Biotinylated HPMA centered polymeric nanoparticles for Bortezomib delivery

Article abstract of DOI:10.1016/j.ijpharm.2020.119173

Bortezomib (BTZ) is a proteasome inhibitor as approved by US FDA for the treatment of multiple myeloma. It exhibits significant anti-cancer properties, against solid tumors; but lacks aqueous solubility, chemical stability which hinders its successful formulation development. The present study is an attempt to deliver BTZ using N-(2-hydroxypropyl) methacrylamide (HPMA) based copolymeric conjugates and biotinylated PNPs in an effective manner. Study describes a systematic synthetic pathway to synthesize functional polymeric conjugates such as HPMA-Biotin (HP-BT) HPMA-Polylactic acid (HPLA) and HPMA-PLA-Biotin (HPLA-BT) followed by exhaustive characterization both spectroscopically and microscopically. Our strategy yielded polymeric nanoparticles (PNPs) of narrow size range of 199.7 ± 1.32 nm. Release studies were performed at pH 7.4 and 5.6. PNPs were 2-folds less hemolytic (p < 0.0001) than pure drug. BTZ loaded PNPs of HPLA-BT demonstrated significant anti-cancer activity against MCF-7 cells. IC₅₀ value of these PNPs was 56.06 ± 0.12 nM, which was approximately two folds less than BTZ (p < 0.0001). Cellular uptake study confirmed that higher uptake of formulations might be an outcome of biotin surface tethering characteristics that enhanced selectivity and targeting of formulations efficiently. In vivo pharmacokinetics evidenced increased bioavailability (AUC_{0 t-∞}) of DL-HPLA-BT PNPs (drug loaded) than BTZ with an improved half-life. Overall the developed PNPs led to the improved and effective BTZ delivery.

Full text of DOI:10.1016/j.ijpharm.2020.119173

International Journal of Pharmaceutics 579 (2020) 119173
Contents lists available at ScienceDirect
International Journal of Pharmaceutics
journal homepage: www.elsevier.com/locate/ijpharm
Biotinylated HPMA centered polymeric nanoparticles for Bortezomib
delivery
a
a
b
b
a,⁎
Sarita Rani , Rakesh K. Sahoo , Kartik T. Nakhate , Ajazuddin , Umesh Gupta
a
Department of Pharmacy, School of Chemical Sciences and Pharmacy, Central University of Rajasthan, Bandarsindri, Ajmer, Rajasthan 305817, India
Rungta College of Pharmaceutical Sciences and Research, Kohka-Kurud Road, Bhilai, Chhattisgarh 490024, India
b
A R T I C L E I N F O
A B S T R A C T
Keywords:
Bortezomib (BTZ) is a proteasome inhibitor as approved by US FDA for the treatment of multiple myeloma. It
exhibits significant anti-cancer properties, against solid tumors; but lacks aqueous solubility, chemical stability
which hinders its successful formulation development. The present study is an attempt to deliver BTZ using N-(2-
hydroxypropyl) methacrylamide (HPMA) based copolymeric conjugates and biotinylated PNPs in an effective
manner. Study describes a systematic synthetic pathway to synthesize functional polymeric conjugates such as
HPMA-Biotin (HP-BT) HPMA-Polylactic acid (HPLA) and HPMA-PLA-Biotin (HPLA-BT) followed by exhaustive
characterization both spectroscopically and microscopically. Our strategy yielded polymeric nanoparticles
HPMA (N-2-hydroxypropyl methacrylamide)
PLA (poly lactic acid)
Biotin (BT)
Polymeric nanoparticles (PNPs)
Bortezomib (BTZ)
Breast Cancer
(PNPs) of narrow size range of 199.7 ± 1.32 nm. Release studies were performed at pH 7.4 and 5.6. PNPs were
2-folds less hemolytic (p < 0.0001) than pure drug. BTZ loaded PNPs of HPLA-BT demonstrated significant
anti-cancer activity against MCF-7 cells. IC50 value of these PNPs was 56.06 ± 0.12 nM, which was approxi-
mately two folds less than BTZ (p < 0.0001). Cellular uptake study confirmed that higher uptake of for-
mulations might be an outcome of biotin surface tethering characteristics that enhanced selectivity and targeting
of formulations efficiently. In vivo pharmacokinetics evidenced increased bioavailability (AUC0 t-∞) of DL-HPLA-
BT PNPs (drug loaded) than BTZ with an improved half-life. Overall the developed PNPs led to the improved and
effective BTZ delivery.
1. Introduction
blood (Elsabahy and Wooley, 2012). PNPs have a high degree of se-
lectivity based on size and can deliver the conjugated molecules to the
In 1970s, Ringsdorf first highlighted the use of polymers as a ther-
site of action.
Breast cancer is the second foremost cause of death worldwide
apeutic agent. Since then, plentiful of literature has been published on
polymeric drug delivery (Ringsdorf, 1975). Polymeric conjugates are an
astounding approach for targeted delivery of drugs at tumorous site.
These can mask and control release of the bio-actives at diseased site.
The breakthrough outcome was witnessed when the first polymer en-
tered in the clinical trials (Vasey et al., 1999). Later on, poly-ethyle-
ne–glycol (PEG) based conjugates of proteins (Baka et al., 2006), an-
tibodies (Veronese and Pasut, 2005) and polymeric micelles of
encapsulated or conjugated drugs also entered in various clinical phases
of evaluation. Conjugated polymer-based drug delivery through a na-
noparticulate approach such as polymeric nanoparticles (PNPs), mi-
celles, liposomes, and dendrimers etc. can efficiently deal with solubi-
lity and instability issues. PNPs are colloidal particles in size range of
among all the subtypes of cancers (Bray et al., 2018). Despite the ad-
vanced chemotherapy that improved the life span of cancer patients,
the mortality rate of cancer had not decreased significantly. The reason
sought to be the vague targeting and cytotoxic effects of drugs, which
limits as well as hampers the positive effects of chemotherapy. ER-po-
sitive subtype is the most commonly diagnosed breast cancer (70%) in
women (Pearce and Jordan, 2004). Bortezomib (BTZ) is a proteasome
inhibitor, approved by US FDA (Food and Drug Administration) in 2003
for the treatment of multiple myeloma and have showed outstanding
results for solid tumor treatment as a single agent or in combination
therapy (Chen et al., 2011; Papandreou et al., 2004). BTZ revealed
some extraordinary outcomes against ER-positive breast cancer recently
(Maynadier et al., 2016; Thaler et al., 2017; Xia et al., 2018), but the
1
0–1000 nm with a potential of prolonged distribution of drugs in the
Abbreviations: HPMA, N-2-hydroxypropyl methacrylamide; PLA, Poly lactic acid; BTZ, Bortezomib; BT, Biotin; PNPs, Polymeric nanoparticles; FITC, Fluorescein
isothiocyanate
⁎
Corresponding author.
E-mail address: umeshgupta@curaj.ac.in (U. Gupta).
https://doi.org/10.1016/j.ijpharm.2020.119173
Received 7 January 2020; Received in revised form 16 February 2020; Accepted 21 February 2020
Available online 22 February 2020
0378-5173/ © 2020 Elsevier B.V. All rights reserved.

