Bilirubin Nanoparticle-Assisted Delivery of a Small Molecule-Drug

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Bilirubin Nanoparticle-Assisted Delivery of a Small Molecule-Drug Conjugate for Targeted Cancer Therapy Soyoung Lee, Yonghyun Lee, Hyungjun Kim, Dong Yun Lee, and Sangyong Jon Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00189 • Publication Date (Web): 30 Apr 2018 Downloaded from http://pubs.acs.org on May 3, 2018

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Bilirubin Nanoparticle-Assisted Delivery of a Small Molecule-Drug Conjugate for Targeted Cancer Therapy Soyoung Lee†, Yonghyun Lee†, Hyungjun Kim†, Dong Yun Lee‡ and Sangyong Jon†,‡,* †

KAIST Institute for the BioCentury, Department of Biological Sciences and ‡Graduate School

of Medical Science and Engineering, Korea Advanced Institute of Science and Technology (KAIST), 291 Daehak-ro, Daejeon 34141, Republic of Korea

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Abstract

Despite growing interest in targeted cancer therapy with small molecule drug conjugates (SMDCs), the short half-life of these conjugates in blood associated with their small size has limited their efficacy in cancer therapy. In this report, we propose a new approach for improving the antitumor efficacy of SMDCs based on nanoparticle-assisted delivery. Ideally, a nanoparticle-based delivery vehicle would prolong the half-life of an SMDC in blood and then release it in response to stimuli in the tumor microenvironment (TME). In this study, PEGylated bilirubin-based nanoparticles (BRNPs) were chosen as an appropriate delivery carrier because of their ability to release drugs in response to TME-associated reactive oxygen species (ROS) through rapid particle disruption. As a model SMDC, ACUPA-SN38 was synthesized by linking the

prostate

membrane-specific

antigen

(PSMA)-targeting

ligand,

ACUPA,

to

the

chemotherapeutic agent, SN38. ACUPA-SN38 was loaded into BRNPs using a film-formation and rehydration method. The resulting ACUPA-SN38@BRNPs exhibited ROS-mediated particle disruption and rapid release of the SMDC, resulting in greater cytotoxicity toward PSMAoverexpressing prostate cancer cells (LNCaP) than toward ROS-unresponsive ACUPASN38@Liposomes. In a pharmacokinetic study, the circulation time of ACUPA-SN38@BRNPs in blood was prolonged by approximately 2-fold compared with that of the SMDC-based micellar nanoparticles. Finally, ACUPA-SN38@BRNPs showed greater antitumor efficacy in a PSMA-overexpressing human prostate xenograft tumor model than SN38@BRNPs or the SMDC alone. Collectively, these findings suggest that BRNPs are a viable delivery carrier option for various cancer-targeting SMDCs that suffer from short circulation half-life and limited therapeutic efficacy.

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Keywords: Bilirubin nanoparticles, Tumor microenvironment, Reactive oxygen species, SN38, Small molecule drug conjugates, Targeted cancer therapy

1. Introduction Small molecule drug conjugates (SMDCs), consisting of a cancer-targeting small-molecule ligand and a chemotherapeutic agent, have attracted increasing research attention. Indeed, several such candidates, including vintafolide,1 a folate-vinblastine conjugate,2 and EC1169, a conjugate targeting prostate-specific membrane antigen (PSMA),3-4 are currently undergoing clinical development. However, because of their small size, SMDCs show an extremely short half-life in the circulation, resulting in much lower tumor uptake compared with long-circulating antibodydrug conjugates; as a consequence, their therapeutic efficacy is limited.5-6 Given this limitation of SMDCs, there is a need to develop a delivery system that is able to improve the pharmacokinetics as well as the therapeutic efficacy of SMDCs in vivo. An ideal SMDC-delivery system would be one that possessed the ability to release the conjugate within the tumor in response to a stimulus present in the tumor microenvironment (TME).7-9 Typical physiological stimuli associated with TME include mildly acidic pH and high oxidative stress, reflecting the abundant production of reactive oxygen species (ROS) within the TME.10-11 In this context, a nanoparticle carrier with ROS-responsive particle-disruption behavior and enhanced circulation in the bloodstream could be a viable option as an SMDC-delivery system.12 Very recently, we published the first report of bilirubin-based nanoparticles (BRNPs) formed by self-assembly from PEGylated bilirubin (PEG-BR) as a nanomedicine platform for antiinflammation therapy.13-16 We have also demonstrated that BRNPs can be used as a dualstimulus (ROS and light)-responsive drug-delivery carrier for cancer therapy, as shown by their

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ability to release encased drugs through rapid particle disruption in response to ROS or 450/650 nm light.17-18 On the basis of these favorable properties, we hypothesized that BRNPs could be used as a TME-responsive delivery carrier for SMDCs. As a proof-of-concept study, we physically loaded a prostate cancer-targeting SMDC into BRNPs. We hypothesize that, by incorporating SMDCs to BRNPs, their circulation time in blood would be similar to that of the nanoparticles, leading to enhanced accumulation in the perivascular region of the tumor through the so-called enhanced permeability and retention (EPR) effect.19 Further, it is expected that SMDCs are released upon disruption process of BRNPs in response to TME-associated ROS,17 penetrate into the tumor tissue, and are finally internalized via target receptor-mediated endocytosis (Figure 1a).

2. Experimental section 2.1. Materials. (ZZ)-bilirubin-IX-alpha and SN38 were purchased from Tokyo Chemical Industry

(Tokyo,

Japan).

