Tannic acid decorated poly(methacrylic acid) micro and nanoparticles with controllable tannic acid release and antioxidant properties

Accepted Manuscript Title: Tannic acid decorated poly(methacrylic acid) micro and nanoparticles with controllable tannic acid release and antioxidant properties Author: Nurettin Sahiner Sultan Butun Sengel PII: DOI: Reference:

S0927-7757(16)30645-8 http://dx.doi.org/doi:10.1016/j.colsurfa.2016.08.014 COLSUA 20900

To appear in:

Colloids and Surfaces A: Physicochem. Eng. Aspects

Received date: Revised date: Accepted date:

26-5-2016 23-7-2016 13-8-2016

Please cite this article as: Nurettin Sahiner, Sultan Butun Sengel, Tannic acid decorated poly(methacrylic acid) micro and nanoparticles with controllable tannic acid release and antioxidant properties, Colloids and Surfaces A: Physicochemical and Engineering Aspects http://dx.doi.org/10.1016/j.colsurfa.2016.08.014 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Research Highlights

-P(MA) microgels from nm to micrometer sizes conjugated with Tannic Acid (TA). -TA release from p(MA)-TA microgel afford antioxidant and antimicrobial properties. -P(MA)-TA colloidal particles spanning from nm to μm offer versatile biomedical use. -P(MA)-TA microgel is made magnetic field responsive for targetable delivery system.

1

Graphical Abstract HO HO HO

HO HO

OH

HO

OH

O

OH

HO

OH

O

C

O

+

HO HO HO

OH

O

OH

O

O

O

O

HO

OH OH

HO

O

HO O HO

O

O

O

O

O

C

OH

O HO

O

O O

O HO

HO OH

HO

OH OH HO

O

C O

OH OH

O

O

O

O

HO HO O O

P(MA) Micro or nanogels

OH

O

O

O

O

HO

OH O

O O

O

O

O

O O

OH

OH

O

O HO

HO

OH

O

O O O OH

OH OH OH

O OH OH

TA P(MA)-TA

2

Tannic acid decorated poly(methacrylic acid) micro and nanoparticles with controllable tannic acid release and antioxidant properties

a,b

Nurettin Sahiner, aSultan Butun Sengel

a

Canakkale Onsekiz Mart University, Faculty of Sciences and Arts, Chemistry Department, Nanoscience and Technology Research and Application Center (NANORAC), Terzioglu Campus, 17100-CANAKKALE-TURKEY. b

*Corresponding Author: Tel: +90-2862180018-2041; Fax: +90-2862181948 [email protected] (N. Sahiner)

Abstract Poly(methacrylic acid), p(MA), particles in the size range of nano and micrometer were prepared via emulsion and reverse suspension polymerization method, respectively. Ethylene glycol dimethacrylate (EGDMA) as a biodegradable cross-linker was used in the preparation of these p(MA) particles. Both nano and micrometer p(MA) particles were decorated with tannic acid (TA) via a direct esterification linkage upon activation of the carboxylic acid with N,N’-carbonyldiimidazole followed out by conjugation with TA to obtain p(MA)-TA. For the characterization of the particles TGA, FT-IR SEM, and light microscope were used. In vitro release of the TA from p(MA)-TA conjugates were investigated at pH 7.4 in phosphate buffered saline (PBS) by UV-Vis spectrometer and result revealed that the amount of the used crosslinker has paramount significance in degradation of TA molecules. For example, p(MA)TA microparticles with 1% of the crosslinking density released 83.24 mg TA per g particles in comparison to nanosizes p(MA)-TA particles with 10% of the crosslinking density that released 35.21 mg per g particles within four days. Additionally, antioxidant properties of all particles were evaluated by Total Phenol Content (TPC) and Trolox Equivalent Antioxidant Concentration (TEAC) methods and found that the p(MA)-TA microparticles has great antioxidant properties with TPC=61.41±1.44 mgL-1 gallic acid equivalent (GAE), TEAC=248.82±1.96 mM trolox g-1 for dry sample. 3

Keywords:

Poly(methacrylic

acid)-Tannic

acid;

microgel/nanogels;

TA conjugate;

antioxidant particles; TA decorated p(MA) colloids.

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1. Introduction Materials with modifiable properties have great application potentials in various areas of medicine. Among the versatile materials, polymer colloids such as micro/nanoparticles represent a promising class of soft materials especially for providing controlled release of bioactive molecules. The usage of polymeric particle as delivery system offers significant advantages owing to their attractive features such as the easy synthesis, tunable particle size, and controllable degradability. Among the most comprehensively studied biomaterials or biofunctional materials are the polymeric materials with micro or nano-sized particles as they provide unique chemical, physical, and biological functions [1-4]. Micro/nanogel based on poly(methacrylic acid), p(MA), has been extensively explored for drug delivery and tissue engineering [5]. Polymers containing methacrylic acid are very useful to produce novel pH responsive systems due to its weak acidic -COOH residues that also futher allow modification of the system via esterification chemistry [6]. Such polymers and copolymers are widely used as dispersants, thickeners, flocculants, superabsorbent materials [7] and in many applications such as medicine, catalysis, adsorption, photonic crystals and in other various nanotechnology fields [6, 8-11]. Tannic acid (TA) is a natural water soluble material and is a typical form of hydrolysable tannin that has containing five gallic acids attached to the center glucose unit, and another five gallic acid are connected this five gallic acids via ester linkage to each other. TA is considered as safe chemical by the Food and Drug administration (FDA) and allowed to be used as direct additive in food products [12]. TA has sufficient hydroxyl and carboxyl groups to form strong complex with a wide range of macromolecules such as carbohydrates, proteins, enzymes and other synthetic polymers [13,14]. TA has been extensively investigated, due to its widespread applications as plant extracts in the leather manufacture, resin production for different metal ion adsorption, and as a polymeric coagulant or flocculants for water treatment. It has applications in medical area as well because of its 5

