Accepted Manuscript

Title: Hyaluronic acid-conjugated polyamidoamine

dendrimers for targeted delivery of 3,4-difluorobenzylidene
curcumin to CD44 overexpressing pancreatic cancer cells

Author: Prashant Kesharwani Lingxiao Xie Guangzhao Mao

Subhash Padhye Arun K. Iyer

PII: S0927-7765(15)30212-5
DOI: http://dx.doi.org/doi:10.1016/j.colsurfb.2015.09.043
Reference: COLSUB 7383

To appear in: Colloids and Surfaces B: Biointerfaces

Received date: 10-8-2015

Revised date: 22-9-2015
Accepted date: 23-9-2015

Please cite this article as: Prashant Kesharwani, Lingxiao Xie, Guangzhao
Mao, Subhash Padhye, Arun K.Iyer, Hyaluronic acid-conjugated polyamidoamine
dendrimers for targeted delivery of 3,4-difluorobenzylidene curcumin to CD44
overexpressing pancreatic cancer cells, Colloids and Surfaces B: Biointerfaces
http://dx.doi.org/10.1016/j.colsurfb.2015.09.043

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Hyaluronic acid-conjugated polyamidoamine dendrimers for targeted delivery of 3,4-

difluorobenzylidene curcumin to CD44 overexpressing pancreatic cancer cells

Prashant Kesharwania, Lingxiao Xieb, Guangzhao Maob, Subhash Padhyec, Arun K.

Iyera,d* arun.iyer@wayn.edu

a
Use-inspired Biomaterials & Integrated Nano Delivery (U-BiND) Systems Laboratory,
Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health
Sciences, Wayne State University, 259 Mack Ave, , Detroit, MI 48201, USA
b
Department of Chemical Engineering and Materials Science, Wayne State University , 5050
Anthony Wayne Drive, Detroit, Michigan 48202, USA
c
ISTRA, Department of Chemistry, MCE Society's Abeda Inamdar Senior College of Arts,
Science and Commerce, University of Pune, Pune 411001, India
d
Molecular Therapeutics Program, Barbara Ann Karmanos Cancer Institute, Wayne State
University, School of Medicine, Detroit, Michigan, 48201, USA
*
Corresponding author at: Department of Pharmaceutical Sciences, Eugene Applebaum College
of Pharmacy and Health Sciences, 259 Mack Ave, Room 3601 Wayne State University. Detroit,
MI 48201. Tel.: 313-577-5875; fax: 313-577-2033. 

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Graphical abstract

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Highlights

 We developed a dendrimer based formulation of CDF for CD44 targeted therapy for

pancreatic cancer.

 The targeting ability of HA facilitated the accumulation of the CDF nanoformulation at

the pancreatic cancer cells.

 HA-PAMAM-CDF nanosystems portend to be highly effective for treating pancreatic

cancers due to efficient cellular uptake in CD44 receptor-over expressing tumors.

 This work offers the prospect of taking highly potent yet less toxic dendrimer

nanosystems for clinical development.

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ABSTRACT

The current study was aimed to develop a targeted dendrimer formulation of 3, 4-

difluorobenzylidene curcumin (CDF) and evaluate its potential in CD44 targeted therapy for
pancreatic cancer. Using amine terminated fourth generation poly(amidoamine) (PAMAM)
dendrimer nanocarrier and hyaluronic acid (HA) as a targeting ligand, we engineered a CD44-
targeted PAMAM dendrimer (HA-PAMAM) formulation of CDF. The resulting dendrimer
nanosystem (HA-PAMAM-CDF) had a particle size and surface charge of 9.3 ± 1.5 nm and

−7.02 ±9.53 mV, respectively. When CD44 receptor overexpressing MiaPaCa-2 and AsPC-1

human pancreatic cancer cells were treated with HA-PAMAM-CDF, a dose-dependent

cytotoxicity was observed. Furthermore, blocking the CD44 receptors present on the MiaPaCa-
2 cells using free excess soluble HA prior to treatment with HA-PAMAM-CDF nano-
formulation resulted in 1.71 fold increased in the IC50 value compared to non-targeted
formulation (PAMAM-CDF), confirming target specificity of HA-PAMAM-CDF.
Additionally, HA-PAMAM-CDF formulation when compared to PAMAM-CDF, displayed
higher cellular uptake in MiaPaCa-2 cancer cell lines as shown by fluorescence studies. In
summary, the novel CD44 targeted dendrimer based nanocarriers appear to be proficient in
mediating site-specific delivery of CDF via CD44 receptors, with an improved therapeutic
margin and safety.
Keywords: Pancreatic cancer; CD44 targeting; PAMAM dendrimer; hyaluronic acid; 3, 4-

difluorobenzylidene curcumin; drug delivery

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1. Introduction

Cancer is the second leading cause of death in the United States, and is anticipated to

surpass heart diseases in the next few years [1]. Pancreatic cancer is one of the most deadly

malignancies among all types of cancers with a five-year survival rate of less than 6% in the

United States [2,3]. Successful translation of potential cancer therapies into clinics will mainly

depend on targeted drug delivery approaches with high safety margin. Surmounting the many

challenges of identifying a successful targeted drug delivery strategy requires an understanding

of events involving transport of drug or drug carrier to a target site after administration as well

as issues relevant for specific target diseases and the body's response toward a drug delivery

system [4]. In this context, polymeric nanocarriers such as micelles, liposomes, nanoparticles,

and dendrimers could be valuable options [5,6].

