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Mitochondria Targeting and Destabilizing Hyaluronic Acid Derivative-Based Nanoparticles for the Delivery of Lapatinib to Triple-Negative Breast Cancer Song Yi Lee, and Hyun-Jong Cho Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b01449 • Publication Date (Web): 19 Dec 2018 Downloaded from http://pubs.acs.org on December 20, 2018

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Mitochondria Targeting and Destabilizing Hyaluronic Acid Derivative-Based Nanoparticles for the Delivery of Lapatinib to Triple-Negative Breast Cancer Song Yi Lee, Hyun-Jong Cho*

College of Pharmacy, Kangwon National University, Chuncheon, Gangwon 24341, Republic of Korea.

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ABSTRACT: CD44 receptor and mitochondria targeting hyaluronic acid-d-α-tocopherol succinate-(4-carboxybutyl)triphenyl phosphonium bromide (HA-TS-TPP)-based nanoparticles (NPs) were designed for the delivery of lapatinib (LPT) to triple-negative breast cancer (TNBC). While LPT is one of dual tyrosine kinase inhibitors for epidermal growth factor receptor (EGFR) and human EGFR2 (HER2), TNBC cells often exhibit EGFR positive and HER2 negative patterns. Along with the HER2-independent anticancer activities of LPT in TNBC, apoptosis-inducing properties of TPP and TS (resulting from mitochondrial targeting and destabilization) were introduced to amplify the anticancer activities of HA-TS-TPP/LPT NPs for TNBC. HA-TS-TPP/LPT NPs with approximately 207-nm mean diameter, unimodal size distribution, spherical shape, negative zeta potential, and sufficient particle stability were prepared in this study. The improved antiproliferation potential, apoptotic efficacy, and mitochondrial destabilizing activity of HA-TS-TPP/LPT NPs, compared with HA-TS/LPT NPs, were demonstrated in TNBC (i.e., MDA-MB-231) cells. The in vivo tumor targeting capability of HA-TS-TPP/LPT NPs was proven in MDA-MB-231 tumor-bearing mouse models using real-time optical imaging. Of note, HA-TS-TPP/LPT NPs exhibited a better tumor growth suppression profile than the other groups after intravenous injection. It is expected that developed HA-TS-TPP NPs can elevate the therapeutic potential of LPT for TNBC.

KEYWORDS: hyaluronic acid, lapatinib, mitochondria targeting, nanoparticle, CD44 receptor


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■ INTRODUCTION Many tumor targeting approaches have been used to try to precisely deliver anticancer drugs to tumor regions.1-4 Macromolecules with over 40-kDa molecular weight were reported to accumulate in the solid tumor tissue due to its leaky vasculatures; this strategy, a so-called enhanced permeability and retention (EPR) effect, has been widely used as a passive tumor targeting method.5,6 As a result, nanocarriers with particle sizes of a certain range can efficiently arrive at the tumor tissue after intravenous administration according to the EPR effect.7 To elevate tumor targeting accuracy after intravenous administration of nanocarriers, active tumor targeting strategies (mainly based on ligand–receptor interactions) have been introduced. Together with an EPR effect, selective targeting the receptors expressed in cancer cells with specific ligand-installed nanocarriers can enhance the tumor targeting efficiency. Various kinds of ligands (i.e., small chemicals, antibodies, and peptides) have been placed on the surface of nanosystems, and their specific interactions were demonstrated in cell culture and animal models.8 Among them, hyaluronic acid (HA) has been used to make nanocarriers for targeting the CD44 receptor expressed in several types of cancer cells.9 Moreover, because HA is biocompatible and biodegradable, it can be used safely in injection formulations for clinical application.10 Several hydrophobic moieties have been introduced to a hydrophilic HA backbone to prepare self-assembled nanoassembly structures for the delivery of drugs with poor water solubility.11,12 In our previous studies,11,13-17 ceramide-conjugated HA-based selfassembled nanostructures were developed, and their tumor targeting capability was demonstrated based on the EPR effect and HA-CD44 receptor interactions. In particular, specialized functional groups (i.e., dopamine and phenylboronic acid) were attached to the amphiphilic HA-based nanostructures to improve tumor targeting and penetration efficiency.14,17



