Biotin functionalized PEGylated poly(amidoamine) dendrimer conjugate for active targeting of paclitaxel in cancer

Article abstract of DOI:10.1016/j.ijpharm.2018.12.069

In the current study, we employed poly(amidoamine) (PAMAM) dendrimers of generation 4 (G4) to deliver paclitaxel (PTX), a poorly soluble anti-cancer agent precisely to cancer cells via its conjugation on dendrimer surface. Further, G4 PAMAM has been PEGyl

Full text of DOI:10.1016/j.ijpharm.2018.12.069

Accepted Manuscript
Biotin Functionalized PEGylated Poly(Amidoamine) Dendrimer conjugate for
Active Targeting of Paclitaxel in Cancer
Sri Vishnu Kiran Rompicharla, Preeti Kumari, Himanshu Bhatt, Balaram
Ghosh, Swati Biswas
PII:
S0378-5173(18)30987-6
https://doi.org/10.1016/j.ijpharm.2018.12.069
IJP 18038
DOI:
Reference:
International Journal of Pharmaceutics
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Revised Date:
Accepted Date:
22 October 2018
5 December 2018
17 December 2018
Please cite this article as: S. Vishnu Kiran Rompicharla, P. Kumari, H. Bhatt, B. Ghosh, S. Biswas, Biotin
Functionalized PEGylated Poly(Amidoamine) Dendrimer conjugate for Active Targeting of Paclitaxel in Cancer,
International Journal of Pharmaceutics (2018), doi: https://doi.org/10.1016/j.ijpharm.2018.12.069
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Biotin Functionalized PEGylated Poly(Amidoamine) Dendrimer conjugate
for Active Targeting of Paclitaxel in Cancer
Sri Vishnu Kiran Rompicharla1, Preeti Kumari1, Himanshu Bhatt1, Balaram Ghosh1,
Swati Biswas1*
1 Department of Pharmacy, Birla Institute of Technology & Science-Pilani, Hyderabad
Campus, Medchal, Hyderabad, Telangana 500078, India.
*Corresponding author:
Swati Biswas, Ph.D.
Associate professor, Department of Pharmacy,
Birla Institute of Technology & Science-Pilani, Hyderabad Campus,
Medchal, Hyderabad, Telangana 500078, India.
Phone: +91-40-66303630,
Email ID: swati.biswas@hyderabad.bits-pilani.ac.in
1

Abstract
In the current study, we employed poly(amidoamine) (PAMAM) dendrimers of generation 4
(G4) to deliver paclitaxel (PTX), a poorly soluble anti-cancer agent precisely to cancer cells
via its conjugation on dendrimer surface. Further, G4 PAMAM has been PEGylated (PEG)
and tagged with Biotin, an essential micronutrient for cellular functions, receptors of which
are overexpressed in certain cancers. The synthesized multifunctional conjugates were
1
characterized by H NMR and zeta potential analysis techniques. In addition, the conjugates
were evaluated in vitro in cell monolayers and 3D spheroids of biotin receptor over-expressed
A549 cell line (human non-small cell lung cancer). G4 PTX PEG-Biotin conjugate penetrated
at significantly higher extent in monolayers as well as spheroids as studied by flow cytometry
and confocal microscopy by visualizing the cells at varied depth. The G4 PTX PEG-Biotin
conjugate demonstrated higher cytotoxicity compared to free PTX and G4 PTX PEG
conjugate as assessed by MTT assay in monolayers and Presto Blue assay in detached
spheroidal cells. G4 PTX PEG-Biotin demonstrated significant inhibition of growth of tumor
spheroids. Therefore, the newly synthesized biotin anchored PTX-conjugated dendrimer
system is promising and could be further explored for efficiently delivering PTX to biotin
receptor overexpressed cancers.
Key words: PAMAM dendrimers; biotin; active targeting; cancer; paclitaxel
2

1. Introduction
Cancer is a leading cause of death worldwide. Chemotherapy, the first line of treatment, poses
deleterious effect on the normal cells in a patient’s body making it a primary obstacle to the
clinical application of otherwise potent anticancer drugs (Tripodo et al., 2014). The efficacy
and tolerability of anticancer agents can be increased by employing target specific drug
delivery systems (DDS) that can limit the associated side effects and improve treatment
prognosis (Ren et al., 2015). Several potent chemotherapeutic agents like taxols, epirubicin,
platinum compounds, methotrexate, doxorubicin (DOX) etc., are clinically used for a variety
of cancer treatments. However, most of the chemotherapies are conventional, and therefore,
pose significant adverse effects (Abraham et al., 2005; Celik et al., 2013; Šimůnek et al.,
2009; Yao et al., 2007). Therefore, DDS with targeting moieties that specifically target cancer
cells could be a promising treatment strategy to overcome such shortcomings (Guo et al.,
2014; Hao et al., 2015; Luo et al., 2014; Quan et al., 2014).
Nanocarriers, by virtue of their size, leaky vasculatures in tumor and poor lymphatic clearance
get accumulated to the tumor microenvironment via the phenomenon, commonly known as
Enhanced Permeability and Retention (EPR) effect. However, a successful cancer treatment
wherein sufficient amount of drug has to reach the site of action cannot solely rely on EPR or
passive targeting. Cancer cells need more micronutrients than normal cells for their
proliferation and survival. As a consequence, they have overexpression of certain receptors
which can be actively targeted using specific ligands (Russell-Jones et al., 2004). Different
ligands, including proteins, hormones, vitamins, and growth factors which are identified by
cancer cells for their active internalization, are attached to the backbone of the DDSkk
(Nateghian et al., 2016). If the circulation times are long enough, effective transport to the site
3