Dendrimers are globular, nano-sized (1–100 nm) macromolecules characterized by high

branching points, three dimensional globular shape, monodispersity and nanometric size range

(1–100 nm). It comprises three distinct domains i.e. core, branches, and various terminal

functional groups, generally located at its surface [7–10]. Among different types of

dendrimers, poly(amidoamine) (PAMAM) dendrimer have been exhaustively investigated and

have received widespread attention for drug as well as gene delivery. It has been reported that

dendrimers facilitate the transdermal delivery of 8-methoxypsoralen [11] and improve the oral

bioavailability of camptothecin [12]. However, the application of plain PAMAM dendrimer to

achieve targeted delivery of anticancer drugs can be restricted by their intrinsic surface

cationic charge, which leads to high cytotoxicity due to nonspecific interactions of PAMAM

with both normal and tumor cells. However, if these problems could be resolved, dendrimers

could possibly evolve as one of the better, if not the best option among the available

5 

 
nanocarriers. A number of strategies including but not limited to PEGylation, coating with

other polymers and, conjugation of targeting ligands are available for surface engineering of

dendrimers that could be used to overcome these limitations [7,13]. Among these strategies,

conjugation with various targeted ligands has been proved to be one of the most effective

approaches. Ligand decoration of dendrimers not only results in protection of surface amine

groups and reduction of the inherent cytotoxicity of dendrimers but also imparts some other

beneficial properties including solubility enhancements, increased drug loading, sustained and

controlled drug release, improved stability as well as favorable bio-distribution and

pharmacokinetic properties [14–17].

Hyaluronic acid (HA) is a naturally occurring mucopolysaccharide composed of

alternating sugar residues of D-glucuronic acid (GlcUA) and N-acetyl D glucosamine (NAG)

units. Of primary importance to pancreatic tumor selective delivery, the HA backbone in itself

is endowed with tumor targeting moieties that specifically recognizes CD44, an integral

membrane glycoprotein over-expressed on several tumors cell surfaces, including pancreatic

cancer and tumor initiating stem like cells [18,19].

We have developed a highly promising derivative of curcumin known as 3,4-

difluorobenzylidene curcumin (CDF) that not only showed 16-fold increase in half-life and

superior anticancer activity but also showed better pancreas accumulation than curcumin

[5,20,21]. More prominently, CDF could inhibit the growth of cancer stem like cells (CSLCs)

are (including but not limited to) through deregulation of multiple miRNAs, induction of

phosphatase and tensin homolog (PTEN), and attenuation of histone methyltransferase EZH2

[22–24]. These findings indicate that CDF could be a good choice for treatment of pancreatic

cancer as well as CSLCs. However, its utility for clinical translation needs to be evaluated.

6 

 
Recently, we have successfully developed styrene-maleic acid (SMA)-CDF nano-micelles that

showed promising anticancer activity for the management of pancreatic cancer. Similarly, Basak

et al. reported that the nude mice xenograft study showed a statistically significant tumor growth

inhibition of UM-SCC-1R cells and a reduction in the expression of CD44, indicating promising

inhibitory effect of liposomal CDF on CSLCs [25]. These reports clearly suggest the potential

for CDF as a therapeutic agent for treatment of pancreatic cancer including CSLCs. However,

poor aqueous solubility of CDF has made its systemic administration problematic [5]. The

present study was aimed at developing a nano-formulation of HA-PAMAM engineered for CD44

mediated targeted delivery of CDF, with the aim to treat human pancreatic cancer including

pancreatic CSLCs (Scheme 1).

2. Material and methods

2.1. Materials

CDF was synthesized as described earlier [5,26]. 4.0G PAMAM dendrimer, N-(3-

(dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride (EDC), and 3-[4,5

dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide (MTT) was purchased from Sigma-

Aldrich (St. Louis, MO). HA (low molecular weight) (Average Mw 10 kDa) was purchased

from Lifecore Biomedical (Chaska, MN). All other chemicals were of reagent grade and used

without any modification

2.2. Synthesis and characterization of HA-PAMAM conjugate

Hyaluronic acid (HA) was conjugated to the peripheral amino group of PAMAM

dendrimer through EDC coupling chemistry. In brief, HA (1mM) was dissolved in minimum

quantity of deionized water and EDC (1.2 mM) was added and kept in 37°C in shaker for 2 h

to activate the COOH group in HA. The activated HA was added drop-wise to PAMAM

7 

 
dendrimer (1.38 mM) in 10 mL of DMSO and stirred for 2 days at room temperature (RT)

(37±0.5°C). The resulting HA-PAMAM conjugate was purified by ultrafiltration using

Millipore tangential flow filtration (TFF) (Molecular weight cut-off 3.5 kDa), (Millipore,

Milford, MA) followed by lyophilization (Scheme 2) and characterization by Fourier

Transform Infrared spectroscopy (FTIR) and proton Nuclear Magnetic Resonance

spectroscopy (1H-NMR).

2.2.1. Transmission electron microscopic analysis

Transmission Electron Microscopy (TEM) was performed to confirm the size of the

PAMAM and HA-PAMAM dendrimer after air-drying of the sample [4 μL aliquot (60 μM)]

onto a Formvar-coated, carbon-stabilized copper grid (400 mesh) for 4 min. The grid was then

rinsed temporarily with distilled water, negatively stained with 2% aqueous uranyl acetate, air-

dried, and examined with a JEOL transmission electron microscope (JEM 2010, Tokyo, Japan) at

an accelerating voltage of 200 kV and 80X magnification.

2.3. Drug loading

CDF was loaded into the PAMAM and HA-PAMAM dendrimer using equilibrium

dialysis method as described earlier [27]. In short, known molar concentration of dendrimer

formulations (PAMAM or HA-PAMAM) and CDF were dissolved in DMSO/phosphate buffered

saline (PBS) pH 7.4 (3:7). The mixed solution was incubated with slow magnetic stirring (50

rpm) using a magnetic bead for 48 h at RT. This solution was dialyzed [cellulose dialysis bag

(MWCO 7000 Da; Sigma)] against DMSO under strict sink conditions for 15 min to remove un-

entrapped drug from the formulations, The un-entrapped drug was then estimated

spectrophotometrically (Jasco 530 UV-Visible spectrometer, Tokyo, Japan) at 447 nm (max of

8 

 
CDF) to determine indirectly the amount of drug loaded within the system. The dialyzed

formulation was lyophilized and used for further characterization.

