Nanomedicine: Nanotechnology, Biology, and Medicine 3 (2007) 239 – 243

www.nanomedjournal.com
Oncology
Chemotherapeutic evaluation of alginate nanoparticle-encapsulated azole
antifungal and antitubercular drugs against murine tuberculosis
Zahoor Ahmad, PhD, a Sadhna Sharma, PhD, b Gopal K. Khuller, PhD b,⁎
a
Center for Tuberculosis Research, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
b
Department of Biochemistry, Postgraduate Institute of Medical Education and Research, Chandigarh, India
Received 3 March 2007; accepted 24 May 2007

Abstract The present study was designed to evaluate the chemotherapeutic potential of alginate nanoparticle-
encapsulated econazole and antitubercular drugs (ATDs) against murine tuberculosis. Alginate
nanoparticles encapsulating econazole and ATDs were prepared by the cation-induced controlled
gelification of alginate and were characterized. Drugs were analyzed by high-performance liquid
chromatography. All the ATDs were detected above minimum inhibitory concentrations for as long
as 15 days and econazole until the day 8 in organs (lungs, liver, and spleen) after administration of
encapsulated drugs, whereas free drugs remained detectable for only 12 to 24 hours. Eight doses of
alginate nanoparticle-encapsulated econazole or 112 doses of free econazole reduced bacterial
burden by more than 90% in the lungs and spleen of mice infected with Mycobacterium tuberculosis.
Econazole (free or encapsulated) could replace rifampicin and isoniazid during chemotherapy of
murine tuberculosis. Alginate nanoparticles reduced the dosing frequency of azoles and ATDs by
15-fold. Alginate nanoparticles are the ideal carriers of azole and antitubercular drugs, which can
reduce dosing frequency of azoles as well as ATDs for the better management of tuberculosis.
© 2007 Published by Elsevier Inc.
Key words: Nanoparticles; Chemotherapy; Econazole; Bioavailability

Azole antifungals have been demonstrated by various
reports to have potent antitubercular activity; these drugs
deserve close attention, because they have already been
assessed for toxicological impact and are in clinical use for
humans [1]. Earlier we have demonstrated the in vitro and ex
vivo potential of azoles against M. tuberculosis H37Rv as
well as the synergism of azole drugs with conventional
antitubercular drugs [2]. Antituberculosis potential of azole
drugs against latent/persistent tuberculosis has also been
demonstrated, and econazole, like rifampicin (RIF), has been
shown to prevent the formation of persistent/latent bacilli in
mice [3]. In addition, econazole has been shown to be more
No conflict of interest was reported by the authors of this paper.
⁎ Corresponding author. Department of Biochemistry, Postgraduate
Institute of Medical Education and Research, Sector-12, Chandrigarh
160012, India.
E-mail address: gkkhuller@yahoo.co.in (G.K. Khuller).
1549-9634/$ – see front matter © 2007 Published by Elsevier Inc.
doi:10.1016/j.nano.2007.05.001
effective than RIF against persistent bacilli [3]. More
recently we have demonstrated that econazole could reduce
bacterial burden by 90% in the lungs and spleen of mice
infected with M. tuberculosis and its chemotherapeutic
potential to be equal to RIF [4]. Further, results of this study
indicated that econazole could replace RIF or isoniazid
(INH), as well as both of them in chemotherapy of murine
tuberculosis [4]. Although azole drugs have been shown to
have strong antimycobacterial potential against latent/
persistent and active murine tuberculosis, their bioavail-
ability is poor through the oral route [5]. Hence, econazole
must be administered with a high dosing frequency-twice
daily [4]. Thus, it seems that even after the incorporation of
azole drugs into a conventional antitubercular regimen, the
problem of patient noncompliance will be of more concern.
Recently, we have demonstrated that bioavailability of the
most potent antimycobacterial azoles (econazole and
clotrimazole) could be enhanced by using alginate/poly
(lactide-co-glycolide) nanoparticles as drug carriers [6].
240 Z. Ahmad et al. / Nanomedicine: Nanotechnology, Biology, and Medicine 3 (2007) 239–243
Hence, the present study was planned with an aim to evaluate
the chemotherapeutic potential of alginate nanoparticle-
encapsulated econazole alone and in combination with
antitubercular drugs against murine tuberculosis.
Methods
Chemicals and drugs
Sodium alginate (medium viscosity, 3500 cps for a 2% w/v
solution), chitosan (minimum 85% deacetylated), econazole,
INH, RIF, ethambutol (EMB), and pyrazinamide (PZA) were
obtained from Sigma Chemical Co. (St. Louis, MO). High-
performance liquid chromatography (HPLC)-grade sol-
vents and water were obtained from Merck Ltd. (Mumbai,
India). All other chemicals used in the study were of
analytical grade.
