Local delivery of minocycline-loaded PEG-PLA nanoparticles for the enhanced treatment of periodontitis in dogs
International Journal of Nanomedicine
local delivery of minocycline-loaded Peg-Pla nanoparticles for the enhanced treatment of periodontitis in dogs
Wenxin Yao 0 3
Peicheng Xu 0 3
Zhiqing Pang 0 1 2
Jingjing Zhao 0 1 2
Menglin Jiang 0 1 2
Bo Zhang 0 1 2
0 Zhiqing Pang Key laboratory of smart Drug Delivery, Ministry of education, Department of Pharmaceutics, school of Pharmacy, Fudan University , 826 Zhangheng road, shanghai 201203, People's republic of china Tel
1 Department of Pharmaceutics, school of Pharmacy, Fudan University
2 Key laboratory of smart Drug Delivery, Ministry of education
3 s hanghai Xuhui District Dental center
4 Department of Pharmacology, school of Pharmacy, Fudan University , shanghai, People's republic of china
8 1 0 2 - l u J - 2 1 n o 7 0 2 . 6 4 . 9 5 . 7 3 y b / m o c . s s e r p e v PowerdbyTCPDF(ww.tcpdf.org) O r I g I N a l r e s e a r c h Xiaoxia li 4 hongbo cheng 2,3 Nengneng cheng 4 Background: Rapid local drug clearance of antimicrobials is a major drawback for the treatment of chronic periodontitis. In the study reported here, minocycline-loaded poly(ethylene glycol)poly(lactic acid) nanoparticles were prepared and administered locally for long drug retention and enhanced treatment of periodontitis in dogs. Methods: Biodegradable poly(ethylene glycol)-poly(lactic acid) was synthesized to prepare nanoparticles using an emulsion/solvent evaporation technique. The particle size and zeta potential of the minocycline-loaded nanoparticles (MIN-NPs) were determined by dynamic light scattering and the morphology of the nanoparticles was observed by transmission electron microscopy. The in vitro release of minocycline from MIN-NPs and in vivo pharmacokinetics of minocycline in gingival crevice fluid, after local administration of MIN-NPs in the periodontal pockets of beagle dogs with periodontitis, were investigated. The anti-periodontitis effects of MIN-NPs on periodontitis-bearing dogs were finally evaluated. Results: Transmission electron microscopy examination and dynamic light scattering results revealed that the MIN-NPs had a round shape, with a mean diameter around 100 nm. The in vitro release of minocycline from MIN-NPs showed a remarkably sustained releasing characteristic. After local administration of the MIN-NPs, minocycline concentration in gingival crevice fluid decreased slowly and retained an effective drug concentration for a longer time (12 days) than Periocline®. Anti-periodontitis effects demonstrated that MIN-NPs could significantly decrease symptoms of periodontitis compared with Periocline and minocycline solution. These findings suggest that MIN-NPs might have great potential in the treatment of periodontitis.
minocycline; nanoparticles; periodontitis; local delivery
open access to scientific and medical research
“Periodontitis” is a common chronic disease that leads to the destruction of
toothsupporting tissues, absorption of alveolar bone, and, finally, tooth loss.1,2
Considerable research has focused on the etiology of human periodontal disease,3 and it has
been generally accepted that chronic periodontitis is induced by microorganisms.
