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Receptor-Mediated Attachment and Uptake of Hyaluronan Conjugates by Breast Cancer Cells Kush Shah, Andrew J Ditto, Douglas Cale Crowder, Jean H. Overmeyer, Hossein Tavana, William A Maltese, and Yang H. Yun Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00636 • Publication Date (Web): 05 Oct 2017 Downloaded from http://pubs.acs.org on October 6, 2017

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Molecular Pharmaceutics

Receptor-Mediated Attachment and Uptake of Hyaluronan Conjugates by Breast Cancer Cells Kush. N. Shah,1,2,3 Andrew. J. Ditto3,4 Douglas. C. Crowder,1,5 Jean. H. Overmeyer,3 Hossein Tavana,1 William. A. Maltese,4 Yang. H. Yun1,* 1

Department of Biomedical Engineering, University of Akron, Akron, OH 44325, USA  2

3

Department of Integrated Biosciences, University of Akron, Akron, OH 44325, USA

Department of Microbial Pathogenesis and Immunology, Texas A&M Health Science Center, Bryan, TX 77807, USA 4

Biochemistry and Cancer Biology, College of Medicine and Life Sciences, University of Toledo, Toledo, OH 43614, USA

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Department of Biomedical Engineering, Case Western Reserve University, Cleveland, OH 44016, USA

ACS Paragon Plus Environment

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KEYWORDS. Hyaluronan, Cancer, Resveratrol, Drug Delivery, Targeted Chemotherapy, Pendant-Chain System

ABSTRACT. Chemotherapy, a mainstay modality for cancer, is often hindered by systemic toxicity and side effects. With the emergence of nanomedicine, the development of drug therapy has shifted toward targeted therapy. Hyaluronan (HA) is an ideal molecule as a targeted delivery system because many carcinomas overexpress HA receptors. We have conjugated resveratrol, a natural polyphenol, and 2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one (MOMIPP), a chalcone, to HA with the goal of enhancing drug bioavailability and targeting triple negative breast cancers. We demonstrate the ability of HA conjugates to accumulate in the tumor interstitium within six hours after tail vein injections. In vitro, these conjugates interact with their target receptors, which are selectively over-expressed by triple negative breast cancer cells under static and physiological flow. These interactions result in enhanced uptake and efficacy of the therapeutic, demonstrated by a reduced IC50 over non-conjugated drugs. Thus, HA offers a platform to solubilize, target, and enhance the efficacy of chemotherapeutics.

1. INTRODUCTION Carcinomas frequently arise from mutations leading to aberrant phenotypes, including rapid cell division, elevated metabolism, and altered expression of cell-surface receptors. For instance, HER2 an overexpressed surface receptor, linked to the degree of invasive nature of breast tumors, has become an important biomarker for diagnosis and target for immunotherapy and nanomedicine.1 Thus, identification of new target receptors that are either over-expressed2 or exclusively expressed2 has been an intensive area of research for both cancer and drug-delivery.


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Drugs that can be modified for receptor-medicated targeting can minimize side effects in contrast to conventional anti-neoplastics, such as paclitaxel. HA receptors are attractive targets since they are highly over-expressed by many carcinomas including breast,3 liver,4 lung,5 colon,6 bladder,7 ovaries,6 and cancer-stem cells8 and multiple pathways are signaled upon binding their ligand. The receptors that bind HA, a major extracellular matrix component, include CD44, RHAMM, LYVE-1, and layilin.9 Of these, CD44 and RHAMM play a key role in formation, progression, and metastasis of tumors10 as well as development of multi-drug resistance.11 Once drugs are conjugated to HA, the pendant-chain system can enhance uptake using these receptors resulting in elevated intracellular drug concentrations. Thus, this strategy has the potential of improving the efficacies of chemotherapeutic drugs with high IC50 concentrations into clinically viable therapies.12 HA is generally a high molecular weight glycosaminoglycan with multiple functional groups available to form hydrogen bonds with water. The high water solubility of HA is an attractive characteristic for conjugating hydrophobic chemotherapeutics or formulating liposomes13 and nanoparticles.14 Pendant-chains with ester and amide bonds have been used for attaching chemotherapeutics.9 In addition to selectivity and solubility, high molecular weight HA lacks an immune response and does not denature.14 Thus, we utilize HA as a targeted drug-delivery platform for resveratrol and MOMIPP. Natural metabolites, such as resveratrol, have captured widespread attention because of several bioactivities including, anti-proliferative and anti-oxidant activities.15 The anticancer activity of resveratrol is a result of multiple mechanisms of action including the activation of p53,16 up-regulation of Fas,17 and the inhibition of AP-1,16 MAPK,18 NFκB,16 MEK,16 and tubulin polymerization.17 Resveratrol can cause cell cycle arrest and induce apoptosis in