Animals
Laca mice of either sex weighing 20-25 g obtained from
the Central Animal House, Postgraduate Institute of Medical
Education and Research (Chandigarh, India) were used in
the study. Animals were housed in biosafety cabinets (Nuaire
Instruments, NU 605-600E, Series 6) and provided with
pellet diet/water ad libitum. The study was approved by the
Institute's Animal Ethics Committee.
Preparation of ATD-loaded alginate nanoparticles
Alginate nanoparticles were prepared by the cation-
induced controlled gelification of alginate [7] with slight
modifications [8]. Briefly, calcium chloride (0.5 mL, 18 mM)
was added to 9.5 mL of sodium alginate solution (0.06%)
containing drugs (ratio of drug to polymer was 7.5:1 w/w %).
Addition of 2 mL of chitosan solution (0.05%) was followed
by stirring for 30 minutes and maintenance at room
temperature (23-25°C) overnight. Drug-loaded nanoparticles
were recovered by centrifugation at 19,000 rpm for
35 minutes and washed thrice with distilled water. Drug-
free nanoparticles were also prepared in the same manner.
Characterization of alginate nanoparticles
Alginate nanoparticles were characterized for their size
and polydispersity index on Zetasizer 1000 HS (Malvern
Instruments, Malvern, UK) as described earlier [8]. The drug
encapsulation efficiency was determined as described earlier
[8]. The drugs were analyzed by HPLC; in the case of RIF/
INH/PZA, the three drugs were separated and quantitated by
using a USP gradient program as described earlier [9].
Because of the ultraviolet transparency of EMB, it was
analyzed by using a USP isocratic program as described
earlier [9]. The sensitivity of the methods was RIF 0.4 μg/
mL, INH 0.2 μg/mL, PZA 1.0 μg/mL, and EMB 0.5 μg/mL
[9]. The econazole was analyzed by using a USP isocratic
program as described earlier with an analytical sensitivity of
0.2 μg/mL [6].
In vivo drug disposition studies
The drug doses used throughout the study were RIF
12 mg/kg, INH 10 mg/kg, PZA 25 mg/kg, EMB 16 mg/kg,
and econazole 3.3 mg/kg body weight according to the
standard adult human doses. Although higher drug doses are
generally recommended for mice (considering that mice
possess a higher surface area-to-body weight ratio as
compared with humans), we have recently demonstrated
the two doses to be equipotent [10]. The dose being different
for each drug, the initial amount of drug taken to prepare the
formulations was calculated as previously described [10].
For the single-dose drug disposition studies, mice were
grouped as follows with six animals in each group: group 1,
oral free econazole; group 2, oral free ATDs; group 3, oral
free econazole + ATDs; group 4, oral econazole-loaded
alginate nanoparticles; group 5, oral ATD-loaded alginate
nanoparticles; group 6, oral econazole + ATD-loaded
alginate nanoparticles; group 7, oral drug-free alginate
nanoparticles (a positive control to explore the influence of
alginate nanoparticles on drug estimation). The animals were
bled at several time points. The plasma obtained from each
mouse was analyzed for the estimation of drug presence as
described earlier [9]. The drugs were analyzed by HPLC and
compared with calibration graphs (obtained by analyzing
pooled blank mice plasma spiked with known drug amounts)
to obtain the plasma drug concentration versus time profile.
The area under the curve for plasma drug concentration in
relation to time was calculated using data analysis tools in
SigmaPlot software (Version 8.0) and further used to
compute the relative bioavailability of each drug.
The animals were sacrificed at different time points. Drug
levels were determined in 20% w/v of tissue homogenates
(lungs, liver, and spleen) by following the same analytical
procedure as described for plasma [9]. It should be noted that
the HPLC analysis in plasma/tissues in the case of mice
dosed with alginate nanoparticles was restricted to free drugs
only (i.e., no attempt was made to lyse the nanoparticles).
