Interpenetrating polymer network (IPN) nanogels based on gelatin and poly(acrylic acid) by inverse miniemulsion technique: Synthesis and characterization

Colloids and Surfaces B: Biointerfaces 83 (2011) 204–213

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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb

Interpenetrating polymer network (IPN) nanogels based on gelatin and poly(acrylic acid) by inverse miniemulsion technique: Synthesis and characterization Veena Koul a,∗ , Raja Mohamed a,c , Dirk Kuckling b , Hans-Jürgen P. Adler c , Veena Choudhary d a

Centre for Biomedical Engineering, Indian Institute of Technology, 110016 New Delhi, India Department of Chemistry, University of Paderborn, Warburger Str. 100, D-33098 Paderborn, Germany c Institute for Macromolecular Chemistry and Textile Chemistry, Dresden University of Technology, D-01069 Dresden, Germany d Centre for Polymer Science and Engineering, Indian Institute of Technology, 110016 New Delhi, India b

a r t i c l e

i n f o

Article history: Received 16 April 2010 Received in revised form 22 October 2010 Accepted 9 November 2010 Available online 23 November 2010 Keywords: Nanogels Interpenetrating polymer networks Gelatin Poly(acrylic acid) Inverse miniemulsion polymerization

a b s t r a c t Novel interpenetrating polymer network (IPN) nanogels composed of poly(acrylic acid) and gelatin were synthesised by one pot inverse miniemulsion (IME) technique. This is based on the concept of nanoreactor and cross-checked from template polymerization technique. Acrylic acid (AA) monomer stabilized around the gelatin macromolecules in each droplet was polymerized using ammonium persulfate (APS) and tetramethyl ethylene diamine (TEMED) in 1:5 molar ratio and cross-linked with N,N-methylene bisacrylamide (BIS) to form semi-IPN (sIPN) nanogels, which were sequentially cross-linked using glutaraldehyde (Glu) to form IPNs. Span 20, an FDA approved surfactant was employed for the formation of homopolymer, sIPN and IPN nanogels. Formation of stable gelatin–AA droplets were observed at 2% surfactant concentration. Dynamic light scattering (DLS) and scanning electron microscopy (SEM) studies of purified nanogels showed small, spherical IPN nanogels with an average diameter of 255 nm. In contrast, sIPN prepared using the same method gave nanogels of larger size. Fourier-transform infrared (FT-IR) spectroscopy, SEM, DLS, X-ray photoelectron spectroscopy (XPS) and zeta potential studies confirm the interpenetration of the two networks. Leaching of free PAA chains in sIPN upon dialysis against distilled water leads to porous nanogels. The non-uniform surface of IPN nanogels seen in transmission electron microscopy (TEM) images suggests the phase separation of two polymer networks. An increase of N/C ratio from 0.07 to 0.17 (from PAA gel to IPN) and O/C ratio from 0.22 to 0.37 (from gelatin gel to IPN) of the nanogels by XPS measurements showed that both polymer components at the nanogel surface are interpenetrated. These nanogels have tailoring properties in order to use them as high potential drug delivery vehicles for cancer targeting. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Polymeric vehicles in the nanometer range have been found useful in the field of engineering and medicine [1–3]. The various challenges faced in the clinical practice include efficient drug loading, sustained/controlled drug release, their stability during circulation in the blood stream, specific targeting to the deceased cells and diffusion through cell membranes. These challenges can overcome by synthesizing multiple polymer component nanogel systems to achieve different goals in biological environment. Such well defined functional nanogels not only provide uniform size and large surface area, their surface functional groups can have a great

∗ Corresponding author at: Centre for Biomedical Engineering, Indian Institute of Technology, 191, Block II, Biosensors Lab, New Delhi, India. E-mail address: [email protected] (V. Koul). 0927-7765/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2010.11.007

potential for efficient binding to various ligands of interest [4–6]. This improves the efficiency and reduces the side effects of the drugs used. Although many processes are available to synthesize polymeric nanogels (nano-hydrogels), miniemulsion (ME)/inverse miniemulsion (IME) [7] is a promising process to synthesize multiple component polymeric particles. It is possible to obtain narrowly distributed submicron sized monomer [8] and polymer [3] droplets in a continuous phase while the inverse miniemulsion is stirred with high shear stress. It provides advantage over nanophase morphology and polymerization of monomers to form network structures. Since the reaction is taking place in biphasic media, it is more challenging than the reactions in homogeneous bulk and solution conditions. Recently, a considerable effort has been focused to develop multicomponent multifunctional nanogels [10,11] using combination of different polymers. Since all the droplets formed in IME process as such duplicate into particles (nano-reactors) upon polymer-