1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide

(EDC)

and

methoxypolyethylene glycol 2,000-NH2 (mPEG2000-NH2) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Maleimide-oligoethyleneglycol-carboxylic acid, Mal-(OEG)6-COOH was purchased from FutureChem (Seoul, South Korea). Cysteine-modified ACUPA was custom synthesized by Peptron (Daejeon, South Korea). Sterile water (water for injection) was purchased from JW Pharmaceutical company (Seoul, South Korea). All other solvents and reagents were purchased from Sigma Chemical. 2.2. Cells and animals. The PSMA-positive human prostate cancer cell line, LNCaP, and PSMA-negative human prostate cancer cell line, PC3, were purchased from the Korean Cell Line Bank (Seoul, Korea). All animals were purchased from Orient Bio Inc. (Seoul, Korea) and

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maintained under pathogen-free conditions in the animal facility at Korea Advanced Institute of Science and Technology (KAIST). Animal experiments were approved by the KAIST Animal Care and Use Committee. 2.3. Synthesis of PEGylated bilirubin. Synthesis of PEGylated bilirubin was described previously.13 Briefly, 0.50 mmol of bilirubin and 0.45 mmol of EDC were dissolved in 5 mL of dimethyl sulfoxide (DMSO). After stirring for 10 min at room temperature (RT), 0.20 mmol of mPEG2000-NH2 and 150 µL triethylamine were added to the mixture, and the reaction was allowed to proceed with stirring for 4 h at RT under a nitrogen atmosphere. Chloroform (200 mL) was added and the reaction mixture was washed with 0.1 M HCl and brine using a separation funnel. The organic layer was collected, concentrated under vacuum, dissolved in chloroform, and purified by column chromatography on silica using chloroform:methanol (85:15) as the mobile phase. The solvents were evaporated to yield PEG-BR, which was subsequently subjected to TLC (thin layer chromatography), MALDI-TOF (matrix-assisted laser desorption/ionization-time of flight) spectrometry, and 1H-NMR spectroscopy. 1H-NMR spectra were obtained using an AVANCE400 system (Bruker Daltonics, Bremen, Germany); chemical shifts represent ppm downfield from tetramethylsilane. MALDI-TOF spectra were obtained using an Autoflex III MALDI-TOF system (Bruker). 2.4. Synthesis of Boc-SN38. As reported previously,20-22 SN38 (500 mg, 1.27 mmol) and di-tertbutyl dicarbonate (360 mg, 1.65 mmol) were dissolved in dichloromethane (DCM; 50.8 mL, 25 mM), to which was added anhydrous pyridine (3.04 mL, 0.05 mmol). After stirring overnight at RT, the reaction mixture was washed with 0.5 N HCl (3 × 250 mL) and saturated NaHCO3 (1 × 250 mL). The organic phase was dried over MgSO4, filtered, and purified by silica gel column

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chromatography to obtain Boc-SN38 with a yield of 96%, as characterized by TLC and MALDITOF spectrometry. 2.5. Synthesis of Boc-SN38-OEG. Maleimide-OEG-COOH (554.3 mg, 0.61 mmol) and EDC (225.7 mg, 1.17 mmol) were dissolved in anhydrous DCM (10 mL, 100 mM). After stirring for 10 min at RT, Boc-SN38 (200 mg, 0.41 mmol) and dimethylaminopyridine (DMAP 15 mg, 0.12 mmol) were added to the mixture, and the reaction was allowed to proceed with stirring for 4 h at RT. The reaction mixture was then washed with 0.5 N HCl (3 × 150 mL) and dried with MgSO4. The organic phase was purified by silica gel column chromatography to give Boc-SN38-OEG with a yield of 55%, as characterized by TLC and MALDI-TOF spectrometry. 2.6. Synthesis of SN38-OEG. Boc groups were deprotected by dissolving Boc-SN38-OEG (100 mg, 0.11 mmol) in 30% trifluoroacetic acid (TFA) in anhydrous DCM (14 mM) and stirring the mixture for 3 h at RT. Thereafter, the solvent was removed under vacuum, and the residue was purified by silica gel column chromatography to give SN38-OEG with a yield of 86%, as characterized by TLC and MALDI-TOF spectrometry. 2.7. Synthesis of ACUPA-SN38. SN38-OEG (8 mg, 9.88 µmol) and cysteine-modified ACUPA (5 mg, 11.9 µmol) were dissolved in anhydrous DMSO (0.1 mL, 100 mM), and the mixture was stirred at RT overnight. ACUPA-SN38 was purified by reverse-phase HPLC (Shimadzu, Kyoto, Japan) on a Nova-Pak C18 column (300 × 3.9 mm; Waters) with acetonitrile/water each containing 0.1 % TFA from 0 to 20 min with acetonitrile 30 % to 40 %. The product fraction was lyophilized to obtain the ACUPA-SN38 conjugate as a yellow solid with a yield of 88%; the product was further characterized using MALDI-TOF spectrometry and 1H-NMR spectroscopy. 2.8. Preparation of ACUPA-SN38–loaded nanoparticles. ACUPA-SN38 loaded liposomes and BRNPs were prepared using a film formation-rehydration method. Briefly, to prepare

