JPR ddi nano

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Original Article

Development, characterization and evaluation ofantiretroviral drug e Didanosine loaded serumalbumin nanocarriers for an antiretroviral therapy

Josephine Leno Jenita Joseph a,*, Vijaya Chockalingam b, Wilson Barnabas a

aDepartment of Pharmaceutics, Dayananda Sagar College of Pharmacy, Bangalore 560078, Karnataka, IndiabDepartment of Pharmaceutics, Ultra College of Pharmacy, Madurai, Tamil Nadu, India

a r t i c l e i n f o

Article history:

Received 6 August 2013

Accepted 15 August 2013

Available online 6 September 2013

Keywords:

Didanosine

Bovine serum albumin

Polysorbate 80

Biodistribution studies

a b s t r a c t

Aim: Didanosine, a nucleoside reverse transcriptase inhibitor, is used to treat HIV infection

in patients with or without acquired immunodeficiency syndrome. The objective of this

study is to prepare didanosine loaded bovine serum albumin nanoparticles by desolvation

technique and coated with 1% polysorbate 80 to improve antiretroviral therapy.

Method: Bovine serum albumin nanoparticles containing didanosine were prepared by des-

olvation techniqueandcross linkedwith8%v/v glutaraldehydesolution. Ethanol andmannitol

were used as a desolvating agent and cryoprotectant respectively. The formulated nano-

particles were characterized, evaluated andwere subjected to stability studies over a period of

threemonths. Biodistribution studies were investigated for the best formulation (D1).

Results: Nanoparticle size was averaged below 270 nmwith 0.2 PDI and zeta potential was in

the range of �23.0 to �36.6. Encapsulation efficiency ranges from 66 to 85.71% and % drug

loading ranges from 9.48 to 28.34. Cumulative percent drug release was in range of 60e80%

and release kinetics suggested that drug release was Fickian diffusion controlled. The sta-

bility studies over period of 3 months confirmed the stability of BSA nanoparticles. Bio-

distribution studiesdemonstrated that thedrug level inmacrophageorgans canbeenhanced

by coating of nanoparticles with 1% polysorbate 80.

Conclusion: The method adopted is simple and able to prepare stable, spherical shaped

nanoparticles which exhibit slow and sustained release.

Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights

reserved.

1. Introduction

Globally each year about 5 million people contract the virus

and over 3 million, including 500,000 children, die of acquired

immune deficiency syndrome (AIDS). HIV is concentrated in

specific anatomic sites such as central nervous system,

lymphoid organs and also testicles, female genital tract.1 Al-

bumin is emerging as a versatile protein carrier for thera-

peutic, diagnostic agent, drug targeting and for improving the

pharmacokinetic profile of drugs. In addition, it is likely that

endogenous albumin and abundant plasma protein, with the

half-life of 19 days in the blood circulation, may play an

* Corresponding author. Tel.: þ91 9448429214.E-mail address: [email protected] (J.L. Jenita Joseph).

Available online at www.sciencedirect.com

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

j o u rn a l o f p h a rma c y r e s e a r c h 7 ( 2 0 1 3 ) 7 1 2e7 1 9

0974-6943/$ e see front matter Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved.http://dx.doi.org/10.1016/j.jopr.2013.08.018


important role for improving the drug targeting properties of

many novel drugs.2 Albumin based nanocarriers drug delivery

systems are superior to traditional medicine with respect to

achieve targeted delivery, steady drug release, reduced dosing

frequency and therapeutic impact. These targeting capabil-

ities of nanocarriers have overcome many of the anatomical

and physiological barriers and deliver the drugs locally at the

HIV-infected sites thereby improving the HIV therapy.3 Even if

not providing a way to cure HIV/AIDS, the ability of a nano-

technology based systems improve drug therapy in infected

patients as demonstrated by in vitro and animal in vivo studies.

Ongoing efforts are being made to develop polymeric nano-

carriers capable of delivering active molecules specifically to

the intended target organ.4 The pharmacokinetic profile of

various therapeutic classes of antiretroviral drugs (ARV’s) can

be modifying through their incorporation into nanodelivery

systems.

