Biomedicine & Pharmacotherapy 144 (2021) 112286

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Biomedicine & Pharmacotherapy

journal homepage: www.elsevier.com/locate/biopha

Development and optimization of paclitaxel loaded Eudragit/PLGA

nanoparticles by simplex lattice mixture design: Exploration of improved
hemocompatibility and in vivo kinetics
Gunjan Jeswani a, b, Lipika Chablani c, **, Umesh Gupta d, Rakesh K. Sahoo d, Kartik T. Nakhate e,
Ajazuddin f, *
a
Rungta College of Pharmaceutical Sciences and Research, Kohka-Kurud Road, Bhilai, Chhattisgarh 490024, India
b
Faculty of Pharmaceutical Sciences, Shri Shankaracharya Technical Campus, Bhilai, Chhattisgarh 490020, India
c
Department of Pharmaceutical Sciences, Wegmans School of Pharmacy, St. John Fisher College, Rochester, NY 14618, USA
d
Department of Pharmacy, School of Chemical Sciences and Pharmacy, Central University of Rajasthan, Bandarsindri, Ajmer, Rajasthan 305817, India
e
Department of Pharmacology, Shri Vile Parle Kelavani Mandal’s Institute of Pharmacy, Dhule, Maharashtra 424001, India
f
School of Pharmacy and Technology Management, SVKM’s NMIMS, Shirpur, Maharashtra 425405, India

A R T I C L E I N F O A B S T R A C T

Keywords: Anemia is the most common hematological abnormality of chemotherapy, which is responsible for poor clinical
Paclitaxel outcomes. To overcome this complication, the present study was aimed for developing a Eudragit/polylactic-co-
Nanoparticle(s) glycolic acid (PLGA) based nanoparticulate system for a model drug paclitaxel (PTX). The study was planned
Eudragit RLPO
using a simplex lattice mixture design. PTX nanoparticles (PTXNp) were evaluated in vitro for physicochemical
Eudragit RSPO
PLGA
properties, hemolytic effects and cytotoxic effects. Further, the nanoparticles were subjected to in vivo screening
Design of experiments (DOE) using rats for hemocompatibility, pharmacokinetic profile, and biodistribution to the vital organs. The PTXNps
Simplex lattice were 65.77–214.73 nm in size, showed more than 60% sustained drug release in 360 h and caused less than 8%
Hemocompatibility hemolysis. The parameters like red blood cell count, activated partial thromboplastin time (aPTT), prothrombin
time (PT) and C3 complement were similar to the negative control. Cytotoxicity results suggested that all the
PTXNp demonstrated drug concentration-dependent cytotoxicity. The in vivo pharmacokinetic study concluded
that PTXNp formulations had significantly higher blood AUC (93.194.55–163,071.15 h*ng/mL), longer half-lives
(5.80–6.35 h) and extended mean residence times (6.05–8.54 h) in comparison to PTX solution (p < 0.05).
Overall, the study provides a nanoparticulate drug delivery system to deliver PTX safely and effectively along
with reducing the associated hematological adverse effects.

1. Introduction
Breast cancer is one of the most common cancers in women. Ac­
cording to the Global Cancer Observatory and the International Agency
for Research on Cancer (IARC), about 2.26 million new breast cancer
cases (11.7% of all new cancer cases) were registered in 2020, and about
6.8 million deaths (6.9% of all deaths due to cancer) were reported due
to breast cancer in 2020. Further, it is projected that the number of cases
will rise from 2.26 to 2.96 million by 2040 [1].
Currently, chemotherapy is the most utilized treatment for breast
cancer. However, it leads to several clinical adverse effects. These
adverse effects can range from but are not limited to hair loss, fatigue,

Abbreviations: AUC, area under curve in ng hr/mL; C0, initial concentration; Cl, Clearance; Clast, concentration corresponding to time of last measurable observed
concentration; Cmax, maximum observed concentration occurring at time Tmax; DLS, dynamic light scattering; EPR, enhanced permeation and retention; FDA, Food
and Drug Administration; Hb, Hemoglobin; HCT, haematocrit percentage; IARC, International Agency for Research on Cancer; MCH, mean corpuscular Hb, and
MCHC, mean corpuscular Hb concentration; MCV, mean corpuscular volume; PBS, phosphate buffered saline; PLGA, polylactic-co-glycolic acid; PLT, platelet count;
PT, prothrombin time; PTT, activated partial thromboplastin time; PTX, paclitaxel; PTXNp, PTX nanoparticle; RBC, red blood cells; T1/2, terminal half life; Vd,
volume of distribution based on the terminal phase; WBC, white blood cells.
* Corresponding author.
** Correspondence to: Department of Pharmaceutical Sciences, St. John Fisher College, 3690 East Ave, Rochester, NY 14618, USA.
E-mail addresses: lchablani@sjfc.edu (L. Chablani), ajazuddin@nmims.edu (Ajazuddin).

https://doi.org/10.1016/j.biopha.2021.112286
Received 31 August 2021; Received in revised form 24 September 2021; Accepted 5 October 2021
Available online 13 October 2021
0753-3322/© 2021 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
G. Jeswani et al.
vomiting, nausea, and clinically remarkable anemia [2]. Hemolytic
anemia predisposes patients to cardiac and neurological diseases.
Approximately 30–90% of patients receiving chemotherapeutics expe­
rience hemolytic anemia. The nadir is reached between 15 and 20 days.
It is a major challenge and is responsible for higher morbidity rates; such
adverse effects often lead to chemotherapeutic dose restrictions [3].
Chemotherapy also can cause antineoplastic multiple drug resistance,
which is a common threat in relapsed tumors [4]. Due to these limita­
tions, chemotherapy needs to be re-evaluated to overcome these chal­
lenges to provide the intended therapeutic outcomes.
The U.S. Food and Drug Administration (FDA) authorized paclitaxel
(PTX) as a first/second-line intervention for various metastatic malig­
nancies, including breast, ovarian, and non-small cell lung cancer [5].
Taxol® is the majorly prescribed formulation of PTX and has been
effective for several patients with metastatic cancer. In 2005, FDA
approved an albumin-bound PTX nanoparticle, Abraxane®, showing
clinical superiority over PTX alone [6]. Currently, 106 interventional
clinical studies are ongoing based on PTX therapies for breast cancer.
Recently, nanotechnology has enabled targeted delivery of chemo­
therapeutics because of the enhanced permeation and retention (EPR)
effect observed in solid tumors. Such applications also limit drug-related
toxicity to healthy cells. Nanoparticles are among the most extensively
researched nanotechnology-based drug delivery systems [7]. The
composition, particle size, and drug release profile of nanoparticles
regulate the efficacy of these drug delivery systems. Often generally
regarded as safe (GRAS), polymers are chosen for the composition of
such nanoparticles. Resomer® RG 502 H, polylactic-co-glycolic acid
(PLGA) is a biodegradable, safe polymer with lactic acid and glycolic
acid as the base monomers. It is completely metabolized via the Krebs
cycle. However, the main drawbacks of the polymer include low circu­
lation time, poor cell permeation, and rapid in vivo clearance [8]. The
combination of PLGA with other ingredients like polyvinyl alcohol,
dextran, d-α-tocopheryl PEG 1000 succinate, chitosan, or hyaluronic
acid, reduces the drawbacks mentioned above.
Eudragit RSPO (ERSPO) and Eudragit RLPO (ERLPO) are referred as
copolymers of ethyl acrylate and methyl acrylate.The average molecular
weight of ERSPO/ERLPO is 32,000 g/mol. Physically, both appear as
white, fine powders and exhibit typical amine like smell. ERLPO con­
tains 10% of functional quaternary ammonium groups whereas ERSPO
contains 5% functional quaternary ammonium groups. They have poor
aqueous solubility but dissolve freely in acetone and alcohols. Because of
the difference in ratio of quaternary ammonium groups, the water
permeability of ERLPO matrix is higher than ERSPO. These properties
make them an ideal candidate for preparing sustained release dosage
forms [9]. They have been used alone or in combination for preparing
customized matrix structures for drug delivery. However, they have not
been evaluated with PLGA to deliver chemotherapeutic drugs to the
specific tumor site so far.
A combination of different grades of Eudragit and PLGA has been
used previously for the formulation of the nanoparticles of heparin [10],
ciprofloxacin [11], diclofenac sodium [12], enteric coating of eluxado­
line PLGA nanoparticles for sustained release [13], and also for intra­
muscular delivery of interleukin-10 [14]. The biocompatibility of PLGA
and Eudragit E nanoparticles has been previously established [14].
Considering the efficacy and broad application of PTX as a chemo­
therapeutic agent, this study aims to formulate an optimized nano­
particulate drug delivery system that limits the hematotoxicity
associated with PTX. We hypothesized that PTX nanoparticles devel­
oped with a blend of polymers would improve hemocompatibility and
enhance antitumor activity. The work utilizes a statistical design of
experiment to formulate and optimize PTX nanoparticle (abbreviated as
PTXNp hereafter) formulation using three polymers (PLGA, Eudragit
RSPO, and Eudragit RLPO). The optimized formulations were then
evaluated in vitro to determine physicochemical properties such as the
size, zeta potential, morphology, drug release profile, and hemolysis.
Further, the in vivo studies were performed in a Wistar rat model to
2
Biomedicine & Pharmacotherapy 144 (2021) 112286
evaluate the hemocompatibility, pharmacokinetic profile, and bio­
distribution of the drug from the optimized formulation.
2. Materials
Paclitaxel (Benzenepropanoic acid, purity ≥103.3%) was received as
a generous gift from Intas Pharmaceuticals Ltd. (Batch No.
684802806004; Exp. Dt. 06/2022). Eudragit RSPO (ERSPO) and
Eudragit RLPO (ERLPO) and Poly D, L Lactide-co-Glycolide 50:50
(PLGA, Resomer® RG 502H, CAS # 26780–50–70) were obtained as a
gift from Evonik India Pvt. Ltd. Phosphate Buffered Saline, pH 7.4 1X
(CAT # TL1101–500 mL), Fetal bovine serum (CAT #RM1112–500 mL),
Streptomycin-penicillin solution (CAT #A007–100 mL) and Dulbecco’s
Modified Eagle Medium, DMEM (CAT #AL007A-500 mL) were bought
from HiMedia, Mumbai, India. Dimethyl sulfoxide (CAT #515505) was
procured from CDH, New Delhi, India. MCF-7 cell line was procured
from National Centre for Cell Science, Pune, India. Polyvinyl alcohol,
acetone, and deionized water (HPLC grade) were bought from Sigma
Aldrich (Mumbai, India). Uniplastin and Liquicellin was the product of
Tulip Diagnostics (P) Ltd, Goa, India. All other chemicals were of
analytical grade.
3. Method
3.1. Experimental design and preparation of nanoparticle
3.1.1. Experimental design
A three-factor, simplex lattice mixture design was constructed to
evaluate the optimum amounts of individual polymers required to yield
a formulation with the lowest hemolysis (less than 10%), smallest par­
ticle size (less than 220 nm) and the desired drug release (more than
70%). Statistical software, JMP® version 15.2.1 (SAS Institute, Cary,
NC), was used to create and evaluate the experimental design. PLGA,
ERSPO, and ERLPO concentrations were included as continuous factors
to develop a mixture design (Table 1).
The design aimed to determine the impact of the concentration of
polymers on the formulation quality. The quality characteristics of the
formulation included particle size, percent hemolysis, and drug release.
Polymer concentrations of all three polymers (PLGA, ERSPO, and
ERLPO) were varied in the range of 0–100% or 0–1 (as coded) (Table 1).
The concentration of PTX was held at 20 mg, such that the drug-polymer
ratio remained constant at 1:4.
3.1.2. Preparation of nanoparticle
The nanoprecipitation method was used to formulate polymeric
nanoparticles [15]. Briefly, PTX (20 mg) was dissolved in 4 mL ethanol.
The solution was stirred on a magnetic stirrer (Remi, India) for 5 min at
100 rpm in a covered beaker to get a 5 mg/mL solution. For individual
formulations, a varying amount of PLGA/ERSPO/ERLPO was taken as
mentioned in Table 1. The organic phase constituted the mixture of the
required amount of PLGA in 2 mL acetone. ERSPO, ERLPO, or both were
dissolved in 2 mL ethanol and the drug solution was also prepared in
ethanol separately. Both polymer solutions were mixed with constant
stirring at 100 rpm on a magnetic stirrer for 2 min. Next, 4 mL of drug
solution was combined with the prepared polymer solution to constitute
the organic phase. The organic phase was subsequently added to 40 mL
of aqueous phase containing phosphate buffer saline (PBS), pH 7.4% and
1% v/v PVA. The addition was performed at a constant rate of 0.5
mL/min using BD Veo™ insulin syringes with BD Ultra-Fine™ 31 G
needle. The addition continued with uninterrupted stirring at 1000 rpm
on a magnetic stirrer at room temperature, 25 ◦ C. Lastly, the stirring was
done at 200 rpm for 12 h in a rotary shaker maintained at 30 ◦ C to
evaporate the organic phase. The resulting PTXNp suspension was
centrifuged at 12,298 xg at 4 ◦ C for 15 min using Eppendorf Centrifuge
5424R (Eppendorf AG, Hamburg, Germany) to get a pellet and further
washed twice with 5 mL of deionized water.
G. Jeswani et al. Biomedicine & Pharmacotherapy 144 (2021) 112286

