Preparation of poly(ethylene imine) particles for versatile applications

Colloids and Surfaces A: Physicochem. Eng. Aspects 433 (2013) 212–218

Contents lists available at SciVerse ScienceDirect

Colloids and Surfaces A: Physicochemical and Engineering Aspects journal homepage: www.elsevier.com/locate/colsurfa

Preparation of poly(ethylene imine) particles for versatile applications Nurettin Sahiner a,b,∗ a

Faculty of Science & Arts, Chemistry Department, Canakkale Onsekiz Mart University, Terzioglu Campus, 17100 Canakkale, Turkey Nanoscience and Technology Research and Application Center (NANORAC), Canakkale Onsekiz Mart University, Terzioglu Campus, 17100 Canakkale, Turkey b

h i g h l i g h t s

g r a p h i c a l

a b s t r a c t

• Single step PEI microgel preparation. • PEI microgels for biomedical application.

• PEI microgels for in situ metal nanoparticle preparation.

• PEI microgels for environmental, energy and catalysis applications.

a r t i c l e

i n f o

Article history: Received 31 March 2013 Received in revised form 4 May 2013 Accepted 8 May 2013 Available online 16 May 2013 Keywords: Hydrogel Crosslinked PEI microgel Nanogel composite Tunable particle

a b s t r a c t Polyethylene imine (PEI) particles were readily prepared via a simple microemulsion polymerization method using AOT as surfactant in commercially available gasoline with moderately high yield (∼75%) depending on the MW of PEI. The aqueous solution of branched PEI crosslinked with divinyl sulfone (DVS), called c-PEI particles were in the size range of tens of nanometers to tens of micrometers and upon filtration the desired size range was readily obtained. The prepared c-PEI particles were highly positively charged depending on the used amounts of crosslinker and the extent of modifying agents such as alkyl halide for different purposes e.g., CH3 I that can conveniently be used for the quaternization reaction. The prepared c-PEI particles were demonstrated to be very useful as antimicrobial materials, drug delivery materials, as a template for metal nanoparticle preparation, as a catalysis medium for the reduction of 4-nitrophenol (4-NP) to 4-amino phenol (4-AP), and for hydrogen generation from the hydrolysis of NaBH4 . © 2013 Elsevier B.V. All rights reserved.

1. Introduction Polymeric particles from various sources have been prepared for different purposes such as tissue engineering, drug delivery, environmental and advanced material design. More recently, materials with tunable charges and sizes are in demand for versatile applications. Especially cationic polymers have attracted great attention for different purposes [1–4]. Moreover, for gene

∗ Correspondence address: Faculty of Science & Arts, Chemistry Department, Canakkale Onsekiz Mart University, Terzioglu Campus, 17100 Canakkale, Turkey. Tel.: +90 286 2180018x2041; fax: +90 286 2181948. E-mail address: [email protected] 0927-7757/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfa.2013.05.029

delivery systems, cationic polymers gain special attention, and amongst these cationic polymers PEI, with various formulations, is the frontrunner for highly efficient and effective delivery systems [5,6]. Polyethylene imines (PEI) offer unique opportunities for advanced material design due to their highly positively charged nature and high transfection efficiency for gene therapy [7–9]. They have been used mostly as DNA compacting materials [10]. PEI is widely used in condensing structures such as DNA and PLA and used in drug delivery [11,12]. Due to this condensing ability and the efficiency of PEI for DNA and siRNA, and some other anionic structures, PEI-based material has been extensively investigated for delivery purposes both in vitro and in vivo [13,8,7,14]. Although PEI-based materials are very effective agents for cell transfection, they suffer from extreme cell death due to cyctotoxicity [8,7,15–19]. To