Experimental infection and chemotherapy
Mice were infected via the lateral tail vein with either 105
to 107 bacilli of M. tuberculosis H37Rv as described earlier
[4]. The confirmation of infection and basal bacterial load
were determined as described earlier [4]. Subsequently, mice
were grouped as follows (eight animals per group): group I,
untreated controls (received phosphate-buffered saline
[PBS]); group II, econazole; group III, RIF; group IV,
INH, PZA, EMB, and RIF; group V, econazole, INH, PZA,
and EMB; group VI, econazole, PZA, EMB, and RIF; group
VII, econazole, PZA, and EMB; group VIII, nanoparticles
containing ,econazole; group IX, nanoparticles containing
RIF; group X, nanoparticles containing INH, PZA, EMB,
and RIF; group XI, nanoparticles containing econazole, INH,
EMB, and PZA; group XII, nanoparticles containing
econazole, RIF, EMB, and PZA; group XII, nanoparticles
containing econazole, EMB, and PZA. Control animals
 Z. Ahmad et al. / Nanomedicine: Nanotechnology, Biology, and Medicine 3 (2007) 239–243 241

Table 1 Table 2
Comparison of organ drug levels following oral administration of alginate- Chemotherapeutic efficacy of econazole in free or alginate nanoparticle-
encapsulated azoles with and without antitubercular drugs to mice encapsulated form against tuberculosis in mice infected with a high dose
(107 bacilli) of M. tuberculosis H37Rv
Drug Time ATDs or econazole ATDs + econazole
analyzed (days) (μg/mL) (μg/mL) Groups Log10 CFU
Lungs Lung Spleen
Econazole 7 0.31 ± .006 0.31 ± 0.01 Untreated controls 6.88 ± 0.035 6.90 ± 0.025
Rifampicin 15 0.69 ± 0.07 0.66 ± 0.04 Encapsulated econazole weekly (six doses) 4.85 ± 0.05 4.90 ± 0.04
Isoniazid 15 0.24 ± 0.03 0.25 ± 0.01 Free econazole twice daily (90 doses) 4.87 ± 0.04 4.89 ± 0.02
Pyrazinamide 15 12.61 ± 2.14 10.66 ± 0.5 Values are mean ± SD of six animals.
Ethambutol 15 1.48 ± 0.15 1.51 ± 11 P b .01 as compared with untreated controls.

Liver
Econazole 7 0.61 ± 0.01 0.29 ± 0.07
RIF, 88% to 95% for EMB, and 70% to 90% for INH and
Rifampicin 15 0.77 ± 0.04 0.77 ± 0.015
PZA. The drug loading capacity of alginate nanoparticles
Isoniazid 15 0.277 ± 0.006 0.27 ± 0.01
ranged from 525 to 730 mg per 100 mg of alginate. The
Pyrazinamide 15 11.43 ± 0.57 10.85 ± 0.41 coefficient of variation was found to be less than 4% with
Ethambutol 15 1.57 ± 0.04 1.64 ± 0.16 batches prepared on the same day and less than 7% in
Spleen batches prepared on different days. Furthermore, the
Econazole 7 0.29 ± 0.070 0.21 ± 0.0 variation remained within the narrow limits irrespective of
the batch size.
Rifampicin 15 0.72 ± 0.025 0.74 ± 0.05
Isoniazid 15 0.26 ± 0.015 0.26 ± 0.01 In vivo drug disposition studies
Pyrazinamide 15 11.13 ± 0.380 11.53 ± 0.09
Econazole was cleared from plasma within 4 hours after
Ethambutol 15 1.52 ± 0.100 1.62 ± 0.04 administration as free drug. In contrast, all the ATDs with or
Values are mean ± SD of six animals. without econazole were cleared from the circulation within
24 hours. In comparison to free drugs, alginate-encapsulated
drugs were detected in plasma from 3 hours onward;
econazole, EMB, RIF, and INH/PZA were observed for as
received PBS only, whereas other groups were administered
long as 5, 7, 9, and 11 days, respectively, both alone or in
the drugs mentioned through the oral route at therapeutic
combination. The bioavailabilities of drugs encapsulated in
doses (INH 10 mg/kg, RIF 12 mg/kg, PZA 25 mg/kg, EMB
alginate nanoparticles were increased significantly (P b .001)
16 mg/kg, and econazole 3.3 mg/kg body weight). Free
in comparison with free counterparts.
