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Sprayable Adhesive Nanotherapeutics: Mussel Protein-Based Nanoparticles for Highly Efficient Locoregional Cancer Therapy Yeonsu Jeong, Yun Kee Jo, Bum Jin Kim, Byeongseon Yang, Kye Il Joo, and Hyung Joon Cha ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b04533 • Publication Date (Web): 27 Jul 2018 Downloaded from http://pubs.acs.org on July 28, 2018

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Sprayable Adhesive Nanotherapeutics: Mussel Protein-Based Nanoparticles for Highly Efficient Locoregional Cancer Therapy Yeonsu Jeong†, Yun Kee Jo†, Bum Jin Kim1, Byeongseon Yang, Kye Il Joo, and Hyung Joon Cha*

Department of Chemical Engineering, Pohang University of Science and Technology, Pohang 37673, Korea

1

Current address: LSK BioPharma, Salt Lake City, UT 84111, USA †

These authors contributed equally to this work *E-mail: [email protected]

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ABSTRACT Following surgical resection for primary treatment of solid tumor, systemic chemotherapy is commonly used to eliminate residual cancer cells to prevent tumor recurrence. However, its clinical outcome is often limited due to insufficient local accumulation and the systemic toxicity of anticancer drugs. Here, we propose a sprayable adhesive nanoparticle (NP)-based drug delivery system using a bioengineered mussel adhesive protein (MAP) for effective locoregional cancer therapy. The MAP NPs could be administered to target surfaces in a surface-independent manner through a simple and easy spray process by virtue of their unique adhesion ability and sufficient dispersion property. Doxorubicin (DOX)-loaded MAP NPs (MAP@DOX NPs) exhibited efficient cellular uptake, endolysosomal trafficking, and subsequent low pH microenvironment-induced DOX release in cancer cells. The locally sprayed MAP@DOX NPs showed a significant inhibition of tumor growth in vivo, resulting from the prolonged retention of the MAP@DOX NPs on the tumor surface. Thus, this adhesive MAP NP-based spray therapeutic system provides a promising approach for the topical drug delivery in adjuvant cancer therapy.

KEYWORDS. mussel adhesive proteins, adhesive nanoparticles, surgical spray, locoregional drug delivery, adjuvant cancer therapy


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The standard treatment for primary cancer includes post-operative chemotherapy to destroy any remaining cancer cells; this treatment reduces the risk of ipsilateral recurrence after tumor resection to achieve long-term remission.1,2 Nanoparticles (NPs) have gained considerable attention as intracellular drug delivery vehicles due to their ability to gain access to cells by endocytosis and passive tumor targeting ability via systemic administration (i.e., intravenous injection) based on the enhanced permeability and retention effects.3,4 However, only 0.7% of intravenously injected NPs have been reported to reach tumor sites, resulting in marginal improvement in a clinical translation.5,6 Thus, the site-directed topical application of NPs may be more advantageous for achieving the desired therapeutic effects, with a lower risk of systemic toxicity, particularly in adjuvant cancer therapy.7 In the field of nanotechnology, the spraying technique, which enables the deposition of suspended particles onto a substrate, has been used frequently for a variety of emerging applications, ranging from solar cells8,9 and electrochemical devices10 to biomedicine,11 due to its simple and easy process and high-efficiency delivery.12 In particular, the spraying technique can ensure high-throughput deposition of NPs, which can be advantageous for topical drug delivery that circumvents metabolic clearance.13 However, the spray deposition of NP suspensions on tissue surfaces has not received much attention for in vivo therapeutic applications due to the lack of underwater adhesion by NPs, which is essential for efficient retention on target tissues in bodily fluids.14,15 In addition, the low dispersion property of NPs, which causes the formation of agglomerates during the spray process, remains a significant challenge because the agglomerated NPs interrupt the stable flow of suspensions and/or clog the narrow orifices of the suspension feeder.16 Thus, an advanced design of NPs that allows NPs to be well dispersed throughout the spray process and exhibit sufficient underwater adhesion properties for stable deposition is required. In this work, we proposed a sticky NP-based spray therapeutic system, inspired by marine mussels, for effective focal cancer treatment. Mussels attach strongly to wet surfaces, even 3 Environment ACS Paragon Plus

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under harsh environments in seawater, by secreting proteinaceous glues.17,18 These specialized proteins, mussel adhesive proteins (MAPs), have received great attention as promising bioadhesives in biomedical applications due to their great underwater adhesion to various surfaces, biocompatibility, and biodegradability.19,20 As a major component of MAPs, 3,4dihydroxy-phenylalanine (DOPA) is known to play a key role in the surface adhesion and cohesion of the byssus. In particular, the multiple bidentate complexes between DOPA and Fe3+ exhibit an extraordinarily stable and reversible bonding capability.21,22 In addition, the stoichiometry of DOPA-Fe3+ complexes can be altered by environmental pH changes to form mono- (at pH < 5.6), bis- (at 5.6 < pH < 9.1), or tris- (at pH > 9.1) crosslinks through protonation or deprotonation of catecholic hydroxyl groups, allowing MAPs to be utilized as pH-responsive biopolymers.23,24 To overcome the limited availability of extracted natural MAPs, bioengineered MAP was previously produced in a bacterial expression system, and its superior adhesion properties, biocompatibility, and biodegradability were sufficiently confirmed.25,26 We expected that bioengineered MAP-based NPs could achieve sufficient adhesiveness and dispersion properties for therapeutic application as a sprayable vehicle system, enabling site-directed and localized anticancer drug delivery, which can be used as an adjuvant treatment during a surgical resection procedure (Figure 1A). Thus, doxorubicin (DOX)-loaded MAP NPs (MAP@DOX NPs) were fabricated based on DOPA-Fe3+ complex coordination, and the in vitro, ex vivo, and in vivo anticancer therapeutic efficacy and potential applicability of these MAP@DOX NPs were evaluated.

