Novel pH-Responsive Polymeric Micelles Prepared through Self-assembly of Amphiphilic Block Copolymer with Poly-4-vinylpyridine Block Synthesized by Mechanochemical Solid-State Polymerization

Abstract

We fabricated polymeric micelles containing 5-fluorouracil (5-FU) or fluorescein using the amphiphilic block copolymer, poly-4-vinylpyridine-b-6-O-methacryloyl galactopyranose. Although the polymeric micelles were stable at pH 7.4, they readily decomposed at pH 5, resulting in near complete release of 5-FU. Uptake of polymeric micelles containing fluorescein by HepG2 and HCT116 cells was also investigated. With both cell types, strong fluorescence was observed after a 12-h incubation, but the fluorescence weakened after 24 h of incubation. The fluorescein incorporated into the polymeric micelles was released into acidic organelles (endosome and/or lysosome), from which it diffused throughout the cell. The cytotoxicity of polymeric micelles containing 5-FU was evaluated against HepG2 cells using a CCK-8 assay. The results suggest that polymeric micelles containing 5-FU are more cytotoxic to HepG2 cells than free 5-FU.

Polymeric micelles formed from amphiphilic block copolymers (which consist of hydrophilic and hydrophobic blocks) have been extensively studied for their potential use as nanocarriers in aqueous solutions. Considerable attention has been paid to the use of polymeric micelles in drug delivery systems because of their ability to solubilize hydrophobic molecules and their nanoscale size, good thermodynamic stability in solution, capacity for extended release of various drugs, and their resistance to rapid clearance by the reticuloendothelial system.^1–9) Stimulus-responsive polymeric micelles have recently emerged as a novel controlled-release system in which drugs can be released by applying an appropriate stimulus, such as a specific temperature, pH, or ultrasound. A pH-sensitive polymeric micelle would be particularly useful for targeting tumor tissues, endosomes, and lysosomes because these sites are more acidic than other parts of the body, such as the blood. Several reports describe the use of various types of pH-sensitive polymeric micelles for acidic pH-triggered drug release of drugs at tumor sites.^10–13)

We previously reported the synthesis and drug release profiles of various polymeric prodrugs prepared by mechanochemical solid-state polymerization carried out through vibratory ball-milling of solid monomers in a metallic vessel under anaerobic conditions.^14–26) A series of studies revealed that this method is applicable to the preparation of a wide variety of vinyl monomers of important classes of bioactive compounds with different physicochemical properties. The method also provides a simple, novel means of synthesizing functionalized polymers in a totally dry process.¹⁹⁾ The mechanochemical solid-state polymerization method we reported has several features: the resulting polymeric prodrugs have a narrow molecular weight distribution, represented by M_w/M_n, where M_w represents the weight average molecular weight and M_n the number average molecular weight; block copolymers can be readily obtained through mechanochemical polymerization of a polymer and a monomer in a non-metallic vessel²¹⁾; and polymerization of a vinyl monomer possessing a drug as a side chain proceeds without detectable side reactions. We have also studied the conventional synthesis and drug release profiles of polymeric micelles formed from amphiphilic block copolymers via mechanochemical polymerization.²⁶⁾

In a previous study, we used mechanochemical solid-state polymerization to synthesize a novel amphiphilic copolymer containing basic and hydrophobic blocks.²⁷⁾ The polymeric micelles prepared from this block copolymer showed pH-dependent micellization and demicellization, which, importantly, occurred over a very narrow pH range. We also prepared polymeric micelles incorporating 5-fluorouracil (5-FU) and confirmed that 5-FU is rapidly released upon demicellization.

In the present study, polymeric micelles containing 5-FU or fluorescein were prepared via the dialysis method using the amphiphilic block copolymer, poly-4-vinylpyridine-b-6-O-methacryloyl galactopyranose (P4VP-PMG). Cellular uptake of the polymeric micelles incorporating fluorescein was confirmed by observation of fluorescence using confocal laser scanning microscopy (CLSM). The cytotoxicity of polymeric micelles containing 5-FU was also evaluated.

Experimental

Materials

P4VP was synthesized by conventional radical-initiated solution polymerization. All chemicals were of reagent grade.

