Formation of calcium phosphates nanoparticles in presence of carboxylate molecules: A time-resolved in situ synchrotron SAXS and WAXS study

Abstract

In this work we have studied in situ the formation and growth of calcium phosphate (CaP) nanoparticles (NPs) in presence of three calcium-binding carboxylate molecules having different affinities for Ca ²⁺ ...

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Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
Formation of calcium phosphates nanoparticles in presence of
carboxylate molecules: A time-resolved in situ synchrotron SAXS
and WAXS study
Dritan Siliqi,a Alessio Adamiano,b Massimo Ladisa,c Cinzia Giannini,a Michele Iafisco,*b and Lorenzo
Degli Esposti*b
In this work we have studied in situ the formation and growth of calcium phosphate (CaP) nanoparticles (NPs) in presence
of three calcium-binding carboxylate molecules having different affinities for Ca2+ ions: citrate (Cit), hydroxycitrate (CitOH),
and glutarate (Glr). The formation of CaP NPs at several reaction temperatures ranging from 25°C to 80°C was followed in
situ through simultaneous Small and Wide X-ray Scattering (SAXS/WAXS) using synchrotron light. SAXS was used to
investigate the first stages of NPs formation where a crystalline order is not yet formed. In this regard we have developed a
new bivariate mesh data analysis method for identifying the SAXS curves associated to the most relevant timeframes for
performing curve modeling. WAXS was used to study the formation of crystalline phases and their evolution over time. The
combined SAXS/WAXS data allowed us to track NPs nucleation, their size and morphology, and their evolution up to mature
hydroxyapatite (HA) nanocrystals. We have assessed that in the first stages of reaction (80 seconds) amorphous, elongated
primary NPs nucleate whose size and morphology depends on temperature and type of carboxylate molecule. Temperature
controls the release of Ca2+ ions from carboxylate molecules, and thus induces the formation of a higher amount of
amorphous particles and increases their size and aspect ratio. As reaction time progresses, amorphous particles evolve into
crystalline ones, whose kinetics of crystal growth are controlled by temperature and carboxylate ions. Stronger Ca-binding
carboxylates (CitOH  Cit  Glr) have a more pronounced inhibiting effect on HA crystallization, retarding the formation and
growth of crystalline domains, while a rise of temperature promotes crystallization. This work allowed us to shed more light
on the formation of HA in presence of growth-controlling molecules, as well as presenting the potentialities of combined
SAXS/WAXS for studying the formation of high relevance NPs for different applications.
Introduction
Calcium phosphate (CaP) nanoparticles (NPs) are widely studied
nanomaterials, as biogenic CaP NPs constitute the mineral
component of mammalian hard tissues (i.e. bones and teeth),
and synthetic ones are extensively used materials for bone
tissue engineering and nanomedicine.1
Their formation in vivo as well as in vitro proceeds through a
complex sequence of steps which involve reactions between
precursor ions, nucleation of ion clusters, and growth processes
that lead to mature NPs.2-4 Understanding CaP NPs formation
and crystallization is of critical importance to comprehend the
processes of bone formation and bone pathogenesis, as well as
to design tailored materials for nanomedicine, bone
regeneration, environment remediation, agriculture, and many
other applications.
With the recent advancement in X-ray scattering techniques, in
situ analysis during NPs formation can be performed, providing
important and new insights into reaction and growth
mechanisms. The use of these techniques allows (i) to probe the
nanomaterials at different size resolutions (i.e. atom scale,
particle scale, etc.), (ii) to investigate pre-nucleation clusters
and other metastable early products, and (iii) to study particle
properties such as size, morphology, and aggregation, as well as
the crystalline order.5
This approach gives the best results in combination with
synchrotron light, as the high radiation brilliance allows to
acquire data with high temporal resolution as well as low-
intensity signals, and thus is perfect for investigating the early
stages of NPs formation or subtle changes that might occur
during crystallization.5
The use of Small Angle X-ray Scattering (SAXS) allows to
evaluate particle size, morphology, and their distribution in the
population. Therefore it enables to follow the formation of
nuclei, their growth into particles, and particle maturation,
especially in the case of nanomaterials.6 Differently, Wide Angle
X-ray Scattering (WAXS) gives information on the atomic
ordering of the material, and thus describes the processes of
a.Institute of Crystallography (IC), National Research Council (CNR), Via Amendola
122/O, 70126, Bari, Italy.
b.Institute of Science and Technology for Ceramics (ISTEC), National Research
Council (CNR), Via Granarolo 64, 48018, Faenza, Italy. E-mail:
michele.iafisco@istec.cnr.it, lorenzo.degliesposti@istec.cnr.it
c. Istituto per le Applicazioni del Calcolo "Mauro Picone" (IAC), National Research
Council (CNR), Via Amendola 122/I, 70126, Bari, Italy.
