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Journal of Pharmacy
Research Paper
And Pharmacology

Isoniazid-gelatin conjugate microparticles containing

rifampicin for the treatment of tuberculosis
Maria L. Mancaa, Roberta Cassanob, Donatella Valentia, Sonia Trombinob, Teresa Ferrarellib,
Nevio Piccib, Anna M. Faddaa and Maria Manconia
a
Department Scienze della Vita e dell’Ambiente, Sezione Scienze del Farmaco, Cagliari and bDepartment Scienze Farmaceutiche, Via P. Bucci,
Arcavacata di Rende (CS), Italy

Keywords
A549 cells; antimicobacterial drugs;
microparticles; pulmonary drug delivery;
toxicity studies
Correspondence
Maria Manconi, Department Scienze della Vita
e dell’Ambiente, Sezione Scienze del Farmaco,
Via Ospedale 72, 09124 Cagliari, Italy.
E-mail: manconi@unica.it
Received January 30, 2013
Accepted May 24, 2013
doi: 10.1111/jphp.12094
Abstract
Objectives In this work, a new polymeric microparticle system based on gelatin
covalently bound to isoniazid (ISN) and containing rifampicin (RFP) was pre-
pared by spray-drying technique. Microparticle aptitude to nebulisation and their
capability of interacting with A549, alveolar basal epithelial cells, were evaluated
in vitro.
Methods Microparticles were obtained by spray drying, and their morphology,
size, zeta potential, thermotropic behaviour and nebulisation ability were evaluated.
Key findings Microparticles were positively charged with a mean size of 4.88 ⫾
0.3 mm. Microspheres were able to incorporate both RFP and ISN: encapsulation
efficiency was 51 ⫾ 6% and 22 ⫾ 1%, respectively. X-ray diffraction study showed
a new extensive and flattened diffraction peak providing evidence that the drugs
were dispersed into the microparticles. Differential scanning calorimetry analysis
confirmed effective interactions between gelatin and drug molecules by the pres-
ence of new transition peaks. Fifty-nine per cent of used microparticles were aero-
solised. In-vitro toxicity studies on A549 alveolar basal epithelial cells showed that
microparticles decreased cytotoxicity in comparison with the RFP solution. Laser
scanning confocal microscopy observation confirmed that fluorescent probes
delivered by microparticles are efficiently internalised in A549 cells.
Conclusions Overall, microparticles based on gelatin covalently bound to ISN
and containing RFP showed a promising behaviour for pulmonary drug delivery.

Introduction
Tuberculosis is the leading cause of infectious disease in the
world today. An important consideration in the treatment
of tuberculosis is the fact that the aetiological agents, Myco-
bacterium tuberculosis (MBT) and mycobacterium avium
complex have the ability to persist intracellularly in the host
macrophages for long periods of time. In fact, they are fac-
ultative intracellular parasites that can multiply primarily in
macrophages, leading to a reduction in the efficacy of anti-
microbial agents that have a poor ability to penetrate into
the phagocytic cells.[1–4] The therapeutic treatment of tuber-
culosis involves the oral administration of rifampicin
(RFP), isoniazid (ISN), ethambutol and pyrazinamide taken
for a period of 6–12 months. These drugs are relatively toxic
and cause several adverse effects. Moreover, drug resistance
can occur when treatment is not followed to completion.
Effective and localised drug administration can lead to a
reduced treatment period and/or dosage, as well as to
increased bioavailability. Thus, an ideal method for treating
tuberculosis would be one that is not only able to safely
deliver drugs systemically for the long term, but would also
be able to deliver the drugs to the intracellular lung epithe-
lial cells and macrophages in which the tubercle bacilli are
found. Microsphere technology is an established technique
that has been used to deliver several different types of drugs
to lungs, including antimicrobial agents like RFP and
ISN.[5–13] Although experience with synthetic polymers is
extensive and encouraging, the recent trend has been to
shift towards natural polymers. The major advantage of
natural polymers (e.g. gelatin) includes their low cost and
compatibility with of a wide range of drugs, and the
minimal use of organic solvents. Gelatin is one of the most
commonly used natural materials, widely employed because

