Polyphosphoester-Camptothecin Prodrug with Reduction-Response

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Polyphosphoester-Camptothecin Prodrug with Reduction-Response Prepared via Michael Addition Polymerization and Click Reaction Xueqiong Du, Yue Sun, Mingzu Zhang, Jinlin He, and Peihong Ni ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b02281 • Publication Date (Web): 05 Apr 2017 Downloaded from http://pubs.acs.org on April 6, 2017

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ACS Applied Materials & Interfaces

Polyphosphoester-Camptothecin

Prodrug

with

Reduction-

Response Prepared via Michael Addition Polymerization and Click Reaction Xueqiong Du, Yue Sun, Mingzu Zhang, Jinlin He, 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-response, Polyphosphoesters, Prodrug, Michael addition polymerization, click reaction ABSTRACT: Polyphosphoesters (PPEs), as potential candidates for biocompatible and biodegradable polymers, play an important role in material science. Various synthetic methods have been employed in the preparation of PPEs such as polycondensation, polyaddition, ring-opening polymerization and olefin metathesis polymerization. In this study, a series of linear PPEs has been prepared via one-step Michael addition polymerization. Subsequently, camptothecin (CPT) derivatives containing disulfide bonds and azido groups were linked onto the side chain of the PPE through Cu(I)-catalyzed azidealkyne cyclo-addition (CuAAC) “click” chemistry to yield a reduction-responsive polymeric prodrug P(EAEP-PPA)-g-ss-CPT. The chemical structures were characterized by nuclear magnetic resonance spectroscopy (NMR), gel permeation chromatography (GPC), fourier transform infrared (FT-IR), ultravioletvisible spectrophotometer (UV-Vis) and high performance liquid chromatograph (HPLC) analyses, respectively. The amphiphilic prodrug could self-assemble into micelles in aqueous solution. The average particle size and morphology of the prodrug micelles were measured by dynamic light scattering (DLS) and transmission electron microscopy (TEM), respectively. The results of size change under different conditions indicate that the micelles possess a favorable stability in physiological conditions and can be degraded in reductive medium. Moreover, the studies of in vitro drug release behavior confirm the reduction-responsive degradation of the prodrug micelles. A methyl thiazolyl tetrazolium (MTT) assay verifies the good biocompatibility of P(EAEP-PPA) not only for normal cells, but also for tumor cells. The results of cytotoxicity and the intracellular uptake about prodrug micelles further demonstrate that the prodrug micelles can efficiently release CPT into 4T1 or HepG2 cells to inhibit the cell proliferation. All these results show that the polyphosphoester-based prodrug can be used for triggered drug delivery system in cancer treatment.

INTRODUCTION Cancer is one of the most serious diseases faced by the humanity, resulting in increasing mortality. Among various cancer treatments, chemotherapy is the most widely used and possesses the optimal efficiency.1 As one of topoisomerase (Top) inhibitors, camptothecin (CPT) has shown potent anticancer efficacy in a broad spectrum of cancers in clinic.2-4 The mechanism of CPT killing cancer cells is to inhibit type I DNA topoisomerase for inducing apoptosis in rapidly dividing tumor cells.5,6 Unfortunately, same as other small-molecular anticancer drugs, there are still some problems, for instance, poor bioavailability, rapid blood clearance and undesirable side effects, etc, which greatly hinder the applications of CPT in cancer therapy.7,8 To overcome these limitations, drug delivery systems based on macromolecular nanoparticles have been developed and demonstrated their superiority in preclinical tumor diagnosis and therapeutic treatments.9-12 Applications of nanoparti-

cles may not only help to decrease systemic toxicity of chemotherapy, but also improve drug uptake into tumors via the enhanced permeation and retention (EPR) effect.13,14 An increasing number of nanocarriers have been considered in drug delivery system, including polymeric micelles,15-17 dendrimers,1820 nanogels,21-23 and vesicles.24,25 Among these, polymeric micelles based on amphiphilic polymers have attracted great interest due to their favorable size distribution and controlled drug release under different conditions. Two kinds of drug loading methods are generally used, that is, encapsulating drug by physical interactions and synthesizing polymeric prodrugs.26,27 The latter can improve the solubility of hydrophobic drugs, prolong the circulation time of drugs and eliminate the premature drug release.28-30 What’s the most important is that polymeric prodrugs can quantitatively control the drug loading content and possess a higher loading efficiency compared to the approach of physical encapsulation of drugs.