S. Rani, et al.
International Journal of Pharmaceutics 579 (2020) 119173
low aqueous solubility, instability, and poor penetration in tumor cells
are the major drawbacks (Wei et al., 2010). Su et al. explored the ca-
techol conjugated polymers for BTZ drug delivery for breast cancer cells
such as MDA MB-231, MCF-7. Results suggested that the prepared ca-
techol system can be beneficial in cancer therapy (Su et al., 2011).
Medel and coworkers prepared curcumin and BTZ loaded nanoparticles
and observed the synergistic effect of curcumin and BTZ nanoparticles
against solid tumor (Medel et al., 2017).
HPMA [N-(2-hydroxypropyl) methacrylamide], co-polymer has
been widely explored for polymeric-drug conjugated delivery by
Duncan in the past (Duncan, 2009). HPMA, a hydrophilic copolymer
was supposed to enhance the aqueous solubility of hydrophobic drugs.
HPMA has gained much attention as its first polymeric conjugate
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide), FITC (fluor-
escein isothiocyanate), DMEM (Dulbecco's Modified Eagle Medium),
FBS (fetal bovine serum), streptomycin-penicillin, trypsin EDTA, dia-
lysis membrane-50 and 150 were purchased from HiMedia. All the
chemicals were used as such without any further purification. N, N-
dicyclohexyl carbodiimide (DCC), 1-ethyl-3-(dimethylaminopropyl)
carbodiimide (EDC), polyvinyl alcohol (PVA), lecithin, disodium hy-
drogen phosphate, potassium dihydrogen phosphate, tetrahydrofuran
(THF), sodium hydroxide, sodium chloride, and ethanol were obtained
from CDH, India. TLC plates were procured from Merck, India.
Whatman filter papers and syringe filters were procured from Rankem,
India. All the other reagents were of analytical grade with highest
purity.
(
FCE28068) entered in the clinical trials (Seymour et al., 2009). Con-
sidering attractive characteristics of HPMA it was selected as a hydro-
philic carrier in the present work. PLA (poly-l-lactic acid) was preferred
because of its ability to encapsulate the hydrophobic drug(s) into the
intended PNPs core. PLA has biocompatibility and biodegradability
properties which are fruitful for drug delivery applications. Barz et al.
reported paclitaxel loaded HPMA polymeric micelles and studied their
intracellular uptake and localization in HeLa cells (Human cervix ade-
nocarcinoma cells) (Barz et al., 2012). Surprisingly, only few reports
based on HPLA conjugation are reported yet (Barz et al., 2012). Hi-
therto, none of those reported a combined ester and amide linkage
approach with biotin as a targeted molecule for developing PNPs. PNPs
have some advantages in drug delivery system. Large sized particles can
be easily engulfed by reticular endothelial cells (Senior et al., 1985) but
this shortcoming can be overcome by developing a tumor specific na-
noparticulate system. Significant drug loading is also expected to meet
the requirement of suppression of cell proliferation and eventually to
kill cancerous cells/tissue. Cancer cells usually have a hunger for some
vitamins and receptors (Maiti et al., 2013). Biotin receptors are highly
over expressed in certain cancer subtypes such as breast, lung, ovarian
etc. in comparison to normal cells (Kue et al., 2016). This made our
basis of using biotin as ligand in the intended nanoparticles of the
present study. Among various vitamins, biotin draws special attention
as it is being uptaken more rapidly by cancer cells than the normal cell
2.2. Synthesis and characterization polymeric conjugates
Synthesis of HPMA (N-2-hydroxypropylmethacrylamide), HPMA-
Biotin (HP-BT), HPMA-PLA (HPMA-poly-L-lactic acid; HPLA) and
HPLA-Biotin (HPLA-BT)
2.2.1. HP-BT (N-2-hydroxypropylmethacrylamide-Biotin or HPMA-Biotin)
The co-polymer, HPMA was synthesized according to a previously
reported literature (Chytil et al., 2010). HPMA (HP) and biotin (BT)
were conjugated using DCC/DMAP coupling reaction (Fig. 1). Briefly,
HPMA (10 mg) was dissolved in methylene chloride, then added in to
the biotin (BT) solution (25.44 mg in DMSO) followed by addition of
DCC. After 20 min, DMAP (dissolved in DCM) was added slowly to the
reaction mixture and reaction time allowed was 24 h. TLC was per-
formed initially to confirm the completion of reaction. The crude pro-
duct was vacuum dried and kept in dialysis bag after dissolving in
distilled water. The whole assembly was stirred in distilled water for
2 days to remove DMAP. The external solvent (water) was replaced
after 12 h timely. A brown colored product was dried and a yield of
86% was calculated. The conjugate was characterized at each step by
FT-IR, and NMR spectroscopy Fig. S1.
2.2.2. HPLA (HPMA-Poly-l-lactic acid)
(
Ren et al., 2015).
Present study is based on the conjugation of different copolymers
HPMA was conjugated to PLA according to the reported procedure
(Upadhyay et al., 2017). 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). After 20 min, DMAP (6.1 mg,
0.05 mmol) was added to the reaction mixture. The reaction was al-
lowed to proceed overnight. The conjugate was dialyzed using dialysis
membrane for 24 h to remove DMAP. HPLA co-polymer was char-
acterized by FT-IR (Fig. 2) using KBr pellet method (Perk, M/s Perkin
Elmer Co., Waltham, Massachusetts, USA) and 1H NMR spectra were
recorded in Bruker Ascend 500 MHz NMR spectrometer (Switzerland)
with biotin as a targeting ligand to examine the effect of biotinylated
conjugates v/s non-biotinylated conjugates against breast cancer cells.
The first objective of this study was to explore the capability of HPMA
to enhance aqueous solubility of poorly soluble drug, BTZ. Therefore
encapsulation of BTZ was achieved in HPMA based PNPs to enhance
drug solubility. The second one was to explore BTZ applicability for
solid tumors as BTZ showed remarkable anti-cancer activity against
human breast cancer cell lines such as MCF-7 (Maynadier et al., 2016;
Thaler et al., 2017; Xia et al., 2018) MDA-MB 231 (Su et al., 2011;
Medel et al., 2017) and MDA-MB 268 (Shen et al., 2015). Third one was
tumor-specific delivery of BTZ through biotinylated HPMA and PLA
based PNPs It was also envisaged that conjugation with HPMA can
potentiate drug payload and enhance the blood residence time of BTZ
which in-turn would improve the overall bioavailability of BTZ.
3
using CDCl as solvent.
2.2.3. HPLA-BT (HPMA-Poly-l-lactic acid-biotin conjugate)
Biotin (BT) (100 mg) was dissolved in dry DCM (5 mL) in RBF and
kept at 0 °C. Thionyl chloride (59.38 μL) was added dropwise into the
above solution. Thereafter, the reaction was transformed at 60 °C for
4–6 h. On completion of reaction, flask was cooled at room temperature
2
. Materials and methods
and a solid yellow brown colored product was obtained. The product
was washed three times with chilled DCM and vacuum dried (Hou
et al., 2017). The product was immediately used for the next reaction.
This biotin acylchloride (BT-Cl) intermediate was vacuum dried and
further conjugated to HPLA. In the next step, HPLA (207 mg) was
dissolved in dry DCM and kept for stirring at 0 °C. Then, tertiary amine
2.1. Materials
Methacryloyl chloride and aminopropanol-2-ol were procured from
Alfa-Aesar Pvt. Ltd. India. Anhydrous sodium carbonate, anhydrous
sodium sulfate, dry dichloromethane (DCM), acetone, methanol
3
(Et N) was added to the reaction dropwise. Later, dry THF was added in
(
MeOH), thionyl chloride (SOCl
2
), chloroform, dimethyl sulfoxide
the reaction mixture and reaction was allowed to proceed in cool
condition and inert atmosphere for 18–20 h (Getlik et al., 2013;
Goodreid et al., 2014). Subsequently, the product was dissolved in cold
DCM (dichloromethane) and dried. The obtained product was washed
with chilled DCM to remove the remaining traces of unreacted/
(
DMSO), tetrahydrofuran (THF), and triethylamine (TEA) were pur-
chased from TCI Chemicals Pvt. Ltd. India. PLA (Mn
1
Biotin, 4-dimethyl aminopyridine (DMAP), MTT (3-(4,5-
−1
0000–18000 g·mol ) was a gift sample from Evonik Industries.
2

