Synergistically Enhanced Antimetastasis Effects by Honokiol-Loaded

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Biological and Medical Applications of Materials and Interfaces

Synergistically Enhanced Antimetastasis Effects by HonokiolLoaded pH-Sensitive Polymer-Doxorubicin Conjugate Micelles Yang Zou, Yuanhang Zhou, Yao Jin, Chuyu He, Yunqiang Deng, Shidi Han, Chuhang Zhou, Xinru Li, Yanxia Zhou, and Yan Liu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b04854 • Publication Date (Web): 11 May 2018 Downloaded from http://pubs.acs.org on May 13, 2018

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ACS Applied Materials & Interfaces

Synergistically Enhanced Antimetastasis Effects by Honokiol-Loaded pH-Sensitive Polymer-Doxorubicin Conjugate Micelles

Yang Zou, Yuanhang Zhou, Yao Jin, Chuyu He, Yunqiang Deng, Shidi Han, Chuhang Zhou, Xinru Li, Yanxia Zhou, and Yan Liu

∗

Beijing Key Laboratory of Molecular Pharmaceutics and New Drug Delivery Systems, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China

*Corresponding author. Phone: 86 10 82801508; e-mail: [email protected]

KEYWORDS: Doxorubicin; honokiol; acid-cleavable imine; polymer-drug conjugate; pH-sensitive polymeric micelles; tumor metastasis; combined delivery

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ABSTRACT：：In an effort to prevent metastasis of breast tumor cells at the same time of inhibiting tumor growth with less toxic side effects, honokiol (HNK) was encapsulated into pH-sensitive polymeric micelles based on the conjugate (PEOz-PLA-imi-DOX) of poly(2-ethyl-2-oxazoline)-poly(D,L-lactide)

(PEOz-PLA)

with

doxorubicin

(DOX).

PEOz-PLA-imi-DOX was successfully synthesized by connecting DOX to the hydrophobic end of PEOz-PLA via acid cleavable benzoic imine linker. HNK-loaded conjugate micelles (HNK/PP-DOX-PM) with a size of 21 nm and homogeneous spherical shape exhibited high drug loading capacity. PEOz-PLA-imi-DOX and HNK/PP-DOX-PM displayed faster release of DOX at pH 5.0 than at pH 7.4. As anticipated, PEOz-PLA-imi-DOX maintained cytotoxicity of DOX against MDA-MB-231 cells. The synergistically enhanced in vitro antitumor effect of HNK/PP-DOX-PM was confirmed by their synergetic inhibition of MDA-MB-231 cell growth. Furthermore, the efficient prevention of tumor metastasis by HNK/PP-DOX-PM was testified by in vitro antiinvasion, wound healing and antimigration assessment in MDA-MB-231 cells, and in vivo bioluminescence imaging in nude mice. The suppression of growth and metastasis of tumor cells by HNK/PP-DOX-PM was attributed to synergistic effect of pH-triggered drug release and HNK-aroused inhibition of MMPs and EMT, respectively. In addition, HNK/PP-DOX-PM exhibited superior biosafety than

physically encapsulated

dual-drug micelles.

Consequently,

the fabricated

HNK/PP-DOX-PM may have great potential for safe and effective suppression of tumor growth and metastasis.


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1. INTRODUCTION Tumor metastasis is considered to be a huge barrier for thoroughly cure cancer and about 90% cancer patients died of cancer metastasis rather than primary tumors.1 It is often the case, however, that metastasis has been already developed while tumor is detected. In this case, simultaneous prevention of tumor metastasis should be brought to the forefront at the time of treating primary tumor, co-delivery of chemotherapeutic drugs with antimetastasis agents by a single vector would be thereby one of the promising strategies. The development of biomedical nanotechnology might offer a hope for this therapy,2,3 and nanosized drug delivery systems including liposomes, nanoparticles, polymeric micelles, and some other nanosystems, have been studied extensively for this purpose.4,5 Mei et al. developed liposomes for co-delivery of doxorubicin (DOX) and Peptide S, a CXCR4 antagonist, and evidenced an enhancement of in vitro and in vivo antitumor and antimetastasis efficiency.6 Tang et al. designed pH-sensitive nanoparticles for co-delivering paclitaxel and two siRNA (Snail siRNA and Twist siRNA) to effectively inhibit metastasis and growth of breast cancer.7 In these reports, drugs were all physically encapsulated in nanocarriers, thus there still existed some challenges such as premature release in circulation system and slow release in cancer cells. We previously reported pH-sensitive

