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Folate-Conjugated Polyphosphoester with Reversible Cross-Linkage and Reduction-Sensitivity for Drug Delivery Youwen Cao, Jinlin He, Jie Liu, Mingzu Zhang, and Peihong Ni ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b18887 • Publication Date (Web): 12 Feb 2018 Downloaded from http://pubs.acs.org on February 13, 2018

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ACS Applied Materials & Interfaces

Folate-Conjugated Polyphosphoester with Reversible CrossLinkage and Reduction-Sensitivity for Drug Delivery Youwen Cao, Jinlin He, Jie Liu, Mingzu Zhang, and Peihong Ni* College of Chemistry, Chemical Engineering and Materials Science, State and Local Joint Engineering Laboratory for Novel Functional Polymeric Materials, Jiangsu Key Laboratory of Advanced Functional Polymer Design and Application, Suzhou Key Laboratory of Macromolecular Design and Precision Synthesis, Soochow University, Suzhou 215123, P. R. China Supporting Information

KEYWORDS: Reduction-sensitivity, Core-crosslinked nanoparticles, Polyphosphoesters, Michael addition polymerization ABSTRACT: To improve the therapeutic efficacy and circulation stability in vivo, we synthesized a new kind of drug delivery carrier based on folic acid-conjugated polyphosphoester via the combined reactions of Michael addition polymerization and esterification. The produced amphiphilic polymer, abbreviated as P(EAEP-AP)-LA-FA, could self-assemble into nanoparticles (NPs) with core-shell structure in water and reversible core cross-linked by lipoyl groups. Using the core cross-linked FA-conjugated nanoparticles (CCL-FA NPs) to encapsulate hydrophobic anticancer drug doxorubicin (DOX), we studied the stability of NPs, in vitro drug release, cellular uptake and targeting intracellular release, compared with both uncross-linked FA-conjugated nanoparticles (UCLFA NPs) and core cross-linked nanoparticles without FAconjugated (CCL NPs). The results showed that under the condition of pH 7.4, the DOX-loaded CCL-FA NPs could maintain stable over 72 h, only a little DOX release (~15%) was observed. However, under the reductive condition (pH 7.4 containing 10 mM GSH), the disulfide-crosslinked core would be broken up and resulted in 90% of DOX release at the same incubation period. The study of methyl thiazolyl tetrazolium (MTT) assay indicated that the DOX-loaded CCL-FA NPs exhibited higher cytotoxicity (IC50: 0.33 mg L-1) against HeLa cells than the DOX-loaded CCL NPs without FA. These results indicate that the core cross-linked FA-conjugated nanoparticles have unique stability and targetability.

INTRODUCTION tensile and shear stresses within the body.15 Otherwise, once injecting intravenously in vivo, unstable nanoparticles will be easily dissociated, which usually result in antedated drug release, broad drug distribution, and a weak targeting ability of the drug. One of the effective strategies is to develop crosslinked nanoparticles. Recently, several methodologies for cross-linking have been developed and applied for drug delivery. For example, through the chemical and physical interaction to make the hydrophilic shell16,17 or hydrophobic core18-20 cross-linked.

Hydrophobic anticancer drugs, such as paclitaxel (PTX)1,2, doxorubicin (DOX)3,4 and camptothecin (CPT)5,6, play a significant role in chemotherapy. However, their high side effects, poor water solubility, nonspecific distribution and inadequate accumulation of therapeutics often limit the clinical applications.7,8 To overcome these limitations, some researchers creatively utilize amphiphilic molecules capable of self-assembling into nanoparticles (NPs) to encapsulate hydrophobic drugs.9,10 In recent years, substantial research work have been paid to introduce multiple functions within the rational design of nanoparticle.11,12

In addition, the nanoparticles with a diameter generally smaller than 200 nm can reach to tumor tissues via passive targeting by means of the enhanced permeation and retention effect (EPR).21-23 To further enhance the efficiency of cell uptake, several approaches are proposed with respect of drug delivery, including active targeting therapy, sensitization of cancer cells to therapeutic modalities and localized controlled

For drug-loaded nanoparticles, there are at least three important issues that need to be considered, i.e. enhancing the stability, increasing the active targeting and controlling drug release on demand.13,14 In general, nanoparticles should have the ability to withstand a subclassification of compressive,

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activation for release of drugs.24,25 For the active targeting therapy, many targeting molecules, such as folic acid (FA),26-28 biotin29,30 and RGD,31-33 can be used as the targets to identify cancer tissues. As a glycosylphosphatidyl-inositol-anchored (GPI-anchored) membrane glycoprotein, folic acid has been widely applied to tumor targeting for its high affinity binding to the folate receptor (Kd ≈ 1×10-10 M), which is restricted distribution in many human cancerous cells (over-expressing in HeLa cells or KB cells, high-expressing in HepG2 cells), but rarely distributed in normal cells.34,35

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dithiothreitol (DTT) was added into above-mentioned system to cross-link the core of nanoparticles. Once the cross-linked NPs were internalized into the cancer tissue, it could be effectively internalized into cancer cells taking advantage of the folate-receptor-mediated endocytosis. Both the cleavage of the cross-linked disulfide bond in a condition of high GSH concentration and the PDE I-accelerated decomposition of polyphosphoester would result in the dissociation of nanoparticles and the release of drug. This study provides a synthesis method for the fabrication of core cross-linked drug-loaded nanoparticles, which has a stable circulating administration and efficient targeting to tumor cells.