N. Sahiner / Colloids and Surfaces A: Physicochem. Eng. Aspects 433 (2013) 212–218

circumvent these problems PEI polymers have been modified with other peptides, polymers (PEG) and structures to increase their responsiveness [12,20–22]. In addition, cationic polymers are the subjects of intense research as non-viral gene delivery systems due to their flexible properties, facile synthesis, robustness, and proven gene delivery efficiency [7,5,23,24,16]. Nevertheless, low transfection efficiency and undesirable cytotoxicity remain the most challenging aspects of these cationic polymers. To overcome these disadvantages, various modifications have been made to improve their gene delivery efficacy. Among them, hydrophobic modifications of the cationic polymers are receiving more and more attention [5,21]. As a cationic polymer, PEI can be employed for advanced materials design for versatile applications e.g., one of the intriguing uses of PEI-based materials is CO2 capture. It was reported that PEI supported on pore-expanded MCM-41 can accommodate PEI loadings of up to 83 wt% with an adsorption capacity of as high as 250 mg/g in the presence of pure CO2 at 75 ◦ C [25]. The resourcefulness of PEI material is so diverse that here, shown for the first time, is a facile PEI particle preparation via microemulsion template crosslinking using a single step by linking amine groups with DVS. It was further demonstrated that c-PEI particles can be applied in biomedical fields as drug delivery devices, antimicrobial materials, as template for metal nanoparticle preparation and in catalysis for reduction of nitro compounds and hydrogen generation. 2. Materials and methods 2.1. Materials Polyethlene imine (PEI) with MWs (Sigma–Aldrich, 50 wt%, Mn: 1200, 60 000, and 600 000), divinyl sulfone (DVS, 98%, Fluka) as a chemical crosslinker, sodium bis(2-ethylhexyl) sulfosuccinate (AOT, 96%, Sigma–Aldrich) as a surfactant, and gasoline as a solvent were used as received. Nickel(II) chloride hexahydrate (NiCl2 ·6H2 O, 97%, Riedel-de Haën), and cobalt(II) chloride hexahydrate (CoCl2 ·6H2 O, 99% Sigma–Aldrich) were used as received in the preparation of metal nanoparticles. NaBH4 (99.9%, Sigma–Aldrich) was used as reduction agent and reagent for 4-nitrophenol reduction and in the hydrolysis reaction for H2 generation. All the solvents, acetone and ethanol were of the highest purity available. All the reagents and solvents (acetone and ethanol, acetonitrile) were of analytical grade or highest purity available, and used without further purification. Ultra pure distilled water 18.2 M cm (Millipore-Direct Q UV3) was used throughout the studies. 2.2. Synthesis of polyethylene imine particles PEI microgels as c-PEI were synthesized by using microemulsion polymerization. To obtain c-PEI microgel, 1 mL of PEI regardless of MW was dispersed in 30 mL of 0.1 M AOT solution in gasoline. The mixture was vortexed until a clear suspension was obtained. Then, the crosslinker, DVS, in varying amounts (50–100 mol% relative to the PEI repeating unit) for Mn 1200 and, 30 ␮L for 1 mL PEI solution with Mn: 60 000 and 600 000 was subsequently added to the mixture and thoroughly mixed to disperse the DVS. The reaction was allowed to proceed for 1 h at ambient temperature with vigorous stirring at 1200 rpm. Then, the obtained particles were precipitated in excess acetone and purified by centrifugation at 10 000 rpm for 10 min at 20 ◦ C several times and dried with a heat gun. The dried PEI particles were stored in a closed container. The yield depending on MW of PEI varied from 30 to 70%; for higher MW PEI e.g., 600 000 higher yield of about 75% was obtained.

213

2.3. Characterization of c-PEI particles Thermal behavior of c-PEI-based particles was investigated with a thermogravimetric analyzer (TGA, Seiko, SII TG/DTA 6300). TGA measurements were carried out by heating samples from 50 to 600 ◦ C under nitrogen flow of 100 mL/min with 10 ◦ C/min heating rate. Approximately 4 mg samples were used and their weight loss against temperature was recorded. The functional group characterizations of the particles were done using an FT-IR (Perkin-Elmer Spectrum 100) instrument with ATR apparatus with resolution between 4000 cm−1 and 650 cm−1 . The SEM (SEM, Jeol, JSM5600) images were obtained by placing the particles onto carbon tape-attached aluminum SEM stubs at ambient temperature after coating with gold to a few nanometer thickness under vacuum, with an operating voltage of 20 kV. The sizes of c-PEI particles were determined with dynamic light scattering (DLS) measurements after filtration. The particle size analyzer (DLS, 90 plus Brookhaven Instrument Crop.) measurements were carried out at 90 ◦ C angle using 35 mW solid state laser detector operating at 658 nm by suspending certain amounts of particles in 10−3 M KCl aqueous solution with 20 s integration time. The zeta potential measurements were conducted by using a ZetaPals Zeta Potential Analyzer BIC (Brookhaven Instrument Corporation) with a diluted aqueous solution of c-PEI-based particles. 2.4. Antimicrobial properties of c-PEI particles Particles derived from PEI were tested against two common bacteria for their antimicrobial properties. Certain amounts of c-PEI particles (100, 50, 25, 5, 1 ␮g/mL) and modified c-PEI particles were contacted with various bacteria such as Escherichia coli ATCC8739 and Staphylococcus aureus ATCC 8739 for 18–24 h, and their MIC (Minimum Inhibitory Concentration) and MBC (minimum bactericidal concentration) were determined using bacterial suspension in nutrient broth. The particles were sterilized under UV irradiation for 2 min for sterilization, and were added to sterile 10 mL glass tubes. The appropriate volume of a solution containing of 9 × 108 CFU/mL of each bacterial suspension in nutrient broth was added. Tubes only containing inoculated broth were used as negative control. Tubes were vortexed and 1/10, 1/100, 1/1000 and 1/1 000 000 diluted samples prepared. From these, 10 ␮l samples were taken and plated on 1% agar and incubated for 18–24 h at 35 ◦ C. Colonies were counted and MIC and MBC values of the c-PEI particles were determined. To confirm c-PEI can be used as drug delivery device, naproxen (NP) was chosen as a model drug. For loading, 200 mg dried c-PEI was placed in 300 ppm 50 mL NP solution in ethanol for 12 h under constant shaking at ambient temperature. For the release studies, 50 mg NP-loaded c-PEI particles were placed in PBS buffer at pH 7.4 at room temperature and the released amounts of NP per gram c-PEI with time were measured using UV–vis spectroscopy. NP has an absorption maximum at 330 nm in both ethanol and PBS solutions and the loading and released amounts were determined from the previously constructed calibration curve of the solvent. The release experiments were repeated three times and the average values with standard deviation are given. 2.5. Metal nanoparticle preparation within c-PEI and catalysis studies To prepare metal nanoparticles within c-PEI particles, about 200 mg c-PEI particles were placed in 50 mL 1000 ppm Co(II) or Ni(II) solution under constant stirring (400 rpm) for 16 h. Then these metal ion-loaded c-PEI microgels were separated by centrifugation at 10 000 rpm and washed with excess DI and were reduced with 50 mL 0.1 M NaBH4 for 4 h at room temperature at 400 rpm