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ACUPA-SN38@BRNP, PEG-BR (1 mg, 0.38 µmol) was dissolved in chloroform (200 µL) and dried under vacuum to yield a film layer. Then the film layer was rehydrated with a solution of ACUPA-SN38 (0.2 mg, 0.14 µmol) dissolved in sterile water (1 mL) and sonicated for 10 min to yield ACUPA-SN38–loaded BRNPs. The resulting suspension was placed on gel filtration and purified using a Sepharose CL-4B column to remove free ACUPA-SN38. For the preparation of ACUPA-SN38@Liposome, all the lipids, including cholesterol (1.95 µmol), DOPE (1.95 µmol) and PEG2000-DSPE (0.1 µmol) were dissolved in chloroform and dried under vacuum to yield a lipid film layer. The remaining steps were as same as those of ACUPA-SN38@BRNPs. The amount of ACUPA-SN38 in each nanoparticle formulation was determined by using HPLC, and the concentrations of BRNPs were determined by measuring bilirubin absorbance at 450 nm using a UV-Vis microplate reader (FL600; Bio-Tek Inc., Winooski, VT, USA). The size and zeta potential of ACUPA-SN38@BRNPs were characterized using a Nanosizer ZS90 (Malvern Instruments, Ltd., Malvern, UK). The morphology of ACUPA-SN38@BRNP was obtained by transmission electron microscopy (TEM) using a Tecnai TF30 ST (FEI Co., Hillsboro, OR). 2.9. In vitro cellular uptake test. Both confocal microscopy and flow cytometry were used to study cellular uptake of ACUPA-SN38@BRNP. For confocal microscopy-based cellular uptake study, LNCaP cells were seeded onto cover glasses at a density of 1 × 105 cells per well, which were placed in a 24-well plate. After reaching 70% confluence, each well was treated with ACUPA-SN38@BRNP (10 µM) for 1 h. Cells were washed with cold PBS, fixed with 4% paraformaldehyde for 15 min, and stained with Hoechst 33342 for 10 min. Images of cellular fluorescence were taken using a confocal laser-scanning microscope (LSM 710 ; Carl Zeiss, Inc., Jena, Germany). For flow cytometry-based cellular uptake study, LNCaP cells were treated for 1 h with free ICG or equivalent amount of ICG-loaded ACUPA-SN38@BRNP. Then, cells were

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harvested, washed three times with cold PBS containing 1% FBS, and analyzed on a LSR II Flow cytometer (BD Biosciences) using FlowJo software (Tree Star Inc., San Carlos, CA, USA). 2.10.

Release

from

ACUPA-SN38@BRNPs.

ROS-responsive

release

of

ACUPA-

SN38@BRNPs was examined by incubating each concentration of ACUPA-SN38@BRNPs (50, 100 and 200 µM) with the peroxy radical precursor 2,2’-azobis(2-amidinopropane) dihydrochloride (AAPH) for 1 h at 37°C. After exposure to peroxy radicals, released ACUPASN38 was separated by Sepharose CL-4B column chromatography at each time point, and then measured by HPLC. 2.11. In vitro cell viability and anti-proliferation test. For in vitro cell viability test, PSMApositive LNCaP human prostate cancer cells and PSMA-negative PC3 human prostate cancer cells were seeded overnight in 96-well plates (5 × 103 cells/well) at 37°C. SN38 and ACUPASN38, dissolved in DMSO and sterile water, respectively, were each diluted 100-fold from their starting concentrations with growth medium and then incubated with cells for 72 h. Cells in other groups were treated for 4 h at 37°C with fresh medium (control) or different concentrations of ACUPA-SN38, BRNPs, ACUPA-SN38@BRNPs, or ACUPA-SN38@Liposomes. Cells were then washed with phosphate-buffered saline (PBS) and incubated with fresh medium for an additional 53 h at 37°C. After the medium was removed, 100 µL of fresh culture medium containing 20 µL of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide] solution (5 mg/mL in PBS) was added and cells were incubated for 3 h. Thereafter, 200 µL of DMSO was added to each well to dissolve the resulting formazan crystals; the contents of each well were mixed by pipetting to ensure complete dissolution of formazan crystals. Finally, the absorbance was measured at 570 nm using a 96-well plate reader. For anti-proliferation test, colony forming assay was carried out. LNCaP cells in growth medium were cultured at 500 cells

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per well in a 12-well plate and treated with BRNPs 25 µg/mL, ACUPA-SN38 2 µg/mL or equivalent concentration of ACUPA-SN38@BRNPs for 24 h. Cells were then allowed to grow for 10 d until colonies were visible. Colonies were stained by incubating with 0.1 % Coomassie Brilliant Blue R-250 for 20 min. 2.12. Competition assay. PSMA-positive LNCaP cells were seeded overnight in 96-well plates (5 × 103 cells/well) at 37°C. Cells were briefly (1 h) treated with ACUPA-SN38, after which media were aspirated from all wells, and wells were washed two times with PBS. Fresh media were added and plates were further incubated for 48 h. For competition assays, a separate group of LNCaP cells was pretreated with ACUPA (10 µM); the remaining steps were the same as described above. At the end of the post-treatment incubation, cell viability was measured as described in the previous section. 2.13. Pharmacokinetic test. Balb/c nude mice were injected intravenously via the tail vein with ACUPA-SN38 or ACUPA-SN38@BRNPs at a dose of 5 mg/kg SN38. Blood was collected into heparinized tubes 0.16, 0.5, 1, 3, 6, and 12 h after injection, and immediately centrifuged at 13,000 rpm for 10 min. ACUPA-SN38 was extracted from 50 µL aliquots of plasma using 100 µL of acetonitrile. The amounts of ACUPA-SN38 in samples were measured after running HPLC. 2.14. Biodistribution test. Balb/c nude mice were subcutaneously inoculated in the dorsal right side with 1 × 106 LNCaP cells. When the tumor volume reached approximately 150 mm3, free ICG or ICG-coloaded ACUPA-SN38@BRNP at an equivalent ICG dose (1 mg/kg) was intravenously injected through the tail vein. At predetermined time points, the biodistribution of free ICG and ICG-coloaded ACUPA-SN38@BRNP were monitored using a Xenogen IVIS Lumina imaging system (Perkin Elmer Inc.) with a built-in ICG filter set (excitation, 710-760