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ization, it allows one to perform a variety of reactions based on the chemical composition and physical/chemical properties of the monomers present in the dispersed droplets to get novel architectures [12,13]. For example, hybrid particles [14–16], nanocapsules [17], metallic particles [18] and nanospheres of polymer blends [3], core–shell and IPN nanogel synthesis by ME/IME process are well reported. Several differently conjugated semi-conducting polymers emulsified to prepare nanospheres of polymer blends for solar cell application were reported by Kietzke et al. [3]. Interpenetrating polymer networks (IPNs) [19,20] are formed when two or more cross-linked polymer networks are at least partially interlaced on a molecular scale but not covalently bonded to each other and cannot be separated unless chemical bonds are broken. IPNs, especially made up of hybrid of synthetic and natural polymers, with altered morphologies, perform better with the synergistic combination of homopolymer properties, which is not possible to achieve with homopolymers (e.g. better mechanical properties [21], sustained/controlled drug delivery etc.). Recently, Owens et al. [22] prepared IPN nanogels composed of poly(Nisopropyl acrylamide) (PNIPAM) and poly(acrylic acid) (PAA) by two step IME sequential polymerization method and evaluated for its temperature sensitivity, swelling, etc. Xia et al. [23] reported physically bonded PAA–PNIPAM IPN nanoparticles as thermoresponsive system by seed and feed precipitation polymerization method. Very recently, Ethirajan et al. have reported the synthesis of gelatin nanogels by inverse miniemulsion process using amphiphilic block copolymer poly[(butylenes-co-ethylene)-blockethylene oxide)] as emulsifier [24]. However, these methods require either two steps or tedious procedure to prepare the nanogels. The present paper describes the synthesis and optimization of IPN nanogels by one pot IME process. Previously our research group have demonstrated the biocompatibility and sustained drug release profile [25,26] from IPN bulk gels made of gelatin and PAA for the treatment of Osteomylitis. However, the bulk gels cannot function as injectable systems because of their method of synthesis and size, nanogels based on those polymers became the research of interest here. Gelatin has appealing biomedical properties such as safety, biocompatibility, and biodegradability. In addition gelatin possesses reactive funtional groups. This biopolymer forms a physical gel network in water below 30 ◦ C which is stabilized by hydrophobic interaction and hydrogen bonding. This process is reversible upon heating. However, upon chemical cross-linking, it forms irreversible network structures. Gelatin has degradable amino acid chains with low antigenicity and is thus widely used in tissue engineering, coating material for pharmaceuticals, wound dressing, adsorbing pad, matrix for drug delivery, etc. [27–31]. The cross-linker glutaraldehyde was introduced in the form of droplets to stabilize the particles. PAA is a biocompatible pH sensitive hydrophilic polymer. There are many reports available for the synthesis of PAA latexes by IME from AA using free radical water/oil soluble initiators [8]. However, to the best of our knowledge, IME was not yet utilized to synthesize IPN nanogels. The main objective of the present investigation was to synthesize IPN nanogels by inverse miniemulsion process using identical components which were used for the preparation of bulk gels mainly because of three reasons. First, such polymers have demonstrated excellent biocompatibility when used as bulk gels. Second, their multiple functionality (with COOH and NH2 groups) is easily accessible for modification to ligands of interest. Third, their size in nano range can penetrate through the pores of the endothelial junctions found in tumor cells. Here a novel synthesis of IPN nanogels composed of gelatin and PAA by one pot inverse miniemulsion method and the characterization of the obtained products by various analytical tools like SEM, TEM, DLS and XPS is being described.

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Scheme 1. Synthesis of sIPN and IPN nanogels by one pot inverse miniemulsion process.

2. Experimental 2.1. Materials Acrylic acid (AA) (Aldrich) was purified by vacuum distillation before use. Gelatin (Sigma) Type A, 175 bloom, N,N-methylene bisacrylamide (BIS) (Carl Roth GmbH), Span 20 (Sigma), glutaraldehyde, 25% solution (Glu) (Sigma), ammonium persulfate (APS) (Sigma), tetramethyl ethylene diamine (TEMED) (Merck), sodium hydroxide (Sigma), cyclohexane (BDH) and dialysis membrane 50,000 MWCO (Spectrum Laboratories) were used as received. 2.2. One-pot synthesis of IPN nanogels The process of synthesis of sIPN and IPN nanogels is depicted in Scheme 1. Three steps are involved in the process. In step A, stable droplets of AA, gelatin and NaOH mixture were obtained by sonication using APS as initiator for radical generation. In step B, accelerator (TEMED) and cross-linker (BIS) has been employed to polymerize and cross-link AA. The sIPN thus formed are further used in step C to make full IPN by the addition of glutaraldehyde as cross-linker for gelatin. A typical synthetic procedure used is explained in the following sections. 2.3. Preparation of stable IME droplets Gelatin (0.5 g) was dissolved in 5 ml of distilled water at 30 ◦ C in a 100 ml beaker. To this purified AA (0.5 g, 6.94 mmol) was added along with 1 g of NaOH and stirred for 15 min till a homogenous mixture was obtained. 2 g of Span 20 was separately dissolved in 100 ml of cyclohexane. Then aqueous monomer/polymer mixture was added to the non-aqueous cyclohexane phase, and to this APS (0.047 g, 3 mol% of AA) was added and the mixture was sonicated for 5 min using Bronson sonicator with an output of 6 and duty cycle of 30% while purging with nitrogen to get stable inverse miniemulsion droplets. 2.4. Preparation of homopolymer nanogels Homopolymer nanogels of PAA and gelatin are designated as Ax and Gx respectively. Ax denotes PAA nanogel cross-linked with BIS and Gx denotes gelatin nanogel cross-linked with Glu (see Table 1). AA homopolymer nanogels were prepared from droplets containing only AA, BIS, APS and TEMED. After the addition of TEMED, the reaction was carried out for 2 h. In case of gelatin homopoly-

