Enhancing the Efficacy and Safety of Doxorubicin against

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Enhancing the Efficacy and Safety of Doxorubicin against Hepatocellular Carcinoma through a Modular Assembly Approach: the Combination of Polymeric Prodrug Design, Nanoparticle Encapsulation, and Cancer Cell-Specific Drug Targeting Li Xu, Shengjun Xu, Hangxiang Wang, Jun Zhang, Zun Chen, Linhui Pan, Jianguo Wang, Xuyong Wei, Hai-Yang Xie, Lin Zhou, Shusen Zheng, and Xiao Xu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b14496 • Publication Date (Web): 09 Jan 2018 Downloaded from http://pubs.acs.org on January 9, 2018

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ACS Applied Materials & Interfaces

Enhancing the Efficacy and Safety of Doxorubicin against Hepatocellular Carcinoma through a Modular Assembly Approach: the Combination of Polymeric Prodrug Design, Nanoparticle Encapsulation, and Cancer Cell-Specific Drug Targeting Li Xu, Shengjun Xu, Hangxiang Wang,* Jun Zhang, Zun Chen, Linhui Pan, Jianguo Wang, Xuyong Wei, Haiyang Xie, Lin Zhou, Shusen Zheng, and Xiao Xu,* Department of Hepatobiliary and Pancreatic Surgery, The First Affiliated Hospital; Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases; Key Laboratory of Combined Multi-Organ Transplantation, Ministry of Public Health, School of Medicine, Zhejiang University, Hangzhou, 310003, PR China. KEYWORDS. Doxorubicin prodrug, polylactide, tumor-specific targeting, modular assembly, cancer chemotherapy

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ABSTRACT. Intervention is urgently required to improve the therapeutic outcome for patients with unresectable hepatocellular carcinomas (HCCs). However, current chemotherapeutics, such as sorafenib and doxorubicin (DOX), provide only limited therapeutic benefits for this devastating disease. In this context, we present a modular assembly approach to the construction of a systemically injectable nanotherapeutic that can efficiently and safely deliver DOX in vivo. To achieve this goal, we covalently attached DOX to a polylactide (PLA) building block (Mw = 2600, n = 36), yielding DOX-PLA conjugate 1. Due to the lipophilicity imparted by PLA, the conjugate 1 co-assembled with an amphiphilic lipid, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N[methoxy (polyethylene glycol) 2000] (DSPE-PEG2000), to form nanoparticles (NPs). To achieve preferential tumor accumulation, we additionally decorated the particle surface with an HCCspecific peptide moiety (i.e., SP94). The resulting HCC-targetable DOX-encapsulating NPs (termed tNP-PLA-DOX) exhibited several unique characteristics, including the feasible fabrication of sub-100 nm NPs, substantially delayed drug release profiles of several weeks, HCC cell-specific uptake and tumor accumulation in an in vivo mouse model, as well as alleviated drug toxicity in animals. Collectively, these results show that the integration of multiple components within a single nanocarrier via modular assembly is cost-effective for the creation of safe anticancer nanotherapeutics. The presented DOX-based nanomedicines have potential for enhancing the therapeutic index in patients.


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INTRODUCTION Hepatocellular carcinoma (HCC), one of the most common malignant tumors, is the second major cause of death among all cancers.1 At present, the overall median survival for patients with advanced HCC is less than 6 months.2 Because there is a lack of early-stage specific symptoms, most patients are not diagnosed until they are in the advanced stages of HCC.3 For these patients with advanced HCC, systemic chemotherapy remains the exclusive choice for palliative treatment.4-5 The current standard-of-care therapeutic agent is sorafenib, an oral multikinase inhibitor; however, this inhibitor provides limited therapeutic outcomes, with evident drug resistance and poor tolerance in patients.6-8 Other chemotherapeutics, such as doxorubicin (DOX) or other combination therapies, have been applied in the treatment of HCC, but the outcomes are still far from satisfactory.9-11 Hence, the development of highly effective therapies for the management of this severe disease is urgently needed. Vectorization of therapeutic drugs in nanocarriers has been of particular interest for efficient cancer therapy due to the improved drug accumulation in tumors and therapeutic index, as well as the mitigated side effects in patients.12-16 In this context, versatile therapies have been formulated into various scaffolds for efficient drug transportation.17-20 However, the therapeutic agents, several of which have entered clinical trials, were frequently encapsulated in nanoparticles by noncovalent interactions.21 For example, Genexol-PM, a polymeric micelle formulation of paclitaxel formed from a biodegradable diblock copolymer of monomethoxy polyethylene glycolblock-poly(D,L-lactide) (mPEG-PLA), showed significant antitumor activity in various solid tumors, such as breast cancer and non-small-cell lung cancer;22-23 however, the intensified doses did not produce better therapeutic efficacy in patients.24 Drug payloads that are physically formulated within vehicles may undergo premature release from the nanocarriers upon systemic


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administration. Consequently, this in vivo behavior could lead to rapid clearance of the majority of the injected doses from the bloodstream and inadequate pharmacokinetic profiles, thereby failing to exploit the enhanced permeability and retention (EPR) effect of the encapsulated therapeutic agent. Prior clinical data have suggested that substantial re-engineering of the current delivery systems is required to deliver a sufficient therapeutic dose to tumor sites. The prodrug strategy can dramatically modulate the physicochemical properties of the parent drug and thereby enhance the compatibility between the drug and the delivery matrix upon encapsulation.25-29 Several groups, including ours, have demonstrated that drug reform strategies can be exploited to construct lipophilic prodrugs by means of directly attaching chemotherapeutics to appropriate hydrophobic modifiers. The augmented association between the prodrug and matrix endows the encapsulated drug with favorable stability and long-term retention within the nanocarrier as well as improved pharmacokinetics upon systemic administration.13, 30 Here, we demonstrate that a combinatorial strategy of rational prodrug design, nanoparticle formulation and cancer cell-specific targeting can be used to construct efficient and safe drug delivery systems (Figure 1A). To validate this concept, we used DOX, a model anticancer drug for the treatment of HCC or other solid tumors,31-34 to generate a DOX-derived polymeric prodrug by tethering DOX to a biodegradable polylactic acid (PLA) segment (Figure S1). The resulting prodrug (PLA-DOX) was amenable to co-assembly with an amphiphilic lipid, distearoyl phosphoethanolamine polyethylene glycol (DSPE-PEG2000), to form systemically injectable nanomedicines. To further install a tumor targeting capacity, we decorated the particle surface with an HCC-specific homing ligand (i.e., a nine-residue peptide, SP94) via a maleimide-thiol reaction (Figure S2 and Figure 1B). The resulting nanodrug exhibited several unique characteristics, including high drug loading, colloidal stability, sustained drug release, and efficient drug


