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Chloramphenicol-incorporated poly lactide-co-glycolide(PLGA) nanoparticles: Formulation, characterization,technetium-99m labeling and biodistribution studiesKamal Krishna Halder a; Bivash Mandal b; Mita Chatterjee Debnath a; Hriday Berab; Lakshmi Kanto Ghosh b; Bijon Kumar Gupta ba Nuclear Medicine Division, Indian Institute of Chemical Biology, Kolkata, Indiab Division of Pharmaceutics, Department of Pharmaceutical Technology, JadavpurUniversity, Kolkata, India

Online Publication Date: 01 May 2008

To cite this Article: Halder, Kamal Krishna, Mandal, Bivash, Debnath, MitaChatterjee, Bera, Hriday, Ghosh, Lakshmi Kanto and Gupta, Bijon Kumar (2008)

'Chloramphenicol-incorporated poly lactide-co-glycolide (PLGA) nanoparticles: Formulation, characterization,technetium-99m labeling and biodistribution studies', Journal of Drug Targeting, 16:4, 311 — 320

To link to this article: DOI: 10.1080/10611860801899300URL: http://dx.doi.org/10.1080/10611860801899300

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Chloramphenicol-incorporated poly lactide-co-glycolide (PLGA)nanoparticles: Formulation, characterization, technetium-99m labelingand biodistribution studies

KAMAL KRISHNA HALDER1, BIVASH MANDAL2, MITA CHATTERJEE DEBNATH1,

HRIDAY BERA2, LAKSHMI KANTO GHOSH2, & BIJON KUMAR GUPTA2

1Nuclear Medicine Division, Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Jadavpur, Kolkata 700032,

India, and 2Division of Pharmaceutics, Department of Pharmaceutical Technology, Jadavpur University, Kolkata 700032,

India

(Received 17 September 2007; revised 29 November 2007; accepted 5 January 2008)

AbstractChloramphenicol-loaded (CHL) poly-D,L-lactic-co-glycolic acid (PLGA) nanoparticles (NPs) were prepared byemulsification solvent evaporation technique either by using polyvinyl alcohol (PVA) as emulsion stabilizer or polysorbate-80 (PS-80) as surfactant and characterised by transmission electron microscopy, zeta-potential measurements. The NPs wereradiolabeled with technetium-99m (99mTc) by stannous reduction method. Labeling conditions were optimised to achievehigh-labeling efficiency, in vitro and in vivo (serum) stability. The labeled complexes also showed very low transchelation asdetermined by DTPA challenge test. Biodistribution studies of 99mTc-labeled complexes were performed after intravenousadministration in mice. The CHL-loaded PLGA NPs coated with PS-80 exhibited relatively high brain uptake withcomparatively low accumulation in bone marrow to that of free drug and CHL-loaded PLGA NPs (PVA, used as emulsionstabilizer) at 24 h post injection time period. This indicates the usefulness of the above delivery system for prolonged use of theantibiotic.

Keywords: Chloramphenicol, chloramphenicol-loaded PLGA nanoparticles, polysorbate-80-coated, technetium-99m,radiolabeling, biodistribution

Abbreviations: CHL, chloramphenicol; PLGA, poly lactic-co-glycolic acid; NPs, nanoparticles; PS-80, polysorbate-80; BBB,blood brain barrier; 99mTc, technetium-99m

Introduction

Nanoparticles (NPs) are solid colloidal particles

ranging in size from 1 to 1000 nm, consisting of

various macromolecules in which drugs can be

adsorbed, entrapped or covalently attached. They

have recently been extensively investigated in biome-

dical and biotechnological areas, especially in drug

delivery systems for drug targeting because their

particle size is acceptable for intravenous injection

(Soppimath et al. 2001; Lockman et al. 2002).

Depending on the desired administration route, the

size of the carriers should be optimized. They serve as

a novel drug delivery system (Hughes 2005). Their

low toxicity and surface characteristics also make them

suitable for scintigraphic imaging after radiolabeling

with suitable g-emitting radionuclide (Wissing et al.

2004). It has been observed that successful delivery of

the drug molecules to the target tissue and its

therapeutic efficacy can be enhanced by targeting the

drug through polymeric NPs as carrier (Reddy et al.

2004a). NPs (200 nm) radiolabeled with 99mTc using

ISSN 1061-186X print/ISSN 1029-2330 online q 2008 Informa UK Ltd.

DOI: 10.1080/10611860801899300

Correspondence: M. C. Debnath, Nuclear Medicine Division, Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Jadavpur,Kolkata 700032, India. E-mail: mitacd@iicb.res.in

Journal of Drug Targeting, May 2008; 16(4): 311–320

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lipophilic chelator DL-hexamethylene propylene

amine oxime can be used as effective colloidal carriers

for lymphoscintigraphy or therapy upon pulmonary

delivery (Videira et al. 2002). Attempts have also been

made to enhance the target specificity of the antic-

ancer drug etoposide by incorporating it in tripalmi-

tin-based NPs, which has been subsequently

radiolabeled with technetium-99m (99mTc) for physi-

cochemical and biological studies in normal and

tumour-bearing mice (Reddy et al. 2004b, 2005).

Recently, NPs have also been gaining interest as a

therapeutic drug carrier across the blood brain barrier

(BBB) to increase central nervous system (CNS) drug

delivery. NPs may cross the tight junction of the

endothelial cell lining at BBB by passive diffusion or

receptor-mediated endocytosis carrying the drug

across the BBB without requiring drug molecular

specificity (Olivier 2005).

Although there are a number of different polymers

that have been investigated for formulating biode-

gradable NPs, of these poly-L-lactic acid and its

copolymers with glycolic acid (PLGA) have been

extensively used in the last decade for controlled drug

delivery systems (Allemann et al. 1998; Dillen et al.