S. Rani, et al.
International Journal of Pharmaceutics 579 (2020) 119173
Fig. 1. Synthesis schemes of; (a) HPMA (b) HPMA-Biotin (HPBT) (c) HPMA-PLA (HPLA), and (d) HPMA- PLA-Biotin (HPLA-BT).
unmodified HPLA. The co-polymeric conjugate (HPLA-BT or HPMA-
2.3. Preparation of HP-BT, HPLA, HPLA-BT polymeric nanoparticles
(PNPs)
PLA-BT conjugate) (Fig. 1) was obtained as yellow color solid. Finally,
1
13
the product was characterized by H and C NMR spectroscopy
Fig. 3).
(
The prepared polymeric conjugates (HPBT, HPLA, HPLA-BT) were
3

S. Rani, et al.
International Journal of Pharmaceutics 579 (2020) 119173
Fig. 2. FT-IR spectrum of prepared conjugates: HPBT, HPLA, and HPLA-BT. The samples were prepared though KBr pellet method.
used for fabrication of BTZ loaded PNPs. Following the o/w single
emulsion method (Prabu et al., 2009), weighed co-polymeric con-
jugates (10 mg) were dissolved in 5 mL of DCM separately. Then, 0.1%
w/v PVA solution was dissolved in distilled water and added to the co-
polymeric solution. BTZ dissolved in methanol and lecithin solution
2.4. Characterization of HPLA-BT PNPs
2.4.1. 1H NMR spectroscopy
1H NMR spectras were recorded to discriminate the blank and drug
2
loaded PNPs Fig. S5. All the samples were dissolved in D O (deuterated)
(
4% ethanol and heated at 65 °C) were added dropwise (1 mL/10 min)
solvent and processed in 500 MHz NMR spectrometer (Bruker Ascend
500 MHz spectroscopy, Switzerland).
with stirring. The reaction vessel was left stirred to evaporate the or-
ganic solvent. At the last, the nanoparticles were vortexed for 5–7 min
and was dialyzed using dialysis membrane (MWCO 10 kDa) for the
entire day. Finally, the PNPs solution was collected, lyophilized and
stored at −20 °C for further studies (Rosca et al., 2004). In total, the
three blank PNPs i.e. HPBT, HPLA, HPLA-BT and three BTZ loaded (or
drug loaded) PNPs i.e. DL-HPBT, DL-HPLA, DL-HPLA-BT were prepared
successfully.
2.4.2. Particle size, zeta potential, PDI (polydispersity index)
The prepared PNPs were characterized by zeta sizer Nano ZS
(
Malvern Instruments, Malvern, UK) for particle size, zeta potential and
PDI. Zeta sizer is based on the principle of photon correlation spec-
troscopy (PCS) where the average particle size was calculated by
measuring the wideness of particle size distribution. The PNPs were
suspended in 1 mL deionized water and filtered the solutions by syringe
filter (0.22 µm) before measurement. Results were expressed as triplet
4

S. Rani, et al.
International Journal of Pharmaceutics 579 (2020) 119173
Fig. 3. 1H NMR spectrum of (a) HPBT (b) HPLA and (c) HPLA-BT. Deuterated solvents were used for the preparation of sample for NMR analysis.
5

S. Rani, et al.
International Journal of Pharmaceutics 579 (2020) 119173
Fig. 3. (continued)
Table 1
Particle size, zeta potential, drug loading capacity (% DLC) and drug entrapment efficiency (% DEE) of the prepared formulations.
Formulation Code
Size (nm)
Zeta potential (mV)
−7.64 ± 1.23
−1.07 ± 1.28
−2.47 ± 0.56
−17.78 ± 0.12
PDI
Entrapment efficiency (% DEE)
Drug loading (% DLC)
HPBT
213.6 ± 0.25
279.7 ± 2.21
356.3 ± 2.21
430.5 ± 2.21
0.150 ± 3.02
0.131 ± 0.22
0.254 ± 0.24
0.269 ± 0.13
–
–
PNPs
DL-HPBT
PNPs
83.59 ± 0.05
–
14.75 ± 0.13
–
HPLA
PNPs
DL-HPLA
PNPs
74.68 ± 0.08
17.93 ± 0.12
HPLA-BT PNPs
DL-HPLA-BT
PNPs
185.3 ± 0.21
199.7 ± 1.32
−6.50 ± 1.12
−3.20 ± 0.22
0.287 ± 0.02
0.196 ± 0.11
–
–
82.20 ± 0.14
19.73 ± 0.03
of mean ± SD (Table 1).
(Table 1). Nanoparticles suspension (2 mL) was filled in dialysis bag
MWCO 5 kDa, Hi-media Laboratories Pvt. Ltd., Bangalore, India) and
(
dialyzed against perfect sink conditions for 2 h to allow the un-
entrapped drug drawn off from the dialysis bag. The amount of un-
entrapped drug was analyzed at 270 nm using HPLC. The % DEE and %
DLC was calculated using the mentioned equations.
2
.4.3. Surface morphology (atomic force microscopy; AFM)
Microscopy was performed using AFM for evaluating surface char-
acteristics (Fig. 4). For AFM analysis, fresh samples were prepared by
dispersion of PNPs in double distilled water. The dried thin film of
samples was spread over the silicon wafer using spin coating phe-
nomenon. Silicon wafer were dried, and excess of water was drawn off
using filter paper. The results were recorded using Bruker nanoscope V
software and images were taken with 256 × 256-pixel camera, and
Total drug feed − Amount of unentrapped drug
%
DEE =
DLC =
× 100
× 100
Total drug feed
Total drug feed − Amount of unentrapped drug
%
3
00 Hz cantilever frequency after measuring the various sections of thin
Total polymer feed
film samples. Ra (average roughness value) and Rq (root mean square
roughness defined the surface texture and kurtosis value told about the
bumpy, spiky and perfectly random surface (Kumar and Rao, 2012).
2
.5.1. HPLC (High performance liquid chromatography)
BTZ estimation during release and other biological studies was
performed using reverse phase HPLC analysis. The reverse phase HPLC
RP-HPLC) analysis was performed at Central University of Rajasthan,
Ajmer. The HPLC system used was Shimadzu LC-2010 CHT (Tokyo,
18
Japan) with PDA detector. Merck HPLC column (RP C ,
2
.5. Drug loading capacity (DLC) and drug entrapment efficiency (DEE)
(
The drug loading capacity of prepared PNPs (i.e. DL-HPBT, DL-
HPLA, and DL-HPLA-BT) was determined by membrane dialysis method
6