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carcinomas.16, 17 MOMIPP, a chalcone-based drug, has a unique mechanism of action that is not dependent on DNA damage or other apoptosis associated triggers.19 Specifically, the exposure of cancer cells to MOMIPP reduces Rab5-GTP and increases Rab7-GTP that arrests recycling of macropinocytic vesicles and locks them in an intermediate stage.20, 21 Down-stream effects are fusion of vesicles forming larger vacuoles, reduction in metabolic activity, and rupture of the cellular membrane.19 This mechanism has been termed ‘methuosis’, which connotes in Greek as drinking to death, and is characterized by extensive accumulation of cytoplasmic vacuoles.19, 22 Resveratrol and MOMIPP are hydrophobic and require organic solvents, such as ethanol and DMSO, for solubility in aqueous solutions. Supplementing cell culture media with small amount of these solvents is common technique in vitro, but this practice cannot be translated to clinical trials. Without these solvents, the bioavailability of these drugs is poor in vivo. In addition, both therapeutics also lack specificity towards cancer cells, which further reduces their bioavailability. Resveratrol also has rapid elimination from the blood stream via glucuronidation and sulfation.15 To enhance solubility of resveratrol and MOMIPP and add targeting, we have conjugated these drugs to HA (HA-R and HA-M, respectively) forming pendant-chain systems. These HA conjugates have been investigated for receptor-mediated attachment and uptake with cancer cells that overexpress HA receptors (HeLa, MDA-MB-231, and MDA-MB-157), a cancer cell that has relatively low expression of HA receptors (MCF7), and control cells (MCF10A and human dermal fibroblasts) in both static and physiological flowing conditions.10,

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Once receptor-

mediated attachment and uptake has been quantified, the efficacy of HA-M as a potential candidate for nanomedicine has been evaluated using an orthotopic tumor model.

2. EXPERIMENTAL SECTION


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2.1 SYNTHESIS AND CHARACTERIZATION OF HA CONJUGATES A mixture of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, SigmaAldrich, St. Louis, MO) and resveratrol (Sigma-Aldrich, St. Louis, MO) or MOMIPP (mole ratio of 1:5 and 1:10 to HA, respectively) dissolved in ethanol was added to a 2.5 mg/mL aqueous HA (6 – 8 x 106 Daltons, Kraeber & Co. GmbH, Waldhofstr, Germany) solution. The reaction (Scheme 1) was allowed to progress overnight at room temperature under an argon blanket and in absence of light. Subsequently, the reaction was dialyzed for 12 hours to remove ethanol, unreacted reagents, and by-products. HA-R was collected and washed with dichloromethane to remove all traces of unreacted resveratrol. For HA-M, the unreacted MOMIPP precipitates were removed by centrifugation. The aqueous solution containing HA conjugates was collected, shell frozen, and lyophilized (Labconco Freezone 4.5, Kansas City, MO) to constant weight. All the HA conjugates were also protected from light and stored in desiccator cabinets after synthesis. The HA conjugates prior to experimentation were reconstituted with sterilized distilled and deionized water to perform characterization studies and determine the degree of conjugation. HA conjugates were dissolved in deuterium oxide at a concentration of 5 mg/mL, and a 1HNMR (Agilent Technologies Varian 500 MHz, Santa Clara, CA) was acquired and analyzed. The degree of substitution of resveratrol or MOMIPP was determined using UV-VIS spectrophotometer (Molecular Devices SpectraMax M2, Sunny Vale, CA). A standard curve of the free drug was generated at 300 nm, and compared to the absorbance from 1.0 mg/mL HA-R or HA-M solution to determine the percentage drug substitution (w/w) for the conjugate. Additionally, TLC was performed on HA-M, HA, and MOMIPP using mobile phase of 1:1 for acetonitrile:water (Sigma-Aldrich, St. Louis, MO), respectively, with and without an anisaldehyde stain to verify covalent conjugation of MOMIPP to HA.


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Scheme 1. Reaction scheme for synthesis of HA conjugates.

2.2 ATTACHMENT OF HA AND HA-R TO BREAST CANCER CELLS HA, HA-R, and resveratrol were tagged with 5-(and-6)-Carboxytetramethylrhodamine, Succinimidyl Ester (TAMRA, Ex/Em: 547/574 nm, ThermoFisher Sci., Waltham, MA) to yield HA-TAMRA, HA-R-TAMRA, and R-TAMRA. These reactions were performed according to manufacturer’s recommended protocol using 20 mg HA or HA conjugates. TAMRA was dissolved in DMF (ThermoFisher Sci., Waltham, MA) at a concentration of 10 mg/ml and placed into a round bottom flask containing a magnetic stir bar. HA, HA-R, or HA-M were dissolved into 0.1M sodium bicarbonate buffer at a concentration of 20 mg/ml. HA or HA conjugates were slowly added to the TAMRA solution while stirring. This mixture was incubated for an hour at room temperature while under constant stirring and protected from light. The reaction stopped by adding 0.1 mL of freshly prepared 1.5 M hydroxylamine (pH 8.5, ThermoFisher Sci., Waltham, MA) and incubated for one hour at room temperature. HA or HA conjugates were purified using dialysis as described in the Section 2.1. The final product was purified using dialysis with a 90% yield. The tagged HA conjugates were reconstituted, aliquoted, and stored as for further use.


