Synthesis and thermo-responsive self-assembly behavior of amphiphilic copolymer β-CD–(PCL–P(MEO₂MA-co-PEGMA))₂₁ for the controlled intracellular delivery of doxorubicin

Abstract

Well-defined amphiphilic β-cyclodextrin (β-CD) star-shaped copolymers with poly(ε-caprolactone)–poly(2-(2-methoxyethoxy)ethyl methacrylate)-co-poly(ethylene glycol)methacrylate) (β-CD–(PCL–P(MEO₂MA-co-PEGMA))₂₁ were synthesized via ring opening polymerization (ROP) and atom transfer radical polymerization (ATRP). These thermo-responsive copolymers exhibited a lower critical solution temperature (LCST) in water, which could be finely tuned by changing the feed ratio of PEGMA and MEO₂MA. The LCST of star-shaped β-CD–(PCL–P(MEO₂MA-co-PEGMA))₂₁ increased from 26 to 62 °C with the increasing PEGMA content, the copolymers were characterized using Fourier transform infrared spectroscopy (FTIR), proton nuclear magnetic resonance (¹H NMR) and gel permeation chromatography (GPC). The star-shaped copolymers could self-assemble into micelles in aqueous solution due to their amphiphilic properties resulting from the hydrophobic β-CD and PCL core and the hydrophilic P(MEO₂MA-co-PEGMA) segments, which was investigated by dynamic light scattering (DLS) and transmission electron microscopy (TEM). The in vitro DOX release studies showed that DOX released from drug-loaded micelles in a thermo-sensitive manner. CCK-8 assays demonstrated that these star-shaped copolymers could possess low cytotoxicity against HeLa cells, and the DOX-loaded micelles exhibited a higher inhibition of the proliferation of HeLa cells in comparison with free DOX. Moreover, the results from confocal laser scanning microscopy (CLSM) revealed that these polymeric micelles could efficiently deliver and release DOX into the nuclei of HeLa cells. This kind of biodegradable, biocompatible and stimuli-responsive copolymer could serve as a promising material for drug delivery.

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1. Introduction

In recent decades, cancers have remained the most disastrous diseases worldwide. However, chemotherapy was usually restricted by the severe cytotoxicity of anticancer drugs to normal tissues and cells.¹ For this purpose, many researchers have paid much attention to various drug formulation micelles to reduce the side effects and improve the therapeutic efficacy of anticancer drugs.² Among them, biocompatible and biodegradable amphiphilic polymeric micelles have become some of the most promising carriers especially for poorly water-soluble anticancer drugs, such as the doxorubicin (DOX).³ This strategy could not only overcome some limitations associated with small molecule chemotherapeutic drugs, but greatly improve drug bioavailability via either passive targeting by the enhanced permeability and retention (EPR) effect^4,5 or active targeting by introducing some specific tumor-homing ligands.⁶

Star-shaped block copolymers have attracted extensive interests during the past decade due to the potential applications as “smart materials”, especially biomedical applications.^7,8 And their self-assembled structures that were able to respond to changes in their environment such as variations in temperature,^9,10 pH^11,12 or light.¹³ Synthesis of star-shaped block copolymers were first realized using ionic polymerization techniques.¹⁴ Beside ring-opening polymerization (ROP),¹⁵ click chemistry,¹⁶ reversible addition fragmentation chain transfer polymerization (RAFT)^17,18 and atom transfer radical polymerization (ATRP)^19,20 could be highlighted in particular. A well-defined star macromolecules could be separated into different categories: the “arm-first” approach²¹ contains the cross-linking of linear arm precursors with a cross-linking agent and the “core-first” approach²² requires the use of a multifunctional initiator (core) to initiate the growth of the polymer chains.

It was well-known that cyclodextrins (CDs) were a range of cyclic oligosaccharides that comprise several glucopyranose linked by α-(1,4) glycosidic bonds. Because of steric effect, α-, β- and r-CD containing 6, 7 or 8 D-glucose units could be produced using genetic engineering techniques.²³ CDs had been widely used as drug carriers to enhance the solubility, stability and thus bioavailability of drug, achieve sustained drug release, as well as make the oily drug solidification.²⁴ Because of their hydrophobic interior cavities CDs were well-known in supramolecular chemistry as hosts capable of including guest molecules in their cavities with high selectivity. The main driving forces behind the formation of these so-called host–guest inclusion complexes were hydrophobic and van der Waals interactions. Due to these capabilities make them form inclusion complexes with low price, non-toxic, good biocompatibility and biodegradation.^25,26 CDs were have frequently been applied in pharmacy, analytical sciences, separation processes and catalysis, as well as in the cosmetic, textile and packaging industry.^27,28