1302 © 2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 65, pp. 1302–1311
Maria L. Manca et al.
of its biocompatibility, biodegradation, non-toxicity, non-
immunogenicity, plasticity and adhesiveness.[14–16] Moreo-
ver, gelatin microspheres have already been used for the
effective delivery of salmon calcitonin to the pulmonary
system.[17–19] Among the various potential uses of gelatin as
drug delivery system, microsphere fabrication and formula-
tions showed several advantages for pulmonary delivery,
such as a large distribution to lung epithelial cells and a
greater availability of the agents to infected cells. Pulmonary
drug administration represents an alternative to oral and
parenteral delivery method as a non-invasive administra-
tion route because this could provide the rapid onset of
action, high bioavailability and large absorptive surface
area. In particular, in this work, we designed a new poly-
meric microparticle system containing RFP and ISN cova-
lently bound to gelatin, which was intended for pulmonary
administration. Microparticles were characterised in term
of size distribution, zeta potential and encapsulation effi-
ciency (E%). X-ray diffraction and differential scanning
calorimetry (DSC) analyses were performed to investigate
interactions between gelatin and drug molecules. Moreover,
in order to investigate the biocompatibility of microparti-
cles, cell viability using adenocarcinomic alveolar basal epi-
thelial cells (A549) incubated with fluorescent formulations
was evaluated for 48 h by the MTT assay, while cell–
microparticle interactions were evaluated by laser scanning
confocal microscopy (CLSM).
Materials and Methods
Materials
Gelatin type A and type B, RFP, ISN, dextrose, cholesterol
and all other products were of analytical grade and were
purchased from Sigma Aldrich (Milan, Italy). Phosphate
buffer solution (PBS) pH 7 was obtained from Carlo Erba
Reagents (Milan, Italy).
Synthesis of isoniazid-gelatin conjugate
The reaction was carried out according to the procedure
reported in literature.[10,14] Briefly, thionyl chloride and pyri-
O H
COOH + 2HN G N H
N O
Gelatin, Sucrose, RFP
Water 40°C
Gelatin-Isoniazid Conjugate
isoniazid and rifampicin
Figure 1 Schematic diagram of the preparation method of gelatin microparticles.
© 2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 65, pp. 1302–1311
Isoniazid-gelatin rifampicin microparticles
dine were added to carboxypolystyrene resin (0.4 g) sus-
pended in N-methyl pyrrolidone. The reaction was
maintained at 25°C under stirring and reflux at 100°C;
gelatin was added after 3 h and then after 4 h; ISN and pyri-
dine were also added. The resin was recovered by using trif-
luoroacetic acid (5%) in dichloromethane, at 60°C and
diethyl ether to allow the precipitation of the gelatin deriva-
tive. Finally, the precipitate was filtered, washed with
ethanol, dried and dissolved in distilled water, frozen and
lyophilised to remove the aqueous solvent.
Preparation of isoniazid-gelatin conjugate
microparticles containing rifampicin
ISN-gelatin conjugate was dissolved under stirring in water
at 40°C and gelatin, RFP and sucrose were slowly added to
the dispersion under stirring and left for 2 h. Then, the
aqueous mixture was spray-dried (SD) using a Minispray
Dryer (Büchi 190, Flawil, Switzerland) with a standard
0.7-mm nozzle to produce microparticles. The inlet tem-
perature, spray flow and compressed spray airflow (repre-
sented as the volume of the air input) were set at 150°C, 3
and 10 ml/min, respectively. SD formulations were stored at
5 ⫾ 1°C in a dry place (Figure 1).
Characterisation of microparticels
Morphology of dried microspheres was evaluated using a
Hitachi S-4800 (Monocomp, Madrid, Spain) scanning elec-
tron microscope (SEM) at 2 kV. Microparticle size was
measured using a Malvern Mastersizer 2000 version 5.1
(Malvern Instrument, Worcestershire, UK) by the laser tech-
nique. Before the measurements, samples were diluted in
Millipore water to avoid multiple light scattering. The size
distribution of each sample was measured at least three
times. Average particle size was expressed in micrometre as
a function of volume mean diameter (d4,3), that is defined
as the diameter of a sphere that has the same volume as
the tested particle.[20] The span was also calculated, and
it is defined as (d(v,90) – d(v,10))/d(v,50), where d(v,90),
Spray-Drying
Dispersion of gelatin- Spray-dried microparticles
1303
Isoniazid-gelatin rifampicin microparticles
d(v,10) and d(v,50) are the diameters at 90%, 10% and 50%
cumulative volume, respectively. Thus, the span gives a
measure of the range of the volume distribution relative to
the median diameter. Zeta potential measured using a Zeta-
sizer Nano ZS (Malvern Instrument, Worcestershire, UK)
was calculated using M3PALS, a second generation Phase
Analysis Light Scattering system, which measures the parti-
cle electrophoretic mobility in a thermostated cell. Each dis-
persion was purified from non-encapsulated drug by
exhaustive dialysis using Spectra-Por membranes (12–
14 000 MW cut-off, 3 nm pore size, Spectrum Laboratories,
Inc., Rancho Dominguez, CA, USA). Dispersions were dia-
lysed at 5°C for 2 h (by replacing water every 30 min),
which were appropriate to allow the dissolution and conse-
