Novel self-assembled amphiphilic mPEGylated starch-deoxycholic acid polymeric micelles with pH-response for anticancer drug delivery

Abstract

A novel amphiphilic polymer (mPEGylated starch-deoxycholic acid, mPEG-St-DCA) was successfully prepared by grafting hydrophobic deoxycholic acid (DCA) into mPEGylated starch. According to the characterization of ¹H NMR and FTIR, mPEG-St-DCA polymers could self-assemble into micelles with spherical core–shell structures. With the degree of substitution (DS) of DCA increasing, the average size of micelles (pH 7.4) decreased to below 200 nm. Correspondingly, the critical micelle concentration (CMC) decreased from 0.048 to 0.022 mg mL⁻¹. And zeta potential values were near −2 mV. But under the condition of pH 6.5, the size and CMC showed an increasing trend. Doxorubicin (DOX), a model anticancer drug, was efficiently loaded into mPEG-St-DCA micelles, and in vitro release exhibited that DOX-loaded micelles had a good in vitro pH-induced drug release. MTT assays confirmed that mPEG-St-DCA micelles were biocompatible with HeLa cells, and DOX-loaded micelles had a relatively better cytotoxicity against HeLa cells with a remarkably high IC₅₀ of 5.74 μg mL⁻¹. Confocal laser scanning microscopy (CLSM) analyses demonstrated that mPEG-St-DCA micelles could be internalized efficiently by HeLa cells to realize intracellular DOX release, which further enhanced the inhibition of cell proliferation. On the basis of the above results, it was indicated that novel amphiphilic mPEG-St-DCA micelles with pH-response could be used as drug delivery carriers for cancer therapy.

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Introduction

In spite of continuous development and systematic study, cancer still remains the second leading cause of death worldwide with approximately 13% of all deaths each year.^1–3 With the discovery of various anticancer drugs, current traditional cancer treatments, involving chemotherapy, surgical intervention and radiation therapy, have still many defects, such as several side effects towards normal tissues and cells, the low tumor selectivity of the drugs and the development of drug resistance.^1,4,5 To address this, the various nanocarriers have been designed and developed in the last two decades,⁶ including micelles,^7,8 nanoparticles,⁹ liposomes¹⁰ and nanogels.^11,12 Among the most promising nanocarrier systems, polymeric micelles as a drug delivery system have attracted great interest in cancer therapy. Polymeric micelles offer an effective and safe platform with several unique features, such as enhancing the aqueous solubility and bioavailability of the hydrophobic drugs, prolonging the circulation time of small molecule drugs, reducing systemic side effects and increasing the preferential accumulation at the tumor site by the enhanced permeability and retention (EPR) effect.^4,9,13,14 And the most effective size of polymeric micelles as outstanding tools for extravasation into tumors by the EPR effect is <200 nm.¹ Moreover, amphiphilic polymeric micelles possess potential core–shell structures with hydrophobic inner core as a depot for hydrophobic drugs and hydrophilic outer shell as a protective interface.^15–19

Recently, many amphiphilic polymeric micelles have been studied to achieve rapid and high drug release inside cells.^6,20 Because the biodegradable and biocompatible properties are vital parameters, thus it is necessary to be taken in account in current cancer therapy.^4,16 Furthermore, as an ideal carrier, polymeric micelles should possess several requisites, including ideal preparation materials, capable of incorporation diverse drugs, controlled drug release and degrading into non-toxic entities to minimize adverse toxicity to out of in vitro.^1,21 Therefore, a lot of well characterized, easily functionalized, biocompatible and biodegradable polymers have been widely explored, including chitosan,²² dextran²³ and starch.²⁴

Starch, an important class of natural polymers, is an attractive substitute for other chemically synthesized polymers due to its non-toxicity, non-immunogenicity, good biocompatible and biodegradable, stability in the air and compatibility with most drugs.^25,26 Besides, the closely related glycoprotein structure makes starch suitable model compounds.²¹ As a result, due to their outstanding advantages, starch has gained increasing attention as a functional material for drug delivery system. In addition, with the high amounts of functional hydroxyl groups along the chains, starch is convenient for facilitating chemical modifications to obtain various desired functional materials.^21,27 Therefore, starch is widely used to prepare drug delivery carriers. Poly(ethylene glycol) (PEG), due to its favorable biocompatibility, non-immunogenicity and high water-solubility, has been extensively selected as a polymeric modifier.²⁸ And PEG has also been approved by Food and Drug Administration (FDA) for the human.²⁹ So PEGylated has been considered as a preferred strategy to prolong circulation time in the bloodstream, avoid the uptake of reticuloendothelial system (RES) as well as increase passive accumulation in tumor tissues for drug delivery system.^18,28 For example, the chemical modifications of chitosan and starch with PEG have been reported.^26,29,30 Deoxycholic acid (DCA), a main component of bile acid, is biologically the most detergent-like molecules, which plays a key role in the emulsification, solubilization and absorption of cholestrol, fat and vitamins in the body.^31,32 In the precious reports, we have prepared a series of hydrophobically modified starch by the introduction of deoxycholic acid to form self-aggregates, but the DS of DCA was obviously low, because starch was not further modified to improve solubility.³³

