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The effect of the oral administration of polymeric nanoparticles on the efficacy
and toxicity of tamoxifen
Amit K. Jain, Nitin K. Swarnakar, Chandraiah Godugu, Raman P. Singh, Sanyog Jain*
Centre for Pharmaceutical Nanotechnology, Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, SAS Nagar (Mohali),
Punjab 160062, India
a r t i c l e i n f o
Article history:
Received 8 July 2010
Accepted 19 September 2010
Available online 8 October 2010
Keywords:
PLGA nanoparticles
Tamoxifen
Oral administration
Nuclear localization
a b s t r a c t
The present investigation reports on the conditions for preparation of tamoxifen loaded PLGA nano-
particles (Tmx-NPs) for oral administration. Tmx-NPs with >85% entrapment efficiency and
165.58 3.81 nm particle size were prepared and freeze dried. Freeze dried Tmx-NPs were found to be
stable in various simulated GIT media (pH 1.2, pH 3.5, pH 6.8, SGF & SIF). No significant changes in
characteristics of Tmx-NPs were observed after 3 months accelerated stability studies. The cell viability
in C127I cells was found to be relatively lower in Tmx-NP treated cells as compared to free Tmx treated
cells. CLSM imaging reveled that nanoparticles were efficiently localized into the nuclear region of C127I
cells. Oral bioavailability of Tmx was increased by 3.84 and 11.19 times as compared to the free Tmx
citrate and Tmx base respectively, when formulated in NPs. In vivo oral antitumor efficacy of Tmx-NPs
was carried out in DMBA induced breast tumor model and tumor size was reduced up to 41.56% as
compared to untreated groups which showed an increase in tumor size up to 158.66%. Finally, Tmx-NPs
showed the marked reduction in hepatotoxicty when compared with free Tmx citrate as evidenced by
histopathological examination of liver tissue as well as AST, ALT and MDA levels. Therefore Tmx-NPs
could have the significant value for the oral chronic breast cancer therapy with reduced hepatotoxicity.
2010 Elsevier Ltd. All rights reserved.
1. Introduction
Breast cancer is the second leading cause of cancer deaths today
after lung cancer and is the most common cancer among women
[1]. For over a quarter of a century, tamoxifen (Tmx) has been
prescribed to treat patients with advanced breast cancer. Tmx
belongs to a class of non-steroidal triphenylethylene derivatives
and is the first selective estrogen receptor modulator (SERM) [2].
The US Food and Drug Administration (FDA) approved Tmx for the
treatment of advanced breast cancer in late 1998 [3]. Tmx shows its
potential effects in patient who possess estrogen receptors (ER)positive cancer cells by competing with estrogen to bind with
estrogen receptor in breast cancer cells [4].
As Tmx therapy is chronic one (3e5 years), oral delivery is the
most preferredroute of administration and its solubility problem in
aqueous milieu has been overcome by forming its salt form,
tamoxifen citrate (Tmx citrate). Commercially, Tmx is available only
as tablet and oral solution containing Tmx citrate in a daily dose of
10e20 mg. However Tmx citrate also showed the poor oral
bioavailability (20e30%) due to its precipitation as free base in the
acidic environment of stomach and also due to extensive hepatic
and intestinalfirst pass metabolism, so as to increase its does [5]. So
in spite of a clinical choice in advanced and metastatic stages of
breast cancer, it suffers from large inter subject variability and
several dose and concentration dependent side effects [6e8]. It
mainly causes oxidative stress mediated hepatotoxicity, i.e. toxic
hepatitis, multifocal hepatic fatty infiltration, sub massive hepatic
necrosis and cirrhosis [9]. Tmx is also having high risk of causing
endometrial cancer which depends mainly upon treatment dura-tion and dose accumulation [10]. Thus, existing therapy renders its
difficult to administer in minimum effective dose, leading to liver
toxicity. Thus an alternate delivery system is essential for optimal
oral chronic therapy of Tmx with improved bioavailability and
reduced side effects especially hepatotoxicity.
Biodegradable polymeric nanoparticles (NPs) have gained
a considerable interest in this regard [11]. Amongst them poly
(lactic-co-glycolic acid) (PLGA) is an approved biodegradable
polymer with good biocompatibility and widely employed for
loading and encapsulation of variety of anticancer drugs [12e14].
When polymeric NPs are administered by oral route, the M-cells* Corresponding author. Tel.: 172 2292055; fax: 172 2214692.
E-mail addresses: [email protected] , [email protected] (S. Jain).
Contents lists available at ScienceDirect
Biomaterials
j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / b i o m a t e r i a l s
0142-9612/$ e see front matter 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biomaterials.2010.09.037
Biomaterials 32 (2011) 503e515
mailto:[email protected]:[email protected]://www.sciencedirect.com/science/journal/01429612http://www.elsevier.com/locate/biomaterialshttp://dx.doi.org/10.1016/j.biomaterials.2010.09.037http://dx.doi.org/10.1016/j.biomaterials.2010.09.037http://dx.doi.org/10.1016/j.biomaterials.2010.09.037http://dx.doi.org/10.1016/j.biomaterials.2010.09.037http://dx.doi.org/10.1016/j.biomaterials.2010.09.037http://dx.doi.org/10.1016/j.biomaterials.2010.09.037http://www.elsevier.com/locate/biomaterialshttp://www.sciencedirect.com/science/journal/01429612mailto:[email protected]:[email protected]7/31/2019 The Effect of the Oral Administration of Polymeric Nanoparticles Efficacy and Toxicity
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(specialized cells staying over mucosa-associated lymphoid tissue)
in Payers patches uptake the nano/microparticle and transport
them from the gut lumen to intra-epithelial lymphoid cells and
afterward through the lymphatic system into the blood stream
[15e17]. NPs follow this special pathway and thus enhance the
bioavailability of encapsulated drug and also avoid the enzymatic
degradation in enterocytes, first pass metabolism in liver thus
decrease the dose and ultimately the drug related toxicity.
In the present work tamoxifen loadedPLGA nanoparticles (Tmx-
NPs) have been prepared,characterized and freezedried. The freeze
dried Tmx-NPs were evaluated for in vitro release characteristics,
GIT stability and accelerated stability study. In vitro antitumor
activity was evaluated on mouse breast cancer cells C127I [18].
Pharmacokinetics, in vivo antitumor efficacy and hepatotoxicity
were also evaluated after oral administration.
2. Materials and methods
2.1. Materials
PLGA 50/50 (inherent viscosity 0.41 dl/g in chloroform at 25 C) was used from
Boehringer Ingelheim (Ingelheim, Germany). Tamoxifen (Z)-2-[4-(1, 2-diphenyl-1-
butenyl)phenoxy]-N,N dimethylethylamine (free base and citrate salt), Didode-cyldimethylammonium bromide (DMAB) (98%), Polyvinyl alcohol (PVA) (MW.
30000e70000), Pluronic F-68, 7, 12-dimethylbenz[a]anthracene (DMBA), Trypsin-
EDTA, MTT (3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide),
coumarin-6, triton X-100 and propidiumiodide (PI)were obtainedfrom Sigma, USA.
Dulbeccos modified Eagles medium (DMEM), fetal bovine serum (FBS), antibiotics
(Antibioticeantimycotic solution) and Hankss balanced salt solution (HBSS) were
purchased from PAA, Austria. Tissue culture plates and 8-well culture slides were
procured from Tarsons and BD Falcon, respectively. Ethyl acetate (LR grade),
Acetonitrile (HPLC grade), methanol (HPLC grade) were purchased from Ranchem
Fine Chemicals, India. Ultra pure water (SG water purification system, Barsbuttel,
Germany) was used for all the experiments. All other reagents used were of
analytical grade.