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nm; emission, 810-875 nm) and an exposure time of 5 s. The ex vivo fluorescence images of major organs and tumor tissues were also taken from the corresponding euthanized mice at predetermined time points using the same imaging system above. 2.15. Anticancer activity evaluation. Anticancer efficacy was investigated in LNCaP xenograft model after tumor volume had reached at least 100 mm3 (day 0), at which point tumor-bearing mice were randomly divided into five groups (n = 5 per group), while minimizing differences in weight and tumor size. Mice in each group were administered 100 µL of PBS (control), ACUPASN38 (5 mg/kg SN38), BRNPs (87 mg/kg), ACUPA-SN38@BRNPs (5 mg/kg SN38), or SN38@BRNPs (5 mg/kg SN38) by intravenous injection five times on predetermined days (day 0, 2, 4, 6, and 8). Tumors in each group were measured at predetermined days using a Vernier caliper, and tumor volume was calculated using the following formula: volume = (length × width × height)/2. Tumor growth inhibition (TGI) on the final day was calculated as TGI = 100% × (TvolControl - TvolTreatment)/TvolControl, where Tvol = final tumor volume – initial tumor volume. Body weights were also monitored at predetermined days. Mice were sacrificed 16 days after the first treatment. 2.16. Statistical analysis. The significance of differences among groups was calculated by oneway analysis of variance (ANOVA) followed by Tukey’s post-hoc test. A p-value < 0.05 was considered statistically significant. XLSTAT Software (Addinsoft, Inc., New York, NY, USA) was used for all statistical analyses.

3. Results and Discussion 3.1. Preparation and characterization of ACUPA-SN38@BRNPs

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As a model SMDC, we designed and prepared an ACUPA-SN38 conjugate. ACUPA is a small molecule that has been used as a cancer-targeting ligand for certain PSMA-overexpressing prostate cancers and other cancers.23-24 SN38, an active metabolite of the anticancer drug camptothecin (CPT), was chosen as a model drug.25-26 Although SN38 is much more potent than CPT (at least ~100-1,000-fold), its poor solubility has hampered its clinical application.20 Thus, we linked ACUPA and SN38 via a cleavable ester bond using a hydrophilic oligo(ethylene glycol) (OEG) linker.20, 22 The overall synthetic scheme for ACUPA-SN38 is shown in Figure 1b. The terminal carboxylic group of a hetero-bifunctional OEG linker containing maleimide at the other end was reacted with the tertiary alcohol of Boc-protected SN38 via an ester bond that can be hydrolyzed in vivo.26 The maleimide group in the resulting OEG-SN38 was subsequently reacted with the sulfhydryl (-SH) group in cysteine-modified ACUPA, yielding the SMDC, ACUPA-SN38, which was characterized by 1H-NMR spectroscopy and MALDI-TOF mass spectrometry, confirming successful synthesis of the SMDC (Figures S1–S3). Next, ACUPASN38–incorporated nanoparticles, ACUPA-SN38@BRNPs and ACUPA-SN38@Liposomes, were prepared using a film-formation and rehydration method, as described in the Experimental Section. ACUPA-SN38@BRNPs had a hydrodynamic size of 83.7 ± 21.8 nm, measured by dynamic light scattering, and a zeta potential of 0.04 ± 3.56; transmission electron microscopy (TEM) revealed spherically shaped nanoparticles with a dehydrated size of 63.2 ± 5.8 nm (Figure S4). The loading capacity and encapsulation efficiency of ACUPA-SN38 into BRNPs was 9.8 ± 3.4 % and 43 ± 4.6 %, respectively (Figure S6a). Interestingly, because of the presence of both hydrophilic ACUPA-OEG and hydrophobic SN38, ACUPA-SN38 itself formed ultra-small

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micelle-like nanoparticles in aqueous medium with a size of 23.2 ± 7.3 nm, as shown in TEM images (Figure S5).

Figure 1. Working hypothesis for BRNP-assisted delivery of a SMDC and preparation of ACUPA-SN38@BRNPs. (a) Illustration of the preparation of ACUPA-SN38@BRNPs in aqueous solution and the working hypothesis for their efficacy in cancer therapy. (b) Synthetic scheme for the synthesis of ACUPA-SN38.

3.2. Cell uptake behavior of ACUPA-SN38@BRNPs

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Cellular uptake of ACUPA-SN38@BRNPs was examined in LNCaP cells using confocal laser scanning microscopy and flow cytometry. For confocal imaging, intrinsic fluorescence of bilirubin was used to detect, while a near infrared dye ICG was embedded to ACUPASN38@BRNPs for flow cytometry analysis. As shown in Figure S9b, the fluorescence signal of bilirubin was observed in the cytosol of the cells after treatment with ACUPA-SN38@BRNPs; in contrast, no fluorescence was seen in the cytosol of non-treated control cells. Interestingly, the fluorescent bilirubin distributed evenly throughout the cytosol of the nanoparticle-treated cells, which was far different from the typical distribution pattern of fluorescent nanoparticles after endocytosis. This suggests that once BRNPs get internalized, they would become dissociated into individual PEGylated bilirubin in response to intracellular ROS and evenly diffuse into the cytoplasm of the cell. A flow cytometry analysis further confirmed that ICG-loaded ACUPASN38@BRNPs could be taken up by LNCaP cells as similar as did free ICG (Figure S9c). This finding suggests that BRNPs may be disrupted first in response to ROS in the cell culture medium and then the released cargo such as ACUPA-SN38 and ICG get internalized into the cells. 3.3. ROS-responsive drug release and anticancer effect of ACUPA-SN38@BRNPs To examine ROS-responsive drug release behavior, we incubated ACUPA-SN38@BRNPs in the presence of 2,2’-azobis(2-amidinopropane) dihydrochloride (AAPH), a peroxy radical precursor. As a comparison control, we also prepared ROS-unresponsive ACUPA-SN38–encapsulated liposome (ACUPA-SN38@Liposome) nanoparticles. The amount of ACUPA-SN38 released from each nanoparticle system was directly measured by HPLC at predetermined times. As shown in Figure 2a, ACUPA-SN38 was rapidly released from ACUPA-SN38@BRNPs in the presence of AAPH, whereas only marginal release of the conjugate from ACUPA-