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It is worth mentioning that the synthetic polymers conjugated with different anticancer drugs require unique physical and chemical properties, for example, higher water solubility, better flexibility, lower toxicity and biodegradability.31 Some biocompatible and multifunctional polymers may be used in biomedicine including poly(ethylene glycol) (PEG), poly (εcaprolactone ) (PCL), polylactic acid (PLA), polyphosphoesters (PPEs) and polypeptides, etc. Of these, a wide range application of PPEs has been reported and reviewed, especially in the field of drug and gene delivery.32-34 Wooly’s group reported a series of nanoparticles with different surface charges and functionalities based on biodegradable PPEs copolymers. The functionality and property were controlled by manipulation of pendant groups on the pentavalent phosphorus atom of cyclic phospholane monomer precursors. In addition, the reactive groups in the side-chain can be taken to make further modifications via “click” chemistry. These copolymers can quickly form micelles in water and show high biocompatibility. 35-37 Our group prepared several polymeric prodrugs8,30 based on PPEs as drug and gene carriers. We synthesized a multifunctional triblock terpolymer containing poly(ethyl ethylenephosphate) (PEEP) for the targeted co-delivery of anticancer drugs and DNA. The polymers possessed reductive and pH dual sensitivity for rapid drug and gene release inside cancer cells.38 It is well known that an ideal nanocarrier usually requires specific design to achieve high tumor accumulation and efficient cellular internalization.39,40 Stimuli-responsive materials can respond to the different environments, including pH, temperature, light, redox, oxidative stress and enzymes.41-44 In general, compared to normal physiological condition (pH~7.4, glutathione (GSH)~2.0 to 20 µM), tumor tissues contain slightly acidic extracellular environment (pH~6.5 to 7.2), more acidic intracellular environment (pH~4.5 to 6.5) and higher GSH level (GSH~2 to 10 mM).45,46 Stimuli-responsive micelles which response to the specific microenvironment are relatively stable under physiological conditions but can be rapidly degraded to release anticancer drugs when reaching into tumor tissues. Up to now, there are a number of pHsensitive drug carriers containing acidic-cleavable groups such as hydrazone,47-49 acetal,50,51 oxime52 and orthoester.53,54 Owing to the different GSH concentrations between the extracellular and intracellular compartments of tumor cells, the study on reduction-sensitive materials as smart drug delivery carriers is also significant.55 In our previous work, we reported a polymer-camptothecin prodrug (PBYP-g-ss-CPT)-b-PEEP, to which a disulfide carbonate linkage was introduced with the reduction-responsive property.8 Very recently, we developed an anticancer drugs co-delivery system with a mixture of reduction-sensitive dextran-ss-camptothecin and pH-responsive dextran-hydrazone-doxorubicin.56 Although many traditional synthetic methods such as polycondensation, polyaddition, transesterification and ringopening polymerization (ROP) have been widely used, the synthesis of PPEs via Michael addition polymerization is less reported. Herein, we put forward a novel divergent approach for gaining the polyphosphoester-based camptothecin prodrug P(EAEP-PPA)-g-ss-CPT via a combination of Michael addition polymerization and CuAAC “click” chemistry. The prodrug can self-assemble into micelles, which reach to tumor tissues via blood circulation and be internalized to tumor cells

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via EPR effect. Because of the higher GSH concentration in tumor tissues as compared to the healthy tissues, disulfide bonds in the prodrug can be reduced and cleaved, resulting in the release of the parent drug CPT. The obtained polymeric prodrug has several advantages: (i) the new type of polyphosphoester is easily prepared by one-step polymerization, to which alkynyl groups can be introduced; (ii) disulfide bonds at the side chain of the polymer have reduction-responsive capacity and allow the micelles to dissociate in tumor cells for controlled CPT release; (iii) compared with parent drug, polymeric prodrug can increase water solubility, decrease side effects and improve drug utilization. EXPERIMENTAL SECTION Materials. Triethylamine (TEA) and pyridine were purchased

from Sinopharm Chemical Reagent and distilled before use. 2,2’-dithiodiethanol (Sigma-Aldrich), α-bromoisobutyryl bromide (98%, Sigma-Aldrich), triphosgene (99%, J&K Chemical), 4-dimethylaminopyridine (DMAP, 99%, Shanghai Medpep), Camptothecin (CPT, 99%, Beijing Zhongshuo Pharmaceutical Technology Development), sodium azide (NaN3, Sinopharm Chemical Reagent), copper(Ⅱ) sulfate pentahydrate (CuSO4⋅5H2O), (+)-sodium L-ascorbate (99%), ethyl dichlorophosphate (98%, J&k Chemical), 2-hydroxyethyl acrylate (96%, Aladdin) and 2-propynylamine (98%, Shanghai Macklin Biochemical) were used without further purification. Dichloromethane (CH2Cl2) and N, N-dimethyl-formamide (DMF) were distilled under reduced pressure before use. Tetrahydrofuran (THF) was dried over KOH for at least two days and then refluxed over a sodium wire with benzophenone as an indicator until the color turned purple. All the other chemicals were analytical reagents and used as received unless otherwise mentioned. Synthesis of Reduction-responsive and Clickable CPT Derivative (CPT-ss-N3). According to the previously

reported methods,8,57 2,2’-dithiobis[1-(2-bromo-2methylpropionyloxy) ethane] (HO-ss-Br) was first synthesized by monoesterfication between 2,2’-dithiodiethanol and αbromoisobutyryl bromide. And then, the transparent viscous liquid of 2,2’-dithiobis[1-(2-azido-2-methylpropionyloxy) ethane] (HO-ss-N3) was prepared. Afterwards, in a three necked flask, CPT (0.67 g, 1.92 mmol), DMAP (0.72 g, 5.85 mmol) and triphosgene (0.19 g, 0.64 mmol) were dissolved using 50 mL dry CH2Cl2 under nitrogen atmosphere and the solution was stirred at room temperature for 1 h. Then, the solution of HO-ss-N3 (0.62 g, 2.34 mmol) was added dropwise into the flask, which was further stirred for 12 h. After filtration, the solution was respectively wasted with 1.0 M HCl solution and sodium chloride (NaCl) aqueous solution for twice. The organic phase was dried over anhydrous Na2SO4. The filtrate was concentrated and further purified by silica gel column chromatography using ethyl acetate as the eluent. Finally, a pale yellow powder was achieved (CPT-ss-N3, 0.39 g, yield: 33.0%). Synthesis of Ethyl-bis(2-(acryloxy)ethyl) Phosphate (EAEP). The unsaturated EAEP monomer was synthesized

according to previous literatures.58 To a dried round-bottomed flask equipped with a magnetic stirring bar, 2-hydroxyethyl

2


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acrylate (4.82 g, 41.51 mmol) and pyridine (3.28 g, 41.47 mmol) were added and dissolved in 50 mL dry CH2Cl2 under nitrogen atmosphere. After that, a solution of ethyl dichlorophosphate (3.07 g, 18.84 mmol) in 20 mL of dry CH2Cl2 was added dropwise into the reactor under 0 °C. The reaction was further stirred overnight at 35 °C. After removing the white precipitate by filtration and the solvent by rotary evaporation, respectively, the crude product was purified by silica gel column chromatography using ethyl acetate/petroleum ether (4/1, v/v) as the eluent. The final product was collected as a clear colorless liquid. (EAEP, 3.75 g, yield: 52.4%).

