Article

pubs.acs.org/molecularpharmaceutics

Click Biotinylation of PLGA Template for Biotin Receptor Oriented

Delivery of Doxorubicin Hydrochloride in 4T1 Cell-Induced Breast
Cancer
Yuvraj Singh,† K.K. Durga Rao Viswanadham,†,# Arun Kumar Jajoriya,‡ Jaya Gopal Meher,† Kavit Raval,†
Swati Jaiswal,§ Jayant Dewangan,∥ H. K. Bora,⊥ Srikanta Kumar Rath,∥ Jawahar Lal,§
Durga Prasad Mishra,‡ and Manish K. Chourasia*,†
†
Pharmaceutics Division, CSIR-Central Drug Research Institute, Lucknow 226031, India
‡
Endocrinology Division, CSIR-Central Drug Research Institute, Lucknow 226031, India
§
Pharmacokinetics and Metabolism Division, CSIR-Central Drug Research Institute, Lucknow 226031, India
∥
Division of Toxicology, CSIR-Central Drug Research Institute, Lucknow 226031, India
⊥
Laboratory animals facility, CSIR-Central Drug Research Institute, Lucknow 226031, India
*
S Supporting Information

ABSTRACT: PLGA was functionalized with PEG and biotin using click
chemistry to generate a biotin receptor targeted copolymer (biotinylated−
PEG-PLGA) which in turn was used to fabricate ultrafine nanoparticles
(BPNP) of doxorubicin hydrochloride (DOX) for effective delivery in 4T1
cell induced breast cancer. However, adequate entrapment of a hydrophilic
bioactive like DOX in a hydrophobic polymer system made of PLGA is not
usually possible. We therefore modified a conventional W/O/W emulsion
method by utilizing NH4Cl in the external phase to constrain DOX in dis-
solved polymer phase by suppressing DOX’s inherent aqueous solubility as
per common ion effect. This resulted in over 8-fold enhancement in
entrapment efficiency of DOX inside BPNP, which otherwise is highly
susceptible to leakage due to its relatively high aqueous solubility. TEM
and DLS established BPNP to be sized below 100 nm, storage stability
studies showed that BPNP were stable for one month at 4 °C, and in vitro release suggested significant control in drug release.
Extensive in vitro and in vivo studies were conducted to propound anticancer and antiproliferative activity of BPNP. Plasma and
tissue distribution study supplemented by pertinent in vivo fluorescence imaging mapped the exact fate of DOX contained inside
BPNP once it was administered intravenously. A comparative safety profile via acute toxicity studies in mice was also generated
to out rightly establish usefulness of BPNP. Results suggest that BPNP substantially enhance anticancer activity of DOX while
simultaneously mitigating its toxic potential due to altered spatial and temporal presentation of drug and consequently deserve
further allometric iteration.
KEYWORDS: nanoparticles, common ion effect, PLGA, doxorubicin hydrochloride, click chemistry, active targeting

■ INTRODUCTION
Doxorubicin hydrochloride (DOX) is a drug of choice in a variety
of cancerous ailments. DOX along with adjuvants is recom-
mended as a first line anticancer drug in NCCN’s 2016 breast
cancer guidelines.1 It interferes in DNA synthesis by binding to
topoisomerase II, damages preexisting DNA via intercalation,
generates reactive oxygen species, induces mitochondrial mem-
brane depolarization by releasing cytochrome c, and obliterates
membranous integrity of treated cells.2 When used as a mono-
therapeutic option, after relapse or failure of combination
therapy, DOX induces response in 40% first-line cases and 20%
second-line cases. Despite these attributes, DOX is connected to
numerous side effects (dose-dependent cardiotoxicity, nausea,
myelotoxicity, gastrointestinal shedding, and acute cramps) which
restrict its utility in a palliative environment.3 DOX consists of
tetracycline quinoid aglycone doxorubicinone and amino sugar
(daunosamine) linked together by a glycosidic linkage (Figure 1),
which is extremely susceptible to acidic degradation prevalent in
gastro intestinal tract.4
This attribute rules out oral administration and necessitates
usage of higher parenteral dose which inadvertently contributes
toward cardiac toxicity. DOX is usually administered intrave-
nously as a hydrochloride salt and therein lays a major delivery
related stumbling block. It is inconceivable to adequately entrap a
hydrophilic moiety (aqueous solubility of DOX is 10 mg/mL) in
Received: April 14, 2017
Revised: June 20, 2017
Accepted: June 21, 2017
Published: June 21, 2017

© 2017 American Chemical Society 2749 DOI: 10.1021/acs.molpharmaceut.7b00310

Mol. Pharmaceutics 2017, 14, 2749−2765
Molecular Pharmaceutics
Figure 1. Chemical structure of DOX. Equilibrium between salt form
and ionized form of drug is rapidly attained in water, yielding to
solubilization.
a hydrophobic polymer like PLGA or its derivative as drug is
subject to leakage in surrounding media during aqueous pro-
cessing of formulation (Figure 2C). Although we, along with
others, have attempted to use base form of DOX to ensure its
entrapment, it all adds to tediousness of formulation develop-
ment causing quantifiable drug loss as the process of convert-
ing DOX to its base form is inefficient.5 To circumvent such
inefficient techniques we have utilized principle of common ion
effect which was theoretically expounded during the last century
itself, but has not been utilized anywhere in pharmaceutics. We
try to increase entrapment of a highly water-soluble salt form of a
drug (i.e., DOX) in a hydrophobic polymer nanoparticle system
(derived from PLGA), despite drug’s tendency to leach out, by
suppressing its aqueous solubility via introduction of a stronger
electrolyte (NH4Cl) which bears a chloride ion common to DOX
in the external phase of formulation. The proposed strategy, if
successful, could work for other bioactives as well (because most
drugs lay in the weak electrolyte category) and potentially open
up a new methodology of entrapping hydrophilic drugs in
polymeric matrices. Selection of PLGA as a delivery construct for
DOX was based on PLGA’s proven industrial and commercial
utility (Zoladex, Lupron, Sandostatin, AtriDox, Buduperon, and
so on). Further advantages of PLGA can be gauged from its
biodegradability, reproducible fabricability, and sheer number of
biomedical devices/formulations undergoing advanced stages of
clinical trials.6 PLGA is famed for producing ultrafine spherical
particles7 and the bi product it generates enter carboxylic acid
cycle, thereby undergoing innocuous elimination or reutilization
in bodily metabolomics.8
Although annals of nanotechnology-assisted anticancer
delivery are heavily interspersed with studies where researchers
have exploited minute dimensions of PLGA nanoparticles to
passively target tumor cells, it is generally agreed that specific
targeting with limited/no off target effects as postulated by
Ehlrich in “magic bullet” concept is only realizable via active
targeting.9 We consequently decorated PLGA with biotinylated-
PEG by utilizing click chemistry to generate a cancer cell homing
copolymer: biotinylated−PEG-PLGA. Presence of PEG in tem-
plate was expected to accord nanoparticles long circulation half-
lives, which in turn would assist in their probability of reaching
tumor microenvironment10 for targeted delivery of potent cyto-
toxic DOX in 4T1 cell induced allogenic breast cancer model.11
It is assumed that these nanoparticles, after accumulation at
intended destination owing to their size, would then be pre-
ferentially internalized by cancer cells due to specific interaction
of their biotin component with overexpressed biotin receptors on
4T1 cells.12 In the direction of scheme depicted in Figure 2,
nanoparticles made of biotinylated−PEG-PLGA containing high
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load of doxorubicin hydrochloride (BPNP) were prepared via
a common ion effect modified W/O/W emulsion method.
Extensive studies were conducted to propound anticancer
activity of developed nanoparticles in 4T1 breast cancer cells.
Pharmacokinetic intonation in DOX brought about by its load-
ing in nanoparticles was observed by both LC-MS and in vivo
imaging. A comparative safety profile via acute toxicity studies in
mice was also generated. In vivo anticancer activity in 4T1 cell
induced breast cancer carrying Balb/C mice model was done to
outrightly establish utility of nanoparticles.
■ EXPERIMENTAL SECTION
Material. Doxorubicin hydrochloride (DOX) was received as
generous gift from Fresenius Kabi, Gurgaon, India. Poly lactide-
co-glycolide (PLGA) with molecular weight 50000 Da (50:50
grade) was bought from DURECT, Birmingham, AL, USA.
Pluronic 127 (PF 127), biotin-PEG-azide conjugate, propargyl
alcohol, copper sulfate, sodium ascorbate, fetal bovine serum
(FBS), antibiotic solution (penicillin/streptomycin, 0.1% v/v),
dialysis membrane (10000−12000 Da), ribonuclease A, and
RPMI 1640 Medium, 3-[4,5-dimethylthiazol-2-yl]-2,5- diphe-
nyltetrazolium bromide (MTT) were purchased from Sigma-
Aldrich, St. Louis, MO, USA. 4′,6-Diamidino-2-Phenylindole,
dihydrochloride (DAPI), JC-1 dye were procured from Invitro-
gen, OR, USA. Three-stage purified water was obtained from
Milli-Q purifier (Millipore, Milli-Q plus 185, Bedford, MA,
USA). All solvents used were of HPLC or LC-MS grade. The
chemicals were used as provided.
Solubility Studies. Excess of DOX was added to 5 mL water
or fixed strength aqueous solutions of different electrolytes (as
mentioned in Table 1) in 10 mL capped vials. The dispersions so
formed were shaken on a benchtop orbital shaker (Thermo
Fischer, India) at room temperature (25 ± 2 °C) for a period of
24 h. A fixed aliquot from the supernatant of each vial was diluted
appropriately using water and subjected to chromatographic
quantification (detailed in Supporting Information). Solubility
experiments were conducted in triplicate.
Development of Biotinylated−PEG-PLGA via Click
Chemistry. Synthesis of Propargyl PLGA Conjugate (3a). A
mixture of PLGA (2a) (1 g, 0.02 mM, 1 equiv), propargyl alcohol
(1a) (0.6 mM, 3eq ), and N,N-dimethylaminopyridine (DMAP)
(0.06 mM 0.3 equiv) was dissolved in 50 mL dry DCM in a
100 mL round-bottom flask and stirred in ice bath for half an
hour. To this mixture, DCC (1 equiv) dissolved in 15 mL dry
DCM was added, and the sample was stirred in an ice bath for 1 h.
The reaction mixture was further stirred for 24 h at room tem-
perature. Reaction was monitored by IR analysis. After comple-
tion, reaction mixture was filtered. Filtrate was washed with water
(2 × 40 mL) and brine (1 × 40 mL), dried over anhydrous
Na2SO4, and evaporated. After removal of solvent under vacuum,
extract was washed with ether and recrystallized with ethyl
acetate and hexane (1:2) to get clickable propargyl PLGA (3a);
85% yield.
Synthesis of Biotinylated-PEG- PLGA via Click Reaction.
Propargyl PLGA conjugate 3a (0.500 mg, 009 mM, 1 equiv) was
dissolved in tBuOH−H2O (1:1, 10 mL) by stirring for 15 min.
Sodium ascorbate (0.0029 mM, 0.3 equiv) was then added and
allowed to stir for 15 min followed by copper(II) sulfate (0.0027
mM 0.1 equiv). After 15 min biotin-PEG-azide (4a) (0.01 mM,
1 equiv) was added to the mixture and it was stirred at room
temperature in nitrogen for 24 h. The reaction conversion was
confirmed by IR analysis. Upon completion, the mixture was
evaporated under vacuum. The crude resultant was diluted with
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Figure 2. Work in brief. (A) Reaction scheme for synthesis of biotinylated−PEG-PLGA using click chemistry. (B) Biotinylated−PEG-PLGA was
employed to generate nanoparticles of DOX via W/O/W emulsion method. Using a strong electrolyte like NH4Cl bearing a common ion to doxorubicin
hydrochloride in the external aqueous phase suppresses aqueous solubility of DOX and traps it in organic phase containing biotinylated−PEG-PLGA.
(D) Once polymer solvent vaporizes, biotin tagged PLGA nanoparticles with multifold higher DOX content are obtained which when tested in vivo are
expected to elicit specific cytotoxic action against biotin receptor overexpressive 4T1 breast cancer cells. Key: 1a, propargyl alcohol; 2a, PLGA; 3a,
clickable alkyne-ended PLGA; 4a, biotin-PEG-azide; 5a, biotinylated−PEG-PLGA; DCC, N,N′-dicyclohexylcarbodiimide; DMAP, 4-dimethyla-
minopyridine; tBuOH, tertiary butyl alcohol; r.t., room temperature.