214

N. Sahiner / Colloids and Surfaces A: Physicochem. Eng. Aspects 433 (2013) 212–218

Fig. 1. (a) The schematic PEI particle preparation from branched PEI, and (b) and (c) the corresponding optical microscopy images of swollen c-PEI particles obtained with Mn 1200 and 60 000, respectively.

mixing rate. Again, c-PEI-M (Ni and Co) composite systems were re-centrifuged at 10 000 rpm and washed with DI water and used for the reduction of 4-NP to 4-AP, and for NaBH4 hydrolysis. The reduction of 4-NP was carried out by preparing in 50 mL 0.01 M 4-NP solution containing 0.4 M NaBH4 using 0.070 g c-PEI-M composite system. As soon as the catalyst was placed in the reaction mixture under constant mixing rate 400 rpm, at certain time intervals (2, 3, 5, 7, 9, 11, 13 and 15 min) 100 ␮L solution was withdrawn and diluted to 10 mL with DI water and their UV–vis spectra were recorded. The reduction of 4-NP at 400 nm was monitored and measured via a previously constructed calibration curve. For the hydrolysis reactions, 200 mg c-PEI-M composite system was used in 50 mL DI water containing 0.0965 g NaBH4 . The amount of produced hydrogen was determined from an inverted cylinder filled with water. The produced hydrogen was passed through concentrated H2 SO4 to capture water vapor for accurate determination of the generated hydrogen from the hydrolysis reaction. 3. Results and discussion Particles of hydrogel, due to their tissue-like resemblance, flexible porous morphology and ample functional groups to render desired tasks, stands as frontrunner for biomedical, environmental and catalyst applications e.g., vehicles for drug delivery and gene therapy, removing toxic species from contaminated environments and/or converting them to more environmentally benign forms, and templates for material synthesis. Therefore, here PEI particles were prepared with a simple crosslinking technique using DVS as crosslinker in AOT emulsion in gasoline for the first time. It is very well known that vinyl sulfones are excellent Michael acceptors due to the electron-poor nature of their double bond and also the sulfone’s electron withdrawing ability makes them good electrophiles. Therefore, DVS has been used as a good crosslinker for

reactions with nucleophilic groups such as hydroxyl and amines hetero atomic nucleophiles [26,27]. The branched PEI chain linked by using DVS as crosslinker to obtain non-soluble PEI microgels is illustrated in Fig. 1(a). As shown, the nucleophile amine groups can easily react with vinyl groups of DVS to generate 3D microgels up to several tens of ␮m from a few of tens of nanometers. The wide size distribution of PEI microgels has great advantages as the smaller sizes can be readily separated by simple filtration, even with a simple filter apparatus. The optical microscopy images shown in Fig. 1(b) and (c) are the c-PEI particles obtained with DVS crosslinking of Mn: 1200 and 60 000 g/mol PEI, respectively. It is important to note that even very small PEI particles can be separated with centrifugal separation at different rates of rpm for particle mixtures of different sizes as they may have different potential uses. To confirm that PEI chains are crosslinked via DVS, FT-IR analysis of PEI and c-PEI were carried out and are given in Fig. 2(a). As the amine groups are linked with DVS, the most obvious peak at 1120 cm−1 for c-PEI, belonging to S O stretching, is clearly seen. Other common peaks such as N H wagging at 813 cm−1 , N H bending about 1658 cm−1 , N H stretching 3287 cm−1 , C H bendings about 1299, 1458, and 1570 cm−1 and C H stretching at about 2817 and 2937 cm−1 are clearly seen for both PEI and c-PEI microgels. To determine whether the crosslinking of PEI induced any thermal stability into branched PEI, thermogravimetric analysis of PEI and c-PEI were carried out and the results are given in Fig. 2(b). As can be seen the PEI starts to degrade about 213 ◦ C with 97.6% weight loss and at 323 ◦ C with 74.2% weight loss and continues to degrade up to 400 ◦ C with almost 99% weight loss. The onset of the degradation of c-PEI is about 300 ◦ C and it continuously degrades up to about 400 ◦ C with 82.6 wt% weight loss. It is obvious that the difference between weight loss of c-PEI and PEI is about 17.44%,

