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Dual-Responsive Polyphosphoester-Doxorubicin Prodrug Containing Diselenide Bond: Synthesis, Characterization and Drug Delivery Guoqing Ma, Jie Liu, Jinlin He, Mingzu Zhang, and Peihong Ni ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00429 • Publication Date (Web): 22 May 2018 Downloaded from http://pubs.acs.org on May 23, 2018

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ACS Biomaterials Science & Engineering

Dual-Responsive Polyphosphoester-Doxorubicin Prodrug Containing Diselenide Bond: Synthesis, Characterization and Drug Delivery Guoqing Ma, Jie Liu, Jinlin He, 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

* To whom correspondence should be addressed. Peihong Ni (E-mail: [email protected]).

ABSTRACT: The development of novel stimuli-responsive and biodegradable polyphosphoester-anticancer prodrugs is of importance in designing water-soluble prodrugs utilized in the field of drug delivery. In this study, the focus is on the synthesis of biocompatible and biodegradable diselenide-containing polyphosphoester [PEEP-b-PBYP-Se]2, using reduction-responsive di(1-hydroxylundecyl) diselenide as an initiator to polymerize 2-(but-3-yn-1-yloxy)-2-oxo-1,3,2-dioxaphospholane (BYP) and 2-ethoxy-2-oxo-1,3,2-dioxaphospholane (EOP). After that, doxorubicin (DOX) derivative

containing

azide

group

was

linked

onto

1

ACS Paragon Plus Environment

the

side

chain

of

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

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[PEEP-b-PBYP-Se]2 via the Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) “click” reaction to yield a pH/reduction-responsive polymeric prodrug, namely [PEEP-b-(PBYP-hyd-DOX)-Se]2. The chemical structures of various polymers were characterized by nuclear magnetic resonance spectroscopy (NMR), ultraviolet-visible spectrophotometer (UV-vis), Fourier transform infrared (FT-IR) spectroscopy and high performance liquid chromatography (HPLC). The self-assemble behavior measured by dynamic light scattering (DLS) and transmission electron microscopy (TEM) clearly supported the formation of the prodrug nanoparticles (NPs). The results indicated that the polymeric prodrug NPs were relatively uniform sphere, which could maintain stability in a physiological condition, but could be cleaved in acidic or reductive medium. Furthermore, the pH- and reduction-responsive properties of the prodrug NPs were investigated via the drug release in vitro in different media. It turned out that the drug was efficiently released in acidic or reductive medium compared with that in a physiological condition. The results of methyl thiazolyl tetrazolium

(MTT)

assay

confirmed

the

favorable

biocompatibility

of

[PEEP-b-PBYP-Se]2. Besides, the cell cytotoxicity and intracellular uptake experiments were carried out to verify the efficient cellular proliferation inhibition. This

finding

contributes

to

the

design

of

novel

polyphosphoester-doxorubicin prodrug.

2


diselenide-containing

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KEYWORDS: Selenium-containing polymer, Prodrug, pH/reduction-responsiveness, Polyphosphoester

INTRODUCTION The conjugation of stimuli-responsive polymers and anticancer drugs to prepare polymeric prodrugs has been widely concerned.1-5 Amphiphilic polymeric prodrugs can enhance the water solubility of hydrophobic antitumor drugs (such as doxorubicin,

paclitaxel

and

camptothecin),

improve

pharmacokinetics

and

biodistribution.6-8 With regard to the design of polymeric prodrug structure, there are three important aspects to be considered. The first is the biocompatibility and biodegradability of polymers; the second is the molds of binding between the hydrophobic drugs and water-soluble polymers, enabling the prodrugs to effectively release the original drug after they enter tumor cells; and the third is the 3



self-assembled nanostructures from prodrugs, which should be small nanoscale sizes (less than 200 nm) with a certain morphology such as sphere, smooth disk, staggered lamellae and so on.9-13 There are many polymers with biocompatibility and biodegradability to be used, including poly(ethylene glycol),14,15 polylactic acid,16,17 poly(ε-caprolactone),18,19 polypeptides,20,21 polyphosphoesters (PPEs),22-24 and so on. In recent years, the structures of main chain PPEs are considered as biodegradable and can be modified via chemical reaction. Wooley et al. reported a polyphosphoester-based degradable paclitaxel (PTX) prodrug (PEO-b-PPE-g-PTX) with ultra-high paclitaxel loading capacity, which formed well-defined micelles in aqueous solution. These micelles possessed positive cell-killing activity against several cancer cell lines.25 Wang et al. developed two drug delivery systems stabilized by zwitterionic polyphosphoesters, which showed favourable stability, anti-protein absorption ability in vitro, prolonged drug circulation half-lives and increased drug accumulation in tumors.26 Very recently, our group prepared two kinds of polyphosphoester-camptothecin prodrugs with reduction-responsiveness.27,28 The disulfide bonds were introduced into polymeric prodrugs, which could be used to link PPE chains and the anti-tumor drug molecules. The prodrug micelles were relatively stable in physiological medium, but could be degraded under the reductive condition, which showed that the camptothecin release from the prodrug micelles was proceeded in a glutathione (GSH)-dependent manner.