drugs (INH, RIF, PZA, and EMB) were administered once
All the ATDs (RIF, INH, EMB, and PZA) were detected
daily, however, econazole and EMB (in the presence of
in tissues (i.e., lungs, liver, and spleen) up to day 1 following
econazole) were administered twice daily. All encapsulated
the administration of the free-ATD combination. However,
ATDs were administered every two weeks, and encapsulated
free EMB was detected only up to 12 hours in all tissues
econazole was administered weekly. Animals were killed on
when administered together with econazole, whereas tissue
day 31, 46, and 58 of chemotherapy; lungs and spleen were
distribution of the other three ATDs remained unchanged in
isolated under sterile conditions and homogenized in 3 mL of
the presence of econazole. Free econazole was detected up to
isotonic saline. Homogenates (100 μL of undiluted, 1:10,
12 hours in tissues after administration alone as well as in
and 1:1000 dilutions) were plated on Middlebrook 7H11
combination with ATDs. All the ATDs were detected in
agar plates supplemented with oleic acid/albumin/dextrose
tissues up to 15 days at above the minimum inhibitory
catalase (OADC) for enumeration of colony-forming units
concentration [11] following the administration of encapsu-
(CFU), and colonies were counted on day 28 after
lated drugs alone or in combination with econazole (Table 1).
inoculation. The CFU data were analyzed by one-way
Econazole was detected as late as day 7 in tissues at above
analysis of variance followed by Student's unpaired t-test.
the minimum inhibitory concentration following the admin-
istration of alginate nanoparticles in the presence or absence
Results of encapsulated ATDs (Table 1).
Physicochemical characterization of alginate nanoparticles Chemotherapeutic efficacy
The alginate nanoparticles had an average size of 229 nm Based on tissue drug distribution, free drugs (INH, RIF,
with a polydispersity index of 0.44. The drug encapsulation PZA, and EMB) were administered once daily; however,
efficiency of alginate nanoparticles for econazole was found econazole and EMB (in the presence of econazole) were
to be 92% to 97.5%, and that of ATDs were 80% to 90% for administered twice daily. All encapsulated ATDs were
242 Z. Ahmad et al. / Nanomedicine: Nanotechnology, Biology, and Medicine 3 (2007) 239–243

Table 3 tuberculosis caused by susceptible, resistant, and latent

Chemotherapeutic efficacy of azoles with or without antitubercular drugs in bacilli. However, the bioavailability of azole antifungal
free or alginate nanoparticle-encapsulated form against tuberculosis in mice
infected with low dose (105 bacilli) of M. tuberculosis H37Rv
drugs through the oral route is a staggering problem. Drug
delivery technology has shown its potential to enhance the
Groups Log10 CFU*
bioavailability of otherwise poor orally bioavailable drugs,
4 weeks 6 weeks and azole drugs are noexception to this fact. The present
Lung Spleen Lung Spleen study aimed to explore the chemotherapeutic potential of
INH, PZA, EMB, b1.0 b1.0 b1.0♦ b1.0♦ nanoencapsulated azoles against murine tuberculosis.
and RIF The size of alginate nanoparticles observed in this present
Eco, INH, EMB, and b1.0 b1.0 b1.0♦ b1.0♦ study is favorable, because particles bearing size less than
PZA 500 nm are known to be suitable for oral drug delivery. The
Eco, RIF, EMB, and b1.0 b1.0 b1.0♦ b1.0♦ drug encapsulation efficiency of alginate nanoparticles was
PZA better than that previously obtained for alginate micro-
Eco, EMB, and PZA 2.3 ± 0.03 2.32 ± 0.05 b1.0♦ b1.0♦ spheres or poly(lactide-co-glycolide) micro/nanoparticles. A
Encap. INH, PZA, b1.0♦ b1.0♦ b1.0♦ b1.0♦
single oral administration of drug-loaded alginate nanopar-
EMB, and RIF ticles to mice maintained therapeutic drug levels in plasma
Encap. Eco, INH, b1.0♦ b1.0♦ b1.0♦ b1.0♦
from 5 to 11 days as compared with less than 1 day the in
EMB, and PZA case of free drugs. Econazole and ATDs were detected in the
Encap. Eco, RIF, b1.0♦ b1.0♦ b1.0♦ b1.0♦
tissues until day 7 and 15, respectively, in the case of alginate
EMB, and PZA nanoparticles (Table 1), whereas free drugs were detectable
Encap. Eco, EMB, 2.3 ± 0.06 2.33 ± 0.03 b1.0♦ b1.0♦
only up to 12 or 24 hours. This formed the basis of the
and PZA chemotherapeutic schedule wherein the alginate formulation
Untreated controls 4.02 ± 0.03 4.1 ± 0.04 4.71 ± 0.04 4.73 ± 0.03
was administered to M. tuberculosis-infected mice on every
eighth/fifteenth day as compared with free drugs adminis-
Eco = econazole; Encap. = nanoparticle-encapsulated drugs.
tered once or twice daily.
*b1.0, ♦ indicates no detectable bacilli. Values are mean ± SD of six animals.