water and extracted with DCM (2 × 20 mL). The extract was
washed with brine solution and dried over anhydrous sodium
sulfate (Na2SO4). After removal of the solvent under vacuum,
extract was washed with ether three times. It was then precip-
itated in DCM and excess ether at low temperature to yield final
compound with 80% yield. The final compound, 5a (biotiny-
lated−PEG-PLGA), was confirmed by 1H NMR.
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Preparation of Nanoparticles. Biotinylated−PEG-PLGA
nanoparticles containing DOX (BPNP) were prepared by a mod-
ified W/O/W double emulsion solvent diffusion technique with
minor changes for trapping hydrophilic molecule DOX.13 Briefly,
as per Table 2, a specified quantity of polymer (biotinylated−
PEG−PLGA) and PF127 (3% w/v) were dissolved in 3 mL ethyl
acetate. To this organic solution, 1 mL triple distilled water
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Molecular Pharmaceutics Article

Table 1. Aqueous Solubility of Doxorubicin Hydrochloride in the Presence of Different Chloride Salts
NaCl (Ki 0.0047) KCl (Ki 0.0059) NH4Cl (Ki 0.0092) ZnCl2 (Ki 0.0259)
NaCl DOX solubility (mg/mL) KCl DOX solubility (mg/mL) NH4Cl DOX solubility(mg/mL) in ZnCl2 DOX solubility (mg/mL)
(mg/mL) in presence of NaCl (mg/mL) in presence of KCl (mg/mL) presence of NH4Cl (mg/mL) in presence of ZnCl2
0 10 ± 0.5 0 10 ± 0.5 0 10 ± 0.5 0 10 ± 0.5
2 8.4 ± 0.3 2 8.1 ± 0.3 2 6.0 ± 0.2 2 3.9 ± 0.3
3 7.8 ± 0.2 3 7.5 ± 0.5 3 3.7 ± 0.3 3 3.1 ± 0.5
5 7.3 ± 0.3 5 7.4 ± 0.4 5 2.6 ± 0.6 5 2.7 ± 0.1

Table 2. Batch Details of BPNP with Selected Physicochemical Characters

batch polymer (mg) drug polymer ratio particle Size (nm) PDI ZP (mV) %EE
F1 10 ∼1:1 48 ± 4 0.18 ± 0.07 −6.1 ± 2.1 3.1 ± 1.3
F2 15 2:3 72 ± 3 0.22 ± 0.11 −7.8 ± 2.0 5.2 ± 1.5
F3 30 1:3 81 ± 3 0.18 ± 0.05 −3.9 ± 0.9 7.4 ± 2.8
F4 40 1:4 98 ± 4 0.14 ± 0.07 −3.7 ± 1.3 12.0 ± 1.6
F5 60 1:6 155 ± 5 0.23 ± 0.14 −8.4 ± 2.7 14.5 ± 2.2