Synthesis of Amphiphilic Block Copolymer via Mechanochemical Polymerization

The amphiphilic block copolymer, P4VP-PMG, was synthesized as described elsewhere.²⁷⁾ P4VP (9.7 mg) and 6-O-methacryloyl-D-galactose (MGP, 90.3 mg) were mechanically fractured under anaerobic conditions (e.g., under nitrogen) using a Type MM 200 mixer mill (Retsch Co., Ltd.) equipped with an agar ball (5.0 mmϕ (e.g., diameter), 190 mg) in an agar twin-shell blender (14 mmϕ, 65 mm in length) at room temperature for 4 h at 30 Hz. Residual oxygen in this system was removed with a Model 1000 oxygen trap (Chromatography Research Supplies). The oxygen concentration was monitored with an LC 750/PC-120 oxygen analyzer (Toray Engineering Co., Ltd.) and kept below 1 ppm.

Preparation of Polymeric Micelles

P4VP-PMG (5 mg) was first dissolved in dimethyl sulfoxide (1 mL). This solution was passed through a 0.45-µm filter (GL Chromatodisc 13N, GL Sciences, Inc.) and into a pre-swollen semi-permeable membrane tube (Spectra/Por^® 3 Dialysis Membrane Standard RC Tubing MWCO: 3.5 kDa, Spectrum Laboratories, Inc.; molecular weight cutoff, 3500 g/mol). The both sides of the tube were sealed with dialysis tubing closures (Dialysis Tubing Closures Standard Closure Type, 35 mm). The solution was then dialyzed against 200 mL of phosphate buffer (pH 7.4) for 24 h to allow for micelle formation. The dialysate was exchanged at 2, 4, 6, 8, and 10 h. Polymeric micelles containing 5-FU or fluorescein were prepared in a similar manner. Briefly, 5-FU (5–80 mg) or fluorescein (10 mg) was added to a solution of P4VP-PMG (5 mg) dissolved in N,N-dimethylformamide (DMF) (1 mL). This solution was passed through a 0.45-µm filter (GL Chromatodisc 13N, GL Sciences, Inc.) and into a pre-swollen semi-permeable membrane tube (Spectra/Por^® 3 Dialysis Membrane Standard RC Tubing MWCO: 3.5 kDa, Spectrum Laboratories, Inc.; molecular weight cutoff, 3500 g/mol). The solution was then dialyzed against 200 mL of phosphate buffer (pH 7.4) for 24 h to allow for micelle formation. The dialysate was exchanged at prescribed intervals (2, 4, 6, 8, 10 h). It was confirmed that no 5-FU or fluorescein eluted from the polymeric micelles following 24 h of dialysis. The amount of 5-FU entrapped in the micelles was determined by measuring the UV absorbance at 265 nm. Drug load (%) was calculated using the following equation:   

Molecular Weight Measurement

The molecular weight of P4VP was determined by gel permeation chromatography (GPC) using a Shimadzu LC-6A system equipped with an RID-6A refractive index detector (Shimadzu), KD-802 and KD-G gel columns (Shodex), and Chromato-PRO-GPC data analyzer (Runtime Instruments Co.). Chromatography conditions were as follows: elution solvent, DMF containing 10 mM LiBr; flow rate, 0.7 mL/min; column temperature, 40°C. The calibration for the molecular weight determination was carried out using a standard specimen of polystyrene (M_w=3114000, 1186000, 523000, 170800, 60450, 30300, 9920, 4490). The molecular weight of P4VP-PMG was determined by static light-scattering measurement using a DLS-5500G Photal dynamic light-scattering spectrophotometer (Otsuka Electronics) equipped with a He/Ne laser. A scattering angle of 90° was used in this study.

Light Scattering

Dynamic light-scattering was measured using a DLS-5500G Photal dynamic light-scattering spectrophotometer (Otsuka Electronics) equipped with a He/Ne laser. A scattering angle of 90° was used in this study. The hydrodynamic diameter and the polydispersity factor of the micelles, represented as μ₂/Γ², were calculated using the Stokes–Einstein equation and the cumulant method. The number-average particle diameter and weight-average particle diameter were determined by histogram method with Marquardt calculation.