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accumulation in the target tumor sites, as well as lower in vivo drug toxicity than its free form. Because HCC is characterized as a typical hyper-vascular tumor, we envision that nanotherapeutics with finely tailored properties could possess enhanced in vivo performance for HCC treatment by exploitation of the EPR effect.35


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Figure 1. (A) Schematic illustration of the self-assembly of the DOX prodrug (PLA-DOX) into single polymeric nanoparticles (NPs) that can release DOX in tumor sites via the enhanced permeability and retention (EPR) effect. (B) Chemical structure and synthetic scheme of PLADOX and DSPE-PEG-SP94, and schematic diagram of the non-targeting, PLA-DOXencapsulating nanoparticles (nNP-PLA-DOX) and the analogous SP94-tethered, targeting nanoparticles (tNP-PLA-DOX).

MATERIALS AND METHODS Materials. DOX was purchased from J&K Scientific (Shanghai, China). Carboxy-terminated polylactides were customized by Advanced Polymer Materials Inc. (Montreal, Canada). 1,2Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol) 2000] (DSPEPEG2000) and maleimide-functionalized DSPE-PEG2000 (termed DSPE-PEG2000-Mal) were purchased from A.V.T. Pharmaceutical Co., Ltd. (Shanghai, China). The peptide SP94 (sequence: CGGSFSIIHTPILPL) was customized by GL Biochem Ltd. (Shanghai, China). All other compounds and solvents were purchased from J&K Scientific (Shanghai, China). Cell Lines and Cell Culture. All human cell lines were purchased from Cell Bank of China Science (Shanghai, China). HCC-LM3 cells were cultured in Eagle’s minimum essential media (MEM) with 10% fetal bovine serum (FBS) and 0.1% antibiotics, while BEL-7402, HL-7702 and NCI-H1299 cells were cultured in RPMI-1640 with 10% FBS and 0.1% antibiotics. All cells were maintained at 37ºC in a humidified atmosphere with 5% CO2. Synthesis of PLA-tethered DOX Prodrug 1. To a solution of PLA (n = 36)-succinic acid (1 g, 0.356 mmol) and DOX (229.4 mg, 0.392 mmol) in 20 mL of anhydrous dichloromethane (DCM) was added 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) (84.4 mg, 0.533 mmol). The reaction mixture was stirred at 50ºC for 4 h and then washed with 5% citric acid, saturated NaHCO3


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and brine. The organic layer was dried over anhydrous Na2SO4, filtered, and evaporated under vacuum. The residue was purified by flash column chromatography on silica gel (DCM:MeOH = 100:1) to give prodrug conjugate 1 (593.7 mg, 49.4%). (1H NMR (500 MHz, CDCl3) δ 8.08 (d, 1H, J = 7.1 Hz), 8.05 (s, 1H), 7.86–7.82 (m, 1H), 7.44 (d, 1H, J = 8.1 Hz), 5.44–5.35 (m, 2H), 5.20 (q, 36H, J = 7.1 Hz), 4.83 (d, 2H, J = 15.3 Hz), 4.35–4.29 (m, 2H), 4.20 (t, 1H, J = 6.7 Hz), 4.13 (s, 3H), 4.12 (s, 1H), 3.67 (q, 7H, J = 3.3 Hz), 3.59–3.57 (m, 2H), 3.41 (s, 3H), 3.31 (dd, 2H, J = 18.5, 2.2 Hz), 3.03 (d, 1H, J = 12.3 Hz), 2.56–2.49 (m, 2H), 2.48–2.41 (m, 2H), 2.21 (dd, 2H, J = 14.7, 4.9 Hz), 1.75 (s, 2H), 1.62 (t, 108H, J = 6.1 Hz), 1.29 (s, 3H)). Preparation of SP94-modified Lipid DSPE-PEG2000 (DSPE-PEG2000-SP94). SP94-modified DSPE-PEG2000 was prepared by a coupling reaction between the cysteine residue in SP94 and a maleimide group at the PEG terminus of DSPE-PEG2000-Mal. The molar ratio of SP94 to DSPEPEG2000-Mal was fixed at 1:1. Briefly, predetermined amounts of SP94 and DSPE-PEG2000-Mal (20 mg) were dissolved in 2 mL of dimethylsulfoxide (DMSO) and stirred overnight at room temperature. The completion of the reaction was confirmed by analytical high-performance liquid chromatography (HPLC). The reaction mixture was lyophilized and used for the next experiments without purification. Preparation

of

PLA-DOX

Prodrug-loaded

Nanoparticles

(NP-PLA-DOX).