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Table 1 Sample designation and composition of reagents used for the synthesis of nanogels [water 5 ml, NaOH 1 g, cyclohexane 100 ml, Span 20 (6 g), APS:TEMED ratio 1:5]. Sample code

Ax Gx AxGx AGx AxG

Composition (g)

Cross-linker (g)

Acrylic acid

Gelatin

BIS

Glutaraldehyde

0.5 – 0.5 0.5 0.5

– 0.5 0.5 0.5 0.5

0.152 – 0.152 – 0.152

– 0.06 0.06 0.06 –

Water (ml)

NaOH (g)

Cyclohexane (ml)

Span 20 (g)

APS:TEMED (mol:mol)

5 5 5 5 5

1 1 1 1 1

100 100 100 100 100

6 6 6 6 6

1:5 1:5 1:5 1:5 1:5

mer nanogels, gelatin was inversely emulsified with cyclohexane to form droplets which were cross-linked with glutaraldehyde and the reaction was carried out for 20 h. 2.5. Preparation of sIPN and IPN nanogels Semi-IPN samples were denoted as AGx and AxG. AGx represents the sample where gelatin is cross-linked with Glu and PAA is free. Whereas, in AxG, PAA was cross-linked with BIS and gelatin chains are left free. IPN samples are designated as AxGx. To prepare sIPN nanogels the droplets from step A prepared by IME were poured into a 250 ml 3-necked round bottom flask fitted with rubber septa. To this TEMED (0.154 ml) and 0.152 g of BIS [IPNs were also prepared with varying cross-linker concentrations to study the effect of cross-linker concentrations on size, morphology, etc. (data not shown)], dissolved in distilled water was added through the septa using a syringe and stirred at room temperature using a magnetic stirrer. Argon was purged throughout the reaction and the reaction was continued for 2 h to get the sIPN nanogels AxG. To prepare AGx, no BIS was added in step B. AA was polymerized into PAA for 2 h followed by Glu addition to cross-link the gelatin chains and this reaction was carried out for 20 h. For the preparation of IPN all three steps were performed as depicted in Scheme 1.

2.8.2. FTIR spectra FT-IR spectra of different nanogel powders were obtained on a “Research Series RS 1000” spectrometer (UNICAM Analytische Systeme GmbH). Freeze dried nanogel powder (approximately 1 mg) was mixed with KBr to make a pellet.

2.8.3. Dynamic light scattering studies (DLS) DLS measurements were performed using commercial laser light scattering equipment (ALV/DLS/SLS-5000) equipped with an ALV-5000/EPP multiple digital time correlator and laser geniometer system ALV/CGS-8F S/N 025 was used with helium neon laser (Uniphase 1145P, output power of 22 mW and wavelength of 632.8 nm) as the light source. Experiments were carried out in the range of scattering angels  = 30–120◦ . All samples were filtered using a 0.45 ␮m membrane filter before measurements. 2.8.4. Zeta potential measurement Zeta potential measurements were performed with a Zetasizer nano ZC instrument (Malvern Instruments). Measurements were performed in distilled water at the concentration of 100 ␮g/ml and the average value of at least ten measurements were adopted on the zeta potential at given pH.

2.6. Template polymerization Ax was taken as template in gelatin solution to synthesize particles. 0.05 g of Ax was dispersed in 50 ml of gelatin solution (2 mg/ml) and left to swell for 2 h. Gelatin chains were cross-linked with Glu (0.012 g) for 20 h and formed nanogels were purified.

2.8.5. Lumifuge Stability measurements of nanogels were performed using Lumifuge 114 (LUM GmbH, Germany) separation analyser. Sample filled glass tubes were accelerated at the rate of 3000 rpm. The slope of the sedimentation curves was used to calculate the sedimentation velocity and to obtain stability of the nanogels.

2.7. Purification of nanogels Homopolymer, sIPN and IPN nanogels were purified by repeated centrifugation and dialysis. The final reaction mixture was transferred into centrifuge tubes in equal amount and centrifuged at 22,000 rpm using Sigma 3K high speed centrifuge for 5 min, supernatent was drained, sediment was redispersed with 20 ml of methanol using a bath sonicator followed by centrifugation. This process was repeated 3 times with methanol followed by water to remove cyclohexane and other unreacted components. In order to remove impurities, the final sediment was redispersed in 7–10 ml of distilled water, dialysed against distilled water using 50,000 MWCO membrane for 5–7 days till free of impurities. It was later freeze dried using Christ-Alpha freeze drier at −52 ◦ C to get nanogel powders. 2.8. Characterization 2.8.1. UV-visible spectrophotometer During dialysis of the nanogels, the dialysate solutions were analysed (at different time intervals) using Perkin-Elmer UV-visible spectrophotometer Lambda 45, to ensure the complete removal of unreacted monomer/cross-linkers from the samples. Scanning of the samples was performed at 200–800 nm.