natural antioxidant, antimicrobial, antimutagenic, hemostatic and anticarcinogenic properties [15-21]. Recently, magnetic nanoparticles with their polymeric composites play an increasingly important role in various biomedical and catalytic applications due to the targetable properties under external magnetic field. The particles have many advantages for diagnostic and therapeutic applications because of their special properties such as super paramagnetic, high dispersible nature, low toxicity and readily surface modification abilities [22-26]. Recent developments in the field of polymer-conjugates provided promising examples that lead new marketable pharmaceutical products [27]. Anti-inflammatory therapies involving the incorporation of low molecular weight drugs into macromolecular systems such as ibuprofen, dapsone, and aspirin polymer-drug conjugates have shown promising advances that enhance the bioavailability and improved treatments efficacies within the body. In addition, chitosan-DNA [28], antibody-drug [29], and dapsone polymer conjugate [30] are among the most recent researched conjugates. Most of these were prepared/synthesized via common esterification or acylation reaction. The acylation procedure involves the activation of acid groups in polymer or monomer by using an activation

agent

e.g.,

esterification

of

polysaccharides

[31-36].

Specifically,

N,N’carbonyldiimidazol was found to be an efficient activator for acylation in non-aqueous systems that offer some advantages such as mild reaction conditions, limited amount of byproducts, and readily commercial availability. The aim of the control release system is to provide the active agent release in effective concentration, and prolong the release of active agents. Similarly, it should not be toxic, and has no side effect to the surrounding environment. It is important to note that the majority of drugs when used directly possess higher cytotoxicity effect in comparison to its’ conjugated form. The stability of the bond between polymer and drug is of ultimate importance in the conjugate system. The common bonds types are generally esters and amides. Therefore, the conjugation of natural materials

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such as TA via conjugation, provide many advantageous due to synergistic effect of combining TA’s antioxidant, antibacterial and anti-cancer properties, and blood clotting properties so on, and with colloidal particles controlled size, additional functional groups, and pH responsive behaviors and so on can contribute many essential properties such as extended drug half-life by prolong resident time in the circulation or in the body, enhanced uptake, and minimum immunologic response, and so on. In the present work, p(MA) particles in micrometer

and

nanometer

sizes

were

synthesized

by

using

ethylene

glycol

dimethacrylate:methacrylic acid (EGDMA:MA) mole ratios to 10:100 and 1.0:100, respectively using emulsion medium and reverse suspension polymerization. These two particles were employed for TA conjugation using N, N’-carbonyldiimidazole to activate carboxylic acid on p(MA) particles. The prepared p(MA) and TA conjugate particles, p(MA)TA, were characterized by using SEM, light microscope, FT-IR, and TGA. TA release studies from p(MA)-TA particles were evaluated by UV-Vis spectrophotometer at 280 nm wavelength in PBS (pH 7.4) at 37 oC. Antioxidant properties of the particles were determined via TPC and TEAC methods.

2. Materials and Methods 2.1. Materials All reagents were used as received without further purification unless otherwise stated. Methacrylic acid (MA, 99%, Aldrich) as monomer, and ethylene glycol dimethacrylate (EGDMA, 99 %, Fluka) as crosslinker, ammonium persulfate (APS, 99%, Sigma–Aldrich) as redox initiator, N,N,N′,N′-tetramethylethylenediamine (TEMED, Merck) as an accelerator and cyclohexane (99.8%,Sigma Aldrich) as solvent were used. Cetyl trimethylammonium bromide (CTAB, 99%, Merck) and span 80 as cationic and nonionic surfactant were used, respectively. Tannic acid (Mw = 1701.2 Da, TA, 99.8 %, Sigma-Aldrich), N, N′7