Electronic Supplementary Information (ESI) available: SAXS and WAXS scattering
curves in function of time during the formation of the HA nanoparticles at 37 °C and
60 °C of reaction temperature (PDF). See DOI: 10.1039/x0xx00000x
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crystallization and crystal growth. Therefore, the combination
of Small and Wide X-ray Scattering (SAXS/WAXS) allows to fully
track the growth kinetics of crystalline NPs such as CaP NPs, thus
obtaining simultaneous structural and chemical insights ranging
from atomic to nanometric scale with high time resolution.5, 7, 8
Synchrotron light combined SAXS/WAXS was previously used to
study biogenic and synthetic CaP, but only as ex situ
measurements. 2, 9
Recently, we have investigated the formation of CaP NPs and
their crystallization into hydroxyapatite (HA, Ca10(PO4)6(OH)2)
nanocrystals in presence of three structurally similar calcium-
binding carboxylate molecules in terms of crystal growth,
chemical composition, and morphology.10 These molecules
were: citrate (Cit), which is a tricarboxylate anion involved in the
growth of HA nanocrystals during bone formation, and two
carboxylate molecules, namely hydroxycitrate (CitOH) and
glutarate (Glr), which share the same structure of Cit but bear
different functional groups in the central region.10
These polycarboxylates molecules complex Ca2+ ions in solution,
and de-complex them with heating, favoring a gradual CaP
particle formation;10, 11 in addition, they also attach to specific
HA NPs surfaces and control their morphology.2, 3, 12, 13
Our ex situ analysis of HA crystallized in presence of Cit, CitOH
or Glr at 80 °C showed that crystallinity, particle dimension and
morphology were controlled by the complexation strength of
carboxylate molecules with Ca2+, where CitOH complexes
calcium ions more strongly than Cit and thus has a higher
inhibition effect on HA crystallization, while Glr has a weaker
interaction and thus a weaker inhibition. Cit and CitOH seem to
induce the same crystallization pathway with the direct
conversion from an amorphous precursor into HA forming
platy, elongated nanocrystals, while Glr leads to the formation
of platelets of octacalcium phosphate (OCP, Ca8H2(PO4)6·5H2O)
as precursor phase that converts into HA nanorods.
Overall, the early steps of HA formation in presence of these
carboxylates remain unclear, as well as their crystallization
pathway at temperatures lower than 80°C. Ex situ
characterization techniques (powder X-ray scattering,
vibrational spectroscopy, etc.) do not allow to study HA
precursors (i.e. pre-nucleation clusters, metastable early NPs,
etc.) nor immature NPs due to their instability.4, 14 Therefore,
there is need of a comprehensive, in situ investigation of the
early formation of HA nanocrystals to understand the role and
mechanism of action of Cit and the other carboxylates during
HA nucleation and growth.
Herein, we present a time-resolved in situ study of the
formation and growth of HA in presence of Cit, CitOH, and Glr
using simultaneous SAXS/WAXS with synchrotron light to
investigate the instable early stages of particle formation and
crystallization, as well as using a new advanced method to verify
the ex situ results. SAXS/WAXS scattering curves were collected
in real time in situ during HA nucleation and growth in an
aqueous solution until particle crystallization was completed.
Several reaction temperatures ranging from 25°C to 80°C were
used to study the influence of temperature on HA nucleation
and crystallization, as well as on thermal decomplexation of
carboxylate-Ca2+ species.
Experimental
Materials
All reagents were purchased from Sigma Aldrich (St. Louis, MO,
USA) and were used as received; these include: calcium chloride
dihydrate (CaCl2·2H2O, ≥ 99.0%), glutaric acid (C5H8O4, ≥99.0%,
H2(Glr)), potassium hydroxycitrate tribasic monohydrate
(K3(C6H5O8)·H2O, ≥ 95.0%, (K3(CitOH)), sodium citrate tribasic
dihydrate (Na3(C6H5O7)·2H2O, ≥ 99.0%, (Na3(Cit)), sodium
hydroxide (NaOH, ≥ 99.0%), and sodium phosphate dibasic
dihydrate (Na2HPO4·2H2O, ≥ 99.0%).
Precursors solutions
Before the experimental data collection, calcium and phosphate
precursors solutions were prepared as reported by Degli Esposti
et al..10 The calcium precursors solution (hereafter called
Ca+carboxylate solution) contained CaCl2 100 mM + X 400 mM
(where X was either Na3(Cit), H2(Glr), or K3(CitOH)), and its pH
was set to 8.5 with NaOH 1M. After preparation Ca+carboxylate
solutions were stored at 4°C to avoid the formation of calcium
carboxylates salts and immediately before use left to
spontaneously warm up to 25°C. The phosphate precursor
solution (hereafter called PO4 solution) contained Na2HPO4 120
mM. After preparation, the solution was heated to 70°C to
completely dissolve Na2HPO4 and subsequently left to cool
down to 25°C before use.
Simultaneous synchrotron light SAXS/WAXS data collection of
real-time HA formation in situ
Simultaneous Small and Wide Angle X-ray Scattering
(SAXS/WAXS) patterns of HA formation in presence of
carboxylate molecules were recorded at the Austrian SAXS
beamline of the ELETTRA Synchrotron (Trieste, Italy), operated
at 2 GeV15 with a custom setup. The setup, which is shown in
Figure 1, allowed to investigate the growth and crystallization
of HA NPs in aqueous suspension in real time. In detail, the
setup consisted in a liquid flow circuit that comprised: (I) a glass
round-bottomed flask containing 12.5 mL of PO4 solution under
magnetic stirring and immersed in a thermostatic oil bath, (II) a
peristaltic pump that collects the liquid from the flask and sends
it to (III) the SAXS/WAXS sample holder, a ⌀ 2 mm special quartz
capillary (WJM-Glas Müller GmbH, Berlin-Pankow, DE) inserted
in a steel frame and placed orthogonally to the X-Ray beam.