Considering the difference in glutathione (GSH) concentration between the normal tissues (GSH ≈ 2.0 to 20 μM) and cancer tissues (GSH ≈ 2 to 10 mM), it is possible to introduce some reduction-sensitive bonds, such as disulfide bond36,37 and selenium bond38 into polymers and further make them selfassemble into “smart” nanoparticles.39,40 Up to now, various kinds of “smart” nanoparticles have been utilized in drug carrier systems. For instance, Zhong et al.18,41 used functional monomers with disulfide bonds to conjugate onto polymers via the substitution reaction or esterification. The produced polymers could readily be formed reversibly stabilized multifunctional nanoparticles in water and showed reductionsensitivity. Our group also prepared a series of copolymers and prodrugs containing disulfide linkers as drug carriers or gene vectors.40,42-44 Recently, a polymeric prodrug P(EAEPPPA)-g-ss-CPT was prepared through the combination of Micheal addition reaction and “Click” chemistry, and then selfassembled into stimuli-responsive nanoparticles.45

EXPERIMENTAL SECTION Materials. The unsaturated phosphate monomer, ethyl-bis[2-

(acryloxy)ethyl] phosphate (EAEP), was prepared according to the literature method and stored under inert atmosphere.45,53 All other chemicals were purchased and can be classified into two groups. One part was used as received without further purification as follows: 3-amino-1-propanol (99%, TCI), 2hydroxyethyl acrylate (96%, Aladdin), ethyldichlorophosphate (98%, J&K Chemical), (±)-α-lipoic acid (98%, Energy Chemical), N, N′-dicyclohexylcarbodiimide (DCC, 99%, Alfa Aesar), 4-(dimethyiamino) pyricline (DMAP, 99%, 9 Ding Chemistry), folic acid (FA, 97%, Sinopharm Chemical Reagen), doxorubicin hydrochloride (DOX·HCl, 99%, Beijing Zhongshuo Pharmaceutical Technology Development), 3-(4, 5dimethylthiazol-2-yl)-2, 5-diphenylte-trazolium bromide (MTT, 98%, Sigma-Aldrich), and dithiothreitol (DTT, 99%, INALCO). The other part including solvents such as pyridine (99.5%, Shanghai Lingfeng Chemical Reagent), dimethylsulfoxide (DMSO, A. R., Enox), triethylamine (TEA, A. R., Enox), and dichlormethane (CH2Cl2, A. R., Enox) were dried over sodium sulfate anhydrous (Na2SO4, 99%, Enox) for 24 h and distilled before use.

Polymers used for drug delivery often require unique physical and chemical properties, such as versatile functionalization, low toxicity, good biocompatibility and biodegradation.46 In the past few years, many polymers have been used in biomedicine, including dextran,18,40 poly(ε-caprolactone) (PCL),47 polyphosphoesters (PPEs)48,49 and polypeptides50,51. Among those polymers, PPEs have been widely studied in drug or gene delivery. It is worth mentioning that PPEs can be degraded into some small phosphates under acidic or basic conditions. In addition, phosphodiesterase I (PDE I), which exists in the cytosome or subcellular regions of cells, can highly accelerate the process of degradation. 42,52

Synthesis of Polyphophoester Precursor P(EAEP45 AP). According to the previously reported method, the unsa-

turated monomer ethyl-bis[2-(acryloxy)ethyl] phosphate (EAEP) was first synthesized. Using this monomer to react with 3-amino-1-propanol via Michael addition polymerization, we obtained an amphiphilic polyphophoester precursor, abbreviated as P(EAEP-AP). In a typical experiment, to a dried flask equipped with a magnetic stirring bar, EAEP (322.00 mg, 1.00 mmol) dissolved in 2 mL of dry DMSO was added carefully. And then 3-amino-1-propanol (75.80 mg, 1.00 mmol) dissolved in 3 mL of dry DMSO was added dropwise into the bottle. The mixture was heated to 60 °C and kept at this temperature for 6 days under the environment of N2. After the reaction was completed, another EAEP/DMSO solution (32.21 mg, 0.10 mmol in 1 mL of dry DMSO) was injected into the above-mentioned mixture, and continued to react at 60 °C for 3 days. Afterwards, DMSO was removed via vacuum evaporation. The residue was then dissolved in 1 mL of CH2Cl2, precipitated two times using a mixed solvent of n-hexane/CH2Cl2 (10/1, v/v) and subsequently precipitated three times using cold diethyl ether. The final product P(EAEP-AP) was collected after dried in vacuum as a sticky liquid (261.74 mg, yield: 65.77%).

In this study, the idea is to create a reversibly cross-linked, reduction-sensitive and biodegradable drug delivery system. We first utilized an unsaturated monomer ethyl-bis[2(acryloxy)ethyl] phosphate (EAEP) to react with 3-amino-1propanol (AP) via Michael addition polymerization, and obtained an amphiphilic polymer P(EAEP-AP). Subsequently, the partial hydroxyl groups at the pendants of P(EAEP-AP) were modified by lipoic acid anhydride (LA) to yield a new polymer P(EAEP-AP)-LA. And the residual hydroxyl groups are further reacted with folic acid (FA) through esterification to produce a FA-conjugated polymer P(EAEP-AP)-LA-FA with lipoyl groups and folate groups in the side chains. The synthetic procedure is shown in Scheme 1. The functional amphiphilic polymer could self-assemble into multifunctional nanoparticles (NPs) in PB 7.4 solution and load anticancer drug DOX inside. The hydrophobic lipoyl groups were wrapped in the core of the nanoparticles, while the hydrophilic polyphosphoester chains formed the corona of the nanoparticles with FA on the surface. After that, a catalytic amount of

Modification of P(EAEP-AP) with Lipoic Acid Anhydride. Lipoic acid anhydride was synthesized following a pre-


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viously published procedure.54 In a dried round bottom flask with a magnetic bar, the amphiphilic polyphophoester precursor P(EAEP-AP) (397.40 mg, 1.00 mmol), lipoic acid anhydride (37.10 mg, 0.18:1 with respect to single 3-amino-1propanol in the P(EAEP-AP) polymer, and DMAP (43.90 mg, 0.36 mmol) were dissolved completely in 10 mL of dry DMSO. The solution was stirred at 25 °C for 48 h. After the reaction was completed, the mixture was filtered out, and solvent was removed via vacuum evaporation to yield the product P(EAEP-AP)-LA, which was used in the next reaction without further purification. (143.36 mg, yield: 33.12%).