Table 1
Simplex lattice mixture design of nanoparticle formulation showing different proportions of polymers used in 21 runs and resulting responses. ERSPO, ERLPO, PLGA
are used in proportion, [0,1]. Responses are represented as mean ± SD (n = 3).
Formulation Code Composition Hemolysis (%) Particle size (nm) Cumulative Release in PBS pH 7.4 (%)

ERSPO (mg) ERLPO (mg) PLGA (mg)

PTXNp1 0 [0] 32 [1] 0 [0] 23.23 ± 1.00 65.77 ± 1.93 82.02 ± 1.74
PTXNp2 6.4 [0.2] 25.6 [0.8] 0 [0] 19.76 ± 2.00 67.74 ± 1.85 80.23 ± 3.01
PTXNp3 6.4 [0.2] 0 [0] 25.6 [0.8] 07.43 ± 1.00 210.73 ± 1.85 66.92 ± 3.01
PTXNp4 0 [0] 19.2 [0.6] 12.8 [0.4] 15.89 ± 2.91 200.92 ± 2.64 72.66 ± 1.47
PTXNp5 12.8 [0.4] 0 [0] 19.2 [0.6] 09.00 ± 1.86 203.67 ± 2.17 68.34 ± 1.72
PTXNp6 12.8 [0.4] 6.4 [0.2] 12.8 [0.4] 11.33 ± 2.65 198.42 ± 3.79 65.58 ± 2.48
PTXNp7 12.8 [0.4] 19.2 [0.6] 0 [0] 16.89 ± 2.86 71.90 ± 1.36 78.28 ± 1.49
PTXNp8 25.6 [0.8] 6.4 [0.2] 0 [0] 18.98 ± 0.92 92.77 ± 0.89 72.65 ± 1.18
PTXNp9 0 [0] 6.4 [0.2] 25.6 [0.8] 07.89 ± 0.98 209.43 ± 1.87 67.32 ± 0.71
PTXNp10 0 [0] 25.6 [0.8] 6.4 [0.2] 21.58 ± 2.36 74.77 ± 1.55 78.51 ± 2.21
PTXNp11 6.4 [0.2] 6.4 [0.2] 19.2 [0.6] 10.21 ± 2.46 206.22 ± 1.22 66.05 ± 1.97
PTXNp12 6.4 [0.2] 12.8 [0.4] 12.8 [0.4] 13.58 ± 1.73 198.27 ± 1.24 65.70 ± 1.96
PTXNp13 19.2 [0.6] 0 [0] 12.8 [0.4] 12.98 ± 1.32 203.13 ± 1.91 69.20 ± 3.38
PTXNp14 0 [0] 12.8 [0.4] 19.2 [0.6] 12.07 ± 1.25 198.36 ± 1.24 69.80 ± 0.72
PTXNp15 0 [0] 0 [0] 32 [1] 02.01 ± 1.27 214.73 ± 4.48 65.00 ± 2.13
PTXNp16 32 [1] 0 [0] 0 [0] 21.00 ± 1.23 111.83 ± 1.77 70.02 ± 1.76
PTXNp17 19.2 [0.6] 12.8 [0.4] 0 [0] 20.89 ± 3.82 88.77 ± 1.68 74.30 ± 1.78
PTXNp18 6.4 [0.2] 19.2 [0.6] 6.4 [0.2] 14.23 ± 3.41 197.27 ± 1.98 78.64 ± 1.35
PTXNp19 19.2 [0.6] 6.4 [0.2] 6.4 [0.2] 13.67 ± 2.00 195.37 ± 1.53 68.51 ± 2.10
PTXNp20 25.6 [0.8] 0 [0] 6.4 [0.2] 18.25 ± 1.91 116.82 ± 3.24 69.84 ± 1.35
PTXNp21 12.8 [0.4] 12.8 [0.4] 6.4 [0.2] 17.36 ± 1.04 125.97 ± 2.58 70.27 ± 2.05
PTX solution in ethanol (200 µg /mL) – – – 100 – –

3.1.3. Freeze-dried nanoparticles India.

The nanoparticle pellet was reconstituted with PBS of pH 7.4 (10 mL)
and 5% w/v mannitol (cryo-preservative). The suspension was main­
tained at − 40 ◦ C for two days in a freezer. Subsequently, PTXNp sus­
pension was transferred to Heto PowerDry® LL 3000 freeze dryer
(Thermo Fisher Scientific, United States) pre-chilled to − 40 ◦ C. Lyoph­
ilization was performed for five days under vacuum (− 20 ◦ C, 100
mTorr). At the end of the process, free-flowing freeze-dried PTXNps
were collected in a 10 mL glass vial and were dessicated at ambient
temperature. Freeze-dried samples were used for all further evaluations.
Collocating with our aim, percent release, hemolysis, and particle size
were used as response variables for the study.
3.2. In vitro characterization
3.2.1. Particle size
The mean particle diameter and size distribution of PTXNp suspen­
3.2.2.1. Hemolysis study. The whole blood was centrifuged (10 min,
867 xg, Eppendorf Centrifuge 5424 R) to obtain a RBC pellet for the
hemolysis experiment [17]. The RBC pellets were washed twice with
0.9% saline. A 3% v/v RBC stock suspension in PBS (pH 7.4) was pre­
pared using 3 mL of packed cell volume. To prepare the sample, 1 mL of
PTXNp suspension in PBS (containing 200 micrograms PTX) was mixed
with 1 mL of 3% v/v RBC suspension. The mixture was incubated for 4 h
at 37 ◦ C [16]. Distilled water (1 mL) was used for 100% hemolysis
(positive control), and 1 mL of PBS was used for 0% hemolysis (negative
control) [18]. After full incubation time, the suspension was centrifuged
for 10 min (867 xg using Eppendorf Centrifuge 5424 R). The supernatant
was examine using UV spectrophotometer (Double beam UV Vis Spec­
trophotometer LT 2800, Labtronics, India) for released hemoglobin (Hb)
at 540 nm [18]. The hemolysis percent was calculated as per Eq. (1).

Absorbance ofblood incubated with PTXNp − Absorbance of negative control

Hemolysis %= ∗ 100 (1)
Absorbanceof positive control − Absorbanceofnegative control

sion was determined by DLS technique on a Nano ZS (Zeta Sizer, Mal­

vern, UK) at 25 ◦ C with an equilibration time of 60 s and 90◦ angle of
detection using DTS0012 transparent disposable cuvette. One milligram
of lyophilized PTXNp formulations were dispersed in 10 mL HPLC grade
water and sonicated for 1 min before measurements. Measurements of
each sample were done in triplicates and reported with standard
deviations.
3.2.2. Hemocompatibility studies
Human blood (10 mL/volunteer) from healthy volunteers (18–50
years) was collected after a written consent. The samples were pooled
and stored in EDTA anti-coagulant blood storage vials at − 6 ± 2◦ C until
used for all the experiments of hemocompatibility [16]. The study
protocol was permitted by the Institutional Research Committee of
Rungta College of Pharmaceutical Science and Research, Bhilai, CG,
3.2.3. In vitro PTX release profile
The dialysis bag diffusion technique was used for the in vitro release
studies, as described by Dong et al. [19]. Lyophilized PTXNp (100 mg)
were incubated with 2 mL of PBS (pH 7.4, receptor fluid to mimic
physiological blood-plasma pH) in a dialysis membrane bag (MW cut off
14000 D). The dialysis bag was secured and placed into a beaker con­
taining 40 mL of PBS with 0.1% Tween 80, maintained at 37 ◦ C in a
shaking water bath. At regular time points (2, 4, 6, 8, 10, 28,50, 74, 168,
360 h) until 15 days, 2 mL of receptor fluid was withdrawn and diluted
to 10 mL with HPLC mobile phase to determine PTX concentration via
HPLC analysis (details provided in the supplementary file, Table S1). In
vitro release data of all PTXNp suspensions were fitted to relevant kinetic
models, and the best fit model was chosen based on the highest

