Content uploaded by Christian I. Nkanga
Author content
All content in this area was uploaded by Christian I. Nkanga on Jan 13, 2020
Content may be subject to copyright.
Copyright © 2020 American Scientific Publishers
All rights reserved
Printed in the United States of America
Article
Journal of
Biomedical Nanotechnology
Vol. 16, 14–28, 2020
www.aspbs.com/jbn
Co-Loading of Isoniazid-Grafted Phthalocyanine-in-
Cyclodextrin and Rifampicin in Crude Soybean
Lecithin Liposomes: Formulation, Spectroscopic
and Biological Characterization
Christian Isalomboto Nkanga1∗, Michael Roth2, Roderick Bryan Walker3,
Xavier Siwe Noundou1, and Rui Werner Maçedo Krause1∗
1Center for Chemico- and Bio-Medicinal Research (CCBR), Department of Chemistry, Faculty of Science,
Rhodes University, Grahamstown 6140, South Africa
2Pulmonary Cell Research & Pneumology Laboratory, Department of Biomedicine, University & University Hospital Basel,
Petersgraben 4, CH-4031, Basel, Switzerland
3Division of Pharmaceutics, Faculty of Pharmacy, Rhodes University, Grahamstown 6140, South Africa
An inclusion complex of isoniazid-grafted phthalocyanine with gamma-cyclodextrin (Complex) was co-encapsulated with
rifampicin (RIF) in crude soybean lecithin liposomes using a heating method. The encapsulation efficiency (%EE) of the
Complex-RIF co-loaded liposomes (Rif-Complex-Lips) was determined using UV-Vis spectrophotometry. Rif-Complex-Lips
formulations were evaluated using dynamic light scattering, transmission electron microscopy (TEM), 1H-NMR, absorption
and emission spectroscopy. Dialysis was used for drug release study in two different media, pH 6.4 and 7.4. HeLa
cells were used to assess potential cytotoxicity, and the uptake by lung fibroblasts and epithelial cells was investigated
using fluorescence microscopy. The particle size and Zeta potential of Rif-Complex-Lips were approximately 594 nm and
−50 mV. Spectroscopic analyses demonstrated molecular distribution of the cargo within the lipid core, and encapsulation
efficiency of 58% for Complex and 86% for RIF. TEM analysis unveiled the existence of spherical nanoparticles in our
samples, indicating the presence of liposomes. Rif-Complex-Lips exhibited much higher release rates for both INH and
RIF at pH 6.4 compared to those tested at pH 7.4. In addition, there was no cytotoxicity on HeLa cells, but remarkable
Rif-Complex-Lips internalization by peripheral lung fibroblasts and epithelial cells. Hence, Rif-Complex-Lips are promising
vehicles for intracellular delivery of antimicrobial drugs.
KEYWORDS: Cyclodextrin, Isoniazid, Liposomes, Phthalocyanines, Rifampicin, Soybean Lecithin.
INTRODUCTION
Tuberculosis (TB), a microbial disease caused by
Mycobacterium tuberculosis, affects the lungs in 90% of
cases and resides mainly inside alveolar macrophages.
In fact, TB infection is initiated by inhalation of M. tuber-
culosis, typically carried in aerosol droplets. In the lungs
the tubercle bacillus is taken up by alveolar macrophages
through phagocytosis [1, 2]. Throughout phagocyto-
sis, alveolar macrophages produce various antimicrobial
∗Authors to whom correspondence should be addressed.
Emails: stianolnkanga@gmail.com, r.krause@ru.ac.za
Received: 8 August 2019
Accepted: 11 November 2019
agents, such as proteases, lipases, reactive oxygen and
nitrogen species as well as a highly acidic environment to
kill the pathogen. Nonetheless, the cell wall of M. tuber-
culosis provides often the necessary protection for the
bacilli to resist the immune attack and survive in the
macrophage’s cytoplasm [3, 4]. The evolution of the infec-
tion to active TB depends on the immune response. About
one-third of the global population have latent TB, a condi-
tion where people host the tubercle bacilli inside alveolar
macrophages asymptomatically, but with high risk of reac-
tivation in case of immune system failure [5].
The histological hallmark of the immune response
to pulmonary tuberculosis is the formation of lung
granuloma, a mass of granulation tissue consisted of
14 J. Biomed. Nanotechnol. 2020, Vol. 16, No. 1 1550-7033/2020/16/014/015 doi:10.1166/jbn.2020.2880
Nkanga et al. Co-Loading of Isoniazid-Grafted Phthalocyanine-in-Cyclodextrin and RIF in Crude Soybean Lecithin Liposomes
monocyte-derived cells (i.e., macrophages) and T lympho-
cytes [6]. A prerequisite for granuloma formation is the
recruitment of these cell lines, a process where many other
cell types are involved, including lung epithelial cells and
fibroblasts located at the periphery of granuloma. The pul-
monary fibroblasts produce chemokines (such as CXCL8)
that affect the intramacrophage survival of the bacilli and
induce chemotaxis, the attraction of cells into the grow-
ing granuloma [7]. As for the lung epithelial cells, they
have been reported to be the first line of defense to inhaled
pathogens. Among the cell types involved in the immune
response, the participation of epithelial cells to the inter-
action between M. tuberculosis and the host has been evi-
denced [8]. The lung epithelial cells have been reported
to be part of the major immune cells involved in TB
infection. Due to their abundance in the lung, epithelial
cells provide potent secretion and high concentrations of
chemokines (including CXCL8 and IL-8), contributing to
the intramacrophage killing of the bacilli and chemotaxis
substantially. In addition to this, it was demonstrated that
virulent M. tuberculosis, strain H37-Rv, can be phagocy-
tosed by the lung epithelial cells [9, 10]. With such a com-
plex immune response to the infection, the fact that TB
disease still holds a devastating status means the immune
system fails often, and hence efforts are to be put on
chemotherapy as a hope for improving TB management.
However, despite the existence of well-established
antimicrobial agents (e.g., first line TB drugs such as iso-
niazid, rifampicin, pyrazinamide and ethambutol), TB has
been ranked as the leading cause of mortality from a single
pathogen over the last five years [11]. The deadly status
of TB reflects the seriousness of the limitations encoun-
tered with the current therapeutic regimens, arising partly
from the self-defensiveness of M. tuberculosis, in addi-
tion to restricted accessibility due to the intra-macrophage
location and poor pharmacological profiles of some of the
existing anti-TB drugs [12–14]. Although reputed to be the
most potent antibiotics for TB, rifampicin (RIF, Fig. 1(a))
and isoniazid (INH, Fig. 1(b)) exhibit widespread biodis-
tribution due to their different solubilities viz., highly and
poorly water soluble respectively [15], which results in fre-
quent side effects associated with low accumulation at the
site of infection [13, 14]. This underlines the need for drug
delivery systems that can target and control simultaneous
release of these drugs for improved combination therapy,
with an associated reduction of off-target side effects.
Liposomes are phospholipid-based vesicular systems
that can load hydrophilic, hydrophobic and amphipathic
compounds within the interior aqueous body, the lipid
bilayers and at the interfaces, respectively [16, 17]. Numer-
ous studies have demonstrated the potential of lipo-
somes for the co-encapsulation of RIF and INH with
enhanced bioavailability [15, 18–24]. Nevertheless, most
of the proposed liposomes are subject to a number of
technological challenges, which might preclude the uni-
versal application of the formulations currently under
development. Some of these challenges include (i) the
use of expensive lipid materials [25, 26], (ii) the poten-
tial loss of small hydrophilic drugs (like INH) [27–29]
due to liposome leakage, which mostly requires incor-
poration of special materials (e.g., pH-sensitive lipids)
for controlled/targeted delivery [30], and (iii) the use of
organic solvents for encapsulation of hydrophobic com-
pounds [31, 32]. To address the critical issue of using cost-
effective lipid materials to prepare liposomal combination
products, our group has previously demonstrated the abil-
ity of crude soybean lecithin to achieve efficient co-loading
of INH and RIF in liposomes [33]. Crude soybean lecithin
is a naturally occuring lipid mixture that was initially
introduced for INH encapsulation due to its cost-effective
status, being readily avaliable and accessible for prod-
uct development [34]. However, crude soybean lecithin
liposomes loaded with INH alone or in cobination with
RIF exhibited a remarkable leaky behaviour, expressed
by burst release of INH. Therefore, in another study, we
have conjugated INH to hydrophobic compounds (para-
hydroxy-benzaldehyde and phthalocyanines) using pH-
labile linkages (e.g., hydrazone, an acid-sensitive bond)
as a potential strategy for stimulus-dependent release of
INH from the liposomes [30, 35]. In this effect, we encap-
sulated the resultant conjugates in crude soybean lecithin
liposomes using a thin film hydration method, and fur-
ther applied pH change (acidification) as the stimulus for
triggering INH release. Since the synthesized hydropho-
bic conjugates were trapped within the liposome’s bilayer,
the hydrolysis of hydrazone bonds on acidification was the
only driving force for the release of INH, which allowed
to avoid the burst release previously observed at neutral
pH [30, 35].
