Inclusion complex from cyclodextrin-grafted hyaluronic acid and pseudo protein as biodegradable nano-delivery vehicle for gambogic acid

Accepted Manuscript Inclusion Complex from Cyclodextrin-grafted Hyaluronic Acid and Pseudo Protein as Biodegradable Nano-Delivery Vehicle for Gambogic Acid Ying Ji, Shuo Shan, Mingyu He, Chih-Chang Chu PII: DOI: Reference:

S1742-7061(17)30550-0 http://dx.doi.org/10.1016/j.actbio.2017.08.036 ACTBIO 5047

To appear in:

Acta Biomaterialia

Received Date: Revised Date: Accepted Date:

20 March 2017 23 August 2017 28 August 2017

Please cite this article as: Ji, Y., Shan, S., He, M., Chu, C-C., Inclusion Complex from Cyclodextrin-grafted Hyaluronic Acid and Pseudo Protein as Biodegradable Nano-Delivery Vehicle for Gambogic Acid, Acta Biomaterialia (2017), doi: http://dx.doi.org/10.1016/j.actbio.2017.08.036

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Inclusion Complex from Cyclodextrin-grafted Hyaluronic Acid and Pseudo Protein as Biodegradable Nano-Delivery Vehicle for Gambogic Acid Ying Jia, Shuo Shanb, Mingyu Hea, Chih-Chang Chua,b* a. Department of Fiber Science and Apparel Design, Cornell University, Ithaca, New York 14853-4401, United States. b. Biomedical Engineering Field. Meinig School of Biomedical Engineering, Cornell University, Ithaca, New York 14853-4401, United States.

* Corresponding author: Chih-Chang Chu, Biomedical Engineering Program, & Department of Fiber Science and Apparel Design, Cornell University, Ithaca, NY 14853-4402, USA. Tel.: +1 607 255 1938; fax: +1 607 255 1093. E-mail address: [email protected] (Chih-Chang Chu)

Abstract β-Cyclodextrin can form inclusion complex with a series of guest molecules including phenyl moieties, and has gained considerable popularity in the study of supramolecular nanostructure. In this study, a biodegradable nanocomplex (HA(CD)-4Phe4 nanocomplex) was developed from βcyclodextrin grafted hyaluronic acid (HA) and phenylalanine based poly(ester amide). The phenylalanine based poly(ester amide) is a biodegradable pseudo protein which provides the encapsulation capacity for gambogic acid (GA), a naturally-derived chemotherapeutic which has been effectively employed to treat multidrug resistant tumor. The therapeutic potency of free GA is limited due to its poor solubility in water and the lack of tumor-selective toxicity. The nanocomplex carrier enhanced the solubility and availability of GA in aqueous media, and the HA component enabled the targeted delivery to tumor cells with overexpression of CD44 receptors. In the presence of hyaluronidase, the release of GA from the nanocomplex was significantly accelerated, due to the enzymatic biodegradation of the carrier. Compared to free GA, GA-loaded nanocomplex exhibited improved cytotoxicity in MDA-MB-435/MDR multidrug resistant melanoma cells, and induced enhanced level of apoptosis and mitochondrial depolarization, at low concentration of GA (1-2 µM). The nanocomplex enhanced the therapeutic potency of GA, especially when diluted in physiological environment. In addition, suppressed matrix metalloproteinase activity was also detected in MDA-MB-435/MDR cells treated by GA-loaded nanocomplex, which demonstrated its potency in the inhibition of tumor metastasis. The in vitro data suggested that HA(CD)-4Phe4 nanocomplex could provide a promising alternative in the treatment of multidrug resistant tumor cells. Keywords: Biodegradable; Poly(ester amide)s; Inclusion complex; Hyaluronic acid; Gambogic acid.

1. Introduction Natural products derived from plants, animals or microbes, have become one of the major sources for developing anti-tumor therapeutics, by virtue of their evolution to preferentially interaction with the molecular targets in biological system [1]. Gambogic acid (GA), a natural compound from gamboge resin that produced by genus Garcinia trees in Asia and Africa, is reported [2] to exhibit significant cytotoxic activity against multiple types of tumor including breast carcinoma, melanoma, leukemia, lung carnicoma, etc [3–6]. GA targets multiple biological events in the inhibition of cancer cells. GA downregulates telomerase activity, inhibits the Bcl-2 family proteins [7] and topoisomerase II, activates cell apoptosis through transferrin receptor and nuclear factor-κB signalling pathway [8]. In addition to the induction of apoptosis, the anti-invasion, anti-metastasis, and the anti-angiogenesis activity of GA is associated with the regulation of matrix metalloproteinase [9], integrin [10], and vascular endothelial growth factor receptor 2 signalling pathway [11]. GA also exhibited enhanced anti-tumor efficiency in several tumor cells with drug resistance, including docetaxel-resistant gastric cancer cells [12], doxorubicin-resistant breast cancer cells [13], 5-fluorouracil-resistant colorectal cancer cells [14], and vincristine-resistant oral squamous cells [15]. However, GA is still facing several challenges, such as low bioavailability due to its poor water solubility, rapid blood clearance, and the lack of adequate tumor targeting due to the wide distribution of GA in normal tissues [16], which can limit its therapeutic efficacy. Nano-delivery vehicles can provide the advantage to solve these challenges associated with the direct administration of GA. Hussain et al. [17] developed poloxamers/D-α-tocopheryl polyethylene glycol succinate (TPGS) mixed micelles for the delivery of GA in MCF-7 breast carcinoma cells, resulting in 1.6-fold higher toxicity compared to free GA. Huang et al.[18] developed a GAdelivery system from polyethylene glycol (PEG)-block-dendritic oligomer of cholic acid and vitamin E, and demonstrated significantly enhanced inhibition effect in HT-29 colon cancer cells both in vitro and in vivo. However, very few research focused on developing biodegradable naoncarriers for GA delivery. It is of particular interest to develop a tumor-specific and biodegradable nano delivery platform for GA, while simultaneously improve its water solubility and bioavailability. Biodegradable amino acid based poly(ester amide)s (AA-PEAs), a new family of synthetic pseudo proteins with alternative ester and amide groups, are synthesized from 3 building blocks: amino acids, dialcohol and diacids. The amino acids contributed to the protein-like characteristics of AA-PEAs, including enzymatic biodegradation. The choice of different amino acids, and the hydrocarbons between the amide and ester groups, makes AA-PEAs tunable in hydrophilicity/hydrophobicity, charge density, and reactive sites for introducing functionality. The current status of AA-PEAs have recently been reviewed [19,20]. AA-PEAs have been studied for various biomedical applications and have also been hybridized with other polymers, such as synthetic aliphatic polyesters and polysaccharides [21,22]. As demonstrated by our previous studies[23–25] , hyaluronic acid (HA) is one of the most promising candidates to form self-assembled nano-structure with AA-PEAs with broadened properties and applications. Identified as the ligand molecule for CD44 receptors, HA has attracted considerable research interests in the field of tumor-targeting delivery, due to the overexpression of CD44 receptors on many types of tumor cells [26]. HA-based nano-carriers exhibited selective binding and uptake by tumor cells, and have been developed to improve the potency of various therapeutic agents [27], such as chemotherapeutics, nucleic acids and proteins.