Over the past few years, biodegradable and biocompatible saccharide-based branched amphiphiles had attracted much attention for their applications as biomaterials. As a U.S. Food and Drug Administration approved biomedical polymer, biodegradable poly(ε-caprolactone) (PCL) and PCL-based biomaterials have been increasingly investigated for pharmaceutical and biomedical applications.^19,29 Poly(ε-caprolactone) (PCL) was hydrophobic, semi-crystalline polymer which was non-toxic, biodegradable and biocompatible. Yuan et al.³⁰ well-defined amphiphilic dendrimer-star copolymer DPCL-b-(P(MEO₂MA-co-OEGMA))₂ with Y-shaped arms were synthesized by the combination of ROP and ATRP. These unique high-branched were amphiphilic copolymers have the potential applications in biomedical field. Drug release was indicated that the release rate of model drug CBL from the micelles can be effectively controlled by changing the external temperatures. Li et al.³¹ synthesized β-CD–PLA–mPEG copolymer by click reaction of β-CD–N₃ and alkyne-terminated β-PLA–mPEG. The β-CD–PLA–mPEG copolymer could self-assemble in aqueous solution to form micelles and loaded with the hydrophobic drug (IND). Compared with formed by PLA–mPEG copolymer, the micelles were formed by β-CD–PLA–mPEG copolymer present higher drug loading efficiency and controlled release profile of IND, especially in the control of its initial burst release. It was suggested that the introduction of β-CD unit could enhance the stability of IND in the core of micelles. Cytotoxicity results demonstrated that β-CD–PLA–mPEG copolymer had low toxicity to cells. Thus the micelles formed by β-CD–PLA–mPEG copolymer could be a promising controlled release system for various hydrophobic drugs. Ni et al.³² developed a novel strategy for the mild and precise modular synthesis of well-defined three-armed star-block copolymers comprising PEG and PCL blocks linked with six acid-cleavable acetal groups, designated as (mPEG-a-PCL-a-)₃. The star-block copolymers could self-assemble into micelles and encapsulate the hydrophobic anticancer drug doxorubicin (DOX) simultaneously. Zhou et al.³³ searched a new targeting therapies for membrane estrogen receptor-positive breast cancer, an estrogen-anchored cyclodextrin encapsulating a doxorubicin derivative Ada-DOX (CDE1-Ada-DOX) had been synthesized and evaluated in human breast cancer MCF-7 cells. It was demonstrated that a targeted therapeutics delivery of CDE1-Ada-DOX to breast cancer cells in a controlled manner and that the drug vector CDE1 could be potentially employed as a molecular tool to differentiate nongenomic from genomic mechanism. Tian et al.³⁴ synthesized the photo-controlled host–guest interaction between azobenzene and β-cyclodextrin as a new strategy to improve the method of core removal to prepare poly((itaconoyloxy)ethyl methacrylate)-block-poly(N-isopropylacrylamide)-based hollow nano-spheres. These results showed hollow nano-spheres displayed a typical “breathing” behavior, which further induced the controlled release of doxorubicin hydrochloride. Lutz et al.³⁵ investigated a new class of water-soluble copolymers based on 2-(2-methoxyethoxy)ethyl methacrylate and oligo(ethylene glycol)methacrylate P(MEO₂MA-co-OEGMA) by ATRP, and P(MEO₂MA-co-OEGMA) copolymers were found to exhibit extremely interesting solution properties in water. Indeed, the random copolymers of P(MEO₂MA-co-PEGMA) were more attractive for their wide and tunable LCST between 26 °C and 90 °C depending on OEGMA content in water,³⁶ which meant that they could undertake a reversible change of swelling and collapse in water at temperature close to physiological ones. And P(MEO₂MA-co-OEGMA) presented good properties, such as water solubility, nontoxicity, anti-mutagenicity and biocompatibility.³⁷

However, there exist some obstacles in cancer chemotherapy including the multiple drugs resistance of the disease causing organism.³⁸ To overcome this drawback and further improve delivery efficiency, a promising method has been developed aiming at achieving synergistic effects by the combination of two or more therapeutic approaches with different mechanisms. For instance, the polymeric micelles self-assembled from amphiphilic block copolymers, which have hydrophobic core to encapsulate some poorly water-soluble drugs and hydrophilic shell to stabilize micelles, hold enormous potential to increase the drug efficacy by means of reducing toxicity, improving the water solubility, prolong circulation time in blood and the enhanced uptake by tumors.^39–41 Therefore, how to prepare a good water-soluble and biocompatible polymeric with stimuli-responsive linker and low cytotoxicity by means of efficient synthesis methods was still a fatal issue.

In this study, we synthesized and well-defined thermo-responsive β-CD–(PCL–P(MEO₂MA-co-PEGMA))₂₁ amphiphilic copolymer via ROP and ATRP with β-CD–PCL–Br as initiator, MEO₂MA and PEGMA as monomers. These thermo-responsive star-shaped copolymers showed critical phase transition temperature in water, which could be finely tuned by changing the feed ratios of PEGMA and MEO₂MA. The star-shaped copolymers could self-assemble into spherical nano-micelles in water, and the tunable thermo-sensitive properties and the morphology of these micelles were observed by UV-vis, DLS and TEM. The release of DOX from DOX-loaded micelles could be effectively controlled by altering the temperature value. In addition, the cellular uptake and cytotoxicity test to HeLa cells were also performed. Hence, such amphiphilic copolymer could be a potential candidate for stimuli-responsive drug delivery vector.