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2.2.1

ATTACHMENT

OF

HA,

RESVERATROL,

AND

HA-R

UNDER

STATIC

CONDITIONS MDA-MB-157 (ATCC, Manassas, VA) seeded in a 24-well tissue culture plate were incubated with HA-TAMRA, HA-R-TAMRA, or R-TAMRA (dissolved in 1% ethanol) for 15, 90, or 180 minutes to quantify their attachment (n = 3). The amount of HA conjugates was based upon 25 µM of resveratrol. After incubation, cells were washed five times with phosphate buffered saline (PBS, pH 7.4), fixed with 0.5% formaldehyde, and imaged using an AxioCam HRm camera (Carl Zeiss, Peabody, MA) on a fluorescence microscope (Carl Zeiss Axiovert 200, Peabody. MA). Cells with a positive signal can be visualized using a rhodamine filter cube and when exposed to the appropriate wavelength. The total cell count was performed for quantifying the positive attachment of HA to its cell surface receptors and determine the targeting efficacy.

2.2.2 ATTACHMENT AND UPTAKE OF HA AND HA-R UNDER PHYSIOLOGICAL FLOW Attachment of HA-TAMRA was investigated with HeLa (ATCC, Manassas, VA), MDAMB157 (ATCC, Manassas, VA), MDA-MB231 (ATCC, Manassas, VA), MCF7 (ATCC, Manassas, VA), MCF10A (ATCC, Manassas, VA), and primary human dermal fibroblasts (HDF) under physiological flow using a previously established protocol.12 Briefly, cells were seeded on a 35 mm tissue culture dish pre-coated with 5.5 µg/mL of fibronectin at a cell density of 200,000 cells per dish (n = 3). Once confluent, the cell culture media was aspirated, and a parallel plate flow chamber with a 0.254 mm gasket was mounted on the cell culture dish. HATAMRA was diluted in Hank’s Balanced Salt Solution (HBSS, Medicatech, Corning, NY) containing 0.5% bovine serum albumin (BSA, ThermoFisher Sci., Waltham, MA) to a


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concentration of 0.25 mg/mL and perfused over the cells at a physiological shear stress of 0.5 dynes/cm2. Afterwards, unattached HA-TAMRA was removed by washing the cells with 5 mL of HBSS with 0.5% BSA. Random images were captured using an AxioCam HRm camera on a Zeiss Axiovert 200 microscope. Cells with a positive fluorescence signal from TAMRA SE were counted and normalized with total number of cells. These studies were repeated with MDA-MB-157 by perfusing HA-R-TAMRA, as previously described, to determine if the presence of the drug hinders the receptor-HA interactions. Afterwards, cells were fixed with 0.5% formaldehyde, mounted with Fluoromount G (SouthernBiotech, Birminghan, AL), and imaged using a Zeiss 510 META laser scanningmodule on an Axiovert microscope (Carl Zeiss, Peabody, MA) with a 543 nm laser excitation and a 100X oil-immersion objective to determine the uptake of HA conjugates. A competitive binding assay was performed with un-labeled HA and HA-TAMRA to verify receptor-ligand interactions. MDA-MB231 and MCF7 were seeded as described above. Confluent monolayers of these cells were then perfused with a 10 mL solution containing a mixture of 0.1% HA-TAMRA and unlabeled HA (0, 0.4, 0.9, 2.4, or 4.9 mg/mL). Cells were analyzed as previously described to determine the inhibition of attachment by unlabeled HA.

2.3 EFFECT OF HA-R ON MIGRATION OF BREAST CANCER CELLS Anti-migratory studies of HA-R using MDA-MB-157 were performed using a polymeric aqueous two phase mediated cell-exclusion patterning technique reported by Ham et al.24 Briefly, cells micro-patterned in a 96 well plate with an initial gap area of 3.33 ± 0.09 mm2 (A1) were incubated with HA-R at concentrations of 0, 1, 5, 10 µM for 48 hours. Cells were then stained with 2 µM Calcein AM (Sigma-Aldrich, St. Louis, MO) for 30 minutes and imaged using


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an inverted fluorescent microscope (Carl Zeiss AxioObserver, Peabody MA) equipped with an AxioCam MRm camera. Pre-migration and post-migration images were computed using a script developed in Matlab (MathWorks, Natick MA) to calculate the initial (A1) and final gap closure area (A2). The gap closure or the ability of cells to migrate was calculated using the equation (A1 – A2)/A1 and plotted against concentration.

