HPMA-PLGA Based Nanoparticles for Effective In Vitro Delivery of Rifampicin

Article abstract of DOI:10.1007/s11095-018-2543-x

Purpose: Tuberculosis (TB) chemotherapy witnesses some major challenges such as poor water-solubility and bioavailability of drugs that frequently delay the treatment. In the present study, an attempt to enhance the aqueous solubility of rifampicin (RMP) was made via co-polymeric nanoparticles approach. HPMA (N-2-hydroxypropylmethacrylamide)-PLGA based polymeric nanoparticulate system were prepared and evaluated against Mycobacterium tuberculosis (MTB) for sustained release and bioavailability of RMP to achieve better delivery. Methodology: HPMA-PLGA nanoparticles (HP-NPs) were prepared by modified nanoprecipitation technique, RMP was loaded in the prepared NPs. Characterization for particle size, zeta potential, and drug-loading capacity was performed. Release was studied using membrane dialysis method. Results: The average particles size, zeta potential, polydispersity index of RMP loaded HPMA-PLGA-NPs (HPR-NPs) were 260.3 ± 2.21?nm, ?6.63 ± 1.28?mV, and 0.303 ± 0.22, respectively. TEM images showed spherical shaped NPs with uniform distribution without any cluster formation. Entrapment efficiency and drug loading efficiency of HPR-NPs were found to be 76.25 ± 1.28%, and 26.19 ± 2.24%, respectively. Kinetic models of drug release including Higuchi and Korsmeyer-peppas demonstrated sustained release pattern. Interaction studies with human RBCs confirmed that RMP loaded HP-NPs are less toxic in this model than pure RMP with (p < 0.05). Conclusions: The pathogen inhibition studies revealed that developed HPR-NPs were approximately four times more effective with (p < 0.05) than pure drug against sensitive Mycobacterium tuberculosis (MTB) stain. It may be concluded that HPR-NPs holds promising potential for increasing solubility and bioavailability of RMP.

Full text of DOI:10.1007/s11095-018-2543-x

Pharm Res
(2019) 36:19
https://doi.org/10.1007/s11095-018-2543-x
RESEARCH PAPER
HPMA-PLGA Based Nanoparticles for Effective In Vitro
Delivery of Rifampicin
Sarita Rani1 & Avinash Gothwal1 & Pawan K. Pandey 1 & Devendra S. Chauhan2 & Praveen K. Pachouri2 & Umesh D. Gupta2
&
Umesh Gupta1
Received: 24 August 2018 /Accepted: 8 November 2018
#
Springer Science+Business Media, LLC, part of Springer Nature 2018
ABSTRACT
were 260.3 2.21 nm, −6.63 1.28 mV, and 0.303 0.22,
respectively. TEM images showed spherical shaped NPs with
uniform distribution without any cluster formation.
Entrapment efficiency and drug loading efficiency of HPR-
NPs were found to be 76.25 1.28%, and 26.19 2.24%,
respectively. Kinetic models of drug release including
Higuchi and Korsmeyer-peppas demonstrated sustained re-
lease pattern. Interaction studies with human RBCs con-
firmed that RMP loaded HP-NPs are less toxic in this model
than pure RMP with (p < 0.05).
Conclusions The pathogen inhibition studies revealed that
developed HPR-NPs were approximately four times more
effective with (p < 0.05) than pure drug against sensitive
Mycobacterium tuberculosis (MTB) stain. It may be concluded
that HPR-NPs holds promising potential for increasing solu-
bility and bioavailability of RMP.
Purpose Tuberculosis (TB) chemotherapy witnesses some
major challenges such as poor water-solubility and bioavail-
ability of drugs that frequently delay the treatment. In the
present study, an attempt to enhance the aqueous solubility
of rifampicin (RMP) was made via co-polymeric nanoparticles
approach. HPMA (N-2-hydroxypropylmethacrylamide)-
PLGA based polymeric nanoparticulate system were pre-
pared and evaluated against Mycobacterium tuberculosis
(MTB) for sustained release and bioavailability of RMP to
achieve better delivery.
Methodology HPMA-PLGA nanoparticles (HP-NPs) were
prepared by modified nanoprecipitation technique, RMP
was loaded in the prepared NPs. Characterization for particle
size, zeta potential, and drug-loading capacity was performed.
Release was studied using membrane dialysis method.
Results The average particles size, zeta potential, polydisper-
sity index of RMP loaded HPMA-PLGA-NPs (HPR-NPs)
KEYWORDS HPMA
.
.
(N-2-hydroxypropylmethacrylamide) nanoparticles (NPs)
.
.
.
PLGA rifampicin (RMP) solubility tuberculosis (TB)
Highlights
• HPMA (N-2-hydroxypropylmethacrylamide) co-polymer was synthesized
and characterized.
• HPMA-PLGA NPs were allowed to self-assemble in order to remove
acetonitrile, organic solvent for safety purposes.
• Solubility of RMP was increased 65 folds via HPMA-PLGA hydrophilic co-
polymer.
• Overall efficacy of the prepared polymeric nanoparticles against the
Mycobacterium tuberculosis was enhanced several folds over the naïve drug
RMP.
