pubs.acs.

org/Biomac Article

Amalgamated Microneedle Array Bearing Ribociclib-Loaded

Transfersomes Eradicates Breast Cancer via CD44 Targeting
Madhu Sharma, Naresh Mittapelly, Venkatesh Teja Banala, Sandeep Urandur, Shalini Gautam,
Disha Marwaha, Nikhil Rai, Neha Singh, Ashutosh Gupta, Kalyan Mitra, and Prabhat Ranjan Mishra*
Cite This: https://doi.org/10.1021/acs.biomac.1c01076 Read Online

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ABSTRACT: HR+/HER2− metastatic breast cancer (MBC) is

one of the most common and life-threatening conditions
diagnosed in women. The endocrine therapy using an orally active
CDK4/6 inhibitor, ribociclib (RB), is the most intriguing approach
for treating HR+/HER2− MBC. However, the repeated three to
six cycles of multiple dosing and non-targeted distribution of RB
led to severe neutropenia; hepatobiliary, gastrointestinal, and renal
toxicities, and QT interval prolongation. Here, a novel organic
solvent-free HA−PVA−PVP (hyaluronic acid−polyvinyl alcohol−
polyvinyl pyrrolidone) composed of a microneedle (MN) array is
formulated to deliver RB, integrated with amphiphilic conjugated
polymer (HA−GMS)-anchored ultradeformable transfersomes.
This unique MN array efficiently crafts microchannels in the
skin, allowing HA-RB-Ts to internalize into the tumor cells through lymphatic and systemic absorption and interact with CD44 both
spatially and temporally with an amplification of drug release time up to 6-folds. The pharmacokinetic and tissue distribution studies
portray drug concentrations within the therapeutic window as long as 48 h, facilitating thrice-a-week frequency with the lower dose,
and rule out severe toxicities, with a significant reduction in 8.3-fold RB concentration in vital organs that ultimately enhances the
survival rate. Thus, the novel MN system pursues a unique embeddable feature and offers an effective, self-administrable,
biodegradable, and chronic treatment option for patients requiring long-term cancer treatments.

1. INTRODUCTION Biopharmaceutical Classification System (BCS) class IV

Breast cancer accounts for approximately 25% of all cancer
cases, which leads to most cancer-related deaths in women
worldwide.1,2 Approximately one-third of patients progress to
the most dangerous and life-threatening stage IV metastatic
breast cancer (MBC).3,4 Among all MBC patients, 73% were
diagnosed as hormone receptor positive (HR+) and human
epidermal growth factor 2 negative with a median overall
survival of approximately 3 years.5 Endocrine therapy using
aromatase inhibitors (AIs), selective estrogen receptor (ER)
modulators, or selective ER downregulators is the key
therapeutic option for HR+/HER2− MBC6,7 but available
with major limitations such as acquired resistance. 8,9
Palbociclib, ribociclib (RB), and abemaciclib are three cyclin-
dependent kinase 4/6 inhibitors (CDK4/6i) approved by the
Food Drug Administration (FDA) to overcome resistance and
reinduce the antiproliferative effect of endocrine therapy.10−13
RB (KISQALI; LEE011), a small molecule, an orally
bioavailable, and selective CDK4/6i, was approved, in
combination with an AI in premenopausal/perimenopausal
or post-menopausal women as an initial endocrine therapy or
with fulvestrant in post-menopausal women as a first- or
second-line treatment for HR+ HER2− MBC.14,15 RB is a
molecule and requires a multiple dose regimen, that is, a
recommended oral dose of 200 mg three times a day.16 The
multiple administration of RB is reported to cause severe
hepatobiliary and renal toxicities, neutropenia, and QT interval
prolongation.17,18 Therefore, to overcome the limitations
associated with oral administration of RB, the development
of alternate delivery systems for non-oral administration is
highly desirable.
Over the past few decades, the MN-based drug delivery
system has attracted the scientific community for application in
several areas.19,20 The uniqueness of the microneedle (MN)
system enjoys the benefits of both hypodermic needles and the
topical transdermal patch.21,22 The MN-assisted drug delivery
avoids first-pass hepatic metabolism and gastrointestinal
degradation while facilitates the rapid onset of action with
Received: August 18, 2021
Revised: November 25, 2021

© XXXX American Chemical Society https://doi.org/10.1021/acs.biomac.1c01076

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Scheme 1. HA-RB-Ts-MNs for Antitumor Spatial−Temporal Release of RB with Enhanced Therapeutic Efficacya

a
First, RB amalgamated into HA ligated amphiphilic transfersomes (HA-RB-Ts) and then incorporated into MN arrays to form HA-RB-Ts-MNs.
The developed biodegradable MN array sustains RB release up to 48 h by dissolving the needles in situ, followed by replenishment through the
backing layer, maintaining the RB level within the therapeutic window, equivalent to multiple oral doses. Afterward, the backing layer of MNs
continuously releases RB in a sustainable manner. Subsequently, targeted transfersomes are further pivoted for ligand−receptor interactions, with
RB internalization, enhancing tumor cell killing (95%) and reducing the toxicity in vital organs of the body.

better patient compliance and minimal adverse effects.23,24 In
market aspects, the application of MNs for drug delivery is
restricted to a few small molecules and vaccines only. The
major hindrance in the translation of anticancer treatment by
MNs is the cost of treatment and dosing frequency. The
dissolvable MNs are intended for rapid or controlled release of
the drug incorporated within the MNs. The release profile
observed with the dissolvable MNs is for 3−4 h, which results
in multiple dosing, thus dose missing, poor compliance, and
overall therapy. In some cases, the MNs cost more, leading to
an extra burden on patients. In some conventional MNs, the
amount of drug that can be mounted is limited, due to which a
sub-therapeutic response is achieved. Further, the MNs deliver
the drugs in systemic circulation but lack targeting to tumor
mass. When MNs are integrated with drug-loaded trans-
fersomes, it provides an efficient strategy to achieve spatial and
temporal delivery in dermal tissues.25−27 Recently, trans-
fersome/MN complex (T/MNs) has been successfully used in
transdermal lymphatic delivery of doxorubicin for tumor
metastasis therapy.28,29 Importantly, tumor targeting can be
enhanced by coating transfersomes with hyaluronic acid (HA)
due to its role in regulating the circulation time and targeting
ability being a major ligand for the cluster of differentiation 44
(CD44) receptors present on cancerous cells.30,31 Despite
significant scientific production in this subject, no dissolvable
MN system has been formulated yet with a targeted long-term
release of chemotherapeutics against cancer.
In this study, we have developed a novel HA−PVA−PVP
(HA−polyvinyl alcohol−polyvinyl pyrrolidone)-amalgamated
biodegradable polymeric dissolvable MN array (HA-RB-Ts-
MNs) system comprising RB-loaded ultradeformable trans-
fersomes (RB-Ts), subsequently anchored with self-assembling
amphiphilic conjugated HA−GMS (HA-RB-Ts) for spatial−
temporal targeting to breast cancer.
We hypothesized that novel HA−PVA−PVP micron-sized
MNs, rendering an amplification of release time up to 6-folds
more than the conventional MN arrays, would initially create
microchannels in the breast skin, allowing RB-loaded trans-
fersomes to get easily transported into the transdermal region
of the breast skin. Now, HA would interact with the CD44
receptor present on the tumor cells to facilitate the entry of
RB-loaded transfersomes inside the target cell. We further
anticipated that transfersomes would release RB inside the
cancer cells in a sustained manner through the backing layer,
and therefore, it would prevent systemic exposure and
associated adverse effects with enhanced therapy. The selected
MNs clearly demonstrated sustained release with a single patch
application on rat skin for 60 h, owing to their polymeric
composition, which distinguishes them from the others, as well
as their mechanical ability to disrupt the skin and complete
insertion, resulting in therapeutic concentrations lasting up to
3 days. This unique combination of ultradeformable trans-
fersomes with novel MNs is likely to provide a viable treatment
option for breast cancer with improved efficacy.
HA-RB-Ts-MNs were prepared and evaluated in vitro/in vivo
to enhance the therapeutic efficacy of RB in spatial−temporal
manner against breast cancer (Scheme 1).
2. MATERIALS AND METHODS
2.1. Materials. All essential chemicals for the study were bought
from Sigma-Aldrich (St Louis, MO, USA) and are represented as
(product-catalogue no.) sodium hyaluronate (HA) (SO780000),
lecithin soya (P3556), sodium deoxycholate (D6750), PVA (341584),
PVP (PVP40), ethyl-3-(3-dimethyl aminopropyl)carbodiimide
(EDC) (341006), N-hydroxysuccinimide (NHS, 99%) (8.51056),
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)
(M2128), propidium iodide (PI) (P4170), and anti-CD44 antibody
(SAB5600074). For cell culture studies, all reagents such as the
Annexin-V apoptosis kit and all other analytical grade chemical
reagents were obtained from commercial sources. All LCMS- and
HPLC-grade solvents were purchased from Merck (India). The Milli-
Q system (Millipore, Bedford, USA) was used to prepare triple
distilled water. RB was purchased in the form of its succinate salt from
Cistron Biotech, and its purity was confirmed by 1H NMR
spectroscopy before use (Figure S1). 1H NMR data were recorded