2.3.1. Zeta potential measurement

The surface charge [zeta potential ()] measurement of PAMAM and HA-PAMAM was

performed using a Beckman Coulter Delsa Nano C DLS Particle analyzer (Beckman Coulter,

Inc., Fullerton, CA) equipped with a 658 nm He-Ne laser. The zeta potentials were evaluated by

measuring the electrophoretic mobility of charged particles under an applied electric field.

2.3.2. Atomic force microscopic analysis

The particle size averages of CDF loaded dendrimer formulations (PAMAM-CDF and

HA-PAMAM-CDF) were determined using the atomic force microscopy (AFM) using

Multimode IIIa atomic force microscope (Digital Instruments/VEECO, Plainview, NY) with an

E-scanner sectional height analysis command (Nanoscope) by manually measuring the lateral

diameters and vertical heights of 80-100 particles on mica. The lateral diameter of the particle

was determined at full width and half-maximum height in order to minimize tip convolution.

2.4. In vitro release studies

Drug release from known weight of CDF loaded plain PAMAM, and HA-PAMAM

dendrimers were determined in acidic (pH 4.0) and physiological buffer (PBS, pH 7.4) using

dialysis membrane diffusion technique [5,28]. Known amount of CDF loaded dendrimer

formulations were separately placed inside the dialysis tubing (3.5 KDa, Sigma, USA),

hermetically sealed and suspended immediately in 50 ml of released media under sink

conditions. The volume of receptor compartment was maintained constant by replenishing it with
9 

 
1 ml of sink solution, after withdrawing of 1 ml aliquot. The drug concentration was detected

spectrophotometrically. This experiment was repeated three times. The results are presented as

mean ± SD (n=3).

2.5. Cell culture

Human pancreatic cancer cell lines MiaPaCa-2 and AsPC-1 were used for our study on

the basis of their sensitivity to CDF, as reported earlier [23]. Both cell lines were maintained in

Dulbecco's Modified Eagle's Medium (DMEM; Fisher Scientific, Waltham MA), supplemented

with 5% FBS (Fisher Scientific, Waltham MA), 2 mmol/L glutamine, 50 units/mL penicillin, and

50 μg/mL streptomycin as standard culture condition. The cell lines have been tested and

authenticated by the core facility of Applied Genomics Technology Center at Wayne State

University. The method used for testing was short tandem repeat profiling using the PowerPlex

16 System from Promega (Fitchburg, WI) [29].

2.6. In vitro cytotoxicity assay

The In vitro cytotoxicity of free CDF, plain PAMAM, HA-PAMAM, PAMAM-CDF, and

HA-PAMAM-CDF were assessed via MTT assay on MiaPaCa-2 and AsPC-1cell lines. Briefly,

cells were seeded in a 96-well culture plate. The control and test formulations were added 24 h

after seeding as freshly prepared solutions in PBS (pH 7.4) between 100 to 1000 nM

concentrations. MTT (0.5 mg/ml) was added at the end of treatment (72 h) and plates further

incubated at 37°C for 2 h, followed by replacement of media with DMSO at RT in a shaker for

30 min. The absorbance was measured at 595 nm using Ultra multi-functional micro plate reader

10 

 
(TECAN, Switzerland) and percent cell viability was determined by comparing the results with

appropriate controls [5,13].

2.7. Receptor blockade assay

The assay is based on the principle of initial blockade of HA receptor by delivery of

excess free HA (10 mg/ml), followed by treatment with the developed formulations (PAMAM-

CDF, HA-PAMAM-CDF, and free CDF) and investigate its effect on cell viability (MTT assay),

as well as on the IC50 shift. The assay was performed according to the approved protocol

reported previously [27]. In brief, MiaPaCa-2 cells were seeded in a 96-well culture plate for 24

h, followed by treatment with excess of HA (10 mg/ml) for 2 h washed thrice with PBS (pH 7.4)

and incubation for 2 h. Laboratory protocol for MTT assay, as stated in previous section, was

performed to determine the cell viability.

2.8. Fluorescence microscopic studies

For the fluorescence microscopic studies, firstly each formulation was labeled with FITC

(5:1 mole: mole; 10 mg/ml in DMSO) after incubation for 8 h at RT (37±0.5°C) with intermittent

mixing. MiaPaCa-2 cells (5 × 104) were seeded in four-well chamber slide and incubated at

37°C under 5% CO2 for 24 h. The medium was replaced with 2 ml of serum-free, antibiotic-free

medium containing various concentrations of labeled formulations, and incubated for different

time intervals. The formulation containing medium was removed, and resulting cells were

washed thrice with PBS and fixed with 2% formaldehyde in the PBS at RT for 10 min, and the

samples were qualitatively analyzed using a fluorescent microscope (Leica, Germany) [27,30].

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2.9. Stability studies

Stability studies were performed to validate the developed formulation in terms of

physical change/stability and drug leakage. For this purpose, the non-targeted and targeted

dendrimer formulations were separately placed in dark in amber colored and colorless glass vials

at 0±2°C (T1), ambient RT (27±2°C) (T2) and 60±2°C (T3) in controlled oven for a period of

five weeks. Samples were analyzed every week for any visual changes like precipitation,

turbidity, crystallization, color and drug leakage (however only terminal results were reported).