of action and substantial uptake of drug via endocytosis can be possible through both - the
EPR effect and active targeting of molecules (Brigger et al., 2002; Byrne et al., 2008; Chen et
al., 2010).
Biotin (vitamin H) is an essential micronutrient, which is vital for normal cellular function
(Livaniou et al., 2000). Owing to low molecular weight, relatively simple biochemical
structure, and high tumor specificity, biotin has attracted great attention from pharmaceutical
research. In order to thrive and multiply rapidly, cancer cells need extra biotin as compared to
normal cells. Rapidly proliferating malignant cells overexpress biotin in order to meet their
biotin uptake. Biotin overexpression is observed in wide types of cancers, including renal
(RENCA, RD0995), lung (A549, M109), ovarian (OV 2008, ID8), mastocytoma (P815) and
breast (4T1, JC, MMT06056) cancer cell lines (Shi et al., 2014). This specific interaction of
biotin and its receptors has been explored to develop various biotin-conjugated nanocarriers to
increase the uptake of anticancer drugs by various tumor cells (Bu et al., 2013; Minko et al.,
2002; Taheri et al., 2011; Tseng et al., 2009; Vadlapudi et al., 2013; Yang et al., 2009; Yang
et al., 2014; Yellepeddi et al., 2009).
Dendrimers are hyperbranched macromolecules having monodispersed three-dimensional
structure with specific molecular weight and available in many generations based on the layer
of branches put on the core (Cheng et al., 2008; Esfand and Tomalia, 2001).
Poly(amidoamine) (PAMAM) dendrimers are the first commercially used class of dendrimers
for drug delivery investigations (Tomalia, 2005). In general, the dendrimers possess vacant
internal pockets which can hold the poorly soluble cargoes (Jansen and Meijer, 1994).
Additionally, the outer shell of dendrimers possess a wide number and variety of surface
functional groups which can be conjugated or anchored with various moieties (Majoros et al.,
4

2006; Thomas et al., 2005). PAMAM dendrimers have been widely explored for delivery of
poorly soluble anti-cancer agents to specific target sites (Asthana et al., 2005; Bhadra et al.,
2005; Bhadra et al., 2003; D'emanuele et al., 2004; Milhem et al., 2000; Najlah et al., 2006,
2007). Further, PAMAM dendrimers find application in gene delivery with specificity to
cancer cells (Li et al., 2018; Liu et al., 2017). Ease of fabrication, nanometer size,
biocompatibility, scalability are some of the advantages that make dendrimers a potential drug
delivery system (Duncan and Izzo, 2005; Svenson and Tomalia, 2012). Previous studies
reported the active targeting of dendrimers by conjugation of ligands such as folic acid
(Thomas et al., 2005), biotin (Xu et al., 2007; Yellepeddi et al., 2009), Lactobionic acid
(Iacobazzi et al., 2017), antibodies (Patri et al., 2004), peptides (Shukla et al., 2005), and
epidermal growth factor (Barth et al., 2004) to dendrimers thereby enhancing the therapeutic
potential of cancer chemotherapeutics.
In the present study, we have developed and investigated the potential of biotin conjugated
PEGylated multifunctional PAMAM dendrimer system to effectively deliver paclitaxel
(PTX), a chemotherapeutic agent which suffers poor physicochemical properties. The unique
globular structure of PAMAM dendrimers allows the spatial arrangement of biotin molecules
on the surface which might contribute to superior internalization of the delivery system into
the cancer cells (Yellepeddi et al., 2009). Following the synthesis, we have characterized the
conjugates and evaluated the delivery system in biotin overexpressed human lung cancer cell
line (A549). A 3D tumor spheroid model which mimics in vivo tumor has been employed to
evaluate the uptake and therapeutic potential of the developed dendrimer system.
5

2. Materials and methods
2.1 Materials
Poly(amidoamine) dendrimer of ethylenediamine core and generation 4.0 with 64 terminal
amino groups (G4 PAMAM) was procured from Dendritech (USA). A gratis sample of
Paclitaxel (PTX) was received from Fresenius Kabi India Pvt., Ltd. (Gurgaon, India).
Methoxy-polyethyleneglycol-succinimidyl carboxymethyl ester of molecular weight 2000 Da
(mPEG-SCM ester) was purchased from Jenkem Technology (USA). N-(3-
Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC. HCl, 98%), Biotin and N-
Hydroxysuccinimide (NHS, 98%) were procured from Sigma Aldrich Chemicals (USA).
Regenerated cellulose dialysis membrane of molecular weight cut-off 2 kDa, 3.5 kDa and 14
kDa was purchased from Spectrum Laboratories, Inc. (USA). N-ethyldiisopropylamine
(DIPEA), NHS-Fluorescein was purchased from Avra Chemicals (India), and Thermo
Scientific (USA) respectively. N,N'-Dicyclohexylcarbodiimide (DCC) was purchased from
Spectrochem Chemicals Ltd. (India). The solvents and reagents utilized in the study were of
analytical grade.
2.2 Methods
2.2.1 Synthesis of multifunctional PAMAM dendrimer
2.2.1.1 Synthesis of fluorescently tagged G4 PAMAM dendrimer
Fluorescently labelled G4 PAMAM dendrimer was prepared following an earlier reported
procedure with a small modification (Biswas et al., 2012). To the DMF solution of G4
dendrimer (100 mg), Fluorescein NHS was added at a mole ratio of 1:1 dropwise under inert
atmosphere. The solution was stirred in dark conditions for 8 h at normal room temperature.
6

