High Loading, Reversible Disulfide Core-Cross-Linked Multifunctional

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High Loading, Reversible Disulfide Core-Cross-Linked Multifunctional Micelles for Triggered Release of Camptothecin Longbing Ling, Muhammad Ismail, Yawei Du, Qing Xia, Wei He, Chen Yao, and Xinsong Li Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/ acs.molpharmaceut.8b00585 • Publication Date (Web): 30 Oct 2018 Downloaded from http://pubs.acs.org on October 31, 2018

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Molecular Pharmaceutics

High Loading, Reversible Disulfide Core-Cross-Linked Multifunctional Micelles for Triggered Release of Camptothecin Longbing Ling, Muhammad Ismail, Yawei Du, Qing Xia, Wei He, Chen Yao, Xinsong Li* School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, PR China *Contact Information of the Corresponding Author Xinsong Li Ph D; Email: [email protected] Abstract Nanomedicines in polymeric therapeutics present a potential treatment for cancers. However, their clinical effectiveness still has room to be improved. Herein, reduction-responsive reversibly core-cross-linked micelles based on the poly(ethylene glycol)-dihydrolipoic acid (MeO-PEG2k-DHLA) conjugate were developed for triggered intracellular release of camptothecin (CPT). Coupling two molecules of dihydrolipoic acid (DHLA) to methyl-terminated PEG (Mw 2000) through a labile ester bond was performed by solution-phase condensation reactions. Due to the amphiphilic property, MeO-PEG2k-DHLA conjugate formed micelles that were readily cross-linked with disulfide formation dispersed in water. These sole cross-linked micelles were 74.9 nm in hydrodiameter, as analyzed by dynamic light scattering (DLS). The nanostructures demonstrated excellent stability against extensive dilution, while rapidly dissociated under 10 mM glutathione (GSH), highlighting their potential for drug delivery. Interestingly, CPT was modified with a disulfide linkage and subsequently conjugated to MeO-PEG2k-DHLA polymer scaffold. Core-cross-linking of the micelles achieved high drug loading of CPT (31.81%, wt%) and demonstrated that CPT release at pH 7.4 was significantly declined by cross-linking (i.e. less than 15% release in 24 h), whereas more than 90% of CPT was released under 10 mM GSH conditions. In vitro cellular uptake and MTT assays showed that CPT-conjugated MeO-PEG2k-DHLA micelles were effectively internalized into tumor cells to induce the cytotoxic effects against HepG-2 and MCF-7 cells. Importantly, in vivo pharmacokinetics analysis demonstrated the nanoscale feature of micelles makes CPT to present longer retention time, resulting in higher accumulation at tumor sites. Taken together, the disulfide core-cross-linked MeO-PEG2k-DHLA multifunctional micelles with high drug loading and excellent stability are potential candidates for tumor-targeting drug delivery. Keywords: Camptothecin; Poly(ethylene glycol)-dihydrolipoic acid Conjugate; Disulfide Core-cross-linked Micelles; GSH-triggered Release.

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Introduction Chemotherapy remains the crucial approach for the treatment of cancer, a major threat to human health and life.1,2 In state-of-the-art chemotherapy, the traditional free antitumor drugs frequently endure several drawbacks, such as lack of aqueous solubility, highly non-specificity and undesirable adverse effects.3,4 Nanomedicines in polymeric therapeutics are an emerging area of opportunity for improving the efficacy of anticancer drugs.5 Covalent conjugation with water-soluble polymeric scaffolds can significantly improve aqueous solubility, extend the blood plasma half-life (t1/2) and decline side effects. Besides, nanoparticles based on the polymer-drug conjugate may enhance the therapeutic index via the “enhanced permeability and retention (EPR) effect” firstly reported by Matsumura and Maeda in 1986.6-8 The EPR effect pronounces the preferential internalization of polymeric prodrugs into tumor tissue due to vascular leakage and subsequent retention in the tumor with poor lymphatic drainage, compared with the tighter vasculature of normal (healthy) tissue. Currently, a considerable amount of effort is directed toward developing the safe 2


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and efficient antitumor polymer-drug conjugate. Examples of utilizing water-soluble linear polymers include poly-N-(2-hydroxypropyl)methacrylamide (HPMA),9,10 poly(vinyl alcohol) (PEG),11 poly(glutamic acid) (PGA),12 and poly(malic acid)13,14 etc. Of note, PEG is one of the extensively investigated chemistries in novel drug delivery system, for which the usage of PEG as a biocompatible polymer was recognized clinically.13 In addition to the traditional linear polymer-drug conjugates, alternative constructions like dendrimers15 and polymeric micelles16-18 have also generated impressive results. Polymeric micelles comprised of block-copolymers with hydrophilic/hydrophobic moieties enabled materials into nanoscale therapeutics in aqueous medium and stabilized the hydrophobic antitumor drugs into core encased by a hydrophilic shell. It should be noted, however, that despite of many advantages, their use still remained prohibitive obstacles for practical applications due to the limited drug loading content and premature burst release properties.19,20 Usually, the drug content encapsulated in polymeric micelles is less than 10%, in order to reduce the initial drug release in blood.21 In polymer-drug conjugates, the drug molecules always occupy small proportion to stay the conjugates water-soluble such as, the content of CPT in its PEG conjugate (Mw 40 kDa) was only 0.86 wt % or 1.72% (conjugated with one or two CPT).22 Very recently, polyprodrug strategy that polymerized block of reductive-responsive CPT prodrug monomer conjugated with hydrophilic PEG was reported by Hu and Liu et al. groups.23-26 These amphiphilic polyprodrugs can self-assemble into different kinds of uniform nanostructures, in which CPT loading content was greatly improved with even higher than 50 wt%. Meanwhile, such high drug loading carriers of CPT were also demonstrated the favorable nanostructure geometry-regulated cell internalization and reductive-responsive release of active CPT at tumor sites, exhibiting the enhanced therapeutic efficacy. Nevertheless, the self-assembled drug delivery systems often expose inadequate in vivo stability below critical micelle concentration (CMC), which leads to the disassociation of the micelles and premature drug release following i.v. injection, thereby causes undesired toxicities.27 Effective strategies to overcome these challenges are highly demanded. Covalent cross-linking is an important approach to stabilize polymeric micelles. Various cross-linking polymeric micelles have been developed in the past decades. Covalent cross-links could be established at the hydrophilic shell or hydrophobic core of the micelles through chemical, photo cross-linking or polymerization technique.28-30 Cross-linking of the coronas was able to form the robust shell cross-linked micelles. Nonetheless, one central issue to such micelles is that the shell cross-linking should be proceeded in a highly diluted condition to prevent unwanted inter-micellar cross-linking. Moreover, these shell cross-linked micelles can cause the loss of the shell fluidity and hydrophilicity, which potentially decreased the stealth effects and thereby, reduced the circulation half-life in the bloodstream.31 By contrast, the ease and flexibility of core-cross-linked strategy make them more attractive for enhancing stability of polymeric micelles with less influence on the nature of surface. Based on the greatly improved stability, the developed corecross-linked micelles could prolong circulation time as well as enhance the 3