S. Rani, et al.
International Journal of Pharmaceutics 579 (2020) 119173
Fig. 4. Atomic force microscopy (AFM) of DL-HPLA-BT PNPs. The scale bar of 1.0 µm in image (a) and (b) and 2.0 µm in image (c) represented.
4
.6 mm × 250 mm) was used throughout the analysis. The mobile
for 10–15 min. The collected RBCs were washed twice using normal
saline (0.9% w/v) and finally, diluted in 1:10 ratio with normal saline.
RBCs suspension (0.8 mL) was mixed with 3.2 mL of the test solutions
to make the final concentration 10, 50, 100 and 150 µg/mL. Distilled
water and normal saline were regarded as the positive and negative
control, respectively. The cell suspension were incubated for 2 h, and
centrifuged. The extracted supernatant was analyzed at 540 nm using
UV–visible double spectrophotometer (Perkin Elmer). The percentage
hemolysis was calculated using the following equation.
phase utilized consisted of HPLC grade acetonitrile (ACN), water and
formic acid (HCOOH) in 70:30:0.1% v/v, respectively. The flow rate
and temperature were maintained to be 1.0 mL/min and 25 C, re-
spectively. The area under curve of the samples was analyzed at 270 nm
°
(
Byrn et al., 2011; Kamalzadeh et al., 2017).
2.6. In vitro release and kinetic models
Time dependent release of BTZ from prepared polymeric nano-
particles was performed in buffer solutions pH 7.4 and 5.6 mimicking
%Hemolysis
Absorbance of test − Absorbance of negative control
the physiological conditions of blood and endosomes, respectively
=
Absorbance of positive control − Absorbance of negative control
× 100
(
Gupta et al., 2018). Briefly, 2 mL suspension of the developed nano-
formulation was filled in a dialysis bag (MWCO 5 kDa, Hi-Media La-
boratories Pvt. Ltd., Bangalore, India) and immersed in 100 mL of PBS
(
pH 7.4) under stirred condition (150 ± 40) rpm. Two milliliters of
2
.8. Cytotoxicity study
aliquots were withdrawn at defined time intervals and the same amount
of PBS was replenished in to the beaker to maintain sink conditions
throughout the release study protocol. Similar protocol was followed to
obtain the in vitro release data at pH 5.6 using buffer solution as release
media. The drug released from formulations was estimated using HPLC
method as mentioned earlier. Further, kinetic models were applied for
the prepared formulations to recognize the best fitted kinetic model
among different non-linear kinetic models such as, zero order, first
order, Higuchi, Korsmeyer-Peppas and Hixson Crowell model.
The cytotoxicity of BTZ, DL-HPBT PNPs, DL-HPLA PNPs, and DL-
HPLA-BT PNPs was evaluated by MTT assay against MCF-7 breast
cancer cell line (Yang et al., 2009). The cell lines were grown in culture
media DMEM (Dulbecco’s Modified Eagle’s Medium) containing 10% v/
v FBS (Fetal bovine serum) and 1% v/v L-Glutamine-Penicillin-Strep-
tomycin solution. The cells were seeded uniformly at a density of
× 104 cells per well into 96-well plates and incubated overnight in
1
2
CO incubator (Hera cell 150i, Thermo Fisher). After 90% confluency,
the cells were treated with different concentration range of formula-
tions and incubated for 24 h (complete media alone was used as con-
trol). Next day, the cells were removed and washed thrice with PBS and
to each well 50 µL of MTT solution was added and left for another 4 h
incubation. Later, 150 µL dimethyl sulfoxide (DMSO, HPLC grade) was
mixed into each well to dissolve the formazan crystals and a violet color
appeared in each well. The absorbance was taken at 570 nm using
ELISA plate reader (Omega Fluostar) after incubation for 10 min. The
2
.7. Hemolytic toxicity study
The hemolytic toxicity study of BTZ, DL-HPBT PNPs, DL-HPLA
PNPs, and DL-HPLA-BT PNPs was performed in concentration depen-
dent manner (Codony-Servat et al., 2006). The whole blood was col-
lected from a healthy volunteer and stabilized by EDTA. The plasma
was removed after centrifugation at 1250 rpm (R-4C DX, REMI, India)
7