3
G. Jeswani et al.
correlation coefficient.
3.3. In vitro analyses of optimized formulations
After evaluating the PTXNp for particle size, hemolysis, and in vitro
release, additional in vitro studies were carried out on selected optimized
formulations only. These formulations included PTXNp3, PTXNp9, and
PTXNp15.
3.3.1. Surface morphology
Electron transmission microscopy (TEM) (Tecnai, G 20, FEI), with
image processing software iTEM, Olympus Soft Imaging System, Ger­
many, was used to study the surface morphology of PTXNps. Before grid
preparation, 1 mL of nanoparticle suspension was diluted with 5 mL of
saline solution and sonicated for 5 min 20 µL of the sample was posi­
tioned onto the non-copper grid and negatively stained with a 0.01% w/
v phosphotungstic acid solution for 50 s [20]. The grid was allowed to
air dry for 5 min and then imaged under the microscope supported with
a CCD camera. TEM analysis was performed at All India Institute of
Medical Sciences (AIIMS), New Delhi, India. Furthermore, for surface
topography, the PTXNps were mounted onto a stub using conductive
carbon adhesive tape and then sputter-coated with gold. The samples
were imaged using a ZEISS EVO series Scanning Electron Microscope
(SEM; Model EVO 18).
3.3.2. Zeta potential
Electrophoretic light scattering technique was used for determining
electrophoretic mobility (zeta potential) using Nano ZS Zetasizer (Zeta
Sizer, Malvern, UK) at 25 ◦ C. The test was carried using a zeta cell,
DTS1060C, with 30 s equilibration time. One milligram of PTXNp for­
mulations were dispersed in 10 mL of HPLC grade water and sonicated
for 1 min before measurements.
3.3.3. FTIR and XRD
The potassium bromide disc method using infrared (IR) spectroscopy
(Shimadzu IR Affinity 1 DRS8000) over wavelength range
4000–400 cm− 1 was used to determine any drug-polymer interaction.
For X-ray diffraction (XRD) studies approximately, 2 mg of the drug,
polymers, and PTXNp were analyzed by high-resolution X-ray diffrac­
tometer (PANalytical 3 kW X′ pert Powder – Multifunctional) in a range
of 5–80◦ (2θ), using Cu-Kα radiation (λ = 1.544 Å) generated at 40 kV
and 30 mA, at a scan step size of 0.026◦ .
3.3.4. Loading and entrapment efficiency
Briefly, 5 mg of each lyophilized PTXNp formulation was suspended
in HPLC grade water (10 mL) and stirred for 15 min on a magnetic
stirrer (Remi, India) at 100 rpm and then sonicated for 5 min (400 W).
The prepared suspensions were centrifuged (Eppendorf Centrifuge
5424R) twice at 1491 xg for 10 min to determine the amount of unin­
corporated drug in the supernatant using UV Spectroscopy (Shimadzu
UV-1800, Labomed USA). The concentration of the PTX was estimated
using the calibration curve. Entrapment efficiency and drug loading
were calculated using the Eqs. (2) and (3) resp.
Total PTX added- PTX in the supernatant
Entrapment Efficiency(%) =
Total PTX added
∗ 100
(2)
Mass of PTX in the nanoparticle
Drug loading(%) = ∗ 100 (3)
The total mass of PTXNp
All the determinations were performed in triplicates, and results are
reported as mean ± standard deviation.
3.3.5. Blood compatibility
Pooled human blood (1 mL) as described in Section 3.2.2 was
4
Biomedicine & Pharmacotherapy 144 (2021) 112286
incubated for a specific time as indicated in the individual tests with
1 mg/mL of all the optimized and placebo nanoparticles for different
hemocompatibility tests in individual EDTA (hemolysis test) or sodium
citrate tubes (coagulation test). The treated sample was used for further
analyses. A 1 mg/mL concentration of the particle was used to achieve
0.77 ± 1.14 mg/mL PTX concentration in vitro. The concentration is
much higher than the concentration used in animal models
(0.06–0.12 mg/mL). Blood incompatibility issues are reported to be
concentration-dependent; hence we selected much higher theoretical
concentration than used for in vivo studies to predict the safety of the
formulation at a higher dose [21].
3.3.5.1. Whole blood clotting time. The whole blood clotting time assay
was executed as per the Lee White method (17). After 30 s of incubation
at 37 ◦ C, the tubes were tilted to 30º to check the blood clotting. After
every 30 s, the tubes were tilted further until they stopped flowing at
90º, and at that instance, the clotting time was recorded. Blood from
blank EDTA test tubes (without formulation) served as the negative
control, and non-EDTA glass tubes were a positive control. All mea­
surements were performed in triplicates.
3.3.5.2. Blood counts. To determine the blood counts, 1 mL of blood
was incubated with 1 mg of formulation (placebo and PTXNp) for
30 min at 37 ◦ C. A double capillary blood cell counter (Model Sysmex
XP-300, Automated hematology analyzer) was used to determine com­
plete blood count, including RBC and WBC count, total Hb concentra­
tion, hematocrit (HCT) percentage, platelet (PLT) count, and differential
counts (lymphocytes, monocytes, and granulocytes). We also deter­
mined the blood indexes [mean corpuscular volume (MCV), mean
corpuscular Hb (MCH), and mean corpuscular Hb concentration
(MCHC)] [16].
3.3.5.3. Activated partial thromboplastin time (aPTT) and prothrombin
time (PT) for coagulation. A 1 mL of sodium citrate blood sample was
mixed with 1 mg of the formulation (placebo and PTXNp) to prepare the
sample for aPTT determination. Then, 50 µL of the sample was mixed
with 50 µL of aPTT reagent (LiquicellinE, Tulip Diagnostics) and incu­
bated with a steel ball for 20 min at 37 ◦ C. After the incubation, 50 µL
calcium chloride was added, and results were recorded with Stago Art4
Coagulomete Diagnostica, France. Similarly, for PT measurements,
50 µL of plasma sample with steel ball were incubated with Calcium
Thromboplastin Reagent (Uniplastin, Tulip Diagnostics), and values
were recorded [22]. Measurements were performed in triplicates.
3.3.5.4. C3 complement. Immunoturbidimetry method using the Com­
plement protein C3 kit (Biobase, China) was used to determine com­
plement activation by optimized PTXNps and respective placebo
nanoparticles [23]. The assay was conducted as per the manufacturer’s
protocol. Briefly, 1 mL of human serum sample was mixed with 1 mg of
formulation (placebo and PTXNp) and incubated for 1 h at 37 ◦ C.
Absorbance was measured thereafter. All measurements were performed
in triplicates [24].
3.4. In vitro cytotoxicity assay
Cytotoxicity assay of PTX solution, PTXNp3, PTXNp9, and PTXNp15
was performed against MCF-7 breast cancer cells using the referenced
MTT assay protocol [25]. Cytotoxic effects of placebo particles corre­
sponding to PTXNp3, PTXNp9, and PTXNp15were also determined.
MCF-7 cells were grown in a T-75 flask using complete Dulbecco’s
modified eagle medium (DMEM) containing fetal bovine serum (10%
v/v) and streptomycin-penicillin (1% v/v). Confluent cells were coun­
ted, and 10,000 cells were seeded into each well of a 96-well plate fol­
lowed by incubation in a CO2 incubator (Thermo-Fisher Heracell150i).
Serial dilutions of PTX (1, 2.5, 5, 7.5, 10, 20, 35, and 50 µg/mL) were
G. Jeswani et al.
formulated in complete DMEM media. Similarly, corresponding to the
PTX concentrations based on the entrapment efficiency, serial dilutions
of PTX loaded and placebo nanoparticles were prepared in the same
media (70, 350, 525, 700, 1400, 2100, 3500, 4900 µg/mL). Further, the
cancer cells were treated with the known concentrations of PTXNp or
PTX to be investigated and incubated for 24 h. Complete DMEM media
served as the negative control. After the incubation period, each well
was washed with pH 7.4 PBS and 50 µL of MTT solution was added.
Subsequently, the cells were kept for 4 h of incubation. To dissolve the
formazan crystals produced due to viable cells, 150 µL dimethyl sulf­
oxide was added into each well [26]. The absorbance was analyzed (λmax
= 570 nm) using a microplate reader (Omega Fluster, BMG Labtech) and
the IC50 values of PTX and nanoparticles were calculated.
3.5. In vivo pharmacokinetic studies
Pharmacokinetic parameters were determined in male Wistar rats
(~250 g) aged 10–12 weeks. The study protocol was approved by the
Institutional Animal Ethics Committee, Rungta College of Pharmaceu­
tical Sciences, Bhilai, CG, India (1189/PO/Re/s/08/CPCSEA/2018/6).
Animals were housed in a temperature and relative humidity controlled
area (25ºC and 50% RH) for 12 h light/dark cycle. The rats were
randomly divided into five groups, P1, P2, P3, P4, and P5 (n = 3/group).
Group P1, P2, and P3 received PTX nanoparticle formulations, PTXNp3,
PTXNp9, and PTXNp15, respectively. The formulations were adminis­
tered via the tail vein as a single dose of 5 mg/kg body weight [27].
Group P4 received pure PTX solution in ethanol (5 mg/mL), and P5
served as the control. Post-treatment, 0.2 mL of blood samples were
collected at the predetermined intervals (0.5, 1, 2, 4, 8, 12, and 24 h) in
heparinized capillaries from retro-orbital plexus. The collected samples
were immediately centrifuged at − 6 ◦ C and 31,304 xg for 5 min
(Eppendorf Centrifuge 5424 R). The plasma was collected in a labeled
microcentrifuge tube and stored at − 20 ºC until further use. Before the
analysis, 200 µL of plasma was mixed with 1 mL acetonitrile to extract
PTX, [28]. The extracted PTX solutions were centrifuged, the superna­
tant was collected and filtered using a 0.25 µm membrane filter, fol­
lowed by analysis through HPLC. Pharmacokinetic parameters, terminal
half-life (T1/2), maximum plasma concentration (Cmax), area under
curve (AUC), volume of distribution based on the terminal phase (Vd),
clearance (Cl), and mean residence time (MRT) were calculated using
Phoenix 64 WinNonlin® version 8.3.3.33 (Pharsight Corporation,
MountainView, CA, USA).
3.6. Biodistribution and hematological studies
Post-treatment, at 24 h, rats were euthanized. Blood was collected by
cardiac puncture, and samples were analyzed for a complete blood
count. Further, the liver, spleen, lungs, kidneys, and heart were isolated.
HPLC grade acetonitrile (10 mL) was used to precipitate out the plasma
proteins and extract the drug from the collected organs. Each organ was
weighed separately and homogenised with a homogenizer (10 min,
10,000 rpm, Ultra-Turrax®, T-25, IKA, Germany). The homogenate was
then centrifuged for 10 min at 2772 xg to extract the drug for its
quantitative analysis. The supernatant was transferred to sample vials,
reconstituted with 1 mL of mobile phase, and vortexed for 2 min. Once
again, the vials were centrifuged for 15 min at 4928 xg. The supernatant
was subsequently collected and filtered using a 0.25 µm membrane fil­
ter, followed by HPLC analysis.
4. Data analysis and statistics
JMP® version 15.2.1 (SAS Institute, Cary, NC) was used to create the
design of experiment. All the statistical analyses were done using
GraphPad Prism v 8.0. Phoenix 64 WinNonlin® version 8.3.3.33
(Pharsight Corporation, MountainView, CA, USA) was used for non-
compartment pharmacokinetic analysis. Results are expressed as mean
5
Biomedicine & Pharmacotherapy 144 (2021) 112286
± standard deviation (SD) of the three replicates. Sample means were
compared by t-test, one-way or two-way ANOVA, whichever applicable,
followed by a Tukey post hoc test. A p-value of < 0.05 was considered
significant.
5. Result and discussion
Recently, advancements in drug delivery techniques have allowed
clinicians to deliver chemotherapeutic agents to cancer patients with
potentially minimum adverse effects. Our research focuses in the same
direction as we utilize a statistical design of experiment to formulate a
PTXNp with the desired physicochemical properties. A mixture design
was prepared with varying Eudragit RSPO/RLPO and PLGA concentra­
tions to study the response variables, including the particle size, percent
hemolysis, and drug release. The overall goal of the study is to benefit
from a small particle size formulation to achieve desirable EPR effect,
minimize hemolysis, and obtain efficient drug release.
5.1. Mixture design and preparation of nanoparticles
PTX is a Biopharmaceutical Classification System class IV drug. It has
an aqueous solubility of 0.25 µg/mL (log D of ~4.95), and the log P
value is 3.66 [29]. Moreover, the major clinical challenge of PTX is
drug-induced hemolytic toxicity. Low solubility, low permeability, and
drug-induced toxicity are the critical limitations of PTX. Nanoparticles
increase the solubility of poorly soluble drugs, enhance permeability,
and help in achieving targeted drug release. Nanoprecipitation method
is the most convenient and feasible formulation technique to prepare
nanoparticles that can help to overcome the above-discussed challenges
[30]. It was hypothesized that composition variables of nanoparticles
significantly affect the major formulation characteristics of PTX (particle
size, drug release, and hemolysis). Hence, modulation of composition
variables can enhance drug delivery, sustain drug release, and minimize
hematotoxicity.
To validate the hypothesis, JMP® version 15.2.1 (SAS Institute, Cary,
NC) statistical software was used to generate a mixture design-based
design of experiment. It generated experimental runs to predict a set
of formulations that will be least hemolytic and have the desired particle
size along with the adequate sustained release. Further, the optimum
combination of Eudragit RSPO/RLPO and PLGA was carefully chosen
with the help of a simplex lattice mixture design, wherein the pro­
portions of constituents were varied from none to whole without the
application of any constraints. In the present investigation, PTX loaded
PLGA, ERSPO, ERLPO, or both nanoparticles were prepared using
nanoprecipitation technique using ethanol and acetone as the organic
phase. Although all the polymers are readily soluble in acetone, ethanol
was used for better tolerability [31]. By varying the proportion of
polymers from 0 to 1 at five different levels (0, 0.2, 0.4, 0.6, 0.8, and 1),
the study attempts to balance the toxicity and the therapeutic efficacy of
PTX with an optimum concentration of biodegradable, hemocompatible
polymer (PLGA), and sustained release polymers (ERSPO/ERLPO).
Table 1 lists all of the 21 experimental runs.
Percent hemolysis, cumulative release in PBS (pH 7.4), and particle
size were measured as three responses. The determination of particle
size correlates with the effect of formulation variables, giving a clear
understanding of the internal phase viscosity containing polymers due to
their concentration, type, and proportion. Drug release is one of the
critical parameters that predict the mechanism of the drug release from
the particle matrix in physiological conditions. A low release may be due
to differences in particle size or the degradation of the polymer. The
conditions of drug release also depends on the tumor environment. The
third and most important variable that was analyzed was the percent
hemolysis. Hemolysis is one of the most challenging drawbacks of the
chemotherapeutics as the RBC depletion occurs within a few weeks
following the treatment leading to an increased risk and requiring RBC
transfusion. Hence, hemolysis was evaluated as one of the vital variables
G. Jeswani et al.
in the study. Summary of fit (R2 coefficient of determination, and
adjusted R2) and analysis of variance (ANOVA) were used to evaluate all
the variables. R2 shows that whether there is a good fit between the
results and the model obtained. Higher is the value, the more variation is
explained, and the better is the model; however, on adding more vari­
ables, adjusted R2 is used to judge the goodness of the model as it only
counts the significantly affected variables. ANOVA shows whether there
is any significant difference between the runs or not [32]. Table 1 lists
the results from all 21 runs. As depicted in Fig. 1, a prediction profiler
95.14 ∗ ERSPO + 47.78 ∗ ERLPO + 202.88 ∗ PLGA + ERSPO(ERLPO ∗ 89.626) + ERSPO(PLGA ∗ 217.09) + ERLPO(PLGA ∗ 287.43)
shows how different formulation variables influence the responses.
The statistical analysis suggests that the DOE factors including the
composition of the formulation significantly influences the studied re­
sponses. An in-depth analysis of all three factors was further done to
define the optimal composition of the formulation.
5.2. In vitro characterization to determine particle size, hemolysis, and
drug release
5.2.1. Particle size
The particle size was determined to observe the impact of polymer
type and its concentration used in the formulation. The mean particle
size of PTXNps ranged between 65.77 ± 1.93 nm to 214.73 ± 4.48 nm
with a relative standard deviation of ≤ 5%, as shown in Table 1. How­
ever, the average particle size of most of the formulations was within
250 nm, ideal for accumulation at the tumor site [33]. Particles in the
range of 50–300 nm in size are reported to accumulate at the tumor site
6
Biomedicine & Pharmacotherapy 144 (2021) 112286
Fig. 1. Mixture profiler with contour grids of
polymeric nanoparticles for particle size less
than 200 nm, cumulative drug release at 15
days greater than 70%, and percent hemolysis
less than 10%. The three contour lines represent
the trend in following response variables:
green=particle size; red: drug release; and
blue=hemolysis. The uncolored white region
denotes the operational design space. Five
nanoparticle formulations: PTXNp3, PTXNp5,
PTXNp9, PTXNp11, and PTXNp15 met the set
criteria for the response variables and appeared
in the uncolored region (PTXNp1-PTXNp21 are
represented as 1–21 in the figure).
owing to easy penetration through leaky vasculature, impaired
lymphatic drainage (EPR effect) and lack of renal clearance [34,35].
ANOVA indicated significant influence of all formulation components
on particle size (p < 0.0001; R2 = 0.83; adjusted R2 = 0.783). All the
polymers impacted the particle size significantly individually ERSPO
(p = 0.0006), ERLPO (p = 0.0462), and PLGA (<0.0001) and their
two-factor interactions ERSPO*PLGA (p = 0.0317) and ERLPO*PLGA
(0.0068). A prediction equation calculated through the best fit model for
particle size is given in Eq. (4).
(4)
According to the equation, all the polymer concentrations exhibited
a positive correlation with particle size. PLGA terms (individual or
interaction terms) had more prominent constants than ERSPO and
ERLPO terms. Thus PLGA, as a polymer, had a significant impact on
particle size than other polymers. PLGA has contributed towards an
increase in the overall particle size. This was evident from the particle
size of PTXNp15 (only PLGA matrix) being 214.73 nm. Whereas, the
mean particle size of PTXNp3 (ERSPO-PLGA combination matrix) was
210.73 nm, and particles devoid of PLGA such as PTXNp1 (ERLPO) and
PTXNp16 (ERSPO) had further smaller sizes of 65.77 nm and 111.83 nm
respectively. Based on the Eq. (4), the numeric constants for PLGA,
PLGA*ERSPO, PLGA*ERLPO are 202.88, 217.09 and 287.43 respec­
tively. This confirms the impact of PLGA and its related terms on overall
particle size. Comparable findings were also reported by Hasan et al. and
Cetin et al. [36,37]. As noted elsewhere, an increase in the PLGA
G. Jeswani et al. Biomedicine & Pharmacotherapy 144 (2021) 112286