4


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Researchers have extensively developed various stimuli-responsive systems to ulteriorly realize controlled drug release and optimal therapeutic effect.29-33 Stimuli-responsive polymers will change their physical or chemical properties when triggered by different stimuli, such as pH,34-36 redox conditions,37-39 temperature,40,41 light,42,43

magnetic

field,44,45

and

so

forth.

Among

those,

pH-

and

redox-responsiveness may be especially explored because they are commonly present in human body.46-48 In comparison with the C-C bond (bond energy, 346 kJ mol-1) and widely used S-S bond (240 kJ mol-1), a Se-Se bond has a weaker bond energy (172 kJ mol-1), which suggests that Se-Se bond is more dynamic.49 Furthermore, the Se-Se bond owns unique redox property that enables it to be oxidized to seleninic acid via oxidants and reduced to selenol via reducers.50 In addition, the Se-Se bond has prominent γ-ray-responsiveness allowing it to be cleaved when exposed to γ radiation.51 Redox-responsive drug carriers containing disulfide bond have the ability to be cleaved easily in the tumor cell environment.52,53 There have been many reports in this field. Due to the diselenide bond has a similar structure with the disulfide bond and lower bond energy, diselenide-containing materials are becoming promising candidates as stimuli-responsive drug vehicles in the field of biological medicine. Zhang et al. prepared two kinds of different block copolymers, PEG-PUSe-PEG and PEG-PUS-PEG, which were composed of polyethylene glycol (PEG) and polyurethane (PU), linked by selenium (Se) and sulfur (S), respectively. They found

5



that PEG-PUSe-PEG nanoparticles were more sensitive to oxidants compared with the sulfide analogue PEG-PUS-PEG.54 Xu et al. reported a diselenide-containing hydrogel showing obvious gel-sol transition as contrast with an analogous disulfide-containing hydrogel without apparent change even after exposure to a higher radiation dose, indicated that diselenide bonds were more sensitive to γ radiation than disulfide bonds.55 Besides, Wang et al. developed a diselenide-linked polycation mPEG-SeSe-PEI for reduction-responsive gene delivery, taking the corresponding stable analog mPEG-PEI [poly(ethylene imine)] and the disulfide-linked polycation mPEG-SS-PEI as controls. The results demonstrated that diselenide bond was more easily cleaved than disulfide bonds at a GSH concentration of 0.3 mM. In other word, diselenide bonds were more sensitive to glutathione compared with disulfide bonds.56 Diselenide-containing polymers attract more and more attention.57 However, to the best of our knowledge, rare concern has been given to the development of diselenide-containing polyphosphoesters, and to the relevant preparation of anticancer prodrugs. Due to the special microenvironment in tumor tissue, stimuli-responsive drug delivery systems (DDSs) can play a significant role in controlling drug release. Moreover, the remarkable reduction-responsiveness of the diselenide bonds allows them to possess prominent effect in stimuli-responsive DDSs. In this study, we synthesized a pH/reduction-responsive diselenide-containing polyphosphoester-DOX prodrug, [PEEP-b-(PBYP-hyd-DOX)-Se]2, via a combination of one-pot sequential

6


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ring-opening polymerization (ROP) of cyclic phosphate monomers and CuAAC “click” chemistry. The polymeric prodrug could self-assemble into nanoparticles (NPs) in aqueous solution, which could keep favourable stability during blood circulation and then reached to neoplastic tissue via enhanced permeability and retention (EPR) effect. Once the NPs was internalized into tumor cells, the diselenide bonds could be cleaved owning to the high GSH concentration, and the hydrazone bonds (-hyd-) could also be cleaved owning to the low pH medium, finally resulting in the disassembly of NPs and the release of parent drug DOX to inhibit tumor proliferation. EXPERIMENTAL SECTION Materials. Selenium powder (99.9%, Aladdin), sodium borohydride (98%, Sinopharm Chemical Reagent), 11-bromoundecanol (98%, Infinity Scientific), 1,8-diazabicyclo