Outside of the circuit, there was (IV) a 25 mL syringe containing
12.5 mL of Ca+carboxylate solution; the syringe was placed in a
syringe pump and connected to the reaction flask. All
components of the circuit were connected to each other by
using ⌀ 1.65 mm PVC tubing. The liquid flow was set to 10 mL
min-1. Incident beam energy was set at 8 keV and allowed to
measure the SAXS range from a q-value of 0.08 to ca. 6 nm−1,
while the WAXS detector allowed to measure the range from a
q-value of 15 to ca. 28 nm−1. The 2D SAXS scattering image was
collected with a Pilatus 1 M detector (Dectris, CH), while a
Pilatus 100k detector (Dectris, CH) was used to simultaneously
acquire the 2D WAXS pattern. As first step, PO4 solution filled
the circuit and was left to recirculate at 10 mL min-1 flow until
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all the fluid in the circuit reached the temperature set in the
reactor, which was either 25°C, 37°C, 60°C, or 80°C. Before the
beginning of HA precipitation experiment, the X-rays beam was
sent to the sample and the SAXS/WAXS 2D scattering images of
the sample holder containing a flow of PO4 solution were
collected to be used for background subtraction. Afterwards,
while SAXS/WAXS 2D scattering images were collected
continuously with a time resolution of 6 seconds (4 s of
exposition time and 2 s interval) the Ca-carboxylate solution
was injected in the reactor with a flow of 25 mL min-1 monitoring
continuously the evolution of SAXS/WAXS curves during particle
precipitation. The data collection continued until no further
evolution of SAXS/WAXS curves was observed, ranging from 1
hour to 14 hours depending on experimental conditions. In the
case of long data collections (> 1.5 h), exposition time was
incremented, and time resolution was decreased to improve
the signal-to-noise ratio. Every data collection was named
based on the carboxylate used (Cit, CitOH, or Glr) and on the
reaction temperature (25°C, 37°C, 60°C, 80°C). In the case of
CitOH, data collection was performed only at 25°C and 80°C due
to limited available beamtime and extremely slow
crystallization kinetics due to strong growth inhibition exerted
by CitOH (see the results reported below).
SAXS/WAXS data analysis and modelling
The 2D SAXS images were normalized by transmission values
and circularly averaged to be converted into conventional SAXS
curves. This step was performed with the software Fit2D
available at the beamline.16 Therefore, the background was
removed with the software Igor Pro (WaveMetrics, Inc., Lake
Oswego, OR, USA). SAXS data analysis and modeling were
performed with ATSAS package V3.0.317 and SasView package
V5.0.3 (www.sasview.org).
SAXS data modeling was performed, first by estimating overall
parameters such as radius of gyration (Rg) and maximum size of
NPs (Dmax), and then using an ellipsoid model18 or a custom
model as a combination of ellipsoid18 and cylinder19 models.
Schulz distribution was used to estimate average particles size
as well as the polydispersity of samples for the selected
structural model.20, 21
The 2D WAXS images were normalized and then circularly
averaged, producing one-dimensional curves, which underwent
background subtraction. In order to improve the signal-to-noise
ratio, WAXS curves collected in short timeframes were
averaged. Data analysis was performed with the software
TOPAS5.22 A full-profile peak broadening analysis was
conducted on the (002) peak of HA, calculating HA c unit cell
parameter and the dimensions of crystalline domains along HA
(002) directions (D(002)). For all analyses, the baseline of the
patterns was calculated through the Chebychev function (16th
order polynomial).
Results and discussion
SAXS
SAXS time-resolved curves were collected continuously before
(frames 0-10, corresponding to 60 s) and after the introduction
of Ca-carboxylate solution into the PO4 solution, which was set
at the defined reaction temperature. Figures 2 and S1 show an
overview of the evolution over time of scattering patterns for
the various carboxylate/temperature combinations. Before Ca-
carboxylate addition there is no scattering except for the
solvent and sample holder, which is removed by background
subtraction, generating a flat curve. After injection of calcium
precursors solution, in all cases there is a sudden increase of the
curve at q < 0.5 nm-1 that indicates the formation of scattering
particles. With time, in all samples there are subtle changes of
curve shape, which suggest variations in particles morphology
and aggregation. An example is the slight decrease of the
scattering intensity at the range near to q ≈ 0.1 nm-1 that is then
recovered with time for all samples, but with different kinetics
depending on the nature of carboxylate and reaction
temperature. This variation might suggest the occurrence of
particle-particle interactions that give rise to a structure factor
contribution to the scattering, whose kinetics is temperature-
and carboxylate-dependent. Another example is the change of
slope at 0.2-0.4 < q < 2 nm-1 that occurs for Cit/60°C, Cit/80°C,
CitOH/80°C, and Glr (all temperatures), which could be
associated to a change in particles morphology and/or the
formation of a polydispersion of particle morphologies (see
below). To gain insight into SAXS data, we choose to organize
the data on a bivariate mesh (Figure 3), where each pixel
represents the integral intensity of the corresponding one-
dimensional plot (as in Figure 2) for a certain time and
temperature (respectively x- and y-axes). A pre-processing step
to merge the different timescales and to normalize the
intensities values was performed.