RESULTS AND DISCUSSION We prepared bioengineered MAP fp-1 which was consisted of 12 tandem repeats of decapeptides [AKPSYPPTYK] inspired from Mytilus edulis with good solubility (>500 g/L) in aqueous solution and high DOPA conversion yield as ~50% (~10 mol%) (Figure S1).24,27-29 4 Environment ACS Paragon Plus

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The MAP NPs were fabricated via electrospraying technique using the DOPA-containing MAP solution in a phosphate-buffered saline (PBS):ethanol (30:70) solvent mixed with FeCl3. The DOPA-Fe3+ complex-containing MAP solution formed NPs by dissociation of the droplets from nozzle via coulombic repulsion under electric field.30 The MAP@DOX NPs were prepared using a mixture of the DOPA-Fe3+ complex-containing MAP and DOX in the same way as synthesis of MAP NPs. After the formation of NPs, they were dropped into PBS (pH 7.4) to achieve structural stability through bis-DOPA-Fe3+ complexation. Spherical NPs with a hydrodynamic diameter of ~218.5 nm were fabricated successfully by exploiting the DOPA-Fe3+ complexes of bioengineered MAP (Figure 1B, C). The drug loading efficiency of MAP@DOX NPs was 74.12 ± 0.03% via π-π, cation-π, and hydrophobic interactions between the aromatic ring of DOX and abundant DOPA, tyrosine, and lysine residues in MAP.31-33 Under the similar mechanism of DOX encapsulation, a myriad of aromatic ring-containing chemotherapeutic agents such as anthracycline (e.g., daunarubicin, epirubicin, idarubicin, and valrubicin) and hydrophobic taxane (e.g., paclitaxel, docetaxel, and tesetaxel) might be loaded into the MAP NPs.33,34 Also, the MAP NPs can load negatively charged therapeutic genes by means of electrostatic interaction with positively charged lysine residues in MAP.35 A suspension of the resultant MAP NPs was spray-coated onto substrates via air-pressured atomization through a nozzle. The MAP NPs were well dispersed in a PBS:ethanol (30:70) solvent; this dispersion was attributed to electrostatic repulsion based on the ionic strength of PBS and the low surface tension of ethanol at the fine nozzle during the spray process.36 The spray process of the MAP NPs was accomplished by hydraulic pressure created in a spray bottle by pushing down an atomizer pump cap, without requirement of compressed carrier gas.37 During the spray process, no notable differences in particle size or shape of the MAP NPs were detected, and no apparent loss of the encapsulated drugs was observed (Figure S2). This retained structural integrity of the MAP NPs under a certain level of fluidic pressure might be due to the stable crosslinking of DOPA-Fe3+ complexes. Moreover, a dense NP 5 Environment ACS Paragon Plus

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structure was deposited on a surface through 10 times of spraying process, while a sparse monolayered surface was formed by one time spraying, indicating that the number of spraying process can modulate the deposition pattern of MAP NPs (Figure 1D). Thus, MAP NP spray system could be properly applied for a focal administration with high-throughput deposition and dose adjustment of NPs. The sprayed MAP NPs exhibited superior adhesion on glass and porcine skin tissue surfaces and maintained their adhesiveness in aqueous conditions for over a month (Figure 2A, S3A, S3B). The MAP NPs (> 90%) were also adsorbed onto a collagen surface within 5 min and were almost retained on the surface, in contrast to bovine serum albumin (BSA)-based fabricated NPs (BSA NPs; Figure S3C, S3D). The adhesion of the MAP NPs was determined quantitatively during flow via quartz crystal microbalance (QCM) analysis and compared with that of BSA NPs. After equilibration (f0) with distilled water (DW), the frequency change (∆f1) of the MAP NPs at a flow rate of 40 µL/min was ~3.7-fold higher than that of BSA NPs (Figure 2B, S4A). The adhesion of MAP NPs was complete within 10 min, while greater than 1 h was needed for the stabilization of BSA NPs. After a DW wash, ~96% of the MAP NPs were retained, whereas BSA NPs were considerably washed out. In addition, the adhesion force of MAP NPs was quantitatively evaluated in a physiological environment by asymmetric MAP NPs coating on a mica surface using surface forces apparatus (SFA; Figure S4B). The adhesion strength of MAP NPs (-12.40 ± 8.65 mN/m) with 10 min contact time was stronger than that of BSA NPs (-2.94 ± 1.50 mN/m) (Figure 2C, S4C). The adhesion property of MAP NPs on mica surface in wet environments could be primarily attributed to electrostatic interaction between positively charged lysine residues (~20 mol%) in MAP and negatively charged mica.38 In addition, hydrogen bonding between unreacted DOPA or unmodified tyrosine (~10 mol%) residues and inorganic surfaces could be also responsible to the adhesion of MAP NPs.39 Meanwhile, the reacted DOPA residues dictate the formation of stably crosslinked structures of MAP NPs,40 exhibiting the recovery of mica-mica separation 6 Environment ACS Paragon Plus