ABBREVIATIONS
ACN
Acetonitrile
Ethambutol
ETB
HPMA
HP-NPs
N-2-hydroxypropylmethacrylamide co-polymer
HPMA-PLGA nanoparticles
HPR-NPs Rifampicin loaded HPMA-PLGA nanoparticles
INH
MABA
MTB
MIC
Isoniazid
* Umesh Gupta
Microplate Alamar Blue Assay
Mycobacterium tuberculosis
Minimum inhibitory concentration
Nanoparticles
Poly-lactic-co-glycolic acid
Polymeric Nanoparticles
Plasma protein binding
Pyrazinamide
umeshgupta175@gmail.com; umeshgupta@curaj.ac.in; https://
www.curaj.ac.in
NPs
1
Department of Pharmacy, School of Chemical Sciences and Pharmacy
PLGA
PNPs
PPB
Central University of Rajasthan, Bandarsindri
Ajmer, Rajasthan 305817, India
2
ICMR-National JALMA Institute for Leprosy and Other Mycobacterial
Diseases, Tajganj, Agra, Uttar Pradesh 282001, India
PYZ

19 Page 2 of 12
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RMP
STP
TB
TEM
WHO
Rifampicin
Streptomycin
Tuberculosis
Transmission electron microscopy
World Health Organization
sites. MTB predominantly resides intracellularly in macro-
phages and phagocytic cells accessed by the systemic vascular
blood supply by phagocytosis (10). From the previous studies,
it was observed that NPs of 1000 nm size, moving in the blood
circulation could be easily phagocytized by the MTB-infected
macrophages. In the nanoparticulate system, drug starts re-
leasing after administration and then moves into the infected
cells in a controlled manner (11). The polymer poly (lactic-co-
glycolic acid) (PLGA) gained the attention of researchers be-
cause of its desirable drug delivery (12). PLGA is one of the
most useful biodegradable polymer which produces two met-
abolic monomer units, i.e., lactic acid and glycolic acid after
hydrolysis. These two monomer units are endogenous and
metabolized by the kreb’s cycle (13). The attractive properties
such as sustained drug release effect, biodegradability, and
biocompatibility make this polymer, a suitable candidate for
drug delivery. The matrix of this polymer prevents degrada-
tion of drugs and maintains release kinetics of the drugs from
NPs.
In the last 30 years, N-(2-hydroxypropylmethacrylamide)
(HPMA) (Fig. 1 (a) co-polymer extensively studied for drug
delivery system. Polymeric conjugates of this co-polymer im-
proved the aqueous solubility, bioavailability, and therapeutic
efficacy of many drugs. HPMA is a potential amide co-
polymer recognized to reduce toxicity and decrease the multi
drug resistance (14). More than two HPMA copolymer-drug
conjugates are in the investigation phase of clinical trials (15).
Safety and lack of immunogenicity are the essential properties
of HPMA copolymer. A conjugate of HPMA with different
therapeutic agents or imaging probes has been reported (16).
HPMA possesses a number of advantages for polymeric drug
delivery system over conventional delivery methods.
Primarily, it prolonged the half-life of low MW payloads with
potential efficacy. It gained much attention in solubilizing the
poor aqueous soluble drugs. Polymer conjugation prevents
the premature metabolism of some drugs carriers before their
distribution to the targeted sites (17–19).
INTRODUCTION
After HIV-AIDS, TB is second most serious infectious disease
comes from a single causative organism, Mycobacterium
tuberculosis (MTB). Global efforts have been successful in re-
ducing the mortality rate of TB patients by 37% and have
saved approximately 53 million lives since 2000 (1). Despite
this achievement, TB remains a severe threat to global health.
According to World Health Organization (WHO) TB report
2017, 10.4 million people were infected with TB in 2016; 5.9
(56%) million men, 3.5 (34%) million women and 1.0 (10%)
million children. Globally, 11% of the 1.2 million new TB
cases in 2016 were HIV-positive. The slow-growing acid-fast
bacilli, MTB infects lungs, spleen, brain, kidneys and other
organs (2). WHO established guidelines that duration of TB
therapy is 9 months for adults and 12 months for children. TB
chemotherapy includes first and second-line drugs. Isoniazid
(INH), rifampicin (RMP), pyrazinamide (PYZ), ethambutol
(ETB), streptomycin (STP) are first-line drugs and kanamycin,
amikacin including with other antibiotics comes in list of
second-line drugs. The daily doses are required in different
phases of treatment. TB chemotherapy consists of two phases
(intensive and continuous). In intensive phase; patients takes
INH, RMP, PYZ and ETB in different doses, while in con-
tinuous phase INH and RMP are given in combination.
Initially, INH is responsible for killing 95% TB bacteria and
then RMP, PYZ replaces its role during the intensive phase of
therapy. The chemotherapy duration prolonged by 12–
18 months if INH or RMP not given to patients. Hence, both
INH and RMP play an important role in the TB chemother-
apy (3–5). When bacteria become multidrug resistant, the
antibiotic therapy competence gradually decreases. The anti-
biotic chemotherapy will be used for six months in case of
multidrug resistance and toxicity of antibiotics will increase
(6). Patient non-compliance adds further complications in
the TB treatment as multiple doses would be administered
on a daily basis or weekly not less than six months according
to WHO guidelines. To enhance the patient compliance,
nanotechnology including polymeric nanoparticles (PNPs) (7)
has been adopted for drug entrapment and release (8).