chromatography (HPLC) (UltiMate 3000, Thermo Fisher Scientific) at 30 °C with acetonitrile/Milli-Q water (75/25, v/v) as the mobile phase at a flow rate of 1.0 mL min-1. The ultraviolet–visible (UV-Vis) absorption spectra were conducted at 365 nm on a UV-Vis spectrophotometer (UV-3150, Shimadzu) and the fluorescence spectra were recorded on a fluorescence spectrophotometer (Cary Eclipse, Agilent Technologies). Self-assembly Behavior of Prodrug. The critical aggregation concentration (CAC) of polymeric prodrug P(EAEPPPA)-g-ss-CPT was measured by the fluorescence probe method on a Agilent Cary Eclipse spectrophotometer using pyrene as the fluorescence probe. Typically, 50 µL of a predetermined pyrene solution in acetone were added into a series of ampoules. After acetone was removed under vacuum evaporation, 5 mL of aqueous polymer solution with different concentrations were added. The mixtures were sonicated for 20 min and further stirred for 48 h at room temperature and analyzed using a fluorescence spectrophotometer. The excitation was set at 335 nm, while emission spectra were recorded with a 2.5 nm slit width over a wavelength from 350 to 550 nm. The intensity ratio (I3/I1) of the third peak (383 nm, I3) to the first peak (373 nm, I1) from the emission spectra was analyzed as a function of the logarithm concentrations of polymeric prodrug. The CAC value was determined from the crossover point in the low concentration range.

Synthesis of Polymer P(EAEP-PPA) via Michael Addition Polymerization. The copolymer P(EAEP-PPA) was

synthesized by Michael addition polymerization.59 In a typical experiment, EAEP (0.65 g, 2.02 mmol) and 2-propynylamine (PPA) (0.11 g, 2.00 mmol) were added into a brown flask, and 8 mL CH2Cl2 was added. The solution was degassed by three exhausting-refilling nitrogen cycles. Polymerization reaction was carried out in the dark at 60 °C for 4 days. Subsequently, 10 mol % excess EAEP (0.065 g, 0.202 mmol ) was added and continued stirring for 2 days at 60 °C to ensure the resulting polymer to have acrylate terminal group on both ends. Afterwards, the solution was concentrated and precipitated three times in cold diethyl ether. The precipitate was dried under vacuum at 37 °C to yield the yellow viscous product (0.38 g, yield: 50.2%).

The average particle size ( Dz ) and size polydispersity index (size PDI) of the prodrug micelles were determined using a dynamic light scattering (DLS) instrument (Zetasizer Nano ZS, Malvern), while the morphology was observed on a transmission electron microscope (TEM) instrument (HT7700, Hitachi) operated at 120 kV. Briefly, 2.5 mg samples were dispersed in 25 mL Milli-Q water by ultrasound and further stirred for 24 h before use. The concentration of all the micellar solution was 0.1 mg mL-1. Normally, using a freeze-drying method to gain the TEM sample, 10 µL of the solution was dripped onto the carbon-coated copper grid and the solvent in its frozen solid state was directly removed without melting in a freeze-drier. The morphology of micelle was then imaged on a normal TEM at room temperature.

Synthesis of Reduction-responsive Prodrug P(EAEP-PPA)-g-ss-CPT. The reduction-responsive prodrug

P(EAEP-PPA)-g-ss-CPT was synthesized via the CuAAC ‘‘click’’ reaction.60 Briefly, CuSO4·5H2O (14.98 mg, 0.06 mmol) and (+)-sodium L-ascorbate (11.89 mg, 0.06 mmol) were first dissolved in 8 mL of anhydrous DMF in a flask under nitrogen atmosphere. After stirring for 5 min, P(EAEPPPA) (0.20 g, 0.49 mmol of alkynyl group) and CPT-ss-N3 (39.45 mg, 0.06 mmol of azido group) were sequentially added into the mixture, which was further degassed through exhausting-refilling nitrogen cycles three times. The reaction was stirred at 35 °C for 24 h. Subsequently, the solution was terminated by exposing to the air, followed by dialysis (MWCO 3500) against DMF for 2 days to remove copper ions. After precipitation by adding the polymer in DMF solution into the diethyl ether three times, the viscous liquid was collected and then dried under vacuum for 12 h to get the product P(EAEPPPA)-g-ss-CPT, (0.13 g, yield: 57.1%). 1

In Vitro CPT Release from P(EAEP-PPA)-g-ss-CPT.

The in vitro CPT release behavior of the prodrug micelles was studied in the following process. Firstly, 7 mg of P(EAEPPPA)-g-ss-CPT prodrug was mixed with 70 mL of phosphate buffer solution (10 mM, pH 7.4) and stirred for 24 h. Secondly, each 5 mL of the prodrug micellar solution was transferred into a dialysis membrane (MWCO 7000), and then the dialysis membranes were placed into a series of tubes with 25 mL of four different buffer solutions, which were phosphate buffer solution (10 mM, pH 7.4), phosphate buffer solution (10 mM, pH 7.4) with 2 µM GSH, phosphate buffer solution (10 mM, pH 7.4) with 5 mM GSH and phosphate buffer solution (10 mM, pH 7.4) with 10 mM GSH. These tubes were placed into a water bath at 37.5 °C with constant shaking. At predetermined intervals, 5 mL of the solution was taken out and replenished with an equal volume of the corresponding fresh buffer solution. Fluorescence measurement was carried out to

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Characterizations. H NMR and C NMR spectra were

recorded on the 400 MHz spectrometer (INOVA-400) using deuterated chloroform (CDCl3) as the solvent and tetramethylsilane (TMS) as internal standard. The number-average molecular weights ( M n ) and molecular weight distributions (PDIs) of P(EAEP-PPA) and P(EAEP-PPA)-g-ss-CPT were analyzed by gel permeation chromatography (GPC) instrument (HLC-8320, Tosoh) using polystyrene as the standard and DMF as the eluent. Fourier transform infrared (FT-IR) spectra were recorded on a Bruker TENSOR-27 FT-IR spectrometer using the KBr disk method. The CPT derivatives and polymeric prodrugs were determined by high performance liquid

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determine the content of the released CPT. The excitation was set at 365 nm while emission spectra were recorded with a 2.5 nm slit width over a wavelength from 390 to 550 nm.