accumulation at the tumor sites.32,33 However, the excessively stabilized micelles would also extensively inhibit drug release at the site of action, thus reducing the therapeutic efficacy. Recently, stimuli-responsive core-cross-linked micelles (SCMs) are proposed to fulfill the contradictory requirements of structural stability in the extracellular environment and drug release at the targeted sites.34 The stated literatures about stimuli factors for drug controlled release including pH, redox, temperature, electrolyte, enzyme, light, ultrasound and magnetic field as well.32, 34-36 In particular, cross-linking with disulfides is increasingly investigated as reduction-responsive linkers to design thiol-responsive polymeric micelles, since the intracellular glutathione (GSH) level in tumorigenic site is much higher (100-1000 fold) than that in the extracellular concentrations.37,38 The disulfide bonds (-S-S-) maintain high stability during circulation in bloodstream and extracellular tissues with less GSH concentration but would rapidly cleavage to release the payload in a highly reductive tumor environment. So far, several disulfide cross-linked micellar systems have been introduced in drug delivery system for therapeutic agents following the pioneer research of Kataoka group, including paclitaxel,39 doxorubicin (DOX),40 methotrexate,41 DNA and siRNA.42 For example, Zhong and coworkers43 prepared a kind of disulfide cross-linked multifunctional dexran-lipoic acid nanostructure for the reduction-sensitive intracellular delivery of DOX. After cross-linking using a catalytic amount of dithiotheitol (DTT), the resultant cross-linked dextran-based nanoparticles showed high drug loading up to 84%. The in vitro release of DOX diminished greatly (< 10%) even upon massive dilution, however, the release was significantly accelerated (> 90% over a period of 11 h) under reductive environment, facilitating it for tumor-targeted delivery. In addition, other studies also verified that such disulfide cross-linked micelles comprised of lipoic acid conjugates could be applied as carriers for drug delivery.44,45 In the present work, we designed CPT-conjugated disulfide core-cross-linked micelles based on MeO-PEG2k-DHLA conjugate that exhibited redox-responsive disassembly and synchronized cleavage of polymer-drug conjugate. Camptothecin (CPT), precisely a pyridyldithio-functionalized CPT was covalently linked to the core of DHLA block via a disulfide bond linker to avoid the undesired leakage and burst release of payloads. With shell PEGylation of and core cross-linking, CPT-conjugated cross-linked MeO-PEG2k-DHLA micelles showed superior stability under the simulated physiological environments. In contrast, upon exposure to the reductive condition, the micellar structure of MeO-PEG2k-DHLA conjugate was disrupted and rapidly released CPT because of disulfide bonds cleavage in both CPT−polyester conjugate and cross-linker. Finally, in vivo blood retention and tissue biodistribution were investigated in detail. This responsive stimulus, together with the inherent passive EPR targeting effect in the polymeric scaffolds, could be beneficial for advancing the outcome of polymeric drug delivery system. Materials and Methods Regents 4


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Camptothecin (CPT, purity ≥ 99%) was provided by Spring&Autumn Biotech Co., Ltd. (Nanjing, China). Poly(ethylene glycol) methyl ether (MeO-PEG-OH, Mw=2000 Da) and lipoic acid (LA, purity ≥ 99%) were obtained from Meryer Chemical Technology Co., Ltd. (Shanghai, China). Succinic anhydride (SA), trifluoroacetic acid (TFA), 1-Ethyl-3-(3-dimethyllaminopropyl)carbodiimide hydrochloride (EDC·HCl), 4-(dimethylamino) pyridine (DMAP), 1-hydroxybenzotriazole (HOBt), N,N-Diisopropylethylamine (DIPEA) and serinol (purity ≥ 98% ) were provided by Aladdin Biochemical Technology Co., Ltd (Shanghai, China). Penicillin−streptomycin, fetal bovine serum (FBS), phosphate-buffered saline (PBS pH=7.4), Roswell Park Memorial Institute (RPMI) 1640 media, and 0.25% trypsin –EDTA were purchased from KeyGen Biotech Co., Ltd. (Nanjing, China), while methyl tetrazolium (MTT) and lysotracker green were supplied by Sigma-Aldrich, USA. Unless stated otherwise, all reagents and chemicals were received from the commercial sources and used without further purification. Synthesis of MeO-PEG2k-DHLA Conjugate MeO-PEG2k-DHLA conjugate was developed by a five-step reaction as stated in Scheme 1. Synthesis and the corresponding characterizations of structures are detailed below. Boc-Serinol: Synthesis of compound Boc-Serinol was performed using the procedure as described previously.46 Briefly, di-tert-butyldicarbonate (5.03 g, 23.04 mmol) in t-BuOH (10.0 mL) was mixed with a suspension of serinol (1.5 g, 16.46 mmol) in a 1:1 mixture of MeOH-t-BuOH (15.0 mL) and stirred at r.t. overnight. Subsequently, the solvent was concentrated to offer a residue, which was further purified by precipitation with cold EtOAc to afford white solid of Boc-Serinol (2.89 g, yield 92%). 1H NMR (500 MHz, CH3OH-d4): δ 1.38 (s, 9H, Boc-CH3), 3.75 (d, 4H, J=5.6 Hz, CH2). 13C NMR (500 MHz, CH3OH-d4, Me4Si): δ 28.4, 58.2, 61.1, 78.5, 155.6. TOF-MS m/z: calculated for C8H17NO4 [M+Na]+, 214.1; found 214.1 [M+Na]+. di-LA-Serinol: Lipoic acid (LA, 2.97 g, 14.42 mmol) was firstly dissolved in anhydrous DCM (40 mL) at r.t., followed by addition of EDC·HCl (3.31 g, 17.43 mmol). The mixture was stirred for 20 min and afterward, Boc-Serinol (1.25 g, 6.54 mmol) and DMAP (2.21 g, 18.11 mmol) were added to the solution. Reaction proceeded for another 12 h at room temperature and monitored by TLC (Eluent: 5% MeOH in DCM, visualized under UV at 254 nm). The resulting mixture was washed with 0.1 M hydrochloric acid (40 mL×3), concentrated and purified by flash chromatography 20:1 (DCM:MeOH, v/v) to yield 3.19 g as a pale-yellow oil (86%). 1H NMR (500 MHz, CDCl , Me Si): δ 1.25-1.48 (m, 17H, H4,4’, H5,5’, Boc-CH ), 3 4 3 1.68 (m, 6H, H3,3’, H7a,7a’), 1.92 (d, J=13.6, 9.2 Hz, 2H, H7b,7b’), 2.32 (m, 6H, H2,2’, H6,6’), 2.47 (td, J=12.4, 6.5 Hz, 4H, H8,8’), 4.13 (m, 4H, H1,1’), 5.04 (s, 1H, H9). 13C NMR (500 MHz, CDCl3, Me4Si): δ 24.55, 28.34, 28.71, 33.82, 34.57, 38.50, 40.24, 48.46, 53.49, 56.30, 62.99, 80.03, 155.12, 173.11. TOF-MS m/z: calculated for C24H41NO6S4 [M+Na]+, 590.2; found 590.2 [M+Na]+. 5



