Conjugated and Entrapped HPMA-PLA Nano-Polymeric Micelles Based Dual Delivery of First Line Anti TB Drugs: Improved and Safe Drug Delivery against Sensitive and Resistant Mycobacterium Tuberculosis

Article abstract of DOI:10.1007/s11095-017-2206-3

Purpose: First line antiTB drugs have several physical and toxic manifestations which limit their applications. RIF is a hydrophobic drug and has low water solubility and INH is hepatotoxic. The main objective of the study was to synthesize, characterize HPMA-PLA co-polymeric micelles for the effective dual delivery of INH and RIF. Methods: HPMA-PLA co-polymer and HPMA-PLA-INH (HPI) conjugates were synthesized and characterized by FT-IR and ¹H–NMR spectroscopy. Later on RIF loaded HPMA-PLA-INH co-polymeric micelles (PMRI) were formulated and characterized for size, zeta potential and surface morphology (SEM, TEM) as well as critical micellar concentration. The safety was assessed through RBC’s interaction study. The prepared PMRI were evaluated through MABA assay against sensitive and resistant strains of M. Tuberculosis. Results: Size, zeta and entrapment efficiency for RIF loaded HPMA-PLA-INH polymeric micelles (PMRI) was 87.64?±?1.98?nm, ?19?±?1.93?mV and 97.2?±?1.56%, respectively. In vitro release followed controlled and sustained delivery pattern. Sustained release was also supported by release kinetics. Haemolytic toxicity of HPI and PMRI was 8.57 and 7.05% (p?

Full text of DOI:10.1007/s11095-017-2206-3

Pharm Res
DOI 10.1007/s11095-017-2206-3
RESEARCH PAPER
Conjugated and Entrapped HPMA-PLA Nano-Polymeric Micelles
Based Dual Delivery of First Line Anti TB Drugs: Improved
and Safe Drug Delivery against Sensitive and Resistant
Mycobacterium Tuberculosis
1
1
1
2
2
Seema Upadhyay & Iliyas Khan & Avinash Gothwal & Praveen K. Pachouri & N. Bhaskar
&
2
2
1
Umesh D. Gupta & Devendra S. Chauhan & Umesh Gupta
Received: 29 April 2017 /Accepted: 8 June 2017
#
Springer Science+Business Media, LLC 2017
ABSTRACT
respectively. MABA assay (cytotoxicity) based MIC values of
PMRI formulation was observed as ≥0.0625 and ≥0.50 μg/
mL (for sensitive and resistant strain). The microscopic analy-
sis further confirmed that the delivery approach was effective
than pure drugs.
Conclusions RIF loaded and INH conjugated HPMA-
PLA polymeric micelles (PMRI) were more effective
against sensitive and resistant M tuberculosis. The devel-
oped approach can lead to improved patient compliance
and reduced dosing in future, offering improved treat-
ment of tuberculosis.
Purpose First line antiTB drugs have several physical and
toxic manifestations which limit their applications. RIF is a
hydrophobic drug and has low water solubility and INH is
hepatotoxic. The main objective of the study was to synthe-
size, characterize HPMA-PLA co-polymeric micelles for the
effective dual delivery of INH and RIF.
Methods HPMA-PLA co-polymer and HPMA-PLA-INH
(
HPI) conjugates were synthesized and characterized by FT-
1
IR and H–NMR spectroscopy. Later on RIF loaded HPMA-
PLA-INH co-polymeric micelles (PMRI) were formulated and
characterized for size, zeta potential and surface morphology
(
SEM, TEM) as well as critical micellar concentration. The
.
safety was assessed through RBC’s interaction study. The pre-
pared PMRI were evaluated through MABA assay against
sensitive and resistant strains of M. Tuberculosis.
KEY WORDS co-polymeric micelles HPMA-PLA
.
.
conjugate MABA tuberculosis
Results Size, zeta and entrapment efficiency for RIF loaded
HPMA-PLA-INH polymeric micelles (PMRI) was
8
7.64 ± 1.98 nm, −19 ± 1.93 mV and 97.2 ± 1.56%, respec-
ABBREVIATIONS
tively. In vitro release followed controlled and sustained deliv-
ery pattern. Sustained release was also supported by release
kinetics. Haemolytic toxicity of HPI and PMRI was 8.57 and
Ar
Aromatic
CMC
DCC
DCM
EDC
ETH
HPI
Critical micelle concentration
N,N′-dicyclohexylcarbodiimide
Dichloromethane
N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide
Ethambutol
HPMA-PLA-INH conjugates
N(2-hydroxypropyl) methacrylamide
Isoniazid
7
.05% (p < 0.01, INH Vs PMRI; p < 0.0001, RIF Vs PMRI),
Electronic supplementary material The online version of this article
doi:10.1007/s11095-017-2206-3) contains supplementary material, which is
available to authorized users.
(
HPMA
INH
*
Umesh Gupta
MDR-TB Multidrug-resistant TB
umeshgupta175@gmail.com; umeshgupta@curaj.ac.in; http://
www.curaj.ac.in
PLA
PMRI
PYZ
RIF
Poly lactic acid
RIF loading in HPMA-PLA-INH conjugates
Pyrazinamide
1
2
Department of Pharmacy, School of Chemical Sciences and Pharmacy,
Central University of Rajasthan, Bandarsindri, Ajmer, Rajasthan 305817,
India
Rifampicin
SEM
TB
Scanning electron microscopy and
Tuberculosis
National JALMA Institute for Leprosy and other Mycobacterial Diseases,
Tajganj, Agra, Uttar Pradesh 282001, India
TEM
Transmission electron microscopy