However, organic solvents and some hazardous chemicals
used in manufacturing, limit the utility of drugs. NPs drug
delivery is a promising approach to improve the solubility of
hydrophobic drugs and their bioavailability (9). The small size
of PNPs carriers may lead to the cellular uptake of drug and
allow efficient drug accumulation in the body at the targeted
In the present study, amide copolymer HPMA and PLGA
(Fig. 1 (b) based NPs were prepared using a surfactant lecithin.
RMP was physically entrapped in the prepared NPs and eval-
uated for anti-TB effectivity against drug sensitive strains of
Mycobacterium tuberculosis and safety as well. The intent of this
research work was to improve the solubility of RMP and to
deliver it safely with improved and sustained effect for the
better treatment of TB and related pulmonary disease.
MATERIALS AND METHODS
Materials
Rifampicin (BP), also called Rifampin (USP) was a gift from
Kwality Pharmaceuticals Ltd., India. Methacryloyl chloride,

Pharm Res
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Page 3 of 12 19
Fig. 1 (a) Chemical structure of
HPMA (N-2-
hydroxypropylmethacrylamide); (b)
Chemical structure of PLGA (x is
the number of lactic acid units and y
is the number of glycolic acid units).
a
b
21
aminopropan-2-ol, and lecithin were purchased from Alfa
Aesar Pvt. Ltd., India. Anhydrous sodium carbonate and
methylene chloride were purchased from TCI Chemicals
Pvt. Ltd. India. PLGA was purchased from Sigma-
Aldrich India. 96 well microplates were purchased from
Tarsons Pvt. Ltd., India. Alamar blue dye and 7H9GC
Middlebrook media were procured from Hi-media
Laboratories Pvt. Ltd., Mumbai, India. Tween-80 was pur-
chased from Spectrochem Pvt. Ltd. Mumbai, India. All other
chemicals and reagents used were of analytical grade.
Bruker Ascend 500.3 MHz NMR spectrometer, Switzerland
(7.269 ppm for 1H-NMR as internal standard). The 1H-NMR
analysis was performed at School of Chemical Sciences and
Pharmacy, Central University of Rajasthan, Bandarsindri,
Rajasthan, India.
Synthesis of RMP Loaded HPMA-PLGA Nanoparticles
HP core-shell NPs were synthesized from HPMA copolymer,
PLGA and soybean phosphatidylcholine lecithin using a mod-
ified nano-precipitation technique (21) Fig. 2 (b). Briefly, 1.0 g
(0.011 mmol) PLGA was dissolved in acetonitrile (ACN).
Lecithin and HPMA (7:3 M ratio) were dissolved in 4% aque-
ous solution of ethanol and heated to 65°C, separately. The
PLGA solution was added to the preheated lipid via syringe
drop-wise (1 mL/min) with gentle stirring. HP-NPs were
allowed to self-assemble for 6 h with continuous stirring
and the organic solvent was evaporated during self-
assembling process. Tomcin et al. also reported that the
solvent evaporation step was followed to avoid hazardous
properties/ harmful effects of organic solvent (22).
Whatman filter paper of size no. 41 was used for further
washing of NPs solution to remove the impurities. The
prepared HP-NPs were stored at 4°C for future use.
Similarly, RMP loaded HP-NPs were prepared following
the similar method and the suitable quantity of the drug was
added to acetonitrile, and the solution was further added to
HPMA-PLGA solution of ACN with stirring at room temper-
ature. In addition, the solution was placed in an end-sealed
dialysis bag (5000 Da, MWCO, Hi-Media, India) and put in
50 mL distilled water for dialysis process. After 36 h of dialysis,
the solution was centrifuged, lyophilized followed by getting a
solid product of HPR-NPs formulation.
Synthesis of N-(2-Hydroxypropyl) Methacrylamide
(HPMA)
HPMA was synthesized using a previously reported method
(20). A quantity of 0.36 mL (0.95 mmol) 1-aminopropanol-2-
ol was dissolved in 5 mL of methylene chloride. An accurately
weighed anhydrous sodium carbonate 100 mg (0.95 mmol)
was suspended in the above solution Fig. 2 (a) at 0°C. Then,
a solution of 98.80 mg methacryloyl chloride in 5 mL methy-
lene chloride was prepared and added drop wise under
cooling with vigorous stirring. The assembly was left for stir-
ring at 15°C for 1 h. Anhydrous sodium sulfate was added to
absorb the moisture from the product and vacuum dried the
solid after filtration. Then the solid product was dissolved in
methylene chloride and kept in the deep freezer at −20°C. At
last, a white crystalline solid product, HPMA was obtained
after recrystallizing with acetone. The synthesized HPMA
was confirmed by FT-IR spectrum and 1H-NMR spectrum.
Characterization of Synthesized HPMA Copolymer
FT-IR Spectroscopy. FT-IR was performed at The Material
Research Centre, MNIT, Jaipur, India for characterization
of the copolymer. Pure RMP FT-IR spectrum was obtained
by using KBr pellet on a Perkin Elmer FT-IR spectrophotom-
eter supported by (PerkinElmer SpectrumTM software (M/s
Perkin Elmer, Inc. Waltham, Massachusetts, USA).
Concentration-Dependent Solubilization Study
In this study, a comparison of solubility of RMP and HPR-
NPs was done. Briefly, RMP (1 mg) was dissolved in 100 mL
water, and a subsequent calibration curve was prepared by
following membrane filtration in the range of 1–10 μg/mL
using methanol as co-solvent. The absorbance was taken by
Proton Nuclear Magnetic Resonance (1H-NMR) Spectroscopy.