quent removal of the non-entrapped RFP (solubility pH
7.3: 2.5 mg/ml) and the non-conjugated ISN. The amount
of drug entrapped (encapsulation efficiency, E%) in micro-
particles was calculated by extracting and quantifying the
amount of RFP and ISN in a known amount of particles
both before and after separation of non-entrapped RFP and
ISN. Briefly, drug-loaded microparticles (200 mg) were dis-
persed in acetonitrile (2 ml); the resulting mixture was
shaken continuously for 24 h. Subsequently, the extracted
drug solution were diluted and analysed spectrophoto-
metrically at 485 nm and 263 nm for RFP and ISN, respec-
tively. A standard calibration curve was built up by using
working, standard solutions (0.01–1 mg/ml). Calibration
curve was plotted according to the linear regression analy-
sis, which gave a correlation coefficient value (R) of 0.999.
Finally, the drug E% was calculated as the percentage of
drug entrapped in microspheres compared with the initial
amount of drug recovered in unpurified samples.
Evaluation of rifampicin and
isoniazid-gelatin conjugate
microparticle interactions
X-ray diffractograms were recorded with Bragg-Brentano
geometry on a Bruker AXS D5005 (Bruker AXS GmbH,
Karlsruhe, Germany) in the 2q range from 5° to 80°, in steps
of 0.02° at 6 s per step. DSC studies were performed using a
DSC model 821e (Mettler Toledo International Inc., Barce-
lona, Spain). The samples (2–5 mg) were scanned in sealed
aluminium pans under nitrogen atmosphere. Thermograms
were scanned in the first heating run at a constant rate of
10°C/min and a temperature range of 0–500°C. DSC ther-
mograms of liposomes, polymers and complexes were
recorded.
Nebulisation studies
Microparticle aerosol was generated by using an efficient
high-output continuous-flow Markos Mefar MB2 air-jet
nebuliser, driven by a Nebula compressor (Markos Mefar,
1304 © 2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 65, pp. 1302–1311
Maria L. Manca et al.
Bovezzo, Brescia, Italy) operating at 7 l/min. A volume of
3 ml of rehydrated samples was used, and the aerosolised
microparticles were collected in PBS using a modified three
stages glass impinger containing 3 ml of PBS in the collect-
ing flask. The aerosol was introduced into the device
through a calibrated glass tube and a critical orifice deliver-
ing the aerosol jet 5 mm above the flask bottom. After aero-
solisation to dryness (10 min), the impinger contents were
collected, the impinger was washed with 2 ml of buffer and
samples were assayed to evaluate the effect of nebulisation
on microparticles and drug content. Total aerosol mass
output (%) was determined by weighing the nebuliser
before and after the nebulisation of the different formula-
tions. The total amount of nebulised formulation (collected
into the apparatus) was determined. The nebulisation effi-
ciency (NE%) of formulation is defined as the total output
of drug collected on the impinger as a percentage of the
total amount of drug submitted to nebulisation. The
amount of drugs still incorporated into the microparticles
after nebulisation was assessed removing the free drug from
the nebulised samples by exhaustive dialysis using Spectra-
Por membranes (as earlier). Dispersions were dialysed at
5°C for 2 h (by replacing water every 30 min), to allow free
drugs removal. Finally, retention of the drugs in the neb-
ulised formulations (NER%) was calculated by the follow-
ing formula:
aerosolised and purified microparticles
(collected-in-flask)
NER % = ×100
total aerosolised microparticles
(collected-in-flask))
Cell culture
A549 cells, adenocarcinomic human alveolar basal epithelial
cells, were used for the experiments at passage 90–100. Cells
were grown as monolayers in 75 cm2 flasks, incubated at
37°C with 100% humidity and 5% CO2. The Dulbecco’s
Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/
F12) was used as culture medium, enriched with 10% fetal
calf serum, 10% penicillin/streptomycin and 10% fungi-
zone. The culture medium was changed every 2 days to
ensure the growth of cells and to avoid the occurrence of
contamination. The cells once they reach the subconfluence
were detached from the bottom of the flask with trypsin
(0.25%), centrifuged (1600 g, 4 min) and subsequently
resuspended in fresh culture medium at a concentration
equal to 2 ¥ 105 cells/plate.
Cell viability studies (MTT assay)
The toxic effect of microparticles has been studied with
the cell viability test (MTT). The test is based on the ability
Maria L. Manca et al.
of the compound MTT (tetrazolium salt, 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to
be metabolised by a mitochondrial enzyme, succinate dehy-
drogenase. The reduction of MTT leads to the formation of
crystals of a product blue/violet formazan, insoluble in
water. The viable cells reduce the MTT, and the amount of
formazan produced is proportional to the number of
present cells. The formed crystals were solubilised in dime-
thyl sulfoxide, and absorbance values were measured at
590 nm using a microplate reader (Synergy 4, Synergy
Multi-Detection Microplate Reader, BioTek Instruments,
AHSI SPA, Bernareggio, Italy). The A549 cells (7500 cells/
well) were seeded in 96-well plates with 250 ml of culture
medium. Sixteen hours after seeding, the cells were treated
