Materials Science and Engineering C 34 (2014) 377–383

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Polyethylenimine-immobilized core–shell nanoparticles: Synthesis, characterization, and biocompatibility test Montri Ratanajanchai a, Sunhapas Soodvilai b, Nuttaporn Pimpha c, Panya Sunintaboon a,d,⁎ a

Department of Chemistry, Faculty of Science, Mahidol University, Phuttamonthon 4 Road, Nakhon Pathom 73170, Thailand Department of Physiology, Faculty of Science, Mahidol University, Bangkok, 10400, Thailand National Nanotechnology Center (NANOTEC), National Science and Technology Development Agency, Pathum Thani 12120, Thailand d Center of Excellence for Innovation in Chemistry (PERCH-CIC), Rama 6 Road, Rajthewee, Bangkok 10400, Thailand b c

a r t i c l e

i n f o

Article history: Received 13 April 2013 Received in revised form 4 September 2013 Accepted 27 September 2013 Available online 8 October 2013 Keywords: Caco-2 Core–shell particle Photo-initiated polymerization Polyethylenimine Surfactant-free

a b s t r a c t Herein, we prepared PEI-immobilized core–shell particles possessing various types of polymer cores via a visible light-induced surfactant-free emulsion polymerization (SFEP) of three vinyl monomers: styrene (St), methyl methacrylate (MMA), and 2-hydroxyethyl methacrylate (HEMA). An effect of monomers on the polymerization and characteristics of resulting products was investigated. Monomers with high polarity can provide high monomer conversion, high percentage of grafted PEI, stable particles with uniform size distribution but less amino groups per particles. All prepared nanoparticles exhibited a core–shell nanostructure, containing PEI on the shell with hydrodynamic size around 140–230 nm. For in-vitro study in Caco-2 cells, we found that the incorporation of PEI into these core–shell nanoparticles can significantly reduce its cytotoxic effect and also be able to internalized within the cells. Accordingly, these biocompatible particles would be useful for various biomedical applications, including gene transfection and intracellular drug delivery. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Polyethylenimine (PEI) is one of amine-containing polymers, widely used in biomedical fields, such as controlled release drug delivery [1], gene therapy [2,3], antimicrobial agents [4], or medical imaging [5]. Amine functional groups on PEI structure provide three main characteristics, suitable to be applied in various biomedical uses. First, the reactivity of amine functionalities is useful for further modifications or covalent couplings with biomolecules [6]. Second, they possess pHresponsive ability, which is useful for controlled release drug delivery Abbreviations: Am-P, amino groups per particle; ATCC, American type culture collection; Cfree, concentration of PEI in diluted supernatant; CLSM, confocal laser scanning microscopy; Cm, concentration of monomer used in the reaction; CPEI, concentration of PEI used in the reaction; CQ, camphorquinone; DC, degree of monomer conversion; Dcv, volume-average diameter of the core; DMEM, Dulbecco's modified Eagle medium; DMSO, dimethylsulfoxide; Dn, number-average diameter; Dv, volume-average diameter; ELS, electrophoretic light scattering; FITC, fluorescein isothiocyanate; FTIR, Fouriertransform infrared spectroscopy; GPEI, percentage of grafted PEI; HEMA, 2-hydroxyethyl methacrylate; M0, molecular weight of PEI repeating unit; MMA, methyl methacrylate; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Np, number of particles per volume; PBS, phosphate buffered saline; PDI, polydispersity index; PEI, polyethylenimine; pI, isoelectric pH; PTA, phosphotungstic acid; RS:C, shell per core ratio; SFEP, surfactant-free emulsion polymerization; St, styrene; TEM, transmission electron microscopy; TBHP, tert-butylhydroperoxide; TNBS, 2,4,6-trinitrobenzene sulfonic acid; WSN, total weight of collected supernatant. ⁎ Corresponding author at: Department of Chemistry, Faculty of science, Mahidol University, Phuttamonthon 4 Road, Nakhon Pathom 73170, Thailand. Tel.: +66 2441 9816x1138; fax: +66 2441 0511. E-mail address: [email protected] (P. Sunintaboon). 0928-4931/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2013.09.037