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Table 1. Morphological and Physicochemical Characteristics of Transfersomesa
a
All values are represented as mean ± SD.
using a Bruker AV 400 MHz in D2O using the residual H2O signal as
the internal standard.
2.2. Methods. 2.2.1. HA-RB-Ts Synthesis and Characterization.
An acidic group of HA esterified with the terminal hydroxy group of
glyceryl monostearate (GMS), ensuing in the conjugation of HA and
GMS (Scheme S1). HA (molecular weight 40 kDa) was esterified
with GMS by using ethyl-3-(3-dimethyl aminopropyl)carbodiimide
(EDC) as a coupling agent in a round-bottom flask containing a
magnetic bead. Initially, the glucuronic acid residue of HA (100 mg)
was activated by reacting with 95.5 mg of EDC (0.5 mol) and 57.5 mg
of NHS (0.5 mol) in 10 mL of phosphate buffer saline (PBS) at pH
4.7, and then the reaction mixture was kept for 2 h to achieve 1:1 ratio
to the glucuronic acid residue of HA. Consecutively, 179.25 mg of
GMS (0.5 mol) was dissolved separately in 5 mL of warm acetone,
followed by dropwise addition of HA to the reaction mixture over a
period of 5 min, and the reaction mixture was continuously stirred for
48 h at 25 °C. Subsequently, the reaction mixture was centrifuged at
18,000 rpm for 30 min at 4 °C until the solution becomes clear. The
resultant mixture was dialyzed using a spectra/pore membrane with
an MW cutoff of 8000 for 3 days, followed by lyophilization for 48
h.32
RB-Ts and HA-RB-Ts were prepared by using the thin-film
hydration method, followed by membrane extrusion. To prepare RB-
Ts, 35 mg of soya phospholipid (Soya PC) and 20 mg of sodium
deoxycholate were solubilized in a mixture of chloroform and
methanol (8:1), respectively. Subsequently, the solvent was
evaporated under vacuum at 40 °C for 30 min using a rotavapor to
obtain thin films. Further, to ensure the removal of trace amounts of
organic solvents, the thin films were kept in a desiccator connected
with a vacuum line for 2 h. The films were hydrated with PBS
containing HA−GMS (5 mg) and RB (2 mg) at a temperature of 55
°C for 30 min. A homogeneous population of dispersion was achieved
by membrane extrusion through a 200 nm polyvinylidene fluoride
membrane (HI media). The size and morphology of developed
transfersomes were confirmed by transmission electron microscopy
(TEM) and atomic force microscopy (AFM). A different formulation
of transfersomes (F-01 to F-07) was prepared and optimized (Table
S1a,b). Similarly, RB-containing transfersomes (RB-Ts) were
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prepared by dissolving RB in PBS and added into the thin film of
lipids (soya phospholipid and sodium deoxycholate), and drug
entrapment and drug loading were calculated.26
The size of transfersomes was determined by the dynamic light
scattering method using a Nano Zs 2000 (Nano ZS, Malvern
instrument, UK), and the data was processed by Zetasizer software
version 7.03.33
The entrapment efficiency (EE) of RB in transfersomes was
calculated using a Centrisart. Free RB was filtered to the recovery
chamber after the centrifugation of aqueous solution of HA-RB-Ts in
the outer chamber. The concentration of RB in both chambers was
determined by RP-HPLC.34 To study the in vitro release profile RB-
Ts, and RB, the formulations, with a concentration equivalent to 2 mg
RB, were incorporated in dialysis bags (Hi media, MWCO: 12 K Da).
These dialysis bags were then enclosed and placed in 200 mL of
dissolution medium at physiological pH 7 with continuous stirring at
100 ± 5 rpm and 37 ± 1 °C temperature maintained throughout the
experiment. Aliquots were gathered at foreordained time points (0,
0.5, 1, 2, 4, 8, 12, 24, and 48 h) and supplanted with an identical
volume of the preheated medium at 37 ± 1 °C for maintaining sink
condition. The collected samples were assessed using the prevalidated
HPLC/PDA method.35,36 The storage stability of HA-RB-Ts was
determined at 25 °C for 90 days. HA-RB-Ts were kept at respective
storage conditions, and samples were withdrawn at predefined (0, 15,
30, 60, and 90 days) time intervals for the evaluation of particle size,
surface zeta potential, PDI, and visual appearance (Figure 1I). The
serum stability of HA-RB-Ts was also monitored in 10% FBS (fetal
bovine serum) after 12 and 24 h.37
2.2.2. Fabrication and Characterization of HA-RB-Ts-MNs.
Fabrication of the MN array patch was carried out via the
micromolding method as shown in Figure S2. A MN mold of 1 cm
× 0.5 cm size arranged in a 16 × 13 array with 600 μm spacing
between tips was used for MN fabrication. A single MN of this mold
was 500 μm in length with 300 μm base diameters and approximately
5 μm with tip diameter. HA (8% w/v), PVA (2% w/v), and PVP (6%
w/v) polymer were dissolved in 1 mL solution of HA-RB-Ts (833.3
μg/mL). Then, the solution was kept at a magnetic stirrer for 25 min
at 4 °C, followed by centrifugation at 12,000 rpm to remove the air
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Table 2. Selected Formulations for the Fabrication of RB-Loaded HA-RB-Ts-MNs
from the mixture. The resulted solution was poured onto a reusable
PDMS mold and kept under vacuum for 15 min and then 1 h at
atmospheric pressure to improve the filling of micropores. This
procedure was repeated until the predefined amount of HA-coated
transfersomes gets loaded in the MN mold. Different formulations
were prepared and optimized (F-05-A1 to F-05-A6) on the basis of
entrapment efficiency and size. After post-casting, a preformed
sustained release gel thin-film backing layer was immediately placed
on top of the MN array. Complete filling of the MN mold was carried
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out by using a 2.5 mL HA-RB-Ts fabrication solution. A single MN
was filled with a 0.0120 mL molding mixture. After completely drying
by desiccation, the MN mold was kept at room temperature for 4−5
h. Adhesive tape (2 cm × 2 cm) was used to extract the MN array
from the MN mold. The MN array was carefully shoring up with the
help of adhesive tape. The morphological evaluation was carried out
by using a Leica EZ4D digital light microscope (Leica Microsystems,
Milton Keynes, UK) and a scanning electron microscope. A mixture
of PVA (10%), PVP (10%), gelatin (5%), and glutaraldehyde solution
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(0.4% v/v) was used to form a sustained release backing layer. A 1
cm2 mold was used to prepare the backing layer.38
For scanning electron microscopy (SEM) analysis, the sample was
put on dual-side sticky tape fixed with aluminum stub and covered
with gold-palladium utilizing a sputter coater (Polaron). Then, at that
point, the MN was examined under an FEI Quanta 250 SEM at 10 kV
utilizing an SE detector.39
HA-RB-Ts-MNs were further evaluated using stereomicroscopy.
Dried drug-loaded MNs were placed on a slide for inspection under a
Leica EZ4D digital light microscope (Figure S3C).40,41
2.2.3. Skin Penetration Study. The permeability of HA-RB-T-
loaded MNs was investigated by utilizing Franz diffusion cell
assembly. A section of the abdominal skin of rat (350 μm thick)
was excised and mounted between donor and acceptor chambers of
the diffusion cell. The permeation profile of RB through the skin was
quantified by comparing HA-RB-Ts-MNs with free drug (RB) (A),
RB patch (B), and RB-Ts-MNs (C). Different groups (equivalent to 2
mg of RB) were incorporated within the layer of the skin section by
applying manual force for 15 s with a flat surface. The acceptor
chamber was filled with a dissolution medium and constantly stirred
at 100 ± 5 rpm at 37 ± 1 °C temperature. At predefined time
intervals (0, 0.5, 1, 2, 4, 8, 12, and 24 h), aliquots were collected
through the sample arm of the Franz cell, with simultaneous
maintenance of sink condition with a replenished equal volume of
PBS. The collected aliquots were centrifuged and filtered with a 220
nm pore size filter membrane. The drug concentration of the collected
sample was analyzed by RP-HPLC.42
The ability of RB-Ts-MNs to disrupt skin was confirmed by the
Parafilm M insertion model.43 The Texture Analyzer Parafilm M test
was performed at 35 N/array for 15 s using eight-layer folded Parafilm
M. When the MN array was completely inserted, the MN array was
removed from the set of Parafilm layers. After the unfolding of
polymeric layers, the number of pores present in each layer was
assessed by utilizing the digital microscope. The in vitro insertion
depth of MNs was determined based on the percentage of cavities
created in each Parafilm.
Ex vivo skin penetration study was performed using confocal
fluorescent microscopy. Confocal laser scanning microscopy (CLSM)
was conducted to visualize the release of fluorescent molecules from
the MNs into the rat skin and their spatial distribution. The freshly
excised rat skin was kept in PBS solution for 15 min, and
subsequently, the MN array was pressed softly over the rat skin for
15 s. The MN array became static on the skin with a manual pressure
of 15 s and completely inserted in the skin. Three skin samples were
taken and fixed in 4% formaldehyde solution after a time interval of 5
min, 24, and 48 h for confocal studies to check the ability of MNs to
disturb the skin layer. The confocal imaging study was performed to
confirm visually the ability of MNs to disrupt the skin layers.44
Optical coherence tomography (OCT) of HA-RB-Ts-MNs was
carried out to assess the mechanical strength and dissolution rate of
MNs in the skin. The swept source Fourier domain OCT system has a
laser center wavelength of 1305.0 ± 15.0 nm, facilitating real-time
high-resolution imaging of the upper skin layers (7.5 μm lateral and
10.0 μm vertical resolution). The skin was scanned at a frame rate of
up to 15 B-scans (2D cross-sectional scans) per second (scan width =
2.0 mm). OCT images were taken at fixed time intervals over a period
of 24 and 48 h experiment.45
2.2.4. Cell Culture Studies. MDA-MB-231 and LA-7 cell lines were
cultured in Dulbecco’s modified Eagle’s medium (DMEM) at 37 °C
in a humidified chamber with 5% CO2. The cells were trypsinized
with 0.01% trypsin to detach the cells from the culture flask.
2.2.4.1. Cell Cytotoxicity Studies. The cell cytotoxicity study of
free RB, RB-Ts, and HA-RB-Ts was assessed using the MTT assay in
MDA-MB 231 and LA-7 cell lines. The respective cell lines were
seeded into 96-well plates (3 × 103 cells/well) in DMEM (100 μL)
with FBS (10%) at 37 °C in the presence of 5% CO2. After reaching
70% confluency of cells, 96-well plates were treated with free RB, RB-
Ts, and HA-RB-Ts (containing an equal amount of RB) in triplicate
at a 1−60 μM concentration range. Two batches of cells were
incubated at 37 °C, the first batch for 24 h and the second batch for
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48 h. Then, the complete medium was removed, and 20 μL of MTT
(5 mg/mL) dye was added to each well. Furthermore, 4 h incubation
resulted in dark purple-colored formazan crystals, and then 100 μL of
DMSO was added to each well to dissolve these crystals. Finally, the
percentage of viable cells was determined by an automated microplate
reader by measuring the absorbance at 570 nm, which is directly
proportional to the number of viable cells. By comparing control cells
(untreated cells) with treated cells, the percentage of viable cells was
calculated and indicates as a means with standard deviation (SD)
(Figure S5). The cytotoxicity and biosafety of HA-RB-Ts and RB
were also evaluated by treating them with J774.2 cells.46
2.2.4.2. Cell Cycle Analysis. RB arrests the cell cycle in the G1
phase by inhibiting the phosphorylation of the retinoblastoma protein
(Rb). MDA-MB 231 cells (1 × 106 cells/well) were seeded into a six-
well plate at 37 °C in 5% CO2 to perform the cell cycle assay based on
PI to assess the cell arrest phase of RB by using a flow cytometer (BD
Biosciences, FACS Calibur, Germany). MDA-MB 231 cells were
treated with free RB, RB-T, and HA-RB-T formulations. After a
specific period of time, chilled ethanol (70%) was utilized to fix the
cells for approximately 2 h. Subsequent centrifugation and
resuspension in the 500 μL PBS (pH 7.4) were followed by
ribonuclease treatments (100 μL) and PI (50 μL/mL) into the dark
for 30 min.47
2.2.4.3. Apoptosis. Metastatic cells (MDA-MB 231) (n = 3) were
cultured in six-well plates (1 × 106/well) for a set time period,
followed by treatment with the inhibitory concentration of RB. After
24 h, the cells were washed with PBS and centrifuged. Subsequently,
the pellet was resuspended by 1× binding buffer (500 μL). Then,
Annexin-FITC (5 μL) and PI (10 μL) were used to stain the cells in
the dark condition for 25 min. Early and late apoptosis was estimated
by utilizing BD CellQuest Pro software, version 3.2.48
2.2.4.4. Mitochondrial Membrane Potential. 5,5-Tetra ethyl
benzimidazolyl carbocyanine iodide (JC-1 dye) (Molecular Probes,
Eugene, Ore) was used to quantify the MMP in MDA-MB 231 cells.
Primarily, cells were incubated in six-well plates (1 × 105cells/well)
and then treated separately with free RB, blank transfersomes, RB-T,
and HA-RB-T formulations, whereas no treatment was given to the
control group. The cells were washed with PBS twice, followed by
treatment with the JC-1 solution (15 μg/mL) (Figure 3C,C1). For
negative control, PBS-treated cells were used. MMP was determined
by measuring the fluorescence using a flow cytometer.49
2.2.4.5. Reactive Oxygen Species Generation. Reactive oxygen
species (ROS) study was conducted with the dichloro-dihydro-
fluorescein diacetate (DCFH-DA) assay. Initially, MDA-MB 231 cells
were cultured in six-well plates at a density of 2 × 105 cells/well and
treated with free RB, RB-T, and HA-RB-T formulations. Sub-
sequently, the cells were treated with DCFH-DA (10 μL) after 12 and
24 h of incubation. After 0.5 h of DCFH-DA treatment, PBS was used
to wash the cells twice, trypsinized, and again resuspended in 500 μL
of PBS. Last, after measuring the fluorescence intensity of DCF with
the aid of the flow cytometer, the ROS production level was
determined (Figure 3D,D1). Untreated cells were used as a control.
ROS study was repeated in triplicate (n = 3).50
2.2.4.6. CLSM Study. To prepare the fluorescent transfersomes, the
post-loading of the drug was carried out with FITC (1:10 ratio of
FITC/Rb) in transfersomes. The FITC-tagged transfersomes were
incubated with MDA-MB 231 cells on poly-lysine-coated sterile
coverslips for 1, 6, and 12 h and then fixed with 10% formaldehyde.
The excess formaldehyde was removed by PBS buffer washing, and
the cells were stained with DAPI. Cellular uptake study was
performed in LA-7 cell lines by treating with RB-TS and HA-RB-Ts
for 12 h. The cellular uptake was analyzed by using a confocal
microscope LEICA TCS SP-8.
CLSM and flow cytometry examination were used for the
determination of expression of CD44 on breast cancer-specific
human MDA-MB 231 and SD rat LA-7 cell lines by treatment with
the free drug and preformed formulations. After set time interval (24
h) incubation, treated cells were washed with the sterile phosphate
buffer, and 1% formaldehyde solution was used to fix the treated cells.
Then, antimouse/human CD44 monoclonal antibody-FITC tagged
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Figure 1. Characterization of HA−GMS conjugates by (A) 1H NMR and (B) FTIR. Morphological characterization of transfersomes by (C) TEM
(scale bar 200 nm) and (D) AFM after dilution and (E) height profile of the AFM image. (F) Geometrical and morphological characterization of
F-05-A4 MN arrays by SEM at high magnification (200×) and low magnification (100×). (G) Percentage of MN height reduction of all selected
formulations tested with a force of 35 N/arrays. (H) Evaluation of percentage of holes created in each Parafilm M layer after the insertion of
selected dissolving MNs. (I) Profiling of physical stability of transfersomes at physiological pH at room temperature for 90 days. (J) Data
representing the serum stability at 12 and 24 h in FBS, mean ± SD (n = 3). (K) Comparative drug release profile of free RB, RB-Ts, HA-RB-Ts, RB
Patch, RB-Ts-MNs, and HA-RB-Ts-MNs.