N. Sahiner / Colloids and Surfaces A: Physicochem. Eng. Aspects 433 (2013) 212–218

215

(a)

(1) PEI

(2) c-PEI particles

A 1101

1051

1658

2937

Naproxene (NP) 813

70

1458

2817

1570

1299

60

Drug release (mg/g)

3287

1120

4000

3500

3000

2500 2000 Wavelength (cm-1)

1500

1000

650

100.0

(b) 80.0

60.0

50 40 30 20

10 0

40.0

0

2

4

(2) c-PEI particles

20.0

%17.44

6

8

10

12

Time (h) Fig. 4. Naproxene (NP) release profile from c-PEI particles in PBS.

a b

(1) PEI

0.0 100.0

200.0

300.0

400.0

500.0

600.0

modifiability with a simple quaternization agent such as CH3 I. As illustrated in Fig. 3(b), the c-PEI microgel zeta potential increased to almost +26 mV from +18.6 mV by chemical modification with CH3 I at room temperature. The zeta potential measurements of c-PEI were carried out in 0.01 M KNO3 as illustrated in Fig. 3(b). To illustrate that c-PEI can even be employed as a drug delivery material, Naproxene, NP a non-steroidal and anti-inflammatory drug was chosen as a model active agent, and used for loading into c-PEI from ethanol, and released into PBS at ambient temperatures. The loading efficiency of NP into c-PEI particles was 83.5% from 300 ppm 50 mL ethanol in NP solution for 200 mg c-PEI particles for 12 h at room temperature. The release experiment was carried out with NP-loaded 50 mg c-PEI particles from a dialysis membrane (MW cut off >12 000) into 25 mL PBS at 400 rpm mixing rate at ambient temperature. The chemical structure and the release profile of NP from c-PEI are shown in Fig. 4. As can be seen NP has an acid group, it was assessed that this group can readily interact with the quaternized amine groups of c-PEI via electrostatic interaction, and this interaction can readily be surmounted in PBS providing efficient release of the loaded NP from c-PEI particles. It is obvious

Temp Cel

Fig. 2. (a) FT-IR spectra and (b) thermogram of (1) PEI and (2) c-PEI particles.

which can be attributed to the croslinking of PEI chains generating more thermal stability. Turning branched PEI into crosslinked microgel form provides advantages in terms of their handling in applications such as environmental and medical fields, as templates for metal nanoparticle preparation, and as antimicrobial agents e.g., it is possible to design new materials for wound dressing materials with antimicrobial properties that can be made by embedding c-PEI into hydrogel films containing different functional groups. To further corroborate that the prepared c-PEI particles can be separated by simple filtration with filter paper or syringe, PEI particles were filtered with filter paper (>2 ␮m) and then syringe filtered with >0.8 ␮m. As illustrated in Fig. 3(a), the particles with sizes about 550 and 1200 nm can be readily obtained by these simple filtration methods or centrifugation. The other validation for the versatility of PEI microgel is their

100 Filtered PEI microgel

80

Not filtered PEI microgel

pEI microgel

0.8

60

Power

Intensity

1

(a)

40 20

m-pEI microgel

(b)

0.6 0.4 0.2

0 0

500

1000

Diameter (nm)

1500

0 0

10

20

30

40

50

Zeta Potenal (mV)

Fig. 3. (a) The sizes of PEI with and without filtration at 558 and 1175 nm respectively, and (b) bare and CH3 I modified PEI microgels with 18.6 and 25.96 mV zeta potential, respectively.