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captured at excitation wavelengths of 560 nm (red) and 380 nm (blue). RESULTS AND DISCUSSION

Cell Culture. Mouse fibroblasts cells (L929 cells), hu-

Synthesis and Characterization of Polymeric Prodrug. In this study, the polyphosphoester-conjugated camptothecin prodrug of P(EAEP-PPA)-g-ss-CPT was prepared by the following steps as shown in Scheme 1. Firstly, an azidofunctionalized reduction-responsive CPT derivative containing disulfide carbonate group (designated as CPT-ss-N3) was synthesized. Secondly, polyphosphoester, abbreviated as P(EAEPPPA), was prepared by Michael addition polymerization. P(EAEP-PPA) is biocompatible and biodegradable copolymer and provide multiple alkynyl groups for facile modification by reacting with azido modified drugs. Finally, P(EAEP-PPA)-gss-CPT prodrugs were obtained via the CuAAC “click” reaction between CPT-ss-N3 and P(EAEP-PPA). Under the intracellular reducing environment, the disulfide carbonate group can be disrupted to allow the parent drug CPT to be released.61

man hepatocellular carcinoma cells (HepG2 cells) and mouse breast cancer cells (4T1 cells) were obtained from American Type Culture Collection (ATCC) and cultured in high glucose DMEM containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin solution. Both the cells were passaged once every 2 days and incubated at 37 °C in an atmosphere containing 5% CO2 and certain humidity. In Vitro Cytotoxicity Test. A methyl thiazolyl tetrazolium (MTT) assay was used to evaluate the cytotoxicity of the P(EAEP-PPA) and P(EAEP-PPA)-g-ss-CPT against HepG2 cells, 4T1 cells and L929 cells using Free CPT as the control. Cells were seeded in 96-well plates at a density of 4×104 cells per well in 100 µL of high glucose DMEM medium, and incubated at 37 °C in a 5% CO2 atmosphere for 12 h. Then, 25 µL of the solutions, that is, the prodrug solutions and Free CPT solutions with different concentrations, were separately added into each well. After incubated for 72 h, 25 µL of MTT stock solution (5 mg mL-1 in PBS) was added to each well. Subsequently, cells were incubated at 37 °C for additional 4 h allowing the viable cells to induce the MTT into purple formazan crystals. DMEM medium was removed and 150 µL of DMSO was added to each well. The optical density (OD) was measured on a microplate reader (Bio Rad 680, USA) at 570 nm. The cell viability was calculated by the formula: Cell viability (%) = (ODtreated/ODcontrol) × 100, where ODtreated and ODcontrol represent the OD values of the treated wells in the presence of samples and the control wells in the absence of samples. The data are presented as the average values with standard deviations.

The chemical structure of CPT-ss-N3 was verified by 1H NMR, FT-IR analysis as shown in Figure S1 and Figure S2 of the Supporting Information. Figure S1 shows the 1H NMR spectra of HO-ss-Br, HO-ss-N3 and CPT-ss-N3, respectively. All the chemical shifts ascribed to the protons of the corresponding chemical structures can be found in the 1H NMR spectra. In addition, FT-IR measurements were also used to characterize the samples as shown in Figure S2. Compared to Figure S2(A), the appearance of the absorption peak at 2108 cm-1 in Figure S2(B) and Figure S2(C) indicates that the azido groups have been successfully introduced onto the CPT structure. All the results confirm the successful synthesis of CPT derivative CPT-ss-N3. Before the alkynyl-functionalized polymer P(EAEP-PPA) was synthesized by Michael addition polymerization, an unsaturated monomer ethyl-bis[2-(acryloxy)ethyl] phosphate (abbreviated as EAEP) was first prepared. Figure 1 displayed the 31PNMR spectrum of the EAEP monomer. It can be found that only a single peak at δ -1.33 ppm appeared, which can be assigned to the phosphorus atoms in the EAEP backbone. In addition, the chemical structure of EAEP was characterized by 13 C NMR and 1H NMR as shown in Figure 2 and Figure 3, respectively. From the results of 1H NMR spectrum in Figure 3, we can find that three new chemical shifts at δ 5.89 ppm (peak 1), δ 6.44 ppm (peak 1) and δ 6.15 ppm (peak 2), respectively, can be ascribed to the protons of acrylate group. The other chemical shifts are corresponding to the protons of EAEP. These results confirm the successful synthesis of EAEP.

Cellular Uptake and Intracellular Release of CPT.

The cellular uptake and intracellular release behaviors of Free CPT and CPT prodrug in HepG2 cells were investigated by the live cell imaging system (CELL’R, Olympus). Typically, HepG2 cells were seeded in a Φ35 mm glass Petri dish at a density of 15 × 104 cells cm-2 and cultured in high glucose DMEM culture medium at 37 °C under a 5% CO2 atmosphere for different times. Afterwards, the culture medium was removed. The cells were washed with PBS three times and stained with Lyso-Tracker Red (1 µL mL-1) for 1 h, followed by washing with PBS three times. The culture medium was then replaced by fresh culture medium containing Free CPT or the CPT prodrug (0.836 mg L-1 of CPT). The images were then

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Scheme 1. Synthetic routes of (1) azide-functionalized reduction-responsive CPT derivative (CPT-ss-N3) and (2) reduction-responsive polymeric prodrug P(EAEP-PPA)-g-ss-CPT.