According to the reports,^6,34 the amphiphilic micelles based on graft polymers could reduce the conformational entropy and lead to novel self-assembled nanostructures, resulting in improving in drug loading efficiency, drug release rate and in vitro circulation. Meanwhile, in order to make polymeric micelles meet all of requirements as an ideal carrier, in this work, deoxycholic acid (DCA) and mPEGylated starch were selected to prepare amphiphilic polymeric micelles by covalently conjugating hydrophobic DCA onto the main chains of mPEGylated starch (mPEG-St). Amphiphilic mPEGylated starch-deoxycholic acid (mPEG-St-DCA) polymers could form self-assembled micelles with core–shell structures, in which DCA acted as the hydrophobic inner core and the mPEGylated starch acted as the hydrophilic outer shell. The physicochemical properties of mPEG-St-DCA polymers were characterized by FTIR and ¹H NMR. And the micellar characteristics were investigated using transmission electron microscopy (TEM), dynamic light scattering (DLS) and fluorescence techniques with pyrene molecular probe. In addition, Doxorubicin (DOX), a model anticancer drug, was encapsulated into the polymeric micelles. In vitro DOX release profiles from DOX-loaded micelles in different pH values were studied. MTT assay demonstrated the mPEG-St-DCA micelles were biocampatible to HeLa cells, and the intracellular release of DOX was investigated with HeLa cells using confocal laser scanning microscopy (CLSM). The results indicated that novel self-assembled amphiphilic mPEG-St-DCA micelles with pH-response are promising potential carriers for anticancer drug delivery.

Experimental

Materials

Soluble starch (M_w = 8.8 kDa) was purchased from Zhejiang Linghu Chemical Reagent Factory, chemical purity grade. Poly(ethylene glycol) monomethyl ether (mPEG, M_n = 1900) was purchased from Alfa Aesar Reagent Inc. (Tianjin, China). 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC·HCl), N-hydroxysuccinimide (NHS) and deoxycholic acid (DCA) were purchased from Aladdin Reagent Inc. (Shanghai, China). 4-Dimethylaminopyridine (DMAP) and succinic anhydride were purchased from Sinopharm Chemical Reagent Co., Ltd. Pyrene and 3-(4,5-diemethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were purchased from Sigma-Aldrich. Doxorubicin hydrochloride (DOX·HCl) was purchased from LSB Biotechnology Inc. (Xi'an, Chian). The other reagents were of analytical grade and used without further purification.

Synthesis of mPEGylated starch (mPEG-St)

The mPEG terminated with carboxyl group (mPEG-COOH) was synthesized using a literature procedure.³⁵ The mPEG (3.8 g, 2 mmol), succinic anhydride (0.6 g, 6 mmol) and DMAP (0.01 g, 0.082 mmol) were dissolved in 100 mL of CH₂Cl₂ at 25 °C for 48 h. After evaporatd a mojority of CH₂Cl₂, the reactant was redissolved in tetrahydrofuran and precipitated for several times in diethyl ether, finally and dried in vacuum. After that, the mPEG-COOH polymer was obtained (yield: 78.2%), and mPEG-SA-mPEG polymer would not be generated.

The mPEGylated starch (mPEG-St) was synthesized by grafting mPEG-COOH into starch according to the published procedure.²⁶ Briefly, starch (0.2 g), mPEG-COOH (0.38 g), EDC·HCl (0.144 g) and DMAP (0.046 g) were dissolved in 15 mL of DMSO. The mixture was stirred at 25 °C for 48 h. Then, the mixture was dialyzed in a dialysis bag (cutoff M_w = 3.5 kDa) against deionized water for three days to remove the solvent and unreacted substances. Finally, the solution was lyophilized to obtain white product mPEGylated starch (mPEG-St) (yield: 76.4%).

Synthesis of deoxycholic acid modified mPEGylated starch (mPEG-St-DCA)

Deoxycholic acid (DCA) was grafted into the mPEGylated starch by the carbodiimide reaction between hydroxyl group of mPEGylated starch and carboxyl group of DCA as described by the literature.^33,36 The synthesis route was shown in Fig. 1. Typically, mPEGylated starch (0.12 g, 0.2 mmol) was dissolved in 15 mL DMSO under stirring at 45 °C. Different amounts of activated DCA (0.4–0.6 mol mol⁻¹ sugar residues of mPEG-St) were added into the DMSO solution containing mPEGylated starch. To activate the carboxyl groups of DCA in the DMSO, equal amounts of EDC (1.2 equiv. DCA) and NHS (0.6 equiv. DCA, 15 min later) were added into DCA solution for 30 min at room temperature. The mixture was reacted under gentle stirring at 45 °C for 48 h. Then, the reactant mixture was cooled to room temperature and dialyzed against the excess amount of distilled water/methanol mixture (1 : 4 v/v) for 5 days using a dialysis bag (cutoff M_w = 3.5 kDa), and the distilled water/methanol mixture was exchanged at intervals of 8 h. Finally, the solution was lyophilized to obtain mPEG-St-DCA polymers. Their three polymers final yields were approximate 52%.

Fig. 1 The Synthetic pathway of mPEG-St-DCA.

Characterization of mPEG-St-DCA

The chemical structure of mPEG-St-DCA polymers was confirmed using a Fourier transform infrared (FTIR) spectrometer (Nicolet 670 FTIR, USA) and ¹H NMR (Bruker Avance III 400, DMSO-d₆). Lyophilized mPEG-St-DCA samples were pressed with KBr and scanned from 4000 to 400 cm⁻¹ with a resolution of 4 cm⁻¹.

The degree of substitution (DS, mol%, expressed as mole DCA/100 sugar residues of mPEGylated starch) was calculated based on the precious report³³ by the UV-vis spectrophotometer (Lambda 35, Perkin Elmer, USA) at 380 nm according to the following equation.

where w is the content of the DCA determined from the corresponding calibration curve, 60 000 is 100 × M_w of glucose unit of mPEGylated starch, and 392.57 is the molecular weight of the DCA residue.

