Synthesis and pH-responsive self-assembly behavior of a fluorescent amphiphilic triblock copolymer mPEG-b-PCL-b-PDMAEMA-g-PC for the controlled intracellular delivery of doxorubicin

Abstract

In this work, well-defined pH-responsive methoxy poly(ethylene glycol)-block-poly(ε-caprolactone)-block-poly[2-(dimethylamino)ethyl methacrylate]-g-7-propinyloxy coumarin triblock amphiphilic copolymers (mPEG-b-PCL-b-PDMAEMA-g-PC) were synthesized using a combination of atom transfer radical polymerization (ATRP), ring opening polymerization (ROP) and click chemistry. The chemical structures and compositions of these copolymers were characterized using Fourier transform infrared spectroscopy (FT-IR) and proton nuclear magnetic resonance (¹H NMR). The molecular weights of the copolymers were obtained using ¹H NMR spectroscopy and gel permeation chromatography (GPC) measurements. Subsequently, the polymers could self-assemble into micelles which were investigated using dynamic light scattering (DLS), transmission electron microscopy (TEM), and fluorescence spectroscopy. The pH-responsive self-assembly behavior of these triblock copolymers in water were investigated at different pH values of 5 and 7.4 for controlled doxorubicin release, the results indicated that the release rate of DOX could be effectively controlled by altering the pH, DOX was sealed in neutral surroundings and DOX release was triggered in acidic surroundings. CCK-8 assays and confocal laser scanning microscopy (CLSM) against HeLa cells indicated that the micelles had no associated cytotoxicity, possessed good biodegradability and biocompatibility, and identified the location of the DOX in HeLa cells. The DOX-loaded micelles possessed high cytotoxicity to HeLa cells and exhibited inhibition of the proliferation of HeLa cells. Moreover, these flexible micelles with an on–off switched drug release may offer a promising pattern to deliver a wide variety of hydrophobic payloads to tumor cells for cancer therapy.

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1. Introduction

Currently, polymeric micelles as promising nanosized antitumor drug carriers are being extensively studied,^1–3 due to their unique properties and structures, such as their nanoscale size, core–shell structure, relatively high stability and prolonged circulation in the blood.^4,5 The ability of amphiphilic block copolymers to self-assemble in selective solvents have been studied widely.^6–8 And much scientific effort have been directed toward engineering ‘smart’ materials that are able to respond to changes such as variations in temperature,^9–11 pH,^12,13 or light in their environment.^14,15 Moreover, some stimuli-responsive polymers can make micelles responsive to a pH value. In contrast to other pH-sensitive chemotherapeutic delivery systems, micelles are highlighted for the following advantages: (i) high circulation half-life via avoiding non-selective uptake by the reticuloendothelial system (RES), (ii) enhanced solvent power to hydrophobic anticancer drugs, and (iii) utilization of the enhanced permeability and retention (EPR) effect for passive targeting.^16–19 For example, cancer tissue is also reported to be acidic extracellularly.^20–22 Polymeric micelles offer many unique advantages, such as passive accumulation in tumors, prolonged circulation time in blood and enhanced uptake by tumors.^23–25 In particular, micelles formed from block copolymers consisting of poly(ethylene glycol) (PEG) and poly(ε-caprolactone) (PCL) have drawn considerable interest.^26,27 PEG with its excellent biocompatibility forms the hydrophilic corona in micelles, thereby it can produce nonspecific adhesion of polymeric micelle surfaces to blood components. PCL, a hydrophobic polymer approved by the FDA for biomedical applications, is biocompatible, biodegradable, and highly permeable to drugs and PCL serves as the hydrophobic core in the micelles, which can be used to load water-insoluble drugs.^22,26,28,29 Poly(2-(N,N-dimethylamino)ethyl methacrylate) (PDMAEMA) is a weak base with a pK_a of about 7. Under its pK_a value, PDMAEMA is hydrophilic as its amine groups are protonated. Above its pK_a value, PDMAEMA is hydrophobic as its amine groups are deprotonated.^30–33 The self-assembly of amphiphilic block copolymers usually requires that copolymers have well-defined structures and narrow polydispersities, which are usually reached by using the “living”/controlled polymerization methods, such as atom-transfer radical polymerization (ATRP),^12,34 ring-opening polymerization (ROP),³⁵ and reversible addition–fragmentation chain transfer polymerization (RAFT).^36,37 Despite the versatility of CRP techniques, it is still a challenge to find feasible means to introduce functional groups onto polymer chains. These functional polymers, for example, fluorescent group labeled polymers, biofunctionalized polymers and drug containing polymers, could find wide applications in chemistry, materials science and biomedical science fields.^38–40 Moreover, a fluorescent coumarin unit could be labeled in vivo in order to detect DOX-loaded micelles. “Click” chemistry is a good choice to synthesize well-defined functional polymers. Chen et al.^41,42 synthesized poly(vinyl acetate) with fluorescence via a combination of RAFT/MADIX and “click” chemistry. Wang et al.⁴³ prepared amphiphilic centipede-like brush copolymers with PCL and poly(ethyl ethylene phosphate) side segments via a combination of ROP and click chemistry using a one-pot syntheses strategy. Ivonne L. et al.⁴⁴ synthesized PDMAEMA-b-PCL-b-PDMAEMA with different compositions via ATRP. The encapsulation ability of AmB depends on micelle dynamics and the length of the hydrophilic segment. Mao et al.³⁴ synthesized PLLA-b-PDMAEMA copolymers via ROP and ATRP. Zhu et al.⁴⁵ demonstrated that compared to stably shielded polyplexes of PDMAEMA-PEG-PDMAEMA analogues, reversibly shielded DNA polyplexes based on low molecular weight bioreducible, PDMAEMA-SS-PEG-SS-PDMAEMA triblock copolymers have excellent colloidal stability under physiological salt conditions. Zhu et al.²⁷ prepared PEG₄₅-b-PBO₉-b-PCL₆₁ triblock and PEG₄₅-b-PCL₆₂ diblock copolymers for comparative studies of micelle formation and relative stability in acidic media. You et al.⁴⁶ prepared two types of fluorescent PDI-core star polyelectrolytes, and unimolecular fluorescent micelles with perylenediimide cores that were sensitive to changes in pH value. Thomas K. et al.⁴⁷ synthesized a set of PEG-PCL-lPEI triblock copolymers, with decreasing hydrophilic/hydrophobic ratios and a transition from partly water-soluble micelle-like assemblies to mainly water-insoluble particle-like precipitates was observed. Dong et al.⁴⁸ synthesized PDMAEMA-g-PEG cationic hydrogel nanoparticles using distillation–dispersion copolymerization of methoxy mPEG and DMAEMA. It was found that the PDMAEMA-g-PEG nanoparticles performed pH, ionic strength, and thermosensitivity in water swelling behavior. Ni et al.⁴⁹ prepared a series of well-defined amphiphilic triblock copolymers mPEG-b-PCL-b-PDMAEMA using a combination of ROP and ATRP, which could self-assemble into micelles or vesicles in PBS buffer solution, depending on the length of PDMA in the copolymer. Agarose gel retardation assays demonstrated that these cationic nanoparticles can effectively condense plasmid DNA. Deng et al.⁵⁰ studied mPEG-b-PCL-b-PDMAEMA nanoparticles as the codelivery vector of hydrophobic drugs and pDNA. Chen et al.³ synthesized a well-defined amphiphilic PCL-b-[PGMA-g-PC]-b-P(PEGMA) with fluorescent units through ROP, RAFT and click chemistry, and the micelles were used as a nano-reservoir for the controlled release of DOX for bladder cancer therapy. Xu et al.⁵¹ developed pH-sensitive PMs self-assembled from amphiphilic poly(ethylene glycol)-copoly(n-butyl methacrylate-ran-methacrylic acid) for DOX release, simultaneously modifying the micellar shell with folate to improve the efficiency of cellular uptake by tumor cells.

