Digest Journal of Nanomaterials and Biostructures

Vol. 7, No. 2, April - June 2012, p. 459 - 467

EVALUATION OF BIOACTIVITY OF POLY(ACRYLIC ACID) HYDROXYAPATITE NANOGOLD COMPOSITES IN IN VITRO CONDITIONS

AGNIESZKA SOBCZAK-KUPIEC, DAGMARA MALINA*, BOENA TYLISZCZAKa, MAREK PITKOWSKIb, KATARZYNA BIALIK-WSa, ZBIGNIEW WZOREK Institute of Inorganic Chemistry and Technology, Cracow University of Technology, 24 Warszawska St, 31-155 Cracow, Poland a Department of Chemistry and Technology of Polymers, Cracow University of Technology, 24 Warszawska St, 31-155 Cracow, Poland b Chair of Biotechnology and Renewable Materials, Cracow University of Technology, 24 Warszawska St, 31-155 Cracow, Poland
In this paper we report the preparation and characterization of ceramic-polymer composites doped with gold nanoparticles. Properties and applications in medicine and dentistry of colloidal gold nanoparticles depend upon their size and shape. The influence of metallic nanoparticles on the in vitro behavior was investigated by pH analyses of simulated body fluid and artificial saliva. The nanocomposites were characterized with the use of X-ray Diffaction (XRD) and Fourier Transformed Infrared Spectroscopy (FT-IR) methods. The results of in vitro tests confirmed that it is possible to produce hydroxyapatite/polymer (HA/polymer) composites doped with gold nanoparticles (AuNPs) for medical applications. Investigations proved that presence of gold nanoparticles in composites had influence on in vitro behaviour of HA/Polymer/AuNPs composites in artificial physiological fluids. Research results indicate that obtained composites demonstrate bioactivity features and could become innovative materials for biomedical and dentist applications. (Recieved November 30, 2011; Accepted March 24, 2012) Keywords: Hydroxyapatite, Poly(acrylic acid), Gold nanoparticles, incubation, SBF, artificial saliva

1. Introduction Polymer materials currently used in implantology have unfavorable mechanical properties such as mechanical strength, Young's modulus and crack resistance. However, it is expected that composite materials might improve these defects of polymers. For example polymer composites (biostable and bioresorbable) with hydroxyapatite as the second phase in the form of particles or fibres offer the possibility of production of materials with controlled mechanical properties and assumed biological behavior [1-5]. Application of resorbable matrices leads to a multifunctional implants, in which after defect repair and resorption of the polymer, hydroxyapatite phase can be the scaffold for bone tissue growth [4-7]. In bone surgery the most frequently used bioresorbable polymers are caprolactam, polylactides and their copolymers with glycolide [8]. The addition of hydroxyapatite particles to biostable polymers reduces the durability of the composite in comparison with starting material. Composites with addition of hydroxyapatite exhibit favorable biological behavior by acting of bioactive particles (originating from HA) as anchors for bone tissue, which provides a good connection with the living tissue implants. In case of biostable composites introduction of particles or fibers to polymer matrix might change the mechanism of biological interaction with
*

