International Journal of Pharmaceutics 565 (2019) 95–107

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International Journal of Pharmaceutics

journal homepage: www.elsevier.com/locate/ijpharm

Atorvastatin loaded PLGA microspheres: Preparation, HAp coating, drug T

release and effect on osteogenic differentiation of ADMSCs
⁎ ⁎
Fatemeh Shokrolahia, , Khosrow Khodabakhshib, Parvin Shokrollahia, , Roya Badiania,
Zohreh Mansoori Moghadama
a
Department of Biomaterials, Iran Polymer and Petrochemical Institute, Iran
b
Department of Process Modeling and Control, Iran Polymer and Petrochemical Institute, Iran

A R T I C LE I N FO A B S T R A C T

Keywords: Polymer/bioceramic composite micro-particles have been used for bone regeneration in order to address weak
PLGA microsphere mechanical properties/bioactivity of polymers and to enable easy filling of irregular bone defects through
Bioactivity minimally invasive injection procedure. The purpose of this study was to determine whether injectable apatite-
Atorvastatin coated atorvastatin (AT) loaded Poly (d,l-lactide-co-glycolide) (PLGA) micro-particles can support osteogenic
Osteogenic differentiation
differentiation of adipose derived mesenchymal stem cells(ADMSCs). Particle preparation conditions (oil-in-
Bone tissue engineering
water (O/W) emulsion), were carefully adjusted to yield uniform particles of about 20–50 µm in diameter.
Taking a solid in oil-in water (S/O/W) emulsion strategy, it became possible to load atorvastatin (10 wt%) in the
micro-particles without deformation. The particles were then coated with HAp by incubation in 10X simulated
body fluid (SBF). The apatite coating layer was similar to apatite in natural bone, as demonstrated by SEM, XRD,
and FTIR analyses. Adipose derived mesenchymal stem cells (ADMSCs), were cultured on the micro-particles and
calcium deposition measurement was performed through Alizarin Red assay. Initial cell adhesion did not differ
significantly between the samples and the control. The strongest osteogenic differentiation was observed on
PLGA-AT-HAp in both the osteogenic and non osteogenic culture media, while PLGA-AT slightly decreased and
PLGA-HAp slightly increased osteogenic differentiation of the cells, indicating suitability of PLGA-AT-HAp as an
injectable tissue engineering system.

1. Introduction medium that, in addition to micro-particles, contains cells and their

Nowadays, replacement and repair of damaged body tissues is of
increasing interest. Among different treatments, bone reconstruction
has attracted much attention. Demand for bone reconstruction, has led
to development of tissue engineering systems (scaffolds, hydrogels…),
that ideally stimulates the injured tissue to self repair (Bonassar and
Vacanti, 1998). Bone tissue engineering is not limited to the use of
composite scaffolds, but the use of bone-making drugs, growth factors
and bioactive proteins alongside scaffolds can encourage bone forma-
tion and improve the healing process efficiently (Nath et al., 2013).
Among various systems that act as carriers of such active substances,
biodegradable polymeric micro-particles incorporated with small mo-
lecule drugs, proteins, and nucleic acids have attracted much attention
and showed noticeable benefits. In addition to topical drug delivery and
reducing the amount of drug used, the active ingredient carrier micro-
particles are easily injected by a syringe (injectable tissue engineering
systems). Injectable micro-particle systems can consist an aqueous
nutrients (Oh et al., 2006; Bose and Tarafder, 2012).
In fabrication of active material carrier micro-particles, polymer
type, molecular weight, degradation mechanism, as well as micro-par-
ticle size and type of drug are among the factors that affect tissue re-
generation. The type of polymer used and the mechanism of degrada-
tion are the first factors to be considered. Polymers with mass
degradation mechanism such as PLGA, allow water penetration into the
polymer matrix and, as a result, hydrolytic degradation easily occurs in
the entire micro-particle matrix (Anderson and Shive, 2012).
The size of micro-particles also affects release rate of the drug. By
decreasing the particle size, surface area and mass transfer increase for
a fixed mass of drug and polymer. Besides that particle size puts a
certain limitation on injectability of the system and must be taken
seriously in tissue engineering applications. Furthermore, needle gauge
will affect the injectability of the system and must be chosen in such a
manner as to effectively deliver the composite to the appropriate ana-
tomical location with a reasonable amount of force needed for

⁎
Corresponding authors.
E-mail addresses: f.shokrolahi@ippi.ac.ir (F. Shokrolahi), p.shokrolahi@ippi.ac.ir (P. Shokrollahi).