systems [7]. Third, the cationic charge of this macromolecule is capable of binding with many negatively charged biomolecules, including DNA, RNA, and proteins for tissue engineering and gene therapy [8,9]. However, its cationic nature could cause cytotoxicity to living cells [10,11], which limits its applicable dose of usage. Consequently, there have been many attempts to improve its biocompatibility. Cytotoxicity of cationic polymers, including PEI, is mainly contributed by their charge density and chain flexibility [11,12]. Using PEI having lower molecular weight [13], linear structure [14], and lower cationic functionality [15] has been found to possibly improve their biocompatibility. However, these could decrease some desired properties for biomedical applications, such as transfection efficiency and buffering capacity. Thus, the methods that could reduce the cytotoxic effect without drastic change on the polymers' molecular weight and functionalities are, therefore, desirable. One plausible approach for such condition is the fabrication in the form of spherical particles, such as intramolecular crosslinked polymers [16], dendrimers [11,17], and immobilized nanoparticles [8,18]. One attractive method for immobilizing PEI onto polymer particles is the one-step surfactant-free emulsion polymerization (SFEP) developed by P. Li and coworkers [19]. Such method provides core–shell particles consisting of covalently grafted PEI as a shell and poly(methyl methacrylate) (PMMA) as a core. These PEI/PMMA particles have been found promising in some biomedical applications, including gene delivery system [18], drug delivery system [7], and antimicrobial agent [20]. In addition to the lower cytotoxicity of PEI as affixed on the particle's surface, the PMMA core can be used as a reservoir for embedding some

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reagents such as drugs [7], or fluorescent dyes [21]. Nevertheless, since drugs or essential reagents have different chemical properties, polarity, or other physical properties, it is quite a challenge to have appropriate core compartment to suitably accommodate those reagents. Herein, PEI-immobilized core–shell particles with three different polymer cores: polystyrene, poly(methyl methacrylate), and poly(2hydroxyethyl methacrylate) were prepared through an alternative one-step SFEP induced by visible light irradiation. The free radicals can be initiated by 3°-amine groups from PEI in the presence of camphorquinone (CQ). By this technique, PEI can be covalently bound as a shell of the particles and a core of vinyl polymers could be designed for specific properties and applications. The effect of three monomers with different polarities on the course of photo-induced SFEP and the properties of colloidal products were investigated. Thereafter, the PEIimmobilized particles with various vinyl polymer cores were also subjected to the biocompatibility test against the Caco-2 cell line. Furthermore, their cellular internalization was also illustrated. 2. Materials and methods

(DLS) method using a laser particle size analyzer (MALVERN instruments) at 25 °C. The measurements were repeated three times. The surface charge of the centrifuged colloidal dispersions was determined using a Zetasizer (Zetasizer 3000, Malvern Instruments, UK) in 1 mM NaCl solution at room temperature. The results reported were the mean of three determinations. The pH-dependent ζ-potential of the nanoparticles was investigated by determining the ζ-potentials of the colloidal dispersions at various pH values (in a range of 3–12) adjusted by HCl–NaOH. The core–shell morphology of PEI-immobilized nanoparticles was observed by transmission electron microscopy (TEM; JEM-1400, JEOL, 100 kV). 20 μL of 500-fold diluted sample was deposited on a copper grid and stained with phosphotungstic acid (PTA, 2 wt.%). Then, the sample was dried under a dust-free ambient environment and visualized by TEM. The information from TEM images was also used to determine volume-average diameter of the core compartment (Dcv) and the number of particles per volume (Np), following Eq. (1) [22]. Np ¼