S. Rani, et al.
International Journal of Pharmaceutics 579 (2020) 119173
−
1
IC50 values were determined using Microsoft Excel.
2925.6 (alkene), and 1630.62 cm (–NH), which indirectly confirmed
−1
synthesis. Peaks at 1271.1 and 1039.9 cm
were of the chemically
2
.9. Cellular uptake study
conjugated moiety ester, which confirmed the conjugation (Fig. 2). In
the H NMR spectra, δ ppm: 8.14 [–NH, HPMA(d)], 3.09 [t, –CH, HPMA
2
(e)], 5.23 [s –CH (b)], 2.21 [m –CH (H)], 1.23 [m –CH (K)], and 2.74
1
In order to investigate the cell uptake potential of prepared for-
mulations, cellular uptake study was performed utilizing fluorescein
isothiocyanate (FITC) labeling of MCF-7 cells (Doerflinger et al., 2018).
The prepared formulations were labelled with FITC (2 mg/mL) via FITC
tagging reaction to examine the cell uptake by different drug loaded
PNPs formulations (i.e. DL-HPBT PNPs, DL-HPLA PNPs, and DL-HPLA-
BT PNPs). Briefly, 2.5 mg of FITC was dissolved in 1 mL of acetone.
Then, in a 25 mL RBF, 1 mL PBS of pH 7.4 were mixed with FITC
solution and stirred. After 30 min, drug loaded formulations were
added. The reaction was carried out in dark conditions for 24 h. For
[–CH2 (M)] were observed (Fig. 3(a)). Further confirmation was en-
sured by 13C NMR (500 MHz, CDCl
) δ ppm: 69.98 [CO (M)], 46.1 [C-N
(N)], 172.0 [CO (P)], 140 [C (R)], 168.5 (OCO, HPBT), 22.5 [CH (K)],
41.0 [CH (e)], 161.85 [CO (C)], and 29.0 [CH (I)] (Fig. S5(a)).
3
2
2
2
3.1.3. HPLA (HPMA-PLA conjugate)
HPMA-PLA conjugation was confirmed by; 1H NMR (500 MHz,
CDCl ) δ ppm: 5.13 [m, CH, PLA (a)], 3.61[CH (g)] 1.10 [s-CH PLA
(c)], 1.50 [t-CH PLA (f)], 8.26 [s-NH (d)], and 1.92 [s-CH (e)] Fig. 3
(b). C NMR (500 MHz, CDCl ) δ ppm: 169.3 [OCO, PLA (a)], 69.0
[CH, PLA (e)], 16.6 [CH , PLA(b)], 30.0 [s-CH (d)], and 52.5 (CH (c)]
(Fig. S5(b)). Likewise, the FT-IR peaks at 1630.98 (alkene), 3339.4 (1°
3
2
3
3
3
3
13
study purpose, MCF-7 cells (4 × 10 cells per well) were seeded in a 6-
well plate and incubated overnight. Next day, the culture media was
removed from each well and then the cells were treated with FITC
3
3
3
2
−1
(
2 mg/mL) tagged formulations diluted with culture media into each
NH), 2930.42 (=CH; alkene), and 1720 cm
(ester) wavenumbers
well for predetermined time interval of 2, 4 and 24 h. FITC treated cells
were considered as control for this experiment. The images were cap-
tured under OLYMPUS CKXF3 fluorescence microscope using 10 and
confirmed peak for conjugation of HPMA and PLA (Fig. 2). Before
conjugation the identity of the procured PLA was also confirmed
through NMR spectroscopy (Fig. S3).
40x magnifications.
3
.1.4. HPLA-BT (HPMA-PLA-biotin)
2.10. In vivo pharmacokinetic study
The conjugation was ensured by 1H and 13C NMR spectroscopy at
1
each step. H NMR (500 MHz, DMSO) δ ppm: 9.64 [–NH, BT (a)], 4.43
[m –CH (b)], 1.13 [m –CH (e)], 1.53 [s CH3 (f)], 3.26 [d CH (d)], and
2.94 [CH2 (c)] Fig. 3(c). 13C NMR δ ppm: 162.99 [OCO (a)], 22.99
[CH3 (b)], 14.13 [CH3 (c)], 23.34 [CH2 (d)], 53.32 [CH2 (e)], and
77.85 [m CH (f)]. FT-IR spectrum peaks at 3453.60 (CONH), 1640. 11
All the animal experimentations performed were approved by the
Institutional Animal Ethical Committee, Rungta College of
Pharmaceutical Sciences and Research, Bhilai, India. In vivo pharma-
cokinetic studies were performed on healthy unisex Sprague Dawley
rats weighing 250–300 gm (Nie et al., 2017). All the animals were kept
on standard diet and water throughout the experiment. The animals
were divided into four groups, in which each group was comprised of 4
animals. The different group of animals were administered in-
travenously with BTZ, DL-HPBT PNPs, DL-HPLA PNPs, and DL-HPLA-
BT PNPs in 1 mg/kg equivalent dose of BTZ in saline. At defined time
intervals, 0.2 mL of blood sample was withdrawn from the retro orbital
plexus and collected in a heparinized (anti-coagulant) vial. The super-
natant was withdrawn after centrifugation of the sample withdrawn at
−
1
(1° NH; biotin), 605.79 (=C-H), 1725.70 and 1005.64 cm
(ester
peak) suggested the conjugation of HPLA with biotin (Fig. S5(c)). The
intermediate biotin chloride (BT-Cl) was also characterized spectro-
scopically by 1H and C NMR analysis to confirm synthesis (Fig. S4).
All the obtained spectral data favored the synthesis of polymeric con-
jugates.
13
3.1.5. UV visible spectroscopic characterization of conjugates
UV Visible spectrophotometry was performed for drug as well as all
the conjugates (Fig. S6). The peaks were clearly visible for biotin
around 350 nm. This characterization step further supported the
synthesis of HPBT and HPLA-BT.
3000 rpm (R-4C DX, REMI, India). In each sample, 0.5 mL of acetoni-
trile was added and centrifuged immediately to denature blood proteins
for better analysis. The concentration of the supernatant was then
evaluated at 270 nm by reverse phase HPLC (Shimazdu LC-2010C,
Japan) methods as explained in earlier paragraphs. The plasma con-
centration–time graph was plotted using the obtained data and various
pharmacokinetic parameters were calculated following one compart-
ment open body model (1 CBM) for iv push.
3.2. Characterization of PNPs
Polymeric nanoparticles (PNPs) were prepared by o/w single
emulsion method particularly to control or optimize the size and drug
efficiency of nano-formulations. Finally, the three different formula-
tions i.e. BTZ loaded HPLA PNPs (DL-HPLA), BTZ loaded HPLA-BT
PNPs (DL-HPLA-BT), and BTZ loaded HPBT PNPs (DL-HPBT) were
prepared, characterized, and evaluated in further steps for different
outcomes and anticancer activities.
3. Results and discussion
3
3
.1. Synthesis and characterization
.1.1. HPMA (N-2-hydroxypropylmethacrylamide)
HPMA co-polymer was synthesized following a previously reported
3.2.1. Drug loading capacity (DLC) and drug entrapment efficiency (DEE)
The DLC of DL-HPLA-BT PNPs, DL-HPLA-PNPs and DL-HPBT was
found to be 19.73 ± 0.03, 17.93 ± 0.12, and 14.75 ± 0.13%, re-
spectively. DEE of DL-HPLA-BT, DL-HPLA and DL-HPBT PNPs was
calculated to be 82.20 ± 0.14, 74.68 ± 0.08, and 83.59 ± 0.05%,
respectively. A slight increase in the DLC was seen in DL-HPLA-BT PNPs
as compared to DL-HPLA-PNPs. In the similar way, DEE of DL-HPLA-BT
PNPs was higher than HPLA-PNPs (Table 1). The observed slight in-
crease in the encapsulation can be seen probably due to surface en-
gineering of the HPLA conjugates with biotin (BT) which might be
providing more room for encapsulation within respective PNPs.
protocol (Chytil et al., 2010). The white crystalline product with per-
cent yield of 82% was confirmed for the synthesis through different
spectroscopic techniques. The FT-IR spectrum revealed peaks at
−
1
1
653 cm for CONH, CH
seen at 3299.74, and 2974.29 cm , respectively Fig. S1. The chemical
shift at 7.7 ppm (s, CONH), OH peak at 5.3 ppm, CH peak at 1.2 ppm
t, –CH ), that further confirmed the synthesis of this co-polymer (Fig.
S1).
3 2
(methyl), and CH (methylene) peaks were
−
1
3
(
3
3
.1.2. HPMA-Biotin (HP-BT)
HPMA was further conjugated to biotin (BT) (Fig. S2) via DCC/
DMAP coupling reaction as shown in synthetic scheme (Fig. 1). In the
3.2.2. UV–Visible spectroscopy
FT-IR spectrum, peaks were seen at 3443.35 (–CONH), 2861.8 (alkane),
Maximum absorption is shifted towards longer wavelength (Fig. S6)
8