In the case of Cit, the first signal occurs within 80 s from the
injection of Ca-carboxylate solution at every temperature
(Figure 3A). The overall intensity at the beginning of particle
formation is directly correlated to increase of reaction
temperature. These results suggest that the nucleation of
primary CaP particles is controlled by the thermal decomplexing
of Cit-Ca species, which gradually increase the concentration of
free Ca2+ available for nucleation.11 Cit/25°C and Cit/37°C
meshes do not show a significant change of the integrated
intensity over time, while for both Cit/60°C and Cit/80°C there
is a strong increase of integrated intensity respectively at 2400
- 3000 s (40 - 50 min) and 600 - 1200 s (10 - 20 min), respectively.
CitOH has a similar behavior to Cit (Figure 3B), without
significant variations of the integrated intensity over time at
25°C, while at 80 °C, changes occurring in the range 900 - 1800
s (15 - 30 min) clearly show the influence of temperature.
Finally, for Glr at all temperatures the first increase of intensity
happens after 80 s similarly to the other carboxylates (Figure
3C). At high temperatures, there is a gradual increase of
intensity that occurs at 900 s (15 min, 60°C) or 300 s (5 min,
80°C), while at lower temperatures the intensity is immediately
higher but decreases after 180 s (3 min). A similar trend of the
intensity can be observed also in Glr/60°C but occurs in a short
timeframe at ca. 90 s (1.5 min) of reaction. This behavior can be
attributed to the fast particles formation, in which Glr-HA NPs
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quickly grow outside SAXS measuring range and thus give a
decrease of integrated intensity (see below for a detailed
explanation). This out-growing phenomenon occurs at every
reaction temperature, but is faster at higher temperatures, and
thus at 80°C is so fast to be non-detectable with the current
time resolution (ca. 10 s).
Based on the bivariate mesh maps, relevant frames
corresponding to significant changes in integrated intensity
were identified and underwent SAXS curve modeling. The
relevant frames were: for Cit, 80 s for all temperatures, 2400
and 3400 s for 60°C and 600 and 1200 s for 80°C. For CitOH, 80
s for both temperatures, 900 and 1800 s for 80°C. For Glr, 80 s
for all temperatures, 150 s for 25°C and 37 °C, 600 s for 60°C,
and 290, 390, and 600 s for 80°C (Figure 4).
Interestingly, for Cit/80°C SAXS curves continue to evolve up to
1200 s (20 min), while for CitOH/80°C the evolution was longer
and concludes at 1800 s (30 min), and for Glr/80°C it ended at
600 s (10 min). This difference in the duration of evolution is a
confirmation of the results of our previous work, in which we
have shown that CitOH is a stronger HA growth inhibitor than
Cit – and hence particle growth requires more time – while Glr
is the weakest.10
For the selected frames reported above, in the first step a
preliminary analysis of NPs size was performed by estimating
the radius of gyration (Rg), which is calculated from the slope of
Guinier plot of SAXS curve,23 and the maximum size of the NPs
(Dmax) which can be obtained by means of the Pair-Distance
Distribution Function [P(r)] by using the Indirect Fourier
Transformation as described by Glatter et al.24 (Tables 1-3).
Both those parameters were evaluated by using AUTORG25 and
GNOM26 programs of the ATSAS package,17 taking into account
a monodisperse system. Afterwards, to estimate NPs shape the
experimental curves were fitted with an ellipsoid model,
refining both model parameters and the associated
polydispersity by using SasView program (Tables 1-3). In this
model the parameters are Rpolar, that is radius along the y-
coordinate, and Requatorial representing the radius along the x-
coordinate. Due to the high shape polydispersity of the NPs at
late stages of maturation at high reaction temperature,
especially for Glr particles (where, in addition, occurs also the
conversion of OCP platelets into HA particles), the classical
ellipsoid model did not allow to obtain satisfactory fits,
therefore we decided to use a custom shape-polydisperse
model made by the combination of an ellipsoid model with a
cylindrical model, which is defined by its radius (Rcyl) and its
length (Hcyl). In this way, we were able to estimate
approximately the relative abundance of each shape in the
mixture of the particles, which is expressed as their normalized
Scale factor (expressed as % in Tables 1-3). The polydispersity of
the refined parameters, reported in the bracket near the data
in Tables 1-3, was evaluated as a ratio between the standard
deviation and the mean value.
In presence of Cit (Table 1), a Rg value of ca. 22 nm for HA NPs
is obtained at every maturation time and temperature,
suggesting that from the first moments of precipitation the Rg
of particles does not change for all the experimental conditions.