In this study, we synthesized well-defined pH-responsive mPEG-b-PCL-b-PDMAEMA-g-PC with fluorescent units via ROP, ATRP and click chemistry with mPEG-Br as a macroinitiator and ε-CL and DMAEMA as the monomers. Incorporation of a block with fluorescent units as the end-capping groups in the shell displayed fluorescence properties as DOX carriers in vitro. The self-assembly and pH responsive properties of the copolymer in water were investigated. The release investigation of DOX from the micelles indicated that the release rate of the drug could be effectively controlled by altering the pH value. Meanwhile, the cellular uptake and cytotoxicity tests against HeLa cells were performed.

2. Experimental

2.1. Materials

mPEG (Alfa Aesar, M_n = 1900 g mol⁻¹), ε-CL (Sigma-Aldrich) was distilled under reduced pressure after being treated with CaH₂. Tin(II)ethylhexanoate (Sn(Oct)₂, Sigma), 2-bromoisobutyryl bromide (BIBB, Aldrich), and N,N,N′,N,N″-pentamethyldiethylenetriamine (PMDETA) were purchased from Sigma-Aldrich. Copper(I) chloride (99.999%, Alfa Aesar) was used without further purification. Doxorubicin hydrochloride (DOX·HCl) was purchased from Beijing Huafeng United Technology Co. Ltd. 2-(Dimethylamino)ethyl methacrylate (DMAEMA 98%, Aldrich) was filtered over aluminum oxide to remove the inhibitor (MEHQ) before being polymerized. Tetrahydrofuran (THF) was refluxed over sodium and distilled twice before use. Dimethylformamide (DMF) and triethylamine (TEA) were dried by refluxing over CaH₂ and distilled before use. 7-Hydroxy coumarin, propargyl bromide and sodium azide were purchased from Aladdin Reagent Company and used without further purification. Enhanced Cell Counting Kit-8 (CCK-8, Shanghai, Beyotime Biotechnology). Copper(I) chloride (99.999%, Alfa Aesar) was used without further purification. HeLa cells (Institute of cells, CAS, Shanghai) were used as received. Dulbecco’s modified eagle medium (DMEM), fetal bovine serum (FBS), and pancreatic enzymes were obtained from biological industries. 4% paraformaldehyde, 4′,6-diamidino-2-phenylindole (DAPI) and Triton X-100 were purchased from Solarbio.