https://doi.org/10.1016/j.ijpharm.2019.05.005
Received 10 January 2019; Received in revised form 22 April 2019; Accepted 4 May 2019
Available online 07 May 2019
0378-5173/ © 2019 Elsevier B.V. All rights reserved.
F. Shokrolahi, et al.
injection.
Kang et al., have prepared PLGA microspheres as an injectable
system for cartilage tissue engineering. They have shown that particles
of 30–80 µm in diameter were injectable with various gauges of nee-
dles, and that the microspheres did not obstruct the needles and no
microsphere size exclusion was reported in their study (Kang et al.,
2005). PLGA micro-particles of 30 to 100 µm (either free or loaded with
active agents), were designed and based on physico-mechanical prop-
erties recommended for application as injectable tissue engineering
substrates (Boukari et al., 2015). A slightly larger range of particle size
of 150–300 µm was used by Kang et al. They have reported versatile
performance of HAp coated PLGA micro-particles as bone tissue en-
gineering constructs (Kang et al., 2008).
Taking all these reports into account, it seems that particles of
20–50 µm in diameter can be considered suitable as injectable sub-
strates.
From an application viewpoint, the complement and macrophage
response to a particulate system is controlled by some parameters in-
cluding surface hydrophilicity (Reddy et al., 2006). For example, it has
been reported that neutral particles coated with hydrophilic polymer
chains such as PEG, trigger less immune response than hydrophobic
systems (Benoit et al., 2006).
One example of injectable PLGA microspheres was introduced by
Kang et al., where the relatively hydrophobic surface of PLGA micro-
particles was coated with highly hydrophilic HAp. Capability of the
apatite-coated poly(lactic-co-glycolic acid) (PLGA) microspheres in
bone regeneration, as an injectable scaffold, has been examined, in
vivo. A mixture of rat osteoblasts with apatite coated PLGA micro-
spheres (150–300 µm) was injected into subcutaneous sites of athymic
mice. Noticeable capability of the apatite coated PLGA microspheres to
bone regeneration was shown through histological analysis of the im-
plants in 6 weeks (Kang et al., 2008). The apatite layer was produced
biomimetically by PBS incubation for 5 days. However, PLGA is prone
to hydrolytic degradation and the relatively long incubation period in
PBS may increase the risk of premature degradation of the PLGA mi-
crospheres. Such protocols are also not applicable for preparation of
drug loaded PLGA microspheres due to early release during SBF in-
cubation period.
A number of drugs are currently used in bone tissue engineering
because they prevent loss of bone mass. Bisphosphonates, such as
alendronate and zoledronate, and statins like simvastatin and atorvas-
tatin have attracted much interest in this regard. These drugs are known
as cholesterol-lowering, but their bone formation capability has been
considered (Nath et al., 2013).
Mechanism of action of atorvastatin is based on expression of BMP2
protein (Mundy et al., 1999), which plays an important role in the
growth of bone and cartilage. This drug stimulates osteogenic differ-
entiation of bone marrow stem cells, and also promotes bone formation
by osteoblasts and inhibits bone resorption by osteoclasts (Bone et al.,
2007). The decrease in the activity of osteoclasts is due to the effect of
statins on HMG-CoA enzyme inhibition, which is a factor involved in
increasing cholesterol levels (Viereck et al., 2005).
Perez-Castrillon et al., studied 83 patients with osteoporosis and
showed that after oral administration of atorvastatin for one year, the
bone density in thigh and spine increased about 1.4% (Perez-Castrillon
et al., 2008).
In an in vitro study and in order to determine the optimal dose of
atorvastatin for treatment of osteoporosis, Viereck et al., have used
different concentrations of the drug in culture of human osteoblasts.
They showed that by increasing concentration of atorvastatin from
1E−9 to 1E−6 M in the culture medium within 24–72 h, expression of
OPG protein, BGLAP protein and alkaline phosphatase also increased.
However, further increase of the drug upto 1E−5 M, has led to death of
osteoblasts (Viereck et al., 2005).
Synergic effect of HAp and drugs such as sodium alendronate and
simvastatin was also studied on osteogenic differentiation of stem cells.
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International Journal of Pharmaceutics 565 (2019) 95–107
For example, Papadimitriou et al., have prepared inorganic bovine bone
graft (BOS), hydroxyapatite combined with calcium sulfate (HACS),
and collagen sponge (COS). They monitored bone regeneration with
time in artificial bone defects in seven groups of rats as passive control
group (CONT), active control groups as BOS, HAC and COS (all without
simvastatin), and groups BOS -SIM), and HACS- SIM (both with sim-
vastatin). The results showed significant differences between groups
depending on the carrier used. The study suggested that at 8 weeks,
more new bone formation was noted for the HACS-SIM group and that
local application of simvastatin, in combination with HACS as carrier,
could promote bone regeneration (Papadimitriou et al., 2015).
In another study, composites of PLGA with hydroxyapatite were
fabricated as microspheres and loaded with the osteoporosis preventing
drug, alendronate (AL) through a solid/oil/water technique. AL release
from the PLGA/HA-AL system showed a sustained profile for 30 days.
Up regulation of ALP was shown in their study (Shi et al., 2009).
Recently, significant alkaline phosphatase activity, a differentiation
marker of MC3T3-E1 cells, was reported when the cells were cultured in
vitro on simvastatin loaded PLGA microspheres/carbonate apatite ce-
ment composite (Terukina et al., 2017).
Ruiz Gaspa S. et al., have compared dose dependant stimulatory
effect of atorvastatin and simvastatin on bone formation, in vitro, using
human osteoblast (hOB) and MG-63 cell line cultures. Analysis of cell
proliferation and collagen polypeptide α1 type 1 (COL1A1) gene ex-
pression, osteocalcin, and BMP2 expression levels after 24 h incubation
with statins in their study showed a statistically significant decrease in
cell proliferation upon addition of both the statins at different con-
centration (10E−9 to 10E−6 M). At the same time, COL1A1, osteo-
calcin, and BMP2 gene expression significantly increased for both the
cell lines. Noticeable that increase of both the osteocalcin and BMP2
gene expression occurred at a remarkably higher concentration of
atorvastatin (about E−6 M) than simvastatin (about E−7 M) (Viereck
et al., 2005).
The aim of the current work was to create an injectable system for
the purpose of bone regeneration. Whilst majority of previous works
has focused mainly on PLGA microsphere (with or without a bioactive
material), we propose a HAp coated atorvastatin loaded system. The
effect of preparation condition and the drug content on microstructure
of the PLGA micro-particles were studied. The drug loaded micro-
particles were then coated with HAp, biomimetically, for 1–2 h, to
avoid premature drug release. Synergistic effect of the drug and the
apatite coating layer on osteogenic differentiation of adipose derived
mesenchymal stem cells was studied in 21 days through Alizarin Red
staining and calcium deposition measurement.
2. Materials and methods
PLGA (G:L ratio of 50:50, molecular weight of 46 kDa, synthesized
and fully characterized at Department of Biomaterials, IPPI, please see
supplementary information)
Atorvastatin, calcium salt (ATC), C66H68CaF2N4O10, Hakim
Chemical Process, Iran; methylene chloride, Sigma-Aldrich; PVA (mo-
lecular weight 13–23 kDa, 87% hydrolyzed), Sigma-Aldrich; and
NaHCO3, Merck were used as received unless mentioned otherwise.
2.1. Fabrication of microspheres
2.1.1. Fabrication of PLGA microspheres (PLGA)
PLGA microspheres were fabricated through a conventional oil-in-
water (O/W) emulsion/solvent evaporation technique. Briefly, PLGA
(100 mg), was dissolved in methylene chloride (2 ml) and added
dropwise into the PVA solutions (0.5, 1.0, 1.5 and 2.0 w/v%). The
emulsion was homogenized (DIAX 900, Heidolf Co., Germany) at
8000 rpm for different time periods (30, 90 and 300 s), followed by
stirring at 500 rpm for 3 h at room temperature until complete eva-
poration of the organic solvent. The hardened microspheres were then
F. Shokrolahi, et al.
Table 1
Composition of the SBF solution (in 1 L).
NaCl KCl NaH2PO4 CaCl2·2H2O MgCl2·6H2O
Weight(g) 58.44 0.37 1.20 3.68 1.02
Concentration(mM) 1000 5 25 5 10
collected by centrifugation (Sigma2-16p, Germany) at 6000 rpm and
washed three times with deionized water to remove residual surfactant,
and finally freeze dried overnight. The particles were stored in fridge
prior to further characterization.
2.1.2. Fabrication of AT loaded PLGA microspheres (AT-PLGA)
AT loaded PLGA microspheres were fabricated similar to PLGA
microspheres with slight changes as follows. PLGA (100 mg) was dis-
solved in methylene chloride (DCM, 2 ml). Carefully weighed amount of
Atorvastatin calcium (5, 10, or 20 mg of the drug fine powder) was