(Elabscience) was used to stain the cells, and a confocal microscope
(Zeiss LSM510 META, Carl Zeiss, Germany) was used to observe the
fluorescence emitted by cells. For the evaluation of the fluorescence
intensity, ImageJ software was used. Similarly, in six-well plates, again
both cell lines were seeded, and free RB and different predefined
formulations were used for treatment. After defined time intervals, the
cells were washed with PBS and stained with 10 μL of FITC
antihuman CD44 antibody in 500 μL PBS. After 15 min of
incubation, a flow cytometer was used to evaluate the expression of
CD44 expression.51
2.2.5. In Vivo Studies. 2.2.5.1. Tumor Induction in Female SD
Rats. Adult Sprague-Dawley female rats of 4 weeks (weight 50−60 g)
were used to develop an LA-7 syngeneic mammary tumor model by
inoculation of LA-7 cells. The animals used were accommodated for a
week before the experiment under light and dark conditions for 12 h.
The female SD rats were inoculated by LA-7 (6 × 105) cells onto the
mammary fat pad and regularly observed. During the study, the tumor
volume was estimated each week using the formula V = (W2 × L)/2,
where W and L indicate the short and long diameters of the tumor
tissue. After 10 days of cell implantation, animals were divided into
four groups (n = 6) constituting tumors with a volume of
approximately 500 ± 100 mm.3,52
2.2.5.3. Tumor Regression Analyses. The animals were divided
into five groups, and each group comprises four animals. Initially, the
animals were observed daily until the tumor volume reached 500 ±
100 mm3. Then, the designed formulation was given through
transdermal and oral routes. The oral formulation was given three
times a day, whereas the transdermal formulations were applied on
the skin after an interval of 3 days. The control group was treated with
phosphate saline through the oral route. The tumor growth was
measured at an interval of 4 days with the help of a Vernier caliper,
and the tumor volume was calculated using the formula mentioned in
the tumor induction study. The animal body weight was also
measured to check the overall toxicity. Finally, the animals were
sacrificed after 28 days, and the tumor weight was calculated.53
2.2.5.2. Pharmacokinetic Study. A pharmacokinetic study was
accomplished in female SD rats having tumors ∼500 mm3. The rats
were split into five groups, with five sub-groups (n = 5 for each sub-
group). RB was given orally, while RB-Ts backing layer, RB-Ts-MNs,
and HA-RB-Ts-MNs were administrated transdermally at a dose of 50
mg/kg. The blood samples (200 μL) were collected under the mild
anesthetic condition through oculi choroideae and subsequently
centrifuged at 2000g for 8 min to separate the plasma. The liquid−
liquid extraction method was used for plasma sample processing and
analyzed by an LC−MS/MS method to evaluate pharmacokinetic
parameters (Table S2).21
2.2.5.4. Survival Analysis. For survival analysis, the female SD rats
were divided into free RB, RB patch, RB-Ts-MNs, and HA-RB-Ts-
MN groups. The tumor was developed by the inoculation of LA-7 (6
× 105) cell lines. The animals were regularly observed until the
desired volume of tumor developed. Once the tumors reached the
volume at approximately 500 mm3, the animals were treated with the