A

nanoprecipitation method was used to construct the DOX prodrug-loaded polymeric nanoparticles. For the preparation of the non-targeting PLA-DOX-encapsulating NPs (nNP-PLA-DOX), the molar ratio of the prodrug (at a DOX equivalent) to the matrix (only DSPE-PEG2000) was fixed at 2:1, whereas in the preparation of the analogous SP94-tethered, targeting nanoparticles (tNP-PLADOX), the mole ratio of drug agent (at a DOX equivalent) to the two matrices (DSPE-PEG2000 and DSPE-PEG2000-SP94) was fixed at 4:1:1. Briefly, predetermined amounts of DSPE-PEG2000-SP94


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and/or DSPE-PEG2000 and the PLA-DOX prodrug (2 mg, at a DOX equivalence) were dissolved in 1 mL of acetone and added dropwise to 8 mL of water while stirring, which gave a final drug concentration of 0.2 mg/mL. After stirring for 30 min, the remaining organic solvent was removed by rotary evaporation at low pressure. The nanoparticle solution was concentrated with a centrifugal filter device (Amicon Ultra-4, 10k MWCO, Millipore Corp.), and the resulting residue was washed with deionized water. Determination of Encapsulation Efficiency and Drug Loading. The drug loading percentages were determined by ultraviolet spectrophotometry. Briefly, lyophilized NP-PLA-DOX were dissolved in acetonitrile, and NaOH was added to the NP solutions. The mixtures were then stirred for 30 min at 37°C to release the DOX molecules. The supernatants were collected after centrifugation of the suspensions, and the DOX contents were quantitatively determined using a UV-Vis spectrometer (Shimadzu, UV-2700) at 488 nm. The encapsulation efficiency (EE) percentages and drug loadings (DL) of DOX in the NPs were calculated via Equations (1) and (2): (1) EE (%) = Wdrug in NP / Winitial drug added × 100% (2) DL (%) = Wdrug / (Wdrug + WDSPE-PEG2000) or Wdrug / (Wdrug + WDSPE-PEG2000 + WDSPE-PEG2000SP94)

× 100%

Characterization of Particle Size by Dynamic Light Scattering (DLS). The hydrodynamic diameters of the NP-PLA-DOX systems were measured through DLS on a Malvern Nano-ZS90 instrument (Malvern, UK) at 25°C, and each sample was tested three times. Particle Morphology Study by Transmission Electron Microscopy (TEM) and scanning electron microscopy (SEM). To characterize the morphologies of the NP-PLA-DOX systems, TEM analysis was performed on a TECNAL 10 instrument (Philips) at an acceleration voltage of 80 kV. A droplet of NP-PLA-DOX at an appropriate concentration was placed on a 400-mesh


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copper grid coated with carbon. Positive staining was performed with a 2 wt% aqueous uranyl acetate solution after the surface of carbon was completely dry. The morphology of nanoparticles was also observed in a Hitachi SU-8010 Cold Field Emission Scanning Electron Microscope. In Vitro DOX Release Kinetics Study. A DOX release kinetics study was carried out with the dialysis method to quantify the drug release behavior. NP-PLA-DOX and NP-DOX solutions with a 0.1 mg/mL DOX equivalent concentration were loaded into dialysis bags and dialyzed against phosphate buffer solutions (PBS, pH 7.4) with 0.2% Tween 80. The dialysis bags were continuously and gently shaken in an orbital shaking water bath at 37°C, and the release media were collected and supplemented with fresh media at predetermined time intervals. The amounts of released DOX were analyzed using a UV-Vis spectrometer (Shimadzu, UV-2700) at 488 nm. In Vitro Cytotoxicity Study by Cell Counting Kit-8 (CCK-8). The in vitro cytotoxicity of the NP-PLA-DOX systems was measured by Cell Counting Kit-8 (CCK-8). Cells were seeded into flat-bottomed 96-well plates with 4000-5000 cells per well and incubated at 37°C under a 5% CO2 atmosphere for 24 h. The cells were treated with serial dilutions of free DOX, nNP-PLA-DOX and tNP-PLA-DOX and then incubated at 37°C for an additional 72 h. At the end of the treatment, 10 μL of the CCK-8 solution was added to each well. After 2 h, the absorbance at 450 nm was measured with a microplate reader (Multiskan FC, Thermo Scientific). Cell Proliferation Study by the EdU Test. HCC-LM3 and BEL-7402 cells were seeded into flat-bottomed 48-well plates with 2×104 cells per well and incubated at 37°C under a 5% CO2 atmosphere for 24 h. Free DOX, nNP-PLA-DOX and tNP-PLA-DOX (5 μmol/L) were then added to the cells and incubated for an additional 24 h at 37°C. DNA synthesis was quantified at the end of the drug treatment by a Click-iT® EdU Alexa Fluor® 488 Assay Kit (Invitrogen) according to the manufacturer’s protocol. Briefly, EdU (5-ethynyl-2’-deoxyuridine) was first added to each well


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and incubated for 2 h at 37°C. Then, the cells were fixed for 15 min at room temperature by adding 4% formaldehyde. Next, 0.5% Triton X-100 was added to the cells and incubated for 10 min. After that, azide-labeled Alexa Fluor® 488 was added to the cells and incubated for 30 min in the dark. After staining the nuclei with Hoechst 33342 (Invitrogen) for 15 min, the cells were imaged using fluorescence microscopy (Olympus, IX71). In Vivo and Ex Vivo Imaging Study. Balb/c nude mice (5-week-old) were purchased from the Shanghai Experimental Animal Center, Chinese Academy of Science. The mice were maintained under the guidelines of the National Institute Guide for the Care and Use of Laboratory Animals. All of the animal studies were approved by the Institutional Animal Care and Use Committee of Zhejiang University. To evaluate the tumor targeting ability of the nanodrugs, a mouse model bearing an HCC-LM3 cell-derived xenograft was established by subcutaneous injection of 5×106 HCC-LM3 cells in the right flank of mice. When the tumor volume reached ~150 mm3, the mice were randomly divided into three groups (n = 3 in each group). A near-infrared (NIR) fluorescence probe, DiR, was coassembled into the NP-PLA-DOX systems (termed DiR@NP-PLA-DOX) to track the in vivo distribution of the nanodrugs. The mice were injected with free DiR, DiR@nNP-PLA-DOX and DiR@tNP-PLA-DOX at a DiR dose of 20 μg per mouse via the tail vein. In vivo imaging was performed on an in vivo imaging system (Clairvivo OPT, SHIMADZU Corporation, Kyoto, Japan) at 1 h, 4 h, 8 h and 24 h post-injection. At 24 h, the mice were sacrificed, and the organs were collected for ex vivo imaging. In Vivo Antitumor Study in a Mouse Model Bearing an HCC-LM3 Cell-derived Xenograft. Cell suspensions containing 5×106 HCC-LM3 cells were subcutaneously injected to the right flank of 5-week-old Balb/c nude mice to establish an HCC-LM3 tumor xenograft-bearing mice model.