added to the solution and dispersed in the solution using bath sonica-
tion for 1 h (100 w, 36 Hz). Next, the process was continued exactly as
was described in 2.1.1. The AT loaded particles were stored in fridge
prior to further characterization (PLGA-AT).
2.2. Biomimetic coating with HAp (AT-HAp-PLGA)
The ten times concentrated simulated body fluid solution
(10 × SBF) was prepared according to Tas and Bhaduri (2004). Briefly,
the stock solution was prepared by dissolving analytical grade chemi-
cals (Merck) listed in Table 1, in a 500 ml volumetric flask in doubly
distilled water (DDW). After complete dissolution of all the chemicals at
room temperature, volume of the solution was made up to 500 ml. The
SBF stock solution (pH = 4.4) is stable at room temperature for several
months. Just before mineralization experiments, an appropriate amount
of NaHCO3 was dissolved in a certain volume of the stock solution to
raise the bicarbonate ion concentration to 10 mM. The solution
(pH = 6.5) was transferred into a centrifuge tube containing a weighed
amount of PLGA or PLGA-AT microspheres and was shaken at 150 rpm
by a mechanical shaker during the mineralization process. At the end of
mineralization process, the apatite coated PLGA/PLGA-ATmicrospheres
were washed with DDW to remove any dissolved inorganic ions and
then dried at room temperature for 24 h, followed by drying under
vacuum for 3 days (PLGA-HAp and PLGA-AT-HAp).
2.3. Characterization of PLGA/AT and HAp coated PLGA/AT
microspheres
Morphology of the microspheres was observed using scanning
electron microscopy (SEM, VEGA 3 SBH, TESCAN, Czech Republic)
after gold coating. Size distribution and average diameter of the mi-
crospheres were determined using SEM images. Ca/P ratio of the
coating layer was evaluated by energy dispersive X-ray analysis (EDX,
Oxford), connected to the SEM system. PLGA, AT and AT loaded PLGA
microspheres were characterized by a fourier transform infrared spec-
trometer (FTIR, EQUINOX55 LS101, Bruker, Germany). Differential
Scanning calorimetry measurements (DSC, Maia-DSC-200-F3, Netzsch,
Germany), were performed to examine physical state of the drug in the
microspheres as well as any potential interaction between the polymer
and the drug. Apatite coated microspheres were characterized by X-ray
diffraction (XRD, D5000, Siemens, Germany), which is a theta/2theta
diffraction instrument operating in reflection geometry, using Cu Ka
radiation = 1.15418 Å, focused by a Ge crystal primary mono-
chromator.
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International Journal of Pharmaceutics 565 (2019) 95–107
2.4. Determination of microsphere formation yield, drug encapsulation
efficiency and in vitro drug release
The microsphere formation yield was estimated using Eq. 1.
mass of the microspheres recovered
%yield = × 100
mass of the polymer and the drug used in formulation
Drug encapsulation efficiency (EE), was determined by an extrac-
tion method. 10 mg of the dried microspheres was dissolved in 0.3 ml
DMSO (a good solvent of the polymer), and subsequently, 10 ml me-
thanol (in which AT is freely soluble) was added (to dissolve all the
drug content and at the same time to precipitate the polymer as a non-
solvent of PLGA). The mixture was vigorously shaken for 2 h at room
temperature and then filtered through a 0.2 µm cellulose filter. AT
content of the solution was analyzed using UV spectroscopy at a wa-
velength of 245 nm. The drug EE was then calculated according to Eq.
2:
mass of the drug in microspheres
%EE = × 100
mass of the drug added
For in vitro release study, to PLGA-HAp-AT (10 mg), in a dialysis
bag (MWCO = 3500), PBS was added (0.5 ml). The bag was placed in a
screwed cap vial containing 2.5 ml PBS and incubated in an incubator
(37 °C, 100 rpm). At definite time intervals, the incubation medium was
replaced with the same volume of fresh PBS. Drug release at each time
point was calculated as according to Eq. (3):
Rd, n = Cn ∗ V − Cp ∗ Vd (3)
where Rd,n is the amount of released drug at each time period, Cn the
drug concentration at current step, Vd volume of the release medium in
the dialysis bag, V the refreshed medium plus volume of the release
medium in the dialysis bag, and CP the drug concentration in previous
step.
For drug concentration calculation, a calibration curve was plotted
first, for absorbance of solutions of different drug concentrations read at
245 nm (R2 = 0.9977).
2.5. Cytotoxicity/bioactivity studies
2.5.1. Cell toxicity and proliferation studies
Toxicity of the PLGA, PLGA -AT, PLGA -HAp and PLGA -AT-HAp
microspheres against L929 mouse fibroblast cells (National Cell Bank-
Pasteur Institute of Iran) was evaluated using MTT assay. Dulbecco's
minimal essential medium (DMEM) supplemented with 10% fetal bo-
vine serum (FBS; heat inactivated), was used as culture medium. About
5.0 × 105 cells were seeded on the samples in the wells of a 24-well
plate (TCP, Swiss) with DMEM containing serum and maintained at
37 °C in a humid incubator (Binder, Germany) supplemented with 5%
CO2. Each group of the microspheres (0.1 mg), were added to the wells
containing L929 fibroblasts and incubated for 24 h. At the end of the
culture period (24 h), the culture medium was carefully removed and
the cells were washed with phosphate-buffered saline (PBS; 0.01 mol/L,
pH = 7.2), followed by addition of 100 μL of MTT (0.5 mg/mL) solu-
tion. The plates were next incubated for 2 h at 37 °C until intracellular
purple formazan crystals became visible under microscope. Then the
MTT solution was removed and 100 μL of isopropanol was added to
each well and incubated at 37C for 2 more hours. Absorbance of the
solution was then read on a microplate reader (ELX808, Biotek, USA) at
570 nm. The cells’ viability was calculated according to Eq. 4.
Cell viability
= (absorbance of the sample/absorbance of the control) × 100
The experiments were carried out in triplicate (n = 3), for which the
average values are reported with standard errors in this paper.
p < 0.01 was considered significant (Morgado et al., 2014; Lou et al.,
F. Shokrolahi, et al.
Fig. 1. Effect of fabrication condition on the size/size distribution of the PLGA particles. All images are of the same magnification. *Particle size is reported as
mean ± SD.
2015; ISO, 2009).
2.5.2. In vitro adipose derived stem cells culture
Adipose derived stem cells (ADSCs, third passage), derived from
C57 mice were re-suspended and seeded in 75 mm flasks at a density of
2 × 103 cells/cm2 with ADSCs medium (L-DMEM, 10% FBS, 100 U/mL
penicillin and 100 μg/mL streptomycin). ADSCs were expanded, and
the cells at the third passage were used for osteogenic differentiation
studies. All cell culture experiments were performed in a humid in-
cubator at 37 °C and 5% CO2 atmosphere.
2.5.3. Osteogenic differentiation
Microspheres were sterilized under UV light for 1 h, and transferred
onto the bottom of a 24-well cell culture plate. The microspheres were
rinsed with ADSCs medium to allow uniform attachment to the bottom
of the wells and air dried for 10 min at room temperature. ADSCs
(1 × 105) were seeded onto surface of the samples and cultured with
0.5 ml of the culture medium for 3 days. At day 3, the medium was
switched to 0.5 ml of osteogenic differentiation medium (DMEM F/12,
10% FBS, 1% penicillin, streptomycin and dexamethasone (0.1 uM), β-
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International Journal of Pharmaceutics 565 (2019) 95–107
glycerophosphate (10 mM), and ascorbic acid (50 uM)) and culture was
continued for 21 days.
2.5.4. ADSCs culture in absence of osteogenic medium
MSCs (1 × 105) were seeded in the 24-well cell culture plate and
cultured with 0.5 ml of MSC medium for 21 days. The medium of all the
three groups was changed on the second day after cell reseeding and
every other day afterward until day 21
2.5.5. Alizarin Red assay
Alizarin Red (AR), reagent is a dye that selectively binds to calcium
salts. Alizarin Red staining (ARS), was used to quantify mineralization
according to a modified published protocol. The cells on all the groups
of microsphere were fixed in paraformaldehyde (PFA), at days 14 and
21 post culture. The fixed samples were placed in the wells of a 24 well
plate and washed 3 times with distilled water. Alizarin Red solution
(%1, pH 4.1, Sigma, UK) was used at 1 ml per sample for 20 min on a
shaker. The dye was removed from the wells and the cells were washed
with distilled water. Next, perchloric acid (0.5 ml of 5% v/v), was
added to each well and incubated for 30 min. Absorbance (optical
F. Shokrolahi, et al.
density, O.D.), was recorded at a wavelength of 405 nm and the data
were corrected by subtracting absorbance of a negative control (blank)
from the readings obtained.
2.6. Statistical analysis
Statistical analysis was performed using one-way ANOVA. All re-
sults are reported as the mean value ± standard deviation. Differences
were considered statistically significant at p < 0.01.
3. Results and discussion
3.1. PLGA and AT loaded microspheres properties
In the emulsion method, several factors have an effect on mor-
phology, size, and size distribution of the resulting micro-particles. In