2.4 ANTI-CANCER EFFICACY OF HA CONJUGATES In vitro anti-cancer efficacy of HA conjugates and free drugs (n =3) was determined with HeLa, MDA-MB-157, MDA-MB-231, MCF7, MCF10A, and HDF using an alamarBlue® Cell Viability Assay (ThermoFisher Sci., Waltham, MA). Cells seeded at a density of 15,000 cells/well in a 96-well plate were incubated with HA-R, HA-M, resveratrol (dissolved in 1% ethanol), and MOMIPP (dissolved in 1% DMSO) at concentrations of 100, 50, 25, 12.5, 6.25, 3.125, and 1.56 µM for 72 hours. Finally, an alamarBlue® Cell Viability Assay was performed to determine the cell viability. Control groups comprising of a mixture of HA and resveratrol (dissolved with 1% ethanol), HA and MOMIPP (dissolved with 1% DMSO), and HA were also tested. These toxicity results were verified using a Live/Dead® Cell Viability Assay (ThermoFisher Sci., Waltham, MA) according to manufacturer’s protocol (Supplementary Information).

2.4.1 CELL RELAPSE WITH HA-M Regrowth of cells following a one-time incubation with HA-M was determined using a Live/Dead® Cell Viability assay. HeLa cells were seeded at a density of 25,000 cells/well in a 24 well plate and allowed to attach overnight. Cells were then incubated with 3.12 µM MOMIPP or


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HA-M. After three days of incubation, the drug solution was aspirated, cells were washed five times with PBS, and drug-free feeding media was added to each well. Cells were incubated for an additional three days, and a Live/Dead® Cell Viability assay was performed. Two control groups of cells incubated for a period of three or six days under identical conditions were also analyzed. C-12 resazurin and SYTOX® stained cells were illuminated and imaged using fluorescence microscopy.

2.5 IN VIVO EFFICACY OF HA-M All animal experiments were performed using a protocol approved by institution animal care and use committee at the University of Toledo (IACUC # 107491). An orthotopic breast cancer model was established by injecting 1 million MDA-MB-231-luc cells suspended in 100 µL matrigel basement membrane matrix, in the mammary fat pad of six-week-old athymic nude mice (Jackson Laboratory, Bar Harbor ME). Progress of tumor growth was determined by injecting the mice with Xenolight Rediject D-Luciferin and visualizing tumors every 6 days using an IVIS imaging system (IVISSPE, Perkin Elmer, Waltham MA).

2.5.1 IN VIVO BIODISTRIBUTION OF HA-M After two weeks, ten animals with a palpable tumor were randomly divided into two groups. Mice were then injected with either 100 µL HA-TAMRA or 100 µL HA-M-TAMRA at a 100 µM drug equivalent via tail vein. At six hours post injection, the animals were sacrificed using carbon dioxide and cervical dislocation. Tumors, kidneys, and livers were harvested and fixed in formalin for 24 hours. The fluorescence intensity of HA-TAMRA or HA-M-TAMRA was measured using an IVIS imaging system.


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2.5.2 IN VIVO TUMOR INHIBITION WITH HA-M Nine days post-inoculation, animals with a palpable tumor were distributed in two groups. Mice were injected directly into the tumor with 100 µL HA or HA-M (50 µM equivalent MOMIPP) on day 9, 11, and 15. The animals were sacrificed on day 17. Kidneys, livers, and tumor were harvested and fixed with formalin. Organs were then imaged for gross representation and weighed to determine the efficacy of HA-M.

2.6 STATISTICAL ANALYSIS The lethal dosages at median cell survival (IC50) were calculated using a non-linear regression curve fit for HA-R, HA-M, resveratrol, MOMIPP, mixture of HA and resveratrol, and mixture of HA and MOMIPP. Statistical differences were determined using an ANOVA with Tukey’s post-hoc test for all in vitro data. For in vivo experiments, an outlier analysis was performed and a p-value determined using Mann Whitney test. Statistical relationship between two quantitative variables was established using linear regression. All results were considered significant for p ≤ 0.05.

3. RESULTS 3.1 SYNTHESIS OF HA CONJUGATES The yield of HA conjugates is typically 90% with 4 - 7% w/w resveratrol or MOMIPP substitution as determined by absorbance.

1

H-NMR (Figure S1) shows characteristic

carbohydrate peaks from HA at δ = 4.09, 3.70, 3.60, 3.40, 3.30, 1.90, and 1.20 for both conjugates. In addition, HA-R (Figure S1A) shows characteristic phenolic (δ = 7.14, 7.12, 6.73,


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and 6.26 ppm) and alkyne (δ = 6.86) peaks corresponding to resveratrol, while HA-M (Figure S1B) shows phenolic (δ = 6.0 and 6.20), methine from pyridine (δ = 8.05), and methyl end groups (δ = 3.8 and 2.05) corresponding to MOMIPP. The water peak is observed at δ = 4.75. These results indicate the conjugation of these hydrophobic therapeutics, resveratrol or MOMIPP to HA result in water-soluble complexes. Finally, TLC demonstrate a lack of migration for HAM, which is analogous to HA, and a result of high molecular weight (Figure S2).