Upadhyay et al.
INTRODUCTION
drug-resistant strains of M. tuberculosis. It is imperative to dis-
cover and develop potent small molecule inhibitors of
M. tuberculosis that have a novel mode of action and are active
against both drug-sensitive and drug-resistant TB (6). All the
above problems can be solved with nano-carrier based deliv-
ery such as using nanoparticles, dendrimers, liposomes and
polymeric micelles etc. Most of the nano-carriers are
nanosized synthetic molecules used to cross the biological bar-
rier. Nano-carriers are used to deliver the pharmaceutical
drugs because of their promising features over conventional
carriers i.e. (i) have ability to encapsulating the drugs without
any effect on it, (ii) release of drugs at target site under appro-
priate condition, (iii) to deliver the drugs actively or passively
to a target site (7). As the polymeric micelles are used as a
novel drug carrier for poorly water soluble drugs and serve
as targetable drug carriers. Polymeric micelles have attracted
significant attention in the field of drug and gene delivery due
to their excellent biocompatibility, low toxicity, enhanced
blood circulation time and ability to solubilize a large number
of drugs in their micellar core (8). Polymeric micelles have
nanometric size (size ranges from 10 to 200 nm) and long
circulation time. Polymeric micelles are an important and
attractive class of drug carriers, useful for the IV administra-
tion of hydrophobic drugs (9).
Micelles were synthesized from two polymers i.e. biocom-
patible and biodegradable block co-polymers and show func-
tional groups on their surfaces for attachment (10). The main
features of co-polymeric micelles are self-assembling of amphi-
philic block co-polymer molecules and encapsulation by
means of physical mixing instead of chemical conjugation
(11). Hydrophobic drug molecules can be incorporated in
the inner core of polymeric micelles and micelles inhibit the
inter-miceller aggregation of hydrophobic cores with a hydro-
philic outer shell layer which works as a barrier against inter-
miceller aggregation, so they maintain water solubility. The
advantage of polymeric micelles is that various chemical spe-
cies can easily binds with the micelles (12).
Tuberculosis (TB) is a lethal infectious respiratory disorder.
Almost 30% of the world population is affected with TB.
According to WHO reports, after the human immune defi-
ciency virus, TB is considered as the second leading cause of
death. TB is caused by different strains of gram negative (−ve)
bacteria i.e. Mycobacteria, in which Mycobacterium tuberculosis
being the most common strain which may affect any part of
the human body. More than 80% of infections are the cases of
pulmonary TB. Extra-pulmonary TB has also been reported
as a result of inhaled M. tuberculosis in the form of bacilli, most
of which exists in Asia and Africa compared to Europe and
American countries. Patients with supressed immunity are
more prone to tuberculosis infection. The main cause of tu-
berculosis is the (due to long course of therapy) poor patient
compliance and failure of uptake of the drug regimen due to
systemic toxicity (1). Worldwide, 9.6 million people are esti-
mated to have fallen ill with TB in 2014, in which 5.4 million
were men, 3.2 million were women and 1.0 million were chil-
dren (2). The first line treatment includes drugs such as
Rifampicin (RIF), Isoniazid (INH), Pyrazinamide (PYZ) and
Ethambutol (ETH). The 6-month regimen includes INH,
RIF, PYZ and ETH given daily or intermittently for 2 month,
followed by RIF and INH for 4 months’ treatment-limiting
side effects fluctuate with minimum severity. Mainly, INH and
RIF are given for the effective management of TB. INH is a
pyridine-4-carbohydrazide, a hydrazide derivative of
isonicotinic acid. Officially, INH is official drug in various
monographs such as, Indian Pharmacopoeia (IP), British
Pharmacopoeia (BP), United States Pharmacopoeia (USP),
Japanese Pharmacopoeia (JP) and European Pharmacopoeia
(EP). INH acts as bactericidal for the actively multiplying mi-
crobe and bacteriostatic for Bresting^ bacilli. INH is
metabolised by mycobacterial catalase-peroxidase into an ac-
tive metabolite, so INH acts as a prodrug. INH exhibits its
activity by inhibiting the bio-synthesis of mycolic acid (3). RIF
is a first line antibacterial drug. It is also a derivative of
rifamycin B, acts as an inhibitor of DNA dependent RNA
polymerase of bacteria by binding strongly to their beta sub-
units (4).
Long term administration of anti TB drugs leads to poor
patient compliance, which is the most common reason for
chemotherapy failure in TB. So, combined delivery of first
line anti TB drugs can help to minimise toxicity as well as to
improve patient compliance. The combined delivery is also a
good strategy, as well as it is aimed to be minimise the dosing
frequency of anti TB drugs through the administration of
multidrug therapy for better patient compliance (5). The
emergence of multidrug resistant TB (MDR-TB) and exten-
sively drug resistant TB (XDR-TB) have been seen from last
two decades. There is an urgent need for drugs that can short-
en the duration of TB chemotherapy and are active against
In the present work, we attempted to formulate HPMA-
PLA co-polymeric micelles of isoniazid and rifampicin (Fig.
1a; schematic representation) for dual delivery at the same
time against different strains of M. tuberculosis. The manuscript
also reported the related evidences of better effectivity of the
prepared formulation over the naïve drugs.
MATERIAL AND METHODS
Materials
INH and RIF were obtained as gift samples from Kwality
Pharmaceutical Limited, Amritsar, Punjab, India. PLA (poly
lactic acid) was also a gift sample obtained from Evonik
Industries, Germany. Dichloromethane (DCM) and sodium