this study, PVA was used as emulsifier because the influential role of
this material was previously examined and reported by different re-
search groups (Garner et al., 2018).
The effect of PVA concentration, aqueous to organic phase’s ratio
(Aq/Org ratio) and the rate of organic phase addition to the aqueous
phase (AddR), on the micro-particles’ size and size distribution were
studied. Based on the data in literature, PVA concentration was
changed at four levels of 0.5, 1.0, 1.5 and 2.0 percent. Aq/Org ratio of
5, 15 and 30 and AddR of 30, 90 and 300 s were also studied.
Micro-particle sizes were controlled by PVA concentration as it is
displayed in Fig. 1A–D. Mean Size of the particles decreased from just
above 10 µm down to below 3 µm as concentration of PVA increased
from 0.5 to 2%. This result is consistence with prior reports (Obayemia
et al., 2016). The higher the concentration of PVA, the more droplets
break and obviously the resulting particles become smaller (Feng et al.,
2013). However, an increase in concentration from 1.5 to 2% did not
reduce the average diameter of the particles. It can be deduced that
increased concentration results in greater stability of the emulsion
droplets and thus prevents droplets from re-joining. But, since PVA is a
polymer, excessive increase in concentration enhances viscosity of the
aqueous phase that results in the breakage of the emulsion droplets into
smaller droplets (Maia et al., 2004).
PVA concentration has an impact on the particle size distribution as
well, as shown in Fig. 1; the narrowest particle size distribution was
recorded at 1% (please see the size distribution histograms in Fig. S1,
Supplementary information).
Aqueous to organic phase ratio (Aq/Org ratio), is another influential
parameter and was studied next. Our results showed that the average
particle size constantly increase with Aq/Org ratio, at constant PVA
concentration of 1%, (Fig. 1E–G). These results are consistent with
Jeffrey et al., who reported a linear relationship between Aq/Org ratio
and particle size (Jeffery et al., 1991).
As it is shown in Fig. 1, the Aq/Org ratio, in addition to size, also
affects the particle size distribution, and the sample with Aq/Org ratio
of 15 exhibits the most uniform size distribution. It is noteworthy that
the excessive increase in Aq/Org ratio causes deviations from spherical
morphology, since larger Aq/Org ratio means more PVA amounts in the
system. As already mentioned, the PVA concentration needs to stand at
a somewhat optimal value, and higher concentrations result in reduced
micro-particles stability (Kemala et al., 2012). Also, solidification rate
of the micro-particles increases with Aq/Org ratio (Mao et al., 2008)
and results in breakage of the micro-particles during the emulsification
process under the stress applied by homogenization.
Finally, impact of the rate of organic phase addition to the aqueous
phase (AddR), on the micro-particles’ size and size distribution was
studied. Since AddR is carried out under homogenization, a slow ad-
dition rate means that the emulsion is placed longer under stress and, as
a result, smaller particles are formed.
Thus, AddR is another factor for controlling particle size in emulsion
method of particle synthesis. According to Fig. 1H–J, with the organic
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International Journal of Pharmaceutics 565 (2019) 95–107
phase addition to the aqueous phase, while the addition period was
increased from 30 to 300 s, the average particle size decreased from
14.5 µm to 660 nm.
Regarding the results of this section, optimum conditions for pre-
paration of micro-particles were determined as PVA concentra-
tion = 1%, Aq/Org ratio = 15, and AddR = 90 s.
3.2. Preparation of atorvastatin containing PLGA micro-particles (LGA-AT)
The choice of the emulsion method for encapsulating the drug in the
polymer microspheres is dependent on hydrophilicity of the drug mo-
lecules. Since atorvastatin is hydrophobic, two methods of O/W (oil in
water) and S/O/W (solid in oil in water) were used to encapsulate it in
the PLGA micro-particles.
3.2.1. O/W method
This requires a solvent that is capable to dissolve both the polymer
and the drug. PLGA is soluble in many organic solvents such as di-
methyl sulfoxide (DMSO), dimethyl formamide, tetrahydrofuran,
acetone, ethyl acetate and chlorinated solvents such as chloroform and
dichloromethane. Atorvastatin calcium is also soluble in dimethyl
sulfoxide and methanol (Ahjel and Lupuleasa, 2009).
Therefore, DMSO is a common solvent for atorvastatin calcium and
PLGA. However, DMSO is not a suitable solvent for emulsion. This
solvent is miscible with water and, as soon as the organic phase is added
to the water phase, it penetrates into the aqueous phase and forms
micro-particles with a wrinkled morphology (Park et al., 1998). To
address this problem, two solvents can be used to dissolve the polymer
and the drug individually and then, by combining these solutions,
achieve a homogeneous organic phase. In the O/W method, DCM and
DMSO were used to dissolve PLGA and the drug, respectively. SEM
images of the micro-particles prepared at atorvastatin calcium to PLGA
ratio of 1/20 through O/W are displayed in Fig. 2. As it is shown in
Fig. 2A–C, presence of DMSO even at small quantities reduced the size
and broadened the size distribution of the particles. Difference between
the solubility of these two solvents in water is the controlling factor of
the micro-particles’ morphology. DCM is only 2% miscible with water
(Park et al., 1998), which made this solvent suitable for emulsion
processes. This is while DMSO is miscible with water in any proportion.
Therefore, when a combination of DCM and DMSI is used, DMSO mi-
grates from the droplets as soon as the organic phase comes into contact
with the aqueous phase, leaving semi-solid particles behind. Due to the
early leave of DMSO from the micro-particles and entry of water, a
counter-current flow is developed. Final morphology of the particles is
controlled by the mass transfer due to DMSO exit and water entering as
well as the exit of DCM (Park et al., 1998; Young and Chen, 1991).
Since in the emulsion method, the solvent exit rate and solidifica-
tion of the droplets control micro-particles morphology, complexity of
using two solvents is increased by changing the solvent/co-solvent
ratio. Increasing the amount of DMSO, on one hand, increases rate of
solidification of the droplets and the drug loading efficacy, and on the
other hand, causes wrinkling and porosity in the morphology and forms
a large accumulation of very small particles (Park et al., 1998;
Bodmeier and McGinity, 1988).
Park et al., have used DMSO and DCM (at different solvent/co-sol-
vent ratios), to encapsulate a protein in PLGA micro-particles. They
showed that by increasing the amount of DMSO, the protein loading
efficiency increased, morphology of the particles turned to porous and
wrinkled and the particle size decreased (Park et al., 1998).
3.2.2. S/O/W Emulsion method
The drug is not dissolved in S/O/W method in an organic phase and
is only dispersed, as powder, uniformly in an organic phase. Therefore,
the powdery particles of the drug should be so small that the polymeric
micro-particles can encapsulate them (Wischke and Schwendeman,
2008). In order to investigate this, hexane, a non-solvent of
F. Shokrolahi, et al.
Fig. 2. SEM images and size information of atorvastatin and the PLGA-AT micro-particles prepared through O/W and S/O/W.
atorvastatin, was used to disperse it for SEM observation. Fig. 2D and E
shows SEM images of the atorvastatin calcium powder used in this
study.
Fine size of the atorvastatin particles (on a scale of tens of nan-
ometers, Fig. 2D and E) indicates its capability for being encapsulated
by PLGA micro-particles (Fig. 2). Microspheres containing the drug at
ratio of atorvastatin/PLGA of 1.20, prepared through S/O/W are dis-
played in Fig. 2. As it can be seen, the drug loaded micro-particles show
a spherical morphology and a small particle size of 0.5 and 11.2 µm
(Fig. 2F).
Since the drug is dispersed as powdery particles in a solvent and
encapsulated in a polymer in S/O/W emulsion method, a part of the
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International Journal of Pharmaceutics 565 (2019) 95–107
drug particles that are present on the surface of the particles, remain
pores behind after washing the particles (Fig. 2G–H, please see the size
distribution histograms for Fig. 2A–D and G–H in Fig. S1,
Supplementary information) (Birnbaum, 2000).
To obtain the highest amount of drug loading, samples were also
prepared at atorvastatin/PLGA ratios of 1/10 and 1/5 in the same way.
SEM images of these samples are shown in Fig. 2G and H. Regarding
Fig. 2F–H, it seems that PLGA microspheres are not able to encapsulate
more than 10% by weight atorvastatin, while maintaining morphology.
The particles became defective and non-spherical with increasing the
drug loading. The reason for this is the small size of atorvastatin mo-
lecules compared to the large polymer chains. When the solvent leaves
F. Shokrolahi, et al. International Journal of Pharmaceutics 565 (2019) 95–107