poly(2-ethyl-2-oxazoline)-poly(D,L-lactide)

(PEOz-PLA)

micelles

for

co-delivery of paclitaxel and honokiol (HNK).8 Although premature drug release in blood circulation was prohibited and slow release of drugs inside tumor cells was accelerated to some extent, the synergistic suppression on cancer metastasis for dual drug-loaded PEOz-PLA micelles was still unsatisfying compared with paclitaxel-loaded PEOz-PLA micelles. Thus, there is an urgent need to develop novel delivery strategies for prevention of cancer metastasis. Recently, polymer-drug conjugate (PDC) has twinkled like a star in which drugs are conjugated with polymers via stimulus responsive linkers such as reduction-responsive disulfide,9,10 pH-responsive hydrozone,11-14 imine15-17 and acetal18,19 due to their relatively higher stability in physiological condition and rapid drug release in tumor cells. For example, hydrazone is cleavable at pH of ∼5.5 while stable at pH 7.4.12 Of these stimulus responsive linkers, acid-cleavable one is the most frequently used due to the fact that



there exist sharp changes in pH value in different tissues and cell organelles. The increased aerobic glycolysis in tumor cells results in lower extracellular pH (6.5-7.2) of tumor cells than that of circulation system and normal tissues. Moreover, there exists a pH gradient from about 5-6 in endosomes to about 4-5 in lysosomes.13 More importantly, PDC can self-assemble into core-shell structured micelles based on its amphipathic character in aqueous medium, thus enhancing accumulation in tumor sites through EPR effect and thereby significantly reducing toxicity to normal tissue.13,20 We previously linked hydrophobic anticancer drug DOX to a pH-responsive hydrophilic polymer PEOz via a hydrazone linkage.21 The conjugate micelles were demonstrated to enhance antitumor efficacy and reduce side effects compared with free DOX, however, the conjugate was more hydrophilic, thus leading to high critical micelle concentration (CMC) value. For the sake of simultaneously inhibiting the growth and metastasis of breast cancer cells, in the present study, a new vehicle that could co-deliver DOX and HNK, which was demonstrated to be effective at suppressing metastases8,22 through inhibition of NF-κB activation,23,24 at the same time was designed. First, a new polymer-drug conjugate (PEOz-PLA-imi-DOX) was synthesized by conjugating a pH-sensitive amphiphilic block copolymer PEOz-PLA, which could endow the conjugate with low CMC value, with DOX via an benzoic imine linker, which is cleavable at pH 5.0-5.5.25 Next, PEOz-PLA-imi-DOX could self-assemble into a nano-micelle structure, by which HNK was encapsulated into the hydrophobic core to form dual drug-loaded micelles. Therefore, full physicochemical characterization was performed for dual drug-loaded conjugate micelles including size and its distribution, drug loading capacity and in vitro release behavior. The cytotoxicity of the designed micelles to MDA-MB-231 cells was also assessed. Furthermore, the feasibility of the designed micelles for preventing cancer metastasis was examined in vitro and in vivo. The designed conjugate micelles were anticipated to efficiently prevent the development of cancer metastasis at the same time of treating cancer while reducing side effects. 2. MATERIALS AND METHODS 2.1. Materials. Doxorubicin hydrochloride (DOX⋅HCl) was purchased from Dalian Meilun Pharmaceutical Technology Co. Ltd. (Dalian, China). HNK was purchased from the