HPMA-PLA Polymeric Micelle for Dual Delivery of Anti- TB Drugs
R Fig. 1 (a) Schematic representation of formation of RIF loaded HPMA-PLA-„
INH (PMRI) co-polymeric micelles. [INH: Isoniazid, RIF: Rifampicin]. (b)
Synthetic scheme of HPMA-PLA co-polymer. (c) Synthetic scheme of
conjugation of INH with HPMA-PLA co-polymer.
hydrogen carbonate were purchased from CDH chemicals,
India. N,N′-dicyclohexylcarbodiimide (DCC), N-(3-
dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) anhy-
drous and sodium sulfate were procured from Hi-Media,
India. DMAP (4-dimethylaminopyriine), dialysis membrane
(
5 kDa, MWCO) were procured from Hi-media
Laboratories, Mumbai, India. 1-aminopropan-2-ol and
methacryloyl chloride were procured from Alfa Aesar. The
inoculum was provided by the Japanese Leprosy Mission for
Asia (JALMA), Agra and 96 well plates were purchased from
Tarsons, India. All the other chemicals, solvents and reagent
were of analytical grade and were used as such without further
purification.
Synthesis and Characterization of [N
(
2-Hydroxypropyl) Methacrylamide] (HPMA)
HPMA was synthesized following previously reported method
13). Briefly, anhydrous sodium hydrogen carbonate (1 g,
1.9 mmol) and 1-aminopropan-2-ol (0.89 g, 11.9 mmol) so-
lution was dissolved in dichloromethane (DCM) and cooled at
°C. Methacryloyl chloride (1.2 g, 11.9 mmol) in DCM was
(
1
0
added drop-wise under cooling and vigorous stirring for 1 h.
The reaction mixture was again stirred for 30 min. at 15°C,
then anhydrous sodium sulfate (1.6 g, mmol) was added, the
mixture was filtered and dried. Under reduced pressure, the
dry filtrate was concentrated approximately to half of its vol-
ume. By crystallization at −20°C, HPMA was procured from
DCM and from acetone the pure HPMA was obtained by
recrystallization. The characterization of HPMA was done
1
by FT-IR and H–NMR spectroscopy. FT-IR analysis was
done by using KBr pellet method (Perkin Elmer spectropho-
tometer, M/s Perkin Elmer Co., Waltham, Massachusetts,
1
USA). Using CDCl as a solvent, proton H–NMR spectros-
3
copy was performed in Bruker Ascend 500 MHz NMR spec-
1
trometer, Switzerland. H–NMR spectrum was recorded con-
1
sidering 7.269 ppm for H–NMR as internal standard.
Synthesis and Characterization of HPMA-PLA
Conjugates
Poly-L-lactic Acid (PLA) (500 mg, 0.05 mmol) was dissolved in
DCM (5 mL) with HPMA (7.1 mg, 0.05 mmol) in the excess of
DCC (10.31 mg, 0.05 mmol). The mixture was stirred for 24 h
in the presence of DMAP (6.1 mg, 0.05 mmol) converted into
HPMA-PLA conjugates (Fig. 1b). The conjugation was
dialysed in 5 kDa membrane for 24 h to remove the uncon-
jugated reactants (14). The characterization of HPMA-PLA