2.2. Preparation of Tmx loaded nanoparticles
Tmx-NPs were prepared by emulsion diffusion evaporation method as reported
earlier in literature [19] with slight modification according the laboratory condi-
tions. Briefly,50 mgof PLGA alongwith5 mgof Tmxwere dissolved in2.5 mlof ethyl
acetate (EA) at room temperature. The organic phase was then added to 5 ml of an
aqueous phase containing the stabilizer. The resulting o/w emulsion was stirred at
1000 rpm for 20 min. The droplet size reduction of resulting emulsion was carried
out either by homogenization (high-speed homogenizer, Polytron PT 4000,
Switzerland) or sonication (Misonix, USA). The resulting emulsion was poured into
25 ml of water with constant stirring to diffuse and finally evaporating the organic
solvent. This resulted in nanoprecipitation and formation of NPs. The NPs suspen-
sion was then centrifuged and washed repeatedly to remove the excess surfactant
and finally dispersed in 2 ml distilled water and freeze dried (FD).
2.3. Optimization of process variables
2.3.1. Effect of droplet size reduction process
Screening of the droplet size reduction processes (i.e. either homogenization or
sonication) was carried out to get the optimum size (below 200 nm). For this, NPswere prepared following the above described process keeping other experimental
parameters like aqueous to organic phase ratio 1:2, final volume of dilution 25 ml
and stabilizer concentration (2% w/v PVA) constant. Different homogenization
speeds and sonication (60% amplitude for 1 min) were employed to prepare NPs
dispersion. Finally particle size and PDI of NPs dispersion was measured using zeta
sizer (Nano ZS, Malvern, UK).
2.3.2. Screening of suitable stabilizer
Tmx-NPs were prepared by using different type and concentration of stabilizers
like DMAB, PVA and Pluronic F-68. The best suitable stabilizer was identified based
on the optimum particle size, zeta potential and entrapment efficiency.
2.3.3. Screening of optimum concentration of stabilizer
The best suitable stabilizer identified as above was then screened for the
optimum concentration of the stabilizer required for the preparation of Tmx-NPs.
The optimum concentration of stabilizer was determined on the basis of particle
size, size distribution and encapsulation effi
ciency.
2.3.4. Optimization of drug loading
Finally, Tmx-NPs were prepared using different Tmx loading i.e. 5%, 10% and 15%
w/w ofpolymerand itseffecton particlesizeand entrapmentefficiency wasstudied.
The other experimental parameters likesonication time (1 cycle at 60% of amplitude
for 60 s), stabilizer concentration (2% PVA) and aqueous to organic phase ratio 1:2
were kept constant.
2.4. Characterization of nanoparticles
2.4.1. Particles size and zeta potential measurementTmx-NPs were evaluated for their mean particle size and polydispersity index
(PDI) by using Zeta Sizer (Nano ZS, Malvern Instruments, UK). All the values were
taken by the average of 6 measurements. Zeta potential was estimated on the basis
of electrophoretic mobility under an electric field, as an average of 30 measure-
ments. Zeta potential was also determined by using Zeta Sizer (Nano ZS, Malvern
Instruments, Malvern, UK).
2.4.2. Entrapment efficiency
The percentage of drug encapsulated in PLGA NPs was determined by using
a validated HPLC method reported in literature with slight modifications [20].
Briefly, Tmx-NPs suspension was centrifuged and the obtained pellet was dissolved
in acetonitrile furthermore analyzed by Waters high-performance liquid chroma-
tography (HPLC) system consisting of 996 Photodiode Array Detector and dVR
Agilent Technologies Lichrospher 100 RP-18e end capped 5 mm column (Lot No. L
54921633) (Germany). Acetonitrile and methanol (containing 0.02% triethylamine)
(70:30) were used as the mobile phase with a flow rate of 0.7 ml/min. The injection
volume was 10 ml and retention time of Tmx was found to be 5.1 min. The detection
wavelength (lmax) for Tmx was 281 nm.
2.4.3. Morphology of nanoparticles
The surface morphology of nanoparticles was analyzed by atomic force micro-
scope (Veeco Bioscope II, USA). The nanoparticles suspension were placed on the
silicon wafer with the help of a pipette and allowed to dry in air. The microscope is
vibration damped and measurements were madeusing commercial pyramidal Si3N4tips (Veecos CA, USA). The cantilever used for scanning was having length 325 mm
and width 26 mm with a nominal force constant 0.1 N/m. Images were obtained by
displaying the amplitude signal of the cantilever in the trace direction, and the
height signal in the retrace direction, both signals being simultaneously recorded.
2.5. Freeze drying of NPs
Tmx-NPs were freeze dried (Vir Tis, Wizard 2.0, New York, USA freeze dryer)
following an optimized freeze dried cycle (Table 1) [21]. The condenser temperature
was 60 C and pressure applied in each step was 200 Torr. 2 ml of washed NPs
suspension was filled in 5 ml glass vials and subjected to freeze drying using 5% w/v
of trehalose. After freeze dying the Tmx-NPs were characterized for the appearance
of the cake, reconstitution time, size after freeze drying, entrapment efficiency,
nature of drug in nanoparticles using DSC and XRD analysis.
2.6. DSC analysis
Differential scanning calorimetry (DSC) thermogram of the freeze dried Tmx-
NPs, physical mixture, pure tamoxifen and trehalose was carried out using a Mettler
Toledo differential scanning calorimeter calibrated with indium standards.
Measurements were performed at heating rate of 10 C/min from 0 to 200 C.
2.7. XRD analysis
The X-ray diffraction patterns of pure tamoxifen, PLGA, blank nanoparticles,
drugloaded freeze dried nanoparticleswere obtainedusing theX-raydiffractometer
Table 1
Optimized freeze drying cycle.
Thermal treatment Primary drying
Step Temperature (C) Time
(Min)
Step Temperature (C) Time
(Min)
1 20 30 1 45 60
2 15 60 2 30 360
3 10 60 3 20 360
4 5 120 4 10 420
5 15 60 5 5 360
6 25 60 6 0 180
7 45 30 7 5 120
Secondary drying 8 10 60
1 25 120 9 15 60
10 20 30
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(Bruker D8 advance, Bruker, Germany). Measurements were performed at a voltage
of 40 kV and 25 mA. The scanned angle was set from 3 2q! 40 , and the scanned
rate was 2 min1.
2.8. In vitro release of Tmx
In vitro release of Tmx form PLGA NPs was performed in phosphate buffer saline
(PBS) pH 7.4, under sinkcondition. The studywas performed usingfreezedried drug
loadedNPs (correspondingto 1 mgof entrapped drug).The freezedried drug loaded
nanoparticles were suspended in dialysis tube bag (MWCS 12000 Da). The bag was
suspended in 10 ml of PBS pH 7.4, containing 1% w/v Tween 80 at 37 C in shaking
water bath at 100 rpm. Aliquots of 200 ml of sample were withdrawn and estimated
by HPLC method for amount of drug released.
2.9. pH dependent stability of freeze dried Tmx-NPs
Freezedried Tmx-NPs were evaluated for their stability in various simulated GIT
fluids (pH 1.2, pH 3.5, pH 7.4, simulated gastric fluids (SGF), and simulated intestinal
fluids (SIF)) to assess the stability of NPs under various GIT pH and enzymatic
conditions that can influence their particle size and drug release characteristics.
Briefly, 10 ml of simulated fluids were added to 2 ml of reconstituted freeze dried
Tmx-NPs. An incubation time of 2 h was employed for pH 1.2, pH 3.5 and SGF while
6 h for pH 7.4 [13,22]. Particle size, PDI and entrapment efficiency were determined
after the incubation of freeze dried Tmx-NPs with different simulated fluids.
2.10. Accelerated stability studies
Freeze dried Tmx-NPs were assessed for accelerated stability studies over
a period of 3 months, according to the some protocols reported in the literature
[13,21,23]. Briefly, freeze dried Tmx-NPs were transferred to 5 ml glass vials sealed
with plastic caps and were kept in stability chamber with temperature of 25 2 C
and RH 60 5%. The different formulations were monitored for changes in particle
size, PDI and entrapment efficiency in addition to for physical appearances and ease
of reconstitution.
2.11. Cell culture experiments
2.11.1. Cells
C127I mouse breast cancer cell line was obtained from National Centre for Cell
Sciences, Pune, India. The cells were maintained in complete medium containing
Dulbeccos modified Eagles medium (DMEM; PAA, Austria), 10% fetal bovine serum
(FBS; PAA, Austria), and antibiotics (Antibiotice
antimycotic solution; PAA, Austria).