Table 2 hemocompatibility [11,16,42]. Most published studies on PLGA nano­

Effect Summary table for 21 observation. particles have reported hemocompatibility of PLGA nanoparticles
Response R2 R2 Root Mean Mean of p value similar to our results [16,17].
adjusted Square Response Taxanes generally cause anemia due to myelosuppressive effects or
Error drug-induced hemolytic anemia [43]. In a study, PTX caused complete
Particle Size 0.837 0.783 27.428 154.881 <0.0001* hemolysis at the concentration of 200 µg, while polymeric blend nano­
Percent 0.942 0.922 1.526 14.670 <0.0001* particles inhibited the drug-induced hemolysis [44]. According to the
Hemolysis hemolysis reports by Namgung et al., Taxol containing 40 µg of PTX
Release in 0.870 0.827 2.179 71.380
exhibited 100% hemolysis (erythrocytic toxicity) at 20 ppm concentra­
<0.0001*
PBS pH 7.4
tion [45]. A much higher concentration was employed in our study to
determine the effect of nanoparticles on hemolysis.
concentration possibly will increase viscosity and diffusion coefficient,
Our study observed that the percent of hemolysis was reduced to
leading to the formation of larger particles [38].
2.01% (PTXNp15). The results of the study indicated the protective ef­
Nanoparticles prepared using ERLPO showed a smaller mean size
fect of nanoparticles on RBCs. The reported reduction in hemolysis can
compared to ERSPO (PTXNp1 Vs. PTXNp16). Similar findings were re­
be credited to the slow-sustained release of PTX from the polymer ma­
ported by Pignatello et al. (2002), Y. Y. Jiao et al.(2001), and Hasan
trix. Comparatively, the mixture of polymers reduced the hemolysis up
et al.(2012) when they encapsulated flurbiprofen, heparin, and dorzo­
to ~20%, which is five times lesser than the pure drug. Placebo nano­
lamide hydrochloride, respectively. They related this effect to higher
particles exhibited a negligible effect on the RBCs.
quaternary ammonium groups in ERLPO [39–41].
The equation related to hemolysis and varying proportion of the
polymers is given below:
5.2.2. Hemolysis study
Table 1 summarizes the percentage of hemolysis on incubation of

