Introduction

Self-assembling water-soluble polymers are a current interest of scientists and technology experts because they are relevant to biological macromolecular systems and various industrial applications.1, 2, 3 Among various type of interactions, hydrophobic interactions are the major driving force for self-organization of amphiphilic polymers in water. A practical approach to synthesizing such polymers is to covalently introduce hydrophobic groups into the water-soluble polymer structures.4 Studies have demonstrated that amphiphilic copolymers with various architectures can self-assemble to minimize unfavorable segment/solvent interactions and maximize favorable segment/solvent interactions.5 Generally, amphiphilic polymers self-assemble into polymeric micelles with a core-shell structure in aqueous media.6 The inner hydrophobic core of micelles can enhance loading efficiency for hydrophobic drugs, the outer hydrophilic shell can provide a stabilizing interface between the hydrophobic cores and the aqueous medium, which inhibits inter-micellar particle aggregation, protects drugs from inactivation in the biological environment.7 Polymeric micelles are drug vehicles that present different and considerable advantages, such as lower side effects from drugs, selective targeting, stable storage and stability under dilution.8 Drug therapy may be temporally controlled through the stimuli-responsive property of hydrophilic segments.

Among biomaterials studies, stimuli-responsive micellar drug carriers constructed from amphiphilic polymers have garnered tremendous attention over the past decade.9 One current trend is to design intelligent nanoparticles that exhibit an ability to target specific sites10, 11 and tunable release kinetics in response to environmental stimuli, such as temperature,12, 13 pH14, 15 and photodynamics.16 Different organs, tissues and cellular compartments have different pH values; thus, pH is a suitable stimulus for releasing therapeutic molecules from micelles. Orally administered drugs undergo pH gradients as they transition from the stomach (pH 1–2) to the duodenum (pH 6) and along the jejunum as well as the ileum (pH 6–7.5).17 An acidic pH at tumor sites has been considered an ideal stimulus for selective release of anticancer drugs to tumors for tumor-targeted drug delivery.18 Therefore, many researchers have developed various pH-sensitive drug carriers to facilitate drug delivery to target tissues or organs.19 Therefore, polymeric micelles that are responsive to pH gradients can be designed to selectively release payloads in tumor tissue or cells. The pendant carboxylic groups can accept protons at low pH values and release protons at high pH values; therefore, the balance between hydrogen bonds and electrostatic repulsion forces produce changes in the hydrophobic/hydrophilic characteristics at different pH values. Furthermore, the pendant carboxylic groups may form multiple hydrogen bonds with the glycoproteins in the mucus, which can generate a bio-adhesive polyelectrolyte outer shell on the micelles.20

Itaconic acid (IA) is a naturally derived unsaturated dicarboxylic acid produced on an industrial scale through cultivating Aspergillus terreus. Owing to two carboxyl groups in IA, two ester groups can be introduced into the molecular structure of IA to modify the hydrophilic/hydrophobic balance of its homopolymer and copolymers.21 Although many papers on preparing acrylate- or methacrylate-based amphiphilic polymeric micelles have been produced,22, 23 little information is available for systematically studying itaconate-based amphiphilic polymeric micelles.24 With its high hydrophobic sterol skeleton, cholesterol can be used to modify certain biomaterials as carriers for hydrophobic drugs or genes.25 On the other hand, these cholesteryl groups are biocompatible and form strong potential interactions with cholesteryl receptors on the cell surface.26

In our previous work, synthesis and characterization of liquid crystalline amphiphilic-modified poly (monomethyl itaconate)s with different quantities of side chain were described.27 We also reported that the novel double hydrophilic poly (monomethylitaconate)-co-poly (N, N-dimethylaminoethyl methacrylate) (PMMI-co-PDMAEMA) and cholesterol-conjugated (PMMI-co-PDMAEMA) are effective pH-responsive nano-carriers for improved cancer therapy.28 Furthermore, novel monomethyl itaconate-based copolymer (PEG-PMMI-Chol-C6) bearing cholesteryl (CholC6) and poly (ethylene glycol) (PEG) side chains were studied in detail as potential pH-responsive nano-carriers for tumor-targeting chemotherapy.29 Regarding the advantages of itaconate-based polymers, the present work is focused on self-assembly of biodegradable amphiphilic PMMI-Chol-C6 polymers with different quantities of cholesteryl side chain (DSchol) for pH-sensitive hydrophobic drug delivery. The influence of DSchol on the pH-phase transition of synthesized polymers was investigated in detail. Moreover, we developed new PMMI-Chol-C6 micelles that contain encapsulated naproxen and evaluated the pH-dependent drug release as well as cytotoxicity of the polymer in HeLa cells. These pH-dependent polymeric micelles are likely promising carriers for pH-responsive drug delivery systems.