the droplets, the polymeric chains penetrate inside and the particles
become smaller increasingly. The polymeric entanglements play a cri-
tical role in the process of chain penetration until the chains solidify
and protect the particles surface (in contact with the aqueous phase)
from early solidification (Festag, 1998). In contrast to polymeric chains
the small drug molecules lack entanglements, thus, lack the ability to
move toward the center in the evaporation process and accumulate on
the surface when the particles solidify. Therefore, increasing con-
centration of the drug has a negative effect on morphology of the
particles (Hong, 2008).
In this study, due to the anomalous morphology of the particle
containing 20% atorvastatin, PLGA-AT20 (Fig. 2-F), PLGA-AT10 with
relatively smooth surface and narrow particle size distribution was
chosen for further studies. Based on the method presented in the
methods section, a drug loading efficiency of about 45.8% (drug
loading about 4.8%), was determined for this sample.

3.3. PLGA-AT characterization

Based on the EDX analysis and the distribution of nitrogen (which is

specifically present in the atorvastatin molecule), the drug was suc-
cessfully encapsulated in the PLGA micro-particles and the relatively
uniform distribution of the drug in the polymer matrix was confirmed
(Fig. 3).
ATIR FTIR spectra of PLGA micro-particles, atorvastatin and PLGA-
AT micro-particles are displayed in Fig. 3. At 1650 cm−1, a peak ap-
pears that was not observed for PLGA micro-particle. The peak at this
region can be related to the aromatic C]C bending vibration of the
NeH group present in atorvastatin (Lin-Vien, 1991).

Fig. 4. TGA/DTGA traces of A; atorvastatin, B: PLGA, and C: PLGA-AT.