Preparation of mPEG-St-DCA micelles

The mPEG-St-DCA polymers (20 mg) were dissolved in 20 mL of phosphate buffered saline (PBS) solution with various pH values under gentle shaking at 25 °C for 8 h, followed by sonication using a probe-type sonifier at 100 W for 10 min. The sonication was repeated three times to obtain optically transparent solution. To protect the solution from heat build-up during sonication, the pulse function was used (pulse on, 5.0 s; pulse off, 2.0 s). The solution of micelles was passed through a 0.45 μm filter (Millipore) and stored at room temperature.

Critical micelle concentration (CMC) measurements of mPEG-St-DCA micelles

The CMC of mPEG-St-DCA micelles were estimated by the probe fluorescence technique in which pyrene was used as a hydrophobic probe. Briefly, a known amount of pyrene solutions (1 mL, 6 × 10⁻⁶ M) in acetone were added to a series of colorimetric tube (10 mL), and followed by evaporation to remove acetone. Then, the various concentrations (1 × 10⁻⁴ to 1.0 mg mL⁻¹) of mPEG-St-DCA solutions were added to each colorimetric tube containing the pyrene residues with the final concentration of 6.0 × 10⁻⁷ mg mL⁻¹. The solutions were sonicated in an ultrasonic bath for 2 h to reach the solubilization equilibrium of pyrene between water phase and micelles, and put overnight at room temperature. The excitation spectra of pyrene were recorded in the range of 300–350 nm using a fluorescence spectrophotometer (LS55, Perkin-Elmer, USA) at the emission wavelength (λ_em) of 390 nm and an integration time of 100 nm min⁻¹. The slit openings for excitation and emission were both set at 15 nm, respectively.

Dynamic light scattering (DLS) and zeta potential measurements

The self-assembled micelle size was determined using DLS at room temperature on a 90 Plus particle size analyzer (Brookhaven Instruments Corporation) at the 90° scattering angle. All sample solutions were filtered using disposable 0.45 μm Millipore filters prior to analysis at the concentration of 1.0 mg mL⁻¹.

The zeta potentials of self-assembled micelles were measured using a Zetasizer (Malvern Instruments Ltd, U.K.). The concentration of micelles was kept constant at 1.0 mg mL⁻¹ in various pH of PBS solution.

Morphology observation of self-assembled micelles

The morphology of micelles was observed using transmission electron microscope (TEM) (JEM-1200EX/S, Hitachi, Japan). A drop of sample solution (1.0 mg mL⁻¹) was placed onto a 300-mesh copper grid coated with carbon. About 2 min after deposition, the grid was tapped with filter paper to remove surface water, followed by air-drying at room temperature and then observed at an acceleration voltage of 200 kV.

Drug loading and in vitro release of DOX from mPEG-St-DCA micelles

The loading of DOX into mPEG-St-DCA micelles was carried out by the dialysis method. Briefly, mPEG-St-DCA micelles were dissolved in DMSO to obtain final concentration of 5 mg mL⁻¹. Meanwhile DOX·HCl was dissolved in DMSO, neutralized with 5.0 equiv. of excess triethylamine, and further mixed with mPEG-St-DCA solution at the different feed weight ratios (1 : 10, 2 : 10 and 3 : 10) by stirring at 25 °C for 2 h. The mixture was dialyzed against deionized water at room temperature for 24 h using a dialysis bag (cutoff M_w = 3.5 kDa). After dialysis, the solution in the dialysis bag was filtrated through 0.45 μm filter (Millipore) to remove the free DOX and further lyophilized. To determine the drug loading content (DLC) and drug loading efficacy (DLE), a certain amount of DOX-loaded micelles was dissolved in DMSO. The drug concentration of DOX was determined by using the UV-vis spectrophotometer at 483 nm based on the standard curve obtained from DOX in DMSO. The DLC and DLE were calculated according to the following equation:

DLC (wt%) = (weight of loaded drug/weight of drug loaded micelles) × 100%

DLE (wt%) = (weight of loaded drug/weight of drug in feed) × 100%

In vitro release of DOX from mPEG-St-DCA micelles was investigated in PBS solution (pH 5.5 and 7.4). Briefly, the DOX-loaded mPEG-St-DCA micelles were dispersed in PBS solution with the final concentration of 1 mg mL⁻¹, and then the solution (3 mL) was transferred into a dialysis bag (cutoff M_w = 3.5 kDa). The dialysis bag was immersed in 50 mL of PBS solution and gently shaken at 37 °C at 100 rpm. At the predetermined time interval, the external buffer solution (5 mL) was taken out with replenishing of the same volume fresh medium. The amount of released DOX was determined by using the UV-vis spectrophotometer at 483 nm according to the following equation:

where M_t is the amount of DOX released at time t and M is the total DOX amount loaded in the micelles.

In vitro cell cytotoxicity assay

In vitro MTT cell proliferation assay was used to evaluate the cytotoxicities of DOX-free micelles, DOX-loaded micelles and free DOX against HeLa cells. HeLa cells were seeded into a 96-well culture plates (5 × 10⁴ cells per well) using Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37 °C in 5% CO₂ for 24 h. The cells were then incubated with DOX-free micelles, DOX-loaded micelles and free DOX for 24 h at 37 °C. The concentration of DOX-free micelles ranged from 20 to 100 μg mL⁻¹. DOX-loaded micelles and free DOX were diluted in complete DMEM with final DOX concentrations from 0.01 to 15 μg mL⁻¹. And they were allowed to incubate for 24 h. At the same time, free DOX was used as the control. Afterwards, MTT reagent (5 mg mL⁻¹ in PBS, 20 μL) was added to each well and incubated for another 4 h. The culture medium was removed and 150 μL DMSO was added. The absorbance of each well was measured using a microplate reader (VICTOR 1420, PE, USA) at 490 nm. The cytotoxicity was expressed as a percentage of the control, and the half maximal inhibitory concentration (IC₅₀) value was determined as the DOX concentration. The cell viability (%) was calculated according to the following equation:where A_sample and A_control represent the absorbencies of the sample and control wells, respectively.