2006). They are biocompatible, have good safety

profile and opened great opportunities for drug

targeting (Mainardes and Evangelista 2005). They

are mainly used for the preparation of biodegradable

medical devices and drug sustained release prep-

aration (Brannon-Peppas 1995). Further, it has been

observed too that the above NPs when coated with

nonionic surfactant polysorbate-80 (PS-80) have their

permeability enhanced through BBB (Koziara et al.

2004; Ambruosi et al. 2005). Thus, the therapeutic

efficacy of CNS active drugs may be increased by

loading them in PS-80-coated, PLGA-based NPs.

This coating with surfactants also reduces the uptake

of NPs by the phagocytic cells of the reticuloendothe-

lial system (RES) located mainly in liver and spleen

(Troster et al. 1990)

Chloramphenicol (CHL) is an antibacterial drug,

extremely lipid soluble and it easily crosses the BBB.

Because of its excellent cerebrospinal fluid (CSF)

penetration, CHL remains the first choice treatment

for staphyloccal brain abscess (McCarroll and Vogel

1988). CHL is also recommended by the World Health

Organisation as the first line treatment of meningitis in

low-income countries and is much cheaper than

ceftriaxone (Wali et al. 1979). However, the most

serious side effect of CHL treatment is bone marrow

suppression and aplastic anaemia (Yunis et al. 1980;

Jimenez et al. 1987). Moreover, it requires several

hours to cross the BBB. In these circumstances, the

above side effects will be much fatal. Nowadays, use of

the drug has been restricted in the West.

Attempt has been made to develop CHL-loaded

NPs to facilitate the CSF penetration of the drug with

subsequent elimination of bone marrow uptake.

CHL-loaded PLGA NPs were fabricated by emulsi-

fication solvent evaporation method using either

polyvinyl alcohol (PVA) as disperse solvent or coated

with PS-80. The prepared NPs formulations were

physicochemically characterized through transmission

electron microscopy (TEM) and zeta-potential

measurements. The free drug (CHL), CHL-loaded

PLGA NPs (containing PVA as emulsion stabilizer)

and CHL-loaded, PS-80-coated PLGA NPs were

radiolabeled with 99mTc, by direct radiolabeling

approach using stannous chloride dihydrate as

reductant. Their physicochemical and biological

properties (in mice) were subsequently evaluated.

The results are discussed in the following sections.

Materials and methods

Poly-D,L-lactic-co-glycolic acid (PLGA), with a copo-

lymer ratio of D,L-lactide to glycolide of 75:25

(molecular weight 15,000 Da, free acid 0.2%, inherent

viscosity 0.166 dl/gm, polydispersion 1.64) and CHL

were gifted by Sun Pharmaceutical Advanced Research

Center (Baroda, Gujarat, India) and Dey’s Medical

Stores (Mfg) Ltd. (Kolkata, India) respectively. PVA

and PS-80 were supplied by S.D. Fine Chemicals Ltd

(Mumbai, India). HPLC grade ethyl acetate was

purchased from Spectrochem (Mumbai, India). All

other chemicals and solvents were of analytical grade.

Preparation of NPs

The NPs loaded with CHL were prepared by an

emulsification solvent evaporation technique. Typically,

a solution of 91 mg of PLGA (75:25) in 3 ml of ethyl

acetate containing 39 mg of CHL (drug polymer ratio

3:7) was slowly poured either into 15 ml of aqueous PVA

(2%, w/v) solution as colloid protectant or into 15 ml of

aqueous PS-80 (1.5–2%, w/v) solution as surfactant.

The mixture was homogenized for 1 min in vortex and

then sonicated using a microtip probe sonicator set at

75–80 Hz of output energy (Misonix Ultrasonicator,

New York, USA) for 1.5 min on ice bath to produce the

oil in water emulsion. The organic phase was evaporated

by slow stirring at room temperature for 4 h. The NPs

were then isolated by ultra centrifugation (21,000 rpm,

25 min, 88C, SORVAIL, RC 5B Plus, Minnesota, USA)

followed by washing twice with double distilled water in

order to remove the adsorbed CHL. The washing

solutions were eliminated by a further centrifugation as

described above. The suspension produced was freeze

dried (VIRTIS, Freeze Mobile, Model -6ES, Cam-

bridge, USA) for 48 h using sucrose (5%, w/w) as

cryoprotectant to obtain fine powder of NPs.

Characterisation of the NPs

The particle size of CHL-loaded PLGA NPs contain-

ing PVA as emulsion stabiliser (CHL-loaded PLGA

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NPs) and CHL-loaded, PS-80-coated PLGA NPs

(CHL-loaded, PS-80-coated PLGA NPs) were

characterized by different physicochemical methods.

Both morphology and particle size distribution of NPs

were determined by TEM on TECNAI SPIRIT

model FE1 electron microscope, The Netherlands. A

drop of the sample was placed on a 400 mesh carbon-

coated copper grid. About 1 min after deposition, the

grid was tapped with a filter paper to remove the

surface water. It was then air dried and subjected to

measurement.

The zeta-potential of CHL-loaded PLGA NPs and

CHL-loaded, PS-80-coated PLGA NPs was

measured by injecting the diluted sample dispersed

in double-distilled water in the electrophoretic cell of

the instrument (Zeta meter 3.0 plus Inc., New York,

USA), which was set at ^150 V at 258C temperature.

The amount of non-entrapped CHL was deter-

mined by spectroscopic method. Optical density of

non-entrapped drug, recovered from the supernatant

obtained after ultracentrifugation of drug-loaded NP

suspension, was measured at 278 nm in U2001

spectrophotometer (Hitachi, Tokyo, Japan) after

appropriate dilution with 0.2 M phosphate buffer of

pH 7.4. The above measurement for each batch of NP

formulation was carried against a suitably diluted

supernatant obtained from the similarly developed NP

formulation without drug.