MeO-PEG2k-COOH: MeO-PEG2k-COOH was synthesized from MeO-PEG2k-OH following a published method.47 Briefly, anhydrous MeO-PEG2k-OH (10 g, 5 mmol) in anhydrous CHCl3 (50 mL) was reacted with SA (1 g, 10 mmol) under the catalytic pyridine (2 mL). After reﬂuxing for 48 h, solvent was removed under vacuum and the residue re-suspended in saturated solution of NaHCO3 (50 mL), followed by extraction with ethyl acetate (2×30 mL). The resulting slurry was finally precipitated in absolute diethyl ether (100 mL) to attain the white product of MeO-PEG2k-COOH (7.8 g, yield 75%). 1H NMR (500 MHz, CDCl3, Me4Si): δ 2.64 (m, 4H, H2,3), 3.38 (s, 3H, -CH3), 3.66 (m, -CH2CH2-), 4.26 (m, 2H, H1). MeO-PEG2k-DHLA: To a solution of Boc-Serinol (1 g, 1.76 mmol) dissolved in DCM (5 mL) pre-cooled to 0 °C was dropwise added trifluoroacetic acid (TFA, 0.41 g, 3.52 mmol) over a period of 30 min. After stirred for 2 h at r.t., the reaction mixture was concentrated and dried in vacuo. The obtained yellow solid without further purification was re-dissolved in dry DCM (20 mL), and subsequently added the MeO-PEG2k-COOH (3.086 g, 1.47 mmol), EDC·HCl (0.42 g, 2.21 mmol), 1-hydroxybenzotriazole (HOBt, 0.19 g, 1.47 mmol) and DIPEA (0.85 g, 6.63 mmol). After reaction completed under N2 atmosphere, purification by column chromatography (SiO2, 20:1, DCM:MeOH) afforded the product of MeO-PEG2k-LA (3.47 g, 92%) as a yellow solid. This solid was dispersed in a 1:4 mixture of MeOH-H2O (15 mL) and stirred at 0 °C. NaBH4 (0.11g, 1.96 mmol) was added and stirred for 2 h or till the mixture solution turned to colorless. The mixture was acidified to pH 3.0 and the product was purified through dialysis (MWCO 1000) versus MeOH-H2O (1:4, v/v) mixture. Lyophilization gave the final product of MeO-PEG2k-DHLA as white solids (2.95 g, yield 86%). 1H NMR (500 MHz, CDCl3, Me4Si): δ 1.25 (s, 4H, H7,7’), 1.32 (dd, J=14.2, 6.6 Hz, -SH), 1.62 (m, 4H, H8,8’), 2.02 (m, 4H, H10,10’), 2.22-2.36 (m, 6H, H2, H5,5’), 2.49 (m, 2H, H1), 2.59-2.64 (m, 6H, H9,9’, H10,10’), 3.38 (s, 3H, -CH3), 3.60 (m, -CH2CH2-), 4.42 (m, 4H, H4,4’). 13C NMR (500 MHz, CDCl3, Me4Si): δ 22.92, 24.80, 26.54, 31.60, 32.30, 33.92, 38.94, 41.43, 43.58, 45.75, 57.85, 62.23, 63.99, 69.54, 70.38, 73.68. Synthesis of camptothecin-pyridyl disulfide31 A mixture of camptothecin (CPT, 0.348 g, 1.0 mmol) and triphosgene (BTC, 0.12 g, 0.4 mmol) was suspended in anhydrous DCM (40 mL). After purged with nitrogen for 20 min, 4-dimethylaminopyridine (DMAP, 0.29 g, 2.4 mmol) was added dropwise and stirred for 3 h at ambient temperature. Then, 2-(pyridyl-disulfanyl)ethanol (0.22 g, 1.2 mmol) in anhydrous DCM (3 mL) was added and the reaction mixture was allowed to stir for 12 h. Washed with deionized (DI) water and evaporated, the crude product was purified by silica column chromatography using EtOAc and DCM (1:1, v/v) to gain the camptothecin-pyridyl disulfide (CPT-SS-Pyr) as a yellow solid (0.31 g, yield 50.01%). 1H NMR (500 MHz, CDCl3, Me4Si): δ 0.87-1.02 (m, 3H, H18), 2.27-2.34 (m, 2H, H19), 3.11 (t, J=6.6 Hz, 6


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2H, H25), 4.33-4.77 (m, 2H, H24), 5.33 (dt, J=22.3, 18.0 Hz, 2H, H5), 5.73 (m, 2H, H17) , 7.09 (dd, J=8.4, 4.8 Hz, 1H, H29), 7.31 (s, 1H, H28), 7.38 (s, 1H, H14), 7.65-7.70 (m, 3H, H27, H28, H10), 7.88 (t, J=7.6 Hz, 1H, H11), 7.98 (d, 8.0 Hz, 1H, H9), 8.27 (d, J=8.5 Hz, 1H, H12), 8.45 (m, 2H, H7, H30). 13C NMR (500 MHz, CDCl3, Me4Si): δ 7.59, 31.92, 36.99, 49.98, 66.41, 67.08, 95.98, 120.01, 120.36, 120.92, 128.00-128.43, 129.71, 130.69, 131.14, 137.27, 145.55, 146.48, 148.90, 149.52, 152.92, 153.37, 157.28, 159.35, 167.20. Measurement of critical aggregation concentration (CAC) The critical aggregation concentration (CAC) of cross-linked MeO-PEG2k-DHLA micelles was checked by fluorescence-probe pyrene. Briefly, MeO-PEG2k-DHLA conjugate was dissolved in PBS (pH 7.4) and diluted from 1000 μg/mL to 1.9 μg/mL, whereas all tubes contained a total volume of 1 mL. After that, 5 μL of pyrene solution in acetone was added to maintain each MeO-PEG2k-DHLA solution with final concentration of pyrene at 6×10-7 M. The samples were equilibrated at ambient temperature for 24 h before test. In the end, HJYFL3-211-TCSPC spectrometer (HORIBA Jobin Yvon, Longjumeau, France) was applied to record the fluorescence emission spectra of all samples under the emission bandwidth of 8 nm at 335 nm. The CAC value was determined by plotting the I3/I1 intensity ratio versus the logarithmic concentration of conjugate. Preparation of MeO-PEG2k-DHLA micelles MeO-PEG2k-DHLA micelles were prepared through a dialysis method. Typically, MeO-PEG2k-DHLA (5 mg) was completely dissolved in DMSO (1 mL) and dropwise added into 5 ml of PBS buffer (pH 7.4) while stirring for 30 min. The solution was then dialyzed in a dialysis tube (MWCO 3500, ThermoFisher Scientific, MA) for 24 h against DI water that was renewed every 3 h intervals. To facilitate the cross-linking via formation of disulfide linkage, the solution was continuously bubbled with air for 12 h at 37 °C, during which Ellman’s test was performed to monitor the loss of free thiol (-SH).48 Size and ζ-potential measurements Particle size and ζ-potential of cross-linked MeO-PEG2k-DHLA micelles were analyzed by DLS measurement (Malvern Instruments Ltd., Worchestershire, UK) at 25 ± 1 °C. The solution was dispersed in PBS (pH 7.4) and sonicated for 3 min, prior the detection at a scattering angle of 173°. Each measurement was performed in triplicate consisting of 10 runs after 5 min equilibrium prior to analysis. Moreover, the colloidal stability and reductive responsivity of micelles against 100-fold dilution, 10% FBS and 10 mM GSH were assessed by recording the change in hydrodynamic diameters using DLS as described above. Transmission electron microscope (TEM) observation Morphology of MeO-PEG2k-DHLA micelles was analyzed by using transmission electron microscopy (TEM). Samples were prepared by dropping 10 μL of micelles 7