for 2, 4, 6, 8, 24 and 48 h with the empty and drug-loaded
formulation (100 mg/ml of RFP and 10 mg/ml of ISN,
respectively). Each sample was tested in triplicate for at least
four times. The untreated cells were used as negative
control. After incubation cytotoxicity assay (MTT) was per-
formed in order to assess the percentage of cell survival.
Cellular uptake of Rho-CF-loaded
microparticles
The internalisation of vesicular systems by A549 cells has
been studied by confocal microscopy. For this purpose,
microspheres were prepared by using a lipophilic fluores-
cent marker (rhodamine B) (0.035 mg/ml Rho) and a
hydrophilic fluorescent marker (6)-carboxyfluorescein
(0.025 mg/ml, CF). A549 cells were maintained in culture
on glass slides of 30 mm in diameter, and the experiments
were performed to the achievement of the subconfluence.
The cells were incubated at 37°C with the different formula-
tions for 2, 4, 8 and 24 h, at the same concentration used for
toxicity studies. At the end of the experiment, the cells were
washed twice with DMEM/F12, fixed with a solution of 4%
paraformaldehyde in PBS (pH 7.4) and treated with Triton
X-100 (0.1%) to increase the permeability of the cell mem-
brane. The fixed cells were washed with PBS and then
stained with Hoechst 33258 blue (Sigma, Milan, Italy) to
display the nucleus. After staining, the cells were again
washed with PBS and allowed to dry for one night. The
filters introduced have allowed almost complete separation
of the emission and the simultaneous observation of the
three fluorescent markers: CF (green), Rho (red) and
Hoechst (blue). The images were obtained using a confocal
Table 1 Mean diameter, span, polydispersity index (PI), zeta potential (ZP), entrapment efficiency (E%) and yield% of ISN-prodrug and microparti-
cles containing RFP and ISN-prodrug (n = 6)
Size ⫾ SD (mm) Span
ISN-gelatin 0.72 ⫾ 0.12
RFP microparticles 4.89 ⫾ 0.25 1.2
© 2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 65, pp. 1302–1311
Isoniazid-gelatin rifampicin microparticles
microscope inverted FluoView FV1000 (Olympus, Barce-
lona, Spain) equipped with a laser to ultraviolet light/
visible; a 60 ¥ objective UPlanSApo was used. The Rho, CF
and the Hoechst were visualised respectively with a wave-
length of excitation and emission of 559 nm and 578 nm,
470 nm and 535 nm, 360 nm and 460 nm.
Statistical analysis of data
Results are expressed as the mean ⫾ standard deviation
(SD). Analysis of variance and Bartlet’s test for homogene-
ity of variance were performed using SPSS version 17.0 for
Windows (SPSS Inc., Chicago, IL, USA). Post-hoc testing (P
< 0.05) of multiple comparisons was performed using the
Scheffe test. Differences were considered significant at the
0.05 level of probability (P).
Results and Discussion
In a previous work, gelatin was suitably derivatised with
ISN to obtain a new ISN-prodrug. ISN-derivatised gelatin
was prepared as previously reported by a heterogeneous
reaction of amidation that allowed the formation of a new
amide bond between the terminal acyl chloride group of
gelatin and ISN hydrazinium group. The effective formation
of ISN-gelatin conjugate was investigated by 1H nuclear
magnetic resonance spectroscopy. Results showed that the
spectrum of gelatin was characterised by the presence of
various peaks characteristic of amino acids forming the
peptide, while the spectrum of ISN-gelatin conjugate dis-
played the presence of ISN benzene rings confirming the
covalent bond between gelatin and ISN.[10] The degree of
substitution was calculated comparing the area of each peak
regarding the same functional groups before and after the
derivatisation. This comparison revealed that the percent-
age of new functional groups (and consequently, the pres-
ence in the spectrum of new signals) introduced in the
derivative was equal to the 75%. ISN-gelatin purity was
90% with a yield of 53%.[10]
Prodrug characterisation is reported in Table 1. ISN-
gelatin conjugates dispersed in water showed a mean size of
around 700 nm and a very high polydispersity index (0.9)
as they are macromolecules and their behaviour in disper-
sion is different from microparticles. Dynamic laser light
scattering measures the hydrodynamic radius, that is, the
radius of a hypothetical hard sphere that diffuses with the
PI ZP ⫾ SD (mV) RFP E% ISN E% Yield%
0.9 11 ⫾ 4 58 ⫾ 9
13 ⫾ 2 51 ⫾ 6 22 ⫾ 1 48 ⫾ 8
1305
Isoniazid-gelatin rifampicin microparticles
same speed as the particle under examination, including
both solvent (hydro) and shape (dynamic) effects. The
hydrodynamic radius is then calculated from the diffusion
coefficient using the Stokes–Einstein equation. However, the
size of macromolecules in solution depends on their spatial
structure, which is commonly described as linear or ellip-
soidal, with two or three different axial radius. This leads to
the obtainment of different scattering values that can
explain the high polydispersity index in the hydrodynamic
radius measurements.[21]
Gelatin is a zwitterionic polymer because of its peptide
molecule; in this work, the prodrug was prepared using a
mixture of gelatin A and B with an isoelectric point of 6,
which, in water (pH 5.5), is positively charged for the
partial dissociation of its amino groups. According to this,
zeta potential of the prodrug was positive (11 mV). The