2.2. Characterization

¹H NMR data were obtained using nuclear magnetic resonance spectroscopy (NMR) using a BrukerDMX-500 NMR spectrometer with CDCl₃ as the solvent. Fourier transform infrared spectroscopy (FT-IR) analysis was measured using an IR-Affinity-1 model spectrophotometer using KBr pellets. The molecular weight and molecular weight distribution of the copolymers were measured using gel permeation chromatography (GPC) using a Viscotek TDA 302 gel permeation chromatograph and THF was used as the eluent. X-ray photoelectron spectroscopy (XPS) was performed using an Axis Ultra spectrometer with a monochromatized Al-Kα X-ray as the excitation source (225 W). The fluorescence spectra were measured using a Hitachi F-2500 fluorescence spectrophotometer at an excitation wavelength of 300 nm. Dynamic light scattering (DLS) measurements were performed using a BECKMAN COULTER Delasa Nano C particle analyzer at a fixed angle of 165°. Before the light scattering measurements, the sample solutions were filtered three times by using Millipore Teflon (Nylon) filters with a pore size of 0.45 µm. All measurements were repeated three times, and the average result was accepted as the final hydrodynamic diameter (D_h) and zeta potential (mV). Samples for the transmission electron microscopy (TEM) images were taken on an H-600 transmission electron microscope (Hitachi, Japan) operating at 120 kV. Confocal laser scanning microscopy images (Zeiss CLSM510) and fluorescence microscope images (OLYMPUS U-RFL-T, Japan) were obtained at an excitation wavelength of 480 nm.

2.3. Synthesis of mPEG-b-PCL-b-PDMAEMA-g-PC with fluorescence triblock amphiphilic copolymer

2.3.1. Synthesis of mPEG-b-PCL diblock copolymer. mPEG-b-PCL was synthesized using ROP with different feed ratios of ε-CL using Sn(Oct)₂ as the catalyst. The typical procedure was as follows: mPEG (3.8 g, 2 mmol), ε-CL (6 g, 52.6 mmol), Sn(Oct)₂ (0.04 g, 0.1 mmol), and anhydrous toluene (50 mL) were added into a fresh flamed and nitrogen purged round-bottomed flask. The flask was then placed in a thermostatted oil bath at 120 °C for 24 h. After the polymerization, the mixture was cooled to room temperature. The product was dissolved in CH₂Cl₂, and precipitated in methanol three times. Finally, the precipitate was dried under vacuum to a constant weight at 35 °C. The different degrees of polymerization (DP) of PCL were synthesised using ROP in the some way, and were named MP1 and MP2 (yield: 90.6% and 95.3%).

2.3.2. Synthesis of mPEG-b-PCL-Br initiator. Typically, mPEG-b-PCL (4 g, 0.2 mmol) and triethylamine (3 mL) were first added into a 100 mL dry flask and after 30 mL of anhydrous CH₂Cl₂ was added to dissolve mPEG-b-PCL under a nitrogen atmosphere. The flask was placed in an ice/water bath. 2.0 mL of 2-bromoisobutyryl bromide was added into the flask dropwise over 1 h, and the reaction mixture was stirred for 48 h at 30 °C. The precipitate was filtered off. Then, the filtrate was washed three times sequentially with an aqueous solution of sodium bicarbonate and water. The product was further concentrated using a rotary evaporator, precipitated three times in methanol, and dried under vacuum to a constant weight at 35 °C (yield: 72.2% and 74.5%).

2.3.3. Synthesis of mPEG-b-PCL-b-PDMAEMA using ATRP. Synthesis of mPEG-b-PCL-b-PDMAEMA: a series of mPEG-b-PCL-b-PDMAEMA were prepared using ATRP of DMAEMA using mPEG-b-PCL-Br as the initiator and CuBr/PMDETA as the catalyst. The reaction procedures are shown in Scheme 1, the products are named MPD1 and MPD2.

Scheme 1 Synthesis of mPEG-b-PCL-b-PDMAEMA-g-PC with fluorescence triblock amphiphilic copolymer.

The typical procedure is described below: the reagents were mPEG-b-PCL-Br (0.2 g, 0.03 mmol), DMAEMA (2.0 g, 9.0 mmol), CuBr (0.151 g, 1.0 mmol), PMDETA (0.337 g, 2 mmol), and THF (30 mL). The flask was degassed with three freeze–evacuate–thaw cycles. Then, the polymerization was performed at 65 °C for 12 h. After being cooled to room temperature, the reaction flask was opened to air, and the crude product was diluted with THF and passed through a neutral alumina column to remove the copper catalysts. Finally the product was precipitated thrice into cold hexane, and dried under vacuum to a constant weight at 40 °C (yield: 50.6% and 52.3%).

2.3.4. mPEG-b-PCL-b-PDMAEMA with sodium azide. mPEG-b-PCL-b-PDMAEMA (1.5 g, 0.2 mmol) was dissolved in DMF (15 mL). Sodium azide (0.225 g, 3.46 mmol) was added to this solution, and the mixture was stirred at 65 °C for 48 h. The final reaction solution was diluted with DMF and purified using dialysis against water to remove any traces of salts and unreacted reactants using a membrane with a molecular weight cutoff of 7000 Da. The azide-containing polymers were collected using freeze-drying.

2.3.5. Synthesis of 7-propinyloxy coumarin (PC). The synthesis procedure was carried out according to the reported methods.^3,42 A mixture of 7-hydroxy coumarin (1.62 g, 10 mmol) in acetone (25 mL), K₂CO₃ (1.38 g, 10 mmol), KI (0.083 g, 0.5 mmol) and propargyl bromide (1.2 mL, 15 mmol) was added to a flask, and the mixture was stirred for 24 h at 80 °C. Then the reaction mixture was cooled to room temperature and extracted with CH₂Cl₂. The combined organic extracts were washed with water, dried over anhydrous MgSO₄ and evaporated to afford a crude product, which was purified using recrystallization from anhydrous ethanol to give a white solid (77.8% yield).