containing known amount of drug was added. The mixture was
then sonicated over an ice bath using a probe sonicator (Sonics,
USA) at 40% amplitude for 3 min to form a W/O emulsion. The
resulting primary emulsion was added to 4 mL aqueous NH4Cl
solution (5 mg/mL), and resonicated over an ice bath at 30%
amplitude for 10 min to form a W/O/W double emulsion. Ethyl
acetate was eliminated by evaporation under reduced pressure,
causing hardening of polymer around drug to form fully ripened
nanoparticles (BPNP). Nanoparticle suspension was dialyzed
against triple distilled water to remove free drug and dissolved
NH4Cl. Optimization studies were conducted to estimate effect
of formulation and processing variables on size and entrapment
efficiency of BPNP. For comparative purposes, blank nano-
particles were similarly developed (PNP or NP) without any
drug. The formulated nanoparticles were stored in cold refrig-
erated conditions until further use. The final coded formulations
were as follows: biotinylated−PEG-PLGA nanoparticles of DOX
(BPNP); PLGA nanoparticles of DOX (DPNP); blank/dummy
biotinylated−PEG-PLGA nanoparticles (NP); blank PLGA
nanoparticles (PNP).
Physicochemical Characterization. Particle size and
polydispersity index were measured by dynamic light scattering.
Zeta potential was evaluated by calculating mean electrophoretic
mobility of particles. Formulations were appropriately diluted
and analyzed at ambient temperature using Malvern zetasizer
(Nano ZS, Malvern Instruments, UK).14 Entrapment efficiency
was determined by employing reverse phase liquid chromatog-
raphy. Briefly, specified volume of formulation was dissolved in
1:1 mixture of DMSO and acetone, and vortexed for 5 min. The
mixture was thereafter diluted with water and analyzed on a
HPLC. Optimized batch was also subjected to storage stability
studies at room temperature (25 ± 2 °C) and cold refrigerated
conditions (4 °C). Samples were drawn on fixed days and
observed for increment/decrement in particle size and entrap-
ment efficiency.15
DSC analysis was performed on synthesized copolymer
biotinylated−PEG−PLGA, dummy nanoparticles made out of
biotinylated−PEG−PLGA, doxorubicin hydrochloride, and
BPNP using a differential scanning calorimeter (PerkinElmer,
USA). Dried samples (lyophilized or otherwise) weighing approx-
imately 5 mg, were placed in hermetically sealed aluminum pans
and heated at uniform rate of 10 °C/min up to 800 °C in an inert
environment maintained under dynamic nitrogen purging. Shape,
size, and surface appearance of nanoparticles were analyzed by
2752
electron microscopy (TEM). Briefly, diluted BPNP suspension
was smeared onto a 300-mesh copper grid in form of a film. The
grid was subsequently coated with 2% w/v uranyl acetate. Excess
solution was blotted off and the grid was dried using liquid nitro-
gen. Dried BPNP were observed at 80 kV by using TECHNAI G2
20 S-TWIN (FEI Netherlands) instrument and photomicro-
graphs of different fields at several appropriate magnifications
were taken.
Dissolution study was conducted using a dialysis tube. BPNP,
DOX solution, and DPNP with equivalent DOX content (2 mg)
were sealed in an activated dialysis bag and submerged in 50 mL
phosphate buffer saline (PBS pH 7.4). The dissolution medium
was magnetically stirred at 100 rpm and temperature was main-
tained within 0.5 °C of 37 °C. Periodic sampling was done and
amount of drug released was analyzed by reverse phase HPLC.
Obtained dissolution profile was fitted in different release models
via linear regression method. This was done to excavate probable
mechanistics of drug release.
Quantitative and Qualitative Cell Uptake Study:
Dependency on Biotin Receptors. Flow cytometry was
used to quantitatively assess cellular uptake of DOX-loaded
nanoparticles (BPNP vs DPNP vs DOX solution). Briefly, 4T1
cells were seeded in six well plates at a density of 0.5 × 105 cells
per well for plate adhesion and priming. Subsequently selected
wells were incubated with media containing BPNP, DPNP, and
DOX solution for 6 h followed by washing with PBS. Cells were
then trypsinized, pelleted, resuspended in PBS, and analyzed for
fluorescence produced by internalized DOX by using flow
cytometer (FACS Calibur, software cell quest Pro). To establish
dependency of BPNP on biotin receptor mediated uptake, an
additional well of cells was first treated with Biotin-Blocking Kit
(E-21390, Thermo-Fischer Scientific, India) as per manufac-
turers protocol to saturate the surface expressed biotin receptors
and subsequently subjected to BPNP for 6 h and analyzed as
above by FACS.
For fluorescence microscopy, 4T1 cells were seeded in 6-well
plates containing poly L-lysine coated coverslips for 24 h.
Thereafter cells were treated with DOX solution, DPNP, and
BPNP for 6 h. We also analyzed time dependent uptake of BPNP.
After staining, cells were rinsed repeatedly in PBS and rein-
cubated with formaldehyde (4% v/v in PBS) at 37 °C for 15 min.
The fixed cells were then aspirated. The coverslip left behind,
containing treated cells, was mounted on slides using ProLong
Gold Antifade Mountant with DAPI (Thermo Fisher Scientific,
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India) and observed under a fluorescence microscope at 60×
(Nikon Eclipse TS100 with fluorescence attachment Nikon-
CHGFI).
Cytotoxicity Study and in Vitro Hemolysis. MTT-based
in vitro assay was performed to compare cytotoxic effects of
different groups on 4T1 cells. Briefly, cells (0.5 × 104 cells per
well) in 96-well culture plates were seeded overnight in a
controlled environment of 37 °C, 5% CO2. Cells were then
treated with DOX solution, BPNP, DPNP, PNP, and NP dis-
persed in media equivalent to different concentrations of DOX.
After 24 h, the treatment media from wells was aspirated off and
replaced with fresh media containing MTT solution (0.5 mg/mL)
for 4 h. The formazan crystals so formed were dissolved by
adding 0.2 mL of DMSO to each well and absorbance of resulting
solution was measured at 570 nm using a multiwell scanning
spectrophotometer (PowerWave XS, Biotek, VT, USA). Non-
linear regression analysis was applied to determine inhibitory
concentration 50% (IC50) for each treatment group.16
Hemo compatibility of nanoparticles was evaluated in
erythrocytes isolated from blood of SD rats by centrifugation
(1000 × g for 10 min). The settled RBCs were suspended in PBS.
Accurately measured 2 mL RBC suspension was mixed with
aliquots of drug and formulation in triplicate to attain a final
DOX concentration of 0.5 to 25 (μg/mL). RBCs dispersed in
PBS only and PBS containing 10% Triton- X were used as
negative and positive control, respectively. The RBC-drug
aliquots were incubated at 37 °C in an atmosphere of 5% CO2
for 2 h. After incubation, samples were centrifuged at 800 × g for
10 min, and supernatant was collected in 96 well plates to be
analyzed spectrophotometrically at 540 nm.
Cell Cycle Distribution. Propidium iodide (PI) was used as
a fluorescent marker for cell cycle analysis. 4T1 cells were grown
in six well plates (106cells/well) and allowed to adhere. Free
DOX, BPNP, and DPNP (each equivalent to 50 nM DOX,
respectively) were added into different wells for a period of 24 h.
After stipulated time, cells were detached, rinsed in PBS, and
fixed in chilled ethanol (70% v/v). Thereafter cells were spun,
pelleted, resuspended in 250 μL PBS, and treated with
ribonuclease A (100 μg/mL) for 15 min followed by staining
with PI (50 μg/mL) for another 30 min. Fluorescence induced
by association of PI and remnant genetic material of perme-
abilized cells was measured employing a flowcytometer (BD
Biosciences, FACS Aria, Germany) and translated into the phase
of cell cycle arrest induced by different treatments.
Apoptosis. To determine extent and type of apoptosis
induced by DOX solution, BPNP, and DPNP we utilized
Annexin V and PI kit (Invitrogen, CA, USA). Protocol prescribed
in the kit was strictly adhered to and 4T1 cells (cultured as in cell
uptake) were treated with different formulations (all equivalent
to 100 nM DOX) for 24 h. Subsequently, cells were trypsinized,
spun, washed thrice with PBS, and dispersed in 500 μL binding
buffer. Thereafter, cells were incubated for 30 min in dark after
adding annexin V-FITC (5 μL) and PI (5 μL). Finally these cells
were subjected to flow cytometry so as to differentiate their
staining pattern.
In Vivo Antitumor Activity. Five to seven week old female
Balb/C mice weighing 15−25 g acted as model for breast cancer.
Murine mammary carcinoma cell line (4T1) was injected sub-
cutaneously into fat pad of mammary gland in lower right
quadrant of mice’s abdomen (1 × 106 cells/mice). After a waiting
period of 10 days post inoculation (tumors became palpable from
7 days on), a careful screening was done to select animals with
adequate tumor volume (100−150 mm3). The selected animals
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were divided into six groups of five each (n = 5). A dosing interval
of 72 h was adapted with DOX solution, BPNP, and DPNP
administered via intravenous entry into mice’s tail on day 0 (day
of treatment initiation), 3, 6, and 9. Another group was dosed
similarly with PBS to form the control group. The study was
ended on 27th day by sacrificing animals and harvesting
their tumors for subsequent processing [sliced into thin sections
(5−8 μm) and embedded in paraffin] which included hemotoxy-
line and eosin staining, immune-staining for PCNA and detec-
tion of apoptotic cells population through a dead end fluorom-
etric TUNEL assay using biotin-dUTP (biotinylated deoxyur-
idine riphosphate) and avidin-FITC as an apoptotic marker
(TUNEL apoptosis detection kit, Millipore). The purported
efficacy of administered treatments was gauged by measuring
tumor dimensions during the study as well as on the day of
termination. A digital Vernier’s caliper was employed to do the
same.
Pharmacokinetics. Pharmacokinetic study of DOX and its