Following the reaction, DMF was removed using rotary evaporator and resulting product was
subjected to dialysis to remove unconjugated fluorescein. After dialyzing for 48 h, the product
(F-G4) was freeze dried and stored until further use.
2.2.1.2 Synthesis of G4-PTX
The OH group at 2` position of paclitaxel (PTX) was modified to its hemisuccinate form
using succinate linker (succinic anhydride) prior to reacting with amine groups of G4
PAMAM dendrimer. In brief, 25 mg of PTX was dissolved in dichloromethane and reacted
with succinic anhydride (4.4 mg) for 3 days under stirring in presence of dry pyridine. The
product was collected by ethyl acetate extraction and dried to yield PTX-2`-hemisuccinate as
white solid. The hemisuccinate –COOH group was reacted with DCC/NHS for 8 h to form
NHS ester of PTX which can easily attach with –NH2 surface groups of dendrimer molecules.
The activated PTX NHS ester was added to 50 mg of G4 dendrimer in DMF at a mole ratio of
1:4 and stirred overnight. Following the reaction, DMF was evaporated and the product was
dialyzed using cellulose ester membrane of MWCO 12-14 kDa against water to remove
impurities. Further, a white fluffy product of G4-PTX was collected by freeze drying.
The schematic representation of synthesis of the multifunctional PAMAM dendrimer is
shown in Figure 1.
2.2.1.3 Synthesis of G4-PTX-PEG
The G4-PTX construct was further attached with PEG using mPEG SCM ester (2 kDa). G4-
PTX was dissolved in DMF, and mPEG SCM was added drop-wise at a mole ratio of G4-
PTX: mPEG-SCM as 1:12. The reaction mixture was stirred overnight in presence of DIPEA
(20 µl). The solvent was removed using rotary evaporator. The product was subjected to
7

dialysis using regenerated cellulose dialysis membranes of MW cutoff 12-14 kDa with
frequent water changes for 48 h. After dialysis, the product was freeze dried to obtain a solid
white product of G4-PTX-PEG. The fluorescently tagged dendrimer conjugates were also
prepared in the same manner. The PEGylation of G4-PTX was confirmed by measuring the
zeta potential changes, and by 1NMR and GPC analysis.
2.2.1.4 Biotinylation of G4-PTX-PEG
Biotinylation of G4-PTX-PEG conjugate was performed by activating the carboxy group of
biotin molecule using EDC/NHS (3 mol equivalent of biotin) in DMF and attaching it with
amine group of G4-PTX-PEG at a mole ratio of biotin: G4-PTX-PEG as 30:1. After reaction,
the solvent was removed using rotary evaporator and the product was subjected to dialysis
against water for 2 days. The final G4-PTX-PEG-Biotin construct was obtained after
lyophilization.
2.2.2 Characterization of multifunctional conjugate
Characterization of the PAMAM dendrimer conjugates G4-PTX, G4-PTX-PEG, and G4-
1
PTX-PEG-Biotin was performed by H NMR (300 MHz, Bruker, USA) and zeta potential
measurements by using dynamic light scattering instrument (Nano ZS, Malvern Instruments,
UK). To determine the relative molecular weights of synthesized conjugates, gel permeation
chromatography (GPC) analysis was performed. Zorbax GF-250 (9.4 mm ID X 25 cm X 4-4.5
µm) size exclusion analytical column was used to run the samples in a HPLC system (Agilent
1100 series). Tris buffer (50 mM) and KCl (100 mM) was used as eluting phase in isocratic
mode at a flow rate of 1 ml/min. A calibration curve was plotted with the standard molecular
8

weight compounds prior to analyzing the samples. Further, to determine the morphology of
the conjugate G4-PTX-PEG-Biotin, TEM analysis was performed.
2.2.3 HABA/Avidin assay
Biotin attachment to the dendrimer was quantitatively estimated using HABA/Avidin assay.
This assay is based on the association of the dye HABA (4'-hydroxyazobenzene-2-carboxylic
acid) to avidin and the ability of biotin molecule to displace HABA in stoichiometric
fractions. HABA binds to avidin to produce a yellow-orange colored complex which absorbs
at 500 nm. Biotin in the sample, if present, would displace the HABA dye and cause the
absorbance to decrease. As per the manufacturer’s protocol, to 900 µl of HABA/Avidin
reagent, 100 µl of sample containing biotin dissolved in deionized water was added. The
absorbance of the test solution was recorded at 500 nm. Further, the biotinylation degree was
calculated from the absorbance values obtained.
2.2.4 Cell studies
The in vitro evaluation of the synthesized conjugates was performed in human lung cancer
cells, A549 which were procured from National Center for Cell Sciences (NCCS, Pune,
India). Cells were grown in Dulbecco’s minimum essential medium (DMEM) containing 10%
FBS and 1% penicillin-streptomycin solution (Himedia Labs, Mumbai). During the period of
study, cell cultures were maintained in an incubator operated at 37°C with 5% CO2.
2.2.4.1 Cellular uptake by confocal microscopy
Cellular internalization of multifunctional dendrimer conjugates was studied using confocal
microscopy. Briefly, A549 cells were harvested from culture flasks at 80% confluence and
counted. The cells were seeded at a cell density of 50,000 cells/well onto round coverslips
9