Preparation of CCL-FA NPs and UCL-FA NPs. The core cross-linked FA-conjugated nanoparticles (CCL-FA NPs) were prepared by the method of solvent exchange. Briefly, P(EAEP-AP)-LA-FA (12.5 mg) was dissolved in 2 mL of DMSO in a round-bottom flask and stirred for 2 h. After that, 15 mL of Milli-Q water was added dropwise under moderate stirring at 25 °C by using an auto-sampler (WZS-FOF) with a velocity of 2 mL h-1. And the solution would be then stirred for 2 h, followed by adding 10 mol% DTT relative to the amount of lipoyl units under nitrogen at 25 °C. Finally, the mixture was stirred for another 22 h, and dialyzed (MWCO 3500) against Milli-Q water for 1 day. The concentration of the nanoparticles in Milli-Q water was kept at 0.51 mg mL-1. The uncross-linked FA-conjugated nanoparticles (UCL-FA NPs) were prepared by the same method as described above without DTT.

Synthesis of P(EAEP-AP)-LA-FA. Folic acid molecule was conjugated onto the polymer P(EAEP-AP)-LA via esterification. In a dried round bottom flask with a magnetic stirring bar, FA (9.00 mg, 0.02 mmol), DMAP (2.44 mg, 0.02 mmol) and P(EAEP-AP)-LA (117.18 mg, 0.25 mmol) were mixed and dissolved in 5 mL of DMSO. And then, a solution of DCC (4.12 mg, 0.02 mmol) was added dropwise into the mixture. The resulting mixture was reacted at 25 °C for 48 h. The solution was filtered, and then the filtrate was precipitated in cold diethyl ether three times to yield the product P(EAEP-AP)-LAFA (59.60 mg, yield: 47.23%). 1

Characterizations. H NMR and

Characterizations of Nanoparticles. The average par-

ticles sizes ( Dz ) and size polydispersity index (size PDI) of the nanoparticles were analyzed using a dynamic light scattering (DLS) instrument (Nano-ZS90, Malvern) with noninvasive backscattering technology detected at 90. Before measurements, the samples were filtered by a Φ 0.45 m microfilter to remove dust particles.

13

C NMR spectra were recorded on a 400 MHz spectrometer (INOVA-400, Varian) using deuterated chloroform (CDCl3) as the solvent and tetramethylsilane (TMS) as the internal standard. The gel permeation chromatography (GPC) curve with the number-average molecular weights (M̅n), weight-average molecular weights (M̅w) molecular weight distributions (PDIs) were analyzed by an instrument (HLC-8320, TOSOH), which was equipped with refractive-index and UV detectors using two TSK gel Super Multipore HZ-N (4.6×150 mm, 3 μm beads size) columns arranged in series, and it could separate polymers in the molecular weight range from 500 ~ 1.9×10 5 g mol-1. The solution of DMF with 0.01 mol L−1 LiBr was used as the eluent. The flow rate was set as 0.60 mL min-1 and the temperature at 40 °C with polystyrene as the standard. An instrument of UVVis spectrophotometer (UV-3150, Shimadzu) was used to measure the UV-Vis absorption.

Transmission electron microscope (TEM, HT7700, 120 kV, Hitachi) was used to observe the morphologies of those samples which were prepared as same as that described in the study of particle size analysis. For this test, a freeze-drying method was used to gain the TEM sample. Specifically, one drop of the corresponding sample solution was added onto the carbon-coated copper grid frozen by liquid nitrogen, and the solvent was then directly removed by lyophilization in freezedrier.55 The morphologies of nanoparticles were imaged by the TEM instrument above-mentioned at room temperature. In Vitro DOX Loading and Drug Release Studies.

DOX·HCl (8 mg) was first dissolved in 0.3 mL of DMSO, and then 3 mL of triethylamine (TEA) was added into the solution. After stirring for 10 min, the upper layer of TEA solution was removed, and DOX/DMSO solution was obtained. This step was repeated three times. Subsequently, DOX-loaded CCL-FA NPs were prepared as follows. Briefly, P(EAEP-AP)-LA-FA (12.5 mg) and DOX/DMSO solution (0.3 mL) were dissolved in 2 mL of DMSO and stirred for 2 h. After that, 15 mL of Milli-Q water was added into the solution by using an autosampler (WZS-FOF) with a velocity of 2 mL h-1. The mixture was then stirred for 2 h, followed by adding 10 mol% DTT relative to the amount of lipoyl units under nitrogen at 25 °C. Finally, the mixture was stirred for another 22 h, and dialyzed (MWCO 3500) against Milli-Q water for 1 day to remove superfluous solvent of DMSO and free DOX. The concentration of the nanoparticles in Milli-Q water was kept at 0.51 mg mL-1. To assess the DOX loading content (DLC) and drug loading efficiency (DLE), 4 mL of DMSO was added into 1 mL of the sample solution. The final solution was measured by fluorescence spectrophotometer (Cary Eclipse, Agilent Technologies) with excitation wavelength at 480 nm over a wavelength range from 500 nm to 650 nm, and the slit width was set at 5 nm. The drug loading content (DLC) and drug loading efficiency

Self-Assembly Behavior of P(EAEP-AP)-LA-FA. The critical aggregation concentration (CAC) of P(EAEP-AP)-LAFA was determined by a fluorescence probe method using pyrene as the hydrophobic probe. Typically, 50 µL of predetermined pyrene solution in acetone (6×10-6 mol L-1) was added into a series of ampoules, and then the solvent of acetone was removed via vacuum evaporation. To each ampoule, different concentrations of the P(EAEP-AP)-LA-FA aqueous solutions (5 mL) were added and stirred for 48 h at 25 °C. After that, the mixture solutions were analyzed using a fluorescence spectrophotometer (Cary Eclipse, Agilent Technologies) to detect the variational fluorescence strength of pyrene, in which the excitation wavelength was kept at 335 nm and emission spectra were collected over a wavelength range from 350 to 550 nm with a 2.5 nm slit width at middle voltage. The data recorded by fluorescence spectrophotometer were drawn by Origin 8 software (Y-axis: the intensity ratio of I383/I373, Xaxis: the emission spectra range) and the CAC value was determined as the concentration of the crossover point in the low concentration range.