[5.4.0]

undec-7-ene

(DBU,

N,N,N′,N′,N″-pentamethyldiethylenetriamine

98%,

(PMDETA,

J&K

98%,

Chemical),

Sigma-Aldrich),

doxorubicin hydrochloride (DOX·HCl, 99%, Beijing Zhongshuo Pharmaceutical Technology

Development),

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-

tetrazoliumbromide (MTT, 98%, Sigma-Aldrich), and reduced glutathione (GSH, ≥99%, Shanghai Yuanye Bio Technology) were used as received. Cuprous bromide (CuBr, 95%, Sinopharm Chemical Reagent) was purified by washing three times with acetone, glacial acetic acid and ethanol in turn, and then drying under vacuum at room temperature. N,N-Dimethylformamide (DMF), dichloromethane (CH2Cl2) and tetrahydrofuran (THF) were purchased from Sinopharm Chemical Reagent with 7



Page 8 of 46

analytical grade. DMF and CH2Cl2 were distilled under vacuum before use. THF was dried over KOH for at least 2 days and then refluxed over a sodium wire with benzophenone as an indicator until the color turned purple. Milli-Q water was generated

using a

water purification

system

(Simplicity

2-(But-3-yn-1-yloxy)-2-oxo-1,3,2-dioxaphospholane

UV,

(BYP)

Millipore). and

2-ethoxy-2-oxo-1,3,2-dioxaphospholane (EOP) were synthesized and purified according to the previously published literatures.58-61 Doxorubicin derivative (abbreviated as DOX-hyd-N3) was prepared according to a previous report.62 The other solvents and chemicals were used as received unless otherwise stated. Synthesis of the Diselenide-Containing Block Copolymer [PEEP-b-PBYP-Se]2. The synthesis of diselenide-containing block copolymer [PEEP-b-PBYP-Se]2 was carried out via ROP initiated by di(1-hydroxylundecyl) diselenide in the presence of DBU as an organic catalyst. Typically, to a 50 mL round-bottom flask equipped with a magnetic stir bar, di(1-hydroxyundecyl) diselenide (0.06 g, 0.12 mmol) dissolved in 8 mL of anhydrous CH2Cl2 was added. Afterwards, BYP (0.95 g, 5.42 mmol) and DBU (0.11 g, 0.75 mmol) were sequentially added into the flask. The mixture solution was stirred at 30 °C for 1.5 h under nitrogen atmosphere. Then, about 2 mL of yellow solution was taken out and concentrated for 31P NMR measurement, which was used to evaluate the conversion rate of the monomer BYP. Subsequently, EOP monomer (3.29 g, 21.60 mmol) was injected into the above-mentioned mixture with further stirred at 30 °C for another 2 h. The crude product was concentrated and

8


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precipitated with a 10:1 (v/v) mixture of diethyl ether and methanol for three times. The precipitate was dried under vacuum to yield the yellow viscous product (3.08 g, yield: 71.7%). Synthesis

of

Diselenide-Containing

Polymeric

Prodrug

[PEEP-b-(PBYP-hyd-DOX)-Se]2. Using CuAAC “click” reaction between the alkynyl groups in the side chain of [PEEP-b-PBYP-Se]2 and the azide groups of DOX derivative which containing a pH-sensitive linker (-hyd-), we prepared the polymeric prodrug [PEEP-b-(PBYP-hyd-DOX)-Se]2. In a typical experiment, to a 50 mL round-bottom flask, CuBr (0.05 g, 0.34 mmol) and PMDETA (0.16 g, 0.94 mmol)

were

added

under

nitrogen

flow.

After

stirring

for

10

min,

[PEEP-b-PBYP-Se]2 (0.55 g, 0.68 mmol of alkynyl group) dissolved in 8 mL anhydrous DMF and DOX-hyd-N3 (0.21 g, 0.29 mmol of azido group) were sequentially added into the mixture, which was purged with nitrogen for 10 min. The reaction was further stirred at 45 °C for 40 h. Subsequently, the solution was purified by dialysis (MWCO: 12∼14 kDa) against Milli-Q water for 2 days. Ultimately, a reddish violet solid product was obtained via lyophilization (0.50 g, yield: 66.0%).