Drug Release from Polymeric Micelles

The pH 7.4 solution (1 mL) of polymeric micelles, in which 5-FU was contained 5 mg, was transferred into a pre-swollen semi-permeable membrane tube (Spectra/Por^® 3 Dialysis Membrane Standard RC Tubing MWCO: 3.5 kDa, Spectrum Laboratories, Inc.). The both sides of the tube were sealed with dialysis tubing closures (Dialysis Tubing Closures Standard Closure Type, 35 mm). The membrane tube containing polymeric micelles was immersed into pH 6.0 or 5.0 phosphate buffer (200 mL) at 37±0.2°C. The outer-layer solution was periodically sampled and the released 5-FU was assayed by measuring the UV absorbance at 265 nm.

Cell Culture

HepG2 cells (Summit Pharmaceuticals International Corporation, Tokyo, Japan) were cultured in Advanced Dulbecco’s modified Eagle’s medium (Advanced DMEM, Life Technologies, Carlsbad, CA, U.S.A.) supplemented with 2% (v/v) fetal bovine serum (FBS, Thermo Fisher Scientific, Waltham, MA, U.S.A.), 100 U/mL penicillin, 100 µg/mL streptomycin, and 1% GlutaMAX (Life Technologies) at 37°C in a humidified atmosphere with 5% CO₂. HCT116 cells (DS Pharma Biomedical, Osaka, Japan) were cultured in McCoy’s 5A medium (Life Technologies) supplemented with 10% FBS, 100 U/mL penicillin and 100 µg/mL streptomycin at 37°C in a humidified atmosphere with 5% CO₂.

Confocal Microscopy

Uptake of P4VP-PMG micelles into cells and degradation of micelles by lysosomes was confirmed by confocal microscopy using human cell lines and the fluorescent dyes fluorescein and LysoTracker Red^® (Life Technologies). In brief, HepG2 or HCT116 cells were seeded at a density of 16000 or 20000 cells/well, respectively, in a chamber slide (chamber slide SCS-008, Matsunami Glass Ind., Ltd.) and allowed to adhere overnight. A solution of P4VP-PMG micelles or fluorescein-containing polymeric micelles (P4VP-PMG-fluorescein) diluted 10-fold in serum-free medium were loaded into the wells and incubated for 1–24 h. After removing the supernatant and washing with phosphate buffered saline (PBS) (pH 7.4), LysoTracker Red^® solution was added to a final concentration of 50 nM and incubated for 30 min. The cells were fixed by adding 3.7% formaldehyde and incubating for 2 h. The cells were then washed twice with PBS and observed for fluorescence using an LSM-700 confocal microscope equipped with Zen 2009 image acquisition software (Carl Zeiss, Oberkochen, Germany). Red emission from LysoTracker Red^® excited at 577 nm was captured using a 590-nm filter set, whereas the red emission from fluorescein excited at 495 nm was captured using a 517-nm filter set. Chloroquine pretreatment was performed by adding chloroquine to the cells at a concentration of 20 µM and incubating for 12 h before the addition of micelles.

Cytotoxicity Assay

The cytotoxicity of polymeric micelles incorporating 5-FU (P4VP-PMG-5-FU) was evaluated using a CCK-8 cell counting assay kit (Dojindo, Kumamoto, Japan). HepG2 cells were seeded at a density of 10000 cells/well in a 96-well plate and allowed to adhere overnight. The cells were then incubated with 10 µM 5-FU, P4VP-PMG micelles diluted 10-fold, and a series of diluted P4VP-PMG-5-FU micelles for 1–72 h. The cells were washed twice with PBS, and cell viability was assessed using the tetrazolium salt-based CCK-8 assay. Formation of the colored formazan product was assessed by monitoring the absorption at 450 nm using a Multiskan JX microplate reader (Thermo Fisher Scientific). Cell viability is presented as the mean (±standard deviation (S.D.); n=6) ratio of the viability of treated cells to that of untreated cells. Chloroquine pretreatment was performed by adding chloroquine to the cells at a concentration of 20 µM and incubating for 12 h before the addition of micelles.