2.11.2. In vitro anticancer activity
C127I cells were harvested from confluent cultures by trypsinization and
adjusted to 50,000 cells/ml in complete medium. The cell suspension was added in
96 well tissue culture plates (0.2 ml/well) and incubated overnight for cell attach-
ment. Following attachment, the medium was replaced with complete medium
(0.2 ml) containing the free Tmx or Tmx-NPs at the desired concentration. The cells
were incubated with free Tmx-NPs for 24 or 72 h and cell viability was determined
by MTT assay. In another set of experiments, the recovery of cells after free Tmx and
Tmx-NPs treatment was determined. The cells were incubated with free Tmx-NPs
for 24 h, washed with Hankss balanced salt solution (HBSS; PAA, Austria) and
furtherincubated in complete medium (withoutdrug/NPs) for 48 h. Thecell viability
was assessed by MTT assay.
2.11.3. MTT assay
Following treatment, the cells were washed with HBSS and incubated with
0.2 ml fresh DMEM containing 0.5 mg/ml MTT (Sigma, USA). The MTT-containingmedium was removed after 3 h incubation. The MTT formazon was dissolved in
0.2 ml dimethylsulfoxide (CDH, India) and opticaldensitywas determined at 550 nm
using a Bio-Tek ELISA plate reader.
2.11.4. Cell uptake studies
Fluorescent NPs were prepared by co-encapsulation of coumarin-6 with Tmx in
PLGA (coumarin-6-Tmx-NPs). The dye was added in the organic phase (100 mg/
50 mg polymer) and coumarin-6-Tmx-NPs were prepared following the optimized
protocol as described earlier. Cumulative dye release from coumarin-6-Tmx-NPs
was determined in phosphate-buffered saline (pH 7.4) by fluorimetry (excitation/
emission458/505nm) after 24h C127Icells wereseededin 8-wellcultureslides (BD
Falcon) and allowed to attach overnight. The cells were incubated with coumarin-6-
Tmx-NPs for 3 h and extracellular particles were removed by washing with HBSS
(5). The cells were fixed with 3% paraformaldehyde (Merck, India) and per-
meabilized with0.2% Triton X-100 (Sigma, USA). The nuclei were stained with10 mg/
ml propidium iodide (Sigma, USA). The cells were observed under the confocal laser
microscope (CLSM) (Olympus FV1000).
2.12. In vivo pharmacokinetic after oral administration
2.12.1. Animals and dosing
Female Sprague Dawley (SD) rats of 220e230 g and 4e5 weeks old were
supplied by the central animal facility (CAF), NIPER, India. All the animal studies
protocols were duly approved by the Institutional Animal Ethics Committee (IAEC),
National Institute of Pharmaceutical Education & Research (NIPER), India. The
animals were acclimatized at temperature of 25 2 C and relative humidity of
50e60% under natural light/dark conditions for one week before experiments. The
animals were randomly distributed into three groups each containing 6 animals.First group of animals received oral free Tmx base (suspension) while another
second group of animals received free Tmx citrate (suspension) and third group
received Tmx-NPs. All the formulations were administered orally at a dose of 10mg/
Kg bodyweight. Theblood samples (approximately 0.25ml) were collected fromthe
retro orbital plexus under the mild anesthesia into the micro centrifuge tubes
containing heparin (40 IU/ml blood). Plasma was separated by centrifuging the
blood samples at 5000 rpm for 5 min at 4 C. To 100 ml of plasma, 200 ml of aceto-
nitrile was added to precipitate proteins and 25 ml of 10 mg/ml of internal standard
(estradiol) was added. The samples were vortexed and centrifuged at 10,000 rpm for
15min. The supernatantswere separatedand analyzedfor drugcontentby validated
RP-HPLC [24].
2.12.2. HPLC quantification of Tmx in plasma samples
Calibration curves were used for the conversion of the Tmx/estradiol chro-
matographic area to the concentration of Tmx. Calibrator and quality control
samples were prepared by adding of appropriate volumes of standard Tmx solution
in acetonitrile to drug free plasma. Calibration curves were designed over the range
of25e1000ng/ml (r2 0.998). Briefly,an aliquot (100 ml) of plasmasample was mixed
with 25 ml of internal standard solution (estradiol 1 mg/ml) and 25 ml of drug solu-
tion. After vortexing for 30 s a protein precipitating agent acetonitrile (100 ml) was
added vortexed for 5 min. The mixture was centrifuged for 10 min at 10,000 rpm.
After centrifugation supernatant was transferred to autosampler vials, capped and
placed in the HPLC autosampler. An 80 ml aliquot of each sample was injected onto
the HPLC column. Mobile phase employed for analysis was the mixture of acetoni-
trile and methanol, containing 0.02% triethylamine (85:15).
2.12.3. Pharmacokinetic data analysis
The pharmacokinetic analysis of plasma concentrationetime data was analyzed
by one compartmental model, using Kinetica software (Thermo scientific). Required
pharmacokinetics parameters like total area under the curve (AUC)0eN, terminal
phase half-life (t1/2), peak plasma concentration (Cmax) and time to reach the
maximum plasma concentration (Tmax) were determined.
2.13. In vivo antitumor efficacy
Female Sprague Dawley (SD) rats of 45e50 day age were used for the induction
of chemical induced breast cancer. 7,12-dimethylbenz[a]anthracene (DMBA) in soya
bean oilwasadministeredorally toratsat 45 mg/kgdoseat weekly intervalfor three
consecutive weeks. Measurable tumor size was observed in animals and tumor
bearing animals were separated and divided randomly into different treatment
groups. Thetumor width( W) and length(L) were recordedwith an electronic digital
caliper and tumor size was calculated using the formula (L W2/2). Drug treatment
was given after 10 weeks of the last dose of DMBA. Animals were treated with
a repeated (once in 3 days) dose of Tmx citrate suspension (group A) and Tmx-NPs
(group B) both in a dose equivalent to 3 mg/Kg body weight, of Tmx. The control
group C received a samerepeated oraladministration of PBS (pH 7.4).The tumor size
was calculated as described above. The tumor size was measured up to 30 days
(during the treatment period). Further, survival rate was observed in another group
of animals up to 60 days.
2.14. Toxicity evaluation
Next day after administration of the last dose the animals were sacrificed and
blood was collected by cardiac puncture. Liver toxicity markers ALT and AST were
estimated in plasma samples by commercially available diagnostic kits (Accurex Pvt.
Ltd., India). Fromthe same groupof animals liverwas isolatedand homogenizedin 5
volumes of PBS (pH 7.4). The total homogenate was used for the oxidative stress
(MDA levels) estimation. Enzyme activities in plasma were evaluated by a UV kinetic
method. Representative liver tissues from each group were excised and fixed in 10%
(v/v) formalin saline and processed for routine histopathological procedures.
Paraffin embedded specimen were cut into 5 mm sections and stained with hema-
toxylin and eosin (H&E) for histopathological evaluations.
2.15. Statistical analysis
All the results were expressed as mean standard deviation (SD). Statistical
analysis was performed with Sigma Stat (Version 2.03) using one-way ANOVA fol-
lowed by TukeyeKramer multiple comparison test. P < 0.05 was considered as
statistically signifi
cant difference.
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3. Results
3.1. Preparation and optimization of Tmx-NPs
3.1.1. Effect of droplet size reduction process
Tmx-NPs were prepared by using both homogenization and
sonication as a tool for droplet size reduction. Two different
homogenization speed10,000 rpmand 15,000 rpmweretested. Fig.1
shows significant reduction in particle size upon increasing the
homogenization speed. Howeverthe desiredparticle size(0.4) and unacceptable. But no significant
difference (p > 0.05) was observed in particle size and PDI with
respect to the 2% and 3% PVA. So 2% w/v PVA was optimized for
Tmx-NPs preparation.
3.1.4. Effect of drug loading
Tmx-NPs were prepared using different theoretical drug loading
i.e. 5%, 10% and 15% w/w of polymer to determine the optimum
percentage of Tmx in PLGA matrix. As shown in the Table 4, theo-
retical drug loading didnt affect the particle size significantly
(p > 0.05) when it was increased form 5e15% but there was
significant change (p < 0.05) in PDI. The entrapment of efficiency
was also increased when drug loading was increased from 5% to
10% but it decreased on further increasing it to 15%.