S. Rani, et al.
International Journal of Pharmaceutics 579 (2020) 119173
in case of DL-HPLA-BT PNPs due to the presence of carbonyl bond in
the polymeric conjugate. BTZ absorption can be observed at 300 and
310 nm wavelengths. However, the blank polymeric conjugates showed
a less intensified absorption and the overlapped wavelengths were not
able to detail any clear indication. The maximum absorption of DL-
HPLA-BT PNPs was an indication of conjugation of HPLA with BT
forming a carbonyl bond elucidated as characteristic peak in UV–visible
graph.
3
.2.3. 1H NMR analysis for BTZ loaded formulations
The 1H NMR spectra (Fig. S8) showed a difference between PNPs
(
blank polymeric nanoparticles) and drug loaded-PNPs (DL-PNPs). A
sharp peak was noticed at a chemical shift of 3.31 ppm in DL-HPLA-BT
PNPs, which is due to the hydrogen atoms attached to the carbon group
near to boron atom in BTZ structure while this peak was absent in B-
HPLA-BT PNPs. These results indicated the encapsulation of BTZ in DL-
HPLA PNPs.
3
.2.4. Particle size, zeta potential, PDI (polydispersity index), and
microscopy
The followed strategy yielded mean particle sizes of formulations
such as DL (drug loaded or BTZ loaded)-HPLA-BT PNPs, HPLA-BT PNPs,
DL-HPLA PNPs, HPLA PNPs, DL-HPBT PNPs and HPBT PNPs
1
2
99.7
±
1.32, 185.3
±
0.21, 430.5
±
2.21, 356.3
±
2.21,
79.7 ± 2.21, and 213.6 ± 0.25 nm, respectively in a narrow size
range (Table 1). The size can have major impact in drug targeting to
tumors as the tumor vasculature cut-off size band varies from 100 to
Fig. 5. In-vitro drug release pattern of BTZ, DL-HPBT PNPs, DL-HPLA PNPs and
DL-HPLA-BT PNPs at pH (a) 7.4 (b) 5.6. Membrane dialysis method was fol-
lowed for the release studies in vitro.
800 nm (Ren et al., 2015). So, the size of the nanocarriers can be
controlled to deliver through the efficacious EPR-mediated approach.
The results obtained for particle size suggested that the DL-HPLA-BT
PNPs size lies in 200 nm range that may be helpful for tumor targeting
and approx. 45% DL-HPLA PNPs, while it was less than 40% for DL-
HPLA-BT PNPs in 4 h. More than 80% drug was released from DL-
HPLA-BT PNPs in 10 h. Conclusively, faster drug release was noticed at
pH 5.6 for the DL-HPLA-BT PNPs than at physiological pH. The pH 5.6
may be favorable for inducing significant anti-cancer effect of BTZ as
tumorous environment is acidic in nature. Different kinetic equations
were tested to get a better fit kinetic model in the prepared formula-
tions at pH 7.4 and 5.6. The results were tabulated in Table S1(a) and
(
Torchilin, 2011). The small size particles can enter in the tumor cells
effortlessly and can produce drug action with a great extent. The size,
zeta potential, PDI, % DLC, and % DEE of formulation were tabulated in
(
Table 1). The prepared PNPs exhibited negative zeta potential which
might be due to the presence of ester groups in HPBT, HPLA and HPLA-
BT.
3.2.5. Surface morphology evaluation by atomic force microscopy (AFM)
(
b), respectively. Incorporating the obtained release data into the
The surface morphology of PNPs was observed by atomic force
mentioned kinetic model, the best fit models at pH 7.4 for DL-HPLA-BT
microscopy (AFM). AFM was performed only for DL-HPLA-BT PNPs
which described the surface topography and texture of these PNPs. The
surface roughness (Ra) 27.2 nm, root mean square roughness (Rq) 20.2,
and skewness 0.168 were observed during analysis. The high kurtosis
value of 4.37 has given the idea of spiky surface or high-pitched peak
surface for DL-HPLA-BT PNPs (Singh et al., 2019). The overall AFM
results concluded that the PNPs showed surface roughness with
heighted peaks (Fig. 4).
2
PNPs was found to be Korsmeyer-Peppas model (R = 0.98), Higuchi
model (R2 = 0.95) and first-order model (R2 = 0.93). In case of DL-
2
2
HPLA PNPs, first-order model (R = 0.92), Higuchi model (R = 0.94),
2
Korsmeyer-Peppas model (R = 0.90) and for DL-HPBT PNPs, first-
order model (R2 = 0.93), Higuchi model (R2 = 0.91), Korsmeyer-
2
Peppas model (R = 0.90) were the best fit models. At pH 5.6 the best
fit models for DL-HPLA-BT PNPs were seen to be Higuchi model
2
2
(
(
R
= 0.91), first-order model (R = 0.90), Korsmeyer-Peppas model
2
0.94) and zero-order model (R = 0.92). For DL-HPLA PNPs the ob-
3.3. In vitro release and kinetic models
2
servations were Higuchi model (R
=
0.94), first-order model
2
(R
(R
(R
(
= 0.92), Korsmeyer-Peppas model (0.91), and zero-order model
Drug release profile is the key determinant factor to know about the
2
2
= 0.96). For DL-HPBT PNPs the observations were Higuchi model
therapeutic performance of a drug in delivery system. At pH 7.4, ap-
prox. 55% and approx. 34% of BTZ was released from DL-HP-BT PNPs
and DL-HPLA-BT PNPs, respectively in 4 h (Fig. 5a). Similarly, greater
than 80% of BTZ was released from DL-HP-BT PNPs, and approx. 76%
drug was released from DL-HPLA PNPs while DL-HPLA-BT PNPs re-
leased more than 65% of BTZ in 24 h of time point. These results
suggested that DL-HPLA-BT PNPs owed slow and controlled release
pattern than DL-HP-BT PNPs and DL-HPLA PNPs. The controlled release
of drug could be beneficial to maintain an appropriate drug con-
centration in the tumor cells for a prolong period of time so as to im-
prove the overall drug action. These results gave an idea that the drug
was encapsulated in the hydrophobic core of nanoparticles and showed
2
= 0.95), first-order model (R = 0.96), Korsmeyer-Peppas model
2
0.94), and zero-order model (R = 0.98).
3.4. Ex-vivo hemolytic toxicity study
Concentration dependent hemolysis toxicity analysis (Fig. 6) was
performed to determine the interaction of erythrocytes with BTZ and
prepared formulations. BTZ displayed 24% hemolysis, whereas, DL-
HPLA-BT PNPs showed only 12% hemolysis against RBCs at a con-
centration of 150 µg/mL indicated that DL-HPLA-BT PNPs were 2-fold
less toxic than BTZ with p < 0.0001. Interestingly, the biotin anchored
HPLA-PNPs (i.e. DL-HPLA-BT PNPs) showed less toxicity propably due
to biocompatible interaction of nanoparticles with erythrocytes. In
a
controlled released slowly at pH 7.4 (Fig. 5a). At pH 5.6
(
Fig. 5b) greater than 50% of BTZ was released from DL-HP-BT PNPs
9