1H-NMR spectrum was recorded in the CDCl3 solvent by

19 Page 4 of 12
Pharm Res
(2019) 36:19
Fig. 2 (a): Schematic
representation of HPMA co-
polymer synthesis and; (b)
Schematic method of preparation of
HPR-NPs.
a
b
UV double beam spectrophotometer at 333 nm. The phase
solubility analysis of HPR-NPs was performed with increasing
co-polymer concentration range. The experiments were per-
formed in triplicate, data was represented as mean, and stan-
dard deviation subsequently subjected to statistical analysis as
described in section 2.11.
distribution of prepared HPR-NPs and HP-NPs. For size
analysis, 1 mL of prepared NPs were diluted with 4 mL de-
ionized water at 25°C by light scattering at 90° angle. Each
data value represents an average of three measurements.
Highly poly-dispersed samples, containing particles with dif-
ferent sizes, will show a high polydispersity index value near to
1. A low PDI close to 0 was desirable. The PDI was deter-
mined by considering the above mentioned concept.
Characterization of HPR-NPs
Size, Zeta Potential and Polydispersity Index (PDI)
Electron Microscopy (Transmission Electron Microscopy)
Zeta sizer (Nano ZS, Malvern, UK) measured the electropho-
resis mobility, average particle size, zeta potential and size
TEM characterized the size and surface morphology of NPs.
A drop of prepared HPR-NPs was placed over a 400-mesh

Pharm Res
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Page 5 of 12 19
carbon-coated copper grid. NPs solution drop was spread on a
degreased glass plate uniformly for sample preparation. Then
negatively stained with phosphotungstic acid solution (3%, w/
v, adjusted to pH 4.7 with KOH) with an accelerating voltage
of 95 kV. The grids were washed twice with distilled water and
air-dried earlier to capture images with the help of the
computer.
(0.22 μ, Rankem, India). The filtrate was collected and quan-
tified by UV-visible spectrophotometer (Double Beam UV-
Vis Spectrophotometer, LT-2800, Labtronics, India) at
333 nm for RMP. Free RMP, was removed by placing the
solution, in the end sealed dialysis membrane bag (MWCO =
5000 Da, Hi-Media, India) in 250 mL distilled water with
overnight stirring. The NPs were purified by dialysis method.
The percentage drug EE and DL was determined by eq. 1
and eq. 2 respectively. A previously reported protocol with
slight modification was used for determining the entrapment
efficiency of HPR-NPs (23). The formulated nanoparticulate
suspension was centrifuged (Remi centrifuge, India) at 4–5°C
for one h. The formulation was freeze-dried after discarding
the supernatant. The NPs yield was calculated by using eq. 3.
Drug Entrapment Efficiency and Percent Drug Loading
For determining entraptment efficiency (EE) and percent drug
loading (DL), 2 mg HPR-NPs were dissolved in ACN (10 mL),
and the solution was centrifuged (Remi 4-RC-DX, India) for
15 min at 1500 RPM and filtered using membrane filter
Amount of drug used in formulation−amount of unbound drug
EE ð%Þ ¼
x 100
ð1Þ
Amount of drug used in formulation
Weight of RMP added
Total amount of polymer and drug added
DL ð%Þ ¼
x 100 ð2Þ
In Vitro Release
Nanoparticles yield
Amount of recovered HP−NPs
Total amount of polymer and drug added
RMP released from the NPs were evaluated by dispensing the
HPR-NPs in PBS buffer 7.4 pH medium containing ascorbic
acid (prevent oxidative degradation of drugs in dialysis meth-
od). Briefly, accurately weighed 2 mg of HPR-NPs solution
was taken in a dialysis bag (MWCO = 5000 Da, Hi-Media,
India) and the dialysis bag was end-sealed and submerged
fully into 50 mL of PBS buffer (pH 7.4) solution at 37°C with
stirring at 120 rpm for 72 h. At predetermined time intervals,
2 mL aliquots were withdrawn filtered through 0.45 μm sy-
ringe filter and replenished with an equal volume of fresh PBS
buffer. The concentrations of RMP in samples were deter-
mined by UV-Vis double spectrophotometer. The
amount of release RMP was analyzed by a UV spectro-
photometer at 333 nm wavelength. Each measurement
was taken three times for better results analysis. The in vitro
release study was further planned by applying in different
kinetic models such as zero, first, Higuchi, Hixon crow-
well, and Korsmeyer-Peppas model, etc.
¼
ð3Þ
Protein Binding Study
Equilibrium retro-dialysis method was performed to evaluate
the plasma protein binding of HPR-NPs (24). Initially, four
mL plasma was added in 2 mL HPR-NPs solution and kept
aside for 30 min. in dialysis (5000 Da; Hi-Media) bag and
placed in a glass beaker with 120 30 rpm magnetic stirring
(Remi, India) at room temperature. Then, 0.5 mL of 5%
glucose filled in dialysis bags and kept into the same glass
beaker then retro-dialysis was done for 6 h with 120
30 rpm magnetic stirring. Volume was made up with distilled
water. Samples of plasma (200 μl) and dialysate were collected
and analyzed using a UV-visible spectrophotometer. The %
PPB of RMP and INH was calculated as:
Hemolysis Toxicity Study
%PPB ¼ 5:2  CoCi=0:5  Ci þ 5:2  Co  100%
The hemolytic study regarding percentage hemolysis was
done to evaluate the HPR-NPs compatibility with RBCs
(erythrocytes) compared to RMP (25). One mL of suitably
diluted HPMA, PLGA, lecithin, HP-NPs, and HPR-NPs were
added separately to 4 mL of normal saline and allowed to
interact with RBCs suspension. Similar step was performed
for drug solution and lecithin solution, mixed with 4 mL of
normal saline, and allowed to interact with RBCs suspension.