Periodontal pockets provide a moist, warm, nutritious, and anaerobic environment
that profits microbial colonization and multiplication.4 Previous research
demonstrated that only 10 to 30 bacteria species, mainly Gram-negative anaerobic bacteria,
live here,5 but it has now been found that approximately 500 bacterial taxa sojourn
here.4 The amounts and species of the bacteria are variable in different parts of the
biofilm and depend on the effectiveness of oral hygiene procedures, depth of pocket,
flow of gingival crevice fluid (GCF), type of interacting microbes, and so on.6,7
A recent study has indicated that periodontal disease not only affects human oral health
but may also induce several systemic diseases.8
The fundamental treatment of periodontitis is to reduce
the pathogenic bacteria by instrumental debridement,9 such as
ultrasonic scaling and root planning. However, instrumental
debridement cannot completely remove pathogens that have
invaded soft tissue and some anatomically inaccessible areas
such as furcation areas and root depressions.10 In view of
81 this, antimicrobial therapy by antibiotics is often executed as
l-20 adjuvant treatment soon after instrumental debridement.11 In
-J2u order to achieve effective inhibitory concentration in the GCF
on1 of the periodontal pockets, large doses must be taken, which
270 may lead to associated side effects and resistance of
antibiot..64 ics.12 Therefore, localized drug-delivery systems containing
.957 antibacterial agents have been developed, including fibers,
y3b strips, films, implants, gels, and so forth, and now many
/om products are commercially available.13–15 These are
admin.ssc istered in the periodontal pocket after ultrasonic scaling and
rep root planning to enhance the local effect. Compared with the
ve conventional systemic administration of antibiotics, these new
.doww l.yno formulations reduce the frequency of administration and keep
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om rse still exist. The vehicles of fibers and strips must be removed
ded roF after complete release of the drug by professionals, and the
loa wound is not conducive to recovery.16 The application of gels is
onw more convenient than fibers and strips, but the issues are burst
iicnedde releBasieodaengdrathdeabralepindalnoocpaalrdtirculgesc(leNaPrasn)cheavoef abneteinmdicervoeblioaplse.d3
onm rapidly over the past few years since they have shown great
faN potential in drug delivery.17 NPs have more advantages than
loa other new formulations and many of the already-mentioned
rnou problems can be avoided. NPs can offer sustained release of
lJa drugs and have the ability to adsorb onto polymer gels and act as
itona a glue to bond pieces of hydrogels and tissues together.18 Based
trne on these properties, NPs can adhere to diseased tissues longer
In and maintain local drug concentration for a long time. Due to
the small particle size, NPs are able to penetrate into alveolar
bone trabeculae, the underlying connective tissue, and even
the periodontal pocket areas below the gum,14,19,20 which can
also significantly improve the antibacterial effect. Successful
periodontal treatment requires a high initial antibiotic
concentration followed by the continued release of the drug at a lower
concentration, both of which can be realized with NPs.21
“Minocycline” belongs to the tetracycline family, but it
has a broader antibacterial spectrum than other tetracyclines
and can be applied in the treatment of periodontitis.21,22 It has
been demonstrated that minocycline can affect the immune
response caused by cell factors and have a beneficial effect
on periodontal health.23 In this study, minocycline was
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encapsulated in poly(ethylene glycol)-poly(lactic acid)
(PEG-PLA) NPs, which are metabolized to nontoxic CO2 and
H O in the body,24 and the pharmacokinetics and
codynamics of this formulation were investigated through a
periodontitis model in beagle dogs.
Materials and methods
Methoxy-poly(ethylene glycol) (MPEG; molecular weight
[MW] 3,000 Da) was supplied by NOF Corporation
(Tokyo, Japan) and dried under vacuum in a desiccator with
P2O5 overnight before polymer synthesis. D,L-lactide (purity:
99.5%) was purchased from Purac Biochem (Gorinchem,
The Netherlands) and purified by twice recrystallizing in
dried ethyl acetate. Stannous octoate [Sn(Oct)2] (95% pure;
Sigma-Aldrich, St Louis, MO, USA) was distilled under
vacuum and was used by dissolving it in dry toluene.
Minocycline hydrochloride (MIN) and tetracycline (both
99.5% pure) were obtained from the HuBei Xing Galaxy
Chemical Corporation Ltd. (Wuhan, People’s Republic of
China). MIN ointment (Periocline®) was purchased from
Sunstar Inc. (Osaka, Japan). Double distilled water was
purified using a Millipore Simplicity System (EMD Millipore,
Billerica, MA, USA). All other chemicals were of analytical
reagent grade and used without further purification.