In Vitro DOX Release from [PEEP-b-(PBYP-hyd-DOX)-Se]2. The free DOX release behavior from the prodrug was investigated via the following process. Firstly, 60 mL of prodrug NPs solution was predetermined in the concentration of 0.5 mg mL-1. In addition, four kinds of phosphate buffer solution (PBS) were also prepared as the following: (1) pH 7.4, (2) pH 7.4 with 10 mM GSH, (3) pH 5.0, and (4) pH 5.0 9



with 10 mM GSH. Secondly, each 4.5 mL of the prodrug NPs solution was transferred into a dialysis membrane (MWCO: 12∼14 kDa), and then all the dialysis membranes were put into tubes with 25 mL corresponding PBS. Every three tubes, as one group, contained the same PBS. After that, these tubes were placed in a water bath at 37 °C under continuous shaking in the dark. At predetermined intervals, 5 mL of the dialysis medium was taken out and replenished with an equal volume of the corresponding fresh PBS. The content of the released DOX was determined using a fluorescence spectrophotometer with excitation at 480 nm while emission spectra from 520 to 620 nm with a 10.0 nm slit width. Cellular Uptake and Intracellular DOX Release. Cellular uptake and intracellular drug release behaviors of free DOX and DOX prodrug in HeLa cells were implemented with a live cell imaging system (CELL’R, Olympus, Japan). Typically, HeLa cells were cultured in a 35 mm glass petri dish at 15×104 cells cm-2, which was mounted in the live cell imaging system at 37 °C under a 5% CO2 atmosphere. Subsequently, the culture medium was removed and cells were carefully washed with PBS for three times, followed by staining with H 33342 (1 µL mL−1) for 1 h and washing with PBS for three times again. Afterwards, the culture medium was replaced with fresh medium containing free DOX or DOX prodrug (65 mg L-1 of DOX). The images were captured at excitation wavelengths of 480 nm (red) and 340 nm (blue) for 6 h.

10


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Scheme 1. Synthetic routes of (1) di-(1-hydroxylundecyl) diselenide, (2) azide-functionalized

pH-responsive

DOX

derivative

DOX-hyd-N3

and

(3)

pH/reduction-responsive polymeric prodrug [PEEP-b-(PBYP-hyd-DOX)-Se]2.

RESULTS AND DISCUSSION Successful Construction of Diselenide-Containing Polymeric Prodrug. The pH/reduction-responsive prodrug [PEEP-b-(PBYP-hyd-DOX)-Se]2 was synthesized by the combination of one-pot sequential ROP and CuAAC “click” chemistry as shown in Scheme 1. First, the reduction-responsive di-(1-hydroxylundecyl) diselenide 11



Page 12 of 46

was synthesized. Second, the block copolymer [PEEP-b-PBYP-Se]2 was prepared by one-pot sequential ROP of BYP and EOP initiated by di-(1-hydroxylundecyl) diselenide

and

catalyzed

by

DBU.

Finally,

the

prodrug

[PEEP-b-(PBYP-hyd-DOX)-Se]2 was synthesized via the CuAAC “click” reaction of the alkynyl groups on the PBYP side chains and azido groups on the DOX-hyd-N3. The 1H NMR spectrum of di-(1-hydroxylundecyl) diselenide was shown in Figure S1 of the Supporting Information (SI). The characteristic chemical shifts were identified as follows: δ 2.91 ppm (peak a, -CH2SeSe-), δ 1.78-1.25 ppm (peak b, HOCH2(CH2)9CH2SeSe-), δ 3.64 ppm (peak c, HOCH2-). The 1H NMR results indicated the successful synthesis of di-(1-hydroxylundecyl) diselenide. The chemical structure of DOX-hyd-N3 was confirmed by 1H NMR and FT-IR analysis, respectively. The 1H NMR spectra of 6-azidehexanohydrazine, DOX, and DOX-hyd-N3 were respectively showed in Figure S2. All of the characteristic peaks ascribed to the protons in the corresponding chemical structure can be found in the 1H NMR spectra. The FT-IR spectra of these samples were shown in Figure S3. In comparison with Figure S3(A), the disappearance of the absorption peak of carbonyl groups at 1732 cm-1 and the appearance of the absorption peak of azido groups at 2098 cm-1 in Figure S3(B) indicated that the azido groups had been successfully linked onto DOX. The results of 1H NMR and FT-IR confirmed the successful synthesis of DOX-hyd-N3. For the block copolymer [PEEP-b-PBYP-Se]2, the chemical structure, the 12


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molecular weight and molecular weight distribution (PDI) were analyzed by 1H NMR, 13

C NMR, 31P NMR and GPC, respectively. The 1H NMR spectrum in Figure 1

showed the characteristic chemical shifts of [PEEP-b-PBYP-Se]2 as followings: δ 2.89

ppm

(peak

a,

-OCH2(CH2)9CH2SeSe-

-CH2SeSe-), and

δ

1.25-1.45

-OCH2CH3),

δ

ppm 3.47

(peak

b

and

ppm

(peak

i, c,

-OCH2(CH2)9CH2SeSe-), δ 4.27 ppm (peak d, -OCH2CH2O-), δ 4.17 ppm (peak e and h, -OCH2CH2- and -OCH2CH3), δ 2.62 ppm (peak f, -CH2C≡CH), δ 2.11 ppm (peak g, -C≡CH).