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distance without a significant breakage of NPs after complete compression, analyzed by a cyclic test in SFA (Figure S4D). Moreover, the encapsulated anticancer drugs in MAP NPs were also successfully retained on the surface due to the stable complex formation of MAP NPs (Figure S5). Thus, we suggest that MAP NPs have great capabilities as a spray therapeutic system, with sufficient underwater adhesiveness and stability for site-directed and localized drug delivery. Stimulus-responsive systems have demonstrated the feasibility of excellent site-specific and on-demand drug delivery in response to changes in the cellular microenvironment.41 In particular, the acidic extracellular and endolysosomal (typically pH 5−6) microenvironments of cancer cells can be exploited to trigger the release of therapeutic cargoes from pHresponsive drug carrers.42 We monitored the DOX release profiles of MAP@DOX NPs in environments simulating blood plasma (pH 7.4) and endosome/lysosome (pH 5.5) to assess the capability of controlled release. The DOX release rate in acidic buffer (pH 5.5) was increased by ~2-fold in the first 3 h of incubation in comparison to release in physiological buffer (pH 7.4), indicating the potential of the MAP@DOX NPs as a tumor microenvironment-specific drug delivery system (Figure 3A). The enhanced release of DOX in acidic condition would be due to the low pH-induced destabilization of DOPA-Fe3+ complexes and loosening of crosslinked networks in the disassembled MAP@DOX NPs via deprotonation of catecholic hydroxyl groups (Figure S6).43-45 The cytotoxicity of the MAP NPs was examined using a cancer cell line (human breast carcinoma, MCF-7) and a normal cell line (mouse pre-osteoblasts, MC3T3-E1) in vitro. No significant cytotoxicity of the DOX-free MAP NPs (>95% viability) was observed in either cell lines (Figure S7). The MAP@DOX NPs exhibited a substantially higher half-maximum inhibitory concentration (IC50) for normal cells (MC3T3-E1 & human umbilical vein endothelial cells (HUVECs)) and a lower IC50 value for cancer cells (MCF-7 & the human breast carcinoma MDA-MB-231) when compared to the values observed for the equivalent 7 Environment ACS Paragon Plus

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dose of free DOX, indicating that the MAP NPs could achieve a more effective therapeutic response toward cancer cells (Figure 3B, S8). Because breast cancer cells have a relatively high intracellular Fe3+ concentration for promoting cell proliferation and an aggressive cancer phenotype,46,47 Fe3+ chelators, such as deferoxamine,48 deferasirox,49 and Dp44mT,50 have been investigated as anticancer agents to induce intracellular Fe3+ depletion and cell cycle arrest, block DNA synthesis, and eventually lead to apoptosis.51 In acidic environments, bisor tris-coordinated DOPA-Fe3+ complexes within MAP NPs are converted to monocoordinated DOPA-Fe3+ complexes with two free DOPA ligands.52 Thus, we surmise that the free DOPA ligands chelate Fe3+ in breast cancer cells, subsequently giving rise to extensive cancer cell death. Based on DOX-associated fluorescence, we found that the MAP@DOX NPs showed a much higher cellular uptake into cancer MCF-7 cells than that into normal MC3T3-E1 cells (Figure 3C). One possible explanation for the improved internalization of the MAP@DOX NPs is the high metabolic rates of cancer cells.53 Moreover, highly positive charges of abundant lysine residues (~20 mol%) in MAP allow the MAP NPs to be more easily attached onto the negatively charged surfaces of cancer cells, leading to higher cellular internalization, whereas the cellular uptake into normal cells is less sensitive to the charge of NPs.54,55 The enhanced cellular uptake can contribute to the selective and efficient therapeutic effects of the MAP@DOX NPs against cancer cells rather than normal cells as opposed to free DOX that can cause side effects to normal cells. Furthermore, the MAP@DOX NPs induced cancer cell death more effectively than free DOX, as indicated by flow cytometry using the Alexa Flour® 488 Annexin V (AV)/7-aminoactinomycin D (7-AAD) apoptosis detection assay (Figure 3D). The early apoptotic ratio (~53%) and the late apoptotic ratio (~42%) of the MAP@DOX NPsprayed cancer cells were conspicuously higher than those (~33% and ~21%, respectively) of free DOX-treated cells. Notably, the MAP@DOX NPs resulted in membrane blebbing of


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cancer cells, which is a distinct feature of apoptosis, implying that the MAP NPs induce enhanced apoptosis, ultimately leading to effective anticancer responses (Figure S9).56 The cellular uptake mechanism of the MAP@DOX NPs was analyzed via treatment with several endocytosis inhibitors.57 The internalization of the MAP@DOX NPs in MCF-7 cells was primarily inhibited by the ice incubation process for energy depletion, indicating that endocytosis – generally an energy-consuming process – is a major pathway of cellular uptake (Figure 4A, B). The cellular uptake of the MAP@DOX NPs was reduced considerably (by more than 50%) when the cells were treated with genistein (an inhibitor of caveolae-mediated endocytosis for blocking tyrosine kinase) and cytochalasin D (an inhibitor of caveolaemediated endocytosis and/or micropinocytosis for depolymerizing actin). Meanwhile, the internalization of the MAP@DOX NPs was slightly reduced in the presence of chlorpromazine (an inhibitor of clathrin-mediated endocytosis for dissociating the clathrin lattice) and wortmannin (a macropinocytosis inhibitor for blocking phosphatidyl inositol-3phosphate) (by 31.55% and 26.06%, respectively). These results suggest that caveolaemediated endocytosis is the main endocytic pathway of the MAP@DOX NPs, and clathrinmediated endocytosis and macropinocytosis are also involved in intracellular entry. In contrast, lipid-raft-mediated endocytosis, which can be inhibited by the cholesterol-depleting inhibitor methyl-β-cyclodextrin (MβCD), is barely involved in the cellular uptake of MAP@DOX NPs. Considering that lipid-raft-mediated endocytosis mediates the intracellular entry of hydrophobic molecules,58 this process might have little impact on the endocytosis of hydrophilic MAP@DOX NPs. To investigate the intracellular fate of the MAP@DOX NPs, their co-localization with early endosome antigen 1 (EEA1; an early endosome marker) and lysosomal-associated membrane protein 1 (LAMP1; a late endolysosomal marker) was analyzed at predetermined time points. The MCF-7 cells exhibited intensive co-localization of the MAP@DOX NPs with both EEA1 and LAMP1 at earlier time points, and this co-localization was reduced with 9 Environment ACS Paragon Plus