In this investigation, D-α-tocopherol succinate (TS) was grafted to an HA backbone to make amphiphilic HA-TS-based nanostructures. TS is a vitamin E analogue and has poor water solubility. Therefore, TS can be used as a segment for designing the hydrophobic cavity of a self-assembled nanostructure. Interestingly, TS can selectively destabilize the mitochondria of cancer cells (avoiding that of normal cells).18 It is reported that TS can induce apoptosis in malignant tumor cells via reactive oxygen species (ROS)-dependent mechanisms.19,20 TS can act as a mitochondria-destabilizing moiety and as an ingredient for the fabrication of the nanoassembly structure in this study. As a mitochondria targeting molecule, (4carboxybutyl)triphenyl phosphonium bromide (TPP) was introduced to HA-TS conjugate. Because TPP is cationic and lipophilic molecule, it can be easily transported across the mitochondrial membrane.21,22 TPP has been widely investigated as a mitochondrial targeting ligand after the endocytosis of nanocarriers. It is surmised that both TPP and TS may provide mitochondria-targeting and destabilizing functions in the nanoparticle (NP)-assisted delivery of anticancer agents in this investigation. Lapatinib (LPT) was encapsulated in HA-TS-TPP-based NPs for the treatment of breast cancers. LPT is a dual tyrosine kinase inhibitor that targets epidermal growth factor receptor (EGFR) and human EGFR2 (HER2).23 LPT has been used for the therapy of HER2-positive advanced or metastatic breast cancers. In this study, the anticancer activities of LPT-loaded NPs were tested in MDA-MB-231 cells, which are triple-negative breast cancer (TNBC) cells. Although TNBC lacks the expression of HER2, EGFR is known to be overexpressed frequently in TNBC.24 The therapeutic potentials of LPT in TNBC were continuously tested and its HER2independent action mechanisms have been elucidated.25-27 In this study, a sequential CD44 receptor targeting and mitochondria-disrupting NP-assisted strategy was devised for elevating the anticancer activities of LPT in TNBC.


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■ EXPERIMENTAL METHODS Materials. LPT was purchased from LC Laboratories (Woburn, MA, USA). HA (molecular weight (MW): 4‒8 kDa) was kindly provided by SK Bioland Co., Ltd. (Cheonan, Korea). Sodium dodecyl sulfate (SDS) and TS were acquired from Tokyo Chemical Industry Co. Ltd. (Tokyo, Japan). N, N´-Dicyclohexylcarbodiimide (DCC), 4-(dimethylamino)pyridine (DMAP), hexadeuterodimethyl sulfoxide (DMSO-d6), and TPP were purchased from Sigma– Aldrich (Saint Louis, MO, USA). Cy5.5-NH2 (FCR-675 amine) was acquired from BioActs (DKC Corp., Incheon, Korea). RPMI 1640 (developed by Roswell Park Memorial Institute), penicillin-streptomycin, and fetal bovine serum (FBS) were provided by Gibco Life Technologies, Inc. (Grand Island, NY, USA). Synthesis and Characterizations of HA-TS-TPP. HA-TS was synthesized via an esterification reaction between the –COOH group of TS and the –OH group of HA. HA (96 mg) was dissolved in dimethyl sulfoxide (DMSO, 20 mL). TS (106.2 mg), DCC (41.2 mg), and DMAP (4.6 mg) were also solubilized in DMSO (10 mL). Those solutions were blended and stirred for 24 h at room temperature. The mixture was transferred to a dialysis bag (molecular weight cutoff (MWCO): 3.5 kDa) and dialyzed against a mixture of methanol and distilled water (DW) for 36 h. The mixture was lyophilized for further use. HA-TS-TPP was synthesized via the esterification reaction between the –COOH group of TPP and the –OH group of HA (in HA-TS). HA-TS (250 mg) was dissolved in DMSO (40 mL). TPP (221.6 mg), DCC (103.6 mg), and DMAP (16 mg) were also solubilized in DMSO (10 mL). Those two solutions were then mixed and stirred for 24 h at room temperature. The mixture was put into a dialysis bag (MWCO: 3.5 kDa) and dialyzed against a mixture of methanol and DW for 36 h. Then, the mixture was freeze-dried for storage. To identify the synthesis of HA-TS and HA-TS-TPP, HA-TS or HA-TS-TPP was dissolved in DMSO-d6 for proton nuclear magnetic resonance (1H-NMR; Varian FT-500 MHz, 5 ACS Paragon Plus Environment


Varian Inc., Palo Alto, CA, USA) spectroscopic analysis. Mixtures of HA and TS at various weight ratios of TS to HA were used as standard samples for 1H-NMR analysis. To determine the content of TS in HA-TS, the regression line between the weight ratios (TS/HA) and the integration ratios of peaks (0.9 ppm/1.8 ppm) was established. To calculate the content of TPP in HA-TS-TPP, mixtures of HA-TS and TPP with different weight ratios between TPP and HA-TS were quantitatively analyzed by 1H-NMR spectroscopy. The regression line between the weight ratios (TPP/HA-TS) and the integration ratios of peaks (7.7‒7.8 ppm/1.8 ppm) was plotted. Preparation and Particle Properties of LPT-Loaded NPs. LPT was loaded to designed NPs as a hydrophobic anticancer drug in this study. To fabricate HA-TS/LPT NPs and HA-TSTPP/LPT NPs, HA-TS or HA-TS-TPP (96 mg) and LPT (12 mg) were dissolved in DMSO (12 mL), added in a dialysis bag (MWCO: 3.5 kDa), and dialyzed against DW for 6 h at room temperature. Residual mixtures in the dialysis bag were lyophilized for further use. The hydrodynamic size, polydispersity index, and zeta potential values of fabricated NPs (at 2 mg/mL) were determined using dynamic light scattering (DLS) and laser Doppler methods (ELS-Z1000; Otsuka Electronics, Tokyo, Japan).14,17 The content of LPT in the prepared nanocarriers was quantitatively measured via high-performance liquid chromatography (HPLC). A reverse-phase C18 column (Kinetex, 250 mm × 4.6 mm, 5 μm; Phenomenex, Torrance, CA, USA) was connected to an HPLC system (1260 Infinity II, Agilent Technologies, Santa Clara, CA, USA) equipped with a pump (1260 Quat Pump VL, Agilent Technologies), an autosampler (1260 Vialsampler) and a UV/Vis detector (1260 VWD, Agilent Technologies) for LPT analysis. The mobile phase was prepared by mixing acetonitrile and water at a 65:35 volume ratio. The sample injection volume was fixed at 20 μL and the flow rate of the mobile phase was maintained at 1 mL/min. The absorbance of eluent was detected at 227 nm. The regression line of the standard samples was drawn at an LPT concentration range of 0.1‒20 6 ACS Paragon Plus Environment