216

N. Sahiner / Colloids and Surfaces A: Physicochem. Eng. Aspects 433 (2013) 212–218

Table 1 MBC and MIC values for PEI-based polymers. MBC values (mg/mL)

E. coli ATCC 8739

S. aureus ATCC 8739

E. coli ATCC 8739

S. aureus ATCC 8739

0.025 0.1 0.05

0.005 0.05 0.05

>0.1 >0.1 >0.05

0.1 0.1 0.1

from Fig. 4 that about 61.6 mg NP, that is about 98.4% of the loaded drug, was released in about 8 h. From the graph, in the first hour, the release is very fast with an almost linear release profile similar to most micro and nanoparticle drug release behavior, and between 1 and 6 h the release slowed down as almost over 90% of loaded NP released. Overall, it can be concluded that c-PEI can even be used for active agent absorption and delivery devices where fast absorption/release is required for certain applications such as removal of toxins or stimulants, etc. Due to the highly charged nature of c-PEI, it was expected that cPEI particles can also be used as antimicrobial materials. Therefore, c-PEI particles antimicrobial behavior against two common bacteria, E. coli and S. aureus, were tested. It was found that there was an observable antibacterial effect for c-PEI and modified c-PEI particles at the used concentrations (1–100 ␮g/mL) against all bacteria tested. Table 1 lists the MIC and MBC values of c-PEI particles. The MIC value is the lowest concentration of the particles where no visible bacterial growth or turbidity could be seen; in other words, a lower MIC value means a higher antibacterial effect. It is obvious from Table 1 that all the particles gave low MIC concentrations above 0.05 mg/mL for two bacteria. The MBC values determine the minimum amount of particle concentration that kills the bacteria, and it is obvious that c-PEI-based particles have MBC values about 0.1 mg/mL for both bacteria. Therefore, it can be concluded that PEI polymer and its modified particles can retain their antimicrobial property and can be used as bactericidal agents for various biomedical applications. Complimentary to the versatility of c-PEI nanoparticles, metal nanoparticles, such Ni and Co, can be readily prepared within the c-PEI network and used as catalyst systems for reduction of 4-nitro phenol and hydrogen production from the hydrolysis of NaBH4 . It is very well known that 4-NP is an environmental concern as a toxic organic pollutant and a carcinogenic material that is discharged to the environment from various industrial sites such as oil refineries, chemical, agriculture and paint industries, as well as various others. Therefore, the conversion of 4-NP to the more useful and less harmful 4-AP form is desirable [28–30]. To demonstrate that c-PEI-M composite system can be used as a catalyst system, the reduction of 4-NP to 4-AP in aqueous media with these catalyst systems was completed. As revealed in Fig. 5, the complete reduction of 4-NP with c-PEI-Ni microgel composite catalyst system is very fast and almost all 4-NP was completely converted to 4-AP in about 15 min from 0.01 M aqueous solution of 4-NP. This demonstrates that c-PEI-Ni particles are very effective in some environmental applications such as the reduction of some organic pollutants. Another potential catalyst application of these prepared c-PEIM composite systems is their usefulness in clean and renewable energy production, such as hydrogen from NaBH4 hydrolysis. Recently, various polymer composites have been paid great and increasing attention for H2 generation from metal hydride hydrolysis [31,32]. Among these materials, the use of microgels is a very novel and original concept. Here, the prepared c-PEI particles were loaded with Ni(II) and Co(II) ions and reduced to their corresponding metal nanoparticles and were used successfully in hydrogen production from the hydrolysis of NaBH4 as illustrated in Fig. 6.

Fig. 5. The reduction of 4-nitro phenol (4-NP) to 4-amino phenol (4-AP) in the presence of c-PEI-Ni catalyst system, and the loss of absorption maximum of 4-NP with time. Reaction conditions: 50 mL 0.01 M 4-NP solution containing 0.4 M NaBH4 using 0.070 g c-PEI-M composite system.

Volume of produced H2 (ml)

PEI c-PEI-NaOH c-PEI-CH3 I

MIC values (mg/mL)

300

(a)

c-PEI-Ni

250

200

c-PEI-Co

150 100 50

0 0

50

100

150

200

250

Time (min) 3000

(b) Volume of produced H2 (ml)

Polymer

c-PEI-Co

2500 2000 1500 1000 500

c-PEI-Ni

0 0

50

100

150

200

250

Time (min) Fig. 6. (a) The hydrolysis reaction of NaBH4 by c-PEI-M (M: Ni and Co) without the use of a base at 30 ◦ C using 0.200 g PEI template (loading capacity of 5.56 mg Co and 70.33 mg Ni per gram of PEI). (b) The same NaBH4 hydrolysis reaction per one of M catalyst. Reaction condition: 0.0965 g M NaBH4 in 50 mL DI.