In addition to ionic interaction and covalent bonding, complexed nanostructure can be built up via complementary stereoelectronic host-guest interaction [28]. β-cyclodextrin (β-CD), composed of 7 glucose units, is a truncated cone-shaped molecule with top and bottom diameters of 0.60 nm and 0.65 nm, respectively [29]. β-CD can form inclusion complex with various guest molecules including poly(propylene oxide) [30], poly(dimethylsiloxane)s [31], aliphatic polyesters [32], adamantyl pendant group [33], and phenyl pendant group [34], etc. β-CD and their derivatives have been successfully employed to construct supramolecular functional materials, the wide range of guest molecules enables the versatile design, and are attractive as the nano-delivery vehicles. Zhang et al. [35] developed a nanostructure assembled from β-CDgrafted-polyethylene glycol and polyaspartatamide as the delivery vehicles for docetaxel. Fan et al. [36] developed a nano-assembly from β-CD grafted polyethyleneimine and adamantinedoxorubicin for the co-delivery of doxorubicin and therapeutic gene pTRAIL. However, very few literatures reported the inclusion complex formed from HA-based nanoparticles. In the work by Kulkarni et al. [37], nanoparticles were prepared from the inclusion complex between PEI grafted with β-cyclodextrin and hyaluronic acid grafted with adamantine groups, as the delivery vehicle for plasmid DNA in HeLa cells. The aim of this study is to develop a biodegradable nanocarrier from the inclusion complex between β-cyclodextrin grafted hyaluronic acid and phenylalanine based pseudo protein, as illustrated in Figure 1. HA component on the surface of the complex facilitated the targeted delivery of GA to CD44 positive tumor cells, while the phenylalanine based pseudo protein provided hydrophobic interaction with GA to improve its encapsulation capacity. MDA-MB435/MDR multidrug resistant melanoma cells with overexpression of CD44 receptors were chosen for the in vitro examination of the GA delivery system. In vitro characterization of the endocytosis and tumor cell inhibition by the GA-loaded nanocomplex was performed. Mitochondrial damage, cell death assay and the activity of matrix metalloproteinases were also investigated to further validate the potency of the GA-loaded nanocomplex.

2.Experimental 2.1 Chemicals and cells Sodium hyaluronate (MW= 10 to 20 kDa) was from Lifecore Biomedical (Chaska, MN). Tetrabutylammonium bromide, β-cyclodextrin (β-CD), 4-toluenesulfonyl chloride, succinic dihydrazide (SDH), N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC·HCl), 1-hydroxybenzotriazole(HOBt), N,N-diisopropylethylamine (DIPEA), 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), hyaluronidase from bovine testes and 1-adamantane carboxylic acid were from Sigma Aldrich (Milwaukee, WI). Amberlite IR120 H+ ion exchange resin was from Alfa Aesar (Ward Hill, MA). Gambogic acid (GA) was from BroadPharm (San Diego, CA). Lysotracker green was from Cell Signaling (Beverly, MA). Dimethyl sulfoxide (DMSO), methanol and other HPLC grade solvents were from JT Baker (Phillipsburg, NJ). JC-1 mitochondrial membrane potential assay kit was from Cayman Chemical (Ann Arbour, MI). Annexin V apoptosis detection kit was from eBioscience (San Diego, CA). Apo-ONE Homogeneous Caspase-3/7 assay kit was from Promega (Madison, WI).

Snakeskin dialysis tubing and NHS-Rhodamine were from Pierce (Rockford, IL). Phenylalanine based pseudo protein (Phe- based poly(ester amide)s), 4Phe4, was synthesized from our previous study [38,39]. MDA-MB-435/MDR multidrug resistant melanoma cells were donated by Dr. Robert Clarke (Georgetown University, Washington, D.C.) and MD Anderson Cancer Center (Houston, TX). MDA-MB-435/MDR cells were maintained at 37 ºC with 5% CO2 in Minimum Essential Medium (Richter’s modification) containing 2mM L-glutamine, phenol red, 10% fetal bovine serum, and 1% penicillin/streptomycin. NIH 3T3 fibroblasts were maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with phenol red, 10% fetal bovine serum, and 1% penicillin/streptomycin. 2.2 Synthesis Synthesis of 6-monotosyl β-cyclodextrin. To introduce amine-reactive functional groups into βcyclodextrin (β-CD), 6-monotosyl β-CD was prepared from previously published study [40]. βCD (17.22 g) was dissolved in 1% NaOH solution (200 mL), and toluene sulfonyl chloride (2.9 g) dissolved in acetonitrile (11 mL) was added dropwise. The reaction was stirred at room temperature for another 2 hrs. The pH of the filtered solution was adjusted to 2-3 with 0.1 M HCl solution and precipitated overnight at 4 °C. The white precipitate was filtered and re-crystalized twice in water to give purified 6-monotosyl β-CD. Synthesis of succinic acid dihydrazide grafted HA. HA (200 mg) was dissolved in DI water (20 mL) followed by successive addition of DIPEA (55 µL), EDC (124 mg) and HOBt (74 mg) [41]. Succinic dihydrazide (SDH, 725 mg, 10 molar equivalent to HA repeating units) was then added and the reaction was stirred at room temperature for 24 hrs. The reaction mixture was dialyzed (MWCO = 3.5 kDa) against DI water for 48 hrs to give the aqueous solution of HA grafted with succinic acid dihydrazide (HA-SDH). To improve the solubility of HA-SDH in organic solvents, HA-SDH was converted to its tetrabutylammonium salt (HA-SDH-TBA) via ion exchange method [41]. Amberlite IR-120 H+ resin was neutralized with NaOH, washed with deionized water and immersed in 0.25 M aqueous tetra-N-butylammonium bromide solution overnight. The resulting ion exchange resin was added to the dialyzed solution of HA-SDH and stirred overnight at room temperature. The resin was removed by filtration, and the filtered solution was lyophilized to give HA-SDH-TBA. Synthesis of β-CD grafted HA. HA functionalized with β-CD was prepared as indicated in Figure 2. HA-SDH-TBA (100 mg) was dissolved in DMSO with triethylamine (30 µL), 6monotosyl β-CD (91 mg) was added and stirred for 24 hrs at 40 °C. The solution was dialyzed against DI water for 48 hrs, the pH of the solution was adjusted to 4.0 with 0.1 M HCl, kept at 4 °C overnight and filtered to remove unreacted 6-monotosyl β-CD. The filtered solution was incubated with TBA loaded ionic exchange resin overnight and lyophilized to give the TBA salt of β-CD grafted HA (abbreviated as HA(CD)-TBA). The 1H-NMR spectrum was examined on a Varian Unity Inova 400 MHz spectrometer (Palo Alto, CA). Synthesis of rhodamine-labeled HA(CD)-TBA. HA-SDH (100 mg) was dissolved in DI water (5 mL) and mixed with NHS-rhodamine (2 mg). The reaction was stirred overnight at room temperature, and dialyzed (MWCO=3.5 kDa) against DI water. The resulted rhodamine-labeled HA-SDH was converted to its TBA salt (rhodamine labeled HA-SDH-TBA) via ionic exchange method. The grafting of β-CD to HA-SDH-TBA labeled with rhodamine was the same as the preparation of HA(CD)-TBA.