21.77 ∗ ERSPO + 23.34 ∗ ERLPO + 2.99 ∗ PLGA + ERSPO(ERLPO ∗ − 18.77) + ERSPO(PLGA ∗ − 5.18) + ERLPO(PLGA ∗ 3.54) (5)

nanoparticle formulation with RBCs. Hemocompatibility of the formu­

lations was the primary aim of the study, and it was evaluated by
determining the hemolytic toxicity. Hemolysis is considered a vital
screening test to perform according to the ISO10993–4 parameters. It
measures the fragility of the RBC membrane when in contact with
foreign materials. The hemolysis of all the formulations ranged from 2%
to 23%. Lower values were obtained for PTXNP15<PTXNp3
<PTXNp9<PTXNp11<PTXNp6 ranging from ~2–11%. Hemolysis was
significantly impacted by the presence of ERSPO and ERLPO in the
formulation (p < 0.0001) along with their cross term (p = 0.0022)
(Table 2). However, the impact of PLGA was comparatively less signif­
icant on hemolysis (p < 0.0274). Increasing the proportion of PLGA in
nanoparticles led to lower hemolysis as PLGA had improved
This equation depicts that the PLGA has a minor cytotoxic/hemolytic
effect on the RBCs as represented by its numeric constant, 2.99. PLGA is
a negatively charged polymer due to carboxyl groups, so it does not form
a complex with negatively charged RBCs [46]. In contrast, ERSPO and
ERLPO are positively charged polymers due to quaternary ammonium
groups present in the molecule, contributing to the formation of the drug
RBC complex [37,47]. Most hemolysis was observed with formulations
containing only ERLPO (constant value of 23.34) and ERSPO (constant
value of 21.77). In contrast, minimum hemolysis was observed with the
PLGA nanoparticles. The hemolysis values of PTXNp1 (ERLPO) was
23.23 ± 1.00, PTXNp 16 (ERSPO) was 21 ± 1.23, and PTXNp15 (PLGA)
was 2.01 ± 1.27. Thus, PLGA as a polymer has a protective impact on

Table 3
Kinetic modeling of PTX release from different nanoparticle formulations. The quantity of PTX released from PTXNp was calculated by means of validated HPLC
method, using a calibration curve (available in supplementary file, Table S1).
Formulation Code R2 Release exponent

Zero Order First order Higuchi Hixon Korsmeyer- Peppas

PTXNp1 0.849 0.961 0.974 0.931 0.976 0.366

PTXNp2 0.852 0.956 0.977 0.926 0.995 0.276
PTXNp3 0.831 0.938 0.958 0.9 0.948 0.344
PTXNp4 0.798 0.904 0.953 0.871 0.992 0.253
PTXNp5 0.834 0.923 0.97 0.896 0.994 0.263
PTXNp6 0.782 0.871 0.947 0.842 0.994 0.289
PTXNp7 0.831 0.952 0.978 0.923 0.994 0.235
PTXNp8 0.61 0.885 0.948 0.99 0.855 0.256
PTXNp9 0.86 0.91 0.96 0.882 0.988 0.255
PTXNp10 0.816 0.896 0.938 0.858 0.986 0.227
PTXNp11 0.829 0.871 0.947 0.846 0.988 0.235
PTXNp12 0.74 0.871 0.944 0.843 0.985 0.254
PTXNp13 0.798 0.89 0.949 0.86 0.991 0.251
PTXNp14 0.682 0.901 0.958 0.873 0.991 0.259
PTXNp15 0.855 0.842 0.931 0.815 0.989 0.257
PTXNp16 0.845 0.86 0.938 0.831 0.989 0.260
PTXNp17 0.628 0.906 0.921 0.868 0.932 0.262
PTXNp18 0.705 0.887 0.919 0.845 0.98 0.213
PTXNp19 0.872 0.881 0.949 0.851 0.991 0.272
PTXNp20 0.808 0.851 0.923 0.817 0.988 0.250
PTXNp21 0.778 0.807 0.905 0.777 0.981 0.256