Experimental Procedure

Material

Acetyl chloride, IA, 1, 6-hexanediol, dicyclohexylcarbodiimide, 4-dimethylaminopyridine, cholesteryl chloroformate and cholesterol were purchased from Merck (Darmstadt, Germany). α, α′-Azobis (isobutyronitrile) was purchased from Acros and purified via recrystallization from ethyl acetate before use. IA and cholesterol were initially purified via recrystallization from acetone and ethanol, respectively, before use. Naproxen and the dialysis membrane (MWCO=10 000) were purchased from Sigma-Aldrich (St Louis, MO, USA). Pyrene, potassium dihydrogen phosphate, sodium hydrogen phosphate and sodium chloride were purchased from Merck. Dimethylformamide (DMF) and tetrahydrofuran (THF) were dried through distillation, and all other chemicals were used as received. The PMMI-Chol-C6 was synthesized using the route outlined in Scheme 1, and further details are provided below.

Synthetic procedure

Polymerization of MMI

Monomethyl itaconate (MMI) was synthesized using the method described by Gargallo et al.20 PMMI was prepared through radical polymerization of MMI in bulk under N2 at 70 °C with 0.2 mol% of α, α′-Azobis (isobutyronitrile) as the radical initiator. The homopolymer produced was purified through dissolution in methanol and precipitation in diethyl ether.27

Synthesis of 6-cholesteryloxycarbonyloxy hexanol

6-Cholesteryloxycarbonyloxy hexanol (Chol-C6) was synthesized using the previously described procedure27 shown in Scheme 1. Thus, a solution of cholesteryl chloroformate (8.9 mmol, 4 g) in dry THF was added drop-wise to a mixture of 1,6-hexanediol (133 mmol, 15.8 g) and 1 ml pyridine in dry THF in a round-bottom flask. The reaction mixture was stirred overnight at room temperature. After the THF was evaporated, the residue was dissolved in 20 ml dichloromethane and washed twice with 50 ml of a solution containing HCl (5 M), 5 wt% NaHCO3, de-ionized water and brine. It was subsequently dried over MgSO4 and filtered, and the solvent was removed. The excess 1,6-hexanediol was removed through washing with water. The residue was purified through recrystallization in boiling n-hexane to yield 2.8 g (90%) of a product. Fourier transform infrared (KBr): 3405 (OH), 2953, 2868 (aliphatic CH2), 1745 (C=O), 1670 (C=C), 1468 (CH2, bending), 1382 (CH3, bending), 1258 (O–CO, bending), 1060 (C–OH) cm−1. 1H NMR (CDCl3): d 0.67 (s, 3H, CH3 from cholesterol), 0.85 (d, 3H, CH3 from cholesterol), 0.87 (d, 3H, CH3 from cholesterol), 0.92 (d, 3H, CH3 from cholesterol), 1.009 (s, 3H, CH3 from cholesterol), 3.64 (t, 2H, CH2OH), 4.12 (t, 2H, CH2–OCOO–), 4.47 (m, 1H, –CH from cholesterol), 5.38 (m, 1H, =CH from cholesterol), p.p.m.