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μg/mL. The particle shape and size of LPT-loaded NPs was observed via transmission electron microscopy (TEM). The dispersion of HA-TS/LPT NPs and HA-TS-TPP/LPT NPs (5 mg/mL) was stained with 2% (w/v) phosphotungstic acid and rinsed with DW. Each sample was put onto a copper grid with film and dried prior to TEM (LEO 912AB OMEGA; Carl Zeiss, Oberkochen, Germany) imaging.14,17 The particle stability of LPT-encapsulated NPs was evaluated by measuring their particle size in DW and FBS (50%, v/v). Freeze-dried NPs were dispersed at 10 mg/mL in each medium and their hydrodynamic size values were measured by the DLS method (ELS-Z1000; Otsuka Electronics) after incubating for 2 and 24 h. A dispersion of NPs (0.15 mL), corresponding to 200 μg of LPT, was put into MiniGeBAflex tubes (MWCO: 14 kDa; Gene Bio-Application Ltd., Kfar Hanagide, Israel). The dialysis tube was immersed in PBS (pH 7.4; 10 mL) including 0.3% SDS and incubated in a shaking water-bath with 50 rpm at 37C. Release media (0.2 mL) were collected at 2, 4, 8, 24, 48, 72, 96, and 168 h, and the equivalent volume of fresh medium was supplemented each time. Collected samples were injected to the HPLC system to analyze the released amounts of LPT from NPs. The previously described HPLC method was applied to determine the concentration of LPT in the release media. Cellular Uptake Tests. MDA-MB-231 cells were used to evaluate the cellular accumulation and localization of developed NPs. MDA-MB-231 cells were supplied by Korean Cell Line Bank (Seoul, Korea). Cells were cultured with RPMI 1640 medium containing FBS (10%, v/v) and penicillin-streptomycin (1%, v/v).17 The cellular accumulation efficiency of nanocarriers in MDA-MB-231 cells was measured by flow cytometry. For the detection of fluorescence signals, Cy5.5-NH2 was covalently bonded to the carboxylic acid group of HA via amide bond formation. HA-TS or HA-TS-TPP (96 mg) was dissolved in DMSO (18 mL). 7 ACS Paragon Plus Environment


EDC (1.15 mg) and NHS (0.69 mg) solubilized in DMSO (1 mL) was added to HA-TS or HATS-TPP solution. Cy5.5-NH2 (2.4 mg) in DMSO (1 mL) was put into that previous mixture and stirred for 24 h; it was then dialyzed against DW for 2 days with a dialysis membrane (MWCO: 3.5 kDa). The final product was acquired by freeze-drying. MDA-MB-231 cells were seeded in six-well plates at a density of 6.0 × 105 cells per well and they were incubated at 37°C for 1 day. Cells were incubated with Cy5.5-HA-TS/LPT NPs or Cy5.5-HA-TS-TPP/LPT NPs (at a Cy5.5 concentration of 25 μg/mL) for 1, 2, and 6 h. Each sample was removed, and the cells were rinsed with PBS (pH 7.4) at least thrice. Cells were harvested, and cell pellets were collected by centrifuging at 13,200 rpm for 5 min. Then, the cells were suspended in FBS solution (2%, v/v) prior to flow cytometry analysis. Cell counts, according to fluorescence intensity, were recorded by a FACSCalibur Fluorescence-activated Cell Sorter (FACSTM) installed with CELLQuest software (BD Biosciences, San Jose, CA, USA). Cellular localization of NPs in MDA-MB-231 cells was tested by confocal laser scanning microscopy (CLSM). Cells, at a density of 1.0 × 105 cells per well (surface area of 1.7 cm2 per well), were seeded onto culture slides (BD Falcon, Bedford, MA, USA) and incubated at 37C for 1 day. Cells were treated with Cy5.5-HA-TS/LPT NPs or Cy5.5-HA-TS-TPP/LPT NPs (at a Cy5.5 concentration of 10 μg/mL) and incubated for 1, 2, and 6 h at 37°C. After eliminating those samples from cells, the cells were rinsed with PBS (pH 7.4) at least thrice. Cells were then fixed with 4% formaldehyde solution for 10 min. After drying, cells were treated with VECTASHIELD mounting medium containing 4',6-diamidino-2-phenylindole (DAPI) (H1200; Vector Laboratories, Inc., Burlingame, CA, USA) to stain nuclei and prevent fluorescence quenching.17 Fluorescence signals in cells were then detected by CLSM (LSM 880, Carl-Zeiss, Thornwood, NY, USA). Antiproliferation Efficacy Assay. Cytotoxic potentials of LPT, blank NPs, and LPTloaded NPs were assessed in MDA-MB-231 cells by the colorimetric assay. MDA-MB-231 8 ACS Paragon Plus Environment