Isoniazid-grafted phthalocyanine (PC-INH, Fig. 1(c))
is one of the hydrazone conjugates we recently devel-
oped for pH-dependent controlled release of INH, with
the possibility for bioimaging evaluation of crude soy-
bean lecithin liposomes [35]. The hydrophobic nature
of the PC-INH was identified as a technological chal-
lenge, due to the imperative use of organic solvents
for successful encapsulation in liposomes using thin film
hydration methods. Hence, our group has investigated
cyclodextrin complexation of PC-INH as a strategic pre-
treatment for liposomal encapsulation with no use of
organic solvents (using a heating method). The inclusion
complex of PC-INH with cyclodextrin showed remark-
able fluorescent behavior, which is useful for potential
image-guided drug delivery studies. The encapsulation
efficiency of liposomes for PC-INH was found to be
doubled when PC-INH/cyclodextrin complex was used
(unlike when PC-INH was used without cyclodextrin), and
the INH pH-dependent release behavior was observed as
expected [36].
Nonetheless, PC-INH/cyclodextrin complex-loaded
liposomes previously developed contain only one type of
J. Biomed. Nanotechnol. 16, 14–28, 2020 15
Co-Loading of Isoniazid-Grafted Phthalocyanine-in-Cyclodextrin and RIF in Crude Soybean Lecithin Liposomes Nkanga et al.
(a)
(b)
(c)
Figure 1. Chemical structures of RIF (a), INH (b), and isoniazid-grafted phthalocyanine (c).
anti-TB drug (i.e., INH), while the current regimen for TB
requires a multi-drug combination to tackle antimicrobial
resistance [11]. It appears thus relevant to consider explor-
ing the co-encapsulation of PC-INH/cyclodextrin complex
with other anti-TB agents to develop liposomes that reflect
the recommended anti-TB multi-drug regimen [37]. Such
a multi-drug composition will permit biological evalu-
ation of crude soybean lecithin liposomes for potential
pulmonary delivery, since it approximates the combination
therapy recommended to address the issue of TB drug
resistance [3, 38, 39]. Therefore, the present work aimed
at formulating and characterizing crude soybean lecithin
liposomes co-loaded with PC-INH/cyclodextrin complex
and RIF. These dual liposomes were prepared without
organic solvents and further investigated for potential
pulmonary biocompatibility and uptake by the peripheral
lung fibroblasts and epithelial cells.
MATERIALS AND METHODS
Chemicals
Rifampicin (RIF), tetraethylammonium bromide
(T-EthBr), gamma-cyclodextrin, Dimethyl sulphoxide
(DMSO), deuterium oxide (D2O), hydrochloric acid
(HCl), orthophosphoric acid, citric acid, tri-sodium citrate,
mono- and di-basic sodium phosphates (NaH2PO4and
Na2HPO4) were sourced from Sigma Aldrich (Germany).
Crude soybean lecithin (lecithin) was purchased from
Health Connection Wholefoods (USA). High performance
liquid chromatography (HPLC) grade Methanol was pur-
chased from Merck (Germany). The inclusion complex
of PC-INH with gamma-cyclodextrin (denoted Com-
plex) was prepared by co-grinding method as described
by Nkanga and Krause [36]. Briefly, A few drops of
Millipore water were added to gamma-cyclodextrin
(455 mg, 0.35 mmol) for kneading and PC-INH (110 mg,
0.07 mmol) was slowly added under constant milling at
RT for 30 min. The obtained paste was milled over 60 min
and dried for 24 hours to constant weight at 70 C. The
obtained complex was fully characterized as previously
reported [36].
Equipment
A Shimadzu UV-2550 spectrophotometer (Shimadzu,
Kyoto, Japan) was used for acquisition of UV-Vis absorp-
tion spectra. Fluorescence emission spectra were col-
lected using a Varian Eclipse Spectrofluorimeter (Varian,
California, USA). Proton nuclear magnetic resonance
(1H-NMR) spectra were recorded on a Bruker AMX
600 MHz NMR spectrometer (Bruker, Rheinstetten,
Germany). Sonication was undertaken using a Digital
Ultrasonic Cleaner (World Precision Instruments, Sarasota/
Florida, USA). Particle size and Zeta potential were deter-
mined on a Zetasizer nano ZEN-3600 (Malvern Instru-
ments, Worcestershire, UK). A Zeiss Libra-120 KV TEM
microscope (Carl Zeiss, Oberkochen, Germany) was used
for microscopic analysis. HPLC was performed on an
HP1100 Agilent LC-MSD (Agilent Technologies, Foster
City, USA) using a ZORBAX Elipse Plus C18 4.6 i.d. ×
150 mm ×5m column and an Agilent HP1100 LC-
MSD equipped with a quaternary pump, in-line degasser,
DAD detector, 1100 MSD and ChemStation acquisition
software.
Liposome Preparation
A full factorial design approach using Minitab 13
(Minitab, Ltd. UK) was used for the preparation of dif-
ferent liposome formulations based on two parameters:
(i) the active pharmaceutical ingredients (mixture of RIF
and Complex, denoted API) to lecithin mass ratios, and
(ii) RIF to Complex mass ratios (Table I).
The liposome formulations were prepared without
organic solvents using a previously described heating
method [36]. A blend of lecithin (100 mg) and appropriate
Tab le I . Formulation parameters for liposome preparation.
Levels API to lecithin (mass ratio) RIF to complex (mass ratio)
1 0.25:1 0.25:1
2 0.5:1 0.5:1
31:1 1:1
Notes: RIF: Rifampicin. Complex: Gamma-CD/PC-INH complex. API: RIF +
complex.
16 J. Biomed. Nanotechnol. 16, 14–28, 2020
Nkanga et al. Co-Loading of Isoniazid-Grafted Phthalocyanine-in-Cyclodextrin and RIF in Crude Soybean Lecithin Liposomes
amounts of API (mixture of RIF and Complex) was
hydrated with Millipore water (10 ml) in the dark at room
temperature (RT) for 60 min. The resultant mixture was
heated under stirring (500 rpm) for another 60 min at
70 C. The suspension was allowed to cool to RT. On
cooling, the preparation was diluted with Millipore water
(5 ml) and subject to two consecutive centrifugations under
different conditions, as follows:
(a) Low-speed centrifugation (LSC): The LSC was done
in order to separate any undissolved particles (macropar-
ticles) of the API from the colloidal dispersion. The
LSC was performed at RT using a MSE Mistral-1000
apparatus set at 4,000 rpm for 5 min [40]. The resul-
tant LSC-pellet (API macroparticles) was isolated by
decantation and stored at RT in the dark prior to fur-
ther encapsulation efficiency (EE) evaluation. The col-
loidal supernatant (liposomes dispersion) was sonicated at
70 C for 20 min. The homogenous dispersion obtained
was subject to another round of centrifugation at higher
speed.
(b) High-speed centrifugation (HSC):TheHSCwas
performed in order to isolate the colloidal particles
(liposomes) from the bulk solution, which contains non-
encapsulated API’s molecules. The HSC was done at
25 C on a Beckman Coulter Allegra 64R set at
20,000 rpm for 20 min [34–36]. The resultant HSC-
supernatant (containing some API in solution) was
decanted and retained for EE study. The HSC-pellet
(liposomes) was freeze-dried using an Apollo Scientific
Lyo Lab-3000 and stored in the dark at 4 C until
further characterizations. Based on the EE data, for-
mulation F1 (subsequently denoted Rif-Complex-Lips)
was selected for further characterizations. Moreover,
three different F1 formulation counterparts were pre-
pared under the same conditions for comparative anal-
yses. These included RIF-loaded liposomes (Rif-Lips),
Complex-loaded liposomes (Complex-Lips) and blank
liposomes containing only gamma-cyclodextrin as control
liposomes (Control-Lips).
Determination of Encapsulation Efficiency
The encapsulation efficiency (EE) of the prepared lipo-
somes was estimated indirectly by the quantitation of
undissolved API’s macroparticles (LSC-pellet) together
with non-encapsulated API’s molecules (API’s molecules
found in the HSC-supernatant). The HSC-supernatant
(containing some API in solution) was added to the LSC-
pellet (undissolved API), and DMSO was added to a col-
umn of 100 ml to ensure complete dissolution. The UV-Vis
absorption spectrum of the solution was recorded over the
wavelength range of 300–800 nm. The maximum absorp-
tion bands at 480 and 681 nm were considered for the
evaluation of EE for RIF and Complex, respectively [41].
In parallel, control samples were prepared using RIF and
Complex in appropriate mass ratios without lecithin but
under the same preparation and dilution conditions as for
the liposome formulations. This was done in order to
provide theoretical absorbance of RIF and Complex that
one should expect in the absence of lecithin (or without
treatment with lecithin). Hence, the comparison between
the absorbance without treatment with lecithin (theoretical
absorbance) and absorbance after treatment with lecithin
(absorbance after encapsulation) allowed for the estima-
tion of the percentage EE (%EE) for each product as
follows [42].
%EE =Theoretical absorbance
−absorbance after encapsulation
/theoretical absorbance ×100 (1)
Particle Size and Zeta Potential
Freeze-dried liposomes (50 mg) were dispersed in
Millipore water (5 ml) and allowed to hydrate at RT for
60 min prior to handshake for 1–2 min. The obtained
dispersion was diluted (1/10) with Millipore water. The
diluted samples were evaluated for particle size and Zeta
potential using dynamic light scattering at a scattering
angle of 173.
Particle Shape Analysis
Samples from dynamic light scattering experiment were
deposited drop wise on copper grids by means of a Pas-
teur pipette. Filter paper was used to absorb the excess
liquid. The grids were air-dried in the dark at RT for 24
hours. Liposomes were visualized by transmission electron
microscopy (TEM).