Corresponding author: dagmaramalina@chemia.pk.edu.pl

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the environment and thus long-term functioning of implants. The most commonly used biostable polymers in bone surgery are: polyethylene, poly(ethylene oxide), poly(methyl methacrylate) [9-11]. It is important to assess the surface properties of implant materials in contact with fluids simulating internal environment of human body, such as SBF (artificial blood plasma), Ringer's solution or artificial saliva. The behavior of composites in the environment of a living organism depends on many factors including chemical and phase composition, crystalline phases, the shape and size of the crystals, as well as the presence of network defects [12-14]. In this paper we synthesized polymer/ceramic composites with addition of nanoparicles, which subsequently were treated with fluids simulating body environment. After incubation composites were characterized with X-ray Diffaction (XRD), Fourier Transformed Infrared Spectroscopy (FT-IR), Gel Permeation Chromatography (GPC) and pH analysis. 2. Experimental part 2.1. Methods FT-IR analyses were carried out with the use of Scimitar Series FTS 2000 spectrophotometer produced by the Digilab company within the basic infrared range of 4004000 cm-1. The sample of 0.0007g was pressed with 0.20000.0011 g KBr into a pellet. The Deuterated Tri-Glycine Sulfate (DTGS) detector was used for mid-IR range measurements. There were 16 scans with the resolution of 4. The phase composition of the samples was analysed with the use of X-ray diffraction with Philips X'Pert diffractometer equipped with a graphite monochromator PW 1752/00, Cu K 1.54 nm, Ni filter (40 kV, 30 mA). The multifunction device CX-742 produced by the Elmetron company was applied in the pH analyses. The weight and number average molecular weight of solution ingredients after degradation were measured by gel permeation chromatography GPC method at 35C, relative to poly(ethylene glycol)/poly(ethylene oxide) standards on POLYSEP-GFC-P4000 column (Phenomenex) and a refractive index detector. The water with a flow rate of 0.5 ml/min was used as the eluent. 2.2. Materials All chemicals were of reagent grade and used without further purification. Hydrogen tetrachloroaurate (III) trihydrate (0.21% solution), trisodium citrate, acrylic acid, ammonium persulfate and potassium hydroxide were purchased from POCh (Poland). Diacrylate ethylene glycol (M = 525) as crosslinking agent was purchased from Sigma Aldrich, however the solution of sodium acrylic copolymer Dispex N40 from Ciba Specialty. 2.2.1. Gold nanoparticles AuNPs The gold nanoparticles were prepared by sodium citrate reduction method [15] with modifications. The synthetic method developed for this experiment consistently produces stable gold nanoparticles, provided that conditions are properly controlled [16]. AuHCl4 was applied as a source of gold ions and trisodium citrate was used both as stabilizing and reducing agent. 2.2.2. Hydroxyapatite HA Hydroxyapatite (HA) applied in our research was animal origin (pork) and was prepared in our original method. The process of obtaining hydroxyapatite consist of three stages: 1. Acid hydrolysis of pork bones using lactic acid under the pressure 0.3 MPa and at temperature 135C. 2. Preliminary calcination at a temperature of 600oC in a chamber kiln with electric heating in air atmosphere within 180 minutes. 3. In the last stage proper calcination the unified material from the second stage was calcined at 950oC while kept at maximum temperature within 150 minutes in the chamber kiln [17]. 2.2.3. Preparation of HA/polymer/AuNPs nanocomposites Preparation process of ceramic/polymer composites modified with gold nanoparticles consists of following stages:

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1. Neutrali ization of acr rylic acid by y 40% potassi ium hydroxide solution, 2. Preparat tion of HA dispersion d in Dispex N40 (dispersing agent), 3. Polymer rization and finally cross slinking of th he product. In connect tion with ne eutralization, which was exothermic reaction, th he rapid increase of temper rature was observed. o The en the mixtu ure was cool led and before prepared HA dispersi ion was added. . Next, the proper am mount (1-5% %) of gold nanoparticl les suspensi ion (AuNPs s) with concen ntration of 50 5 ppm, initiator and cro osslinking ag gent were ad dded. After h homogenizat tion the mixtur re was micr rowave irrad diated for 3 minutes to enhance po olymerization n and cross slinking reactio ons [18-21]. lts and dis scussion 3. Resul 3.1. FT-IR R analysis of f composites s Spectral analyses a were made fo or all ceram mic-polymer composites doped wit th gold nanopa articles.

Fig. 1. FT-IR spectra of f composites

Figure 1 demonstrates d the FT-IR s spectrum of composite with w content of 5% AuN NPs and sample e without go old nanoparti icles. All spe ectra of samp ple with AuN NPs were sim imilar. On th he basis of thes se investigat tions it was found that v vibration cha aracteristic for fo functiona al groups pre esent in polym mer matrix, were w observe ed. The broa ad absorption band at th he range of 3500 300 00 cm-1 corresp ponded to st tretch vibrati ions of hydro oxide group was observe ed - the band d broadening g was a result o of water molecules abso orbed into the e polymer matrix. m In the range of 29 970 2930 cm c -1 the observ ved valence vibrations v we ere from the e CH2 group, , however ba ands in the ra ange of 1150 0 350 -1 cm came from to orsional vibr rations of thi is functional l group. The e absorption band at 164 40 cm-1 was co orresponded to tensile vib brations of C C=O group. The band in the range o f 1580 147 75 cm-1 was a result of de eforming vib bration of N NH and CN N groups. However ba ands from CH C 2-CO groups s were visible in the rang ge of 1435 1390 cm-1. The T bands in n the range o of 1100 120 00 cm-1