Intracellular drug release

The intracellular drug release behaviors of DOX-loaded mPEG-St-DCA micelles were visualized by confocal laser scanning microscopy (CLSM) in HeLa cells. HeLa cells were cultured with culture medium containing 10% FBS in the 96-well plates at density of 5 × 10⁴ cells per well for 24 h. Then the culture medium was replaced by DOX-loaded mPEG-St-DCA micelles with DOX concentration of 30 μg mL⁻¹. Then the cells were incubated for 0.5, 1 or 2 h at 37 °C in a humidified 5% CO₂-containing atmosphere. After each time point the culture medium was removed and the cells were washed three times with PBS. Thereafter, the cells were fixed 4% formaldehyde for 30 min at room temperature. Finally, the cells nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI, blue). The images of the cells were obtained using CLSM (Olympus Fluoview 1000).

Results and discussion

Synthesis and characterization of mPEG-St-DCA

The synthetic pathway of mPEG-St-DCA polymers was shown in Fig. 1. Firstly, mPEGylated starch (mPEG-St) was synthesized according to the published procedure.²⁶ Next, mPEG-St-DCA polymers with different DS of the deoxycholic acid (DCA) moiety were synthesized by grafting DCA into mPEGylated starch backbones. Because the hydroxyl groups of mPEGylated starch were able to react with carboxyl groups of hydrophobic DCA in the presence of EDC and NHS to form active ester linkages, therefore, novel kinds of amphiphilic polymers were achieved.

The chemical structure of mPEG-St-DCA polymers was confirmed by ¹H NMR and FTIR spectroscopy. The FTIR spectra of mPEG-St, DCA and mPEG-St-DCA were shown in Fig. 2A, which could evidence the formation of ester linkage between DCA and mPEGylated starch. After chemical modification, the FTIR spectrum of mPEG-St-DCA exhibited significant differences. In comparison with the FTIR spectra of DCA and mPEG-St, a new absorption peak at 1742 cm⁻¹ (Fig. 2A(c)) assigned to the ester linkage was obviously observed. The characteristic peaks of 2924 and 2889 cm⁻¹ could be assigned to the stretching vibration of DCA,³³ and the typical absorptions of ester linkage of mPEG-St at 1108 cm⁻¹ were also observed.²⁶ And with increasing of the feed ratio of DCA to mPEGylated starch (from 40 to 60%), the intensity of vibration of ester linkage became obviously different in Fig. 2B. The structure of mPEG-St-DCA was further verified by ¹H NMR spectrum. As shown in Fig. 3, the obvious four peaks located between 4.5 and 5.6 ppm consisting of the C₂–OH (a, 5.44 ppm), C₃–OH (b, 5.43 ppm), C₁–H (c, 5.10 ppm) and C₆–OH (d, 4.60 ppm) could be clearly observed, which were contributed to the hydroxyl groups from glucose unit of starch.³⁷ And the characteristic peaks appearing at 3.51 ppm (–CH₂–CH₂–, 2, 3) and 3.23 ppm (–O–CH₃, 1) were attributed to the methylene protons and terminal methoxyl protons of mPEG. Moreover, due to the appearance of the characteristic peaks of DCA from 0.6 to 2.5 ppm including 18-CH₃ (0.60 ppm), 19-CH₃ (0.90 ppm), 21-CH₃ (0.99 ppm) and methylene–methine envelope (1–2.5 ppm) in Fig. 3,^33,38 it could further indicate that hydrophobic molecular DCA was successfully grafted into mPEGylated starch.

Fig. 2 (A) FTIR spectra of (a) mPEG-St, (b) DCA and (c) mPEG-St-DCA and (B) FTIR spectra of mPEG-St-DCA derivatives: (a) mPEG₃₂-St-DCA₁₀, (b) mPEG₃₂-St-DCA₁₁, and (c) mPEG₃₂-St-DCA₁₃; 32 and 10 are the degree of substitution (DS) of mPEG and DCA, respectively.

Fig. 3 ¹H NMR spectrum of mPEG-St-DCA polymers in DMSO-d₆.

It is well known that the DS is a key parameter to determine the hydrophobic and hydrophilic properties of mPEG-St-DCA polymers, which finally affects the size and CMC of self-assembled micelles.³⁸ As shown in Fig. S1,† the proton signals corresponding to starch and mPEG could be found. The DS of mPEG was calculated according to the equation: DS = [(A₁)/2]/A₂ × 100%, where A₁ and A₂ were the integral area of peak 1 and 2, respectively. The DS of mPEG was 23, and the mean molecular weight of glucose unit of mPEG-St was 600 (M_w = 162 + 1900 × 0.23). The DS of DCA is also defined as the number of DCA per 100 glucose units of mPEG-St-DCA and could be easily controlled by adjusting the feed ratio of DCA to mPEG-St. It was calculated through DCA contents in mPEG-St-DCA polymers by the UV absorbance based on quantification (Fig. S2†). With increasing the feed ratio of DCA in glucose units of mPEG-St from 40 to 60%, the DS values were 10.42, 13.06 and 11.66 in Table 1, respectively. Compared with the previous work,³³ it could be found that even though at the same feed ratio of DCA there was a higher DS value in mPEG-St-DCA than that in St-DCA, which suggested that introduction of mPEG was beneficial to improve DCA contents in the starch backbone due to possibly increasing the solubility of starch and improving the reactive collision chances between DCA and starch. Simultaneously, the DS was much larger when the feed ratio of DCA was 50%. It implied that the amounts of DCA grafted into mPEG-St chains have achieved saturation under 50% feed ratio of DCA. So with increasing the feed ratio of DCA, the DS could continuously decrease. Because under the condition of complete saturation, the residual DCA molecules made the environment of reactive system became acid. And the acidic environment further influenced the hydrolysis of the ester linkages. As a result, the DS would decrease. In order to verify this guess, this reactive system was tested by the pH test papers as shown in Fig. S3.† It was found that when the feed ratio of DCA was 60%, the result of pH test paper was close to 6.