The samples were analyzed for drug leakage by UV spectrometry (Jasco 530 UV-Visible

spectrometer, Tokyo, Japan) [28].

2.10. Statistical analysis

The statistical analysis of data was performed using analysis of variance (ANOVA)

followed by Tukey's multiple comparison test. The results are expressed as mean ± standard

deviation and n showing the number of repeats. A difference of p < 0.05 was considered as

statistically significant.

3. Result and discussion

3.1. Synthesis and characterization of HA-PAMAM conjugate

HA-PAMAM conjugate was synthesized by formation of an amide linkage between the

peripheral –NH2 groups of 5.0G PAMAM dendrimer and –COOH groups of HA in the

presence of EDC. The structural characterization of HA-PAMAM conjugate was confirmed

by 1H-NMR and FTIR spectroscopy. The 1H NMR spectra of HA-PAMAM in D2O showed the

specific peaks of methyl group (NHCOCH3) and methenyl group (CHOH) of HA at 1.9 ppm
12 

 
and 3.1 – 4.6 ppm, respectively. Peaks of PAMAM corresponding to NCH2CH2CO,

CONHCH2CH2N, CONHCH2CH2N, CONHCH2CH2NH2 and NCH2CH2NHCO at 2.2, 2.4,

2.6, 3.1 and 3.25 ppm, respectively confirmed the formation of HA-PAMAM conjugate. The

FTIR spectra of HA-PAMAM showed stretching vibration peak of the –OH group of HA at

3445.22 cm−1 that further confirmed the formation of HA-PAMAM conjugate (Fig. 1).

The electron microscopic analysis of PAMAM and HA-PAMAM dendrimers confirmed

their nanometric size. TEM data revealed higher size of HA-PAMAM conjugate (9.3 ± 1.5 nm)

as compared to PAMAM dendrimers (7.6 ± 1.7 nm), respectively that further confirmed the

conjugation of HA to PAMAM (Fig. 2a).

3.2. Drug loading

CDF loaded dendrimer formulations were synthesized by equilibrium dialysis method.

PAMAM-CDF dendrimers revealed 17.26% ± 2.88% w/w CDF entrapment efficiency;

however HA-PAMAM-CDF showed significantly higher CDF encapsulation efficiency of

24.54% ± 3.51% w/w. The conjugation of the HA moiety resulted in 1.42 times increased CDF

encapsulation compared with 4.0G PAMAM dendrimer. A significantly (P < 0.05) better

entrapment efficiency was observed due to augmented architecture supplemented by additional

crowding of ligand (HA molecules) on the surface of HA-PAMAM dendrimer. When a larger

number of bulky HA moieties were conjugated to the PAMAM periphery, they created more

space, which can accommodate more number of drug molecules. Our results are in accordance

with the previous reports, which showed similar pattern of drug entrapment in surface

conjugated dendrimers [13,31,32].

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3.2.1. Zeta potential measurement

The zeta potential of PAMAM-CDF and HA-PAMAM-CDF was found to be

12.8 ± 8.66 and −7.02 ±9.53 mV, respectively. As compared to plain formulation, HA-

PAMAM-CDF showed significantly negative zeta potential due to coupling of the HA. This

may be attributed to masking of the cationic surface charge by the negatively charged

hyaluronan coating. The negative surface of the HA-PAMAM dendrimer is less prone to cause

adverse side effects when used for drug delivery in vivo which is one of the key criteria for

developing safe and effective drug delivery systems (Fig. 2b).

3.2.2. Atomic force microscopic analysis

AFM analysis was performed by immobilizing PAMAM-CDF and HA-PAMAM-CDF

dendrimers on freshly cleaved mica to further verify the size of drug loaded dendrimer

formulations. As observed in the AFM images, small dendrimer aggregates appear to be

embedded in the matrix, which also indicate nano-metric size range, in concordance with TEM

results (Fig. 3) [32].

3.3. In vitro release studies

In vitro release of CDF from various dendrimer formulations was studied at pH 4.0 and

pH 7.4 to evaluate CDF release under acidic tumor environment and physiological conditions

(blood) respectively. The outcome of the investigation clearly displayed pH responsive and slow-

sustained CDF release patterns from all the nano-formulations compared to free CDF. Plain

PAMAM based formulation (PAMAM-CDF) released more than half of the drug

(52.37 ± 2.72%@pH 4.0 and 47.56 ± 2.88%@pH 7.4) in just 2 h, while at the same time point

14 

 
HA engineered formulation (HA-PAMAM-CDF) released only one fourth of the drug

(26.64 ± 1.94%@pH 4.0 and 22.58 ± 2.98%@pH 7.4). Similar pattern of drug release was

observed until the conclusion of the study, indicating that the conjugation of HA on dendrimer

surface results in sustained and delayed CDF release (Fig. 4). The possible reason for delayed

release pattern by HA-PAMAM-CDF may possibly be due to steric hindrance endowed by HA

residues conjugated on the surface of dendrimer, which acts as a hydrophilic protective coating

that may impede drug diffusion and release. The results are in concordance with existing reports

wherein hindered drug release was observed in case of different ligand (folate, dextran and

galactose) conjugated dendrimer compared to unmodified dendrimer counterparts [33].

Chandrasekar et al. have also found similar trend when they compared the release rate for folate

modified versus plain PAMAM dendrimer based formulation for delivery of an anti-arthritic

drug, indomethacin [34].

As we shift from acidic pH (pH 4.0) towards basic pH (pH 7.4), a relatively much slower

and delayed CDF release pattern was observed. At acidic pH, the interior tertiary amine groups

of dendrimer were protonated, causing repulsion of charges. This results in an “extended

conformation” of the dendrimer. However, at alkaline pH the tertiary amines remain

deprotonated, causing a collapse of the dendrimer on itself, known as ‘back folding’. Therefore,

alkaline pH resulted in compact, globular dendrimer structure leading to slowed and controlled

release of drug from dendrimer formulations [35,36].