2.1. Materials Styrene (St), methyl methacrylate (MMA), 2-hydroxyethyl methacrylate (HEMA), branched polyethyleneimine (b-PEI, 50 wt.% in water, Mn 60,000 g/mol) and camphorquinone (CQ) were all obtained from Aldrich. MMA and St were purified by distillation under reduced pressure after removing inhibitor by extraction with NaOH solution, while HEMA was purified using a column packed with aluminum oxide adsorbents (pH 7 and 9.5) obtained from Fluka. 2.2. PEI-immobilized core-shell nanoparticles via photo-induced SFEP The procedure for the surfactant-free emulsion polymerization was described as follows. First, 5 g of PEI solution (10 wt.%) was mixed with a predetermined amount of distilled water in water-jacketed flask equipped with nitrogen inlet–outlet, water-circulating thermostat, and magnetic stirrer. The mixture was stirred with a magnetic stirrer at 600 rpm, under nitrogen gas purge for 30 min. Thermostat-controlled water (25 °C) was pumped through the jacketed flask. After that, 2 mL of purified monomer (St, MMA, or HEMA) was added and then followed by 1 mL of CQ (0.02 M, predissolved in ethanol). A total volume of reaction mixture was 50 mL. All experimental steps involving CQ were accomplished without the exposure to light before polymerization. To trigger the polymerization, the mixture was then irradiated by visible light from the 300 W light source (SP. Electric halogen floodlight with a Sylvania tubular lamp; the distance from the lamp to the center of the reactor was 25 cm) for 3 h. The colloidal products prepared from the photo-induced SFEP using St, MMA, and HEMA as monomers were abbreviated as PEI/PS, PEI/PMMA, and PEI/PHEMA, respectively. The degrees of monomer conversion (DCs) were determined gravimetrically. 2.3. Characterization of PEI-immobilized nanoparticles Unbound PEI from the synthesized nanoparticle dispersions was removed by repeated centrifugation–redispersion cycle at 25,000 rpm for at least 2 cycles (45 min each cycle). The supernatant from each centrifugation–redispersion cycle was collected for determining the unbound PEI and a percentage of grafted PEI through TNBS assay. Chemical functional groups of dried nanoparticles after purified by centrifugation were characterized by Fourier-transform infrared (FTIR, Perkin Elmer, PE 2000) spectroscopy using KBr disks. The spectra were recorded at a resolution of 4 cm−1 and 32 scans. The scanning for each spectrum was attained in a range of 4000 to 370 cm−1 with KBr powder as a reference background. Number- and volume-average (Dn and Dv) hydrodynamic diameters of cleaned nanoparticles were then acquired by dynamic light scattering

Total volume of core polymer per volume mp =ρp ¼ Volume of core compartment per particle π6ðDcv Þ3

ð1Þ

where mp is the total mass of polymerized monomer and ρp is the density of each core polymer assumed to be the density of the corresponding bulk polymer [23,24]. 2.4. Quantitative measurement of grafted PEI The 2,4,6-trinitrobenzene sulfonic acid (TNBS, Aldrich) assay [25,26] was slightly modified to assess the amount of free PEI in the supernatant solutions collected from the above centrifugal cleaning process. Generally, this method has been used to determine a concentration of proteins or amino acids through the reaction between the TNBS reagent and free amine groups, which yields an observable chromogenic product. First, 50 μL of the 20-fold diluted supernatant (in 0.1 M bicarbonate solution pH8.5) was mixed with 50μL of TNBS solution (0.02%w/v in 0.1M bicarbonate solution at pH 8.5) in a 96-well plate. The plate was instantaneously placed in an incubator shaker (150 rpm, 37 °C) for 2 h. Finally, the reaction was quenched by 20 μL of HCl solution (1 M). The absorbance of the solution in each well in the 96-well plate was then read by an automated microplate reader (Perkin Elmer, Wallac Victor 1420) at 355 nm. The concentration of free PEI in the supernatant was determined with reference to the calibration curve of PEI solution (10–120 μg/mL) (as shown as Fig. S1 in Supporting Information). The percentage of grafted PEI (GPEI) was calculated using Eq. (2) by comparing between weights of grafted PEI and total PEI used. The shell per core ratio (RS:C), indicating the weight ratio between grafted PEI and polymer core, was determined by Eq. (3). The amino groups per particle (Am-P) was calculated by Eq. (4). GPEI ¼