On the other hand, at higher temperatures the Dmax values
become progressively higher, indicating a temperature-
controlled particle growth. Together, these two parameters
suggest that NPs population is constituted on average by
particles comprised between 20 and 70 nm. Regarding particles’
morphology, the ellipsoid model for Cit/25°C, Cit/37°C, and the
early stages of Cit/60°C SAXS curves (shown in Figure 4A)
describes a particle long ca. 34 nm along one axis and 62 nm
along the other axis, i.e. an elongated particle with a ratio
between major and minor dimension of ca. 2, in agreement with
our previous ex situ electron microscopy data, which described
Cit-HA as elongated platy NPs ca. 70 nm in length, 30-40 nm
wide, with a thickness of ca. 3-4 nm.10 Unfortunately, it was not
possible to model NPs thickness, as in this experimental setup
SAXS is an ensemble measurement of free-moving particles in a
flowing suspension, and thus all dimensions are averaged
together and the small contribution of thickness is negligible. At
higher maturation times for Cit/60°C and for Cit/80°C there is a
progressive evolution in curve shape (Figure 4A) that is modeled
as a decrease of Rpolar parameter up to values of 1-2 nm together
with the introduction of shape polydispersity to have a suitable
fit. These changes suggest that particles become progressively
thinner in function of time at high reaction temperatures. For
these cases, our previous ex situ study proved that NPs grow
preferentially along their main axis incrementing their aspect
ratio (i.e., length/width ratio). These modifications are reflected
in SAXS modeling parameters, as particle length goes outside
the measured SAXS range, i.e. below a q-value of 0.08 nm−1
corresponding to ca. 100 nm. Consequently, the contribution of
particles’ thickness to scattering becomes more relevant, and
thus particle width and thickness are modeled as ellipsoids and
cylinders. On the other hand, Rg and Dmax values remain
unaffected as they depend only on the average measurable
particle dimensions. The similarity between ellipsoidal and
cylindrical model parameters (Rpolar and Requatorial vs Rcyl and Hcyl,
see Table 1) suggests that in the polydisperse particle
population the overall morphology is similar (i.e. elongated
particles), but individually the particle size may vary.
In conclusion, particle morphology modeling proved that at low
reaction temperatures particle shape is in agreement with
average dimensions estimated by Rg and Dmax, while at higher
temperatures the formation of highly anisotropic thin and
elongated particles occurs. This change of morphology is almost
concomitant with crystallization (see below), suggesting that
the progressive increase of temperature which induced the
particle growth is associated with the formation of the
crystalline phase.
Crystallization of HA NPs in presence of CitOH was studied at 25
and 80 °C (Table 2). All Rg and Dmax values are comparable to Cit,
indicating a similar particle size distribution on average, while
polydisperse shape modeling parameters are different. Indeed,
their Requatorial values are independent from time and reaction
temperature and are similar to Cit values, while Rpolar ones are
shorter – indicating a diameter of ca. 20 nm – and only the
cylindrical sub-population decreases at 80°C. This difference
between CitOH and Cit confirms that for CitOH particles’ growth
is more inhibited, with smaller particles that do not elongate at
higher reaction temperatures as also proved by the more
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limited change in CitOH/80°C SAXS curves reported in Figure 4B.
It must be considered that all values here reported for
CitOH/80°C are bigger than the ones previously reported by ex
situ analysis. This discrepancy might be due to post-synthesis
product work-up necessary for the ex situ characterizations.
SAXS modelling parameters of HA crystallization in presence of
Glr are reported in Table 3. For Glr/25°C and Glr/37°C both Rg
and Dmax as well as Rpolar and Requatorial values are comparable to
Cit (as also shown by similar SAXS curves reported in Figure 4C).
At higher reaction temperatures curve shape changes (Figure
4C), which leads to a lower Rg and the need to use polydispersity
of shapes in the fitting. Similarly to Cit, also for Glr at 60 and 80
°C particle’s length falls outside probed SAXS range and
particle’s thickness is modeled with the Rpolar/Rcyl values. This
suggests an influence of temperature on particle evolution
similar to Cit, where higher reaction temperatures induce the
formation of highly anisotropic particles in concomitance to
crystallization. However, the ex situ characterization pointed
out that at 80°C and < 300 s of maturation the sample is
constituted by sub-micrometric OCP platelets, whose
dimensions fall outside SAXS scattering range.15 Therefore, it is
likely that values modeled by in situ SAXS curves correspond
only to the fraction of smaller particles in the system.
WAXS
Complementary to SAXS data analysis, WAXS patterns were
collected to study the crystallization of the particles observed
by SAXS (Figures 5 and S2). In the case of precipitation in
presence of Glr at any temperature and for Cit/80°C several
diffraction peaks appear with time, which were all indexed as
HA (powder diffraction file 00-009-0432). Our previous work
evinced that in presence of Glr also OCP phase (powder
diffraction file 00-026-1056) forms at short crystallization times
as a precursor of HA,10 so we hypothesize that the same occurs
here but the low intensity of WAXS peaks and the high
superposition between HA an OCP peaks does not allow to
confirm it. For Cit/25°C and CitOH/25°C there are no signals of
crystallization at every time point, in the case of Cit/37°C,
Cit/60°C, and CitOH/80°C the formation of a single diffraction
peak can be observed at 18.1 nm-1, which was indexed as the
(002) peak of HA. This peak commonly has a high relative
intensity and is well resolved; its position is associated with HA
crystallographic c-axis and its broadness is directly proportional
to the main axis of elongated HA nanocrystals.10 The
appearance of this peak suggests that in presence of a weak
inhibitor such as Glr or at higher precipitation temperatures – T
≥ 37°C (Cit) or T ≥ 80°C (CitOH) – particles crystallize into HA,
while in presence of strong inhibitors and at low temperatures
particles remain amorphous or form nanocrystals that are too
small and poorly crystalline to be detected by WAXS analysis.