(DLE) of the DOX-loaded CCL-FA NPs were calculated according to Eqs (1) and (2): DLC 

Weight of DOX loaded in nanoparticles 100 Weight of polymer

(1)

DLE 

Weight of DOX loaded in nanoparticles 100 Weight of DOX in feed

(2)

without nanoparticle sample, respectively. The data are gathered and processed as the average values with standard deviations. Cellular Uptake and Intracellular DOX Release. The cellular uptake and intracellular release in HepG2 and HeLa cells of free DOX and DOX-loaded CCL-FA NPs were investigated by a live cell imaging system (CELL‟R, Olympus). Typically, cells were seeded in a glass Petri dish (Φ=35 mm) with a density of 5×104 cells cm-2 and cultured in high glucose DMEM culture medium at 37 °C with an atmosphere containing 5% CO2 for different times. After that, removed the culture medium from the glass Petri dish, and washed the cells three times using PBS and stained them with Lyso-Tracker Red (1 μL mL-1) for 1 h, followed by washing the cells three times using PBS. After that, the culture medium was then replaced by samples containing free DOX or the DOX-loaded CCL-FA NPs (0.5 mg L-1 of DOX). Every 30 min, the images were caught at excitation wavelengths of 480 nm (red) and 340 nm (blue).

The in vitro release of DOX from DOX-loaded CCL-FA NPs with a concentration of 1 mg mL-1 was investigated by a dialysis method. Three different media were prepared to simulate different human body environments. They were individually made up of (i) phosphate buffer (PB) 7.4, (ii) PB 7.4 containing 2 µM GSH, and (iii) PB 7.4 containing 10 mM GSH. Typically, each 5 mL of DOX-loaded CCL-FA NPs in Milli-Q water was transferred into a dialysis membrane (MWCO 3500), and the dialysis membrane was then placed in a centrifuge tube containing 20 mL of the corresponding PB solution. Those centrifuge tubes were kept constantly shaking with a speed of 160 rpm at 37 °C. At the preset time interval, partial solution (5 mL) was extracted and replaced with 5 mL of the corresponding PB solution. Fluorescence spectrophotometer with excitation at 480 nm and slit width at 5 nm over a wavelength range from 500 nm to 650 nm was carried out to determine the content of the released DOX.

Flow Cytometry Analysis. The flow cytometry analysis were recorded by a flow cytometry (Cytomics FC500, Beckman Coulter). Typically, HeLa cells were cultured in a 35 mm culture dish with the density of 5×104 cells cm-2 and adhered for 12 h. After that, 1 mL of fresh medium (either FAconjugated or not conjugated with the DOX-loaded CCL NPs, the final DOX concentration of was kept at 0.3 mg L-1) was prepared to replace the culture medium. At the designed time, the culture medium was removed and the cells were washed three times with PBS. Subsequently, the cells were then digested with trypsin, each culture dish was added with 2 mL of new culture medium. After that, the solutions were centrifuged at 1200 rpm for 5 min. At last, the residual cells were dispersed by adding with 0.5 mL of PBS and the data were obtained via the flow cytometry analysis.

Cell Culture. Mouse fibroblasts cells (L929 cells), Hela human cervical carcinoma cell (HeLa cells) and human hepatocellular carcinoma cells (HepG2 cells) were purchased from American Type Culture Collection (ATCC). These cells were cultured in high glucose DMEM, in which 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution were contained. The cells were passaged once every 2 days, and both those cells were incubated at 37 C in an atmosphere containing 5% CO2 and certain humidity. In Vitro Cytotoxicity Test. The cytotoxicity of nanoparticles against L929 cells, HeLa cells and HepG2 cells were evaluated individually by a methyl thiazolyl tetrazolium (MTT) assay using free DOX as the control. The cells were seeded in 96-well plates, in which each well was added 100 L of high glucose DMEM medium, and cells were incubated at 37 C in a 5% CO2 atmosphere for 12 h. The density of cells was set at 4104 cells per well. Then, 25 L of the nanoparticle solutions were added into each well to achieve different concentrations. The solutions of these nanoparticles included free DOX, DOX-loaded CCL-FA NPs, CCL-FA NPs without DOX, and DOX-loaded CCL NPs without FA. After 48 h incubation, each well was added with 25 L of MTT stock solution (5 mg mL-1) in PBS. After incubated at 37 C for 4 h, DMEM was removed and each well was added with 150 L of DMSO to dissolve the purple formazan. The absorbance of each well at 570 nm was measured by a microplate reader (Bio Rad 680, USA) to get the optical density (OD) values. The cell viability was calculated by Eq (3): Cell Viability (%) 

ODtreated 100 ODcontrol

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RESULTS AND DISCUSSION Synthesis and Characterization of P(EAEP-AP)-LAFA. In this study, a functional polyphosphoester P(EAEP-AP)-

LA-FA were prepared through three steps and the synthetic routes are shown in Scheme 1. Firstly, the polyphosphoester precursor, abbreviated as P(EAEP-AP), was prepared by Michael addition polymerization. Secondly, lipoic acid anhydride was conjugated to P(EAEP-AP) via esterification. Thirdly, the targeting molecule folic acid (FA) was connected onto P(EAEP-AP)-LA to yield the final product P(EAEP-AP)-LAFA. The functional polymer P(EAEP-AP)-LA-FA could selfassemble into nanoparticles and further form reversible core cross-linked and folate-targeted nanoparticles (CCL-FA NPs) because the disulfide bonds in lipoyl groups could be broken up by a catalytic concentration of dithiothreitol (DTT) and then reconnected to form a complex network. The anti-cancer drug DOX was also encapsulated into the CCL-FA NPs. Finally, the cross-linked core could be disrupted under an intracellular reductive environment, leading to the disassociation of nanoparticles and a rapid release of DOX.