Proton Nuclear Magnetic Resonance (1H-NMR)
The interactions between cargo molecules and lipid com-
ponents were evaluated by 1H-NMR. Freeze-dried lipo-
somes (25 mg) were hydrated in D2O(0.5ml)atRTfor
60 min. The resultant mixture was shaken by hand for
1–2 min and the suspension transferred to the specialized
NMR glass tube. Samples were subjected to 32 scans for
acquisition of 1H-NMR spectra at RT using tetramethylsi-
lane (TMS) as internal reference.
Absorption and Emission Spectroscopy
An experimental trial was performed using different Com-
plex to RIF molar ratios to investigate possible interac-
tions between the two aromatic compounds in solution.
Two stock solutions (0.048 mole/ml) of Complex and
RIF were separately prepared in DMSO and mixed up in
1:1, 1:2 and 1:3 molar ratios. Different portions of fresh
solvent were added accordingly for volume adjustment and
the resultant mixtures were stirred at 70 C for 24 hours,
alongside with single component solutions (controls)
appropriately diluted with DMSO. On cooling, samples
were subjected to UV-Vis absorption spectroscopic mea-
surements. In parallel, freeze-dried liposomes (Rif-Lips,
J. Biomed. Nanotechnol. 16, 14–28, 2020 17
Co-Loading of Isoniazid-Grafted Phthalocyanine-in-Cyclodextrin and RIF in Crude Soybean Lecithin Liposomes Nkanga et al.
Rif-Complex-Lips and Complex-Lips) were dissolved in
DMSO, and UV-Vis absorption measurements were taken
for evaluation of electronic transition behaviour of RIF
from liposomal samples. For selective exploration of PC-
INH structure, samples were subjected to fluorescence
emission spectroscopy using the excitation wavelength of
PC-INH (605 nm) [36].
Dual Release Study
The analysis of drug content in liposomes was performed
prior to drug release studies. The concentrated dispersion
of formulation F1 (1 ml) prepared for dynamic light scat-
tering experiment was mixed with methanol (4 ml) in
a 10 ml volumetric flask and HCl 32% (3.928 ml) was
slowly added using a micropipette. Methanol was care-
fully added for volume adjustment to yield a 4 N HCl
solution that was sonicated at 60 C for 30 min. This
was undertaken to promote the disruption of liposomes
and cause effective hydrolysis of hydrazone linkages in
PC-INH, which permitted content determination of INH.
Since RIF is known to undergo molecular degradation in
acidic media [43–46], RIF content analysis was achieved
without addition of HCl. On cooling to RT, the volume
was readjusted with methanol and samples were filtered
using 0.22 m syringe filters. Aliquots were withdrawn
for simultaneous quantification of RIF and INH using the
reversed-phase HPLC method developed by Mariappan
et al. [47] with slight modifications. Gradient elution was
done using methanol (sol A) and NaH2PO4 0.01 M con-
taining 0.05% w/v T-EthBr, which was adjusted to pH 3.5
using orthophosphoric acid (sol B). The column was equi-
librated over 15 min with a mixture of sol B 90% v/v and
sol A 10% v/v. After 2 min of sample injection, sol A
was gradually increased by 3% per minutes for 15 min.
Then sol B was decreased at the rate of 1% per minute
for remaining time of the experiment (40 min). The DAD
detection was set at 254 nm and the flow rate for the sol-
vent was 1 ml/min. The drug content (% C) in liposomes
was calculated as follows.
%C=Drug amount
/Freeze dried Liposomes amount ×100 (2)
Furthermore, another aliquot of liposome F1 (1 ml) was
transferred to the dialysis bag (Membra-Cel MD 10–14 ×
100 CLR, Sigma Aldrich). The release study was con-
ducted according to the experimental procedure described
in Nkanga et al. [34] with a few modifications. The release
media considered for this study were constituted of two
phosphate buffers of pH 6.4 and 7.4, which respectively
correspond to the pH of early formed endosomes and
extracellular fluid [48, 49]. The loaded dialysis bag was
soaked in 20 ml of the release medium placed in a glass
vial that was kept at 37 C under stirring at 240 rpm. Sam-
ples (5 ml of the release medium) were withdrawn at 0.5;
1; 1.5; 2; 3; 4; 5; 7; 9 and 12 hours, and fresh buffer (5 ml)
was replaced at each sampling point. Aliquots were quan-
titatively analyzed by HPLC and calibration curves were
prepared daily [34].
In Vitro Cytotoxicity Assay
The liposomes of formulation F1 that showed the high-
est %EE (Rif-Complex-Lips) was selected for cell toxic-
ity assay together with liposomal counterparts (Rif-Lips,
Complex-Lips and Control-Lips) and raw materials (RIF
and Complex) for comparative purposes. Each liposomal
sample (25 mg) was dispersed in Millipore water (0.5 ml)
as previously described for liposome hydration, while RIF
and Complex raw materials were dissolved in DMSO.
The medium for HeLa (human cervix adenocarcinoma)
cells (Cellonex) culture was Dulbecco’s Modified Eagle
Medium (DMEM)–(Lonza), which was supplemented with
10% fetal calf serum and a mixture of antibiotics (peni-
cillin/streptomycin/amphotericin B). The culture was done
at 37 Cina5%CO
2incubator. Cells were placed in 96-
well plates at the density of 6.7×104cells/well. Cells were
allowed to grow overnight. Serial dilutions (50 g/ml)
of the samples were incubated with the cells for another
48 hrs. The viability of cells was assessed in the wells
after addition of 20 L of 0.54 mM resazurin in PBS
for another 2–4 hrs. Individual wells with the sample
were subject to fluorometric measurements (at excitation
560 nm and emission 590 nm) and the recorded intensities
were converted to % cell viability in conjunction with the
fluorometric measurements done on the untreated control
wells.
Airway Cell Uptake Studies
Cell Isolation
Endoscopic bronchoscopy was used to obtain airway wall
tissue samples from patients with chronic obstructive
lung diseases. The procedure was approved by the ethics
committee of North-western Switzerland (EKNZ2016-
01057). Tissue samples were collected after having written
informed consent from the patients for tissue harvesting
for additional research biopsy. Biopsies were monitored
under a stereo-microscope at a magnification of 5×and the
epithelium was separated from smooth muscle cell bundles
and connective tissue. Different tissue compartments were
used for isolating airway epithelial cells and fibroblasts as
described in the following paragraphs.
Airway Epithelial Cells. The epithelium was cut into
2×2×2 mm pieces. Epithelium pieces were placed
into a 25 cm flask (Sarstedt, cat# 83.3911.302), which
was pre-wetted with 1 ml of epithelial cell selective
medium (CellnTec, Bern, Switzerland, cat# Cnt-PR-A).
The medium was replaced every second day until epithelial
cells became confluent. Epithelial cells were characterized
by positive staining for E-Cadherin, and cytokeratin 13,
as well as negative staining for fibronectin [50]. Epithelial
18 J. Biomed. Nanotechnol. 16, 14–28, 2020
Nkanga et al. Co-Loading of Isoniazid-Grafted Phthalocyanine-in-Cyclodextrin and RIF in Crude Soybean Lecithin Liposomes
cells were expanded by 3×washes with phosphate
buffered saline, followed by treatment with trypsin/EDTA
(Biological Industry, Cromwell, CT, USA, cat#: 03-079-
1C) for 5 minutes at 37 C. The detached cells were mixed
with 1 ml fetal bovine serum and 2 ml CnT-PR-A and
seeded into three 25 cm flasks, three 96-well plates or
cover slips. The adherence of the cells was monitored after
24 hours by microscopy, and the medium was replaced
every second day until confluence.
Peripheral Lung Fibroblasts. Connective tissue pieces
(2 ×2×2 mm) were placed into 25 cm flasks, which
were pre-wetted with fibroblast selective medium (Cell-
nTec, cat#: CnT-PR-F). The medium was exchanged every
third day and fibroblast outgrowths were monitored by
microscopy every second day. Fibroblasts were charac-
terized by negative staining for E-Cadherin and cytoker-
atin 13, and positive staining for fibronectin and -smooth
muscle actin [50]. Fibroblasts were expanded after 3×
washes with phosphate buffered saline, followed by
trypsin/EDTA treatment for 1 minute at RT. Trypsin/EDTA
was removed and the cells were further incubated for
5 minutes until complete detachment. Fibroblasts were
re-suspended in RPMI1640 supplemented with 10% fetal
bovine serum, 20 mM HEPES, 8 mM glutamine and
10 mM sodium pyruvate.
Cell Uptake Experiment
Liposomes Sample Preparation. Three different liposomes
formulations (F1, F4 and F7) prepared with increasing
API to lecithin mass ratios (Table I) were used to assess
the potential effect of the dual cargo on the airway cells
in addition to the fluorescent-imaging uptake study. For
each sample, a 1.5 ml Eppendorf tube containing 15 mg
of the formulation was filled to the mark with phosphate
buffer saline. The resultant mixture was incubated at RT
for 60 min and vortex mixed for 2 min to yield a homoge-
nous stock dispersion (10 mg/ml).
Cell Uptake Assay. Cells were seeded onto 8-well glass
slides and grown to 80% confluence. Confluent cells were
treated with increasing concentrations of the selected lipo-
somes (0.1–1 mg/ml) for 24 hours. The cells were visu-
alized in a microscope at 40×magnification (EVOS FL
cell imaging system, Thermofisher Scientific, Switzer-
land) in fluorescence mode and the presence of green
dots in the cytosolic media was considered as positive
(liposomes in cells). In addition, a ready-to-use assay
kit, viz., ReadyProbes®Cell Viability Imaging Kit (Invit-
rogen, Thermofisher Scientific, Basel, Switzerland), was
used to differentiate live from dead lung fibroblasts fol-
lowing internalization of liposomes. For this purpose,
two 2 drops/ml medium of each test-dye was added to
the cell medium for 30 minutes at RT. NucBlue®Live
reagent that stains the nuclei of all cells was detected
with a standard DAPI (blue) filter (at excitation/emission
maxima: 360/460 nm). NucGreen®Dead reagent that
stains only the nuclei of dead cells with compromised
plasma membranes was detected with standard FITC/GFP
(green) filter (at excitation/emission maxima: 504/523 nm).