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When the tumor volume reached ~150 mm3, mice were randomly divided into four groups (n = 5 in each group). The mice were intravenously injected with saline, free DOX, and the solutions containing nNP-PLA-DOX and tNP-PLA-DOX. The tumor growth and body weight were monitored. The tumor volume was evaluated by measuring the length (L) and width (W) with calipers and calculated as V = (L × W2)/2. On day 21 post-treatment, the mice were sacrificed, and the tumor tissues and other major organs were collected. After being fixed with 4% formaldehyde, the tissues were embedded in paraffin and cut into 5 μm slices. These slices were then stained with hematoxylin and eosin (H&E, Sigma) for histological analysis. Statistical Analysis. All quantitative data are shown as the mean ± SD. Student’s t-test was used to assess the statistical significance. Statistical significance was defined as a p-value below 0.05, and high significance was defined as a p-value below 0.01.

RESULTS AND DISCUSSION Rational Design of a DOX Prodrug and the Construction of SP94 Peptide-Decorated Nanoparticles. PLA is a well-studied biodegradable material that has been widely used to create various nanoparticulate drug delivery systems. Prior studies have demonstrated that PLA provides a useful class of skeletons for chemical drug derivatization.36-37 Motivated by these studies, we conjugated a PLA segment to DOX through an amide bond. In the presence of EDC and dimethylaminopyridine (DMAP), carboxyl-terminated PLA (Mw = 2800, n = 36) was condensed with the anticancer agent DOX in DCM. The resulting reaction mixture was purified by silica gel chromatography to obtain the product in high yield (52.1% ± 5.8% from three independent experiments). 1H and 13C NMR spectra unambiguously confirmed the successful synthesis of the desired PLA-DOX conjugate (Figure 2 and Figure S3). In addition, the 1H NMR results indicated


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that nearly one DOX molecule was conjugated to each PLA segment. The PLA fragment made the overall PLA-DOX construct lipophilic, providing the opportunity for the system to be integrated into the hydrophobic core of polymeric micelles. To improve the pharmacokinetic properties of this prodrug, DSPE-PEG2000 was used as an amphiphilic lipid to cloak the prodrug-assembled nanostructures. This lipid is readily miscible with a diverse variety of hydrophobic drugs and has been widely exploited as a synthetic drug delivery matrix that protects the vehicle from phagocytosis and thereby prolongs drug circulation in the blood. To facilitate HCC tumor-specific drug targeting, we attached an SP94 peptide ligand (peptide sequence: SFSIIHTPILPL; characterization of SP94 peptide was shown in Figure S4 and Figure S5) to the particle surface. SP94 was originally identified by the phage display technique to show an affinity toward unconfirmed receptor(s) expressed on HCC cells.38 The enhanced delivery of a drug payload into HCC cells by decoration with the SP94 peptide was demonstrated by several groups, including ours.21, 39-40 To achieve surface modification with the SP94 ligand, the thiolated peptide was conjugated to maleimide-terminated DSPE-PEG2000 via a thiol-maleimide coupling reaction. The formation of a covalent adduct (termed DSPE-PEG2000-SP94) following the reaction between a 1:1 molar ratio of the SP94 peptide and DSPE-PEG2000-Mal at room temperature for 12 h was verified by HPLC analysis (Figure S6 and Table S1) and real-time 1H NMR spectra (Figure S7). Especially, the singlet peak at 7.01 ppm 1H NMR spectra that can be assigned to the proton of maleimide group was clearly observed. However, after 12-h reaction, this peak disappeared, indicating the completion of thiol-maleimide coupling. All data suggest that SP94 ligand was successfully coupled to DSPE-PEG2000 lipid.


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With these three components (i.e., PLA-DOX prodrug, DSPE-PEG2000, and DSPE-PEG2000SP94) in hand, we then utilized a nanoprecipitation method to construct an HCC-targeting nanodevice containing DOX (termed tNP-PLA-DOX) by co-assembly. In addition, the analogous non-targeting nanodevice composed of PLA-DOX and DSPE-PEG2000 was also readily prepared (termed nNP-PLA-DOX). The EE and DL values for nNP-PLA-DOX were 84.98% ± 1.62% and 10.35% ± 0.15%, respectively, whereas the EE and DL values for tNP-PLA-DOX were 88.77% ± 3.79% and 9.95% ± 0.42%, respectively. Characterization of the NP-PLA-DOX Systems. The primary properties of nNP-PLA-DOX and tNP-PLA-DOX, such as the hydrodynamic diameter (DH), zeta potential and polydispersity index (PDI), are presented in Table 1 and Figure 3A and B. The DLS analysis revealed that the mean DH of nNP-PLA-DOX was 122.6 ± 3.8 nm and the PDI was 0.091 ± 0.017, while the mean DH of tNP-PLA-DOX was 75.3 ± 9.6 nm and the PDI was 0.150 ± 0.011. Furthermore, TEMbased morphology studies (Figure 3A and B) and SEM-based morphology studies (Figure 3C and D) showed that both nanomedicines exhibited monodispersed and well-defined spherical nanostructures. Prior studies have demonstrated that the EPR effect remarkably influences the penetration of nanoparticles into solid tumors since the tumor vascular endothelium has a larger barrier gap than a normal vascular endothelium.41-43 The size of our nanoparticles were in the range suitable for accumulation in tumor sites (below 200 nm) according to the traditional concept of the EPR effect. The long-term stabilities of the nanomedicines were assessed over 9 days by storing the corresponding NP solutions at room temperature. No obvious variation in the particle size was observed for either nanomedicine during the DLS analysis (Figure 3C), suggesting an excellent stability and long-term storage ability for future clinical applications.