S. Rani, et al.
International Journal of Pharmaceutics 579 (2020) 119173
MCF-7 breast cancer cell lines through MTT assay (Fig. 7a and Fig. S9a).
BTZ showed variable cytotoxicity against breast cancer cell line MCF-7
(
IC50 = 100 ± 8.4 nM), MDA-MB-231 (IC50 = 7 ± 2.7 nM), MDA-
MB-468 (IC50 2.4 nM) and MDA-MB- 453
IC50 = 100 ± 12.1 nM) (Doerflinger et al., 2018). The IC50 values
calculated for DL-HPBT-PNPs was 56.06 ± 0.12 nM, which was 1.97
approx. 2-fold) less than BTZ with p < 0.0001 as per the experimental
=
5
±
(
(
results while blank PNPs of HPLA-BT were found to be non-toxic to
MCF-7 cells (Fig. S9B). Results inferred that significant reduction in
IC50 values was observed for biotin conjugated PNPs (DL-HP-BT PNPs
and DL-HPLA-BT PNPs). However, significant reduction in IC50 values
was seen for DL-HPBT PNPs and DL-HPLA-BT PNPs as compare to DL-
HPLA PNPs which indicates the role of biotin in ascertaining enhanced
anticancer activity of biotin conjugated formulations against MCF-7
breast cancer cells. The biotinylated formulations acted efficiently than
non-biotinylated one. The drug loaded PNPs (DL-HP-BT PNPs, DL-HPLA
PNPs, and DL-HPLA-BT PNPs) exhibited higher cytotoxicity at lower
concentration of drug as compared to pure BTZ against MCF-7 breast
cancer cells (Fig. 7b). These results revealed that drug loaded PNPs
possessed significant anti-cancer effect.
Fig. 6. Ex vivo hemolysis of BTZ and drug loaded (DL) PNPs of HPBT, HPLA,
and HPLA-BT at different concentrations (10, 50, 100, and 150 µg/mL); Values
represents mean ± SD (n = 3). **** p < 0.0001 indicates the extremely
significant difference between various concentrations of drug and drug loaded
formulations (Two-way ANOVA; Bonferroni multiple comparisons test).
comparison to DL-HPLA-PNPs, the DL-HPBT PNPs exerted a minimal
hemolytic toxicity on erythrocytes at similar concentration. At lower
concentration of 10 µg/mL, a considerable decrease in hemolytic toxi-
city was noticed for DL-HPLA-BT PNPs than BTZ. The hemolytic toxi-
city for PNPs was 2-fold less than BTZ at 50 µg/mL, similarly 2-fold
decrease was seen in case DL-HPBT PNPs than DL-HPLA PNPs. Per-
ipheral neuropathy and thrombocytopenia are considered as dose lim-
iting toxic effects of BTZ as per literature (Chen et al., 2011). This is also
one reason that the study was planned and conducted.
3.6. Cellular uptake study
The intracellular uptake of free BTZ and BTZ loaded formulations
(
DL-HPBT PNPs, DL-HPLA PNPs, and DL-HPLA-BT PNPs) was in-
vestigated by fluorescence microscopy using FITC (fluorescence iso-
thiocyanate) dye. Here, we used biotin which acts as a cell targeting
molecule and can be taken up by tumor cells selectively as per reported
literature (Yang et al., 2009). Biotin receptors are overexpressed in
cancerous cells and biotin can be used in tracking of tumor surface via
prepared nanoparticles tagged with polymers as cargo (Vineberg et al.,
3.5. Cytotoxicity assay
2014). Therefore, now-a-days targeting ligand-based therapeutics has
become a choice of drug targeting that can directly enter into tumor
cells/surface. It was inferred that higher uptake of biotinylated PNPs
than non-biotinylate HPLA-PNPs was seen in 2 h time (Fig. 8). This may
be due to the targeting potential of biotin attached to HPLA-BT PNPs.
Similarly, HPBT PNPs exhibited more uptake than HPLA PNPs (Fig. 8).
Higher uptake of drug loaded PNPs was observed in the cells in a time
dependent manner. This plausible effect ensured more surface binding
affinity of biotin to tumor cells surface in case of HPLA-BT nano-
particles than non-biotinylated HPLA PNPs. Similarly, biotin con-
jugated HPMA PNPs (HPBT PNPs) showed more intensity than non-
biotin conjugated HPLA-PNPs after 24 h. This may be due to the ap-
propriate biotin tethering on the surface of HPLA-BT PNPs. From these
findings, it was concluded that developed HPLA-BT PNPs could be used
for effective breast cancer targeting using biotin as a ligand.
Cytotoxicity of drug-loaded formulations was examined against
BTZ
1
1
50
00
a
DL-HPBT PNPs
DL-HPLA PNPs
DL-HPLA-BT PNPs
50
0
0.5
1
10
30
50
70
100
Concentration (nM)
3
.7. In vivo pharmacokinetic study
*
***
b
****
BTZ
1
1
50
00
The BTZ calibration curve was prepared via RP-HPLC method op-
*
***
***
DL-HPBT PNPs
DL-HPLA PNPs
DL-HPLA-BT PNPs
timized by ACN: Methanol: Water (70: 30: 0.1) as mobile phase to
obtain a sharp peak of drug (Fig. S7) (Kamalzadeh et al., 2017). The
positive and encouraging results obtained from in vitro release studies,
cellular uptake, and cytotoxicity study impelled us for the in vivo
pharmacokinetic studies in Sprague Dawley rats. Time dependent
plasma drug concentration of BTZ, DL-HPBT PNPs, DL-HPLA PNPs, and
DL-HPLA-BT PNPs were plotted in Fig. 9. The desired pharmacokinetic
parameters were estimated by indicating the data into one compart-
mental open body (1 CBM) i.v. push model. The high elimination rate
constant of drug and relatively low K values supports the intended
objectives of the study. The elimination rate constant of DL-HPLA-BT
PNPs was observed to be less than pure drug. The bioavailability
(AUC0−∞) of DL-HPLA-BT PNPs was enhanced by 8.5 folds than BTZ. A
*
*
****
5
0
0
Drug/Formulations
Fig. 7. (a) Percent cell viability of BTZ and DL-HPBT PNPs, DL-HPLA PNPs, and
DL-HPLA-BT PNPs. The data obtained was part of the MTT assay performed at
different concentrations. (b) IC50 of BTZ and DL-HPBT PNPs, DL-HPLA PNPs,
and DL-HPLA-BT PNPs. **** indicates the extremely significant difference with
0.0001, while * indicates less significant difference with p
Newman-Keuls multiple comparison test).
d
reduction in V was calculated for DL-HPBT PNPs (Table 2). The half-
life of DL-HPLA-BT PNPs was increased by approximately 2.52 times
than BTZ. The increase in bioavailability, half-life and decrease in
p
(
<
< 0.05
10