3.4. In vitro cytotoxicity assay

PAMAM dendrimers are known to be highly cytotoxic due to presence of positively

charged amine groups on the periphery. HA was selected to be conjugated to the surface of
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PAMAM dendrimers to shield the positive charge of PAMAM and to actively target CD44

receptors, which is highly expressed on the surface of many types of tumor cells including

pancreatic cancer cells [8,19,31,37]. The ability of dendrimer formulations to inhibit the growth

of MiaPaCa-2 and AsPC-1 human pancreatic cancer cells (CD44-positive tumor cell line) was

evaluated by in vitro MTT cytotoxicity assay between 250-1000 nM concentrations. Fig. 5

shows that all the formulations displayed dose-dependent killing of both MiaPaCa-2 and AsPC-

1cells. HA-PAMAM-CDF exhibited lowest percentage cell viability compared to other

formulations as well as free drug itself. The outcome of the investigation revealed an IC50 of

860 ± 2.43 nM, 750 ± 1.96 nM and 390 ± 2.28 nM for CDF, PAMAM-CDF and HA-PAMAM-

CDF, respectively in MiaPaCa-2 cells. Similar pattern was found in AsPC-1 cells, for which the

IC50 values for free CDF, PAMAM-CDF and HA-PAMAM-CDF were 975 ± 3.42 nM, 860 ±

1.79 nM, and 580 ± 1.55 nM, respectively (Fig. 5). Higher uptake in case of the HA-PAMAM-

CDF was most possibly due to the CD44 receptor specific targeting of dendrimers due to surface

conjugation with HA. Plain HA-PAMAM (drug free) showed no apparent cytotoxicity in both of

the cells. These results indicate that on systemic administration the targeted dendrimer nano-

formulation may not cause any adverse side-effects. However the results need to be verified

using animal tumor models.

Additionally, it can be noted that CDF containing formulations showed better anticancer

activity in MiaPaCa-2 cells than AsPC-1 cells. The observed results may be attributed to

overexpression of comparatively more CD44 receptors in MiaPaCa-2 cells, which allow more

amount of CDF delivered by the HA conjugated formulation (HA-PAMAM-CDF) within the

cells and displayed better cell killing. Our results are in agreement with reported literature [5,26].

16 

 
3.5. Receptor blockade assay

The objective of this assay was to reconfirm that the activity of HA-SMA-CDF was due

to specific targeting of CD44 receptor. For this, MTT assay was also performed in presence of

excess free HA (10 mg/ml) to recognize the fate of HA targeting upon blockade of CD44

receptor. Before blockade of HA receptors, the IC50 values were found to be 814 ± 10.58 nM,

719 ± 7.48 nM and 405 ± 6.32 nM for CDF, PAMAM-CDF and HA-PAMAM-CDF,

respectively. However, after blockade of HA receptors, the IC50 values was found to be 826 ±

4.71 nM, 727 ± 9.34 nM and 694 ± 8.36 nM for CDF, PAMAM-CDF and HA-PAMAM-CDF,

respectively. For free CDF and PAMAM-CDF treated cells, no significant difference in the

cytotoxicity curve was observed; however, in case of HA-PAMAM-CDF, a clear upward shift of

cytotoxicity curve was observed, inferring CD44 receptor to be a prime pathway for the uptake

of targeted HA-PAMAM-CDF formulation (Fig. 6).

3.6. Fluorescence microscopic studies

MiaPaCa-2 cell line was selected for fluorescence microscopic studies based on the MTT

results, which revealed better response to the CDF loaded dendrimer formulation compared with

AsPC-1. To examine whether HA-PAMAM-CDF enters MiaPaCa-2 cells mainly through CD44

receptor-mediated trans-membrane transport, we firstly labeled them with FITC and incubated

with MiaPaCa-2 cells for 4 h. As shown in Fig. 7, free FITC did not show any fluorescence in

cells; however the fluorescence intensity of FITC-labeled dendrimer formulations was obvious.

The high uptake of dendrimer formulations could be attributed to efficient endocytosis followed

by release of high dose of FITC dye in the cells. HA-PAMAM-FITC displayed highest

fluorescence intensity. The higher uptake was possibly due to the presence of hyaluronate
17 

 
residue on the surface of the dendrimer compared to non-targeted formulation (PAMAM-FITC)

(Fig. 7). The results are in agreement that the HA anchored dendrimers entered into the cancer

cell by receptor mediated endocytosis as CD44 is well known to be overexpressed in MiaPaCa-2

cell line [38]. Similarly Luo et al. reported that HA-modified and DOX conjugated N-(2-

hydroxypropyl) methacrylamide (HPMA) polymer (HA-HPMA-DOX) is capable of selectively

targeting CD44-overexpressing tumor cells compared to non-targeted HPMA-DOX conjugates.

Authors form this study reported that the uptake of HA-HPMA-DOX conjugates into CD44

overexpressed tumor cells increased quickly with time while tumor cell uptake of non-targeted

HPMA-DOX increased slowly with incubation time [39].

3.7. Stability studies

Stability study data revealed that the dendrimer formulations were most stable at RT in

dark. No visual changes were observed in the formulation stored at RT of dendrimer formulation

in dark condition. However, after storage at T1 (light and dark), T3 (light and dark) and T2

(light), slight turbidity was observed in dendrimer formulations, which might be due to

aggregation of dendrimers. At T3, formulations showed higher turbidity, which may possibly be

due to formation of larger aggregates (Table 1).