(3)

where, ODtreated and ODcontrol stand for the OD values of the wells treated with nanoparticle samples and the control wells


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Scheme 1. Synthesis routes of the FA-conjugated PPEs of P(EAEP-AP)-LA-FA.

The actual chemical structure of EAEP was verified by 1H NMR analysis, which was shown in Figure 1(A). Figure 1(B) shows the 1H NMR spectrum of polymer P(EAEP-AP), in which some new chemical shifts appear at δ 2.50 ppm (signal i), δ 2.80 ppm (signal j), δ 2.60 ppm (signal k) and δ 4.30 ppm (signal h). Figure 1(C) shows the new chemical shifts of P(EAEP-AP)-LA at δ 1.48 ppm (signals 3, 4), δ 1.68 ppm (signals r, 2, 3, 4), δ 1.92 ppm (signal 6), δ 2.32 ppm (signal 1), δ 2.46 ppm (signal 6), δ 3.20 ppm (signal 7), δ 3.58 ppm (signal 5) and δ 4.06 ppm (signal q), respectively. In addition, the chemical structures of EAEP, P(EAEP-AP) and P(EAEP-AP)LA were also characterized by 13C NMR spectra as shown in Figure S1 of Supporting Information. All these indicate that LA have been conjugated onto the side chains of P(EAEP-AP) via esterification, and the functional polymers P(EAEP-AP)LA has been successfully prepared.

AP)-LA-FA and P(EAEP-AP) in their peak shapes ranging from 335 nm to 400 nm, which could be attributed to the existence of LA and FA moieties. According to the 1H NMR spectrum of P(EAEP-AP) in Figure 1(B), the number of repeat unit (n) was calculated by the Eq (4), where Ag and Aa represent the integral values of the peak g and peak a, respectively. Every repeat unit is composed of EAEP monomer conjugated with 3-amino-1-propanol, and the both ends of the polymer chain were capped by C=C double bonds. The molecular weight of P(EAEP-AP) ( M n,NMR ) could be obtained according to Eq (5), where, 397.5 is the theoretic molecular weight of each repeating unit of P(EAEPAP); 322.5 is the molecular weight of EAEP, respectively. n

As shown in Figure S2 of Supporting Information, the chemical structure of P(EAEP-AP)-LA-FA was analyzed by UV-Vis. There was a notable difference between P(EAEP-

2Ag 1 3 Aa

(4)

M n  397.5  n+322.5 (5)



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molecular weights obtained from GPC are much smaller than those from 1H NMR. This is because that the structure of polyphosphoester containing many tertiary amines in the backbone is different from that of polystyrene in GPC standard. In our recent research, we have found that the polyphosphoesters with many tertiary amines in the main chain may have strong interaction with column material, leading to extended elution time and reduced molecular weight during GPC analysis.45 Herein, the pendent hydroxyl groups in the polymer backbone of P(EAEP-AP) would reinforce this interaction and result in the large differences between molecular weights from the different analyses. Self-Assembly of Polymer and Core Cross-Linking.

In aqueous solution, the polymer P(EAEP-AP)-LA-FA can self-assemble into nanoparticles with LA segments as the hydrophobic core and P(EAEP-AP) as the hydrophilic shell. The value of critical aggregation concentration (CAC) of P(EAEPAP)-LA-FA was detected by the pyrene fluorescence probe. The CAC value can be used to indicate the stability of nanoparticles in aqueous solution. As shown in Figure S4, the CAC value of the P(EAEP-AP)-LA-FA nanoparticles is 63 mg L-1. The morphologies of nanoparticles were observed by TEM analysis, while the average particle diameters ( Dz ) and size PDIs were determined by DLS. Figure 2(A) and (C) show the morphologies of uncross-linked FA-conjugated nanoparticles (UCL-FA NPs) and core cross-linked FA-conjugated nanoparticles (CCL-FA NPs), respectively. Figure 2(B) and (D) show the histograms of corresponding particle size distributions to the samples in (A) and (C), respectively. There are different particle diameters and PDIs between CCL-FA NPs and UCLFA NPs, which can be mainly ascribed to the rebuilding of disulfide bonds in the hydrophobic core of CCL-FA NPs.

Figure 1. 1H NMR spectra of (A) EAEP, (B) P(EAEP-AP) and (C) P(EAEP-AP)-LA in CDCl3.

Table 1. Characterization data of molecular weights and PDIs of a series of P(EAEP-AP) polymers. Sample

Mn

a)

Mn

b)

Mw

b)

PDI

b)

(g mol-1)

(g mol-1)

(g mol-1)

P(EAEP-AP)-1

9300

2700

5600

2.08

P(EAEP-AP)-2

10700

2600

4900

1.84

P(EAEP-AP)-3

10300

2700

5100

1.85

P(EAEP-AP)-4

10300

2100

3100

1.50

P(EAEP-AP)-5

8800

1800

2300

1.34

P(EAEP-AP)-6

9400

2500

3700

1.47

P(EAEP-AP)-7

9900

2400

4000

1.69

P(EAEP-AP)-8

9800

2300

4000

1.81

a)

1

Calculated from H NMR spectra. (solvent: CDCl3).

b)

Determined by GPC (eluent: DMF; standard: polystyrene).