Radiolabeling of CHL-loaded NPs

Free CHL, CHL-loaded PLGA NPs and CHL-

loaded, PS-80-coated PLGA NPs were labeled with99mTc by standard stannous reduction method as per

the equation given below. Nitrogen-purged water was

used for the preparation of aqueous 99mTcO42 solution

and stannous chloride solution. Briefly, aqueous99mTcO4

2(2mci/ml) was mixed with either (a)

0.02 ml or (b) 0.03 ml of freshly prepared stannous

chloride solution (1 mg/ml) and pH of the solution

was adjusted to 6.5 with 0.5 M sodium bicarbonate

buffer solution. To (a) 1 ml of CHL solution

(4.5 mg/ml) and (b) 1 ml of drug-loaded NP, suspen-

sion (equivalent to 4.5 mg of CHL/ml) was added and

each mixture was incubated separately for 15 min at

room temperature.

free drug=drug loaded NPs þ99m TcO24 !

Reduction

ðSnþþÞ

99mTc labeled ðfree drug=drug loaded NPsÞ:

The effect of pH on the labeling efficiency of 99mTc-

labeled CHL and drug-loaded PLGA NPs was

thoroughly studied to determine the optimum pH

for radiolabeling. Similarly, the effect of stannous

chloride concentration on the labeling efficiency was

studied to obtain the optimum concentration needed

for maximum labeling. After adding the drug (free or

NP forms) to the mixture of 99mTcO42 and SnCl2

adjusted at desired pH level, the solution was

incubated for various time periods to observe the

effect of incubation time on labeling yield.

The labeling efficiency of free CHL, CHL-loaded

PLGA NPs and CHL-loaded PS80-coated PLGA

NPs was determined by ascending thin layer

chromatography (TLC) using 2.5 £ 10 cm silica gel

strips as stationary phase and either acetone or

pyridine:acetic acid:water (3:5:1.5) as mobile phase.

The test sample (2–3ml) was applied 1 cm from the

base of the TLC plate and dried at room temperature.

The plates were then developed in appropriate solvent

systems. Acetone is used for the determination of free

pertechnetate, whereas pyridine:acetic acid:water

(3:5:1.5) is used for determination of radiocolloid.

After developing, the plates were dried and radioac-

tivity distribution was determined by cutting the

portion of the strips and counting it in a gamma

scintillation counter (Electronic Corporation of India,

Model LV4755, Hyderabad, India) at 140 keV.

Stability studies

The stability of 99mTc-labeled, drug-loaded PLGA

NPs was determined in vitro using 0.9% sodium

chloride and serum by ascending TLC technique. The

labeled complex (0.5 ml) was mixed with 1.5 ml of

normal saline or rat serum and incubated at 378C. The

samples were withdrawn at regular intervals up to

24 h, monitored by ITLC and analysed in gamma

counter.

Transchelation with DTPA

This study was performed to check the stability and

strength of binding of 99mTc with the drug-loaded

NPs. Radiolabeled preparations of 0.5 ml were

challenged against three different concentrations (10,

30 and 50 mM) of DTPA in 0.9% saline by incubating

at 378C for 2 h. The effect of DTPA on labeling was

measured by TLC on silica gel plate using normal

saline and acetone as mobile phase that allowed the

separation of free pertechnetate and DTPA-chelate

(Rf ¼ 0.9) from that of the 99mTc-labeled CHL-

loaded NPs which remains at the point of application

(Rf ¼ 0).

Biodistribution studies

All animal experiments were carried out in compli-

ance with the relevant national laws relating to the

conduct of animal experimentation. Male Balb/c mice

(25–30 gm) after 12 h fasting were well hydrated

by intra-peritoneal administration of saline (0.9%,

2 ml) for 1 h. After another 1 h, the 99mTc-chelate of

free CHL, CHL-loaded PLGA NPs and CHL-loaded

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PS-80-coated PLGA NPs in a total volume 0.03 ml

(5–8mCi) were injected through the tail vein in each

mouse. The mice were sacrificed either at 1, 4 and

24 h post injection; blood and urine samples were

obtained by puncture of heart and urinary bladder.

Other desired organs (heart, brain, liver, lung, spleen,

bone marrow, muscle, stomach, intestine and kidney)

were dissected, washed with normal saline, made free

from adhering tissues, weighed and counted in

gamma scintillation counter against suitably diluted

aliquots of the injected solution as standard.

The results were expressed either as percent dose

per gram of tissue or percent dose per organ.

Results

CHL-loaded PLGA NPs were prepared by emulsifi-

cation solvent evaporation technique using either PVA

or PS-80 as emulsion stabilizer and surfactant,

respectively. The powdered NPs were finally obtained

by lyophilisation of the nanodispersion. The technique

yielded spherical powder particles (Figure 1) with

diameter of 10–70 nm (PVA used as emulsifier) and

40–120 nm (PS-80 used as surfactant) with excellent

redispersibility in aqueous media. The drug-loaded

PLGA NPs could incorporate 27.9% CHL with 65–

66% entrapment efficiency as measured by spectro-

scopic method. The results of zeta-potential measure-

ments reveal that the CHL-loaded PLGA NPs

containing PVA as emulsifier exhibited negative zeta

potential of 220.80 ^ 2.30 mv, whereas this value

became 217.20 ^ 2.10 mv in CHL-loaded PS-80-

coated PLGA NPs. To prevent aggregation, organic

solvent from the nanodispersion was evaporated by

slow stirring at room temperature instead of quick

rotative evaporation under partial vacuum.