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prolonged incubation, as observed by confocal laser microscopy (Figure 4C, D). Upon lysosomal escape of the MAP@DOX NPs, the encapsulated DOX was released and delivered to the nucleus (Figure S10). These results indicate that MAP NPs are trafficked through the endo-lysosomal pathway for pH-responsive drug release triggered by the acidic environment of endocytic organelles. The therapeutic applicability of the MAP NP-based spray system was evaluated using the MCF-7 tumor xenograft model ex vivo. Rhodamine B isothiocyanate (RITC)-labeled MAP NPs or RITC-labeled BSA NPs (as negative control) were sprayed onto the surfaces of tumor biopsies (~60 mm3) harvested from mice with xenograft tumors. While the MAP NPs successfully adhered to tumor surfaces 24 h after spraying, the BSA NPs were quickly washed from the tumor sites, as observed in fluorescence images (Figure 5A). The adhesion of MAP NPs to tissue surfaces might be attributed to lysine, unreacted DOPA, and unmodified tyrosine residues, similar to the adhesion on mica surfaces. Lysine residues contribute to the adhesion onto biological substrates by forming ionic bonding with negatively charged collagen and acidic polysaccharides,59 as well as the synergistic adhesion with the aromatic rings from unreacted DOPA for inhibiting DOPA oxidation.60 Unreacted DOPA residues can interact with biological surface by hydrogen bonds with H-bonding groups and covalent bonds with nucleophilic groups (e.g., -SH, -NH2, -COOH, and hystidyl groups) that are abundant in tissue surface and cation-π/π-π interactions of DOPA also can contribute to adhesion on biological tissues.61,62 In addition, aromatic rings of tyrosine residues enable the interaction with the extracellular matrix (ECM) proteins on tissue substrates via hydrogen bonding, cation-π/π-π stacking under physiological conditions, regardless of pH.59,63,64 Importantly, the MAP@DOX NPs exerted a significantly stronger ex vivo anti-proliferative effect on the tumor biopsies than the PBS or free DOX groups, which is attributed to the successful delivery of DOX through the sufficient adhesion of NPs to the biopsy surfaces (Figure 5B). 10 Environment ACS Paragon Plus

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To investigate the retention of the MAP NPs on tumors in vivo, RITC-conjugated MAP NPs were sprayed onto the surfaces of tumor tissues (~200 mm3) and the localization of these MAP NPs was evaluated using the IVIS live animal imaging system. For the entire treatment period, the sprayed MAP NPs exhibited sufficient retention on tumor sites, and the rhodamine-associated fluorescence signals of the MAP NPs decreased gradually as time progressed, indicating the biodegradability of the MAP NPs (Figure 5C, D). The anticancer effects of MAP@DOX NPs were also evaluated in vivo, by surgical spraying process on tumor sites directly after surgical incision (Figure 6A). The growth of tumor in MAP@DOX NPs group was suppressed effectively, while no observable inhibition of tumor growth was detected in the negative control free DOX and PBS groups (Figure 6B-D). We surmise that the notable therapeutic effects observed in the MAP@DOX NPs group could be attributed to the successful tumor localization of the drugs based on their peculiar adhesiveness on the tumor surfaces after the spray process. The free DOX group failed to exert therapeutic efficacy due to a significant loss of DOX molecules from the sprayed tumor sites in bodily fluids. Notably, we can conclude that the MAP@DOX NPs exhibited no toxicity because there was no significant difference in body weights (Figure 6E). Next, we performed histological analyses through the visualization of tumor sites using hematoxylin and eosin (H&E) staining, the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, and cleaved caspase-3 immunohistochemical staining 1 month after the spraying of the MAP@DOX NPs (Figure 6F). In the H&E-stained sections, a large number of necrotic cells exhibiting shrinkage/fragmentation of the nucleus and reduced nuclear density were clearly observed in the MAP@DOX NP group. However, a necrotic area and the normal morphology of cells were not detected in the PBS and the free DOX groups. Based on the images for the TUNEL assay and cleaved caspase-3 staining, the MAP@DOX NPs group showed apoptotic regions around the sprayed sites, in contrast to the PBS and free DOX groups, indicating that the tumor growth inhibition of the MAP@DOX NPs was caused 11 Environment ACS Paragon Plus

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by both necrosis and apoptosis. Therefore, we concluded that our proposed MAP NP spray system achieves site-directed administration of drugs onto a target site in a simple and convenient way, subsequently improving therapeutic efficiency with a reduced risk of systemic toxicity.

CONCLUSION Here, we proposed sprayable, adhesive NPs fabricated using DOPA-Fe3+ complexation of bioengineered MAP for the site-directed and local delivery of anticancer drugs. This bioinspired spray system allowed the easy and successful deposition of MAP NPs onto a target surface with the proper dispersion ability and sufficient adhesive properties, and the sprayed MAP NPs exhibited retained adhesion under aqueous conditions. The MAP NPs demonstrated potential as an anticancer therapeutic delivery platform through successful cellular uptake by the endocytic pathway as well as cancer-triggered drug release caused by alterations of the DOPA-Fe3+ complexation. Our findings highlight that the spraying of MAP NPs provides localized delivery and controlled release of drugs, minimizing systemic side effects and maximizing therapeutic impact. Thus, we anticipate that MAP NPs can be successfully employed to inhibit ipsilateral recurrence in adjuvant therapy for the focal treatment of cancer.