carbonyldiimidazole was purchased from Sigma-Aldrich. Iron (III) chloride hexahydrate (FeCl3.6H2O, 99%, Acros), iron (II) chloride tetrahydrate (FeCl2.4H2O, 99 %, Fluka), aqueous ammonia (NH4OH, 25%, Merck), and sodium chloride (NaCl, 99.8 %, Sigma-Aldrich) were used for the preparation of the magnetic nanoparticles. Dimethyl sulfoxide (DMSO, 99.5%) as solvent, and Folin Ciocalteau’ phenol reagent and 2,2’-azinobis-(3-ethylbenzothioazoline-6sulfonic acid (ABTS, Sigma-Aldrich) were used in the determination of the antioxidant properties of the particles. All aqueous solutions were prepared using ultrapure water (Millipore Direct-Q3 UV). 2.2. Synthesis of p(MA) nanoparticles P(MA) nanoparticles were synthesized by microemulsion polymerization as described in the literature with some modifications [37,38]. Briefly, polymerization and simultaneous crosslinking were accomplished in a 50 mL flask equipped with a magnetic stirring bar containing 30 mL 0.01 M CTAB solution in water. In a typical p(MA) nanoparticle synthesis: 0.486 mL MA was dispersed in the 0.01 M 30 mL CTAB solution together with 10 mole % EGDMA (110.4 μL) with respect to monomer amount. The mixture was vortexed 2 min in order to obtain a clear isotropic solution, and then this mixture was placed in a temperaturecontrolled oil bath at 75 °C. The stirring rate was adjusted to 600 rpm and left for 20 minutes to equilibrate the temperature of the system to 75 °C. The polymerization was initiated by addition 1 mL of 0.0129 g mL-1 APS solution in water. The polymerization was carried out for 8 h, and the obtained nanoparticles were purified by centrifugation at 10000 rpm for 20 min at 20 °C, followed by removal of the supernatant solution and re-dispersing in DI water and re-centrifugation at least five times. The products were dried by using a freeze dryer (Christ Alpha 2-4 LSC) over night and saved in a closed vial for further use. Magnetic p(MA) particles were synthesized by the same method by adding previously prepared Fe3O4 particles to the emulsion before addition of the initiator for the polymerization and crosslinking of 8

p(MA) particles.

The magnetic Fe3O4 particles were synthesized in accord with the

previously reported method with some modifications [37]. In brief, FeCl2.4H2O (0.430 g) and FeCl3.6H2O (1.168 g) were dissolved in 20 mL DI water in centrifuge tube and vortexed. Then, 0.5 ml aqueous ammonia (25%) was added into the mixture and vortexed. This addition was repeated till the total volume of the ammonia reaches to 1.5 mL. The stirring was stopped when the color of the solution is turned from orange to black immediately upon the initial addition of ammonia. The magnetite precipitates were washed with DI water and 0.02 M sodium chloride by magnetic decantation. After last decantation, the ferrite magnetite particles were dispersed in 10 ml DI water with ultrasonic bath and 100 µL of this ferroliquid was added to the polymeric particle precursor (MA, EGDMA in CTAB solution) at 75 °C before the addition of initiator in order to prepare magnetic-p(MA) composite particles. The same procedures as in p(MA) particle preparation was applied for the magnetic composite p(MA) particles. 2.3. Synthesis of p(MA) microparticles P(MA) microgels were prepared by inverse suspension polymerization with some modifications from the earlier reported method [9]. Briefly, in a 100 mL round bottom flask, 50 mL cyclohexane containing 160 μL span 80 was added. This mixture was homogenized by stirring and purged with N2 for 30 minutes to remove oxygen. The initiator, 54 mg of APS (2 mol % of MA) dissolved in 1 mL distilled water (DI), and 22.7 µL EGDMA (1.0 mol % of MA) and 1 mL MA were mixed in a vial, and added into reaction flask containing cyclohexane and span 80 mixture. This new reaction mixture was stirred continuously and purged with N2 throughout the experiment. The reaction was initiated by the addition of 250 μL TEMED and allowed to proceed for 5 hours at 40 °C in an oil bath. Then, the prepared microgels were collected by decantation of cyclohexane. The microgels were washed with ethanol and then with DI by centrifugation at 10000 rpm at 20 °C for 10 minutes followed by 9

removal of the supernatant solution and re-dispersing in DI and re-centrifugation at least five times for cleaning purpose. Finally, p(MA) microgels were dried in freeze dryer, used for characterization and TA conjugation study. 2.4. Synthesis of p(MA)-TA conjugates In order to conjugate TA onto p(MA) particles, firstly, 0.250 g of N, N′-carbonyldiimidazole was added to 8.75 mL DMSO solution containing 0.280 g p(MA) particles. This reaction mixture was stirred at room temperature for 2 h. Subsequently, 0.386 g of TA was added to this mixture and the reaction was allowed to progress for 24 h at 80 °C under constant stirring. After it is allowed to reach to room temperature, the prepared p(MA)-TA particles were separated by centrifugation at 10000 rpm at 20 °C for 10 minutes followed by the removal of the supernatant solution and re-dispersing in DMSO and re-centrifugation two times for cleaning purpose. The precipitates were washed several times with water-ethanol mixture and dried under vacuum (freeze-dryer) to obtain p(MA)-TA conjugates. 2.5. Characterization The SEM images of the freeze dried particles were obtained by placing the p(MA) particles and p(MA)-TA conjugates onto carbon tape that is attached on aluminum SEM stubs, and after coating with gold to a few nanometers thickness under vacuum using SEM (Jeol JSM5600 LV) operating at 20 kV. Optic microscope (BX 52 Olympus, Cameram 21 attached) was used for imaging the water swollen gel. Particle size distribution were determined via Dynamic Light Scattering (DLS) technique by using particle size analyzer (Brookhaven Ins. & cor. 90 plus) from the particle solutions that were obtained by the dilution with 0.01 M KCI solution in water.