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National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). 2-Ethyl-2-oxazoline obtained from Sigma-Aldrich (St Louis, MO) was dried by molecular sieve desiccant. D,L-Lactide was supplied by Daigang Biological Technology Co. Ltd. (Jinan, China). 4-Carboxybenzaldehydec (CBA) was purchased from Sigma (USA). 4-dimethylaminopyridine (DMAP) was purchased from Adamas-Beta (Shanghai, China). Dicyclohexylcarbodiimide (DCC) was purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Sulforhodamine B sodium salt (SRB) and Matrigel were obtained from Sigma-Aldrich (St Louis, MO, USA). Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum (FBS), trypsin-EDTA, penicillin-streptomycin solution, were supplied by M&C Gene Technology (Beijing, China). 25 and 75 cm2 plastic culture flasks, 6-well and 96-well tissue culture plates, and Transwell of 12-well plate were purchased from Corning (USA). All other reagents and chemicals were of analytical grade or better.

Scheme 1. Synthesis routes of PEOz-PLA-imi-DOX.



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2.2. Synthesis and Characterization of the Conjugate of PEOz-PLA with DOX. The polymeric conjugate of PEOz-PLA with DOX was synthesized via a series of reactions as depicted in Scheme 1.

2.2.1. Synthesis and Characterization of Poly(2-Ethyl-2-Oxazline)-Poly(D,L-Lactic Acid) (PEOz-PLA). PEOz-PLA was prepared as our published report.26 The obtained product was confirmed by chromatography (GPC).

1

1

H NMR and then characterized by gel permeation

H NMR spectra was recorded on a Bruker MSL2300

spectrometer (400 MHz, Germany) using tetramethylsilane (TMS) as an internal reference following dissolution of the product in CDCl3. The molecular weight (Mn), weight-averaged molecular weight (Mw) and polydispersity index (PDI) were determined by GPC (Waters 1515) equipped with a refractive index detector (Waters 2414) and column (Styragel HT4-HT3-HT2, 10 µm, 7.8×300 mm) using polystyrene as standard. THF was adopted as the mobile phase at a flow rate of 1.0 mL/min.

2.2.2.

Synthesis

and

Characterization

of

Aldehyde-Terminated

Poly(2-Ethyl-2-Oxazoline)-Poly(D,L-Lactic Acid) (PEOz-PLA-CHO). PEOz-PLA-CHO was synthesized by esterification of PEOz-PLA with CBA according to the previous report with a little modification.16 In brief, the resultant PEOz-PLA (10.0 g) together with CBA (1.49 g, 10 mmol), DCC (2.06 g, 10 mmol) and DMAP (0.122 g, 1 mmol) were added into a 250 mL flask with 120 mL of strictly anhydrous THF under continuous stirring at room temperature. The reacting system must be isolated from air and strictly anhydrous. A few minutes later, white sediment of DCU was formed. The resultant mixture was left to react for 24 h followed by filtration, and the solvent THF was removed by rotary evaporation under vacuum. The obtained crude product was purified by dissolving in dichloromethane (DCM), filtration, and then precipitation in cooled diethyl ether (800 mL) for four times. A white solid was finally obtained through vacuum drying. Thin layer chromatography (TLC) was used to determine whether DCC, DMAP and CBA were all removed (developing solvent: methanol : DCM : acetic acid (HAc) (10:50:0.1)). 1H NMR spectra was used to confirm the successful synthesis of PEOz-PLA-CHO.

2.2.3.