Figure 1. 1H NMR spectrum of [PEEP54.5-b-PBYP13.5-Se]2 in CDCl3.

The chemical structure of block copolymer [PEEP-b-PBYP-Se]2 was further confirmed via 13C NMR as shown in Figure S4. Furthermore, the 31P NMR spectra of [PBYP-Se]2 reaction mixture and purified [PEEP-b-PBYP-Se]2 displayed in Figure 2 were analyzed. With a comparison of chemical shift of the monomer BYP at δ 17.84 ppm and that of the homopolymer [PBYP-Se]2 at δ -1.74 ppm as shown in Figure 2(A), it can be evaluated that more than 97% of BYP was consumed, revealing that the block copolymer was obtained. In addition, the strong peak appearing at δ -1.38 13



ppm in Figure 2(B) can be assigned to the phosphorus atoms in the PEEP repeat units. Moreover, the GPC elution curve in Figure S5 showed unimodal distribution of the block copolymer [PEEP-b-PBYP-Se]2. These results confirmed the successful synthesis of the block copolymer.

Figure 2. 31P NMR spectra of (A) [PBYP13.5-Se]2 reaction mixture and (B) purified [PEEP54.5-b-PBYP13.5-Se]2 in CDCl3.

The molecular weights and molecular weight distributions (PDIs) of two samples of the [PEEP-b-PBYP-Se]2 block copolymers were summarized in Table S1. Based on the 1H NMR spectrum in Figure 1, the polymerization degrees (m and n) of PBYP and PEEP were respectively calculated by eqs (1) and (2), Af Aa 1  2 A i+b  n=  -18  3  Aa 

m=

(1) (2)

where Aa is the integral area of the protons of the -CH2SeSe- group (peak a), Af is the integral area of the protons of methylene group adjacent to alkynyl (peak f) in the PBYP chain, and Ai+b is the integral area of the protons of methyl group (peak i) in the 14


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PEEP chain and -OCH2(CH2)9CH2SeSe- group (peak b). Then, the molecular weight of [PEEPn-b-PBYPm-Se]2 could be calculated according to eq (3), _ M n,NMR =m × 176.10+n × 152.09+500.51

(3)

where 176.10 and 152.09 are the molecular weights of one repeating unit of PBYP and PEEP, respectively; 500.51 is the molecular weight of di-(1-hydroxylundecyl) diselenide. The difference of molecular weights calculated by 1H NMR and measured by GPC can be found, which may be ascribed to that relative molecular weights were provided via GPC measurement using polystyrene as standard. In order to ensure the stability of prodrug NPs, it is necessary to consider the hydrophilicity of the block copolymer

precursor

[PEEP-b-PBYP-Se]2.

Therefore,

the

sample

[PEEP54.5-b-PBYP13.5-Se]2 was selected to further conduct click reaction with DOX-hyd-N3.

Table 1. The drug contents of [PEEP-b-(PBYP-hyd-DOX)-Se]2. Theory mole ratio

DLC a)

[PEEP-b-PBYP-Se]2: DOX-hyd-N3

(wt%)

Number

1

4.30:1

4.53

2

6.96:1

6.57

3

11.45:1

12.90

The prodrug was obtained by the highly efficient CuAAC “click” reaction of the alkynyl groups on the PBYP and azido groups on the DOX-hyd-N3 as shown in the Step (3) of Scheme 1. UV-vis and HPLC were used to confirm the successful 15



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preparation of the prodrug. As shown in Figure 3, compared with free DOX, the ultraviolet absorption peak of DOX in [PEEP54.5-b-(PBYP13.5-hyd-DOX5.8)-Se]2 reveals obvious red shift. The reason can be attributed to that when DOX was conjugated to the main chain of the polymer to form the prodrug, the carbonyl group on the DOX was reacted to form the hydrazone bond and the original structure was changed, which led to the red shift of the UV absorption peak in Figure 3. The subscript 5.8 is the half of the average number of DOX molecules linked onto the block polymer, which was calculated by DOX contents (CDOX). The CDOX was measured by UV-vis spectroscopy via standard method and the results were listed in Table 1. In addition, the HPLC measurement was carried out as shown in Figure S6, where

the

elution

time

of

DOX,

DOX-hyd-N3

and

[PEEP54.5-b-(PBYP13.5-hyd-DOX5.8)-Se]2 were 5.23 min, 1.65 min and 1.13 min, respectively. The different elution time indicated that the prodrug was successfully prepared.