3.2 BINDING OF HA-R TO BREAST CANCER UNDER STATIC CONDITIONS Binding of HA-R-TAMRA, HA-TAMRA, and R-TAMRA to MDA-MB-157 under static conditions are shown in Figures 1A and 1C. The attachments for HA-R-TAMRA and HATAMRA (99% and 95%, respectively) at 15-minute exposure time are significantly higher (p < 0.01) as compared to resveratrol (9.5%). The binding of R-TAMRA increases (16% and 37%) as the incubation increases to 90 and 180 minutes, respectively; however, the interactions are significantly lower (p < 0.01) than HA-R-TAMRA and HA-TAMRA. These results indicate enhanced attachment of HA and HA-R to MDA-MB-157 due to the over-expression of HA receptors (see Figure S3 for CD44 staining).10, 23 Additionally, these results show resveratrol conjugation does not hinder interactions between HA and its receptors.

3.3

ATTACHMENT

AND

UPTAKE

OF

HA-R

BY

CANCER

CELLS

UNDER

PHYSIOLOGICAL FLOW Attachment of HA to breast cancer cells was evaluated using a perfusion chamber under conditions that mimic physiological flow conditions. Images captured after perfusion of HA over breast cancer cells show robust binding to all three-breast cancer cell lines as compared to non-


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cancerous controls. Upon quantification (Figures 1D and S4), 95%, 97%, and 97% binding is observed for MDA-MB-157, MDA-MB-231, and MCF7s, respectively, which is significantly higher (p < 0.01) than MCF10A and HDF (66% and 1%, respectively). In contrast, only 44% HeLa exhibit attachment of HA, which is significantly lower (p < 0.05) compared to breast cancer cells. These results indicate that HA exhibits high affinity towards these cancer cells under physiological flow conditions. Additionally, conjugation of resveratrol does not interfere (p > 0.05) with the binding efficacy for MDA-MB-157 (Figure S5). MDA-MB-157 shows 95% positive binding when perfused with either HA-R or HA (Figure S5). Confocal microscopy images of MDA-MB-157 after perfusion with HA-R (Figure 1B) or HA show a fluorescence signal intensifying between 1.0 and 6.0 µm. The fluorescence attenuates thereafter. These results show positive uptake as opposed to non-specific binding on the cell surface. Similar results were observed with HeLa incubated with HA-M-TAMRA (Figure S6). Confocal images show abundance of vacuole formation and localization of HA-M-TAMRA within these structures as indicated by the intensity of the fluorescence signal. This morphology is characteristic of cells undergoing methuosis. The receptor-mediated binding is also supported by perfusing HA-TAMRA mixed with excess of un-labeled HA (Figure 1E and S7). MDA-MB-231 has been chosen for this study because of the their ability to metastasize24 and compared with MCF7, which generally lack this ability.25 In absence of competition, 97% attachment is observed for both cell lines. The attachment of HA-TAMRA is not significantly (p > 0.05) different at concentrations lower than 0.9 mg/mL of un-labeled HA. MDA-MB-23 shows 82% binding of HA-TAMRA when 4.9 mg/mL of un-labeled HA is in competition. Although the number of cells that show positive binding is decreased, a statistically significant (p < 0.05) inhibition is observed. MCF7 cells


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show a dramatic (p < 0.05) reduction in HA-TAMRA binding (28% and 18%) when competing with 2.4 and 4.9 mg/mL of un-labeled HA, respectively. These results denote the bioavailability of HA receptors.

Figure 1. Binding of HA and HA conjugates under static and flowing conditions and uptake of HA. (A) Attachment of HA-R-TAMRA, HA-TAMRA, and R-TAMRA to MDA-MB-157 after a 15 minute incubation (scale bars are 100 µm), (B) Uptake of HA-R-TAMRA by MDA-MB-157 (optically sliced Z-stack using confocal microscopy, scale bars are 50 µm), (C) MDA-MB-157 with positive attachment after 15, 90, and 180 minute incubation under static conditions, (D) Binding of HA-TAMRA to MCF7, MDA-MB-157, and MDA-MB-231, HeLa, MCF10A, and HDF under physiological flow rate of 0.5 dynes/cm2, and (E) HA-TAMRA in competition with un-labeled HA at a concentration of 0.4, 0.9, 2.4, and 4.9 mg/mL with MDA-MB-231 and MCF7 under physiological flow of 0.5 dynes/cm2. The results for D and E are shown for positive signals after a wash step to remove unattached HA-TAMRA. HA and HA-R were dissolved in cell culture media. R-TAMRA was dissolved in cell culture media with 1% ethanol.