2.3 Preparation of GA-loaded HA(CD)-4Phe4 nanocomplex Inclusion complex from HA(CD)-TBA and phenylalanine based biodegradable poly(ester amide)s (4Phe4) was prepared via the host-guest interaction between β-CD and the phenyl groups of 4Phe4, as illustrated in Figure 1. 4Phe4 was synthesized based on published procedures [38,39], and had an average molecular weight of 20.6 kDa and PDI of 1.27; the data are consistent with the prior published studies [39,40]. To prepare GA-loaded HA(CD)-4Phe4 nanocomplex, HA(CD)-TBA and 4Phe4 with various weight ratio (1:2, 1:1, 2:1, 4:1) was dissolved in DMSO and stirred overnight. GA (GA to polymers in feed = 1:4 w/w)) was then added and stirred for another 4 hrs. The resulting solution was dialyzed (MWCO = 10 kDa) against DI water for 48 hrs, filtered with 0.45 µm PVDF microfilter to remove unloaded GA and repeatedly concentrated in Macrosep centrifugal filter (MWCO=100 kDa, Pall Life Sciences, Ann Arbor, MI) to remove any free HA. To examine the loading content (LC) and encapsulation efficiency (EE) of GA, GA was extracted from the nanocomplex in ethanol [42]. The concentration of GA was determined by the absorbance at 360 nm on Lambda Bio40 UV-Visible spectrophotometer (Perkin-Elmer, Norwalk, CT), calibrating with a standard GA curve. The LC and EE were calculated by the following equation. EE = LC =

Mass of loaded GA in nanocomplex × 100% Mass of GA in feed

Mass of loaded GA in nanocomplexs × 100% Total mass of GA loaded nanocomplex

The morphology of GA-loaded HA(CD)-4Phe4 nanocomplex was observed on FEI Tecnai Spirit T12 TEM (FEI Co., Hillsboro, OR) at operating voltage of 120 kV. The morphology of HA(CD)-4Phe4 nanocomplex was also observed after the nanocomplex was incubated with 1adamantane carboxylic acid sodium salt (2 molar equivalent to β-CD units) in aqueous solution for 24 hrs [43]. Zeta potential and size of the nanocomplex were characterized at room temperature on Zetasizer NanoZS system (Malvern, UK). 2.4 In vitro release profile of GA-loaded HA(CD)-4Phe4 nanocomplex The GA-loaded HA(CD)-4Phe4 nanocomplex (10 mg) was dissolved in phosphate buffer (pH 7.4 , 5mL) containing 0.5% Tween 80 with or without the presence of 120 unit/mL hyaluronidase (Hyal). The solution was placed in dialysis tubing (MWCO = 10 kDa) and dialyzed against pH 7.4 phosphate buffer (30 mL) containing 0.5% Tween 80 at 37 °C under shaking (100 rpm). At predetermined time intervals, 5 mL of the solution outside the dialysis tubing was retrieved and replaced by fresh media. The concentration of GA in each sample was determined by the absorbance at 360 nm in UV-Vis spectra and calibrated against a standard GA calibration curve. The percentage of released GA was plotted vs. time. Each test was performed in triplicate. 2.5 Endocytosis and subcellular distribution of blank HA(CD)-4Phe4 nanocomplex To study the accumulated endocytosis of the blank HA(CD)-4Phe4 nanocomplex, MDA-MB435/MDR (CD44 positive) cells or NIH 3T3 cells (CD44 negative) were incubated with blank