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Fourier Transform Infrared Spectrometry (FT-IR, Perkin–Elmer) was employed to confirm the functional groups of MA and TA in the spectral range from 650 to 4000 cm-1. The data were collected by using attenuated total reflectance (ATR) with 4 cm−1 resolutions. Thermal analysis was conducted via thermogravimetric analyzer (TGA, Seiko, SII TG/DTA 6300). For TG analysis, about 3 mg samples were heated on a ceramic pan from 50 oC to 900 o

C at a heating rate of 10 oC min−1 under nitrogen flow (100 mL min−1) and the weight loss

against temperatures were recorded. UV-Vis spectrometer (T80+ UV/VIS Spectrometer, PG Ins. Ltd.) was used for in-vitro release study to determine released amounts of TA from p(MA)-TA particles. And p(MA)-TA was used to determine the antioxidant activity of the particles. For the titration of the particles with NaOH, 100 mg p(MA) and p(MA)-TA conjugate were placed into 50 mL 0.01 M KCI solution in a baker, and 0.1 N NaOH solution was added dropwise while the mixture was stirred at 500 rpm under N2 purge, and the pH values were recorded after certain volume of NaOH addition upon reaching to a stable reading, and NaOH addition was continued within pH range of 3-11 using a pH-meter (Sartorius). 2.6. In vitro release study of conjugates Dried p(MA)-TA particles (0.05 g) were re-suspended in 1 mL of PBS at pH 7.4, and transferred to a dialysis membrane (molecular weight cut off ≥12000 Da, Aldrich), and placed in a container with 29 mL of phosphate buffered saline (PBS) at 37 °C. The released amount of TA into the PBS was evaluated as a function of time by using an UV-Vis spectrometer from the corresponding calibration curves constructed for the same release pH medium at 280 nm. 2.7. Antioxidant activity

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To determine antioxidant activity of p(MA) and p(MA)-TA, two methods were employed. One of them is the total phenol content (TPC) by using colorimetric Folin-Ciocalteu, and the other one is Trolox Equivalent Antioxidant Capacity (TEAC) by using scavenging capability of the ABTS radical. All particles were dispersed in PBS at 1.0 mg.mL-1 concentration, and sonicated for 2 minutes, and this solution is used for TPC and TEAC tests. 2.7.1. Total Phenol Content (TPC) In the evaluation of TPC value of all the particles, the reported method in the literature was followed by slight modification [16, 39]. P(MA) based particles weighing 1.0 mg.mL-1 was prepared for the test, and 100 µL volume was added to 1.25 mL of 0.2 N Folin-Ciocalteau phenol reagent solution, and the solution was vortexed. After 4 minutes, 1.0 mL of 0.7 M NaHCO3 solution was added to this mixture. After 2 h incubation at room temperature, the absorbance was measured at 760 nm by using UV-Vis spectrophotometer. Each particle sample was measured three times, and the result was given as mg L-1 Gallic Acid Equivalent (GAE) with standard deviation. 2.7.2. Trolox Equivalent Antioxidant Capacity (TEAC) In order to determine TEAC value of p(MA) particles and p(MA)-TA, ABTS scavenging assay was used as reported in literature with a little modification [16,27]. The ABTS·+ (radical cation solution) was prepared by incubating 2.45 mM potassium persulfate with 7.0 mM ABTS in water for 12-16 h in the dark. The stock solution was diluted with PBS (pH 7.4) until the absorbance of about 0.7±0.02 at 734 nm was observed using an UV-Vis spectrometer. Then, 0.02-0.20 mL (0.05-0.20 mL, 0.02-0.08 mL) of sample at four concentrations were added to 3 mL ABTS·+ solution and incubated for 6 min. The decrease in absorbance was monitored at 734 nm after 6 min. ABTS radical scavenging capacity was calculated using the following equation:

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Inhibition % = [(AB-AS)/AB] x100

(1)

Where AB is the absorbance of the ABTS·+ solution without an antioxidant, and AS is the absorbance of the ABTS·+ solution in the presence of the sample. The samples were diluted with PBS so as to give 20-80 % reduction of the blank absorbance. Trolox standard solution (0-15 mM) was prepared in PBS and assayed under the same conditions. The TEAC values were calculated from the slopes of the plots and expressed as “mM trolox g-1” dry sample. 3. Results and discussion 3.1. Synthesis of p(MA) micro and nano particles and their TA conjugates Two different polymerization techniques were used in the preparation of the p(MA) particles; one of them is inverse suspension polymerization, and the other one is emulsion polymerization. For both particle preparations, a biodegradable cross-linker, EGDMA was used. As shown in the Fig. 1(a), these two different polymerization techniques allowed to prepare micrometer and nanometer sized p(MA) particles. Three different amounts of crosslinker (1, 5 and 10 %) were used for these two polymerization techniques. The measured hydrodynamic diameter (dH) and polydispersity index (PDI) results were given in Table 1. 1 % cross-linked particles’ size were not given in Table 1, yet the optic image of 1% EGDMA crosslinked p(MA) particles were provided in Fig 1. The DLS results of particles prepared via emulsion polymerization were found as 585±2.2 and 337±14.3 nm for 5 and 10 % crosslinked particles, respectively. On the other hand, DLS result of the particles that were prepared by reverse suspension polymerization were found as 1439±166.6 and 652±31.2 nm for 5 and 10 % cross-linked particles, respectively. For the progress of this research only two samples (max. and min. sized particles) were chosen e.g., 1 % cross-linked p(MA) particles prepared by reverse suspension polymerization, and 10 % cross-linked p(MA) particles prepared via emulsion polymerization particles were used for TA conjugation and release and