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Biomacromolecules
Figure 2. Real-time monitored OCT images of F-05-A4, showing dissolution kinetics after 5 min, 24 h, and 48 h (complete dissolution). (A) RB-
Ts-MNs and (B) HA-RB-Ts-MNs. (C) Confocal images after insertion of HA-RB-Ts-MNs in rat skin, showing insertion depth and dissolution
kinetics at predefined time intervals (scale bar 100 μm).
same procedure as defined in the tumor regression study. The survival
study was carried out for up to 45 days. Finally, the dignified survival
rate was evaluated with the use of the GraphPad Prism 5.0 software.54
2.2.5.5. Histopathology Study. After the tumor regression study,
the rats were sacrificed, and the tumor parts along with major organs
such as heart, liver, spleen, lungs, and kidney were collected. The
collected organs and the tumor were separately fixed in 10%
paraformaldehyde (PBS buffer) for 12 h. The tissues of these organs
and the tumor were kept in wax blocks and subsequently inserted into
the melted wax. Further, the wax block sectioning was carried out by
using a microtome (Leica) to obtain 5 μm thick sections. Finally,
these sections were exposed to hematoxylin and eosin staining in the
appropriate organs for visualizing histological changes.55
2.3. Statistical Analysis. Statistical significance of the data was
examined by utilizing t-test (non-parametric), one-way ANOVA, and
two-way ANOVA with probability <0.001 or <0.05 as significant. All
the factual computation was performed utilizing the GraphPad Prism
version 8.0 for Windows (GraphPad Software, San Diego, CA, USA).
3. RESULTS AND DISCUSSION
The results of all the evaluated parameters are discussed in
detail in the following section.
3.1. HA-RB-Ts Synthesis and Characterization. HA was
coupled to hydrophobic GMS (glycerol monostearate)
through EDC-assisted esterification using a 1:1 ratio of each
HA and GMS at pH 4.5 with a 90% yield. The conjugation and
degree of substitution (DS) were confirmed through a 1H
NMR spectrum that shows a signal at 1.0 ppm (terminal
methyl of the alkyl chain of GMS) with 23% of DS calculated
as a ratio of integration of signal for methyl of the acetylamino
group at 2.0 ppm and terminal methyl of the alkyl chain at
around 1.0 ppm (Figure 1A). The synthesized HA−GMS
conjugate was further characterized by FTIR spectroscopy and
found that in the FT-IR spectrum, a characteristic IR signal of
HA gets sharper after covalent attachment of GMS (Figure
1B).56
HA-RB-T formulation was prepared using phospholipids
and sodium deoxycholate through the thin-film hydration
method. Upon addition of RB and amphiphilic HA in the
medium, RB sneaks inside the transfersomes, whereas
amphiphilic HA gets oriented on the surface on hydration.
pubs.acs.org/Biomac Article
HA-RB-Ts were subjected to surface charge computation in
order to depict the electrokinetic potential (Table 1). HA-RB-
Ts (F-05) were found to be highly negatively charged (−34.6
± 5.72 mV) with respect to RB-Ts and blank-Ts. This negative
charge is due to HA and helps in the stabilization of particles
by electrostatic interaction.
The average hydrodynamic diameter of HA-RB-Ts was
found at 81.81 ± 7.47 nm. The particle size was also similarly
congruent obtained with TEM (Figure 1C) and AFM
dimensions (Figures 1D,E and S3A). This particle size is
suitable for amassing in the tumor cells due to enhanced
permeation and retention effect, and HA present on the surface
facilitates receptor-mediated targeting.36,57 The drug loading
and entrapment efficiency were found at 9.5 and 98%,
respectively. Subsequently, the free RB-Ts and RB were
subjected to dissolution study using the dialysis bag technique.
The results showed a sustained profile exhibition by the
transfersomes as compared to free RB (Figure S3E). The HA-
RB-Ts storage stability was examined at 25 °C for 90 days. The
serum stability of HA-RB-Ts in 10% FBS (fetal bovine serum)
was also assessed after 12 and 24 h. No discernible change in
the characteristics measured was observed (Figure 1J).
3.2. Fabrication and Characterization of HA-RB-Ts-
MNs MNs. Among different combinations, PVP and PVA
combined with HA were found to be most promising in
developing an MN array matrix with a 60 h dissolution profile,
resulting in mechanically strong and skin-compatible arrays
(Table 2).
The MN matrix solution was uniformly mixed and blended
with HA-RB-Ts to get finally HA-RB-Ts-MNs. The morphol-
ogy of HA-RB-Ts-MN arrays was confirmed through SEM
imaging of the HA-RB-Ts-MN (Figure 1F) photograph,
stereomicroscopy, and SEM image (RB-Ts-loaded MNs)
(Figure S3B−D). The mechanical strength of HA-RB-Ts
MNs was determined using a texture analyzer at 35 N/array to
ensure its ability that could facilitate insertion into the skin
without force failure. The original height of the MNs was
reduced to 10, 6, 2, 7, and 4% with insertion of F-05-A1, F-05-
A2, F-05-A3, F-05-A5, and F-05-A6 formulations, respectively
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Figure 3. Cell cycle arrest, apoptosis, mitochondrial membrane potential, and reactive oxygen study in MDA-MB 231 cells. (A,A1) Percentage of
cells in the G1 phase significantly increased in all the three groups after 48 h, HA-RB-Ts (96.34%) showed much higher cell cycle arrest in
comparison to free RB (50.05%) and RB-Ts (76.65%). (B,B1) Quantitative analysis of apoptosis optimizing MDA-MB 231 cells treated with
formulations containing RB. (C,C1) Comparative MMP graph. HA-RB-Ts showing the highest depolarization of mitochondrial membrane after 24
h in MDA-MB-231 cell lines. (D,D1) Graph showing comparative ROS generation after 24 h in MDA-MB-231 cell lines. HA-RB-Ts show the
highest ROS generation. (E) Cytotoxicity of blank-Ts and blank HA-Ts in (J774.2) cells for 48 h.

(Figure 1G). F-05-A4 showed (3%) a significant reduction in
the height as compared to other formulations. The ability of
skin disruption and in vitro insertion depth of MNs were
confirmed by analyzing the percentage of holes created in 126
± 7 μm average thicknesses of a Para films M (Figure 1H).
The MNs were inserted up to 348 μm of the total 500 μm
height, which is approximately 69.6% of the tip height of MNs.
The percentage of total MN length inserted was nearly the
same as reported previously, which is about 60% insertion
(35). The comparative release profile of RB from F-05-A1, F-
05-A2, F-05-A3, F-05-A4, F-05-A5, and F-05-A6 was found to
be 50.40 ± 1.39, 70.47 ± 1.9, 70.53 ± 2.303, 94.76 ± 2.34,
62.11 ± 2.18, and 50.43 ± 1.26%, respectively, after 48 h
(Figure S3F). F-05-A4 showed higher and sustained
permeability for more than 48 h as compared to other
formulations with a maximum release of the drug from the
backing layer, which may be due to the ability of skin
disruption and complete insertion depth combined with the
swelling property of HA, resulting in the increased diffusional
flux of RB-Ts through the skin in a controlled manner, and
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therefore responsible for delivering a therapeutic concentration
of the RB with a single patch for 3 days without dissipating of
RB present in the backing layer.
The RB release from the RB solution, RB patch, RB-Ts, HA-
RB-Ts, RB-Ts-MNs, and HA-RB-Ts-MNs was found to be 24
± 1.4, 13 ± 1.9, 32.98 ± 2.30, 23.662 ± 3.18, 48.21, and 91 ±
2.4%, respectively, after 48 h (Figure 1K). HA-RB-Ts-MNs
showed higher permeability as compared to other formula-
tions, which may be due to the increased diffusional flux of HA
through the skin, and therefore responsible for delivering a
relatively higher amount of the drug into the cavernous layer of
skin.
3.3. Skin Penetration Study. The F-05-A4 formulation
was selected for ex vivo permeation studies to investigate the
insertion depth of the MN array. However, the formulations F-
05-A1, F-05-A2, F-05-A3, F-05-A5, and F-05-A6 were dropped
for further study as these formulations remained adhered in
Parafilm M after the insertion test (Figure S4). The selected
MN array was inserted into the rat skin with the manual
pressure, and the skin was imaged with the help of confocal
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Figure 4. CELL uptake study of FITC-tagged HA-RB-Ts, RB-Ts, and RB was examined using CLSM and flow cytometry. (A,A1) Flow cytometry
in MDA-MB-231 cell lines at 4 and 12 h. Quantitative analysis of cell uptake by CLSM in MDA-MB231 and (B,B1) quantitative analysis of cell
uptake by CLSM LA-7 (C,C1) cell lines.
microscopy, which shows 341.87 μm (approx. 68.37%)
insertion depth (Figure 2C). This in vitro insertion study is
consistent with the results obtained from the ex vivo study.
Interestingly, the mechanical strength of the formulation was
duly maintained when stored at 19 °C for more than 3 weeks.
OCT was used to evaluate the mechanical strength and
dissolution rate of HA-RB-Ts-MN and RB-Ts-MN formula-
tions in the skin (Figure 2A,B). The OCT images showed that
the MN array of F-05-A4 effectively pierced the rat skin with
precise insertion strength, and MNs start dissolving within 5
min and get completely dissolved after 48 h.
3.4. Cell Culture Studies. Cell cytotoxicity was
determined through the MTT assay on MDA-MB-231 and
LA-7 cell lines as these show good expression of CD44
receptors. We performed cellular studies to demonstrate and
verify the specificity and efficacy of this novel CD44-specific
targeting system and investigated the underlying tumor cell
killing frequency. CD44 can promote uncontrolled growth,
evasion of apoptosis, angiogenesis, cell motility, and invasion,
which are hallmarks of cancer progression. CD44 receptors are
expressed more than 30% in MDA-MB 231 cell lines as
compared to the other 13 breast cancer cell lines.58,59 The loss
of cell viability was measured in a dose-dependent manner in
both cell lines, and the IC50 value of HA-RB-Ts was observed
to be 2.5- and 1.67-fold less than that of free RB and RB-Ts,
respectively, after 24 h of treatment with MDA-MB 231 cell
lines (Figure S5A,A1). A parallel scenario was also observed in
pubs.acs.org/Biomac Article
LA-7 cell lines, although it was more sensitive toward RB, and
the half-inhibitory concentration of HA-RB-Ts was found to be
4.7- and 2.7-fold lower than that of free RB and RB-Ts,
respectively (Figure S5B,B1). HA-RB-Ts showed low cytotox-
icity and higher therapeutic effect as compared to RB and RB-
Ts. Further, the J774.2 cell lines were used to assess the
biosafety of blank transfersomes (Figure 3E) and found to be
safe for normal cells, and therefore, HA-RB-Ts show antibreast
cancer activity selectively.
The effect of free drug, RB-Ts, and HA-RB-Ts as compared
to placebo was tested in MDA-MB 231 cell lines (Figure
3A,A1). There was 96% of cell cycle arrest when treated with
HA-RB-Ts, which is significantly higher as compared to RB-TS
(76%) and RB (50%) after 48 h. These studies demonstrate
that our designed HA-RB-Ts significantly reduced cell
proliferation with enhanced selectivity in comparison to free
drugs.
The quantitative analysis of apoptosis in MDA-MB 231 cells
was revealed to be ≈75% of the apoptotic effect by HA-RB-Ts
compared to 61 and 55% by RB-Ts and free RB after 24 h of
treatment, respectively (Figure 3B,B1). The HA-RB-Ts
significantly showed enhanced apoptosis in contrast to free
RB and RB-Ts owing to the selective targeting ability of HA on
CD44 receptors. Since HA has the ability to bind the CD44
receptors on tumor cells, HA-functionalized RB-Ts elevated
the concentration of RB in tumor cells, resulting in increased
apoptosis. The mitochondria-dependent potential further
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Biomacromolecules pubs.acs.org/Biomac Article