N. Sahiner / Colloids and Surfaces A: Physicochem. Eng. Aspects 433 (2013) 212–218

217

as DNA condensing, as antimicrobial agents, and for gene therapy purposes. The bigger sizes (<1 ␮m), on the other hand, provide easy handling for in situ metal nanoparticle preparation and catalysis in addition to environmental applications such as the removal of toxic species. As proven here these catalyst systems can also be used in the reduction of toxic species such as 4-NP and even hydrogen production from the hydrolysis of NaBH4 . As demonstrated here that a versatile material such as PEI can be made in particle form in one-step readily and can have many applications. Our current investigation is focused on the use of different metal nanoparticleembedded PEI in catalysis, environmental applications, and in the biomedical field for drug targeting and antimicrobial applications.

Fig. 7. Digital camera images of (a) PEI microgels, (b) magnetic ferrite-containing PEI microgel, and (c) and their behavior under an externally applied magnetic field.

Fig. 6(a) reveals that the Ni amount (70.33 mg) is much more than Co (5.56 mg) per gram of c-PEI composite catalyst system. Their use in NaBH4 hydrolysis showed that Ni-containing particles of c-PEI produced the same amount of H2 (252 mL) faster than the co-containing c-PEI composite system. This is due to the higher Ni content of c-PEI particles. If the same graphic as shown in Fig. 6(b), is redrawn for per mole metal catalyst that requires a greater amount of PEI Co template, the H2 production rate for Co is much faster than Ni nanoparticles. This is in accordance with the literature [33–36]. One of the most important findings of the c-PEI-M system reported here is that these composite catalyst systems do not require the basic medium that almost all the NaBH4 catalysis reactions in the presence of metal nanoparticles, including noble metals such as Au, Pt, Ru and Rh, need [35,31,37]. The presence of the amine group in c-PEI could be accommodating for the lack of basic medium as the reaction is taking place about the periphery of metal nanoparticles that are sited within c-PEI matrices. All these are extraordinary and appealing; in fact; this phenomena is currently under investigation by our group. The use of c-PEI microgel as template for metal nanoparticles is captivating as even magnetic ferrite particles can also be prepared in situ within the macro, micro and even nanogels [38]. Therefore, as a proof of concept, here it is also shown that magnetic ferrite particles can be prepared in situ by loading Fe(II) and Fe(III) into c-PEI from the corresponding aqueous solution of the metal ions, and precipitating by NaOH. As the digital camera images show in Fig. 7, the magnetic-PEI microgels can be readily separated from an aquatic environment by a simple externally applied magnetic field [39]. This type of multipurpose material, such as magnetic fieldresponsive c-PEI microgel, has great potential in various applications e.g., removal or enrichment of certain species of DNA, gene or toxic species from various media or control of different reactions such reduction and hydrolysis of various organic reagents as demonstrated earlier [40,41]. 4. Conclusion With this investigation, a facile method for PEI microgel preparation was reported in a single step using an AOT microemulsion system. The obtained PEI particle size distribution spanned from tens of nanometers to tens of micrometers. The prepared PEI particles were demonstrated to be versatile as antimicrobial agents, drug delivery devices and templates for metal nanoparticle preparation. It was proven that PEI particles are resourceful as they can be further modified to increase positive charges for potential applications in microbiology as antimicrobial materials. The wide size distribution of PEI particles is another advantage as the smaller sizes of PEI particles (>1 ␮m) can be used in biomedical fields such