MATERIALS AND METHODS Preparation of MAP NPs. The bioengineered MAP, which is comprised of 12 tandem repeats of the Mytilus foot protein type 1 (fp-1) consensus decapeptide (AKPSYPPTYK), was expressed in Escherichia coli and purified as described previously.24 To convert the tyrosine residues of the bioengineered MAP into DOPA, MAP was dissolved in a modification buffer (100 mM sodium phosphate dibasic, 20 mM boric acid, and 25 mL ascorbic acid; pH 6.8) at a final concentration of 1.5 mg/mL, reacted with mushroom tyrosinase (100 µg/mL; Sigma, St


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Louis, MO, USA) for 1 h and then dialyzed with 1% (v/v) acetic acid. The yield of DOPAmodified bioengineered MAP was determined via amino acid composition analysis after hydrolysis in 6 N HCl with 5% water-saturated phenol at 156 °C using an amino acid analyzer (S4300; SYKAM, Eresing, Germany). The MAP NPs were fabricated through electrospraying of the DOPA-containing MAP, as reported previously.39 Briefly, the MAP solution was mixed with FeCl3 at a DOPA:Fe3+ molar ratio of 3:1 to generate DOPA-Fe3+ complexes and then electrosprayed via an electrospray system (ESR200R2D; NanoNC, Seoul, Korea) using a 5 mL syringe at a flow rate of 1 mL/h with a high voltage (15−20 kV). The synthesized NPs were collected in PBS (pH 7.4). For the fabrication of MAP@DOX NPs, 1 mM of DOX was added to the MAP solution containing the DOPA-Fe3+ complexes prior to electrospraying. The prepared MAP@DOX NPs were dialyzed 3 times with PBS using a membrane with a molecular weight cut-off (MWCO) of 3500 to remove unloaded DOX molecules. Characterization of MAP NPs. The spray process of the MAP NPs in a PBS:ethanol (30:70) solvent was performed using a commercial air-pressured atomizer. The suspension of the MAP NPs was sprayed through a fine nozzle via air-fluid pressure. The particle sizes before and after the spray process were measured by using dynamic light scattering (DLS; ELSZ-1000; Photal Otsuka Electronics, Osaka, Japan) equipment. The morphologies of the NPs before and after spraying were observed via FE-SEM (XL30S FEG; Philips, Eindhoven, Netherlands) at an accelerating voltage of 5 kV after gold sputtering. The morphologies of NPs-deposited surfaces with a different number of spraying were investigated by atomic force microscopy (AFM; VEECO Dimension 3100; VEECO, Plainview, NY, USA). MAP NPs were coated on glass surfaces by 1 and 10 times of spraying process and left to air dry. Images were analyzed using a Nanoscope V (Bruker, Billerica, MA, USA). To visualize NPs, FITC (Sigma)-conjugated MAP and RITC (Sigma)-conjugated MAP were used for the fabrication of NPs in the same electrospraying method. The FITC was 13 Environment ACS Paragon Plus

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mixed with an MAP solution and then incubated for 3 h in a 0.1 M sodium bicarbonate buffer (pH 9.0). The unreacted FITC was removed via dialysis with a membrane (MWCO 3500). To analyze the co-localization of MAP and DOX in the MAP@DOX NPs, the FITC-labeled MAP@DOX NPs were sprayed onto a glass surface, and fluorescence images were obtained via fluorescence microscopy (Olympus, Tokyo, Japan). Surface adhesion of MAP NPs. To analyze the adhesion properties of the MAP NPs, the RITC-labeled MAP NPs were sprayed onto a glass surface and stained with a Coomassie-blue solution (Sigma). The Coomassie-stained glass surface was incubated in PBS under shaking and observed via optical microscopy (Olympus) at a predetermined time. The RITC-labeled MAP NPs were sprayed onto a porcine skin tissue surface (Stellen Medicine, St Paul, MN, USA), and half of the surface was blocked by taping to divide the non-blocked ‘sprayed surface’ and blocked ‘non-sprayed surface’. The porcine skin tissue surface was incubated in PBS under shaking for 24 h at 37 °C and the adhesion of MAP NPs was analyzed using fluorescence microscopy after PBS washing. The adhesion efficiency of the MAP NPs was evaluated after spraying and compared with BSA-based NPs (Promega, Madison, WI, USA) as a control. The NPs were sprayed onto a collagen surface (Genoss, Suwon, Korea) and then incubated in PBS for 1, 5, 10, and 30 min. The unattached NPs were collected in the buffer solution, and the attached NPs on the collagen surface were further incubated in fresh buffer solution for 1, 2, 4, 8, and 24 h. Each buffer solution was sampled at predetermined time points, and the amounts of unattached and released NPs were analyzed using the Bradford assay (Bio-Rad, Hercules, CA, USA), with BSA as a protein standard. The absorbance at 595 nm was measured using a microplate absorbance spectrophotometer (Perkin Elmer, Waltham, MA, USA). The surface adhesion of the MAP NPs and BSA NPs (as a control) were quantified using a QCM (QCM922A; SEIKO EG&G, Tokyo, Japan). A gold-coated quartz sensor crystal (8.9 MHz) was washed with ethanol, followed by rinsing with DW and incubation under UV 14 Environment ACS Paragon Plus