Upadhyay et al.
co-polymer was done by FT-IR using KBr pellet method
Perk, M/s Perkin Elmer Co., Waltham, Massachusetts,
USA). H–NMR spectroscopy was performed in Bruker
PMRI was done by size analysis, polydispersity index, zeta
potential and surface morphology (SEM, TEM)
(
1
Ascend 500 MHz NMR spectrometer, Switzerland using
1
CDCl as a solvent. H–NMR spectrum was recorded con-
3
1
sidering 7.269 ppm for H–NMR as internal standard.
CHARACTERIZATION
Size, Zeta Potential and Polydispersity Index
Synthesis and Characterization of HPMA-PLA-INH
(
HPI) Conjugates
Photon correlation spectroscopy (Nano ZS, Malvern, UK)
was used to determine the mean particle size, zeta potential
and polydispersity index of PMRI. Briefly, the samples were
diluted appropriately using deionized water, measurement of
the samples was performed in triplicate with fixed angle of 90°
at 25°C temperature with the help of electrophoretic cell (18).
HPMA-PLA (100 mg, 0.009 mmol) was dissolved in DCM
5 mL) in the presence of DMAP (1.099 mg, 0.009 mmol)
(
followed by the addition of INH (1.23 mg, 0.009 mmol)
dropwise under nitrogen atmosphere at room temperature,
then EDC (1.39 mg, 0.009 mmol) was added and mixture
was stirred for 24 h. The conjugated INH with co-polymer
was synthesized (Fig. 1c) and characterized (15). The conju-
gation of INH with HPMA-PLA co-polymer was character-
ized by FT-IR using KBr pellet method (Perk, M/s Perkin
Elmer Co., Waltham, Massachusetts, USA) and H–NMR
spectroscopy using CDCl as a solvent, proton NMR spectros-
copy was performed in Bruker Ascend 500 MHz NMR spec-
trometer, Switzerland. H–NMR spectrum was recorded con-
Surface Morphology & Electron Microscopy
1
Scanning electron microscopy (SEM) and Transmission elec-
tron microscopy (TEM) techniques were used to determine the
shape, size and morphology of the prepared PMRI micelles.
SEM analysis was performed at Malaviya National Institute of
Technology (MNIT), Jaipur, Rajasthan, India (Nova Nano
SEM 450, FEI) (19). TEM analysis was performed using cop-
per grids stained with phosphotungstic acid solution (2%). A
drop of diluted sample was placed on this copper grids for
3
1
1
sidering 7.269 ppm for H–NMR as internal standard.
Critical Micellar Concentration (CMC)
3
0 s and dried at room temperature and observed in electron
To determine the CMC, iodine was used as a hydrophobic
2
microscope (FEI, Japan, Model techani G ) at Sophisticated
Advanced Instrument Facility, All India Institute of Medical
Science (SAIF, AIIMS), New Delhi, India (20).
probe. Briefly, standard solution of KI/I was prepared by
2
dissolving iodine (250 mg) and potassium iodide (500 mg) in
deionized water (25 mL). A set of dilutions of HPMA-PLA co-
polymer in deionized water were prepared. In each dilution,
2
5 μL of KI/I standard solution was added, the mixture was
2
Entrapment Efficiency and Drug Loading
kept into the dark for 12 h at room temperature, than absor-
bance of dilutions was recorded in UV-Visible spectropho-
tometer (Labtronics, India) at 366 nm. The graph was plotted
between absorption intensity and polymer-mass concentra-
tion. The concentration of the polymer where there is sharp
increase in absorbance was detected and considered as CMC
value (16).
Drug entrapment efficiency and drug loading were deter-
mined by UV-Vis spectroscopy. Weighed amount of PMRI
was dissolved in phosphate buffer (PBS pH 7.4) and kept
overnight for continuous shaking at room temperature follow-
ed by centrifugation. After centrifugation, supernatant liquid
was absorbed at 262 and 334 nm by UV-Vis spectroscopy.
Drug entrapment efficiency and drug loading efficiency were
expressed as the percentage of drug amount in co-polymeric
micelles to the initial feed drug quantity (21,22).
Rifampicin Loading in HPMA-PLA-INH Conjugates
(PMRI)
Rifampicin loaded HPMA-PLA-INH (PMRI) micelles were
prepared by direct dissolution method. Briefly, the formation
is induced by adding two solutions to suitable drug-polymer
ratio. This process involves dissolving the HPI conjugates
along with the drug in distilled water (10 mL). RIF (10 mg)
dissolved in acetonitrile and was added dropwise in aqueous
solvent, the mixture of solutions was stirred for 24 h in round
bottom flask at room temperature (17). Characterization of
%
%
Entrapment efficiency
h
.
i
¼
Mass of drug in micelles Mass of feed drug  100
Loading efficiency
h
.
i
¼
Weight of drug in micelles Weight of micelles  100

HPMA-PLA Polymeric Micelle for Dual Delivery of Anti- TB Drugs
In Vitro Drug Release and Mathematical Kinetic
Modelling
Leprosy Mission for Asia (JALMA) Institute of Leprosy and
Other Mycobacterial Diseases, Agra, India.
In this study, to minimize evaporation of the medium, ster-
ile deionized water (200 μL) was added all outer-perimeter
wells of sterile 96-well plates during incubation. 7H9GC broth
(100 mL) was added in rows B to G in columns 3 to 11 of wells.
Drug solutions (100 μL) were added to the wells in rows B to G
in columns 2 and 3. From column 3 to column 4, by using a
multichannel pipette, 100 mL was transferred and mixed
properly. Through column 10, serial dilutions (1:2) were con-
tinued and excess medium from column 10 was withdrawn.
By using an eppendorf repeating pipette, M. tuberculosis inoc-
ulum (100 μL) was added in B to G rows of columns 2 to 11.
Now, column 11 was worked as drug-free (inoculum-only)
controls. With the help of parafilm, the plates were sealed
and incubated for 5 days at 37°C. Freshly prepared mixture
of Alamar Blue (Accumed International, Westlake, Ohio) re-
agent and 10% Tween 80 (50 μL) was added to B11.
Reincubation of plates was done for more 24 h at 37°C, then
the colour change of wells was observed, if the well showed
pink colour from blue, in microplate, all the wells were filled
with reagent mixture and again incubated for 24 h at room
temperature. After incubation, the colour change of wells was
evaluated. Pink colour indicated the the growth while the blue
colour showed no growth of the respective MTBs. If a well
showed violet colour, it turned pink after another day of incu-
bation. The lowest drug concentration which prevented a col-
our change from blue to pink, was considered as MIC and the
obtained results were further confirmed through microscopic
examination (24).
The in-vitro release of INH and RIF from PMRI micelles was
performed in phosphate buffer at pH 7.4 using membrane
dialysis method. Briefly, 2 mL of drug loaded micelles were
suspended in dialysis membrane (MWCO; 50 kDa, Hi-media)
and kept in 100 mL of buffer at room temperature with con-
tinuous stirring at 100–150 rpm (Remi, India). After a
predetermined time interval, 2 mL of PBS solution was with-
drawn and replaced with the equal volume of fresh PBS buff-
er. Withdrawn samples were scanned at 262 and 334 nm, the
amount of released INH and RIF was determined based on
the absorbance. The release of drugs (INH and RIF) was
compared using UV-Vis spectroscopy graphs (15,19).
Five different kinetic models like zero order, first order,
Higuchi, Hixon Crow-well and Korsmeyer-Peppas model
were used to study the release kinetic model. The obtained
in-vitro drug release data was fitted in five different kinetic
models.
Ex-Vivo Hemolytic Toxicity
Hemolytic toxicity of prepared PMRI micelles and drugs were
executed. Briefly, the whole blood was collected from a
healthy volunteer (5 mL) and stored in EDTA anticoagulant
storage vial. Then, RBCs were separated using centrifugation
at approximately. 1350 rpm (R-4C DX, REMI, India) and
suspended in normal saline (0.9% w/v). In normal saline,
different-different samples (20 ppm) were prepared, each sam-
ple in 1 mL quantity was added to the RBC’s. The mixture
was stirred continuously for 1 h and samples were centrifuged
and supernatant was taken and diluted with saline.
Absorbance was observed at wavelength of 540 nm. As a
control, distilled water was used to RBC’s suspension for
which hemolytic value was considered as 100%.
Statistical Data Analysis
All the experiments were performed at least three times.
Student’s t test was used for statistical treatment of data using
GraphPad Prism (version 7.0, CA, USA) software, with
p < 0.05 considered to be significant differentiate.
h
.
i
%
Hemolysis ¼ Ab AB100 Â 100
s
RESULTS AND DISCUSSION
Where Ab is the absorbance of the sample and AB is the
s
100
absorbance of control without formulation (23). Microscopic
study of hemolysed RBCs was performed using fluorescence
microscope at 40x (Olympus Inverted Fluorescence
Microscope CKX53, Japan).
Synthesis and Characterization of HPMA
HPMA was synthesized and characterized by FT-IR and pro-
1
ton ( H) NMR spectroscopy. The obtained spectra confirmed
the formation of HPMA. In FT-IR, a peak of amide bond
−
1
Cytotoxicity and Microscopy
detected at 1653.04 cm . Another peak was observed at
−
1
3
3
299.74 cm for sp carbon (alkyl/methyl group), and at
−1
3
Cytotoxicity studies of prepared PMRI micelles were per-
formed to check the anti-TB potential of prepared nano for-
mulations. The cytotoxicity of drug loaded formulation was
evaluated through MABA (Microplate-Based Alamar Blue
Assay). The study was performed at National Japanese
2974.29 cm for sp carbon (alkenes). HPMA showed the
characteristic shift for proton of amide at 7.7 ppm (s,1H,
−CO-NH-), 5.3 ppm of alcohol (s, 1H, −OH) and confirmed
the presence of methyl at 1.2 ppm (t, 3H, −C-CH ) protons,
respectively (Supporting data Fig. S1, Fig. S2).
3