As shown the Step 2 in Scheme 1, P(EAEP-PPA) was synthesized by Michael addition polymerization between EAEP and PPA. Afterwards, CPT-ss-N3 was conjugated to the side chain of P(EAEP-PPA) by efficient CuAAC “click” reaction. Figure 4 shows the 1H NMR spectra of P(EAEP-PPA) and P(EAEP-PPA)-g-ss-CPT, from which all the chemical shifts assigned to the protons of the copolymer and prodrug can be observed. Compared with the 1H NMR spectrum of EAEP in Figure 3, the new proton signals appears at δ 2.25 ppm (peak 10), δ 2.52 ppm (peak 7), δ 2.86 ppm (peak 8) and δ 3.43 ppm (peak 9) in the Figure 4(A), which can be attributed to the introduction of the units of 2-propynylamine (PPA). According to the Figure 4(B), some new chemical can be ascribed to the protons of CPT. Furthermore, a new peak (10′) attributed to the proton of the triazole ring is clearly detected at δ 7.68 ppm and the proton of methyl [-(CH3)2C-] of CPT-ss-N3 at δ 1.44 ppm has shifted to δ 1.92 ppm after click reaction. These results verified that the CPT had been successfully grafted onto the PPA segment of P(EAEP-PPA).

Figure 1.

31

P NMR spectrum of EAEP in CDCl3.

Figure 2. 13C NMR spectrum of EAEP in CDCl3.

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The molecular weights ( M n ) and molecular weight distributions (PDIs) of P(EAPE-PPA) are listed in Table 1, which verify the successful synthesis of the functionalized polyphosphoesters. Figure 3. 1H NMR spectrum of EAEP in CDCl3.

Table 1. Molecular weights and molecular weight distributions of P(EAEP-PPA) in different mole ratio conditions.

Based on the 1H NMR spectrum in Figure 4(A), the polymerization degree (n) of P(EAEP-PPA) was calculated by using the following eqn (1), where A2 is the integral area of the protons of CH2=CH- group (peak 2) in acrylate terminal group, and A6 is the integral area of the protons of methyl group (peak 6) in the EAEP. In addition, based on the 1H NMR spectrum in Figure 4(B), the number (x) of CPT derivative was calculated by eqn (2), where Af represents the integral area of methyl protons (peak f) of CPT. The molecular weights of P(EAEP-PPA) and P(EAEPPPA)-g-ss-CPT were calculated according to the 1H NMR analysis by the following eqn (3) and eqn (4), where 377.32 is the molecular weight of one repeating unit of P(EAEP-PPA), 322.25 is the molecular weight of EAEP, and 639.7 is the molecular weight of CPT-ss-N3.

n= x=

2 A6

Number

EAEP:PPA

a b

(1)

3 A2 2 Af

M n,NMR(P(EAEP-PPA)) =377.32 n+322.25

(3)

M n, NMR(P(EAEP-PPA)− g −ss−CPT) =M n, NMR(P(EAEP-PPA)) +639.7 x

Mn

a

Mn

-1

b -1

(g mol )

Mw

b -1

(g mol )

(g mol )

PDI

b

1

1.06:1

4050

3130

5140

1.64

2

1.04:1

5680

6600

10310

1.56

3

1.02:1

4460

2530

4340

1.72

4

1.01:1

10950

4310

7620

1.77

5

1:1

13380

2340

3650

1.56

6

1:1.02

7300

2810

4290

1.52

1

Calculated by eqn (1) and (3) based on H NMR analysis (solvent: CDCl3). Determined by GPC with DMF as the eluent and polystyrene as the standard.

Furthermore, Figure 5 shows the GPC curves of P(EAEPPPA) and P(EAEP-PPA)-g-ss-CPT, in which the curve of the polymeric prodrug shifts to the higher molecular weight side compared with P(EAEP-PPA), indicating the successful preparation of CPT prodrug. Table 2 shows the information of three different P(EAEP-PPA) copolymers and their corresponding polymeric prodrugs measured by GPC and 1H NMR. Among these, the sample 3 and sample 4 are selected to be used for the later in vitro tests. The different molecular weights determined by GPC and calculated by 1H NMR may be attributed to some deviations using polystyrene as the standard in the GPC measurement. In addition, there are intermolecular interactions and aggregations of PPEs chains in DMF during GPC analysis.62

(2)

3 A2

Mole ratio

(4)

Figure 5．GPC traces of P(EAEP-PPA)13.6 ( M n = 3670 g mol-1, PDI = 1.36) and P(EAEP-PPA)13.6-g-ss-CPT1.5 ( M n = 5550 g mol-1, PDI = 1.43) (Sample 3 and 4 as listed in Table 2).

1

Figure 4. H NMR spectra of (A) P(EAEP-PPA)13.6 and (B) P(EAEP-PPA)13.6-g-ss-CPT1.5 in CDCl3.

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Table 2. Molecular weights and molecular weight distributions (PDIs) of P(EAEP-PPA) and P(EAEP-PPA)-g-ss-CPT with the different degrees of polymerization. Number 1

a b

Mn

a

Mn

b

Mw

b

PDI

Sample

(g mol )

(g mol )

(g mol )

P(EAEP-PPA)14.5

5790

3620

5460

-1

-1

addition, the CPT contents (CCPT) can be determined by UVVis spectroscopy and all the results are listed in Table 3. These results confirm the successful synthesis of P(EAEP-PPA)-g-ssCPT.

b

Table 3. The drug contents of P(EAEP-PPA)-g-ss-CPT

-1

1.51

Number

Feed mole ratio of P(EAEP-PPA):CPT-ss-N3

CPT content a (wt%)

2

P(EAEP-PPA)14.5g-ss-CPT1.8

6940

5530

7820

1.42

1

2:1

21.8

3

P(EAEP-PPA)13.6

5450

3670

5010

1.36

2

7:1

11.9

4


6410

1.43

3

8:1

8.36

4

10:1

6.83

5

P(EAEP-PPA)24.4

9210

6050

9910

1.64

6


10680

6600

10310

1.56

5550

7940

a

Calculated by CCPT (wt%)=(CUV-Vis/CP(EAEP-PPA)-g-ss-CPT) × 100，where CUV-Vis represents the concentration of CPT measured by UV-Vis, while CP(EAEP-PPA)-gss-CPT is the concentration of prodrug.