HA(CD)-4Phe4 nanocomplex labeled with rhodamine for 1, 2, 4, 8, 12 or 24 hrs. The cells were washed with PBS, harvested and the cellular fluorescence intensity was analyzed on FACSAria fusion fluorescence activated cell sorter (BD Biosciences, Franklin Lakes, NJ). Subcellular distribution of blank HA(CD)-4Phe4 nanocomplex was visualized on Zeiss LSM710 confocal microscope (Carl Zeiss MicroImaging, Thornwood, NY). MDA-MB435/MDR cells were incubated with blank HA(CD)-4Phe4 nanocomplex labeled with rhodamine (0.1 mg/mL) for 4 hrs. Cells were then stained with Lysotracker green as per manufacturer’s instruction, washed with PBS and imaged by confocal microscopy. To study the pathway of endocytosis, MDA-MB-435/MDR cells were first treated with inhibitors that block different endocytic pathways (nystatin, 50 µg/mL; chloropromazine, 30 µM; amiloride hydrochloride, 50 µM; methyl-β-cyclodextrin, 3 mg/mL or anti-CD44 antibody as per manufacturer’s instruction) for 0.5 hr, before rhodamine labelled blank HA(CD)-4Phe4 nanocomplex was added and incubated for another 4 hrs. The cells were then washed with PBS, harvested and analysed by flow cytometry. The effect of low temperature (4°C) on endocytosis was also studied. Cells incubated with the blank nanocomplex at 37°C but not treated with any inhibitor were tested as control. The mean fluorescence intensity for each sample was recorded and normalized to control. The tests were performed in triplicate. 2.6 Cytotoxicity of GA-loaded nanocomplex Free GA was dissolved in DMSO (1 mM) and diluted to the testing concentrations in cell culture media. The GA-loaded nanocomplex was directly dispersed in PBS and diluted by cell culture media. MDA-MB-435/MDR cells were incubated with blank nanocomplex, free GA or GA-loaded HA(CD)-4Phe4 nanocomplex (c(GA) =0.1 to 20 µM) for 24 hrs. To validate the drug resistance of MDA-MB-435/MDR cells, toxicity of doxorubicin(c(DOX) =0.1 to 20 µM) was also detected in the same procedure. The viability of cells was determined by MTT assay, and each experiment was run in six replicates. The absorbance at 570 nm was recorded and normalized to the results of the untreated cell to give the percentage of cell survival. Each test was run in 6 replicates. 2.7 Mitochondrial membrane potential assay MDA-MB-435/MDR cells were incubated with blank nanocomplex, free GA or GA-loaded HA(CD)-4Phe4 nanocomplex (c(GA) =1 µM) for 24 hrs. The cells were stained with JC-1 probe as per manufacturer’s instruction and analyzed by flow cytometry. The experiments were performed in triplicate. The percentage of cells stained with higher degree of JC-1 monomer and lower degree of J-aggregates was recorded. Each test was run in triplicate. 2.8 Apoptosis study To study the apoptotic behaviour, MDA-MB-435/MDR cells were incubated with blank nanocomplex, free GA or GA-loaded HA(CD)-4Phe4 nanocomplex (c(GA) =1 µM) for 24 hrs. The cells were washed and harvested. For the annexin-V assay, annexin-V/PI double staining was performed as per manufacturer’s instruction. The percentage of dead/apoptotic cells were analysed by flow cytometry. Each experiment was performed in triplicate. For the assay of caspase 3/7 activities, the cells were further incubated with Apo-ONE Homogeneous Caspase3/7 assay reagents as per manufacturer’s instruction. The fluorescence intensity was recorded (λex = 485 ± 20 nm/λem = 528 ± 20 nm). The untreated cells were tested as control. The

fluorescence for each sample was normalized to control to give the fold of caspase 3/7 activities. Each experiment was performed in triplicate. 2.9 Gelatin zymography MDA-MB-435/MDR cells were incubated in serum free medium with free GA, blank HA(CD)-4Phe4 nanocomplex, or GA-loaded HA(CD)-4Phe4 nanocomplex (c(GA) =1 µM) for 24 hrs. The media then collected and centrifuged (400 rcf, 5 min, 4 ºC), and gelatin zymography for the supernatant was performed as described previously [42,44]. In brief, SDS-PAGE gels were prepared containing 1% type B gelatin (w/v). After electrophoresis, the gels were washed with 2.5% Triton X-100 to remove SDS and incubated with developing buffer (50 mM Tris-HCl, 10 mM CaCl2, 0.02% NaN3, pH 7.6) at 37 °C for 16 hrs. The gels were stained with 0.25% Coomassie Brilliant Blue R-250 solution and destained (acetic acid/ethanol/water =1/4/5, v/v). Regions digested by matrix metalloproteinases (MMP) were observed as white bands against dark background. 2.10 Statistical analysis The data are presented as mean values with standard deviations (SD). All data were analyzed using one-way ANOVA, followed by Tukey’s multiple comparison tests, and p < 0.05 or lower was used for statistical significance.

3. Results and discussion 3.1 The characterization of polymers β-Cyclodextrin-grafted HA (abbreviated as HA(CD)) was prepared from the reaction between 6-monotosyl β-CD and succinic dihydrazide functionalized HA (HA-SDH). Purification was performed by precipitation of unreacted 6-monotosyl β-CD in mildly acidic environment and filtration. The 1H NMR characterization for 6-monotosyl β-CD and HA-SDH was shown as Figure S1and Figure S2 (supporting information). As indicated in the 1H NMR spectrum of HA(CD) in Figure 3, the typical peak for the methyl groups in HA was observed at 1.73 ppm, and the typical peak for the H-1 proton from the glucose residue in β-CD was observed at 4.75 to 5.05 ppm. Based on the integral of these two peaks, the grafting ratio of β-CD onto HA, defined as the molar amount of β-CD divided by the total molar amount of HA repeating units, was calculated to be 13%, which is in line with the reported grafting ratio [41]. The 1H-NMR spectrum suggested the effective grafting of β-CD onto HA, and the β-CD pendant groups provides available sites to form inclusion complex with the phenylalanine moieties from 4Phe4, with an average molecular weight of 20.6 kDa and PDI of 1.27. The chemical structure of 4Phe4 was shown in Figure 1.

3.2 The characterization of GA-loaded HA(CD)-4Phe4 nanocomplex The scheme for the formation of HA(CD)-4Phe4 nanocomplex was illustrated in Figure 1. In aqueous environment, HA provided the hydrophilic component as the outer layer of the nanocomplex, and 4Phe4 provided the hydrophobic component in the interior. The hydrophobic

and hydrophilic segments were held together via the host-guest interaction between β-CD side groups on HA(CD) and phenyl side groups on 4Phe4. The impact of the feed ratio between HA(CD) and 4Phe4 on the yield and size of the nanocomplex was shown in Table 1. When the weight ratio between HA(CD)/4Phe4 was 1:2, significant precipitation was observed after dialysis, which can be attributed to the excessive amount of the hydrophobic 4Phe4 component that didn’t form the inclusion complex with HA(CD). As the feed ratio between HA(CD)/4Phe4 increased, the HA(CD)-4Phe4 nanocomplex was effectively formed, and the average diameter of the nanocomplex decreased with an increase in the HA(CD) component, i.e. 732.6 ± 38.2 nm (yield 13.6%) for HA(CD)/4Phe4=1:1 (w/w), and 148.5 ± 21.2 nm (yield 65.1%) for HA(CD)/4Phe4=2:1 (w/w). When the HA(CD)/4Phe4 ratio further increased to 4:1, no significant change in diameter was detected, but the yield reduced to 27.5%, since that the excessive amount of HA(CD) which didn’t form the nanocomplex was removed in the purification process. Negative zeta potential ranging from 19.5 ± 2.7 mV to -26.23 ± 3.2 mV was detected for the nanocomplex, which can be attributed to the presence of HA at the surface of the nanocomplex. Nano-carriers of smaller size (< 200 nm) were found to be more effective in drug delivery because they are less likely to be captured by reticuloendothelial system than larger particles during systemic circulation [45]. The hydrophobic interior of the HA(CD)/4Phe4 nanocomplex provided the loading capacity for GA due to the hydrophobic interaction. The loading content (LC) and encapsulation efficiency (EE) was determined by extraction of GA from the nanocomplex by ethanol with an extraction efficiency of 91.8%. The nanocomplex was dissociated by adding 1-adamantane carboxylic acid sodium salt (2 molar equivalent to β-CD units). The dissociation of nanocomplex lead to the precipitation of hydrophobic GA and 4Phe4 phenylalanine PEA. The GA content in the precipitate was determined by UV absorbance at 360 nm and calibration with a standard curve of GA (Figure S3, supporting information). LC and EE for GA in the nanocomplex (HA(CD) to 4Phe4 weight ratio in feed = 2:1) were 13.5% and 50.6%, respectively. As indicated in Table S1 (supporting information), for HA(CD)TBA/4Phe4 feed ratio 1:1 to 4:1, LC was in the range from 13 wt% to 18 wt%, while EE was in the range from 50 wt% to 67 wt%. The LC and EE were not significantly affected when the HA(CD) to 4Phe4 weight ratio in feed varies from 1:1 to 4:1. The loading of GA did not significantly impact the size (diameter = 184.16 ± 16.3 nm, PDI = 0.169 ± 0.042) and surface charge (-20.2 ± 2.8 mV) of the HA(CD)-4Phe4 nanocomplex (HA(CD)/4Phe4=2:1), when compared to the blank counterpart. The best balance among size, yield as well as the encapsulation efficiency was used to optimize the nanocomplex formulation. As indicated in Table 1, the nanocomplex from the HA(CD)/4Phe4 at the feed ratio 2:1 was selected for the following studies.