Acknowledgements N. Sahiner is grateful to the Turkish Academy of Science for partial support under the 2008 TUBA-GEBIP program. The support from Sahin Demirci, Selin Sagbas, and Duygu Alpaslan for repetition of some of the experiments and antibacterial measurements is greatly appreciated. References [1] T. He, V. Chan, Covalent layer-by-layer assembly of polyethyleneimine multilayer for antibacterial applications, J. Biomed. Mater. Res. A 95A (2010) 454–464. [2] B.A. Lander, K.D. Checchi, S.A. Koplin, V.F. Smith, T.L. Domanski, D.D. Isaac, S. Lin, Extracytoplasmic stress responses induced by antimicrobial cationic polyethylenimines, Curr. Microbiol. 65 (2012) 488–492. [3] M.A. Barakat, N. Sahiner, Cationic hydrogels for toxic arsenate removal from aqueous environment, J. Environ. Manage. 88 (2008) 955–961. [4] N. Sahiner, Hydrogels of versatile size and architecture for effective environmental applications, Turkish J. Chem. 32 (2008) 113–123. [5] Z.H. Liu, Z.Y. Zhang, C.R. Zhou, Y.P. Jiao, Hydrophobic modifications of cationic polymers for gene delivery, Prog. Polym. Sci. 35 (2010) 1144–1162. [6] M.N. Collins, C. Birkinshaw, Investigation of the swelling behavior of crosslinked hyaluronic acid films and hydrogels produced using homogeneous reactions, J. Appl. Polym. Sci. 109 (2008) 923–931. [7] D.M. Xu, S.D. Yao, Y.B. Liu, K.L. Sheng, J. Hong, P.J. Gong, L. Dong, Size-dependent properties of M-PEIs nanogels for gene delivery in cancer cells, Int. J. Pharm. 338 (2007) 291–296. [8] L. Dong, H. Xu, Y.B. Liu, B. Lu, D.M. Xu, B.H. Li, J. Gao, M. Wu, S.D. Yao, J. Zhao, Y.J. Guo, M-PEIs nanogels: potential nonviral vector for systemic plasmid delivery to tumor cells, Cancer Gene Ther. 16 (2009) 561–566. [9] R. Sunasee, P. Wattanaarsakit, M. Ahmed, F.B. Lollmahomed, R. Narain, Biodegradable and nontoxic nanogels as nonviral gene delivery systems, Bioconjugate Chem. 23 (2012) 1925–1933. [10] G. Cheng, Y.Y. He, L. Xie, Y. Nie, B. He, Z.R. Zhang, Z.W. Gu, Development of a reduction-sensitive diselenide-conjugated oligoethylenimine nanoparticulate system as a gene carrier, Int. J. Nanomed. 7 (2012) 3991–4006. [11] T. Yang, A. Hussain, S. Bai, I.A. Khalil, H. Harashima, F. Ahsan, Positively charged polyethylenimines enhance nasal absorption of the negatively charged drug, low molecular weight heparin, J. Control. Release 115 (2006) 289–297. [12] D. Mishra, H.C. Kang, Y.H. Bae, Reconstitutable charged polymeric (PLGA)(2)-bPEI micelles for gene therapeutics delivery, Biomaterials 32 (2011) 3845–3854. [13] N.C. Bellocq, S.H. Pun, G.S. Jensen, M.E. Davis, Transferrin-containing, cyclodextrin polymer-based particles for tumor-targeted gene delivery, Bioconjugate Chem. 14 (2003) 1122–1132. [14] S. Kawakami, Y. Higuchi, M. Hashida, Nonviral approaches for targeted delivery of plasmid DNA and oligonucleotide, J. Pharm. Sci. 97 (2008) 726–745. [15] C.W. Lin, M.S. Jan, J.H.S. Kuo, L.J. Hsu, Y.S. Lin, Protective role of autophagy in branched polyethylenimine (25 K)- and poly(l-lysine) (30–70 K)-induced cell death, Eur. J. Pharm. Sci. 47 (2012) 865–874. [16] A.K. Larsen, D. Malinska, I. Koszela-Piotrowska, L. Parhamifar, A.C. Hunter, S.M. Moghimi, Polyethylenimine-mediated impairment of mitochondrial membrane potential, respiration and membrane integrity: implications for nucleic acid delivery and gene therapy, Mitochondrion 12 (2012) 162–168. [17] Y. Gao, L.L. Chen, Z.W. Zhang, W.W. Gu, Y.P. Li, Linear cationic click polymer for gene delivery: synthesis, biocompatibility, and in vitro transfection, Biomacromolecules 11 (2010) 3102–3111. [18] M. Thomas, Q. Ge, J.J. Lu, J.Z. Chen, A.M. Klibanov, Cross-linked small polyethylenimines: while still nontoxic, deliver DNA efficiently to mammalian cells in vitro and in vivo, Pharm. Res. 22 (2005) 373–380. [19] A. Beyerle, M. Irmler, J. Beckers, T. Kissel, T. Stoeger, Toxicity pathway focused gene expression profiling of PEI-based polymers for pulmonary applications, Mol. Pharm. 7 (2010) 727–737. [20] D. Velluto, S.N. Thomas, E. Simeoni, M.A. Swartz, J.A. Hubbell, PEG-b-PPS-b-PEI micelles and PEG-B-PPS/PEG-b-PPS-b-PEI mixed micelles as non-viral vectors

218

[21]

[22]

[23]

[24]

[25]

[26]

[27]

[28]

[29]

[30]