Supernatants were diluted with equal volume of normal saline
and an absorbance was seen at 540 nm wavelength. Distilled
Here,
Co drug concentrations in the plasma outside the dialysis
bags.
Ci dialysate inside the dialysis bags (mg/mL).
The values 0.5 and 5.2, represents inside solution volume
and outside the dialysis bags respectively (mL).

19 Page 6 of 12
Pharm Res
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water was used as a control with hemolytic value as 100%.
Percent hemolysis was thus determined for each sample, using
the following equation
wells in column 10. Now, the final concentration range was
as: 0.4 to 0.0015 μg/mL for HPMA solution, same for the
PLGA and RMP in HPR-NPs 1 to 0.0078 μg/mL in each
well of plates. After that, 100 μl of sensitive M. tuberculosis strain
(tested by 1% proportion method) was added to the test wells.
The well B11 was taken as control/drug-free. Plates were left
for incubation after resealing at 37°C for 5 days. At 5th day,
50 μl of a freshly prepared 10x Alamar Blue reagent was
added to well B11. Then plates were again incubated at
37°C for 24 h. If B11 turned pink after that reagent, mixture
was added to the wells of microplate. After adding Alamar dye
in all the test wells, microplate was allowed to incubate for
next 24 h at 37°C, and recorded the colors of all wells of
microplate. A blue colour was an indication of no growth
and pink colour indicated as growth. Moreover, the
M. tuberculosis was examined under microscope without any
treatment and at treated MIC of both RMP and HPR-NPs
by fluorescence microscope (Olympus Inverted Fluorescence
Microscope CKX53, Japan).
%Hemolysis ¼ ½AbsŠ=AB100X 100
Abs
Sample absorbance.
AB100 Absorbance of control without formulation.
Anti-TB Assay
Micro Plate Alamar Blue Assay (MABA)
MABA was performed with slight modification in the previ-
ously reported method (26). In outer peripheral wells of sterile
96-well plates, sterile deionized water (200 μL) was added to
minimize the loss of medium during incubation. In each plate,
100 μl of 7H9GC broth was added in rows B to G in columns
2 to 11 (7H9 broth). The broth was prepared by this method:
2.35 g 7H9 broth was dissolved in deionized-autoclaved water
followed by adding 2 mL of glycerol and autoclaved at 15 lbs.,
121°C for 10 min and cooled at 45°C. At the end, 1 mL
Middle brook ADC was added aseptically and mixed well
before dispensing. The 100 μl of RMP solution was added
from B to G rows (column 2, 3) to investigate the minimum
inhibitory concentration (MIC) of pure drug. Then, 100 μl
was transferred from column 3 to 4 and mixed the wells using
a multichannel pipette. Similar serial dilutions (1:2) were done
in columns 3 to 10, and simultaneously discarded the excess
medium (100 μL) from wells (column 10) to get a final con-
centration range for RMP 4 μg/mL to 0.015 μg/mL in each
96 well plate. After that, 100 μl of sensitive M. tuberculosis strain
(tested by 1% proportion method) were added in rows B to G
(columns 2 to 11). Column 11 was considered as a control.
Plates were sealed with parafilm and kept for incubation at
37°C for 5 days. On 5th day, 50 μl of a freshly prepared
mixture of 10x Alamar Blue reagent (1:1) was added to well
B11 and left to incubate at 37°C for 24 h after resealing with
parafilm. All the wells would receive the Alamar dye if well
B11 turned pink. The microplates were resealed with parafilm
after adding Alamar dye in each test well and incubated for
24 h. A blue color was an indication of no growth and pink
color indicated as growth. For the determination of MIC of
the formulations, same procedure was applied. The wells, in
rows B to G (columns 2 to 11) filled with 100 μl of 7H9GC
broth. In rows, B to C 1000 μl of 2x HPMA solution was
added and PLGA solution in D to E and HPR-NPs solutions
in F to G in columns 2 and 3 respectively. Then, transferred
100 μl from column 3 to 4 and 4 to 5 through column 10 for
the identical serial 1:2 dilution and mixed the wells content
nicely and excess medium (100 μl) was discarded from the
Microscopy
Microscopy was performed to investigate the growth of MTB,
the heat fixed slides were prepared for growth control and
determined MIC, stained with carbol-fuschin and again heat-
ed after 5 min, washed with water and decolorized with 25%
sulphuric acid and kept for 2 min. In the last, slides were
stained with 0.1% methylene blue and studied under the mi-
croscope (CX21FS1, Olympus Corporation, Tokyo, Japan) at
different magnifications.
Statistical Treatment of Data
All data were expressed as means standard deviation (SD)
from at least three values. Student-tests, one way ANOVA
Dunnett’s Multiple Comparison tests and One way
ANOVA Newman-Keuls Multiple Comparison Test were
used for the statistical treatment of data using Graph Pad
Prism (version 7.0, CA, USA) software, with p < 0.05 consid-
ered as significant.