Table 1. Characterization of HA(CD)-4Phe4 nanocomplex with different HA(CD) to 4Phe4 ratios. HA(CD) to 4Phe4 / (w/w) ratio in feed 1:2 1:1

DLS diameter c /nm Precipitate b 732.6 ± 38.2

PDI NA 0.171 ±

Zeta potential (mV) NA -26.2 ± 3.2

Yield a NA 13.6%

2:1

161.5 ± 11.2

4:1

148.5 ± 21.2

0.061 0.176 ± 0.053 0.181 ± 0.021

-21.4 ± 1.7

65.1%

-19.5 ± 2.7

27.5%

a. Yield was calculated from the mass of purified nanocomplex divided by the total mass of HA(CD) and 4Phe4 in feed. b. Formation of precipitate; the PDI, zeta potential and yield were non-detectable. c. The average DLS diameter was the mean from 3 different measurement of the same batch of samples.

The morphology of GA-loaded HA(CD)-4Phe4 nanocomplex was shown in Figure 4A. Spherical assembled structure was observed for GA-loaded HA(CD)-4Phe4 nanocomplex in aqueous solution. However, the assembled structure disappeared when a competing guest, 1adamantane carboxylic acid sodium salt (2 molar equivalent to cyclodextrin moieties), was incubated with aqueous solution of HA(CD)-4Phe4 nanocomplex for 24 hrs. Significant precipitation was formed, and no assembled structure was observed in the filtered solution, as suggested in Figure 4B. The replacement of phenyl groups of 4Phe4 with the competing guest molecule lead to the dissociation of the nanocomplex, which further confirmed that the nanocomplex was formed from the host-guest inclusion complex between HA(CD) and 4Phe4. After 48 hrs incubation with hyaluronidase, a specific enzyme that breaks down HA into oligosaccharide [46], the destabilization of the HA(CD)-4Phe4 nanocomplex was evidenced in Figure 4C, as no spherical assembled structure was observed.

3.3 Solubility of GA-loaded HA(CD)-4Phe4 nanocomplex and in vitro release profile The solubility of GA-loaded nanocomplex was tested and the result was listed in Table S2 (supporting information). The solubility of GA loaded in nanocomplex was which was 1.7 mg/mL (in PBS) and 2.6 mg/mL (in PBS-0.5% Tween 80), which was almost 1000-fold compared to free GA. The increased solubility of GA enhanced its bioavailability, and is expected to further improve the therapeutic efficacy of GA. In vitro release of GA was tested in PBS+0.5% Tween 80. No precipitation was observed, the DLS size of the nanocomplex after incubation was 189 nm, with a PDI of 0.179, which was comparable to the size of nanocomplex before incubation as listed in Table 1. Due to the hostguest interaction between hydrophilic HA segment and hydrophobic 4Phe4, the nanocomplex was not sensitive to surfactant. The consistent DLS size of the nanocomplex before and after 48 hrs incubation in PBS+0.5% Tween 80 indicated its stability. The in vitro release profiles of GA were shown in Figure 4D. At the end of 48 hrs, 64% of total GA was released in the presence of hyaluronidase (black curve), while 23% cumulative release was detected in PBS (red curve). The release profiles of GA correlated directly with the morphological changes of HA(CD)-4Phe4 nanocomplex after incubation with hyaluronidase (Figure 4C), and the accelerated GA release can be attributed to the disintegration of the nanocomplex induced by enzymatic biodegradation.