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cells (at a density of 5.0 × 103 cells per well) were seeded onto a 96-well plate and incubated at 37C for 1 day. LPT solution, blank NPs (HA-TS NPs and HA-TS-TPP NPs), and LPTloaded NPs (HA-TS/LPT NPs and HA-TS-TPP/LPT NPs), at 1, 2.5, 5, 7.5, 10, and 20 μg/mL corresponding LPT concentrations, were applied to cells and further incubated at 37°C for 24, 48, and 72 h. Then, cells were treated with CellTiter 96® AQueous One Solution Cell Proliferation Assay Reagent (Promega Corp., Fitchburg, WI, USA) according to the manufacturer’s protocols.14,17 The absorbance was scanned at 490 nm with an EMax Precision Microplate Reader (Molecular Devices, Sunnyvale, CA, USA). Cell viability was calculated by dividing the absorbance of the tested sample with that of control (no treatment) group. Apoptosis Assay. The apoptosis induction capabilities of NPs were tested in MDA-MB231 cells. MDA-MB-231 cells (at a density of 1.0 × 105 cells per well) were seeded on 6-well plates and incubated for 1 day at 37°C. Cells were treated with LPT solution, blank NPs (HATS NPs and HA-TS-TPP NPs), and LPT-loaded NPs (HA-TS/LPT NPs and HA-TS-TPP/LPT NPs) at an LPT concentration of 10 μg/mL and incubated at 37°C for 24 h. Cells were rinsed with PBS (pH 7.4) at least thrice after the elimination of samples. Cells were harvested by centrifugation at 13,200 rpm for 5 min and suspended in the reaction buffer of FITC Annexin V Apoptosis Detection Kit (BD Pharmingen, BD Biosciences, San Jose, CA, USA) for staining with Annexin V-FITC and PI.28 The cellular fluorescence intensity of both reagents (Annexin V-FITC and PI) was detected by different channels of FACSCalibur fluorescence-activated cell sorter (FACSTM) installed with CellQuest software (BD Biosciences, San Jose, CA, USA). Mitochondrial Membrane Potential Assay. The mitochondria-related biofunctions of the TS and TPP in HA-TS-TPP were tested via JC-1 mitochondrial membrane potential assay kit (Abcam, Cambridge, UK). MDA-MB-231 cells (at a density of 2.0 × 105 cells per well) were seeded onto six-well plates and incubated at 37°C for 1 day. Cells were incubated with LPT solution, HA-TS/LPT NPs, HA-TS-TPP/LPT NPs, HA-TS, HA-TS-TPP, and HA-TPP 9 ACS Paragon Plus Environment


(10-μg/mL LPT concentration) for 24 h at 37°C. Those cells were harvested and rinsed with dilution buffer. Then, cells were stained with JC-1 (10 μM) in dilution buffer for 30 min at 37°C. After rinsing cells with the dilution buffer, the cellular fluorescence signals were detected by a FACSCalibur Fluorescence-activated Cell Sorter (FACSTM) installed with CELLQuest software (BD Biosciences, San Jose, CA, USA). Near-Infrared Fluorescence (NIRF) Imaging. The in vivo tumor targeting capability of the designed NPs was tested via real-time NIRF imaging in tumor-bearing murine models. Cy5.5-conjugated NPs were prepared for NIRF imaging in this study. The contents of Cy5.5 in Cy5.5-HA-TS/LPT NPs and Cy5.5-HA-TS-TPP/LPT NPs were determined according to the absorbance values at 675 nm. BALB/c nude mice (female, 5 weeks old; Charles River, Wilmington, MA, USA) were used to make MDA-MB-231 tumor xenografts. The experimental protocols of animal studies were approved by the Animal Care and Use Committee of the Kangwon National University. Animal studies were carried out according to the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978). MDA-MB-231 cell suspension (2.0 × 106 cells in 0.1 mL) was injected into the dorsal side of the mouse. The tumor volume (V, mm3) was estimated according to the following formula: V = (1/2) × longest diameter × (shortest diameter)2.14,17,28 Upon reaching approximately 150–200 mm3, the dispersion of NPs (at a Cy5.5 dose of 200 µg/kg) was injected to the tail vein of mouse models. NIRF images of the whole body were scanned at 0 (pre) and 24 h postinjection using a fluorescence in vivo imaging system (FOBI; NeoScience Co., Ltd., Suwon, Korea) equipped with a red laser source. Ex vivo images of excised tumor (at 24 h postinjection) were also attained, and fluorescence signals were quantitatively analyzed.28 In Vivo Anticancer Efficacy Tests. The in vivo anticancer activities of LPT solution and LPT-loaded NPs were tested in MDA-MB-231 tumor xenograft murine models. MDA-MB10 ACS Paragon Plus Environment

