Synthesis and characterization of pluronic-block-poly(N,N-dimethylamino-2-ethyl methacrylate) pentablock copolymers for drug/gene co-delivery systems

Abstract

We synthesized three pluronic-based cationic pentablock copolymers with different hydrophilic/lipophilic balance (HLB) values using atom transfer radical polymerization (ATRP), including PF127-block-poly(N,N-dimethylamino-2-ethyl methacrylate) (PF127-b-pDMAEMA), pluronic P123-block-poly(N,N-dimethylamino-2-ethyl methacrylate) (PP123-b-pDMAEMA), and PL121-block-poly(N,N-dimethylamino-2-ethyl methacrylate) (PL121-b-pDMAEMA). The copolymers self-assembled into core–shell structures, which could be used to co-deliver a plasmid DNA (pEGFP) and a hydrophobic drug (epirubicin, EPI). The physicochemical properties of the copolymers and drug-loaded micelles were thoroughly characterized. The micelles had a high EPI encapsulation efficiency, ∼6%, and the EPI-loaded micelles exhibited a similarly cytotoxic effect to free EPI. Among the three copolymers, the gene transfection efficiency of PL121-b-pDMAEMA was the highest, indicating that the greater the hydrophobic effect the greater cellular internalization was. The co-delivery effect of pEGFP and EPI was directly visualized using a confocal laser scanning microscope. Thus, the pluronic-b-pDMAEMA micelles are a promising co-delivery system for therapeutic pDNA and hydrophobic anticancer drugs.

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Introduction

The rapid development of amphiphilic copolymers is mainly due to their promising characteristics in drug delivery systems, including micelles,¹ nanogels,² and polymersomes.³ Among them, micelles have smaller hydrodynamic dimensions, usually spherical core–shell construction, in which the hydrophobic core provides a depot for accommodation of hydrophobic drugs and the hydrophilic shell maintains water solubility. Although the micelles have many advantages for biomedical applications,⁴ the advancement of micellar drug formulations for the clinic has been challenging. Major barriers include difficulty in transport through the cell membrane and optimum drug delivery necessary for therapeutic effect.⁵ Therefore, novel amphiphilic copolymers were designed with not only various hydrophobic and hydrophilic segments, but also multiple functions.

Recently, amphiphilic copolymers containing cationic segments have been extensively used as non-viral gene carriers including polyethylenimine (PEI),^6,7 poly(N,N-dimethylamino-2-ethyl methacrylate) (PDMAEMA),^8,9 poly(lysine) (PLL),¹⁰ and polyamidoamine (PAAM).¹¹ The cationic amphiphilic copolymers self-assembled into dispersive and stable micelles with a cationic shell and a hydrophobic core. These novel carriers condensed gene drugs via electrostatic interactions and loaded hydrophobic drugs in the hydrophobic core.

Due to the molecular complexity of cancer, the combination therapy of chemo and gene drugs becomes important for better long-term prognosis with fewer side effects.¹² To further increase therapeutic effects, advanced drug delivery systems (DDSs), capable of simultaneously delivering drugs and genes to the site of action with specific time-programmed release profiles, are important for drug development. Nanocarriers based on cationic amphiphilic micelles for the simultaneous co-delivery of drugs and genes in combination therapy have been studied. Biodegradable cationic micelles were prepared from PDMAEMA–PCL–PDMAEMA triblock copolymers and applied for the delivery of siRNA and paclitaxel (PTX) into cancer cells. The PTX-loaded PDMAEMA–PCL–PDMAEMA displayed higher drug efficacy than free PTX and the co-delivery of VEGF siRNA and PTX showed an efficient knockdown of VEGF expression in PC3 cells.¹³ A co-delivery system was prepared from pluronic85-PEI/D-α-Tocopheryl polyethylene glycol1000 succinate (TPGS)/PTX/survivin shRNA (shSur) complex nanoparticles (PTPNs). The experimental results showed PTPNs could facilitate drug entry into cells and induce shSur into nuclei in both A549 and A549/T cells.¹⁴ Three amphiphilic star-branched copolymers comprising polylactic acid (PLA) and PDMAEMA with AB₃, (AB₃)₂, and (AB₃)₃ molecular architectures were synthesized to co-deliver microRNA and doxorubicin (DOX). The (AB₃)₃ architecture micelles exhibited the highest transfection efficiency.⁸ When delivering DOX and miR-21 inhibitor (miR-21i) into LN229 glioma cells, the micelles could mediate escaping miR-21i from lysosome degradation and the release of DOX into the nucleus.