Synthesis

and

Characterization

of

the

Conjugate

of

Poly(2-Ethyl-2-Oxazoline)-Poly(D,L-Lactic Acid) with DOX via an Benzoic Imine Linker


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(PEOz-PLA-imi-DOX). DOX was conjugated to PEOz-PLA-CHO through a benzoic imine bond formed between the amino of DOX and the aldehyde of PEOz-PLA-CHO (Scheme 1) according to the previous report with a little modification.16 Briefly, DOXHCl (587.5 mg, 1 mmol), triethylamine (840 µL, 6 mmol) and strictly anhydrous methanol (400 mL) were put into a 500 mL flask under continuous stirring at room temperature in the dark. After stirring for 8 h, the solvent was eliminated by rotary evaporation. Then PEOz-PLA-CHO (4.3 g) and strictly anhydrous chloroform (400 mL) was put into the flask. The reaction mixture must be kept from air and the light, and strictly anhydrous. After the mixture was left to react for 24 h at room temperature, the resultant dark orange solution was concentrated to about 20 mL by rotary evaporation and filtered to remove most of the unconjugated DOX. The filtrate was evaporated, and the residue was solubilized in 10 mL methanol followed by filtration with a 0.22 µm filter, then the filtrate was applied onto a Sephadex LH-20 gel (GE Healthcare, Sweden) column with methanol as eluent to separate the residual unbound DOX from PEOz-PLA-imi-DOX. The red powdery PEOz-PLA-imi-DOX conjugate was obtained by precipitating of the collected eluate in cooled diethyl ether and drying under vacuum. TLC was used to confirm both the conjugation of DOX to PEOz-PLA-CHO and absence of free DOX (developing solvent: methanol : DCM : HAc (10:50:0.1)). 1H NMR spectra was used to examine the chemical structure of the product. HPLC was used to determine the DOX content as described later. The hydrolysis of PEOz-PLA-imi-DOX at different pH values was conducted and qualitatively characterized using TLC. Briefly, 1 mg PEOz-imi-PLA-DOX was dissolved in 1 mL buffer solution with different pH values (acetate buffer (ABS) with pH 3.4 and pH 5.0, and PBS with pH 7.4), respectively. 5 min (for pH 3.4) or 24 h (for pH 5.0 and pH 7.4) later, 10 µL sample solution was taken out and analyzed with TLC (developing solvent: methanol : DCM : HAC (10:50:0.1)). The PEOz-PLA-CHO in methanol (1 mg/mL), PEOz-PLA-imi-DOX solution in methanol (1 mg/mL) and DOXHCl solution in methanol (50 µg/mL) were also applied for comparison, respectively. 2.3. Determination of Critical Micelle Concentration. The critical micelle concentration (CMC) of PEOz-PLA-imi-DOX conjugate was determined by fluorescence spectroscopy using pyrene as a hydrophobic probe as reported previously.27,28 The final



concentration of PEOz-PLA-imi-DOX ranged from 2×10-6 to 2 mg/mL. 2.4. Preparation of Polymeric Micelles. Film hydration method was utilized to prepare all micelles such as blank polymeric micelle (PP-PM), single drug-loaded micelles (HNK/PP-PM, PP-DOX-PM), and dual drug-loaded conjugate micelles (HNK/PP-DOX-PM) as reported earlier.8 In brief, 40 mg of PEOz-PLA-CHO and 2 mg of HNK was dissolved in 10 mL of methanol and methanol was then removed by rotary evaporation at 25°C to obtain a thin film. 10 mL of PBS (pH 7.4) at 60°C was then added to hydrate the thin film followed by vortexing for 5 min. The mixture was filtered through a membrane filter (0.22 µm) to remove the non-encapsulated HNK, and then a clear, transparent and homogeneous HNK/PP-PM solution was obtained. The resultant micelle solution was freeze-dried for further characterizations. PP-DOX-PM and PP-PM were prepared as described above except that no HNK was added. Dual drug-loaded micelle HNK/PP-DOX-PM was prepared as described above except that PEOz-PLA-CHO (40 mg) was replaced with PEOz-PLA-imi-DOX (40 mg) and the amount of HNK ranged from 0.5 mg to 2.5 mg. The prepared HNK/PP-DOX-PM with different mass ratio of HNK to DOX was denoted as HNK1/PP-DOX1-PM for 1:1 of HNK to DOX, HNK2/PP-DOX1-PM for 2:1 of HNK to DOX, HNK3/PP-DOX1-PM for 3:1 of HNK to DOX, and HNK5/PP-DOX1-PM for 5:1 of HNK to DOX, respectively. 2.5. Physicochemical Characterization of Polymeric Micelles. Dynamic light distribution (DLS) (Zetasizer nano series, Malvern, UK) was used to determine the size and size distribution of all prepared micelles as our published report.8 The morphology of micelles was observed by using transmission electron microscope (TEM, JEM-1230, JEOL, Japan) as previously reported.28 Before observation, the lyophilized powder of micelles was dispersed in distilled water to obtain a micelle solution with concentration of 40-80 µg/mL. The mobile phase pumped at a flow rate of 1.0 mL/min was a mixed solution of methanol : acetonitrile : acetate buffer (pH 3.4, 20 mM) (3:6:1, v/v/v). The encapsulation efficiency (EE, w/w, %) and drug loading content (LC, w/w, %) of