formulations was carried out in 4T1 cell induced tumor bearing
Balb/C mice. DOX solution, BPNP, and DPNP were admin-
istered intravenously to conscious mice (via the caudal vein) at a
dose of 4 mg/kg. Blood (approximately 100 μL) was collected by
puncturing retero-orbital plexus with heparinised capillaries at
0.25, 0.5, 0.75, 1, 1.5, 2, 4, 6, 8, 10, 24, 48, and 72 h post dosing.
Sparse sampling was applied for blood collection and the total
volume of blood collected from each mouse was not more than
3% of total body volume. Blood samples were centrifuged at
5000 rpm for 10 min at 4 °C and plasma was separated into clean
and neatly labeled tubes. All samples were stored at −80 °C until
analysis. A different set of animals which had received the same
treatment as above were sacrificed at 2, 6, 10, 24, 30, and 72 h
post dosing and organs, such as spleen, kidney, and heart, and
tumor were collected. Protein precipitation approach was
employed for sample cleanup. To the plasma sample or tissue
homogenate (30 μL; blank, spiked or test), 170 μL acetonitrile
containing 30% ammonium acetate buffer (pH 3.5, 0.01M) was
added. The samples were vortex-mixed for 5 min followed by
centrifugation for 10 min at 10,00 rpm. Clear supernatant
(80 μL) was transferred into HPLC vials and 20 μL was injected
into LC-MS/MS system (refer to Supporting Information). Win
Nonlin 6.0 (Pharsight Co., Mountain View, CA, USA) was
utilized to determine various pharmacokinetic parameters.
In Vivo Fluorescent Imaging. To complement pharmaco-
kinetic studies, distribution and localization of DOX was
measured by using live in vivo imaging system IVIS Spectrum
(Caliper Life Sciences/PerkinElmer). Noninvasive whole body
fluorescent images of DOX solution, BPNP, and DPNP (equiv-
alent to 4 mg/kg DOX) treated mice (bearing 4T cell inuced
tumor) were generated by subjecting them to frontal examina-
tion at 535 nm. Subsequently generated emission was recorded at
560 nm. Images were acquired for 1 min at pre fixed time points
(5 min, 6 and 24 h after dosing) and emission emanating from
mice was analyzed using Living Image Software 4.4. In order to
facilitate accurate imaging, mice were preanaesthetized using
ketamine (20 mg/kg i.p.) before their placement inside imaging
chamber of instrument.
Toxicity Study. Acute toxicity study was performed on
six week old Balb/C mice (weighing ∼20 g) according to insti-
tutionally approved protocol. Mice were divided into four groups
of five animals each. The first group acted as control and was
treated with PBS via tail vein whereas the second, third, and
fourth group received DOX solution, DPNP, and BPNP, respec-
tively at a dose equivalent to 10 mg/kg DOX. This dosing cycle
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was repeated thrice, with 72 h between each dose. After 14 days,
all animals were sacrificed and major organs, such as liver, heart,
and kidney, were harvested. For histological examination excised
organs were fixed in formalin (10% in normal saline), rooted in
paraffin blocks, sliced to very thin sections using automatic micro
tome (Leica, model- 2155), and stained with hemotoxyline and
eosin. The sections were observed under an optical microscope
to grossly map their cellular architecture (Eclipse 50i, Nikon,
Japan). For biomarker assay, heart of each animal was purged in
liquid nitrogen at the moment of excision, thereafter cut into
weighted pieces and homogenized in PBS (weight to volume
ratio of 1:5) at 20000 rpm for 3 min using an ULTRA TURRAX
T-10 basic (Ika, Germany). The tissue homogenate was used for
estimation of catalase (CAT), malondialdehyde (MDA), and
superoxide dismutase (SOD). All three enzymes were estimated
as per protocols prescribed in their respective estimation kits
(Sigma-Aldrich, St. Louis, MO, USA)
Statistical Evaluation. Statistical appraisal of stability batches
was carried out by applying one way analysis of variance
(ANOVA) followed by Dunnett’s Multiple Comparison Test
using readings of day 1 as control. Results of in vivo efficacy
studies were compared by two way ANOVA followed by Bon-
Feroni post-test. Point to point comparison required in experi-
ments, such as IC50, were done with unpaired t tests. p < 0.05,
p < 0.01, and p < 0.001 acted as different significance levels. All
calculations were carried out using Graph pad Instat software 5.0
(Graph Pad Software Inc., CA, USA).
■ RESULTS AND DISCUSSION
Synthesis of Biotinylated−PEG-PLGA Using Click
Chemistry. The first premise of our work was to synthesize a
ligand tagged PLGA copolymer: biotinylated−PEG-PLGA (5a).
We proceeded as per scheme in Figure 2A. In order to make
PLGA (1a) clickable, we introduced an alkyne group in its struc-
ture by exploiting its terminal carboxylate residue. To do so, an
esterification reaction was carried out with hydroxyl group of
propargyl alcohol (2a) using DCC coupling. Thereafter, formed
propargyl PLGA conjugate (3a) was subjected to click condensa-
tion with commercially available Biotin-PEG-azide (4a). The
reaction underwent completion at room temperature under
appropriate catalysis (sodium ascorbate and copper sulfate).17
Refer to Supporting Information for IR spectrum (Figure S1).
Product formation was confirmed by 1H NMR.
NMR Results. PEG-Biotin Azide (4a). Purchased from Sigma-
Aldrich. White solid; 1H NMR (500 MHz, CDCl3) δ = 4.51−4.48
(m, 1H, NHCHCH), 4.32−4.29 (m, 1H, NHCHCH2),
3.68−3.61 (m, 8H, 4 × O-CH2), 3.57−3.55 (m, 2H, CH2-O),
3,45−3,38 (m, 1H, S-CH), 3.16−3.12 (m, 2H, CH2NH),
2.91−2.87 (m, 1H, CHaHbS), 2.72−2.71 (m, 1H, CHaHbS) 2.22
(t, J = 7.39 Hz, 2H, H2C − CO), 1.72−1.65 (m, 6H, 2 × CH2);
1.47−1.41 (m, 2H, CH2). Refer to Supporting Information for
spectrum (Figure S2).
Propargyl PLGA Conjugate (3a). White solid; 1H NMR
(500 MHz, CDCl3) 5.30−5.17 (m, CH, PLG) 4.89−4.37
(m, CH2-PGA+ CH2−O-CO) 2.46O−CH2 (t, J = 7.26),
1.72−1.55 (m, PLA- CH3). Refer to Supporting Information for
spectrum (Figure S3).
Biotinylated−PEG-PLGA (5a). White solid; 1H NMR (500 MHz,
CD3OD) δ = 7.36 (s, 1H triazole proton), 5.25−5.16 (m, CH,
PLA) 4.90−4.76 (m, CH2-PGA+ CH2−O-CO), 4.45−4.43
(m, 1H, NHCHCH), 4.29−4.26 (m, 1H, NHCHCH2),
3.60−3.64 (m, 8H, 4 × O-CH2) (m, 6H), 3.54−3.52 (m, 2H,
CH2-O), 3.42−3.39 (m, 1H, S-CH), 3.38−3.20 (m, 2H
2754
Article
N−CH2), 2.90−2.86 (m, 2H, CHaHbS), 2.74−2.72 (m,
CHaHbS) 2.60−2.59 (m, 2H, O−CH2), 2.19 (t, J= 7.45 Hz,
2H, CH2-CO) 1.72−1.55 (m, PLA- CH3+ 6H, 3 × CH2);
1.46−1.41 (m, 2H, CH2) (Figure S3).
Exploring Common Ion Effect and Its Influence on DOX
Solubility. In our earlier effort, we had tried to circumvent low
entrapment of DOX by hyodrophobicizing DOX into its base
form: this involved subjecting DOX to triethylamine treatment
followed by extraction with chloroform. However, extraction
was never absolute and there was perpetual drug loss even
before delivery system was fabricated.5a Trying to find alternate
methods of solubility suppression we came across a study where
Miyazaki et al. had suggested precaution in oral use of hydro-
chloride salts (papaverine and demeclocycline hydrochlorides)
as their solubility was heavily suppressed due to abundant
chloride ion (common ion) present in gastric secretions.18
Taking note we tried common ion effect to reduce aqueous
solubility of DOX. We did so by tuning solubility of DOX in the
presence of chloride containing strong electrolytes like NaCl,
NH4Cl, ZnCl2, and KCl. All solubility experiments were
conducted at room temperature. Saturation solubility of DOX
in water as per HPLC was 10 ± 0.5 mg/mL and modulation
attained in its solubility in the presence of different salts is shown
Table 1.
There was almost 5-fold reduction in solubility of DOX when
it was codissolved with 5 mg/mL NH4Cl or ZnCl2. Exact order
of solubility reduction was as follows: NH4Cl (2.6 ± 0.6 mg/mL)
∼ ZnCl2 (2.7 ± 0.1 mg/mL) ≫ NaCl (7.3 ± 0.3 mg/mL) ∼ KCl
(7.4 ± 0.4 mg/mL). Obtained DOX concentration in the pre-
sence of different salts (Sb) is plotted as a function of salt con-
centration according to empirical Setschenow equation in
Figure S4 of Supporting Information.19
log S0/S b = K i[chloride salt]
Where S0 is saturation solubility of DOX alone, Sb is solubility of
DOX in the presence of chloride salt, and Ki is salting out
constant attributable to that common chloride ion which reduces
solubility of DOX. Consistent with results in Table 1, ZnCl2
(0.0259 mM−1) and NH4Cl (0.0092 mM−1) had highest salting
out constants. A higher salting out constant can be interpreted as
greater capacity of the selected salt to suppress solubility of DOX.
Based on solubility data and estimated salting-out constants,
NH4Cl and ZnCl2 were selected for further investigation of
common ion effect on drug entrapment. A diagrammatic repre-
sentation of how common ion effect reduces aqueous solubility
of DOX when an electrolyte bearing a common counterion
(chloride) is added to its solution is shown in Figure 2. Upon
solubilization DOX yields a positively charged doxorubicin ion
and a chloride ion. In accordance with Le Chatelier’s principle if
concentration of any one of the ions is increased (chloride in our
case by addition of a chloride bearing strong electrolyte), ion
product of dissolved ions will exceed solubility product of DOX,
shifting ionic equilibrium toward left which in turn forces DOX
to precipitate out of solution by combining with some dissolved
chloride ion.20 Precipitation of DOX will continue until ion
product is equal to solubility product and as a result overall
solubility of DOX is reduced many folds. Practically, it would be
possible to reduce solubility of DOX even further by raising
dissolved chloride ion concentration. However, increasing salt
content beyond 5 mg/mL (for all the selected salts) led to
cracking of emulsion in downstream formulation step. Also,
formulation manufactured using ZnCl2 at any concentration
experienced phase separation and we consequently used NH4Cl
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Molecular Pharmaceutics Article