Beagle dogs weighing 12–15 kg were obtained from the
Shanghai Xingang Laboratory Animal Co Ltd. (Shanghai,
People’s Republic of China). The animals used for
experiments were treated according to protocols evaluated and
approved by the Experimental Animal Ethical Committee
of Fudan University.
Methoxy-poly(ethylene glycol) poly(lactic acid) (MPeg-Pla) copolymer synthesis and characterization
MPEG-PLA block copolymer was synthesized by
ringopening polymerization of D,L-lactide, using stannous
octoate as the catalyst and MPEG as the initiator. Briefly,
0.4 g of MPEG, 4 g of D,L-lactide, and 50 mg of stannous
octoate in dry toluene were added to a round-bottomed flask.
The reactants were dried at 70°C under vacuum for 1 hour
then polymerized at 160°C under vacuum for 4 hours. The
cooled product was dissolved in dichloromethane and
precipitated into an excess solvent mixture of ethyl ether and
petroleum ether. The precipitant was then redissolved in
acetone and precipitated into excess methanol. Finally, the
purified MPEG-PLA copolymer was vacuum-dried at 40°C
for 24 hours and stored in an electronic dryer. Proton nuclear
magnetic resonance (1H NMR) spectroscopy of MPEG-PLA
was recorded in CDCl3 with a Varian Mercury Plus-400 MHz
(Agilent Technologies, Santa Clara, CA, USA) apparatus
operating at 400 MHz at 25°C. Chemical shifts in parts per
million (δ) were determined using the chloroform signals
at 7.26 ppm as a reference. The integrals of the peaks
corresponding to the poly(lactic acid) (PLA) methane protons
(δ 5.18 ppm) and the poly(ethylene glycol) (PEG) methylene
protons (δ 3.65 ppm) were used to determine the weight
ratio of PLA to PEG and to calculate the average number
molecular weight (Mn) of the PLA moiety. The molecular
weight and molecular distribution of the polymer were also
determined by gel permeation chromatography (GPC) as
previously described25 using an Agilent 1,100 GPC (Agilent
Technologies) with tetrahydrofuran as the solvent.
Preparation and characterization
of minocycline-loaded NPs
Preparation of minocycline-loaded NPs
Minocycline-loaded nanoparticles (MIN-NPs) were prepared
using an emulsion/solvent evaporation method. In brief,
40 mg of MPEG-PLA and 8 mg of MIN were dissolved in
1 mL of dichloromethane then were added into 5 mL of 0.6%
sodium cholate aqueous solution. The mixture was
intensively emulsified by sonication (200 W, 5 seconds) 15 times
in ice water using a JY92-II probe sonicator (Ningbo Scientz
Biotechnology Co Ltd., Ningbo, People’s Republic of China).
After evaporating dichloromethane off with a RE-5205 rotary
evaporator (Shanghai Yarong Biochemistry Instrument
Factory, People’s Republic of China) at 37°C, the obtained
NPs were concentrated by centrifugation at 21,000 g for
45 minutes using a TJ-25 centrifuge (Beckman Coulter Inc.,
Brea, CA, USA). After discarding the supernatant, the NPs
were resuspended in 0.3 mL of deionized water. Blank NPs
were prepared using the same procedure without the addition
of MIN in dichloromethane.
Morphology and particle size
The particle size and zeta potential of the NPs were determined
by dynamic light scattering using a Nano-ZS Malvern
Zetasizer Nano analyzer (Malvern Instruments, Malvern, UK). The
morphology of the NPs was observed using an H-600
transmission electron microscope (Hitachi, Tokyo, Japan) after negative
staining with 2% sodium phosphotungstate solution.
Drug encapsulation efficiency and loading capacity
The drug encapsulation efficiency (EE) and drug loading
capacity (LC) of the MIN-NPs was investigated as previously
described.25 Briefly, the minocycline concentration in the
supernatant was determined by high-performance liquid
chromatography (HPLC) using a Waters® e2695 Separations
Module (Waters Corporation, Milford, MA, USA) equipped
with an Agilent Zorbax SB analytical column (150×4.6 mm,
pore size 5 μm; Agilent Technologies). The mobile phase
was a mixture of methanol, acetonitrile, and 0.01 M
KH2PO4 (23:5:22 v/v/v) containing 0.03 mM Na2EDTA
and 60% HClO4 (2.9 mL), adjusted to pH 2.5 with 10 M
KOH.26 The flow rate was 1.2 mL/min. The sample injection
volume was 20 μL, and the detector wavelength was 350 nm.