3.4 Endocytosis and subcellular distribution of blank HA(CD)-4Phe4 nanocomplex MDA-MB-435/MDR cells, with overexpression of CD44 receptor [47], were selected as the in vitro model to study the endocytosis and subcellular distribution of the blank HA(CD)-4Phe4 nanocomplex. 3T3 fibroblast, which is CD44 negative, was also included as the negative control. The subcellular distribution of the blank HA(CD)-4Phe4 nanocomplex after 4 hrs incubation was studied and shown in Figure 5A. The red fluorescence from rhodamine-labelled nanocomplex in 3T3 fibroblasts was weaker than in MDA-MB-435/MDR cells, which correlated well with the accumulated endocytosis in Figure 5B. In MDA-MB-435/MDR cells, the red fluorescence of the rhodamine-labelled HA(CD)-4Phe4 nanocomplex was identified to be colocalized with endolysosomes, as yellow pixels (overlay from rhodamine-labelled nanocomplex with Lysotracker green) were observed. The results suggested that HA(CD)-4Phe4 nanocomplex was entrapped inside endolysosomes which have a series of digestive enzymes to break down the nanocomplex and accelerate the release of GA, as indicated in Figure 4. The endocytosis of HA(CD)-4Phe4 nanocomplex as a function of time in both MDA-MB435/MDR cell (CD44 positive) and 3T3 fibroblast (CD44 negative) were given in Figure 5. A significant difference in the intracellular level of the rhodamine-labelled nanocomplex between MDA-MB-435/MDR cells and 3T3 cells was observed throughout 24 hrs as demonstrated in Figure 5B. The HA(CD)-4Phe4 nanocomplex exhibited significantly enhanced endocytosis by CD44 overexpressed MDA-MB-435/MDR cells, and could be developed as the tumor-targeted delivery vehicles. To identify the pathway for the entry of HA(CD)-4Phe4 nanocomplex into MDA-MB435/MDR cells, inhibition study was performed and the results were shown in Figure 5C. The uptake of the HA(CD)-4Phe4 nanocomplex was decreased by 21% in MDA-MB435/MDR cells pre-treated with anti-CD44 antibody, indicating that the entry of HA(CD)4Phe4 nanocomplex was mediated by the CD44 receptor. Incubation of MDA-MB435/MDR cells at 4 °C resulted in a reduction of 38% in the intracellular accumulation of the nanocomplex, indicating that the internalization of the nanocomplex is energydependent. Inhibition of clathrin-mediated pathway by chloropromazine resulted in 17% reduction in intracellular fluorescence, which suggested the endocytosis is also dependent on clathrin pathway. However, the inhibition of macropinocytosis, caveolae-mediated or lipid-raft mediated pathways by amiloride, nystatin or methyl- β-CD showed no significant reduction in the transmigration of HA(CD)-4Phe4 nanocomplex, which suggested that the internalization was independent of these pathways. In general, the CD44-mediated endocytosis of the HA(CD)-4Phe4 nanocomplex in MDA-MB-435/MDR cells lead to the selectivity between CD44 positive cells versus CD44 negative cells, which provide the rationale for the tumor-targeting delivery of GA. The endocytosis inhibition study of the HA(CD)-4Phe4 nanocomplex in CD44 negative 3T3 fibroblasts was also included in Figure 5D. In contrast to Figure 5C, preincubation with antiCD44 antibody didn’t result in any significant reduction in the uptake of nanocomplex, suggesting that the process was not mediated by CD44-recepter. However, significantly reduced uptake of nanocomplex was observed when the NIH 3T3 cells were incubated at 4 °C or treated with chloropromazine, suggesting the entry of nanocomplex in 3T3 cells were dependent on clathrin-mediated endocytosis pathway as well as ATP-consumption. Hussain et al. [17] reported enhanced internalization rate of GA when loaded in Poloxamer 407/TPGS micelles compared to free GA. The internalization of free GA involved binding with transferrin receptor [48], and is less competent as the endocytosis of GA-loaded nano-delivery

system [17]. The HA(CD)-4Phe4 nanocomplex is expected to improve the intracellular availability of GA, and further impact its therapeutic efficiency.

3.5 Cytotoxicity of GA-loaded HA(CD)-4Phe4 nanocomplex Cytotoxicity of free GA, blank HA(CD)-4Phe4 and GA-loaded HA(CD)-4Phe4 nanocomplex in MDA-MB-435/MDR cells was shown in Figure 6A. No significant cytotoxicity from the blank nanocomplex was observed at the concentration range which correlated to equivalent c(GA) between 0.1 to 20 µM. Therefore, the cytotoxicity of GA-loaded HA(CD)-4Phe4 nanocomplex can be attributed to the loaded GA, instead of the carrier. Free GA and GA-loaded HA(CD)-4Phe4 nanocomplex exhibited concentration-dependent toxicity in MDA-MB-435/MDR cells. Significantly higher toxicity from GA-loaded nanocomplex than free GA was detected at c(GA) =1 and 2 µM. At the rest of the tested concentrations, GA-loaded nanocomplex exhibited equivalent cytotoxicity as free GA on MDAMB-435/MDR cells. The IC50 was calculated as 1.14 µM for GA-loaded nanocomplex, and 1.63 µM for free GA. It’s worth mentioning that, due to its poor solubility in aqueous solution, free GA was first dissolved in DMSO and diluted to the testing concentrations, but the presence of organic solvent is unfavorable for the drug administration in vivo. GA-loaded nanocomplex, on the other hand, can be directly dispersed in aqueous solution, which significantly improved the solubility and availability of GA. Cytotoxicity of free GA and GA-loaded HA(CD)-4Phe4 nanocomplex was also detected on MDA-MB-435/WT cells (without multidrug resistance) as shown in Figure 6B. After 24 hrs incubation, the IC50 on MDA-MB-435/WT cells were 0.37 µM for free GA and 0.45 µM for GAloaded HA(CD)-4Phe4 nanocomplex. Comparing the IC50 values between wild type vs. multidrug resistant phenotype, MDA-MB-435/MDR cells did not exhibit significant resistance in the case of both free GA and GA-loaded HA(CD)-4Phe4 nanocomplex. In contrast, the IC50 of free doxorubicin on MDA-MB-435/WT and MDA/MB-435/MDR cells were tested as 0.4 µM and 10 µM, respectively. The therapeutic potency of GA was also reported by Wang et al. [49], in which GA effectively inhibited docetaxel resistant BGC-823 gastric cancer cells. Since the presence of drug efflux pump on MDA-MB-435/MDR cells did not adversely affect the therapeutic performance of GA-loaded HA(CD)-4Phe4 nanocomplex, this delivery system could provide the potential as a complementary treatment for multi-drug resistant tumors.

3.6 Mitochondrial depolarization Studies were then performed to characterize the interaction between GA-loaded HA(CD)4Phe4 nanocomplex and MDA-MB-435/MDR cells. The change in the mitochondrial membrane potential of MDA-MB-435/MDR cells was detected after incubated with blank HA(CD)-4Phe4 nanocomplex, GA-loaded HA(CD)-4Phe4 nanocomplex or free GA (c(GA) = 1 µM) for 24 hrs. The percentage of cells with depolarized mitochondria can be identified as the population that stained with a higher level of JC-1 monomerand lower level of J-aggregates (quadrant 3) from Figure 7A. The quantitative results were also shown in Figure 7B. In the case of blank HA(CD)-4Phe4 nanocomplex, only 3.9% of total population were observed in quadrant 3 (with depolarized mitochondria), which was consistent with the untreated MDA-MB-435/MDR cells (i.e. 3.8% in quadrant 3). The result demonstrated the biosafety of the blank nanocomplex.

A significant loss of mitochondrial membrane potential was observed in MDA-MB-435/MDR cells treated by both free GA and GA-loaded HA(CD)-4Phe4 nanocomplex. At c(GA) =1 µM, the GA-loaded HA(CD)-4Phe4 nanocomplex induced significantly greater level of mitochondrial depolarization, as 16.6% of cells were detected with depolarized mitochondria in the nanocomplex treatment when compared to 10.3% of cells when treated by free GA. These results suggested that the cytotoxicity induced by either free GA or GA-loaded HA(CD)-4Phe4 nanocomplex was related to mitochondria, and was also consistent with the study by Qiang et al. [50], in which 21% of Rat C6 glioma cells were detected with depolarized mitochondria when treated by free GA (1 µM). As demonstrated by Guizzunti et al. [51], GA directly targets mitochondria, and induces intrinsic pathway in the inhibition of tumor cells. The results in Figure 7 correlated well with the enhanced cytotoxicity of GA-loaded nanocomplex in Figure 6.