Figure 3. Physicochemical characteristics, stability profile, and in vitro release of BPNP. (A) Particle size, (B) PDI and zeta potential, and
(C) entrapment efficiency of different batches of BPNP. (D) Effect of NH4Cl concentration on entrapment efficiency of DOX. (E) Measurements of
particle size and entrapment efficiency at different storage conditions during the course of stability study of F4. (n = 3; * p < 0.05, ** p < 0.01, *** p < 0.001,
n.s. not significant). Refer to Table 2 for further batch details. (F) Characterization of biotinylated−PEG-PLGA, dummy nanoparticles, DOX and BPNP
using differential scanning calorimetry. (G) TEM images of BPNP. Scale bar represents 100 nm. (H) Comparative dissolution profile of DOX solution,
DPNP, and BPNP in PBS pH 7.4. (I) Controlled release behavior offered by BPNP kicks in straightaway as suggested by the inset of initial time points.

at its highest permissible concentration of 5 mg/mL to exert
common ion effect.
Formulation Dynamics in the Presence of a Common
Ion: Why Entrapment Is Favored. We utilized common ion
2755
effect modified W/O/W double emulsion method to manu-
facture BPNP (Batches: F1 to F5). It involved emulsifying a
primary W/O emulsion with drug containing aqueous phase
in a secondary external aqueous phase containing dissolved
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Molecular Pharmaceutics
NH4Cl (5 mg/mL). The oil phase consisted of ethyl acetate and
polymer (biotinylated−PEG-PLGA), whereas surfactant, plur-
onic 127 (PF 127) was expected to be at its most convenient site
of residence, i.e., oil−water interface.14 If we comprehend an
infinitesimally small duration which follows introduction of drug
containing primary W/O emulsion droplet into NH4Cl con-
taining external aqueous phase, under ultrasonic stimulation, two
concurrent dynamic processes responsible for raised entrapment
efficiency of BPNP can be visualized.
(1) W/O droplet is disrupted into many smaller droplets due
to intense energy produced by implosion of cavitation
bubbles created by ultrasonic probe.21 This raises inter-
facial energy of W/O/W system exponentially destabiliz-
ing the emulsion system despite it having simultaneously
attained increment in overall entropy.22 The only plausible
way to counter this instability is recoalescence of O/W
droplets to reduce surface area. However, steric hindrance
offered by interfacially located PF 127 implies that distance
of closest approach between two emulsified droplets is
substantially increased.23 O/W droplets are therefore in a
state of suspended animation inside W/O/W emulsion,
whereby they are constantly trying to reattach.
(2) Both internal (DOX at 10 mg/mL) and external phase
(NH4Cl at 5 mg/mL) consist of dissolved solutes. How-
ever, for fixed 1 mL internal and 4 mL external phase
volumes, the osmotic pressure exerted by NH4Cl in
external phase (2.22 atm) is roughly 5 fold greater than
that exerted by DOX in the internal phase (0.41 atm).
Osmotic pressure was calculated by employing van’t Hoff’s
law; molar concentrations of solute in the internal and exter-
nal phases, respectively, at room temperature, i.e., 298 K
with universal gas constant of 0.08206 L atm mol−1 K−1.
This osmotic pressure gradient effectively drags out water
from innermost phase toward the bulk.24 Intuitively it
would seem that DOX will also be carried out with this
hydrostatic drag to solubilize freely in bulk aqueous phase,
but existence of common chloride ion in external phase
due to dissolved NH4Cl massively reduces doxorubicin
hydrochloride’s water solubility and constrains it in the
oil phase away from aqueous bulk. Once ethyl acetate is
evaporated under vacuum, dissolved polymer congeals in
shape of its ancestral oil droplet to form regularly shaped
spherical nanoparticles trapping DOX which in actuality
had nowhere else to go due to influence of common ion
effect in the external phase.
Preliminary Optimization of BPNP. Size plays a pivotal
role in passive targeting of cancerous site due to EPR effect.
Retrospective analysis reveals that in order to capitalize on EPR
effect, a carrier must be sized between 10 and 100 nm. Entities
smaller than 10 nm are either washed away by glomerular
filtration or are extravasated at unwanted sites and those larger
(∼100 nm upward) are trapped by reticuloendothelial system.25
Therefore, one of our objectives was to keep BPNP within
touching distance of above prescribed size range (10−100 nm),
so as to maximize any advantage offered by biotinylation of
PLGA. BPNP were subjected to routine physicochemical char-
acterization like zeta potential, PDI, particle size, and entrap-
ment efficiency as shown in Figure 3A, B, and C. Particle size of
different batches was found in range of 40 to 150 nm, whereas
PDI was below 0.3 suggesting particle uniformity. Zeta potential
as expected was slightly negative between −1 to −8 mV due to
biotinylated−PEG-PLGA backbone which might have residual
2756
Article
carboxylate charge like in studies conducted on PLGA nano-
particles elsewhere.26
Particle size and entrapment efficiency were given highest
priority during formulation optimization. It was found that
increasing content of polymer [drug polymer ratio (DPR) from
1:1 to 1:6] had a direct influence on both of these parameters.
Best formulation with compliant size was F4 (DPR 1:4; size
98 ± 4.58 nm). It had 12.0 ± 1.6% drug entrapment which was
significantly greater (p < 0.01) than F3 (DPR 1:3) with drug
entrapment of 7.4 ± 2.8%. Entrapment did not increase any
further (p > 0.05) when DPR was increased to 1:6 in F5 (14.50 ±
2.2%), but there was substantial size increase (from 98 ± 4.5 nm
for F4 to 155 ± 5 nm for F5). Keeping size and entrapment in
mind we did not iterate drug polymer combinations any further
and selected F4 as the optimum batch. Once size and loading
protocols were optimized for BPNP, entrapment was maximized
by manufacturing F4 in the presence of varying concentration of
NH4Cl.
Effect of Ammonium Chloride on Drug Entrapment:
Storage Stability of Formulation. As suggested by Figure 3D,
entrapment efficiency of BPNP increased in direct correlation
to NH4Cl concentration in external aqueous phase of W/O/W
emulsion. Maximum entrapment (90 ± 3.2%) was obtained
when 5 mg/mL NH4Cl was used which is more than 8-fold
greater than what was obtained in batches manufactured in
absence of NH4Cl. This ability to vary entrapment efficiency
favorably establishes the second premise of our work, i.e., to
adequately capture a hydrophilic salt like DOX in an optimally
sized carrier system derived from a robust and safe polymer like
PLGA. Batch F4 was taken forward for further in vitro and in vivo
testing. BPNP were tested for storage stability at room tem-
perature (25 ± 2 °C) and in a refrigerator (4 °C). Particle size
and entrapment efficiency of BPNP after 30 days at 4 °C did
not register any significant alterations. However, particle size
at 25 ± 2 °C increased rapidly to 135 ± 4.3 nm after 30 days
(Figure 3E). One way ANOVA followed by comparative post-
test against readings of day 1 revealed significantly greater incre-
ment (p < 0.001) in particle size when BPNP were stored beyond
4 days at room temperature. Drug entrapment was also substan-
tially reduced (75.6 ± 3.5%) at the end of 30 day testing period
(p < 0.001). Storage at room temperature increases kinetic
energy of BPNP suspension, promoting collisions, which escalates
chances of particle aggregation and subsequent growth.13 To sum
up it was found that BPNP were stable enough in refrigerator
conditions for a period of one month negating chances of any false
positives or negatives creeping into the study due to usage of a
stored formulation during that one month window.
DSC. An overlay of DSC curves for BPNP, biotinylated−PEG-
PLGA, dummy nanoparticles made from biotinylated−PEG-
PLGA (NP) and DOX is provided in Figure 3F. DOX showed an
endothermic peak at 241.76 °C which was in close agreement to
its reported melting point of 230 °C.27 An endothermic peak at
375.37 °C indicative of thermal degradation was observed for
biotinylated−PEG-PLGA. DSC thermogram of blank nano-
particles of biotinylated−PEG-PLGA and BPNP had almost
coincident endothermic peaks between 310 and 315 °C. How-
ever, both of these peaks were less sharply defined than their
parent copolymer i.e. biotinylated−PEG-PLGA. Shifting of
thermal degradation peak from 375.37 °C to 310−315 °C
suggests: biotinylated−PEG-PLGA when formulated into nano-
particles degrades faster due to exposition of larger surface area
and/or it has been reduced to a comparatively more amorphous
state.28
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Figure 4. In vitro cell culture assays. (A) Quantitative cellular uptake of BPNP in 4T1 cells. 4T1 Cells were exposed to media containing (i) blank
nanoparticles, (ii) DOX solution, (iii) DPNP, and (v) BPNP at a concentration equivalent to 100 nm DOX for 6 h.(iv) A seeded well presaturated with
biotin was also used to investigate whether blockade of biotin receptor induces variation in BPNP uptake. Standard deviation in
MFI values were calculated for cells from 3 tests. (C) Results from MTT assay. BPNP (91.5 ± 13.7 nM) exhibit 2 fold reduction in IC50 of DOX
solution (179.5 ± 11.8 nM) whereas NP and PNP were inert. (C) BPNP cause lysis of less than 10% incubated erythrocytes at 10 μg/mL. Hemolysis was
nonexistent for NP or PNP. (D) Apoptosis and (E) cell cycle analysis was conducted at concentration equivalent to 100 nM and 50 nM DOX,
respectively, for a treatment period of 24 h. BPNP cause maximum early and late apoptosis (59% cells) and G2-M phase arrest (93.44%).