Co-delivery of chemo and gene drugs has a potential to efficaciously treat human diseases because of their synergetic effects. Construction of a highly efficient multifunctional drug carrier combining chemotherapy and gene therapy attracts many researchers' attention. Pluronic copolymers consist of hydrophobic poly(propylene) (PPO) segments and hydrophilic poly(ethylene oxide) (PEO) segments. They self-assemble into micelles in water with a hydrophobic core by PPO and a hydrophilic shell by PEO.¹⁵ Pluronic copolymers were used to modify cationic polymers like PEI,¹⁶ PLL,¹⁷ and PDMAEMA¹⁸ to improve gene transfection as well as to reduce their intrinsic cytotoxicity. The presence of the hydrophobic PPO block in the pluronic-modified PDMAEMA enhanced the association with DNA.¹⁸ Moreover, pluronic polymers promote cellular uptake of polyplexes because they inhibit P-glycoprotein (Pgp) to overcome multidrug resistance (MDR), subsequently enhancing transgene expression.¹⁹

PDMAEMA is a well-known cationic polymer, easily condensing nucleic acids and efficiently transfecting them. The high transfection efficiency is attributable to the positive charge of PDMAEMA forming a complex with nucleic acids and facilitating a proton sponge effect. Compared with other cationic polymers, PDMAEMA's macromolecular architecture is the easiest to manipulate, including star, liner, graft, and block.²⁰

In our previous work, we found PF127 modified PDMAEMA (PF127-b-pDMAEMA) reduced the cytotoxicity of PDMAEMA as a gene vector and its transfection efficiency mainly depended on the PDMAEMA chain length.²¹ The higher block length of PDMAEMA indeed showed higher transfection efficiency but resulted in higher cytotoxicity as well. Since no one has used pluronic-b-pDMAEMA to co-deliver chemo and gene drugs and compared their performance using various pluronic polymers with different hydrophilic/lipophilic balance (HBL) values. In this study, we synthesized a series of pentablock copolymers consisting of pluronic polymers and a small length of PDMAEMA (∼35) to minimize the cytotoxic problem. We selected three pluronic polymers, PF127, PP123, and PL121, with different lengths of PEO and similar lengths of PPO. The effect of the pluronic polymers used in preparation of pentablock copolymers on particle size, drug encapsulation, release behavior, cell viability, and gene expression were thoroughly studied.

The pentablock copolymers were characterized using Fourier transform infrared (FTIR) spectroscopy and ¹H-nuclear magnetic resonance (¹H-NMR) spectroscopy. Hydrodynamic diameters and zeta potentials were measured using dynamic light scattering (DLS). Morphologies were obtained using a transmission electron microscope (TEM). To test the potency of pentablock copolymers as gene carriers, the gene transfection efficiencies of polyplexes were studied using 293T cells. To examine the potency of pentablock copolymers as drug carriers, the cell viabilities of free epirubicin (EPI) and EPI-loaded micelles were studied using 293T and KB cells. The co-delivery effect of EPI and pDNA was visualized using a confocal laser scanning microscope (CLSM).

Experimental

Materials

Pluronic F127, copper(I) bromide (CuBr), 3-(4,5-dimethyl-thiazol-2yl)-2,5-diphenyl-tetrazolium bromide (MTT), deuterium oxide (D₂O) and deuterium chloroform (CDCl₃) were purchased from Sigma (St. Louis, MO). Epirubicin hydrochloride was purchased from Zhejiang Hisun Pharmaceutical (Zhejiang, China). Pluronic L121 and P123 were purchased from BASF (Ludwigshafen, Germany). 2,2′-Bipyridine (Bpy), 2-bromo-2-methylpropionyl bromide, Amberlite^® IR120, N,N-dimethylamino-2-ethyl methacrylate (DMAEMA) and pyrene were purchased from Acros (Morris Plains, NJ). Aluminum oxide neutral (Al₂O₃) was obtained from Seedchem Company PTY. LTD (Melbourne, Australia). Fetal bovine serum (FBS) was purchased from Biological Industries (Beit Haemek, Israel). Roswell park memorial institute medium (RPMI-1640) was purchased from Invitrogen (Carlsbad, CA).

Preparation of macroinitiator (pluronic-Br)

In a two-neck round-bottom flask, each pluronic polymer (1 mmol) was dissolved in 20 mL dichloromethane at room temperature. The solution was cooled to 0 °C and triethylamine (5 mmol) was added with stirring. After 20 min, 2-bromoisobutryl bromide (5 mmol) was slowly added with a syringe that had been purged with argon. Following 48 h reaction at room temperature, the product was obtained by precipitation in excess n-hexane and dried under vacuum. The yield of PF127-Br, PP123-Br and PL121-Br were approximately 70.3, 42.8 and 25.6%, respectively.

Preparation of pentablock copolymers

Pentablock copolymers (pluronic-b-pDMAEMA) were synthesized using a molar feed ratio of [DMAEMA] : [macroinitiator] :[CuBr] : [Bpy] at 40 : 1 : 2 : 2. Briefly, macroinitiator (1 mmol), DMAEMA (40 mmol), 2-propanol (1.6 mL), and double-deionized (DD) water (0.4 mL) were added to a two-neck round-bottom flask. The reaction was degassed by five consecutive standard freeze–pump–thaw cycles. CuBr (2 mmol) and Bpy (2 mmol) were quickly added to the mixture under an argon atmosphere. Following 4 h reaction at room temperature, the reaction was stopped by diluting with DD water. The product was purified by dialysis against DD water using M_w cut-off 3500 membrane (Spectrum Labs, Rancho Dominguez, CA) for 2 days. The freeze-dried product was dissolved in toluene and passed through aluminum oxide column and Amberlite^® IR120 to remove catalyst complexes. The purified solution was further precipitated in excess n-hexane and dried under vacuum.