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231 tumor-bearing mouse models were prepared as described in the previous section. After reaching approximately 300 mm3, mice were randomly divided into four groups. LPT solution was prepared by dissolving LPT in a mixture of ethanol, propylene glycol, and DW (15:45:40, v/v/v). LPT solution, HA-TS/LPT NPs, and HA-TS-TPP/LPT NPs (at an LPT dose of 20 mg/kg) were injected intravenously into the mouse model on days 0, 4, 7, and 11; body weight was measured when tumor size was measured. At the final day, tumor tissues were separated and fixed in formaldehyde (4%, v/v) solution for further histological staining. Sliced tumor tissues were deparaffinized and hydrated with ethanol. They were stained with hematoxylin and eosin (H&E) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) reagents with standard protocols.17,28 3,3’-Diaminobenzidine (DAB) was used for color development in a TUNEL assay. Data Analysis. Statistical analysis of experimental data were carried out with a two-tailed t-test and analysis of variance (ANOVA). Each experiment was repeated at least thrice. Data are presented as the mean ± standard deviation (SD).

Figure 1. Schematic illustration of anticancer action strategy of HA-TS-TPP/LPT NPs. 11 ACS Paragon Plus Environment


■ RESULTS AND DISCUSSION Synthesis and Characterizations of HA-TS-TPP. TS and TPP were covalently bonded to the HA backbone as mitochondria destabilizing and targeting moieties in this study. Hydrophobic TS was introduced to HA to make the nanoassembly structure based on HA-TS. Then, HA-TS was further modified with TPP to enhance mitochondrial targeting efficiency after entry into the cancer cell. In this investigation (Figure 1), LPT was loaded into the hydrophobic internal cavity of HA-TS NPs and HA-TS-TPP NPs as an anticancer agent. Although LPT is a dual tyrosine kinase inhibitor for EGFR and HER2, it may principally aim at EGFR expressed in MDA-MB-231 cells with the lack of HER2. LPT is known to bind to the ATP-binding pocket of protein kinase domain, which is located inside the cell. Therefore, cellular uptaken LPT may bind to the protein kinase domain as shown in Figure 1. It is expected that cancer cell targeting properties and anticancer functions of all components of NPs (HA, TS, TPP, and LPT) may contribute to the improvement of anticancer activities.


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Figure 2. Synthesis and verification of HA-TS-TPP. (A) Synthetic scheme of HA-TS-TPP. (B) 1H-NMR

spectra of HA-TS and HA-TS-TPP. The chemical shifts 1 (1.8 ppm), 2 (0.9 ppm),

and 3 (7.7–7.8 ppm) belong to HA, TS, and TPP, respectively. HA-TS and HA-TS-TPP were dissolved in DMSO-d6 for 1H-NMR analysis.

HA-TS was synthesized via an esterification reaction between the hydroxyl group of HA and the carboxylic acid group of TS. A DCC/DMAP-catalyzed ester bond formation strategy was adopted to prepare HA-TS conjugate. A graft of hydrophobic TS to hydrophilic HA backbone may produce a micellar structure.18 To verify the substitution of TS to HA, integrated peaks of N-acetyl group (peak 1 at 1.8 ppm) for HA and terminal methyl group for TS (peak 2 at 0.9 ppm) were used in 1H-NMR assay (Figure 2 and Figure S1). The weight ratio of TS in HA-TS determined by the 1H-NMR spectroscopic method was 3.6% in this study. 13 ACS Paragon Plus Environment


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Table 1. Particle characterization of developed NPs. Composition

Mean diameter (nm)

Polydispersity index

Zeta potential Encapsulation (mV) efficiency (%)

HA-TS NPs

641 ± 39

0.38 ± 0.03

-32.7 ± 0.8

-

HA-TS/LPT NPs

411 ± 14

0.24 ± 0.01

-29.1 ± 2.9

78.4 ± 10.9

HA-TS-TPP NPs 378 ± 11 0.22 ± 0.02 -26.4 ± 0.6& HA-TS-TPP/LPT 207 ± 3 0.19 ± 0.03 -24.2 ± 3.0# NPs Each freeze-dried material was dispersed in DW at 2 mg/mL.

83.6 ± 10.1

Data are presented as the mean ± standard deviation (SD) (n = 3‒6).

Εncapsulation efficiency (%) 

actual amount of drug in NPs 100 input amount of drug in NPs

&p

< 0.05: compared with the HA-TS NPs group.

#p

< 0.05: compared with the HA-TS/LPT NPs group.

To provide mitochondrial targeting efficiency, the TPP group was additionally attached to HA-TS conjugate (Figure 2). The DCC/DMAP-mediated esterification reaction between the hydroxyl group of HA and carboxylic acid group of TPP-COOH was used to synthesize HATS-TPP. Due to the relative polar property of TPP (compared to TS), TPP might be mainly located in the outer surface of nanoassembly in the aqueous environment. The introduction of TPP in HA-TS was also verified by 1H-NMR assay (Figure 2). The phenyl group of TPP (peak 3 at 7.7–7.8 ppm) and the N-acetyl group of HA (peak 1 at 1.8 ppm) were used to calculate the weight ratio of TPP in HA-TS-TPP (Figure 2 and Figure S1). The weight ratio of TPP in HATS-TPP was 1.8% according to the result of 1H-NMR spectroscopic method.