placed in 12-well plates. On the day of study, cells were treated with conjugates F-G4-PEG
and F-G4-PEG-Biotin (20 µg/ml) and incubated at 37 °C for 1 h and 4 h. Also, to assess the
mechanism of uptake of biotin tagged dendrimer conjugates, the cells were pre-incubated with
excess free biotin (300 µm for 30 min) to saturate the biotin receptors. Further, the wells
containing medium with free biotin was removed and F-G4-PEG-Biotin was added to the
cells. Following incubation, cells were washed three times with sterile PBS, stained with
DAPI (1 µg/ml) for 5 min, washed and fixed with 4% para-formaldehyde for 15 min. Using a
mounting medium (Fluoromount-G), the coverslips containing cells were placed on the
microscopic slides. At 40 X magnification, cells were visualized under confocal microscope
(Leica DMi8, Leica Microsystems, Germany), Photographs of cells were taken in FITC and
DAPI fluorescence filters. The obtained data were processed using Image J software.
2.2.4.2 Cellular uptake by flow cytometry
To quantitatively determine the cellular association of dendrimer conjugates, flow cytometry
experiments were performed in A549 cells. The 6-well microplates were seeded with A549
cells at a cell population of 0.8 × 106 cells/well and allowed to attach overnight. Similar to the
confocal microscopy study, biotin receptors were saturated by pre-incubating the cells with
excess of free biotin (300 µm for 30 min) which receive F-G4-PEG-Biotin treatment to
observe if the cellular uptake was mediated via biotin receptors. Cells were treated for 1 h and
4 h with F-G4-PEG and F-G4-PEG-Biotin at a concentration of 20 µg/ml. After the
incubation period, cells were washed with sterile fresh PBS and then trypsinized. Cells were
centrifuged and the obtained cell pellet was re-dispersed in PBS before analyzing the samples
by flow cytometer (Amnis, EMD Millipore, USA). Control cells did not receive any
10

treatment. At least 10,000 events were collected for each sample. The data was captured as
geometric mean fluorescence and was calculated using the IDEAS software V6.0.
2.2.4.3 Cell viability study
The cytotoxic activity of free PTX, G4-PTX-PEG, and G4-PTX-PEG-Biotin was studied by
MTT colorimetric assay. A549 cells were seeded in 96-well plates at a population of 10,000
cells/well and allowed to attach overnight. On the day of the study, cells were treated with
free PTX and PTX conjugates (0-50 µg/ml) and incubated for 24 h and 48 h at 37°C.
Following incubation, formulations were removed and 50 µl of MTT reagent (5 mg/ml
solution) was added to each well. The plates were further incubated for another 4 h, and MTT
reagent was discarded. The purple colored formazan crystals formed in the wells were
dissolved by adding DMSO (150 µl) to each well. The absorbance of the wells was measured
at 570 nm using a microplate reader (Spectramax™, Molecular Devices, USA) with a
reference wavelength of 630 nm. Control cells did not receive any treatment containing PTX.
The cytotoxicity caused due to various treatments was calculated as percentage cell viability
and is represented as a bar graph against the concentration of PTX.
2.2.5 Evaluation of Biotin tagged PAMAM Dendrimer conjugates in multicellular tumor
spheroids
2.2.5.1 Formation of A549spheroids
Multicellular spheroids are 3D structures which mimic the in vivo tumors. In the current
study, A549 tumor spheroids were prepared by liquid overlay method (Perche and Torchilin,
2012; Sarisozen et al., 2014). In brief, 1.5% (w/v) agar solution was prepared in serum free
DMEM medium and autoclaved. A volume of 50 µL of the agar solution was added to each
11

well of a 96-well plate at the bottom to prevent cell adhesion. Care should be taken so as to
not let the agar solution solidify before adding to wells. For confocal microscopy study, 8-
chambered glass slides were used and to each well 150 µl of agar solution were dispersed on
the inner bottom.
From the culture flasks, A549 cells were detached using trypsin and cell pellet was collected
by centrifugation. To each well, cells were seeded at a density of 8,000 cells. Culture plates
were centrifuged at room temperature for 15 min. After centrifugation, plates are left in the
incubator. The spheroid formation was constantly supervised using an inverted microscope
(Leica DMi8, Leica Microsystems, Germany). For further studies, 3 – 5 days old spheroids
were used.
2.2.5.2 Penetration efficiency in multicellular tumor spheroids
The internalization of targeted and non-targeted dendrimer conjugates into the spheroids was
studied using confocal microscopy. F-G4-PEG-Biotin and F-G4-PEG were added to the
spheroids grown in 8-well glass chamber slides and incubated for 1 and 4 h. After that, the
cancer cell spheroids were gently washed with PBS and visualized by laser scanning confocal
microscope (Leica DMi8, Leica Microsystems, Germany) at 10X magnification. Z-stack
images were captured to see the penetration, from the surface where fluorescence is observed
to the center of spheroid at every 10 μm thickness. Image J software was used to process the
captured images.
2.2.5.3 Flow Cytometry analysis of spheroid uptake of dendrimer conjugates
Flow cytometry study was performed to quantitatively determine the uptake of targeted and
non-targeted dendrimer conjugates in 3D cell spheroids. Five-day-old spheroids were
12