CHL and CHL-loaded PLGA NPs were radiolabeled

by 99mTc with high-labeling efficiency. The pertechne-

tate was first reduced to its lower valency state using

stannous chloride dihydrate as reductant and then pH

was adjusted to a desired level followed by the addition

of either drug or drug-loaded NPs. The major

radiochemical impurities are free 99mTcO42 and reduced

hydrolysed 99mTc (nanocolloids). Radiolabeling was

optimised under different experimental conditions

involving the pH of the complex, concentration of

stannous chloride dihydrate and concentration of the

drug (CHL). The labeling efficiency and stability of

labeled complex were ascertained by ascending TLC.

As the pH increased from 4 to 6.5, the radiolabeling

efficiency also increased from 82.7 to 97.7% for free

CHL and from 85.9 to 95.9% for CHL-loaded PLGA

NPs, whereas for CHL-loaded PS-80-coated PLGA

NPs this value was increased from 88.3 to 96.9%

(Table I). However, further increase in pH resulted in

subsequent reduction in the labeling efficiency.

The maximum labeling achieved for drug-loaded

PLGA NPs was at pH 6.5.

Radiolabeling efficiency at a different concentration

of SnCl2, 2H2O varying from 10 to 40mg/ml was

studied (Table II), keeping the amount of CHL both

in free and in drug-loaded NP formulations fixed

(4.5 mg/ml) and at a constant pH level of 6.5. It was

observed that the optimum amount of stannous

chloride resulting in high-labeling efficiency and low

amount of radiocolloids was found to be 20mg for free

CHL and 30mg for drug-loaded PLGA NPs. Lower

amount of stannous chloride led to poor labeling

efficiency, while higher amounts led to the formation

of undesirable radiocolloids.

Radiolabeling efficiency of drug-loaded NPs at pH

6.5 was also monitored with a different concentration

of drug incorporated in the nano matrix using a

desired amount of SnCl2, 2H2O (30mg) required for

complexation. Highest labeling yield was observed at

the drug concentration of 4.5 mg/ml (Table III).

After the addition of stannous chloride to the

preparations containing 99mTcO42, either free CHL or

CHL-loaded NPs were incubated for various time

periods and the effect of incubation time on labeling

efficiency was determined keeping other variables

constant (Table IV).

DTPA challenge study was performed to measure

the strength of the binding of technetium to CHL-

loaded NPs. It was observed (Figure 2) that with

increase in the concentration (10–50 mM) of DTPA,

there was not much change in labeling efficiency.

At a 50 mM concentration of DTPA, the amount

Figure 1. Transmission electron micrograph of CHL-loaded PLGA NPs. (a) PVA used as emulsifier and (b) PS-80 used as surfactant.

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Table I. The effect of pH on the labeling efficiency during the chelation of CHL and CHL-loaded NPs with 99mTc.

Free CHL CHL-loaded PLGA NPs CHL-loaded, PS-80-coated PLGA NPs

pH Colloid percentage Percentage labeled Percentage free Colloid percentage Percentage labeled Percentage free Colloid percentage Percentage labeled Percentage free

4.0 15.96 ^ 0.09 82.72 ^ 0.09 1.32 ^ 0.03 11.53 ^ 0.08 85.96 ^ 0.09 2.51 ^ 0.06 9.50 ^ 0.09 88.30 ^ 0.10 2.20 ^ 0.06

4.5 7.00 ^ 0.13 90.30 ^ 0.10 2.70 ^ 0.08 6.17 ^ 0.10 91.71 ^ 0.08 2.11 ^ 0.08 5.41 ^ 0.09 92.28 ^ 0.08 2.30 ^ 0.05

6.0 4.56 ^ 0.09 92.68 ^ 0.10 2.76 ^ 0.15 5.21 ^ 0.10 93.02 ^ 0.10 1.77 ^ 0.08 4.07 ^ 0.06 93.87 ^ 0.08 2.06 ^ 0.05

6.5 1.81 ^ 0.03 97.76 ^ 0.09 0.42 ^ 0.03 3.05 ^ 0.05 95.94 ^ 0.06 1.01 ^ 0.04 1.82 ^ 0.03 96.97 ^ 0.08 1.21 ^ 0.03

6.8 3.23 ^ 0.11 95.78 ^ 0.10 1.06 ^ 0.05 3.90 ^ 0.07 94.31 ^ 0.08 1.79 ^ 0.09 3.28 ^ 0.06 94.62 ^ 0.09 2.10 ^ 0.08

7.0 17.53 ^ 0.08 80.09 ^ 0.06 2.37 ^ 0.07 5.83 ^ 0.08 91.92 ^ 0.08 2.25 ^ 0.04 4.43 ^ 0.08 93.11 ^ 0.09 2.46 ^ 0.05

8.0 20.47 ^ 0.09 77.85 ^ 0.09 1.68 ^ 0.08 8.33 ^ 0.07 90.01 ^ 0.08 1.66 ^ 0.07 7.22 ^ 0.06 91.19 ^ 0.09 1.58 ^ 0.07

Each value is the mean of four results.

Table II. Influence of the amount of stannous chloride on the labeling efficiency of CHL, CHL-loaded PLGA NPs and CHL-loaded PS-80-coated PLGA NPs.