Characterization of copolymers

The chemical structure of copolymers was determined using proton nuclear magnetic resonance (¹H-NMR) and Fourier-transform infrared (FTIR) spectroscopy. ¹H-NMR spectra were obtained from a Varian Mercury plus-200 spectrometer (Palo Alto, CA), using D₂O and CDCl₃ as a solvent. FTIR spectra were acquired using a Perkin-Elmer System 2000 (Norwalk, CT). The molecular weights of copolymers were measured by gel permeation chromatography (GPC) using an Agilent 1100 series (Santa Clara, CA) equipped with a Shodex-KF804 column. Samples were dissolved in tetrahydrofuran (THF) at a concentration of 5 mg mL⁻¹ and filtered through a 0.45 μm filter prior to injection. THF was used as an eluent at a flow rate of 1 mL min⁻¹. Polystyrene standards were used to generate a calibration curve.

Acid-base titration was carried out using a PC-controlled system assembled with a 702 SM Titroprocessor, a 728 stirrer, and a PT-100 combination pH electrode (Metrohm, Herisau, Switzerland). Approximately 20 mg of each copolymer was dissolved in 20 mL of 150 mM NaCl solution. The pH value of the solution was adjusted to 2 using 0.1023 N HCl followed by back-titration to pH 11 using 0.0998 N NaOH.

Preparation and characterization of micelles

Micelles were prepared using a precipitation/solvent evaporation technique. Each pentablock copolymer (5 mg) in THF (200 μL) was dropwise added into DD water (5 mL). The solution was sonicated for 3 min. The organic THF solvent was removed by rotary vacuum evaporation and the rest was freeze-dried. Fluorescence spectra were recorded on a Cary Eclipse fluorescence spectrophotometer (Cary, Varian, CA). Pyrene was used as a fluorescence probe.²² Pyrene excitation spectra were recorded using an emission wavelength at 390 nm. Emission and excitation slit widths were set at 2.5 and 2.5 nm, respectively. A critical micelle concentration (CMC) value was determined from the ratios of pyrene intensities at 339 and 334 nm and calculated from the intersection of two tangent plots of I₃₃₉/I₃₃₄ versus the concentrations of copolymers.

Epirubicin encapsulation (EPI-loaded micelle) and in vitro release

In this preparation, EPI HCl was solubilized in THF at a concentration of 1 mg mL⁻¹, containing 3 molar ratio of triethylamine relative to EPI.²³ To encapsulate epirubicin (EPI), THF (500 μL) containing a pentablock copolymer (5 mg) and EPI (0.5 mg) was dropwise added to DD water (5 mL). The mixture was sonicated for 3 min. THF was removed by rotary vacuum evaporation and a 0.45 μm microporous filter was used to eliminate any unencapsulated EPI. The obtained micellar solution was lyophilized and properly stored before further characterization. To evaluate EPI loading efficiency, a dried sample was dissolved in THF and the concentration of EPI was measured using a fluorescence spectrometer at 590 nm. The EPI amount was calculated based on a standard calibration curve in EPI concentrations ranging of 0.8–12.5 μg mL⁻¹. The loading efficiency (LE) and encapsulation efficiency (EE) were calculated using the following equations:

EE (%) = (amount of drug in micelle/amount of drug in feed) × 100 (1)

LE (%) = {amount of drug in micelle/(amount of polymer + amount of drug in micelle)} × 100 (2)

The in vitro release of EPI was carried out in 0.1 M phosphate-buffered saline (PBS) buffers at pH 7.4. The EPI-loaded micelle (30 mg) was placed in a dialysis membrane with MWCO of 3500 Da. The tube was then immersed in a beaker containing 30 mL of PBS, which was shaken at a speed of 150 rpm and incubated at 37 °C. At predetermined time intervals, 2 mL of solution was withdrawn and the EPI content was determined using a UV-vis spectrophotometer at 480 nm. The same amount of fresh PBS was replaced back to the beaker. The released EPI amount was calculated from a standard calibration curve of pure EPI in the range of 2–40 μg mL⁻¹.

Preparing plasmid DNA

pEGFP-C₁ plasmid driven by a cytomegalovirus (CMV) promoter and pGL3-control plasmid with a Hind III/Xba I firefly luciferase cDNA fragment cloned into the pCDNA vector were introduced into the E. coli strain DH5α (Gibco-BRL, Gaithersbury, MD) and purified using a kit (Maxi-V500 plasmid kit; Viogene, Sunnyvale, CA). The purity of the pDNA was certified by the absorbance ratio at OD₂₆₀/OD₂₈₀ and by distinctive bands of DNA fragments at corresponding base pairs in gel electrophoresis after restriction enzyme treatment of DNA. The pDNA was stored at −20 °C until used.

Preparing copolymer/pDNA polyplexes

All copolymer stock solutions were prepared at 2 mg mL⁻¹ in DD water and the pH was adjusted to 5. The pDNA concentration was fixed at 3 μg/100 μL in DD water to measure pDNA binding assay and 1 μg/100 μL for other studies. Equal volumes of pDNA and copolymer solution with different N/P ratios ranging of 1–20 were immediately vortexed at a high speed for 60 s. Polyplexes were kept at room temperature for 10 min for complete complexation before analysis.