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irradiation for 30 min. To stabilize the baseline frequency f0, DW was allowed to flow through the QCM flow cell. Then, the NPs (1 mg/mL) were injected into the flow module for adhesion to the gold-coated surface. After the f shift (∆f1) was stable, the quartz sensor crystal was washed with DW to remove unattached NPs, and the frequency shift of the washed crystal (∆f2) was measured. Each solution was injected into the QCM flow cell at a flow rate of 40 µL/min. The surface adhesion forces of MAP NPs and BSA NPs were quantitatively measured using a SFA (SFA2000; SurForce LLC, Goleta, CA, USA). NPs (7 mg/mL) in 10 mM PBS (pH 7.4) were placed onto a freshly cleaved mica surface for 10 min followed by rinsing with the same buffer. The deposition of NPs on a mica surface was confirmed by AFM. The adhesion force between the asymmetric NPs-coated mica surface and a bare mica surface was measured in 10 mM PBS (pH 7.4) with a compression time of 5 min and a contact time of 10 min. To examine the retention of DOX in the MAP@DOX NPs, FITC-labeled MAP@DOX NPs were sprayed onto a glass surface and incubated in PBS under shaking. At predetermined time points, fluorescence images were obtained via fluorescence microscopy after PBS washing. In vitro DOX release profiles of the MAP@DOX NPs. The solution of MAP@DOX NPs in PBS was dialyzed (MWCO 3500) in different pH environments (pH 5.5 and 7.4) under shaking at 37 °C. Each solution was sampled at predetermined time points, and the removed volume was replaced with the same volume of fresh buffer solution. The DOX released in each buffer was measured using a fluorescence spectrometer (Perkin Elmer) with an excitation wavelength of 485 nm and an emission wavelength of 580/10 nm. Cell culture. Human breast carcinoma cells MCF-7 (American Type Culture Collection (ATCC) No. HTB-22) and MDA-MB-231 (ATCC No. HTB-26) were cultured in Dulbecco’s modified eagle medium (DMEM; HyClone, Logan, UT, USA). Mouse pre-osteoblast cells 15 Environment ACS Paragon Plus

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(MC3T3-E1; RIKEN Cell Bank, Tsukuba, Japan) were cultured in α-minimal essential medium (α-MEM; HyClone), and HUVECs (Lonza Verviers, Belgium) were cultured in Endothelial Cell Growth Medium-2 (EGM-2) medium (Lonza). All the media were supplemented with 10% (v/v) fetal bovine serum (FBS; HyClone) and 1% (v/v) penicillin/streptomycin (HyClone), and the cells were incubated at 37 °C in a humidified atmosphere of 5% CO2. The cells were routinely tested for mycoplasma contamination. In vitro cytotoxicity analyses. The cancer cells (MCF-7 and MDA-MB-231) and normal cells (MC3T3-E1 and HUVEC) were seeded on 24-well tissue culture plates at a density of 5 × 104 cells per well and then incubated overnight. The cells were treated with MAP@DOX NPs or the equivalent amount of free DOX. After 1 day of incubation, an aliquot of the CCK8 (Dojindo Laboratories, Tokyo, Japan) reagent was added to each well, and the cells were incubated for 3 h. The absorbance at 450 nm was measured using a microplate absorbance spectrophotometer. To investigate the cytotoxicity of the MAP NPs, MCF-7 and MC3T3-E1 cells were treated with different concentrations of MAP NPs 1 day after seeding, and CCK-8 assays were performed after a further incubation of 1 day. For apoptosis analyses, MCF-7 cells were seeded at a density of 1 × 106 cells per well and incubated for 1 day. After 8 h of treatment with the MAP@DOX NPs or the equivalent amount of free DOX, the cells were stained with Alexa Fluor® 488-conjugated AV (Invitrogen, Carlsbad, CA, USA) and 7-AAD (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s protocol, after harvesting cells by trypsinization. The stained cells were analyzed by flow cytometry (FACS Calibur; Becton Dickinson San Jose, CA, USA). The cells positive for AV were recorded as early apoptosis, the cells positive for 7-AAD were recorded as necrosis, and the cells double positive for AV and 7-AAD were recorded as late apoptosis. Non-treated cells were used as a control, and 10,000 cells were analyzed for each measurement. 16 Environment ACS Paragon Plus

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For morphological analysis of cells after apoptosis, the cells were incubated with the MAP@DOX NPs for 1 day and then fixed with 2% glutaraldehyde (Sigma). Subsequently, the cells were dehydrated via sequential treatment with increasing concentrations of ethanol (25−100%; Sigma) for 5 min each. After gold sputtering, the morphology of the cells was analyzed via SEM imaging. Cellular uptake and intracellular trafficking. MCF-7 and MC3T3-E1 cells were seeded at a density of 1 × 106 cells per well and then cultured for 1 day. Then, the cells were incubated with the MAP@DOX NPs (final DOX concentration of 0.36 µg/mL) for 1, 3, 6, and 9 h. After washing 3 times with PBS, the cells were trypsinized, centrifuged, and resuspended in cold Dulbecco’s phosphate-buffered saline (DPBS; Hyclone). The mean fluorescence intensity (MFI) was measured by flow cytometry with an excitation wavelength of 488 nm and an emission wavelength of 585/42 nm. Non-treated cells were used as a control, and a total of 10,000 cells were counted for each experiment. To assess the cellular uptake mechanism of the MAP@DOX NPs, MCF-7 cells were seeded at a density of 1 × 106 cells per well and incubated for 1 day. The cells were incubated with endocytosis inhibitors, including 500 nM wortmannin (Sigma), 50 µM cytochalasin D (Sigma), 200 µM genistein (Sigma), 5 mM MβCD (Sigma), and 7 µg/mL of chlorpromazine (Sigma) for 30 min at 37 °C in serum-free medium. Then, the MAP@DOX NPs (final DOX concentration of 1 µg/mL) were added to the culture, and the cells were further incubated at 37 °C for 1 h. Alternatively, to inhibit energy-dependent endocytosis, the cells were preincubated in serum-free medium at 4 °C for 30 min and then further incubated at 4 °C for 1 h in the presence of the MAP@DOX NPs. The cells were washed with PBS, trypsinized, and resuspended in cold DPBS. The samples were analyzed by flow cytometry with an excitation wavelength of 488 nm and an emission wavelength of 585/42 nm. To determine the intracellular pathway of the MAP@DOX NPs, MCF-7 cells were seeded at a density of 5 × 104 cells per well. After incubation for 24 h, the cells were treated with 17 Environment ACS Paragon Plus