  grafted PEI 0:5−ð20C free  WSN Þ  100 ¼  100 total PEI 0:5

ð2Þ

RS:C ¼

ðG =100Þ  ðC PEI Þ wt of PEI GPEI ¼ ¼ PEI 4  DC ðDC=100Þ  ðC m Þ wt of PMMA

ð3Þ

Am−P ¼

total amine groups ½ðGPEI =100Þ  ðC PEI =100Þ  NA ¼ M0  Np number of particles

ð4Þ

where the total PEI of all reactions is 0.5 g, Cfree is the concentration of PEI in diluted supernatant (g/mL), CPEI are the concentrations of PEI (1 wt.%) in the reaction, Cm are the concentrations of monomer (4wt.%) in the reaction, WSN is the total weight of collected supernatant, NA is Avogadro's number, and M0 is the molecular weight of PEI repeating unit (43 g/mol).

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2.5. Cell culture Caco-2 cells (human colon carcinoma, American Type Culture Collection (ATCC) no. HTB-37) were maintained in Dulbecco's modified Eagle medium (DMEM) high glucose (Gibco) at a pH value of 7.4, supplemented with 15% (v/v) fetal bovine serum (FBS, Hyclone), 1% L-glutamine (Gibco), 1% non-essential amino acid (Sigma), and 100 U/mL penicillin–streptomycin (Gibco). The cells were propagated in 75-cm2 T flasks (Corning) under a humidified atmosphere containing 5% CO2 at 37 °C and subcultured as described in the ATCC protocol.

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incubated at 37 °C for 2 days in a humidified atmosphere, containing 5% CO2. For treatment, the cells were washed twice with PBS and followed by 0.1 mg/mL of FITC-labeled nanoparticles in DMEM without a supplement. After 2 h of incubation at 37 °C in 5% CO2 atmosphere, the cells were washed twice with PBS and fixed with 4 wt.% paraformaldehyde (Merck) in PBS for 30 min. After a wash with PBS, the cell nuclei were stained by TO-PRO-3 iodide (1:500; Invitrogen) for an hour and then washed with 1 wt.% Tween20 in PBS. Finally, the cover slip was mounted on a glass slide using an antifade (Fluorpreserve, Calbioscience). The slide was observed by an Olympus FV1000 confocal microscope using a 60× objective oil with additional 2× zoom at resolution of 640 × 640 pixel resolution.

2.6. Biocompatibility assay Biocompatibility of synthesized latexes was evaluated as the cell viability though 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma) assay. The confluent Caco-2 monolayer (passage 35–40) was prepared by seeding the cells in a density of 2×104 cells per well on a 96-well plate and maintained with the DMEM-containing supplement. After 2 days of incubation at 37 °C in a humidified atmosphere containing 5% CO2, the medium was removed and washed with phosphate buffered saline (PBS). The cells were then treated with 100 μL sample dispersion (varying by 5-fold dilution) in DMEM without a supplement and incubated for 3 h or 24 h. Thereafter, the solution was removed and the cells were washed with PBS. 100 μL of freshly prepared solution of 0.5 mg/mL MTT in DMEM without any additives was added and the cells were incubated for 3 h at 37 °C and 5% CO2. Subsequently, the wells were emptied. 150 μL of dimethylsulfoxide (DMSO) was used to dissolve the formed formazan crystals and the absorbance was recorded by the microplate reader (Thermo Labsystems, Multiscan EX) at 540 nm. Percentage of viable cells was calculated by comparing with the control DMEM. The cell viability test of each sample was done by at least three independent experiments (n ≥ 3). 2.7. Internalization of the particles The amine-functionalized nanoparticles were first labeled with fluorescein isothiocyanate (FITC, Fluka). In the labeling reaction, an isothiocynate group of FTIC reacted with primary or secondary amines of PEI segments at the particles' surface to form stable thiourea bond. Firstly, 2.5 mg/mL of nanoparticles was mixed with 250 μg/mL of FITC in 0.1 M NaHCO3 pH 8.5 on a shaker for 2 h. The unreacted FITC was removed by repeated centrifugation–redispersion at 30,000 rpm ultracentrifugation for 30 min 4 times. To confirm the complete removal of FITC (as indicated by an absence of FITC emission in Fig. S2), the supernatants from each centrifugation cycle were taken into 96-well black opaque plate. The fluorescent emission at 518 nm (excitation wavelength is 492 nm) was monitored by an automated microplate reader (Perkin Elmer, Wallac Victor 1420). All steps involving FITC were carried out in a dark room. Then, the internalization of the particles was illustrated through a confocal laser scanning microscopy (CLSM). Caco-2 cells (5 × 104 cells/well) were seeded on a 22 × 22 mm2 cover slip placed in a 6-well plate and