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were corresponde ed to valen nce vibration ns of CN N groups. No absorp rption bands s from hydrox xyapatite we ere observed d. The lack of the mos st intensive band at 12 200-1000 cm m-1 was corresp ponded to as symmetric st tretch vibrati ions of P-O bond, b as wel ll as absorpti ion at 964 cm m-1 was from s symmetric vi ibrations of P-O bond. Vibrations of o O-P-O bo onds assigned ed to the absorption -1 bands within wave e numbers ra ange of 604 -563 cm were w not noticed. In the s spectra of obtained compo osites the low w intensive bands b at 145 57 and 881cm m-1 correspon nded to stret tching vibrat tions of CO32- w were not obs served. After r comparing of these two o spectra it was w observed d that peaks at a 1551, 1439 a and 814 cm-1 were strengthened and s shifted to 155 58, 1423, 835 5 cm-1 respec ctively. 3.2. XRD analysis of composites c The XRD analyses of ceramic/poly c ymer composites modifie ed with gold nanoparticle es were carried d out. Figure 2 demonstra ates the typic cal XRD pat ttern. The am morphous pol lymer matrix x on the XRD pattern wa as observed d as backg ground nois ses and it t covered p peaks came e from hydrox xyapatite (HA A). The cont tent of ceram mic particles in composite e materials am mounted to 1% and it could be near the e limit of pha ase determin nation by roen ntgenographic method.

F 2. Typical XRD pattern of ceramic/po Fig. olymer compo osite with AuN NPs

3.3. Invest tigation in si imulated bo ody fluid SBF S The immer rsion study of o PAA/HA/ /AuNPs was carried out in a solution n of simulate ed body SBF prepared according to Kokubo et al. [22]. The assessm ment of in vi itro bioactivi ity was fluid S lastic container in 100 ml carried d out by soa aking the sam mples in a pl m of SBF w with pH = 7. .34 and mainta ained at 37C. The SBF F solution ha ad a composition and concentration c n similar to that of human n plasma. The ion concen ntration of SB BF (mmol) is s given in tab ble 1.
T Table 1. The comparison c of f ion concentr ration and the pH value of SBF S and huma an plasma [17 7]

Conc centation [m mmol/dm3] pH 2Na+ K+ Mg2+ C Ca2+ ClHCO3- HPO H 42- SO4 4 7.2human n 142.0 5. .0 1.5 3 3.5 103.0 27.0 1.0 1 0.5 7.4 plasma a 142.0 5. .0 1.5 3 3.5 147.8 4.2 1.0 1 0.5 7.4 SBF

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Time o of soaking was w 14 days. After soakin ng, the specim mens were removed r from m the fluid, washed w with di istilled water r and dried to o a constant weight.

Fig. 3. Th he pH changes of simulated d body fluid in n the course of f composites in incubation

The chemi ical stability and biologi ical activity were evaluat ted on the b basis of pH changes c recorded with time e of immersio on in physio logical fluid - Simulated Body Fluid (SBF) - rele evant to natural l human env vironment (F Fig. 3). The initial pH value of SBF F was 7.34 a at 25C. Sign nificant effect of gold nan noparticles co ontent on the e pH value of SBF was observed. F For sample without w nanopa articles incre ease of pH value v was ob bserved in th he first perio od of immers sion. On the e fourth day of f soaking the pH value sta abilized at le evel of ~7.65 5 and was kept to the end d of incubatio on. The growth h of gold nan noparticles co oncentration n caused decr rease of pH value. v The lo owest pH val lue was observ ved for samp ple containin ng 5% AuNP Ps and amou unted 7.28 after a second day of incu ubation. Next, p pH value gra adually increased for all i immersed samples.
Table 2. Results of gel per rmeation chro omatography analyses a (GPC C) of Simulate ed Body Fluid d

Sample

Mw Weight W avera age mo olecular wei ight 241 317 286 309 301 267

Mn umber avera age Nu mo olecular weight 249 325 294 317 309 276

P olecular weight Mo distribution n 0.97 0.98 0.97 0.97 0.97 0.97

0% Au 1% Au 2% Au 3% Au 4% Au 5% Au

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In samples s with SBF as a the degrad dation mediu um no polym meric or oligo omeric comp pounds, t medium in ngredients, were w found. Therefore polymeric p materials m bas sed on poly( (acrylic except acid), h hydroxyapat tite and gold d nanoparticle es are consid dered to be stable s in Sim mulated Body y Fluid. Howev ver, no signif ficant differe ences correla ated with gol ld concentrat tion changes were visible e.