2.3.6. Synthesis of mPEG-b-PCL-b-PDMAEMA-g-PC using click chemistry. The synthetic pathway is shown in Scheme 1. Azide-containing polymers (1.0 g, 0.057 mmol), CuBr (0.151 g, 1.0 mmol), and PC (80 mg, 0.40 mmol) were purged with nitrogen to remove the dissolved oxygen, PMDETA (0.337 g, 2 mmol) was added under a nitrogen atmosphere. Then the ampoule was sealed and stirred at 60 °C in the absence of oxygen for 24 h. The reaction mixture was exposed to air, and the mixture was diluted using DMF, and purified using dialysis against water to remove unreacted reactants using a membrane with a molecular weight cutoff of 7000 Da. The resultant polymers mPEG-b-PCL-b-PDMAEMA-g-PC were collected using freeze-drying, and were named MPDP1 and MPDP2 (yield: 65.5% and 66.8%).

2.4. Self-assembly of mPEG-b-PCL-b-PDMAEMA-g-PC in aqueous solution

Samples for UV-vis, DLS and TEM were prepared as follows: mPEG-b-PCL-b-PDMAEMA-g-PC (20 mg) was dissolved in THF (4 mL) and subsequently, deionized water (1 mL) was added dropwise over a period of 30 min under high-speed stirring at room temperature. After 4 h, 8 mL of water was added to quench the micellar assembly, the mixture was subsequently dialyzed (molecular weight cut-off: 3500 Da) against distilled water for 36 h, and micelles with different concentrations could be obtained by diluting with distilled water. During this dialysis process, the hybridized copolymers self-assembled into micelles with PCL cores and mPEG, PDMAEMA shells, and were named MPDP1 and MPDP2.

2.5. DOX encapsulation and release studies

100 mg of MPDP1/MPDP2 and 10 mg of DOX·HCl were dissolved in DMF (4 mL) separately, and the two solutions were mixed in a vial and stirred for 30 min, a 3-fold excess of TEA was added in order to obtain DOX base. Then the mixture was added dropwise into water (80 mL) using a syringe pump under high-speed stirring. The DOX-containing suspension was then equilibrated under stirring at room temperature for 4 h, followed by thorough dialysis (molecular weight cut-off: 3500 Da) against deionized water for 2 days to remove any unloaded DOX. The obtained DOX-loaded micelles were named D-MPDP1 and D-MPDP2.

The DOX loading content (DLC) and loading efficiency (DLE) were determined using UV-vis spectrophotometry at 480 nm. To determine the drug loading level, a small portion of the DOX-loaded micelles was withdrawn and diluted with DMF to a volume ratio of 9/1 (DMF/H₂O). The amount of DOX encapsulated was quantitatively determined using UV-vis. The calibration curve used for drug loading characterization was established using the intensity of DOX with different concentrations in DMF/H₂O (9/1, v/v) solutions. The DLC was defined as the weight ratio of entrapped DOX to that of the DOX-loaded micelles. The DLE of DOX was obtained as the weight ratio between the DOX incorporated in the assembled micelles and that used in fabrication.

The in vitro DOX release profiles from the MPDP1/MPDP2 assembled micelles were evaluated using buffer solutions with pH values 5 and 7.4 in a dialysis bag (molecular weight cut-off: 3500). The whole bag was placed into 35 mL of PBS or acetate buffer and shaken (200 rpm) at 37 °C.

At specified time intervals 4 mL (V_e) samples were taken and an equal volume of fresh buffer added to maintain the total volume. The concentrations of DOX in the different samples were analyzed using UV-vis spectrophotometry at 480 nm. The cumulative percent drug release (E_r) was calculated using

where m_DOX represents the amount of DOX in the micelle, V₀ is the volume of the release medium (V₀ = 70 mL), C_i represents the concentration of DOX in the i_th sample and C_n represents the concentration of DOX in the n_th sample. The in vitro release experiments were carried out in triplicate at each pH and the reported results are the average values with standard deviations.

2.6. Cytotoxicity test

The cytotoxic effects of the polymers, free DOX or DOX-loaded mPEG-b-PCL-b-PDMAEMA-g-PC micelles were evaluated against HeLa cells using the standard CCK-8. To perform the cytotoxicity assay, HeLa cells were seeded at a density of 5000 cells per well on a 96-well plate and cultured for 24 h. The samples were prepared at a series of desired concentrations. Every experimental well was treated with the samples for 24 h and others were added with fresh medium as the control. After incubation for 24 h, CCK-8 was added into each well to dissolve the formazan by pipetting in and out several times. The absorbance of each well was measured at a test wavelength of 450 nm. The cell viability of the samples was calculated as follows:^18,52,53
where A_test and A_control represent the intensity determined for cells treated with different samples and for control cells, respectively, and A_blank is the absorbance of the wells without cells.