3.2. Shape and morphology of Tmx-NPs
AFM image of nanoparticles showed distinct spherical particles
with smooth surface (Fig. 2). A good correlation was obtained in the
particle size as observed by both zeta sizer and AFM.
3.3. Freeze drying of Tmx-NPs
The Tmx-NPs were freeze dried using optimized stepwise freezedrying cycle developed previously by our group [21]. A 5% w/v
trehalose was added to 2 ml of NPs suspension. After freeze drying
the obtained cake was redispersed in 2 ml distilled water and
particle size along with PDI after freeze drying was analyzed using
zeta sizer. Different properties of freeze dried NPs like physical
appearance, reconstitution nature and size ratio (before and after
freeze drying) are given in Table 5. It is clear form Table 5 that
lyophilization using 5% trehalose as a lyoprotectant produced intact
fluffy cake which was easily redispersed to form NPs by mere
manual shaking (upside down for 20 s). No significant changes in
particle size, PDI and entrapment efficiency were observed after
freeze drying. Moreover, Sf/Si (ratio of particle size after and before
freeze drying) remained almost unity (1.005) when Tmx-NPs were
lyophilized in presence of 5% trehalose. In contrast, particle size ofNPs was increased significantly (p < 0.05) with unacceptable PDI
for freeze dried Tmx-NPs without trehalose. No significant differ-
ence in percentage entrapment efficiency (p > 0.05) was observed
before and after freeze drying in all cases.
Fig. 1. Effect of droplet size reduction process on particle size and PDI.
Table 2
Effect of stabilizer type on particle size, PDI, entrapment efficiency and zeta
potential.
Surfactants Size (nm) PDI Entrapment
efficiency (%)
Zeta potential
2% PVA 165.58 3.81 0.085 0.07 86.20 1.450 3.26 0.95
1% DMAB 120.00 4.10 0.015 0.04 15.30 0.312 45.57 4.68
PF-68 130 5.60 0.136 0.08 38.56 0.245 3.45 0.67
Values are in mean
SD (n
6)
Table 3
Effect of PVA concentration on particle size, PDI and entrapment efficiency.
Surfactants
concentration
Size (nm) PDI Entrapment
efficiency (%)
1% PVA 195.50 9.20 0.425 0.90 86.20 1.550
2% PVA 165.58 3.81 0.079 0.07 85.78 2.540
3% PVA 168.00 3.10 0.055 0.05 84.94 1.550
Values are in mean SD (n 6)
Table 4
Effect of initial drug loading on particle size and entrapment efficiency.
Drug loading
(% w/w)
Particle
size (nm)
PDI Zeta
potential (mV)
Entrapment
efficiency (%)
5% 153.23 4.35 0.034 0.056 3.26 0.95 65.32 2.23
10% 165.58 3.81 0.085 0.070 3.50 0.50 86.20 1.45
15% 190 1.58 0.198 0.011 3.34 0.89 60.45 1.38
Values are in mean
SD (n
6)
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3.4. DSC analysis
Fig. 3 shows DSC thermograms of pure Tmx, Tmx-NPs, PLGA,
trehalose and their physical mixture. Tmx-NPs showed no melting
peak indicating absence of crystallinity. Whereas, Tmx in pure form
exhibited a melting peak around at 98 C indicating crystalline
nature of the drug.
3.5. XRD analysis
To study the characteristic of drug inside the NPs, X-ray diffrac-
tion (XRD) pattern of pure Tmx, PLGA, Tmx-NPs, physical mixture
andtrehalose were studied. ThecharacteristicXRD pattern of Tmxis
shown in Fig. 4, while in the case of freeze dried nanoparticles, no
characteristic peaks of Tmx were observed.
3.6. In vitro release of Tmx
The release profile of optimized Tmx-NPs is shown in Fig. 5. The
formulation exhibited sustained release profile over a period of
time. The Tmx released from PLGA nanoparticles showed the
biphasic release pattern with 23.65% of drug released in 24 h fol-
lowed by sustained release up to 23 days (94.25%). No initial burst
release was obtained from the formulation. The cumulative drug
release was fitted into different release models namely zero order,
first order, Higuchis square root plot and Hixson Crowell cube root
plot and the model giving a correlation coefficient close to unity
was taken as order of release. An initial rapid release was found in
formulation, followed by Higuchis square root pattern with r2
0.989 values and zero order patterns with r2 0.951 values.
3.7. pH dependent stability of freeze dried Tmx-NPs
Table 6 shows the change in particle size and PDI after theincubation of Tmx-NPs in different simulated GIT fluids. It is clear
form the Table 6, no significant change (p > 0.05) in the particle
size, PDI and entrapment efficiency of Tmx-NPs were observed
upon incubation with the various GI fluids.
3.8. Accelerated stability studies
Freeze dried nanoparticles containing trehalose as a lyopro-
tectant were used for accelerated stability studies. After 3 months
of storage in accelerated conditions, freeze dried Tmx-NPs with 5%
trehalosewere found to be stable without any collapse or shrinkage
of dried cake. No changes in the physical appearance as well as
encapsulation efficiency, particle size and PDI before and after
storage were observed with freeze dried Tmx-NPs (Table 7).
3.9. Cell culture experiments
3.9.1. Cell cytotoxicity assay (MTT assay)
The results demonstrate that free Tmx and Tmx-NPs showed
similar effect on cell viability after 72 h of incubation (Fig. 6A).
Regression analysis showed similar trends in concentration
dependent cytotoxicity and IC50 values were also similar (w0.1 mg/
ml) in both cases. However, in recovery experiments, the cell
viability was relatively lower in Tmx-NP treated cells as compared
to free Tmx treated cells (Fig. 6B). The regression analysis showed
higher activity of Tmx-NPs as compared to free drug. Further, the
IC50 value of Tmx-NPs was lower as compared to free Tmx by
w0.2 mg/ml.
3.9.2. Cell uptake studies by confocal laser microscopy
The cellular uptake of Tmx-NPs was evident within 3 h of
incubation with cells (Fig. 7AeD). Further, the Tmx-NPs showed
good nuclear co-localization after 3 h of incubation (Fig. 8AeD).
Nearly 80% of the green fluorescence (coumarin-6) co-localized
with the red fluorescence (propidium iodide) (Fig. 8E) indicating
rapid internalization and nuclear transport of Tmx-NPs. The
nuclear co-localization was further confirmed by line series anal-
ysis (Fig. 8FeI).
3.10. In vivo pharmacokinetics after oral administration
The plasma concentration profiles of Tmx after a single oral
administrationof the Tmx-NPs,free Tmxcitratesuspension,and free
Tmx base at 10 mg/kg are shown in Fig. 9. Table 8 summarizes the
pharmacokinetic parameters estimated with one compartmental
Fig. 2. AFM image of Tmx-NPs.
Table 5
Particle size and PDI of Tmx-NPs before and after freeze drying.
Formulation Before freeze drying After freeze drying Ratio
(Sf/Si)
Physical
Appearance
Reconstitution
ScoreParticle size
(nm)
PDI Entrapment
efficiency
Particle
size (nm)
PDI Entrapment
efficiency
NPs with trehalose 168.45 1.54 0.045 0.002 86.20 1.45 172.65 2.78 0.135 0.052 84.30 1.65 1.005 Intact floppy cake a
NPs without trehalose 165.60 2.56 0.069 0.014 86.20 1.45 228.34 3.56 0.45 0.127 85.56 2.65 1.564 Collapsed cake b
Values are in mean SD (n 6); Sf/Sieratio of particle size after freeze drying to particle size before freeze drying;a Reconstitution in 1 ml water and cake is easily redispersed within 20 sec by mere shaking.b
Not reconstitute.