7
G. Jeswani et al.
RBCs compared to the other polymers.
5.2.3. In-vitro drug release kinetics
Table 1 presents the percent cumulative release of 21 PTXNp for­
mulations. The release studies were executed to establish the influence
of the composition of the nanoparticles on the release kinetics and their
in vivo performance. Release data were processed and mapped according
to different release kinetic equations accompanied by regression ana­
lyses to explain the drug release mechanism of from various formula­
tions. Additionally, the in-vitro performance indicates the in-vivo
therapeutic efficacy of the formulation. Defining the exact mechanism,
order of release for formulations is challenging due to different polymer
Fig. 2. (A) Transmission electron microscopic (TEM) images of. Scale bar = 1 µm (B) Scanning electron microscopic images (SEM) Scale bar = 2 µm.
8
Biomedicine & Pharmacotherapy 144 (2021) 112286
blends and their varying degradation and release mechanisms. There­
fore, the best fit model was selected based on the goodness of fit (R2
values). For each plot, slope and release rate constants were determined.
The same procedure was adopted for each model. The release rate
mechanism was also inferred from the same. The correlation coefficient
(R2) was calculated using different release model equations.
Table 3 presents the release profiles of the drug from 21 PTXNp
formulations. About 9–20% of drug release was detected in the first 2 h.
The burst release may be caused by two reasons: firstly, the fraction of
the unincorporated drug weakly bound to the vicinity of the nano­
particle matrix or its surface, and secondly, owing to the existence of
quaternary ammonium functional group present in the Eudragit
G. Jeswani et al.
molecule, which interacts with water molecules leading to swelling of
the polymeric matrix. These results also corroborate with the diffusion-
based drug release pattern, as discussed further in this section. In the
nanoparticles, some drugs may be dispersed in the amorphous state in
the polymer matrix, as reported in FTIR studies. These results are similar
to those reported by Dillen et.al regarding the drug being in amorphous/
semicrystalline state in the nanoparticles [11]. As the amorphous form
has better solubility, it can contribute to the burst release of drug mol­
ecules from the nanoparticle. As observed in the release study, beyond
2 h, the release rates were meager. The required time to release more
than 65% of the drug from PTXNp is too long (360 h). ANOVA showed
that the proportion of polymers significantly impacted the drug release
(p < 0.0001, Table 2). The R2 obtained for the model was 0.870, and the
R2 adjusted was 0.827. The equation modeling related to drug release
and a different proportion of ERSPO, ERLPO, and PLGA is given in Eq.
(6):
70.83 ∗ ERSPO + 84.13 ∗ ERLPO + 66 ∗ PLGA + ERSPO(ERLPO ∗ − 8.928) + ERSPO(PLGA ∗ − 5.610) + ERLPO(PLGA ∗ − 19.568)
This equation indicates that a negative impact on drug release is
exerted by combining ERLPO and PLGA as indicated by the lowest
interaction coefficient (− 19.568). This trend was followed by the
negative impact of ERSPO and PLGA combination (coefficient =
− 5.610). The highest release was obtained from PTXNp1 containing
only ERLPO. This data was corroborated by the highest coefficient of
84.13 associated with ERLPO in Eq. (6). ERSPO/ERLPO polymers have a
quaternary ammonium functional group that leads to quick disintegra­
tion of ERSPO/ERLPO containing nanoparticles. The disintegration of
the particles facilitates the drug release through diffusion, whereas
PLGA being less hydrophilic, limits the drug release. Consequently, drug
release from nanoparticles happens due to diffusion and surface/bulk
erosion of PLGA matrix. The difference in the release profile may also be
credited to the variation in particle size of PTXNp. An increase in the
particle size results in an increased length of the diffusional pathways
and reduced total exposed surface area. Notably, the mixture of the
polymers decreases the drug release [48]. Overall, an increase in the
quantity of ERLPO influenced the dissolution profile due to more
permeability than ERSPO and PLGA.
The average in vitro release data was processed for zero order, first
order, Higuchi, Hixon, and Korsmeyer-Peppas release kinetics. Higuchi
kinetics showed the best fit compared to others (Table 3). These findings
are consistent with the literature where Eudragit and PLGA nano­
particles were used [49]. It was observed that the Korsmeyer-Peppas
model also depicted the data fit for drug release profiles (as seen in
Table 3). The Higuchi model described that the drug on the surface is
released at the beginning, followed by that in the core. The drug in the
center needs to diffuse first to the surface and then solubilize from there.
A decrease in drug diffusion results in a proportionately lower release
rate. Korsmeyer-Peppas allows exponential analyses of release rate. It
was also observed that all the PTXNps followed Fickian diffusion (n less
than 0.5). Conclusively, the release rate of PTX was influenced by the
drug diffusion rate, leading to a sustained drug release.
5.3. Screening of best formulation from mixture profiler
A two tier screening approach was followed to select the optimized
formulation(s) with minimal toxicity. In the first step, five formulations
were selected from the design. The white/unshaded region in Fig. 1
portrays the operational design space for the selected formulations. The
9
Biomedicine & Pharmacotherapy 144 (2021) 112286
desirable values were set as following: particle size less than 220 nm,
cumulative drug release greater than 50% at 15 days, and percent he­
molysis less than 10%. Five nanoparticle formulations that met these
criteria were PTXNp3, PTXNp5, PTXNp9, PTXNp11, and PTXNp15.
In the second step, out of the five formulations, three formulations
(PTXNp3, PTXNp9, PTXNp15) were selected based on their low hemo­
lysis values and clinical significance of hemolysis. The hemolysis values
of these three formulations ranged between 2% and 8%.
5.4. In vitro and in vivo characterization of selected nanoparticle
formulations: PTXNp3, PTXNp9, and PTXNp15
5.4.1. Physical appearance and morphology
Nanoparticles obtained through the use of nanoprecipitation tech­
nique displayed a uniform appearance. The particles appeared as
distinct entities as seen on transmission electron microscopy images and
(6)
also supported by polydispersity index (Fig. 2A) (data available in
supplementary file Table S2). However, as seen in SEM images, the
particles had a rough beaded/spiked surface (Fig. 2B). This potentially
supports them being polydispersed and avoid the adherence of the
particles to each other. Uneven surfaces have been reported to sustain
the drug release. Niu Y et al., 2015 reported improved sustained release
profile from irregular surfaced silica nanoparticles [50]. Similarly, Sun
et al. reported prolonged blood circulation time for the rough-surfaced
nanoparticles compared to smooth doxorubicin nanoparticles [51].
5.4.2. Zeta (ζ) potential
The negative surface charge of the pure PLGA nanoparticles was
provided by the carboxyl groups present on the polymer chain endings,
while the positive zeta potential of Eudragit nanoparticles was due to
quaternary ammonium groups of Eudragit RSPO and RLPO. For inject­
ables, positive surface charge on particles increases the cellular uptake
by interacting with the negatively charged mucin, sialic acid, and
phospholipid present on the cell surface. In contrast, a negative charge is
appropriate for the EPR effect [52]. However, in the present studies, a
blend of Eudragit RSPO, RLPO, and PLGA have provided more neutral
particles due to the shielding effect of the carboxyl group of PLGA on the
quaternary ammonium groups of Eudragit. Moreover, some studies
report that neutral particles are best suited for matrix transport as they
do not undergo any repulsive interactions with biomacromolecule fibers
present in the tumor’s extracellular matrix [53]. Data presented in
Fig. 4C and supplementary data file, Table S2.
5.4.3. Excipients compatibility by Fourier transform infrared and XRD
FTIR and XRD analyses were performed for free PTX, PLGA, ERSPO/
ERLPO, physical mixture, and the prepared nanoparticle (PTXNp18)
(Fig. 3 A). The major infrared peaks assigned to PLGA were observed at
2995 cm− 1 (C–H), 1775 cm− 1 (C− O), 1420 cm− 1 (–CH2), and
1253 cm− 1 (CH3). The PTX spectrum showed characteristic peaks at
2954 cm− 1 (C–H), 1649 and 1726 cm− 1 (CO carbonyl ketone group),
1649 cm− 1 (amide group), 1254 cm− 1 (C–O–O bending vibrations),
1085 cm− 1 (C–O stretch vibration), and 704 cm− 1 (aromatic C–H).
ERSPO peaks were observed at 2980 cm− 1 (C–H), 1729 cm− 1 (CO),
1461 cm− 1 (–CH2), and 1374 cm− 1 CH(CH3) (Peak intensity was 29.8).
ERLPO peaks were observed at 2980 cm− 1 (C–H), 1728.29 cm− 1 (C-O),
1463 cm− 1 (–CH2), and 1374 cm− 1 CH(CH3) (Peak intensity was 36.84
because of higher electronegativity due to 10% of functional quaternary
ammonium group compared to 5% in ERSPO). A rise in peak intensity
G. Jeswani et al.
Fig. 3. (A) Excipient compatibility by Fourier transform infrared (acronym PM represents PTXNp18). Fig. 3. (B) XRD patterns of (a) PLGA, Eudragit RSPO, and
Eudragit RLPO (b) PTXNp3, PTXNp9, PTXNp15, and PTX. The graph of FTIR is between %Transmittance (Y axis) and wavelength (X axis) and XRD between In­
tensity, counts (Y Axis) and 2 Theta (◦ ), (X Axis).
signifies an increase in the number of the functional group associated
with the molecular bond (per unit volume). Noteworthy, all the char­
acteristic peaks of drug and polymers were noticeable in the physical
mixture spectra, confirming the identity. Some shifts and broadening
bands due to overlapping peaks of similar functional groups of both drug
and polymer were observed in the nanoparticle. No new peaks were
observed, indicating that no chemical change or interaction has taken
place and the drug has been physically encapsulated in the polymeric
nanoparticles.
XRD analysis was used to investigate the crystallinity changes within
the drug molecule during nanoparticle formulation. As depicted in
Fig. 3B, PTX lyophilized powders exhibited a few distinctive diffraction
peaks at 8.9, 12.6, 16.8, and 34.1 scattered angles (2θ), indicating the
crystalline structure of molecules. The broad peak pattern of ERSPO,
ERLPO, and PLGA indicate an amorphous state of polymers (Fig. 3B,a).
Meanwhile, in the nanoparticle diffractogram, the characteristic peaks
of PTX were present at the same location as that in PTX, representing
that PTX in the mixture still exists in crystalline form predominantly
(Fig. 3B,b). Nevertheless, the intensity of PTX diffraction peaks
10
Biomedicine & Pharmacotherapy 144 (2021) 112286
decreased, which might be attributed to some PTX molecules present in
the amorphous state. The amorphous form allows easy solubility, which
allowed burst release from nanoparticles in vitro release studies. The
diminished peak for PTX was observed in PTXNp15 formulation as
compared to PTXNp3 and PTXNp9. Therefore, despite the bigger par­
ticle size of the PTXNp15 nanoparticle, the results were comparable to
PTXNp3 and PTXNp9 having smaller particle sizes. Furthermore, the
decrease in the crystallinity of polymers assists in the release of drugs
from polymeric matrices due to its degradation. These findings are
similar to those of Zhang et al., corresponding to PTX loaded PLGA
microspheres [54].
5.4.4. Loading and entrapment efficiency
All the optimized lyophilized nanoparticle formulations were
assessed for entrapment efficiency and drug loading. The entrapment
efficiency (%w/w) for all the formulations was > 70%w/w, and loading
was in the range 8–11%w/w (Fig. 4 A and B). Among the optimized
formulations, the entrapment efficiency was in the following order
PTXNp15 (78.56 ± 5.25) >PTXNp3 (77.05 ± 4.07) >PTXNp9
G. Jeswani et al.
Fig. 3. (continued).
(76.23 ± 5.33). Although the ANOVA for entrapment efficiency was
insignificant (p = 0.85), pure PLGA nanoparticles (PTXNp15) had the
highest entrapment efficiency because of their significant size as
compared to other nanoparticles (PTXNp3 and PTXNp9); therefore,
entrapment was enhanced. However, an attraction between carboxyl
groups (negative charge) on PTX and the quaternary ammonium group
(positive charge) on ERSPO/ERLPO resulted in substantial entrapment
efficiency. Minor differences between the PTXNp3 and PTXNp9 can be
explained based on the particle size. These findings are consistent with
those reported by Cetin et al., who related the difference with the mo­
lecular weight and available charge on the polymer [12]. Similarly,
there was an insignificant difference in the drug loading for different
nanoparticle formulations (p = 0.94) (Fig. 4A).
5.4.5. Blood compatibility
Histological challenges are the major limitation of the chemo­
therapy; hence it is vital to evaluate the hemocompatibility of the
optimized formulation. The shape and size of the nanoparticles
contribute to reduced blood compatibility [55]. The characteristic fea­
tures responsible for hemocompatibility challenges are reactive groups
on the particle surface and a greater surface/volume ratio [55]. For
adequate hemocompatibility, the nanoparticles should not activate
platelet cells or the coagulation pathway. The formulation should avoid
any damage to the endothelial cells, resulting in thrombus formation. It
should maintain the normal level of the plasma factors for the coagu­
lation pathway without creating any imbalance. These ideal features are
11
Biomedicine & Pharmacotherapy 144 (2021) 112286
required to prevent any hemorrhagic phenomenon. Furthermore, the
deleterious acts of cytotoxic drugs on RBCs’ membrane results in their
destruction causing hemolytic anemia. The activation of the comple­
ment system is responsible for cell inflammation and hemolysis.
Thereby, an assessment of the bio and hemocompatibility of placebo and
loaded nanoparticle helps to establish the safety and efficacy of the
nanoparticles. The results presented in Table 4 describe the effects of
nanoparticle composition on different hematological parameters.
5.4.5.1. Blood clotting time. Lee white method, as described in Section
3.3.5.1, was used to assess polymer ratio impact on the whole blood
clotting time [56]. The mean clotting time (in min) for different for­
mulations were 5.33 ± 0.29 (PTXNp3); 5.76 ± 0.21 (PTXNp9);
5.67 ± 0.34 (PTXNp15); 5.06 ± 0.19 (PTXNp3 Placebo); 5.70 ± 0.18
(PTXNp9 Placebo); 5.80 ± 0.15 (PTXNp15 Placebo). The difference
between clotting time for PTXNp3, PTXNp9, and PTXNp15 was insig­
nificant (p = 0.16) and differed from the positive control by 266 s
(significant; p = 0.05). The mean clotting time for positive control was
1.4 ± 0.32 min (Fig. 5). The clotting time of the nanoparticle treated
samples was similar to the negative control (p = 0.07). The normal
value of clotting time is 5–10 min and our study results complied with
this range [57]. This study indicates that the nanoparticles did not
interfere with the blood clotting mechanism.
5.4.5.2. Blood counts and hemolysis. Table 4 summarizes the values of
blood counts obtained with 1 mg of PTX loaded and corresponding
G. Jeswani et al. Biomedicine & Pharmacotherapy 144 (2021) 112286

Fig. 4. Graphical representation of drug loading and entrapment efficiency of PTXNp3, PTXNp9, and PTXNp15. (A) Drug loading; (B) Drug entrapment efficiency;
and (C) Zeta(ζ) potential values (mean ± SD; n = 3). (**p < 0.05).

Table 4
The effect of PTXNp on hematological parameters. Each value represents the mean of three determinations ± standard deviation, n = 3. Values in parenthesis indicate
normal value range. PLT (150–400 × 103 /µL); WBC (4–11 × 103 / µL); RBC (4.7–6.1 × 106 / µL); Hb (12–17 g/dL), HCT (37–52%); MCV (77–100 fL); MCH
(27–33 pg); MCHC (33.4–35.5 g/dL). Negative control: phosphate buffer saline.
Formulation Code PLT (103 /µL) WBCs (103 /µL) RBC (106 /µL) Hb (g/dL) HCT (%) MCV (fL) MCH (pg) MCHC (g/dL)
(ERSPO:ERLPO: PLGA)

PTXNp3 (0.2:0:0.8) 337.33 ± 10.01 12.18 ± 1.22 5.72 ± 0.92 11.39 ± 0. 93 37.51 ± 2.25 54.89 ± 3.74 18.67 ± 0.90 37.53 ± 0.73
PTXNp3Placebo 332.90 ± 9.25 10.02 ± 2.08 5.98 ± 0.79 11.68 ± 0.96 37.82 ± 1.10 56.66 ± 3.23 19.23 ± 0.62 36.25 ± 0.50
PTXNp 9(0:0.2:0.8) 357.66 ± 21.78 10.55 ± 1.24 5.54 ± 0.70 11.42 ± 1.25 37.82 ± 1.04 55.16 ± 1.97 18.3 ± 0.65 37.08 ± 0.70
PTXNp 9 Placebo 341.33 ± 8.02 10.34 ± 1.26 5.65 ± 0.61 13.88 ± 1.62 37.12 ± 1.59 55.16 ± 1.55 18.6 ± 0.80 35.73 ± 1.04
PTXNP15 (0:0:1) 365.33 ± 12.66 11.62 ± 1.84 5.51 ± 0.83 12.97 ± 0.41 37.65 ± 0.71 55.6 ± 3.35 18.48 ± 1.05 35.1 ± 2.06
PTXNp15 Placebo 334.66 ± 5.54 12.02 ± 1.53 4.54 ± 0.56 11.75 ± 0.96 37.9 ± 1.43 52.1 ± 1.53 19.17 ± 0.81 35.06 ± 1.71
Negative control 344.33 ± 11.01 11.50 ± 1.07 5.8 ± 0.94 13.38 ± 1.47 38.19 ± 1.57 55.3 ± 0.25 19.4 ± 1.04 34.96 ± 1.09

placebo nanoparticles. When compared to the negative control, none of
the placebo or PTX-loaded nanoparticles had any effect on blood count
(PLT, WBC, RBC, MCH, MCV, MCHC, HCT and Hb) (p > 0.05).
Fig. 6 shows the hemolysis values of selected PTXNps and corre­
sponding placebo nanoparticles. All placebo nanoparticles did not show
any significant hemolysis compared to the negative control (p > 0.001).
The values obtained for PTXNp3, PTXNp9, and PTXNp15 were 7.43%,
7.89%, and 2.01%, respectively. The results showed that pure PLGA
nanoparticles (PTXNp15) displayed the least hemolysis values while
others surpassed the 5% limit recommended by ISO-10993. However,
the difference is marginal, and further improvements can be made to
reduce hemoncompatibility. The obtained value is less than the percent
hemolysis caused by Taxol®, as reported by Ran Namgung et. al [45].
The group observed hemolysis greater than 40% for 10, 20, and 40 µg of
PTX. The results suggest that PTX nanoparticles have better biosafety
than Taxol®. Materials with more than 5% hemolysis are classified as
hemolytic, those with 2–5% hemolysis as slightly hemolytic, and those
with less than 2% hemolysis as nonhemolytic [58]. Overall, blood count

