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

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: DOI: Reference:

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

To appear in:

Colloids and Surfaces B: Biointerfaces

Received date: Revised date: Accepted date:

10-8-2015 22-9-2015 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 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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

Prashant Kesharwania, Lingxiao Xieb, Guangzhao Maob, Subhash Padhyec, Arun K. Iyera,d* [email protected] 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. 

1   

Graphical abstract

2   

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.

3   

ABSTRACT The current study was aimed to develop a targeted dendrimer formulation of 3, 4difluorobenzylidene 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 CD44targeted 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 MiaPaCa2 cells using free excess soluble HA prior to treatment with HA-PAMAM-CDF nanoformulation 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, 4difluorobenzylidene curcumin; drug delivery

4   

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 3,4difluorobenzylidene 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 SigmaAldrich (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, airdried, 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 unentrapped 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 (PAMAMCDF, 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].

11   

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].

13   

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, HAPAMAM-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 slowsustained 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 15   

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 AsPC1cells. 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-PAMAMCDF, 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-PAMAMCDF 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 nanoformulation 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-(2hydroxypropyl) 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 drugdelivery 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. 

19   

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 tumortargeted 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) 30423053. [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., Structureactivity 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 selfassembling 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.  

24   

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 nanoformulations at different pH are shown (n=3). Fig. 5. Percent cell viability as measured by MTT assay observed at 72 h after treating MiaPaCa2 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 Stability

Dark

Parameter

HA-PAMAM-CDF Light

Dark

Light

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