2. Results and discussion

2.1 Synthesis and characterization of β-CD–PCL and β-CD–(PCL–P(MEO₂MA-co-PEGMA))₂₁

The β-CD, β-CD–PCL and β-CD–(PCL–P(MEO₂MA-co-PEGMA))₂₁ were characterized by FTIR. The structure of β-CD–PCL and β-CD–(PCL–P(MEO₂MA-co-PEGMA))₂₁ respectively showed that there was a wide peak round about 1728 cm⁻¹, which indicated that the stretch vibration absorption of C–O groups; the peaks at 3400 cm⁻¹ and 2931 cm⁻¹ was ascribed to C–H stretching vibrations; a strong broad band peak observed at about 1158 cm⁻¹ results from C–O–C stretching. In addition, peaks at 1650 cm⁻¹, 1150 cm⁻¹ and 1027 cm⁻¹ corresponded to C–O, C–O–C glucose units and C–O–C of rings CD were observed. It can be seen that the absorption peak of C–C and C–O of β-CD–P(MEO₂MA-co-PEGMA)₂₁ at 1039 and 1105 cm⁻¹ was much weaker than that of C–H in β-CD–Br due to the high content of methyl and methylene groups in P(MEO₂MA-co-PEGMA) segments (Fig. 1).

Fig. 1 FTIR spectra of β-CD, β-CD–PCL and β-CD–(PCL–P(MEO₂MA-co-PEGMA))₂₁.

The chemical structure of β-CD, β-CD–PCL and β-CD–PCL–Br were also characterized by ¹H NMR was spectroscopy in DMSO-d₆ and CDCl₃ (Fig. 2 and 3). The signals located at broad chemical shifts in the region of 3.35–3.90 ppm were mainly associated with the inner methylidyne and methylene protons between the oxygen and carbon moieties (H-4, 5, 6, O–CH–C and O–CH₂–C) on the glucose units of β-CD. The peak located at a chemical shift of δ = 4.88 ppm was attributable to the inner methylidyne protons between the oxygen moieties (H-1, O–CHO). The signals located at the chemical shifts in the region of δ = 4.19–4.40 ppm were mainly attributable to the hydroxyl protons adjacent to the methylene moieties (OH-6, CH₂–OH). The peak at δ = 5.75 ppm corresponds to the hydroxyl protons adjacent to the methylidyne moieties (OH-2, 3, CH–OH) of glucose units. The structural characteristics of the obtained star-shaped β-CD–PCL and β-CD–(PCL–P(MEO₂MA-co-PEGMA))₂₁ have been determined by ¹H NMR analysis in Fig. 3 and 4. The resonance at 4.07 ppm (a), 2.32 ppm (b), 1.67 ppm (c), and 1.39 ppm (d) were the characteristic signal of the methylene protons at different positions respectively of PCL group, whereas the peak located at a chemical shift of δ = 1.92 ppm was associated with the methyl protons (a, C(Br)–CH₃) of the 2-bromoisobutyryl groups. In the upper diagram β-CD–(PCL–P(MEO₂MA-co-PEGMA))₂₁ some new peaks from MEO₂MA and PEGMA units can be seen in Fig. 4, the protons in P(MEO₂MA-co-PEGMA) can be observed: 0.890 ppm (a, c, e: CH₂C(CH₃)), 1.802 ppm and 1.926 ppm (b, d: CH₂C(CH₃)), 3.412 ppm (h: CH₂CH₂OCH₃), 3.661 ppm (g: CH₂CH₂O), and 4.118 ppm (f: CH₂CH₂O). It was confirmed that the copolymer have been produced. As shown in Fig. 4, the peaks of the protons in β-CD–PCL were overlapped by the signals of protons in P(MEO₂MA-co-PEGMA). Therefore, it was calculate the molecular weight of the copolymers according to ¹H NMR spectrum. From the data in Table 1, the clearly identified signals of β-CD provide us with a chance to calculate molecular weight (M_n,NMR) of star polymers, the molecular weights obtained from ¹H NMR spectra.⁴⁴ The ratio of MEO₂MA to PEGMA in copolymer calculated by the integral ratio of peak g to peak h (Fig. 4) were similar with the feed ratio of MEO₂MA to PEGMA. The GPC traces of β-CD–(PCL–P(MEO₂MA-co-PEGMA))₂₁ copolymers were shown in Fig. 5. All the curves of β-CD–(PCL–P(MEO₂MA-co-PEGMA))₂₁ shifted to lower elution volume compared to that of β-CD–PCL. The detailed information about the molecular weights and PDI of the copolymers were listed in Table 1.

Fig. 2 The ¹H NMR spectrum of β-CD.

Fig. 3 The ¹H NMR spectra of (a) β-CD–PCL and (b) β-CD–PCL–Br.

Fig. 4 The ¹H NMR spectrum of β-CD–(PCL–P(MEO₂MA-co-PEGMA))₂₁ copolymer (MEO₂MA : PEGMA = 90 : 10).