employed in the study and incubated with F-G4-PEG and F-G4-PEG-Biotin for 1 h and 4 h.
After the treatment period, 10 spheroids each of every treatment were pooled up to achieve
required cell population for flow cytometry analysis. The spheroids were gently washed with
sterile PBS and dissociated by adding Accutase cell detachment solution. Accutase solution is
used to create single cell suspensions from a clump of cells. It contains proteolytic and
collagenolytic enzymes. The obtained cell suspension after Accutase treatment was
centrifuged and the single cell suspension was prepared by adding PBS. Further, the samples
were run using flow cytometer and atleast 10,000 events were collected for every sample. The
fluorescence intensity histograms were captured in the FITC channel for each sample. A bar
diagram was plotted with geometric mean fluorescence exhibited by biotin modified and
unmodified dendrimer conjugates.
2.2.5.4 Growth inhibition of multicellular tumor spheroids
The inhibitory effect of PTX formulations on the growth of A549 cancer cell spheroids was
studied to evaluate the potential of synthesized conjugates in delivering the drug to tumors.
The cancer cell spheroids were incubated in complete media added with free PTX, G4-PTX-
PEG, and G4-PTX-PEG-Biotin at a PTX concentration of 25 μg/mL. After 24 h, the growth
medium with formulations was carefully discarded and fresh growth medium was added. The
magnitude of spheroidal growth inhibited by dendrimer drug conjugates was visualized using
inverted microscope (Leica DMi8, Leica Microsystems, Germany). Brightfield images were
captured at 10X magnification. The diameter of the spheroids at regular intervals was
measured and represented as a line graph.
13

2.2.5.5 In vitro cytotoxic activity in multicellular tumor spheroids
To evaluate the cytotoxic activity of targeted and non-targeted dendrimer conjugates in
multicellular tumor spheroids, Presto Blue assay was performed as per the manufacturer's
instructions. Presto Blue reagent is a cell permeable resazurin-based solution which turns to a
red fluorescent complex upon reduction by viable cells. The fluorescence/absorbance of the
sample is measured and the cell viability is calculated. On the day of study, 3-5 day old
spheroids were selected and treated with free PTX, G4-PTX-PEG, and G4-PTX-PEG-Biotin
at a PTX concentration of 25 μg/ml. For each treatment, 10 individual spheroids were used as
a group. Following the incubation for 24 h, the medium in the wells was carefully discarded,
washed with PBS and then disaggregated by adding 50 μL of Accutase solution. The cell
suspension resulted by mild shaking was subjected to centrifugation to obtain cell pellet. The
samples for analysis were prepared by adding 10 µl of Presto Blue reagent to the cell pellet
dispersed in 90 μl of growth medium. After a brief incubation for 2 h at 37°C, the absorbance
of the samples was measured at 570 nm with a reference wavelength of 600 nm.
2.2.5.6 Live/Dead cell assay in multicellular tumor spheroids
The amount of live and dead population developed as a result of various PTX treatments to
A549 cell spheroids was assessed by live/dead cell assay using Calcein Blue AM reagent. In
brief, dendrimer conjugates and free PTX were added to individual spheroids at a
concentration of PTX equivalent to 50 µg/ml and incubated for 24 h. Following the treatment
period, spheroids were washed with PBS and stained with Calcein Blue AM reagent and
Propidium iodide (PI). This assay works on the principle of diffusion of Calcein Blue AM
ester passively into the cells and cleaves to calcein blue in presence of cellular esterases
present in viable cells. The resultant calcein blue remains inside the cytoplasm and emits
14

bright blue fluorescence. Further, PI dye stains the dead population. The fluorescence emitted
was observed using a fluorescence microscope (Leica DMi8, Leica Microsystems, Germany)
with blue and red filters. The images were processed using Image J software.
2.2.6 Statistical analysis
Data were represented as a mean of atleast three individual experiments with standard
deviation. Student’s t-test (GraphPad Software, Inc.; San Diego, CA) was performed to
establish significance between groups. A p value of less than 0.05 was considered statistically
significance. The representation of *, ** or ##, *** or ### corresponds to p values < 0.05,
0.01 and 0.001, respectively.
15