The drug release from the dendrimer formulation was used to evaluate their integrity. It is

possible that the presence of HA on the dendrimer surface imparts steric stabilization. In general,

dendrimer formulations (PAMAM-CDF and HA-PAMAM-CDF) were found to be most stable at

RT in dark. Comparatively, drug leakage was higher in presence of light than in dark, which may

be ascribed to chemical degradation due to exposure to higher temperature and light. Higher drug

leakage observed at T3 may probably be due to increased solution kinetics; however at 0°C
18 

 
shrinking of dendrimer formulations may be the possible reason leading to decreased cavity

enclosing drug molecules and hence higher leakage. The lesser drug leakage from HA

engineered formulation might be ascribed to surface conjugation of HA, which imparts rigidity

to the dendrimers. In this case the dendrimer architecture is a rigid structure with a tightly

hydrogen-bonded compact periphery. Thus, HA conjugated dendrimers can display identical

properties in which the periphery is almost sealed by bulky groups [27,28,40]. Similar findings

were found in our previous report, wherein folate conjugated PPI dendrimers showed better

stability profile compared to plain dendrimer formulations [27,28].

4. Conclusions

In summary, our current work is a step forward in utilizing dendrimer based drug-

delivery strategy exploiting HA as targeting ligands to specifically deliver CDF to pancreatic

cancer cells. In addition, we were successful in reducing the cationic surface charge of native

PAMAM increasing the prospect of its clinical translation.

Acknowledgements

The authors wish to acknowledge partial support for this work by Wayne State University

Start-up funding to AKI. Authors also wish to acknowledge Dr. Fazlul H. Sarkar's group at

Barbara Ann Karmanos Cancer Institute, Wayne State University, for providing cancer cells

lines used in this study.

Disclosures: There is no conflict of interest and disclosures associated with the manuscript. 

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References

[1] R.L. Siegel, K.D. Miller, A. Jemal, Cancer statistics, 2015., CA. Cancer J. Clin. 65 (2015)
5–29.

[2] S.K. Bunt, A.M. Mohr, J.M. Bailey, P.M. Grandgenett, M.A. Hollingsworth,
Rosiglitazone and Gemcitabine in combination reduces immune suppression and
modulates T cell populations in pancreatic cancer, Cancer Immunol. Immunother. 62
(2013) 225–236.

[3] J. Long, Y. Zhang, X. Yu, J. Yang, D.G. LeBrun, C. Chen, et al., Overcoming drug
resistance in pancreatic cancer, Expert Opin. Ther. Targets. 15 (2011) 817–828.

[4] Y.H. Bae, K. Park, Targeted drug delivery to tumors: myths, reality and possibility, J.
Control. Release. 153 (2011) 198–205.

[5] P. Kesharwani, S. Banerjee, S. Padhye, F.H. Sarkar, A.K. Iyer, Parenterally administrable
nano-micelles of 3, 4-difluorobenzylidene curcumin for treating pancreatic cancers,
Colloids Surfaces B Biointerfaces. 132 (2015) 138–145.

[6] S. Kommareddy, S.B. Tiwari, M.M. Amiji, Long-circulating polymeric nanovectors for
tumor-selective gene delivery, Technol. Cancer Res. Treat. 4 (2005) 615–625.

[7] P. Kesharwani, K. Jain, N.K. Jain, Dendrimer as nanocarrier for drug delivery, Prog.
Polym. Sci. 39 (2014) 268–307.

[8] P. Kesharwani, R.K. Tekade, N.K. Jain, Dendrimer generational nomenclature: the need to
harmonize, Drug Discov. Today. 20 (2015) 497-499.

[9] P. Kesharwani, S. Banerjee, U. Gupta, M.C.I. Mohd Amin, S. Padhye, F.H. Sarkar, et al.,
PAMAM dendrimers as promising nanocarriers for RNAi therapeutics, Mater. Today.
(2015) In press. doi:10.1016/j.mattod.2015.06.003.

[10] S. Jain, P. Kesharwani, R.K. Tekade, N.K. Jain, One platform comparison of
solubilization potential of dendrimer with some solubilizing agents, Drug Dev. Ind.
Pharm. 41 (2015) 722-727.

20 

 
[11] K. Borowska, S. Wołowiec, K. Głowniak, E. Sieniawska, S. Radej, Transdermal delivery
of 8-methoxypsoralene mediated by polyamidoamine dendrimer G2.5 and G3.5--in vitro
and in vivo study, Int. J. Pharm. 436 (2012) 764–770.

[12] S. Sadekar, G. Thiagarajan, K. Bartlett, D. Hubbard, A. Ray, L.D. McGill, et al.

Poly(amido amine) dendrimers as absorption enhancers for oral delivery of camptothecin.,
Int. J. Pharm. 456 (2013) 175–185.

[13] P. Kesharwani, R.K. Tekade, V. Gajbhiye, K. Jain, N.K. Jain, Cancer targeting potential
of some ligand-anchored poly(propylene imine) dendrimers: A comparison,
Nanomedicine Nanotechnology, Biol. Med. 7 (2011) 295–304.

[14] R. Duncan, L. Izzo, Dendrimer biocompatibility and toxicity, Adv. Drug Deliv. Rev. 57
(2005) 2215–2237.

[15] K. Jain, P. Kesharwani, U. Gupta, N.K. Jain, Dendrimer toxicity: Let’s meet the challenge,
Int. J. Pharm. 394 (2010) 122–142.

[16] P. Kesharwani, V. Mishra, N.K. Jain, Generation dependent hemolytic profile of folate
engineered poly(propyleneimine) dendrimer, J. Drug Deliv. Sci. Technol. 28 (2015) 1–6.

[17] P. Kesharwani, A.K. Iyer, Recent advances in dendrimer-based nanovectors for tumor-
targeted drug and gene delivery, Drug Discov. Today. 20 (2015) 536-547.