2.8.6. Scanning electron microscopy (SEM) SEM images were taken with Gemini microscope DMS-982 (Zeiss, Germany). Samples were prepared in the following manner. 3 cm × 3 cm aluminium foils were cut and placed on a cleaned glass plate of size 10 cm × 10 cm then cleaned with dust free tissue paper moistured with acetone. Diluted dispersions of nanogels in distilled water (100 ␮g/ml) were prepared, sonicated for 5 min using Branson sonicator at room temperature and filtered through a 0.45 ␮m cellulose acetate filter. The filtrate was placed on the aluminium foil to form a bigger droplet of aqueous dispersion and covered with a glass petri-dish, dried at 37 ◦ C over night. Small pieces of dried films were cut and placed on the SEM sample stage and coated with gold to increase the contrast and quality of the images.

2.8.7. Transmission electron microscopy (TEM) TEM images were recorded using Phillips CM12 instrument. Images were taken in the imaging mode at 100 kV. Stock dispersion of nanogel in distilled water (100 ␮g/ml) was prepared, sonicated for 5 min using Branson sonicator at room temperature and filtered through 0.45 ␮m cellulose acetate syringe filter. 100 ␮l of the filtrate was placed on the copper grid and dried in an air circulated incubator for 24 h at 37 ◦ C before taking the images.

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2.8.8. X-ray photoelectron spectroscopy (XPS) XPS studies were performed using Physical Electronics ESC 5700 at Ae Kapha E = 1486.6 eV and Epass = 29.35 eV. The results were analysed using “Multipak” programe fitting provided with the software. Samples were scanned at an angle of 45◦ and at a depth of 3–5 nm. The raw data obtained were treated with Multipak software to get the resolved spectrum. The percent atomic concentrations were also determined. 3. Results and discussion The main objective of the present work was to synthesize and characterize tailorable IPN nanogels composed of gelatin–PAA, which could later be used as targeted carriers of therapeutic agents for the treatment of solid tumors. Gelatin is a natural biopolymer and derived from the alkaline or acidic hydrolysis of collagen. It possesses a backbone with branched amino acid chains of varying length [32–34]. It has been found that gelatin and PAA form reversible complex in aqueous solution due to electrostatic interactions and hydrogen bonding [35]. Polymerization of AA in the presence of gelatin in aqueous solution indeed led to the formation of sIPN network bulk gels [36]. When the aqueous solution was emulsified in a non-aqueous continuous phase (cyclohexane), one can get aqueous droplets containing both gelatin and AA and further polymerization of AA monomer would lead to the formation of sIPN nanogels. This was achieved by IME polymerization technique. Table 1 shows the compositions of reagents used for the preparation different nanogels by IME process. Methanol and water were used to remove various unreacted components such as cyclohexane, unreacted AA, initiator, promotor and surfactant by centrifugation. The nanogels were further dispersed and dialysed against distilled water for a period of 7 days. Although stable IME were obtained with 2% Span 20, increasing the Span 20 concentration to 6% led to the formation of particles of smaller size. It was found that larger aggregates of the gelatin homopolymer nanogels were observed at the bottom of the reaction vessel. This could be due to the formation of nanogel aggregation because of unreacted free gelatin chains. However, PAA homopolymer nanogel inverse miniemulsions were stable even after several hours of reaction. 3.1. Synthesis of homopolymer nanogels Synthesis of poly(acrylic acid) PAA nanogels by IME process is well established using cyclohexane or liquid paraffin as continuous phase [37]. For the synthesis of nanogels, NaOH (liphophobe) to monomer ratio was kept constant (2:1 ratio) [8]. Liphophobe not only reduces AA’s solubility in cyclohexane, it also increases hydrophilicity of the AA and thus avoids Ostwald ripening. As summarized in Table 2, AA IME droplets showed excellent colloidal stability with the surfactant concentration of 2%. In the present study the concentration of BIS, Glu, initiator and promotor were kept constant. Various types of gelatin were utilized to make nanogels of size less than 200 nm by IME [24]. However, the cross-linker glutaraldehyde was introduced through IME droplets which require an extra step and high cross-linker concentration. Since glutaraldehyde is water soluble and has high affinity to free amino groups the simple mixing is enough to cross-link the droplets. Although one can find several reports to prepare gelatin particles by emulsification technique [38,39], for the present study a non-ionic, biocompatible surfactant, Span 20 has been preferred because it almost posses no interaction with proteins [40–42]. The average size of the gelatin nanogel was found to be 249 ± 20 nm which is almost equal to the size of gelatin nanogels obtained by Ethirajan et al. [24] with a lower PDI (0.111) in water.