2.7. Intracellular release of DOX

Confocal laser scanning microscopy (CLSM) was used to visualize the subcellular localization and intracellular release behavior of the DOX-loaded micelles and free DOX for various lengths of time (0.5 h, 4 h and 24 h). First, the HeLa cells were seeded in a glass base dish with a coverslip at a density of 5000 cells and cultured in DMEM supplemented with 10% FBS for 24 h. Then the DOX-loaded micelles and free DOX were added, and the cells were cultured for 0.5 h, 4 h and 24 h under a humidified 5% CO₂-containing atmosphere. Finally, the location of intracellular fluorescence was validated using a CLSM imaging system (Zeiss CLSM510) at an excitation wavelength of 480 nm.

3. Results and discussion

3.1. Synthesis and characterization of the polymers

The overall experiment is illustrated in Scheme 1. mPEG-b-PCL-b-PDMAEMA-g-PC was synthesized with different hydrophobic segments using ROP, ATRP and click chemistry. The azide-terminated diblock copolymer was prepared using a nucleophilic substitution reaction between mPEG-b-PCL-b-PDMAEMA-Br and NaN₃. Subsequently, the obtained azido functionalized copolymer was involved in “click” chemistry with 7-propinyloxy coumarin to prepare the fluorescent polymers.

The FTIR spectra of the mPEG, mPEG-b-PCL, mPEG-b-PCL-b-PDMAEMA-N₃ and mPEG-b-PCL-b-PDMAEMA-g-PC are shown in Fig. 1. The intensive absorption peak at 1730 cm⁻¹ of (B)–(D) was assigned to the carbonyl band of PCL in comparison with mPEG (A). mPEG-b-PCL was confirmed using ¹H NMR (Fig. 2(B)) and FT-IR (Fig. 1(B)). Moreover, the success of the “click” reaction could also be confirmed using FT-IR spectroscopy from Fig. 1(C). It was clearly observed that the absorption peak at 2103 cm⁻¹ attributed to the azide group, disappeared after the click reaction in Fig. 1(D). All the results demonstrated that the click reaction had been successfully achieved.

Fig. 1 FT-IR spectra of mPEG (A), mPEG-b-PCL (B), mPEG-b-PCL-b-PDMAEMA-N₃ (C) and mPEG-b-PCL-b-PDMAEMA-g-PC (D).

Fig. 2 The ¹H NMR spectra of mPEG-b-PCL-Br (A) and mPEG-b-PCL (B).

Fig. 2 shows a comparison of the ¹H NMR spectra of mPEG-b-PCL and mPEG-b-PCL-Br with peak assignments in CDCl₃. In Fig. 2, the small peak at 3.396 ppm (peak a) and the sharp singlet at 3.650 ppm (peak b) respectively correspond to the protons of the CH₃O– and –CH₂CH₂O– units of the mPEG block, whereas the signals of the –CH₂– units of the PCL block could be found in peak c, d, e, and f at 2.319 ppm, 1.657 ppm, 1.394 ppm and 4.072 ppm. Moreover, as observed in Fig. 2(A), compared with the spectrum of mPEG-b-PCL, the ¹H NMR spectrum of mPEG-b-PCL-Br displayed new chemical shifts attributed to the protons of –COC(CH₃)₂– at 1.940 ppm (peaks g). The structural characteristics of mPEG-b-PCL-b-PDMAEMA had been determined using ¹H NMR analysis in Fig. 3. We found the characteristic signals of the protons in the PDMAEMA block in Fig. 3, the protons of –CH₂N– and –CH₃ of the PDMAEMA block corresponded to the peaks k and i at 2.602 ppm and 0.909 ppm, which could be observed at 2.602 ppm and 0.959 ppm. Furthermore, the overlapped signals at 4.076 ppm, 2.322 ppm and 1.816 ppm belonged to the protons of –COOCH₂CH₂–, –N(CH₃)₂ and –COOCH₂CH₂– in the PDMAEMA block. Synthesis of the 7-propinyloxy coumarin (PC) was carried out using a similar procedure to the one described in previous publications.^3,42 The resonance at 4.773 ppm of the ¹H NMR analysis of PC was the characteristic signal of the methylene protons of the propargyl group, while resonance of the alkenyl proton was observed at 2.602 ppm in Fig. 4.

Fig. 3 The ¹H NMR spectrum of mPEG-b-PCL-b-PDMAEMA (MPD2).

Fig. 4 The ¹H NMR spectrum of PC.

The obtained azido-functionalized copolymers were subsequently involved in “click” chemistry with the 7-propinyloxy coumarin to prepare fluorescent copolymers. The copper(I) and its ligands were reported to be very efficient for catalyzing the 1,3-dipolar cycloaddition of organic azides with the terminal alkynes. Herein, CuBr/PMDETA is used as the catalytic system,^54,55 and DMF as the solvent for the 1,3-dipolar cycloaddition of the azido-functionalized copolymers and 7-propinyloxy coumarin. Fig. 5 shows the ¹H NMR spectrum recorded for mPEG-b-PCL-b-PDMAEMA-g-PC. The characteristic signals at around 7.680 ppm, 7.407 ppm, 6.963 ppm and 6.315 ppm were assigned to the protons of the coumarin group and the signals of the alkenyl group of the PC units at 2.602 ppm disappeared completely, which further confirmed the formation of mPEG-b-PCL-b-PDMAEMA-g-PC.

Fig. 5 The ¹H NMR spectrum of mPEG-b-PCL-b-PDMAEMA-g-PC.