RESULTS AND DISCUSSION
In recent years, PLGA with chitosan and alginate widely used
for preparing a nanoparticulate formulation for reducing the
dose frequency, patient non-compliance as well as the
sustained release of TB frontline drugs. Terry et al. (27) also
reported the sustained release formulation of RMP using
PLGA polymer. HPMA co-polymer has unique characteris-
tics to enhance the solubility of hydrophobic drugs and the
overall efficacy of drugs too (28). Results from the present

Pharm Res
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Page 7 of 12 19
study revealed that the solubility of RMP was increased sev-
eral folds using HPMA.
1H, -OH) and methyl at 1.2 ppm (t, 3H, -C-CH3), respectively
Fig. 3 (b). The noticed peaks in FT-IR and H-NMR con-
1
firmed the synthesis of HPMA. The synthetic pathway was
simple, easy with high yield product during synthesis (Table I).
Synthesis and Characterization of HPMA
HPMA co-polymer was synthesized by previously reported
protocol (20). The calculated percent product yield was 85%
with melting point 69–70°C. The obtained product was crys-
talline and white in colour with good yield after complete
removal of residual organic solvents. The synthesis of
HPMA was confirmed at each step with thin layer chroma-
tography (TLC) and spectroscopic techniques (Fig. 3).
Preparation and Characterization of HP-NPs
RMP Loaded HP-NPs
HPMA provide electrostatic and steric stabilizations with lon-
ger circulation half-life of therapeutics in vivo. Functional-end
group’s attachment of targeting ligand such as antibodies,
peptides and aptamers is the significant characteristic of
HPMA. PLGA offer the hydrophobic core to encapsulate
hydrophobic drugs. Lecithin provided a monolayer in the
region of the hydrophobic core and act as emulsifying agent
as well. The prepared HP-NPs were novel regarding the con-
stitution and widely used HPMA co-polymer NPs were select-
ed first time for this study. The percent nanoparticle yield of
blank HP-NPs and HPR-NPs was calculated as 66.8 1.24
and 64.16 2.11 respectively. RMP loaded NPs were soluble
in aqueous solution as observed in the solubilization study.
FT-IR and 1H NMR Spectroscopy of HPMA
The synthesized HPMA was characterized by FT-IR and 1H-
NMR spectroscopy. In FT-IR spectrum, amide bond peak
was seen at 1653.04 cm−1. The sp3 carbon (alkyl/methyl
group) and sp2 carbon (alkenes) peak were appeared at
3299.74 cm−1, and 2974.29 cm−1 respectively in Fig. 3 (a).
HPMA showed the characteristic shift for a proton of
amide at 7.7 ppm (s, 1H, -CONH), of alcohol at 5.3 ppm (s,
Fig. 3 (a) FT-IR spectra of co-
a
polymer HPMA; (b) 1H-NMR
spectra of co-polymer HPMA
b

19 Page 8 of 12
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Tab le I Size, Zeta Potential, Entrapment Efficiency and Percent Yield for HP-NPs and HPR-NPs
Formulation Code Morphology
Size (nm)
Zeta potential (mV) PDI
Entrapment
Drug loading (%) Percent Yield (%)
efficiency (%)
HP-NPs
–
184.4 2.03 −7.37 1.23
0.265 0.02
–
–
66.8 1.24
HPR-NPs
Spherical shape NPs 260.3 2.21 −6.63 1.28
0.303 0.22 76.25 1.28 26.19 2.24
64.16 2.11
The solubility studies results were explained in the section
concentration dependent solubilization study.
PEGylated PLGA-bendamustine NPs was 52.3% (29) sug-
gesting that the encapsulation of drug was substantial.
Concentration-Dependent Solubilization Study
Analysis of Size, Zeta Potential and PDI
RMP is a hydrophobic drug with 0.014–0.018 mg/mL solu-
bility in distilled water. RMP bioavailability is unpredictable
as compared to other first-line TB therapy drugs such as INH,
PYZ, and ETH. Pelizza et al. reported that many factors
affecting the solubility of RMP. One of the reason is that
RMP shows polymorphism (30) and this affects the solubility
of the drug, as many types of hydrates were seen during crys-
tallography of RMP. Jindal et al. (31) studied the effect of
particle size on the bioavailability and dissolution of RMP. It
has been reported that the HPMA co-polymer increases the
solubility of some hydrophobic drugs (17–19). Solubility study
results revealed that solubility of RMP enhanced in a
concentration-dependent manner of HPMA-PLGA co-poly-
mer. In water, the maximum obtained solubility for RMP was
0.017 0.002 mg/mL only, while the highest solubility at
50 μg/mL concentration of HPMA-PLGA in HPR-NPs was
1.056 0.024 mg/mL (Fig. 5). There was an increase of al-
most 65 fold which was a highly significant result (p < 0.05).
Therefore, the HPR-NPs were able to increase the aqueous
solubility of RMP that might be due to the hydrophilic behav-
ior of HPMA in HPMA-PLGA co-polymeric structure of the
prepared HPR-NPs and might be the result of the formation
of H-bond with HPMA co-polymer.
