*Article* **Tumor-Homing pH-Sensitive Extracellular Vesicles for Targeting Heterogeneous Tumors**

#### **Jaeduk Park <sup>1</sup> , Hyuk Lee <sup>1</sup> , Yu Seok Youn <sup>2</sup> , Kyung Taek Oh <sup>3</sup> and Eun Seong Lee 1,4,\***


Received: 31 March 2020; Accepted: 15 April 2020; Published: 17 April 2020

**Abstract:** In this study, we fabricated tumor-homing pH-sensitive extracellular vesicles for efficient tumor treatment. These vesicles were prepared using extracellular vesicles (EVs; BTEVs extracted from BT-474 tumor cells or SKEVs extracted from SK-N-MC tumor cells), hyaluronic acid grafted with 3-(diethylamino)propylamine (HDEA), and doxorubicin (DOX, as a model antitumor drug). Consequently, HDEA/DOX anchored EVs (HDEA@EVs) can interact with origin tumor cells owing to EVs' homing ability to origin cells. Therefore, EV blends of HDEA@BTEVs and HDEA@SKEVs demonstrate highly increased cellular uptake in both BT-474 and SK-N-MC cells: HDEA@BTEVs for BT-474 tumor cells and HDEA@SKEVs for SK-N-MC tumor cells. Furthermore, the hydrophobic HDEA present in HDEA@EVs at pH 7.4 can switch to hydrophilic HDEA at pH 6.5 as a result of acidic pH-induced protonation of 3-(diethylamino)propylamine (DEAP) moieties, resulting in an acidic pH-activated EVs' disruption, accelerated release of encapsulated DOX molecules, and highly increased cell cytotoxicity. However, EV blends containing pH-insensitive HA grafted with deoxycholic acid (HDOC) (HDOC@BTEVs and HDOC@SKEVs) showed less cell cytotoxicity for both BT-474 and SK-N-MC tumor cells, because they did not act on EVs' disruption and the resulting DOX release. Consequently, the use of these tumor-homing pH-sensitive EV blends may result in effective targeted therapies for various tumor cells.

**Keywords:** tumor-homing extracellular vesicles; pH-sensitive extracellular vesicles; doxorubicin; tumor therapy

#### **1. Introduction**

Extracellular vesicles (EVs) are nanosized cellular vesicles released from various types of tumor cells [1–5]. To achieve quick and extensive intercellular communication between tumor cells, EVs are secreted out of the cells so that they can enter the recipient cells [4–7]. These EVs perform various biological functions, such as the disposal of cellular waste products, release of foreign invaders, control of gene expression, and activation of the immune system [8–10].

In addition, EVs intrinsically express various membrane proteins, cell adhesion molecules, and tumor specific ligands, thereby enabling the homing of EVs to origin cells [4,11–16]. These properties of EVs enable tumor-homing ability and render them potential candidates as tumor-recognizing drug carriers. In particular, recent studies suggest that these EVs have the ability to interact with their

released parental cells, and this property has been used to target tumor cells [4,11–16]. This means that these EVs are suitable as tumor-targeting and tumor-penetrating drug carriers, as they can be selectively homed to their parent tumor cells [4,13–22]. Furthermore, the immunogenicity of these EVs is relatively low; therefore, they exhibit excellent body safety for biomedical applications [4,10,19,20].

In this study, we fabricated tumor-homing pH-sensitive EVs. These EVs were prepared using EVs (BTEVs extracted from BT-474 tumor cells or SKEVs extracted from SK-N-MC tumor cells), hyaluronic acid grafted with 3-(diethylamino)propylamine (HDEA), and doxorubicin (DOX) [3]. In particular, we prepared EV blends using HDEA/DOX anchored EVs (HDEA@BTEVs and HDEA@SKEVs) to target different tumor cells. The EV blends are expected to yield efficient cellular uptake for parent BT-474 and SK-N-MC tumor cells. Therefore, we hypothesize that these different EVs can target their origin tumor cells, owing to their homing ability to origin cells, allowing their efficient accumulation into heterogeneous tumor cells. Furthermore, 3-(diethylamino)propylamine (DEAP) moieties present in HDEA can be protonated at endosomal pH and induce the destabilization of EVs, owing to DEAP-mediated vesicle destabilization, followed by the release of encapsulated DOX [3,23–31]. In this study, we investigated the tumor targeting ability, pH-sensitive properties, and antitumor efficacy of EV blends against BT-474 and SK-N-MC tumor cells.

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

#### *2.1. Materials*

Hyaluronic acid (HA, Mw = 4.8 kDa), 3-(diethylamino)propylamine (DEAP), N-hydroxysuccinimide (NHS), N,N'-dicyclohexylcarbodiimide (DCC), triethylamine (TEA), deoxycholic acid (DOCA), 4-dimethylaminopyridine (DMAP), pyridine, dimethyl sulfoxide (DMSO), sodium tetraborate, adipic acid dihydrazide (ADH), doxorubicin hydrochloride (DOX), paraformaldehyde, heparin, and Triton X-100, 40 ,6-diamidino-2-phenylindole dihydrochloride (DAPI) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Chlorin e6 (Ce6) was purchased from Frontier Scientific Inc (Logan, UT, USA). RPMI-1640 medium, DMEM medium, fetal bovine serum (FBS), phosphate buffered saline (PBS), ethylene diamine tetra-acetic acid (EDTA), penicillin, trypsin, and streptomycin were purchased from Welgene Inc (Seoul, Korea). EV-depleted FBS was purchased from System Biosciences Inc. (Palo Alto, CA, USA). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies Inc. (Santa Clara, CA, USA). Wheat Germ Agglutinin Alexa Fluor® 488 Conjugate (WGA-Alexa Fluor® 488), fluorescein isothiocyanate (FITC) were purchased from Life Technologies (Carlsbad, CA, USA).

#### *2.2. Synthesis of HA-g-DEAP*

The detailed synthesis method of HA grafted with DEAP (HA-g-DEAP: HDEA) was described in our previous report [3,29–31]. Briefly, HA (200 mg) was reacted with DEAP (55 mg) in DMSO (10 mL) containing DCC (110 mg), NHS (60 mg), and TEA (500 µL) at 25 ◦C for 3 days, to produce HDEA (Figure S1). HA grafted with DOCA (HA-g-DOCA: HDOC) was prepared as a pH-insensitive control group against pH-sensitive HDEA. The detailed synthesis method of HDOC was described in our previous report [3,29–31]. Briefly, HA (200 mg) was reacted with DOCA (650 mg) in DMSO (10 mL) containing DCC (340 mg), DMAP (20 mg), and pyridine (500 µL) at 25 ◦C for 3 days, to produce HDOC (Figure S2). In addition, HDEA (100 mg) or HDOC (100 mg) were reacted with Ce6 (15 mg) in DMSO (10 mL) containing DCC (10 mg), ADH (9 mg), NHS (6 mg), and TEA (200 µL) at 25 ◦C for 2 days, to produce Ce6-labeled HDEA or Ce6–labeled HDOC [3,30,31]. The non-reacted chemicals were removed via dialysis against fresh DMSO for 3 days, and then deionized water for 3 days using a pre-swollen dialysis membrane (Spectra/Por®6 MWCO 2 kDa, Spectrum Laboratories Inc, Rancho Dominguez, CA, USA). The dialyzed solution was freeze-dried; subsequently, the final product was obtained [3,29–31].