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Figure 3. Particle properties of LPT-loaded NPs. (A) Particle size distribution and particle morphology (TEM image) of HA-TS/LPT NPs and HA-TS-TPP/LPT NPs. The length of the scale bar in the TEM image is 500 nm. (B) Particle stability of HA-TS/LPT NPs and HA-TSTPP/LPT NPs after incubation in DW and FBS (50%, v/v). Mean diameters according to the incubation time (for 24 h) are plotted. Each point represents the mean ± SD (n = 3). (C) Drug



release profiles of HA-TS/LPT NPs and HA-TS-TPP/LPT NPs. Released amounts of LPT at pH 7.4 are plotted according to the time. Each point represents the mean ± SD (n = 3).

Preparation and Particle Properties of LPT-Loaded NPs. The particle characteristics of HA-TS and HA-TS-TPP-based NPs were tested (Figure 3 and Table 1). It is expected that the combination of the hydrophilic property of HA and the hydrophobic property of TS can produce a self-assembled nanostructure. The mean diameter of HA-TS NPs was 641 nm; however, it did not show unimodal size distribution. After TPP conjugation to HA-TS, its mean diameter was reduced to 378 nm, and it presented a unimodal size distribution. Installed TPP appears to affect the production of more compact NPs. After LPT loading into the NP formulations, their hydrodynamic size was lower than that of blank NPs. Encapsulation of a hydrophobic drug into the internal hydrophobic space of self-assembled nanostructure appears to result in smaller particulates. The zeta potential value of HA-TS-TPP NPs was closer to neutral value rather than that of HA-TS NPs (p < 0.05), indicating the contribution of cationic property of TPP. LPT loading to each NP lead to slight change of zeta potential value, which may be induced by the adsorption of LPT on the outer surface of NPs. As shown in Table 1, the zeta potential value of HA-TS-TPP/LPT NPs was closer to zero value rather than that of HA-TS/LPT NPs (p < 0.05). These data suggest that TPP may be mainly located on the outer surface of NPs rather than internal space. HA-TS-TPP/LPT NPs exhibited a 207-nm mean diameter, unimodal size distribution, negative zeta potential, and approximately 84% drug encapsulation efficiency. Observed particle size and its size distribution pattern may be appropriate for passive tumor targeting based on the EPR effect after intravenous injection.29,30 Corresponding particle size and round particle shape were also shown in TEM images (Figure 3A). The particle stability of HA-TS/LPT NPs and HA-TS-TPP/LPT NPs was tested in DW 16 ACS Paragon Plus Environment

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and FBS (50%) (Figure 3B). Both HA-TS/LPT NPs and HA-TS-TPP/LPT NPs maintained their initial particle size after incubation for 24 h in DW. However, an inclining pattern was observed in the particle size of HA-TS/LPT NPs incubated in FBS. In case of HA-TS-TPP/LPT NPs, although the hydrodynamic size was changed from 240 ± 10 nm (0 h) to 348 ± 84 nm (24 h), there was no formation of particle aggregates after incubation for 24 h (Figure S2). The interactions between the exposed TPP group in the outer shell of HA-TS-TPP/LPT NPs and the serum components may contribute to the maintenance of initial particle distribution pattern. Drug release patterns from NPs were also evaluated in PBS (pH 7.4) (Figure 3C). Drug release profile may govern the pharmacokinetic profile and in vivo anticancer activity after intravenous injection of developed NPs. In general, sustained drug release (without initial burst release) has been regarded as an important factor for reducing dosing frequency in clinical application of injection formulation. For 24 h, 7.3% and 9.8% of encapsulated LPT were released from HA-TS/LPT NPs and HA-TS-TPP/LPT NPs, respectively. Higher drug release rate was present in HA-TS-TPP/LPT NPs group, compared with HA-TS/LPT NPs. Smaller particle size of HA-TS-TPP/LPT NPs (202 nm at pH 7.4), compared to HA-TS/LPT NPs (476 nm at pH 7.4), might partially contribute to its higher drug release. Polar characteristic of TPP installed in HA-TS-TPP/LPT NPs also seems to affect the release kinetics. Of note, sustained drug release profiles (for 7 days) were observed in both HA-TS/LPT NPs and HA-TS-TPP/LPT NPs. Observed findings regarding the particle properties of HA-TS-TPP/LPT NPs suggest their efficient in vitro and in vivo applications for cancer therapy. Cellular Uptake and Localization. The cellular internalization efficiency of fabricated nanostructures was assessed by flow cytometry and CLSM imaging (Figure 4). To trace the cellular fate of developed NPs with a fluorescence detector, Cy5.5 was attached to the synthesized conjugates (HA-TS and HA-TS-TPP) as a fluorescence dye. The EDC/NHScoupled amide bond reaction was used to introduce Cy5.5-NH2 to the HA backbone (in HA17 ACS Paragon Plus Environment