3.7 Annexin-V and caspase 3/7 assay The loss of mitochondrial membrane potential marks the dysfunction of mitochondria [52], and can be associated with the release of apoptogenic factors in the signaling of cell death. Apoptosis studies were then performed to probe the cell death induced by GAloaded HA(CD)-4Phe4 nanocomplex. Results for annexin V/PI assay are shown in Figure 8A and 8B. The percentages of cell population in each quadrant were obtained as: live cells (PI-/annexin V-, quadrant 4), dead cells (PI+/annexin V-, quadrant 1), late apoptotic cells (PI+/annexin V+, quadrant 2) and early apoptotic cells (PI-/annexin V+, quadrant 3). The blank HA(CD)-4Phe4 nanocomplex didn’t induce significant cell death, as 81.5% of MDA-MB-435/MDR cells was alive. In both untreated group and blank nanocomplex treated group, minor population of cells were detected as early apoptotic. For MDA-MB-435/MDR cells treated by GA-loaded HA(CD)-4Phe4 nanocomplex (c(GA)=1 µM), a significantly higher population of dead cells was observed in quadrants 1 and 2, and only 51.0% of cells were identified as alive. The percentage of live cells treated by GA-loaded nanocomplex was also significantly lower than cells treated by free GA (70.5%). In addition, 33.2% of cells treated by GA-loaded HA(CD)-4Phe4 nanocomplex were subjected to early apoptosis, which was also significantly greater than cells treated by free GA (16.2%). In addition to annexin V staining, assay of caspase 3/7 activities was performed (Figure 9A). The activation of caspase marks the execution phase of apoptosis, and subsequently result in the disassembly of cells. In MDA-MB-345/MDR cells treated by GA-loaded nanocomplex (c(GA) = 1 µM) for 24 hrs, significantly increased activities of caspase 3 and caspase 7 were observed, comparing to free GA. The detection of caspase activities was in line with the results from annexin V/PI assay in Figure 8A, which suggested that, at a relatively low GA concentration (1 µM), the GA-loaded nanocomplex was more potent in inducing apoptotic cell death to MDAMB-435/MDR cells than free GA. Apoptotic death induced by GA treatment was reported in multiple tumor cells with drug resistance, such as doxorubicin resistant leukemia P388 cells [2], docetaxel-resistant gastric carcinoma BGC-823 cells [49], doxorubicin-resistant breast carcinoma cell line MCF-7/ADM [15], etc. In those researches, GA inhibited the growth of tumor cells which were resistant to common chemotherapeutics, and further provided the potential to reverse the chemo-resistance. Compared to free GA, the GA-loaded HA(CD)-4Phe4 nanocomplex exhibited an enhanced level of apoptosis and cytotoxicity in MDA-MB-435/MDR cells at lower concentrations. Huang et al. [18] suggested that free GA dissolved in DMSO may precipitate after dilution, which decreased

the stability and availability of GA in cell culture. At higher concentrations, the impact from the precipitation and destabilization of free GA was not significant, as the remaining GA was sufficient to induce cell death. However, at a relatively lower concentration, HA(CD)-4Phe4 nanocomplex enhanced the dispersion of GA in aqueous environment, and exhibited improved potency in killing tumor cells. In addition, the enhanced toxicity of GA-loaded nanocomplex can be related to the efficient endocytosis (Figure 5) and enzyme-accelerated release of GA (Figure 4) as we demonstrated previously. The efficiency to treat tumor at lower concentrations of drug, is of importance in in vivo application, as the potency of drug is greatly challenged due to the dilution of in physiological environment. The GA-loaded nanocomplex could be employed at a diluted dose, without the expenses of its efficient anti-tumor effect and simultaneously reduces the risk of side effects to normal tissues.

3.8 Gelatin zymography Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases that are involved in tumor invasion and metastasis. Due to the metastatic characteristics of MDA-MB435 cells [46], it’s important to address the impact of the GA-loaded HA(CD)-4Phe4 nanocomplex on the MMP activities and further inhibiting the metastasis and invasion of tumor cells. Gelatin zymography was performed to examine the activities of MMP-2 and MMP-9 in MDA-MB-435/MDR cells treated by free GA or GA-loaded HA(CD)-4Phe4 nanocomplex (c(GA) = 1 µM). As shown in Figure 9B, the zymography of either untreated cells or cells treated by the blank HA(CD)-4Phe4 nanocomplex showed broader bands digested by MMP, indicating a high level of MMP activity and that MDA-MB-435/MDR cells maintained a strong tendency of metastasis. However, after being treated by free GA or GA-loaded HA(CD)-4Phe4 nanocomplex, smaller band area than the untreated control was observed, suggesting significantly suppressed MMP activities. No difference was observed between the free GA and GA-loaded HA(CD)-4Phe4 nanocomplex treated groups, and it can be concluded that the delivery of GA by HA(CD)-4Phe4 nanocomplex didn’t hinder the pharmaceutical potency of GA in controlling the invasion of tumor cells. In addition to exhibiting significant cytotoxicity, inducing mitochondrial depolarization and apoptotic cell death in MDA-MB-435/MDR melanoma cells, the GA-loaded HA(CD)-4Phe4 nanocomplex also retarded the invasiveness of MDA-MB-435/MDR cells. As we demonstrated in vitro, the HA component of the HA(CD)-4Phe4 nano-carrier introduced the targeted delivery to CD44-overexpressed tumor cells, which could further improve the therapeutic potency of this delivery system. To extend the current in vitro study to in vivo models, and investigate the antitumor performance of GA-loaded HA(CD)-4Phe4 nanocomplex, would also be the focus of our future work.

4. Conclusions In this study, biodegradable nano-delivery vehicle for GA was developed from the inclusion complex between cyclodextrin grafted HA and phenylalanine based pseudo protein. The 4Phe4 pseudo protein in the nanocomplex provided the hydrophobic interaction and loading capacity

for GA, while the HA component targeted the overexpressed CD44 receptor and improved the selective endocytosis in MDA-MB-435/MDR melanoma cells. The biodegradability of the HA(CD)-4Phe4 nanocomplex enabled the enzyme-accelerated release of GA. Additionally, the nanocomplex enhanced the solubility and availability of GA in aqueous environment, and could improve the therapeutic potency of GA which is originally of poor solubility in conventional pharmaceutical solvents. Compared to free GA at lower drug concentration, GA-loaded HA(CD)-4Phe4 nanocomplex induced significantly enhanced mitochondrial depolarization and apoptotic cell death in MDA-MB-435/MDR cells, and effectively inhibited the MMP activities which are responsible for tumor metastasis. Therefore, the GA-loaded HA(CD)-4Phe4 nanocomplex could provide a promising alternative to the traditional chemotherapeutic treatment of multidrug resistant tumor cells.