Next, we extended our studies to thermal properties of the micro-

particles and first focused on thermal stability of atorvastatin and its
variation upon encapsulation in PLGA micro-particles (Fig. 4). The
PLGA particles degraded between 250 and 310 °C, where the particles
experienced a 90% weight loss. Degradation of atorvastatin containing
micro-particles (PLGA-AT), occurred in one stage (300–350 °C), which
implies that thermal stability of the drug somehow increased upon
encapsulation within the PLGA particles.
In order to examining physical state of atorvastatin calcium en-
capsulated in the micro-particles, DSC experiment was carried out on
PLGA-AT, PLGA, and the pure atorvastatin. The DSC thermogram of
PLGA micro-particles (Fig. 5) showed a glass transition temperature of
about 49.5 °C and no melting peak for PLGA, which is obvious due to its
amorphous nature.
A relatively broad endothermic transition was recorded in the DSC
trace of calcium atorvastatin in the temperature range of 120–140 °C,
which is related to the loss of moisture. Also, a melting transition ap-
peared at 166.5 °C, which is in close match with those in the references
(Kulthe and Chaudhari, 2013; Choudhary, 2012). Atorvastatin calcium
is a polymorph drug and at least 60 different polymorphic forms have
been reported from it (Censi and Di Martino, 2015). Various forms of
this drug can be converted into one another through processing or even
during storage (Skorda and Kontoyannis, 2008).
Fig. 3. Up: EDXA analysis reveals successful encapsulation and uniform dis-
Different amorphous and crystalline forms of atorvastatin calcium
persion of AT within the PLGA microspheres, Down: ATIR FTIR spectra of PLGA
have different physical properties due to differences in the hydrogen
micro-particles, atorvastatin and PLGA–AT micro-particles.

101
F. Shokrolahi, et al.
Fig. 5. Polymorphism study of atorvastatin in PLGA-AT using; Up: DSC and
Down: XRD.
bonds, the arrangement and interaction of molecules, and their density
within the crystalline network. This is why no specific melting point is
attributed to the drug but instead it shows more than one melting point
(Kulthe and Chaudhari, 2013). After the drug encapsulation, Tg of the
PLGA particles remained 49.5 °C. Since the amount of drug loading is
relatively small in comparison with the polymer matrix, the melting
temperature region of the drug was expanded to examine absence or
presence of a melting peak for it in PLGA-AT (Fig. 5).
Observation of weak melting transitions in the studied range may
indicate presence of crystalline form of the drug in the micro-particles.
As mentioned above, atorvastatin is a polymorphic drug and may had
undergone a change in form during the process. Therefore, wide angle
X-ray diffraction (XRD) experiment was performed to monitor any
possible changes in the crystalline form of the drug, next (Fig. 5).
No crystalline peak was observed for PLGA-AT which indicates
mainly amorphous structure of the drug in the micro-particles. This
form change might have happened during particle fabrication (Fig. 5).
The weak melting transitions in the DSC trace of AT in PLGA-AT, can be
possibly because a major part of the drug exists in amorphous form in
PLGA-AT while a minor part of it was crystalline (less than that of the
detection limit of DSC).
3.4. Biomimetic coating
SBF has been widely used to apply bone-like apatite on various
biomaterials. Ion concentration of SBF and soaking time are the most
extensively studied parameters that affect biomimetic formation of
calcium phosphate coatings on biomaterials (Tas and Bhaduri, 2004). In
this study, we used different volumes of 10 × SBF to coat a certain
amount of micro-particles (54.8 ± 6 mg) at room temperature.
It has been previously reported that nucleation of calcium phos-
phate occurs within one hour soaking in 10 × SBF and after 2 h, the
deposition gradually grows (Tas and Bhaduri, 2004). Fig. 6A–F shows
SEM images of PLGA microspheres after 1 and 2 h incubation in 5, 10,
and 15 ml of 10 × SBF. A direct relationship is clearly seen between the
amount of calcium phosphate precipitation and the volume of 10 × SBF
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International Journal of Pharmaceutics 565 (2019) 95–107
(Fig. 6A–C and D–F) and incubation time (Fig. 6A–F). However, pre-
sence of precipitation around microspheres showed unwillingness of
calcium ions to interact with the polymer surface (Fig. 6D–F). Pre-
viously surface modification methods have been developed to improve
bonding of calcium ions to the substrate in order to nucleate on the
surface. Reportedly, when SBF with an ion concentration up to three
times higher than that in human blood plasma is used to coat polyesters
such as PLLA and PLGA, it is not necessary to modify the surface. These
biodegradable polymers are partially hydrolyzed during the lengthy
incubation time in SBF that generates considerable numbers of COOH
groups. Negative charge on the COO− groups at surface is able to at-
tract calcium ions with positive charge and nucleation becomes possible
(Yang et al., 2008). However, 10 × SBF shortens the times for calcium
phosphate deposition to hours, which is obviously not enough to make
the above mentioned mechanism efficient, as seen in SEM images
(Fig. 6A–C).
PLGA is a linear copolymer of hydrophobic PLA and hydrophilic
PGA. Therefore, the copolymer chains contain both hydrophobic and
hydrophilic ends randomly. Considering the short-range mobility of
polymer chains in solid state, they may orient themselves differently in
the bulk than at surface depending on the surrounding phase, e.g. air or
water. As it was discussed earlier, the PLGA-AT micro-particles were
freeze dried after preparation. The micro-particles remained in contact
with air during and after drying process. Therefore, it can be imagined
that the hydrophobic ends oriented themselves towards air, and that
such hydrophobic surface is not appropriate for nucleation of calcium
phosphate and needs surface modification.
In obvious contrast to air, hydrophilic segments of PLGA copolymer
can orient themselves towards surface in aqueous medium. Therefore,
in this study, we anticipated that soaking micro-spheres in SBF just after
emulsion process is a possible way of coating PLGA micro-spheres
without any further surface treatment step. SEM images of the coated
micro-particles (Fig. 6G–I), confirmed the hypothesis. The hydrophilic
ends of PLGA oriented themselves towards the water phase of emulsion
system and provided favorable sites on the surface of the micro-spheres
for nucleation and growth of calcium phosphate. OH groups on the
surface can bind to the calcium ions in SBF, and thus making the nu-
cleation possible. Fig. 6 also shows the effect of SBF volume on calcium
phosphate nucleation. It was observed that an optimum volume of SBF
is required to coat the microspheres completely, while 5 ml and 10 ml
SBF were not enough to coat all the microspheres (Fig. 6G and H), using
20 ml SBF caused formation of microspheres clusters due to filling the
space between microspheres by the excess amount of calcium and
phosphate ions (Fig. 6J and K).
At the same time, the microspheres immersed in 15 ml SBF revealed
a uniform coating consisting of micrometer range plates oriented ran-
domly (Fig. 6I and L), and was selected for further studies. Substituting
carbonate ion into calcium phosphate is the reason of changing the
growth morphology from needles to plate-like (Wopenka and Pasteris,
2005).
Previous findings indicate that randomly roughened surfaces induce
greater osteoblast attachment and proliferation compared to smooth
surfaces.
XRD analysis was performed on the PLGA-AT coated in 10XSBF
(PLGA-AT-HAp), at the end of emulsification step for two hours and the
result was compared with that of the unspecific CaP deposition in SBF
solution (not on the particles, CaP). The data obtained showed sig-
nificant structural and compositional differences between the CaP
coating on the microspheres (PLGA-AT-HAp), and the precipitates ob-
tained from SBF during 2 h (CaP, Fig. 6D–F). Characteristic peaks of
hydroxyapatite (HAp) appeared in XRD spectrum of PLGA-AT-HAp
(Fig. 7A). Two peaks at 2θ = 25.8° and 32° corresponding to (2 1 1) and
(1 1 2) planes, resemble poorly crystallized bone-like apatite (Drouet,
2013).
In addition, a small peak at 2θ = 11.7° can be attributed to presence
of brushite (DCPD) as a minor secondary phase beside HAp.
F. Shokrolahi, et al.
Fig. 6. SEM images of PLGA microspheres after 1 and 2 h incubation in 5, 10, and 15 ml of 10 × SBF; Up: for dried particles, and Down: for the as prepared particles.
Furthermore, it is well known that amorphous calcium phosphate (ACP)
is always present in deposited calcium phosphate coatings. However, it
appears as large halos, which are sometimes overlooked in XRD pat-
terns. XRD spectra of CaP exhibited a characteristic peak at 2θ = 11.7°
corresponding to diffraction plane (0 2 0) of DCPD of a monoclinic
structure. (Drouet, 2013) A possible explanation for the observed dif-
ferences between the CaP deposition and PLGA-AT-HAp can be pro-
vided based onto the mechanism of apatite formation in SBF. As it was
mentioned before, presence of hydrophilic groups such as COOH and
OH on the surface is the main factor responsible for apatite formation.
Dissociation of the carboxyl groups in aqueous solution renders the
surface negatively charged, thereby attracting positive calcium ions
electrostatically. This electrostatic force effectively enhances the local
concentration of calcium and phosphate ions around the microspheres
and causes apatite nucleation to preferentially occur at surface. It must
be noted that the least stable phases such as ACP and DCPD form first
due to less thermodynamic driving force. In fact, these kinetically
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International Journal of Pharmaceutics 565 (2019) 95–107
favorable forms of calcium phosphate, which have fast nucleation rates,
are precursor phases of apatite formation. At longer time scales, ACP
and DCPD act as nucleating sites for the more thermodynamically stable
form of calcium phosphate, allowing apatite crystals to gradually grow
on the surface. According to this mechanism, prerequisite condition of
increased local concentration of calcium and phosphate ions is not sa-
tisfied in CaP formation. In fact, there is no substrate surface with
special affinity to attract calcium ions and raise local concentration of
the ions in the SBF. Therefore, apatite crystals cannot be expected to
form during 2 h of precipitation from SBF. However, DCPD is the most
kinetically favorable phase because it has the highest nucleation rates
among all calcium phosphate forms. Particular attention must be paid
to the influence of SBF recipes on the nucleation rates of different
calcium phosphate forms. In this regard, precipitation of DCPD is not
possible in normal SBF, while it is the most likely phase to precipitate
when the calcium and phosphate ion concentrations increase above the
normal level (Drouet, 2013).
F. Shokrolahi, et al. International Journal of Pharmaceutics 565 (2019) 95–107

