O4 MNPs were prepared in an autoclave by
the decomposition of acetylacetonate iron(III) and manganese(II) as
described in case of AmMNPs, while in the amount of ODA
(0.998 mmol) and DPE (7 mL) was constant. The temperature of the
oven was elevated with a steady rate (4 °C/min) to 200 °C and
remained stable for 24 h. After the 24 h reaction the autoclaves
were left to cool down to room temperature with a rate of
5 °C/min and MnFe2O4 MNPs were isolated after repeated washing
cycles with EtOH and centrifugation (5000 rpm).
added in each tube, mixed well and heated in a water bath at
100 °C for 5 min. After a short heating period (5 min) the color of
the solution changed (formation of Ruhemann's purple) and the
content was transferred to a 5 mL volumetric flask and was diluted
with DMF for the UV/Vis absorbance measurement. A suspension
of MNPs in DMF (0.25 mg mL–1, stock) was prepared accordingly for
all the samples (AmMNPs, Non-AmMNPs before and after function-
alization) and 0.4 mL of each stock solution was pipetted into boil-
ing tubes with 0.7 mL of ninhydrin solution following the same
procedure described above. The absorbance of the formed complex
(Ruhemann's purple) was recorded at ≈ 600 nm.
Loading of Acetylsalicylic Acid, Mefenamic Acid, and Naproxen
on Non-AmMNPs: A 3 mL of solution of Non-AmMNPs in CHCl3
(5 mg mL–1) was added dropwise in a 25 mL of solution of PEG8000
in H2O (3.2 mg mL–1), the two-phase mixture was sonicated in a
cold sonication bath (25–30 °C) for ≈ 2 h until a stable emulsion
was formed (high temperature prevents the formation of a stable
emulsion). After ≈ 2 h, 40 mg of PEG8000 were added in the emul-
sion and sonication was applied for 30 min. The CHCl3 was evapo-
rated from the system with elevation of temperature of the sonica-
tion bath and then the solution was left to cool to room tempera-
ture. 120 mg of PEG8000 were added in the solution a new followed
by the addition of 2 mL of NSAIDs (8 mg of ASA, 10.5 mg of MEF,
10 mg of NAP) in CHCl3 (5 mg mL–1). The emulsion was sonicated
again for 2 h and CHCl3 was evaporated. The Non-AmMNPs@PEG-
NSAIDs was isolated through washing and centrifugation cycles
with EtOH and was dried under vacuum.
Evaluation of Anti-Inflammatory Activity
1. Evaluation of Lipoxygenase Activity: The reaction mixture con-
tained (final concentration) the MNPs (AmMNPs, Non-AmMNPs be-
fore and after functionalization with NSAIDs), dissolved in water
at concentrations of 2–50 μg/mL, or the solvent (control), soybean
lipoxygenase, dissolved in 0.9 % NaCl solution (250 mg mL– 1 final
concentration) and sodium linoleate (100 μM), in Tris/HCl buffer,
pH 9.0. The reaction was monitored for 9 min at 28 °C, by recording
the absorbance of a conjugated diene structure at 234 nm, due to
the formation of 13-hydroperoxy-linoleic acid. The performance of
the assay was checked using aspirin and naproxen as reference
drugs.
2. Albumin Denaturation Experiments: To evaluate the anti-in-
flammatory activity of MNPs and functionalized MNPs with ASA, a
modified assay studying bovine serum albumin denaturation was
implemented.[54] In detail, MNPs were mixed with 1 % w/v PBS solu-
tion of bovine albumin to reach final concentration of 5, 10, 25, 50
and 100 μg/mL. The mixtures were incubated at 37 °C for 20 min,
Drug Release: For the drug release study, ≈ 3 mg of the functional-
ized MNPs were placed into a Pur-A-Lyzer Midi 1000 dialysis mem-
brane. The membrane floated in 45 mL of phosphate buffer solution
(PBS pH 6.4 and 7.4) or buffer HCl/NaCl (pH 2.2) and was shaken
(200 rpm) at 35 °C. In certain time intervals 2 mL of the medium
were removed and replaced with fresh PBS solution. This procedure
was followed for both AmMNPs-NSAIDs and Non-AmMNPs@PEG-
NSAIDs, as well as for ASA, MEF and NAP. UV measurements were
recorded at 265 nm and the cumulative release in percentage (Q%)
and subsequently 200 μL of HCl 6
M were added to each sample,
while the temperature was raised at 51 °C and kept for 2 min to
prop up the denaturation of albumin. The turbidity of the solutions
was measured spectrophotometrically at 660 nm. ASA was used as
the reference drug. Denaturation of albumin in the presence of dis-
tilled water is considered as 100 % denaturation. The protection of
protein denaturation (%), hence the anti-inflammatory activity of
MNPs was calculated according to Equation (3):
was calculated and plotted against time according to Equation (1)16
:
(3)
Where OD refers to the turbidity values at OD660 nm and control
refers to the aqueous solution of 1 % bovine albumin in distilled
water.
(1)
where, A1 is the absorption of the first medium removal, A2 the
absorption of the second medium removal, An is the absorption of
the nth medium removal, As (absorption of the standard) for each
sample, V is the total volume of the medium (mL), C concentration
of the standard (mg mL–1), W weight of the active compound [mg
(label claim)], R removed medium volume (mL) and n is the removal
number. The release profiles were estimated by using Ritger–
Peppas equation [Equation (2)][50–52]
Acknowledgments
The authors would like to thank Dr. D. Sakellari for her assist-
ance in TEM imaging.
Keywords: Manganese · Magnetic properties ·
Nanoparticles · Biological activity · Functionalized
nanoplatforms · Drug delivery
(2)
where Q is the cumulative drug release in percentage, t is the re-
lease time in hours, k is a rate constant reflecting the structural
and geometric characteristics of the carriers, and n is the release
component that corresponds to the release mechanism of the drug.
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Nanoparticles for Drug Delivery
Magnetic nanoparticles (MNPs) func-
tionalized with nonsteroidal anti-in-
flammatory drugs (NSAIDs) can be
used for the combined diagnosis and
treatment of inflammation-correlated
diseases.
K. Giannousi, E. Koutroumpis,
V. Georgiadou, V. Karagkounis,
C. Dendrinou-Samara* .................. 1–10
Nanoplatforms of Manganese Fer-
rite Nanoparticles Functionalized
with Anti-Inflammatory Drugs
DOI: 10.1002/ejic.201801539
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