Acknowledgements This work was partially supported by the Rebecca Q. Morgan Foundation, and made use of the Cornell Center for Materials Research Shared Facilities which are supported through the NSF MRSEC program (DMR-1120296). Imaging data was acquired through the Cornell University Biotechnology Resource Center, with NIH S10RR025502 funding for the shared Zeiss LSM 710 confocal microscopy. We would like to thank Dr. David Putnam from Cornell University for generously providing the stock of MDA-MB-435/MDR and MDA-MB-435/WT cell lines.

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Figure Legends: Figure 1. Chemical structures of β-cyclodextrin grafted HA (HA(CD)), phenylalanine based poly (ester amide)s (4Phe4), and gambogic acid (GA). HA(CD)-4Phe4 was formed from the inclusion complex between β-cyclodextrin moieties on HA(CD) and the phenyl groups from 4Phe4.

Figure 2. Synthesis scheme for tetrabutyl ammonium salt of β-CD grafted hyaluronic acid (HA(CD)-TBA).

Figure 3. 1H NMR spectra of β-cyclodextrin-grafted HA (HA(CD)).

Figure 4. A) TEM image of GA-loaded HA(CD)-4Phe4 nanocomplex in aqueous solution. B) TEM image of GA-loaded HA(CD)-4Phe4 nanocomplex incubated with 1-adamantane carboxylic acid sodium salt for 24 hrs. C) TEM image of GA-loaded HA(CD)-4Phe4 nanocomplex after 48 hrs incubation with 120 unit/mL hyaluronidase. D) Percentage of accumulated release of GA from HA(CD)-4Phe4 nanocomplex with or without the presence of 120 unit/mL hyaluronidase in phosphate buffer (pH = 7.4). Statistical significance: *** p< 0.001.

Figure 5. A) The confocal image of MDA-MB-435/MDR cells (CD44 positive) or NIH 3T3 fibroblasts (CD44 negative) incubated with blank HA(CD)-4Phe4 nanocomplex (0.1 mg/mL) for 4 hrs. Green (Lysotracker); red (rhodamine-labelled nanocomplex); the overlay of green and red pixels; and bright field (BF) image of the cells. Scale bar represented 20 µm. B) Accumulated endocytosis of blank HA(CD)-4Phe4 nanocomplex (0.1 mg/mL) in MDA-MB-435/MDR cells or NIH 3T3 cells. The average cellular fluorescence from rhodamine-labelled nanocomplex was determined via flow cytometry. (n=3). C) Endocytosis inhibition studies on MDA-MB435/MDR cells, pre-treated with various endocytosis inhibitors or low temperature, were incubated with rhodamine labelled HA(CD)-4Phe4 nanocomplex for 4 hrs. D) Endocytosis inhibition studies on NIH 3T3 fibroblast cells. Mean cellular fluorescence was recorded by flow

cytometry and normalized to control (cells incubated with the nanocomplex but not treated by any inhibitor). Values represent the average ± SD (n=3). Statistical significance: *, p < 0.05; *** p< 0.001.

Figure 6. Viability of A) MDA-MB-435/MDR cells (with multidrug resistance) and B) MDAMB-435/WT cells (wild type) incubated with blank HA(CD)-4Phe4 nanocomplex, free GA or GA-loaded HA(CD)-4Phe4 nanocomplex with various GA concentrations for 24 hrs. Statistical significance was compared between free GA and GA-loaded nanocomplex treated groups: *, p < 0.05.

Figure 7. A) Mitochondrial membrane potential in MDA-MB-435/MDR cells after incubation with blank HA(CD)-4Phe4 nanocomplex, free GA or GA-loaded HA(CD)-4Phe4 (c(GA) = 1 µM) for 24 hrs. B) The average percentage of MDA-MB-435/MDR cells with depolarized mitochondria (quadrant 3) in JC-1 assay. Statistical significance: *, p < 0.05.

Figure 8. A) Annexin-V/PI staining of MDA-MB-435/MDR cells incubated with blank HA(CD)-4Phe4 nanocomplex, free GA or GA-loaded HA(CD)-4Phe4 nanocomplex (c(GA) = 1 µM) for 24 hrs. B) The percentage of live cells, early apoptotic cells and dead cells was recorded. Statistical significance: **, p < 0.01.

Figure 9. A) Caspase 3/7 activities of MDA-MB-435/MDR cells incubated with GA-loaded HA(CD)-4Phe4, free GA, or blank HA(CD)-4Phe4 nanocomplex for 24 hrs (c(GA) = 1 µM). Statistical significance: *, p < 0.05. B) Gelatin zymography of MDA-MB-435/MDR cells incubated with blank HA(CD)-4Phe4 nanocomplex, free GA or GA-loaded HA(CD)-4Phe4 (c(GA) = 1 µM) in serum-free media for 24 hrs. The area of bright band correlated to the activities of matrix metalloproteinases MMP-9 and MMP-2.

Statement of Significance Authors are now required to submit a brief statement about the significance of their research, which should be written to a broad audience at an undergraduate level, and limited to 120 words. These statements will be peer-reviewed alongside the article and should hence be included in the initial submission. This Statement of Significance should be prepared to address (1) the novelty and significance of the work with respect to the existing literature, and (2) the scientific impact and interest to our readership. Statement of Significance. Gambogic acid (GA), naturally derived from genus Garcinia trees, exhibited significant cytotoxic activity against multiple types of tumors with resistance to traditional chemotherapeutics. Unfortunately, the poor solubility of GA in conventional pharmaceutical solvents and non-targeted distribution in normal tissues greatly limited its therapeutic potency. To overcome the challenges, we develop a nanoplatform from the supramolecular assembly of β-cyclodextrin grafted hyaluronic acid (HA) and phenylalanine based pseudo protein. The pseudo protein in the nanocomplex provided the hydrophobic interaction and loading capacity for GA, while the HA component targeted the overexpressed CD44 receptor and improved the selective endocytosis in multidrug resistant melanoma cells. The supramolecular nanocomplex provide a promising platform for the delivery of hydrophobic chemotherapeutics to improve the bioavailability and efficiency.