Synthesis of PMMI-Chol-C6

PMMI-Chol-C6 with different quantities of cholesteryl side chain was previously synthesized in our laboratory using poly (monomethyl itaconate) (PMMI) and 6-cholesteryl-1-hexanol (Chol-C6) (Table 1).27 P-77 synthesis is described as a representative case. Dicyclohexylcarbodiimide (3.4 mmol, 0.7 g) was added to a reaction flask containing a solution composed of PMMI (3.42 mmol of the –COOH group, 0.5 g) in 5 ml dry N, N-dimethylformamide (DMF). The reaction mixture was stirred for 4 h at room temperature. Next, a solution of 4-dimethylaminopyridine (1 mmol, 0.1 g) and Chol-C6 (3.4 mmol, 1.8 g) in 5 ml dry DMF was added drop-wise to the reaction flask for almost 2 h. The reaction mixture was stirred for 48 h. The precipitated dicyclohexyl urea was removed through filtration, and the filtrate was poured into an n-hexane/ethanol mixture (8/2; v/v) (diethyl ether for P-5) to yield a white precipitate composed of P-77. Fourier transform infrared (KBr): 3446 (OH, carboxylic acid), 2957 (aliphatic CH2), 1741 (ester C=O), 1670 (carboxylic acid C=O), 1439 (CH2, bending), 1364 (CH3, bending) cm−1. 1H NMR (CDCl3): d 0.70 (s, 3H, CH3 from cholesterol), 0.87 (d, 3H, CH3 from cholesterol), 0.89 (d, 3H, CH3 from cholesterol), 0.93 (d, 3H, CH3 from cholesterol), 1.03 (s, 3H, CH3 from cholesterol), 2.41 (s, 2H, –CH2– polymer backbone), 3.13 (s, 2H, –CH2COOCH3), 3.5 (s, 3H, –COOCH3), 3.66 (t, 2H, –CH2OCO–), 4.14 (t, 2H, –CH2OCOO–), 4.48 (m, 1H, CH from cholesterol), 5.41 (m, 1H, =CH from cholesterol), 7.29 (1H, –COOH), p.p.m.

Table 1 Degrees of substitution and solubility of the PMMI-Chol-C6 polymers
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Preparation of the PMMI-Chol-C6 micelles

Blank polymeric micelles were prepared based on co-solvent evaporation and dialysis methods.

(1) Membrane-dialysis method: P-5 (30 mg) was dissolved in 4 ml of DMF and then dialyzed against 250 ml of water by adjusting the solution pH to 4 using 0.10 M HCl for 24 h at room temperature using a dialysis membrane (MWCO=10 000).

(2) Evaporation method: P-77 (and/or P-63; 30 mg) was dissolved in 2 ml of THF; the polymeric solution was then added drop-wise into 10 ml of water by adjusting the solution pH to 6. THF was evaporated by stirring overnight at room temperature. The micellar solutions were filtered using a microfilter (pore size: 0.4 μm) to eliminate the aggregated particles.

Phase-transition pH determination

The phase-transition pH values of PMMI-Chol-C6 polymers were determined by monitoring the transmittance changes. The transmittance of the polymer solutions (3 mg ml−1) at different pH values, ranging from 2 to 11, was measured at 500 nm. The phase-transition pH values were determined at the pH with a 50% optical transmittance.

The effect of ionic strength on micelle stability

Different concentrations of NaCl aqueous solutions in the range 0.1–2.9 mol l−1 were used to measure solution turbidity through ultraviloet-visible spectrophotometry at 500 nm.

Fluorescence measurements

To further evaluate pH-dependent phase transitions, the transitions were also determined using the fluorescence method and pyrene as a fluorescence probe. Pyrene was dissolved in acetone at 5 × 10−5 mol l−1 and then diluted by adding 10 ml PMMI-Chol-C6 polymer solution. Two milliliters of polymer solution with the same concentration (3 mg ml−1) but a different pH value (by adjusting the solution pH using 0.10 M HCl or NaOH aqueous solutions) was added to the pyrene solution. After acetone evaporation, the final pyrene concentration in the polymer solution was adjusted to 6 × 10−7 mol l−1. The mixed solution was equilibrated at room temperature for 24 h before measurement. Fluorescence measurements were performed using the excitation wavelength λ=339 nm, a 2.5-nm slit width for excitation and a 2.5-nm slit width for emission. The emission wavelengths were scanned from 350 to 600 nm.

Transmission electron microscopy

The morphology of self-assembled PMMI-Chol-C6 polymer structures in an aqueous solution (3 mg ml−1) near the transition pH was visualized through transmission electron microscopy (TEM; PHILIPS SM10 TEM (Amsterdam, The Netherlands) and an EPSON HP8300 Photo flat-bed scanner operated at the accelerating voltage 150 keV). The samples were prepared by dropping the micelle suspensions on a carbon-coated copper grid and then drying in a vacuum. Finally, the samples were coated with carbon before observation.