Eight doses of encapsulated econazole (administered
weekly) or 112 doses of free econazole (administered twice
administered every two weeks, and encapsulated econazole daily) proved to bear equal therapeutic potential (Table 2).
was administered weekly. Eight weeks of chemotherapy These data clearly demonstrate the sustained-release
with econazole alone either in free form (112 doses, potential of alginate nanoparticles (which can reduce dosing
administered twice daily) or in encapsulated form (8 doses, frequency of azole drugs 15-fold without compromising
administered weekly) resulted in approximately 90% therapeutic efficacy) as well as the antimycobacterial
clearance of bacilli from lungs and spleen of animals potential of azole drugs. The equipotency of four different
infected with 1 × 107 cells of M. tuberculosis as compared drug combinations (INH, PZA, EMB, and RIF/econazole;
with untreated controls (Table 2). Further chemotherapeutic INH, EMB, and PZA/econazole; RIF, EMB, and PZA) in
potential of econazole was comparable to that of RIF, free or in alginate nanoparticle-encapsulated form reflects
because both these drugs (free or encapsulated) decreased the potential of alginate nanoparticles to be suitable carriers
bacterial burden from 6.88 log CFU and 6.9 log CFU to for azoles as well as ATDs. This also highlights the
approximately 4.87 log CFU and 4.89 log CFU in the potential of econazole to be a good substitute for RIF/INH
lungs and spleen, respectively. or both of these drugs in free or encapsulated form during
The administration of four drug combinations (INH + murine tuberculosis chemotherapy. The role of econazole
PZA + EMB + RIF or econazole + INH + EMB + PZA, or in reducing bacterial burden from lungs and spleens of
econazole + RIF + EMB + PZA) either free or encapsulated infected mice in the presence of other ATDs (INH + PZA +
(administered as per tissue distribution described above) for EMB; RIF + PZA + EMB; PZA + EMB) is supported by
30 days to M. tuberculosis-infected mice resulted in the observation that all of these combinations without
undetectable CFU of tubercle bacilli in undiluted homo- econazole cleared half the bacterial burden in comparison-
genates of lungs and spleen as compared with ~4 log CFU in with untreated controls (Table 3). Furthermore, that
untreated controls (Table 3). However, it took 45 days for the econazole played a significant role in reducing bacterial
three-drug combination (econazole, EMB, and PZA) in free burden from lungs and spleens of infected mice in the
or encapsulated form (dosing strategy same as above) to presence of other ATDs is also supported by the previous
yield the same results (i.e., undetectable CFU). studies wherein the more potent ATD combination (INH,
PZA, and RIF) failed to yield undetectable levels of
colony-forming units in 4 weeks [12]. The observed potent
Discussion
antimycobacterial potential of econazole can be explained
Azole drugs have proven their antimycobacterial potential on the basis of multiple targets of econazole in M.
under in vitro, ex vivo and in vivo conditions against murine tuberculosis [13]. Furthermore, encapsulated as well as
 Z. Ahmad et al. / Nanomedicine: Nanotechnology, Biology, and Medicine 3 (2007) 239–243
free econazole alone or in combination with antitubercular
drugs did not produce hepatotoxicity in normal or M.
tuberculosis-infected mice based on biochemical para-
meters, indicating their safe use.
So far, attempts to enhance the oral bioavailability of
azoles have focused on the use of cyclodextrins, which had
limited success [5]. Additionally, alginate poly-1-lysine
microparticles have been used to encapsulate azole
antifungal ketoconazole with encapsulation efficiency of
71.5% and the particle size being 80 to 130 μm [14]. Our
results clearly demonstrate the superiority of alginate
nanoparticles over cyclodextrins as well as alginate poly-
1-lysine microparticles, as azole carriers with respect to
encapsulation, formulation size, polymer consumption, and
use of chitosan in place of poly-1-lysine. The present study
also emphasizes that nanotechnology is a powerful tool that
can be used not only to improve the oral bioavailability of
azole drugs but to retain their full chemotherapeutic
potential. Not only ATDs and azoles have been success-
fully encapsulated in alginate, but a large number of drugs
such as indomethacin [15], sodium diclofenac [16],
nicardipine [17], dicoumarol [18], gentamicin [19], vitamin
C [14], and amoxicillin [20] have been successfully
incorporated in alginate. This study reports for the first
time the in vivo potential of alginate nanoparticle-
encapsulated econazole alone or in combination with
ATDs against experimental tuberculosis.
Acknowledgments
Z. A. P. thanks the Council of Scientific and Industrial
Research, New Delhi, India, for the award of a Senior
Research Fellowship.
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