An amorphous substance has less ordered structure and
consequently requires lesser energy to undergo thermal degrada-
tion. Since BPNP (265 mJ) requires lesser energy compared to
blank biotinylated−PEG-PLGA nanoparticles (316 mJ) and
biotinylated−PEG-PLGA (395 mJ) it might have greater amor-
phous character. Dissolution of polymer in an organic solvent
followed by its co-precipitation with DOX into aqueous phase
during formulation processing might be responsible for such a
shift in phase transition. Also disappearance/merging of endo-
thermic peak of DOX with the main endothermic peak of BPNP
suggest that it might have been rendered completely amorphous
and is uniformly dissolved in BPNP.29
In Vitro Drug Release. Comparative in vitro dissolution
study for DOX solution, DOX loaded regular PLGA nano-
particles (DPNP), and BPNP was conducted in PBS pH 7.4 and
the results were plotted versus time in Figure 3H,I. Equilibration
of DOX solution with dissolution media was seamless with
46.0 ± 2.6% drug content available within first 3 h followed by
net release of 86.3 ± 1.0% in 24 h. In contrast, BPNP and DPNP
released only 30.00 ± 2.9% and 32.8 ± 3.2% of their respective
drug contents in first 12 h of release study. Two-way ANOVA
revealed significant difference (p < 0.05) in drug release pattern
2757
between BPNP and DOX solution at all-time points, suggesting
modulation in drug release was induced by usage of biotinylated−
PEG-PLGA. Obtained dissolution data for BPNP was fitted into
release kinetic models like Hixson-Crowell (r2 = .95), Korsmeyer
peppas (r2 = .96, n = 0.70), Higuchi (r2=.98), zero order (r2 = .92),
and first order (r2 = .95), to gain an insight into mechanism of
drug release. Since r2 values did not differ much and most of the
models had comparable fit we could not select any mechanism
which out rightly dominated drug release. Literary sources also
lend weight to our findings as they propose multiple com-
binatorial mechanisms (like diffusion through particle matrix
either independently or following matrix disintegration, desorp-
tion of surface-adhered drug, diffusion through polymer wall,
surface and bulk degradation of polymer exposing housed drug to
dissolution media) responsible for drug release in case of PLGA-
derived nanoparticles.30 Therefore, it will not be an over exag-
geration to assume BPNP, which are made up of a derivatized
form of PLGA, also behave similarly.
Quantitative Cell Uptake. DOX is an inherently florescent
molecule which enables direct analysis of its uptake by 4T1 cells
using flow cytometry (Figure 4A). Results revealed that BPNP
had adequate uptake in 4T1 cells. Median florescence intensity
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Figure 5. Pictorial evidence of time dependent BPNP uptake in 4T1 cells. The drug seems to localize completely in nuclear region at 6 h after BPNP
treatment. However, comparative fluorescence from DPNP and DOX solution treated cells at 6 h is much lesser than that produced by BPNP. Scale bar
represents 30 μm.
(MFI) counted in 10000 cells after BPNP treatment (214 ± 19) dimension for an entity to undergo endocytosis (a specialized
was 20-fold higher (p < 0.001) than untreated control cells uptake mechanism existent in almost all cell types,) should be
(9.8 ± 2.1), 7-fold higher than DOX solution (36.9 ± 3.0), and below 100 nm.31 Since average particle size of DPNP and BPNP
2-fold greater than DPNP (113.4 ± 10.4) treated cells. Usually fall in this range, expected cell uptake should have been the same.
2758 DOI: 10.1021/acs.molpharmaceut.7b00310
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Molecular Pharmaceutics
But surface decoration of PLGA with biotin, lends BPNP a
recognizable epitope which causes its greater internalization by
4T1 cells due to receptor mediated facilitatory uptake. We
suspect biotinylation of PLGA leads to advertent targeting and
rapid cell uptake of carrier.32 Relatively high uptake of BPNP, in
comparison to cells treated with DOX solution and DPNP, is
expected to be of substantial significance, as it would prefer-
entially ensure higher payload delivery inside cell improving
expected anticancer activity. To showcase involvement of
biotin receptors on uptake of BPNP we conducted a differential
uptake experiment where 4T1 cells were pretreated with biotin
(Figure 4A (iv). It was observed that uptake of BPNP and DPNP
in biotin-treated cells was indistinguishable. Biotin treatment
here acted as a competitive blocker of biotin receptors expressed
on 4T1 cell surface and thereby precluded biotin receptor
mediated endocytic uptake of BPNP, reinforcing our earlier
statement.
Cytotoxicity Assay and Hemolysis. MTT assay was
performed in order to ascertain in vitro cytotoxic activity of
BPNP. Inhibitory concentration which induced cell death in 50%
population (IC50) was calculated using nonlinear regression
(Figure 4B). BPNP was 1.44-fold more cytotoxic than DPNP
[P < 0.05 (n = 3)]. IC50 values of DOX solution, DPNP, and BPNP
were 179.5 ± 11.8 nM, 131.8 ± 17.2 nM, and 91.5 ± 13.7 nM,
respectively, after 24 h. BPNP inducing a 2-fold reduction in IC50
[P < 0.05 (n = 3)] compared to DOX solution might be a trickle-
down effect of enhanced biotin receptor mediated intracellular
drug uptake. Once inside the cell, whatever drug is released by
BPNP is available in vicinity of its action site thereby exerting
greater cell death. Dummy nanoparticles (NP and PNP) were
found to be inert and did not cause any unwanted cell death
suggesting excipients employed in formulation did not have any
unwarranted cytotoxic activity of their own.
Hemolysis study was performed on resuspended erythrocytes.
It was observed that both BPNP and DPNP were safe on
hemolytic front (Figure 4C). They caused lysis in 7.8 ± 2.4% and
6.9 ± 2.1% erythrocytes at concentration equivalent to 10 μg/mL
DOX. However, at same concentration, pure drug solution
caused lysis in 16.8 ± 2.6% cells (p < 0.01). Lowering of DOX’s
inherent hemolytic potential by BPNP might be due to altered
drug release offered by the carrier system, which reduces direct
exposition of erythrocytes to drug. Low hemolytic potential of
BPNP will bode well for its amicability toward intravenous
administration.
Cell Cycle and Apoptosis. Free DOX solution, BPNP, and
DPNP were incubated with 4T1 cells for 24 h and a propidium
iodide-based assay was performed to assess phase in which
treatments induce cell arrest, if any (Figure 4E). Results
suggested that BPNP caused massive cell arrest (93.44%) in
G-2 M phase which was greater than DPNP (89.42%), DOX
solution (76.49%), and untreated control cells (13.19%). This
incremental trend can be attributed to rapid and selective uptake
of BPNP, which causes sustained doxorubicin delivery at
intracellular locations. However, significance of above statistic
becomes apparent only if we ponder into DOX’s mechanism of
action. Doxorubicin hydrochloride has high affinity toward DNA
causing its intercalation and subsequent distortion. Doxorubicin
specifically inhibits progression of topoisomerase II, which
ultimately results in deformed DNA during S-phase or synthetic
phase of cell cycle.33 Each cell has a DNA damage check point in
G2-M phase which ensures that cells do not initiate mitosis
before they have had a chance to repair damaged DNA syn-
thesized in S-phase. If cells have a defective G2-M checkpoint
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they proceed with damaged DNA leading to cell death (mostly
by apoptosis) after division.34
Since BPNP cause overt G-2 M phase arrest there is high
probability its DOX content has profusely damaged DNA of
almost all the cells which necessitates activation of G-2 M
checkpoint for DNA repair. BPNP, DPNP, and DOX solution
were also quantitatively evaluated for their apoptotic potential
by using dual annexin V and propidium iodide staining. Early
apoptotic cells were captured in the lower right quadrant of dot
plot, while the late apoptotic cells were seen in the upper right
quadrants (Figure 4D). BPNP enhanced early and late apopto-
sis (59.63%) compared to DPNP (38%) and DOX solution
(36.44%). These results were substantiated qualitatively by fluo-
rescence microscopy described next.
Time Dependent Cell Uptake of BPNP by Fluorescence
Microscopy. Encouraged by results of quantitative uptake and
in vitro cytotoxicity experiments we attempted to visually map
fate of BPNP both with respect to time and in comparison to
DPNP and DOX solution (Figure 5). 4T1 were cells cultured in
6 well plates and incubated with BPNP dispersion equivalent to
100 nM DOX. At prefixed times of 1, 3, and 6 h following BPNP
treatment; cells were counterstained with nuclear dye DAPI and
visualized. At 1 h it was found that doxorubicin signal was
observable (indicated by arrows) specifically in cytoplasmic
domain only, away from an independently discernible and
regularly shaped nuclear region. This suggests successful
transmigration of drug across cellular membrane via BPNP and
its rapid intracellular availability. At 3 h, intensity of doxorubicin
signal was clearly enhanced signifying increase in its concen-
tration causing co-localization with DAPI, due to DNA inter-
calating ability of drug. It can be assumed that DOX contained
inside BPNP has moved to its natural site of action, i.e., DNA
housed in nuclear region. At 6 h nuclear migration of payload
seems complete as BPNP produces selective co-localization with
DAPI. However, at same time point, intracellular levels of DOX
after incubation of 4T1 cells with DPNP and DOX solution are
significantly lesser, as suggested by low intensity of nuclear
fluorescence, and it becomes easier to comprehend why BPNP
is much more cytotoxic. This study further substantiates the
observations made by flow cytometry based assays and proves
that decoration of PLGA template with biotin leads to both rapid
and quantitatively greater intracellular delivery of DOX than
what was possible with native drug solution or a simpler carrier
loaded with drug.
Since cytotoxicity experiments had already shown that dummy
nanoparticles constructed out of biotinylated−PEG-PLGA or
PLGA did not produce any activity of their own, it was concurred
that dynamics of cellular killing were dictated by DOX alone, and
any advantage which had been obtained thus far by BPNP must
be a manifestation of altered intracellular DOX presentation
vindicating our purported hypothesis of decorating PLGA with
biotin at least on the in vitro scale.
In Vivo Pharmacokinetics. DOX levels attained in plasma
and in different tissues after intravenous administration of DOX
solution, DPNP, and BPNP (4 mg/kg) in tumor bearing Balb/C
mice were monitored using LC-MS. Noncompartmental ana-
lysis was applied on obtained measurements to estimate pharma-
cokinetic parameters for formulations in plasma and differ-
ing tissues, namely tumor, heart, kidney, spleen, and lung
(Figure 6A). DOX solution in correspondence to its dissolution
pattern attained Cmax of 758.84 ± 57.3 ng/mL within 5 min of
dosing. Since DOX has high aqueous amicability, intravenously
applied bolus dose of DOX solution undergoes instantaneous
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Figure 6. Pharmacokinetics and tissue distribution. (A) Comparative pharmacokinetic profile attained by DOX solution, DPNP and BPNP in plasma
and different organs after intravenous dose equivalent to 4 mg/kg in tumor bearing Balb/C mice. All plasma values have been plotted as mean ± s.d. for
three animals each. Tissue distribution has been plotted using mean values only for sake of clarity. (B) In vivo images of 4T1 tumor bearing Balb/C mice
treated intravenously with DOX solution, DPNP, and BPNP, respectively, at different time points.