Size and size distribution measurements

The sizes of the freshly prepared micelles in an aqueous solution near the transition pH were measured using a Zetasizer Nano ZS (Malvern Instruments, Malvern, UK, MAL 1001767) with a He–Ne laser beam at 511 nm and 25 °C. The average values were obtained from three repeated measurements for each sample.

Drug loading

Drug-loaded micelle formulations were prepared using naproxen, which has low aqueous solubility, as a model drug. Naproxen-loaded P-5 micelles were prepared using dialysis as follows. Briefly, 30 mg of polymer and 6 mg of naproxen were dissolved in 2 ml of DMF. The solution was then dialyzed against 500 ml near the phase-transition pH (the point of stable micelle formation) for 24 h using a dialysis tube (MWCO=10 000). After dialysis, the micelle solution was filtered using a microfilter (pore size: 0.4 μm) to eliminate the aggregated particles. The dialysate was collected, and the absorbance (A) of the unencapsulated naproxen was analyzed using a ultraviolet-visible spectrophotometer at 330 nm. The standard solutions were prepared at concentrations ranging from 0.01 to 0.10 mg ml−1 at the desired pH. The correlation coefficient (R2) was at least 0.995.

Naproxen-loaded P-77 (and/or P-63) micelles were prepared by dissolving 30 mg of polymer and 6 mg of naproxen in 2 ml of THF, which were then added drop-wise into a 10-ml solution near the related phase-transition pH. The micelle-dispersed solutions were obtained after removing THF through evaporation. The suspensions were centrifuged at 7 000 r.p.m. for 10 min, and the supernatant that contained naproxen-loaded micelles was collected. Precipitate containing the unloaded drug was dissolved in 3 ml THF, and the unloaded naproxen levels were analyzed through ultraviolet-visible spectrophotometry. The drug-loading efficiency and capacity were calculated as follows (Table 2):

Table 2 Characterization of PMMI-Chol-C6 micelles
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where A is the total weight of the naproxen used, B is the weight of the unloaded naproxen in the precipitate after centrifugation and C is weight of the polymeric sample.

In vitro drug release test

The pH-dependent model drug release profiles from P-63 micelles were determined. Three milliliters of drug-loaded PMMI-Chol-C6 micellar solutions were placed into a dialysis membrane (MWCO=10 000). The membrane was immersed in 25 ml of phosphate-buffer solution (pH 7.4, 5.5) and 1 M HCl solution (pH 1.2) in a shaking water bath at 37 °C to produce sink conditions. Three ml of the outer buffer solution of membrane with the released naproxen was collected at predetermined time points and replaced with 3 ml of a fresh buffer solution. The level of naproxen released was measured using ultraviolet-visible spectroscopy at 330 nm and calculated using a calibration curve with naproxen solutions at various concentrations, ranging from 0.01 to 0.10 g l−1, for the three pH values 1.2, 5.5 and 7.4. The experiment was performed in triplicate. The percentage of naproxen released was calculated using the following equation:

where Wt is weight of the naproxen released at time t, and Wtotal is the total naproxen absorbed in the polymeric micelle structure. Wtotal was calculated based on the free drug levels (that is, the total drug levels used herein, 6 mg (A) minus the quantity of unloaded drug (B).

MTT assay and cell treatment

A cellular toxicity assay was performed to evaluate the effect of different micelle concentrations on cell survival. When cell growth reached 70–80% confluency, the cells were washed in phosphate-buffer saline and passaged with 0.05% trypsin-EDTA, the number of live cells was evaluated through Trypan blue staining, and 5000 live cells were seeded onto 96-well flat-bottomed culture plates and incubated at 37 °C in a humidified gas environment of 5% CO2 in air for 24 h. The day of treatment, the medium was removed, and the cells were washed with phosphate-buffer saline. Next, 200 μl of conditioned media was added to each well and incubated 24 h in the incubator. In addition, the culture without treatment was used as a control group. After 24 h, the cell viability was determined using the MTT assay; briefly, the cell treatment medium was removed from each well, and a 50 μl ml−1 of MTT (Sigma, St Louis, MO, USA) solution with 150 μl medium was added to each well and incubated for 4 h at 37 °C. After incubation, the medium was removed, and the mixture of 200 μl dimethylsulfoxide and 25 μl Sorenson buffer (0.1 glycine plus 0.1 NaCl equilibrated to pH 10.5 with 0.1 N NaOH) was added to each well to dissolve the blue formazan crystals. Next, the optical density (OD) was measured at 570 nm using a Microplate Reader (Awareness Technologic, Palm City, FL, USA). Each experiment was repeated in triplicate.