obtained prodrug was entrapped together with RFP in
gelatin microparticles. Microparticles were prepared by a
spray-drying method adding gelatin, sucrose and RFP to
the ISN-gelatin conjugate. Sucrose was used to improve the
SD yield and avoid microparticle sticking because it facili-
tates the formation of the backbone structure of the solid
particles during the spray-drying process. The mean diam-
eter of microparticles was 4.89 mm with span values > 1,
indicating a poorly homogeneous system (Table 1). In fact,
the size distribution of microparticles revealed the presence
of different population of particles. These results are prob-
ably the consequence of an aggregation process that
occurred during the spray-drying process; in addition, the
effect of the solution viscosity during the atomisation step
may influence particle size and size distribution. The parti-
cle size is a critical factor affecting the site of their deposi-
tion because it determines operating mechanisms and the
extent of penetration into the lungs. The upper airways
(nose, mouth, larynx and pharynx) and the branching
anatomy of the tracheobronchial tree acts as a series of
filters for inhaled particles. Thus, aerosol particles, larger
than 10 mm, will not penetrate the tracheobronchial tree.
Particles must generally be 5 mm maximum, as are our
microparticles, in order to reach the alveolar space.[22] On
the other hand, particles smaller than 0.5 mm in diameter
penetrate the lung deeply, but have a high tendency to be
exhaled without deposition.
Microparticles, as well as prodrugs, were positively
charged (zeta potential 13 ⫾ 2) because of the dissociation
of the gelatin amino groups at pH 5.5 that occurs below the
isoelectric point.[23] In fact, microparticles were prepared
with the same mixture of gelatin A and B used to synthesise
the prodrug. The zeta potential of microparticles is com-
monly used to evaluate their stability because surface charge
prevents their aggregation, and a zeta potential far from
zero has been shown to increase the suspension stability. In
addition, the zeta potential could influence the particles
1306 © 2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 65, pp. 1302–1311
Maria L. Manca et al.
ability to interact with biological membranes (mucoadhe-
sion). The positively charged gelatin would be expected to
strongly interact with a negatively charged surface, such as
the mucus of the lungs, increasing the interaction between
microparticles and pulmonary epithelium, favouring the
drug internalisation into lung cells and consequently
improving drug bioavailability. During tuberculosis, mucus
is produced by the lungs as a defence response to the infec-
tion, carrying out the function of air filter, similarly to
intestinal mucosa. To overcome this additional barrier,
mucoadhesive properties of microparticles may increase the
interaction with the mucin layer of the respiratory epithe-
lium through hydrogen bonding, electrostatic, hydrophobic
or van der Waals interactions. The positively charged gelatin
should strongly interact with a negatively charged surface,
such as the mucus of the lungs, increasing the interaction
between the microparticles and pulmonary epithelium,
favouring the drug internalisation into lung cells and conse-
quently improving drug bioavailability.
Microspheres showed an encapsulation efficiency (E%)
value higher for RFP (51%) than for ISN (22%), probably
due to the large molecule of ISN-derivatised gelatin leading
a reduced incorporation of the same into the microparti-
cles. The yield of the spray-drying process was about 53%
because of the high hygroscopic nature of the components
that adhere to the walls of the instrument and are responsi-
ble for the loss of the product during the process of powder
atomisation. SEM images of the SD microparticles are
shown in Figure 2. SEM images show that microparticles
have an irregular shape and a rough surface, which,
however, might improve the contact between a bioadhesive
polymer, such as gelatin, and the biological mucus.[24]
X-ray spectrum of ISN and RFP presented characteristic
intense peaks at 10–40° and 5–25° q respectively because of
their crystalline nature (Figure 3). The non-crystalline
structure of gelatin is characterised by a diffraction pattern
with a flattened and diffuse peak. In the spectrum of micro-
particles, all of the above diffraction peaks (5–40) overlap
with a new extensive and flattened diffraction peak with a
relative sharp peak at 5–10°2q, providing evidence that the
drug-loaded microparticles are in an amorphous state and
are dispersed into microparticles.
ISN showed a characteristic endothermic peak at 170°C
while RFP showed an endothermic peak at 180°C and an
exothermic peak at 270°C (Figure 4). Gelatin presented a
broad peak at 100°C corresponding to the loss of water and
others at 200–280°C assigned to glass transition of the
polymer. In our previous study, ISN-gelatin derivatisation
was confirmed by DSC spectrum of the gelatin derivative
revealing the absence of the pure ISN transition peak
(170°C) and the appearance of a high endothermic peak
centred at 85°C, narrower and higher than that of gelatin
alone (60–180°C).[25] Thermal curves of sucrose showed a
Maria L. Manca et al. Isoniazid-gelatin rifampicin microparticles