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analysis of the experimental data obtained after the oral adminis-
tration of Tmx-NPs, Tmxcitrate and Tmx base to rats. The data were
adjusted by one compartmental model. The peak plasma concen-
tration (Cmax) ofTmx citrate was about48.34ng/ml. Themeanvalues
obtained for AUC0eN andhalf-life(t1/2) were 891.87ng ml1 h1 and
11.29 h, respectively. Where as Tmx base showed the Cmax about
23.56 ng/ml with AUC0eN andhalf-life (t1/2) 306.55ng ml1 h1 and
7.58 h respectively. On the contrary, when Tmx was loaded in PLGA
NPs formulation, sustained plasma Tmx levels for at least 72 h were
observed (Fig. 9). The plasma level curve obtained by the adminis-
tration Tmx-NPs could be described as divided into two parts; the
first part involves the absorption phase until the achievement of
Cmax (18 h) followed by maintenance of plasma concentration up to
72 h. Upon comparing the AUC0eN it was observed that the nano-particles formulation increased the bioavailability of Tmx by 3.84
and 11.19 times for Tmx citrate and Tmx base respectively.
3.11. In vivo antitumor efficacy
Fig. 10 shows the antitumor activity of Tmx-NPs and Tmx citrate
suspension after oral administration. Orally administered Tmx-NPs
suppressed tumor growth significantly (p < 0.05) as compared to
untreated control group and free Tmx citrate solution. After 30
days, tumor size was reduced up to 41.56% with Tmx-NPs whereas
untreated groups showed an increase in tumor size up to 158.66%
as compared to tumor volume before the start of treatment which
was considered to be 100%. Fig. 11 represents the tumor burden on
rats after 30 days after the start of treatment. Significant reduction
(p< 0.05) in tumor burdenwas observed after 30 days for Tmx-NPs
group. The survival of animals was monitored for 60 days after the
start of the treatment in another group of animals. Kaplane
Meiersurvival curve (Fig.12) was plotted for survival analysis of Tmx-NPs.
The Tmx-NPs enhanced the survival of 83.33% of animals up to 58th
Fig. 3. Overlay of DSC thermograms of Tmx, PLGA, trehalose, physical mixture and freeze dried Tmx-NPs.
Fig. 4. Overlay of X-ray diffraction pattern of Tmx, PLGA, trehalose, physical mixture and freeze dried Tmx-NPs.
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day as compared to the free drug where almost complete loss of
animal took place at end of the study.
3.12. Toxicity evaluations
Tmx-NPs showed a marked reduction in hepatoxicity as
compared to free Tmx citrate. AST, ALT levels in plasma while MDA
level in liver are normally increased in case of hepatotoxicity. As
observed in Fig. 13AeC, the levels of these markers increased
significantly in the case of Tmx citrate given orally while no
significant (p< 0.05) increase wasobserved in the case of Tmx-NPs.
Conventional histopathological examinations were carried out
to determine the possibility of Tmx induced hepatotoxicity in rats,
treated chronically with Tmx-NPs and Tmx Citrate solutions. As
evident from the control group liver sections, normal parenchymal
cells, portal system, and blood sinusoids were observed (Fig. 14A).
Fig. 14B shows the liver section of rats treated with Tmx citrate
orally. The liver sections clearly indicate the appearance of edema
and swelling of hepatocytes, necrosis, kupffer cells showed hyper-
plasia and apoptosis. Liver sections of rats treated with oral Tmx-
NPs showed normal histopathological appearance (Fig. 14C).
4. Discussion
Different process variables like droplet size reduction process,
stabilizer type/concentration and % of theoretical drug loading for
the preparation of Tmx-NPs were optimized [19]. The NPs formu-
lation involves the formation of initial o/w emulsion. Further, the
droplet size of emulsion has to be reduced toget a proper size range
of the NPs. Two methods were employed for reducing the droplet
size i.e. homogenization and sonication. Firstly homogenization
was carried out at twodifferent speeds (10,000 and 15,000 rpm) for
5 min. Reduction in the particles size was observed upon increasing
the homogenization speed from 10,000 rpm to 15,000 rpm. This
could be due to increased shear provided at higher speed of
homogenization. The effect of homogenization and sonication on
particles size and PDI is previously discussed. Upon comparison of
both the methods, it was observed that sonication for 1 min, half
cycle at 60% amplitude, produced the NPs of lesser sized and better
PDI as compared to homogenization (165.6 4.67 nm with PDI
0.029 0.001). Moreover, sonication provides high energy as
compared to homogenization in shorter period of time. Therefore
the sonication was selected for the droplet size reduction of o/w
emulsion. While screening of different stabilizers i.e. PVA,
DMAB and PF-68, it was observed that DMAB produced the
smallest size particles as compared to PVA and PF-68 but at the
same time entrapment efficiency was considerably reduced
(15.28 0.425%). The lower entrapment of Tmx in PLGA NPs couldbe due the increased aqueous solubility in presence of DMAB
(0.45 0.056 mg/ml in 1% w/v DMAB solution at 25 C). To prove
this hypothesis we have also prepared Tmx-NPs with different
concentration of DMAB ranging from 0.2% w/v to 1% w/v. It was
observed that, as the concentration of DMAB was increased form
0.2%e1% w/v, entrapment efficiency decreased very significantly
(p < 0.01) (complete data are not shown). This could be attributed
tothe higher affinity of Tmx for DMAB as compared toPLGA matrix.
DMAB is cationic surfactant having a positively charged amino
group and a long hydrophobic alkyl chain. If we consider the
orientation of DMAB on the PLGA nanoparticles, hydrophobic alkyl
chain is more oriented or penetrated inside the PLGA matrix and
positive charged amino group is present on the surface of nano-
particles and imparts the cationic charge to nanoparticles. Due tothe excessive penetration or high affinity of hydrophobic chain of
DMAB inside the PLGA matrix, Tmx was unable to retain inside the
PLGA matrix and thrown to outer environment where DMAB was
present and due to its higher solubility in DMAB, it showed a lesser
entrapment in the PLGA nanoparticles. On the other hand PVA has
short hydrocarbon chain as compared to DAMB and Tmx was also
having significantly lesser solubility (25.45 4.56 mg/ml in 1% w/v
PVA solution at 25 C) in PVA as compared to DMAB. So due to the
higher solubility of Tmx in DMAB, lesser entrapment was observed
in PLGA.
Effect of theoretical drug loading on the particles size, PDI and
entrapment efficiency was also studied. At 15% drug loading due to
the saturation of the polymer matrix with drug the entrapment
efficiency was not increased upon increase in drug loading. More-
over, the total amount of the drug inside the NPs, at 10% and 15%
drug loading was found to be almost constant. Taking this into
consideration 10% w/w theoretical drug loading was optimized.
Fig. 5. In vitro release of Tmx-NPs in PBS (pH 7.4).
Table 6
Initial and final particle size/PDI of Tmx-NPs after exposure to simulated GIT media.
Medium Particle size PDI Entrapment ef ficiency
Initial Final Initial Final Initial Final
pH 1.2 171.65 3.78 175.34 4.65 0.135 0.052 0.171 0.067 84.30 1.65 85.90 1.30
pH 3.5 171.65 3.78 176.78 3.23 0.135 0.052 0.178 0.098 84.30 1.65 83.56 2.01
pH 7.4 171.65 3.78 180.12 4.83 0.135 0.052 0.167 0.034 84.30 1.65 86.23 3.10
SGF 171.65 3.78 179.89 2.45 0.135 0.052 0.198 0.023 84.30 1.65 83.76 3.78
SIF 171.65 3.78 181.73 2.45 0.135 0.052 0.213 0.056 84.30 1.65 86.12 2.40
Values are in mean
SD (n
6)
Table 7
Characterization of formulation after 3 months of accelerated stability studies.