TS and HA-TS-TPP). Cy5.5-conjugated HA-TS/LPT NPs and HA-TS-TPP/LPT NPs were incubated for 1, 2, and 6 h and their cellular accumulation efficiency and intracellular localization were tested by flow cytometry and CLSM imaging, respectively. According to the data of flow cytometry analysis (Figure 4A), the mean fluorescence intensity values of Cy5.5HA-TS-TPP/LPT NPs were 2.5-, 1.9-, and 1.2-fold higher than those of Cy5.5-HA-TS/LPT NPs at 1, 2, and 6 h, respectively (p < 0.05). The cellular accumulation efficiency of the HATS-TPP/LPT NPs group was significantly higher than that of HA-TS/LPT NPs group at the observed incubation period (p < 0.05). In CLSM images (Figure 4B), a stronger red fluorescence signal was detected in the Cy5.5-HA-TS-TPP/LPT NPs group than the Cy5.5HA-TS/LPT NPs group. That superior cellular internalization capability of HA-TS-TPP/LPT NPs over HA-TS/LPT NPs can be explained by the existence of TPP molecule on the outer surface of HA-TS-TPP/LPT NPs. Cationic and lipophilic properties of TPP seem to improve cellular accumulation efficiency of HA-TS-TPP/LPT NPs compared with HA-TS/LPT NPs (Table 1). Electrostatic interactions between positively charged TPP and negatively charged cell membrane and lipophilic characteristic of TPP could increase the transport of HA-TSTPP/LPT NPs across the cell membrane. Improved cellular internalization efficiency of developed HA-TS-TPP/LPT NPs might secure their pharmacological efficacies after arriving in the tumor tissue via intravenous administration.


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Figure 4. Cellular internalization and distribution tests of developed NPs in MDA-MB-231 cells. (A) Cellular accumulation efficiency of NPs measured by flow cytometry. Cy5.5-HATS/LPT NPs and Cy5.5-HA-TS-TPP/LPT NPs were incubated for 1, 2, and 6 h. Each point represents the mean ± SD (n = 3). #p < 0.05, compared with Cy5.5-HA-TS/LPT NPs group. 19 ACS Paragon Plus Environment


(B) Cellular location of NPs measured by CLSM imaging. Cy5.5-HA-TS/LPT NPs and Cy5.5HA-TS-TPP/LPT NPs were incubated for 1, 2, and 6 h. Blue and red indicate DAPI and Cy5.5, respectively. The length of the scale bar (white) in the CLSM image is 20 μm.

Figure 5. In vitro anticancer efficacy tests in MDA-MB-231 cells. (A) Antiproliferation test of LPT, HA-TS NPs, HA-TS/LPT NPs, HA-TS-TPP NPs, and HA-TS-TPP/LPT NPs. The cell viability according to LPT concentration after incubating 24, 48, and 72 h is shown. Each point represents the mean ± SD (n = 3). (B) Annexin V-FITC and PI-based assay for measuring apoptotic efficacies of LPT, HA-TS NPs, HA-TS/LPT NPs, HA-TS-TPP NPs, and HA-TSTPP/LPT NPs. The population percentage in each panel after 24 h incubation is plotted. UL, UR, LL, and LR mean upper left, upper right, lower left, and lower right, respectively. Each point represents the mean ± SD (n = 3). *p < 0.05: compared with the control group; +p < 0.05: 20 ACS Paragon Plus Environment

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compared with the LPT group; &p < 0.05: compared with the HA-TS NPs group; #p < 0.05: compared with the HA-TS/LPT NPs group; %p < 0.05: compared with the HA-TS-TPP NPs group. (C) Mitochondrial membrane potential measurement by JC-1 assay. The population percentages in UL and UR panels are shown. Each point represents the mean ± SD (n = 3). *p < 0.05: compared with the control group; +p < 0.05: compared with the LPT group; #p < 0.05: compared with the HA-TS/LPT NPs group.

In Vitro Anticancer Potentials. In vitro anticancer potentials of LPT-included NPs were investigated by antiproliferation assay, apoptosis test, and mitochondrial membrane potential assay (Figure 5). LPT (as one of dual tyrosine kinase inhibitors) has been used for the treatment of breast cancer by interrupting EGFR and HER2 pathways.23 LPT was incorporated into the nanocarrier based on HA-TS-TPP for targeting CD44 receptor and mitochondria in this study. As shown in Figure 5A and Table S1, cell viability after application of LPT, HA-TS/LPT NPs, and HA-TS-TPP/LPT NPs for 24, 48, and 72 h was measured by MTS-based assay. Interestingly, the IC50 values of HA-TS-TPP/LPT NPs were lower than those of HA-TS/LPT NPs at 24, 48, and 72 h of incubation (Table S1) (p < 0.05). The existence of TPP in HA-TSTPP/LPT NPs may improve the antiproliferation potentials of LPT by efficient cellular entry and the subsequent destabilization of the mitochondrial membrane. The anticancer potentials of LPT-entrapped NPs were also identified by Annexin V-FITC and PI-based apoptosis assay (Figure 5B). The induction of apoptosis by LPT treatment was already reported in breast cancer cells.26 In addition, apoptosis can be induced by the modulation of mitochondria dynamics in cancer cells.31,32 The TPP and TS included in HATS-TPP NPs may exert mitochondria targeting and disrupting activities and may contribute to the apoptosis of cancer cells.33,34 In Annexin V-FITC and PI-based apoptosis assay, the sum of the UR and LR panels was used to count the population of the late and early apoptosis stages. 21 ACS Paragon Plus Environment