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Since the stability of the droplets plays a major role in formation of particles, formed IME droplets were analysed using Lumifuge centrifuge for their stability to obtain optimum stable emulsion. This step was performed after the sonication procedure. In order to get a stable emulsion, initially the coarse dispersion was made by stirring the emulsion for atleast half an hour [till the mixture became intense turbid]. It has been found that faster sedimentation velocity indicates lower stability and slower sedimentation velocity indicates higher stability. Table 2 shows the sedimentation velocity data obtained at 3000 rpm for the IME droplets. Very low sedimentation velocity (0.46 ␮m/s) for AA emulsion indicates its excellent stability. From the initial studies we found that longer sonication time and lower Span concentration immediately separates the gelatin droplets from the continuous phase. But when mixed with AA an increase in stability (0.67 ␮m/s) of the AA–gelatin IME droplets were obtained (Table 2). The higher value of sedimentation velocity (1.12 ␮m/s) is indicative of lower colloidal stability of gelatin droplets. 3.2. Synthesis of IPN nanogels Our initial experiments demonstrate compatibility of Span 20 in preparing AA, gelatin and gelatin–AA droplets (see Table 2) in terms of stability of the emulsion. The stability and size of the IME droplets is a critical factor and is influenced by the surfactant concentration [43] (high concentration and low aqueous phase volume forms stable IME) and sonication parameters. Hence, various concentrations of Span 20 (0.05, 0.08, 0.12, 0.15, 0.5, 0.6, 0.675, 2, 3.2, 4.0 and 6.0%) and sonication parameters (3, 6 and 8 relative sonication output) were tried to achieve smaller and stable droplets. Formed miniemulsions were stable at shelf for 24 h at a minimum concentration of 2 g Span 20 and with ultrasonication output of 3 for 5 min. Since the single pot methodology has been adopted for the synthesis of IPN nanogel, the discussion regarding the stability and depletion is being elaborated below. 3.3. Stability Stability of the emulsion is of concern when multicomponent nanogel systems are synthesized from monomers by IME. Earlier reports on IME process showed coalescence (phase separation) of the droplets during reaction due to the change in the thermodynamics of the reaction process which led to the varying droplet size, depletion of the first formed chains, etc. [44,45]. The fusion of the droplets may increase the particle size and would influence the cross-linking process. In order to avoid this researchers are exploring newer methods. For example Holtze et al. [46] found a novel route to synthesize composite polymer. To avoid the fusion of droplets and to improve stability of the emulsion they adopted gelation of continuous phase to immobilize the droplets. Thus, we initially focused to prepare stable IME droplets and evaluated for their stability in emulsion by Lumifuge centrifuge. From Table 2 it can be seen that AA–G (acrylic acid–gelatin) droplets were stable at shelf atleast for 24 h which is enough for complete polymerization and cross-linking of both the polymers. Further, the same droplets demonstrated sedimentation velocity of 0.67 ␮m/s. Emulsion with such sedimentation velocity are generally regarded as stable emulsion [47]. 3.4. Depletion It is well known fact that flexible polymer chains (for example PAA chains) formed at the interface would try to extend away from the nanogel surface into the solution but semi-flexible or

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Table 2 Optimized formulation composition and parameters used to obtain stable droplets along with stability studies (Span 2 g). Sample code (IME droplets)

Composition

Span 20 (%)

Sonication time (min)

Observation by naked eye at shelf

Sedimentation velocity (␮m/s) at 3000 rpm, 20 ◦ C

5 10 20 5 10 20

Stable for atleast 24 h Stable for atleast 24 h Stable for atleast 24 h Stable for atleast 16 h Stable for atleast 12 h Forms 2 phases in few hours but partially dispersed Stable for atleast 24 h Stable for atleast 20 h Stable for atleast 10 h

0.46

Acrylic acid (g)

Gelatin (g)

AA

0.5

–

2.0

G

–

0.5

2.0

AA–G

0.5

0.5

2.0

5 10 20

1.12

0.67

rigid chains can form dense and compact adsorption layers [45]. However, in the present method, growth of PAA by radical polymerization was carried out in the presence of gelatin (a biopolymer) which did not allow the early formed PAA chains to go into the continuous phase (depletion) due to the presence of bulky side chains, as we speculated. It was further supported by SEM and TEM (Figs. 5 and 6 respectively) [no ill-formed IPN nanogel morphology after extensive dialysis] which confirm the above speculation. It indicates the choice of suitable monomers or polymers would improve the usefulness of the method to synthesize other polymers.

3.5. Interaction of carboxylate group of PAA with amino group of gelatin There is a possibility of interaction (non-covalent and pH dependent) between carboxylic groups of PAA and amino groups of gelatin chains [48–50] which might affect the successive crosslinking of gelatin by glutaraldehyde. It is reported by Bigi et al. [51] that 0.05% glutaraldehyde cross-link 60% of the free amino groups present in the gelatin films. Considering the report of Bigi et al., we hypothetise that 0.06% of glutaraldehyde (used in our preparation) will be sufficient for complete cross-linking, however in the present system it was observed that 0.06% of Glu was not sufficient to react with all the amino groups present in the gelatin as analysed using TNBS (Trinitro Benzene Sulfonic acid) analysis which showed 2781 ␮mol of free amino groups per gram of IPN nanogels (data shown in part II).