S. Rani, et al.
International Journal of Pharmaceutics 579 (2020) 119173
1
0 X
40 X
2
h
4 h
24 h
2 h
4 h
24 h
Fig. 8. Cellular uptake study of (a) FITC (fluorescein isothiocyanate) as control; (b) DL-HPBT PNPs; (c) DL-HPLA PNPs; (d) DL-HPLA-BT PNPs performed against
MCF-7, breast cancer cell line. FITC tagged formulations were used for the cellular uptake studies of drug loaded formulations.
6
4
2
0
0
0
0
behavior of DL-HPLA-BT PNPs.
DL-HPLA-BT PNPs
DL-HPLA PNPs
DL-HPBT PNPs
BTZ
This study is a mix of polymeric synthesis exhaustively supported by
the extensive characterization as well as the exploration of these de-
veloped conjugates for targeted delivery of BTZ. However, polymeric
conjugation is a complex task to achieve, but present attempt was
successful in synthesizing and characterizing polymeric conjugates
through 1H NMR, C NMR, FT-IR and UV–visible spectroscopy. In the
previous reports, HPMA based drug encapsulation and conjugation has
been reported which did enter in the clinical trials too. The char-
acterstics properties of HPMA enforces to utilize it for improving BTZ
delivery in breast cancer cells. HPMA-PLA (HPLA) block copolymers
were synthesized following the mechanism relied on an active ester and
amide functionality approach. Then, the polymeric conjugates were
fabricated into PNPs by o/w emulsionsification. The obtained results
can be clearly correlated with the effect of size and biotinylation. The
cytotoxicity data is indicative to emphasize on significant aspects of
HPMA over pure drug delivery. BTZ is relatively new molecule for es-
tablishing delivery strategy. In this regard the study can be significant
contribution. The prepared PNPs were able to demonstrate the influ-
ence of polymeric conjugation in the form of nanoparticles to in-
vestigate hemolytic toxicity, cytotoxicity, cellular uptake, and in vivo
13
0
5
10
15
20
25
Time (h)
Fig. 9. Plasma concentration–time profile of DL-HPLA-BT PNPs, DL-HPLA
PNPs, DL-HPBT PNPs and BTZ obtained from in vivo pharmacokinetic study in
Sprague-Dawley rats followed by intravenous administration of formulations
with BTZ equivalent dose of 1 mg/mL. Values represents mean ± SD (n = 4).
clearance rate confirmed the increment in the retention time of the
formulations. Overall the pharmacokinetic study reflected the results
obtained in in vitro release study. The higher retention time in terms of
improved bioavailability is an indirect evidence of the sustained
Table 2
Pharmacokinetics Parameters of BTZ, DL-HPBT PNP, DL-HPLA PNPs and DL-HPLA-BT PNPs by Intravenous Administration in Sprague Dawley Rats. Values represent
mean ± S.D, AUC = area under the curve, K(h) elimination rate constant, t1/2 = plasma half-life, Cl = clearance, Vd = volume of distribution.
PK parameters
BTZ
DL-HPBT
PNPs
DL-HPLA PNPs
DL-HPLA-BT PNPs
AUC 0-t
102.74 ± 0.28
112.90 ± 0.32
391.60 ± 0.022
546.27 ± 0.02
493.21 ± 0.01
755.38 ± 0.021
596.95 ± 0.15
955.51 ± 0.23
(
µg mL-1h−1
)
AUC 0-∞
(
µg mL-1h−1
)
)
−1
k (h
1/2 (h)
(L)
Cl (L/h)
0.110 ± 0.01
6.28 ± 0.04
18.65 ± 0.024
2.058 ± 0.03
0.054 ± 0.011
12.91 ± 0.14
8.40 ± 0.02
0.451 ± 0.12
0.049 ± 0.001
14.19 ± 0.011
6.70 ± 0.24
0.33 ± 0.20
0.044 ± 0.02
15.84 ± 0.01
5.90 ± 0.31
0.258 ± 0.03
t
V
d
11

S. Rani, et al.
International Journal of Pharmaceutics 579 (2020) 119173
pharmacokinetics. BTZ loaded HPLA-BT PNPs were also less hemolytic
than the drug. It is also noticed in the previous literature that, BTZ
exhibited hemolytic toxicity behavior as a side-effect of chemotherapy.
The cell-line based study inferred that BTZ loaded PNPs were more
cytotoxic than BTZ. From the cellular uptake studies, it was concluded
that DL-HPLA-BT PNPs can enter into the MCF-7 breast cancer cells
more efficiently than the pure drug. The enhanced cellular uptake may
be due to the biotinylation of HPMA in HPMA-BT and HPMA-PLA-BT
polymeric conjugates. Biotin overexpression on receptors in breast
cancer cells may be one of the reasons why the uptake was enhanced in
case of biotinylated PNPs in comparison to non-biotinylated one. Of-
course the better outcome can be evaluated only with the in vivo studies
performed in breast cancer induced animal model, but we decided to
carry out the in vivo studies first in normal healthy rats. The objective
was to ensure, what kind of pharmacokinetic profile is displayed by the
prepared PNPs. The obtained results were encouraging and supported
the hypothesis that the prepared PNPs were able to exert the stealth
nature as per as plasma drug concentration is concerned.
Acknowledgement
The authors acknowledge the financial support from the University
Grants Commission, New Delhi, India and Department of Science and
Technology, New Delhi, India through DST Start up Research Grant
(Young Scientists) to Dr. Umesh Gupta. We are thankful to Fresenius
Kabi Pvt. Ltd. for providing Bortezomib as a gift sample.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.ijpharm.2020.119173.
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