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3.4 EFFECT OF HA-R ON MIGRATION OF BREAST CANCER CELLS HA-R at sub-lethal concentrations inhibits the migration of MDA-MB-157 (Figure 2). Incubation with 5 and 10 µM HA-R limits the ability of these cells to migrate into the open area. The gap closure is 50% and 20%, respectively, which is significantly different (p < 0.05) than untreated controls. In contrast, a 10 fold higher concentration of resveratrol is required to achieve similar inhibition as demonstrated previously.24

Figure 2. Effect of HA-R on migration of invasive MDA-MB-157 cells. (A) Images captured after 24-hour exposure to HA-R and live cells stained with Calcein AM (Scale bars are 500 µm), and (B) Gap closure area measured using a script developed in MATLAB.

3.5 ANTI-CANCER EFFICACY OF HA CONJUGATES The activity of HA conjugates was compared to free drug (resveratrol or MOMIPP dissolved using organic solvents) and drug-HA mixtures (unreacted). The IC50 values are shown in Figures 3B and 4B. Conjugation of resveratrol to HA results in significantly higher anti-cancer activity (p < 0.05) then the free drug, especially towards MDA-MB-157 and MDA-MB-231 (Figure 3), which overexpresses HA-receptors.26-29 The IC50 decreases as much as 14- to 25- folds. However, HeLa and MDA-MB-157 show limited improvement of 1.0- and 1.4- fold upon incubation with HA-M over MOMIPP, respectively. MCF7 breast cancer cells, despite their lower receptors expression as compared to MDA-MB-32126-28 show a significant increase (p
0.05) from free drug. We have observed an increase in cell viability upon incubation with low concentrations of resveratrol or resveratrol mixed with HA with several cell lines (Figure 3A). These trends in cell viability were then verified using a Live/Dead® Cell Viability Assay (Figure S9 and S10). HeLa cells were tested to determine their ability to regrow after the initial exposure to MOMIPP because of their high sensitivity to the chemotherapeutic. The results were then analyzed qualitatively using a Live/Dead® Cell Viability assay (Figure 5). Third or sixth day incubation with HA-M or MOMIPP results in similar toxicity profiles. The advantage of conjugation is evident when cells are incubated with the therapeutic for 3 days followed by a three-day incubation with drug-free medium. Under these conditions, cells incubated with MOMIPP show signs of regrowth with greater cell viability compared to third or sixth day of MOMIPP or HA-M treatment. Meanwhile, cells exposed to HA-M continue to die, as demonstrated by similar cell viability to sixth day HA-M and lower cell viability to third or sixth day of MOMIPP exposure.


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Figure 3. Toxicities studies of HA-R. (A) Cellular viability after 72-hour incubation with ○ resveratrol, ● a mixture of HA and resveratrol with ethanol (HA + Resveratrol), and ■ HA-R for MCF7, MDA-MB-231, MDA-MB-157, HeLa, MCF10A, and HDF. (B) Lethal doses at median cell viability for resveratrol, HA + resveratrol, and HA-R. *The non-linear regression curve fit does converge onto a IC50 value.


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Figure 4. Toxicities studies of HA-M. (A) Cellular viability after 72-hour incubation with ○ MOMIPP, ● a mixture of HA and MOMIPP with DMSO (HA + MOMIPP), and ■ HA-M for MCF7, MDA-MB-231, MDA-MB-157, HeLa, MCF10A, and HDF. (B) Lethal dose at median cell for MOMIPP, HA + MOMIPP, and HA-M. *The non-linear regression curve fit does converge onto a IC50 value.


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Figure 5. Cellular relapse studies. HeLa cells incubated with (A) no drug, (B) 3.12 µM HA-M, and (C) 3.12 µM MOMIPP dissolved in DMSO for 3 days, (D) no drug, (E) 3.12 µM HA-M, and (F) 3.12 µM MOMIPP dissolved in DMSO for 6 days, and (G) no drug, (H) 3.12 µM HA-M, and (I) 3.12 µM MOMIPP dissolved in DMSO for 3 days followed by a 3-day incubation with drug free medium, and stained with Live/Dead® Cell Viability assay reagent. Live cells are stained red and dead cells are stained green. Scale bars are 100 µm. 3.6 IN VIVO EFFICACY OF HA CONJUGATES Organs harvested from animals injected with HA-TAMRA or HA-M-TAMRA via tail-vein (Figure 6A) show at least 35-fold higher accumulation in the tumors over the kidneys and livers (p < 0.05, Figure 6B). Moreover, the conjugation of MOMIPP does not alter HA accumulation in the tumors as demonstrated by the fluorescence signal from various organs (p > 0.05, Figure 6B). Palpable tumors when injected with three HA-M-TAMRA does show a significant reduction in tumor mass (p < 0.05, Figure 6C). Treatment with HA-M-TAMRA do not affect kidneys and


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livers, and the mass of these organs are comparable to the HA-TAMRA group (p > 0.05, Figure 6C).