Free CHL CHL-loaded PLGA NPs CHL-loaded PS-80 coated PLGA NPs

SnCl2, 2H2O (mg) Colloid percentage Percentage labeled Percentage free

Percentage

colloid Percentage labeled Percentage free

Percentage

colloid Percentage labeled Percentage free

10 1.03 ^ 0.15 88.87 ^ 0.38 10.09 ^ 0.12 0.65 ^ 0.09 84.60 ^ 0.06 14.75 ^ 0.13 0.46 ^ 0.06 85.14 ^ 0.09 14.44 ^ 0.08

20 1.81 ^ 0.09 97.76 ^ 0.18 0.42 ^ 0.08 0.82 ^ 0.1 89.99 ^ 0.09 9.11 ^ 0.10 1.62 ^ 0.09 89.74 ^ 0.09 8.63 ^ 0.08

30 3.10 ^ 0.09 95.84 ^ 0.18 1.06 ^ 0.09 3.05 ^ 0.09 95.94 ^ 0.10 1.01 ^ 0.09 1.82 ^ 0.07 96.97 ^ 0.10 1.21 ^ 0.07

40 11.83 ^ 0.11 87.21 ^ 0.18 0.86 ^ 0.09 4.39 ^ 0.06 94.65 ^ 0.09 0.96 ^ 0.09 4.92 ^ 0.08 94.19 ^ 0.09 0.88 ^ 0.08

50 17.40 ^ 0.13 82.10 ^ 0.09 0.50 ^ 0.09 5.69 ^ 0.09 93.54 ^ 0.10 0.76 ^ 0.05 5.31 ^ 0.07 93.60 ^ 0.09 1.09 ^ 0.05

60 23.54 ^ 0.08 76.16 ^ 0.09 0.30 ^ 0.07 7.45 ^ 0.05 92.06 ^ 0.08 0.49 ^ 0.10 7.32 ^ 0.08 91.76 ^ 0.06 0.91 ^ 0.09

Each value is the mean of four experiments.

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of transchelate was found to be nearly 4% for CHL-

loaded NPs, indicating the in vitro stability of the

radiolabeled complexes.

The stability of the 99mTc-labeled CHL-loaded

PLGA NPs and CHL-loaded, PS-80-coated PLGA

NPs with time was also studied in saline and in serum

(rat) at 378C (Table V). It was observed that after

incubation for a period of 24 h, there was only 4–6%

decrease in labeling efficiency, indicating the stability

of the complex for in vivo use.

The distribution of radioactivity in different organs

after 1, 4 and 24 h of intravenous injection of 99mTc-

labeled free CHL, CHL-loaded PLGA NPs and

CHL-loaded, PS-80-coated PLGA NPs in Balb/c

mice is shown in Tables VI and VII. Various organs

like brain, liver, lung, spleen, kidney, stomach,

intestine and bone marrow were removed and

analysed for radiolabeled drug. Both in the case of

free drug and drug-loaded NPs, a major portion of the

injected dose was found to accumulate in the liver.

The biodistribution results (Table VII) indicate that

hepatic uptake was 60.11, 49.64 and 47.05% at 1 h,

55.8, 58.9 and 52.62% at 4 h, and 34.84, 42.21 and

43.62% at 24 h for 99mTc-CHL, CHL-loaded PLGA

NPs and CHL-loaded PS-80-coated PLGA NPs,

respectively. The hepatic uptake of the 99mTc-labeled

free drug was higher than that of the drug-loaded NPs

both at 1 and 4 h time period. However, hardly any

activity was found to clear by intestine. Similarly,

kidney and heart uptake was not very significant.

Urinary clearance was low initially, at 1 h post

injection it was 2.19, 7.95 and 8.28% per organ for99mTc-labeled CHL, CHL-loaded PLGA NPs and

CHL-loaded PS-80-coated PLGA NPs respectively,

where as this value was moderately increased with

time. At 24 h post injection, the urinary clearance

of CHL, CHL-loaded PLGA NPs and CHL-loaded

PS-80-coated PLGA NPs was 31.45, 14.5 and 16.17%

dose per organ respectively (Table VII). The urinary

clearance of 99mTc-labeled free drug was much higher

than that of the drug-loaded NPs at this time point.

Activity in spleen and lungs was moderately high.

The percentage of injected dose accumulated per

gram of spleen was 9.12, 9.09 and 6.37 for 99mTc-

labeled CHL, CHL-loaded PLGA NPs and CHL-

loaded PS-80-coated PLGA NPs at 1 h post injection

(Table VI), whereas this was reduced to 3.61, 5.92 and

2.04 as for the above-labeled drugs at 24 h post

injection period. Similarly, the percentage of injected

dose per gram of lung was also high initially. This was

8.93, 6.78 and 4.20 at 1 h post injection for 99mTc-

labeled CHL, CHL-loaded PLGA NPs and CHL-

loaded, PS-80-coated PLGA NPs respectively and

gradually decreased with time. The radioactivity in the

whole blood was found to be much less. This pointed

towards the rapid blood clearance of 99mTc-labeled

Figure 2. The effect of DTPA on transchelation of 99mTc-labeled

complexes of CHL, CHL-loaded PLGA NPs and CHL-loaded, PS-

80-coated PLGA NPs.

Table III. The effect of drug concentration on the labeling efficiency during the chelation of CHL-loaded NPs with 99mTc.

Drug concentration (mg/ml) Colloid percentage Percentage labeled Percentage free

2.50 4.03 ^ 0.72 91.48 ^ 0.52 4.48 ^ 0.28

4.50 1.81 ^ 0.18 97.76 ^ 0.42 0.42 ^ 0.12

7.50 2.89 ^ 0.22 91.42 ^ 0.48 5.68 ^ 0.30

Table IV. The effect of incubation time on the labeling efficiency of CHL and CHL-loaded NPs.