Table 1 Characterization of β-CD–(PCL–P(MEO₂MA-co-PEGMA))₂₁ copolymers^a

Samples	[MEO2MA]/[PEGMA] (mol%/mol%)		Mn,GPC^b (g mol^−1)	Mn,NMR^c (g mol^−1)	PDI^b
	Feed ratio	Actual ratio
a Polymerization conditions [monomer]0/[β-CD–PCL–Br]0/[CuBr]0/[PMDETA]0 = 100/1/1/2.b Measured by GPC calibrated with PS standards. THF was used as eluent.c Calculated from ^1H NMR data.
β-CD–PCL	—	—	26 003	27 500	1.19
C0	100/0	100/0	78 265	28 900	1.18
C1	95/5	94/5	76 601	76 800	1.55
C2	90/10	91/9	73 851	74 000	1.38
C3	85/15	87/14	75 821	75 600	1.29
C4	80/20	79/21	73 699	74 500	1.45

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Fig. 5 Evolution of GPC chromatograms of β-CD–PCL and β-CD–(PCL–P(MEO₂MA-co-PEGMA))₂₁ copolymers with different molecular weights.

2.2 Formation and characterization of the blank and DOX-loaded β-CD–(PCL–P(MEO₂MA-co-PEGMA))₂₁ (PCD1 and PCD2) copolymers micelles

As an amphiphilic copolymers, when the concentration was above the LCST, β-CD–(PCL–P(MEO₂MA-co-PEGMA))₂₁ could self-assemble into micelles in selective solvent. The hydrophilic P(MEO₂MA-co-PEGMA) arm chains were mainly in the corona of the micelles, whereas the hydrophobic β-CD and PCL chains in the star-shaped copolymer were mainly in the core of the micelles. The hydrophobic of PCL as cores have been extensively used for drug delivery system, which were named PCD1 and PCD2 micelles. DOX was physically incorporated into PCD1 and PCD2 micelles, which were named D-PCD1 and D-PCD2. The physico-chemical properties of the blank and DOX-loaded micelles were characterized by DLS analysis, respectively. The average particle sizes, polydispersity index (PDI) of the blank and DOX-loaded micelles were summarized in Table 2.

Table 2 Hydrodynamic diameter (D_h) and size distributions (PDI) of the blank and DOX-loaded β-CD–(PCL–P(MEO₂MA-co-PEGMA))₂₁ micelles

Micelle	Blank		DOX-load
	Dh (nm)	PDI	Dh (nm)	PDI	DLC (wt%)	DLE (wt%)
D-PCD1	83.0	0.243	113.9	0.225	5.75	57.5
D-PCD2	57.8	0.216	90.1	0.124	5.43	54.3

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As shown in Fig. 6. The hydrodynamic particle size, particle size distribution and polydispersity index (PDI) of the β-CD–(PCL–P(MEO₂MA-co-PEGMA))₂₁ polymeric micelles were evaluated by DLS and TEM. The combination of TEM and DLS results were confirmed that a spherical morphology with the respective average radii around 83.0 and 57.8 nm, respectively (Fig. 7 and Table 2). Fig. 7 indicated that TEM images obtained from aqueous solutions of β-CD–(PCL–P(MEO₂MA-co-PEGMA))₂₁ (C1 and C2). It was strange that, the self-assembly aggregates of β-CD–(PCL–P(MEO₂MA-co-PEGMA))₂₁ were small spherical. For all the samples (C1, a and b and C2, c and d), the spherica of polymeric micelles formed at room temperature (30 °C). A close view of the enlarged spherical in Fig. 7(a and b) showed that some β-CD aggregates connected to each other, tending to a layered irregular structure. According to this observation, we believed that the capability of the β-CD aggregates to form layered crystal were the main driving force in the formation of spherical. At room temperature, the micelles were relatively diameter of about 85 nm in Fig. 7(a and c) (bar = 100 nm) and Fig. 7(b and d) (bar = 500 nm). As a comparison, the values increased drastically to approximately 450 nm. The change tendency of diameter values of the micelles were consistent with DLS results, however, the diameter of the ellipsoid measured by DLS were larger than that in TEM images, these were due to the DLS results directly reflected the dimension of micelles in solution, where the P(MEO₂MA-co-PEGMA) chains as the corona were highly stretched in aqueous solution. And we sum up the self-assembly process of β-CD–(PCL–P(MEO₂MA-co-PEGMA))₂₁ in Scheme 2.

Fig. 6 The particle size distribution curves corresponding to the samples in (a) PCD1 and D-PCD1, (b) PCD2 and D-PCD2.

Fig. 7 TEM images of β-CD–(PCL–P(MEO₂MA-co-PEGMA))₂₁ polymeric micelles (PCD1 (a, b) and PCD2 (c, d)).

As shown in Fig. 8, a stability assay in terms of transmittance and particle size of the β-CD–(PCL–P(MEO₂MA-co-PEGMA))₂₁ polymeric micelles were investigated in water for (a) and (b). The β-CD–(PCL–P(MEO₂MA-co-PEGMA))₂₁ (PCD1 and PCD2) (a) micelles solution were presented reversible transformation of transparency and turbidity during the reversible heating and cooling cycles. Apparently, the phase transition of the micelles were reversible, which showed that the β-CD–(PCL–P(MEO₂MA-co-PEGMA))₂₁ copolymers micelles solution were stable and could undertake a reversible change of swelling and collapse. As shown in Fig. 8(b), the particle size of the D-PCD1 and D-PCD2 polymeric micelles in water, no apparent change of the particle size. This was indicated that the polymeric micelles have a well long-term stability without the presence of precipitation and phase separation. The results demonstrated that the PCD1 and PCD2 micelles could potentially be used in efficient drug delivery.