Positive controls were cells treated with ZnCl2(10 g/ml)
as a cytotoxic standard (yielding green nuclei). The epithe-
lial cell uptake was investigated without staining for viabil-
ity checkup, due to the intrinsic poor viability observed for
the untreated cells (negative controls) under actual experi-
mental conditions. Each condition was performed in repli-
cate (n=5) for each cell type.
Statistical Analysis
Unless indicated, all the experiments were undertaken
in triplicate and numerical data is expressed as mean ±
standard deviation (SD). Calculations were done by means
of software Minitab 13 (Minitab, Ltd. UK). Where appli-
cable, data were subjected to one-way ANOVA and the
significance level was set at p-value <0.05.
RESULTS AND DISCUSSION
Encapsulation Efficiency for the Dual Cargo
The data for encapsulation efficiency (%EE) are reported
in Table II. The %EE for the co-loaded liposomes were
within the range of 52–86% for RIF and 13–58% for
Complex, while the mono-drug loaded liposomes exhib-
ited approximately 96 and 61%, respectively. As shown
in Figure 2(a), the %EE of RIF tends to decrease signifi-
cantly as API to lecithin mass ratio increases, while only
a slight decrease can be observed when RIF to Complex
mass ratio increases. Surprisingly, no significant change in
%EE for Complex was observed on increase of API to
lecithin mass ratio, compared to increase in RIF to Com-
plex mass ratio (which caused remarkable decrease, as
depicted in Fig. 2(b)). This suggests the existence of poten-
tial competition between the two components when loaded
simultaneously in liposomes. A possible explanation for
this observation could be due to saturation of the lipo-
some membrane when accommodating some of the RIF
and Complex molecules simultaneously.
A similar observation was reported by Rodrigues
et al. [20] These authors reported the presence of RIF
molecules within the lipid bilayers and intermolecular
interactions between liposome membrane and INH, which
is a hydrophilic drug. The higher %EE of the mono-loaded
liposomes also supports the hypothesis that the two API
components could potentially compete for the liposomal
system. The formulation F1 showed the highest %EE and
was therefore used for further analysis, since %EE is an
essential parameter in the development of cost-effective
drug carriers [34].
Particle Size and Zeta Potential
The mean particle size, size distribution (expressed by
PDI) and Zeta potential of the liposome formulations are
J. Biomed. Nanotechnol. 16, 14–28, 2020 19
Co-Loading of Isoniazid-Grafted Phthalocyanine-in-Cyclodextrin and RIF in Crude Soybean Lecithin Liposomes Nkanga et al.
Table II. Characteristics of the liposome formulations.
EE ±SD (%)
F-code API-lecithin ratio RIF-complex ratio PS ±SD (nm) PDI ±SD ZP±SD(mV) RIF Complex
F1 0.25:1 0.25:1 593.7 ±12.1 0.32 ±0.04 −49.9 ±0.4 86.4 ±0.8 58.4±0.5
F2 0.25:1 0.5:1 530.9 ±7.4 0.31 ±0.00 −50.3 ±1.2 84.5 ±2.2 41.6 ±4.8
F3 0.25:1 1:1 533.9 ±7.3 0.31 ±0.00 −55.2 ±1.4 83.2 ±7.2 26.4 ±1.2
F4 0.5:1 0.25:1 554.1 ±6.2 0.32 ±0.02 −50.6 ±1.0 75.2 ±0.9 55.5 ±1.9
F5 0.5:1 0.5:1 568.4 ±2.3 0.33 ±0.04 −52.1 ±0.9 69.2 ±2.1 46.5 ±2.3
F6 0.5:1 1:1 504.4 ±11.1 0.33 ±0.05 −52.3 ±0.6 49.5 ±5.4 16.6±0.9
F7 1:1 0.25:1 602.2 ±7.6 0.39 ±0.00 −50.5 ±0.8 62.4 ±7.2 47.9±2.3
F8 1:1 0.5:1 539.5 ±7.0 0.32 ±0.04 −55.3 ±1.0 58.2 ±8.7 19.2±3.3
F9 1:1 1:1 520.3 ±4.9 0.27 ±0.00 −48.5 ±1.2 52.3 ±1.1 13.3 ±1.5
CPL 0.25:1 0:1 326.4 ±8.4 0.39 ±0.03 −66.1 ±1.8 NA 61.8 ±2.5
RL 0.25:1 0.25:0 198.7 ±1.5 0.36 ±0.00 −63.0 ±1.9 96.6 ±5.5 NA
CL 0:1 NA 177.3 ±2.9 0.33 ±0.03 −69.6 ±0.8 NA NA
Notes: F-code: Formulation code. API: RIF+complex. PS: Particle size. SD: Standard deviation. PDI: Polydispersity index. ZP: Zeta potential. Complex:
CD/PC-INH complex. NA: Not applicable. CPL: Complex-Lips. RL: Rif-Lips. CL: Control-Lips.
reported in Table II. All mean sizes for the dual liposomes
(F1 to F9) ranged between 504 and 602 nm. There was no
correlation between the particle size and the mass ratios
of API to lecithin and RIF to Complex (data not shown)
investigated. However, the outcome of one-way ANOVA
demonstrated that the ratio of API to lecithin significantly
affects the mean liposome size while the RIF to Complex
ratio appeared to have no influence (with similar mean
sizes reported). In addition, the co-loaded liposomes exhib-
ited larger mean sizes than both mono-loaded and blank
liposomes. This supports the hypothesis regarding possible
saturation of liposomes based on %EE data when load-
ing RIF and Complex simultaneously. Interestingly, most
of the PDI values for the co-loaded liposomes were found
to be <0.35, which indicates homogeneous particle size
distributions [51]. All dual liposome formulations exhib-
ited highly negative Zeta potential (surface charge), from
−49 to −55 mV, and only a slight difference was observed
within a series (Table II). However, Zeta potential values
for the blank and mono-loaded liposomes were found to
be greater than those of the dual liposomes. This repre-
sents further evidence that RIF and Complex are embed-
ded to a greater extent in liposome membranes when
loaded simultaneously, resulting in potential disruption of
lipid bilayers. Nevertheless, the density of surface charges
for all these liposomes looks promising for shelf stabil-
ity [31] and potential macrophage uptake via scavenger
receptors [2, 52].
Particle Morphology
Microscopic analyses of liposome samples unveiled the
existence of spherical and individual particles, suggest-
ing the presence of liposomal systems. As illustrated in
Figure 3, both mono-loaded and dual liposomes exhib-
ited well dispersed particles and no particle aggregates
were observed. This might be due to highly negative sur-
face charges, since the charge on the surface prevents par-
ticles from aggregating. This enhances the reliability of
dynamic light scattering data, regarding the mean particle
size recorded, as particle aggregation leads to false read-
ings in size measurements [53].
Proton Nuclear Magnetic Resonance
In general, NMR analysis of liposomal dispersions is
different to that of solutions, since liposomes are col-
loidal aggregates that are neither homogeneous liquids
nor solids [54]. 1H-NMR is one of the most powerful
techniques frequently used to study the structural char-
acteristics of liposomes and to elucidate the location of
encapsulated materials [20, 55]. Therefore, 1H-NMR spec-
tra of the liposomes co-loaded with RIF and Complex
(Rif-Complex-Lips) were analyzed and compared to those
of mono-loaded (Rif-Lips and Complex-Lips) and blank
liposomes (Control-Lips). Figure 4 compares portions of
the 1H-NMR spectra of these liposomes. The grey frames
in this figure demonstrate the more remarkable changes
occurring in the 1H resonance signals of the dual lipo-
somes F1 (Rif-Complex-Lips) compared to the other lipo-
somal counterparts. In the presence of RIF and Complex,
one of the peaks around 2.5–2.6 ppm, which are assigned
to –CH or –CH2– protons near a double bond of acyl
chains of lipids, showed a noticeable increase in intensity
while the same peak tends to disappear in Rif-Lips.
Similar changes were observed together with promi-
nent broadening of the peaks assigned to –CH2–close
to a carbonyl group at 3.4–3.5 ppm [54]. This is indica-
tive of strong molecular interactions between loaded mate-
rials and lipid components of liposomes. Interestingly,
some of the cyclodextrin protons at 3.65–3.70 ppm exhib-
ited much broader peaks in the dual liposomes com-
pared to Complex-loaded liposomes (Complex-Lips) and
cyclodextrin-loaded liposomes (Control-Lips). This sug-
gests disruption of Complex in the bilayers of Rif-
Complex-Lips, since this peak broadening occurred in the
signals assigned to protons H2 and H6 located outside
20 J. Biomed. Nanotechnol. 16, 14–28, 2020
Nkanga et al. Co-Loading of Isoniazid-Grafted Phthalocyanine-in-Cyclodextrin and RIF in Crude Soybean Lecithin Liposomes
90
80
70
60
50
API-Lecithin * RIF-CP
Interaction Plot for RIF EE%
Fitted Means
(a)
0.25:1 0.5:1 1:1
API–Lecithin ratio
0.25:1 0.5:1 1:1
API–Lecithin ratio
1
2
3
RIF-CP
0.25:1
0.5:1
1:1
60
50
40
30
20
10
API-Lecithin * RIF-CP
Mean of RIF EE%Mean of CP EE%
Interaction Plot for CP EE%
Fitted Means
(b)
1
2
3
RIF-CP
0.25:1
0.5:1
1:1
Figure 2. Effects of API-lecithin and RIF-CP mass ratios on
%EE of RIF (a) and CP (b). With CP corresponding to complex
(CD/PC-INH complex).
cyclodextrin cavity [56]. Surprisingly, all spectral changes
in the 1H resonance signals of the co-loaded liposomes
were found to be dependent on the API to lecithin mass
ratio, with formulations F1 and F7 showing the lowest
and highest spectral variations respectively (Fig. 5). This
observation appears consistent with the fact that the mean
size of the dual liposomes was found to be significantly
affected by the mass ratio of API to lecithin.