Calculated by eqn (1)-(4) based on 1H NMR analysis (solvent: CDCl3). Determined by GPC with DMF as the eluent and polystyrene as the standard.

Self-assembly of the Polymeric Prodrug. The value of the critical aggregation concentration (CAC) indicates the thermodynamic stability of micelles in aqueous medium. When the concentration of prodrug is higher than the CAC value, the P(EAEP-PPA)-g-ss-CPT prodrug can self-assemble into micelles with CPT as core and hydrophilic P(EAEP-PPA) parts as the shell in aqueous solution. The micellization behavior was studied by the steady-state fluorescence probe method using pyrene as the probe. In the Supporting Information, Figure S3 shows the CAC value of the prodrug micelles is 23.86 µg L-1. In general, drug-loaded nanocarriers with the sizes less than 200 nm can extravasate into the tumor tissues via EPR effect and release drugs into the vicinity of the tumor cells.63

In order to prove the CPT-ss-N3 had been conjugated to P(EAEP-PPA), HPLC and UV-Vis measurements were used. The HPLC traces are shown in Figure 6, where the CPT-ss-N3 elutes at 7.65 min while P(EAEP-PPA)-g-ss-CPT elutes at 3.03 min. There is no trace at 7.65 min from Figure 6(B), which illustrates the polymeric prodrug has been purified without residual CPT-ss-N3.

The morphology, average particle size ( Dz ) and size polydispersity index (size PDI) of the prodrug micelles were respectively investigated by TEM and DLS measurements. From the results as shown in Figure S4(A) of the Supporting Information, we can find that the TEM images of micelles are mainly spherical and the average size is less than 200 nm. Moreover, the corresponding particle size distribution curve measured by DLS displays an average diameter of 141 nm as shown in Figure S4(B). These results indicate that the prodrug can form micelles in aqueous solution. In order to demonstrate the reduction-responsive degradation of prodrug micelles, DLS measurement was carried out to monitor the size change of micelles with 2 µM GSH and 10 mM GSH at different time intervals. Figure 8(A) and (B) show a control experiment without GSH, in which there were unconspicuous changes of the particle size over 48 h, indicating the favorable stability of micelles. Furthermore, the size of micelles has a slight change at 2 µM GSH medium over 48 h in Figure 8(A). Because of the low GSH concentration, micellar swelling can be observed, which may make the hydrophobic core of micelles loose but not enough to dissociate the micelle structure. In contrast, in Figure 8(B), an obvious size change of prodrug micelles can be observed in the presence of 10 mM GSH with the extension of time, which is attributed to the cleavage of disulfide linker. Meanwhile, DLS measurement was also used to verify the degradation of polyphosphoester backbone in the presence of phosphodiesterase I (PDE I, 0.25 mg mL-1). From Figure 8(C), it shows the similar results that the micelles keep stable under pH 7.4 for 48 h without PDE I, while the degradation occurs in the presence of PDE I.

Figure 6. HPLC analyses results of (A) CPT-ss-N3 and (B) P(EAEP-PPA)13.6-g-ss-CPT1.5. HPLC analyses were performed with acetonitrile/water (75/25, v/v), as the mobile phase at 30 °C with a flow rate of 1.0 mL min-1.

Figure 7. UV-Vis spectra of Free CPT, P(EAEP-PPA)13.6-g-ssCPT1.5 and P(EAEP-PPA)13.6.

The UV-Vis spectroscopy shows that both of the Free CPT and P(EAEP-PPA)-g-ss-CPT have peaks at 365 nm in Figure 7, while no signal was present for P(EAEP-PPA). In

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These results are in agreement with our previous reports. 56,26 Therefore, during the process of degradation, the CPT can be cleaved from backbone at high GSH concentration and the PDE I accelerates the degradation of PPEs to make the micelle loose, which would further form large aggregates due to inferior stability.

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sulfide bond, which generates a thiol intermediate that was expected to be followed by intramolecular cyclization and cleavage of the neighboring carbonate bridge, thus releasing native CPT molecules from prodrug micelles.8 Considering the premature CPT release would lead to the unfavorable drug leakage in the blood circulation, these prodrug micelles display its great potential to load CPT in the drug delivery system.

Figure 9. In vitro CPT release curves from P(EAEP-PPA)13.6-g-ssCPT1.5 micelles recorded at pH 7.4 and 37.5 °C under different reductive conditions. In Vitro Cytotoxicity. As we all know that biocompatibility is one of the most important properties to be considered in the application of polymeric materials in drug delivery.64 Herein, the cytotoxicity of P(EAEP-PPA) micelles without CPT against L929 cells, 4T1 cells and HepG2 cells was studied by MTT assays. Figure 10 shows the cell viabilities of L929 cells, 4T1 cells and HepG2 cells after 72 h incubation with polymer P(EAEP-PPA) at different concentrations. For three kinds of cells, the polymer has a negligible impairment in the cell viability, even after increasing the concentration to 0.2 mg mL-1, indicating the polymer P(EAEP-PPA) possesses a good biocompatibitity as a drug carrier. Figure 8. Reduction- and enzyme-induced size change of P(EAEP-PPA)13.6-g-ss-CPT1.5 under different conditions of (A) pH 7.4 with 2 µM GSH, (B) pH 7.4 with 10 mM GSH and (C) pH 7.4 with PDE I as determined by DLS. In Vitro Drug Release. The reduction-responsive polymers are generally used in triggered drug delivery systems. In our study, to investigate the effects of various media on the release behavior of prodrug micelles, in vitro cumulative CPT release data were measured at 37.5 °C. Four different media were selected: (i) pH 7.4, (ii) pH 7.4 with 2 µM GSH, (iii) pH 7.4 with 5 mM GSH and (iv) pH 7.4 with 10 mM GSH. As shown in Figure 9, in the presence of 10 mM GSH, approximately 90% of CPT was released from the micelles after incubation for 105 h, whereas the release of CPT was about 50% in the presence of 5 mM GSH. In comparison, for prodrug micelles in the absence of GSH or in the presence of 2 µM GSH, only 30% of CPT release was observed after incubation for 105 h. These results indicate that the release of the parent drug can be attributed to the GSH triggered disassembly of prodrug micelles. The highly reduction-responsive release behavior of micelles could be ascribed to the reduction of di-

Figure 10. Cell viability of L929 cells, 4T1 cells and HepG2 cells treated with P(EAEP-PPA)13.6 at different concentrations for 72 h of incubation.