The drug EE was calculated as EE = (MINtotal - MINsupernatant)/
MINtotal ×100% and the drug LC was calculated as LC =
(MINtotal - MINsupernatant)/materials ×100%.
In vitro release study
The in vitro release profile of minocycline from the
MINNPs was investigated by a dialysis method using
phosphatebuffered saline (PBS; 0.01 M, pH 7.4) as the release medium.
Briefly, 1 mL of MIN solution or MIN-NP suspension in
PBS (containing 100 μg of MIN) was introduced into a
dialysis bag (MWCO 8,000 Da; Greenbird Inc., Shanghai,
People’s Republic of China) and incubated in 10 mL of
release medium at 37°C at the shaking speed of 100 rpm.
At each setting time point, a 0.2 mL aliquot was withdrawn,
and, immediately, an equal volume of fresh release medium
was added. The released samples were analyzed by HPLC
as already described. Samples stored were away from light
throughout the experimental procedure. For the release study,
these experiments were performed in quadruplicate.
In vivo pharmacokinetics
Periodontitis modeling in beagle dogs
Periodontitis in beagle dogs was modeled by tying ligatures
around the cervical region of the tooth using dental ligature
wire (ClassOne Orthodontics, Carlsbad, CA, USA).27 Briefly,
2 weeks after scaling, dogs were anesthetized by intravenous
injection of pentobarbital sodium at a dose of 30 mg/kg.
Experimental periodontitis was induced on the left upper
second premolar (PM2), third premolar (PM3), and fourth
premolar (PM4) as well as the left lower PM3, PM4, and
first molar (PM1). Prior to ligation, the gingival attachment
was incised, and the periodontal ligaments were undermined
until a periodontal pocket depth of up to 3 mm was reached
with a straight elevator (Osung MND Co Ltd., Gyeonggi-do,
Korea). After undermining, a shallow notch was made in the
mesial and distal region of each tooth with a round bur to act
as a retentive groove for the ligature. After ligature placement,
tramadol (4 mg/kg, intramuscular injection) was administered
twice a day for 3 days for pain control. To promote plaque
formation, soft moistened food was given for the following
8 weeks. The ligatures were checked weekly and any missing
ligatures were replaced immediately. To evaluate periodontal
status, the clinical periodontal parameters of plaque index (PI),
gingival index (GI), periodontal pocket depth (PPD), clinical
attachment level, and bleeding on probing were recorded.3
Pharmacokinetics experiments were performed as previously
described.3 In brief, 8 weeks after dental ligature, dogs were
anesthetized by intravenous injection of pentobarbital sodium.
Fifty microliters of either minocycline solution in
N-methyl2-pyrrolidone (NMP) (2%), MIN-NPs, or Periocline were
injected into the periodontal pocket of each beagle dog by a
disposable syringe with a 21-gauge needle. No periodontal
.doww l.yno dressing or adhesive was used to aid in retention of the
mino//w se cycline formulations. At a set time point after administration,
ttsph launo the dogs were anesthetized and GCF samples were collected by
om rse positioning an absorbent paper point (Meta, Tianjin, People’s
ded roF Republic of China) at the orifice of the sulcus for 30 seconds.
loa To avoid contamination with blood and saliva, the paper strips
now were manipulated gently. The paper strips were weighed and
cwuarsvceatlhcautlawteads.cGalCibFravtoeldumuseinwgabsomveinaenom serum. Minocycline was immediately eluted from the paper
faN strips by 0.1 M glycine buffer and stored at -80°C. GCF
loa minocycline levels were calculated by dividing the content
rnuo of each sample pool by total volume. All GCF samples were
lJa processed with minimal delay. Minocycline concentrations in
itnao GCF were plotted with time and processed by DAS software
trne (v 2.0; Professional Committee of Pharmacomtrics of Chinese
In Pharmacological Society, People’s Republic of China) to
calculate the pharmacokinetics parameters.