From the variations in broadness and position of (002) peak at
18.1 nm-1 we have calculated the time evolution of D(002)
crystalline domain as well as the variation of HA c cell axis
(Figure 6) for the conditions where HA formation was detected.
It must be mentioned that the (002) peak has an anomalous
broadness, as it appears to be very narrow and with a peak
broadness almost comparable to instrumental peak
broadening. It is likely that in the capillary sample holder the
high liquid flow rate, and thus shear rate, oriented the
anisotropic HA NPs aligning their main axis (c-axis) with the flow
direction. The consequence of this effect is that the scattering
of (002) peak is enhanced by preferential orientation; thus, the
instrumental response function of the experimental apparatus
could not be calculated and the broadness of the (002) peak
could not be correlated to a numeric value of crystalline domain
size. Therefore, D[002] crystalline domain values are expressed as
arbitrary units and only their trends over maturation time are
discussed.
The size of D(002) crystalline domains of the samples and their
evolution over time and temperature are reported in Table 4
and Figure S3. Crystalline domain size depends both on the
nature of the carboxylic molecule as well as on the temperature.
For Cit, (Table 4 and Figure S3A,D) the appearance of the
crystalline phase occurs at progressively shorter times in
function of temperature, ranging from no crystallization over
170 min at 25°C to the formation of HA phase after 3 min of
reaction at 80°C. In addition, also the evolution of the crystalline
domain size is directly correlated to the reaction temperature.
Indeed, at 80 °C there is a burst growth of D(002) in less than 50
min, while at 60°C the crystalline domain is ca. half smaller than
that at 80 °C and has no change over time. At 37°C the D(002)
crystalline domain size is again the half in comparison to the one
at 60 °C and slowly grows in ca. 100 min. These results are in
agreement with SAXS data on the formation of Cit-HA
nanocrystals. Indeed, SAXS proved that Cit-HA nucleation is
controlled by the thermal decomplexation of Cit-Ca species in
solution, while WAXS proved that reaction temperature
accelerates the conversion of the first amorphous NPs into
nanocrystals, and the subsequent crystal growth into bigger
particles.
A similar situation occurs for CitOH, which is a stronger
crystallization inhibitor than Cit, as HA is formed only at 80 °C
after ca. 40 min of maturation, and the D(002) crystalline domain
is ca. from two to four-fold smaller than D(002) of Cit at the same
reaction temperature (Table 4 and Figure S3B,E). During
maturation CitOH-HA crystalline domain size doubles in ca. 110
min, and after 150 min of reaction no changes occur anymore.
For Glr, which is the weakest crystallization inhibitor,
temperature has less influence as HA/OCP phase was always
detected in the first moment of reaction independently from
reaction temperature, and always with similar values of
crystalline domain size (Table 4 and Figure S3C,F). In details, the
evolution of D[002] crystalline domain size at 80 °C is the same
for Glr and Cit systems (curves are superimposed), while at 60°C
and 37°C there is almost no change over time. The
independence of HA formation from reaction temperature
suggests a lesser relevance of thermal decomplexation for the
crystallization in presence of Glr. It is particularly interesting the
evolution of Glr/25°C, as the growth of crystalline domain
progresses in discrete steps, suggesting a different evolution
mechanism, probably related to OCP formation and subsequent
transformation into HA.
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The variations of HA c cell axis over time (Table 4 and Figure S4)
are less correlated to carboxylic acid and precipitation
temperature than the D(002) crystalline domain. For all the
carboxylates at 80 °C a cell expansion from ca. 6.92-6.93 Å to
6.945 Å occurs over time. These values are slightly higher than
the ones calculated by ex situ crystallization (ca. 6.87 Å), but this
difference is not so relevant as a slight expansion of the c cell
parameter with maturation in the reaction solution has been
reported previously.10, 27 At lower reaction temperatures this
cell parameter remains constant at ca. 6.93 Å for Cit and CitOH,
while in presence of Glr it decreases to ca 6.91 Å, and the speed
of this decrease is directly correlated with temperature. This
suggests that for Glr at temperatures < 80°C a different process
occurs in comparison to the other carboxylates. A tentative
interpretation for the decrease of the c-axis (i.e., the shift of the
(002) peak at higher q) can be due to a progressive formation
and evolution of OCP phase at lower reaction temperature,
while at 80°C OCP quickly transforms into HA and thus the peak
shifts in the opposite direction. In fact, the (002) peak situated
at 18.2 nm-1 for the Glr system could be composed by the
overlapping of HA (002) peak (theoretical position 18.07 nm-1)
and OCP (002) peak (theoretical position 18.35 nm-1).
Overall, considering our SAXS/WAXS data and the previous
works on the crystallization of HA in presence of Cit, we
hypothesize that HA nanocrystals formation follows a 4-step
process: (i) Cit anions bind partially calcium ions, forming Ca-Cit
complexes. Then, (ii) when put in contact with phosphate ions
there is an immediate nucleation of non-crystalline CaP
clusters/nuclei independently from reaction temperature.