3. Results and discussion 3.1. Preparation of PEI-immobilized core–shell nanoparticles by the photoinduced SFEP In this work, PEI-immobilized core–shell nanoparticles were prepared via SFEP using a CQ/3°-amine photo-redox system (Fig. 1). The particle nucleation and growth mechanism for the polymerization process in this work is proposed, which is similar to the previous reports [22,27]. A visible-light irradiation to CQ in the presence of PEI molecules, possessing 3°-amine, could generate active radicals on the PEI chains [28,29]. These radicals are able to polymerize vinyl monomers, yielding amphiphilic grafted copolymer chains, which are subsequently selfassembled to form micelle-like microdomains. These domains have hydrophilic PEI as a periphery, enwrapping relatively hydrophobic grafted chains, and could be the sites for further polymerization. Eventually, the mature colloidal product was formed and had the core–shell structure with PEI as the shell and vinyl polymers as the core. Accordingly, PEI in this photo-induced SFEP plays the important roles as a co-initiator by providing 3°-amine groups and as a colloidal stabilizer for the resulting nanoparticles. The SFEP of three vinyl monomers: St, MMA, and HEMA, with different polarities, was conducted. From Table 1, DCs were higher than 80% for MMA (83%) and HEMA (91%), but was low for St (16%). It was obvious that the system was effective to polymerize both HEMA and MMA but not effective for styrene. It is postulated that the difference in polarity of these vinyl monomers might cause this observation. Because the monomers used have different polarities, their diffusion capability to the PEI macro-radicals might be different. MMA and HEMA are polar monomers that might have higher capability of accessing to these radical sites, leading to higher DCs. In contrast, St is a non-polar and hydrophobic monomer. It might have a high extent of difficulty to access to the radical sites for polymerization, especially through an aqueous medium and the impeding hydrophilic branched PEI. The covalent grafting between vinyl polymer core and PEI shell components was analyzed using FTIR spectroscopy. Fig. 2 shows FTIR spectra of pure PEI and the three polymer nanoparticles after purified by centrifugation. The spectrum of branched PEI (Fig. 2a) provided the broad peak around 3200–3500 cm−1 (N\H stretching), two peaks at 1570 and 1645 cm−1 (amine scissoring), and strong absorption band

Fig. 1. Mechanistic scheme of the particle formation resulting PEI-immobilized core–shell nanostructure in the photo-induced SFEP using CQ/PEI initiating system.

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Table 1 Product characteristics of PEI-immobilized nanoparticles prepared via the photo-induced SFEP. Product characteristics Monomer conversion, DC (%) Density of core polymer, ρp (g·cm−3)a Total polymerized monomer, mp (mg·mL−1) Volume-average core diameter, Dcv (nm) Number of particles per volume, Np (1013 mL−1) Percentage of grafted PEI, GPEI (%) Shell per core weight ratio, RS:C Amine groups per particle, Am-P (105) Number-average diameter, Dn (nm) Particle size distribution, Dv/Dn ζ-potential (mV)b