Characterization of nanoparticles

In this experiment, micelles were solubilized in DD water at a concentration of 1 mg mL⁻¹. The hydrodynamic diameters and zeta potentials of nanoparticles were measured using laser Doppler anemometry with a Zetasizer Nano ZS instrument (Marlvern Instruments, Worcestershire, UK). Light scattering measurements were done with a laser at 633 nm and a 90° scattering angle. Particle sizes and zeta potentials were measured three times. The morphologies of micelles (or polyplexes) were observed using TEM (JEM-2000 EXII, JEOL, Japan). A carbon coated 200 mesh copper specimen grid (Agar Scientific Ltd. Essex, UK) was glow-discharged for 1.5 min. The solution of micelles or polyplexes at N/P = 9 was placed on the copper grid and allowed to dry for 5 days at room temperature. The images of samples were analyzed using Image J software (NIH, Bethesda, MD).

Gel retardation assay

The DNA binding ability of polyplexes was evaluated using an agarose gel electrophoresis. The stability of copolymer/pDNA polyplexes with and without 10% FBS was evaluated using a gel electrophoresis with 0.8% agarose in Tris acetate–EDTA (TAE) with EtBr (1 μg mL⁻¹). A current of 100 V was applied to the gels for 35 min, and DNA retention was visualized under ultraviolet illumination at 365 nm.

Cell experiments

HEK 293T cells (human embryonic kidney 293T cell line) and KB cells (human nasopharyngeal epidermoid carcinoma cell line) were cultivated at 37 °C under humidified 5% CO₂ in DMEM and RPMI-1640, supplemented with 10% FBS and 100 μg mL⁻¹ penicillin-streptomycin. The medium was replenished every three days and the cells were sub-cultured after they had reached confluence.

Cytotoxicity

The cytotoxicities of copolymers were measured using the MTT assay in 293T and KB cells. 293T cells or KB cells (5 × 10³ cells per well) were seeded in 96-well tissue culture plates at 37 °C under 5% CO₂ for 24 h in DMEM and RPMI-1640 medium containing 10% FBS. The cytotoxicity was evaluated by determining the viability after 24 h incubation with various concentrations of copolymers (2.5–100 μg of copolymer per milliliter).

The cytotoxicities of free EPI and EPI-loaded micelles were measured using the MTT assay in KB cells. The cells were seeded in 96-well tissue culture plates at a density of 5 × 10³ cells per well in RPMI-1640 medium containing 10% FBS at 37 °C under 5% CO₂ for 24 h. The equivalent EPI concentrations of EPI-loaded micelles were controlled within 0.43–9.28 μM. After 24 h, the cells were washed trice with PBS and incubated for another 48 h. The number of viable cells was measured by the estimation of their mitochondrial reductase activity using the tetrazolium-based colorimetric method. The half maximal inhibitory concentration (IC₅₀) was analyzed using SigmaPlot software.

In vitro transfection

The transfection assay was evaluated using a pGL-3 plasmid in 293T cells. The transfection efficiencies of polyplexes were compared with those of naked DNA as a negative control, Lipofectamine 2000 (LIPO, Invitrogen), and branched PEI (25 kDa, N/P = 10) as positive controls. The 293T cells were seeded at a density of 1 × 10⁵ cells per well in 12-well tissue culture plates and incubated in DMEM medium containing 10% FBS for 24 h before transfection. When the cells were at 50–70% confluence, the culture medium was replaced with 1 mL of DMEM with or without 10% FBS. Polyplexes with N/P ratios ranging of 1–20 were prepared using different amounts of copolymers and a fixed pDNA amount of 1 μg to a final volume of 100 μL. After left to stand for 10 min, polyplexes were added to each well containing the cells and incubated for 4 h. The medium was replaced with 1 mL of fresh DMEM and the cells were incubated for 44 h post-transfection. The transfected cells were rinsed gently with 1 mL of 0.1 M PBS (twice) and added to a 200 μL per well of lysis buffer (0.1 M Tris–HCl, 2 mM EDTA, and 0.1% Triton X-100, pH 7.8). The luciferase activity was monitored using a microplate scintillation and luminescence counter after mixing the contents of a 50 μL per well of supernatant with the contents of 50 μL per well of luciferase assay reagent (Promega). The total protein content of the cell lysate was examined using a BCA protein assay kit (Pierce, Rockford, IL).

Confocal laser scanning microscopy (CLSM)

The cells were seeded at a density of 1 × 10⁵ cells per well in 12-well plates containing one glass coverslip per well in RPMI-1640 supplemented with 10% FBS and incubated for 24 h. Subsequently, the culture medium was replaced with 1 mL of RPMI-1640 without 10% FBS. The EPI-loaded micelle (PL121-b-pDMAEMA)/pDNA polyplex was added and incubated for 4 h. The medium was replaced with 1 mL of fresh RPMI-1640 and the cells were incubated for 24 h post-transfection. The coverslips were removed and washed three times with PBS. The cell nuclei were stained using Hoechst 33342 (5 μg mL⁻¹) for 15 min. Next, the cells were fixed with 3.7% paraformaldehyde for 30 min and the cells on the coverslips were washed 3 times with PBS and mounted with fluorescent mounting medium on glass slides. A CLSM (Fv 1000; Olympus, Tokyo, Japan) was used for cell imaging.