Figure 5. Downregulation of CD44 expression evaluated by flow cytometry after 24 h in (A,A1) LA-7 and (B,B1) MDA-MB-231 cell lines after
saturation with HA-RB-Ts as compared to control, RB, and RB-Ts (n = 3). (C,C1) Digital images of MDA-MB-231 cell lines showing the
downregulation of CD44 expression after treatment with HA-RB-Ts-MNs in comparison with control, RB and RB-Ts in 24 h (scale bar 75 μm).
Statistical analysis with significance: ****p ≤ 0.001, ***p ≤ 0.01, and *p ≤ 0.05.

confirms the level of apoptosis, and it was found to be 14.77,
36, and 57.98% for free RB, RB-Ts, and HA-RB-Ts,
respectively (Figure 3C,C1).
The intracellular-generated ROS species were quantitatively
detected by the DCFH-DA assay. Considerably, the high mean
fluorescence intensity (97%) of the HA-RB-T-treated cell
group indicates an enhanced ROS level production, leading to
higher apoptosis, as compared to free RB (48%) and RB-Ts
(67%) after 24 h (Figure 3D,D1).
The intracellular trafficking of FITC-tagged HA-RB-Ts and
RB-Ts was examined in MDA-MB-231 cell lines with the aid of
CLSM and FACs. FITC-tagged HA-RB-Ts showed a higher
quantum of fluorescence in the cytoplasm as compared to
FITC-RB-Ts after 4 and 12 h of treatment in the CLSM study.
The presence of HA at the surface of HA-RB-Ts makes a
significant difference in the fluorescence intensities of the
cytoplasm and the nucleus, which indicates receptor-mediated
uptake in MDA-MB cells (Figure 4B,B1). On the other hand,
RB-Ts did not participate in receptor-mediated uptake in
MDA-MB 231 cells due to the lack of HA at the periphery and
therefore showed low fluorescence intensities. Flow cytometry
was also used for further evaluation of intercellular trafficking
of HA-RB-T and RB-T formulations (Figure 4A,A1). The
cellular uptake study was performed in LA-7 cell lines by
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treatment with RB-TS and HA-RB-Ts for 12 h via CLSM
(Figure 4C,C1). CD44 downregulation was assessed by flow
cytometry in the MDA-MB 231 cell lines resulted from RB-T
and HA-RB-T treatment. Control cells show the highest CD44
expression, that is, 96.2%, while RB-, RB-T-, HA-RB-T-treated
cells show 50.07, 42.60, and 29.21%, respectively (Figure
5B,B1). These results clearly indicate that the ability of CD44
expression was significantly reduced after treatment with HA-
RB-Ts as compared to free drug and RB-Ts. The CLSM study
also confirmed the downregulation of CD44 expression with
an immunofluorescence staining technique. These results
strongly support the flow cytometry statistics with a high
signal of CD44, as observed in control cells; however, there
was a significant decrease in the CD44 expression in HA-RB-
Ts in contrast to RB-Ts and RB (Figure 5C,C1). This data
clearly demonstrates the role of HA in enhancing the
intracellular trafficking of HA-RB-Ts mediated by the CD44
ligand−receptor interaction. The LA-7 cell lines were also used
to study CD44 downregulation (Figure 5A,A1). To our
delight, HA-RB-Ts also showed significantly less mean
fluorescence intensity as compared to RB-Ts and showed the
significance of HA in the intracellular spatial distribution of
transfersomes.
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Figure 6. (A) Photography image of an SD rat at 0 and 72 h after insertion, (B) tumor weights, (C) morphology of tumors harvested at the end of
the study, and (D) pharmacokinetic parameter of free RB, RB patch, RB-Ts-MNs, and HA-RB-Ts-MNs in the serum at different time intervals.
Each formulation contains 50 mg/kg (RB) equivalent dose (n = 5). In vivo antitumor activity against LA-7 cell-transplanted mammary tumor model
in female SD rats (RB dose 50 mg/kg body weight). (E) Tumor volume vs time graph and (F) body weight changes during the study period. (G)
Survival rates of LA-7 tumor-bearing SD rats after treatment with free RB, RB patch, RB-Ts-MNs, and HA-RB-Ts-MNs as compared to control (n
= 4). In survival studies, the “day 0” represents the day when the tumor volume reached ∼500 mm3. After transdermal application, the effect of RB,
RB patch, RB-Ts-MNs, and HA-RB-Ts-MNs on the biodistribution at different time intervals on body tissues was evaluated. These graphs
represent the (H) liver, (I) heart, (J) kidney, and (K) tumor. (****p < 0.001 represents control vs HA-RB-Ts-MNs.).