16


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Figure 3. UV-vis spectra of free DOX, [PEEP54.5-b-PBYP13.5-Se]2 and [PEEP54.5-b-(PBYP13.5-hyd-DOX5.8)-Se]2.

Self-Assembly of the Polymeric Prodrug. In general, the amphipathic polymers can self-assemble into NPs in aqueous solution with hydrophobic segments as core while hydrophilic segments as corona. The value of the critical aggregation concentration (CAC) is a very important parameter to evaluate the stability of NPs. Hence, the CAC value of the prodrug [PEEP54.5-b-(PBYP13.5-hyd-DOX5.8)-Se]2 was measured with pyrene as the fluorescence probe. As shown in Figure S7, the CAC value of the prodrug is 0.23 mg mL-1. According to reports in the literature, drug-loaded NPs with sizes less than 200 nm can effectively extravasate into the tumor tissues by EPR effect, and then release drugs around tumor cells. DLS and TEM measurements were applied to analyze the morphology, average particle size ( Dz ) and size polydispersity index (size PDI) of the prodrug NPs. As shown in Figure S8(A), the TEM imagine is relatively spherical while the size is less than 200 nm. In addition, the corresponding nanoparticle size distribution curve measured by DLS displays unimodal distribution with a Dz of 138 nm shown in Figure S8(B). The prodrug NPs can disassemble in acidic or reductive medium for pH-sensitive hydrazone

bond

and

reduction-sensitive

diselenide

bond.

To

demonstrate

pH/reduction-induced drug release mechanism, the DLS analysis was carried out to monitor the size change of the prodrug NPs in PB 5.0 and PB 7.4 with 10 mM GSH 17



(PB 7.4 + 10 mM GSH) at predetermined time intervals, respectively. In Figure 4(A), no obvious change of particle size over 48 h in PB 7.4, indicating the favourable stability of the prodrug NPs. Under the condition of PB 5.0, the particle sizes gradually increased with the increasing stirred time (5, 10, 24, and 48 h), as shown in Figure 4(B), which could be attributed to the cleavage of hydrazone bonds and the aggregates of loose NPs. Furthermore, Figure 4(C) shows the trend of particle size enlargement in the medium of PB 7.4 + 10 mM GSH, which may be concluded that the cleavage of diselenide of the prodrug NPs and the formation of large aggregates.

Figure 4. The particle size distribution histograms of [PEEP54.5-b-(PBYP13.5-hyd-DOX5.8)-Se]2 under different conditions of (A) PB 7.4, (B) PB 5.0 and (C) PB 7.4 + 10 mM GSH, as determined by DLS.

In Vitro Release of DOX. To intuitively verify the responsive release behavior of 18


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the prodrug NPs in tumor microenvironment, in vitro cumulative DOX release data were measured under different pH and reductive conditions at 37.5 °C (Figure 5). On one hand, compared with the less than 20% of cumulative DOX release in PB 7.4, about 35% of DOX was released from prodrug NPs after incubation for 108 h in the presence of 10 mM GSH, revealing the reduction-responsive drug release behavior. It is likely due to the decrease in the stability of prodrug NPs resulting by the cleavage of diselenide bonds. On the other hand, compared to DOX release in PB 7.4, a rather quick release behavior in PB 5.0 with about 50% of DOX release could be observed, exhibiting pH-responsive drug release behavior. The release of the parent drug can be ascribed to the breakage of hydrazone bonds. Notably, the DOX release rate was the fastest when the prodrug NPs were simultaneously triggered by acid and GSH with up to 85% of DOX cumulative release after incubation for 108 h. These results indicated that the prodrug NPs can be promising drug carriers for controlling drug release.

Figure 5. In vitro DOX release curves from [PEEP54.5-b-(PBYP13.5-hyd-DOX5.8)-Se]2 NPs at different pH and reductive conditions with a speed of 160 r min-1 at 37.5 °C.