There are 7 classes of FDA-approved antiretroviral agents

(ARV) and more than 25 individual drugs.5 Majority of the ARV

drugs are marketed as conventional dosage forms such as

tablets, capsules and suspensions are not able to deliver the

drug to brain due to thenature of the bloodebrain barrier (BBB).

It contains some significant drawbacks like short half-life, low

bioavailability, poor permeability and undesirable side effects.6

Didanosinewas theseconddrugapprovedbytheUSFDAfor the

treatment of patients infected with the human immunodefi-

ciency virus (HIV) in 1991. It has chosen as a model drug and

which act as chain terminators to HIV reverse transcriptase.

The most serious adverse events associated with didanosine

treatmenthavebeenperipheralneuropathy,pancreatitis, lactic

acidosis7 and also have poor gastrointestinal tolerability, un-

dergoes hepatic first pass metabolism, low oral bioavailability

(35e40%), short biological half-life (30 min e 4 h), low plasma

protein binding and narrow therapeutic index. These problems

can be overcome by formulating nanoparticles for sustained or

prolonged and targeted drug delivery.

Hence considering the importance of treating HIV, an

attempt was made to prepare didanosine loaded albumin

nanoparticles in a particular range which is suitable for the

drug delivery system that will increase bioavailability, dosing

frequency and also allow sustained drug delivery. The effect

of manufacturing conditions such as pH, BSA concentration

and agitation speed was also extensively investigated.

2. Materials and methods

2.1. Materials

Bovine serum albumin (BSA) (fraction V, with purity of 98%)

was purchased from Himedia laboratories Ltd. (Mumbai,

India). Didanosine (ddi) was received as a gift sample from the

Strides Arcolabs Ltd. (Bangalore, India). Mannitol, polysorbate

80, sodium hydroxide and glutaraldehyde and all other

chemicals were commercially supplied by Sigma Aldrich.

2.2. Method of preparation

Albumin nanoparticles were prepared by a desolvation

method.8 Different ratio of BSA powder (1%, 1.5%, 2%, 2.5% &

3%) was dissolved in distilled water; subsequently, pH was

adjusted to 8 by 0.1 M NaOH using digital pH meter under

constant stirring at 500 rpm. Didanosine was mixed, incu-

bated for 1 h at room temperature. Ethanol was added drop-

wise at a rate of 1 ml/min into the BSA solutions as a

desolvating agent until the solutions became just turbid.

Thereafter 30min of the desolvation process, 100 ml of an 8% v/

v aqueous solution of glutaraldehyde was added to induce

particle cross linking. This process was performed during

stirring over a time period of 3 h at room temperature. The

nanosuspensions were purified by two cycle centrifugation at

20,000 rpm for 30min and then subjected to freeze drying after

adding 2% (w/v) mannitol as a cryoprotectant for 8 h to obtain

fine powder of nanoparticles. The dried nanoparticles ob-

tained were then transferred to vials and were stored at 4 �C.Coating was done for D1 immediately after cross linking by

adding 1% polysorbate 80 andwas incubated for 30min, as per

the procedure described by Amit Bansal et al. Finally the

nanosuspensions was centrifuged and lyophilized with 2%

mannitol.

3. Nanoparticle characterization

3.1. Compatibility study

Compatibility of ddi and BSA, were analyzed using FT-IR

(Fourier transform infrared) spectroscopy, Shimadzu Corpo-

ration, Japan by the potassium bromide disc method (1:100).

The DSC (differential scanning calorimetry, MettlereToledo

star 822 systems, Switzerland) thermogram of drug and

lyophilized nanoparticles gives information regarding the

physical properties and melting point of the drug.

3.2. Surface morphology

Scanning electron microscopy was performed to characterize

the surface morphology of the prepared nanoparticles to

detect their morphological character of nanoparticles. This

was done by placing freeze dried nanoparticles on brass stub

then were gold-coated to render them electrically conductive

and examined under the Scanning Electron Microscope at

20 kV (JSM 6100 JEOL, Tokyo, Japan).