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micelles was determined by dissolving the lyophilized powder of drug-loaded micelles in mobile phase (methanol : acetonitrile : acetate buffer (pH 3.4, 20 mM), 3:6:1, v/v/v), and then the drug content was determined by HPLC at the wavelength of 294 nm according to our previous report.8 In order to evaluate the stability of HNK5/PP-DOX1-PM in PBS (pH 7.4) at 37°C against time, the particle size and its distribution, and morphology of the micelles were monitored during the storage period of 72 h. 2.6. In Vitro Release Study. Dialysis diffusion technique was used to evaluate in vitro release behavior of the drugs loaded in the micelles according to our previous report

8

except that the release medium was substituted with PBS (pH 7.4, 20 Mm) and ABS (pH 5.0, 20 mM) with 0.2% Tween 80 (w/v). 2.7. Cell Culture. MDA-MB-231 breast cancer cells, and the luciferase and green fluorescent protein-labeled MDA-MB-231 (MDA-MB-231-luc-GFP) cells were obtained from the China Infrastructure of Cell Line Resources (Beijing, China) and cultured in DMEM supplemented with 10% FBS at 37°C in a 5% CO2 humidified atmosphere. 2.8. In Vitro Cytotoxicity Assessment. The cytotoxicity of various micellar formulations (PP-PM, PP-DOX-PM, HNK/PP-PM and HNK/PP-DOX-PM) was determined by use of the SRB assay.8 Briefly, MDA-MB-231 breast cancer cells (1×104 cells/well) in exponential phase were cultured in 96-well plates to allow the cells to overspread the bottom plates about 80-90%. After the medium was removed, 200 µL of the tested micelle solution in DMEM with 10% FBS or negative control (fresh DMEM with 10% FBS) was added and incubated for 48 h. The cells were fixed with 10% trichloroacetic acid, dried, and stained with SRB in 1% (v/v) acetate solution for 30 min, washed with 1% (v/v) acetate solution followed by drying at 37°C. Subsequently, 150 µL Tris (10 mmol/L) was added and the plates were shaken at 37°C for 30 min, the optical density (OD) was assayed at the wavelength of 540 nm by use of a 550 model microplate reader from Bio-Rad laboratory (California, USA). 2.9. Evaluation of Cell Invasion. Transwell chambers (8 µm pore size, Corning, 3422, USA) were applied to evaluate the ability of the micelles to inhibit cell invasion.8,29 The membrane was covered with 80 µL Matrigel with a concentration of 12.5 µg/mL in