PEI/PHEMA

PEI/PMMA

PEI/PS

91.3 1.15 39.2 72.6 17.0

82.9 1.20 31.0 86.6 7.61

15.6 1.05 5.67 87.4 1.54

83.8 0.214 6.89 158 1.24 36.0

69.2 0.223 12.7 136 1.11 43.0

51.1 0.900 46.3 127 1.26 41.4

chains, especially for the non-polar St. In case of PEI/PHEMA, highest GPEI was, therefore, observed probably due to its hydrophilic nature. Size and size distribution of PEI-immobilized particles are also important parameters for the polymer nanoparticles prepared by the SFEP system. Hydrodynamic size (Dn) and size distribution (Dv/Dn) of prepared nanoparticles determined using DLS technique are shown in Table 1. The sizes of prepared nanoparticle dispersions were about 120–160 nm in diameter. Furthermore, it seemed that the monomer

a

The parameter was referred to bulk densities of the homopolymers [23,24]. To assess shelf-life of native products, the measurement was performed without pH adjustment. b

around 2900 cm−1 (C\H stretching). The spectra of three dried nanoparticles (Fig. 2b–d) showed the same characteristic signals as those of PEI. Moreover, they also exhibited additional signals that corresponded to the grafted vinyl polymers, including carbonyl stretching peaks in PEI/PHEMA (1728 cm−1) and PEI/PMMA (1732 cm−1) spectra, aromatic C\H bending (755 and 698 cm−1) and aromatic C\H stretching (around 3030 cm−1) peaks in PEI/PS spectrum. As can be seen, the spectra of all nanoparticles still showed characteristic peaks identical to pure PEI. This evidence suggested that PEI component was covalently attached to the core–shell nanoparticles. Next, TNBS assay was performed to evaluate the grafting performance, which relates to the attachment of PEI onto the nanoparticles' surface. It was found that GPEI (Table 1) calculated from TNBS assay was 84%, 69%, and 51% for PEI/PHEMA, PEI/PMMA, and PEI/PS, respectively. The lower GPEI extent may be mainly contributed by the higher difficulty of hydrophobic monomers to graft from the hydrophilic PEI

Fig. 2. FTIR spectra of a) branched PEI; b) PEI/PHEMA after centrifuged; c) PEI/PMMA after centrifuged; and d) PEI/PS after centrifuged.

Fig. 3. TEM images (10k magnifications) of three synthesized nanoparticles; a) PEI/PHEMA, b) PEI/PMMA, and c) PEI/PS. Insets are higher magnified images.

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type can influence the particle size of prepared nanoparticles. PEI/PS provided the smallest size (127 nm) whereas PEI/PMMA and PEI/ PHEMA are larger in size (136 and 158 nm, respectively). In general, SFEP of monomer with more hydrophobic character provides higher number of particles (more reaction sites) [30]. The SFEP of hydrophobic St, therefore, led to smaller particle size than the SFEP of MMA and HEMA. The morphology of the prepared nanoparticles was investigated by TEM with selective staining. According to the TEM images (Fig. 3) of the dried particles stained with PTA, the core–shell morphology was observed. In contrast to the polymer core segment, the PEI segment, containing amine groups, could interact with anionic metal complex and resulted in high electron density regions. Therefore, the contrast between vinyl polymer core and PEI shell regions can be clearly observed. Moreover, TEM images can provide the information on the sizes, which were in a sub-micron range (about 100–200 nm). The uniform size distributions were obtained in PEI/PHEMA and PEI/PMMA, but PEI/PS provided broader distribution, which were consistent to the results from the DLS method. In addition, it was observed that the PEI shell layer in PEI/PS particles was thicker than those of PEI/PMMA and PEI/PHEMA. Based on the following values: DC; Dcv (from TEM images); and GPEI (from TNBS assay), two additional important parameters can then be derived; the shell per core weight ratio (RS:C) and amine groups per particle (Am-P) (Table 1). The first parameter is useful and related to the amount of PEI in a total solid content for each product. It was observed that RS:C values of PEI/PHEMA and PEI/PMMA nanoparticles were about 0.2 (shell:core ≈ 1:5), whereas that of PEI/PS was almost equal to 1 (shell:core ≈ 1:1). The second parameter, Am-P, is also an essential value to assess an amount of amine functionalities on the particle surface. It was found that Am-P value of PEI/PS was the highest (~4.6 × 106 groups/particles). For PEI/PMMA, its Am-P was ~1.3 × 106 groups/particles, while PEI/PHEMA had the lowest Am-P (~6.9 × 105 groups/particles). Based on our proposed mechanism, this evidence might be related to the type of monomers. The lower polarity of monomer would have a lower capability of diffusing to PEI radicals in this polymerization system, and then lead to lower DC. In the same regard, the fewer formation of self-assembled micelles can be occurred as indicated by Np values (Table 1), which were the lowest for PEI/PS (~1.5×1013 particles/mL) and the highest for PEI/PHEMA (1.7 × 1014 particles/mL). The less formation of precursor micelles would subsequently lead to the more PEI on each particle. 3.2. Surface characteristics of nanoparticles The surface charge of PEI-immobilized core–shell nanoparticles was assessed by measuring ζ-potentials of the samples (Table 1). It was found that all prepared nanoparticles exhibited positive ζ-potentials with magnitudes higher than 30mV. Furthermore, to assure that the colloidal products can be used in physiological condition, pH-dependent surface charges were also acquired as shown in Fig. 4. It was found that