The molecules weights ( M n,NMR , M n,GPC , M w,GPC ) and moFigure 2. TEM images of the nanoparticles: (A) UCL-FA nanoparticles, (C) CCL-FA nanoparticles. (B) and (D) are the particle

lecules weight distributions (PDIs) of P(EAEP-AP) are listed in Table 1. P(EAEP-AP)-8 sample was selected for the following research. Its GPC curve is shown in Figure S3, from which one can find that the polymer exhibits a unimodal distribution with an acceptable PDI value, indicating the successful synthesis of P(EAEP-AP). From Table 1, it is noteworthy that the

size distributions histograms of the samples in (A) and (C), respectively.


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ACS Applied Materials & Interfaces mM GSH was added into the medium, the particle size had an obvious change, as shown in Figure 3(B). It could be ascribed to the rupture of the nanoparticle core in the presence of GSH. Under the confluence of mechanical forces, the unstable NPs would be constantly broken up and recombined. Furthermore, polyphosphoesters could be degraded into phosphates and dihydric alcohol in the presence of phosphodiesterase I (PDE I), which may decrease the ratio of the hydrophobic and hydrophilic segments. This would result in the fast disintegration of NPs and formation of aggregates with larger diameter.56 As shown in Figure 3(C), multimodal size distribution and larger aggregates were observed under the condition of PDE I treatment. These results suggest that the CCL-FA nanoparticles can be of both reduction-sensitive degradation and enzyme degradation. To further verify the stability of CCL-FA NPs, the nanoparticles were dissolved in DMF and analyzed by TEM and by DLS analysis. Specifically, Figure S5(A) shows the image of CCL-FA NPs obtained from TEM, from which one can see that the nanoparticles were swelled with a broad size PDI. Also, the histograms of the corresponding size distribution in Figure S5(B) displays a broad dispersity, while the UCL-FA nanoparticles were completely dissolved in DMF, since no obvious nanoparticles can be observed, as shown in Figure S5(C). In Vitro Encapsulation and Release of DOX. The hydrophobic DOX was loaded into the hydrophobic core of nanoparticles via a dialysis method. In this study, the DLC and DLE values were calculated as 11.89% and 57.16%. Figure 4 shows the morphology, average particle diameters and size PDI of the DOX-loaded CCL-FA NPs in Milli-Q water. One can find that the spherical nanoparticles with a narrow particle size distribution were mainly formed.

Figure 3. Reduction- or enzyme-induced size changes of CCL-FA NPs as determined by DLS under various conditions of (A) pH 7.4 (B) pH 7.4, 10 mM GSH, and (C) pH 7.4, 10 mM GSH with PDE I.

Figure 4. (A) TEM image of the DOX-encapsulated CCL-FA nanoparticles in PB 7.4 solution (scale bar = 200 nm), (B) the particle size distribution histogram of the sample used for TEM analysis.

The formation of new covalent bonds in the hydrophobic cores has been considered as an important strategy to stabilize nanoparticles and prevent their dissociation during the blood circulation process. To verify the stability of CCL-FA NPs, three kinds of PB media, including pH 7.4, pH 7.4 containing 10 mM GSH, and pH 7.4 with both 10 mM GSH and PDE I, were used to monitor the size change of the CCL-FA nanoparticles by DLS measurement. In Figure 3(A), as a control at pH

To further compare the effect of core cross-linkage on the nanoparticle stability, two DOX-loaded samples were prepared, named DOX-loaded CCL-FA NPs and UCL-FA NPs. Their in vitro releases of DOX were studied. As shown in Figure 5(A), after dialysis of 100 h at pH 7.4 without GSH, approximately 89% of DOX was released from the UCL-FA NPs, much higher than that of the DOX-encapsulated CCL-FA NPs (15%), which can be attributed to the strong stability of core crosslinked nanoparticles.

7.4, the D z of CCL-FA nanoparticles changed little with the increasing time, until over 48 h, indicating that the nanoparticles were relatively stable in the pH 7.4 condition. When 10



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DOX-encapsulated CCL-FA NPs for 48 h and assessed by the MTT assays, using DOX-loaded CCL NPs without FA and free DOX as the references. As can be found in Figure 7, the DOX-encapsulated CCL-FA NPs (IC50: 0.33 mg L-1) exhibited higher cytotoxicity against HeLa cells at 48 h than the DOXloaded CCL NPs without FA (IC50: 1.5 mg L-1), demonstrating that FA enhanced the targeting ability of NPs on internalization of HeLa cells.

Figure 6. Cell viability of three kinds of cells treated with the blank CCL-FA NPs with different concentrations. All the incubation time was 48 h.

Figure 5. (A) In vitro release of DOX of UCL-FA NPs (Black) and CCL-FA NPs (Red) in PB at 37 °C. (B) In vitro release of DOX of CCL-FA NPs in PB at 37 °C in the presence of 10 mM GSH (Blue), 2 μM GSH (Black) and without GSH (Red). All nanoparticle concentrations were set at 1 mg mL-1.

Considering the different glutathione concentrations between the normal tissues and cancer tissues, in this study, we selected three systems to evaluate the capacity of the drug release for the DOX-loaded CCL-FA NPs. The results are shown in Figure 5(B). Under the condition of PB 7.4 with 2 µM GSH, which was similar to the normal human body environments, approximately 20% of DOX was released. However, when the GSH concentration was increased to 10 mM, nearly 90% of DOX loaded in the CCL-FA NPs was released, which further verified the reduction-sensitive disulfide bond scission.

Figure 7. Cell viability of HeLa cells treated with free DOX, DOX-loaded CCL-FA NPs, and DOX-loaded CCL NPs with different DOX dosages for 48 h incubation.