FBS-free DMEM medium for 90 min at 37°C, then MDA-MB-231 breast cancer cells suspended in 100 µL of DMEM with 1% FBS (5×104 cells/well) were added into the top chamber followed by addition of 100 µL tested micelle solution in fresh DMEM containing 1% FBS with different concentration of drugs or negative control (fresh DMEM with 1% FBS). 500 µL DMEM with 10% FBS was added to the bottom chamber. After incubation in a 5% CO2 humidified atmosphere at 37°C for another 48 h, the chambers were washed twice with PBS, the remaining tumor cells on the top chamber were eliminated by a swab, and the cells invaded to the bottom surface of the membrane were fixed with 500 µL 4% paraformaldehyde for 30 min at room temperature. Following staining with 0.4% crystal violet solution for 30 min, the cells were washed with PBS for five times to remove unbound crystal violet, dried at 37°C and then observed using an inverted fluorescence microscope (Olympus, IX-71). After that, 600 µL of 33% (v/v) acetic acid aqueous solution was used to dissolve the crystal violet in the stained cells and the optical density (OD) of the solution at 560 nm was then determined by the microplate reader. The cell invasion (%) was calculated in accordance with the following formula:

Cell invasion % =

OD × 100 1 OD

2.10. Cell Migration Assay. Wound healing was used to observe the cell migration visually and evaluate the cell migration qualitatively.6 In short, MDA-MB-231 breast cancer cells in exponential phase were seed in 6-well plates at a density of 6×104 cells/well in 2 mL medium and cultured for 48 h in a 5% CO2 humidified atmosphere at 37°C. After removal of the medium, the cell layer was scratched using a 200 µL micropipette tip followed by washing for three times with PBS to remove loose cells. Then 2 mL tested micelle solution in fresh serum-free DMEM with different concentration of drugs or fresh serum-free DMEM used as negative control was added and cultured. The cell wound was photographed by inverted fluorescence microscope (Olympus, IX-71) at 0 h, 24 h, and 48 h, respectively. To simultaneously evaluate the cell migration qualitatively and quantitatively, transwell chambers (8 µm pore size, Corning, 3422, USA) were also applied. MDA-MB-231 cells in exponential phase suspended in 100 µL of DMEM with 1% FBS


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were placed into the top chamber (5x104 cells/well) followed by addition of 100 µL tested micelle solution in fresh DMEM containing 1% FBS with different concentration of drugs or negative control (fresh DMEM with 1% FBS). Then 500 µL DMEM with 10% FBS was added to the bottom chamber. After incubation for 48 h in a 5% CO2 humidified atmosphere at 37°C, the cells migrated to the bottom surface of the membrane were fixed, stained, photographed and counted as mentioned in cell invasion assay. The cell migration (%) was calculated as follows:

Cell migration % =

OD × 100 2 OD

2.11. Assessment of Pulmonary Metastasis Suppression. 6-8 weeks old female BALB/c nude mice were purchased from Animals Center of Peking University Health Science Center. All animal experiments were carried out under the guideline approved by Institutional Authority for Laboratory Animal Care of Peking University. The efficacy of pulmonary metastasis suppression for the designed micelles was assessed and analyzed as previously reported. 8,30,31 The tested micelles were intravenously administered to mice on the day 2, 4, 6, 8,10 and 12 after injection of MDA-MB-231-luc-GFP cell suspension (1.2×106 cells) through the tail vein at a dose of 4 mg/kg for DOX and 20 mg/kg for HNK, respectively. At day 14 following the xenograft, all mice were imaged after luciferin injection. 2.12. Assessment of In Vivo Safety. To assess the toxicity of the micelles to the blood, at 4 h before living images, 20 µL blood sample was obtained from the orbit of each mouse and then diluted for routine blood test. For the hematological assessment, 18 common blood routine index such as white blood cell (WBC), red blood cell (RBC) and platelet (PLT), etc. were determined with MEK-6400K Automated Hematology Analyzer (Nihonkohden, Shinjuku-ku, Japan). To evaluate the toxicity of the micelles to heart and kidney, after the mice were sacrificed, the hearts and kidneys were isolated, fixed with paraformaldehyde for 24 h, and their histologic sections were stained by hematoxylin and eosin (H&E) for histopathological examination.