Upadhyay et al.
1
Synthesis and Characterization of HPMA-PLA
Conjugates
ester bond and at 3383 cm− that confirmed the presence of
amide (N-H). Amide (C = O) and alkene (C = C) peaks were
−1
observed at 1660 and 1624 cm , respectively, (Fig. 2a). In FT-
IR spectra, all the observed peaks confirmed the conjugation of
HPMA-PLA. Like FT-IR spectroscopy, the conjugation of
HPMA-PLA co-polymer was further confirmed through proton
HPMA-PLA co-polymer was synthesized and characterized by
1
FT-IR and proton H–NMR spectroscopy. FT-IR spectroscopy
−1
conforms the conjugation, 1747 cm , showed the presence of
1
nuclear magnetic resonance ( H–NMR) spectroscopy. The con-
jugation of HPMA-PLA signified the characteristic shift at
8
.262 ppm (s, 1H, −CO-NH-) which reveals that proton in am-
ide group on structural moiety. Chemical shift at 4.194 ppm (m,
H, −CH) indicates the presence of methine group which con-
1
jugated with α -O-C = O (ester group) that conforms formation
of ester bond (Fig. 2b). Chemical shift of amide proton of
HPMA group was deshielded might be coupling with PLA and
formed an ester bond in HPMA-PLA conjugation.
Synthesis and Characterization of HPMA-PLA-INH
(
HPI) Conjugates
The conjugation of INH with HPMA-PLA co-polymer was
1
synthesized and characterized by FT-IR and proton H–
NMR spectroscopy. Major peak of conjugated formulation ob-
−1
served at 1648 cm i.e. a weak peak, refers to C = N (imines)
−1
and at 2931 cm that confirmed the presence of C-H (alkanes)
Fig. 2a). Further confirmation of conjugation of INH with co-
polymer was done through proton nuclear magnetic resonance
(
1
1
(
H–NMR) spectroscopy. The H–NMR proton spectrum of
HPI conjugation showed chemical shift at 8.262 ppm (s, 1H,
CO-NH-) for amide proton of HMPA-PLA conjugate and
−
chemical shift at 4.194 ppm (m, 1H, -CH-) exhibiting the CH
proton of HPMA and moreover, characteristics chemical shift
at 7.526 (d, 2H, −Ar CH) (J = 5.5)Hz and 7.714 (d, 2H, −Ar
CH) (J = 5.5)Hz for the -CH group of 4-pyridine moiety (an
aromatic ring) revealed confirmation for the conjugation of
INH to the HMPA-PLA co-polymer (Fig. 2c). INH also con-
1
tains an amide group, hence H–NMR spectrum displayed two
chemical shifts for amidic proton, while in HMPA-PLA conju-
gation, it shows chemical shift only for -NHCO- group.
Critical Micellar Concentration (CMC)
CMC of the co-polymeric micelles was determined by iodine
method in which iodine was used as a hydrophobic probe.
CMC of the co-polymeric micelle affect its in vivo and in vitro
stability. Conversion of I into I (in the excess of KI) felicitat-
3
2
ed I solubilization in the hydrophobic microenvironment of
2
the synthesized HPMA-PLA co-polymer. It maintained the
concentration of I and the graph of absorption intensity of
2
I2 was plotted between concentration (μg/mL) and absor-
bance in Fig. 3 and the CMC value was obtained 35 μg/mL
and at this concentration, the micelles were formed (16,25).
The result of critical micellar concentration of HPMA-PLA
co-polymeric micelles was reported for the first time.
Fig. 2 (a) FT-IR spectra of; A naïve INH, B HPMA-PLA conjugates, C
1
HPMA-PLA-INH (HPI) conjugates. (b) H–NMR spectrum of HPMA-PLA
1
conjugate. (c) H–NMR spectrum of HPMA-PLA-INH (HPI) conjugate.