Figure 6. In vivo uptake and efficacy of HA-M. (A) Gross images of explanted tumor, kidney, and liver captured using an IVIS live mice imaging system from animals 6-hour post-injection with HA-TAMRA labeled HA, and HA-M. (B) Quantified fluorescence signal and (C) the mass of explanted organs after 3 treatments with HA or HA-M over a period of 17 days after palpable tumors were established in an orthotopic breast cancer model using MDA-MB-231-luc cells.

4. DISCUSSION


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HA offers a versatile platform for targeted delivery of many chemotherapeutics that lack adequate blood solubility. Conjugating these drugs to a water-soluble polymer effectively addresses the use of organic solvents, which are common in in vitro settings and in mice studies, as well as solvents such as Cremophor EL, known to cause several side-effects in patients30 and has the added benefit of targeting specific set of cellular receptors. For example, targeting triplenegative breast cancers are problematic since they are not sensitive to hormone or Her2 antibody based therapies.10 However, these cells overexpress HA binding receptors;26, 27 therefore, the application of HA as a delivery system could be promising for developing alternative therapies for these tumors. The efficiency of HA conjugates depends on their ability to accumulate in the tumor interstitium and transport the therapeutic across the cellular membrane. Although the uptake mechanisms of HA are well established, 35-fold higher accumulation of the HA conjugate in the MDA-MB-231 tumor as compared to the kidneys and liver validates the strategy of using HA as a targeting molecule. The reduction in tumor mass correlates with the HA-M accumulation and is also consistent with literature.7, 31 The water solubility imparted by conjugation as well as the targeting ability improves the bioavailability of therapeutics and is responsible for tumor attrition. The affinity of tumors to bind HA depends on their microenvironment,9, 32 expression levels, activation states of transmembrane receptors,9, 33 and the amount of shear stress. These receptors are often constitutively active in cancers,34 which results in positive interaction with invasive breast cancer cells as early as 15 minutes. The receptors are saturated within 15 minutes as demonstrated by 100% cellular attachment that is maintained for the duration of the experiment. This binding then leads to internalization of HA conjugates.6 In contrast, the interactions of


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resveratrol (free drug) with breast-cancer cells are significantly lower (p < 0.05), and the binding results could be attributed to non-specific binding. For HA to be an effective targeted-delivery device, it must interact with the target receptors under flowing conditions. The convection, as determined by the shear stress, in the tumor core ranges between 0.007 and 0.015 dynes/cm2 but increases to 0.5 dynes/cm2 at the periphery.12, 35 Successful targeting to a tumor requires binding and endocytosis under these conditions. Triple negative breast cancers exhibit nearly 100% HA binding under physiological flow. Surprisingly, similar results are also observed for MCF7. Although over 90% of individual cells in MCF7 tumors could express CD44 receptors,36 approximately 9-fold lower expression of CD44 mRNA has been documented10,

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and should result in lower receptor density as compared to triple

negative breast cancers. HDF express HA receptors at lower levels or in low affinity states,32 which significantly reduces and even prevents the binding of HA. Unlike HDF, MCF10A demonstrate ~6-fold higher expression of CD44 receptors over non-aggressive MCF7 cells,23 which likely contributes to increased HA-TAMRA attachment. Reduction in HA attachment under static conditions upon pre-incubation with HA has been reported,6, 9, 37 but the washout dynamics could alter these interactions under perfusion. A lower saturation threshold for MCF7 is observed as compared to MDA-MB-231, which may be attributed to the expression patterns of HA binding receptors. The HA binding receptors for MDA-MB-231 are always available as indicated by HA competition study. The perfusion of unlabeled HA up to 4.9 mg/mL did not saturate the bioavailability of HA binding receptors even though the attachment of HA-TAMRA decreases from 97 to 82%. For MCF7, the binding of HA-TAMRA is significantly decreased when in competition with unlabeled HA. This outcome also confirms lower HA receptor density expressed by MCF7.


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The competition results are indicative of classic receptor-ligand interactions. Upon binding with receptors, HA targeted devices may be internalized, or degrade to release the therapeutic in the vicinity of target cells. The first situation is more likely since post-perfusion confocal images show positive uptake of both, fluorescent dye tagged HA and HA-R. Thus, HA targeted devices provide a direct pathway for uptake by multiple receptors including endocytosis. The expression levels of HA synthase (HAS1/2) and hyaluronidase (HYAL1) is responsible for HA rich tumor microenvironment and influences HA attachment.11,