Parameters Initial Final
Particle size 175.65 3.78 178.45 4.34
PDI 0.235 0.042 0.305 0.056
Entrapment efficiency 85.30 2.55 84.23 3.45
Physical appearance Intact cake Intact cake
Ease of reconstitution By mere shaking By mere shaking
Values are in mean SD (n 6)
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Tmx-NPs formulation was freeze dried by an optimized step-
wise freeze drying cycle (Table 1) using 5% trehalose as a cryopro-
tectant. When NPs are subjected to rapid freezing, a large
iceewater interface is formed thus producing small ice crystals
which determines the size of the formed pores after the sublima-
tion of ice crystals [25]. On the other hand a small interface is pro-
duced by slow freezing. Cryoprotectants form a glassy amorphous
matrix around the NPs and also form the hydrogen bonds with the
polar groups of nanoparticles at the end of the drying process
[26,27]. It isclearfrom Table 5, that size of Tmx-NPs after the freeze
drying without the trehalose changed very significantly (p > 0.05)
as compared to the NPs freeze dried with the trehalose, as the later
provided a barrier for the formation of aggregates of NPs after the
process. Entrapment efficiency of NPs after freeze drying were also
Fig. 6. Concentration dependent effect of Tmx and Tmx-NPs on cell viability. (A) Cell viability of Tmx and Tmx-NPs after 72 h of incubation; (B) Cell viability in recovery exper-
iments; the dashed and solid lines show regression lines for Tmx and Tmx-NPs, respectively.
Fig. 7. Cellular uptake of Tmx-NPs. (A) A single cell with Tmx-NPs; (B) Nuclear staining with propidium iodide; (C) Differential interference contrast image of the same cell; (D) 3D
image of (A) obtained by Z-stacking.
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not changed significantly (p < 0.05) and NPs was easily dispersed
after reconstitution due to the formation offlaccid cake.
DSCstudieswereperformed to study thephysical state of drug in
nanoparticles whether it was present in amorphous or crystalline
state [28] and also to confirm the possible interaction between the
drug and the polymer within the matrix [29]. The disappearance of
endothermic peak of the free drug at 98 C confirmed the entrap-
ment of Tmx in the amorphous form inside PLGA nanoparticles.
PLGA and trehalose showed endothermic peak at around 56 C and
102 C. All the three endothermic peaks i.e. of free drug, trehalose
and PLGA were clearly observed in DSC thermogram of physical
mixture as above mentioned. The DSC thermogram of physical
mixture clearly indicated the absence of the physical interaction
between the drug and polymer when it was entrapped in PLGA
nanoparticles.
In the XRD pattern (Fig. 4), no characteristic peaks of Tmx wereobserved in case of freeze dried nanoparticles. That could be due to
entrapment of Tmx in molecular form in the PLGA nanoparticles
during the preparation of nanoparticles. Our finding was also
supported by the DSC analysis of freeze dried Tmx-NPs which
clearly indicated the presence of amorphous nature of drug while
encapsulated in nanoparticles. The present finding was further
supported by earlier reports in literature [21,29,30].
In vitro drug release from the freeze dried Tmx-NPs (10% drug
loading) was estimated in phosphate buffer saline (pH 7.4) under
sink condition. The Tmx-NPs exhibited Higuchi release pattern and
showed sustained drug release for 20 days. Although there is no
clear defined drug release mechanism is proposed for PLGA nano-
particles. Several release mechanisms could be involved including
surface and bulk erosion, disintegration, diffusion, and desorption.
Fig. 8. Nuclear co-localization of Tmx-NPs. (A) Tmx-NPs uptake by cells; (B) shows the nucleus; (C) Differential interference contrast of the same field; (D) Overlay offigure A, B and
C. (E) Scatter plot of overlap between green (A) and red (B) channel fluorescence. (FeI) Line series analysis offluorescence. (FeH) show the same fields as AeC along with the line
(red line) along which the analysis was performed. (I) Results of line series analysis showing co-localization of green and red fluorescence.
Fig. 9. Plasma concentration time profiles of Tmx after oral administration to SD rats at
10 mg/kg dose formulated in the PLGA NPs, compared with the oral administration of
Tmx free base and Tmx salt.
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The best described release mechanism of Tmx from the PLGA NPs is
by diffusion and in the initial phases followed by the diffusion and
degradation in later phase of release [19,21].
Data presented in Table 6 confirms the stability of formulated
Tmx-NPs in simulated GIT fluids. The present work mainly focuses
on the design of Tmx loaded PLGA for oral administration so it is
necessary that NPs would resist to their aggregation in presence of
various GIT environment so as to facilitate their intestinal absorp-
tion [31].
As a final exercise of characterization we have evaluated accel-
erated storage stability of freeze dried Tmx-NPs for 3 months at
temperature of 25 2 C and RH 60 5%. The study demonstrated
the stable nature of nanoparticles in presence of trehalose afteraccelerated stability studies (Table 7).
Free Tmx and Tmx-NPs were further evaluated for their in vitro
cellular viability assay on C127I cell line by MTT assay [32]. After
72 h of incubation, both Tmx and Tmx-NPs showed concentration
dependent reduction in cell viability to similarextent. These results
suggest that Tmx retained its antitumor efficacy even after its
encapsulation in polymeric NPs. Further, in recovery experiment,
the Tmx-NPs resulted in significantly lower cell viability at 1 mg/ml
as compared to Tmx. In general, the recovery of cells after an initial
exposure to Tmx-NPs was lower as compared to free Tmx. The
lower recovery in NPs-treated cells may be attributed to internal-
ization followed by retention of NPs attributed to retention of NPs
inside the cell. Thus, the drug is slowly released inside the cells
even when extracellular NPs have been washed away.Further, the NPs accumulated in cell nucleus which is also the
site for pharmacological action of the drug as the estrogen receptor
is a nuclear receptor. When applied to in vivo systems, this indi-
cated that the cellular uptake and nuclear delivery of NPs in cancer
cells leads to retention of NPs while the free drug is cleared by
metabolism or excretion. This could ultimately lead to a reduction
in both dose as well as dosing frequency of drug loaded NPs as
compared to free drug.
In vitro cellular uptake of NPs was carried out by co-encapsu-
lation of hydrophobic fluorescent marker, coumarin-6 with Tmx in
PLGA (coumarin-6-Tmx-NPs) with the same procedure employed
as for the Tmx-NPs preparation [19,33] with slight modification. It
is clear from the Fig. 7AeD that the Tmx-NPs incubated with C127I
cells showed the significant internalization after 3 h. Fluorescence
after 3 h, inside the cells is an indication of rapid internalization of
Tmx-NPs which were co-encapsulated with the coumarin-6. In
vitro release of coumarin-6 from the PLGA NPs showed the negli-
gible release (
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inside the cells for longer period of time as evident by recovery cell
viability experiments.The Tmx-NPs and free Tmx base were evaluated for their phar-
macokinetics after oral administration. The market availability of
Tmx is in a tamoxifen citrate tablets (10 mg, 20 mg) which are
administered for 2e3 years. Thus theoral pharmacokinetics of Tmx-
NPs was compared with both the freebase and salt form of the drug
in the same dose (10 mg/Kg). The plasma level of Tmx after the
administration of free Tmx base was detected only up to 12 h of the
oraladministrationwith the Cmax 23.56ng/ml. The Cmax and AUC0eNof thedrug were further increasedto 48.34 ng/ml,when salt form of
the drug was administered with the same dose. The Cmax and
AUC0eN of the drug were obtained in line with the data reported in
theliterature forTmx citrate salt [24]. On the other hand, whenTmx
was loaded in PLGA NPs and orally administered to rats, the drug
plasma concentrations dramatically increased (Fig. 10). Thus, the
AUC0eN for Tmx-NPs was drastically increased to 3431.11 ng/ml h
from 891.87 ng/ml h that was obtained when Tmx citrate salt was
administered orally (Table 8). So administration of Tmx by loadinginto the PLGA nanoparticles leads to enhancement of oral bioavail-
ability of Tmx by 3.84 and 11.19 times as compared to the free Tmx
citrate and free Tmx base respectively. The increased bioavailability
of Tmx was observed with O/W microemulsion by Araya et al. when
they employed medium chain fatty acid triglyceride (MCT), digly-
ceryl monooleate (DGMO-C), polyoxyethylene hydrogenated castor
oil 40 (HCO-40) for the formulation Tmx O/W microemulsion [37].
Here we have reported the oral bioavailability enhancement of
poorly water soluble drug Tmx by its encapsulation in the PLGA
nanoparticulate matrix. When nanoparticles are administered by
oral route, they are absorbed through specialized M-cells of the
peyers patches in the small intestine [15]. Absorption through the
M-cells directly drains into the lymphatics thus prevents the drug
fromthefirst passmetabolism in the hepatic tissue and thereforethe
chances of drug induced hepatotoxicity are also reduced.