equilibration with blood and yields high systemic concentration.
However, rapid disposition takes hold simultaneously and drug
levels thereafter start falling with a terminal half-life of 5.39 ±
0.76 h. Drug levels attributable to BPNP and DPNP had an almost
5-fold lower Cmax of 154.3 ± 21.4 ng/mL and 162 ± 11.69 ng/mL,
respectively. This stemmed from control exerted by polymeric
carriers, i.e., biotinylated−PEG-PLGA and PLGA. For BPNP,
elimination changes drastically, resulting in 7-fold prolongation
2760
of half-life (35.18 ± 0.36 h) compared to DOX solution and
2-fold prolongation in comparison to DPNP (17.71 ± 1.27 h).
Half-life extension for BPNP might be a resultant of two
overlapping phenomena: (1) avoidance of phagocytic uptake due
to PEG component of BPNP prolonging systemic circulation of
intact nanoparticles providing sustained drug levels35 and/or (2)
accumulation of nanoparticles in large perforated organs (like
spleen, liver) juxtaposed with secondary redistribution of drug
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Molecular Pharmaceutics Article

Figure 7. BPNP show higher antiproliferative activity. (A) Photographs were taken to visually demonstrate discernible reduction in tumor caused by
BPNP in comparison to DPNP and DOX solution. (B) BPNP produced highly significant (p < 0.001) reduction in tumor volume when compared to
DOX solution and DPNP treated tumors. (C) Tumor weight was measured at the end of study and expectedly BPNP treated mice exhibited lower
tumor burden. Tumor section of BPNP treated animal had several areas of gross cellular degeneration and matrix abnormalities which were rare in DOX
solution treated animals. Number of PCNA carrying cells (appear brown) was substantially more in DOX solution treated tumor than in BPNP treated
tumor. Green fluorescence produced by apoptotic cells in TUNEL assay was also higher in BPNP treated group. These results cumulatively suggest
multifactorial improvement in in vivo efficacy of doxorubicin hydrochloride when it is administered as BPNP (n = 5; *p < 0.05, **p < 0.01, ***p < 0.001,
n.s. = not significant). Scale bar in tumor sections, PCNA stained sections, and TUNEL assay sections are equal to 100 μm.

2761 DOI: 10.1021/acs.molpharmaceut.7b00310

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Molecular Pharmaceutics
from these temporary reservoirs back into plasma.36 Long circula-
tion times raises probability of BPNP reaching their intended site
of action (i.e., tumor). Lower clearance (1505 ± 38.2 mL/h/kg),
extended MRT (22.56 ± 3.8 h), and higher AUC0‑∞ (2657.2 ±
180.4 h*ng/mL) of BPNP when compared to DPNP and
DOX solution further substantiate consequences of above
description. It was also noted that BPNP elicited significantly
greater (p < 0.01) Cmax (49.4 ± 8.2 ng/mg) and AUC0‑∞ (1588 ±
218.4 h*ng/mg) than DOX solution (Cmax 11.45 ± 4.7 ng/mg,
AUC0‑∞ 367 ± 58.4 h*ng/mg) and DPNP (Cmax 22.45 ±
6.9 ng/mg, AUC0‑∞ 763 ± 80.4 h*ng/mg) in tumor tissue. This
was a strong evidence showcasing effectiveness of active
targeting. Raised drug concentration in tumor vicinity will elicit
a more potent response. Further complementing favorable tumor
accumulation of BPNP were its low levels in heart (AUC0‑∞
2255 ± 35.6 ng/g*h, Cmax 36.7 ± 8.3 ng/g). Contrastingly if
we note statistically greater accumulation of drug in cardiac
tissue (AUC0‑∞ 3961 ± 43.8 ng/g*h, Cmax 149.4 ± 14.6 ng/g)
following administration of DOX solution, it becomes clear as to
why doxorubicin hydrohloride elicits a cardiotoxic response.
Several favorable distinctions exist at critical junctures for
BPNP which are attributable to its specific size as well as mate-
rial of construction, i.e., biotinylated−PEG-PLGA. Exploiting
(1) size (below 100 nm), which has been promulgated to be
suited toward infiltrating leaky vasculature in and around tumor,
and (2) biotin component which facilitates rapid uptake inside
4T1 cancer cells, BPNP possesses capability to deliver drug speci-
fically to tumor site away from cardiac tissue which might tilt risk-
benefit ratio of doxorubicin hydrochloride favorably. Please refer
to Supporting Information sheet for detailed listing of pharma-
cokinetic parameters. Drug was not detected in brain.
In Vivo Imaging. Current work professes delivering doxoru-
bicin hydrochloride in vicinity of tumor for eliciting maximum
possible therapeutic response, without burdening body with
undue toxicity. To further strengthen this hypothesis, we sub-
stantiated quantitative pharmacokinetic data with live in vivo
images to accurately portray exact fate of BPNP (Figure 6B). We
did so by employing above-described pharmacokinetic model, in
which three distinct mice were treated intravenously with DOX
solution, DPNP, and BPNP via tail vein. Intensity of signal prod-
uced was related to amount of fluorescent moiety, i.e., doxoru-
bicin hydrochloride.
At 5 min post dosing, fluorescence emanating from doxoru-
bicin hydrochloride was visible in tail region of all animals. One
might argue isolated fluorescence in tail region contradicts
plasma pharmacokinetic data (an indicator of systemic drug
presence) where Cmax was recorded for all groups at 5 min;
however (1) superficial nature of tail vein, (2) time required for
drug to undergo adequate distribution, and (3) lack of
surrounding connective tissue in mice’s tail might be responsible
for this selective signal. At 6 h owing to drug distribution,
fluorescence signal was observable throughout animal body for
DOX solution, DPNP, and BPNP; however spatial disposition
for each was different. DOX’s inherent affinity toward cardiac
tissue dictated DOX solution and produced little localization in
tumor associated regions. Since PLGA controls drug release,
cardiac sequestration of DPNP was slightly lesser than DOX
solution at 6 h, though it was still more than what was seen with
BPNP at same point. In case of BPNP, we saw selective localiza-
tion of drug in tumor region. Armed with its sub 100 nm size and
surface decorated biotin moiety, BPNP spontaneously gravitates
toward tumor, releasing substantial drug at required site of
action. At 24 h DOX solution owing to its elimination does not
2762
Article
produce detectable signal in most regions apart from cardiac
tissue whereas DPNP seems sparsely distributed and it is difficult
to draw any conclusion. Contrarily BPNP at same time point is
housed exclusively in and around tumor (encircled). BPNP’s
distribution pattern suggests that drug carried by it is not only
targeted specifically toward tumor due to active targeting, but
chances of it producing any unwanted off target effects like
cardiotoxicity in comparison to DOX solution are also minimal.
In Vivo Antitumor Efficacy. Balb/C mice carrying 4T1
breast cancer were treated intravenously with DOX solution,
BPNP, and DPNP at a dose equivalent to 4 mg/kg. A separate
group of cancerous mice was treated intravenously with PBS to
act as negative control. Results are summarized in Figure 7.
Relative efficacy of treatments was assessed by (1) measuring
tumor volume during and after the study, (2) documenting
tumor burden, (3) examining tumor histology, (4) staining for
proliferating cell nuclear antigen (PCNA), and (5) counting
apoptotic cells in tumor sections via TUNEL assay. Photographs
in Figure 7A visually demonstrate discernible reduction in tumor
caused by BPNP in comparison to DPNP and DOX solution.
Two way ANOVA followed by Bon Feroni post-test (Figure 7B)
revealed that BPNP produced significantly greater (p < 0.001)
tumor volume reduction (from 99.36 ± 4.23 to 29.74 ±
9.17 mm3) when compared to DOX solution (from 100.43 ±
5.20 to 76.38 ± 6.24 mm3) and DPNP (from 102.72 ± 6.80 to
68.43 ± 8.42 mm3). BPNP also caused maximum reduction in
tumor burden (123.5 ± 20 mg). It approximately caused 10-fold
and 3-fold reduction in tumor burden when compared to PBS
and DOX solution (Figure 7C). Interestingly there was insig-
nificant difference in antitumor efficacy of DOX solution and
DPNP. If we look at the pointed portions in H and E stained
tumor sections of BPNP, there are several regions where cellular
matrix has undergone major degeneration. Spindle shaped
regular 4T1 cells which are most prominent in PBS treated group
and to some extent in DOX solution and DPNP treated tumors
seem to have given way to constricted dots which might be a
manifestation of pyknosis associated with apoptotic cells.
Considering the DNA specific mechanism of action of DOX,
we also gauged PCNA expression in tumor sections. It was found
that BPNP treated tumor section had much less PCNA expres-
sion (seen as brown spots in Figure 7) in comparison to DOX
solution treated tumor. PCNA is a DNA immobilizer and acts as
a functional co factor for DNA polymerase activity in replication.
PCNA recruits proteins involved in DNA repair and chromatin
remodelling.37 Doxorubicin causes covalent cross-linking of
PCNA trimers which raises probability of DNA damage.38
PCNA’s down regulation after treatment with BPNP is an indi-
cator that cellular proliferation is stalled more effectively by it in
comparison to DOX solution or DPNP. TUNEL assay showed
that BPNP induced greater apoptotic activity compared to DOX
solution and DPNP against 4T1 cells (as suggested by number of
green fluorescent spots in Figure 7). These results cumulatively
corroborate improved efficacy of BPNP, and strengthen the basis
of lending drug loaded PLGA nanotemplate an active targeting
ligand biotin so as to increase in vivo antitumor efficacy of DOX.
Toxicity Study. Results of toxicity study are summarized in
Figure 8. At the forefront it was observed that BPNP treated
animals did not exhibit any behavioral modulation like irritancy,
hair fall, necrosis at the site of injection, or morbidity. It was also
found that BPNP induced roughly 2-fold greater (p < 0.05)
catalase activity (1.24 ± 0.20 μmol/mL/min/mg) than that of
PBS treated control mice (0.65 ± 0.16 μmol/mL/min/mg),
whereas DOX solution caused more than 3-fold (p < 0.001)
DOI: 10.1021/acs.molpharmaceut.7b00310
Mol. Pharmaceutics 2017, 14, 2749−2765
Molecular Pharmaceutics Article