solution on a 200-mesh carbon film copper grid, followed by air-drying and negatively staining with phosphomolybdic acid (2%, w/v). Observation was recorded by JEM-2100 system (JEOL, Japan) that operated at an acceleration of 200 kV. Preparation of CPT-conjugated MeO-PEG2k-DHLA micelles For the preparation of CPT-conjugated MeO-PEG2k-DHLA micelles, MeO-PEG2k-DHLA (0.06 g, 0.047 mmol DHLA) and CPT-SS-Pyr (0.1 g, 0.188 mmol) were co-dissolved in a 2:3 mixture of methanol/DMSO (2 mL). After vigorously stirred for 48 h at 37 °C, the solution was dialyzed versus water containing Tween 80 (0.1%, v/v) for 12 h to induce cross-linked micelle formation. Micelles solution was then filtered thrice via a syringe filter (0.22 µm, Corning Incorporated, NY) to remove any free CPT. To determine drug loading content of CPT, CPT-conjugated MeO-PEG2k-DHLA solutions were lyophilized and re-dissolved in methanol and subsequently tested with UV/vis spectroscopy at the wavelength of 365 nm (Waters, MA, USA). The standard calibration curve was attained with CPT/methanol solutions at different CPT concentrations as prepared, while drug loading contents (DLC) and drug conjugation efficiency (CE) were calculated based on the following formula:

DLC (%) =

Weight of drug conjugated 100% Weight of polymer + drug conjugated

CE (%) =

Weight of conjugated drug 100% Weight of input drug

Reduction-triggered release of CPT Release profile of CPT from cross-linked MeO-PEG2k-DHLA micelles was studied by a dialysis method (MWCO 3500) under sink condition. Typically, lyophilized CPT-conjugated polymer micelles were dissolved in PBS (pH 7.4, 10 mL) and dialyzed against 100 mL of PBS solution, containing or not 10 mM GSH at 37 °C. At prearranged time points, the amount of released CPT was quantified by withdrawn 10 mL of release medium and replaced with fresh PBS to maintain the sink condition and finally, measured by HPLC (Agilent 1100, CA) using gradient elution within 18 min. Gradient elution: acetonitrile in water (0.1 % TFA), 0–4 min, 20-20%; 4–16 min, 20–65%; 16–18 min; 65–65%. Flowrate: 1.0 mL/min. Temp.: 25 °C. Detection: 365 nm. Column: ZORBAX SB-C18 (Analytical, 4.6×150 mm, 5 μm). Cell culture Human liver carcinoma cell line HepG-2 and human breast adenocarcinoma cell line MCF-7 were gifts from the Department of Public Health in Southeast University. For HepG-2 cells, they were cultured in RPMI-1640 media containing 10% FBS and penicillin (100 U/mL) and streptomycin (100 µg/mL), while MCF-7 carcinoma cells were cultured in DMEM medium. Both the tumor cells were incubated in an incubator (Thermo Scientific, MA) at 37 ºC under 5% CO2 humidified atmosphere. 8


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Hemolysis study In vitro hemolysis analysis was employed to assess the blood compatibility of CPT-conjugated cross-linked MeO-PEG2k-DHLA micelles. Human red blood cells were gifted from the Affiliated Zhongda Hospital of Southeast University (Nanjing, China) and the comprehensive procedure is outlined in the hemolytic ASTM standard E2524-08. Briefly, 200 μL of CPT-conjugated micelles at three different concentrations was added into the tubes, following addition of 1.0 mL red blood cells (RBCs, 2%, v/v) suspension and 800 μL normal saline. After incubated in water bath at 37 ºC for 4 h, an aliquot of the mixture was centrifuged at 1500 rpm for 15 min and the supernatants were collected for analysis of the extent of hemolysis by recording the absorbance of the haemoglobin at 540 nm by UV/vis spectrometer (Waters, MA, USA). Distilled water was selected as positive control with 100% hemolysis and normal saline were used as negative control with 0% hemolysis, respectively. The percent hemolysis ratio (HR%) lower than 5% was considered nontoxic. All the tests were repeated three times, while the HR% was calculated as follows:

HR (%) =

As  An 100% Ap  An

whereas, the absorbance of samples was labelled as As, and the positive and negative controls were Ap and An, respectively. Cellular uptake and subcelluar localization Qualitative cellular uptake behavior of CPT-conjugated cross-linked MeO-PEG2k-DHLA micelles in vitro was observed by confocal laser scanning microscopy (CLSM, Olympus FV1000, Japan) against HepG-2 cells. During the experiment, Approximately 2×105 cells/well were seeded onto the glass bottom dishes containing RPMI-1640 medium, incubated at 37 ºC overnight and then CPT-conjugated cross-linked micelles (10 µg/mL) were added for additional 1 h or 4 h. After that, the cells were fixed with 4% paraformaldehyde for 30 min and stained with 100 µL of propidium iodide (PI) solution for 15 min. Finally, fluorescence images were obtained via Leica TCS SP5 CLSM with CPT excitation at 405 nm (blue channel) and 543 nm for PI (red channel). For preliminary analysis of the uptake mechanism, subcellular localization of micelles was further assayed by using fluorescence of CPT and lysotracker green (Molecular Probes). After incubation with CPT-conjugated micelles for 1 h at 37 ºC as described above, HepG-2 cells were treated with lysotracker green (2 nM) for additional 1 h following the suppliers’ protocols. Cells were then washed with PBS three times, fixing with 4% paraformaldehyde and observed via CLSM. The fluorescence signals were detected with λexc = 405 nm for CPT and λexc= 505 nm for lysotracker green at 100×magnification. MTT assays The in vitro cytotoxicity of CPT-conjugated cross-linked MeO-PEG2k-DHLA 9



micelles against HepG-2 and MCF-7 was investigated by MTT method, while free CPT was applied as the positive control. In detail, the logarithmic growth of HepG-2 cells were seeded into a 96-well plate (5×103 each well) using RPMI-1640 medium supplemented with 10% FBS, while MCF-7 cells were cultured using DMEM medium. The medium was aspired after 24 h attachment and substituted with 200 µL of fresh medium containing free CPT and CPT-conjugated micelles with the final concentrations (CPT equivalent) of 32, 16, 8, 4, 2, 1, 0.5 and 0.25 µg/mL (n=6) for additional 48 h. Afterward, MTT solution (20 μL, 5 mg/mL) was added to each well and further incubated 4 h at 37 °C. The produced formazan crystals were dissolved by addition of 150 µL DMSO and finally, the absorbance of individual samples was examined using a microplate-680 reader (Bio-Rad, CA) at 570 nm. The percent viability of MCF-7 and A549 cells after different treatments were evaluated compared with the blank group and the value of half maximal inhibitory concentration (IC50) were calculated by GraphPad Prism software (version 6, GraphPad Software, Inc., CA). Each experiment was repeated in triplicate to calculate the cell viability as follows: ODtreated  ODblank Cell viability (%) = 100% ODcontrol  ODblank whereas, ODtreated indicates the optical density (OD) from the carcinoma treated with free CPT or micelles, while ODcontrol represents the averaged OD from the control (culture medium only) group and ODblank is the averaged OD of MTT without cells. Animals and tumor models Based on the guidelines and policies approved by the Institutional Animal Care and Use Committee of Southeast University, female BALB/c nude mice (5-7 old and weighting 16-18 g) were received from Qinglong Mountain Animal Breeding Ground (Nanjing, China) and housed under sterile conditions as per standard protocols. 150 µL of MCF-7 cells suspension holding 1×106 cells were subcutaneously injected into the nude mice in the right flank region and allowed the tumors to grow upto about 100-150 mm3 before use. Pharmacokinetics and biodistribution analysis MCF-7 tumor (100-150 mm3) bearing female BALB/c nude mice were randomly grouped into irinotecan and CPT-conjugated MeO-PEG2k-DHLA micelles (n=3). After post-intravenous injection of irinotecan and CPT-conjugated micelles (both equivalent to 5 mg/kg CPT) by tail vein, blood samples were taken from eye socket in a heparin containing tubes and centrifuged at 3,000 rpm for 15 minutes to collect plasma at specific time intervals. The amounts of irinotecan and unhydrolyzed CPT-conjugated MeO-PEG2k-DHLA conjugate in the plasma were extracted using acetonitrile. Briefly, the blood plasma samples (100 μL) were transferred into a centrifuge tube (2 mL), followed addition of 1 mL ice-cold acetonitrile containing 0.5% acetic acid. The samples were centrifuged at 11,000 rpm for 15 min and the obtained supernatant was subjected to HPLC analysis (Agilent 1100, CA) under 10