Fig. 8 Plots of transmittance as a function of temperature for β-CD–(PCL–P(MEO₂MA-co-PEGMA))₂₁ copolymer micelle solution (a), particle size of the β-CD–(PCL–P(MEO₂MA-co-PEGMA))₂₁ in water at room temperature (b).

Thermo-responsive polymers were completely miscible in the solvent in all proportions at temperature below the LCST but tend to collapse and become insoluble above the LCST. The obtained four samples of β-CD–(PCL–P(MEO₂MA-co-PEGMA))₂₁ were used to self-assemble into micelles in aqueous solution and the tunable thermo-responsive property was surveyed. Fig. 9 showed the transmittance curves of β-CD–(PCL–P(MEO₂MA-co-PEGMA))₂₁ micelles in water with different molar ratio of MEO₂MA and PEGMA. It could be seen that the transmittance curves showed sharp transition during heating process. The LCST values were increased from 25, 35, 42 and 56 to 61 °C with the increase of molar ratio of PEGMA in polymers from 0%, 5%, 10% and 15–20%, which were indicated that the LCST values of micelle solutions could be easily adjusted through altering the ratios of MEO₂MA and PEGMA in the twenty-one hydrophilic arms.

Fig. 9 Transmittance versus temperature for 1 mg mL⁻¹ β-CD–(PCL–P(MEO₂MA-co-PEGMA))₂₁ aqueous solution at the wavelength of 590 nm and the optical photographs of β-CD–(PCL–P(MEO₂MA-co-PEGMA))₂₁ aqueous solution at different temperature values. (a) C0, (b) C1, (c) C2, (d) C3, and (e) C4 (concentration: 1 mg mL⁻¹).

As shown in Fig. 10 the plots of the hydrodynamic diameters (D_h) of C0–C4 in water as a function of temperature. When the temperature were relatively lower, the D_h values were small the change slightly. On the contrary, the values were increased significantly in the higher temperature ranges due to the aggregation among micelles. For instance, the D_h value of β-CD–(PCL–P(MEO₂MA-co-PEGMA))₂₁ (C2) was about 105 nm at 37 °C, and the D_h value increased significantly to 300 nm when temperature was raised to 42 °C. The schematic process of micelles aggregation with the increase of temperature was shown in Fig. 10. At low temperature range, the P(MEO₂MA-co-PEGMA) chains existed in random coil conformation owing to the hydrogen-bonding interaction between the copolymers and the water molecules. When the temperature increased to a critical value, P(MEO₂MA-co-PEGMA) chains will shrink into a spherical structure since the hydrogen bonds between the ether oxygen of P(MEO₂MA-co-PEGMA) and water molecules collapsed and become hydrophobic. Therefore, the intermolecular hydrophobic attractions were thermodynamically favored and the micelles aggregation will be occurred, which resulted in the increase in D_h and visible turbidity.

Fig. 10 Effect of temperature on the hydrodynamic radium (D_h) of the micelles of β-CD–(PCL–P(MEO₂MA-co-PEGMA))₂₁ in aqueous solution measured at 1 mg mL⁻¹. (a) C0, (b) C1, (c) C2, (d) C3, and (e) C4.

2.3 In vitro cytotoxicity study

PCL were well-known polymers as their low cytotoxicity and good biocompatibility, making them suitable for various applications in biomedical fields such as drug carriers, tissue engineering as well as stimuli-responsive materials. Fig. 11 showed the in vitro cytotoxicity of β-CD–(PCL–P(MEO₂MA-co-PEGMA))₂₁ (PCD1 and PCD2), against HeLa cells were evaluated by the CCK-8 assays. Fig. 11 showed the cell viabilities of HeLa cells after 48 h incubation with two kinds of copolymers with different concentrations. Both copolymers were showed low toxicity against HeLa cells at various concentrations, and the cell viabilities were still higher than 90% even at the concentration up to 200 μg mL⁻¹. The results suggested that the copolymers micelle were nontoxic and biocompatible, which were used as a delivery system for anticancer agents.

Fig. 11 In vitro cell viability of the β-CD–(PCL–P(MEO₂MA-co-PEGMA))₂₁ micelles. Concentration-dependent cell viability of HeLa cells treated with the PCD1 and PCD2 after incubation of 48 h.

The in vitro cytotoxicity of free DOX and DOX-loaded micelles against HeLa cells were also investigated by CCK-8 assay. As shown in Fig. 12, DOX-loaded micelles exhibited a slightly lower cytotoxicity to HeLa cells compared with free DOX at the same dosages. These results could be by the prolonged release of DOX from micelles as demonstrated by the in vitro DOX release profile shown in Fig. 12. The IC₅₀ values (inhibitory concentration to produce 50% cell death) of DOX-loaded micelles were 0.434 μg mL⁻¹, 0.987 μg mL⁻¹, 1.220 μg mL⁻¹. In contrast, the HeLa cells were incubated with thermo-responsive β-CD–(PCL–P(MEO₂MA-co-PEGMA))₂₁ (90 : 10) micelles showed that could be largely caused by the slower release of DOX from the thermo-responsive micelles.