In Vitro Cytotoxicity. Good cytocompatibility is a significant requirement for NPs used for drug delivery. In this study, MTT assays were used to study the cytotoxicity of the CCLFA nanoparticles against normal cells (L929 cells) and cancer cells (HeLa cells, HepG2 cells). In Figure 6, the CCL-FA nanoparticles showed minimal cytotoxicity against the three cells, even if the concentrations of CCL-FA nanoparticles were up to 250 mg L-1, indicating that the CCL-FA NPs possess good biocompatibility as a drug carrier.

Cellular Uptake. HeLa cells and HepG2 cells were used to evaluate the cellular uptake behavior for the DOX-loaded CCL-FA nanoparticles since folate receptor was overexpressed in HeLa cells but high-expressed in HepG2 cells.34,35. The process was monitored by a live cell imaging system and the results are shown in Figure 8. Figure 8(A) belongs to the HeLa cells treated with DOX-loaded CCL-FA NPs for different times. The DOX fluorescence looks stronger because there were over-expressed folate receptors on the HeLa cells surface. By comparison, DOX-loaded CCL-FA NPs were slowly internalized into HepG2 cells (see Figure 8(B)). In addition, we

According to the design, the DOX-loaded CCL-FA NPs can be internalized by cells and then release DOX to inhibit the growth of tumor cells. HeLa cells possess over-expressed folate receptors on the cell surface. They were treated with the


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also carried out the competition experiments under the same condition as described in Figure 8(A) by adding additional free folic acid (10 mM) simultaneously. As a result, the endocytosis of DOX-loaded CCL-FA NPs was significantly reduced and weak fluorescence was detected as shown in Figure 8(C). As an additional control, HeLa cells were incubated with free DOX for 6 h, as shown in Figure 8(D, iv). In order to better illustrate the difference of the DOX fluorescence intensity, we combined this image with the other three images that cells were incubated for the same time, as can be observed in Figure 8(D). The DOX fluorescence in HeLa cells, which were incubated with DOX-loaded CCL-FA NPs for 6 h (see Figure 8(D, ii)), was obviously stronger than the other three images. This result further demonstrates that DOX-loaded CCL-FA nanoparticles possess good targeting ability.

lized DOX-encapsulated CCL-FA NPs by HeLa cells increased with different treatment times (1 h, 3 h, and 5 h), marked F-1 h, F-3 h and F-5 h, respectively. Compared with DOX-loaded CCL NPs without folate group for 5 h of incubation (N-5 h), the DOX-loaded CCL-FA NPs had significantly higher celluar uptake, implying that FA could enhance the phagocytosis of DOX-loaded CCL-FA NPs by FA receptor-overexpressing HeLa cells.

Figure 9. Flow cytometry curves of HeLa cells treated with (F) DOX-loaded CCL-FA NPs and (N) DOX-loaded CCL NPs for different times with a certain concentration of DOX (0.3 mg L-1).

CONCLUSIONS In this study, a combination of Michael addition polymerization and esterification was utilized to synthesize a polyphosphoester-based FA-conjugated copolymer P(EAEP-AP)-LAFA. The amphiphilic copolymer can form nanoparticles in water with hydrophobic lipoyl side chains as the core and hydrophilic P(EAEP-AP) as the shell. The CCL-FA nanoparticles were prepared by rebuilding disulfide bonds with DTT and exhibited DLC and DLE values of 11.89% and 57.16%, respectively. The DOX-loaded CCL-FA nanoparticles possessed high stability and good reduction-cleavable property. They could keep stability in PB 7.4 but fast dissociated in the environment of PB 7.4 with high concentration of GSH, and finally resulted in the released of DOX. In addition, in vitro cytotoxicity and degradation results indicated that the CCL-FA nanoparticles based on polyphosphoesters had favorable biocompatibility and biodegradability. By cellular uptake and flow cytometry analyses, we demonstrated that the DOXloaded CCL-FA nanoparticles had targeting ability and effective release of DOX into HeLa cells due to the presence of large amount of folate receptor on the HeLa cells surface. We believe that the new kind of DOX-loaded CCL-FA nanoparticles will be highly promising for targeted cancer chemotherapy.

Figure 8. Images obtained from live cell imaging system: (A) HeLa cells treated with the DOX-encapsulated CCL-FA NPs, (B) HepG2 cells treated with the DOX-encapsulated CCL-FA NPs, (C) HeLa cells treated with the DOX-encapsulated CCL-FA nanoparticles plus 10 mM FA, (D) combination of (i) HepG2 cells treated with the DOX-encapsulated CCL-FA NPs for 6 h, (ii) HeLa cells treated with the DOX-encapsulated CCL-FA NPs for 6 h, (iii) HeLa cells treated with the DOX-encapsulated CCL-FA NPs plus 10 mM FA for 6 h, (iv) HeLa cells treated with free DOX for 6 h. For each group, blue color shows the cell nuclei stained with H 33342, red color shows the DOX fluorescence in cells, and the rightmost show overlays of the other two images. Each scale bar in the image is 50 μm.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami. xxxx

Flow cytometry analysis was utilized to further study the effect of FA in cellular uptake. Figure 9 reveals that the relative geometrical mean fluorescence intensities of the interna-



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The 13C NMR spectra of EAEP, P(EAEP-AP) and P(EAEPAP)-LA; UV-Vis spectra of LA, FA, P(EAEP-AP), P(EAEPAP)-LA-FA; the GPC curve of P(EAEP-AP)-8; the CAC, TEM and DLS measurements of the nanoparticles

AUTHOR INFORMATION Corresponding Author * Tel: +86 512 65882047; E-mail: [email protected]