(a) (b) (c)
Figure 3. Typical TEM micrographs of the liposomes F1 (a), complex-lips (b) and Rif-lips (c).
Since the area under the resonance signal is related to
the number of protons inducing the signal, the increase in
peak intensity from the lipid acyl chains in the presence
of RIF and Complex suggests possible reorganization of
the lipid molecules within the liposome membranes. The
rearrangement of the acyl chains might have exposed more
protons to the applied magnetic field, leading therefore to
enhanced signal intensity with increased API to lecithin
mass ratio (F1 <F4 <F7).
Absorption and Emission Spectra
The evaluation of liposomal cargoes at molecular level
is crucial for characterization of the quality and proper-
ties of the final product, considering the direct impact of
drug stability on the clinical performance of therapeutic
liposomes. In the case of Rif-Complex-Lips particularly,
molecular investigations hold paramount importance due
to the complexity of the encapsulated cargoes (RIF and
Complex), which are both large aromatic molecules with
high electronic density (Fig. 1). Since remarkable interac-
tions between aromatic compounds via –stacking have
been observed before [57], we have considered exploring
the absorption and emission spectra of Complex and RIF
to provide insights into their electronic states and molecu-
lar stability following liposomal encapsulation.
The UV-Vis absorption spectroscopy of the mixture of
Complex and RIF in DMSO was performed to verify any
molecular interactions between the two species, since vari-
ations in their electronic transitions would have an impact
on UV-Vis spectral features [58]. Interestingly, the spec-
trum of Complex remained unchanged regardless of the
amounts of RIF used at different molar ratios (1:0 to 1:3
for Complex:RIF). Similar to the behaviour of Complex,
RIF absorption bands did not exhibit any noticeable shifts
(graphical data not shown). However, the UV-Vis absorp-
tion behaviour of Rif-Lips and Rif-Complex-Lips appeared
clearly different from that of raw RIF alone (Fig. 6).
While raw RIF showed a characteristic single absorption
band centred at 480 nm, the liposomal samples (Rif-Lips
and Rif-Complex-Lips) exhibited a distinctive blue-shift
band with a fine vibrionic ‘shoulder-like’ structure. This
behaviour was previously correlated to solvent effects [59],
but could indicate in this case a change in solubility of
the species arising from interactions between RIF and the
lipids used. Rif-Lips and Rif-Complex-Lips have shown
J. Biomed. Nanotechnol. 16, 14–28, 2020 21
Co-Loading of Isoniazid-Grafted Phthalocyanine-in-Cyclodextrin and RIF in Crude Soybean Lecithin Liposomes Nkanga et al.
Rif-Lips
Rif-Complex-Lips
Complex-Lips
Control-Lips
f1 (ppm)
4.1 4.0 3.9 3.8 3.7 3.6 3.5 3.4 3.3 3.2 3.1 3.0 2.9 2.8 2.7 2.6 2.5 2.4 2.3
4
3
2
1
Figure 4. 1H-NMR spectra of Rif-complex-lips and its liposomal counterparts.
similar electronic absorption spectra, suggesting absence
of noticeable aromatic interactions between RIF and Com-
plex co-encapsulated.
In addition, fluorescence emission properties of the
liposome samples were investigated to selectively verify
F7
F4
F1
3
2
1
f1 (ppm)
4.1 4.0 3.9 3.8 3.7 3.6 3.5 3.4 3.3 3.2 3.1 3.0 2.9 2.8 2.7 2.6 2.5 2.4 2.3
Figure 5. 1H-NMR spectra for Rif-complex-lips with different API-lecithin ratios (Table II).
the aromatic structure of Complex. Figure 7 shows that
Complex-Lips and Rif-Complex-Lips exhibit almost the
same fluorescence emission spectra, which confirms the
absence of major interactions between the co-encapsulated
species (Complex and RIF). However, the maxima of the
22 J. Biomed. Nanotechnol. 16, 14–28, 2020
Nkanga et al. Co-Loading of Isoniazid-Grafted Phthalocyanine-in-Cyclodextrin and RIF in Crude Soybean Lecithin Liposomes
350 375 400 425 450 475 500 525 550 575 600
0
1
2
Absorbance
Wavelength (nm)
480
RIF
Rif-Complex-Lips
Rif-Lips
Figure 6. Comparison of UV-Vis absorption spectra of raw
RIF and liposomal samples in DMSO.
emission bands for the two liposomal samples were found
to have shifted to lower wavelengths compared to that of
Complex raw material. Such blue-shift fluorescence spec-
tra for zinc phthalocyanines was previously correlated to
material concentration and solvent effects [60]. Similar
to the variations in the UV-Vis absorption exhibited by
liposomal RIF, the spectral changes in fluorescence emis-
sion of Complex from liposomal samples may be due to
its modified solubility in DMSO, because of molecular
interactions with the lipids. These photophysical observa-
tions strongly support the 1H-NMR data, suggesting that
at least some of RIF and Complex are embedded within
the lipid core of the liposomes. In sum, the outcome from
the absorption and emission spectroscopy of Complex and
RIF revealed some electronic variations due to the interac-
tions between the cargoes and lipids, confirming the prox-
imity between the encapsulated species and the liposome’s
membrane. Nevertheless, since all the characteristic bands
were observed in both UV-Vis and fluorescence spectra,
620 640 660 680 700 720 740 760 780 800
0.0
0.2
0.4
0.6
0.8
1.0
Normalized intensity
Wavelength (nm)
Complex
Rif-Complex-Lips
Complex-Lips
Figure 7. Comparison of emission spectra of raw complex
and liposomal samples in DMSO.
Time (hr)
02468101214
Cumulative release (%)
INH% in pH 6. 4
INH% in pH 7. 4
RIF% in pH 6.4
RIF% in pH 7.4
(a)
0
20
40
60
80
100
Time (hr)
02468101214
Cumulative release (%)
0
10
20
30
40
50
60
RIF% in pH 6. 4
RIF% in pH 7. 4
(b)
Figure 8. Drug release profiles of Rif-complex-lips (a) and
Rif-lips (b) in buffers of different pH.
it appears that there was no remarkable change in the
molecular structures of the cargoes upon liposomal encap-
sulation. Further molecular studies are underway in our
labs to better understand the chemical fate of large aro-
matic compounds in the liposomes.
Dual Drug Release
Drug release from Rif-Complex-Lips was monitored over
12 hours in media of two different pH specifically phos-
phate buffer at pH 6.4 and 7.4. These pH media were
selected for simulation of physiological conditions to ver-
ify whether the concept of liposomal pH-dependent release
of INH previously introduced [36] was maintained in the
Table III. Data from in vitro cytotoxicity assay.
Sample designation Viability ±standard deviation (%)
Complex 114.52 ±1.62
Complex-lips 111.60 ±4.31
Rif-complex-lips 109.37 ±11.94
Rif-lips 100.91 ±10.38
RIF 86.16 ±6.81
Control-lips 98.89 ±7.38
J. Biomed. Nanotechnol. 16, 14–28, 2020 23
Co-Loading of Isoniazid-Grafted Phthalocyanine-in-Cyclodextrin and RIF in Crude Soybean Lecithin Liposomes Nkanga et al.
presence of RIF. Figure 8(a) compares the release profiles
of INH and RIF from Rif-Complex-Lips in different pH
media. The two drugs have both shown higher release rate
in pH 6.4 than what observed in pH 7.4 media. Rapid
release of INH in acidic medium may be related to the
higher extent of cleavage of hydrazone bond in PC-INH
molecule [61], while faster release of RIF was likely due
to increased solubility in acidic conditions [62]. Such a
pH-dependent release is promising for possible targeted
intra-macrophage delivery throughout phagocytosis, where
engulfed liposomes are subsequently exposed to variable
acidic conditions, with pH 6.5–4 [63].
Surprisingly, the release profiles of RIF from Rif-Lips
at pH 6.4 and 7.4 appeared similar during the first 5 hours
of the experiment. After 7 hours of the experiment, a
distinctive burst release was observed in buffer of pH
6.4 (Fig. 8(b)). Nevertheless, the overall release kinet-
ics of Rif-Lips was found to be much slower than that
of Rif-Complex-Lips irrespective of pH of the media
used. This suggests that RIF was more deeply embedded
within the bilayers of Rif-Lips than in Rif-Complex-Lips,
which appears consistent with %EE data where competi-
tion between RIF and Complex was raised up in the co-
loaded liposomes (Rif-Complex-Lips).