50.0 μM 3.0 μM

Figure 2 Scanning electron microscope (SEM) images of isoniazid (ISN)-gelatin conjugate microparticles containing rifampicin (RFP).

10 000 10 000 10 000

Intensity (au)

5000 5000 5000

Isoniazid Sucrose
Rifampicin
0 0 0
0 20 40 60 80 0 20 40 60 80 0 20 40 60 80
Degrees 2 theta Degrees 2 theta Degrees 2 theta

1000 1000
Intensity (au)

500 500

Gelatin Microparticles

0 0
0 20 40 60 80 0 20 40 60 80
Degrees 2 theta Degrees 2 theta

Figure 3 X-ray powder diffraction patterns of isoniazid (ISN), rifampicin (RFP), sucrose, gelatin and gelatin microparticles containing RFP and
ISN-prodrug.

peak at the temperature corresponding to its melting point
(T = 180°C). The calorimetric analysis of microparticles
revealed the presence of an endothermic peak of ISN-
gelatin conjugated at 85°C and the appearance of a new
peak around 200°C because of the interactions between
polymer, sucrose and drugs that allowed a new transition
peak. Moreover, the disappearance of endothermic peaks of
ISN and RFP in the microparticle spectrum confirmed the
effective ISN-gelatin derivatisation and RFP incorporation
into gelatin formulation and the formation of a new system,
thus minimising the individual peaks of drugs.
Aerosol is an effective method to deliver therapeutic
agents to the respiratory tract. Nebulisers are commonly
used for this purpose, especially in patients with specific
pulmonary diseases like cystic fibrosis, chronic pulmonary
infections or lung cancer.[26] To this purpose, and in order to
evaluate microparticles stability during nebulisation, differ-
ent analyses were carried out. These studies are of particular
importance in order to determine the capability of micro-
particles to be aerosolised and also to retain the entrapped
drug during the process. Rehydrated microparticles were
nebulised for 10 min; after this time, some residual fluid
remains in the nebuliser reservoir. For this reason, the total
mass output calculated during this study, which represents
the percentage of the average delivered dose, was 59%. The
total mass output value is a function of the humidity of
the gas source, the driving pressure and the flow rate, but
also the viscosity of the sample has an important effect on
this parameter.[27] Aerosolised particles were collected in the
three stages of the glass impinger, and successively, the
amount of drug nebulised compared with that inserted in
the nebuliser (NE%) was measured. As can be seen from