19



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In Vitro Cytotoxicity. As is well known, the biocompatibility of polymers is an important argument to evaluate if they can be used for drug carriers. Hence, the cytotoxicity of [PEEP54.5-b-PBYP13.5-Se]2 NPs against L929 cells, HeLa cells, and HepG2 cells was studied by MTT assays. As shown in Figure 6, the cell viabilities of L929 cells, HeLa cells and HepG2 cells are all above 85% after incubating with the block copolymer [PEEP54.5-b-PBYP13.5-Se]2 for 48 h at different concentrations, even if the concentration is up to 1.2 mg mL-1. It indicated that the polymer [PEEP54.5-b-PBYP13.5-Se]2 own excellent biocompatibility as a drug carrier.

Figure 6. Cell viability of L929 cells, HeLa cells and HepG2 cells treated with [PEEP54.5-b-PBYP13.5-Se]2 at different concentrations for 48 h of incubation.

The

anti-proliferative

activity

of

the

polymeric

prodrug

[PEEP54.5-b-(PBYP13.5-hyd-DOX5.8)-Se]2 against HeLa cells and HepG2 cells were also investigated by MTT assays using free DOX as control. As shown in Figure 7, the cell viability of HeLa cells and HepG2 cells gradually decreased with the increase of DOX concentration, which showed the dose-dependent anti-proliferative activity. 20


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In addition, the half max-imal inhibitory concentration (IC50) values of the prodrug NPs with equal DOX concentration against HeLa cells and HepG2 cells were much higher than that of free DOX, which can be attributed to the following reasons. As previously mentioned, the backbone [PEEP54.5-b-PBYP13.5-Se]2 has favorable biocompatibility, which largely decreases the cytotoxicity. In ad dition, the disparate mechanism into cells is the key point. The prodrug NPs are internalized into tumor cells and gradually released DOX, while free DOX is transported into tumor cells through diffusion. In general, it is anticipated that the prodrug NPs possess good antitumor activity and relatively lower cytotoxicity.

21



Figure 7. Cell viability of (A) HeLa cells and (B) HepG2 cells treated with [PEEP54.5-b-(PBYP13.5-hyd-DOX5.8)-Se]2 and free DOX with different DOX dosages for 48 h of incubation.

Cellular Uptake. To real-time monitor the endocytosis process of the prodrug NPs, the cellular uptake behavior of [PEEP54.5-b-(PBYP13.5-hyd-DOX5.8)-Se]2 NPs was investigated against HeLa cells by a live cell imaging system. The cells were incubated with 10% prodrug NPs of DMEM using free DOX with the same concentration as a control. The emission of DOX can be directly used to visualize cellular uptake without fluorescence probes for its own fluorescent. As shown in Figure 8(A), there appeared slight DOX fluorescence in the cytoplasm of HeLa cells after incubation with [PEEP54.5-b-(PBYP13.5-hyd-DOX5.8)-Se]2 NPs for 1.5 h, which indicated that the prodrug NPs were successfully internalized into HeLa cells and released the parent DOX. Whereafter, the DOX fluorescence intensity became gradually stronger with the increase of incubation time from 0 h to 5.5 h. Just for 5.5 h incubation, the strong DOX fluorescence appeared inside of HeLa cells, demonstrating the efficient release of DOX from the prodrug NPs for the special microenvironment in HeLa cells. By contrast, the fluorescence intensity of free DOX in HeLa cells was relatively weak in 5.5 h incubation time as shown in Figure 8(B). This is due to the different ways of cellular internalization of free DOX and the prodrug NPs. Free DOX is transported into tumor cells through free diffusion according to the concentration gradient across the cell membrane, while prodrug NPs

22


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are internalized into tumor cells via endocytosis. And the prodrug needs to be triggered by the intracellular reductive and acidic environment, which will take some time to complete as the in vitro DOX release shown. All the above-mentioned results indicated that the prodrug NPs can be internalized by HeLa cells and accumulate more active DOX in HeLa cells.

Figure 8. The cellular uptake behavior of (A) [PEEP54.5-b-(PBYP13.5-hyd-DOX5.8)-Se]2 NPs following different incubation times in HeLa cells and (B) free DOX in HeLa cells. The DOX dosage was 64.5 mg L-1. For each panel, images from left to right show cell nuclei stained by Hoechst 33342 (blue), DOX (red) and overlays of two images. All the images were observed by a confocal laser scanning microscopy and the scale bars correspond to 50 µm in all images.