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the PEI/PMMA nanoparticles showed maximum positive ζ-potentials around +50 to +55 mV in acidic and neutral conditions (at pHs below 7), because of the protonation on amine groups of PEI. Increasing the pH values led to the decrease in the ζ-potentials across the isoelectric points at about 10 and then negative values. In general, a physiological pH was about 7.4 in which the ζ-potentials of all nanoparticle dispersions were still around +50 mV, which implies their possible utilization in physiological environment without a loss of colloidal stability. Moreover, pH-dependent ζ-potential of pure PEI polymer was also compared with the colloidal products. It was obvious that the prepared nanoparticles exhibited the pH-dependent ζ-potentials similar to the native PEI polymer [31,32]. This result strongly suggests that the particle surfaces be covered by PEI. The slightly difference in pIs might be due to the less presence of amine groups on the polymer surface when incorporated in a form of nanoparticle. In addition, the presence of PEI on the surface could provide a buffering characteristic to resulting particles, which could protect encapsulated species (e.g. drug or nucleic acids) from the degradation by lysosomal enzymes in acidic environment and also lead to ‘sponge mechanism’ for escaping from endolysosome to cytoplasm [8], which is very essential for applying them as intracellular delivery carriers. 3.3. Evaluation of biocompatibility of synthesized nanoparticles It has been well known that a major problem limiting the use of amine-containing polymers, especially PEI, in biomedical applications is its high cytotoxicity caused by its cationic characteristic [11,12,33,34]. One of the key factors controlling this cytotoxic effect is the flexibility of the molecules that is necessary for multiple attachments of polymers to the cell membrane [11,12]. The three monomers were used not only for evaluating the polymerization process, but also for producing various colloidal materials being used in biomedical applications. PHEMA is a well-known polymer used as hydrogels for biomedical applications due to its hydrophilicity and biocompatibility. PMMA is one of mostly used polymers, which has been reported in various biomedical aspects. While, an incorporation of PS into the particle as a core compartment would be effective for encapsulation of hydrophobic agents. Herein, the prepared core–shell nanoparticles together with the native PEI polymer were subjected to biocompatibility evaluation by MTT assay in the Caco2 cell line, the widely used in-vitro representative human intestinal epithelium at 3 h (Fig. 5a) and 24 h (Fig. 5b) in order to observe the acute and late toxic effects, respectively. It was found that PEI solution caused cytotoxicity in a dose-dependent manner and showed significant cytotoxic effect at 40 μg/mL, where 76% and 29% cell viability values were observed for 3 h and 24 h treatments, respectively. Furthermore, it can be seen that an applicability of native PEI in this cell line without any cell death was only 8 μg/mL. Surprisingly, all synthesized nanoparticle dispersions provided 100% cell viability for 24h treatment at the concentrations up to 200 μg/mL, which contained PEI about 35, 36, and 95 μg/mL for PEI/PHEMA, PEI/PMMA, and PEI/PS, respectively (estimated using RS:C from Table 1). It is clearly shown that the cytotoxicity of PEI-shell