© 2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 65, pp. 1302–1311 1307
Isoniazid-gelatin rifampicin microparticles
Sucrose
Gelatin
Microparticles
0 100 200 300 400
Temperature (°C)
Figure 4 Differential scanning calorimetry of sucrose, rifampicin
(RFP), isoniazid (ISN), gelatin and gelatin microparticles containing RFP
and ISN-prodrug.
Table 2, the greater amount of RFP was found in the first
stage of the impinger (46%) while only 9% and 4% were
recovered in the second and in the third stage, respectively.
Similar results were shown, previously, in a study concern-
ing aerosolisation of liposomes coated with chitosan-
xanthan gum.[28] This behaviour is probably related to the
droplet formation in an air-jet nebuliser; compressed air is
forced through an orifice over the open end of a capillary
tube creating a region of low pressure. The liquid formula-
tion is drawn through the tube to mix with the air jet and
forms droplets, whose size is dependent on the pressure of
the compressed air, the architecture of the nebuliser and the
viscosity of dispersion. Indeed, during atomisation with air-
jet nebuliser, the amount of water progressively reduces by
evaporation leading to more viscous dispersions. Conse-
quently, as viscosity increases, the production of large drop-
lets also increases, causing a reduction of particles that are
able to reach the second and the third stage of the impinger.
Moreover, microparticles may have been broken up during
passage through the nebuliser, resulting in a net loss of
1308
Maria L. Manca et al.
entrapped material. This process could also explain why
such a low amount of drug was found in the two last stages
of the impinger device. In order to better understand the
deposition pattern of RFP-ISN microparticles, fine particle
dose (FPD, 1229 mg) and fine particle fraction (FPF, 12%)
were also evaluated (Table 2). The three collection stages of
the home-made impinger device can mimic the whole
human respiratory system; for this reason, FPF and FPD
were evaluated in the last two stages of the impinger that
represent the lower airway region.[29,30]
RFP
The ability of microparticles to retain the drug during the
nebulisation process was also evaluated. Nebulised disper-
ISN sion was separated by dialysis from the RFP that was
released during the process, and the retention of the drug
(NER%) was successively evaluated. It was not possible to
measure this parameter in each stage of the impinger, and
for this reason, at the end of the experiment, the nebulised
dispersion, recovered on the three stages, was combined,
purified and subsequently analysed for the determination of
NER%. About 42% of the drug was retained within the par-
ticles; this is a good amount, confirming that these systems
are stable and therefore suitable for lung delivery by aerosol
therapy. The size of the nebulised microparticles decreased
from the first to the third stage of the impinger device. At
the end of this process, three distinct samples with different
size distribution were obtained. Mean diameter reduction
500 might be the consequence of fractionation and/or deaggre-
gation of the particles during air-jet nebulisation (data not
shown).[7,30–32] Nebulised samples were assayed also for ISN
content, but it was not possible to determine the quantity of
ISN nebulised and retained inside the microparticles during
the nebulisation process, probably because the drug
remained in the form of a prodrug and did not permit
analysis with the used methods. Nevertheless, in previous
work, the ISN-gelatin conjugate exhibited an excellent
in-vitro antimicobacterial activity against MTB complex,
showing a minimum inhibitory concentration value
(0.125 mg/ml) close to that of the free ISN (0.1 mg/ml).[14]
Moreover, the presence of amidases in both lungs and
plasma ensures the cleavage of the amide bond thus releas-
ing the free ISN in vivo.
M. tuberculosis, inhaled into the respiratory tract, is gen-
erally ingested by alveolar macrophages and subsequently
transported across the alveolar wall and the bloodstream. It
was also demonstrated that this mycobacterium invades and
survives within alveolar epithelial cells that can be used as
in-vitro model to predict the in-vivo behaviour. During this
study, A549 basal alveolar epithelial cells, obtained from
pulmonary adenocarcinoma, were chosen for cell viability
and uptake experiments.[33,34] Moreover, in nature, these
cells are squamous and responsible of the spread of
different substances, such as water and electrolytes through
the alveoli of the lungs. A549 cells are able to synthesise
© 2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 65, pp. 1302–1311
Maria L. Manca et al.
Table 2 NE%, FPD, FPF and NER% of gelatin microparticles containing RFP and ISN-prodrug
RFP-ISN-microparticles Stage 1
Stage 2
Stage 3
100
90
80
70
% Viability
60
50
40
30
20
10
0
2 4
Empty-Microparticles
Figure 5 In-vitro cytotoxic effect of empty and isoniazid (ISN)-prodrug containing rifampicin (RFP) microparticles compared with RFP and ISN-
prodrug solution on A549 cells at different incubation times.
lecithin and to contain high amounts of fatty acids, which
increase the stability of the membrane of the cells.
In-vitro toxicity studies were carried out using the A549
cells at step 90–100. Cells were incubated with empty and
ISN-gelatin conjugate containing RFP microparticles for 2,
4, 6, 8, 24 and 48 h and compared with RFP solution and
ISN-gelatin conjugate dispersion. At the end of each experi-
ment, the toxicity was measured by MTT cell viability test.
As shown in Figure 5, empty and drugs-loaded microparti-
cles statistically showed the same toxicity (P > 0.05), which
increased over time. In fact, until 8 h, the vitality was 58%
and 54% for empty and drug-loaded-particles, respectively,
and gradually decreased, reaching 60% and 63% of mortal-
ity after 48 h compared with untreated control cells (100%
viability). On the contrary, the cytotoxicity induced by RFP
solution was always substantially higher (P < 0.01) than that
of empty and loaded carriers, in particular from 8 to 48 h
incubation, where the mortality reached 60%, 70% and
80%, respectively; this was probably because microparticles
first interact with the cells and thereafter release the drug.
The behaviour of the ISN-gelatin prodrug dispersion was
different; in this case, the toxicity was similar to empty and
drug-loaded microparticles, reaching the 60% of mortality
after 48 h. This was probably due to the effectiveness of the
prodrug that linked the ISN, and was able to modulate its
toxicity in the cells. Moreover, because of its high molecular
© 2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 65, pp. 1302–1311
Isoniazid-gelatin rifampicin microparticles
NE% ⫾ SD FPD (mg) FPF (%) NER% ⫾ SD
46 ⫾ 2.6
9 ⫾ 0.4 42 ⫾ 3.2
1229 ⫾ 48 12 ⫾ 1.3
4 ⫾ 0.9
6 8 24 48
Time (h)
RFP-ISO-Microparticles RFP Sol Isoniazid gel
weight, the ISN prodrug was used at a concentration 10
times less than that of RFP. Drug toxicity is strictly depend-
ent of drug concentration, as already reported by others.[35]
In order to evaluate the possibility of microparticle inter-
nalisation, A549 cells were incubated with RFP-ISN micro-
particles containing two fluorescent markers: one lipophilic,
rhodamine (0.035 mg/ml Rho) and one hydrophilic,
(6)-carboxyfluorescein (0.025 mg/ml, CF). CF was chosen
because it is a membrane impermeable probe that is espe-
cially used for investigating membrane integrity and perme-
ability.[36,37] Internalisation experiments were carried out on
sterile 24 mm slides until the achievement of the subconflu-
ence; successively, fluorescent microparticles were incubated
for 2, 4, 8 and 24 h. At the end of the experiments, the cells
were fixed, and the Hoechst dye, which allows the visualisa-
tion of the nucleus, was added. The slides were observed
using a confocal microscope (FluoView FV1000, Olympus).
The images obtained for each marker were superimposed
to simultaneously display the location of each marker
inside the cells. Figure 6 shows images obtained after each
incubation time, in which it is clear that the two markers
were internalised. In particular, after 2 and 4 h uptake
experiments, both markers were able to interact with cells
but the fluorescent intensity was lower compared with 8
and 24 h exposure, in which the fluorescent intensity
increased significantly. Images do not give any evidence of
1309
Isoniazid-gelatin rifampicin microparticles Maria L. Manca et al.