HPMA-PLA Polymeric Micelle for Dual Delivery of Anti- TB Drugs
Entrapment Efficiency and Drug Loading
RIF entrapment efficiency of PMRI was 97.2 ± 3.56% while
loading was 32.4 ± 1.56% and INH conjugation efficiency
was observed 71.54 ± 1.23%. These results are supported
by the earlier reported studies of polymeric micelles (26), as
the RIF is a hydrophobic drug and it is good to have higher
content of PLA in co-polymer for entrapment of RIF in mi-
celles. RIF can be easily loaded in the hydrophobic core due to
hydrophobic interactions. The compatibility of polymeric mi-
celles hydrophobic core and hydrophobic drug RIF was
depended on PLA side chain.
Fig. 3 CMC plot as a function of copolymer concentration (HPMA-PLA) in
μg/mL.
Size, Polydispersity Index and Zeta Potential
In Vitro Drug Release and Kinetic Modelling
The mean size and zeta potential of drug loaded co-polymeric
micelles (PMRI) were 87.64 ± 1.98 nm and −19 ± 1.93 mV
and mean size and zeta potential of HPMA-PLA-INH conju-
gates (HPI) was 111.2 ± 2.34 nm and −45.3 ± 2.30 mV, re-
spectively. The size of RIF loaded co-polymeric micelles can
be observed in Table 1. The PMRI and HPI conjugates
showed a larger size.The findings indicated the conjugation/
encapsulation of both the bio-actives within the micelles.
Polydispersity index of PMRI and HPI were obtained
In vitro release study of prepared formulations (HPI and
PMRI) and pure drugs (INH and RIF) was performed by
membrane dialysis method. The results of the study revealed
that, pure INH and RIF were released in a burst manner in
approximately 2 h and after that concentration was constant
while the release of conjugated INH and loaded RIF from
micelles was observed more than 36 h and after that concen-
tration was constant (Fig. 5a). Loaded RIF released faster
from micelles in comparison to the conjugated INH in
HPMA-PLA-INH. INH have hydrazine group which was co-
valently conjugated with PLA. INH release will lead to bond
breakage (15), while loaded RIF was released as burst from co-
polymeric micelles. The comparative study is shown in Fig.
0
.374 ± 0.17 and 0.546 ± 0.10 which represents that how
broad the distribution is around the mean particle size (26).
Surface Morphology & Electron Microscopy
5b. Result of release of RIF was supported by the previous
The morphology of PMRI was observed by SEM and TEM.
The micelles were formed to have uniform surface
morphology with spherical shape. SEM images confirmed
about the homogenous nature of particles with a uniform
distribution (19) (Fig. 4a). TEM micrographs further con-
firmed the size and distribution as well as morphology of mi-
celles (Fig. 4b). The results were supported by the earlier
studies, it explained that the size of RIF loaded co-polymeric
micelles was less than RIF free co-polymeric micelles (27). The
average particle size of HPI and PMRI were observed around
reported results (19) that explained release rate of RIF was fast
in the initial stage and the rate of release of RIF slowed down
at later time in the encapsulated form. The rate of release was
also reduced due to increased PLA contents.
The in vitro release of drug from PMRI micelles was easily
explained by Korsmeyer-Peppas model (Fig. 5c), the mathe-
matical linear kinetic modelling presented highest linearity
2
[r = 0.8197, INH) & 0.8803, RIF], described in Table S1
Korsmeyer-Peppas model described the magnitude of release
exponent BN^ which explained that the release mechanism
was case-II relaxational release having stresses in hydrophilic
polymers which swell in water or biological fluids (28).
1
00 nm.
Ex-Vivo Hemolytic Toxicity
Tab le 1 Comparative data for the particle size, zeta potential, polydisper-
sity index, and entrapment efficiency of optimized formulations [HPI: HPMA-
PLA-INH conjugates, PMRI: Rifampicin loaded HPMA-PLA-INH co-polymeric
micelles]
Hemolytic toxicity of the prepared co-polymeric micelles, con-
jugates and pure drugs was performed. The results obtained
showed that the toxicity of PMRI was reduced in comparison
with free drugs (INH, RIF). The hemolytic toxicity of RIF and
INH were observed to be 19.6% and 14.1% (p < 0.005, INH
vs HPI), respectively, while the toxicity of HPI and PMRI were
observed 8.57% and 7.05% (p < 0.01, INH vs PMRI;
p < 0.0001, RIF vs PMRI), respectively, which is less than half
the toxicity of pure drugs (Fig. 6a). To reduce the toxicity of
Formulation Average
Zeta Potential Polydispersity Entrapment
Code
Particle Size
nm)
(mV)
Index (pdi)
Efficiency
(%)
(
HPI
111.2 ± 2.34 −45.3 ± 2.30 0.546 ± 0.10 –
PMRI
87.64 ± 1.98 −19.0 ± 1.93 0.374 ± 0.17 97.2 ± 1.56
micelles