In Vitro Cytotoxicity Data
Although most of the components of Rif-Complex-Lips
are known to be biocompatible, the presence of PC-INH
as a new molecule triggered the need to assess cytotoxicity
on HeLa cells using emetine as a positive control. At the
experimental concentration used (50 g/ml), none of the
liposomal formulations and raw products reduced cell via-
bility to <50% (Table III). This indicates the absence of
noticeable cytotoxicity and suggests good biocompatibility
of Rif-Complex-Lips, which was expected since phthalo-
cyanines are known to be nontoxic in the dark [64].
Figure 9. Micrographs of fibroblasts (A) and epithelial cells
(B) stained with NucBlue®and NucGreen®, indicating live and
dead cells with blue and green nuclei respectively.
Uptake of Liposomes by Airway Cells
Liposomal systems are well known for their potential to
ferry drug molecules to target cells and relevant subcel-
lular compartments. This is an essential prerequisite for
liposome cargoes to exhibit biological activities against
intracellular pathogens [65], like M. tuberculosis that often
resides inside alveolar macrophages [2]. In line with pH-
sensitive liposomes discussed in this work, cell uptake
is desired to take advantage of phagocytotic acidification
for pH triggered release [63]. Therefore, two human air-
way cell lines viz., peripheral lung fibroblasts and epithe-
lial cells, were investigated for potential internalization of
the developed dual liposomes (Rif-Complex-Lips). Though
alveolar macrophages (i.e., TB host cell) could have been
a better cell type in this case, the use of peripheral lung
fibroblasts and epithelial cells appears to be appropriate
since all three cell types respond similarly in vivo [66], and
they collectively represent the first line of cellular defense
against inhaled particles [8, 67].
Prior to actual cell uptake experiments, the airway cells
were treated with and without ZnCl2(10 g/ml) over
Figure 10. Images of airway epithelial cells without staining
reagents. The green dots correspond to the liposomes incu-
bated together with cells in the culture media over 24 hours.
24 J. Biomed. Nanotechnol. 16, 14–28, 2020
Nkanga et al. Co-Loading of Isoniazid-Grafted Phthalocyanine-in-Cyclodextrin and RIF in Crude Soybean Lecithin Liposomes
24 hours and visualized for intrinsic viability, as posi-
tive and negative viability controls respectively. As illus-
trated in Figure 9, there was significant cell death (green
nuclei) in positive control fibroblasts (5% viability) while
untreated cells showed negligible cell death (97% viabil-
ity), which confirmed the intrinsic viability of the fibrob-
lasts (blue nuclei). However, the epithelial cells showed
remarkable cell death in the presence and absence of the
cytotoxic standard (ZnCl210 g/ml), with about 0.5 and
61% of viability observed, respectively. Because of the
presence of dead cells (green nuclei), epithelial cells were
investigated for uptake without viability staining to avoid
visual interference with liposomal green dots.
Following incubation with increasing concentrations of
liposomal samples over 24 hours, microscopic obser-
vation revealed the presence of green dots inside the
cytosol of the airway cells used, suggesting success-
ful uptake of fluorescent liposomes [68, 69]. Figures 10
and 11 are representative micrographs of epithelial cells
and fibroblasts containing liposomes (green dots) in
Figure 11. Images of peripheral lung fibroblasts stained with
NucBlue®and NucGreen®, indicating live and dead cells with
blue and green nuclei respectively. The green dots correspond
to the liposomes incubated together with cells in the culture
media over 24 hours.
the cytosol irrespective of the sample concentration
used.
Since the fibroblasts were stained with live and dead
reagents, the microscopic visualization of the cell nuclei
(blue or green area) facilitated the ability to distinguish
live from dead cells in the presence of liposomes. Formu-
lations F1 and F4 showed no significant cell death in a
range 0.1 to 0.8 mg/ml and only a negligible decrease in
viability at 0.9 mg/ml (96% viability) or 1 mg/ml (94%
viability). However, the viability of cells treated with F7
dropped to <50% at concentrations between 0.7–1 mg/ml,
while the viability was >90% at concentrations between
0.1–0.6 mg/ml (Fig. 12). This could be related to the dif-
ference in Complex content between these liposomes as
they were made of increasing API to lecithin mass ratios
(Table II), which resulted in 10.8, 17.4 and 25.9% of
Complex content in F1, F4 and F7 respectively. Based on
the singlet oxygen quantum yield of Complex previously
reported [36], it stands to the reason that F7 exhibits some
cytotoxicity at high concentrations following illumination
under the microscope [41, 70]. This suggests the potential
of these liposomes for further “photodynamic antimicro-
bial chemotherapy,” which could be explored at a later
stage.
Nevertheless, this observation places formulation F1
(which also demonstrated the highest %EE, Table II) at
the forefront of the liposomal systems reported herein.
In addition to good cell viability, the observed uptake
by airway cells represents a promising feature for effi-
cient intracellular delivery of the liposomal cargo [65].
Although macrophages were not investigated, these pre-
liminary data permit rational prediction of the potential of
F1 liposomes to reach the TB infected sites (i.e., intra-
cellular media), since the cell lines used in this study are
known to exhibit much lower liposome internalization than
alveolar macrophages [71].
Liposomes concentration (mg/ml)
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1
Fibroblasts viability (%)
-10
0
10
20
30
40
50
60
70
80
90
100
110
F1
F4
F7
Figure 12. Estimated viability of peripheral lung fibroblasts
following incubation over 24 hours with liposome formulations
(F1, F4 and F7) at various concentrations (0.1–1.0 mg/ml).
J. Biomed. Nanotechnol. 16, 14–28, 2020 25
Co-Loading of Isoniazid-Grafted Phthalocyanine-in-Cyclodextrin and RIF in Crude Soybean Lecithin Liposomes Nkanga et al.
CONCLUSIONS
In summary, the inclusion complex of isoniazid-grafted
phthalocyanine with gamma-cyclodextrin (Complex) was
successfully co-encapsulated with rifampicin (RIF) in
crude soybean lecithin liposomes without the use of
organic solvents. The co-loaded liposomes (Rif-Complex-
Lips) demonstrated good %EE for such large and com-
plex molecules (58% for Complex and 86% for RIF).
In addition, Rif-Complex-Lips exhibited potentially use-
ful pH-dependent controlled release of the two drugs
incorporated (INH and RIF), which is worthy notic-
ing for potential targeted drug delivery to macrophages
following phagocytosis [48, 72]. Furthermore, prelimi-
nary bioassays demonstrated that Rif-Complex-Lips hold
promise for good biocompatibility and excellent pene-
tration through the biological membranes of lung cells.
This suggests that dual liposomes are promising for
effective and potentially safe controlled delivery of TB
drugs following pulmonary administration. However, fur-
ther studies exploring inhalable dosage forms based on
Rif-Complex-Lips would be necessary. Furthermore, it
would be interesting to interrogate other potential medic-
inal applications of Rif-Complex-Lips, most specially the
photodynamic antimicrobial chemotherapy of local infec-
tions such as cutaneous TB. This work illustrates the
potential of concomitant loading of large/complex com-
pounds with different hydrophobicity in liposomes under
organic solvent-free conditions. The findings suggest that
the full potential of crude soybean lecithin in liposo-
mal encapsulation processes is promising. Nevertheless,
extensive investigations exploring encapsulation of other
large or complex molecules using similar methods with
other types of lecithin (i.e., from rice or eggs) would pro-
vide greater insights and further development of versatile
biomedical liposomes at low cost.
Acknowledgments: The authors are grateful to the
National Research Foundation (NRF) of South Africa,
the South African Medical Research Council (MRC) with
funds from National Treasury under its Economic Com-
petitiveness and Support Package, and Rhodes University
Sandisa Imbewu for their financial support. The NGO
Förderverein Uni Kinshasa e. V.-BEBUC/Else-Kroener-
Fresenius Stiftung and Holger-Poehlmann foundation are
also sincerely acknowledged for their valuable advice.
REFERENCES
1. Maretti, E., Rossi, T., Bondi, M., Croce, M.A., Hanuskova, M.,
Leo, E., Sacchetti, F. and Iannuccelli, V., 2014. Inhaled solid lipid
microparticles to target alveolar macrophages for tuberculosis. Inter-
national Journal of Pharmaceutics, 462(1–2), pp.74–82.
2. T˘
ab˘
aran, A.F. and Catoi, C., 2014. Macrophages targeted drug deliv-
ery as a key therapy in infectious disease. Biotechnology, Molecular
Biology and Nanomedicine, 2, pp.2330–9326.
3. Hawn, T.R., Alastair, I.M., Stephen, N.M. and Omar, V., 2013.Host-
directed therapeutics for tuberculosis: Can we harness the host?
Microbiology and Molecular Biology Reviews, 77(4), pp.608–627.
4. Houben, E., Nguyen, L. and Pieters, J., 2006. Interaction of
pathogenic mycobacteria with the host immune system. Current
Opinion in Microbiology, 9(1), pp.76–85.
5. Zuniga, J., Torres-Garcıa, D., Santos-Mendoza, T., Rodriguez-Reyna,
T.S., Granados, J. and Yunis, E.J., 2012. Cellular and humoral mech-
anisms involved in the control of tuberculosis. Clinical and Devel-
opmental Immunology, 2012, pp.1–18.
6. Wickremasinghe, M.I., Thomas, L.H. and Friedland, J.S., 1999.Pul-
monary epithelial cells are a source of IL-8 in the response to
mycobacterium tuberculosis: Essential role of IL-1 from infected
monocytes in a NF-kB-dependent network. Journal of Immunology,
163, pp.3936–3947.