Fig. 12 Cell viability of HeLa cells treated with free DOX and DOX-loaded β-CD–(PCL–P(MEO₂MA-co-PEGMA))₂₁ micelles (D-PCD1, D-PCD2) at different concentrations.

2.4 In vitro DOX loading and release

The thermo-responsive property of the micelles of β-CD–(PCL–P(MEO₂MA-co-PEGMA))₂₁ with star-shaped copolymer, the micelles could be used as the thermo-controllable drug release carrier. DOX was a kind of hydrophobic anticancer agent and used as the model drug to investigate the controlled property of the micelles. The drug release of the thermo-responsive micelles was investigated in PBS pH 7.4, and the data were shown in Fig. 13. The drug release profile from micelles was showed greater changes with the temperature alternations around the LCST of copolymer. Under the LCST (30 °C and 37 °C) (D-PCD1 and D-PCD2), the highly hydrated P(MEO₂MA-co-PEGMA) segments stabilize the hydrophobic–hydrophilic core–shell structure of micelles of copolymer with star-shaped arms, and only small amount of drug could diffuse out from the micelles. As a result, the drug release was slow and about 75% drug still remains in the core of the micelles after 48 h. But, when the temperature was raised above the LCST (37 °C and 50 °C), the drug release was accelerated owing to the temperature-induced structural changes of the micelles. That was to say, the P(MEO₂MA-co-PEGMA) copolymers shell become hydrophobic, and the micellar core–shell structure was deformed. Therefore, the hydrophobic DOX incorporated in core diffused out quickly and about 40.2% and 53.6% drug was released from the micelles. Obviously, the release rate of DOX from the micelles could be effectively controlled by changing the external temperatures. Therefore these results showed that the copolymer micelles could have potential applications for the selective release of drugs under intracellular environments (Scheme 2).

Fig. 13 In vitro release profile of DOX from various DOX-loaded micelles incubated in pH 7.4 buffer solutions at 30, 37 and 50 °C.

To evaluate the intracellular drug release of β-CD–(PCL–P(MEO₂MA-co-PEGMA))₂₁ micelles, the Confocal Laser Scanning Microscopy images was used. The release of DOX from DOX-loaded copolymer micelles could be estimated by observing the red fluorescence intensity of DOX inside the cells. Fig. 14 showed the fluorescence images of HeLa cells were after 0.5, 4 and 24 h of incubation with D-PCD2 using free DOX as a control. One can observed that the fluorescence intensity of DOX became stronger with the increase of incubation time, indicated that DOX was gradually released. Moreover, the fluorescence could be apparently observed at the cytoplasm after 0.5 h incubation (Fig. 14(A)). As shown in Fig. 14(B), DOX was dispersed into the partly cells including nuclei and cytoplasm after 4 and 24 h incubation, more than half of the blue fluorescence was overlapped by red fluorescence and became weaker, it were suggested that DOX was delivered into nuclei and successfully inhibited the proliferation of HeLa cells. While for the DOX-loaded copolymer micelles with much larger sizes, it was likely that the prolonged circulation and passive tumor-targeting delivery process caused by the EPR effect will enhance the delivery of hydrophobic drugs into the tumor cells, and once the micelles were internalized, it was not easy for them to escape from the cells. In the case of thermo-responsive micelles, as shown in Fig. 14(B), the intracellular uptake of DOX loaded micelles were showed a much less efficient intracellular release of DOX. The results indicated that free DOX were taken up by diffusion through the cell membrane and the DOX loaded micelles were taken up by the nuclei of cells via the endocytosis process. In addition, the self-assembled micelles of the amphiphilic copolymers were showed a great potential as anti-tumor drug carriers for cancer therapy.

Fig. 14 Comparison of the fluorescence images of HeLa cells incubated with (A) free DOX and (B) DOX-loaded (D-PCD2) for different times. The dosage of DOX was 5 μg mL⁻¹. For each panel, images from up to down show cell nuclei stained by HeLa (blue), DOX fluorescence in cells (red) and overlays of two images. The scale bars correspond to 20 μm in all the images.

3. Experimental

3.1 Materials

β-Cyclodextrin (β-CD, >98%, purified by recrystallization from water twice prior to use) was purchased from Aladdin. 2,2′-Azobisisobutyronitrile (AIBN) were purchased from Aladdin, MEO₂MA and PEGMA (M_n = 500 g mol⁻¹) (Aldrich) were passed through a column of activated basic alumina to remove the inhibitors. ε-CL (Sigma-Aldrich) was distilled under reduced pressure after being treated with CaH₂. Tin 2-ethylhexanoate (Sn(Oct)₂, Aldrich) was distilled under reduced pressure. 2-Bromoisobutyryl bromide (BIBB, Aldrich), N,N,N′,N′,N′′-pentamethyldiethylenetriamine (PMDETA) was purchased from Sigma-Aldrich, copper(I)chloride (99.999%, Alfa Aesar) was used without further purification. Tetrahydrofuran (THF) was refluxed over sodium and distilled twice before use. Dimethylformamide (DMF), toluene and triethylamine (TEA) were dried by refluxing over CaH₂ and distilled just before use. Doxorubicin hydrochloride (DOX·HCl) was purchased from Beijing HuaFeng United Technology Co. Ltd. HeLa cells (Institute of Cells, CAS, Shanghai) were used as received, Cell Counting Kit-8 (CCK-8, Shanghai, Beyotime Biotechnology), Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS), pancreatic enzymes were obtained from biological industries. 4% paraformaldehyde, 4′,6-diamidino-2-phenylindole (DAPI) and TritonX-100 were purchased from Solarbio.