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gradient elution in 18 min. Standard curves of CPT in blood were performed via adding different concentrations of free CPT to the whole blood, followed by extraction and quantification as mentioned above. The pharmacokinetics parameters were determined by a non-compartment model using the drug and statistics (DAS) software (version 2.0.1). To assess the tissue biodistribution of drug formulations, MCF tumor-bearing BALB/c nude mice were sacrificed at 0.5 h, 2 h and 6 h after irinotecan and CPT-conjugated MeO-PEG2k-DHLA micelles (5 mg CPT/kg, n=3 for each time point) administrations. Major organs (e.g. heart, liver, spleen, lung and kidney) and tumors were excised, rinsed with normal saline (0.9%) and weighted before being homogenized. CPT was extracted from tissue homogenates with the same procedures from the plasma extraction. The drug concentrations were determined using HPLC and the corresponding CPT concentrations in tissues were calculated accordingly. Statistical analysis Numerical data are shown as the mean ± standard deviation (SD). One-way ANOVA analysis by GraphPad Prism software (version 6, GraphPad Software, Inc., CA) was applied to statistical evaluation in the mean. p < 0.05 is considered as statistical significance and p < 0.01 is highly significance. Results and Discussion Synthesis of MeO-PEG2k-DHLA conjugate MeO-PEG2k-DHLA conjugate with two DHLA molecules linked to methyl-terminated PEG (Mw 2000) through labile ester bonds, was prepared via condensation reaction as shown in Scheme 1. First, PEG monomethyl ether terminated with carboxyl headgroup (MeO-PEG2k-COOH) was synthesized through a facile conjugation from MeO-PEG2k-OH with succinic anhydride (SA) according to a published protocol. To acquire two lipoic acid terminated PEG monomethyl ether (MeO-PEG2k-LA), di-LA-Serinol, which was prepared and verified by TOF-MS, 1H NMR and 13C NMR (see Supporting Information, Figure S2, S3), was deprotected in trifluoroacetic acid (TFA) and then covalently conjugated with the carboxylic group of MeO-PEG2k-COOH using EDC·HCl and 1-hydroxybenzotriazole (HOBt) as coupling agents in anhydrous DCM for 24 h. The obtained MeO-PEG2k-LA polymer was isolated as a light yellow solid after precipitation into ice-cold ether three times. As a final step, MeO-PEG2k-LA was converted into free thiol form after treated with 2 molar equivalents of NaBH4 relative to lipoic acid under N2 environment followed by dialysis. Lyophilization afforded the desired MeO-PEG2k-DHLA conjugate as a white solid and their structure was verified by 1H NMR spectroscopy. As revealed in Figure S5, the characteristic proton signals appeared in 1H NMR spectrum at δ 3.63 ppm can be attributed to the methylene groups of PEG, while the peaks at δ 1.32 ppm accredited to the sulfhydryl (-SH) moiety of DHLA, clearly indicating the successful synthesis of the MeO-PEG2k-DHLA conjugate with well-defined structure.

11



Scheme 1. Synthetic procedure of MeO-PEG2k-DHLA conjugate.

Characterization of cross-linked MeO-PEG2k-DHLA micelles With the presence of the hydrophobic (DHLA) and hydrophilic (PEG) domains, the inherent amphiphilicity of MeO-PEG2k-DHLA conjugate provides an opportunity for itself to rapid assembly in aqueous and the coupled thiols in the DHLA block are able to further enhance the colloidal stability of micelles through disulfide formation. To evaluate its self-assembly behavior of MeO-PEG2k-DHLA conjugate in water, pyrene fluorescent probe technique was used to measure the critical aggregation concentration (CAC), whereas I1 (labeled as 1) and I3 (labeled as 3) are the first and third emission band intensities in the fluorescence spectrum. Resulted from the polarity of medium surrounding pyrene molecules, pyrene exhibits a sensitive shift in emission intensity ratio of I3/I1 peak and thereby, CAC value is accomplished by plotting the peak intensity ratio I3/I1 versus logarithmic concentration of the polymer. As illustrated in Figure 1, at low MeO-PEG2k-DHLA concentration, there is no significant change in I3/I1 value, suggesting the characteristics of pyrene in aqueous environment. As MeO-PEG2k-DHLA concentration increased, the ratio of I3/I1 rapidly increased which is ascribed to the accumulation of pyrene in a hydrophobic microenvironment. According to the emergence of sharp change in the slope curve, CAC value was examined to be at about 15.8 µg/mL, for which revealed the formation of micelles. The low CAC of MeO-PEG2k-DHLA conjugate shall offer a good stability for the micelles upon extensive dilution in blood after i.v. administration. Thiol-containing micelles could spontaneously cross-link through disulfide bonds formation under air atmosphere. The content of disulfide formation was estimated by Ellman’s test, which was carried out through the dissolution of MeO-PEG2k-DHLA conjugate in water above its CAC value and bubbled with air. At the determined time intervals, 100 µL aliquots were collected and added to a reaction buffered medium of Ellman’s reagent. The residual thiol groups are quantitated in 12