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2.13. Statistical Analysis. The statistical significance of differences among more than two groups was analyzed using one-way ANOVA. A statistically significant difference was set at p≤0.05. 3. RESULTS

Figure 1．．1H NMR spectra of PEOz-OH (A), PEOz-PLA (B), PEOz-PLA-CHO (C) in CDCl3 and CBA (D), PEOz-PLA-imi-DOX (E), DOX (F) in DMSO-d6. (G) The gel permeation chromatograms of PEOz-OH (G1), PEOz-PLA (G2) and PEOz-PLA-imi-DOX (G3). 3.1 Synthesis and Characterization of PEOz-PLA-imi-DOX Conjugate. The conjugate of DOX with PEOz-PLA via an benzoic imine linker was synthesized through a series of reactions as shown in Scheme 1.26 The 1H NMR spectra confirmed the chemical structure of intermediates and titled product (Figure 1). The peaks at 3.04 and 1.10 ppm belonged to protons of the methyl in the terminal and side chains of PEOz-OH, respectively. While the peaks at 3.40 and 2.33 ppm were attributed to protons of the methylene groups in the backbone and side chains, respectively (Figure 1A). PEOz-PLA was prepared by anionic ring-opening polymerization of D,L-LA with PEOz-OH as a macroinitiator. Besides the characteristic peaks of PEOz-OH, the peaks at 1.50 and 5.10 ppm which were assigned to protons of the methyl and methine in PLA chains were identified, respectively (Figure 1B). These results were in line with previous reports.


26, 28

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The number-average molecular weight (Mn) of the synthesized PEOz-OH and PEOz-PLA measured by GPC was 2036 g/mol with 1.25 of PDI (Figure 1G1) and 3229 g/mol with 1.22 of PDI (Figure 1G2), respectively, indicating that the synthesized polymers exhibited a narrow molecular weight distribution. Afterwards, to conjugate DOX to the end of PLA, CBA was first linked to the end of PLA by esterification of the carboxyl of the CBA with terminal hydroxyl of PEOz-PLA to form aldehyde-terminated PEOz-PLA (PEOz-PLA-CHO) in the presence of DCC and DMAP as a condense agent and catalyst, respectively. The successful synthesis was confirmed by the TLC (Supporting Information (SI) Figure S1A) and 1H NMR spectra (Figure 1C). Firstly, the absence of free CBA in the product was supported by TLC (Supporting Information (SI) Figure S1A). The characteristic peaks of CBA at 10.15, and 7.80 and 8.20 ppm corresponding to aldehyde and phenyl protons (Figure 1D), respectively, were found. Besides, the carboxyl proton peak from CBA at 13.42 ppm disappeared. According to the ratio of peak area of aldehyde proton to that of the terminal methyl protons, the conjugation efficiency was determined to be up to about 97%. Finally, the conjugation of DOX with PEOz-PLA-CHO was achieved through a Schiff's base reaction of the aldehyde group of PEOz-PLA-CHO with the amino group of DOX. TLC (Supporting Information (SI) Figure S1B) suggested the successful synthesis of PEOz-PLA-imi-DOX, in which the orange fluorescence of PEOz-PLA-imi-DOX, being similar to that of DOX, was observed at the same position of PEOz-PLA-CHO, while no orange fluorescence was found for PEOz-PLA-CHO. Furthermore, only trace of free DOX was present in the desired product after being applied to Sephadex LH-20 column. The conjugation was further supported by 1H NMR spectra. As shown in Figure 1E, besides the characteristic peaks for PEOz-PLA-CHO (1.10 and 3.04 ppm, 2.33 and 3.40 ppm, 1.50 and 5.10 ppm, 7.80 and 8.20 ppm) (Figure 1C), the peaks at 7.66 and 7.92 ppm also observed which were attributed to the phenyl protons of DOX (Figure 1F). The existence of the new proton peak at 8.40 ppm (Figure 1E), which belonged to the proton of benzoic imine,32 strongly suggested the successful synthesis of the conjugate. The successful conjugation of DOX to PEOz-PLA-CHO was also proved by GPC (Figure 1G3). The content of DOX in the conjugate, which was determined by HPLC after treatment of