3.2 Characterization

¹H NMR data were obtained by Nuclear Magnetic Resonance Spectroscopy (NMR) using a BrukerDMX-500 NMR spectrometer with DMSO-d₆ and CDCl₃ as solvent. Fourier transform infrared spectroscopy (FTIR) analysis was measured by Nicolet Avatar 360 using KBr pellets. The molecular weight and molecular weight distribution of copolymers were measured by Gel Permeation Chromatography (GPC) using a Viscotek TDA 302 gel permeation chromatograph and THF was used as eluent. The transmittances of copolymers aqueous solutions at various temperatures were measured at a wavelength of 500 nm on a UV-visible spectrophotometer (UV-vis). The LCST value of the copolymer solution was defined as the temperature producing a 50% decrease in transmittance. Dynamic Light Scattering (DLS) measurements were performed by 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 results were accepted as the final hydrodynamic diameter (D_h). Samples for transmission electron microscopy (TEM) images were taken on an H-600 transmission electron microscope (Hitachi, Japan) operating at 120 kV. Confocal Laser Scanning Microscopy (CLSM) images (Zeiss CLSM510) was operated.

3.3 Synthesis of β-CD–(PCL–P(MEO₂MA-co-PEGMA))₂₁ with star-shaped amphiphilic copolymers

3.3.1 Synthesis of the β-CD–PCL. β-CD–PCL was synthesized by ROP of ε-CL with β-CD as initiator and Sn(Oct)₂ as catalysis. A typical procedure was as follows: β-CD (1 g, 0.88 mmol), ε-CL (10 g, 88 mmol), Sn(Oct)₂ (0.04 g, 0.1 mmol), and anhydrous toluene (40 mL) were added into a fresh flamed and nitrogen purged round-bottomed flask and the flask was then placed in a thermostated oil bath at 135 °C for 24 h. After the polymerization, the mixture was cooled to room temperature, the product was dissolved in dichloromethane, and precipitated three times in methanol. Finally, the precipitate was collected and dried under vacuum to a constant weight at 50 °C.

3.3.2 Synthesis of β-CD–PCL–Br initiator. A solution of β-CD–PCL (4 g, 0.2 mmol) and triethylamine (3 mL) in CH₂Cl₂ (30 mL) was stirred 0.5 h under nitrogen atmosphere in a 100 mL three-necked flask, then the flask was placed in an ice/water bath. 2.0 mL of 2-bromoisobutyryl bromide was added dropwise via constant pressure funnel over 0.5 h, and the reaction mixture was stirred 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 by a rotary evaporator and precipitated three times in methanol, and dried under vacuum to a constant weight at 35 °C.

3.3.3 Synthesis of β-CD–(PCL–P(MEO₂MA-co-PEGMA))₂₁ by ATRP. Synthesis of β-CD–(PCL–P(MEO₂MA-co-PEGMA))₂₁: a series of β-CD–(PCL–P(MEO₂MA-co-PEGMA))₂₁ were prepared by ATRP of MEO₂MA and PEGMA using β-CD–PCL–Br as initiator and CuBr/PMDETA as catalyst. The reaction procedures were shown in Scheme 1. In order to adjust the temperature responsivity, the feed ratio of MEO₂MA/PEGMA was varied (mol%/mol%: 100/0, 95/5, 90/10, 85/15, 80/20). And it named C0, C1, C2, C3 and C4. For example star-shaped block copolymers β-CD–(PCL–P(MEO₂MA-co-PEGMA))₂₁ by ATRP of MEO₂MA and PEGMA with multifunctional β-CD–PCL–Br as the initiator was described below. β-CD–PCL–Br (0.1 g, 0.03 mmol), MEO₂MA (3.86 g, 9.0 mmol), PEGMA (1.14 g, 1.0 mmol), CuBr (0.151 g, 1.0 mmol), PMDETA (0.173 g, 1 mmol) and THF (30 mL). The flask was degassed with three freeze–evacuate–thaw cycles. Then, the polymerization was performed at 60 °C for 24 h. After being cooled to room temperature, the reaction flask was open to air, and the crude product was diluted with THF and passed through a neutral alumina column to remove the copper catalysts. Finally it was precipitated thrice into cold hexane, and dried under vacuum to a constant weight at 50 °C.

Scheme 1 Synthesis of thermo-responsive star-shaped β-CD–(PCL–P(MEO₂MA-co-PEGMA))₂₁ copolymers.

Scheme 2 Illustration of thermo-responsive self-assembly of the β-CD–(PCL–P(MEO₂MA-co-PEGMA))₂₁ copolymer for the efficient intracellular release of anti-cancer drugs triggered by the acidic microenvironment inside the tumor tissue.