Fig. 13. (A) ALT levels in plasma after one month of the treatment using Tmx formulations. (*p < 0.05 a Vs control, b Vs Oral Tmx citrate group). (B) AST levels in plasma after one
month of the treatment using Tmx formulations. (**p < 0.01,*p < 0.05 a Vs control, b Vs Oral Tmx citrate group). (C) Lipid Peroxidation products (MDA) in liver homogenates after
one month of the treatment. (**p < 0.01.*p < 0.05 a Vs control and b Vs oral Tmx citrate group).
Fig. 14. Liver microscopic sections after treatment of different formulations.
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Since pharmacokinetics studies of Tmx-NPs revealed significant
enhancement of bioavailability of Tmx as compared to free Tmx
base and Tmx citrate. So they were further evaluated for their in
vivo antitumor efficacy in breast cancer induced animal model. The
in vivo antitumor tumor efficacy of Tmx-NP offered a proof of
concept of their effectiveness as appropriate delivery carriers for
breast cancer. Since, the bioavailability of Tmx base was found
significantly lower (p < 0.05) as compared to Tmx citrate and only
salt form of the drug is commercially available the later only was
employed for evaluation of antitumor efficacy of Tmx and for
comparison purpose. A chemical mutagen (7,12-dimethylbenz-
anthracene) (DMBA) was employed as a suitable breast cancer
animal model because of its structural resemblance to the human
breasts cancer [38e40]. Oral administration of Tmx-NPs at an
interval of 2 days for 28 days significantly reduced the tumor
burden as compared to the Tmx citrate. On the contrary, continue
increase in the tumor volumewas observed in DMBA control group.
The enhanced efficacy of Tmx-NPs could be attributed to increased
bioavailability of Tmx by Tmx-NPs. The increased absorption of
particulate carriers through a specific reign of GIT (Payers patches)
lead to their increased availability in the central compartment [40].
Since the Tmx-NPs showed the sustained pharmacokinetic pattern,
thus had the longer circulation time in the blood compartment soas to have greater tumor accumulation. The greater tumor accu-
mulation of Tmx-NPs could be attributed to their enhanced
permeation and retention (EPR) [41]. The extensive metabolism of
free Tmx citrate in the liver lead to reduced antitumor efficacy as
compared to Tmx-NPs and subsequent enhanced hepatotoxicity.
KaplaneMeier survival curve (Fig. 12) showed enhanced survival
time of tumor bearing rats following oral administration of Tmx-
NPs as compared to Tmx citrate administration. These observations
are also reliable dueto the higher buildup of the drug concentration
in a discriminating manner in tumor tissue when animal were
treated with Tmx-NPs, compared to free Tmx-NPs.
Tmx increases some hepatotoxicity markers levels like ALT, AST
and MDA in the plasma as well as in liver homogenate [8,42,43].
AST, ALT and MDA level were not significantly increased (p > 0.05)when Tmx-NPs were administered orally repetitively for 30 days
whereas these levels increased for free Tmx citrate administered in
similar manner. The increased hepatotoxicity marker levels for Tmx
citrate could be attributed to oxidative reactions that takes place
during its metabolism in the liver [44]. It is acting as an uncoupling
agent and powerful inhibitor of mitochondrial electron transport
chain. This ultimately results in oxidative stress mediated damage
of mitochondria [9]. Decreased hepatotoxicty of Tmx after the
encapsulation in PLGA NPs could be attributed to its escape from
first pass metabolism in the liver as NPs are absorbed viaa different
route. Certainly a tissue biodistribution study would be required to
determine the actual concentration of Tmx in hepatic tissues after
administration of Tmx-NPs and Tmx citrate formulations.
The reduced hepatotoxicity associated after the chronic oraladministration of Tmx-NPs was further established by histopa-
thology of the liver tissues after one month of oral administration.
Tmx citrate showed marked changes in cellular integrity leading to
the hepatotoxicty (Fig. 14) whereas the Tmx-NPs ameliorated the
changes in hepatic ultra structure by preserving cellular integrity
and preventing oxidative stress and ultimately inhibited the
hepatic inflammation.
5. Conclusions
In summary, Tmx-NPs can be prepared by solvent-diffusion
evaporation method with high encapsulation efficiency with 2%
PVA as a stabilizer with probe sonication method. After freeze
drying of Tmx-NPs with 5% trehalose, NPs were found to be stable
in simulated GIT medium hence are suitable for oral administra-
tion. In vitro release study showed Higuchis release pattern for
more than 20 days. Freeze dried Tmx-NPs were also stable in
accelerated stability condition after 3 months. Tmx-NPs can be
efficiently delivered, retained and localized in the nuclear region
when tested in mouse breast cancer cells. In vivo pharmacokinetics
of freezedried Tmx-NPs showed 3.84 and 11.19 times enhancement
in oral bioavailability as compared toTmx citrate and free Tmx base.
Further enhanced accumulation of Tmx-NPs in tumor tissue might
have taken place due to combined effect of enhanced oral
bioavailability and enhanced permeation and retention (EPR)
effect. Increased Tmx-NPs accumulation in tumor cells led to
enhanced antitumor activity with reduced Tmx related heapto-
toxicity as compared to the marketed Tmx citrate. Therefore Tmx-
NPs can be of significant value in chronic Tmx therapy for the
treatment of breast cancer with reduced side effects.
Acknowledgement
Authors are thankful to Director, NIPER for providing necessary
infrastructure facilities and Department of Science & Technology
(DST), Government of India, New Delhi, India, for financial support.Authors are also thankful forhelp and co-operation renderedby Mr.
Dinesh Singh and Rahul Mahajan. The histopathological examina-
tion carried out at Dr. Vijay Malhotras Lab, Chandigarh is also duly
acknowledged.
Appendix
Figures with essential colour discrimination. Figs. 1,2,7e14 in
this article have parts that are difficult to interpret in black and
white. The full colour images can be found in the online version, at
doi:10.1016/j.biomaterials.2010.09.037.
References
[1] Jemal A, Siegel R, Ward E, Hao Y, Xu J, Murray T, et al. Cancer statistics. CACancer J Clin 2008;58:71e96.
[2] Osborne CK, Fuqua SAW. Selective estrogen receptor modulators: structure,function, and clinical use. J Clin Oncol 2000;18:3172e86.
[3] Jordan VC. Tamoxifen: a most unlikely pioneering medicine. Nat Rev DrugDiscov 2003;2:205e13.
[4] Desai PB, Nallani SC, Sane RS, Moore LB, Goodwin BJ, Buckley DJ, et al.Induction of cytochrome P450 3A4 in primary human hepatocytes and acti-vation of the human pregnane X receptor by tamoxifen and 4-hydrox-ytamoxifen. Drug Metab Dispos 2002;30:608e12.
[5] Shin SC, Choi JS. Effects of epigallocatechin gallate on the oral bioavailabilityand pharmacokinetics of tamoxifen and its main metabolite, 4-hydrox-ytamoxifen, in rats. Anticancer Drugs 2009;20:584e8.
[6] McVie JG, Simonetti GP, Stevenson D, Briggs RJ, Guelen PJ, de Vos D. Thebioavailability of tamoplex (tamoxifen) part 1 a pilot study. Methods Find ExpClin Pharmacol 1986;8:505e12.
[7] Tukker JJ, Blankenstein MA, Nortier JW. Comparison of bioavailability in man
of tamoxifen after oral and rectal administration. J Pharm Pharmacol1986;38:888e92.
[8] Hard GC, Iatropoulos MJ, Jordan K, Radi L, Kaltenberg OP, Imondi AR, et al.Major difference in the hepatocarcinogenicity and DNA adduct forming abilitybetween toremifene and tamoxifen in female Crl: CD (BR) rats. Cancer Res1993;53:4534e41.
[9] Parvez S, Tabassum H, Rehman H, Banerjee BD, Athar M, Raisuddin S. Catechinprevents tamoxifen-induced oxidative stress and biochemical perturbationsin mice. Toxicol 2006;225:109e18.