The average particle size, zeta potential of HP-NPs were cal-
culated as 184.4 2.03 nm, −7.37 1.23 mV while for HPR-
NPs the obtained values were 260.3 2.21 nm, −6.63
1.28 mV, respectively (Table I). PDI of HP-NPs and HPR-
NPs was determined 0.265 0.02, 0.303 0.22. PDI values
were less than 1 which suggests that the prepared NPs have
similar or uniform size particles with homogeneity. Zeta po-
tential had minimal change after RMP loading which might
be because of the state of polymers used in the research
work. The slight increase in size was indirect evidence or
indication for drug loading in HPR-NPs. Tomcin et al.
reported NPs synthesized from HPMA with size range of
250–300 nm and described a method in which PNPs were
stabilized by HPMA co-polymer for sustained drug re-
lease of poorly soluble drug (21).
Electron Microscopy
Transmission electron microscopy (TEM) was used to evalu-
ate the morphology of prepared HPR-NPs. TEM images re-
vealed that the PNPs have uniform spherical shell (Fig. 4) with
size similar as obtained with particle size analysis. TEM im-
ages correspond to NPs with no agglomeration or collapse
suggesting that particles of prepared formulations were
remained unaggregated. The prepared PNPs were observed
for the particle size and other properties to achieve the desired
results in vitro.
Entrapment Efficiency and Drug Loading Efficiency
The percent EE and DL efficiency of HPR-NPs were 76.25
1.28 and 26.19 2.24 (Table I), respectively. The results sug-
gested that the HPR-NPs were able to encapsulate drug in an
excellent quantity. Results are supported by our previously
reported work, in which EE of NPs can be more than 70%.
In one of our earlier work, we reported PLGA-PEG copoly-
mer nanoparticles as promising nanocarriers for anti-cancer
drug-bendamustine. The entrapment efficiency exhibited by
Fig. 4 Transmission electron microscopy (TEM) of RMP loaded HP-NPs.

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Page 9 of 12 19
case-II relaxation release having stresses in hydrophilic poly-
mers, which swell in water or biological fluids (35).
1.2
1
Hemolysis Toxicity Studies
0.8
0.6
0.4
0.2
The importance of safety and toxicity was addressed by
accessing the compatibility and interactions of the developed
nano-formulations with human RBCs. Ex-vivo hemolytic tox-
icity studies was done for HPMA, PLGA, RMP, lecithin, HP-
NPs, and HPR-NPs. Percent hemolytic toxicity of RMP and
HPR-NPs were observed as 33.4 0.45% and 8.9 0.25
(Fig. 7), respectively (p < 0.05). HPR-NPs showed four times
less toxicity as compared to RMP which means the prepared
NPs formulation reduced the toxicity of RMP. Additionally,
the percent hemolytic toxicity of the synthesized HPMA was
3.62 0.24 with (p < 0.05) which suggested that HPMA co-
polymer has less hemolytic toxicity in comparison to RMP.
PLGA, HP-NPs and lecithin percent hemolysis were observed
as 4.87 0.17, 4.41 0.16 and 5.62 0.25% (p < 0.05), re-
spectively. The pure drug showed more toxicity as compared
to PNPs formulation probably because of the reason that the
hydrophobic drug RMP was completely encapsulated in the
NPs and there was non-availability of the free drug for RBCs
interaction.
0
0
10
20
30
40
50
60
Concentration of HPMA-PLGA (µg/mL) in HPR-NPs
Fig. 5 Concentration dependent aqueous solubility of RMP using HPR-NPs.
Values represents mean SD (n = 3)
% Plasma Protein Binding (PPB)
The protein binding study was carried out using previous-
ly reported literature with slight modifications (21). %PPB
of HPR-NPs was found to be 68 2.46% which showed
that the drug PPB rate was relatively high as the pure
RMP (57–70%) (32). Briefly, equilibrium retro-dialysis
was used to calculate the PPB of RMP encapsulated in
HP-NPs. The binding efficiency depends on the structure,
loading and physicochemical properties of the drug mol-
ecule (33). Moreover, the clearance from the body is also
affected by protein binding (34). Hence, it can be hypoth-
esized that the drug bioavailability, residence time of the
drug will be higher and it will result in improving the
overall effectivity.
Anti-TB Studies
Microplate Based Alamar Blue Assay (MABA) is one of the
robust method to determine the inhibitory concentration of
test formulations against M. tuberculosis bacterial (MTB) strain.
The handing of the M. tuberculosis needs a sophisticated facility
to carry out the experimentation, and hence, this was per-
formed in the designated laboratory of Government of
India, i.e., ICMR-National JALMA Institute for Leprosy
and Other Mycobacterial Diseases, Tajganj, Agra, India.
After 6 days, M. tuberculosis strain incubation tests results were
recorded. After 5 days of incubation, the Alamar blue dye was
added to the B11 (Growth control), MTB growth was con-
firmed by the change in color from blue to pink Fig. 8 (1). Dye
was added simultaneously to rest of the wells and incubated
for the next 24 h. Results were recorded on the following day.
MIC of RMP for sensitive strain was recorded as 0.5
0.036 μg/mL. The MIC value of HPR-NPs was found
≥0.125 0.02 μg/mL for the sensitive strain (p < 0.05), which
was significantly lesser than the pure drug and showed four
times more effective against the MTB strain Fig. 8 (2).