3.4 Self-assembly of β-CD–(PCL–P(MEO₂MA-co-PEGMA))₂₁ in aqueous solution

Samples for UV-vis, DLS and TEM were prepared as follows: β-CD–(PCL–P(MEO₂MA-co-PEGMA))₂₁ (20 mg) was dissolved in THF (2 mL) and subsequently, deionized water (2 mL) was added dropwise from an additional funnel over a period of 0.5 h. After 4 h quick stirring, 8 mL water was added to quench the micellar assembly. Subsequently dialyzed (molecular weight cut-off: 7000 Da) against distilled water for 48 h. During this dialysis process, the hybridized copolymers self-assembled into micelles with β-CD, PCL core and star-shaped P(MEO₂MA-co-PEGMA) shell. Micelles solutions with different concentrations could be obtained by diluting with distilled water and equilibrating at room temperature for 48 h, which were named PCD1 and PCD2.

3.5 DOX encapsulation and release studies

The encapsulation, 50 mg of PCD1/PCD2 and 10 mg of DOX·HCl, were dissolved in 2 mL of DMF separately and the two solutions were mixed in a vial and stirred for 60 min, then, a 3-fold excess of TEA in 2 mL DMF overnight to obtain DOX base. Then the mixture was added dropwise using a syringe pump to water (30 mL) 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: 500 Da) against deionized water for 48 h to remove unloaded DOX, and it were named D-PCD1 and D-PCD2.

The DOX loading content (LC) and entrapment efficiency (EE) were determined by UV-vis spectrophotometry at 480 nm. To determine the drug loading level, a small portion of DOX-loaded micelles was withdrawn and diluted with DMF to a volume ratio of DMF/H₂O = 9/1. The amount of DOX encapsulated was quantitatively determined by a UV-vis spectrophotometer and the calibration curve used for drug loading characterization was established by 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 DOX incorporated in assembled micelles and that used in fabrication.

The in vitro DOX release profiles from the PCD1/PCD2 assembled micelles were evaluated using buffers (PB, 10 mM) solution with pH values 7.4. In each experiment, 4 mL of DOX-loaded nanoparticle solution was transferred into a dialysis bag (molecular weight cut-off: 500 Da), and immersed into a tube containing 60 mL of buffer solution and shaken (200 rpm) at 30, 37 and 50 °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 concentration of DOX in different samples was analyzed by 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 was the volume of the release medium (V₀ = 60 mL), C_i represents the concentration of DOX in the ith sample and C_n represents the concentration of DOX in the nth sample. The in vitro release experiments were carried out in triplicate at each temperature and the reported results were the average values with standard deviations.

3.6 Cytotoxicity test

The cytotoxic effects of polymers, free DOX or DOX-loaded β-CD–(PCL–P(MEO₂MA-co-PEGMA))₂₁ micelles were evaluated against HeLa cells by the standard Cell Counting Kit-8 (CCK-8). To perform 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 control. After 48 h incubation, 10 μL of the CCK-8 stock solution was adder into each well. After incubation for another 1–4 h, the absorbance of each well was measured at a test wavelength of 450 nm. The cell viability of samples was calculated as follow:^42,43where A_test and A_control represent the intensity determined for cells treated with different samples and for control cells, respectively, and A_blank was the absorbance of wells without cells.

3.7 Intracellular release of DOX

Confocal laser scanning microscopy (CLSM) (Zeiss CLSM510) was used to visualize the subcellular localization and intracellular release behavior of 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 DOX-loaded micelles and free DOX were added, and cells were cultured medium after 0.5 h, 4 h and 24 h at 37 °C under 5% CO₂ atmosphere of incubation, and the cells were washed with PBS and stained with DAPI (10 mg L⁻¹) for 0.5 h. Afterwards, the culture medium was replaced with a DMEM medium containing DOX-loaded micelles. Finally, the location of intracellular fluorescence was validated using a CLSM imaging system at the excitation wavelength of 480 nm.

4. Conclusions

In summary, a novel star-shaped copolymers β-CD–(PCL–P(MEO₂MA-co-PEGMA))₂₁ has been designed and synthesized by a combination of ROP and ATRP. These block copolymers could self-assemble into spherical nanoscale micelles comprising of β-CD and PCL core and P(MEO₂MA-co-PEGMA) coronas in aqueous solution. The self-assembly behavior of the copolymers were investigated by UV-vis, DLS, and TEM. The LCST of the thermo-responsive micelles was well controlled from 25, 35, 42 and 56 to 61 °C by adjusting the content of PEGMA, these results showed that the copolymers micelles diameters (D_h) were increased from 100 nm to 450 nm in different temperature. And TEM and DLS confirmed the formation of spherical particles micelles and large aggregates at temperature below and above the LCST, respectively. The biocompatible block copolymer could be self-assemble with the anti-cancer drug DOX into DOX loaded micelles with a size of less than 200 nm. It should be noted that these copolymers possess relatively low cytotoxicity, decent drug loading levels, and fast and maximum drug release inside cancer cells. In addition, the cytotoxicity tests showed that these star-shaped block copolymers possessed good biocompatibility. The DOX-loaded micelles exhibited a stimuli-responsive release manner, and they could efficiently release DOX into tumor cells and significantly enhance drug efficacy.

Acknowledgements

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

Notes and references

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