Upadhyay et al.
Fig. 4 (a) Scanning electron (SEM)
photomicrograph of; A HPMA-PLA
conjugates, B HPMA-PLA-INH
a
b
(
HPI) conjugates, C rifampicin
loaded HPMA-PLA-INH (PMRI)
co-polymeric micelles (The scale
bar represents 1 μm). (b)
Transmission electron (TEM) pho-
tomicrograph of; A HPMA-PLA
conjugates, B HPMA-PLA-INH
(
HPI) conjugates, C Rifampicin
loaded HPMA-PLA-INH (PMRI)
co-polymeric micelles (scale bar
=
200 nm).
drug, HPMA acted as a shell forming segment because
of its physicochemical properties including high water
solubility, low toxicity as well as having multiple func-
tional groups. As per previous results of nanoparticulate
formulations of antineoplastic drugs the hemolytic toxic-
ity can be reduced because of reduced direct contact of
the drugs with RBCs. The results revealed that RIF is
more hemolytic than the INH and block copolymer as
well, but in the micellar form RIF was masked within the
hydrophobic environment of micelles leading to the re-
duced hemolytic toxicity (18,29,30).
Microscopic Analysis RBC Hemolysis
The results obtained were further confirmed through mi-
croscopic examination at 10 and 40X magnification
c
a
1
00
9
8
7
6
5
4
3
2
1
0
0
0
0
0
0
0
0
0
0
INH-MICELLE
RIF-MICELLE
Pure INH
RIF
0
4
8
12
16
20
24
28
32
36
40
Time (h)
b
(
INH, 262nm)
(
RIF, 334nm)
Fig. 5 (a) In vitro drug release from drug loaded co-polymeric micelles (HPI & pure drug) at PBS buffer pH 7.4 (n = 3). [INH: Isoniazid, RIF: Rifampicin, HPI:
HPMA-PLA-INH conjugates, PMRI: Rifampicin loaded HPMA-PLA-INH co-polymeric micelles]. (b) Comparative in vitro of INH and RIFin PBS buffer at pH 7.4 in
UV-Vis spectroscopy. (c) Graphical representation of Korsmeyer-Peppas kinetic release model for;(A) INH and (B) RIF.

HPMA-PLA Polymeric Micelle for Dual Delivery of Anti- TB Drugs
Fig. 6 (a) Percent hemolysis of
a
represents mean ± SD (n = 3).
25
20
various formulations. Values
****
[
INH: Pure Isoniazid, RIF: Pure
**
Rifampicin, HPI: HPMA-PLA-INH
conjugates, PMRI: Rifampicin
**
1
5
loaded HPMA-PLA-INH co-
polymeric micelles]. * indicates less
significant difference with p
value = 0.0122 (HPI Vs PMRI). **
indicates significant difference with p
value = 0.0014 (INH Vs HPI). **
indicates significant difference with p
value = 0.0098 (INH Vs PMRI).
*
10
5
0
RIF
INH
HPMA-PLA
HPI
PMRI
*
*** indicates extremely significant
difference with p value = <0.0001
RIF Vs PMRI). (b) Microscopic
Drug/Formulation
(
images of drugs as well as
b
formulation effected RBC’s at 40X.
A Pure RBC’s B Distilled water
treated RBC’s C INH treated RBC’s
D RIF treated RBC’s E HPI treated
RBC’s & F PMRI treated RBC’s.
(A)
(B)
(C)
[
INH: Pure Isoniazid, RIF: Pure
Rifampicin, HPI: HPMA-PLA-INH
conjugates, HPI: HPMA-PLA-INH
conjugates, PMRI: Rifampicin
loaded HPMA-PLA-INH co-
polymeric micelles].
(D)
(E)
(F)
under microscope. The microscopic study of samples (pre-
pared for hemolytic study) was done under Olympus
Inverted Fluorescence Microscope CKX53, Japan. Pure
drugs (INH and RIF) ruptured the RBC’s and distorted
RBC’s shape. It led to increased toxicity in blood while
drug loaded co-polymeric micelles showed very less toxic-
ity because of polymer (HPMA) which is more biocom-
patible can be seen (Fig. 6b).
sensitive and 0.50 ± 0.08 μg/mL for resistant strain
(p < 0.0001, sensitive MIC Vs resistant MIC in MABA)
i.e. less than the pure drugs. In sensitive, PMRI was 2
folds more effective than pure INH and 8 folds than pure
RIF, while in resistant, PMRI was 4 folds more effective
than pure INH and 2 folds than pure RIF and the com-
parison shown in Fig. 7b. The obtained results were fur-
ther analysed with the help of microscopic examination of
M. tuberculosis at treated MIC of pure drugs (INH and
RIF) as well as HPI and PMRI by fluorescence micro-
scope (Olympus Inverted Fluorescence Microscope
CKX53, Japan). The results showed that the growth of
M. tuberculosis was inhibited by the HPI and PMRI signif-
icantly (Fig. 7c) in comparison to pure drugs because of
polymeric micellar nanocarrier.
The microscopic results clearly showed that the pre-
pared formulations were more effective in comparison to
pure anti TB drugs (Fig. 7d). It was clearly observed from
the cytotoxicity assay that potency of micelles were higher
than pure INH and RIF. This result confirmed that drug
delivery through multidrug therapy will be beneficial
along with reduce dosing frequency for patient compli-
ance. Hence, it can be proved that co-delivery of drugs
through same polymeric micelles can be an effective strat-
egy to deliver anti-TB drugs.
Cytotoxicity Studies, Microscopy and Anti-TB Assay
MABA test was performed for determination of MIC val-
ue of INH, RIF and PMRI micelles. MABA is a quick as
well as efficient method in comparison to others (24).
After 6 days of incubation of M. tuberculosis, colorimetric
test results were noted for all strains. Alamar blue dye was
added to the column B11 and incubated for 24 h, growth
of M. tuberculosis was confirmed by change in colour from
blue to pink. Alamar blue dye was added to rest of the all
wells, plates were incubated for another 24 h and results
were analysed (Fig. 7a). MIC of INH and RIF was re-
corded 0.1 ± 0.02 and 0.5 ± 0.04 μg/mL respectively for
sensitive strain. The MIC value of prepared formulation
(
PMRI) was observed to be 0.0625 ± 0.02 μg/mL for