PEOz-PLA-imi-DOX with acetate buffer (pH 3.40), was about 1.67%. To validate the cleavage of benzoic imine bonds in an acid environment, PEOz-PLA-imi-DOX conjugate was incubated in aqueous solution with different pH values, and the hydrolysate was analyzed by TLC. The release of DOX from the conjugate was profoundly accelerated at pH 5.0 compared with that at pH 7.4 within 24 h (Supporting Information (SI) Figure S1C) Further, DOX was completely released from the conjugate within 5 min at pH 3.4 (Supporting Information (SI) Figure S1D), which was consistent to previous report.25 These evidenced the acid sensitivity of the conjugate. The CMC of the synthesized PEOz-PLA-imi-DOX was determined to be 14.84±3.85 mg/L (Supporting Information (SI) Figure S2), indicating that the conjugate micelles might exhibit better dilution stability in systemic circulation.

Figure 2. (A) Schematic diagram of the formation for dual drug-loaded conjugate micelles. Size for PP-DOX-PM (B), HNK/PP-PM (C) and HNK5/PP-DOX1-PM (D) and transmission


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electron microscope images of HNK5/PP-DOX1-PM (E). 3.2. Physicochemical Characterization of Polymeric Micelles. Single drug-loaded micelles and dual drug-loaded micelles were prepared by film hydration method and then characterized in terms of size and its distribution, morphology, LC and EE. All the micelles had an relatively smaller size ranging from 20 nm to 22 nm with a narrow distribution (Figure 2 and Table 1), indicating that they were inclined to permeate through the vascular space to the tumor site.33 TEM images (Figure 2E) directly evidenced that HNK5/PP-DOX1-PM exhibited spherical shape and roughly homogeneous diameter. For HNK/PP-PM, the LC and EE were 3.7% and 94.1%, respectively (Table 1). For dual drug-loaded micelles, the LC of HNK/PP-DOX-PM for DOX was around 1.29-1.39%, which was lower than that of PP-DOX-PM (1.67%). As the mass ratio of DOX to HNK was decreased from 1:1 to 1:5, the LC of HNK/PP-DOX-PM for HNK was increased from 1.44% to 6.64%, while the EE was decreased from 86.6% to 57.9%, which could be explained by the limited encapsulation capacity of the conjugate micelles. Table 1. Physicochemical Characteristics (n=3), IC50 and CI (n=5) of Various Formulations. DOX

HNK

Diameter Formulation

PDI

LC

LC

EE

IC50

(nm)

CI

(%)

IC50（µg/mL)

(%)

(%)

（µg/mL)

DOXHCl

--

--

--

1.16±0.18

--

--

--

--

HNK/PP-PM

20.09±0.42

0.18±0.04

--

--

3.70±0.15

94.12±3.94

24.30±0.63

--

PP-DOX-PM

21.39±1.14

0.29±0.02

1.67±0.05

1.61±0.20

--

--

--

--

HNK1/PP-DOX1-PM

21.08±0.44

0.27±0.01

1.29±0.03

1.01±0.18b

1.44±0.04

84.41±2.24

1.12±0.21c

0.67

HNK2/PP-DOX1-PM

20.83±0.60

0.23±0.03

1.33±0.01

0.80±0.07

a

2.52±0.02

86.60±0.59

1.50±0.09

c

0.56

HNK3/PP-DOX1-PM

20.38±0.19

0.16±0.04

1.39±0.01

0.62±0.09

a

3.76±0.01

82.33±0.13

1.74±0.17

c

0.46

HNK5/PP-DOX1-PM

20.86±0.46

0.23±0.01

1.39±0.01

0.43±0.03a

6.64±0.02

57.90±1.90

2.06±0.17c

0.36

d

a: p

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