*Article* **Albumin-EDTA-Vanadium Is a Powerful Anti-Proliferative Agent, Following Entrance into Glioma Cells via Caveolae-Mediated Endocytosis**

**Itzik Cooper 1,2,3,\*, Orly Ravid <sup>1</sup> , Daniel Rand <sup>1</sup> , Dana Atrakchi <sup>1</sup> , Chen Shemesh <sup>1</sup> , Yael Bresler 1,4 , Gili Ben-Nissan <sup>5</sup> , Michal Sharon <sup>5</sup> , Mati Fridkin <sup>6</sup> and Yoram Shechter <sup>5</sup>**


**Abstract:** Human serum albumin (HSA) is efficiently taken up by cancer cells as a source of carbon and energy. In this study, we prepared a monomodified derivative of HSA covalently linked to an EDTA derivative and investigated its efficacy to shuttle weakly anti-proliferative EDTA associating ligands such as vanadium, into a cancer cell line. HSA-S-MAL-(CH<sup>2</sup> )2 -NH-CO-EDTA was found to associate both with the vanadium anion (+5) and the vanadium cation (+4) with more than thrice the associating affinity of those ligands toward EDTA. Both conjugates internalized into glioma tumor cell line via caveolae-mediated endocytosis pathway and showed potent anti-proliferative capacities. IC<sup>50</sup> values were in the range of 0.2 to 0.3 µM, potentiating the anti-proliferative efficacies of vanadium (+4) and vanadium (+5) twenty to thirty fold, respectively. HSA-EDTA-VO++ in particular is a cancer permeable prodrug conjugate. The associated vanadium (+4) is not released, nor is it active antiproliferatively prior to its engagement with the cancerous cells. The bound vanadium (+4) dissociates from the conjugate under acidic conditions with half maximal value at pH 5.8. In conclusion, the anti-proliferative activity feature of vanadium can be amplified and directed toward a cancer cell line. This is accomplished using a specially designed HSA-EDTA-shuttling vehicle, enabling vanadium to be anti-proliferatively active at the low micromolar range of concentration.

**Keywords:** albumin; conjugates; vanadium; cancer; prodrug

#### **1. Introduction**

Intensive studies have been carried out on the insulin-like effects of vanadium salts. Vanadium mimics the action of insulin in insulin responsive tissues and in diabetic rodents via insulin-independent pathways [1–3], which is reviewed in [4,5]. Vanadium belongs to a family of metals, which interferes with cellular redox homeostasis [6], and as such was investigated also for its anti-cancer efficacy. Vanadium is an element with a wide range of effects on the mammalian organism. In recent years, many studies were published regarding its various organic complexes in view of their application in medicine and the fact that the bioactive complexes/compounds of this metal can be therapeutically active at low

**Citation:** Cooper, I.; Ravid, O.; Rand, D.; Atrakchi, D.; Shemesh, C.; Bresler, Y.; Ben-Nissan, G.; Sharon, M.; Fridkin, M.; Shechter, Y. Albumin-EDTA-Vanadium Is a Powerful Anti-Proliferative Agent, Following Entrance into Glioma Cells via Caveolae-Mediated Endocytosis. *Pharmaceutics* **2021**, *13*, 1557. https://doi.org/10.3390/ pharmaceutics13101557

Academic Editor: Katona Gábor

Received: 4 July 2021 Accepted: 20 September 2021 Published: 25 September 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

concentrations [7]. With its physiological duality, vanadium is essential in trace amounts and toxic at concentrations above 10 µM. Its biological activities include anti-viral, antibacterial, anti-parasitic, anti-fungal, anti-cancer, anti-diabetic, anti-hypercholesterolemic, cardio-protective, and neuroprotective activity [8]. Moreover, in vivo studies reported chemo-preventive effects of vanadium complexes, whereas observations regarding therapeutic activities were limited [9–14]. Indeed vanadium can act in two opposing directions: as a metabolic factor, it might promote proliferation, and on the other hand due to its ability to generate ROS and/or to inhibit a large variety of phosphatases and hydrolases, it can act as an anticancer agent [6]. Consequently, the question arose whether these two opposing effects can be dissociated to permit conversion of vanadium exclusively into an anticancer agent.

In this study, we initially turned vanadium into a prodrug, capable of uptake preferentially by cancerous cells. A derivative of EDTA was covalently linked to HSA in a monomodified fashion. Albumin is largely taken by malignant tissues as a source of carbon and energy [15]. Albumin is also a natural transport protein with long circulatory half-life, which promotes it as an attractive candidate for half-life extension and targeted intracellular delivery of drugs attached by covalent conjugation or association [16].

Both vanadium (+4) and vanadium (+5) associate with EDTA at physiological pH [17]. We assumed that the resultant HSA- EDTA-vanadium conjugates will be inactive extracellularly, but will release bound vanadium both in the cytosol and even more efficiently at the acidic pH of the lysosome, following internalization.