Fig. 4. ζ-potentials of PEI solutions and PEI/PMMA nanoparticles at different pHs; Isoelectric pHs can be defined as x-intercept values.

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many cationic polymer vectors, including PEI, have significant cytotoxicity to human cells [2,11,35]. The highest biocompatible concentrations (with no less than 100% cell viability) of the PEI/PHEMA, PEI/ PMMA, and PEI/PS nanoparticles for 24 h treatment were 5.0, 1.0, and 0.2 mg/mL, respectively. The RS:C and Am-P parameters from Table 1 show that more RS:C (PEI content) and/or Am-P (cationic charge density) on the particle surface probably led to higher cytotoxicity. Therefore, PEI/PS that exhibited the highest RS:C (~ 0.9) and Am-P (~ 4,600,000 groups/particle) values seemed to be the most cytotoxic. In fact, the nature of the core polymers might also contribute to the cytotoxicity. PEI/PHEMA provided the core-shell nanoparticles with the lowest cytotoxicity, because of PEI affixed in a nanostructure as mentioned above, and also PHEMA, a well-known biocompatible polymer. 3.4. Intracellular uptake of core–shell nanoparticles

Fig. 5. Relative cell viability (mean ± SD; n = 3) compared with control of various concentrations (5-fold) of PEI and PEI-immobilized nanoparticles evaluated by MTT assay for a) 3 h and b) 24 h treatment. Cytotoxicity indicated as asterisks (p b 0.05), was evaluated by the cell viability significant less than 100% (control).

nanoparticles was greatly reduced, compared to that of native PEI polymer. This result was close to the result reported by J. Zhu and coworkers (2005) [18]. The much lower cytotoxicity of PEI when in the form of nanoparticles might be resulted from the alteration of PEI's threedimensional structure, causing a low accessibility of cationic groups to the cell surface, especially for multiple attachments. This evidence is important in the gene and drug delivery systems, indicating that

As found in MTT assay, the prepared nanoparticles have a potential to be used in biomedical applications (especially oral drug delivery) instead of the native PEI due to their much improved biocompatibilities. Next, the investigation on their internalization capability to the cells was conducted since PEI has been reported as a non-viral vector for gene transfection and a carrier for intracellular protein delivery [2,36,37]. In this work, CLSM was used as a qualitative analysis for monitoring the intracellular uptake of synthesized particles into Caco-2 cells (Fig. 6). All images were taken at the section within the cell, implied by the presence of nuclei. It was shown that the FITC-labeled particles: PEI/PMMA, PEI/PHEMA, and PEI/PS, were taken up into the cells within 2 h of incubation. This could be implied that our materials could be the candidates for intracellular carriers useful in biomedical applications such as gene, protein, and drug delivery systems. 4. Conclusions PEI can be covalently immobilized onto different polymer nanoparticles via the photo-induced SFEP using CQ/3°-amine initiating system.

Fig. 6. Intracellular uptake of PEI-immobilized core–shell particles (0.1 mg/mL, 2 h treatment) into Caco-2 cells verified by an appearance of the FITC labeled-nanoparticles around the area within the cells (assured by locating of nuclei labeled with TOPRO-3). Boundary of the cells could be clearly seen by differential image contrast (DIC). The scale bars at the right corner of DIC images indicate 20 μm.

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Polyethylenimine-immobilized core-shell nanoparticles: synthesis, characterization, and biocompatibility test.

Herein, we prepared PEI-immobilized core-shell particles possessing various types of polymer cores via a visible light-induced surfactant-free emulsio...
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