Fig. 4. FT T-IR spectra o of composites after incubati ion in SBF

FT-IR spectra of samp ples after inc cubation in simulated s body fluid pres sents Figure 4. The ed spectra ar re similar to each other. After compa arison of spectra of samp ples before an nd after obtaine immer rsion distinct t peak at 104 40-1032 cm-1 , which corresponds to vibration v of P PO42- groups, , can be observ ved. The PO42- group orig ginated from m hydroxyapa atite incorpor rated in poly ymer matrix as well as hyd droxyapatite growing on the samples s surface. The hydroxya apatite agglo omerates formed of SBF io ons indicate that t the comp posite sampl les exhibit bi iological acti ivity. 3.4. Invest tigation in artificial a sali iva In order to evaluation n of applicat tion of hydr rogel/hydrox xyapatite/AuN NPs compos sites in stomat tology, incu ubation in artificial a sali iva was rea alized. The artificial sa aliva was pr repared accord ding to ISO standard [23] and its pH v value was be etween 5.2 an nd 5.5. Comp position of artificial saliva applied in in n vitro tests presents p Tabl le 2. Due to o assessment t of composi ites behavior r in condition ns of simula ated oral cavi vity the pH value v of artifici ial saliva was s investigated during 14 days at 37C. C

Ta able 3. Compo osition of artif ficial saliva [2 23]

Component t NaCl KCl CaCl2H2O NaH N 2PO4H2 O Na2SH2O urea

Quantity Q [g/d dm3] 0.400 0.400 0.795 0.780 0.005 1.000

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Fig. 5. The pH chan nges of artifici ial saliva in th he course of co omposites incu ubation

Figure 5 presents p chan nges of pH v values of arti ificial saliva in function of incubatio on time. For all l composite samples s rapi id decrease o of pH value was w noticed after second d day of incu ubation, and am mounted from m 5.08 (5% Au) to 5.29 (0% Au). All A samples were w charact terized with similar tenden ncy in pH value changes. . For sample e contained th he highest am mount of Au uNPs the low west pH values was observe ed and next gradual g and constant gro owth of pH was w noticed. S Samples con ntaining 1% to 4% AuNPs s caused sim milar growth of pH value of artificia al saliva. Ho owever, for sample withou ut gold nanop particles the highest incre ease of pH value was not ted.
Table 4. Results R of gel permeation p ch hromatograph hy analyses (G GPC) of artific cial saliva.

Sample

Mw Weight W avera age mo olecular wei ight 2 500 29 2 700 31 2 900 30 3 900 30 2 900 30 2 500 26

Mn Nu umber avera age mo olecular weight 2 400 33 2 600 35 2 800 34 3 600 33 2 800 34 2 400 29

P Mo olecular weight distribution n 1.06 0.86 1.06 0.89 1.06 0.89 1.07 0.89 1.06 0.89 1.06 0.89

0% Au 1% Au 2% Au 3% Au 4% Au 5% Au

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In samples s with artificial saliva use ed as the deg gradation me edium only o oligomeric fr ractions of poly ymer and lo ow molecula ar solution in ngredients were w found. Therefore c composite materials m based on poly(acry ylic acid), hy ydroxyapatite e and gold na anoparticles are considere red to be unst table in artifici ial saliva. The T most int teresting is sample with h AuNPs co oncentration of 3%, where the degrad dation rate of f polymer ma atrix was the e lowest.

Fig. 6. FT-IR R spectra of f samples incu ubated in art tificial saliva a The FT-IR R analyses were w realized d for cerami ic/polymer composites c d doped with AuNPs after im mmersion in n artificial sa aliva at 37C C (Figure 6). The research h results ind dicated that 14-days 1 degrad dation period d caused insig gnificant dec composition of polymer matrix. The FT-IR spect tra after immer rsion in artif ficial saliva for all com mposite samp ples containi ing Au were e of similar shape. Howev ver some pe eaks (at 797 7, 1078, 109 99, 1260 cm m-1) were str rengthened f for sample without w AuNPs s. Infrared investigation i ns confirmed d that presen nt of gold nanoparticles n s had consid derably effect o on composit tes structure. 4. Concl lusion vironment of f the living o organism the e behavior of o polymer/c ceramic com mposites In the env with th he addition of o gold nanop particles dep pends on chem mical and ph hase composi ition, shape and the size of crystallites s, presence of network defects, as well as bio ological con nditions arou und the implan nt. The resea arch on the influence of simulated bo ody fluids on n composite e materials provides interes sting informa ation about stability in the environm ment of the living orga anism, but th hey are limited d comparing g with tests on cell line es or in vivo o conditions s. An undou ubted advant tage on incuba ative experim ments in flui ids simulatin ng living org ganism is easiness of co onducting the em and lack of f surgeries on n tested anim mals. The resu ults of in vitro tes sts confirm med that it i is poss sible to produce p hydrox xyapatite/pol lymer compo osites doped with gold nanoparticles n for medical l applications s. Tests proved d that conte ent of gold d nanopartic cles in com mposites had d influence on behavi iour of HA/Po olymer/AuNP Ps in artific cial saliva an nd simulated body fluid d. Measurem ments of pH H value confirm med that com mposite mate erials charact terises differe ent in vitro behaviour. b

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