Incubation time (min) Free CHL CHL-loaded PLGA NPs CHL-loaded, PS-80-coated-PLGA NPs

Radiolabeling percentage

0 94.82 ^ 0.65 92.92 ^ 0.24 93.16 ^ 0.21

10 95.35 ^ 0.72 94.78 ^ 0.41 95.06 ^ 0.21

15 97.76 ^ 0.32 95.94 ^ 0.26 96.97 ^ 0.20

20 95.81 ^ 0.29 94.14 ^ 0.23 95.76 ^ 0.19

25 95.27 ^ 0.31 94.09 ^ 0.28 94.89 ^ 0.23

30 95.20 ^ 0.33 93.84 ^ 0.26 94.18 ^ 0.25

35 94.25 ^ 0.37 93.12 ^ 0.32 93.39 ^ 0.24

Results are the mean of four separate experiments.

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Table V. Stability studies of CHL and CHL-loaded PLGA NPs in physiological saline and serum in vitro at 378C.

In saline In serum

Time (h) Free CHL CHL-loaded PLGA NPs CHL-loaded, PS80-coated PLGA NPs Free CHL CHL-loaded PLGA NPs CHL-loaded, PS80-coated PLGA NPs

Radiolabeling percentage

Initial (0 h) 97.76 ^ 0.32 95.94 ^ 0.26 96.97 ^ 0.20 97.76 ^ 0.32 95.94 ^ 0.26 96.97 ^ 0.20

0.5 97.56 ^ 0.09 95.89 ^ 0.08 96.77 ^ 0.08 97.62 ^ 0.12 95.73 ^ 0.10 96.71 ^ 0.09

1 97.32 ^ 0.11 95.69 ^ 0.08 96.59 ^ 0.09 97.25 ^ 0.12 95.59 ^ 0.10 96.60 ^ 0.11

2 97.11 ^ 0.12 95.69 ^ 0.10 96.39 ^ 0.10 97.09 ^ 0.13 95.43 ^ 0.11 96.45 ^ 0.11

4 96.86 ^ 0.15 95.41 ^ 0.11 95.91 ^ 0.10 96.36 ^ 0.16 95.34 ^ 0.13 95.36 ^ 0.11

8 95.62 ^ 0.15 94.83 ^ 0.12 95.13 ^ 0.11 95.93 ^ 0.18 95.13 ^ 0.13 95.16 ^ 0.12

24 93.90 ^ 0.18 94.16 ^ 0.13 94.81 ^ 0.12 93.78 ^ 0.20 93.96 ^ 0.15 94.91 ^ 0.14

Table VI. Biodistribution of 99mTc-labeled CHL, CHL-loaded PLGA NPs and CHL-loaded, PS-80-coated PLGA NPs after intravenous injection in Balb/c mice.

1 h 4 h 24 h

Tissue

(gm) Free CHL

CHL-PLGA

NPs

CHL-PLGA-PS-80-

coated NPs Free CHL

CHL-PLGA

NPs

CHL-PLGA-PS-80-

coated NPs Free CHL

CHL-PLGA

NPs

CHL-PLGA-PS-80-

coated NPs

Liver 37.87 ^ 0.92 29.90 ^ 1.10 26.23 ^ 1.46 34.04 ^ 0.45 41.64 ^ 0.41 33.50 ^ 0.77 28.04 ^ 0.06 26.13 ^ 2.15 30.83 ^ 0.51

Lung 8.93 ^ 0.19 6.78 ^ 0.37 4.20 ^ 0.15 7.16 ^ 0.13 6.08 ^ 0.22 4.17 ^ 0.06 0.86 ^ 0.02 4.07 ^ 1.00 1.32 ^ 0.10

Spleen 9.12 ^ 0.29 9.09 ^ 0.67 6.37 ^ 0.11 7.67 ^ 0.25 8.38 ^ 0.09 5.54 ^ 0.29 3.61 ^ 0.34 5.92 ^ 0.88 2.04 ^ 0.10

Blood 0.26 ^ 0.007 1.04 ^ 0.04 1.28 ^ 0.07 0.06 ^ 0.01 0.45 ^ 0.03 0.27 ^ 0.03 0.16 ^ 0.04 0.20 ^ 0.015 0.18 ^ 0.007

Kidney 1.08 ^ 0.08 3.60 ^ 0.02 1.84 ^ 0.08 0.52 ^ 0.07 3.05 ^ 0.04 2.16 ^ 0.15 0.60 ^ 0.06 1.11 ^ 0.05 0.82 ^ 0.02

Intestine 0.06 ^ 0.013 0.55 ^ 0.02 0.21 ^ 0.02 0.08 ^ 0.004 0.41 ^ 0.03 0.52 ^ 0.014 0.07 ^ 0.01 0.40 ^ 0.06 0.16 ^ 0.01

Stomach 0.30 ^ 0.15 0.73 ^ 0.03 0.62 ^ 0.04 0.51 ^ 0.05 0.62 ^ 0.03 0.66 ^ 0.02 0.23 ^ 0.02 0.34 ^ 0.05 0.24 ^ 0.007

Brain 0.024 ^ 0.01 0.12 ^ 0.008 0.185 ^ 0.02 0.05 ^ 0.008 0.13 ^ 0.007 0.24 ^ 0.01 0.036 ^ 0.008 0.09 ^ 0.007 0.27 ^ 0.01

Bone 0.166 ^ 0.03 0.08 ^ 0.01 0.067 ^ 0.004 0.34 ^ 0.06 0.13 ^ 0.008 0.11 ^ 0.01 0.53 ^ 0.08 0.09 ^ 0.007 0.055 ^ 0.004

Heart 0.06 ^ 0.02 0.28 ^ 0.008 0.17 ^ 0.008 0.06 ^ 0.02 0.23 ^ 0.008 0.165 ^ 0.02 0.06 ^ 0.03 0.15 ^ 0.05 0.057 ^ 0.003

Muscle 0.04 ^ 0.02 0.17 ^ 0.02 0.047 ^ 0.004 0.04 ^ 0.02 0.13 ^ 0.008 0.06 ^ 0.007 0.03 ^ 0.02 0.15 ^ 0.05 0.032 ^ 0.004

Results are expressed in percent-injected dose per gram of tissue (each value is ^SEM of six mice).