3.3. Determination of particle size and zeta potential

The particle size and zeta potential of didanosine albumin

nanoparticles was determined by dynamic light scattering,

using a Malvern system, with vertically polarized supplied by

Helium/Neon laser (red laser) operated at 4 mM, 633 nm. The

samples were dispersed in distilled water and taken in clear

disposable zeta cell. The experiments were performed with

non-invasive backscatter technology at a temperature of

25.0 � 0.1 �C at a detection angle of 173� to the incident beam.

3.4. Determination of % drug entrapment efficiency and% drug loading

Freshly prepared nanosuspensions were centrifuged at

20,000 rpm for 30 min and the amount of unincorporated

j o u r n a l o f p h a rm a c y r e s e a r c h 7 ( 2 0 1 3 ) 7 1 2e7 1 9 713


didanosine in supernatant liquid was measured. The %

entrapment efficiency (EE) and % drug loading were calculated

according to the following formula.

% EE ¼ ½Amount of drug actually present in nanoparticles=

amount of drug actually added� � 100

% Drug loading¼½ðTotal amount of drug added� amount of

unbound drug in supernatant liquidÞ=total amount of drug added� � 100

3.5. In vitro drug release studies

The dug release studies were carried out by dialysis method. A

knownquantity of nanoparticles equivalent to 10mgof the drug

was taken in a cellulose dialysis bag (molecular weight cut off

5kDa,Himedia, India)andadded5mlofpH7.4phosphatebuffer.

Thedialysisbagwas thensuspended inaflaskcontaining100ml

of phosphate buffer (pH 7.4) on amagnetic stirrer at 37� 0.5� at100 rpm. 5 ml quantity of sample was withdrawn at different

time periods and same volume of dissolution medium was

replaced in the flask tomaintain sink condition. Thewithdrawn

samples were filtered and then the filtrate was diluted with

phosphate buffer (pH 7.4). The samples were analyzed for drug

releasebymeasuringtheabsorbanceat249nmusingUVevisible

spectrophotometer.The invitrodrugreleasestudieswerecarried

out in triplicate for each formulation.

3.6. Release kinetics

The in vitro release data of all the formulation were fitted with

various kineticsmodels such as zero order, first order, Higuchi

model and KorsmeyerePeppas,9 in order to predict kinetics

and mechanism of drug release. The release constant was

calculated from the slope of plots and regression coefficient

(r2), diffusion exponent (n) was determined.

3.7. Stability studies

The stability study of freeze dried nanoparticles was carried

out for D1 (1:2) to assess the stability of drug in nanoparticles.

For this purpose the samples were taken in borosilicate vials

and sealed and the vials were stored in room temperature

(25�e30 �C) and refrigerator (3�e5 �C) over a period of 3

months. After specified period 0, 1, 2 and 3 months, the

samples were checked for their physical appearance and drug

content by UV spectrophotometer, as well as chemical sta-

bility by Fourier transform infrared (FTIR) studies.

3.8. Biodistribution studies

The biodistribution studies8 of ddi loaded albumin nano-

particles were carried out on healthy adult Wistar rats weigh-

ing 200e250 g and after obtaining approval from the local

animal ethics committee and CPCSEA (DSCP/PH.D PHARM/

IAEC/49/2010-2011). All animals were provided with proper

care, food, water ad libitum and were maintained under well

ventilated in large spacious cages throughout the study. The

rats were divided randomly into three groups with three ani-

mals per group and they were fasted at least 12 h before

experimentation. Group 1 was injected with ddi (which was

dispersed inwater for injection) into the tail vein of rats, Group

2 was received ddi loaded albumin nanoparticles and Group 3

was administered polysorbate 80 coated albumin nano-

particles. All the formulations were given in a dose level

equivalent to 20mg/kg body weight.7 One hour after injection,

the ratswere sacrificedby euthanized andorgans such as liver,

lung, kidney, lymph nodes, spleen, brain and blood were iso-

lated. The organs were washed with clean buffer saline and

absorbed dry with filter paper and then weighed. Prior to the

analysis organs homogenateswere prepared andwas digested

with 10% v/v trichloroacetic acid andwas treatedwith 10ml of

acetonitrile to extract didanosine. Didanosine content in the

various organswas estimated by reverse-phase HPLCmethod.