Table 1 The physical characterization of mPEG-St-DCA micelles in PBS solution

Sample	Feed ratio^a	DS^b	pH	Size^c (nm)	ζ^d (mV)	CMC^e (mg mL^−1)
a Mole ratio of DCA and sugar units of mPEGylated starch.b Degree of substitution of DCA per 100 glucose units of mPEG-St-DCA.c Mean diameter measured by dynamic light scattering. The data are presented as mean ± SD (n = 3).d The ζ-potential of mPEG-St-DCA micelles in PBS solution at 1 mg mL^−1. The data are presented as mean ± SD (n = 3).e Critical micelle concentration determined by fluorescence spectroscopy.
mPEG32-St-DCA10	0.4	10.42	7.4	193.79 ± 2.33	−2.13 ± 0.35	0.048
mPEG32-St-DCA13	0.5	13.06	7.4	130.70 ± 2.29	−2.06 ± 0.15	0.022
			6.5	177.82 ± 5.82	−0.42 ± 0.26	0.058
mPEG32-St-DCA11	0.6	11.66	7.4	142.05 ± 1.51	−2.16 ± 0.24	0.031

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Self-assembly behavior of mPEG-St-DCA micelles

The amphiphilic polymers could spontaneously self-assemble into micelles, vesicles or other assemblies in a selective solvent.²⁵ Liking amphiphilic polymers, mPEG-St-DCA polymers composed of hydrophilic mPEGylated starch and hydrophobic deoxycholic acid could also self-assemble into nano-sized micelles in PBS solution. And the hydrophobic DCA groups could spontaneously aggregate, acting as physical cross-linkers between polymers.¹⁷ The micellar solutions were prepared by a process of direct dissolution and sonication to obtain optical transparent solutions. The formation of mPEG-St-DCA micelles could be attributed to the hydrophilic and hydrophobic properties between mPEGylated starch chains and DCA molecules. In order to minimize the system energy of mPEG-St-DCA polymers, the brush-like structured core of micelles was formed by hydrophobic effects of DCA molecules, while the hydrophilic mPEGylated starch backbone comprised the shell of micelles, as illustrated in Scheme 1.

Scheme 1 Schematic illustration of preparation and intracellular drug release of DOX-loaded mPEG-St-DCA micelles.

The critical micelle concentration (CMC) is a vital evidence to prove the formation of micelles by self-assembly and also a key parameter to evaluate the stability of micelles in the blood circulation system post-administration.¹⁶ The self-assembly behavior of mPEG-St-DCA polymers in PBS media was confirmed by the fluorescence probe technique using pyrene as a fluorescence probe. Pyrene has very low fluorescence intensity due to its poor solubility and self-quenching.³⁹ With the formation of self-assembled micelles, pyrene molecules are transferred from hydrophilic to hydrophobic microenvironment, the fluorescent intensity ratio of pyrene increases dramatically under a certain concentration, which could be defined as the critical micelle concentration (CMC). So the CMC of mPEG-St-DCA micelles could be determined from the intensity ratio (I₃₃₂/I₃₃₀) of the pyrene excitation spectra versus the logarithm of concentration ranged from 1 × 10⁻⁴ to 1 mg mL⁻¹. Fig. 4 showed the change of the intensity ratio of mPEG₃₂-St-DCA₁₃ micelles in PBS solution (pH 7.4 and 6.5). The CMC was obtained from the crossover point of two straight lines with the base line and the tangent of the rapid rising curve. The influence of DS on the CMC of mPEG-St-DCA micelles was shown in Table 1. It is well known that the CMC values are able to be controlled by the DS.⁴⁰ The CMC values of mPEG-St-DCA micelles decreased from 0.048 to 0.022 mg mL⁻¹ with the increase of the DS in the range of 10 to 13. It was arised from the enhanced hydrophobicity by introducing large amount of hydrophobic DCA moieties into the mPEG-St, and resulted in strong self-assembled ability. The results indicated that mPEG-St-DCA micelles were thermodynamically stable at low concentration in PBS solution. However, the CMC values of mPEG₃₂-St-DCA₁₃ micelles decreased with increasing pH values because of the effective hydrophobic interaction enhancing, which agreed with results of the previous work.³³ The hydrogen-bond interactions between the hydroxyl groups of mPEGylated starch and DCA became reduced and the ester linkages were hydrolyzed partially under the acidic condition to decrease the hydrophobic ability.^25,33 As a result, it led to the loose self-assembled structure of micelles to get large CMC values. All the above results suggested the formation of mPEG-St-DCA micelles.

Fig. 4 Intensity ratios (I₃₃₂/I₃₃₀) from pyrene excitation spectra as a function of mPEG₃₂-St-DCA₁₃ concentrations in PBS solution with different pH values: (A) pH 7.4 and (B) pH 5.5.