Results and Discussion

Characterization of Polymeric Micelles Containing 5-FU

The amphiphilic block copolymer P4VP-PMG was synthesized by mechanochemical block copolymerization of poly-4-vinylpyridine (M_n=815000 g/mol and M_w/M_n=3.36) and 6-O-methacryloyl galactopyranose (Fig. 1). Here, M_n and M_w denote the number average molecular weight and the weight average molecular weight, respectively. The M_n of P4VP-PMG determined by static light-scattering measurements was 100000 g/mol. The molar ratio of P4VP/PMG was 1/4. This ratio was calculated on the assumption that all PMG block was conjugated to the P4VP block. Polymeric micelles containing 5-FU were prepared with P4VP-PMG by dialysis in the presence of various amounts of 5-FU (see Experimental section). Figure 2 shows a plot of the drug load against the weight ratio of 5-FU to that of polymeric micelles. The drug load increased in a parabolic manner as the weight ratio of 5-FU to polymeric micelles increased. As the polymeric micelles exhibited the highest drug load (approximately 50%) under the experimental conditions used, these micelles were used in subsequent experiments. In the previous paper,²⁷⁾ we prepared the P4VP-PMG polymeric micelle of which drug load was about 60%. Although the M_n of the previous P4VP-PMG block copolymer was 56000 g/mol, the particle diameter of the previous polymeric micelle (approximately 200 nm) was greater than that of the present study (vide infra). It was assumed that the difference of the drug load between the previous and present polymeric micelle could be ascribed to the size of particle diameter which might relate to the association number of block polymers.

Fig. 1. Structure of the Amphiphilic Block Copolymer (P4VP-PMG) Synthesized by Mechanochemical Polymerization

Fig. 2. Plot of the Weight Percent of 5-FU in Polymeric Micelles versus the Ratio of 5-FU to P4VP-PMG

Figure 3A shows the change in the number average particle diameter of polymeric micelles containing 5-FU (P4VP-PMG-5-FU) against pH. The average particle diameter at pH 7.4 was approximately 150 nm. It was separately confirmed that these polymeric micelles possessed a narrow particle diameter distribution (weight average particle diameter/number average particle diameter was less than 1.15). The pH of the solution was stepwisely changed to acidic values, so that a sudden decrease in average diameter was observed between pH 6 and 5. At pH 5, the average particle diameter of the P4VP-PMG-5-FU micelles was less than 10 nm, and particles with larger diameters were not observed. It was indicated that the polymeric micelle was completely destroyed. The drug release profile from P4VP-PMG-5-FU at pH 6.0 and 5.0 was also examined (Fig. 3B). The 5-FU contained within the P4VP-PMG-5-FU micelles was completely released within 30 min at pH 5, whereas no release of 5-FU was detected at pH 6.0 up to 5h. These results indicate that pH-induced demicellization leads to the release of 5-FU. These results were similar to those previously reported.²⁷⁾

Fig. 3. Change in Average Particle Diameter of P4VP-PMG-5-FU Micelles against pH (A) and Its Drug Release Profile at pH 6.0 and 5.0 (B)

Cellular Uptake of Polymeric Micelles Containing Fluorescein

Cellular uptake of polymeric micelles was assessed using CLSM and micelles containing the fluorescent dye fluorescein. The results of CLSM analysis of the uptake of P4VP-PMG-fluorescein micelles by HepG2 and HCT116 cells are shown in Fig. 4. LysoTracker Red^® solution was used to label acidic organelles in the live cells.

Fig. 4. Confocal Microscopic Images (×200) of HepG2 (Left) and HCT-116 (Right) Cells Treated with P4VP-PMG Micelles, P4VP-PMG-Fluorescein Micelles, and P4VP-PMG-Fluorescein Micelles with Chloroquine

Fluorescence from fluorescein (green color) was slightly detected after only 1 h in both cell lines incubated with P4VP-PMG-fluorescein micelles. In HepG2 cells, the fluorescein and LysoTracker Red^® (red color) CLSM images overlapped at 1 h and the green fluorescence partially separated from red fluorescence after 3 h of incubation. Similar alteration was observed in HCT116 cells; the separated fluorescein image was detected from 6 h. Strong fluorescence was exhibited after 12 h of incubation in both cell types. In the case of blank (P4VP-PMG) micelles, however, no fluorescence was observed, even after 12 h of incubation. Preincubation with chloroquine, a known inhibitor of lysosomes function, before P4VP-PMG-fluorescein treatment, suppressed the fluorescein-derived fluorescence in both cell lines. These results suggest that fluorescein is released from P4VP-PMG-fluorescein micelles into acidic organelles (endosomes and/or lysosomes) and then diffuses within the cell. The distinct fluorescence was not observed after 24 h of incubation but green fluorescence was remained to detect. This fluorescence might be due to intrinsic fluorescence from cells or leaked fluorescence from LysoTracker Red^®.