The pharmacodynamics experiments were performed as
described in the “Pharmacokinetics experiments” section with
a little modification. Eight weeks after dental ligature, 12 dogs
were randomly assigned to four groups: saline, free
minocycline, MIN-NPs, and Periocline. After anesthetization with
pentobarbital sodium, dogs were treated with 50 μL of saline,
minocycline solution in NMP (2%), MIN-NPs, or Periocline by
injection into the periodontal pocket of each beagle dog with a
disposable syringe. The clinical periodontal parameters of PI,
GI, PPD, and clinical attachment level were recorded every
3 days to evaluate the therapeutic efficacy of the MIN-NPs.
The pharmacokinetics and clinical parameters for each group
are expressed as the mean ± standard deviation. Statistical
differences were determined by one-way analysis of
variance, followed by Dunnett post-hoc analysis for multi-group
comparison. Data within the 95% confidence level were
MPeg-Pla copolymer synthesis and characterization
Block copolymer MPEG-PLA was synthesized with
molecular weight of 33,126 Da, as determined by 1H NMR.
The typical 1H NMR spectra and chemical shifts (Figure 1)
were in agreement with previously published data on
MPEG-PLA copolymers. Peaks at 3.38 ppm (a), 3.65 ppm
(b), 5.18 ppm (c), and 1.59 ppm (d) confirmed the block
nature of the MPEG-PLA copolymer. GPC measurements
indicated that the polydispersity index and number-based
molecular weight of MPEG-PLA were 1.25 and 28,932 Da,
respectively. MPEG-PLA was selected and synthesized
for preparing NPs due to its biodegradability. Unlike
other nonbiodegradable polymers, PEG-PLA NPs should
be fully biodegradable, leaving no potentially toxic
byproducts upon their degradation, after in vivo application
for a long time.
Preparation and characterization of MIN-NPs
Morphology, particle size, and zeta potential
As shown in Figure 2, transmission electron
microscopy showed that the minocycline-loaded PEG-PLA
NPs were spherical with a uniform size and well
dispersed without any adhesion or aggregation. The mean
Size distribution by intensity
Size (d. nm)
Zeta potential distribution
particle size of the MIN-NPs was 98±12 nm (Figure 3),
which was slightly increased compared with that of blank NPs
(90±16 nm). In a previous report,21 poly(lactic-co-glycolic
acid) (PLGA)-based NPs were prepared using different
methods, but the particle sizes of these PLGA NPs ranged
from 85 nm to 7,070 nm, and most of them were larger than
200 nm. Moreover, PLGA NPs without a surface coating or
PEG or another hydrophilic component are not stable and
prefer to aggregate in physiological conditions.28 Therefore, the
local delivery of PLGA NPs in periodontal pockets is limited.
Considering the relationship between particle size and tissue
absorption and penetration, smaller-size stable PEG-PLA
NPs were prepared in this study. A negative surface charge
was detected for the MIN-NPs (-24 to -20 mV), which was
similar to that of the blank NPs (-23 to -20 mV) (Figure 3).
No significant changes in the zeta potential of the NPs were
observed after MIN loading.
Drug ee and lc
The drug LC and EE of the MIN-NPs were detected by
HPLC, and the values of LC and EE were 9.3%±0.2% and
46.5%±0.9%, respectively. The highest LC and EE for
PLGA-based NPs prepared using different methods were
1.92%±0.19% and 29.95%±2.47% respectively,21 and both of
these two values are lower than those obtained in our study,
indicating the superiority of our MIN-NP design.