Blank HA-TS NPs and HA-TS-TPP NPs also exhibited elevated apoptotic inducing capability compared to the control (no treatment) group (p < 0.05). Attached TS and TPP groups in the HA backbone appear to work on the functions of mitochondria and further induce apoptosis in cancer cells. The (UR+LR) population percentage of the HA-TS-TPP/LPT NPs group was significantly higher than those of the LPT and HA-TS/LPT NPs groups (p < 0.05). Installment of TPP to HA-TS/LPT NPs also increased the apoptotic events in MDA-MB-231 cells. Mitochondria-related biofunctions of developed HA-TS-TPP-based NPs were further evaluated by mitochondrial membrane potential assay (Figure 5C). Together with TS and TPP, encapsulated LPT can also induce an apoptosis accompanied with the reduction in mitochondrial transmembrane potential.35 Alteration of membrane potential and oxidationreduction potential in the mitochondria can trigger disruption, which may be connected to the early stage of programmed cell death. JC-1 dye is specifically for the mitochondrial membrane rather than the plasma membrane, and it may be accumulated in healthy mitochondria. JC-1 aggregates (red to orange color) are observed at high concentration, implying hyperpolarization. The opening of the mitochondrial permeability transition pore (MPTP) may bring about the reduction of mitochondrial membrane potential and show weak red/orange color (JC-1 aggregates) and strong green color (JC-1 monomer).36,37 In this study, the increment in the population percentage of the UR panel indicates the increase in JC-1 monomer. The HA-TSTPP/LPT NPs group displayed a higher population percentage in the UR panel than in the LPT and HA-TS/LPT NPs groups (p < 0.05), which means that HA-TS-TPP/LPT NPs can give rise to the more efficient opening of MPTP and the drastic subsequent diminution of the mitochondrial membrane potential, relative to the other groups. Influences of TS and/or TPP (attached forms to HA) on the mitochondria membrane potential were also investigated by JC-1 assay (Figure S3). The percentage of UR panel in HA-TS-TPP group was similar with the sum of HA-TS and HA-TPP groups. It implies the collaboration of TS and TPP for mitochondria22 ACS Paragon Plus Environment

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related functions. However, those percentages were obviously lower than those of LPT-loaded NPs (Figure 5C). The mitochondria-related roles of the TS and TPP installed in HA-TSTPP/LPT NPs were appropriately demonstrated by these findings. After endocytosis of HATS-TPP/LPT NPs, TPP and TS might transfer to mitochondria as a conjugate form (i.e., HATS-TPP) and released LPT from HA-TS-TPP/LPT NPs can bind to ATP-binding pocket of the EGFR protein kinase domain. The integration of TS, TPP, and LPT into the single nanocarrier and its efficient endocytosis into MDA-MB-231 cells may amplify apoptosis-related anticancer activities.

Figure 6. Real-time NIRF imaging study in MDA-MB-231 tumor xenograft model. Cy5.5HA-TS/LPT NPs and Cy5.5-HA-TS-TPP/LPT NPs were intravenously injected to the mouse model. (A) Whole body NIRF images at 0 (pre) and 24 h. The yellow dashed circle indicates the tumor region. (B) NIRF image of dissected tumor mass. (C) Integrated intensity values of fluorescence signals in tumor tissue. Each point represents the mean ± SD (n = 3). #p < 0.05: compared to the Cy5.5-HA-TS/LPT NPs group.



Tumor Targeting. The in vivo tumor targeting capability of the designed NPs was tested in an MDA-MB-231 tumor-bearing mouse model after intravenous injection (Figure 6). For the detection of NIRF signals, Cy5.5-labeled HA-TS/LPT NPs and HA-TS-TPP/LPT NPs were prepared and injected intravenously into the tumor-bearing mouse model. The fluorescence signal produced from the body implies the in vivo movement of the injected NPs. A stronger fluorescence signal was shown in the tumor region of the Cy5.5-HA-TS-TPP/LPT NPsinjected group than in the Cy5.5-HA-TS/LPT NPs-injected group (Figure 6A). Also in the ex vivo scanned image of the excised tumors, a higher fluorescence signal was detected in the Cy5.5-HA-TS-TPP/LPT NPs-injected group than the Cy5.5-HA-TS/LPT NPs-injected group (Figure 6B). Then, ex vivo data of dissected tumor tissues were presented as an integrated intensity, which can be calculated by multiplying the mean intensity by the area (Figure 6C). The integrated intensity of the tumor tissue in the Cy5.5-HA-TS-TPP/LPT NPs-injected group was 2.1-fold higher than that of the Cy5.5-HA-TS/LPT NPs-injected group (p

Mitochondria Targeting and Destabilizing ... - ACS Publications

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