According to the design, the CPT prodrug micelles can be internalized into cells and release the parent drug to inhibit the proliferation of tumor cells. Therefore, the anti-proliferation activity of P(EAEP-PPA)-g-ss-CPT prodrug micelles against 4T1 cells and HepG2 cells was investigated using MTT assays. As shown in Figure 11(A), the cell viabilities of 4T1 cells were decreased gradually when the CPT concentration increases from 0.035 mg L-1 to 4.5 mg L-1. And the prodrug micelles

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show a higher IC50 value compared with the Free CPT. The result of Figure 11(B) is similar to that of Figure 11(A). It shows that the viability of cells incubated with the Free CPT was lower than the prodrug system with equal CPT concentration after 72 h incubation. It is due to the decrease of cytotoxicity by P(EAEP-PPA), which would not impair normal cell before reaching cancer cell. In addition, there is timeconsuming CPT release from prodrug micelles in comparison to Free CPT, proved by the in vitro CPT release.65,66 The micelles self-assembled by polymeric prodrug can improve the stability in blood circulation and enhance the CPT passive accumulation at tumor tissue via EPR effect.67

HepG2 cells was weak as shown in Figure 12(B) and there was not CPT fluorescence in the lysosome. This is because of the different cellular internalization mechanisms between the CPT-loaded micelles (endocytosis) and Free CPT (diffusion).60,68,69 We further do a quantitative measurement of the CPT fluorescence intensity by fluorescence microscopy, and the results are provided in Figure S5 of the Supporting Information. It shows that the CPT fluorescence intensity of prodrug micelles in HepG2 cells have an increased tendency from 5 h to 22 h, which is much higher than Free CPT at 22 h. All these results show that the prodrug micelles can keep longer period to accumulate more CPT in the tumor cells.

Figure 12. The cellular uptake behavior of (A) P(EAEP-PPA)13.6g-ss-CPT1.5 micelles following different incubation times in HepG2 cells and (B) Free CPT in HepG2 cells. The dosage of CPT was 0.836 mg L-1. For each panel, images from left to right show cell cytoplasm stained by Lyso-Tracker Red, CPT fluorescence in cells (blue) and overlays of two images. All the images were observed by fluorescence microscopy and the scale bars correspond to 100 µm in all the images.

Figure 11. Cell viability of (A) 4T1 cells and (B) HepG2 cells, treated with P(EAEP-PPA)13.6-g-ss-CPT1.5 and Free CPT with different CPT dosages for 72 h of incubation.

Cellular Uptake. To demonstrate whether the prodrug micelles could be efficiently internalized into tumor cells and increase intracellular drug accumulation, the cellular uptake behavior of P(EAEP-PPA)-g-ss-CPT micelles was investigated against HepG2 cells by fluorescence microscopy of the live cell imaging system. The cells were incubated with 10% CPT prodrug micelles of DMEM for different time using the same concentration of Free CPT as a control. Before measurement, the cytoplasm was stained with Lyso-Tracker Red, since CPT is a fluorescent molecule whose emission can directly use to visualize cellular uptake. As shown in Figure 12(A), when HepG2 cells were treated with the prodrug micelles for 5 h, a slight blue fluorescence in the cytoplasm can be observed. After 16 h incubation, the CPT fluorescence existed in the lysosome. With the increase of incubated time, the stronger CPT fluorescence appeared in the nucleus of HepG2 cells at 22 h. All these results demonstrated the efficient intracellular CPT delivery from prodrug micelles via endocytosis. However, for the Free CPT case, the fluorescence intensity of CPT in

CONCLUSIONS In this study, a facile method based on the combination of Michael addition polymerization and CuAAC “click” chemistry was utilized to develop a new reduction-responsive polyphosphoester-based camptothecin prodrug P(EAEP-PPA)-gss-CPT. Compared to the method of the physical encapsulation of drugs, these polymeric prodrug nanoparticles can improve the drug loading content and loading efficiency. The amphiphilic prodrug can self-assemble into micelles in aqueous solution with hydrophobic CPT segment as the core and hydrophilic P(EAEP-PPA) as the corona, and the size of micelles is 141 nm. The results of DLS verified that these micelles were relatively stable under physiological conditions, but could be rapidly degraded in the presence of 10 mM GSH medium. About 90% of CPT was released from the prodrug micelles in the reductive medium, indicating that the polyphosphoestercamptothecin prodrug possesses the reduction-responsive. By MTT assays, it can be known that P(EAEP-PPA) is a favorable biocompatibility material and the CPT prodrug micelles effi-