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in the determination of antioxidant properties. The Synthesized particles optic microscope and SEM images were also given in the Fig. 1 (b). It is clear that from the optical microscope images that p(MA) microgels can swell up to a few hundred micrometer sizes, whereas the p(MA) nanogels are about few hundred nm as can be seen from the SEM images. It is obvious that swelling capacity of the particle is increased with the decrease in used amount of crosslinker during particles preparation. A possible way for the preparation of grafting or conjugating onto polymer is through a direct esterification linkage between the carboxylic acids of the polymer backbone and the hydroxyl groups of polyphenol. The effective esterification of TA could be achieved via the activation of the carboxylic acid with N,N-carbonyldiimidazole. The synthetic route of the TA conjugate onto p(MA) is given in Fig 2(a). Firstly, the activation of the carboxylic acid with N,Ncarbonyldiimidazole avoids the formation of acidic by-products during the reactions. Subsequently, the TA was added to the reaction solution and reaction continued 24 h at 80 °C in DMSO. The measured hydrodynamic diameter of TA conjugate for 10 % cross-linked p(MA) nanogels is 888 nm as given in Table 1, whereas the same particle diameter before TA conjugation was measured as 337 nm. The synthesized p(MA)-TA conjugate particles photograph, SEM and optical images are given in Fig. 2(b), as (1), (2) and (3), respectively. Color of the particles changed from white/cream to brownish upon the realization of TA conjugation. As can be seen in Fig. 2(b) (2), the p(MA)-TA particles seemed nanosized in the SEM image as the images are required in dry form; however, optic images clearly revealed that p(MA)-TA particles are in micrometer size range upon swollen in basic medium. After conjugation, hydrophilic properties increased significantly, due to attachment TA molecule that have 25 -OH groups in one molecule of TA in addition to the -COOH groups of MA. Therefore, therefore the increase in the particle size is rational. Comparison of the optic images, it is evident that p(MA) particles are decorated with TA molecules by chemical 14

conjugation is bigger or high swelling degree than the undecorated p(MA) particles because of the more hydrophilic and pH responsiveness of TA molecules that are bonded to polymeric network chemically. FT-IR spectra of p(MA) particle (10 % cross-linked), TA and p(MA)-TA conjugated particles is presented in Fig. 3(a). As can be seen, p(MA) particles exhibited wide stretching vibration band for -OH coming from -COOH group at about 3600-3200 cm-1. And at 2988 cm-1, 2927 cm-1 and 2855 cm-1 for the C-H stretching vibrations, and a strong band at 1708 cm-1 for C=O belonging to the cross-linker (EGDMA) and acid group are clearly seen. The peaks at 1252 cm-1 for C-O-C, and 1168 cm-1 for -CH2 and at 961 cm-1 can be attributed to bending vibration for -CH3. In the same figure for TA spectrum, the Ar-OH, Ar-COOH and C=O vibration bands are observed at 3600-3100 cm-1, and 1716 cm-1, respectively. A small absorption band near 2922 cm−1 is assigned to aromatic C-H stretching vibration. The peaks at about 1613 cm-1 1537 cm-1, 1448 cm-1, 1320 cm-1 and 1200 cm-1 belong to aromatic C=C and phenolic C-O stretching vibrations of TA. The spectra of the p(MA)-TA conjugate exhibit characteristic changes in the functional group frequencies by comparing with the spectrum of bare TA and p(MA) particles. In the conjugated particles, p(MA)-TA, the change in the aromatic peaks intensity, and the shifts in position of the bands in the spectrum are due to the interaction of the hydroxyl groups of TA with the carboxylic acid of p(MA). Compared with the FT-IR band frequencies, the broader peaks at 3600-3150 cm-1 can be attributed to -OH stretching frequencies of -COOH, Ar-OH and Ar-COOH groups for p(MA)-TA particles. The band occurring at 1714 cm-1 in p(MA)-TA spectra can be assigned to the carbonyl band in comparison to at 1716 cm-1 band for free TA molecules. The clear peak at 1600 cm-1, and 1538 cm-1 for conjugated from p(MA)-TA is shifted due to TA peaks at 1612 cm-1 and 1537 cm-1 for bare TA. The p(MA)-TA peaks come from each of the components are clearly seen,