7. O’Kane, C.M., Boyle, J.J., Horncastle, D.E., Elkington, P.T. and
Friedland, J.S., 2007. Monocyte-dependent fibroblast CXCL8 secre-
tion occurs in tuberculosis and limits survival of mycobacteria within
macrophages. Journal of Immunology, 178(6), pp.3767–3776.
8. Bermudez, L.E., Sangari, F.J., Kolonoski, P., Petrofsky, M. and
Goodman, J., 2002. The efficiency of the translocation of Mycobac-
terium tuberculosis across a bilayer of epithelial and endothelial cells
as a model of the alveolar wall is a consequence of transport within
mononuclear phagocytes and invasion of alveolar epithelial cells.
Infection and Immunity, 70(1), pp.140–146.
9. Bermudez, L.E. and Goodman, J., 1996. Mycobacterium tuberculosis
invades and replicates within type II alveolar cells. Infection and
Immunity, 64(4), pp.1400–1406.
10. Mehta, P.K., King, C.H., White, E.H., Murtagh Jr., J.J. and Quinn,
F.D ., 1996. Comparison of in vitro models for the study of mycobac-
terium tuberculosis invasion and intracellular replication. Infection
and Immunity, 64(7), pp.2673–2679.
11. World Health Organization, 2018. Switzerland, WHO Report: Global
Tuberculosis Report.
12. Kaur, I.P. and Singh, H., 2014. Nanostructured drug delivery for
better management of tuberculosis. Journal of Controlled Release,
184,pp.36–50.
13. Mehanna, M.M., Mohyeldin, S.M. and Elgindy, N.A., 2014.Res-
pirable nanocarriers as a promising strategy for antitubercular drug
delivery. Journal of Controlled Release, 187, pp.183–197.
14. Costa-Gouveia, J., Aınsa, J.A., Brodin, P. and Lucıa, A., 2017.How
can nanoparticles contribute to antituberculosis therapy? Drug Dis-
covery Today, 22(3), pp.600–607.
15. Truzzi, E., Meneghetti, F., Mori, M., Costantino, L., Iannuccelli, V.,
Maretti, E., Domenici, F., Castellano, C., Rogers, S., Capocefalo,
A. and Leo, E., 2019. Drug/lamellae interface influences the inner
structure of double-loaded liposomes for inhaled anti-TB therapy:
An in-depth small-angle neutron scattering investigation. Journal of
Colloid and Interface Science, 541, pp.399–406.
16. Lila, A.S. and Ishida, T., 2017. Liposomal delivery systems: Design
optimization and current applications. Biological and Pharmaceuti-
cal Bulletin, 40(1), pp.1–10.
17. Nisini, R., Poerio, N., Mariotti, S., De Santis, F. and Fraziano, M.,
2018. The multirole of liposomes in therapy and prevention
of infectious diseases. Frontiers in Immunology, 9, p.155, DOI:
10.3389/fimmu.2018.00155.
18. Deol, P., Khuller, G.K. and Joshi, K., 1997. Therapeutic effi-
cacies of isoniazid and rifampicin encapsulated in lung-specific
stealth liposomes against mycobacterium tuberculosis infections
induced in mice. Antimicrobial Agents and Chemotherapy, 41(6),
pp.1211–1214.
19. Labana, S., Pandey, R., Sharma, S. and Khuller, G.K., 2002.
Chemotherapeutic activity against murine tuberculosis of once
weekly administered drugs (isoniazid and rifampicin) encapsulated
in liposomes. International Journal of Antimicrobial Agents, 20(4),
pp.301–304.
20. Rodrigues, C., Paula, G., Prieto, M. and Baltazar de, C., 2003.Inter-
action of rifampicin and isoniazid with large unilamellar liposomes:
Spectroscopic location studies. Biochimica et Biophysica Acta, 1620,
pp151–159.
26 J. Biomed. Nanotechnol. 16, 14–28, 2020
Nkanga et al. Co-Loading of Isoniazid-Grafted Phthalocyanine-in-Cyclodextrin and RIF in Crude Soybean Lecithin Liposomes
21. Justo, O.R. and Moraes, A.M., 2003. Incorporation of antibiotics
in liposomes designed for tuberculosis therapy by inhalation. Drug
Delivery, 10(3), pp.201–207.
22. Gürsoy, A., Kut, E. and Özkırımlı, S., 2004. Co-encapsulation of
isoniazid and rifampicin in liposomes and characterization of lipo-
somes by derivative spectroscopy. International Journal Pharmaceu-
tics, 271(1–2), pp.115–123.
23. Karki, R., Subramanya, G. and Udupa, N., 2009. Formulation and
evaluation of co-encapsulated rifampicin and isoniazid liposomes
using different lipids. Acta Pharmaceutica Sciencia, 51, pp.177–188.
24. Attar, A., Ceren Bakir, C., Yuce-Dursun, B., Demir, S.,
Cakmakci, E., Danis, O., Birbir, M. and Ogan, A., 2018.Prepa-
ration, characterization, and in vitro evaluation of isoniazid and
rifampicin-loaded archaeosomes. Chemical Biology & Drug Design,
91(1), pp.153–161.
25. Li, J., Wang, X., Zhang, T., Wang, C., Huang, Z., Luo, X. and
Deng, Y., 2015. A review on phospholipids and their main appli-
cations in drug delivery systems. Asian Journal of Pharmaceutical
Sciences, 10(2), pp.81–98.
26. Yokota, D., Moraes, M. and Pinho, S.C., 2012. Characterization
of lyophilized liposomes produced with non-purified soy lecithin:
A case study of casein hydrolysate microencapsulation. Brazilian
Journal of Chemical Engineering, 29(2), pp.325–335.
27. Rojanarat, W., Changsan, N., Tawithong, E., Pinsuwan, S., Chan,
H.K. and Srichana, T., 2011. Isoniazid proliposome powders for
inhalation-preparation: Characterization and cell culture studies.
International Journal of Molecular Sciences, 12(7), pp.4414–4434.
28. Randles, E.G. and Bergethon, P.R., 2013. A photodependent switch
of liposome stability and permeability. Langmuir, 29(5), pp.1490–
1497.
29. Zariwala, G.M., Bendre, H., Markiv, A., Farnaud, S., Renshaw, D.,
Taylor, K.M. and Somavarapu, S., 2018. Hydrophobically modified
chitosan nanoliposomes for intestinal drug delivery. International
Journal of Nanomedicine, 13, pp.5837–5848.
30. Nkanga, C.I., Walker, R.B. and Krause, R.W., 2018. pH-dependent
release of isoniazid from isonicotinic acid (4-hydroxy-benzylidene)-
hydrazide loaded liposomes. Journal of Drug Delivery Science and
Technology, 45, pp.264–271.
31. Pattni, B.S., Chupin, V.V. and Torchilin, V.P., 2015.Newdevel-
opments in liposomal drug delivery. Chemical Reviews, 115(19),
pp.10938–10966.
32. Maherani, B., Arab-Tehrany, E., Mozafari, M.R., Gaiani, C. and
Linder, M., 2011. Liposomes: A review of manufacturing techniques
and targeting strategies. Current Nanoscience, 7 (3), pp.436–452.
33. Nkanga, C.I., Noundou, X.S., Walker, R.B. and Krause, R.W.M.,
2019. Co-encapsulation of isoniazid and rifampicin in liposomes
using crude soybean lecithin. South African Journal of Chemistry,
72(1), pp.80–87.
34. Nkanga, C.I., Krause, R.W., Noundou, X.S. and Walker, R.B., 2017.
Preparation and characterization of isoniazid-loaded crude soybean
lecithin liposomes. International Journal of Pharmaceutics, 526(1),
pp.466–473.
35. Nkanga,C.I.andKrause,R.W.,2018. Conjugation of isoniazid to a
zinc phthalocyanine via hydrazone linkage for pH-dependent liposo-
mal controlled release. Applied Nanoscience, 8(6), pp.1313–1323.
36. Nkanga, C.I. and Krause, R.W., 2019. Encapsulation of
isoniazid-conjugated phthalocyanine-in-cyclodextrin-in-liposomes
using heating method. Scientific Reports, 9(1), p.11485,
DOI:10.1038/s41598-019-47991-y.
37. Walvekar, P., Gannimani, R. and Govender, T., 2018. Combina-
tion drug therapy via nanocarriers against infectious diseases. Euro-
pean Journal of Pharmaceutical Sciences, 127, pp.121–141, DOI:
10.1016/j.ejps.2018.10.017.
38. Yuen, C.M., Tolman, A.W., Cohen, T., Parr, J.B., Keshavjee, S.
and Becerra, M.C., 2013. Isoniazid-resistant tuberculosis in children:
A systematic review. Pediatric Infectious Disease Journal, 32(5),
pp.e217–e226.
39. Karantzas, C.A. and Jacobs, W.R.J., 2017. Origins of combi-
nation therapy for tuberculosis: Lessons for future antimicro-
bial development and application. mBio, 8(2), p.e01586-16, DOI:
10.1128/mBio.01586-16.
40. Patel, G., Chougule, M., Singh, M. and Ambikanandan, M., 2009.
Nanoliposomal dry powder formulations. Methods in Enzymology,
464, pp.167–191.
41. Zeballosa, N.C.L., García, Viorb M.C., Awruch, J. and Dicelio,
L.E., 2012. An exhaustive study of a novel sulfur-linked adaman-
tane tetrasubstituted zinc (II) phthalocyanine incorporated into lipo-
somes. Journal of Photochemistry and Photobiology A: Chemistry,
235, pp.7–13.