Chloram

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free drug as well as drug-loaded NPs. The insignif-

icant accumulation of radioactivity in the stomach also

ruled out the in vivo decomposition of the radiolabeled

drug and drug-loaded NPs.

The concentration of 99mTc-labeled CHL-loaded

PS-80-coated PLGA NPs was relatively higher

(almost nine times) in brain than that of free drug

both in 1 and 24 h post injection and at 4 h post

injection it was five times higher than that of free drug

(Table VI). However, the brain uptake of CHL-loaded

PLGA NPs (containing PVA as emulsion stabilizer) in

the above time periods was only 2–2.5 times higher

than that of the free CHL. Although the overall brain

uptake of technetium-labeled, CHL-loaded NPs was

not very high, the significantly higher concentration of

CHL-loaded, PS-80-coated PLGA NPs reaching the

brain at 1 and 24 h post injection compared to that of

CHL-loaded PLGA NPs suggesting the greater brain

transport of the former.

Four samples of bone (femur, tibia) per animal were

taken and bone marrow was scrapped out with the

help of fine metallic needle on a piece of pre-weighed

filter paper and counted. The bone marrow uptake of99mTc-labeled, CHL-loaded NPs was much less than

that of radiolabeled free CHL at all studied time

points. At 1 h post the injection period, the

concentration of 99mTc-labeled free drug in the bone

marrow was 2–2.5 times higher than that of the drug-

loaded NPs, whereas at 24 h, the injected counts of99mTc-CHL in the bone marrow was 6- and 9.6-fold

higher than that of the CHL-loaded PLGA NPs and

CHL-loaded, PS-80-coated PLGA NPs, respectively

(Table VI).

Discussion

Attempts were made to formulate CHL in NPs form

by loading it into PLGA biodegradable polymers just

to enhance the brain permeability of the antibiotic and

to observe whether this formulation could minimize

the bone marrow uptake of the drug simultaneously.

The coating of the NPs with surfactant during

formulation is essential to prohibit the engulfment of

NPs formulation by mononuclear phagocytic system.

Thus, during the preparation of CHL-loaded PLGA

NPs by emulsion solvent evaporation technique,

either PVA or PS-80 was used as surfactant. Based

on several reports (Alyautidin et al. 1997; Gulyaev

et al. 1999; Ambruosi et al. 2005, 2006) that PS-80-

coated NPs facilitate the transport of the entrapped or

adsorbed drugs across BBB, we introduce PS-80

coating to modify the brain uptake of the drug

formulated as PLGA-based NPs forms. We have tried

to investigate the influence of PS-80 coating on PLGA

NPs loaded with CHL on brain accessibility and bone

marrow uptake.

Emulsification was achieved by sonication prior to

freeze drying, producing spherical NPs with diameter

Tab

leV

II.

Bio

dis

trib

uti

on

of

99m

Tc-

lab

eled

CH

L,

CH

L-l

oad

edP

LG

AN

Ps

an

dC

HL

-load

ed,

PS

-80-c

oate

dP

LG

AN

Ps

aft

erin

traven

ou

sin

ject

ion

inB

alb

/cm

ice.