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SN38@Liposomes was observed. Moreover, neither ACUPA-SN38@BRNPs nor ACUPASN38@Liposomes showed drug release in the absence of an AAPH ROS source. Disruption of BRNPs in response to ROS was also monitored by measuring UV-vis absorption of ACUPASN38@BRNPs at 450 nm, which showed a rapid decrease in absorbance corresponding to bilirubin (Figure S6b). These findings clearly indicate that BRNPs rapidly release ACUPA-SN38 in response to ROS, a TME-associated stimulus. It has been generally accepted that both cancer cells and their surrounding TME present high oxidative stress compared with normal cells and tissues.12 To assess the relative levels of ROS released by cancer cells, we cultured three prostate cancer cell lines, LNCaP, PC3 and DU145, and murine fibroblast NIH3T3 cells (control), collected the conditioned medium from each culture, and incubated it with the ROS-detecting dye, Acridan Lumigen PS-3 reagent.27 LNCaP cell conditioned medium showed the highest extracellular ROS levels (Figure S7). Moreover, LNCaP cells showed higher expression of PSMA, the target antigen of the ACUPA ligand, than DU145 cells, whereas PC3 only marginally expressed the antigen (Figure S8a). These findings suggest that LNCaP is a suitable cancer cell line for exploring the therapeutic potential of ACUPA-SN38@BRNPs. Next, we assessed the targeting ability and cytotoxicity of ACUPASN38 and ACUPA-SN38@BRNPs in PSMA-positive LNCaP and PSMA-negative PC3 cells. Whereas SN38 exhibited a similar level of cytotoxicity against both cancer cell lines, ACUPASN38 showed much higher cytotoxicity against LNCaP cells than PC3 cells (Figure 2b). Pretreatment of LNCaP cells with an excess of free ACUPA (10 µM) significantly reduced (~3fold) the cytotoxicity of ACUPA-SN38, suggesting competition for PSMA binding of the ACUPA ligand (Figure 2c). Given that ROS detection assays confirmed high-level production of extracellular ROS by LNCaP cells, the prediction is that, upon incubation with LNCaP cells,

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ACUPA-SN38@BRNPs would release a native form of ACUPA-SN38, which would, in turn, become internalized by these cancer cells through PSMA-mediated endocytosis. As expected, ACUPA-SN38@BRNPs exhibited much higher cytotoxicity against LNCaP cells than ACUPASN38@Liposomes (IC50 value: 1.39 µM versus 7.64 µM); unexpectedly, ACUPASN38@BRNPs showed higher cytotoxicity than ACUPA-SN38 (IC50 value: 1.39 µM versus 3.40 µM) (Figure 2d). Furthermore, to confirm anti-proliferation of BRNPs, ACUPA-SN38 and ACUPA-SN38@BRNPs, colony forming assay was performed. As anticipated, ACUPA-SN38 and ACUPA-SN38@BRNPs showed higher anti-proliferation property through PSMA-mediated active and nanoparticle-mediated passive process. While BRNPs also have a little cancer cell growth inhibition because of bilirubin physiological function by modulating intracellular antioxidant system (Figure S10).28-29

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Figure 2. In vitro release and cytotoxicity of ACUPA-SN38@BRNPs. (a) Release of ACUPASN38 from ACUPA-SN38@BRNPs in the presence (●) and absence (▼) of 2,2’-azobis(2amidinopropane) dihydrochloride (AAPH), and from ACUPA-SN38@Liposomes in the presence (○) and absence (△) of AAPH. Data are expressed as means ± s.e.m. (n = 3). (b) MTT assay of PSMA-positive LNCaP and PSMA-negative PC3 cell lines incubated with SN38 for 72 h at 37°C or with ACUPA-SN38 for 4 h, followed by further incubation in fresh medium for 48 h at 37°C. (c) The in vitro-targeting ability of ACUPA-SN38 was evaluated by competition assay. LNCaP cells were treated with ACUPA-SN38 for 1 h, washed, and incubated for 48 h. For competition assays, LNCaP cells were pretreated with free ACUPA (10 µM), and processed as described above. (d) Viability of LNCaP cells after a 4-h incubation with different concentrations of ACUPA-SN38, BRNPs, ACUPA-SN38@BRNPs, or ACUPA-SN38@Liposomes or culture medium (control), followed by further incubation for 53 h. Data are expressed as means ± s.e.m. (n = 6).