Statistical analysis

Means and standard deviations (SD) of data were calculated. Comparison between groups was tested using Student's t-test and P < 0.05 was considered as significant.

Results and discussion

Preparing and characterizing copolymers

In this work, an ATRP technique was applied to obtain pentablock copolymers. The central triblock copolymers were pluronic F127, P123 and L121, which were reacted with 2-bromoisobutyl bromide to form ATRP macroinitiators followed by copolymerization with DMAEMA. The chemical structure of PF127-Br was confirmed from the ¹H-NMR spectrum at the peak intensity of 1.92 ppm (b, C(CH₃)₂–Br of 2-bromo-2-methylpropionyl groups) and 1.18 ppm (a, methyl protons of the PPO block) (Fig. 1a). The degree of halogenation was determined to be ∼95%, indicating the terminal hydroxyl groups were approximately substituted. The PF127-Br was further characterized using GPC. The number-averaged molecular weight (M_n) and polydispersity index (PDI) of PF127-Br were ∼13 000 g mol⁻¹ and 1.27 (data not shown). The synthesis of a series of pluronic macroinitiators was well-controlled.

Fig. 1 (a) ¹H-NMR and (b) FTIR spectra of PF127, PF127-Br and PF127-b-pDMAEMA.

The pentablock copolymers composed of DMAEMA were further prepared via ATRP using pluronic-Br as a central block (Scheme 1). Fig. 1b shows the ¹H-NMR spectrum of PF127-b-pDMAEMA. The chemical shifts in the region of 2.25–2.63 ppm were mainly associated with the methyl (b, N–CH₃) and methylene (c, N–CH₂) protons of the DMAEMA segments. The chemical shift at 4.12 ppm was attributed to the methylene protons adjacent to the oxygen moieties of ester linkages (d, H₂C–O–C=O). The number of repeating units of DMAEMA in copolymers could be determined from ¹H-NMR spectra. The integration areas of the methyl (–CH₃) protons at 1.18 ppm (peak a) of the pluronic segments and the methyl (N–CH₃) protons of the DMAEMA segments at 2.25 ppm (peak b) were used to calculate the final copolymer composition. The number of the DMAEMA segment of the three copolymers was ∼35 units (Table 1). This value could be correlated to the monomer/macroinitiator ratio in feed. The ¹H-NMR spectra of PP123-b-pDMAEMA and PL121-b-pDMAEMA were shown in ESI Fig. S1.†

Scheme 1 Synthesis of pluronic-b-pDMAEMA pentablock copolymers.

Table 1 Molecular weights and properties of copolymers

Sample	Mn,theo^a (g mol^−1)	Mn^b (g mol^−1)	DP^c (DMAEMA)	DP^d (DMAEMA)	DP^e (EO)	DP^f (PO)	HLB^d	PDI^e	Conversion^g	Yield (%)
a Theoretical Mn calculated as (target DPn × 157 g mol^−1) × actual fractional conversion achieved and includes initiator residue.b The number-averaged molecular weight (Mn) of copolymers was done by GPC.c The number of DMAEMA per copolymer chain was estimated by ^1H-NMR.d The number of DMAEMA per copolymer chain was estimated by GPC.e The number of EO and PO segments and Hydrophilic/Lipophilic Balance (HLB) were obtained from ref. 15.f PDI = Mw/Mn measured by GPC.g The conversion was estimated by ^1H-NMR.
PF127-b-pDMAEMA	17 800	14 370	33	23	200	65	22	1.22	0.83	53.2
PP123-b-pDMAEMA	12 080	7150	34	22	40	70	8	1.20	0.85	39.2
PL121-b-pDMAEMA	10 680	6000	38	23	10	68	1	1.27	0.95	12.4

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The FTIR spectrum of PF127-Br peaked at 1726 cm⁻¹, which was attributable to characteristic C=O stretching. In addition, the enhanced stretching bands of PDMAEMA at 1726 cm⁻¹ (C=O), 2767 cm⁻¹ and 2819 cm⁻¹ (C=N), implied the successful polymerization of the pentablock copolymer (Fig. 1c). The FTIR spectra of PP123-b-pDMAEMA and PL121-b-pDMAEMA were shown in ESI Fig. S2.† The molecular weights and their corresponding MW distribution were measured by GPC and listed in Table 1. The number of the DMAEMA segments estimated from the MW measurements was ∼23 units, which was lower than the value calculated by NMR. This might be due to the fact that the MW measurement by GPC is relative to polystyrene standards, where its chemical structure is quite different from that of the pentablock copolymers. The narrow polydispersity (PDI) of 1.20–1.30 suggested the polymerization kinetics of DMAEMA with pluronic-Br was successfully controlled by ATRP.²⁴

Fig. 2 shows the titration profiles of PF127-b-pDMAEMA, PP123-b-pDMAEMA, and PL121-b-pDMAEMA. The copolymers showed a similar buffer capacity. The apparent pK_a values of the amino groups were 7.29, 7.33, and 7.30, for PF127-b-pDMAEMA, PP123-b-pDMAEMA, and PL121-b-pDMAEMA, respectively. These values were close to that reported on pluronic L92-pDMAEMA (pK_a = 7.1)¹⁸ and independent of the central pluronic polymers used.