1h

4h

24

h

Tota

l

org

an

Fre

eC

HL

CH

L-P

LG

A

NP

s

CH

L-P

LG

A-P

S-8

0-

coate

dN

Ps

Fre

eC

HL

CH

L-P

LG

A

NP

s

CH

L-P

LG

A-P

S-8

0

coate

dN

Ps

Fre

eC

HL

CH

L-P

LG

A

NP

s

CH

L-P

LG

AP

S-8

0-

coate

dN

Ps

Liv

er60.1

1^

1.0

649.6

4^

0.4

847.0

5^

1.0

555.8

0^

2.2

758.9

0^

1.6

252.6

2^

1.0

634.8

4^

2.5

842.2

1^

2.2

043.6

2^

0.7

6

Lu

ng

1.6

5^

0.0

91.9

5^

0.0

10.8

1^

0.0

31.3

7^

0.0

81.6

4^

0.0

31.3

1^

0.0

20.1

64^

0.0

91.6

0^

0.2

80.3

1^

0.0

3

Sp

leen

1.4

9^

0.0

81.2

7^

0.0

51.0

8^

0.0

50.8

5^

0.0

61.6

2^

0.0

51.2

6^

0.0

60.4

2^

0.0

31.2

3^

0.0

80.3

6^

0.0

2

Blo

od

0.4

5^

0.0

21.9

5^

0.0

12.2

1^

0.1

80.3

0^

0.0

30.7

4^

0.0

50.4

2^

0.0

80.2

5^

0.0

40.3

8^

0.0

30.3

2^

0.0

3

Kid

ney

0.5

9^

0.0

71.5

0^

0.0

40.6

1^

0.0

20.2

2^

0.0

41.1

5^

0.0

50.8

2^

0.0

80.1

8^

0.0

10.3

6^

0.0

30.3

2^

0.0

2

Inte

stin

e0.2

1^

0.0

31.6

3^

0.0

50.8

2^

0.0

80.2

6^

0.0

41.2

0^

0.0

91.9

8^

0.0

80.2

2^

0.0

31.2

9^

0.3

20.4

7^

0.0

3

Sto

mach

0.1

8^

0.0

30.2

9^

0.0

07

0.2

9^

0.0

20.3

6^

0.0

30.3

6^

0.0

16

0.5

3^

0.0

30.0

9^

0.0

10.2

0^

0.0

40.1

25^

0.0

05

Bra

in0.0

09^

0.0

03

0.0

36^

0.0

02

0.0

37^

0.0

02

0.0

2^

0.0

04

0.0

52^

0.0

08

0.0

87^

0.0

04

0.0

14^

0.0

07

0.0

34^

0.0

08

0.1

0^

0.0

1

Hea

rt0.1

0^

0.0

04

0.0

3^

0.0

16

0.0

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0.0

00

0.0

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0.0

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0.0

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00.0

22^

0.0

02

0.0

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0.0

03

0.0

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0.0

00.0

09^

0.0

Uri

ne

2.1

9^

0.1

57.9

5^

0.6

38.2

8^

0.2

815.3

2^

0.2

813.5

1^

0.3

712.2

3^

0.7

531.4

5^

1.8

314.5

0^

0.4

716.1

7^

0.4

8

Res

ult

sare

expre

ssed

inper

cen

t-in

ject

edd

ose

/org

an

(each

valu

eis^

SE

Mof

six

mic

e).

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of 10–70 nm (PVA used as emulsifier) and 40–120 nm

(PS-80 used as surfactant) with excellent redispersi-

bility and did not require any sort of energy at the time

of dispersion. Zeta-potential measurements revealed

that CHL-loaded PLGA NPs were negatively charged.

However, this value was slightly higher when PVA was

used as emulsifier. The amount of surfactant added has

been well standardized, as it plays an important role in

the emulsification process and in the protection of the

droplets from agglomeration.

Several experiments were carried out to achieve

optimal preparation conditions including PLGA

content, drug–polymer ratio, time of sonication,

surfactant content in the formulation and evaporation

rate of organic solvents. To minimize aggregation,

solvents were evaporated from nanodispersion by slow

stirring at room temperature. However, this process

might cause leaching of the drug from the matrix,

reducing entrapment efficiency.99mTc labeling with a different amount of stannous

chloride (SnCl2, 2H2O) was performed to determine

the optimum concentration of reductant required to

prevent the formation of radiocontaminant-like

pertechnetate (99mTcO42) and radiocolloid (Sn-Tc).

The amount of SnCl2 above the desired level results in

radiocolloid formation and subsequent accumulation

in the organs of RES due to macrophage uptake.

However, the concentration of SnCl2 less than the

optimum level results in pertechnetate formation due

to incomplete reduction.

The stability of 99mTc-labeled, CHL-loaded PLGA

NPs and CHL-loaded PS-80-coated PLGA NPs with

time was studied in saline and in serum (rat) at 378C;

results revealed substantial stability of the chelate.

Even after a period of 24-h incubation, the labeling

efficiency was in the range of 94–95%, indicating that

there was hardly any decomposition of the radiolabel

and also the suitability of the complex for in vivo

studies. Similarly, the transchelation was found to be

1–2% at 10 mM concentration of DTPA, which

became 4–5% at 50 mM concentration of DTPA.

This low degree of transchelation observed for 99mTc-

labeled, CHL-loaded NPs reveals the strong binding

of 99mTc with drug-loaded NPs.

The tissue uptake of 99mTc-labeled free CHL was

comparatively much higher than that of drug-loaded

NPs in the different organs of RES, e.g. liver, lung,

spleen owing to its affinity for those organs. Compara-

tively, less uptake in stomach and intestine suggests the

in vivo stability of 99mTc-labeled complexes. Urinary

clearance of 99mTc-labeled free drug was moderately

higher than that of CHL-loaded NPs. Drug-loaded NPs

accumulated in liver and cleared slowly from there.

Though overall brain uptake of drug-loaded PLGA NPs

was not very high, the brain accumulation of NPs was

significantly higher than that of free CHL.

Replacement of PVA with PS-80 resulted in the

difference in uptake and distribution profile in some

organs. Higher brain accumulation of CHL-loaded,

PS-80-coated PLGA NPs, compared with that of free

CHL and CHL-loaded PLGA NPs (containing PVA

as emulsion stabilizer), can be attributed to their

enhanced brain transport. Additionally, the bone

marrow uptake of the antibiotic has been found to be

much reduced (almost 10 times) when formulated as

PS-80-coated, PLGA-based NPs. Nowadays the

usefulness of the antibiotic has been much reduced

due to its fatal bone marrow suppression. From the

biodistribution studies (in mice), it has been

confirmed that by incorporating the drug in PS-80-

coated, PLGA-based nanomatrix, bone marrow

toxicity of CHL can be minimized with concomitant

rise in brain uptake. Thus PS-80-coated PLGA

nanomatrix can be used as a promising drug delivery

carrier for CHL with low bone marrow toxicity.

Conclusion

Emulsion solvent evaporation technique led to the

development of CHL-loaded PLGA NPs that were

radiolabeled efficiently with 99mTc. The labeled

complex showed excellent in vitro stability (deter-

mined by DTPA challenge test) as well as in vivo

serum stability. The significantly higher brain uptake

and simultaneously lower bone marrow uptake

suggest that the CHL-loaded, PS-80-coated, PLGA-

based NPs could be an advantageous drug delivery

system that could minimise the toxicity of the drug by

reducing the fatal bone marrow uptake of the

antibiotic; hence it is expected that prolonged use of

this life-saving drug could be recommended. Further

investigations to increase the entrapment efficiency of

the NP formulation are underway.

Acknowledgements

The authors are thankful to Mr Sailendra Nath Dey

for his moral support during experiments with TEM

study. The technical assistance of Dr Abhijit Das

Sharma (CGCRI, Kolkata, India) is gratefully

acknowledged for Zeta-potential measurement. We

are also thankful to the Council of Scientific and

Industrial Research for providing financial assistance

to carry out this work.

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