Physical characterization of mPEG-St-DCA micelles

The polymeric micelles in a size range <200 nm can reduce non-selective reticuloendothelial system (RES) scavenge and show EPR effect at solid tumor sites for passive targeting.^39,41 The size of mPEG-St-DCA micelles with different DS in PBS solution (pH 7.4) was determined by DLS in Table 1. The mean size was in the range of 130.70 to 193.79 nm. Accompanying with the DS values increasing, the size decreased, indicating the formation of more compact hydrophobic cores. It could be ascribed that the hydrophobic interaction was much stronger as the DCA contents in mPEG-St-DCA micelles increased, which resulted in smaller size of micelles. Simultaneously, to evaluate the stability of mPEG-St-DCA micelles with different DCA amounts, we incubated these micelles in PBS solution (pH 7.4) for 24 h or 48 h and measured the size using DLS. In Fig. S4,† the data showed that the size of mPEG-St-DCA micelles had no significant change for 2 days. For example, mPEG₃₂-St-DCA₁₃ micelles showed a small increase in size from 130.70 nm to 140.62 nm over 48 h. It indicated that mPEG-St-DCA micelles were stable under physiological conditions.

Simultaneously, the effects of pH on the morphology of mPEG-St-DCA micelles were investigated by TEM. The TEM results of mPEG₃₂-St-DCA₁₃ micelles in the different pH values were shown in Fig. 5, indicating that mPEG-St-DCA micelles were spherical. However, it should be noted that when pH decreased from 7.4 to 5.5, the spherical mPEG₃₂-St-DCA₁₃ micelles underwent a dramatic process of structural change from clearly complete core–shell structure (pH 7.4, Fig. 5a) to fuzzy core–shell structure (pH 6.5, Fig. 5b), finally to complete structural collapse and aggregation (pH 5.5, Fig. 5c). The phenomenon is caused by gradually weakening hydrogen-bond interactions between the hydroxyl groups of mPEG-St-DCA under the acidic condition, which made the structure of micelles become loose to finally appear structure collapse in PBS solution (pH 5.5). On the other hand, according to the reports,^33,42,43 the structural dramatic changes should be attributed to a different hydrolysis degree in different pH values from 7.4 to 5.5, because the acidic environment could promote the hydrolysis of the ester linkages. As the result of hydrolysis, the contents of obtained DCA also further influence the hydrolysis of the ester linkage. In addition, the pK_a of DCA is 6.58.⁴² So when pH decreased to 5.5, the DCA molecule was protonated.⁴³ As shown in Fig. S5,† it was found that mPEG₃₂-St-DCA₁₃ micelles solution was transparent in pH 7.4 condition (left vial), but micelles solution showed turbidity in PBS solution of pH 5.5 (right vial), which was attributed to the hydrophobicity of DCA. Because the ester bonds were hydrolyzed partially to obtain free DCA under the acidic condition, which was in accordance with our previous report.³³ In order to confirm the above explanation, the pH as a function of the volume of HCl (0.1 M) added in mPEG₃₂-St-DCA₁₃ micelles solution (0.5 mg mL⁻¹) was shown in Fig. S6.† When pH was above 5.87, pH decreased slowly with the addition of HCl solution, it was the fact that ester linkages of mPEG₃₂-St-DCA₁₃ micelles were hydrolyzed gradually. But pH decreased quickly in the range from 5.87 to 2.76, it was because that DCA obtained by hydrolysis was protonated. With the continuous addition of HCl, pH decreased slowly, the protonation was mostly complete to appear the collapse of core–shell structure. Therefore, the hydrolysis of ester linkage and protonation of DCA under the acidic condition could have a common effect on core–shell structure. These results also indicated that mPEG-St-DCA micelles had a pH-responsive property. In view of the morphology under different pH values, it assumed that mPEG-St-DCA micelles were beneficial to the rapid drug release from the carriers in tumor environment.

Fig. 5 TEM images of mPEG₃₂-St-DCA₁₃ micelles at (A) pH 7.4, (B) pH 6.5, and (C) pH 5.5.

The zeta potential of mPEG-St-DCA micelles was also investigated. As shown in Table 1, the zeta potentials of micelles with different DS (pH 7.4) were almost near to −2 mV. But with pH value decreasing to 6.5, the zeta potential of mPEG₃₂-St-DCA₁₃ micelles decreased to −0.42 mV, which was contributed to the partial hydrolysis of the ester linkages and uncomplete protonation of DCA under the acidic condition. Thus, it indicated that the mPEG-St-DCA micelles with low absolute zeta potential values could avoid the unspecific conjugation with proteins and enzymes by electrostatic interaction, reducing rate of macrophage phagocytosis system (MPS) uptake and keeping in vivo long blood circulation time.^16,24

In vitro DOX loading and triggered drug release

As one of the most potent anticancer drugs, DOX is widely applied in the treatment of several types of solid malignant tumors, which interacts with DNA by interaction and inhibition of macromolecular biosynthesis.^4,23 But its use is frequently limited by toxic side effects due to its indiscriminate distribution after systemic administration.⁴⁴ To solve this problem, a new strategy, loading DOX into polymeric micelles, has been studied.