Upadhyay et al.
a
Fig. 7 a Cytotoxicity studies for determination of MIC value of drugs and„b
formulation by MABA (Microplate-Based Alamar Blue Assay) A effect of pure
INH & pure RIF B Effect of HPI & PMRI on Sensitive strain & C Effect of HPI &
PMRI on resistant strain. [HPI: HPMA-PLA-INH conjugates, PMRI: Rifampicin
loaded HPMA-PLA-INH co-polymeric micelles]. b Minimum inhibitory
concentration of INH, RIF, PMRI against A sensitive M. tuberculosis (purple);
B resistant M. tuberculosis (blue). Values represents mean ± SD (n = 3). [INH:
Isoniazid, RIF: Rifampicin, PMRI: Rifampicin loaded HPMA-PLA-INH co-
polymeric micelles]. c Microscopic study of cytotoxicity studies of drugs and
formulation [RIF: Pure Rifampicin, INH: Pure Isoniazid, HPI: HPMA-PLA-INH
conjugates, PMRI: Rifampicin loaded HPMA-PLA-INH co-polymeric
micelles]. The number of M. tuberculosis decreases as the affectivity
increases from left to right. d Microscopic images of M. TUBERCULOSIS at
different concentration of formulation as well as pure drugs at 40X. (A)
Growth of Mycobacterium tuberculosis. B pure INH treated. C & D pure RIF
treated and (E) & (F) PMRI treated. [INH: Pure Isoniazid, RIF: Pure Rifampicin,
PMRI: Rifampicin loaded HPMA-PLA-INH co-polymeric micelles].
(
A)
(
B)
CONCLUSIONS
RIF loaded HPMA-PLA-INH co-polymeric micelles were
successfully synthesized and characterized. In the present
work, we aimed to enhance the effectiveness of INH and
RIF with the help of polymeric micelles as a nanocarrier.
The polymeric micelles are a novel drug carrier. In present
study, HPMA acted as a hydrophilic shell and PLA as a hy-
drophobic core. After conjugation of HPMA-PLA, INH was
further conjugated to develop HPMA-PLA-INH conjugate.
RIF is a hydrophobic drug, because of the higher content of
PLA in co-polymer, RIF was entrapped in micelles. RIF was
easily loaded in the hydrophobic core due to hydrophobic
interaction. The micellar size appears as the most crucial
property. The formation of co-polymeric micelles enhances
the effects of drugs, enhances the solubility of drug and coun-
ter the drug resistance developed by anti-TB drugs. Co-
polymeric micelles enhanced the solubility of RIF and in ad-
dition reduced the overall hemolytic toxicity.
(
C)
In vitro effectivity testing against strains of M. tuberculosis is a
strenuous job. The culture of this gram negative (−ve) bacteria
necessitates special equipment and facility and that is the rea-
son the reported studies were conducted at the National
Japanese Leprosy Mission for Asia (JALMA) Institute of
Leprosy and Other Mycobacterial Diseases, Agra, India.
The institute is among one of few institute designated by
Government of India to study the genetic framework of
M. tuberculosis as well as other diseases. The assay revealed that
the effect of INH and RIF was enhanced when tested with the
co-polymeric micelles. The prepared formulation has resulted
into minimal hemolytic toxicity. The release data shows that
the breaking of hydrazone bond leads to release of INH from
co-polymeric micelles, while loaded RIF is released as a burst
from co-polymeric micelles. RIF release was fast in the initial
stage and slowed down at later time. The rate of release was
reduced because the PLA content was increased. This ap-
proach of co-delivery was novel and promising and has
immense potential of safe and effective delivery of multiple
anti-TB drugs.
Multiple drug delivery is prerequisite in the mycobacteri-
um induced infections such as pulmonary tuberculosis as well
as others. The available chemotherapy is unpopular and is not

HPMA-PLA Polymeric Micelle for Dual Delivery of Anti- TB Drugs
Sensitive (MIC)
Resistant (MIC)
b
1
1
1
0
0
0
0
.50
.25
.00
.75
.50
.25
.00
**
**
**
INH
RMP
Formulation/Drug
PMRI
c
Growth Control
At MIC
RIF
INH
HPI
PMRI
d
(A)
(B)
(C)
(D)
(E)
(F)
Fig. 7 continued.

Upadhyay et al.
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ACKNOWLEDGMENTS AND DISCLOSURES. The authors
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Conflict of Interest The authors declare no competing financial
interest.
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