4. Results and discussions

4.1. Nanoparticle preparation

BSA nanoparticles were prepared and loaded with didanosine

by desolvation techniques with ethanol as it does not require

an increase in temperature. Initially this method was opti-

mized with different parameters like stirring speed, pH and

volume of glutaraldehyde solution to get an optimized for-

mula (data not given). This optimized method was able to

produce smooth, spherical, stable, white colored free flowing

nanoparticles. Furthermore the drug loaded nanoparticles

were characterized and evaluated.

4.2. Compatibility study

The FT-IR spectra illustrated that the characteristic peaks of

ddi, BSA and nanoparticles whereas the characteristic peaks

of nanoparticles (Fig. 1) remain samewith slightmodifications

due to other excipients present in the formulations. The DSC

thermogram of drug and lyophilized nanoparticles are shown

in Fig. 2. DSC curves showed that endothermic peak at

193.8 �C, 282.9 �C in didanosine and 77.6 �C, 193.6 �C in

nanoparticles and represented the didanosine melting point.

From DSC profiles, it was concluded that the didanosine was

present in the formulated nanoparticles in the amorphous

state and might have dispersed uniformly in the polymer.

4.3. % EE and % drug loading

% EE and % drug loading depending on the drug polymer ratio

are shown in Table 1. The % EE was decreased with respect to

drug polymer mass ratio due to limited affinity of the drug

molecule to the macromolecular material. In a nanocarrier

system the drug loading is important to determine the

amount of drug substance required for the injection. The %

drug loading was found to be high to low with increase con-

centration of BSA due to the concentration of ddi was kept

constant and was 28.34 � 0.23 to 9.48 � 0.83.

4.4. Particle size and zeta potential

The morphological properties and surface appearance of ddi

loaded BSA nanoparticles has observed using scanning



electron microscopy and demonstrated that nanoparticles

were spherical, smooth surface. Fig. 3a and b depicts the SEM

image and particle size distribution of ddi loaded nano-

particles. The mean particle size of ddi loaded nanoparticles

were found to be ranged between 194.8 and 268 nm with

polydispersity index was in the range of 0.121e0.281.The

mean zeta potential was found to be �23.0 to �36.6 which

indicates high degrees of stability due to inter particle re-

pulsions and are shown in Table 2.

4.5. In vitro drug release

Fig. 4 shows the comparative graph of cumulative percentage

ddi release profiles from nanoparticles and was observed

Fig. 1 e FTIR spectra of didanosine, BSA, physical mixture and formulated nanoparticles.

Fig. 2 e DSC thermograms of didanosine and formulated nanoparticles.



burst release of ddi within 1 h from nanoparticles due to the

dissociation of entrapped drug close to the surface layer of

nanoparticles. Later the drug release was observed the slow

and sustained manner over 24 h. In D1% cumulative ddi

release was found to be high due higher drug loading and

lower polymer concentration than in D5 which showed %

cumulative ddi release was low and also observed lesser burst

effect.

Table 1e Percent EE and% drug loading of ddi loaded BSAnanoparticles.

Formulation code D:P % EE % Drug loading

D1 1:2 85.71 � 0.39 28.34 � 0.23

D2 1:3 79.81 � 0.58 19.95 � 0.35

D3 1:4 76.56 � 0.68 15.37 � 0.61

D4 1:5 75.75 � 0.19 12.62 � 0.29

D5 1:6 66.38 � 0.33 9.48 � 0.83

Ps 80 coated D6 1:2 83.29 � 1.53 26.4 � 0.05

Fig. 3 e a. SEM image of ddi loaded BSA nanoparticles (D1). b. Particle size distribution of formulated ddi loaded

nanoparticles (D1).

Table 2 e Physicochemical characterization of ddi loadedBSA nanoparticles.

Formulation code Particle size PDI Zeta potential

D1 194.8 0.281 �36.6

D2 201.4 0.121 �23.0

D3 211.5 0.197 �32.9

D4 223.6 0.276 �36.1

D5 268.0 0.243 �30.8

Ps 80 coated D6 196.7 0.387 �40.2



4.6. Release kinetics

The drug release mechanism characterized by applying the

in vitro release data to various kinetic models and results

of n and r2 values of different kinetic model represent in

Table 3. Diffusion controlled drug release was observed

with higher r2 in Higuchi model. The diffusion exponent (n)

value is used to characterize different release mechanism

in KorsmeyerePeppas model. The n value was found to

be less than 0.45 and suggested that drug release from

nanoparticles followed Fickian diffusion controlled mech-

anism.