ORCID Jinlin He: 0000-0003-3533-2905 Peihong Ni: 0000-0003-4572-3213

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS We gratefully thank for the financial supports from the National Natural Science Foundation of China (21374066), the Major Program of the Natural Science Project of Jiangsu Higher Education Institutions (15KJA150007), Natural Science Foundation of Jiangsu Province (BK20171212), a Project Funded by the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions, and Soochow-Waterloo University Joint Project for Nanotechnology from Suzhou Industrial Park. We are also grateful to Professor Jian Liu (FUNSOM, Soochow University) for his valuable help in the cell-related tests. REFERENCES (1) Nehate, C.; Jain, S.; Saneja, A.; Khare, V; Alam, N.; Dubey, R. D.; Gupta, P. N. Paclitaxel Formulations: Challenges and Nnovel Delivery Options. Curr. Drug Deliv. 2014, 11, 666-686. (2) Wani, M. C.; Taylor, H. L.; Wall, M. E.; Coggon, P.; McPhail, A. T. Plant Antitumor Agents. VI. The Isolation and Structure of Taxol, a Nnovel Antileukemic and Antitumor Agent from Taxus Brevifolia. J. Am. Chem. Soc. 1971, 93, 2325-2327. (3) Wei, L.; Cai, C. H.; Lin, J. P.; Chen, T. Dual-Drug Delivery System Based on Hydrogel/Micelle Composites. Biomaterials 2009, 30, 2606–2613. (4) Li, J. F.; Yang, H. Y.; Zhang, Y. J.; Jiang, X. T.; Guo, Y. B.; An, S.; Ma, H. J.; He, X.; Jiang, C. Choline Derivate-Modified Doxorubicin Loaded Micelle for Glioma Therapy. ACS Appl. Mater. Interfaces 2015, 7, 21589-21601. (5) Hertzberg, R. P.; Caranfa, M. J.; Hecht, S. M. On the Mechanism of Topoisomerase I Inhibition by Camptothecin: Evidence for Binding to an Enzyme-DNA Complex. Biochemistry 1989, 28, 4629-4638. (6) Opanasopit, P.; Yokoyama, M.; Watanabe, M.; Kawano, K.; Maitani, Y.; Okano, T. Block Copolymer Design for Camptothecin Incorporation into Polymeric Micelles for Passive Tumor Targeting. Pharm. Res. 2004, 21, 2001-2008.


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This article focuses on the synthesis of folic acid-conjugated polyphosphoester and its core cross-linking nanoparticles as drug carrier with stability, targetability, and anticancer effect. 295x124mm (300 x 300 DPI)

ACS Paragon Plus Environment


Scheme 1. Synthesis routes of the FA-conjugated PPEs of P(EAEP-AP)-LA. 234x242mm (300 x 300 DPI)


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Figure 1. 1H NMR spectra of (A) EAEP, (B) P(EAEP-AP) and (C) P(EAEP-AP)-LA in CDCl3. 215x237mm (300 x 300 DPI)



Figure 2. TEM images of the nanoparticles: (A) UCL-FA nano-particles, (C) CCL-FA nanoparticles. (B) and (D) are the histograms of particle size distributions corresponding to the samples in (A) and (C), respectively. 199x181mm (300 x 300 DPI)


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Figure 3. Reduction- or enzyme-induced size change of CCL-FA NPs under different conditions of (A) pH 7.4 (B) pH 7.4 with 10 mM GSH, and (C) pH 7.4, 10 mM GSH with PDE I as determined by DLS. 249x540mm (300 x 300 DPI)



Figure 4. (A) TEM image of the DOX-encapsulated CCL-FA nanoparticles in PB 7.4 solution (scale bar = 200 nm), (B) the particle size distribution histogram of the sample used for TEM analysis. 197x90mm (96 x 96 DPI)


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Figure 5. (A) In vitro DOX release profiles of UCL-FA NPs (Black) and CCL-FA NPs (Red) in PB (pH 7.4, 37 °C). (B) In vitro DOX release profiles of CCL-FA NPs in PB (pH 7.4, 37 °C) in the presence of 10 mM GSH (Blue), 2 µM GSH (Black) and without GSH (Red). All nanoparticle concentrations were kept at 1 mg mL-1. 250x365mm (300 x 300 DPI)



Figure 6. Cell viability of L929 cells, HeLa cells and HepG2 cells, treated with the blank CCL-FA NPs at different concentrations. All the incubation time was 48 h. 232x170mm (96 x 96 DPI)


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Figure 7. Cell viability of HeLa cells treated with free DOX, DOX-loaded CCL-FA NPs, and DOX-loaded CCL NPs with different DOX dosages for 48 h incubation. 239x183mm (300 x 300 DPI)



Figure 8. Images obtained from live cell imaging system: (A) HeLa cells treated with the DOX-encapsulated CCL-FA NPs, (B) HepG2 cells treated with the DOX-encapsulated CCL-FA NPs, (C) HeLa cells treated with the DOX-encapsulated CCL-FA nanoparticles plus 10 mM FA, (D) combination of (i) HepG2 cells treated with the DOX-encapsulated CCL-FA NPs for 6 h, (ii) HeLa cells treated with the DOX-encapsulated CCL-FA NPs for 6 h, (iii) HeLa cells treated with the DOX-encapsulated CCL-FA NPs plus 10 mM FA for 6 h, (iv) HeLa cells treated with free DOX for 6 h. For each group, blue color shows the cell nuclei stained with H 33342, red color shows the DOX fluorescence in cells, and the rightmost show overlays of the other two images. Each scale bar in the image is 50 µm. 250x306mm (300 x 300 DPI)


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Flow cytometry curves of HeLa cells treated with (F) DOX-loaded CCL-FA NPs and (N) DOX-loaded CCL NPs for different times with a certain concentration of DOX (0.3 mg L-1). 247x180mm (300 x 300 DPI)


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