Moreover, the microscopic examination of MTB revealed
that HPR-NPs inhibited the MTB growth more significantly
Fig. 8 (2) with respect to pure RMP, which might be due to the
nanoparticulate delivery of RMP. Interestingly, we also ob-
served the anti-TB activity for HPMA (MIC=2.0 0.09 μg/
mL). We were unable to understand the obtained anti-TB
activity in case of HPMA for which mechanism was
In Vitro Drug Release
Membrane dialysis method in phosphate buffer saline pH 7.4
was used to study the in vitro release. This study revealed that
pure RMP was released in a burst manner within an initial
first hour and approximately 90% in 4 h. While the encapsu-
lated RMP release was slower in case of HPR-NPs and con-
tinued up to 70 h and more Fig. 6 (a). In vitro release of RMP
from HPR-NPs showed controlled release fashion up to 70 h
while the pure drug released within 6 h (approximately 98%).
To ensure that the release pattern was sustained or not it was
decided to study the best-fit non-linear kinetics models (Fig. 6b
and c). The obtained release kinetics was confirmed by
Higuchi and Korsmeyer-Peppas model. The in vitro release
of drug from HPR-NPs was easily explained by Korsmeyer-
Peppas model Fig. 6 (b). The mathematical linear kinetic re-
lease modeling presented the highest linearity and was de-
scribed as Korsmeyer-Peppas model and Higuchi Fig. 6 (c)
model with r2 = 0.905 (HPR-NPs) and r2 = 0.920 (HPR-
NPs), respectively. It also described the magnitude of release
exponent N that explained that the release mechanism was

19 Page 10 of 12
Pharm Res
(2019) 36:19
Fig. 6 In vitro drug release of RMP
and HPR-NPs in PBS buffer
(pH 7.4). The values represents
mean SD (n = 3). (a); Graphical
representation of Korsmeyer-
Peppas kinetic release model (b);
Higuchi model for HPR-NPs (C).
120
100
80
60
40
20
0
a
RMP
HPR-NPs
0
10
20
30
40
50
60
Time (h)
b
c
unpredictable Fig. 8 (2). Additionally, it can be clearly
observed that the initial four wells of HPMA treated
group in Fig. 8 (1b) were blue, indicating no growth
suggesting that HPMA may have some anti-TB effect
against the sensitive MTB strain. The previous reports
also supports that the HPMA has some anti-infective
activity (36). In the fluorescence images, it is clear that
the prepared formulations were more effective than
pure anti-TB drug Fig. 8 (3). Microscopy also supported
the results that the HPR-NPs showed higher and effec-
tive pathogen inhibition against MTB strain. In our
previous report, we have reported that nanoparticulate
delivery of bioactive agents enhances the effectivity due
to slow and sustained release (26).
Fig. 7 Ex-vivo hemolytic study of
polymers (HPMA, PLGA), lecithin,
pure RMP, HP-NPs and HPR-NPs.

Pharm Res
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Page 11 of 12 19
a
b
a
b
c
Fig. 8 (1) Pathogen inhibition studies for determination of MIC value of drugs and formulations by MABA (Microplate-Based Alamar Blue Assay) [A] effect of pure
RMPon sensitive stain and [B] Effect of HPMA co-polymer, PLGA and HPR-NPs. (Pink colour showed the growth of bacteria and blue colour showed no growth
of bacteria). (2) Minimum inhibitory concentration of RMP (red), HPMA co-polymer (green), RMP loaded HPMA-PLGA NPs (blue) against a sensitive
M. tuberculosis. Values represents mean SD (n = 3) [RMP, HPMA, HPR-NPs]. (3) Microscopic images of M. Tuberculosis at different concentration of
formulation as well as pure drugs at 40X. [(a) Growth of M. tuberculosis without treatment (b) pure RMP treated (c) HPR-NPs].
CONCLUSIONS
nano-constructed PNPs may improve the delivery of the
anti-TB agent RMP.
This study reports a novel approach for delivering anti-TB
drug RMP by HP-NPs. The prepared HPR-NPs were not
only able to solve the solubility issue of RMP but equally able
to reduce the toxicity and to impart boosted cytotoxicity
against drug sensitive MTB strain. The drug loading as well
as entrapment efficiency were high enough that the robust
method can be transformed into pilot batches in future.
Interestingly, apart from increasing 65 fold the aqueous solu-
bility; initial limited biocompatibility studies the nanoparticles
were safe enough to be delivered in biological milieu in future
studies. In-vitro MABA study revealed four fold higher anti-TB
activity against MTB strain, which was an effective outcome
of our study. Hence, it can be proved that co-polymeric NPs
drug delivery can be an effective strategy to deliver hydropho-
bic anti-TB drug RMP. Additionally, use of HPMA as an
established polymer for controlled release, also showed the
potential for anti-TB drug delivery. This is one of few studies
in which, the delivery of anti-TB drug has been attempted
using HPMA copolymer. Conclusively, we may say that
ACKNOWLEDGMENTS AND DISCLOSURES
The authors acknowledge the financial support from the
Rajasthan Department of Science and Technlogy, Jaipur
India and Department of Science and Technology, New
Delhi, India through DST Start up Research Grant (Young
Scientists) to Dr. Umesh Gupta. Authors would also like to
acknowledge Indian Council of Medical Research (ICMR) for
providing facilities at JALMA, Agra, India. The authors de-
clare no competing financial interest.
Publisher’s Note Springer Nature remains neutral with regard to jurisdic-
tional claims in published maps and institutional affiliations.
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