12
G. Jeswani et al.
Fig. 5. Clotting Time of PTXNp3, PTXNp9, and PTXNp15 compared to their
respective placebos (mean ± SD; n = 3).
Fig. 6. Hemolysis of PTXNp3, PTXNp9, and PTXNp15 compared to their
respective placebo particles (mean ± SD; n = 3) (****p < 0.0001; ns =
not significant).
studies suggested that the formulation constituents were biocompatible.
The reason behind the biocompatibility cannot be clearly stated at this
stage, other than the restricted exposure of blood components to the
drug and the use of nontoxic polymers.
5.4.5.3. Activated partial thromboplastin time (aPTT) and prothrombin
time (PT) for coagulation. Fig. 7A shows the aPTT values for all nano­
particle formulations lie within the range 31–36 s Fig. 7B shows the PT
values for all nanoparticle formulations ranging between 13 and 15 s.
The aPTT and PT measurement exhibits an effect of nanoparticles
interaction with the coagulation cascade. Results showed that after 4 h
of incubation with a defined nanoparticles concentration (1 mg), there
was no substantial reduction in prothrombin activity relative to the
negative control, PBS (p = 0.99). Basal aPTT values vary between
32− 36 s. At the same time, PT values range from 12 to 15 s for healthy
adults aged 18–50 years [59]. Mittal et al. [72] reported similar results
where the PLGA nanoparticles of PTX demonstrated hemocompatibility
in terms of hemolysis, aPTT and PT measurement.
13
Biomedicine & Pharmacotherapy 144 (2021) 112286
The clinical etiology of extended clotting times in PT values indicates
reduced levels of factors VII, X, V, prothrombin, or fibrinogen [60].
Although aPTT is considered a sensitive version of the PTT, many factors
influence the results of these tests, like coagulant type, test compounds,
method of sample storage, and the time difference between collection,
processing and analysis. Therefore, all aPTT tests were carried out
within 2 h of collection of a blood sample.
Nanoparticles can have an impact on coagulation time. Shorter pe­
riods of coagulation may lead to toxicity issues due to thrombus for­
mation that could partly or entirely occlude blood vessels, whereas
longer cycles of coagulation might cause bleeding. The incompatibility
of the nanoparticles with the coagulation system may be due to the
biocompatibility issues of the polymers and the negative surface charge
on the nanoparticles. The surface charge on nanoparticles plays an
important role in the aggregation of PLT and hence thrombus formation.
The association between oppositely charged particles and platelets re­
duces the surface charge of PLT and allows them to form aggregates
leading to thrombocytopenia that occurs after injection [61]. Therefore,
it can be concluded that the neutral particles are safer than the charged
particles in terms of hemocompatibility and transport to tumors [53].
Hence, the lower the surface charge of the particles better is the
hemocompatibility. The values of zeta potential of the nanoparticles
PTXNp3, PTXNp9, and PTXNp15 were − 5.56 ± 0.34, − 5.37 ± 0.47,
and − 7.44 ± 0.41 mV, respectively. The statistical analysis revealed a
significant difference between the zeta potential of the three formula­
tions (p = 0.0014). Pure PLGA particles are highly negatively charged
(− 7.44 ± 0.41 mV) and pure Eudragit RLPO/RSPO particles are highly
positively charged (11.15 ± 0.23/10.5 ± 0.4), the zeta potential of the
developed nanoparticles as a result of the combination of both polymers
is close to neutral.
5.4.5.4. C3 complement. Fig. 8 shows the concentration of C3 protein
complement after incubating the PTXNp and corresponding placebo
nanoparticles with 1 mL of human serum. The expression of comple­
ment C3 protein indicates the association with the coagulation cascade.
Hence, it also indicates the hemolytic nature of nanoparticles. In gen­
eral, nanoparticles form a protein corona upon contact with blood,
which is followed by conformational changes in protein C3 in the
presence of complement factors B and D, resulting in the induction of
enzyme C3 convertase and consecutively splitting of protein C3 into C3a
and C3b, thereby activating the alternative pathway. Hence, C3 deple­
tion in the blood indicates an indirect measurement of complement
activation. Nanoparticles of size less than 100 nm activate the comple­
ment system strongly [62]. However, as the size of the obtained nano­
particles was greater than 100 nm, the values of the C3 complement
component after incubation for 1 h at 37 ◦ C (108–135 mg/dL)were
within the normal range. The normal range for a complement C3 blood
test is 80–178 mg/dL [63]. Also, the results of all the formulations were
very close to negative control values (114 mg/dL), showing that the
formulations had no effect on complement activation at the particular
concentration (1 mg) used in this experiment (p = 0.27).
Therefore, all formulations tested for hemocompatibility as per ISO-
10993 indicated strong hemo-tolerance to blood components. Addi­
tionally, all the research methods used in this section did not require
animals as used for in vivo tests.
5.4.6. In vitro cytotoxicity assay
Colorimetric MTT assay was performed against MCF-7 blood cancer
cells to evaluate the cytotoxicity of PTX and the developed nano­
particles. The results obtained from the in vitro cytotoxicity assay were
represented as a percent cell viability vs. concentration graph (Fig. 9).
The outcomes revealed that all the placebo nanoparticles were non-
cytotoxic against MCF-7 breast cancer cells. PTX loaded nanoparticles
exhibited cell viability ranging between 14% and 99%. Results suggest
that all the PTXNp demonstrated concentration-dependent cytotoxicity.
G. Jeswani et al. Biomedicine & Pharmacotherapy 144 (2021) 112286

Fig. 7. Interaction of nanoparticles with the coagulation cascade for selected formulation PTXNp3, PTXNp9, PTXNp15 compared with placebo (A) aPTT (B) PT
(mean ± SD; n = 3). Basal values of the aPTT range between 32 and 36 s while PT ranges between 12 and 15 s for healthy adults of age between 18 and 50 years.
Tests carried out within 2 h of blood sample collection.

Fig. 9. Percent cell viability vs concentration plot obtained from MTT assay of
PTX, PTXNp drug loaded and respective placebo particles against MCF-7 breast
Fig. 8. C3 protein complement activation for PTXNp3, PTXNp9, and PTXNp15
cancer cells after 24 h incubation (mean ± SD; n = 3).
compared to their respective placebo particles (mean ± SD; n = 3).

m2 via the IV route for a duration of three hours. The administered PTX
For the entire concentration range, the cell viability of the respective
dose for this study was chosen at a much lower concentration than the
placebo nanoparticles was significantly higher than the drug-loaded
human equivalent therapeutic dose. Pure PTX solution at the same dose
formulations (p < 0.0001). The lack of cytotoxicity of the placebo
was also administered to allow for pharmacokinetic comparisons. The
nanoparticles confirmed the biocompatibility of matrix polymers,
mortality rate was zero percent for the study.
Eudragit RSPO/RLPO, and PLGA. The IC50 value of PTX was
Fig. 10A depicts log mean plasma drug concentration versus time
40.26 ± 2.70 µg/mL (supplementary data in Fig. S1), and PTXNp3 and
curve for all the PTXNPs and pure PTX solution. In vivo pharmacoki­
PTXNp15 were 3453.30 ± 111.10 and 3382.30 ± 95.32 µg/mL,
netics of PTX for all the treatments was investigated using Wistar rats at
respectively. PTXNp9 exhibited the minimum IC50 value among all the
a dose of 5 mg/kg. A non-compartment analysis was performed to
formulations (1913.15 ± 64.12 µg/mL) (supplementary data in
determine the pharmacokinetic parameters of interest including area
Table S3). The reason behind the enhanced cytotoxicity could be due to
under the curve (AUC); terminal half-life, (T1/2); total body clearance
the presence of positively charged polymers, ERLPO and ERSPO [64].
(Cl), and volume of distribution based on the terminal phase (Vd)
(Table 5). The calculated R2 and R2 adjusted values for all the formu­
5.4.7. In-vivo pharmacokinetic studies
lations were close to 1, indicating a perfect fit to the model. The phar­
As established through the cytotoxicity study results, all three for­
macokinetic curves of the log PTX concentration in the plasma versus
mulations (evaluated within the concentration range of 70–4900 µg/
time are shown in Fig. 10A. The mean AUC of PTX upon IV adminis­
mL) were suitable for the in vivo pharmacokinetic and biodistribution
tration of PTXNp was significantly higher than free PTX (p < 0.0001).
study. All three formulations were individually evaluated using 10–12
For pure PTX solution, the drug was rapidly cleared from the plasma
week old male Wistar rats. The drug loaded formulations were admin­
after bolus administration. On the contrary, all the PTXNps exhibited
istered via the intravenous route at an equivalent PTX dose of 5 mg/kg.
delayed clearance/ higher Clast when compared to PTX solution
Clinically, PTX is generally administered at a dose of 135 and 175 mg/
(PTXNp9 (267 times) > PTXNp3 (213 times) > PTXNp15 (79 times)).

14
G. Jeswani et al. Biomedicine & Pharmacotherapy 144 (2021) 112286

Fig. 10. (A) log Plasma concentration (ng/mL) over time (h) after IV administration of pure drug solution, PTXNp 3, PTXNp9, and PTXNp15 at a dose of 5 mg/kg (B)
Bio-distribution in Wistar rat after 24 h at single dose of 5 mg/kg (C) In vivo hematological parameters observed aat 24 h after a single dose of 5 mg/kg (mean ± SD;
n = 3) (****p < 0.0001).