Here, we wished to determine whether vanadium (+4) or vanadium (+5) generate intracellular cytotoxicity, if shuttled into a cancerous cell line with this HSA-EDTA carrier. Likewise, we attempted to identify conditions that eliminate the proliferative effects of vanadium and preserve solely its anti-proliferative efficacy. Our efforts in those directions are presented here in detail.

#### **2. Materials and Methods**

#### *2.1. Materials*

Dulbecco's Modified Earl's medium (DMEM) was purchased from Gibco (Life Technologies, Carlsbad, CA, USA). Gentamicin, glutamine, fetal calf serum (FCS), and penicillin/streptomycin were obtained from Biological Industries (Kibbutz Beit Haemek, Galilee, Israel). Human serum albumin (HSA), diethylenetriamine pentaacetic dianhydride (EDTAdianhyride), *N*(2-aminethyl) malemide, 4.40 dithiodipyridine (4.4DTDP), dithiothreitol (DTT), phosphatase acid from potato (#P-3762), pNitrophenyl phosphate (pNPP), phenylarsine oxide (PAO), indomethacin (IND), nystatin, methyl β cyclodextrin (MCD), and bafilomycin A1 (BAF) were purchased from Sigma Aldrich (Jerusalem, Israel). Sodium metavanadate (NaVO3) and Vanadyl chloride (VOCl2) were from BDH Chemicals Ltd. Poole England. Ethylenediaminetetraacetic acid (EDTA) from Baker Analyzed A.C.S Reagents, and PEG30-SH (M-SH-30K) were purchased from Jenkem Technology (Plano, TX, USA). All other materials used in this study were of analytical grade.

#### *2.2. Preparation of Mercaptoalbumin*

About one third of cysteine-34 of HSA is disulfide-bonded to glutathione or cysteine and this can be reversed by mild reduction with dithioerithritol [18]. This allows us to obtain conjugates containing 0.56 mole EDTA per mole of human serum albumin (Supplementary Table S1). HSA (1.4 g, 20 µmol) dissolved in 0.1 M Hepes buffer (pH 7.3) followed by the addition of one equivalent of dithiothreitol (20 µmol). The reaction was carried out for 1 h at 0 ◦C and dialyzed over a period of two days with several changes of H2O and lyophilized. This procedure removes mixed disulfide bonded glutathione or cystine from the cysteinyl moiety of HSA [19]. Mercapto-HSA prepared by this procedure contains 0.7 + 0.05 mole-SH per mole human serum albumin, as determined with 4.4 dithiodipyridine (4.40 DTDP) using ε<sup>324</sup> = 19,800 [20].

## *2.3. Preparation of EDTA-NH-(CH2)2-Maleimide (EDTA-Maleimide)*

EDTA-Dianhydride (38 mg, 100 µmol) suspended in 1.0 mL DMSO and transferred to a tube containing 110 µmol (27 mg) MAL-(CH2)2-NH2. DIPEA (*N*,*N*-Disopropylethylamine) was then added in aliquots to achieve neutral pH value, upon 100 times dilution of aliquots in H2O. Following one hour, the product EDTA-maleimide was obtained by centrifugation, washed twice with DMSO and stored at −70 ◦C until used.

#### *2.4. Preparation of HSA-S-MAL-EDTA*

*N*-(2 aminoethyl) maleimide (2.5 mg, 10 µmol) was dissolved in 1.0 mL of 1 M Hepes buffer (pH 7.3) and transferred immediately to a glass tube containing 38 mg (100 µmol) of EDTA-dianhydride. The reaction mixture was stirred for 30 min and combined with a solution of Mercapto-HSA (210 mg/3.0 mL H2O, 3 µmol). Following 1 h, the product (HSA-S-MAL-(CH2)2-NH-CO-EDTA) was dialyzed against H2O over a period of three days with several changes of H2O and lyophilized.

#### *2.5. Characterization of HSA-S-MAL-(CH2)2-NHCO-EDTA by Reversed Phase Liquid Chromatography, Coupled to Mass Spectrometry (LC-MS) Analysis*

The different protein samples were diluted to 0.17 µM in 100 mM ammonium acetate, pH 6.8. A total of 5 µL from each sample were loaded onto a monolithic reversed phase column [21] and eluted over a gradient of 10–60% acetonitrile, during 15 min, at column temperature of 60 ◦C. The HSA proteins eluted at 43% acetonitrile and were directly sprayed into a modified Q Exactive Plus EMR Orbitrap mass spectrometer [21], for intact mass measurements. The instrument was operated using the HESI source, at a flow rate of 15 µL/min, using sheath gas 10 and auxiliary gas 3. The inlet capillary was set to 320 ◦C, capillary voltage 4.3 kV, fore vacuum pressure 1.54 mbar, and trapping gas pressure 0.8, corresponding to HV pressure of 3.0 <sup>×</sup> <sup>10</sup>−<sup>5</sup> mbar and UHV pressure of 2.2 <sup>×</sup> <sup>10</sup>−<sup>10</sup> mbar. The source was operated at a constant energy of 2 V in the flatapole bias and interflatapole lens. Bent flatapole DC bias and gradient were set to 1.7 and 10 V, respectively, and the HCD cell was operated at 15 V. Measurements were performed at inject time of 250 and resolution of 10,000. Masses were calculated by the computational suite UniDec v. 4.1.1 [22] (2019, University of Arizona, Tucson, AZ, USA).