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tested solution by comparison to a standard curve comprised of known concentrations of a thiol-containing cysteine compound at 412 nm. As shown in Figure 2, the regular decline of free thiol from MeO-PEG2k-DHLA micelles over time was observed and after bubbled with air in 24 h, samples achieved more than 90% conversion based on the loss of free thiol. Cross-linked MeO-PEG2k-DHLA micelles were obtained through dialysis followed by bubbling with air for 24 h to facilitate cross-linking as described above. Dynamic light scattering (DLS) measurement exhibited that MeO-PEG2k-DHLA conjugate formed cross-linked micellar nanoparticles having average hydrodiameter of 74.9 nm and narrow size distribution as well as a PDI of 0.201 (Figure 3A). The surface charge of the cross-linked micelles was also investigated by DLS and the data showed that the surface is charged negatively with a ζ-potential of -21.7 mV in PBS solution, indicating promising stability. Transmission electron microscopy (TEM) illustrated that the nanoparticles are homogeneously distributed with spherical morphology and the size is close to that measured by DLS (Figure 3C). Furthermore, TEM of cross-linked micelles exhibited similar average diameter compared with un-cross-linked micelles (Figure S9), suggesting no disruption of MeO-PEG2k-DHLA micelles occurred during cross-linking. To further prove sufficient crosslinking, the lyophilized sample of the resultant crosslinking micelles was dissolved in DMF and checked by DLS. As indicated in Figure 3B, the average size of cross-linked MeO-PEG2k-DHLA micelles in DMF was 78.5 nm with a PDI of 0.145, which displayed no substantial difference for the average size of the micelles in water and organic solvent. The result convincingly demonstrated the structure of the micelles remains intact, resulting from the core crosslinking of MeO-PEG2k-DHLA micelles. The in vitro colloidal stability of cross-linked MeO-PEG2k-DHLA micelles versus extensive dilution and PBS solution (pH 7.4) containing 10% FBS was studied by DLS. The corresponding un-cross-linked MeO-PEG2k-DHLA micelles were used as a control. As seen in Figure 4A, 0.1 mg/mL of cross-linked micelles after 100-fold dilution (C < CAC) or incubation with PBS solution (pH 7.4) containing 10% FBS maintained similar distribution of hydrodynamic size compared with the parent micelles. But for un-cross-linked MeO-PEG2k-DHLA micelles under the identicial dilutive conditions, the size significantly reduced from 68.2 nm to 10.8 nm (Figure S7), that is ascribed to the disruption of micelles to polymeric unimers. The combined results indicated that these cross-linked MeO-PEG2k-DHLA micelles greatly enhanced the colloidal stability in vitro, thereby might arrive to tumor sites with the intact form in passively tumor-targeted drug delivery. The resultant cross-linked MeO-PEG2k-DHLA micelles have reduction-sensitive disufide crosslinks sequestered in their hydrophobic cores. As displayed in Scheme 3, the pendant disufide linkages could readily be cleaved into thiol groups (-SH) in response to cellular reductive triggers. Here, the reduction-sensitivity of cross-linked MeO-PEG2k-DHLA micelles was examined by measuring the variation of micelle size with response to GSH (10 mM) in PBS buffer (pH 7.4) at 37 °C. As shown in Figure 4B, almost no signal was detected after 12 h treatment with 10 mM GSH, whereas, 13



the DLS measurements of micelles exhibited a little size change in the absent of GSH under the same environment (data not given). These results obviously suggested that the disruption of disulfide linkages in hydropholic core caused the destabilization and disruption of degraded micelles. Thus, we can conclude that cross-linked MeO-PEG2k-DHLA micelles with good colloidal stability in vitro are liable to dissociate quickly under reductive environment.

Figure 1. The critical aggregation concentration (CAC) of MeO-PEG2k-DHLA micelles determined via pyrene fluorescence probe method.

Figure 2. Percent loss of free thiol over time for MeO-PEG2k-DHLA micelles cross-linking.

14


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Figure 3. Size distribution of core-cross-linked MeO-PEG2k-DHLA micelles in PBS solution (pH 7.4) (A) and in DMF (B) determined by DLS and TEM micrograph (C).

Figure 4. In vitro colloidal stability of cross-linked MeO-PEG2k-DHLA micelles versus 100-fold dilution, 10% FBS (A) and GSH cleavage of sample after 12 h incubation measured by DLS (B). Loading and GSH-triggered release of CPT Based on the promising results mentioned above, MeO-PEG2k-DHLA core corss-linked micelles can provide a potential and most suitable drug delivery platform for cancer therapy, in which the behavior of reductive-sensitive release can boost the specific on demand delivery of therapuetic molecules, due to the 100-1000 times higher reductive envoirnments in the cellular cytosol and nuclei than extracellular milieu (0.5-10 mM versus 2-20 µM GSH).49 So, to preliminarily evaluate the cross-linked MeO-PEG2k-DHLA micelles as a reduction-sensitive delivery system, a pyridyl disulfide-functionalized CPT derivative was designed to covalently conjugate to polymer MeO-PEG2k-DHLA through disulfide linker (Scheme 2). The camptothecin-pyridyl disulfide (CPT-SS-Pyl) was prepared according to the previously reported literature.50 Its chemical structure was characterized by TOF-MS (calculated 561.1; found 562.1 [M+H]+) and NMR spectra (Figure S6). Then, CPT-SS-Pyl was conjugated to the core of MeO-PEG2k-DHLA via a disulfide bond linker. After stirred in methanol/DMSO (2:3, v/v) for 48 h at 37 °C, the solution was dialyzed in water containing 0.1% (v/v) Tween 80 for 12 h to remove unreacted derivative as well as induce the cross-linked micelle formation. HPLC technique was 15



further used to check the removal of residual unreactive CPT derivative. It was found that no significant peak of free CPT-SS-Pyr at 6.025 min was detected compared to that of standard CPT sample, indicating the total removal of the residuals (Figure S8). The loading level of CPT was investigated by UV/vis spectroscopy at the wavelength of 365 nm (Figure S11), compared with a standard CPT curve. As examined, the CPT content in the PEG-drug conjugates is 31.81% while the conjugation efficiency is 45.37 %. It’s reported that either nanoparticles or liposomes generally possess less than 10% drug content to diminish drug release at the early stage in the blood circulation.13 Herein, the fixed CPT loading content is significantly improved without premature drug release, which is a great advantage of these prodrug micelles in drug delivery. The in vitro release behaviors of CPT from MeO-PEG2k-DHLA micelles were studied by a dialysis method under sitimulated physiological (pH 7.4) environment in the absence and presence of GSH (10 mM) at 37 °C. Parent CPT was applied as a control, which dialyzed against PBS media with Tween 80 (0.1%, v/v) under the same condition. As shown in Figure 5, free CPT was able to completely diffuse out of the cartridge within 10 h and notably, more than 95% CPT was released in the first two hours, indicating that diffusion from the dialysis cartridge is not influenced to assess the performance of micelles. Meanwhile, the cross-linked MeO-PEG2k-DHLA micelles exhibited obvious reduction-responsive release behavior of CPT drug. Under pH 7.4 without GSH, release of CPT was considerably reduced and approximately less than 15% drug was released over 72 h. The result revealed that the MeO-PEG2k-DHLA micelles still maintained a fairly higher stability under simulated physiological enviornment. But importantly, the introduction of GSH remarkably enhanced the CPT release rate at pH 7.4. Specifically, the collective release of CPT was up to 90% after 24 h incubation, which is probably due to the GSH-induced breakage of disulfide crosslinks in micelles. The morphology and size change of cross-linked MeO-PEG2k-DHLA micelles during the release of CPT were monitored using TEM and DLS, respectively. As displayed in Figure 6A and 6C, the morphology of CPT-conjugated cross-linked micelles exhibited no significant change after 48 h incubation under pH 7.4. However, within 10 mM GSH, swelling micellar aggregates were evidently observed after 4 h (Figure 6B). It can be ascribed to partial breakdown of disulfide bonds in the dense core of micelles inducing swelling of the loosed core. After 24 h incubation, irregular micellar debris were formed (Figure 6D), indicating de-crosslinking of the loose core, resulting in GSH-responsive release of CPT. Furthermore, DLS analysis of CPT-conjugated MeO-PEG2k-DHLA micelles (Figure 6E) and concomitant de-crosslinking-based swelling and CPT release after 48 h incubation also were in good agreement with TEM observation. It was found that the size of cross-linked micelles enhanced from 74.9 nm to around 480 nm for 12 h and even larger after 48 h incubatory period within 10 mM GSH. The result remarkably demonstrated that the -SS- linkages in the micellar core can effectively be cleaved under a highly reductive condition, thereby leading to swelling and aggregation of hydrophobic segments and further rupture of the micelles to form 16


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debris and release CPT. Conclusively, CPT-conjugated MeO-PEG2k-DHLA prodrug micelles are reasonably stable under neutral condition, and can exploit the reduction-sensitive properties at the intracellular environment in tumor sites. Scheme 2. Synthesis of CPT-pyridul disufide and conjugation to MeO-PEG2k-DHLA.