3.5. In Vivo Studies. 3.5.1. Tumor Inhibition Study and
Tumor Regression Analyses. The in vivo antitumor efficacy of
HA-RB-Ts-MNs was tested, and the comparative data of
tumor volume versus time curve and tumor photographs are
shown in Figure 6C,E, respectively. The tumor inhibition rate
was significantly higher in the HA-RB-Ts-MN patch as
compared to other tested formulations. The calculated tumor
volume for the HA-RB-Ts-MN patch group was 22.00-, 7.50-,
14.56-, and 4.41-fold lower than control, free RB solution, RB
patch, and RB-Ts-MNs, respectively (p < 0.0001) (Figure 6E),
and the tumor weight of the HA-RB-Ts-MN patch group was
13.50-, 7.08-, 7.15-, and 5.41-fold lower as compared to that of
free RB solution, RB patch, and RB-Ts-MNs (p < 0.0001)
(Figure 6B). Altogether, these data clearly demonstrate that
the HA-RB-Ts-MN group showed significantly higher
antitumor efficacy compared to free RB solution, RB patch,
and RB-Ts-MNs. This enhanced antitumor efficacy of the HA-
RB-Ts-MN patch group may be attributed due to the
combination of three basic mechanisms, that is, improved
pharmacokinetics and biodistribution, ligand−receptor-medi-
ated targeted delivery, and spatial−temporal release of RB.
The assessment of RB deposition on body tissues was
carried out at different time intervals. Free RB and RB-Ts-MN
K https://doi.org/10.1021/acs.biomac.1c01076
patches were rapidly distributed in the kidney and heart in the
early stage, which gradually decreased with time. In contrast,
HA-RB-Ts-MNs and RB patches render a lower deposition of
RB in the kidney (Figure 6J) and heart (Figure 6I). The first-
pass hepatic metabolism is avoided by the developed
formulation, and therefore, the hepatobiliary toxicity of RB is
ruled out (Figure 6H). Consequently, a lower amount of RB
from HA-RB-Ts-MNs as compared to other formulations was
deposited in the cardiac tissues and other drug toxicity-
susceptible organs.
The HA-RB-Ts-MN patch group exhibited a maximum drug
concentration at the tumor site as compared to the free RB, RB
patch, and RB-Ts-MNs. These results indicate that HA renders
the ligand receptor-mediated active targeting and prolonged
blood circulation time. After 48 h of treatment, the HA-RB-Ts-
MN patch and RB-Ts-MNs showed 6- and 3.8-fold higher drug
concentration in contrast to the free drug, while the RB patch
results 1.8-fold lower concentration than free RB (Figure 6K).
3.5.2. Survival Analysis. The survival rate of the HA-RB-Ts-
MN-treated group was significantly increased as compared to
other groups (Figure 6G). The median survival time of rats
treated with saline, free RB, RB patch, RB-Ts-MNs, and HA-
RB-Ts-MNs was 23, 32, 26, 36, and >45 days, respectively. As
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Figure 7. Histologic assessments of major organs isolated from rats after tumor regression study using H&E stain (magnification 20×; scale bar 100
μm).
compared to control, the free RB-treated group increased the
survival by 48%, while in the case of transdermally applied HA-
RB-Ts-MNs, the survival rate gets increased by 96%, probably
due to its lower toxicity and high efficacy. There is no
significant change in the body weight of rats during the study
(Figure 6F).
3.5.3. Pharmacokinetics and Biodistribution Studies. The
pharmacokinetics and biodistribution studies were performed
on SD female rats. After acclimatization for 10 days, SD female
rats were shaved, and transdermal patch loaded with free RB
solution, RB patch, RB-Ts-MNs, and HA-RB-Ts-MNs
containing 50 mg/kg RB equivalent were applied the next
day. A sustained release was observed in the plasma over a long
period of time, even after reaching C-max. This data provides
evidence that RB continues to release from the MN patch and
cleared from the animal’s body simultaneously. RB-Ts loaded
in MN tips achieved an efficient drug level in a short period of
time similar to IV, and the backing layer (patch) maintained a
sustained release profile of RB through the pore of the MN.
Therefore, MNs facilitate sustained release as observed by
AUC obtained after MN application. The profile of HA-RB-
Ts-MN formulation over a period of 48 h shows a higher
distribution of RB in the serum as compared to vital organs.
The area under the curve of the HA-RB-Ts-MN patch was
found to be 5.93, 8.67, and 6.85 times higher with respect to
pure RB, RB patch, and RB-Ts-MNs, respectively, indicating a
higher amount of RB diffused in the serum from the HA-RB-
Ts-MN patch (Figure 6D). Further, the mean retention time of
the HA-RB-Ts-MN patch was also found to be 1.6-, 1.4-, and
2.8-fold higher as compared to RB-Ts-MNs, RB patch, and
pure RB, respectively (Table S2). These results clearly indicate
that HA-conjugated transfersomes promote the RB delivery
with the longer residence of the drug at the tumor site by
enhancing the penetration and receptor-mediated transport.
pubs.acs.org/Biomac Article
3.5.4. Histopathological Study. In histopathological anal-
ysis, the control group shows a well-defined anatomy with the
regular shape of cells with spherical and spindle-shaped large
blue nucleus; however, in the case of RB and RB-formulation-
treated groups, the cells showed condensed nucleus and
shrunken anatomy. The HA-RB-Ts-MN- and RB-Ts-MN-
treated groups showed significantly higher apoptosis in tumor
histopathological section in contrast to RB due to the direct
delivery of RB at the tumor site. Hepatobiliary toxicity is a
major concern with oral administration of RB, whereas
transdermal application of RB formulations, bypass first-pass
hepatic metabolism. Major apoptotic hepatocytes and
cardiomyocytes were observed in the liver and heart section,
respectively, upon RB treatment. This is clear evidence that
transdermal application of RB formulation significantly reduces
the toxicity in vital organs as compared to the oral
administration of the drug. Histological examination of the
kidney, lung, and spleen revealed significantly no major
morphological differences in the HA-RB-Ts-MN- and RB-Ts-
MN-treated groups compared to the control group, although
minor disruption of organs was found in the free RB-treated
group (Figure 7). These results are consistent with the
biodistribution study, where free RB was found to be more
amassed in vital organs in comparison to transdermally applied
formulations.
4. CONCLUSIONS
In the present study, the developed novel MN system was
exploited to deliver RB disguised in the form of HA-coated
transfersomes. This unique concoction enjoys two-tier benefits
in having both intravenous and transdermal approaches to
deliver RB both spatially and temporally to tumor cells via
CD44 interaction. This system rules out any hepatobiliary
toxicity and significantly reduces the toxicity in vital organs.
L https://doi.org/10.1021/acs.biomac.1c01076
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Biomacromolecules
We have developed and optimized different HA-RB-Ts-MN
formulations to achieve enhanced sustainability and skin
disruption ability with improved efficacy. The outcomes
established a novel sustained and biodegradable dissolvable
MN (HA-RB-Ts-MNs) system with receptor-mediated target-
ing to CD44 receptors, which helps in regulating cell
progression and invasion of tumor cells. Importantly, in vitro
and in vivo studies prove that HA-RB-Ts-MNs inhibited tumor
growth significantly in tumor-bearing SD rats with improved
therapy. The hepatobiliary and cardiac toxicities, which are
associated with the oral delivery of RB, get reduced
prominently by HA-RB-Ts-MN formulation. A single-patch
application maintains sustained therapeutic concentrations
lasting up to 60 h owing to amalgamated polymeric
composition, rendering reduction in dosing frequency from
thrice a day to thrice a week. This novel delivery system not
only enhances the therapeutic efficacy but also avoids the
multiple dosage regimens with an improved survival rate.
These outcomes suggest that HA-RB-Ts-MNs are a potential
tool for spatial and temporal targeting of RB and can deliver
other chemotherapeutic drugs in an efficient way that can be a
lifesaving aid for outpatients.
■ ASSOCIATED CONTENT
* Supporting Information
sı
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.biomac.1c01076.
Details of experimental materials and methods; RB
structure standardization; synthesis of the HA−GMS
conjugate: composition of formulations, particle size,
and zeta potential of transfersomes; drug loading and
entrapment efficiency (EE %); fabrication and character-
ization of HA-RB-Ts-MN; in vivo studies; 1H-NMR
spectra, schematic representation of HA-RB-Ts-MNs,
morphological characterization of transfersome, micro-
scopic images of the parafilm, and comparative growth
inhibition of MDA-MB-231; and quantitative composi-
tion and pharmacokinetic parameters for LC-MS/MS
analysis (PDF)
■
AUTHOR INFORMATION
Corresponding Author
Prabhat Ranjan Mishra − Division of Pharmaceutics and
Pharmacokinetics, CSIR−Central Drug Research Institute,
Lucknow 226031 Uttar Pradesh, India; orcid.org/0000-
0002-7418-8283; Phone: 91-522-2772450 (4537);
Email: prabhat_mishra@cdri.res.in; Fax: 91-522-2771941
Authors
Madhu Sharma − Division of Pharmaceutics and
Pharmacokinetics, CSIR−Central Drug Research Institute,
Lucknow 226031 Uttar Pradesh, India; Academy of Scientific
and Innovative Research (AcSIR), Ghaziabad 201002, India
Naresh Mittapelly − Division of Pharmaceutics and
Pharmacokinetics, CSIR−Central Drug Research Institute,
Lucknow 226031 Uttar Pradesh, India
Venkatesh Teja Banala − Division of Pharmaceutics and
Pharmacokinetics, CSIR−Central Drug Research Institute,
Lucknow 226031 Uttar Pradesh, India
Sandeep Urandur − Division of Pharmaceutics and
Pharmacokinetics, CSIR−Central Drug Research Institute,
pubs.acs.org/Biomac Article
Lucknow 226031 Uttar Pradesh, India; orcid.org/0000-
0001-5803-077X
Shalini Gautam − Division of Pharmaceutics and
Pharmacokinetics, CSIR−Central Drug Research Institute,
Lucknow 226031 Uttar Pradesh, India
Disha Marwaha − Division of Pharmaceutics and
Pharmacokinetics, CSIR−Central Drug Research Institute,
Lucknow 226031 Uttar Pradesh, India
Nikhil Rai − Division of Pharmaceutics and Pharmacokinetics,
CSIR−Central Drug Research Institute, Lucknow 226031
Uttar Pradesh, India
Neha Singh − Division of Pharmaceutics and
Pharmacokinetics, CSIR−Central Drug Research Institute,
Lucknow 226031 Uttar Pradesh, India
Ashutosh Gupta − Division of Pharmaceutics and
Pharmacokinetics, CSIR−Central Drug Research Institute,
Lucknow 226031 Uttar Pradesh, India
Kalyan Mitra − Electron Microscopy Division, CSIR−Central