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Figure 2. 1H NMR characterization of the PLA-DOX prodrug conjugate. Each number designates different protons in the PLA-DOX prodrug conjugate. The 1H NMR spectrum was measured in deuterated DMSO.


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Figure 3. Morphology image and hydrodynamic diameter distribution (DH) determined by transmission electron microscopy (TEM), scanning electron microscopy (SEM) and dynamic light scattering (DLS). (A) TEM image and DH data, and (C) SEM image for nNP-PLA-DOX. (B) TEM image and DH data, and (D) SEM image for tNP-PLA-DOX. (E) Diameter changes of nNP-PLADOX and tNP-PLA-DOX over the course of 9 days, indicating the stability of the NPs. (F) In vitro accumulative release profiles of DOX from NP-DOX, nNP-PLA-DOX and tNP-PLA-DOX


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determined by dialysis against PBS (pH 7.4) at 37°C over a 20-day period. The data are presented as the mean ± SD (n = 3). Table 1. Zeta Potentials and Sizes of the Drug-Loaded Nanoparticles

[a]

Size (nm)

PDI[a]

Material

Zeta Potential (mV)

nNP-PLA-DOX

-14.83 ± 0.61

122.6 ± 3.8 0.091 ± 0.017

tNP-PLA-DOX

-3.10 ± 1.51

75.3 ± 9.6

0.150 ± 0.011

PDI: polydispersity index Kinetics of DOX Release from the Drug-loaded Nanoparticles. We next evaluated the drug

release kinetics to validate whether the covalent conjugation constrained DOX within the nanoparticle core. This behavior could inhibit the premature release of the active chemotherapeutic from the nanoparticle reservoir upon systemic circulation, ultimately enhancing the accumulation of the active drug in tumor sites. Previous reports indicated that the parent DOX agent can assemble with DSPE-PEG2000 lipid to form nanoparticles.33 Therefore, we prepared a DSPEPEG2000 micelle that physically encapsulated the parent DOX agent as a reference. The in vitro release of DOX from the NPs was performed by dialysis against PBS buffer (pH 7.4) at 37°C, and the amounts of released DOX agents were determined by UV spectrophotometry (Figure S8). The DSPE-PEG2000 micelles that encapsulated the parent DOX showed a quick drug release profile with over 75.38 ± 2.96% of the total DOX liberated within 24 h (Figure 3D). By sharp contrast, only negligible amounts of DOX were released from nNP-PLA-DOX and tNP-PLA-DOX after one day. After 20 days, the amount of the total DOX released increased to 30.09 ± 2.53% and 32.55 ± 0.48% for nNP-PLA-DOX and tNP-PLA-DOX, respectively. These data clearly suggest that NP-PLA-DOX systems possessed a remarkably sustained release property, preventing the premature release of the DOX anticancer drug before reaching the tumor region. Thus, we envision


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that both NP-PLA-DOX systems might be able to accumulate a larger therapeutic dose in tumors than simple physical encapsulation of the parent DOX in DSPE-PEG2000 micelles. Cell-Specific Uptake of Nanoparticles. The cell-specific uptake of the drug-loaded nanoparticles was evaluated in various cells using confocal laser scanning microscopy (CLSM). Since the SP94 ligand possess affinity toward the overexpressed unidentified receptors on the HCC cell surface, we initially tested whether the decoration of the nanoparticles with SP94 enhanced the uptake in HCC cells. Both HCC cells (i.e., HCC-LM3 and BEL-7402 cells) were treated with nNP-PLA-DOX and tNP-PLA-DOX and subjected to CLSM observation at time points of 1, 3, and 6 h post-treatment. As shown in Figure 4A and C, strong red fluorescence derived from DOX was observed in both the HCC-LM3 and BEL-7402 cells following treatment with tNP-PLA-DOX. However, only a negligible fluorescence signal was found at all time points in the cells treated with nNP-PLA-DOX. To validate whether the fluorescence signal was derived from nanoparticleformulated DOX, we additionally performed CLSM images in HCC-LM3 cells after incubation with tNP-PLA-DOX and free DOX. As shown in Figure S9, we found that the red fluorescence was averagely distributed in the cytosol and most of them rapidly diffused into nucleus after free DOX treatment. However, for nanoparticles, the fluorescence signal exhibited point-like distribution in the cytosol and negligible signal was observed inside nucleus. This kind of distribution is typical for nanoparticles when they are internalized by cells through the endocytosis/lysosome pathway. These results indicate that DOX agents are still constrained in nanoparticles upon internalization. Next, we used flow cytometry to determine whether the cellular uptake of DOX could be augmented by the use of targetable nanocarriers. Consistently, both the HCC-LM3 and BEL-7402 cells exhibited time-dependent uptake of the nanodrugs, as evidenced by the flow cytometry analysis (Figure 4B and D). More importantly, the targetable NPs (tNP-


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PLA-DOX) displayed superior drug accumulation compared to the non-targetable NPs (nNP-PLADOX) in both HCC cells. To demonstrate the cell-specificity, we further included a normal liver cell line, HL-7702, and a lung cancer cell line, NCI-H1299, as references. As shown in Figure 4E and F, we interestingly found that the difference in the cellular drug accumulation between nNP-PLA-DOX and tNP-PLADOX was abolished in these two non-HCC cells, as evidenced by the flow cytometry analysis. This result indicates that the HCC-specific peptide, SP94, was not capable of targeting normal liver cells or other types of cancer cells, such as the lung cancer cells NCI-H1299. Therefore, we concluded that the present HCC-specific nanodrugs effectively accumulate in the target HCC tumors, while sparing the normal tissues. Ultimately, this active targeting effect could reduce the in vivo toxicity of cancer drugs.