Table 5
Pharmacokinetic parameters from the in vivo study using pure drug solution, PTXNp3, PTXNp9, and PTXNp15 at a dose of 5 mg/kg (mean ± SD, n = 3). Multiple
comparisons were performed using one-way ANOVA and Tukey post hoc test.
Parametrs PTXNp3 PTXNp9 PTXNp15 Pure drug solution

Avg. SD Avg. SD Avg. SD Avg. SD

R2 0.97 0 0.98 0 1 0 1 0
R2 adjusted 0.96 0 0.97 0 1 0 1 0
Cmax ng/mL 20,482.04 9.97 24,228.96 4.55 22,381.57 7.62 18,317.91 1.52
C0 ng/mL 23,852.72 16.83 31,078.55 18.51 31,921 1.43 29,869.4 58.19
AUC h*ng/mL 141,260.24 128.39 163,071.15 86.68 93,194.67 52.03 39,589.31 238.23
Cl mL/h/kg 32.89 0.03 28.25 0.03 51.59 0.15 126.24 0.77
MRT h 8.29 0.02 8.54 0.02 6.05 0.09 2.04 0.05
Vd mL/kg 291.01 0.19 258.98 0.26 431.41 0.99 438.85 12.57
T1/2 h 6.13 0.36 6.35 0.01 5.88 0.14 2.41 0.08
Clast ng/mL 1216.60 2.60 1521.21 7.58 445.74 18.26 5.69 1.11

Rapid clearance of PTX in the solution group is also confirmed with the
declining PTX concentration for that group. In 24 h, its concentration
decreased from peak to below minimum therapeutic effective level
(43 ng/mL) [65]. Further, the total area under the curve (AUC), which
determines the total systemic exposure to the drug from PTXNps, was
also higher than pure PTX solution {PTXNp9 (4.12 times) > PTXNp3
(3.57 times) > PTXNp15 (2.35 times)}. The difference in the results can
be attributed to the polymer characteristics. As both PLGA (50:50) and
Eudragit RSPO/RLPO are hydrophobic polymers, they resist the drug
release from the polymer matrix. Consequently, the combination of
PLGA with Eudragit RSPO/RLPO further reduced the release of PTX
from the formulation of PTXNp9 and PTXNp3 as compared to PTXNp15
(only PLGA). Hence, the in vitro drug release results corroborate the in
vivo pharmacokinetic study results.
The terminal half-life (T1/2), was found to be highest for PTXNp9,
which is 2.64 times longer than for pure PTX solution (p < 0.0001). The
volume of distribution is an essential clinical pharmacokinetic param­
eter useful in discussing drug disposition processes. Smaller values imply
swift elimination of the drug from the blood. The PTXNps exhibited low
clearance (Cl), and volume of distribution (Vd) compared to pure PTX
solution.
The volume of distribution was lowest for PTXNp9 (258.01 mL/kg)
which was 0.59 times of 438.85 mL/kg for pure PTX solution
(p < 0.0001). Similarly, PTXNp9 exhibited the lowest clearance
(28.25 mL/h/kg) which was 0.22 times of 126.24 mL/h/kg for pure PTX
solution (p < 0.0001), which validates the half-life analysis result.
Reduced clearance leads to increased plasma half-life, as expected.
PTXNp9 increased the MRT of PTX in plasma by 4.19 times. Overall, the

15
G. Jeswani et al.
results confirmed improvement in pharmacokinetic profiles when PTX
was formulated as a nanoparticle PTXNps when compared to PTX
solution.
The in vivo results were corroborated by the in vitro drug release
studies where all the formulations released only upto 20% of the drug
within 2 h. After 360 h, the cumulative drug released was in the range of
65–82% w/w, confirming the slow, sustained release of the drug.
Similarly, the in vivo results support the prolonged circulation times of
the nanoparticles in vivo along with a longer plasma half-life. Also, the in
vivo results confirmed the significant safety and efficacy of the nano­
particle formulations versus the pure PTX solution. Similarly, the safety
profile was also evident via the in vitro cytotoxicity studies mentioned
earlier. Based on the in vitro and in vivo study results, PTXNp9 is the most
optimum formulation among the three optimized formulations
(PTXNp3, PTXNp9, and PTXNp15). With further modifications, it can
further be effectively used to gain site-specific drug delivery.
5.4.8. Biodistribution and hematological studies
A biodistribution study was conducted to quantify the amount of
drug deposited in different organs. Five cardinal organs were selected
for the study: heart, spleen, kidney, lung, and liver. Fig. 10B shows that
the nanoparticulate formulations led to a substantially higher level of
drug delivery to all the isolated organs than the pure drug solution
correlating to the sustained drug release of PTX from the nanoparticles.
PTX was majorly distributed to the liver, spleen, and heart.
A significantly higher amount of drug was observed in the liver in the
group receiving PXTNp formulations (p < 0.0001) as compared to the
group receiving pure PTX solution. Similar results have been reported by
De Jong et al., where the distribution of gold nanoparticles to the liver
occurred preferentially irrespective of the size of the nanoparticles [66].
Li et al. also reported significant levels of PTX in the liver, spleen, kid­
ney, heart, and lung [67]. The study reported an eight-fold higher PTX
levels in the liver than the other organs. The reason for this selective
accumulation is the fact that plasma proteins rapidly coat nanoparticles
after an intravenous injection. Protein-coated nanoparticles easily
accumulate in the liver while those present in the bloodstream diffuse
into the tumor upon circulation [68]. Overall, we infer that all three
PTXNp formulations had a significant effect on the biodistribution of the
drug, and the extent of biodistribution of PTX was organ-dependent with
the maximum accumulation in the liver.
The hematological analysis, Fig. 10C, showed that the concentration
of PLT in the group that received PTXNps was substantially higher
compared to the group treated with pure PTX solution (p < 0.0001).
Also, the RBC count for the PTX solution group was lower (3.81 × 106
/µL) compared to PTXNp9 treated group (5.54 × 106 /µL). RBCs are
vital for haemoglobin levels and to transport oxygen. Thus, lack of RBCs
can be detrimental clinically. Reduced RBC counts indicate toxic effects
of the treatment [69,70]. The WBCs counts were 11.62 ± 1.84 × 103
per µL for the PTXNp15 treated group, 10.55 ± 1.24 × 103 per µL for
the PTXNp9 treated group, 12.18 ± 1.22 × 103 per µL for the PTXNp3
treatment, 3.47 ± 0.62 × 103 per µL for pure drug solution, and
11.50 ± 1.079 × 103 per µL for the control group. Hb levels were close
to the values of control group (13.38 ± 1.4 g /dL), in PTXNp treated
groups (PTXNp3 11.39 ± 0.93 g /dL, PTXNp9 11.42 ± 1.2 and
PTXNp15 12.97 ± 0.41) as compared to the pure drug solution
(7.95 ± 0.08 g/dL). The HCT value for the PTXNp3, PTXNp9 and
PTXNp15 were 27.39 ± 0.78, 29.69 ± 0.75, 30.44 ± 0.72,
31.75 ± 1.74 respectively. All PTXNps showed HCT values < 45%
which is within the reference value and with no significant differenece
when compared with control, 31.74 ± 1.72 (p > 0.05) [71]. HCT mea­
sures the percentage of red blood cells in the sample of blood. It is an
indirect method of measuring Hb. Thus, the normal values obtained for
the PTXNp treated groups are indicative of healthy RBC counts and no
blood disorder. The MCV value for the PTXNp3, PTXNp9, PTXNp15 and
control were 55.48 ± 1.45, 55.05 ± 1.50, 55.44 ± 1.27, and
51.00 ± 1.26 respectively. Moreover, there was no statistical difference
16
Biomedicine & Pharmacotherapy 144 (2021) 112286
between MCV values for all formulations and the control (p > 0.05).
Higher MCV value indicate abnormal size of RBCs.
The MCH value for the PTXNp3, PTXNp9, PTXNp15 and control
were 19.19 ± 1.57, 19.09 ± 2.55, 19.74 ± 1.72, 19.22 ± 3.56 respec­
tively. All PTXNps showed MCH values < 25 pg with no significant
differenece when compared with control (p > 0.05). Higher MCH values
indicate abnormal/large size of RBCs. Similarly, MCHC values of
PTXNp3, PTXNp9, PTXNp15 and control were 38.39 ± 1.67,
38.01 ± 1.35, 40.24 ± 1.06, and 37.22 ± 2.37 g/dL respectively. All
PTXNps showed MCHC values < 40 g/dL with no significant differenece
when compared with control (p > 0.05). Low MCHC values indicate less
amount of Hb in the RBCs. The hematological parameters were used to
diagnose the effect of nanoparticles on the RBCs. Since all the polymers
were used below their LD50 values, no toxicity or adverse effects were
observed. The values of all haematological tests [as shown in Fig. 10(C)]
for all groups treated with nanoparticles were close to the control, thus
confirming the safety and biocompatibility of the formulations.
6. Limitation
To get an idea of the off-target effects of the developed nanoparticles
the present study has been limited to blood compatibility studies and
biodistribution studies in detail. However, the microtubule stability is
also important activity to be noted as microtubules are important for cell
division, specifically in the segregation of chromosomes.
7. Conclusion
The study utilizes a statistical design to formulate a nanoparticulate
sustained release drug delivery systems for PTX. This is the first study to
evaluate the role of such formulations on hemocompatibility parameters
as specified by the ISO-10993. The study evaluates the formulated
nanoparticles via both in vitro and in vivo analysis. The in vitro drug
release profiles corroborated the in vivo pharmacokinetic profiles of the
nanoparticles. Additionally, the biodistribution studies provided a
unique understanding of drug distribution to five cardinal organs and
the impact of PTX delivery to those sites. Overall, the formulation and
evaluation approaches offered in this study are promising to deliver PTX
in a safe, efficient, and reliable manner to enhance its clinical outcomes
and reduce the associated adverse effects.
CRediT authorship contribution statement
Gunjan Jeswani: Conceptualization, Investigation, Methodology,
Formal analysis, Data curation, Writing − review & editing. Lipika
Chablani: Conceptualization, Methodology, Formal analysis, Data
curation, Writing − review & editing, Supervision. Umesh Gupta:
Investigation, Methodology, Data curation, Writing. Rakesh Kumar
Sahoo: Investigation, Methodology, Data curation, Writing, Formal
analysis. Kartik T. Nakhate: Investigation, Methodology. Ajazuddin:
Supervision, Conceptualization, Methodology.
Conflict of interest statement
The authors declare that there are no conflicts of interest.
Acknowledgment
All the research studies were done under the joint supervision of Dr.
Ajazuddin and Dr. Lipika Chablani. We acknowledge Intas Pharmaceu­
ticals Ltd. for providing Paclitaxel as a gift and Evonik India Private Ltd.
for providing ERSPO, ERLPO, and PLGA samples. Special thanks to Dr.
Naveen Verma, M.D Pathology, for his support during the hemo­
compatibility studies.
G. Jeswani et al.
Appendix A. Supporting information
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.biopha.2021.112286.
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