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3.4. Pharmacokinetics and biodistribution of ACUPA-SN38@BRNPs The pharmacokinetics of ACUPA-SN38@BRNPs and ACUPA-SN38 self-assembled micellar nanoparticles were assessed after tail vein injection of a 5-mg SN38/kg equivalent of each formulation (Figure 3a). The amount of ACUPA-SN38 released into blood as a function of time was measured by HPLC. Plasma clearance curves for ACUPA-SN38@BRNPs and ACUPASN38 micellar nanoparticles are shown in Figure 3b, and the major pharmacokinetic parameters are summarized in Figure 3c. Using a one-compartment model, we calculated the clearance halflife (t1/2) of ACUPA-SN38 released from ACUPA-SN38@BRNPs to be 4.91 h, which is similar to that of BRNPs17 and approximately 2-fold longer than that of micellar ACUPA-SN38, which showed a half-life of 2.45 h. Thus, the area-under-the-curve (AUC0-∞ ) value of ACUPASN38@BRNPs was also ~2-fold greater than that of the micellar ACUPA-SN38. Considering that the clinically available anticancer drug camptothecin (CPT), the parent drug of SN38, has a half-life of 0.44 h in blood,30 the improvement in the pharmacokinetic profiles of ACUPASN38@BRNPs is significant. These findings suggest that BRNPs are viable carriers with the potential to improve the pharmacokinetics of ACUPA-SN38. We next carried out biodistribution study after tail vein injection of free ICG or ICG-loaded ACUPA-SN38@BRNPs at a 1-mg ICG/kg equivalent of each group. Figure 4 shows the realtime in vivo and ex vivo biodistribution at the predetermined time points. At 1 h post injection, while both groups showed a similar pattern of distribution in the liver and kidneys, weak but apparent fluorescence signal was observed in the tumor from ICG-loaded ACUPASN38@BRNPs only (Figure 4a). Such trend became more apparent at 6 h post injection. It seems that free ICG was mostly excreted from the body; however, much increased fluorescence signal

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was observed in the tumor and at the same time, the highest signal was found in the liver of mice treated with ICG-loaded ACUPA-SN38@BRNPs (Figure 4b). Considering that liver is the organ of excretion and metabolism for both ICG and bilirubin, it is natural to see the highest fluorescence signal of ICG. This biodistribution results suggest that ACUPA-SN38@BRNPs could be localized into the tumor via so-called EPR effect.

Figure 3. Plasma concentration-time curves of ACUPA-SN38 micellar nanoparticles and ACUPA-SN38@BRNPs. (a) Balb/c nude mice were intravenously injected with ACUPA-SN38 and ACUPA-SN38@BRNPs at designated time points, and (b) the plasma concentration of ACUPA-SN38 was measured over time. Data are expressed as means ± s.e.m (n = 5). (c) Pharmacokinetic parameters for ACUPA-SN38 and ACUPA-SN38@BRNPs.

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Figure 4. In vivo and ex vivo biodistribution images of free ICG and ICG-loaded ACUPASN38@BRNPs in tumor bearing mice as a function of time: (a) 1 h and (b) 6 h post intravenous injection of free ICG or ICG-loaded ACUPA-SN38@BRNPs at a 1-mg ICG/kg equivalent.

3.5. Antitumor efficacy of ACUPA-SN38@BRNPs in vivo The antitumor efficacy of ACUPA-SN38@BRNPs was assessed in the ROS-producing, PSMApositive LNCaP human prostate cancer xenograft model. When tumors reached ~100 mm3 in size, mice in each experimental group were injected intravenously with the corresponding formulation, and tumor growth and changes in body weight were monitored. As shown in Figure 5a, ACUPA-SN38@BRNPs (5 mg SN38/kg equivalent) showed much greater antitumor activity than other formulations, exhibiting tumor growth inhibition (TGI) of 53% relative to the control saline-treated group compared with 30% for ACUPA-SN38 micellar nanoparticles, 35% for SN38@BRNPs, and 21% for BRNP vehicle (TGI: 21%). Interestingly, BRNP vehicle alone showed appreciable antitumor efficacy, presumably owing to the known intrinsic anticancer activity of bilirubin, an observation in agreement with our previously reported results17. Moreover, changes in body weight were similar between each regimen and the control group (Figure 5b). These findings suggest that BRNPs are able to simultaneously act as a ROS-

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responsive drug-delivery carrier for the model SMDC, ACUPA-SN38, and as therapeutic agent, thereby exerting greater antitumor efficacy compared with the SMDC alone.

Figure 5. ACUPA-SN38@BRNPs exert improved anticancer activity in vivo. Mice bearing LNCaP tumors (size, ~100 mm3) were intravenously administered PBS (vehicle control), ACUPA-SN38 (5 mg/kg SN38 equivalent), BRNPs (87 mg/kg), SN38@BRNPs (5 mg/kg SN38 equivalent), or ACUPA-SN38@BRNPs (5 mg/kg SN38 equivalent). Drugs (5 mg/kg) were injected intravenously at the indicated times (arrows). (a) Tumor volume; (b) body weight. Data are expressed as means ± s.e.m. (n = 5; ***p < 0.05 vs. control group; **p < 0.05 vs. CPT-11 group; *p < 0.05 vs. ACUPA-SN38@BRNPs group).

4. Conclusions In this study, we demonstrate that ROS-responsive BRNPs are viable delivery carriers for the model SMDC, ACUPA-SN38, that serve to enhance the antitumor efficacy of the drug cargo. BRNP-assisted delivery extended the half-life of the SMDC in the bloodstream, increased uptake by prostate cancer cells and enhanced cytotoxicity, ultimately producing greater therapeutic efficacy than the parent SMDC in a tumor-bearing mouse model. Moreover, BRNPs, composed of the biocompatible, biodegradable and endogenous bilirubin exhibited rapid release of payloads in response to a TME-associated ROS stimulus, and possessed intrinsic anticancer

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activity. Therefore, it is anticipated that BRNPs with these favorable delivery-carrier characteristics could be used to improve therapeutic outcomes of other SMDC candidates currently at various stages of development.