Results and Discussion

In our previous work, cholesteryl-modified poly(monomethyl itaconate)s (PMMI-Chol-C6)s were prepared through an esterification reaction between the O–H groups of Chol-C6 and carboxylic acid groups of the homopolymer (PMMI) using dicyclohexylcarbodiimide/4-dimethylaminopyridine coupling agents (Scheme 1).27 Different samples with different DSchol were synthesized and characterized through Fourier transform infrared and 1H NMR spectroscopy (Table 1). The DSchol of the samples were determined through conductivity measurements and 1H NMR analyses. P-5, P-63 and P-77 with DChol=5, 63 and 77, respectively, were used herein.

PMMI-Chol-C6 micelle preparation

Compared with PMMI, the synthesized PMMI-Chol-C6 showed increased solubility in organic solvents, such as chloroform and THF. PMMI-Chol-C6 polymers with different DSchol values exhibited different solubilities (Table 1). For example P-77, which had the most cholesterol side chains, was easily dissolved in organic solvents, such as THF, while P-5 with the lowest DSchol levels was not soluble in THF. Therefore, the P-77 and P-63 micelles were prepared using the co-solvent evaporation method, and for the P-5 micelles, the dialysis method was applied. These amphiphilic polymers were assembled as micelles in an aqueous solution. It is reasonable that the hydrophobic Chol-C6 was mainly in the core, whereas the hydrophilic PMMI main chains were in the micelle shell.

pH-responsive micelle behavior

Chol-C6 is a hydrophobic side chain, while PMMI is a highly hydrophilic and water-soluble backbone. Therefore, PMMI-Chol-C6 formed micelles in an aqueous solution with hydrophobic Chol-C6 as the core, and the hydrophilic PMMI main chain as the shell. To evaluate the pH-responsive properties, polymeric micelles were treated with different pH values. Figure 1 shows the pH-dependent properties of the P-77, P-63 and P-5 micelle solutions, which were measured through ultraviolet-visible transmittance and should reflect macroscopic changes in polymeric micelles in water. By raising the pH, the solution changed from opaque to transparent, which indicates an obvious pH-responsive property of PMMI-Chol-C6 polymers. On the other hand, by lowering the pH, transmittance decreased due to disrupted hydrogen bonds among carboxylic acid groups on the outer shell. Micro particles from micelle aggregation appeared rapidly at the pH transition points. The phase-transition pH values for the P-77, P-63 and P-5 micelles in water were 5.3, 4.9 and 3.3, respectively. The transition process is reversible through tuning the pH value. The pH response was sharp and rapid.

Figure 1
figure 1

The pH dependence of transmittance at 500 nm for polymeric micelles in water at 25 °C; the concentrations of the PMMI-Chol-C6 polymers were 3 mg ml−1. A full color version of this figure is available at Polymer Journal online.

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The pH-responsive behaviors of the PMMI-Chol-C6 polymers were also studied using pyrene as a fluorescence probe. Pyrene has a low fluorescence intensity in water, and it increases when transferred into a hydrophobic environment. In this study, the fluorescence intensity of pyrene emission spectra were used to indicate the polarity of the pyrene environment. For P-5, the fluorescence intensity values were low due to sample precipitation at a low pH. The I1 fluorescence intensity of PMMI-Chol-C6 versus pH is shown in Figure 2. For the P-63 and P-77 polymeric solutions, a sharp increase in fluorescence intensity (I1) was observed upon increasing the pH to the phase-transition pH values 4.9 and 5.3, respectively, which indicates micelle formation. By further increasing the pH, the intensity decreased due to micelle destruction from the repulsion forces among the carboxylate ions on the individual micelle shells. The critical pH values of the PMMI-Chol-C6 polymer solution were specifically determined by the mid-points of the fluorescence intensity change plots.