3.6. Possible presence of free radical species, monomer conversion and final chemical composition In the present system ammonium persulfate (APS) and TEMED have been used as combination of redox initiators. TEMED accelerates the decomposition of APS at room temperature to form the radicals. The life time of free radical formed being short [52] and thus it is expected that free radical would not exists in the final sIPN or IPN nanogels. ESR spectroscopy and radical traps have been used by researchers to investigate the presence of free radicals, however it was not possible in the present system as radicals are short lived and are destroyed by radical recombination or disproportionation. Percentage yield of IPN nanogel was determined and it was found to be 98% i.e. almost all the feed component have been converted into product. It was not possible to determine the chemical composition while reaction was proceeding due to the formation of crosslinked polymers. However, the chemical composition of final nanogels was determined using XPS data and presented in Table 4. It was found to be in correlation with the initial feed.

Fig. 1. UV/vis scans obtained for dialysates and 0.1 M monomer solutions.

3.7. Purification of nanogels Nanogels for medical applications must be free from toxic substances. The presence of feed reagents influences the drug loading, drug release, biocompatibility and physico-chemical behaviour of nanogels. In order to avoid the cytotoxicity of unreacted glutaraldehyde, Gx (gelatin nanogel) and AxGx (IPN nanogel) samples were treated with 5 ml 0.01 M solution of glycine for 5 min to block the free gluteraldehyde groups present and dialysed. Dialysis of nanogel dispersion for several days is a well known process to completely remove unreacted water soluble monomers [53,54], salts, oligomers from the prepared gels. Purification by this method also improves the quality of morphological images of samples. Since the reagents used in the reaction system show characteristic absorption peak in UV-visible spectroscopic range, it was possible to confirm the removal [48] of unreacted monomers by above mentioned technique. Fig. 1 shows the scanning profile of the reference solutions and dialysates. Monomer solutions (0.1 M) show absorption peaks, where as dialysate after 24 h did not show any absorption for the different monomers. Table 3 Particle size determined by DLS and percent yield after dialysis and freeze drying. Nanogel code

Nanogel diameter (nm)

PDI

% Yield

Ax Gx AxGx AGx AxG

312 ± 5 249 ± 20 255 ± 25 48, 373, 4957 376

0.221 0.111 0.088 0.478 0.175

85 85 98 80 96

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Fig. 2. Size distribution graph by DLS measurement: (a) for sIPN AGx and (b) for IPN AxGx.

Higher percent yield (98%) suggests all the monomers are converted into polymer. The loss is only 2% which could be due to the processing. Moreover, acrylic acid and bisacrylamide (BIS) being water soluble are removed by repeated centrifugation and dialysis. In addition, the aldehyde groups of partially reacted glutaraldehyde were blocked with glycine [55] (by 5 ml of 0.01 M solution for 5 min). Research on implants and injectables in recent years indicate that the glutaraldehyde cross-linking has been clinically acceptable at lower concentrations [56,57]. Furthermore, these nanogels were evaluated for cytotoxicity by MTT assay on J774 cell lines for 48 h. It was found that these nanogels were not cytotoxic and demonstrated increasing cell viability with Ax, Gx, AxGx (data shown in part II) in comparison to Triton X (cytotoxic molecule) which demonstrated only 2% cell viability. This further confirms that the samples prepared are free from monomers and other unreacted components.

Fig. 4. Morphology of IPN nanogels at different stages of process and treatment: (a) SEM image of IPN nanogel immediately after reaction, (b) IPN after centrifugation and (c) IPN after 7 days of dialysis.

3.8. Analytical studies by FT-IR

Fig. 3. Zeta potential values for different nanogel samples in dependence of pH values.

FT-IR spectra of dialysed and freeze dried nanogel powder were recorded in the range of 400–4000 cm−1 . Mixed absorption bands in case of AxGx confirmed the presence of both polymers. N–H stretching vibration at 3429 cm−1 can be assigned to BIS in Ax nanogels. This stretching band shifts to 3401 cm−1 in AxGx. However, though PAA was not cross-linked in sIPN AGx, observation of peak at 3420 cm−1 was probably due to amine group [58] present in the gelatin chain. The gelatin backbone in Gx showed absorption due to amide group at 1534 cm−1 . This absorption shifted to 1548 cm−1 in case of AxGx. This increase in wave number could be due to the presence of carboxyl group of poly(acrylic acid) as neigh-

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Fig. 5. Morphology of sIPN and IPN nanogels after different treatment times: (a) SEM images of sIPN AGx after 7 days of dialysis. Porous structure evidents the leaching of free PAA chains, (b) broken sIPN AGx nanogels after extensive dialysis (11 days) and (c) intact spherical IPN (AxGx) nanogels even after 11 days of dialysis.