ASSOCIATED CONTENT Supporting Information Detailed methods for some experiments; Synthesis of PEG-BR (Figure S1) and ACUPA-SN38 (Figure S2 and S3); Characterization of ACUPA-SN38@BRNPs (Figure S4) and ACUPA-SN38 (Figure S5); Sepharose CL-4B column chromatography for purification of ACUPASN38@BRNPs; In vitro ROS-responsiveness ACUPA-SN38@BRNPs determined by UV-Vis absorbance and color change; In vitro stability of ACUPA-SN38 in reactions with AAPH (Figure S6); ROS levels in culture media of various cancer cell lines (LNCaP, PC3, DU145) (Figure S7).27 Quantification of PSMA mRNA levels in each cell line; Viability of J774A1 and NIH3T3 cells (Figure S8); In vitro cellular uptake of ACUPA-SN38@BRNPs (Figure S9); Colony forming assay for anti-proliferation test in LNCaP cells (Figure S10).

AUTHOR INFORMATION Corresponding Author Prof. Dr. Sangyong Jon, KAIST Institute for the BioCentury, Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST)

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291 Daehak-ro, Daejeon 34141, Republic of Korea. Tel: (+82)-42-350-2634 Fax: (+82)-42-350-4450 E-mail: [email protected] Author Contributions All authors contributed to writing the manuscript and have given their approval of the final version. Funding Sources National Research Foundation of Korea Notes The authors declare no competing financial interests. ACKNOWLEDGMENT This research was supported by Global Research Laboratory (GRL) Program (NRF2012K1A1A2045436) and the Bio & Medical Technology Development Program (NRF2018M3A9B5023527) through the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT and the Korea Health Technology R&D Project (grant no. HI13C21810301) through the Korea Health Industry Development Institute (HHIDI) funded by the Ministry of Health & Welfare, Republic of Korea.

ABBREVIATIONS

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TME, tumor microenvironment; SMDC, small molecule drug conjugate; ROS, reactive oxygen species; PEG-BR, polyethylene glycol-bilirubin; BRNPs, bilirubin nanoparticles; TEM, transmission electron microscopy; AAPH, 2,2’-azobis(2-amidinopropane) dihydrochloride; TLC, thin layer chromatography; MALDI-TOF, matrix-assisted laser desorption/ionization-time Of flight; NMR, nuclear magnetic resonance.

REFERENCES This article references 30 other publications. (1) Vergote, I.; Leamon, C. P. Vintafolide: A Novel Targeted Therapy for the Treatment of Folate Receptor Expressing Tumors. Ther Adv Med Oncol 2015, 7 (4), 206-218. (2) Okamatsu, A.; Motoyama, K.; Onodera, R.; Higashi, T.; Koshigoe, T.; Shimada, Y.; Hattori, K.; Takeuchi, T.; Arima, H. Design and Evaluation of Folate-Appended Alpha-, Beta-, and Gamma-Cyclodextrins Having a Caproic Acid as a Tumor Selective Antitumor Drug Carrier in Vitro and in Vivo. Biomacromolecules 2013, 14 (12), 4420-4428. (3) Huang, B.; Otis, J.; Joice, M.; Kotlyar, A.; Thomas, T. P. Psma-Targeted Stably Linked "Dendrimer-Glutamate Urea-Methotrexate" as a Prostate Cancer Therapeutic. Biomacromolecules 2014, 15 (3), 915-923. (4) Fuchs, A. V.; Tse, B. W.; Pearce, A. K.; Yeh, M. C.; Fletcher, N. L.; Huang, S. S.; Heston, W. D.; Whittaker, A. K.; Russell, P. J.; Thurecht, K. J. Evaluation of Polymeric Nanomedicines Targeted to Psma: Effect of Ligand on Targeting Efficiency. Biomacromolecules 2015, 16 (10), 3235-3247. (5) Kumar, A.; Mastren, T.; Wang, B.; Hsieh, J. T.; Hao, G.; Sun, X. Design of a SmallMolecule Drug Conjugate for Prostate Cancer Targeted Theranostics. Bioconjug Chem 2016, 27 (7), 1681-1689. (6) Chari, R. V.; Miller, M. L.; Widdison, W. C. Antibody-Drug Conjugates: An Emerging Concept in Cancer Therapy. Angew Chem Int Ed Engl 2014, 53 (15), 3796-3827. (7) Liu, Z.; Xiong, M.; Gong, J.; Zhang, Y.; Bai, N.; Luo, Y.; Li, L.; Wei, Y.; Liu, Y.; Tan, X.; Xiang, R. Legumain Protease-Activated Tat-Liposome Cargo for Targeting Tumours and Their Microenvironment. Nat Commun 2014, 5, 4280. (8) Singh, S. R.; Rameshwar, P.; Siegel, P. Targeting Tumor Microenvironment in Cancer Therapy. Cancer Lett 2016, 380 (1), 203-204. (9) Shi, J.; Kantoff, P. W.; Wooster, R.; Farokhzad, O. C. Cancer Nanomedicine: Progress, Challenges and Opportunities. Nat Rev Cancer 2017, 17 (1), 20-37. (10) Khandrika, L.; Kumar, B.; Koul, S.; Maroni, P.; Koul, H. K. Oxidative Stress in Prostate Cancer. Cancer Lett 2009, 282 (2), 125-136. (11) Trachootham, D.; Alexandre, J.; Huang, P. Targeting Cancer Cells by Ros-Mediated Mechanisms: A Radical Therapeutic Approach? Nat Rev Drug Discov 2009, 8 (7), 579-591.