In order to evaluate the potential of mPEG-St-DCA micelles as a controlled drug delivery carrier, DOX could be loaded into the inner core of mPEG-St-DCA micelles. As shown in Fig. S7,† when the UV wavelength was over 380 nm, mPEG-St-DCA micelles had no UV absorption. However, the maximum UV absorption of DOX was 483 nm. It was confirmed that mPEG-St-DCA micelles have no effect on UV absorption of DOX. Considering that the smaller size and CMC value were beneficial to intracellular drug delivery, the mPEG₃₂-St-DCA₁₃ micelles was selected to evaluate in vitro drug loading and release. In this work, the influence of the weight ratio of DOX to mPEG₃₂-St-DCA₁₃ micelles (1 : 10, 2 : 10 and 3 : 10) was investigated by UV-vis spectrophotometer in the drug encapsulation process. With the weight ratio of DOX to micelle increasing from 1 : 10 to 3 : 10, the DLC increased significantly with 2.05%, 7.04% and 15.73%, respectively. It was indicated that mPEG-St-DCA micelles could encapsulate DOX easily. As the results, the weight ratio of DOX to micelles (3 : 10) was chosen to investigate in vitro drug release, and its corresponding DLE was 50.66%, which was attributed to the hydrophobic interactions between DOX and micelles.

In vitro DOX release profiles from mPEG₃₂-St-DCA₁₃ micelles were investigated in PBS solution (pH 7.4 and 5.5) to evaluate the influence of pH-response to the release profiles of DOX-loaded micelles. As shown in Fig. 6A, it was obviously found that DOX-loaded mPEG₃₂-St-DCA₁₃ micelles displayed a clearly pH-dependent drug release. Either under pH 7.4 or 5.5 conditions, it showed a steady sustained DOX release due to the more dense and compact hydrophobic cores of mPEG₃₂-St-DCA₁₃ micelles. Besides, in the pH 5.5 condition, stimulating biologic environment of tumor tissues (pH 5.0–6.5), DOX-loaded mPEG₃₂-St-DCA₁₃ micelles exhibited more or less faster drug release rates than that in pH 7.4. The only 32% of the drug was released from DOX-loaded mPEG₃₂-St-DCA₁₃ micelles in 71 h at pH 7.4. In contrast, when the pH value decreased to 5.5, about 59% of the drug was released. This was because the acidic environment could promote the hydrolysis of the ester linkages. Moreover, DCA obtained from the hydrolysis could further increase the pH value of around microenvironment, and DCA molecules could be protonated under acidic condition.⁴² As a consequence, the complete core–shell structural collapse of micelles (Fig. 5c) would facilitate the drug release.

Fig. 6 (A) In vitro release profiles of DOX from mPEG₃₂-St-DCA₁₃ in PBS solutions with pH 7.4 and 5.5; (B) plots of log(M_t/M_∞) against log t at pH 7.4 and 5.5 for DOX in mPEG₃₂-St-DCA₁₃ micelles.

The release mechanism studies

As we all know, the mechanism of drug release from polymeric micelles is a complex process. It could be simply classified as diffusion or permeation of drugs through polymeric network, the erosion of polymeric core or shell and the combination of these mechnisms.^21,45 According to the physicochemical data of micelles, mPEG₃₂-St-DCA₁₃ micelles were stable and had no swelling in pH 7.4 because the size of micelles had no clear change in 48 h in Fig. S4.† However, the size of mPEG₃₂-St-DCA₁₃ micelles increased from 130.70 nm to 177.82 nm in pH 6.5, and the size would increase continuously in pH 5.5, which was similar to results of TEM. So the mPEG₃₂-St-DCA₁₃ micelles were not stable and had swelling under the acidic environment due to the cleavage of ester bonds, causing size increase and swelling. In addition, according to the results of in vitro drug release, it was assumed that the drug release from mPEG-St-DCA micelles was a diffusion controlled release process in pH 7.4, and the drug from DOX-loaded mPEG₃₂-St-DCA₁₃ micelles was released by the rapid diffusion after the swelling in pH 5.5.

To confirm and understand the mechanism of DOX release, a classical semi-empirical power equation was used by Peppas et al.^21,46 The equation is written as:

where M_t and M_∞ are the cumulative amount of drug released at time t and infinite time, respectively; k is a constant incorporating structural and geometric characteristic of the device, and n is the release exponent, indicative of the mechanism of drug release. In the Peppas's equation, for spherical micelles, the special case of n = 0.43 is called Fickian diffusion, indicating diffusion-controlled drug release, and the special case of n = 0.85 is called case-II transport, indicating swelling-controlled drug release. When n has a value between 0.43 and 0.85, the drug release behavior can be regarded as the superposition of both phenomena, which is called anomalous transport.

According to the equation, the plots of log(M_t/M_∞) against log t of the pH 7.4 and 5.5 for DOX in mPEG₃₂-St-DCA₁₃ micelles were shown in Fig. 6B. Through the plots, the values of n, k and R² for mPEG₃₂-St-DCA₁₃ micelles under the pH 7.4 condition were 0.367, 0.062 and 0.987. And corresponding values of n, k and R² in pH 5.5 were 0.451, 0.092 and 0.986. Due to the good linearity in Fig. 6B, it indicated that the Peppas's equation was suitable to in vitro drug release of mPEG-St-DCA micelles. And the value of n at pH 7.4 was much lower than 0.43, indicating the release was a diffusion-controlled drug release mechanism (Fickian diffusion). Moreover, the value of n at pH 5.5 was slightly greater than 0.43, indicating the release was the anomalous transport, and Fickian diffusion was a major factor. Through the release mechanism in different pH values, DOX from mPEG-St-DCA micelles was mainly released by the Fickian diffusion, which was in accordance with the assumption.

In vitro cell cytotoxicity study

As an ideal drug carrier for biomedical applications, the biocompatibility or cytotoxicity of polymeric micelles is a very important parameter. To evaluate the biocompatibilty of the obtained mPEG-St-DCA micelles, the cell viability experiment was carried out by MTT assay against HeLa cells with incubating blank mPEG₃₂-St-DCA₁₃ micelles at the different concentrations in the range from 20 to 100 μg mL⁻¹ for 24 h. Fig. 7A showed the viabilities of HeLa cells were all above 75% at the test concentration up to 100 μg mL⁻¹, which indicated that blank mPEG₃₂-St-DCA₁₃ micelles had a low toxicity and good compatibility to HeLa cells and are promising biomaterials for biomedical application.