CF Rho Merged reducing side effects and increasing efficacy. Microcarriers

may ensure sustained drug delivery to the lungs, extended
duration of action, reduced therapeutic dose and, in par-
ticular, improved patient compliance. For this reason,
2h gelatin was derivatised with ISN to obtain a prodrug that
can be incorporated in gelatin microparticles also contain-
ing RFP. X-ray analysis provided evidence that the drugs
were in an amorphous state and dispersed into the micro-
particles. The disappearance of endothermic peaks of ISN
and RFP in the microparticle DSC spectrum confirmed the
4h effective ISN-gelatin derivatisation and RFP incorporation
into the gelatin microcarriers. Microparticles were able to
incorporate suitable amounts of RFP and ISN in a good
yield (~60%). Results also showed that microparticles are
good candidates as pulmonary delivery systems of antitu-
bercular drugs, as they showed good nebulisation efficiency
8h
(NE~59%) and also a good capability of retaining the RFP
during the nebulisation process (NER~42%). Cell viability
tests performed by using A549 cells demonstrated the low
toxicity of both microparticles and prodrug. Internalisation
studies performed using CLSM demonstrated the effective
internalisation of both hydrophilic and lipophilic markers,
24 h
encapsulated into the microcarrier. Therefore, on the whole,
ISN-gelatin RFP containing microparticles showed a prom-
ising behaviour for pulmonary drug delivery.

Figure 6 Images of A549 cells incubated for 2, 4, 8 and 24 h with

Rho and CF loaded microparticles. The localisation and intensity of
dyes are displayed in red for Rho, in green for CF.
microparticle internalisation but there was no apparent dif-
ference in the pattern and intensity of fluorescence at
uptake time of 8 or 24 h. Indeed, both Rho and CF are
located throughout the cytoplasm and around the nucleus.
The localisation of two markers is very similar, as confirmed
by the yellow colour of the merged images, allowing us to
suppose the possible presence of microparticles within cells.
Conclusion
Targeted therapy of drugs to the lungs may provide a better
deposition of suitable therapeutic agents at the lung surface,
Declarations
Conflict of interest
The Author(s) declare(s) that they have no conflicts of
interest to disclose.
Acknowledgements
Sardegna Ricerche Scientific Park (Pula, CA, Italy) is
acknowledged for free access to facilities of the Nanobio-
technology Laboratory. Dr. M.L. Manca was financed by
Fondazione Banco di Sardegna (2011–2012). This work was
partially supported by MIUR grants (PRIN 2010-2011, Prot.
2010H834LS_004).

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