Drug Research Institute, Lucknow 226031 Uttar Pradesh,
India
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.biomac.1c01076
Notes
The authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
The authors would like to express their deepest gratitude to
CSIR, New Delhi, and AcSIR, Ghaziabad, for providing the
research fellowship and the CSIR-CDRI for the research
facilities and the necessary resources. They would also like to
thank Dr. Himanshu Kathuria of NUSMETIC Pvt. Ltd. for
providing MN molds as a gift sample. They also thank A.L.
Vishwakarma and Madhu Chaturvedi for their assistance in
flow cytometry studies and Garima Pant to contribute to SEM
facilities, Electron Microscopy Division, CSIR-CDRI. They
would like to thank Rima Ray Sarkar’s contribution to CLSM,
Molecular & Structural Biology Division, CSIR-CDRI. The
authors would also like to appreciate Sushil Mishra’s
contribution to OCT, Prakash Netra Kendra, Lucknow. The
CDRI communication number for this manuscript is 10326.
■ REFERENCES
(1) Bray, F.; Ferlay, J.; Soerjomataram, I.; Siegel, R. L.; Torre, L. A.;
Jemal, A. Global Cancer Statistics 2018 : GLOBOCAN Estimates of
Incidence and Mortality Worldwide for 36 Cancers in 185 Countries.
Cancer J. Clin. 2018, 68, 394−424.
(2) Pernas, S.; Tolaney, S. M.; Winer, E. P.; Goel, S. CDK4/6
Inhibition in Breast Cancer : Current Practice and Future Directions.
Ther. Adv. Med. Oncol. 2018, 10, 175883591878645.
(3) Wang, R.; Zhu, Y.; Liu, X.; Liao, X.; He, J.; Niu, L. The
Clinicopathological Features and Survival Outcomes of Patients with
Different Metastatic Sites in Stage IV Breast Cancer. BMC Cancer
2019, 19, 1091.
(4) American Cancer Society. Breast Cancer Facts & Figures 2019−
2020; Atlanta Am. Cancer Soc. Inc., 2019, 2020.
(5) Goel, S.; DeCristo, M. J.; McAllister, S. S.; Zhao, J. J. CDK4/6
Inhibition in Cancer: Beyond Cell Cycle Arrest. Trends Cell Biol.
2018, 28, 911−925.
(6) Cardoso, F.; Senkus, E.; Costa, A.; Papadopoulos, E.; Aapro, M.;
André, F.; Harbeck, N.; Aguilar Lopez, B.; Barrios, C. H.; Bergh, J.;
Biganzoli, L.; Boers-Doets, C. B.; Cardoso, M. J.; Carey, L. A.; Cortés,
J.; Curigliano, G.; Diéras, V.; El Saghir, N. S.; Eniu, A.; Fallowfield, L.;
Francis, P. A.; Gelmon, K.; Johnston, S. R. D.; Kaufman, B.; Koppikar,
M https://doi.org/10.1021/acs.biomac.1c01076
Biomacromolecules XXXX, XXX, XXX−XXX
Biomacromolecules
S.; Krop, I. E.; Mayer, M.; Nakigudde, G.; Offersen, B. V.; Ohno, S.;
Pagani, O.; Paluch-Shimon, S.; Penault-Llorca, F.; Prat, A.; Rugo, H.
S.; Sledge, G. W.; Spence, D.; Thomssen, C.; Vorobiof, D. A.; Xu, B.;
Norton, L.; Winer, E. P. 4th ESO − ESMO International Consensus
Guidelines for Advanced Breast Cancer (ABC 4). Ann. Oncol. 2018,
29, 1634−1657.
(7) Giuliano, M.; Schiff, R.; Osborne, C. K.; Trivedi, M. V.
Biological Mechanisms and Clinical Implications of Endocrine
Resistance in Breast Cancer. Breast 2011, 20, S42−S49.
(8) Johnston, S. R. D. New Strategies in Estrogen Receptor −
Positive Breast Cancer. Clin. Cancer Res. 2010, 16, 1979−1987.
(9) Sutherland, R. L.; Sergio, C. M.; Mcneil, C. M.; Anderson, L. R.;
Inman, C. K.; Butt, A. J.; Musgrove, E. A. Estrogens, Cell Proliferation
and Breast Cancer. Hormonal Control of Cell Cycle, Research and
Perspectives in Endocrine Interactions; Springer: Berlin, Heidelberg,
2008; pp 123−138.
(10) Eggersmann, T. K.; Degenhardt, T.; Gluz, O.; Wuerstlein, R.;
Harbeck, N. CDK4/6 Inhibitors Expand the Therapeutic Options in
Breast Cancer: Palbociclib, Ribociclib and Abemaciclib. BioDrugs
2019, 33, 125−135.
(11) Thangavel, C.; Dean, J. L.; Ertel, A.; Knudsen, K. E.; Aldaz, C.
M.; Witkiewicz, A. K.; Clarke, R.; Knudsen, E. S. Therapeutically
Activating RB : Reestablishing Cell Cycle Control in Endocrine
Therapy-Resistant Breast Cancer. Endocr. Relat. Cancer 2011, 18,
333−345.
(12) OLeary, B.; Finn, R. S.; Turner, N. C. Treating Cancer with
Selective CDK4/6 Inhibitors. Nat. Publ. Gr. 2016, 13, 417−430.
(13) Polk, A.; Kolmos, I. L.; Kümler, I.; Nielsen, D. L. Specific
CDK4/6 Inhibition in Breast Cancer: A Systematic Review of Current
Clinical Evidence. ESMO Open 2016, 1, No. e000093.
(14) Burris, H. A., III Expert Review of Anticancer Therapy
Ribociclib for the Treatment of Hormone Receptor- Positive , Human
Epidermal Growth Factor Receptor 2-Negative Advanced Breast
Cancer Growth Factor Receptor 2-Negative Advanced Breast Cancer.
Expert Rev. Anticancer Ther. 2018, 18, 201−213.
(15) Sobhani, N.; D’Angelo, A.; Pittacolo, M.; Roviello, G.; Miccoli,
A.; Corona, S. P.; Bernocchi, O.; Generali, D.; Otto, T. Combinations
in Breast Cancer. Cells 2019, 8, 1−24.
(16) Papich, M. G.; Martinez, M. N. Applying Biopharmaceutical
Classification System (BCS) Criteria to Predict Oral Absorption of
Drugs in Dogs: Challenges and Pitfalls. AAPS J. 2015, 17, 948−964.
(17) Laderian, B.; Fojo, T. Seminars in Oncology CDK4 / 6
Inhibition as a Therapeutic Strategy in Breast Cancer : Palbociclib ,
Ribociclib , and Abemaciclib. Semin. Oncol. 2017, 44, 395−403.
(18) Vasiliki, T. C.; Aristeidis, G. T.; Christos, A. K.; Timotheadou,
E. T. The Pharmacological Profile of Cyclin-Dependent Kinase
(CDK) 4/6 Inhibitors: Clinical Management of Toxicity and Drug
Interactions Related to CDK 4/6 Inhibitor-Based Treatment in
Advanced/Metastatic Breast Cancer. Forum Clin. Oncol. 2019, 10, 2−
14.
(19) Kommareddy, S.; Baudner, B. C.; Oh, S.; Kwon, S.-y.; Singh,
M.; O’hagan, D. T. Dissolvable Microneedle Patches for the Delivery
of Cell-Culture-Derived Influenza Vaccine Antigens. J. Pharm. Sci. Res.
2012, 101, 1021−1027.
(20) Kim, Y.-C.; Park, J.-H.; Prausnitz, M. R. Microneedles for Drug
and Vaccine Delivery. Adv. Drug Deliv. Rev. 2012, 64, 1547−1568.
(21) He, X.; Sun, J.; Zhuang, J.; Xu, H.; Liu, Y. Microneedle System
for Transdermal Drug and Vaccine Delivery: Devices, Safety, and
Prospects. Dose-Response 2019, 17, 155932581987858.
(22) Vicente-Perez, E. M.; Larrañeta, E.; McCrudden, M. T. C.;
Kissenpfennig, A.; Hegarty, S.; McCarthy, H. O.; Donnelly, R. F.
Repeat Application of Microneedles Does Not Alter Skin Appearance
or Barrier Function and Causes No Measurable Disturbance of Serum
Biomarkers of Infection, Inflammation or Immunity in Mice in Vivo.
Eur. J. Pharm. Biopharm. 2017, 117, 400−407.
(23) Larrañeta, E.; Lutton, R. E. M.; Woolfson, A. D.; Donnelly, R.
F. Microneedle Arrays as Transdermal and Intradermal Drug Delivery
Systems : Materials Science , Manufacture and Commercial
Development. Mater. Sci. Eng., R 2016, 104, 1−32.
pubs.acs.org/Biomac Article
(24) Courtenay, A. J.; Mccrudden, M. T. C.; Mcavoy, K. J.;
Mccarthy, H. O.; Donnelly, R. F. Microneedle-Mediated Transdermal
Delivery of Bevacizumab. Mol. Pharm. 2018, 15, 3545−3556.
(25) Lee, J. W.; Park, J.; Prausnitz, M. R. Dissolving Microneedles
for Transdermal Drug Delivery Jeong. Biomaterials 2009, 29, 2113−
2124.
(26) Jiang, T.; Wang, T.; Li, T.; Ma, Y.; Shen, S.; He, B.; Mo, R.
Enhanced Transdermal Drug Delivery by Transfersome-Embedded
Oligopeptide Hydrogel for Topical Chemotherapy of Melanoma. ACS
Nano 2018, 12, 9693−9701.
(27) Dudhipala, N.; Mohammed, R. P.; Adel, A.; Youssef, A. Effect
of Lipid and Edge Activator Concentration on Development of
Aceclofenac-Loaded Transfersomes Gel for Transdermal Application :
In Vitro and Ex Vivo Skin Permeation. Drug Dev. Ind. Pharm. 2020,
46, 1334.
(28) Rajan, R.; Vasudevan, D.; Biju Mukund, V.; Jose, S.
Transferosomes − A Vesicular Transdermal Delivery System for
Enhanced Drug Permeation. J. Adv. Pharm. Technol. Res. 2011, 2,
138−144.
(29) Waghule, T.; Singhvi, G.; Dubey, S. K.; Pandey, M. M.; Gupta,
G.; Singh, M.; Dua, K. Microneedles: A Smart Approach and
Increasing Potential for Transdermal Drug Delivery System. Biomed.
Pharmacother. 2019, 109, 1249−1258.
(30) Yang, X. Y.; Li, Y. X.; Li, M.; Zhang, L.; Feng, L. X.; Zhang, N.
Hyaluronic Acid-Coated Nanostructured Lipid Carriers for Targeting
Paclitaxel to Cancer. Cancer Lett. 2013, 334, 338.
(31) Yang, J.; Liu, X.; Fu, Y.; Song, Y. Recent Advances of
Microneedles for Biomedical Applications: Drug Delivery and
Beyond. Acta Pharm. Sin. B 2019, 9, 469−483.
(32) Kong, M.; Chen, X.; Park, H. Design and Investigation of
Nanoemulsified Carrier Based on Amphiphile-Modified Hyaluronic
Acid. Carbohydr. Polym. 2011, 83, 462−469.
(33) Duangjit, S.; Opanasopit, P.; Rojanarata, T.; Ngawhirunpat, T.
Characterization and In Vitro Skin Permeation of Meloxicam-Loaded
Liposomes versus Transfersomes. J. Drug Deliv. 2011, 2011, 1−9.
(34) Wu, P. S.; Li, Y. S.; Kuo, Y. C.; Tsai, S. J.; Lin, C. C.
Preparation and Evaluation of Novel Transfersomes Combined with
the Natural Antioxidant Resveratrol. Molecules 2019, 24, 1−12.
(35) Marwah, H.; Garg, T.; Rath, G.; Goyal, A. K. Development of
Transferosomal Gel for Trans-Dermal Delivery of Insulin Using
Iodine Complex. Drug Deliv. 2016, 23, 1636−1644.
(36) González-vázquez, P.; Larrañeta, E.; Mccrudden, M. T. C.;
Jarrahian, C.; Rein-weston, A.; Quintanar-solares, M.; Zehrung, D.;
Mccarthy, H.; Courtenay, A. J.; Donnelly, R. F. Transdermal Delivery
of Gentamicin Using Dissolving Microneedle Arrays for Potential
Treatment of Neonatal Sepsis. J. Controlled Release 2017, 265, 30−40.
(37) Kim, N. W.; Kim, S.-Y.; Lee, J. E.; Yin, Y.; Lee, J. H.; Lim, S. Y.;
Kim, E. S.; Duong, H. T. T.; Kim, H. K.; Kim, S.; Kim, J.-E.; Lee, D.
S.; Kim, J.; Lee, M. S.; Lim, Y. T.; Jeong, J. H. Enhanced Cancer
Vaccination by In Situ Nanomicelle-Generating Dissolving Micro-
needles. ACS Nano 2018, 12, 9702−9713.
(38) An, M.; Liu, H. Dissolving Microneedle Arrays for Transdermal
Delivery of Amphiphilic Vaccines. Small 2017, 13, 1700164.
(39) Moreira, A. F.; Rodrigues, C. F.; Jacinto, T. A.; Miguel, S. P.;
Costa, E. C.; Correia, I. J. Poly (Vinyl Alcohol)/Chitosan Layer-by-
Layer Microneedles for Cancer Chemo- Photothermal Therapy. Int. J.
Pharm. 2020, 576, 118907.
(40) Larrañeta, E.; Stewart, S.; Fallows, S. J.; Birkhäuer, L. L.;
Mccrudden, M. T. C.; Woolfson, A. D.; Donnelly, R. F. A Facile
System to Evaluate in Vitro Drug Release from Dissolving
Microneedle Arrays A Facile System to Evaluate in Vitro Drug
Release from Dissolving Microneedle Arrays. Int. J. Pharm. 2016, 497,
62−69.
(41) Alkilani, A.; Mccrudden, M. T.; Donnelly, R. Transdermal Drug
Delivery: Innovative Pharmaceutical Developments Based on
Disruption of the Barrier Properties of the Stratum Corneum.
Pharmaceutics 2015, 7, 438−470.
(42) Donnelly, R. F.; Morrow, D. I. J.; Mccarron, P. A.; Woolfson, A.
D.; Morrissey, A.; Juzenas, P.; Juzeniene, A.; Iani, V.; Mccarthy, H. O.;
N https://doi.org/10.1021/acs.biomac.1c01076
Biomacromolecules XXXX, XXX, XXX−XXX
Biomacromolecules pubs.acs.org/Biomac Article