Fig. 4 e In vitro drug release profile of ddi loaded BSA nanoparticles in pH 7.4 PBS. (Data shown as mean ± SD, n [ 3;

ddi [ didanosine).

Table 3 e Release kinetics of ddi loaded BSA nanoparticles.

Formulation code Drug release kinetics, correlation coefficients (r2) KorsmeyerePeppas

Zero order First order Higuchi HixsoneCrowell r2 n

D1 0.8836 0.9581 0.9724 0.9471 0.9758 0.3189

D2 0.8964 0.9536 0.9677 0.9439 0.9658 0.3243

D3 0.9194 0.9647 0.9744 0.9568 0.9698 0.3458

D4 0.9185 0.9637 0.9754 0.9543 0.9737 0.3555

D5 0.9132 0.9602 0.9814 0.9489 0.9858 0.3704

Ps 80 coated D6 0.8846 0.9559 0.9700 0.9428 0.9727 0.3169

Table 4 e Stability studies of ddi loaded BSA nanoparticles (D1).

Temperature Evaluation parameters Observation (months)

0 1 2 3

3e5 �C Physical appearance White in color No change No change No change

% Drug content 28.34 � 0.85 28.14 � 0.36 28.09 � 0.74 28.02 � 0.51

FTIR Done No significant change No significant change No significant change

15e20 �C Physical appearance White in color No change No change No change

% Drug content 28.34 � 0.17 28.04 � 0.86 27.97 � 0.55 27.75 � 0.56

FTIR Done No significant change No significant change No significant change



4.7. Stability studies

The results of stability studies are shown in Table 4. The

physical as well as chemical characteristics of the formulation

were not affected at both temperature 3e5 �C and 15e25 �Cduring 3months storage. Therewere no significant changes in

drug content and FTIR spectra. From the above results the

developed nanoparticles are stable at various temperatures.

4.8. Biodistribution studies

From this study, concentration of didanosine (ng/ml) from

polysorbate 80 coated, uncoated formulationwasmeasured in

various organs of Wistar rats and compared with free drug of

didanosine in solution. Fig. 5 shows that the mean concen-

tration (ng/ml) of ddi in blood, liver, spleen, kidneys, lungs,

lymph nodes and brain from polysorbate 80 coated, uncoated

and free drug solution after 1 h of i.v administration. In

almost, higher concentration of ddi reached in macrophage

rich organs from group which has received polysorbate 80

coated nanoparticles than group 2 (uncoated nanoparticles),

group 1 (the free drug solution). The concentration of ddi in

brain, spleen and lymph nodes from polysorbate 80 coated

nanoparticles was found in 12.38, 8.15, 9.51 fold in comparison

with the free ddi solution after 1 h of intravenous injection due

to opsonization of albumin nanoparticles.

5. Conclusion

In this study BSA nanoparticles were used as a carrier for

antiretroviral and can be concluded that it is possible to pre-

pare by desolvation technique. In vitro studies were evaluated

to confirm the Fickian diffusion controlled drug release

mechanism. Based on biodistribution studies polysorbate 80

coated nanocarriers play a specific role to extend the half-life

of therapeutically active drugs with reduced dose related

adverse effects and also able to deliver higher drug levels in

HIV reservoir sites which can provide better viral suppression

by terminating HIV reverse transcriptase. From the results,

human serum albumin can be substituted by bovine serum

albumin to prepare nanoparticles containing antiretroviral

drugs in further experiments.

Conflicts of interest

All authors have none to declare.

r e f e r e n c e s

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Fig. 5 e Biodistribution of ddi in rat’s organs after i.v injection of BSA nanoparticles. ddi: didanosine, ddi Np: ddi loaded

nanoparticles, Ps 80-ddi Np: polysorbate coated ddi loaded nanoparticles. (Mean ± SD, n [ 3).



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