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

#### **Jaeduk Park 1 , Hyuk Lee 1 , Yu Seok Youn 2 , Kyung Taek Oh 3 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].


*3.1. Precipitation Rates*

A highly significant interactive effect of Mg ion concentration and Ca:CO3 concentration was observed (Figure 3; Table 2; F[2,24] = 150.924, *p* < 0.001). When Mg was included into the aquatic solution, neither a significant weight change of the CaCO3 seed nor a difference between the two Ca:CO3 scenarios were observed (Table 2; Table A1). Conversely, both Mg-free Ca:CO3 scenarios had significant weight increases. The Mg-free 47:1 Ca:CO3 scenario had a calcification rate four times that of the Mg-free 2.8:1 Ca:CO3 scenario (Figure 3, points labelled A and B). The average weight increase (± SD) in the Mg−free 47:1 Ca:CO3 treatment was 1.017 (± 0.130] mg d−1 and 0.229 (± 0.061) mg d−1 in the Mg-free 2.8:1 Ca:CO3 treatment. The high amount of newly formed CaCO3 measured in the Mg-free 47:1 Ca:CO3 2− is partly explained by spontaneous nucleation, which was only observed in this scenario. Slight dissolution was observed under the intermediate (26.5 mM] and high (53 mM) Mg scenario with a 47:1 Ca:CO3 concentration (−0.001 mg d−1 ± 0.001 and −0.002 mg d−1 ± 0.003, respectively). The intermediate and high Mg scenario with a 2.8:1 Ca:CO3 concentration had no significant weight changes (0.000 mg d−1 ± 0.001 and 0.002 mg d−1 ± 0.007, respectively), the Ca:CO3 concentration had no effect on the growth rate when Mg ion concentration was equal to or half that found in the ambient ocean, indicating that in this situation Mg has a stronger inhibiting effect towards calcification than Ca:CO3 concentrations.

**Figure 3.** Scatter plot showing the standardized total weight change per day [mg d−1] of each experimental scenario. The data shows a clear inhibiting effect of Mg and 2.8:1 Ca:CO3 stoichiometry towards inorganic mineralization. Treatments not connected by the same alphabetical symbol (A, B, or C) are significantly different as shown in Table 2; F[2,24] = 150.924, *p* < 0.001.


5. Graphene-based metamaterial reflectarray for orbital angular momentum (OAM) vortex wave

The electromagnetic waves carry both linear and angular momentums. Angular momentum comprises spin angular momentum (SAM) and orbital angular momentum (OAM). The SAM is associated with the circular polarization states of electromagnetic beams. The OAM arises from spatial variations of amplitude and phase that render the beam asymmetric around its propagation axis [55, 56]. In 1992, Allen et al. found that light beam with an azimuthal phase dependence of exp.(ilϕ) carries an OAM, in which ϕ represents the azimuthal angle and l is the topological charge. For any given l, the OAM vortex wave has l interwinded helical phase fronts and a phase singularity with zero intensity on the beam axis. With a theoretically unlimited range of orthogonal eigenstates, OAM offers new degrees of freedom in communication in addition to traditional linear momentum and polarization degrees of freedom [57–60].

An attractive feature of graphene is that its conductivity is changeable by controlling voltage applied to graphene via an external gate. With this characteristic, a graphene-based metamaterial reflectarray is designed for the generation of the wideband OAM vortex waves with tunable modes in this section. As shown in Figure 16, the designed reflectarray with a size of 10 10λ comprises 12 regions, each of which has the same azimuthal angle. Here, λ is wavelength in free space at the frequency of 2.3 THz. In each region, a same graphene-based metamaterial structure is designed. By tuning the conductivities of the graphene sheets in the jth region, the reflection phase of πlj/6 (j = 1, …,12) is achieved such that the whole reflectarray can generate a helical profile of exp.(ilϕ). To guarantee independently adjustable conductivities

Figure 14. Simulated variation of absorption with frequencies for different azimuth angles: (a) TM mode and (b) TE

Figure 15. Variation of the absorption with the chemical potential μc: (a) TE mode and (b) TM mode. Reprinted from

Figure 13. Simulated absorption performance at different incidence angles: (a) TM mode and (b) TE mode. Reprinted

mode. Reprinted from Zhang et al. [38], with permission from the Optical Society of America.

Zhang et al. [38], with permission from the Optical Society of America.

from Zhang et al. [38], with permission from the Optical Society of America.

182 Metamaterials and Metasurfaces

Figure 16. Schematic diagram of the designed reflectarray. (a) The whole array divided into 12 regions, each of which is filled by the same metamaterial unit cells. (b) The side view of the reflectarray. Reprinted from Shi et al. [43], with permission from the IEEE.

Figure 17. The graphene-based metamaterial unit cell and its equivalent circuit model: (a) the unit cells and (b) the equivalent circuit model. Reprinted from Shi et al. [43], with permission from the IEEE.