Fig. 2 Titration profiles of PF127-b-pDMAEMA, PP123-b-pDMAEMA and PL121-b-pDMAEMA.

Micellar properties

The amphiphilic copolymers of pluronic-b-pDMAEMA, consisting of the hydrophilic PDMAEMA and PEO segments and the hydrophobic PPO segments, provided an opportunity to examine their self-assembly behavior in aqueous solutions (Scheme 2). As listed in Table 1, the numbers of the PPO block were 65, 70, and 68, and those of the PEO block were 200, 40, and 10 for PF127, PP123, and PL121, respectively. To measure the self-assembled ability of copolymers, the CMC values of micelles were determined using pyrene as a fluorescence probe (Fig. 3a). The CMC values were 4.99 × 10⁻², 3.64 × 10⁻², and 3.54 × 10⁻² mg mL⁻¹ for PF127-b-pDMAEMA, PP123-b-pDMAEMA, and PL121-b-pDMAEMA, respectively. Compared the values with PF127, PP123, and PL121 of 3.53 × 10⁻², 2.53 × 10⁻², and 4.40 × 10⁻³ mg mL⁻¹,¹⁵ the introduction of PDMAEMA to the pluronic polymers increased water-solubility,²⁵ leading to an increase in CMC value. In addition, the CMC values of the copolymers showed the same trend as their nascent pluronic polymers, showing the higher the hydrophobicity the smaller the CMC value was.

Scheme 2 Preparation of pluronic-b-pDMAEMA micelle to co-deliver hydrophobic drugs and gene drugs.

Fig. 3 (a) Critical micelle concentration measurements of pluronic-b-pDMAEMA; transmission electron microscope (TEM) images and dynamic light scattering (DLS) diagrams of PF127-b-pDMAEMA micelle (b) without EPI and (c) with EPI.

The hydrodynamic diameters of micelles were measured by DLS and summarized in Table 2. The hydrodynamic sizes of micelles decreased as increasing the PEO block in a copolymer. The sizes were also estimated from TEM images, ∼175 nm, 180 nm, and 250 nm for PF127-b-pDMAEMA, PP123-b-pDMAEMA, and PL121-b-pDMAEMA, respectively. These particle sizes were smaller than the values measured by DLS because of the drying effect. The average core diameters of PF127-b-pDMAEMA (Fig. 3b), PP123-b-pDMAEMA (ESI Fig. S3a†), and PL121-b-pDMAEMA (ESI Fig. S3c†) were in the order of ∼80, 112, and 188 nm, respectively. The sizes strongly depend on the ratio of hydrophilic and hydrophobic segments in the backbone. The largest HLB value of 22 for PF127 forced the self-assembly of the hydrophobic PPO core the easiest, resulting in the smallest particle size with the clearest corona layer.¹⁵ In contrast, the lowest HLB value of 1 for PL121 formed the biggest core area. The zeta potential values of the three micelles were similar, ranging from 18.7 to 21.5 mV, due to the analogous number of cationic DMAEMA segments.

Table 2 Hydrodynamic diameters, zeta potentials, and EPI encapsulation of micelles^a

Sample	Dh (nm)	PDI	Zeta (mV)	E.E (%)	L.E (%)
a E.E: encapsulation efficiency; L.E: loading efficiency.
PF127-b-pDMAEMA	206.9 ± 9.5	0.29 ± 0.02	19.1 ± 0.5	68.1 ± 11.3	6.2 ± 1.0
PP123-b-pDMAEMA	271.0 ± 30.0	0.33 ± 0.02	21.5 ± 0.6	78.7 ± 10.6	7.2 ± 0.9
PL121-b-pDMAEMA	350.9 ± 24.9	0.32 ± 0.03	18.7 ± 0.7	71.8 ± 5.7	6.8 ± 0.5

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Drug encapsulation and release

EPI was encapsulated via a precipitation/solvent evaporation method. All three micelles showed a similar loading capacity of ∼70% (Table 2). As seen in TEM images, all three drug-loaded micelles showed a spherical shape with a nice size distribution. The sizes of the EPI-loaded PF127-b-pDMAEMA (∼330 nm, Fig. 3c) and EPI-loaded PP123-b-pDMAEMA micelles (∼355 nm, ESI Fig. S3b†) increased dramatically as compared with those without EPI (Fig. 3b and ESI Fig. S3a†). In the highest hydrophobic PL121-b-pDMAEMA, both the DLS and TEM results showed the size decreased after the drug had been loaded (compared the image of ESI Fig. S3d† with that of S3c). The size of PL121-b-pDMAEMA decreased from 250 nm to 200 nm when EPI was encapsulated. This might be explained by the enhanced hydrophobic effect in the drug and the core.²⁶ In addition, the hydrophilic corona layer of the micelles faded when the drug was loaded (see Fig. 3c, ESI Fig. S3b and S3d†) because of the high electron density of the drug.