3. Results and Discussion
3.1 Synthesis and characterization of multifunctional dendrimer conjugate
In the NMR spectrum, the aromatic groups of PTX resulted in peaks at δ (ppm) 7-8.5 and
aliphatic protons at δ (ppm) 1.3. The protons of PEG chains were observed at δ (ppm) 3.2-4.1
whereas the protons corresponding to the PAMAM dendrimer molecule were observed at δ
(ppm) 1.5-3.5 as shown in figure 2A. In figure 2B, the peak at δ (ppm) 4.4 and 4.6
corresponds to the ring juncture protons of Biotin. This data confirms the successful
conjugation of biotin to the dendrimer surface to form G4-PTX-PEG-Biotin. Attachment of
fluorescein moiety was confirmed by the shift in absorption maxima as determined by UV-
Visible spectroscopy (Figure S1).
In addition, from the GPC analysis relative mass of each conjugate was identified. From the
mass value, the approximate number of molecules anchored to each dendrimer molecule was
calculated. It was observed that approximately 2.76 molecules of PTX, 10.9 molecules of
PEG were attached on the dendrimer surface following the synthesis procedure mentioned
above (Table S1). Further, characterization by TEM analysis illustrates that the synthesized
conjugate was spherical in morphology and has size in nanometer range as depicted in Figure
2C.
Furthermore, the HABA/Avidin assay revealed the degree of biotinylation of the dendrimer
molecules. The number of biotin molecules attached to each dendrimer was calculated to be
20.98 (See Table S1). Also, a variation in the zeta potential as presented in Table 1 following
each synthetic step supports the successful construction of the conjugates.
16

3.2 Cell Studies
3.2.1 Cellular uptake by confocal microscopy
The cellular internalization of F-G4-PEG and F-G4-PEG-Biotin by the A549 cells after 1 h
and 4 h treatment was observed by confocal microscopy. A bright green fluorescence was
noticed in the cells treated with biotin anchored dendrimer conjugate in comparison to the
cells treated with the non-targeted conjugate. The green fluorescence caused by F-G4-PEG-
Biotin in cells saturated with free biotin was seen to be less in intensity than the cells without
excess biotin. This suggests that the biotin targeted dendrimers internalize actively by the
biotin receptors present in higher concentration on the surface of the A549 cells. The less
significant uptake occurring in the presence of free biotin also suggests that the dendrimer
conjugates underwent charge based adsorptive endocytosis. The adsorptive endocytosis of
positively charged moieties and the biotin receptor mediated uptake together significantly
improved the cellular association of the synthesized dendrimer conjugates. Further, the results
of confocal microscopy corroborated with the flow cytometry analysis indicating a higher
cellular uptake in cells which received F-G4-PEG-Biotin treatment compared to F-G4-PEG,
in a time dependent manner. The images captured at 1 h and 4 h incubation were represented
in Figure 3.
3.2.2 Cellular uptake by flow cytometry
Quantitative assessment of cellular association was performed using flow cytometry. Similar
to the microscopy studies, influence of free biotin on the uptake of targeted dendrimers was
also studied. The saturation of the biotin vitamin transporters with addition of excess free
biotin resulted in decreased intensity of the fluorescence in the cells treated with F-G4-PEG-
17

Biotin. Flow cytometry data revealed a time dependent increase in the internalization at the
end of 1 h and 4 h treatment. An increase in the geometric mean fluorescence was observed
with time resulting in 1.53 fold and 1.94 fold higher uptake at 1 h and 4 h respectively for
biotin tagged dendrimer conjugate. The results also indicated that the Biotin tagged dendrimer
conjugates were promptly internalized via the biotin receptors. The role of biotin receptors in
uptake of actively targeted dendrimer conjugates was understood and the data was in
accordance with the confocal microscopy results. A bar graph of geometric mean fluorescence
was plotted from the values obtained at 1 h and 4 h and represented along with the histograms
of events collected by flow cytometer in Figure 4.
3.2.3 Cytotoxicity Study
The cytotoxicity exhibited by the dendrimer-drug conjugates compared to free drug in A549
cells was studied by MTT assay. For the experiment, cells were treated with free PTX, G4-
PTX-PEG, and G4-PTX-PEG-Biotin at a concentration range of 0-50 µg/ml and placed the
well plates in incubator for 24 and 48 h. From the MTT assay, it was observed that there was
an overall decrease in the cell viability with increase in treatment time and concentration of
the drug given to the cells (Figure 5). Of all the treatments, G4-PTX-PEG-Biotin was found to
be more active in killing the cells compared to other treatments. After 24 h, cells treated with
G4-PTX-PEG-Biotin exhibited 32.7±5.14% cell viability against 51.62±3.9% for G4-PTX-
PEG and 62.3±3% for free PTX, respectively. Furthermore, the cell viability declined to
14.5±3.44%, 29.8±3.2%, and 43.2±2.8% for G4-PTX-PEG-Biotin, G4-PTX-PEG, and free
PTX, respectively after 48 h. Dendrimer-drug conjugates were more active at all the time
points compared to free PTX. Moreover, G4-PTX-PEG-Biotin had shown superior cell killing
which can be ascribed to its capability of internalizing actively into the cells via the biotin
18