15

and the FT-IR result confirms that the TA conjugation onto p(MA) particles are done successfully. Thermal degradation profiles of p(MA) and p(MA)-TA particles is shown in Fig. 3(b). In thermogram, p(MA) particles decomposes in two steps shown as dashed lines in the figure. The first step degradation occurred between 164-278 °C with a total weight loss of 10 %. The second step degradation occurred at 278-466 °C with a total weight loss of 82 wt %. The p(MA)-TA particles possess three thermal degradation steps. The first step is slow degradation in the range 164-275 °C resulted in 12 wt %. The second step degradation occurred sharply between 275-438 °C with a total weight loss of 72 wt %. The third step degradation occurred slow degradation in the range of 438-544 °C with a total weight loss of 88 wt %. A flat region with no weight loss was obtained after heating above 466 °C and 544 °C for p(MA) and p(MA)-TA, respectively. There is a 6 % difference that is the remained weight at the end of the 900 °C. It is obvious that with conjugation of TA molecules onto p(MA) microgels the degradation profile of p(MA) is changed conforming the structural change occurred. To determine the equivalent pH values, certain amount of p(MA) and p(MA)-TA particles put into 0.01 M 50 mL KCI solution and stirred continuously under N2 purge. The particles were titrated with 0.1 N NaOH, and the obtained titration curves are given in the Fig. 4(a). In this figure, the shape of the titration curves for the two samples are similar and the base neutralizing capacities of p(MA) and p(MA)-TA is very close. Upon conjugation of p(MA) carboxylic acid groups with new acidic molecules such as TA, the equivalent point that is pH value of the medium measured by the addition of NaOH that is added to neutralize p(MA)-TA was found to shift to 4.64 from 4.81 that is the equivalent point pH value unconjugated p(MA) as illustrated in Fig. 4(b). The TA molecules have many hydroxyl groups that can also ionize after addition certain amount of NaOH, that is the reason for the observation of 16

multiple equivalent points value at pH 8.57 and 8.97 due to the presence of phenolic functional groups on p(MA)-TA particles. This titration results also confirms the conjugation of TA molecule on p(MA) particles. 3.2. Degradation of TA from p(MA)-TA particles The degradation study of p(MA)-TA particles was performed in PBS (pH 7.4) at 37 °C. The released amount of TA was determined by withdrawing the sample from degradation medium with certain time intervals up to 100 h, and measuring the absorbance values of the solutions by UV-Vis spectrometer. TA profile of the p(MA)-TA conjugate is given in Fig. 5. The degradation result was shown only up to 4 days in Fig 5. As can be seen, TA releases were increased linearly in first 33 h releasing 64.14 mg and 22.07 mg TA per g particles for 1 % and 10 % cross-linked p(MA)-TA, respectively. It is clearly seen that there is no initial burst release of TA from p(MA)-TA particles where this is generally the case for the drug loadings from the corresponding drug solutions into the carriers. The release time of between 33-97 h, the degradation rate was decreased slowly, and the amount of released TA was calculated as 83.24 mg and 35.21 mg per g particles end of the 97 h. The measured amounts of TA after 17 day of degradation in PBS was found as 40.3 mg as cumulative TA from 10 % cross-linked particles p(MA)-TA particles (data is not shown). In comparison to similar system in the literature (using EC50), it can be assumed that this released amount of TA from p(MA)-TA conjugate is enough to treatment neurologic disease such as Alzheimer [40], and also to reduce hypertension [41]. Additionally, due to well-known properties such as speeding up of blood clotting, reduction of blood pressure, deceleration in liver necrosis and modulation of immune responses, therefore, the p(MA)-TA conjugate can be used in many biomedical areas. As shown in Fig. 5, the release rates depend on the used amounts of cross-linker, and the particle dimensions. It is also apparent that the release rate of TA was found to increase with the decrease in the amounts of the used cross-linker, EGDMA. This is a great advantage that a 17

controllable release profile of TA can be designed or obtained by preparation p(MA) particles with different dimension and by using different amounts of cross-linkers. As magnetic field responsive particles are very important due to known targetable nature under and applied magnetic field, these types of composite attracted a lot attention for many potential use in biomedical application such as targeted release studies and hyperthermia applications [42,43]. In this study, we also prepared magnetic p(MA)-TA composites particles. The photograph images of p(MA), mag-p(MA) and mag-p(MA)-TA particles in the dried and suspended in DI water, and before and after an externally applied magnetic field is given Supporting Fig. S1(a). Mag-p(MA) particles color was changed from brown to darkbrown upon conjugation with TA as can be seen for p(MA)-TA molecules by in the supporting Fig. S1(a). Due to magnetic ferrite particle in p(MA) based particles, composite particles gives very fast response (few seconds) to the applied magnetic field as shown in supporting Fig. S1(a) in (1) and (2). It is known that TA can be used in treatment of various cancers due to very well-known the antioxidant, anti-cancer, and anti-mutagenic properties of TA [21]. The TA conjugated p(MA) particles with magnetic properties can be targetable to a specified region in the body that required medical treatment. In addition to the magnetic properties of the particles, the controlled release of conjugated TA molecules provides additional advantageous for treatment of various diseases or discomfort zones of the body. Therefore, many properties of TA such as antimicrobial properties, blood clotting, blood pressure, immune response regulations and so on can also be introduced to the regions of interest in the body for the treatment purposes. As illustrated in supporting Fig. S1(b), the release profile of TA from mag-p(MA)-TA composites can also be accomplished. The calculated amounts of the released amounts of TA was found as 32 mg and 8 mg g-1 from p(MA)-TA and mag-p(MA)-TA composites end of the 71st h, respectively. It is apparent that