FTIR spectra (Fig. 7B) corroborated previous XRD results. PLGA-AT-

HAp exhibited characteristic absorption peaks for phosphate in apatite
at 565 cm−1 and 604 cm−1 for PO ν4 mode, 960 cm−1(ν1) and
1050 cm−1(ν3) (Wopenka and Pasteris, 2005).
Carbonate incorporation in the biomimetic apatite was confirmed
by bands at 870 cm−1 (ν2) and 1650 cm−1 (ν3). (Costa, 2012).
Carbonate has four vibrational modes that two of them, ν2 and ν3
can be detected in FTIR spectrum of carbonated apatite, which are due
to occupancy of hydroxyl and phosphate sites respectively (Costa,
2012).
In the case of CaP, the OH band at 3569 cm−1 with a decreased
intensity compared to commercial HAp is difficult to detect by FTIR
spectroscopy. Low crystallinity is probably the reason for covering
weak OH signals due to enlarging vibrational bands. The other reason
could be attributed to the substitution of two hydroxyl ions by one
carbonate ion leading to a significant decrease in the OH absorption
intensity. FTIR spectra of CaP showed characteristic peaks of DCPD
crystals, confirming the XRD observations. The absorptions between
3500 and 2400 cm−1 are assigned to OeH stretching vibration and also
the HeOeH bending is present as a single intense peak at 1648 cm−1.
The absorption peaks centered at 1214 cm−1, 1132 cm−1 and
1065 cm−1 are due to PO stretching vibrations. Intense and broad
peaks at 983 cm−1, 870 cm−1 and 788 cm−1 are assigned to (H)PO
groups. Additionally, three bands corresponding to (HOe), PO are ob-
served at 661 cm−1, 579 cm−1 and 526 cm−1. (Binitha and
Pradyumnan, 2013)
According to EDX analysis results, the Ca/P atomic ratio of the
coatings turned out to be 1.36 ± 0.05, which is noticeably lower than
that of stoichiometric HA (1.67). The apatite structure has the ability of
hosting different substituents in cationic and OH sites that can explain
Fig. 7. XRD (A) and FTIR (B) spectra of the calcium phosphate deposits formed
the low Ca/P value.
in 10XSBF.

Fig. 8. L929 mouse fibroblasts behavior/viability on PLGA, PLGA-AT, PLGA-HAp, and PLGA-AT-H. Ap. Scale bars represent 200 µm.

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F. Shokrolahi, et al. International Journal of Pharmaceutics 565 (2019) 95–107

Fig. 9. A: Protocol of ADMSCs culture on the micro-particles. B: Alizarin Red S staining of ADMSCs cultured on PLGA, PLGA-AT, PLGA-HAp, and PLGA-AT-HAp
micro-spheres in osteogenic medium (OsM) and normal medium (NOsM) for 14 and 21 days. After 21 days incubation, PLGA–AT-HAp showed significantly more
intense and remarkably higher uniformity in dispersion of the ARS staining compared to all the other sample groups. C: comparative calcium deposition on PLGA,
PLGA-AT, PLGA-HAp, and PLGA-AT-HAp micro-spheres.