42. Costa, A.P., Xu, X. and Burgess, D.J., 2014. Freeze-anneal-thaw
cycling of unilamellar liposomes: Effect on encapsulation efficiency.
Pharmaceutical Research, 31(1), pp.97–103.
43. Shishoo, C.J., Shah, S.A., Rathod, I.S., Savale, S.S., Kotecha, J.S.
and Shah, P.B., 1999. Stability of rifampicin in dissolution medium
in presence of isoniazid. International Journal of Phar maceutics,
190(1), pp.109–123.
44. Singh, S., Mariappan, T.T., Shankar, R., Sarda, N. and Singh, B.,
2001. A critical review of the probable reasons for the poor variable
bioavailability of rifampicin from anti-tubercular fixed-dose com-
bination (FDC) products, and the likely solutions to the problem.
International Journal Phar maceutics, 228(1–2), pp.5–17.
45. Singh, S.A.R.A., Mariappan, T.T., Sharda, N.I.S.H. and Singh,
B.A.L.J., 2000. Degradation of rifampicin, isoniazid and pyrazi-
namide from prepared mixtures and marketed single and combina-
tion products under acid conditions. Phar macy and Pharmacology
Communications, 6(11), pp.491–494.
46. Sankar, R., Sharda, Singh and S., 2003. Behavior of decomposition
of rifampicin in the presence of isoniazid in the pH range 1–3. Drug
Development and Industrial Pharmacy, 29(7), pp.733–738.
47. Mariappan, T.T., Baljinder, S. and Saranjit, S., 2000. A validated
reversed-phase (C18) HPLC method for simultaneous determina-
tion of rifampicin, isoniazid and pyrazinamide in USP dissolution
medium and simulated gastric fluid. Pharmacy and Pharmacology
Communications, 6(8), pp.345–349.
48. Hwang, A.A., Lee, B.Y., Clemens, D.L., Dillon, B.J., Zink, J.I. and
Horwitz, M.A., 2015. pH-responsive isoniazid-loaded nanoparticles
markedly improve tuberculosis treatment in mice. Small, 11(38),
pp.5066–5078.
49. Zhang, Y., Gao, M., Chen, C., Wang, Z. and Zhao, Y., 2015. Residue
cytotoxicity of a hydrazone linked polymer-drug conjugate: Implica-
tion for acid-responsive micellar drug delivery. RSC Advances, 5(44),
pp.34800–34802.
50. Lambers, C., Roth, M., Jaksch, P., Muraközy, G., Tamm, M.,
Klepetko, W., Ghanim, B. and Zhao, F., 2018. Treprostinil inhibits
proliferation and extracellular matrix deposition by fibroblasts
through cAMP activation. Scientific Reports, 8 (1), p.1087.
51. Cavalcanti, I.M., Mendonça, E.A., Lira, M.C., Honrato, S.B.,
Camara, C.A., Amorim, R.V., Filho, J.M., Rabello, M.M.,
Hernandes, M.Z., Ayala, A.P. and Santos-Magalhães, N.S., 2011.
The encapsulation of b-lapachone in 2-hydroxypropyl-b-cyclodextrin
inclusion complex into liposomes: A physicochemical evaluation and
molecular modeling approach. European Journal of Pharmaceutical
Sciences, 44, pp.332–340.
52. Kelly, C., Jefferies, C. and Cryan, S., 2011. Targeted liposomal drug
delivery to monocytes and macrophages. Journal of Drug Delivery,
2011, pp.1–11, DOI 10.1155/2011/727241.
53. Fissan, H., Ristig, S., Kaminski, H., Asbach, C. and Epple, M.,
2014. Comparison of different characterization methods for nanopar-
ticle dispersions before and after aerosolization. Analytical Methods,
6(18), pp.7324–7334.
54. Timoszyk, A.. 2017. Application of nuclear magnetic resonance
spectroscopy (NMR) to study the properties of liposomes, liposomes,
edited by Angel Catala, InTech, DOI: 10.5772/intechopen.68522.
J. Biomed. Nanotechnol. 16, 14–28, 2020 27
Co-Loading of Isoniazid-Grafted Phthalocyanine-in-Cyclodextrin and RIF in Crude Soybean Lecithin Liposomes Nkanga et al.
55. Gabrielska, J., Gago´
s, M., Gubernator, J. and Gruszecki, W.I., 2006.
Binding of antibiotic amphotericin B to lipid membranes: A 1H
NMR study. FEBS Letters, 580(11), pp.2677–2685.
56. Pessine, F.B.T., Calderini, A. and Alexandrino, G.L., 2012.Review:
Cyclodextrin inclusion complexes probed by NMR techniques, mag-
netic resonance spectroscopy, edited by DongHyun Kim, ISBN: 978-
953-51-0065-2, InTech, Available from: http://www.intechopen.com/
books/magneticresonance-spectroscopy/review-study-ofinclusion-
complexes-with-cyclodextrins-by-mrs.
57. Hunter, C.A. and Sanders, J.K.M., 1990. The nature of –interac-
tions. Journal of the American Chemical Society, 14(14), pp.5525–
5534.
58. Achadu, O.J., Uddin, I. and Nyokong, T., 2016. Fluorescence behav-
ior of nanoconjugates of graphene quantum dots and zinc phthalo-
cyanines. Journal of Photochemistry and Photobiology A: Chemistry,
317, pp.12–25.
59. Hoeben, F.J.M., Jonkheijm, P., Meijer, E.W. and Meijer, A.P.H.J.,
2005. About supramolecular assemblies of -conjugated systems.
Chemical Reviews, 36(32), pp.1491–1546.
60. Durmus, M. and Nyokong, T., 2007. Synthesis and solvent effects
on the electronic absorption and fluorescence spectral properties of
substituted zinc phthalocyanines. Polyhedron, 26(12), pp.2767–2776.
61. Kalia, J. and Raines, R.T., 2008. Hydrolytic stability of hydrazones
and oximes. Angewandte Chemie International Edition, 47(39),
pp.7523–7526.
62. Gohel, M.C. and Sarvaiya, K.G., 2007. A novel solid dosage form of
rifampicin and isoniazid with improved functionality. AAPS Pharm-
SciTech, 8(3), pp.E133–E139.
63. Karanth, H. and Murthy, R.S.R., 2007. pH-sensitive liposomes—
Principle and application in cancer therapy. Journal of Phar macy
and Pharmacology, 59(4), pp.469–483.
64. Young, J., Yee, M., Kim, H., Cheung, J., Chino, T., Düzgüne¸s,
N. and Konopka, K., 2016. Phototoxicity of liposomal Zn- and
Al-phthalocyanine against cervical and oral squamous cell carci-
noma cells in vitro. Medical Science Monitor Basic Research, 22,
pp.156–164.
65. Hillaireau, H. and Couvreur, P., 2009. Nanocarriers’ entry into the
cell: Relevance to drug delivery. Cellular and Molecular Life Sci-
ences, 66(17), pp.2873–2896.
66. Takeshi, F., Shizu, H., James, C.H., Hiroshi, M., Tatsushi, S.,
Goto, Y., Vincent, R. and van Eeden, S.F., 2002. Interaction of alve-
olar macrophages and airway epithelial cells following exposure to
particulate matter produces mediators that stimulate the bone mar-
row. American Journal of Respiratory Cell and Molecular Biology,
27(1), pp.34–41.
67. Brandenberger, C., Rothen-Rutishauser, B., Mühlfeld, C.,
Schmid, O., Ferron, G.A., Maier, K.L., Gehr, P. and Lenz, A.G.,
2010. Effects and uptake of gold nanoparticles deposited at the
air-liquid interface of a human epithelial airway model. Toxicology
and Applied Pharmacology, 242(1), pp.56–65.
68. Usman, F., Khalil, R., Ul-Haq, Z., Nakpheng, T. and Srichana, T.,
2018. Bioactivity, safety, and efficacy of Amphotericin B nanomicel-
lar aerosols using sodium deoxycholate sulfate as the lipid carrier.
AAPS PharmSciTech, 19(5), pp.2077–2086.
69. Wang, Y., Zheng, K., Xuan, G., Huang, M. and Xue, J., 2018. Novel
pH-sensitive zinc phthalocyanine assembled with albumin for tumor
targeting and treatment. International Journal of Nanomedicine, 13,
pp.7681–7695.
70. Nombona, N., Maduray, K., Antunes, E., Karsten, A. and
Nyokong, T., 2012. Synthesis of phthalocyanine conjugates with
gold nanoparticles and liposomes for photodynamic therapy. Journal
of Photochemistry and Photobiology B: Biology, 107, pp.35–44.
71. Togami, K., Miyao, A., Miyakoshi, K., Kanehira, Y., Tada, H.
and Chono, S., 2015. Efficient delivery to human lung fibroblasts
(WI-38) of pirfenidone incorporated into liposomes modified with
truncated basic fibroblast growth factor and its inhibitory effect on
collagen synthesis in idiopathic pulmonary fibrosis. Biological and
Pharmaceutical Bulletin, 38(2), pp.270–276.
72. Bhardwaj, A., Grobler, A., Rath, G., Goyal, A.K., Jain, A.K. and
Mehta, A., 2016. Pulmonary delivery of antitubercular drugs using
ligand anchored pH-sensitive liposomes for the treatment of pul-
monary tuberculosis. Current Drug Delivery, 13(6), pp.909–922.
28 J. Biomed. Nanotechnol. 16, 14–28, 2020