Fig. 4 exhibits the in vitro drug release profiles of EPI-loaded micelles. The initial burst release of EPI might be attributed to that EPI molecules located within the corona or at the interface between the corona and core. Such EPI does not need to diffuse through the large segments of the core and the drug release rate was rapid. Following the burst release, EPI was continually released from the EPI-loaded micelles over 50 h without a lag time. Interestingly, the release rate of PP123-b-pDMAEMA was slightly faster than those of PF127-b-pDMAEMA and PL121-b-pDMAEMA. This result implied the PL121-b-pDMAEMA with the lowest HLB enhanced the interactions between EPI and the hydrophobic PPO core and sustained the drug release. On the other hand, the PF127-b-pDMAEMA had the highest HLB, leading to a difficulty in drug diffusion because of a thicker hydrophilic layer on the surface.²⁷ Hence, choosing a pluronic polymer with a suitable HLB is possible to facilitate a controllable drug release rate.

Fig. 4 In vitro EPI released profiles of EPI-loaded micelles done in 0.1 M PBS at pH = 7.4 at 37 °C (n = 3).

Cytotoxicity

The in vitro cytotoxicities of micelles were measured using an MTT method in KB (Fig. 5a) and 293T cells (Fig. 5b). The cell viability was dose dependent in both the KB and 293T cells. The cell viability dramatically dropped at a concentration of >30 μg mL⁻¹ in KB cells but gradually decreased in 293T cells. The impact of different pluronic polymers on cell viability was trivial at a low concentration. However, at a high concentration of 100 μg mL⁻¹, it seems PF127-b-pDMAEMA had higher cell viability than other two pentablock copolymers.

Fig. 5 Relative cell viabilities of (a) KB and (b) 293T cells exposed to pluronic-b-pDMAEMA copolymers for 24 h (n = 8); (c) relative cell viabilities of KB cells exposed to free EPI or EPI-loaded pluronic-b-pDMAEMA micelles at various EPI concentrations for 24 h followed by 48 h post incubation (n = 8).

To examine the cell viability of drug-loaded micelles, the cells were exposed to free EPI or the EPI-loaded micelles at various concentrations for 48 h (Fig. 5c). The IC₅₀ values were 0.20, 0.45, 0.79, and 0.55 μg mL⁻¹ for free EPI, PF127-b-pDMAEMA/EPI, PP123-b-pDMAEMA/EPI, and PL121-b-pDMAEMA/EPI, respectively. Free EPI exhibited the best cytotoxic effect as compared with the EPI-loaded micelles because of it having the most rapid diffusion of drug inside the cells to perform its cell-inhibiting effect. No significance was observed among the EPI-loaded micelles with different pluronic polymers in the cytotoxic study.

Characterization of copolymer/pDNA polyplexes

The gel retardation assay was performed to examine the DNA binding ability of copolymers and pDNA at various N/P ratios. Since no exposed pDNA was stained by EtBr at every N/P ratio ranging of 1 −12, PF127-b-pDMAEMA, PP123-b-pDMAEMA, and PL121-b-pDMAEMA had excellent binding ability with pDNA both with (Fig. 6b) and without 10% FBS (Fig. 6a).

Fig. 6 Gel electrophoresis to test pDNA retention in copolymers/pDNA polyplexes at various N/P ratios (a) without 10% FBS, and (b) with 10% FBS. Naked pDNA was used as a reference, and numerals of each graph indicate an N/P ratio.

Three copolymers/pDNA polyplexes showed stable hydrodynamic diameters at an N/P of ≥6 and their hydrodynamic diameters were ∼100 nm (ESI Fig. S4a†). The polyplexes showed positive charges with zeta potentials of 7–14 mV (ESI Fig. S4b†), slightly increasing with an increase in N/P ratio. The copolymers/pDNA polyplexes at N/P = 9 formed spheroid and their sizes were approximately 95 nm, 102 nm, and 108 nm for PF127-b-pDMAEMA, PP123-b-pDMAEMA, and PL121-b-pDMAEMA/pDNA, respectively measured by TEM (ESI Fig. S5†). The strong electrostatic interactions between the positively-charged pluronic-b-pDMAEMA and the negatively-charged pDNA magnificently reduced the particle size of the polyplexes. In addition, pluronic polymers assisted the polyplexes to form a core–shell structure, in which the core of PPO segments could stabilize PDMAEMA/pDNA polyplexes.¹⁸ Thus, a micelle-based gene delivery system could facilitate cellular uptake and enhance stability, leading to improved gene expression.²⁸