Cytotoxicity of Polymeric Micelles Containing 5-FU

The cytotoxicity of P4VP-PMG-5-FU micelles against HepG2 cells was evaluated using a CCK-8 assay. A 1-mL solution of P4VP-PMG-5-FU micelles containing 1 µmol of 5-FU was prepared and then diluted with PBS to obtain various micelle concentrations. A dose-dependent effect of the P4VP-PMG-5-FU micelles on the viability of HepG2 cells was observed after 24 h of incubation (Fig. 5). Blank P4VP-PMG micelles were shown to be nontoxic to HepG2 cells, as evidenced by ca. 130% viability after 24 h of incubation. Although the viability of HepG2 cells incubated with P4VP-PMG-5-FU micelles in the 1/10 to 1/2 dilution range was high, the viability of cells incubated with undiluted P4VP-PMG-5-FU micelles (1/1) clearly decreased, to 57% of the control. The viability of cells incubated with 1 µmol/mL 5-FU was about 80% of the control, suggesting that P4VP-PMG-5-FU micelles are more cytotoxic to HepG2 cells than free 5-FU. The cell viability of the blank P4VP-PMG micelle increased and that of 5-FU decreased. Therefore, these conflicting properties of P4VP-PMG micelle and 5-FU might affect on the cell viability in 1/2 dilution of P4VP-PMG-5-FU micelle. The cell viabilities in 1/3 and 1/2 dilution were 150% and 130%, respectively. This result suggested that the cytotoxicity in 1/2 dilution might be stronger than that in 1/3 dilution, although the cell viability in 1/2 dilution increased against blank. It was supposed that longer treatment time in 1/2 dilution of P4VP-PMG-5-FU micelles could give the lower cell viability.

Fig. 5. Viability of HepG2 Cells Treated with Various Dilutions of P4VP-PMG-5-FU Micelles

Data are presented as the mean±S.D. (n=6). * p<0.05, compared with blank group (one-way ANOVA with Tukey’s test).

Undiluted P4VP-PMG-5-FU micelles were used to investigate the effect of micelles on the viability of HepG2 cells over time (Fig. 6). Although no decrease in the viability of HepG2 cells was observed up to 6 h of incubation, the viability of cells incubated with P4VP-PMG-5-FU micelles for 12, 24, and 72 h decreased to 87, 42, and 33% of the control, respectively. Pretreatment of HepG2 cells with chloroquine suppressed the cytotoxic effect of the micelles, suggesting that chloroquine inhibits the release of 5-FU from P4VP-PMG-5-FU micelles by suppressing demicellization.

Fig. 6. Viability of HepG2 Cells over Time When Treated with P4VP-PMG-5-FU Micelles

Data are presented as the mean±S.D. * p<0.05, compared with blank group (one-way ANOVA with Tukey’s test).

Conclusion

Polymeric micelles containing 5-FU (P4VP-PMG-5-FU) were prepared via dialysis with amphiphilic block copolymer (P4VP-PMG) and 5-FU. The drug load increased in a parabolic manner as the weight ratio of 5-FU to polymeric micelles increased. Complete release of 5-FU contained within P4VP-PMG-5-FU micelles occurred within 30 min at pH 5, whereas no 5-FU was released from micelles at pH 7.4. Fluorescent dye-containing polymeric micelles (P4VP-PMG-fluorescein) were used to evaluate cellular uptake. The fluorescein contained within the P4VP-PMG-fluorescein micelles was released into acidic organelles (endosome and/or lysosome) and then diffused within the cell. The cytotoxicity of P4VP-PMG-5-FU micelles against HepG2 cells was evaluated using a CCK-8 assay. The results suggest that P4VP-PMG-5-FU micelles are more cytotoxic to HepG2 cells than free 5-FU. We are currently investigating other types of stimulus-responsive polymeric micelles.

Conflict of Interest

The authors declare no conflict of interest.

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