receptors which in turn delivers greater concentrations of PTX to cancer cells than non-
targeted dendrimer or free drug. The uptake of the dendrimer conjugates by receptor
mediated/adsorptive endocytosis could avoid the drug efflux by P-glycoprotein. A reason for
lesser cytotoxicity noticed in free PTX treated cells could be due to the drug being pushed out
of the cell by P-glycoprotein transporter system.
3.3 Evaluation of Biotin tagged PAMAM Dendrimer conjugates in multicellular tumor
spheroids
3.3.1 Penetration efficiency in multicellular tumor spheroids
Multicellular tumor spheroidal models simulate the in vivo tumors in terms of tumor structure
and microenvironment. 3D spheroids retain cancer cells in a natural morphology in presence
of extracellular matrix, pH and oxygen gradients resembling actual tumors. Also, spheroid
models mimic drug resistance in solid tumors to a greater extent compared to 2D monolayer
cell system. (van den Brand et al., 2017; Yang et al., 2017). These features make 3D
spheroidal cancer cell model an attractive alternative to understand the movement of
molecules into the cancer tissue.
From the Z-stack images captured using confocal microscopy, it was observed that the
fluorescence was deeper in spheroids treated with biotin tagged dendrimers in comparison to
the non-targeted conjugate. Further, the conjugates moved into the core of the spheroids as
evidenced by the bright green fluorescence at depths with increase in the time of incubation
from 1 h to 4 h (Figure 6). Biotin receptor mediated uptake of F-G4-PEG-Biotin resulted in
superior diffusion of dendrimer conjugates into the 3D tumor spheroids compared to non-
19

targeted dendrimers. This observation ascertains the advantage of active targeting to tumors
by biotinylation of the delivery systems.
3.3.2 Uptake of dendrimer conjugates in multicellular spheroids by flow cytometry
The internalization of biotin targeted and non-targeted dendrimers in the 3D multicellular
cancer spheroids was quantified by flow cytometry. The fluorescence intensity was observed
to be higher with F-G4-PEG-Biotin conjugate treatment compared to F-G4-PEG. The biotin
tagged dendrimer conjugate exhibited a 1.63 fold and 1.7-fold higher geo mean fluorescence
value compared to non-targeted dendrimer conjugate following 1 h and 4 h of incubation,
respectively.
The flow cytometry data supports the confocal microscopy results, where time-dependent
intensification of the green fluorescence due to accumulation in the spheroids was seen. It can
be affirmed that the biotin conjugation on the surface remarkably improved the uptake of the
dendrimer conjugates. Data collected from flow cytometer was represented in Figure 7 as
fluorescence intensity histogram and a bar graph.
3.3.3 Growth inhibition of multicellular tumor spheroids
Growth inhibition caused due to exposure of multicellular cancer spheroids to free PTX, G4-
PTX-PEG, and G4-PTX-PEG-Biotin was evaluated by measuring the diameter of the tumor
spheroids over a period of time. The spheroids were examined for 6 days and the microscopic
images were captured on Day 0, Day 3, and Day 6 using brightfield microscope. The diameter
of the spheroids was noted down to analyze their growth. As depicted in Figure 8A, the
diameter of the spheroids was significantly less on treatment with PAMAM dendrimer-drug
conjugates compared to free drug. Moreover, the diameter of control spheroids increased
20

drastically up to 1000 µm diameter during the study. In the treatment groups, the average
diameter of spheroids at the end of 6 days was measured to be 531.3 ± 13.5 µm, 647 ± 32.6
µm, 791.6 ± 38.2 µm, and 927.3 ± 10 µm for G4-PTX-PEG-Biotin, G4-PTX-PEG, free PTX,
and complete media (control), respectively. The plot of diameters of spheroids is illustrated as
a line graph in Figure 8B.
The observation can be explained based on the cellular association data where active targeting
via biotin receptors promoted the delivery system to internalize efficiently in-turn leading to
localization of PTX in the spheroids obstructing their growth. This was supported by the
uptake studies performed in the 3D spheroid model, where the Biotin-dendrimer conjugate
significantly internalized into the spheroids, making more amount of drug available for
therapeutic action at the target location. Since 3D spheroid model mimics in vivo solid tumors
to a great extent, it could be extrapolated that the Biotin anchored PAMAM dendrimers could
significantly reduce the tumor growth in vivo.
3.3.4 In vitro cytotoxic activity in multicellular tumor spheroids
The reducibility of Presto Blue reagent upon treatment with PTX formulations was used as a
measure to quantify the cytotoxic activity exhibited by the formulations in the A549
multicellular spheroids. Treatment of spheroids with G4-PTX-PEG-Biotin and G4-PTX-PEG
exhibited superior cytotoxicity over free drug after incubation for 24 h at all the tested
concentrations. Highest degree of cytotoxicity was observed with G4-PTX-PEG-Biotin with a
cell viability of 39.35 ± 2.6% whereas, G4-PTX-PEG and free PTX displayed 50.5 ± 3.7%
and 68.9 ± 1.48% viability respectively (Figure 9). The cytotoxic activity in spheroids was
slightly reduced in comparison to cell monolayers as the penetration of the delivery system
21