Figure 2
figure 2

The fluorescence intensity (I1) of the cholesteryl-modified PMMI-Chol-C6 solution as a function of pH; the concentration of the PMMI-Chol-C6 polymers was 3 mg ml−1.

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Ionic strength-dependent stability of the polymeric micelles

The P-77, P-63 and P-5 micelle stabilities at different ionic strengths were studied (Figure 3). In dilute ionic solutions with concentrations below the transition point, ions were randomly distributed throughout the liquid phase, and the micelles were stable and well-dispersed due to the electrostatic repulsion forces among the deprotonated carboxyl groups on the outer shells. While at high ionic strengths, the charges on the micelle outer shell were neutralized by electrolytes, which decreased the electrostatic repulsion forces among the ionized carboxyl groups such that the micelles became unstable and aggregated.30 For P-77 and P-63, by increasing the ionic strength of the media, the micelles aggregated to form microparticles. However, even in solutions with low ionic concentrations, the P-5 micelles were unstable due to the high levels of carboxylic acid groups on the polymer backbone as well as low DSchol values, which effectively neutralized the micelles even at low salt concentrations.

Figure 3
figure 3

The effect of ionic strength (reflected the NaCl concentration) on the stability of cholesteryl-modified PMMI-Chol-C6 micelles. The concentration of the PMMI-Chol-C6 polymers was 3 mg ml−1.

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Micelle morphology and size

The polymeric micelle morphology and size distribution was investigated using TEM and dynamic light scattering (DLS) near the transition pH values (Figure 4). The increased levels of cholesterol side chains yielded the smaller size micelles25 (Table 2). Under acidic conditions, irregular aggregation of P-5 micelles (not shown) appeared due to hydrogen bonding among the carboxylic acid groups between the individual micelle shells. Large aggregates were confirmed using DLS (780 nm) (Figure 4a). Further, the P-63 TEM images showed well-dispersed micelles with regular spherical shapes near the transition pH (Figure 4b). The corresponding micelles had a diameter at approximately 250 nm (DLS). Further, two other small peaks were observed; one was assigned to the average size of a small micelles (approximately 72 nm), and the other indicated large aggregates (>1 μm). The polydispersity index of P-63 micelles determined using DLS was low (0.27), which verified successful micelle formation (Figure 4b). P-77 micelles showed aggregated spherical micelles (not shown). DLS data near pH 5.3 showed a bimodal distribution with a smaller micelle diameter (approximately 100 nm) and a clear, well-resolved aggregated peak at approximately 340 nm (Figure 4c). The average hydrodynamic diameter was 510 nm with the polydispersity index 0.49. The results suggest that the P-77 micelles were unstable, which led to aggregation due to the high levels of Chol-C6. The smaller size and higher stability of the P-63 micelles compared with P-5 and P-77 might be ascribed to the balance between the hydrophilic and hydrophobic sections.

Figure 4
figure 4

(a) Dynamic light scattering (DLS) results for P-5, (b) DLS results for P-63 and a typical transmission electron microscopy image for P-63 near the pH transition. (c) DLS results for P-77.

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The discrepancy in micelle size between TEM and DLS measurements was attributed to the methodology; the DLS method measures the hydrodynamic diameter of micelles in water, whereas TEM measures the morphology size of the freeze-dried micelles in a solid state.28 Nevertheless, both methods exhibited the same trend in the micelle particle sizes.

In vitro drug-loading and release profiles for naproxen under sustained release

To assess the potential of supramolecular copolymeric micelles based on PMMI-Chol-C6, naproxen was used as a model drug to evaluate the drug-loading and pH-sensitive release properties. Naproxen is a non-steroidal anti-inflammatory drug commonly used to reduce mild-to-moderate pain, fever and inflammation.31 A sustained-release drug delivery system is designed to produce prolonged therapeutic effects through continuous discharge of drug over an extended period of time from a single dose.32 In addition, naproxen has been highly effective in preclinically preventing colon, urinary bladder and skin cancer as well as in clinical trials of colon adenoma formation with the lowest cardiovascular events of the common non-steroidal anti-inflammatory drugs, except aspirin.33 Targeting drugs to the colon offers several potential therapeutic advantages, such as increasing systemic absorption of poorly absorbed drugs and effective treatment of colonic diseases, including colorectal cancer. One available approach to selectively targeting drugs to the colon includes pH-sensitive polymers.34 Physical entrapment of hydrophobic drugs in polymeric micelles is mainly driven by hydrophobic interactions between the drug and hydrophobic segments of the polymers.35