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RITC-labeled MAP@DOX NPs for a predetermined time period and washed with PBS. For immunohistochemical analysis, the cells were fixed with 4% formaldehyde (Sigma), rinsed 3 times with PBS, and permeabilized with 0.5% Triton X-100 (Sigma). Primary antibodies for EEA1 (Santa Cruz Biotechnology, Dallas, TX, USA) and LAMP1 (Santa Cruz Biotechnology) were added to the cells for 1 h at room temperature and washed with PBS. Subsequently, the cells were treated with secondary antibodies conjugated with Alexa Fluor® 488 (Thermo Fisher Scientific) for 1 h, and the nuclei were stained with 0.5 µg/mL DAPI (Sigma). After 3 PBS washes, the cells were observed using confocal laser scanning microscopy (CLSM; TCS SP5 II; Leica, Wetzlar, Germany). For morphological analysis of MCF-7 cells, actin filaments were labeled with FITC-conjugated phalloidin (Sigma) and nuclei were stained with 0.5 µg/mL of DAPI. The cell images were obtained using fluorescence microscopy. Animals and tumor xenograft models. All the animal studies were performed in accordance with the guidelines approved by the Institutional Animal Care and Use Committee of POSTECH (POSTECH IACUC-2016-0044-R1). To establish the tumor xenograft model, 1 × 107 MCF-7 cells were subcutaneously injected into the flank of a female BALB/c nude mouse (weight ~18 g, 5 weeks old; Orient Bio, Seongnam, Korea). Ex vivo experiments. Tumor tissues were extracted from the MCF-7 tumor-bearing mice when the tumor reached 200 mm3 and washed 3 times with cold PBS. Using a surgical blade, the tissues were dissected to 5 × 5 mm2 tumor blocks, transferred to 12-well plates and incubated in DMEM supplemented with 10% (v/v) FBS and 1% (v/v) penicillin/streptomycin for 1 day at 37 °C in a humidified 5% CO2 atmosphere. After the media were removed, the tumor tissue blocks were treated with the RITC-labeled MAP NPs or RITC-labeled BSA NPs via spraying and incubated for 5 min. Subsequently, fresh media were added to each well, and the tissue blocks were further incubated for 1 day. After 3 washes with PBS, adhesive properties were analyzed using fluorescence microscopy. 18 Environment ACS Paragon Plus

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To measure ex vivo cytotoxicity, the metabolic activities were evaluated using the CCK-8 assay before and after spraying PBS, free DOX, and MAP@DOX NPs (final DOX concentration of 0.15 µg/mm3). The samples were sprayed onto the surface of the tumor tissue blocks and incubated for 1 day. The tissue blocks were washed 3 times with PBS and further incubated for 3 days following replacement with fresh media. The anti-proliferative activities were measured based on the difference in CCK-8-associated absorbance at 450 nm before and after spraying. In vivo retention assay. The tumor sites of MCF-7 tumor-bearing mice were surgically treated with the RITC-labeled MAP NPs or RITC-labeled BSA NPs via the spraying procedure when the volume of the tumors reached ~200 mm3. In vivo retention of NPs was monitored using an IVIS live animal imaging system (Perkin Elmer) at 1, 10, and 20 days after administration. The fluorescence intensity in the region of interests (ROI) around the tumor was analyzed with an excitation wavelength of 535 nm and an emission wavelength of 580 nm. In vivo anticancer efficacy. The MCF-7 tumor-bearing mice were randomly divided into three groups (six mice per group) when the volume of the tumors reached ~200 mm3. The mice were anesthetized with isoflurane and administered PBS, free DOX, and MAP@DOX NPs (final DOX concentration of 0.15 µg/mm3) locally on the tumor via the surgical spraying process. Tumor length (L) and width (W) were measured daily using an electronic caliper, and tumor volume was calculated using the following formula: Tumor volume = L × W2 /2 Body weight was monitored throughout the healing period, and the mice were euthanized on day 28. The tumors were resected, weighed, and fixed with 10% formalin (Sigma) for histological analyses. For histological analyses, the fixed tumor tissues were dehydrated, embedded in paraffin, and cut into 5 µm thick sections. The tissue sections were stained with H&E (Sigma) and 19 Environment ACS Paragon Plus

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stained immunohistochemically with a cleaved caspase-3 antibody (Cell Signaling Technology, Danvers, MA, USA). To evaluate the apoptotic activity of the tumor, the TUNEL assay (Roche, Basel, Schweizerland) was performed according to the manufacturer’s protocol. Histology images were collected using a BX-41 microscope (Olympus) and analyzed using the CaseViewer 2.0 software (3DHISTECH, Budapest, Hungary). Statistical analysis. In vitro experiments were performed independently at least in triplicate, and 3 samples were examined for each experiment. In vivo data were obtained from 6 individual mice. The normality of the data distribution was examined using the ShapiroWilk test. The significance of the data was analyzed statistically using the unpaired t-test (for a normal distribution) or the Wilcoxon rank-sum test (for a non-normal distribution). Significance was designated with the notation ‘*’ (*p < 0.05, **p < 0.01, and ***p < 0.005). All the data were processed using the R software (v.3.2.1; R Development Core Team, Vienna, Austria).