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However, the high concentration of

unlabeled HA required for inhibition suggests that indigenous HA, which is often secreted in picogram quantities per cell by MDA-MB-231,39 is unlikely to prevent the conjugate-receptor interactions. HA upon binding with its receptors plays a critical role in metastasis and wound healing by initiating cell migration.40, 41 The expression levels of HA receptors have been directly correlated to the invasiveness of cancers.9 The conjugated therapeutic, resveratrol inhibits ERK1/2 phosphorylation by 55% in MDA-MB-157 cells, which is a part of the MAPK signal transduction pathway, a key regulator of cell migration.24 Recently, Ham et al.24 demonstrated a 70% reduction in cell migration upon incubation with 100 µM resveratrol. Under similar conditions, the amount of drug required to achieve similar results for HA-R is 10-fold less in dosage. The anti-cancer activity of resveratrol also improved significantly upon conjugation to HA, and these results are analogous to cisplatin. Similarly, HA conjugation improves anticancer activity for MOMIPP. Enhanced activity is observed for MDA-MB231, MDA-MB-157, and HeLa cells for HA-R, and MDA-MB-231 and MCF7 for HA-M. The potential difference in activity can be attributed to the mechanism of action, conjugation strategy as well as properties of the therapeutic including solubility in water and release rate. For example, a key mechanism


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of action for resveratrol is the p53 pathway,17 and diminished activity against MCF7s can be expected since these cells over-express MDM2, a protein that rapidly degrades p53 (a tumor suppressor gene).42 The conjugation of MOMIPP to HA forms a pro-drug in which the drug must be hydrolyzed after internalization. However, the formation of prodrug for HA-M provides extended release of MOMIPP, which is evident by cellular relapse studies. Cells incubated with HA-M for 3 days, followed by a 3-day incubation with drug-free media do not show signs of cellular regrowth. In contrast, solvated therapeutics obtained by mixing HA with resveratrol or MOMIPP (dissolved in organic solvents) shows initial efficacy but cells regrow to their original cellular density after drug-free media exchange. Although solvation has been utilized to deliver several hydrophilic therapeutics,9,

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our results demonstrate the shortcoming of this approach for delivery of

hydrophobic drugs. Thus, covalent conjugation is desired to achieve enhanced bioavailability, targeting, and therapeutically relevant efficacy with hydrophobic agents. HA also provides a customizable drug-delivery platform that can employ various conjugation strategies and to alter the drug release rates.

5. CONCLUSION We report here HA as a platform to delivery hydrophobic therapeutics. Once systemically administered, these conjugates could accumulate in the tumor interstitium via enhanced permeation and retention effect, selectively bind to their target receptors, and result in receptormediated uptake. The enhanced uptake results in increased intracellular concentration of the desired therapeutic drugs and their efficacy. In addition, this platform provides an economical


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solution to solubilize hydrophobic chemotherapeutics. Thus, HA has the potential to change the current approach for treating triple negative breast cancers.

ASSOCIATED CONTENT Supporting Information. Supporting information contains a list of all the materials and their suppliers utilized to conduct these experiments, as well as standard cell culture and Live/Dead Cell Viability assay® used for part of these experiments. Additional supporting data included in the supplementary information are: NMR spectra for HA-R and HA-M; TLC results for HA-M; Confocal images of HeLa cells incubated with HA-M; Comparison of HA and HA-R attachment to breast cancer MDA-MB-157 cells under physiological flow conditions; Representative images of TAMRA tagged HA attachment to breast cancer cells, MCF7 and MDA-MB-157, in competition with HA; Cell viability of cell lines upon incubation with HA; and Representative images of cells incubated with HA, HA-conjugates, or free drug, and stained with Live/Dead® Cell Viability reagent.

AUTHOR INFORMATION Corresponding Author * Dr. Yang H. Yun Department of Biomedical Engineering University of Akron 302 Buchtel Common Akron, OH, 44325-0302 Tel: + 1-330-972-6619 Email: [email protected]


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Funding Sources The authors are grateful for the funding of this research, which was made possible in part through the National Science Foundation (CAREER Award, CBET-0954360).

ACKNOWLEDGMENT The authors would like to thank Drs. Wiley Youngs. The University of Akron, for the gift of MDA-MB-157, Dr. Anirban SenGupta of Case Western Reserve University for his gift of MCF7 cells, and Dr. Judy Fulton of Akron General Medical Center for her gift of HDF.

ABBREVIATIONS EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; Her2, Human epidermal growth factor receptor 2; HA, Hyaluronan; HA-R, Hyaluronan-resveratrol conjugate; HA-M, Hyaluronan-MOMIPP conjugate; HDF, human dermal fibroblast; LYVE-1, Lymphatic Vessel Endothelial HA Receptor 1; MOMIPP, 3-(5-methoxy, 2-methyl-1H-indol-3-yl)-1-(4- pyridinyl)2-propen-1-one; NMR, nuclear magnetic resonance; PBS, phosphate buffered saline; RHAMM, Receptor for HA Mediated Motility; TAMRA, 5-(and-6)-Carboxytetramethylrhodamine, Succinimidyl Ester; TLC, thin layer chromatography.

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