The Effect of the Oral Administration of Polymeric Nanoparticles Efficacy and Toxicity

7/31/2019 The Effect of the Oral Administration of Polymeric Nanoparticles Efficacy and Toxicity

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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]


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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.



9/13

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.



10/13

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 (


11/13

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.



12/13

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.

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