The degrees of polymerization (DP) for the PCL could be calculated from the integration ratio between the methylene protons (2.32 ppm) in the repeat units and those (3.70 ppm) in the terminal unit based on the ¹H NMR spectrum.

The molecular weights (M_n, NMR) of MP1, MPD1, MP2 and MPD2 were calculated according to the ¹H NMR analysis using the following equations:

The number-average molecular weight (M_n) of the copolymer was obtained using the following formula:

M_n of copolymer (MP1 and MP2) = 1900 + 114.14 × N_CL;

M_n of copolymer (MPD1 and MPD2) = 1900 + 114.14 × N_CL + 157.2 × N_DMAEMA

where A_b, A_c and A_f+j were the integral values of the peaks b, c and f, j in Fig. 2 respectively; 114.14 was the molecular weight of one repeating unit of the PCL block, 157.2 was the molecular weight of the DMAEMA, and 43 is the polymerization degree of mPEG.

The GPC traces of the MP1, MPD1, MPDP1, MP2, MPD2 and MPDP2 block copolymers are shown in Fig. 6. Of all the curves of MPD1, MPDP1 and MPD2, MPDP2 is shifted to a lower elution time compared to that of MP1 and MP2. The detailed information about the molecular weights and PDI of the triblock copolymers are listed in Table 1. The GPC results were almost consistent with the theoretical values and ¹H NMR analysis, which suggested that the MPDP block copolymers were synthesized and characterized successfully.

Fig. 6 Evolution of the GPC chromatograms of the mPEG-b-PCL, mPEG-b-PCL-b-PDMAEMA and mPEG-b-PCL-b-PDMAEMA-g-PC block copolymers.

Table 1 Compositions of the mPEG-b-PCL-b-PDMAEMA-g-PC block copolymers

Samples	M n,Th ^a	M n,NMR ^b	M n,GPC ^c	M w/Mn^c
a Calculated using theory analysis from the feed ratio of monomers to initiator. b Calculated from ^1H NMR data. c Polymerization conditions [monomer]0/[mPEG-b-PCL-Br]0/[CuBr]0/[PMDETA]0 = 100/1/1/2, measured using GPC calibrated with PS standards. THF was used as the eluent.
MP1	7942	10 035	8990	1.29
MPD1	16 320	17 940	15 980	1.13
MPDP1	—	—	16 567	1.15
MP2	10 450	13 440	12 757	1.09
MPD2	18 327	19 482	23 250	1.16
MPDP2	—	—	25 258	1.18

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3.2. Formation and characterization of the blank and DOX-loaded mPEG-b-PCL-b-PDMAEMA-g-PC (MPDP1 and MPDP2) block copolymer micelles

As an amphiphilic block copolymer, when the concentrations were above the critical micelle concentration (CMC), mPEG-b-PCL-b-PDMAEMA-g-PC could self-assemble into micelles in a selective solvent. The hydrophilic mPEG and PDMAEMA chains were mainly in the corona of the micelles, whereas the hydrophobic PCL side chains were mainly in the core of the micelles. Hydrophobic PCL as micelle cores has been extensively used for drug delivery systems. DOX was physically incorporated into MPDP1 and MPDP2 polymeric micelles, which were named D-MPDP1 and D-MPDP2. The physico-chemical properties of the blank and DOX-loaded micelles were characterized using DLS analysis. The hydrodynamic diameter (D_h), polydispersity index (PDI), and the zeta potentials of the blank and DOX-loaded micelles are summarized in Table 2.

Table 2 Hydrodynamic diameter (D_h), size distributions (PDI) and zeta potentials of the blank and DOX-loaded mPEG-b-PCL-b-PDMAEMA-g-PC micelles

Micelle	Blank			DOX-loaded
	D h (nm)	PDI	Zeta (mV)	D h (nm)	PDI	Zeta (mV)	DLC (wt%)	DLE (wt%)
MPDP1	78 ± 1.3	0.280 ± 0.035	24.67 ± 0.32	102 ± 2.5	0.135 ± 0.073	2.50 ± 0.22	4.58	45.8
MPDP2	95 ± 2.2	0.156 ± 0.021	26.05 ± 0.24	115 ± 3.1	0.216 ± 0.035	5.35 ± 0.45	5.43	54.3

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As shown in Fig. 7, the D_h, PDI and zeta potentials of the micelles were evaluated using DLS and TEM. The combination of TEM and DLS data showed a spherical morphology with a respective average radii around 78 and 95 nm (Fig. 7, and Table 2). In contrast, D_h was measured using TEM in Fig. 8, and the smaller observed sizes were probably due to the differences in the two measuring methods. The dehydration of the micellar shell during the TEM sample preparation process may have led to a decrease in the size of the micelles. Further, discrepancies were expected because DLS reported an intensity-average diameter, whereas TEM reported a number-average diameter. Thus, for a given size distribution of finite polydispersity, TEM images will always undersize relative to DLS data. When DOX was loaded into the polymeric micelles in the core and the drug adsorbed on the surface, it could also be found that the zeta potentials of the drug-loaded micelles were lower than those of the blank micelles for both of the polymers, resulting from the decreased charge density because of the larger particle sizes in Table 2 and Fig. 7. As shown in Fig. 9, a stability assay in terms of transmittance and particle size of the mPEG-b-PCL-b-PDMAEMA-g-PC micelles was investigated in water for (A) and (B). The MPDP1 (A) micelles presented reversible transformation of transparency and turbidity during the reversible cooling and heating cycles, when the temperature increases, hydrogen bonding could not be weakened but the micelles started to contract because of the decrease in electrostatic repulsion between the PDMAEMA chains, indicating that the coronas are hydrophobic to some extent. Obviously, the phase transitions of the micelles were reversible, which indicated that the mPEG-b-PCL-b-PDMAEMA-g-PC micelles were stable. As shown in Fig. 9(B), no obvious change of the particle size of the D-MPDP1 and D-MPDP2 micelles over 50 h indicated that the micelles had a good long-term stability without the presence of precipitation and phase separation. The results revealed that the MPDP1 and MPDP2 micelles could offer the protection of drugs from untimely structure disintegration and premature drug release before arriving at a disease site.