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(12) Shim, M. S.; Xia, Y. A Reactive Oxygen Species (Ros)-Responsive Polymer for Safe, Efficient, and Targeted Gene Delivery in Cancer Cells. Angew Chem Int Ed Engl 2013, 52 (27), 6926-6929. (13) Lee, Y.; Kim, H.; Kang, S.; Lee, J.; Park, J.; Jon, S. Bilirubin Nanoparticles as a Nanomedicine for Anti-Inflammation Therapy. Angew Chem Int Ed Engl 2016, 55 (26), 74607463. (14) Kim, J. Y.; Lee, D. Y.; Kang, S.; Miao, W.; Kim, H.; Lee, Y.; Jon, S. Bilirubin Nanoparticle Preconditioning Protects against Hepatic Ischemia-Reperfusion Injury. Biomaterials 2017, 133 (Supplement C), 1-10. (15) Kim, D. E.; Lee, Y.; Kim, M.; Lee, S.; Jon, S.; Lee, S. H. Bilirubin Nanoparticles Ameliorate Allergic Lung Inflammation in a Mouse Model of Asthma. Biomaterials 2017, 140, 37-44. (16) Kim, M. J.; Lee, Y.; Jon, S.; Lee, D. Y. Pegylated Bilirubin Nanoparticle as an AntiOxidative and Anti-Inflammatory Demulcent in Pancreatic Islet Xenotransplantation. Biomaterials 2017, 133, 242-252. (17) Lee, Y.; Lee, S.; Lee, D. Y.; Yu, B.; Miao, W.; Jon, S. Multistimuli-Responsive Bilirubin Nanoparticles for Anticancer Therapy. Angew Chem Int Ed Engl 2016, 55 (36), 1067610680. (18) Lee, D. Y.; Kim, J. Y.; Lee, Y.; Lee, S.; Miao, W.; Kim, H. S.; Min, J. J.; Jon, S. Black Pigment Gallstone Inspired Platinum-Chelated Bilirubin Nanoparticles for Combined Photoacoustic Imaging and Photothermal Therapy of Cancers. Angew Chem Int Ed Engl 2017, 56 (44), 13684-13688. (19) Moghimi, S. M.; Hunter, A. C.; Murray, J. C. Long-Circulating and Target-Specific Nanoparticles: Theory to Practice. Pharmacol Rev 2001, 53 (2), 283-318. (20) Zhao, H.; Rubio, B.; Sapra, P.; Wu, D.; Reddy, P.; Sai, P.; Martinez, A.; Gao, Y.; Lozanguiez, Y.; Longley, C.; Greenberger, L. M.; Horak, I. D. Novel Prodrugs of Sn38 Using Multiarm Poly(Ethylene Glycol) Linkers. Bioconjug Chem 2008, 19 (4), 849-859. (21) Liu, Y.; Piao, H.; Gao, Y.; Xu, C.; Tian, Y.; Wang, L.; Liu, J.; Tang, B.; Zou, M.; Cheng, G. Comparison of Two Self-Assembled Macromolecular Prodrug Micelles with Different Conjugate Positions of Sn38 for Enhancing Antitumor Activity. Int J Nanomedicine 2015, 10, 2295-2311. (22) Xie, J.; Zhang, X.; Teng, M.; Yu, B.; Yang, S.; Lee, R. J.; Teng, L. Synthesis, Characterization, and Evaluation of Mpeg-Sn38 and Mpeg-Pla-Sn38 Micelles for Cancer Therapy. Int J Nanomedicine 2016, 11, 1677-1686. (23) Laidler, P.; Dulinska, J.; Lekka, M.; Lekki, J. Expression of Prostate Specific Membrane Antigen in Androgen-Independent Prostate Cancer Cell Line Pc-3. Arch Biochem Biophys 2005, 435 (1), 1-14. (24) Zhang, H.; Liu, X.; Wu, F.; Qin, F.; Feng, P.; Xu, T.; Li, X.; Yang, L. A Novel ProstateSpecific Membrane-Antigen (Psma) Targeted Micelle-Encapsulating Wogonin Inhibits Prostate Cancer Cell Proliferation Via Inducing Intrinsic Apoptotic Pathway. Int J Mol Sci 2016, 17 (5). (25) Van Rensburg, C. E.; Slabbert, J. P.; Bohm, L. Influence of Irinotecan and Sn-38 on the Irradiation Response of Who3 Human Oesophageal Tumour Cells under Hypoxic Conditions. Anticancer Res 2006, 26 (1A), 389-393. (26) Ramesh, M.; Ahlawat, P.; Srinivas, N. R. Irinotecan and Its Active Metabolite, Sn-38: Review of Bioanalytical Methods and Recent Update from Clinical Pharmacology Perspectives. Biomed Chromatogr 2010, 24 (1), 104-123.

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(27) Uy, B.; McGlashan, S. R.; Shaikh, S. B. Measurement of Reactive Oxygen Species in the Culture Media Using Acridan Lumigen Ps-3 Assay. J Biomol Tech 2011, 22 (3), 95-107. (28) Qaisiya, M.; Coda Zabetta, C. D.; Bellarosa, C.; Tiribelli, C. Bilirubin Mediated Oxidative Stress Involves Antioxidant Response Activation Via Nrf2 Pathway. Cell Signal 2014, 26 (3), 512-520. (29) Jansen, T.; Daiber, A. Direct Antioxidant Properties of Bilirubin and Biliverdin. Is There a Role for Biliverdin Reductase? Front Pharmacol 2012, 3, 30. (30) Kim, H.; Lee, Y.; Kang, S.; Choi, M.; Lee, S.; Kim, S.; Gujrati, V.; Kim, J.; Jon, S. SelfAssembled Nanoparticles Comprising Aptide-Sn38 Conjugates for Use in Targeted Cancer Therapy. Nanotechnology 2016, 27 (48), 48LT01.

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