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Figure 4. Cellular uptake of nNP-PLA-DOX and tNP-PLA-DOX in HCC-LM3 (A) and BEL7402 (C) cells at several time points, as shown by confocal laser scanning microscopy (CLSM). Flow cytometry analysis of (B) HCC-LM3, (D) BEL-7402, (E) HL-7702 and (F) NCI-H1299 cells. The cells were incubated with nNP-PLA-DOX (brown) and tNP-PLA-DOX (green) at a DOX equivalent concentration of 10 μM.


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In Vitro Cytotoxicity against HCC Cells. We next assessed the cytotoxicity of free DOX, nNPPLA-DOX and tNP-PLA-DOX against HCC-derived cancer cells using a CCK-8 assay. Human HCC-LM3 and BEL-7402 cells were exposed to different concentrations of drugs for 72 h, and the cell viability was quantified by CCK-8. The concentrations required to inhibit 50% of the cells (IC50) were extrapolated from the dose response curves. The results are shown in Figure 5A and Table 2. We observed a higher cytotoxicity for tNP-PLA-DOX than for nNP-PLA-DOX in both distinct HCC cell lines, indicating that the attachment of the SP94 ligand augmented the potency of the NPs. For example, the IC50 values of tNP-PLA-DOX in HCC-LM3 and BEL-7402 cells were 6.571 ± 0.855 and 3.709±0.702 μM, respectively, whereas the IC50 values of nNP-PLA-DOX in HCC-LM3 and BEL-7402 cells were 9.283 ± 0.422 μM and 6.921 ± 0.841 μM, respectively. These results are consistent with the enhanced intracellular uptake of tNP-PLA-DOX in cells observed by confocal studies. Meanwhile, we found that both nanomedicines were less potent than the free form, which could be attributed to the markedly delayed release of the therapeutically active DOX agent from the assembled NPs.

Table 2. In vitro cytotoxicity after 72-h incubation with different drug formulations as determined by CCK-8 assay (expressed as IC50 ± SD) IC50 (μM) Treatment Group HCC-LM3

BEL-7402

Free DOX

0.231 ± 0.036

0.126 ± 0.013

nNP-PLA-DOX

9.283 ± 0.422

6.921 ± 0.841

tNP-PLA-DOX

6.571 ± 0.855** 3.729 ± 0.702**

**p < 0.01 versus nNP-PLA-DOX


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To verify whether HCC cell proliferation could be inhibited by the DOX encapsulated in the nanostructures, an EdU cell proliferation assay was performed in HCC cells. As illustrated in Figure 5B and C, cell proliferation was obviously inhibited by incubation with the DOX-loaded NPs. Notably, tNP-PLA-DOX showed a higher inhibitory effect on proliferation than nNP-PLADOX in both cells (p 3 regions; *p < 0.05, **p < 0.01, and ***p < 0.001. To further determine whether the targetable DOX nanodrug, tNP-PLA-DOX, was more potent than the non-targetable nNP-PLA-DOX to HCC, we designed a new in vitro cytotoxicity study process (Figure 6A). After incubation of the cells (HCC-LM3, BEL-7402 and NCI-H1299) with each nanodrug for 24 h, the cell culture media containing the drugs were replaced with fresh culture media. After an additional 72 h of culture, the cell viability was determined using a CCK-8 assay. We interestingly observed a higher anti-proliferation activity for tNP-PLA-DOX than for nNPPLA-DOX in both tested HCC cell lines (e.g., HCC-LM3 and BEL-7402) (Figure 6B and C). However, such a variation in cytotoxicity between the nanomedicines was not observed in the lung cancer cell line, NCI-H1299 (Figure 6D). This difference might result from the decoration of the tNP-PLA-DOX surface with SP94, which is an HCC-specific small peptide motif that was identified by phage display technology.38 Due to the high affinity of this ligand to HCC cells while sparing healthy tissues, SP94 has been used for the targeted delivery of various cargoes in HCC therapy.21 The cell surface receptors of SP94 remain unknown; however, in this study, we confirmed that the SP94-decorated NPs did not show selectivity toward lung cancer cells (i.e., NCI-H1299).


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Figure 6. (A) Schematic illustration of the in vitro cytotoxicity analysis of a 24-h culture with the different NPs and an additional 72-h culture with fresh medium. Cell viabilities in (B) HCC-LM3, (C) BEL-7402, (D) NCI-H1299 cells treated with 5 μM and 10 μM nNP-PLA-DOX and tNP-PLADOX for 24 h (with NPs) plus 72 h (with fresh medium). In Vivo Tumor Targeting and Nanoparticle Distribution. Inspired by the in vitro results, we subsequently investigated whether decoration of the particle surface with SP94 could give the NPs the ability to preferentially accumulate in HCC-LM3 tumors. To track the nanoparticles in vivo, we co-assembled a NIR probe (i.e., DiR) into the inner core of PLA-DOX.44 DiR is a lipophilic, NIR fluorescent cyanine dye, rendering its ability for membrane insertion, and has been widely used for cell labeling. This dye also can be exploited to label various nanomaterials for in vivo noninvasive imaging due to the NIR property with the maximum excitation and emission wavelength at 750 nm and 780 nm, respectively.45-46 Whole body fluorescence imaging was conducted following injection of solutions containing free DiR, DiR-loaded nNP-PLA-DOX (DiR@nNP-PLA-DOX) and tNP-PLA-DOX (DiR@tNP-PLA-DOX). As shown in Figure 7A, we


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observed a strong fluorescence signal derived from DiR in the tumor region in both groups of nanoparticle-injected mice. The distribution was observed by an in vivo imaging system 1 h, 4 h, 8 h, and 24 h after injection. The fluorescence signal of free DiR in the tumor site was much lower than that of DiR@nNP-PLA-DOX and DiR@tNP-PLA-DOX (Figure 7A), which implied excellent tumor site accumulation of the NPs. In contrast, the fluorescence signal in the mice that received free DiR quickly decayed after the early time points, while no accumulation in the tumors was observed.