bouring group which confirms the formation of IPN. The presence of aldimine absorption peak at 1450 cm−1 in Gx, AxGx and AGx [59,60] further confirm the Glu cross-linked gelatin chains and thus the formation of sIPN and IPN (for FT-IR graphs see supportive information). The percent yield of nanogels obtained after freeze drying was given in Table 3. 3.9. Particle size analysis by DLS Table 3 shows the nanogel diameter as obtained by DLS. Gelatin nanogels were found to be smaller than the PAA nanogels. However, sIPN AGx were larger than homopolymer and IPN nanogels. The reason could be due to the increase in hydration and mobility of non-cross-linked (free) PAA chains in sIPN. This was confirmed from the DLS data. Semi-IPN AGx showed 3 different and distinct peaks (Fig. 2a) with an average diameter of 48 nm, 373 nm and 4957 nm. The peak at 48 nm can be attributed to the leached free PAA chains from the nanogels [35]. However, it is worth to mention here that the other sIPN where gelatin chains are not cross-linked (AxG) did not show any peak at 48 nm (data not shown). The reason is attributed to high molecular weight gelatin chains, get interlaced with formed PAA chains and unable to leach from the system. The peak at 373 nm was due to the formed sIPN nanogels. The third peak at 4957 nm was due to the nanogel aggregates formed due to the adhesion of free PAA chains of the adjacent sIPN nanogels and it was further confirmed by TEM (Fig. 6c) and light microscope (data not shown). The aggregation of adjacent sIPN nanogels was due to free PAA chains. However, IPN nanogels where both the chains (gelatin and PAA) were cross-linked showed smaller size with a single peak (Fig. 2b) of 255 nm. As shown in Table 3, DLS experiments showed process reproducibility with average size of ±20 nm.

3.10. Stability of prepared nanogels at different pHs All samples were diluted in distilled water (pH 5.7). Results obtained by zeta potential measurements in distilled water showed good stability of the samples and further demonstrated the formation of interpenetration. Zeta potentials of homopolymers and IPN samples posses negative values. Because of the contribution of small amount of acid groups in Gx, zeta value of −32 mV in distilled water was observed, whereas, an increased zeta value of −48 mV in Ax was due to the presence of large number of acid groups. One would expect zeta values for AxGx close to Ax or Gx if it forms a core–shell structure [35]. However, the zeta value of −38 mV with AxGx was obtained remains in between the zeta value of Ax and Gx. This could be due to the interpenetration of networks which further support the preparation of IPN nanogels using one pot method. Furthermore in order to prove this, nanogels were synthesized by template polymerization. Ax was taken as template in gelatin solution to synthesis the particles. Zeta potential measurement of the formed nanogels showed a zeta value of −24 mV which was far away from the zeta value of Ax (−48 mV), but closer to Gx (−32 mV) supporting the formation of core–shell structure. This result supports that the one-pot synthesis would be the better option to synthesize IPN nanogels. Comparative stability of the nanogels at varying pH range was studied using zeta potential measurement. Working universal buffer solutions (0.1 M) of varying pH were prepared from the stock buffer solution obtained from Sigma. Nanogels were dispersed in different pH solutions (100 ␮g/ml). The pH was adjusted to 3.0, 5.6, 6.6, 7.4 and 9.0 using 0.1 M NaOH or 0.1 M HCl solutions and zeta potentials were measured. Fig. 3 shows the comparative stability data obtained for different nanogel samples. Ax showed highest stability, which can be deduced from larger zeta values, whereas Gx

Fig. 6. TEM images of individual nanogels: (a) Ax spread out on the surface completely, (b) AxGx shows uneven boundary because of interpenetration and (c) AGx shows aggregation because of free PAA chains, large and transparent boundary.

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Fig. 7. Resolved C1s scans by XPS: (a) Ax, (b) Gx, (c) AxGx and (d) AGx.

showed lowest stability. IPN nanogel (AxGx) showed zeta potential value close to Ax indicating that combination of PAA with gelatin increases the stability of IPN nanogels. AxGx, AGx and AxG are showing better stability at pH 6.6 compared to pH 7.4. At pH 3, all the samples were less stable because of the aggregation, which was observed by naked eye. 3.11. Scanning electron microscopy SEM is an efficient tool to study the morphology of submicron sized materials. We have evaluated the formation and stability of the nanogels during process and treatment (namely after crosslinking, centrifugation and dialysis). Fig. 4a depicts SEM image of IPN nanogel in cyclohexane immediately after the reaction. Nanogels appear bright, separate and smooth. Fig. 4b shows the

image after centrifugation, which shows particles stick to each other. However, after 7 days of dialysis they were devoid of unreacted substances (Fig. 4c) and it can be seen that the prepared nanogels are not monodispersed (see PDI values in Table 3). Earlier studies from our lab on sIPN bulk gels [35] composed of gelatin and PAA showed leaching of PAA chains on hot water treatment. In the present work, similar behaviour has been demonstrated with nanogels too. When sIPN nanogels AGx were extensively dialysed (7 days), resulting nanogels became highly porous (Fig. 5a and inset). Further dialysis for extended period of 11 days led breaking of the sIPN (Fig. 5b). Due to the high rate of swelling, chains initially become weak and finally leachout. The broken part of the sIPN samples can be observed as white powder like substance in the backround of Fig. 5a (shown by arrows). In contrast to sIPN, IPN nanogels retain their structure even after 11