The loaded drug levels and encapsulation efficiency of the polymeric micelles are listed in Table 2. The drug-loading capacities of the micelles indicate that the higher drug-loading capacity (DLC %) and drug-loading efficiency (DLE %) were attributed to the higher DSchol (Table 2).

Among these polymers, P-63 with moderate Chol-C6 content, a good micelle size, well-defined stability and polydispersity was used to study drug release behavior. Hydrophobic naproxen was well-encapsulated into the hydrophobic inner core with a loading efficiency DLE % of approximately 35% and a drug-loading capacity (DLC %) at 7% (Table 2). The naproxen release behavior was evaluated at pH 7.4, 5.5, and pH 1.2 at 37 °C to assess the feasibility of using PMMI-Chol-C6 micelles as a pH-sensitive drug-delivery carrier. As illustrated in Figure 5, the release profiles show that naproxen was quickly released from micelles in the first stage; drug release was then sustained over a prolonged time. Release at pH 5.5 was clearly much faster than at pH 7.4 and 1.2, which indicates a pH-dependent drug release profile. At pH 5.5, the naproxen release rate was high; more than 48% was released in 30 h. A notably lower release rate was observed at pH 7.4 and 1.2; less than 20 and 5% of the naproxen was released within 30 h, respectively. Slow release in an alkaline environment may be attributed to complete deprotonation of the PMMI carboxylic acid groups (pka∼3.8) and naproxen molecules (pka∼4.2), which would prohibit drug release due to repulsion forces between the carboxylate groups in the shell and the drug molecules. At pH 5.5, due to both natural and anionic forms of PMMI carboxylic acid groups in the shell and the naproxen molecules, such repulsion forces decreased, which enhanced drug release. While at pH 1.2, strong hydrogen bonds among the carboxylic acid groups in the shell prevented considerable levels of drug release. Therefore, PMMI-Chol-C6 micelles might represent a highly promising approach to rapid, controlled drug release.

Figure 5
figure 5

In vitro release profiles of naproxen from P-63 micelles at 37 °C at () pH 7.4, () pH 5.5 and () pH 1.4.

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Cell viability testing

Statically significant differences were not detected in the viability of treated cells compared with a control group; thus, the different P-63 micelle concentrations (0.06–1 mg ml−1) after a 24-h treatment did not produce a cytotoxic effect on HeLa cell viability (Figure 6).

Figure 6
figure 6

The cell viability of HeLa cells for different concentrations of P-63 micelle samples following a 24-h treatment. The experiment was performed in triplicate.

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Conclusions

We successfully developed a new class of pH-responsive micelles that self-assemble from amphiphilic polymers based on poly (monomethyl itaconate)s with different levels of DSChol to release naproxen in a pH-responsive manner. Owing to these amphiphilic polymers’ pH-responsive properties, the micelle stability, size and morphology could be changed through altering the solution pH and ionic strength. The micelle morphology was spherical, and the mean diameter was between 250 and 510 nm. Naproxen-loaded P-77 polymeric micelles exhibited successful and high hydrophobic drug-loading efficiency. Compared with the polymers containing different DSChol values, P-63 formed more stable micelles than P-5 and P-77 polymers. This observation is attributed to the appropriate hydrophobic/hydrophilic balance in the amphiphilic P-63 polymer with the optimum levels of Chol-C6 side chain. The naproxen release from these micelles was pH-responsive, as expected. The cytotoxicity measurements using the MTT assay indicate that PMMI-Chol-C6 has low toxicity in HeLa cells, which suggests that this polymer could be used as a safe candidate for pH-responsive drug delivery. Therefore, these micelles show a pH-responsive release, which renders these micelles potential candidates for oral administration and controlled drug release in the gastrointestinal tract by responding its pH changes.

scheme 1

Synthetic route of PMMI-Chol-C6 polymers.