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publication website at DOI: Additional figures related to composition of MAPs determined by amino acid analysis, characterization and adhesion properties of the sprayed MAP NPs and MAP@DOX NPs, the dissociation of MAP NPs, in vitro cellular responses, and cytotoxicity of MAP NPs and MAP@DOX NPs. Supplementary figures S1-S10 (PDF)

AUTHOR INFORMATION Corresponding Author 20 Environment ACS Paragon Plus

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*E-mail: [email protected] Author Contributions Y.J., Y.K.J., and H.J.C. designed the study, analyzed the data, and wrote the paper; Y.J., Y.K.J., and B.Y. performed experiments. Y.K.J., B.J.K., K.I.J., and H.J.C. advised on the interpretation of the experiments. H.J.C. directed the overall project. Y.J. and Y.K.J. contributed equally to this work. All authors discussed the results and edited the manuscript. All authors have given approval to the final version of the manuscript. ORCID Yeonsu Jeong: 0000-0001-9232-8510 Yun Kee Jo: 0000-0003-4240-7821 Hyung Joon Cha: 0000-0003-4640-189X

ACKNOWLEDGMENT Financial support was provided by the Marine Biomaterials Research Center grant from Marine Biotechnology Program of the Korea Institute of Marine Science & Technology Promotion funded by the Ministry of Oceans and Fisheries, Korea.

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Figure 1. Characterization of MAP NP spray therapeutic system. (A) Schematic illustration of the MAP NP spray therapeutic system. A suspension of MAP NPs was sprayed onto target cancer using an air-pressured atomizer and adhered to target cancer, enabling site-directed and local drug delivery. The NPs were internalized to cancer cells through endocytosis, releasing anticancer drugs in an acidic environment of cancer cells. (B) FE-SEM (field emissionscanning electron microscopy) images of MAP NPs before and after spraying. Scale bars, 500 nm. (C) Hydrodynamic diameter of MAP NP before and after spraying. (D) AFM images of MAP NPs-deposited glass surface by 1 time (left) and 10 times (right) of spray process.


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Figure 2. Adhesiveness of MAP NP spray therapeutic system. (A) Optical images of Coomassie-stained glass surfaces 1 day, 1 week, and 1 month after spraying of MAP NPs. (B) QCM analysis of MAP NPs at a flow rate of 40 µL/min. BSA NP was used as a comparative control. (C) Schematic representation (top) and measurement (bottom) of the surface adhesion force of MAP NP against a bare mica surface in PBS (pH 7.4) by SFA analysis.


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Figure 3. In vitro anticancer therapeutic effect of MAP@DOX NPs. (A) DOX release profiles under acidic (pH 5.5) and physiological (pH 7.4) conditions. (B) IC50 values of MAP@DOX NP and free DOX for breast cancer cells (MCF-7 and MDA-MB-231) and normal cells (MC3T3-E1 and HUVEC). (C) Cellular uptake profiles for MCF-7 cancer cells and MC3T3E1 normal cells. (D) Flow cytometry analyses for apoptosis and necrosis in MCF-7 cells. The cells were stained with AV and 7-AAD. AV+/7-AAD-, early apoptosis; AV-/7-AAD+, necrosis; AV+/7-AAD+, late apoptosis.


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Figure 4. Cellular uptake and intracellular fate of MAP@DOX NPs. (A) Flow cytometry analysis for cellular uptake and (B) normalized cellular uptake with endocytosis inhibitor treatments. (C) Fluorescence images of the intracellular co-localization of RITC-labeled MAP@DOX NP (red) with early endosomes and (D) late endosome/lysosomes. EEA1 and LAMP1 were stained with fluorescein isothiocyanate (FITC)-conjugated antibodies (green), and nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI; blue). The insets indicate the magnified views of the white boxed sections. Scale bar is 10 µm.


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Figure 5. Ex vivo and in vivo retention of MAP NP spray system using a tumor xenograft model. (A) Ex vivo fluorescence images of MCF-7 tumor biopsies after spraying of RITClabeled MAP NPs. BSA NP was used as a comparative control. Scale bars, 2 mm. (B) Ex vivo anti-proliferative effects of tumor biopsies analyzed using the Cell Counting Kit-8 (CCK-8) assay. (C) In vivo fluorescence images of subcutaneous MCF-7 tumor-bearing mice at predetermined time points after spraying of RITC-labeled MAP NPs. BSA NP was used as a comparative control. (D) Time-course radiant efficiency of RITC-labeled MAP NPs and BSA NPs in the tumor region of MCF-7 tumor-bearing mice after spraying.


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Figure 6. In vivo therapeutic effect of MAP NP spray system using a tumor xenograft model. (A) Schematic illustration of therapeutic mechanism of MAP NP spray system. MAP@DOX NPs were surgically sprayed on a tumor site, adhered on a surface, and exposed the encapsulated DOX, inducing a focal cancer cell death. (B) Optical images of excised tumors after in vivo spray therapy. (C) Tumor volume change of mice treated with PBS, free DOX, MAP@DOX NPs at a dosage of 0.15 µg/mm3 DOX concentration (n = 6 per group). (D) Tumor weight at the end of the study (week 4). (E) Body weight over time. (F) H&E-stained images, microscopic images from the TUNEL assay, and cleaved caspase-3-stained tumor images. Scale bar is 100 µm.


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BRIEFS A sprayable adhesive nanoparticle-based therapeutic system exploited by using a bioengineered mussel adhesive protein exhibits a remarkable adhesion ability, an efficient cellular uptake, and cancer-triggered drug releasing property, thereby leading to excellent anticancer responses for site-directed and local delivery of anticancer drugs.

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