Table 4 Percent atomic concentration, elemental ratio and resolved C1s components of prepared samples by XPS. Sample code

Ax Gx AxGx AxG AGx

Percent atomic concentration (by XPS)

Calculated elemental ratio

Resolved C1s scan (% area contribution of C1s components)

C1s

N1s

O1s

N/C

O/C

C–C (281.01 eV)

C–O (286.03 eV)

C O (287.61 eV)

COOH (288.59 eV)

65.0 71.0 63.6 64.6 65.5

4.7 11.3 10.5 8.8 9.9

22.2 16.1 23.8 24.7 18.7

0.07 0.16 0.17 0.13 0.15

0.34 0.22 0.37 0.38 0.28

75.05 63.92 49.32 71.36 58.18

3.63 12.67 14.59 5.64 14.04

10.9 18.64 12.39 19.47 10.52

10.42 2.03 12.92 13.66 3.52

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days of dialysis (Fig. 5c). The above results thus confirm that the IME technique can be used for the synthesis of interpenetrating polymer network nanogels. Porous bulk with non-uniform surface and larger size (>500 nm, SEM images) of sIPN (AGx) (Fig. 5a) supports the formation of sIPN structure. Another notable point is that DLS size determination of sIPN gives smaller values than SEM images. The average diameter of the nanogel in SEM images was about 1.5 times larger than the size determined by DLS. The difference in size was due to the flattening of the nanogels during drying process [9].

multicomponent nanogel system may be used as a targeted drug delivery system in the treatment of solid tumors.

3.12. Transmission electron microscopy

Appendix A. Supplementary data

TEM study was carried out to see the morphology of the individual nanogels to confirm the formation of IPN. Fig. 6 shows the TEM images of nanogels of homopolymer (Fig. 6a), IPN (Fig. 6b) and sIPN (Fig. 6c) (for Ax, AxGx and AGx). After dialysis and freeze drying, images obtained for the different samples gave detailed insight on the morphology of the nanogels. PAA (Fig. 6a) spread over the surface with non transparent periphery and, thus, appears like a flattened disc with smooth boundary. The highly flexible chains and low mechanical strength led to complete spreadening and smooth surface of the Ax nanogels after drying. However, as expected IPN nanogels did not spread completely. IPN appear with partially transparent, non-uniform periphery which fades towards the boundary (Fig. 6b). This is because of the higher cross-linked density and complex interlaced structure of PAA and gelatin networks. Interestingly sIPN nanogels (AGx) appear with a smooth, large and completely transparent (fades for a large distance) periphery (Fig. 6c). They spread out completely, because of the hydrated free PAA chains and the surface was completely transparent and large.

Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.colsurfb.2010.11.007.

3.13. X-ray photoelectron spectroscopy Fig. 7 depicts the resolved C1s scans of homopolymer, sIPN and IPN nanogels. Signals from both gelatin and PAA molecules in AxGx sample confirm the formation of IPN networks. The characteristic signal at 284.94 eV in Gx can be attributed to aliphatic and aromatic rings, which were found absent in Ax. A small intensity peak at 401.95 eV (lost peak) in case of Ax was due to the burried COOH group because of cross-linking. Percentage atomic concentration at the nanogel surface and data obtained from resolved C1s peak are presented in Table 4. A significant difference of N/C ratio in Ax and AxGx (0.07–0.17, Table 4) and O/C ratio (0.22–0.37) in Gx and AxGx indicates the formation of interpenetrating polymer networks. One would have expected N/C and O/C values equal to homopolymer gels, if the system would not have been interpenetrated. Since both the networks are cross-linked in AxGx, it shows higher N/C ratio and a loss of free PAA chains led to lower O/C ratio (0.28) in sIPN (AGx) after dialysis. The area corresponding to COOH signal in sIPN AGx decreased (to 3.52%) significantly after dialysis (Table 4). This indicates the leaching of PAA chains and supports the results obtained by DLS and SEM studies. 4. Conclusion IME polymerization technique was successfully exploited for the preparation of homopolymer, sIPN and IPN nanogels. The formation and stability of the droplets was optimized to get smaller sized nanogels. Results of different characterization techniques such as FT-IR, SEM, TEM and zeta potential measurements confirmed the formation of IPN network. The results represent an applicable strategy for the synthesis of multifunctional nanogels but restricted to the suitable interplay of system components. Such

Acknowledgements The fellowship (DAAD-Sandwich) to Mr. Raja Mohamed offered by DAAD to carryout the research work in Germany is greatly acknowledged. Special thanks to Frau Kern for SEM and Dr. Barbara Adolphi for the useful discussions regarding measurement and interpretation of XPS.

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