Figure 18. Reflection phase range for all possible chemical potentials of three graphene layers in a wide frequency band from 1.8 THz to 2.8 THz. Reprinted from Shi et al. [43], with permission from the IEEE.

Figure 19. The OAM beams with the different modes: (a) l = 1, (b) l = 2, (c) l = 3, (d) l = 1, (e) l = 2, and (f) l = 3.

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185

Reprinted from Shi et al. [43], with permission from the IEEE.

Figure 17. The graphene-based metamaterial unit cell and its equivalent circuit model: (a) the unit cells and (b) the

Figure 18. Reflection phase range for all possible chemical potentials of three graphene layers in a wide frequency band

from 1.8 THz to 2.8 THz. Reprinted from Shi et al. [43], with permission from the IEEE.

equivalent circuit model. Reprinted from Shi et al. [43], with permission from the IEEE.

184 Metamaterials and Metasurfaces

Figure 19. The OAM beams with the different modes: (a) l = 1, (b) l = 2, (c) l = 3, (d) l = 1, (e) l = 2, and (f) l = 3. Reprinted from Shi et al. [43], with permission from the IEEE.

of the graphene sheets in each region, a small height difference Δh is introduced to ensure the insulation between two adjacent regions, as shown in Figure 16(b).

vortex waves with l = 1, l = 2, and l = 3 modes and the doughnut-shaped intensity distributions. Note that the radiation patterns with singularity in the center greatly reduce the coupling between the reflectarray and the horn antenna. With the reflection phase range of

In sum, several characteristics of graphene including the conductivity model and equivalent circuit model have been presented. Two graphene-based devices, i.e., metamaterial absorber and metamaterial reflectarray, have been designed. By varying graphene's chemical potential, the wideband tunable absorption for the designed absorber and the broadband tunable OAM modes for the developed reflectarray have been demonstrated, respectively. Graphene pro-

This work is supported by National Natural Science Foundation of China under Contract 61771359 Fundamental Research Funds for the Central Universities (No. JBF180202), and

[1] Caloz C, Itoh T. Electromagnetic Metamaterials: Transmission Line Theory and Micro-

wave Applications. New Jersey: Wiley; 2005. 376 p. DOI: 10.1002/0471754323

vides more degrees of freedom for the design of the tunable metamaterial systems.

in a wide frequency band from 1.8 to 2.8 THz, as shown in Figure 18, the OAM vortex waves can be generated by the proposed reflectarray in the wide frequency band, as shown in Figure 20. It is observed that the desirable spiral phase distributions can be obtained in the

Design of Graphene-Based Metamaterial Absorber and Antenna

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360

whole frequency band.

Acknowledgements

Conflict of interest

Author details

References

Yan Shi\* and Ying Zhang

Technology Innovation Research Project of the CETC.

No conflict of interest was reported by the authors.

\*Address all correspondence to<EMAIL_ADDRESS>
School of Electronic Engineering, Xidian University, Xi'an, Shaanxi, China

6. Conclusion

To obtain a desirable reflection phase in each region, a metamaterial unit cell composed of a three-layer sandwich structure has been designed, as shown in Figure 17. Each sandwich structure comprises graphene/Al2O3/SiO2 materials from the top to the bottom. An insulating layer of Al2O3 material is inserted between two adjacent sandwich structures. A ground consisting of an Au material is placed at the bottom of the unit cell. The designed unit cell periodicity is P = 20 μm, and the thicknesses of SiO2, Al2O3, and Au materials are 12 μm, 10 nm, and 5 μm, respectively. In each sandwich structure, an external DC voltage is applied between the graphene layer and the SiO2 layer to control the conductivity of graphene. For convenience, the chemical potentials of the graphene layers from the top to the bottom are denoted as "μc1," "μc2," and "μc3," respectively. With the equivalent circuit model given in Figure 17(b), the maximum reflection phase range of the proposed unit cell can be obtained. As shown in Figure 18, the reflection phase range of the proposed unit cell can cover 360o in a wide frequency band from 1.8 to 2.8 THz, when three chemical potentials independently vary from 1 to 1 eV.

A wideband horn antenna as the excitation is used to generate a wave incident on the reflectarray. Figure 19 shows the OAM vortex waves with different modes generated by the reflectarray at 2.3 THz. We can clearly observe the spiral phase distributions of the OAM

Figure 20. Simulated OAM beams with l = 1 mode at different frequencies. Reprinted from Shi et al. [43], with permission from the IEEE.

vortex waves with l = 1, l = 2, and l = 3 modes and the doughnut-shaped intensity distributions. Note that the radiation patterns with singularity in the center greatly reduce the coupling between the reflectarray and the horn antenna. With the reflection phase range of 360 in a wide frequency band from 1.8 to 2.8 THz, as shown in Figure 18, the OAM vortex waves can be generated by the proposed reflectarray in the wide frequency band, as shown in Figure 20. It is observed that the desirable spiral phase distributions can be obtained in the whole frequency band.