#### *2.6. Evaluating the Affinities of HSA-EDTA to Vanadium: Reversal of Vanadium-Evoked Inhibition of Acid Phosphatase*

This assay was carried out essentially according to reference [4] with slight modifications. It evaluates the efficacy of vanadium chelators toward vanadium, by determining their potency to reverse vanadium-evoked inhibition of acid phosphatase at pH 7.3. Each tube contains 0.5 mL of 0.05 M Hepes buffer pH 7.3, 1.0 M KCl, p-nitophenylphosphate, (0.2 mM) either NaVO<sup>3</sup> or VOCl<sup>2</sup> (5 µM), increasing concentration of the studied chelator and acid phosphatase (50 µg/tube). Following 40 min at 25 ◦C, NaOH (20 µL from 4 M NaOH) was added and the absorbance corresponding to the formed p-nitrophenolate was determined at 410 nm. IC<sup>50</sup> is defined here as the concentration of the vanadium chelator that reversed half maximally vanadium (+4) or vanadium (+5) evoked inhibition of acid phosphatase. It should be noticed that since this is an in-direct method, direct measurements of released vanadium using procedures such as ICP-MS, should be conducted in future studies.

#### *2.7. Preparation of HSA-EDTA-Vanadium*

HSA-EDTA (25 mg, 0.37 µmol) dissolved in 0.2 mL H2O and VOCl2, or NaVO3, 4 molar excess was then added. The reaction mixture was loaded on a Sephadex G-50 column (12 × 1.7 cm) pre-equilibrated and run with 0.01 M NaHCO<sup>3</sup> (pH 8.22). The peak corresponding to the protein fraction was pooled and lyophilized.

#### *2.8. Preparation of Rhodamine-Labeled HSA-EDTA*

Rhodamine-labeled HSA and HSA-EDTA were prepared by dissolving 17 mg of each (~0.25 µmol) in 0.2 mL of 0.1 M Na2CO<sup>3</sup> (pH 10.3). Rhodamine B isothiocyanate 0.9 mg (2.5 molar excess over HSA) was then added and the reaction was carried out for 1 h at 25 ◦C. The reaction mixture was loaded on a Sephadex G-50 column (1.7 × 14 cm) equilibrated and run in the same buffer. The tubes containing rhodamine-labeled HSA were pooled, dialyzed against water, and lyophilized. Rhodamine-HSA and rhodamine-HSA-EDTA prepared by this procedure contain 0.95 ± 0.1 mole of rhodamine/mole of HSA as determined by its absorbance at 550 nm using ε<sup>550</sup> = 11,400.

#### *2.9. Growth Inhibitory Effects of EDTA and Vanadium Containing Conjugates*

The glioma cell line CNS-1 (obtained from Mariano S. Viapiano [23]) was grown in 96 well plates in DMEM containing 10% fetal calf serum; 2 mM L-glutamine, penicillin (100 units/mL), and streptomycin (0.1 mg/mL) under humidified atmosphere containing 5% CO2. Cells were seeded at 1000 cells/well. Twenty-four hours later, the EDTA and vanadium containing conjugates were added to each plate to give concentrations as indicated in the text. Control experiments using non-cancer cells were conducted with primary bovine brain pericytes and CD34+ human endothelial cells (both obtained and characterized at the Artois University, France [24–26]) treated with the HSA-EDTA-VO++ conjugate. These cells were seeded at 15,000 cells/well in ECM medium (Sciencell, Carlsbad, CA, USA), which was composed as follows: 5% fetal calf serum (Gibco, Gaithersburg, MD, USA), ECGS supplements, and 50 mg/mL gentamicin (Biological industries, Beit-Haemek, Israel). Cells were treated the day after. Cell viability was measured after 72 h using a standard MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay as described before [27]. Experiments were repeated at least 3 times in quadruplicate. IC<sup>50</sup> values were calculated from the dose response curves using a median-effect plot.

#### *2.10. Immunocytochemistry*

CNS-1 cells (60,000/well) were seeded on cover slips in 24 well-plates. After 24 h, growth medium (10% FCS, penicillin (100 U/mL) streptomycin (0.1 mg/mL) and Lglutamine (2 mmol/L) dissolved in DMEM was replaced with fresh medium containing 5 µM rhodamine-labeled HSA or HSA-EDTA-VO++. After 5 min, 1 h, or 24 h, the cells were washed with cold PBS and fixed with 4% paraformaldehyde for 15 min. The cells were then stained with Alexa fluor 488-phalloidin (Thermo Fisher Scientific, Waltham, MA, USA) for 20 min and 2 min with Hoechst (Sigma, Burlington, MA, USA). Cells were rinsed with PBS and coverslips were mounted and observed with Olympus IX43 fluorescence microscope.