Moan, J. Microneedle-Mediated Intradermal Delivery of 5-Amino- Proliferation and Invasive Potential and Activating the Immune
levulinic Acid : Potential for Enhanced Topical Photodynamic Response in Mammary Cancer Xenografts. BMC Cancer 2019, 19,
Therapy. J. Controlled Release 2008, 129, 154−162. 261.
(43) Larrañeta, E.; Moore, J.; Vicente-pérez, E. M.; González- (59) Zucchi, I.; Astigiano, S.; Bertalot, G.; Sanzone, S.; Cocola, C.;
vázquez, P.; Lutton, R.; Woolfson, A. D.; Donnelly, R. F. A Proposed Pelucchi, P.; Bertoli, G.; Stehling, M.; Barbieri, O.; Albertini, A.;
Model Membrane and Test Method for Microneedle Insertion Scholer, H. R.; Neel, B. G.; Reinbold, R. A.; Dulbecco, R. Distinct
Studies. Int. J. Pharm. 2014, 472, 65−73. Populations of Tumor-Initiating Cells Derived from a Tumor
(44) Coulman, S. A.; Birchall, J. C.; Alex, A.; Pearton, M.; Hofer, B.; Generated by Rat Mammary Cancer Stem Cells. Proc. Natl. Acad.
O’Mahony, C.; Drexler, W.; Považay, B. In Vivo, in Situ Imaging of Sci. U.S.A. 2008, 105, 16940−16945.
Microneedle Insertion into the Skin of Human Volunteers Using
Optical Coherence Tomography. Pharm. Res. 2011, 28, 66−81.
(45) Verma, A.; Sharma, S.; Gupta, P. K.; Singh, A.; Teja, B. V.;
Dwivedi, P.; Gupta, G. K.; Trivedi, R.; Mishra, P. R. Vitamin B12
Functionalized Layer by Layer Calcium Phosphate Nanoparticles: A
Mucoadhesive and PH Responsive Carrier for Improved Oral
Delivery of Insulin. Acta Biomater. 2016, 31, 288−300.
(46) Li, T.; Xiong, Y.; Wang, Q.; Chen, F.; Zeng, Y.; Yu, X.; Wang,
Y.; Zhou, F.; Zhou, Y. Ribociclib (LEE011) Suppresses Cell
Proliferation and Induces Apoptosis of MDA-MB-231 by Inhibiting
CDK4/6-Cyclin D-Rb-E2F Pathway. Artif. Cells, Nanomed., Biotechnol.
2019, 47, 4001−4011.
(47) Mittapelly, N.; Thalla, M.; Pandey, G.; Banala, V. T.; Sharma,
S.; Arya, A.; Mishra, S.; Mitra, K.; Shukla, S.; Mishra, P. R. Long
Acting Ionically Paired Embonate Based Nanocrystals of Donepezil
for the Treatment of Alzheimer ’ s Disease : A Proof of Concept
Study. Pharm. Res. 2017, 34, 2322−2335.
(48) Urandur, S.; Banala, V. T.; Shukla, R. P.; Mittapelly, N.;
Pandey, G.; Kalleti, N.; Mitra, K.; Rath, S. K.; Trivedi, R.; Ramarao,
P.; Mishra, P. R. Anisamide-Anchored Lyotropic Nano-Liquid
Crystalline Particles with AIE E Ff Ect : A Smart Optical Beacon
for Tumor Imaging and Therapy. ACS Appl. Mater. Interfaces 2018,
10, 12960−12974.
(49) Sharma, S.; Verma, A.; Mittapelly, N.; Mishra, P. R.
Investigating the Role of Pluronic-g-Cationic Polyelectrolyte as
Functional Stabilizer for Nanocrystals: Impact on Paclitaxel Oral
Bioavailability and Tumor Growth. Acta Biomater. 2015, 26, 169.
(50) Pandey, G.; Mittapelly, N.; Banala, V. T.; Mishra, P. R.
Multifunctional Glycoconjugate Assisted Nanocrystalline Drug
Delivery for Tumor Targeting and Permeabilization of Lysosomal-
Mitochondrial Membrane. ACS Appl. Mater. Interfaces 2018, 10,
16964−16976.
(51) Moore, B. P.; Hayden, T. J.; Forsyth, I. A. Mammary-Tumour
Incidence in Sprague-Dawley Rats Treated with 7,12-Dimethylbenz-
(a)Anthracene: Effect of Pregnancy and Lack of Effect of Unilateral
Lactation. Br. J. Cancer 1981, 44, 451−455.
(52) Oyenihi, O. R.; Krygsman, A.; Verhoog, N.; de Beer, D.;
Saayman, M. J.; Mouton, T. M.; Louw, A. Chemoprevention of LA7-
Induced Mammary Tumor Growth by SM6Met, a Well-Characterized
Cyclopia Extract. Front. Pharmacol. 2018, 9, 650.
(53) Tham, H. P.; Xu, K.; Lim, W. Q.; Chen, H.; Zheng, M.; Thng,
T. G. S.; Venkatraman, S. S.; Xu, C.; Zhao, Y.; Zhao, Y. Microneedle-
Assisted Topical Delivery of Photodynamically Active Mesoporous
Formulation for Combination Therapy of Deep-Seated Melanoma.
ACS Nano 2018, 12, 11936−11948.
(54) Golombek, S. K.; May, J.-N.; Theek, B.; Appold, L.; Drude, N.;
Kiessling, F.; Lammers, T. Tumor Targeting via EPR : Strategies to
Enhance Patient Responses. Adv. Drug Deliv. Rev. 2018, 130, 17−38.
(55) Nune, S. K.; Gunda, P.; Majeti, B. K.; Thallapally, P. K.;
Forrest, M. L. Advances in Lymphatic Imaging and Drug Delivery.
Adv. Drug Deliv. Rev. 2011, 63, 876.
(56) Cai, S.; Yang, Q.; Bagby, T. R.; Forrest, M. L. Lymphatic Drug
Delivery Using Engineered Liposomes and Solid Lipid Nanoparticles.
Adv. Drug Deliv. Rev. 2011, 63, 901.
(57) Kim, K.; Choi, H.; Choi, E. S.; Park, M.-H.; Ryu, J.-H.
Hyaluronic Acid-Coated Nanomedicine for Targeted Cancer
Therapy. Pharmaceutics 2019, 11, 301.
(58) Mendieta, I.; Nuñez-Anita, R. E.; Nava-Villalba, M.; Zambrano-
Estrada, X.; Delgado-González, E.; Anguiano, B.; Aceves, C.
Molecular Iodine Exerts Antineoplastic Effects by Diminishing

O https://doi.org/10.1021/acs.biomac.1c01076
Biomacromolecules XXXX, XXX, XXX−XXX