[18] J.-H. Park, H.-J. Cho, H.Y. Yoon, I.-S. Yoon, S.-H. Ko, J.-S. Shim, et al., Hyaluronic acid
derivative-coated nanohybrid liposomes for cancer imaging and drug delivery., J. Control.
Release. 174 (2014) 98–108.

[19] Q. Zhao, H. Geng, Y. Wang, Y. Gao, J. Huang, Y. Wang, et al., Hyaluronic acid
oligosaccharide modified redox-responsive mesoporous silica nanoparticles for targeted
drug delivery, ACS Appl. Mater. Interfaces. 6 (2014) 20290–20299.

[20] S. Padhye, S. Banerjee, D. Chavan, S. Pandye, K.V. Swamy, S. Ali, et al.,

Fluorocurcumins as cyclooxygenase-2 inhibitor: molecular docking, pharmacokinetics and
tissue distribution in mice, Pharm. Res. 26 (2009) 2438–2445.

21 

 
[21] S. Padhye, H. Yang, A. Jamadar, Q.C. Cui, D. Chavan, K. Dominiak, et al., New Difluoro
Knoevenagel Condensates of Curcumin, Their Schiff Bases and Copper Complexes as
Proteasome Inhibitors and Apoptosis Inducers in Cancer Cells, Pharm. Res. 26 (2009)
1874–1880.

[22] L. Li, F.S. Braiteh, R. Kurzrock, Liposome-encapsulated curcumin: in vitro and in vivo
effects on proliferation, apoptosis, signaling, and angiogenesis., Cancer. 104 (2005) 1322–
1331.

[23] B. Bao, S. Ali, D. Kong, S.H. Sarkar, Z. Wang, S. Banerjee, et al., Anti-tumor activity of a
novel compound-CDF is mediated by regulating miR-21, miR-200, and PTEN in
pancreatic cancer., PLoS One. 6 (2011) e17850.

[24] B. Bao, S. Ali, S. Banerjee, Z. Wang, F. Logna, A.S. Azmi, et al., Curcumin analogue
CDF inhibits pancreatic tumor growth by switching on suppressor microRNAs and
attenuating EZH2 expression, Cancer Res. 72 (2012) 335–345.

[25] S.K. Basak, A. Zinabadi, A.W. Wu, N. Venkatesan, V.M. Duarte, J.J. Kang, et al.,
Liposome encapsulated curcumin-difluorinated (CDF) inhibits the growth of cisplatin
resistant head and neck cancer stem cells, Oncotarget. 6 (2015) 18504-18517.

[26] P. Kesharwani, S. Banerjee, S. Padhye, F.H. Sarkar, A.K. Iyer, Hyaluronic acid
engineered nano-micelles loaded with 3, 4-difluorobenzylidene curcumin for targeted
killing of CD44+ stem-like pancreatic cancer cells, Biomacromolecules. 16 (2015) 3042-
3053.

[27] P. Kesharwani, R.K. Tekade, N.K. Jain, Generation dependent safety and efficacy of folic
Acid conjugated dendrimer based anticancer drug formulations, Pharm. Res. 32 (2015)
1438–1450.

[28] P. Kesharwani, R.K. Tekade, N.K. Jain, Formulation development and in vitro-in vivo
assessment of the fourth-generation PPI dendrimer as a cancer-targeting vector,
Nanomedicine (Lond). 9 (2014) 2291-308.

22 

 
[29] S. Banerjee, A.S. Azmi, S. Padhye, M.W. Singh, J.B. Baruah, P.A. Philip, et al., Structure-
activity studies on therapeutic potential of Thymoquinone analogs in pancreatic cancer,
Pharm. Res. 27 (2010) 1146–58.

[30] Y. Huang, L. He, W. Liu, C. Fan, W. Zheng, Y.-S. Wong, et al., Selective cellular uptake
and induction of apoptosis of cancer-targeted selenium nanoparticles, Biomaterials. 34
(2013) 7106–7116.

[31] X. Qi, Y. Fan, H. He, Z. Wu, Hyaluronic acid-grafted polyamidoamine dendrimers enable
long circulation and active tumor targeting simultaneously., Carbohydr. Polym. 126
(2015) 231–239.

[32] M. Han, Q. Lv, X.-J. Tang, Y.-L. Hu, D.-H. Xu, F.-Z. Li, et al., Overcoming drug
resistance of MCF-7/ADR cells by altering intracellular distribution of doxorubicin via
MVP knockdown with a novel siRNA polyamidoamine-hyaluronic acid complex, J.
Control. Release. 163 (2012) 136–144.

[33] P. Kesharwani, R.K. Tekade, V. Gajbhiye, K. Jain, N.K. Jain, Cancer targeting potential
of some ligand-anchored poly(propylene imine) dendrimers: a comparison,
Nanomedicine. 7 (2011) 295–304.

[34] D. Chandrasekar, R. Sistla, F.J. Ahmad, R.K. Khar, P. V Diwan, The development of
folate-PAMAM dendrimer conjugates for targeted delivery of anti-arthritic drugs and their
pharmacokinetics and biodistribution in arthritic rats, Biomaterials. 28 (2007) 504–12.

[35] P. Kesharwani, R.K. Tekade, N.K. Jain, Generation dependent cancer targeting potential
of poly(propyleneimine) dendrimer, Biomaterials. 35 (2014) 5539–5548.

[36] Y. Liu, V.S. Bryantsev, M.S. Diallo, W.A. Goddard, PAMAM dendrimers undergo pH
responsive conformational changes without swelling, J. Am. Chem. Soc. 131 (2009)
2798–2799.