18

magnetic composites of p(MA)-TA can also be prepared and used for magnetically targetable TA releasing systems. 3.3. Antioxidant activity of the particles and conjugates TA is known for its antioxidant properties, and the strength of antioxidant capacity is dependent on the concentration phenolic groups and the free radical source. Therefore, the antioxidant properties of p(MA) based particles were investigated using FC, and ABTS assay methods and their results are summarized in Table 2. As given in Table 2, The total phenol content and antioxidant properties of p(MA) particles and TA conjugates were determined from their suspension in PBS at the concentration of 1.0 mg.mL-1. P(MA) particles prepared by 10 % crosslinking, and its p(MA)-TA conjugates have antioxidant equivalent capacity of 3.75±0.36, 3.75±0.36 and 37.04±1.98 mgL-1 GA for p(MA), mag-p(MA)-TA, and p(MA)TA, respectively. P(MA) particles prepared by 1% cross-linker, and its p(MA)-TA conjugates have antioxidant equivalent capacity of 32.20±1.26, 17.91±0.18, and 61.41±1.44 mgL-1 GA for p(MA), mag-p(MA)-TA, and p(MA)-TA respectively. Additionally, the antioxidant effects of the p(MA) particles, and p(MA)-TA were determined by ABTS radical scavenging methods. TEAC values for p(MA) based materials were also measured with various concentrations of the samples to determine the % scavenging capability of certain ABTS radicals. The micro/nano p(MA) particles with mag-p(MA)-TA conjugates have no antioxidant activity, whereas p(MA)-TA conjugates demonstrated very good antioxidant activity. The TEAC value of p(MA)-TA conjugates were determined as 108.85±0.55 and 248.82±1.96 mM trolox equivalent g-1 dry sample for p(MA)-TA nano and micro particles, respectively. The obtained result from both antioxidant tests confirm that p(MA)-TA conjugate has considerable antioxidant properties. 4. Conclusions

19

Here, p(MA) particles were prepared in nano and micro dimensions with the various degree of crosslinking via emulsion, and reverse suspension polymerization techniques, respectively. The prepared p(MA) particles were decorated successfully with TA molecules as p(MA)-TA via direct esterification linkage between the carboxylic acid of p(MA) with N, N’carbonyldiimidazole and then their reaction with TA [31,39,44]. The TA conjugation onto various sizes and different amounts of crosslinked p(MA) particles were confirmed by structural analysis via FT-IR, and thermal stability studies via TG analysis. Earlier, interpenetrating polymer network (IPN) of TA in acrylamide film gel or bulk and even microgel of TA were reported for prolonged TA release via degradation [16]. Additionally, our last study showed that poly(Tannic Acid) nanoparticles can be prepared at various size (400-800 nm) by using different cross-linker to have controllable TA release [45]. Here, we prepared conjugated forms of TA into nano and micron size p(MA) particles to avoid extensive use of TA as well as to bring new functional groups (-COOH) from p(MAc) particle. From the degradation studies in PBS (pH 7.4), it was demonstrated that p(MA)-TA microparticles with 1% crosslinking density released 83.24 mg TA per g particles in comparison to p(MA)-TA nanoparticles with 10% the crosslinking density that released 35.21 mg per g particles within four days without any burst release. The magnetic field responsive p(MA)-TA composites were also successfully prepared by the inclusion of magnetic Fe3O4 particles within p(MA) particle during synthesis and within the TA conjugated p(MA) particles readily. Magnetic particles stabilized with carboxylic acid with electrostatic interaction provide targetable characteristic for many potential applications. Moreover, it was found that the p(MA)-TA microparticles (1% cross-linked) has great antioxidant properties with TPC=61.41±1.44 mgL-1 GAE, TEAC=248.82±1.96 mM trolox g-1 values for dry samples suggesting that these p(MA)-TA particles have many potential applications in biomedical field and food industry. For example, p(MA)-TA particles with tunable sized and

20

controllable degradation capabilities may be useful as an additive in the wound dressing materials that allow the protection of the wound from inflammation. Furthermore, the use of these kind of materials in different pharmaceuticals to capture radicals, to reduce oxidative stress caused by free radicals are also some of the other added gains. Moreover, this TA based materials can also be used as food seasonings to reduce spoilage and extent self-life of food products.

Acknowledgements This work is supported by the Scientific and Technological Research Council of Turkey (113Z238).

21

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Figure captions Fig. 1 (a) Schematic illustration of the preparation of p(MA) particle (1) micrometer sizes prepared by 1% EGDMA, (2) nanometer size particles prepared by 10% EGDMA, and (b) optical image of the 1% cross-linked microparticles prepared by method (1) and SEM image of the 10 % cross-linked nanoparticles prepared by method (2).