N. Sahiner / Colloids and Surfaces A: Physicochem. Eng. Aspects 433 (2013) 212–218 for plasmid DNA: tumor immunotoxicity in B16F10 melanoma, Biomaterials 32 (2011) 9839–9847. D. Dey, M. Inayathullah, A.S. Lee, M.C. LeMieux, X.X. Zhang, Y. Wu, D. Nag, P.E. De Almeida, L. Han, J. Rajadas, J.C. Wu, Efficient gene delivery of primary human cells using peptide linked polyethylenimine polymer hybrid, Biomaterials 32 (2011) 4647–4658. Y.Y. Liu, Z.L. Liu, Y. Wang, Y.R. Liang, X.J. Wen, J.Y. Hu, X.Y. Yang, J. Liu, S.H. Xiao, D. Cheng, Investigation of the performance of PEG-PEI/ROCK-II-siRNA complexes for Alzheimer’s disease in vitro, Brain Res. 1490 (2013) 43–51. W. Shao, A. Paul, S. Abbasi, P.S. Chahal, J.A. Mena, J. Montes, A. Kamen, S. Prakash, A novel polyethyleneimine-coated adeno-associated virus-like particle formulation for efficient siRNA delivery in breast cancer therapy: preparation and in vitro analysis, Int. J. Nanomed. 7 (2012) 1575–1586. R.E.B. Fitzsimmons, H. Uludag, Specific effects of PEGylation on gene delivery efficacy of polyethylenimine: interplay between PEG substitution and N/P ratio, Acta Biomater. 8 (2012) 3941–3955. A. Heydari-Gorji, Y. Belmabkhout, A. Sayari, Polyethylenimine-impregnated mesoporous silica: effect of amine loading and surface alkyl chains on CO2 adsorption, Langmuir 27 (2011) 12411–12416. N. Sahiner, X.Q. Ja, One-step synthesis of hyaluronic acid-based (sub)micron hydrogel particles: process optimization and preliminary characterization, Turkish J. Chem. 32 (2008) 397–409. S. Butun, F.G. Ince, H. Erdugan, N. Sahiner, One-step fabrication of biocompatible carboxymethyl cellulose polymeric particles for drug delivery systems, Carbohydr. Polym. 86 (2011) 636–643. N. Sahiner, O. Ozay, N. Aktas, Aromatic organic contaminant removal from an aqueous environment by p(4-VP)-based materials, Chemosphere 85 (2011) 832–838. S. Wunder, F. Polzer, Y. Lu, Y. Mei, M. Ballauff, Kinetic analysis of catalytic reduction of 4-nitrophenol by metallic nanoparticles immobilized in spherical polyelectrolyte brushes, J. Phys. Chem. C 114 (2010) 8814–8820. J.Y. Yuan, F. Schacher, M. Drechsler, A. Hanisch, Y. Lu, M. Ballauff, A.H.E. Muller, Stimuli-responsive organosilica hybrid nanowires decorated with metal nanoparticles, Chem. Mater. 22 (2010) 2626–2634.

[31] S. Sagbas, N. Sahiner, A novel p(AAm-co-VPA) hydrogel for the Co and Ni nanoparticle preparation and their use in hydrogel generation from NaBH4 , Fuel Process. Technol. 104 (2012) 31–36. [32] O. Ozay, N. Aktas, E. Inger, N. Sahiner, Hydrogel assisted nickel nanoparticle synthesis and their use in hydrogen production from sodium boron hydride, Int. J. Hydrogen Energy 36 (2011) 1998–2006. [33] S. Butun, N. Sahiner, A versatile hydrogel template for metal nano particle preparation and their use in catalysis, Polymer 52 (2011) 4834–4840. [34] S. Sagbas, N. Sahiner, Tunable poly(2-acrylamido-2-methyl-1-propan sulfonic acid) based microgels with better catalytic performances for Co and Ni nanoparticle preparation and their use in hydrogen generation from NaBH4 , Int. J. Hydrogen Energy 37 (2012) 18944–18951. [35] N. Sahiner, S. Butun, T. Turhan, p(AAGA) hydrogel reactor for in situ Co and Ni nanoparticle preparation and use in hydrogen generation from the hydrolysis of sodium borohydride, Chem. Eng. Sci. 82 (2012) 114–120. [36] N. Sahiner, O. Ozay, E. Inger, N. Aktas, Superabsorbent hydrogels for cobalt nanoparticle synthesis and hydrogen production from hydrolysis of sodium boron hydride, Appl. Catal. B: Environ. 102 (2011) 201–206. [37] N. Sahiner, S. Sagbas, The preparation of poly(vinyl phosphonic acid) hydrogels as new functional materials for in situ metal nanoparticle preparation, Colloids Surf. A: Physicochem. Eng. Aspects 418 (2013) 76–83. [38] N. Sahiner, Colloidal nanocomposite hydrogel particles, Colloid Polym. Sci. 285 (2007) 413–421. [39] O. Ozay, S. Ekici, Y. Baran, S. Kubilay, N. Aktas, N. Sahiner, Utilization of magnetic hydrogels in the separation of toxic metal ions from aqueous environments, Desalination 260 (2010) 57–64. [40] A. Elaissari, F. Ganachaud, C. Pichot, Biorelevant latexes and microgels for the interaction with nucleic acids, Colloid Chem. II 227 (2003) 169–193. [41] N. Sahiner, P. Ilgin, Multiresponsive polymeric particles with tunable morphology and properties based on acrylonitrile (AN) and 4-vinylpyridine (4-VP), Polymer 51 (2010) 3156–3163.