[10] Shin SC, Choi JS, Li X. Enhanced bioavailability of tamoxifen after oraladministration of tamoxifen with quercetin in rats. Int J Pharm 2006;313:144e9.
[11] Peer D, Karp JM, Hong S, Farokhzad OC, Margalit R, Langer R. Nanocarriers asan emerging platform for cancer therapy. Nat Nanotech 2007;2:751e60.
[12] Astete CE, Sabliov CM. Synthesis and characterization of PLGA nanoparticles.J Biomater Sci Polym Ed 2006;17:247e89.
[13] Kalaria DR, Sharma G, Beniwal V, Ravi Kumar MNV. Design of biodegradablenanoparticles for oral delivery of doxorubicin: in vivo pharmacokinetics and
toxicity studies in rats. Pharm Res 2009;26:492e
501.
A.K. Jain et al. / Biomaterials 32 (2011) 503e515514
7/31/2019 The Effect of the Oral Administration of Polymeric Nanoparticles Efficacy and Toxicity
13/13
[14] Govender T, Stolnik S, Garnett MC, Illum L, Davis SS. PLGA nanoparticlesprepared by nanoprecipitation: drug loading and release studies of a watersoluble drug. J Control Release 1999;57:171e85.
[15] Florence AT. The oral absorption of micro-and nanoparticulates: neitherexceptional nor unusual. Pharm Res 1997;14:259e66.
[16] Clark M, Jepson MA, Hirst BH. Exploiting M cells for drug and vaccine delivery.Adv Drug Deliv Rev 2001;50:81e106.
[17] Hussain N, Jaitley V, Florence AT. Recent advances in the understanding ofuptake of microparticulates across the gastrointestinal lymphatics. Adv DrugDeliv Rev 2001;50:107e42.
[18] Kogure K, Hama S, Manabe S, Tokumura A, Fukuzawa K. High cytotoxicity of[alpha]-tocopheryl hemisuccinate to cancer cells is due to failure of theirantioxidative defense systems. Cancer Lett 2002;186:151e6.
[19] Hariharan S, Bhardwaj V, Bala I, Sitterberg J, Bakowsky U, RaviKumar MNV. Design of estradiol loaded PLGA nanoparticulate formula-tions: a potential oral delivery system for hormone therapy. Pharm Res2006;23:184e95.
[20] Fried KM, Wainer IW. Direct determination of tamoxifen and its four majormetabolites in plasma using coupled column high-performance liquid chro-matography. J Chromatogr B Biomed Sci Appl 1994;655:261e8.
[21] Jain S, Mittal A, Jain AK, Mahajan RR, Singh D. Cyclosporin A loaded PLGAnanoparticle: preparation, optimization, in vitro characterization and stabilitystudies. Curr Nanoscience 2010;6:422e31.
[22] Zimmermann E, Mller RH. Electrolyte-and pH-stabilities of aqueous solidlipid nanoparticle (SLN (TM)) dispersions in artificial gastrointestinal media.Eur J Pharm Biopharm 2001;52:203e10.
[23] Abdelwahed W, Degobert G, Fessi H. Investigation of nanocapsules stabiliza-tion by amorphous excipients during freeze-drying and storage. Eur J PharmBiopharm 2006;63:87e94.
[24] Buchanan CM, Buchanan NL, Edgar KJ, Little JL, Malcolm MO, Ruble KM, et al.Pharmacokinetics of tamoxifen after intravenous and oral dosing of tamox-ifenehydroxybutenylecyclodextrin formulations. J Pharm Sci 2006;96:644e60.
[25] Willemer H. Measurements of temperatures, ice evaporation rates andresidual moisture contents in freeze-drying. Dev Biol Stand 1992;74:123e36.Discussion.
[26] Ford AW, Dawson PJ. The effect of carbohydrate additives in the freeze-dryingof alkaline phosphatase. J Pharm Pharmacol 1993;45:86e93.
[27] Crowe JH, Crowe LM, Carpenter JF. Preserving dry biomaterials: the waterreplacement hypothesis, part 1. Biopharm-Eugene 1993;6:28.
[28] Layre AM, Gref R, Richard J, Requier D, Chacun H, Appel M, et al. Nano-encapsulation of a crystalline drug. Int J Pharm 2005;298:323e7.
[29] Chawla JS, Amiji MM. Biodegradable poly (-caprolactone) nanoparticles fortumor-targeted delivery of tamoxifen. Int J Pharm 2002;249:127e38.
[30] Kojima T, Katoh F, Matsuda Y, Teraoka R, Kitagawa S. Physicochemical prop-erties of tamoxifen hemicitrate sesquihydrate. Int J Pharm 2008;352:146e51.
[31] Jain AK, Goyal AK, Mishra N, Vaidya B, Mangal S, Vyas SP. PEGePLAePEG blockcopolymeric nanoparticles for oral immunization against hepatitis B. Int JPharm 2010;387:253e62.
[32] Westedt U, Kalinowski M, Wittmar M, Merdan T, Unger F, Fuchs J, et al. Poly(vinyl alcohol)-graft-poly (lactide-co-glycolide)nanoparticles for local deliveryof paclitaxel for restenosis treatment. J Control Release 2007;119:41e51.
[33] Panyam J, Sahoo SK, Prabha S, Bargar T, Labhasetwar V. Fluorescence andelectron microscopy probes for cellular and tissue uptake of poly (D, L-lactide-
co-glycolide) nanoparticles. Int J Pharm 2003;262:1e
11.[34] Htun H, Holth LT, Walker D, Davie JR, Hager GL. Direct visualization of the
human estrogen receptor alpha reveals a role for ligand in the nucleardistribution of the receptor. Mol Biol Cell 1999;10:471e86.
[35] Gupta R, Mishra P, Mittal A. Enhancing nucleic acid detection sensitivity ofpropidium iodide by a three nanometer interaction inside cells and in solu-tions. J Nanosci Nanotechnol 2009;9:2607e15.
[36] Razandi M,PedramA, GreeneGL, Levin ER.Cell membraneand nuclearestrogenreceptors (ERs) originate from a single transcript: studies of ERa and ERbexpressed in Chinese hamster ovary cells. J Mol Endocrinol 1999;13:307e19.
[37] Araya H, Tomita M, Hayashi M. The novel formulation design of O/W micro-emulsion for improving the gastrointestinal absorption of poorly watersoluble compounds. Int J Pharm 2005;305:61e74.
[38] Kaufmann Y, Kornbluth J, Feng Z, Fahr M, Schaefer RF, Klimberg VS. Effect ofglutamine on the initiation and promotion phases of DMBA-inducedmammary tumor development. JPEN J Parenter Enteral Nutr 2003;27:411e8.
[39] Rehm S. Chemically induced mammary gland adenomyoepitheliomas andmyoepithelial carcinomas of mice. Immunohistochemical and ultrastructuralfeatures. Am J Pathol 1990;136:575e84.
[40] Bhardwaj V, Ankola DD, Gupta SC, Schneider M, Lehr CM, Ravi Kumar MNV.PLGA nanoparticles stabilized with cationic surfactant: safety studies andapplication in oral delivery of paclitaxel to treat chemical-induced breastcancer in rat. Pharma Res 2009;26:2495e503.
[41] Maeda H. The tumor blood vessel as an ideal target for macromolecularanticancer agents. J Control Release 1992;19:315e24.
[42] Recknagel RO, Glende Jr EA, Dolak JA, Waller RL. Mechanisms of carbontetrachloride toxicity. Pharmacol Ther 1989;43:139e54.
[43] Albukhari AA, Gashlan HM, El-Beshbishy HA, Nagy AA, Abdel-Naim AB. Caffeicacid phenethyl ester protects against tamoxifen-induced hepatotoxicity inrats. Food Chem Toxicol 2009;47:1689e95.
[44] Elefsiniotis IS, Pantazis KD, Ilias A, Pallis L, Mariolis A, Glynou I, et al.Tamoxifen induced hepatotoxicity in breast cancer patients with pre-existingliver steatosis: the role of glucose intolerance. Eur J Gastroenterol Hepatol2004;16:593e8.
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