3.5. Biological activity of PLGA-AT-HAp
In the next step, the PLGA-AT-HAp micro-particles were subjected
to biological evaluation for possible application as injectable system for
bone tissue engineering.
(according to ISO 10993). Also, Viability of the cells was evaluated
using MTT assay. Adhesion, growth and spreading of the cells on the
negative control (TCP), as well as in proximity of the micro-particles,
are evident in Fig. 8, and reveals non-toxic effect of the micro-particles
on the cells.

3.5.1. Cytotoxicity assay 3.5.2. Alizarin Red staining

Effect of the fabrication steps on biocompatibility was evaluated
through culture of L929 mouse fibroblasts on the PLGA-AT micro-par-
ticles. The optical microscopic images in Fig. 8 compare the L929 fi-
broblast cells behavior on PLGA, PLGA-AT, and PLGA-AT-HAP with the
cells behavior on the culture plate (TCP), 24 h after cell seeding
To monitor impact of atorvastatin on the cells’ differentiation, drug
release experiment (Fig. 8C), and Alizarin Res Staining were performed.
Mineralization of Adipose Derived Mesenchymal Stem Cells (ADMSCs)
into osteoblastic phenotype on the blank control (tissue culture plate,
TCP) and the micro-spheres was measured using the positive staining of

105
F. Shokrolahi, et al.
Alizarin Red in osteogenic medium (Osteogenic Medium, OsM) as well
as in normal culture medium (Non-Osteogenic Medium, NOsM), as
presented in Fig. 9. Minerals deposition is a late stage marker of os-
teogenic differentiation and it was employed in this study to confirm
that ADMSCs entered into the mineralization phase and that the process
of ECM mineralization was started 14 and 21 days post cell seeding.
After ADMSCs were incubated with PLGA, PLGA-AT, PLGA-HAp, and
PLGA-AT-HAp micro-particles for 14 days, the mineralization of ADSCs
was hardly observed in the NOsM plates. At the same period, the ARS
staining showed slight reddish dots on Control, PLGA-HAp, and PLGA-
AT-HAp micro-particles, but almost no positive stains were found over
PLGA and PLGA–AT samples. A sharp increase in the intensity of
staining was observed at day 21, for all the micro-particle groups stu-
died. At this time point, the AT-incorporated HAp coated group
(PLGA–AT-HAp) showed significantly more intense and remarkably
higher uniformity in dispersion of the ARS staining compared to PLGA,
PLGA–AT, and PLGA–HAp particles, indicating that PLGA–AT-HAp can
stimulte calcium mineralization of ADSCs at a higher rate.
Effect of statin family of drugs was the subject of a few studies. For
example, in an interesting study, Yu et al., incorporated simvastatin(S)
into mesoporous hydroxyapatite microspheres(S-MHMs) and prepared
composites of collagen with S-MHMs (C-S-MHMs). By culture of rat
bone marrow mesenchymal stem cells on C-S-MHMs for 21 days, they
have shown that the composites enhanced expression of osteogenic
markers in rBMSCs accompanied with improved neovascularization in
the composites, in a concentration-dependent manner (Wei-Lin and
Tuan-Wei, 2017).
As shown in Fig. 9C, the total calcium content was found to increase
with time. At day 21, total calcium content in the deposited minerals on
PLGA–AT-HAp was significantly higher than that of TCP, PLGA and
PLGA–AT. Furthermore, after culturing for 21 days, the calcium mineral
deposition in the PLGA–AT-HAp micro-particles was also greater than
that on the PLGA-HAp. Interestingly, similar trend was observed in
osteogenic and non osteogenic media, that provides extra proof to sy-
nergistic effect of AT and HAp on osteogenic differentiation of ADMSCs.
Osteogenic effect of statin family drugs was also studied by a few
research groups. For example, Magan-Fernandez et al., have reported
that simvastatin treatment of MG63 human osteosarcoma cells pro-
vided a pathway for enhancing the differentiation degree of the cells
while reducing their proliferation at concertations well above 1 µM.L−1
(Magan-Fernandez and Fernández-Barbero, 2018). This report is in
close agreement with that by Pagkalos et al., who have shown that
simvastatin at concentrations about 0.1 nM induced osteogenic differ-
entiation of mESC while micromolar concentration of the drug was
toxic to the cells. Atorvastatin release data in our study showed a sus-
tained release of about 160 nM, and our differentiation evaluation re-
sults agree well with the previous reports (Fig. 8C) (Jae Min Cha and
Pagkalos, 2010). Sustained release of statin family of drugs (simvas-
tatin), from PEG-PLGA copolymers was also reported by et al. (Wang
and Liu, 2017).
4. Conclusion
Recent reports on bone anabolic effects of statins have directed a
great deal of interest to potential applications of statin family drugs
such as simvastatin and atorvastatin in bone tissue engineering.
Anabolic effect of statins on stem cells or bone cells including human
osteoblast like cells, and embryonic stromal cells (ESCs) have been in-
dicated by monitoring expression of differentiation and osteogenesis
markers. The osteoblast-differentiating effect of statins has been ex-
plained by a BMP2 mediated action as according to the present litera-
ture.
In this study, an aptly designed injectable PLGA micro-particulate
system that was loaded with atorvastatin (10 wt%) and coated (bio-
mimetically) with hydroxyapatite was prepared. Osteoinductive prop-
erties of atorvastatin loaded PLGA micro-particles in synergy with
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International Journal of Pharmaceutics 565 (2019) 95–107
hydroxyapatite was studied on adipose derived mesenchymal stem cells
(ADMSCs) and compared with those on the pure PLGA micro-particles,
as well as the PLGA micro-particle loaded with the drug. Strong os-
teogenic differentiation was observed on PLGA–AT-HAp as demon-
strated by the increased S Alizarin Red staining/calcium deposition
measurements. Combination of a small quantity of atorvastatin (in the
order of about a few nM, according to drug release measurements) in
the culture medium could induce calcium deposition in both the os-
teogenic and non osteogenic media as evidenced by our results. To sum
up, an injectable atorvastatin loaded PLGA micro-particulate system
was designed and HAp was coated on the surface of the particles
without any initial surface treatment. The coating period was decreased
to 1–2 h to avoid drug loss during SBF incubation and also to minimize
polymer degradation prior to sue. The system facilitates osteogenic
differentiation of ADMSCs in both osteogenic and non osteogenic media
as evidenced by Alizarin Red staining and calcium deposition mea-
surements. Our results confirm the important role that a combination of
atorvastatin loading and HAp coating can play in a novel and injectable
PLGA micro-particulate system in bone tissue engineering applications.
Declaration of Competing Interest
None.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.ijpharm.2019.05.005.
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