In vitro release of MIN-NPs
The release profiles of minocycline solution and MIN-NPs
in PBS (0.01 M, pH =7.4) at 37°C showed that almost 100%
of the minocycline solution was released within 24 hours,
while the cumulative release of MIN from the MIN-NPs
Pharmacokinetics of different formulations of MIN in gcF
The concentration of MIN in GCF was determined by HPLC,
and the baseline separation of MIN and tetracycline was
achieved with the chromatographic conditions outlined.
The retention time (RT) for MIN and tetracycline was
7.768 minutes and 9.801 minutes, respectively (Figure 5).
The standard curve (y =0.158 x +0.359, R²=0.997) was
obtained by the line regression of MIN concentration with the
peak area ratio of MIN and tetracycline, while the linearity
was given in the range of 0.1–20.0 μg/mL.
that the concentration of MIN in GCF declined rapidly after
the administration of MIN solution and was lower than
the median effective concentration (1 μg/mL) after 2 days.
The concentration of MIN in GCF declined slowly after
administration of Periocline, and the action of the drug
lasted 8 days (1 μg/mL), suggesting good sustained
release of Periocline. However, after administration of
the MIN-NPs, the concentration of MIN in GCF declined
slowly and was still higher than 1 μg/mL (1.28 μg/mL)
12 days later, representing the longest action time among
three groups. The long action time of the MIN-NPs may be
related to either the drug’s sustained release or the adherence
and penetration of MIN-NPs into inflammation tissues. The
pharmacokinetics parameters of MIN in GCF, as
calculated using DAS pharmacokinetics software, are shown in
Table 1. The elimination of MIN in each group was well fitted
to two-compartment open models. There were significant
differences in the area under the curve (AUC) from zero to infinity
(0–∞), mean retention time (MRT) (0–∞), half-life (t1/2),
elimination rate constant (k), and clearance (Cl) between groups
(P0.01). The AUC(0–∞), MRT(0–∞), and t1/2 of the MIN-NP
group were 4.62-fold, 2.21-fold, and 3.74-fold, respectively,
those of the MIN solution group. The AUC(0–∞) and MRT(0–∞) of
the MIN-NP group were 1.42-fold and 1.21-fold, respectively,
higher than those of the Periocline group, indicating higher
local bioavailability and the better effect of the former.
cen ) 8
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Pharmacodynamics of MIN-NPs
As shown in Figure 7, there were no significant differences
in clinical periodontal parameters between the MIN
solution group and saline group after the local delivery of MIN
solution in the dog periodontitis models, indicating the MIN
solution had no obvious treatment efficacy for periodontitis.
However, for Periocline and MIN-NPs, 6 days after local
administration, clinical periodontal parameters (PPD, PI,
GI) were significantly improved, and significantly better than
those of the MIN solution group. In addition, 9 days after
administration, the PPD, PI, and bleeding on probing of the
Periocline group increased again, which may be because
an effective drug concentration of Periocline could only
be maintained for 8 days (Figure 6). Twelve days after the
administration, the PPD and PI of the MIN-NP group were
still significantly lower than those of the MIN solution and
Periocline groups, suggesting that MIN has a long treatment
efficacy, which agreed well with pharmacokinetics results.
In order to maintain the effective concentration of MIN
in GCF for a longer period, MIN-NPs were prepared for
evaluation in the study. Different forms of MIN were placed
into the periodontal pockets of beagle dogs with periodontitis.
GCF samples were collected after drug delivery and
quantified by HPLC. After local administration of MIN-NPs, the
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minocycline concentration in GCF decreased slowly and
an effective drug concentration was retained for a longer
time than with Periocline. The MIN-NPs demonstrated
anti-periodontitis effects that MIN-NPs could significantly
decrease the symptoms of periodontitis, compared with
Periocline and MIN solution. These findings suggest that MIN-NPs
might have great potential in the treatment of periodontitis.
This work was supported by the Shanghai Municipal Public
Health Bureau Research Grant, National Science and
Technology Major Project no. 2012ZX09304004, National Natural
Science Foundation of China no. 81001404, the Doctoral Fund
of the Ministry of Education of China no. 20100071120050,
and the Zhuoxue Plan of Fudan University.
The authors declare no conflicts of interest in this work.
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