Fig. 7 The viability of HeLa cells after incubation with (A) blank mPEG₃₂-St-DCA₁₃ micelles and (B) DOX-loaded mPEG₃₂-St-DCA₁₃ micelles and free DOX at different concentrations for 24 h. The data represent the means ± SD (n = 6).

Simultaneously, the anticancer activity of DOX-loaded mPEG₃₂-St-DCA₁₃ micelles was also investigated by MTT assay against HeLa cells, and free DOX was used as a control. The cell growth inhibitions of HeLa cells incubated with DOX-loaded micelles and free DOX were shown in Fig. 7B. Compared with blank micelles after DOX was loaded into micelles, the cell viability sharply decreased. It was found that with the DOX concentration increased to 15 μg mL⁻¹, the cell viability incubated DOX-loaded mPEG₃₂-St-DCA₁₃ micelles decreased to 45%, the IC₅₀ was 5.74 μg mL⁻¹. While the cell viability incubated with free DOX under the same concentration decreased to 40%. As a comparison, IC₅₀ of DOX-loaded micelles were about 6-times higher than IC₅₀ of free DOX. The free DOX showed higher activity than DOX-loaded mPEG₃₂-St-DCA₁₃ micelles at the same concentration of DOX, probably because of the time-consuming drug release from the micelles and thus delayed nuclear uptake.²²

Intracellular drug release of DOX-loaded micelles

The cellular uptake and intracellular release behaviours of DOX-loaded mPEG-St-DCA micelles were investigated in HeLa cells by confocal laser scanning microscopy (CLSM). Fig. 8 was the CLSM images of HeLa cells incubated with DOX-loaded mPEG₃₂-St-DCA₁₃ micelles for 0.5, 1 and 2 h, respectively. The dose of DOX was 30 μg mL⁻¹. By taking advantage of the blue fluorescence from DAPI and red fluorescence from DOX, the intracellular location of drug was observed. It was clearly found that the red DOX fluorescence intensity gradually increased with prolonging incubation time from 0.5 h to 2 h. And the cells did not displayed red fluorescence for 0.5 h. After 1 h, the cells presented weak red fluorescence in cytoplasm. When the incubation time was extended to 2 h, the strong red fluorescence was observed in cytoplasm mainly and in nucleus with a small amount. Therefore, the results confirmed that DOX-loaded micelles were effectively internalized by cancer cells. The internalization of DOX-loaded micelles was carried out through endocytosis pathway, and the drug release in cells was realized by responding to the acidic environment for cancerous cells by passive accumulation due to the tumors' enhanced permeation and retention (EPR) effect. Based on the above results, it could be summarized that firstly, DOX-loaded micelles were easily internalized into tumor cells through endocytosis into the cytoplasm. Secondly, DOX was released from micelles by diffusion after the swelling with the low pH (pH 5.0–6.5) in tumor tissues due to the core–shell structural collapse. Finally, DOX was uptaken by the cell nucleus to take effect through interaction with DNA, as shown in Scheme 1.

Fig. 8 CLSM images of HeLa cells incubated with DOX-loaded mPEG₃₂-St-DCA₁₃ micelles for 0.5, 1 and 2 h. DOX dosage was 30 μg mL⁻¹. For each panel, the images from left to right showed cell nucleus stained by DAPI (blue), DOX fluorescence in cells (red), and overlays of the above two images.

Conclusion

In summary, a novel kind of amphiphilic mPEG-St-DCA polymers was successfully prepared by introducing hydrophobic DCA molecules grafted into the hydrophilic mPEGylated starch. The obtained polymers could self-assemble into micelles, whose size was <200 nm. With the increasing DS, the CMC decreased in the range from 0.048 to 0.022 mg mL⁻¹. Besides, the effect of pH value on the morphology of mPEG-St-DCA micelles was very obvious. When the pH values decreased from 7.4 to 5.5, the spherical mPEG₃₂-St-DCA₁₃ micelles underwent a dramatic process of structural change from clearly complete core–shell structure to complete structural collapse. DOX could be easily loaded into mPEG-St-DCA micelles. And the results of in vitro DOX release showed that the DOX release from mPEG-St-DCA micelles was a slow and steady process. It could be accelerated to release DOX under the acidic environment (pH 5.5). The results of MTT assay confirmed that the blank mPEG-St-DCA micelles had a good biocompatibility, and the cell viability remained higher than 75% with the concentration up to 100 μg mL⁻¹ after incubation for 24 h. And DOX-loaded micelles showed a significant cytotoxicity against HeLa cells, the IC₅₀ was 5.74 μg mL⁻¹. The CLSM measurement indicated that DOX-loaded mPEG-St-DCA micelles could be effectively internalized by HeLa cells. As a consequence, the novel kind of amphiphilic mPEG-St-DCA micelles has a good pH-response and could be a promising carrier for anticancer drug delivery.

Acknowledgements

The authors gratefully acknowledge the financial support of the National Natural Science Foundation of China (grant no. 51273086) and Special Doctorial Program Fund from the Ministry of Education of China (grant no. 20130211110017).

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Footnote

† Electronic supplementary information (ESI) available: ¹H NMR spectrum of mPEG-St polymer, UV scanning spectra of mPEG-St-DCA polymer, pH values of three reactive systems monitored using the pH test papers, the pH as a function of the volume of HCl and the UV scanning spectra of DOX·HCl and micelles. See DOI: 10.1039/c4ra07315k

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