Scheme 3. Illustration of reversible core-cross-linked MeO-PEG2k-DHLA micelles for loading and reduction-responsive release of CPT.

17



Figure 5. Reduction-triggered release of CPT at pH 7.4 with or without 10 mM GSH from MeO-PEG2k-DHLA micelles at 37 °C.

Figure 6. TEM images of CPT-conjugated cross-linked micelles under different environments: (A) before and (C) after incubation in PBS at pH 7.4 for 48 h; after incubation in PBS with 10 mM GSH for (B) 4 h and (D) 48 h, respectively. (E) Time-dependant size change of CPT-conjugated crosslinked micelles responsing to 10 mM GSH as measured by DLS. Hemolysis study Hemocompatibility is an important parameter to evaluate the safety of the developed formulation in human body. Therefore, the hemolysis activities of CPT-unconjugated and CPT-conjugated cross-linked MeO-PEG2k-DHLA micelles were analyzed at the concentrations of 1000 µg/mL, 100 µg/mL and 10 µg/mL by incubation with normal red blood cells as an in vitro toxicity model. As shown in Figure 7, negligible percentage (less than 5%) of hemolysis was found in terms of blank and CPT-conjugated micelles, indicating that these developed micelles were compatible with RBC cells and safe for i.v injection. This was an expected result as both the components of the micelles, lipoic acid and PEG are extensively used as 18


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matrices for biological applications in drug delivery.

Figure 7. Hemolysis of CPT-unconjugated and CPT-conjugated cross-linked MeO-PEG2k-DHLA micelles. Data are given as the mean ± standard deviation (n=3). Cellular uptake Cellular uptake analysis and intracellular release profile of CPT-conjugated cross-linked MeO-PEG2k-DHLA micelles were investigated against HepG-2 cells for 1 h and 4 h incubation period. The HepG-2 cells were first rinsed, fixed by 4% paraformaldehyde and their nuclei were stained by propidium iodide (PI, red), following determined the inherent fluorescence intensity of CPT by confocal laser scanning microscope (CLSM). As revealed in Figure 8, a significant CPT fluorescence in the cytoplasmic region of HepG-2 cells in 1 h incubation with CPT-conjugated MeO-PEG2k-DHLA micelles at 37 °C was observed and the fluorescence bacame much stronger after 4 h treatment. The above-mentioned results remarkably suggseted that the MeO-PEG2k-DHLA micelles allowed for the outstanding intracellular uptake and quickly release the CPT possibly due to the GSH-triggered destruction of micelles. It is well-known that the endocytosis is usually considered as one of the key entry mechanisms for various drug deliveries. More importantly, exploiting the basic principles of intracellular processing fate of drug carriers can offer the key insights for improving the efficiency of drug delivery.51 Therefore, cellular uptake mechanism of CPT-conjugated MeO-PEG2k-DHLA micelles in HepG-2 cells was studied by counterstaining with lysotracker green. As illustrated in Figure 9, CPT-conjugated MeO-PEG2k-DHLA micelles were uptaken by HepG-2 cells (blue) while lysotracker uptaken by the cells was observed as green fluorescence. The co-localization of CPT-conjugated micelles and lysotracker obviously confirmed the retention of micelles in the lysosomes. Moreover, the intracellular fluorescence intensity of CPT became insignificant even after 4 h incubation at 4 °C (Figure 8), further demonstrating the cellular uptake of MeO-PEG2k-DHLA micelles by 19



energy-dependant endocytosis and then localized to lysosomes of HepG-2 cells.

Figure 8. CLSM images of HepG-2 cells after treatment with CPT-conjugated MeO-PEG2k-DHLA micelles at 37 °C or 4 °C for 1 h and 4 h. Cell nuclei were stained by PI (red). Scale bar: 40 µm.

20


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Figure 9. Endo-lysomal uptake of CPT-conjugated MeO-PEG2k-DHLA micelles in HepG-2 cells after 1 h incubation, as observed by CLSM. Lysosomes were stained by lysotracker (green). Scale bar: 20 µm. Cell cytotoxicity To assess the inhibitory performance of cellular proliferation of CPT-conjugated MeO-PEG2k-DHLA micelles against HepG-2 cells and MCF-7 cells, their cellular viability was evaluated by typical MTT protocol. Cells were treated with free CPT and CPT-conjugated MeO-PEG2k-DHLA micelles at a series of concentratins (0.25, 0.5, 1, 2, 4, 8, 16, 32 equivalent CPT µg/mL) for 48 h incubation. Control groups included a DMSO solution of CPT and MeO-PEG2k-DHLA conjugate itself in sterile distilled water. As shown in Figure 10, CPT-conjugated micelles exhibited dose-response cytotoxic effect against the tested tumor cells, which is likely due to the released CPT resulted from the cleavage of disulfide linkage under reductive condition. Additionally, the MeO-PEG2k-DHLA conjugate itself observed no cytotoxicity in a series of concentrations even up to 2 mg/mL (Figure S12), indicating its good biocompatibility of blank MeO-PEG2k-DHLA as a nanocarrier. Furthermore, the half-maximal inhibitory concentration (IC50) values of free CPT and CPT-conjugated micelles against HepG-2 cells and MCF-7 cells were calculated. The data showed that the polymer micelles (HepG-2 and MCF-7 cells were 1.89 µg/mL and 2.06 µg/mL, respectively) with equivalent CPT dose have a slight decrease of inhibition effect than free CPT (HepG-2 and MCF-7 cells were 1.57 µg/mL and 1.05 µg/mL, respectively). This is as-expected for polymer prodrug arising from the gradual release of CPT from the polymer chain over time. Altogether, the combination of reduction-sensitive release behavior and the feasible cytotoxicity will benefit the controlled release in vivo evaluation, while manipulating the high drug loadings for enhancing drug efficacy and minimizing toxicity.