Fig. 2 (a) The schematic of the synthesis Tannic Acid decorated poly(methacrylic acid) micro and nanoparticles, (b) The photograph (1), SEM (2) and optical (3) images of the micro p(MA)-TA conjugate particles.

Fig. 3 (a) FT-IR spectrum of p(MA), TA, and p(MA)-TA and, (b) TGA thermogram of the p(MA) particles (dash line) and TA conjugated p(MA) particles as p(MA)-TA (straight line).

Fig. 4 (a) Titration curves of 100 mg p(MA), and 100 mg p(MA)-TA particle suspension in 50 mL 0.01 M KCl solution with 0.1 M NaOH, (b) differential plots of the titrations curves.

Fig. 5. In vitro release profile of tannic acid conjugated p(MA) micro and nanoparticles in o PBS (pH 7.4) at 37 C.

26

Table Legends Table 1. The size of p(MA) the particles obtained by different polymerization technique using different amount of cross-linker.

Crosslinker % (nano)*

dH (nm)

Crosslinker % (micro)*

5

10

5

10

585±2

337±14 # (888±41)

1439±166

652±31

0.01

0.29 and (0.11)

0.07

0.41

PDI

#

*with respect to monomer mole amount . # Tannic acid conjugated p(MA)-TA particles

27

Table 2. The comparison of antioxidant capability of p(MA) and TA conjugates to determine their Total Phenol Content (TPC), and Trolox Equivalent Antioxidant Capacity (TEAC) value of p(MA) based particles.

10 % crosslinked (nano)

TPC

1 % crosslinked (micro)

p(MA)

magp(MA)-TA

p(MA)-TA

p(MA)

magp(MA)-TA

p(MA)-TA

3.75±0.36

3.75±0.36

37.04±1.98

32.20±1.26

17.91±0.18

61.41±1.44

-

-

108.85±0.55

-

248.82±1.96

TEAC -

28

(a) H2C

CH3

CH3

H2C

(1) C

O

APS, Span 80, Cyclohexane, 40 °C

O

O

OH

MA

EGDMA

(2) APS, CTAB, O

Water, 75 °C

O

H3C

CH2

(1)

(b)

(2)

Fig. 1 (a) Schematic illustration of the preparation of p(MA) particle (1) micrometer sizes prepared by 1% EGDMA, (2) nanometer size particles prepared by 10% EGDMA, and (b) optical image of the 1% cross-linked microparticles prepared by method (1) and SEM image of the 10 % cross-linked nanoparticles prepared by method (2).

(a)

HO HO

C

O

N

N

N

N

DMSO

C

1 h, RT

OH

HO

O

24 h, 80 oC

DMSO HO O

HO

OH

O O

HO

O

O

O

O

O

C

O HO

O

O O

O HO

HO

HO

O

O HO

O

OH OH OH

OH

OH O

O

C O

OH OH

O

HO OH

O O

O HO

OH

HO

OH

O

HO

O O HO

O

OH O

HO

OH

OH

O

O

HO

N

HO

O

O

O

O

HO

O O

O

O

HO

N

O

OH

HO

O

OH

O

OH

HO

OH

O

HO

O

O O O OH

OH OH OH

O OH OH

p(MA)-TA conjugate

(b)

1

2

3

Fig. 2 (a) The schematic of the synthesis of Tannic Acid decorated poly(methacrylic acid) micro and nanoparticles, (b) The photograph (1), SEM (2) and optical (3) images of the micro p(MA)-TA conjugate particles.

% Transmittance

p(MA)

(a)

TA

P(MA)-TA

4000

3500

3000

2500

2000

1500

1000

Wavenumbers (cm-1) 100.0

(b)

Weight loss ( %)

80.0

P(MA)

60.0

P(MA)-TA

40.0

20.0 0.0 100

200

300

400

500

600

700

800

900

Temperature (oC) Fig. 3 (a) FT-IR spectrum of p(MA), TA, and p(MA)-TA and, (b) TGA thermogram of the p(MA) particle (dash line) and TA conjugated p(MA) particles as p(MA)-TA (straight line).

12

(a)

11 10

pH

9 p(MA)

8

p(MA)-TA

7 6 5 4 3 0

2

4

6

8

10

12

14

16

Volume of 1.0 N NaOH, mL

20

p(MA)

(b)

18

p(MA)-TA

16

∆PH/∆ML

14 12 10 8 6 4 2 0 0

2

4

6 PH

8

10

12

Fig. 4 (a) Titration curves of 100 mg p(MA), and 100 mg p(MA)-TA particle suspension in 50 mL 0.01 M KCl solution with 0.1 M NaOH, (b) differential plots of the titrations curves.

Released amount of TA (mg/g)

100

p(MA)-TA nano

p(MA)-TA micro

80 60 40 20 0 0

20

40 Time (h)

60

80

100

Fig. 5. In vitro release profile of tannic acid conjugated p(MA) micro and nanoparticles in PBS (pH 7.4) at 37 oC.