becomes difficult due to structural complexity than the monolayers. The efficiency of biotin
tagged dendrimers to enter the cells actively promoted in superior buildup of PTX in cells to
exhibit therapeutic action.
3.3.5 Live/Dead cell assay in multicellular tumor spheroids
The calcein blue AM following internalization by living cells yields emits a strong blue
fluorescence due to cleavage of the ester by the intracellular esterases. The dead cells lack the
ability to convert the calcein blue AM reagent yielding no fluorescence. Further, dead
population in the spheroids was located in red color due to Propidium iodide staining.
From the fluorescence images of the spheroids (Figure 10), highest extent of dead population
seen as bright red fluorescence was induced by biotin anchored dendrimer followed by non-
targeted conjugate and free PTX. No significant dead population was observed in control
spheroids.
In free PTX treated spheroids, dead cells were majorly observed towards the boundary
suggesting limited penetration of free drug deeper into the spheroid mass. On the other hand,
dead cell population was observed clearly on both the boundary and within the central regions
of spheroid with G4-PTX-PEG and G4-PTX-PEG-Biotin treated spheroids. The study
indicated active targeting and as a result of that, higher penetration of biotin modified
conjugate in tumor spheroids compared to unmodified conjugate.
22

4. Conclusion
In this study, we have developed a G4 PAMAM dendrimer system to deliver paclitaxel (PTX)
specifically to the cancer cells by following active-targeting approach. Biotin was anchored
on to the surface of the dendrimers to target the vitamin uptake receptors overexpressed on the
cancer cells. PTX was covalently linked to the surface of the G4 PAMAM dendrimer using a
succinate linker. Further, to avoid the toxicity of dendrimers due to their cationic nature and to
prolong the systemic circulation of the conjugate, PEG has been attached. The presence of
biotin on the dendrimer surface was quantified using HABA/Avidin assay. G4-PTX, G4-
PTX-PEG and G4-PTX-PEG-Biotin had attachments of approximately 2.76 molecules of
PTX, 10.9 molecules of PEG and 20.98 molecules of biotin per molecule of G4 PAMAM
dendrimer as indicated by GPC analysis of G4-PTX, and G4-PTX-PEG and HABA assay for
G4-PTX-PEG-Biotin. TEM analysis revealed a spherical morphology and nanometer size
range of the conjugates. Cytotoxicity study by MTT assay and, cellular uptake studies
performed on A549 cells revealed that dendrimer–drug conjugates were efficient in cellular
internalization and cell killing activity. Biotin saturation studies demonstrated the role of
biotin receptor in mediating cellular uptake. Further, in 3D cancer cell spheroids, biotin
tagged conjugate displayed superior penetration, cytotoxicity, and inhibition of growth in
comparison to the treatment with non-targeted conjugate and free drug. Considering the
results, it can be affirmed that the newly developed active targeted dendrimer system could be
a promising treatment strategy for solid tumors and warrants further exploration for clinical
translation.
23

Conflicts of Interest
Authors declare no conflicts of interest
Acknowledgements
This work was supported principally by the Department of Biotechnology (Bio-CARe
scheme, BT/BioCARe/07/10003/2013-2014) to author Swati Biswas.
24

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27

List of Tables
Table 1 Zeta potential values of dendrimer conjugates after each step of synthesis (Mean±SD,
n=3).
Dendrimer conjugate
G4
Zeta potential (mV)
+18.1±2.2
G4-PTX
+13.3±1.8
G4-PTX-PEG
G4-PTX-PEG-Biotin
+6.3±1.26
+4.8±1.35
28

List of Figures
Figure 1 Schematic illustration of synthesis of G4 dendrimer conjugates
Figure 2 1H NMR spectrum of (A) G4-PTX-PEG and (B) G4-PTX-PEG-Biotin in D2O at 300
MHz, (C) TEM image of G4-PTX-PEG-Biotin. Scale bar indicates 50 nm.
Figure 3 Confocal microscopy images of A549 cells after (A) 1 h and (B) 4 h of incubation
with F-G4-PEG and F-G4-PEG-Biotin and, Biotin+F-G4-PEG-Biotin. Scale bar indicates 50
µm.
Figure 3 Cellular uptake of fluorescently tagged G4 conjugates in A549 cells after 1 h and 4 h
incubation as assessed by flow cytometer and represented as histograms and bar graph of geo
mean fluorescence (Mean±SD, n=3).
Figure 4 Percentage cell viability of A549 cells treated with different concentrations of PTX,
G4-PTX-PEG, and G4-PTX-PEG-Biotin at 24 h and 48 h (Meanꢀ±ꢀSD; nꢀ=ꢀ3).
Figure 5 Penetration of G4 PAMAM dendrimer conjugates in multicellular spheroids at
various depths after 1 h and 4 h incubation captured as Z-stacks by confocal microscopy.
Figure 6 Quantitative assessment of cellular uptake in multicellular tumor spheroids treated
with F-G4-PEG and F-G4-PEG-Biotin by flow cytometry (Meanꢀ±ꢀSD; nꢀ=ꢀ3).
Figure 7 (A) Brightfield images of tumor spheroids after different PTX treatments at the end
of Day 0, Day 3, and Day 6 using bright field microscope. Magnification: 10X. (B) Line
graph of spheroid diameters depicting growth inhibition. Data is represented as Meanꢀof
diameter in µm with SD; nꢀ=ꢀ3.
Figure 8 In vitro cytotoxicity induced by free PTX, G4-PTX-PEG, and G4-PTX-PEG-Biotin
in tumor spheroids as evaluated by Presto Blue assay after 24 h treatment (Meanꢀ±ꢀSD; nꢀ=ꢀ3).
29