Cytotoxicity and in vitro gene transfection of polyplexes

The gene transfection efficiencies and cytotoxicities of copolymers/pDNA polyplexes at various N/P ratios were tested and compared with those of LIPO/pDNA, PEI (25K)/pDNA at N/P = 10, and naked pDNA in 293T cells. In Fig. 7, polyplexes exhibited higher cell viability than LIPO/pDNA and PEI/pDNA. As the N/P ratio increased, cell viability gradually decreased. At an N/P ratio of ≥15, the highest hydrophilic PF127-b-pDMAEMA/pDNA showed the lowest cytotoxicity as compared with PP123-b-pDMAEMA/pDNA and PL121-b-pDMAEMA/pDNA. This result agreed with the previous study using the copolymers alone (Fig. 5). Thomas et al.²⁹ studied the cytotoxicity of unalkylated and alkylated PEIs, and found the alkylated PEIs displayed no or reduced cytotoxicity, compared with the parent PEI. Tian et al.³⁰ also reported reduced cytotoxicity of PEI after introducing a biocompatible hydrophobic poly(γ-benzyl-L-glutamate) moiety. In contrast, some authors have reported negative effects of hydrophobic modifications in comparison with parent polymers.^31,32 Thus, the hydrophobic effect of polycations on cytotoxicity remains controversial.

Fig. 7 Relative cell viabilities of copolymers and their copolymers/pDNA polyplexes in 293T cells as a function of polyplexes at various N/P ratios (n = 3, *P < 0.05). The N/P ratio of PEI (25K)/pDNA was 10.

The transfection ability of copolymers was assayed by pGL3-control plasmid for luminescence measurement and conducted in with and without 10% FBS culture medium. Without 10% FBS (Fig. 8a), PP123-b-pDMAEMA/pDNA and PL121-b-pDMAEMA/pDNA exhibited higher gene expression than did PF127-b-pDMAEMA/pDNA at a high N/P ratio of ≥6 (p < 0.05). The higher hydrophobic characteristics of PP123-b-pDMAEMA/pDNA and PL121-b-pDMAEMA/pDNA facilitated higher cellular uptake of the polyplexes with increasing interactions between the cell membrane and the polyplexes. On the other hand, the serum-containing environment caused the aggregation between the protein and polyplexes, reducing the gene transfection. At an N/P ratio of ≥15 with 10% FBS (Fig. 8b), the gene transfection efficiency of pluronic-b-pDMAEMA/pDNA decreased but was still higher than that of PEI/pDNA. The PL121-b-pDMAEMA/pDNA showed significantly higher gene expression than the other two polyplexes at the N/P ratio of ≥15. According to the previous report,³³ the more hydrophobic PL121 supplied less protection to the polyplex than the more hydrophilic-formed polyplexes against serum aggregation. On the other hand, the highest hydrophobic effect of PL121 provided the highest possibility for cellular uptake because of hydrophobic interactions. Based on the best gene expression observed in the PL121-b-pDMAEMA/pDNA, it seems the impact of hydrophobic property on cellular uptake is a dominating factor in this study.

Fig. 8 In vitro gene expression of copolymers/pDNA polyplexes in 293T cells using various N/P ratios (a) without 10% FBS, and (b) with 10% FBS (n = 3, *P < 0.05). The N/P ratio of PEI (25K)/pDNA was 10.

Co-delivery

To verify the co-delivery effect of PL121-b-pDMAEMA, EPI and EGFP plasmid were used as the model drug and gene. The solvent evaporation method was used for EPI encapsulation followed by condensation with pDNA. The EPI loading concentration of the PL121-b-pDMAEMA in this study was 0.625 μg mL⁻¹ and the EPI-loaded micelle/pDNA polyplex was prepared at an N/P ratio of 15. The combination effect of the EPI-loaded micelle/pDNA was observed using CLSM in KB cells (Fig. 9). After 24 h of incubation, the CLSM image showed EPI was delivered into the cells and released in the cytoplasm. The similar co-delivery result was observed using PL121-b-pDMAEMA as a delivery vector (ESI Fig. S6†). The presence of EPI did not affect gene transfection. The GFP protein was highly expressed inside the cells. Thus, we concluded that the pluronic-b-pDMAEMA micelles seem a potential carrier for drug/gene co-delivery.

Fig. 9 Confocal images of the EPI-loaded PL121-b-pDMAEMA/pDNA polyplex at an N/P ratio of 15.

Conclusions

This study successfully synthesized PF127-b-pDMAEMA, PP123-b-pDMAEMA, and PL121-b-pDMAEMA copolymers with similar DMAEMA chain lengths via ATRP. The copolymers could easily self-assemble into core–shell micelles in aqueous solutions to co-deliver EPI and pDNA into cancer cells. The micelles had high capacity for EPI encapsulation and the EPI-loaded micelles had similar cell-killing effects to free EPI. The copolymers were capable of carrying pDNA, showing high gene transfection efficiencies. Among the three copolymers, the PL121-b-pDMAEMA showed the highest gene transfection efficiency as compared with the PF127-b-pDMAEMA and PP123-b-pDMAEMA in both with and without 10% FBS culture medium. The CLSM result demonstrated the PL121-b-pDMAEMA delivered the EPI molecules and pEGFP inside the cells. The PL121-b-pDMAEMA micelles displayed a combinational property.

Acknowledgements

We are grateful for the financial support from the Ministry of Science and Technology of Taiwan under grant numbers of NSC-98-2221-E037-001-MY3 and NSC-102-2325-B037-005. This study is also supported by “Aim for the Top Journals Grant” under grant number KMU-DT103007 from Kaohsiung Medical University.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra04308a

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