Figure 8. Acute toxicity study. DOX solution, BPNP, and DPNP were administered at a dose of 10 mg/kg via tail vein of healthy BALB/C mice and
toxicity was evaluated 14 days post injection by analyzing (A) tissue histology and measuring relative (B) catalase activity, (C) MDA content, and
(D) SOD levels in heart. Saline treated animals displayed regular tissue architecture. DOX solution caused massive cardiac degeneration. The striations
which typify cardiac tissue seem completely obliterated. Additionally there was substantial parenchymal thinning of liver and distortion of glomerulus
(pointed). Animals treated with DPNP and BPNP however conserved cardiac architecture but there were foci of tissue breakdown. Mild portal
congestion and tubular infiltration was also visible. (n = 3; * p < 0.05, ** p < 0.01, *** p < 0.001, n.s. not significant).

catalase activation (1.92 ± 0.29 μmol/mL/min/mg). SOD levels
in treated mice varied as DOX solution (42.13 ± 4.18 U/mg
cardiac tissue) > DPNP (32.89 ± 2.10 U/mg cardiac tissue) >
BPNP (29.11 ± 2.23 U/mg cardiac tissue) > PBS (21.65 ±
1.89 U/mg cardiac tissue). MDA levels also followed a similar
trend: DOX solution (3.82 ± 0.10 nM/mg cardiac tissue) > DPNP
(3.45 ± 0.2 nM/mg cardiac tissue) > BPNP (3.01 ± 0.23 nM/mg
cardiac tissue) > PBS (2.2 ± 0.05 nM/mg cardiac tissue).
2763
DOX is infamous for its cardiotoxic potential. Several studies
have shown that microsome present in organs, including heart,
convert DOX into highly reactive semiquinone intermediate
which facilitates generation of superoxide anion (O2− and
H2O2−) and causes lipid peroxidation and cell damage.39 There-
fore, lower levels of above-described pathological markers
[catalase, superoxide dismutase (SOD), lipid peroxidation
(MDA)] in heart of BPNP treated animals in comparison to
DOI: 10.1021/acs.molpharmaceut.7b00310
Mol. Pharmaceutics 2017, 14, 2749−2765
Molecular Pharmaceutics
DOX solution and DPNP are indicative of relative safety of
BPNP. Histological analysis was conducted to map toxic
implication of treatments in vital organs (liver, kidney, and
heart) using an optical microscope. It was seen in photomicro-
graphs that DOX solution produced areas of severe parenchymal
damage, obliteration of myofibrils, and infiltration of inflam-
matory cells in cardiac tissue. There was also gross hepatic
parenchymal degeneration and dilatation of intertubular sinus-
oidal spaces and glomerular degeneration. Contrarily BPNP and
DPNP produced only mild interfibrillar thinning and moderate
portal vein congestion along with traces of intertubular infiltra-
tion. Tissue sections of kidney, heart, and liver displayed regular
architecture when animals were treated with PBS. In light of
toxicity results it would be appropriate to assume that BPNP
is safer than DOX solution and DPNP in terms of cardio-
compatibility and overall effect on morphology of vital organs.
BPNP’s altered biodistribution and control exerted in drug
release by polymeric vehicle might be responsible for this
favorable safety profile when compared to drug alone.
■
CONCLUSIONS
BPNP displayed substantial merit in enhancing anticancer
activity of DOX while simultaneously mitigating its toxic
potential due to altered spatial and temporal presentation of
drug. Since the nanoparticle template possesses capability to
undergo click reactions, we assume that in future it can be
decorated with a variety of counter click ligands which in turn will
allow clickable drug loaded template to be theoretically used for
targeting different cancer cells expressing different receptors. We
have already applied the principle of common ion effect to tailor
entrapment of another hydrophilic drug diethyl cabamazine
citrate in conventional PLGA nanoparticles and believe this
technique is ripe for others to exploit. In future endeavors we
wish to come up with a one-step fabrication technique, where in,
an optimally synthesized and highly drug-loaded nanoparticle
template is decorated with a ligand in situ via an easy to carry out
click reaction after all the pharmaceutical characterization tools
have been applied.
■
ASSOCIATED CONTENT
*
S Supporting Information
The Supporting Information is available free of charge on the ACS
Publications website at DOI: 10.1021/acs.molpharmaceut.7b00310.
HPLC method, LC-MS method, FTIR spectra with inter-
pretation, NMR spectra, macrophage evasion study via
flow cytometry, differential uptake studies of BPNP in
4T1 cells, effect of BPNP on apoptotic and antiapoptotic
proteins via Western blotting, induction of mitochondrial
membrane potential disturbance (qualitative and quanti-
tative) and tables detailing pharmacokinetic parameters
(PDF)
■ AUTHOR INFORMATION
Corresponding Author
*Telephone: +91 522-2772450, 2772550; Fax: +91 522
2623405; E-mail: manish_chourasia@cdri.res.in.
ORCID
Manish K. Chourasia: 0000-0002-8318-9325
Present Address
#
Faculty of Pharmaceutical Sciences, University of British
Columbia, Vancouver, British Columbia V6T 1Z3, Canada.
2764
Article
Notes
The authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
We are thankful to DBT grant BT/PR14769/NNT/28/996/
2015 for providing financial assistance in smooth conduction of
this study. We appreciate facilitation of flowcytometry experi-
ments by Dr. A.L. Vishwakarma and Mrs. Madhu who are
members of SAIF Division, CDRI. We are also thankful to
Dr. Chauhan from CSIR-IITR, Lucknow, for providing TEM
facility. CSIR CDRI Communication 9507.
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