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(6) Huang, Q. Q.; Wang, L.; Lu, W. Evolution in Medicinal Chemistry of E-Ring-Modified Camptothecin Analogs as Anticancer Agents. Eur. J. Med. Chem. 2013, 63, 746-757. (7) Kaur, S.; Prasad, C.; Balakrishnan, B.; Banerjee, R. Trigger Responsive Polymeric Nanocarriers for Cancer Therapy. Biomater. Sci. 2015, 3, 955-987. (8) Zhang, Q. Q.; He, J. L.; Zhang, M. Z.; Ni, P. H. A Polyphosphoester-Conjugated Camptothecin Prodrug with Disulfide Linkage for Potent Reduction-Triggered Drug Delivery. J. Mater. Chem. B 2015, 3, 4922-4932. (9) Wong, P. T.; Choi, S. K. Mechanisms of Drug Release in Nanotherapeutic Delivery Systems. Chem. Rev. 2015, 115, 33883432. (10) Cheng, R.; Meng, F. H.; Deng, C.; Klok, H. A.; Zhong, Z. Y. Dual and Multi-Stimuli Responsive Polymeric Nanoparticles for Programmed Site-Specific Drug Delivery. Biomaterials 2013, 34, 3647-3657. (11) Wang, S.; Huang, P.; Chen, X. Y. Hierarchical Targeting Strategy for Enhanced Tumor Tissue Accumulation/Retention and Cellular Internalization. Adv. Mater. 2016, 28, 7340-7364. (12) Tai, W. Y.; Mo, R.; Lu, Y.; Jiang, T. Y.; Gu, Z. Folding Graft Copolymer with Pendant Drug Segments for Co-delivery of Anticancer Drugs. Biomaterials 2014, 35, 7194-7203. (13) Fang, J.; Nakamura, H.; Maeda, H. The EPR effect: Unique Features of Tumor Blood Vessels for Drug Delivery, Factors Involved, and Limitations and Augmentation of the Effect. Adv. Drug Delivery Rev. 2011, 63, 136-151. (14) Maeda, H.; Sawa, T.; Konno, T. Mechanism of TumorTargeted Delivery of Macromolecular Drugs, Including the EPR Effect in Solid Tumor and Clinical Overview of the Prototype Polymeric Drug SMANCS. J. Controlled Release 2001, 74, 4761. (15) Wei, H.; Zhuo, R. X.; Zhang, X. Z. Design and Development of Polymeric Micelles with Cleavable Links for Intracellular Drug Delivery. Prog. Polym. Sci. 2013, 38, 503-535. (16) Huang, M. M.; Zhao, K. J.; Wang, L.; Lin, S. Q.; Li, J. J.; Chen, J. B.; Zhao, C. G.; Ge, Z. S. Dual Stimuli-Responsive Polymer Prodrugs Quantitatively Loaded by Nanoparticles for Enhanced Cellular Internalization and Triggered Drug Release. ACS Appl. Mater. Interfaces 2016, 8, 11226-11236. (17) Wang, Y.; Luo, Q. J.; Zhu, W. P.; Li, X. D.; Shen, Z. Q. Reduction/pH Dual-Responsive Nano-Prodrug Micelles for Controlled Drug Delivery. Polym. Chem. 2016, 7, 2665-2673. (18) Sun, Q. H.; Ma, X. P.; Zhang, B.; Zhou, Z. X.; Jin, E. L.; Shen, Y. Q.; Van Kirk, E. A.; Murdoch, W. J.; Radosz, M.; Sun, W. L. Fabrication of Dendrimer-Releasing Lipidic Nanoassembly for Cancer Drug Delivery. Biomater. Sci. 2016, 4, 958-969. (19) Zolotarskaya, O. Y.; Xu, L. Y.; Valerie, K.; Yang, H. Click Synthesis of a Polyamidoamine Dendrimer-Based Camptothecin Prodrug. RSC Adv. 2015, 5, 58600-58608. (20) Tian, W. D.; Ma, Y. Q. Theoretical and Computational Studies of Dendrimers as Delivery Vectors. Chem. Soc. Rev. 2013, 42, 705-727. (21) Li, J. Y.; Mooney, D. J. Designing Hydrogels for Controlled Drug Delivery. Nat. Rev. Mater. 2016, 1, 16071. (22) Zhang, Y. F.; Wang, R.; Hua, Y. Y.; Baumgartner, R.; Cheng, J. J. Trigger-Responsive Poly(β-amino ester) Hydrogels. ACS Macro Lett. 2014, 3, 693-697. (23) Yang, H.; Wang, Q.; Huang, S.; Xiao, A.; Li, F. Y.; Gan, L.; Yang, X. L. Smart pH/Redox Dual-Responsive Nanogels for On-

ciently inhibit the cell proliferation of 4T1 cells and HepG2 cells. Moreover, the prodrug micelles could be internalized into HepG2 cells to deliver the active CPT by endocytosis. This kind of reduction-responsive polymeric prodrug is highly potential in cancer chemotherapy. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami xxxx The 1H NMR spectra and FT-IR analysis of HO-ss-Br, HO-ssN3 and CPT-ss-N3; the CAC, TEM and DLS measurements of the prodrug micelles; a quantitative measurement of the CPT fluorescence intensity by fluorescence microscopy. AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]

ORCID Peihong Ni: 0000-0003-4572-3213 ACKNOWLEDGMENTS We gratefully acknowledge 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), 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) Chen, S. Z.; Yang, K. N.; Tuguntaev, R. G.; Mozhi, A. B.; Zhang, J. C.; Wang, P. C.; Liang, X. J. Targeting Tumor Microenvironment with PEG-based Amphiphilic Nanoparticles to Overcome Chemoresistance. Nanomedicine 2016, 12, 269-286. (2) Wall, M. E.; Wani, M. C.; Cook, C. E.; Palmer, K. H.; McPhail, A. T.; Sim, G. A. Plant Antitumor Agents. I. The Isolation and Structure of Camptothecin, a Novel Alkaloidal Leukemia and Tumor Inhibitor from Camptotheca Acuminata. J. Am. Chem. Soc. 1966, 88, 3888-3890. (3) Thomas, C. J.; Rahier, N. J.; Hecht, S. M. Camptothecin: Current Perspectives. Bioorg. Med. Chem. 2004, 12, 1585-1604. (4) Adams, D. J.; Silva, M. W.; Flowers, J. L.; Kohlhagen, G.; Pommier, Y.; Colvin, O. M.; Manikumar, G.; Wani, M. C. Camptothecin Analogs with Enhanced Activity Against Human Breast Cancer Cells. I. Correlation of Potency with Lipophilicity and Persistence in the Cleavage Complex. Cancer Chemother. Pharmcol. 2006, 57, 135-144. (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.

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Polyphosphoester-Camptothecin Prodrug with Reduction-Response

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