These amorphous particles (observed within 1 min after mixing)
are anisotropic and are ca. 50-60 nm long. With time (iii)
particles grow and elongate, preferentially along their main
axis. Finally, (iv) HA crystalline phase nucleates and grows within
the amorphous particles, as described by Delgado-Lopéz et al.
and Iafisco et al..2, 28 Depending from reaction temperature,
steps (iii) and (iv) might be consequential (< 60 °C) or
simultaneous (80 °C). The temperature influences nucleation
rate, crystal formation rate, and particles/crystals growth
speeds. Regarding the influence of carboxylate molecules on
the formation of nanoparticles, a stronger complexation (CitOH)
does not change the development of early particles, but the
process is slowed down, smaller, and less anisotropic particles
are formed, and the formation of the crystalline phase is
inhibited. The weaker Ca-carboxylate interaction of Glr gives
fewer clear results, due to the simultaneous occurrence of
several phenomena, i.e. particle precipitation, OCP formation,
and OCP conversion into HA.
Conclusions
We have studied in situ the formation and growth of CaP NPs in
presence of calcium-binding dicarboxylate molecules at several
reaction temperatures through simultaneous SAXS/WAXS using
synchrotron light.
SAXS investigation was focused on the early stages of particle
formation, proving that both the nature of carboxylate
molecules and reaction temperature had a strong influence on
the amount, size, and morphology of first-nucleated NPs. The
structural features of the molecules controlled the Ca2+-
carboxylate complexation and thus ion supersaturation and
association to nanoparticles, while temperature influenced the
decomplexation rate.
In particular, SAXS data and its modeling showed that: (i)
nucleation of amorphous particles is almost immediate
independently from reaction temperature and carboxylate
molecules; (ii) at low temperatures the first-formed particles
have similar morphology and dimensions (elongated particles
ca. 50-60 nm in length and 20-30 nm in width) for Cit and Glr,
while with CitOH morphology is similar but width is smaller; (iii)
at high temperatures particles quickly elongate and become
polydisperse and with heterogeneous morphology.
WAXS data gave complementary insights on the transformation
of the amorphous particles in the HA nanocrystals, showing that
the formation of crystalline order is temperature- and
carboxylate-dependent. In presence of Cit and CitOH the
crystallization to HA occurs between 180 s and 7200 s (120 min)
at 80° and 37°C, respectively, while at 25°C crystallization does
not occur. Moreover, the combination of SAXS and WAXS data
shows that crystallization is simultaneous to particle elongation.
Finally, the CaP formation in presence of Glr follows a different
mechanism as at reaction temperatures < 60°C the OCP phase
dominates and grows in a temperature-dependent fashion,
while at 80°C there is the conversion of OCP into HA phase.
To the best of our knowledge, the present study is the first
report on the use of simultaneous SAXS/WAXS with synchrotron
light for studying the formation of CaP NPs. By using custom
data analysis tools, we have shed more light on the influence of
growth inhibitor molecules on HA nucleation and growth,
showing the relations between carboxylate-Ca2+ interaction,
temperature, and NPs formation. This study proves the
potentialities of SAXS/WAXS as an innovative approach for the
detailed characterization of nanomaterials, which could be
extended to several types of biomimetic and bio-inspired
nanomaterials for advanced applications.
Author Contributions
D. Siliqi: Investigation, Writing - original draft, Writing - review
& editing. A. Adamiano: Investigation, Writing - review &
editing. M. Ladisa: Investigation, Visualization. C. Giannini:
Investigation, Writing - original draft, Writing - review & editing.
M. Iafisco: Conceptualization, Writing - original draft, Writing -
review & editing, Supervision. L. Degli Esposti: Investigation,
Visualization, Writing - original draft, Writing - review & editing,
Funding acquisition.
Conflicts of interest
There are no conflicts to declare.
Funding Sources
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This work was supported by the CERIC-ERIC Consortium for
financial support and for giving access to ELETTRA SAXS
beamline (proposal CERIC-ERIC-20175403).
Acknowledgements
The authors acknowledge Dr. Heinz Amenitsch for the scientific
and technical support during SAXS data collection.
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Table 1. Overall particle size parameters (Rg and Dmax) and fitted ellipsoid-cylinder model parameters for HA NPs synthesized in presence of Cit at the most significant timepoints
Temperature (°C)
Time
(s)
Rg | Dmax (nm)
Scale (%)
Model parameters
Ell. | Cyl.