[37] M. Gary-Bobo, D. Brevet, N. Benkirane-Jessel, L. Raehm, P. Maillard, M. Garcia, et al.

Hyaluronic acid-functionalized mesoporous silica nanoparticles for efficient
photodynamic therapy of cancer cells, Photodiagnosis Photodyn. Ther. 9 (2012) 256–260.

23 

 
[38] S. Ganesh, A.K. Iyer, D. V. Morrissey, M.M. Amiji, Hyaluronic acid based self-
assembling nanosystems for CD44 target mediated siRNA delivery to solid tumors,
Biomaterials. 34 (2013) 3489–3502.

[39] Y. Luo, N.J. Bernshaw, Z.-R. Lu, J. Kopecek, G.D. Prestwich, Targeted delivery of
doxorubicin by HPMA copolymer-hyaluronan bioconjugates., Pharm. Res. 19 (2002)
396–402.

[40] B. Birdhariya, P. Kesharwani, N.K. Jain, Effect of surface capping on targeting potential
of folate decorated poly (propylene imine) dendrimers., Drug Dev. Ind. Pharm. (2014) 1–
7.

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Figure Captions

Fig. 1. a) FTIR and 1H-NMR spectrum of HA, PAMAM and HA-PAMAM conjugate are shown.

Fig. 2. a) TEM images of PAMAM and HA-PAMAM conjugates, which are highly
homogeneous with an average size of 7.6 and 9.3 nm, respectively; b) Zeta potential of
PAMAM-CDF and HA-PAMAM-CDF dendrimer nano-formulations are shown.

Fig. 3. a) AFM images of PAMAM-CDF and HA-PAMAM-CDF dendrimer nano-formulations

are shown.

Fig. 4. In vitro CDF release profile of PAMAM-CDF and HA-PAMAM-CDF dendrimer nano-
formulations at different pH are shown (n=3).

Fig. 5. Percent cell viability as measured by MTT assay observed at 72 h after treating MiaPaCa-
2 and AsPC-1 pancreatic cancer cells with free drug and various nano-formulations are shown
(n=8).

Fig. 6. Percent cell viability as measured by MTT assay observed at 72 h after HA receptor
blocking and treating MiaPaCa-2 cells with free drug and dendrimer nano-formulations are
shown (n=8).

Fig. 7. Fluorescence microscopic images (40X) of MiaPaCa-2 cells incubated with FITC tagged
CDF loaded HA conjugated PAMAM dendrimer (FITC-PAMAM-CDF), and FITC tagged CDF
loaded HA conjugated PAMAM dendrimer (FITC-HA-PAMAM-CDF) at 4 h are shown.

Scheme 1. Schematic depicting the method used to synthesize CDF-loaded HA conjugated

PAMAM dendrimer nano-system and their uptake in pancreatic cancer cells over-expressing
CD44 receptors. The HA-PAMAM-CDF targeted nano-systems are selectively taken up by
tumor cells over-expressing CD44 via receptor-mediated endocytosis. The CDF is released at
acidic endolysosomes, followed by its release into the cytoplasm for therapeutic action.

Scheme 2. Synthesis of HA-PAMAM conjugates using EDC coupling method.

 

25 

 
 Figure 1
26 

 
 Figure 2

27 

 
 Figure 3

28 

 
 Figure 4

29 

 
 Figure 5

30 

 
 Figure 6

31 

 
 Figure 7

32 

 
 Scheme 1

33 

 
 Scheme 2

34 

 
Tables

Table 1. Accelerated stability studies on CDF loaded Plain PAMAM and HA conjugated
dendrimers formulations [Results are represented as MeanSD (n = 3)].

PAMAM-CDF HA-PAMAM-CDF
Stability
Dark Light Dark Light
Parameter
T1 T2 T3 T1 T2 T3 T1 T2 T3 T1 T2 T3

Turbidity + + ++ ++ - +++ + - + + - +

Precipitation + + ++ + - ++ + - - + - -

Color - - + + + +++ - - - - - +

Crystallization + - + + + +++ - - - - - +

Percent drug leakage (after weeks)

1 1.9±0.2 0.9±0.03 3.3±0.1 2.1±0.5 1.1±0.1 6.5±0.3 1.2±0.2 0.2±0.2 1.4±0.2 1.4±0.3 0.4±0.05 1.7±0.1

2 2.3±0.3 1.8±0.1 3.5±0.4 2.5±0.4 2.1±0.4 9.4±0.5 1.4±0.1 0.5±0.1 1.6±0.1 1.8±0.8 0.6±0.2 2.1±0.2

3 2.7±0.1 2.2±0.2 6.9±0.3 3.1±0.6 2.5±0.5 13.9±0.6 1.9±0.3 0.7±0.2 2.3±0.4 2.4±0.5 0.9±0.1 2.5±0.3

4 3.1±0.4 2.5±0.2 8.6±0.6 3.9±0.5 2.8±0.2 18.7±0.8 2.3±0.5 0.9±0.1 2.8±0.3 3.1±0.7 1.2±0.1 3.2±0.4

5 3.5±0.5 3.1±0.3 12.5±0.2 4.1±0.2 3.3±0.1 24.5±0.2 2.6±0.6 1.2±0.1 4.2±0.4 3.5±0.3 1.4±0.2 9.1±0.6

T1, T2 and T3 represent 0, 27 and 60°C temperatures, respectively. All the values represent mean ± SD (n=3); “-”
indicates no change; “+” indicates small change; “++” indicates considerable change; “+++”indicates major change;
PAMAM-CDF = Difluorobenzylidene curcumin loaded 4.0 G PAMAM dendrimer; HA-PAMAM-CDF =
Difluorobenzylidene curcumin loaded hyaluronic acid conjugated 4.0 G PAMAM dendrimer.

35