Figure 7. (A) In vivo fluorescence images of the xenograft model of HCC-LM3 1 h, 4 h, 8 h, and 24 h after the injection of DiR, DiR@nNP-PLA-DOX and DiR@tNP-PLA-DOX via a tail vein. (B) Ex vivo fluorescence image of the heart, lungs, liver, spleen, kidneys and tumor of the xenograft


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model of HCC-LM3 24 h post-injection. (C) Average fluorescence signal of DiR in the excised organs after 24 h. *p < 0.05. The concentration of DiR was 0.1 mg/mL. (n = 3 in each group) After 24 h post-administration, the mice were sacrificed, and the major organs were excised for ex vivo imaging. The results showed that most of the DiR probe accumulated in the lungs and spleen in the mice treated with free DiR, whereas DiR was observed in the tumors in both groups of mice that received the nanodrugs (Figure 7B). We further quantified the fluorescence intensity in the organs and found that the mean fluorescence signal in the DiR@tNP-PLA-DOX-treated mouse tumors was significantly larger than that in the DiR@nNP-PLA-DOX-treated mouse tumors (Figure 7C). These results clearly confirmed the tumor targeting capability of the nanomedicines obtained through proper exploitation of the EPR effect, which was further augmented by the active targeting property endowed by the decoration with tumor-specific ligands (e.g., SP94 peptide). In Vivo Antitumor Efficacy. Finally, the in vivo therapeutic potential of these DOX prodrugintegrated nanoparticles was evaluated in comparison with free DOX in an HCC-LM3 xenograftbearing nude mice model (n = 5 per group). When the tumors reached a volume of ~150 mm3, solutions containing both nanodrugs and free DOX were intravenously injected at a DOX equivalent dose of 10 mg/kg three times. Compared to the group treated with free DOX, the tumor growth in the nanodrug-treated groups was significantly inhibited (Figure 8A). In particular, treatment with the targetable NPs resulted in more prominent tumor suppression than treatment with the non-targetable NPs. This therapeutic effect was further validated by determining the weights of representative tumor samples after excision from the mice at the end of the study (Figure 8B and C). For instance, the determined excised tumor weights (TWs) showed that free DOX (TW = 0.92 ± 0.16 g) produced a valid but limited therapeutic effect when compared to the


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saline group (TW = 1.28 ± 0.11 g, p < 0.05). To our delight, the weights of the tumors from mice that received tNP-PLA-DOX were significantly lower than those from mice that received nNPPLA-DOX (TW = 0.43 ± 0.16 versus 0.59 ± 0.18 g, p < 0.05). This therapeutic effect is consistent with the histological analysis of the tumor sections. H&E analysis revealed that the NP therapy, especially with tNP-PLA-DOX, induced intensive intratumor necrosis (Figure 9). Furthermore, to confirm whether polymeric prodrug strategy shows superiority compared with free-drugformulated nanomedicines, we additionally conducted the comparative therapeutic study in the same mice model bearing HCC-LM3 xenograft. As shown in Figure S10, the results suggest that PLA-DOX prodrug-encapsulated NPs (e.g., nNP-PLA-DOX) was more potent to inhibit tumor growth than the nanoparticles that physically encapsulate free DOX (e.g., NP-DOX), validating the rationale of our prodrug design and sequential nanoparticle formulation.


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Figure 8. (A) Tumor volume (mm3) and (B) tumor weight (g) of the xenograft model of HCCLM3 for different treatment groups (10 mg/kg DOX equivalent dose). (C) Representative tumors in each group at the end point of the study. (D) The average body weight of mice was used to evaluate the toxicity of the different treatments. The tumor volume, tumor weight and average body weight are described as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 (n = 5 in each group).


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Free DOX

nNP-PLA-DOX

tNP-PLA-DOX

Lung

Spleen

Liver

Heart

Tumor

Control

Kidney

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 9. Representative H&E staining of the tumor, heart, liver, lung, spleen and kidney from different treatment groups. The drug toxicity and side effects of free DOX, primarily its cardiotoxicity, greatly restricts its therapeutic outcomes and hampers its dose intensification in the clinic.47-48 We hypothesize that


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this combinatorial strategy of polymeric prodrug design, nanoparticle formulation, and tumor cellspecific drug delivery could substantially alleviate the drug toxicity in animals. To validate this assumption, we additionally performed histological analyses on the organs excised from mice that received the various formulations. The histological analysis of the hearts revealed that less cardiomyocyte injury was induced by the DOX-encapsulating NPs than by free DOX (Figure 9). Additionally, no anomalous changes in the other major organs were observed in the mice receiving nanoparticle therapy (Figure 9). More interestingly, as shown in Figure 8D, the body weight of the mice clearly decreased after receiving free DOX, whereas the NP therapy did not produce a change in body weight, reflecting the lower systematic toxicity of our approach.

CONCLUSIONS In summary, we have established a novel formulation to enhance the therapeutic potential of the DOX anticancer agent for HCC chemotherapy while alleviating its systemic toxicity and damage to normal organs. Our approach combined the rational design of a PLA-based DOX prodrug, subsequent nanoassembly with a biocompatible matrix (i.e., DSPE-PEG2000), and surface decoration of the particles with an HCC-specific ligand (i.e., SP94 peptide). The covalent linkage of the anticancer agent DOX to PLA facilitated extended drug retention in the NP reservoirs and thereby impeded the burst release of the drug payloads upon systemic circulation. In addition, the described targetable PLA-DOX-NPs possessed long-term stability and high selectivity toward cancer cells while leaving normal cells and tissues untouched. As a consequence, the accumulation of the therapeutics in the target tumor sites was augmented by proper exploitation of the EPR effect and accumulation of the active nanoparticle. Finally, as a result of the modular assembly strategy, the individual building blocks can be easily varied. We therefore envision that many traditional


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chemotherapeutics could be adapted into this nanoplatform to target other cancers and improve their therapeutic outcomes.

ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.XXXXXX. Synthetic scheme and characterization data for PLA-DOX conjugate 1 and DSPE-PEG2000-SP94. Tables showing the successful synthesis of DSPE-PEG2000-SP94. AUTHOR INFORMATION Corresponding Authors *Hangxiang Wang ([email protected]) and Xiao Xu ([email protected]) Funding Sources This work was supported by the National Natural Science Foundation of China (Grants 81571799, 81773193, 31671019, 81421062, 91542205 and 81625003) and the Major Science and Technology Project of Zhejiang Province (Grant 2014C03043-2). Notes The authors declare no potential conflicts of interest. REFERENCES (1) Torre, L. A.; Bray, F.; Siegel, R. L.; Ferlay, J.; Lortet-Tieulent, J.; Jemal, A. Global Cancer Statistics, 2012. CA Cancer J. Clin. 2015, 65, 87-108.


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Table of Contents


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Figure 1. (A) Schematic illustration of the self-assembly of the DOX prodrug (PLADOX) into single polymeric nanoparticles (NPs) that can release DOX in tumor sites via the enhanced permeability and retention (EPR) effect. (B) Chemical structure and synthetic scheme of PLA-DOX and DSPE-PEG-SP94, and schematic diagram of the non-targeting, PLA-DOX-encapsulating nanoparticles (nNP-PLA-DOX) and the analogous SP94-tethered, targeting nanoparticles (tNP-PLA-DOX).



Figure 2. 1H NMR characterization of the PLA-DOX prodrug conjugate. Each number designates different protons in the PLA-DOX prodrug conjugate. The 1H NMR spectrum was measured in deuterated DMSO.


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Figure 3. Morphology image and hydrodynamic diameter distribution (DH) determined by transmission electron microscopy (TEM), scanning electron microscopy (SEM) and dynamic light scattering (DLS). (A) TEM image and DH data, and (C) SEM image for nNP-PLA-DOX. (B) TEM image and DH data, and (D) SEM image for tNP-PLA-DOX. (E) Diameter changes of nNP-PLA-DOX and tNP-PLA-DOX over the course of 9 days, indicating the stability of the NPs. (F) In vitro accumulative release profiles of DOX from NP-DOX, nNP-PLA-DOX and tNP-PLA-DOX determined by dialysis against



PBS (pH 7.4) at 37°C over a 20-day period. The data are presented as the mean ± SD (n = 3).

Figure 4. Cellular uptake of nNP-PLA-DOX and tNP-PLA-DOX in HCC-LM3 (A) and BEL-7402 (C) cells at several time points, as shown by confocal laser scanning microscopy (CLSM). Flow cytometry analysis of (B) HCC-LM3, (D) BEL-7402, (E)


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HL-7702 and (F) NCI-H1299 cells. The cells were incubated with nNP-PLA-DOX (brown) and tNP-PLA-DOX (green) at a DOX equivalent concentration of 10 µM.



Figure 5. (A) In vitro cytotoxicity analysis of HCC-LM3 and BEL-7402 cells after 72h treatment with free DOX, nNP-PLA-DOX and tNP-PLA-DOX. The cell viabilities were determined by Cell Counting Kit-8. (B and C) Representative images of proliferation in the HCC-LM3 and BEL-7402 cells treated with various drugs (10 μM DOX equivalent concentration) for 48 h. The degree of cell proliferation was determined by EdU assay. The data are presented as the mean ± SD for n > 3 regions; *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 6. (A) Schematic illustration of the in vitro cytotoxicity analysis of a 24-h culture with the different NPs and an additional 72-h culture with fresh medium. Cell viabilities in (B) HCC-LM3, (C) BEL-7402, (D) NCI-H1299 cells treated with 5 μM and 10 μM nNP-PLA-DOX and tNP-PLA-DOX for 24 h (with NPs) plus 72 h (with fresh medium).


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Figure 7. (A) In vivo fluorescence images of the xenograft model of HCC-LM3 1 h, 4 h, 8 h, and 24 h after the injection of DiR, DiR@nNP-PLA-DOX and DiR@tNP-PLADOX via a tail vein. (B) Ex vivo fluorescence image of the heart, lungs, liver, spleen, kidneys and tumor of the xenograft model of HCC-LM3 24 h post-injection. (C) Average fluorescence signal of DiR in the excised organs after 24 h. *p < 0.05. The concentration of DiR was 0.1 mg/mL. (n = 3 in each group)



Figure 8. (A) Tumor volume (mm3) and (B) tumor weight (g) of the xenograft model of HCC-LM3 for different treatment groups (10 mg/kg DOX equivalent dose). (C) Representative tumors in each group at the end point of the study. (D) The average body weight of mice was used to evaluate the toxicity of the different treatments. The tumor volume, tumor weight and average body weight are described as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 (n = 5 in each group).


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Figure 9. Representative H&E staining of the tumor, heart, liver, lung, spleen and kidney from different treatment groups.



Table of Contents


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Table 1. Zeta Potentials and Sizes of the Drug-Loaded Nanoparticles

[a]

Material

Zeta Potential (mV)

Size (nm)

PDI[a]

nNP-PLA-DOX

-14.83 ± 0.61

122.6 ± 3.8

0.091 ± 0.017

tNP-PLA-DOX

-3.10 ± 1.51

75.3 ± 9.6

0.150 ± 0.011

PDI: polydispersity index

Table 2. In vitro cytotoxicity after 72-h incubation with different drug formulations as determined by CCK-8 assay (expressed as IC50 ± SD) IC50 (µM) Treatment Group HCC-LM3

BEL-7402

Free DOX

0.231 ± 0.036

0.126 ± 0.013

nNP-PLA-DOX

9.283 ± 0.422

6.921 ± 0.841

tNP-PLA-DOX

6.571 ± 0.855**

3.729 ± 0.702**

**p < 0.01 versus nNP-PLA-DOX


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