Fig. 7 The particle size distribution curves corresponding to the samples in MPDP1 and D-MPDP1 (A), MPDP2 and D-MPDP2 (B) and zeta potentials of DOX-loaded polymeric micelles in MPDP1 and D-MPDP1 (C), MPDP2 and D-MPDP2 (D).

Fig. 8 TEM images of the mPEG-b-PCL-b-PDMAEMA-g-PC polymeric micelles (MPDP1 (A and B) and MPDP2 (C and D)).

Fig. 9 Plots of transmittance as a function of temperature for the mPEG-b-PCL-b-PDMAEMA-g-PC copolymer micelle solution (A), particle size of the mPEG-b-PCL-b-PDMAEMA-g-PC in H₂O at room temperature (B).

The UV-vis spectrum of the MPDP1 micelles showed a characteristic UV-vis absorption peak of the coumarin moiety at 300 nm in Fig. 10(A). The fluorescence spectra of mPEG-b-PCL-b-PDMAEMA-g-PC are displayed in Fig. 10(B). It can be observed that the triblock copolymer, obtained by the coupling of the azide-containing polymers with 7-propinyloxy coumarin, exhibited a strong fluorescence peak at about 425 nm. Fig. 11 shows the fluorescence images of the PC (A), MPDP1 (B), MPDP2 (C), D-MPDP1 (D) and D-MPDP2 (E) micelles. We could see the bright blue of PC, MPDP1 and MPDP2 in Fig. 11(A)–(C). The DOX-loaded micelles displayed the bright blue fluorescence corresponding to the coumarin dye implying that the fluorescent micelles had been effectively internalized.

Fig. 10 (a) UV-vis spectrum of the MPDP1 micelles and (b) fluorescence spectra of the mPEG-b-PCL-b-PDMAEMA-g-PC micelles solution.

Fig. 11 Fluorescence images of the PC (A), MPDP1 (B), MPDP2 (C), D-MPDP1 (D) and D-MPDP2 (E) micelles. PC (blue), DOX (red).

3.3. In vitro release of DOX from the micelles

As expected, the mPEG-b-PCL-b-PDMAEMA-g-PC micelles exhibited a pH-responsive characteristic. In vitro drug release performances of the micelles were studied under physiological conditions (PBS, pH = 5 and 7.4) at 37 °C in Fig. 12. It could be observed that the drug release rates of DOX from the micelles were obviously changed by pH values as well as time. With regard to a pH of 7.4 at 37 °C, the micelles stayed compact and the loaded DOX was released slowly. After 5 h, less than 20% of the DOX (8.6% and 15.1% for D-MPDP1 and D-MPDP2, respectively) was released. Even after 24 h, only about 26.1% and 35.1% for D-MPDP1 and D-MPDP2 was released, respectively. In contrast, when the pH was lower at 37 °C (pH = 5), the drug release was accelerated. After 24 h, the cumulative release was 83.1% and 88.7% for D-MPDP1 and D-MPDP2, respectively. Although the mechanism of drug release from the polymeric matrices was very complex and is still not completely understood, it could be simplistically classified as either pure diffusion, erosion controlled release or a combination of the two mechanisms.^56,57 In this study, the results were due to the swollen drug-loaded micelles, attributing to the protonation of the amino groups in the PDEAEMA segments at weakly acidic conditions. Furthermore, the accelerated micelles rate might be related to the swollen hydrophobic core, which could cause the drug molecules close to the surface to diffuse into the medium. Meanwhile, the DOX molecules were not only encapsulated inside the micellar core, but also absorbed on the PDMAEMA shell due to the electric action, while only that loaded by the hydrophobic effect could be released comparatively fast, so it might require an extended period to achieve complete release. These MPDP1 and MPDP2 micelles were just like on–off switching nanocarriers in release kinetics with changing pH values. Therefore, they are highly interesting for intracellular anti-cancer drug delivery.

Fig. 12 In vitro release of DOX from various DOX-loaded polymeric micelles at 37 °C under different pH conditions.

3.4. Cytotoxicity test

The cytotoxic effects of the polymers, free DOX or DOX-loaded micelles in HeLa cells were determined using a CCK-8 assay. The cell viability of the blank micelles and DOX-loaded micelles against HeLa cells was evaluated. The cell viability of the blank micelles was measured after 48 h incubation. The blank micelles with different concentrations were nontoxic to HeLa cells and the cell viability was over 95% at all concentrations (12.5–200 µg mL⁻¹) tested in Fig. 13, which indicated that the blank micelles could be used as a delivery system for anticancer agents.