CONCLUSIONS In summary, we have successfully prepared a new pH/reduction-responsive diselenide-containing prodrug [PEEP-b-(PBYP-hyd-DOX)-Se]2 via a combination of 23



Page 24 of 46

one-pot sequential ROP of cyclic phosphate monomers and CuAAC “click” chemistry. The prodrug could self-assemble into NPs in aqueous solution with spherical morphology and Dz of 138 nm. The prodrug NPs kept rather stable in physiological medium while degradable in reductive and/or acidic condition. In vitro drug release results showed more efficient DOX release with maximum accumulative release of 85% from the prodrug NPs under the simulated tumor environment in compare with physiological condition, which indicates the favorable pH/reduction-responsiveness of the diselenide-containing polyphosphoester-DOX prodrug. Moreover, the MTT assay demonstrated that the prodrug NPs had good inhibition efficacy against HeLa cell and HepG2 cell, which could entered into HeLa cells via endocytosis to release the parent drug DOX and further to induce cell apoptosis. Overall, the pH/reduction-responsive polymeric prodrug could be a potential candidate to improve the efficiency of cancer treatment as a biocompatible drug carrier.

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

procedures

of

the

initiator

di-(1-hydroxylundecyl)

diselenide;

characterizations of NMR, GPC, FT-IR, UV-vis and HPLC; measurements of CAC value, TEM and DLS; method of cell culture; assay of in vitro cell cytotoxicity; 1H 24


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NMR spectrum of di-(1-hydroxylundecyl) diselenide;

1

H NMR spectra of

6-azidehexanohydrazine, DOX and DOX-hyd-N3; FT-IR spectra of DOX, DOX-hyd-N3,

[PEEP54.5-b-PBYP13.5-Se]2

and

[PEEP54.5-b-(PBYP13.5-hyd-DOX5.8)-Se]2; GPC curve of [PEEP54.5-b-PBYP13.5-Se]2; HPLC

analysis

results

of

DOX,

DOX-hyd-N3

and

[PEEP54.5-b-(PBYP13.5-hyd-DOX5.8)-Se]2; the result of CAC value; the morphology of prodrug NPs and corresponding particle size distribution.

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 25



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.

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Scheme 1. Synthetic routes of (1) di(1-hydroxyethylene) diselenide, (2) azide-functionalized pH-responsive DOX derivative DOX-hyd-N3 and (3) pH/reduction-responsive polymeric prodrug [PEEP-b-(PBYP-hyd-DOX)Se]2. 174x167mm (300 x 300 DPI)



Figure 1. 1H NMR spectrum of [PEEP54.5-b-PBYP13.5-Se]2 in CDCl3. 215x131mm (300 x 300 DPI)


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Figure 2.31P NMR spectra of (A) [PBYP13.5-Se]2 reaction mixture and (B) purified [PEEP54.5-b-PBYP13.5-Se]2 in CDCl3. 216x151mm (300 x 300 DPI)



Figure 3. UV-vis spectra of free DOX, [PEEP54.5-b-PBYP13.5-Se]2 and [PEEP54.5-b-(PBYP13.5-hyd-DOX5.8)-Se]2. 121x94mm (300 x 300 DPI)


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Figure 4. The particle size distribution histograms of [PEEP54.5-b-(PBYP13.5-hyd-DOX5.8)-Se]2 under different conditions of (A) PB 7.4, (B) PB 5.0 and (C) PB 7.4 + 10 mM GSH, as determined by DLS. 207x220mm (300 x 300 DPI)



Figure 5. In vitro DOX release curves from [PEEP54.5-b-(PBYP13.5-hyd-DOX5.8)-Se]2 NPs at different pH and reductive conditions with a speed of 160 r min-1 at 37.5 °C. 186x143mm (300 x 300 DPI)


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Figure 6. Cell viability of L929 cells, HeLa cells and HepG2 cells treated with [PEEP54.5-b-PBYP13.5-Se]2 at different concentrations for 48 h of incubation. 173x134mm (300 x 300 DPI)



Figure 7. Cell viability of (A) HeLa cells and (B) HepG2 cells treated with [PEEP54.5-b-(PBYP13.5-hyd-DOX5.8)Se]2 and free DOX with different DOX dosages for 48 h of incubation. 117x167mm (300 x 300 DPI)


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Figure 8. The cellular uptake behavior of (A) [PEEP54.5-b-(PBYP13.5-hyd-DOX5.8)-Se]2 NPs following different incubation times in HeLa cells and (B) free DOX in HeLa cells. The DOX dosage was 64.5 mg L-1. For each panel, images from left to right show cell nuclei stained by Hoechst 33342 (blue), DOX (red) and overlays of two images. All the images were observed by a confocal laser scanning microscopy and the scale bars correspond to 50 µm in all images. 148x149mm (300 x 300 DPI)



Graphic for Table of Contents 251x172mm (300 x 300 DPI)


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