Figure 10. In vitro cytotoxic activity of CPT-conjugated MeO-PEG2k-DHLA micelles. (A) HepG-2 cells and (B) MCF-7 cells after incubation with micelles over 48 h period. Data are given as the mean ± standard deviation (n=6). Pharmacokinetics and biodistribution studies 21



As previously reported, polymeric micelles with suitable size (100~200 nm) can effectively extend the circulation half-life of conjugated or loaded drugs in the blood and thereby, accumulate specifically in the tumor tissue through EPR effects.52 Hence, the in vivo pharmacokinetics of CPT-conjugated MeO-PEG2k-DHLA micelles was studied by the administration of equivalent dose of 5 mg CPT/kg to BALB/c mice. For evaluation, BALB/c mice were randomly distributed into the respective experimental groups (n=3) and injected with the developed CPT-conjugated micelle formulation. Irinotecan, a clinically approved formulation of CPT, was chosen as a control in the following experiments. The plasma concentration−time profiles of both irinotecan and CPT-conjugated micelles are presented in Figure 11A, and their relevant pharmacokinetic parameters calculated by a non-compartmental model are provided in Table 1. As anticipated, free irinotecan was notably cleared from the blood circulation within 4–8 h of drug administration. This result was in contrast with CPT-conjugated cross-linked MeO-PEG2k-DHLA micelles, which potentially extended the blood circulation time period of drug with 66.2 magnitudes even after 24 h injection versus positive control of CPT. As reveled in Table 1, the overall AUC0-∞ of CPT-conjugated micelles, which indicates the presence of pharmaceutical ingredient in the body, is 3.22-fold higher than free irinotecan (22.72 mg/L·h). The elimination half-life (t1/2) of CPT-conjugated micelles demonstrated that their blood circulation was significantly extended with the value of 5.79 h, while free irinotecan was 2.36 h. Moreover, CL of micelles (0.015 L/h/kg) was notably lower than that of free irinotecan (0.033 L/h/kg). The enhanced in vivo performance of CPT-conjugated cross-linked MeO-PEG2k-DHLA micelles could be explained by few aspects. First, nanosized and uniformly distributed micelles were beneficial to avoid the rapid uptake of the reticular endothelial system (RES). Second, the polymeric conjugate assembled as a nanocarrier sustained its good stability without disassembled in the physiological environment. Third, the presence of PEG would further enhance the blood circulation as a result of minimizing recognition of micelles by RES and thus declining the plasma elimination rate of the prodrug. The afore-mentioned results were in accordance with the previous report, where a manifold upsurge in the plasma concentrations was detected after PEGylation.2 To further examine the accurate amount in the tumors and other organs targeted by drug formulations, difference in organs distribution of CPT-conjugated MeO-PEG2k-DHLA micelles compared to free irinotecan were analyzed in the MCF-7 tumor-bearing BALB/c nude mice and the obtain results at 0.5, 6 and 12 h post i.v. administration of the micellar formulations were illustrated in Figure 11B. In principle, administration of CPT-conjugated MeO-PEG2k-DHLA micelles to tumor-bearing mice resulted in a biodistribution model significantly different from mice dosed with irinotecan alone. The micelles were primarily distributed to RES organs, such as lung, spleen, and particularly liver after 2 h i.v. injection, whereas the corresponding level of irinotecan was lower. The larger accumulation of CPT-conjugated micelles in these organs is likely because of the nanoscale features of the formulation, which might provide a long-term therapeutic effect. More importantly, the group treated with CPT-conjugated MeO-PEG2k-DHLA micelles exhibited much higher CPT 22


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concentration in tumors (p < 0.01) than those with free irinotecan treatment. This is true for both 6 h and 12 h time-points, which further supports the hypothesis that the PEGylated CPT-conjugated micelles may decline the quick arrest by RES system and passively accumulate in the tumors due to the EPR effect. Therefore, the cross-linked CPT-conjugated MeO-PEG2k-DHLA micelles assembled from the polymer-CPT conjugate can prolong the plasma half-life and remarkably enhance in vivo delivery to tumor tissues as compared to irinotecan alone. Encouraged by the reduction-responsive release behavior mentioned above, the combined effects could promote the antitumor activity of CPT-conjugated micelles than irinotecan that will be performed in the preclinical studies.

Figure 11. Pharmacokinetics and biodistribution of CPT-conjugated MeO-PEG2k-DHLA micelles. (A) Plasma concentration−time curves and (B) tissue distribution of free irinotecan and CPT-conjugated micelles after i.v. injection into rats with a CPT dose of 5 mg/kg. Data are presented as mean ± standard deviation (n = 3), and the statistical significance level is **P < 0.01. Table 1. Pharmacokinetic parameters of free irinotecan and CPT-conjugated micelles after a single i.v. injection. Parametersa Unit Irinotecan CPT-conjugated Micelles AUC0-t mg/L·h 22.68 67.63 AUC0-∞

mg/L·h h-1

Kel

22.72

73.15

0.29

0.11

CL

L/h/kg

0.033

0.015

Cmax

mg/L

16.51

19.61

t1/2

h

2.36

5.79

Vd

L/kg

0.76

0.52

a

Parameters: AUC, area under the curve from zero to time t; Kel, elimination rate constant; CL, plasma clearance; Cmax, peak plasma concentration; t1/2, elimination half-life; Vd, apparent volume of distribution. 23



Conclusions In summary, we developed a novel redox-responsive cross-linked micelle based on the conjugate of PEG containing dihydrolipoic acid and exploited their potential as a drug delivery platform for tumor therapy. In the designing of prodrug, hydrophobic DHLA was applied to provide post-polymerization and cross-linking reaction by disulfide formation. Camptothecin conjugated in this micellar system via the DHLA block exhibited high drug loading efficiency (31.81% of free CPT, w/w), favorable colloidal stability caused by cross-linking and effective release with the controlled manner triggered in a reductive condition. The CPT-conjugated micelles taken up by HepG-2 cells induced the proliferation of tumor cells effectively. More significantly, in vivo pharmacokinetics validated that the nanoscale features of the micelles make CPT possess longer plasma retention time, which consequently results in superior accumulation in tumor tissue. The combination of cross-linked and redox-responsive release behavior of these micelles could be applied to design new nanocarriers to overcome the existed challenges in polymeric micelles, such as in vivo stability and burst, non-specific release of their payloads. Supporting Information Structure characterization of intermediates and product MeO-PEG2k-DHLA as confirmed by TOF-MS and 1H NMR; Hydrodynamic particle size change of cross-linked MeO-PEG2k-DHLA micelles dispersed in water before and after lyophilization; UV/Vis spectroscopy of CPT-conjugated cross-linked polymer micelles at the weight percentage of CPT; Cell viability of MeO-PEG2k-DHLA micelles against HepG-2 cells. Author Information Corresponding Author *E-mail: [email protected]. Notes The authors declare no competing financial interest. Acknowledgement The current research is funded by the project approved from Major National Science and Technology Program of China for Innovative Drug (2017ZX09101002-001-004). It is also supported by National Natural Science Foundation of China (Project 51373034) and Department of Science & Technology of Jiangsu Province, China under Projects No: BA2013037 and BY2015070-11 and the Priority Academic Program Development of Jiangsu Higher Education Institutions. References (1) Joralemon, M. J.; McRae, S.; Emrick, T. PEGylated polymers for medicine: from conjugation to self-assembled systems. Chemi. Commun. 2010, 46, 1377-1393. (2) Pasut, G.; Veronese, F. M. PEG conjugates in clinical development or use as anticancer agents: an overview. Adv. Drug Delivery Rev. 2009, 61, 1177-1188. 24


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High Loading, Reversible Disulfide Core-Cross-Linked Multifunctional

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