Fig. 13 In vitro cell viability of the mPEG-b-PCL-b-PDMAEMA-g-PC micelles. Concentration-dependent cell viability of HeLa cells treated with the MPDP1 (A) and MPDP2 (B) after incubation for 48 h.

Fig. 14 shows the results of samples treated with free DOX and DOX-loaded micelles for 48 h. The DOX dosages required for the inhibitory concentration to produce 50% cell death (IC₅₀) were 0.633 µg mL⁻¹, 1.208 µg mL⁻¹, 1.092 µg mL⁻¹ for 48 h for free DOX, D-MPDP1 and D-MPDP2 against HeLa cells, respectively. The slight difference between the two DOX-loaded micelles could be explained in that the latter contained more PCL segments, leading to a higher drug loading level and more sensitivity. Both of the DOX loaded micelles showed slightly lower cytotoxicity than free DOX due to the time-consuming DOX release from the micelles in comparison to free DOX at the same concentration. All of the DOX-loaded micelles had a similar capacity for killing tumor cells as free DOX for 48 h, which indicated that the DOX-loaded micelles might not inhibit the ability for killing cells although it slowed down the release of DOX. Moreover, the results revealed that DOX released from the micelles could exploit as potent a drug efficacy as free DOX after entry into the HeLa cells. It could produce the desired pharmacological action and minimize the side effects of free DOX.

Fig. 14 Cell viability of HeLa cells incubated with free DOX, DOX-loaded micelles (D-MPDP1 and D-MPDP2) for 48 h at different concentrations.

3.5. In vitro cellular uptake studies

To evaluate the intracellular uptake efficiency, confocal laser scanning microscopy was used to identify the location of the DOX in the HeLa cells. The CLSM images of the HeLa cells after 0.5, 4 and 24 h of incubation with free DOX and DOX-loaded micelles (D-MPDP2) are presented in Fig. 15. After incubation for 0.5 h, and staining with DAPI, the nuclei and cytoplasm of the pretreated cells are observed using CLSM. By comparison with the control in Fig. 15(B), the observation revealed that free DOX was slightly accumulated in the cell nuclei of the HeLa cells in Fig. 15(A). After incubation for 4 h and 24 h, a larger amount of free DOX had accumulated than DOX-loaded micelles (D-MPDP2). While DOX released from D-MPDP2 was mainly located in the cytoplasm, and DOX was released into the cytoplasm and nuclei of the cells under acid conditions in the lysosomes in Fig. 15(A) and (B). The results also indicated that free DOX was taken up by diffusion through the cell membrane and that the DOX loaded micelles were taken up by the nuclei of the cells via the endocytosis process. Moreover, the self-assembled micelles showed great potential as anti-tumor drug carriers for cancer therapy (Scheme 2).

Fig. 15 Confocal laser scanning microscopy images of HeLa cells incubated with (A) free DOX and (B) DOX-loaded (D-MPDP2) over different times. The DOX dosage was 10 µg mL⁻¹. For each panel, images from left to right show cell nuclei stained by DOX fluorescence in cells (red), bright field of cells, HeLa (blue), and overlays of the blue and red images. The scale bars are 20 µm in all images.

Scheme 2 Illustration of pH-responsive self-assembly of the mPEG-b-PCL-b-PDMAEMA-g-PC copolymer for the efficient intracellular release of anti-cancer drugs triggered by the acidic microenvironment inside the tumor tissue.

4. Conclusion

In the current work, we have reported pH-responsive mPEG-b-PCL-b-PDMAEMA-g-PC copolymers, synthesised using click chemistry, ROP, and ATRP, with fluorescence units for cancer therapy. The hydrodynamic diameter, polydispersity index (PDI) and zeta potential of the mPEG-b-PCL-b-PDMAEMA-g-PC micelles were evaluated using DLS and TEM, and the sizes of the MPDP1 and MPDP2 micelles with spherical shapes were determined using TEM to be about 78 nm and 95 nm. These copolymers could markedly improve micellar stability and extend the range of applications of micelles in controlling drug delivery with increasing PCL segments. In addition, the DOX-loaded micelles had a fluorescence, which were investigated using a fluorescence spectrophotometer and fluorescence microscope in order to label the location of the drug in vivo.

The release behaviors of DOX from the micelles exhibited pH-responsive characteristics in vitro, the DOX loading content became higher as the PCL segments increased. And the release of DOX from the micelles was significantly accelerated by decreasing pH from 7.4 to 5.0 at 37 °C. After 50 h for the D-MPDP2 micelles, the cumulative release was about 88.7% (w/w), which could provide sustained drug delivery behavior after the DOX-loaded micelles entered into blood circulation by endocytosis. In addition, the cytotoxicity tests revealed that these MPDP micelles possessed good biodegradability and biocompatibility for the HeLa cells and the DOX-loaded micelles showed a much higher toxic effect, which was almost similar to free DOX. Moreover, the DOX loaded micelles were found to be taken up by the nuclei of the cells via the endocytosis process using CLSM. Furthermore, the applicability of these micelles toward tumor-targeting delivery applications in vivo is highly promising for chemotherapy in the future.

Acknowledgements

The authors gratefully acknowledge financial support from the National Natural Science Foundation of China (21367022) and the Bingtuan Innovation Team in Key Areas (2015BD003).

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