Ellipsoid model
Cylinder model
Rpolar | Requatorial (nm)
Rcyl | Hcyl (nm)
25
80
22.5 | 65.1
100
15.9 (0.2) | 31.5 (0.0)
-
37
80
23.4 | 73.9
100
17.2 (0.2) | 31.7 (0.7)
-
80
23.2 | 67.9
100
17.3 (0.2) | 31.9 (0.1)
-
2400
19.9 | 65.2
67 | 33
2.2 (0.1) | 32.3 (0.1)
4.4 (0.1) | 54.4 (0.0)
60
3400
22.9 | 71.4
64 | 36
4.6 (0.2) | 42.2 (0.1)
22.2 (0.1) | 63.9 (0.0)
80
23.3 | 77.3
100
0.2 (0.1) | 35.1 (0.0)
-
600
21.8 | 74.7
67 | 33
0.9 (0.5) |33.4 (0.1)
1.5 (0.0) | 55.8 (0.4)
80
1200
21.3 | 63.5
79 | 21
0.9 (0.0) |32.5 (0.0)
1.4 (0.0) | 54.9 (0.0)
Table 2. Overall particle size parameters (Rg and Dmax) and fitted ellipsoid-cylinder model parameters for HA NPs synthesized in presence of CitOH at the most significant timepoints
Temperature (°C)
Time
(s)
Rg | Dmax (nm)
Scale (%)
Model parameters
Ell. | Cyl.
Ellipsoid model
Cylinder model
Rpolar | Requatorial (nm)
Rcyl | Hcyl (nm)
25
80
20.5 | 67.3
64 | 36
9.9 (0.1) | 29.7 (0.0)
2.9 (0.0) | 50.9 (0.0)
80
20.1 | 66.0
70 | 30
3.5 (0.1) | 26.7 (0.0)
4.8 (0.0) | 30.2 (0.0)
900
21.4 | 65.3
42 | 58
10.4 (0.1) | 30.5 (0.0)
1.2 (0.0) | 53.1 (0.0)
80
1800
21.6 | 65.1
32 | 68
12.5 (0.1) | 29.1 (0.0)
3.5 (0.0) | 51.9 (0.0)
Table 3. Overall particle size parameters (Rg and Dmax) and fitted ellipsoid-cylinder model parameters for HA NPs synthesized in presence of Glr at the most significant timepoints
Temperature (°C)
Time
(s)
Rg | Dmax (nm)
Scale (%)
Model parameters
Ell. | Cyl.
Ellipsoid model
Cylinder model
Rpolar | Requatorial (nm)
Rcyl | Hcyl (nm)
80
23.1 | 67.6
100
14.3 (0.3) | 33.4 (0.0)
-
25
150
23.2 | 68.6
100
15.4 (0.4) | 34.0 (0.0)
-
80
24.4 | 74.0
100
15.6 (0.2) | 33.1 (0.2)
-
37
150
22.7 | 66.1
100
12.5 (0.5) | 32.4 (0.0)
-
80
19.8 | 60.6
71 | 29
10.2 (0.1) |38.4 (0.1)
10.4 (0.0) | 260.0 (0.0)
60
600
16.2 | 54.5
92 | 8
1.1 (0.0) | 24.5 (0.3)
7.6 (0.0) | 62.3 (0.0)
80
19.1 | 65.9
35 | 65
3.9 (0.1) | 28.2 (0.0)
2.2 (0.0) | 18.3 (0.0)
290
16.8 | 56.9
91 | 9
2.6 (0.0) | 24.6 (0.0)
8.6 (0.0) | 14.3 (0.0)
390
17.2 | 60.2
76 | 24
2.1 (0.0) | 24.4 (0.0)
2.4 (0.0) | 89.1 (0.1)
80
600
17.4 | 59.4
79 | 21
2.4 (0.2) | 24.7 (0.0)
2.4 (0.0) | 35.5 (0.0)
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Table 4. Crystalline domain D[002] and unit cell parameter c of the precipitate in function of carboxylate, temperature, and time.
Sample
Temperature (°C)
Appearance of
(002) peak (min)
Time evolution of D[002]
Initial c parameter
(Å, min)
Final c parameter
(Å, min)
25
-
-
-
-
37
115
155% increase from 115 to 195m
6.930, 150m
6.935, 500m
60
60
20% increase from 60 to 130m
6.933, 60m
6.933, 130m
Cit
80
3
100% increase from 3 to 120m
6.934, 3m
6.945, 120m
25
-
-
-
-
CitOH
80
37
60% increase from 37 to 150m
6.931, 40m
6.943, 150m
25
4
150% increase from 4 to 800m
6.918, 4m
6.902, 800m
37
4
10% increase from 4 to 50m
6.921, 4m
6.910, 50m
60
2
15% increase from 2 to 50m
6.923, 2m
6.913, 50m
Glr
80
4
100% increase from 4 to 120m
6.923, 4m
6.944, 200m
Figure 1. Schematic representation of the setup employed for real-time in situ SAXS/WAXS measurements during the formation of HA nanoparticles. The setup measures SAXS and
WAXS simultaneously in the same sample volume.
Figure 2. Examples of stack-plots of the measured SAXS curves as a function of time during the formation of the HA nanoparticles in presence of different carboxylate molecules and
reaction temperatures.
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Figure 3. Bivariate mesh of the integrated SAXS intensity as function of time and temperature for each carboxylate molecule used. (A) Cit, (B) CitOH, and (C) Glr.
Figure 4. SAXS curves of the selected timeframes corresponding to significant changes in integrated intensity used for SAXS curve modelling for (A) Cit, (B) CitOH, and (C) Glr
crystallization.
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Figure 5. Examples of stack-plots of the measured WAXS curves as a function of time during the formation of the HA nanoparticles in presence of different carboxylate molecules
and reaction temperatures.
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