PMMA-multigraft copolymers derived from linseed oil, soybean oil, and linoleic acid: Protein...

PMMA-Multigraft Copolymers Derived from Linseed Oil,Soybean Oil, and Linoleic Acid: Protein Adsorption andBacterial Adherence

Birten Cakmaklı,1 Baki Hazer,1 Serefden Acıkgoz,2 Murat Can,2 Fusun B. Comert3

1Department of Chemistry, Faculty of Arts and Sciences, Zonguldak Karaelmas University, 67100 Zonguldak, Turkey2Department of Biochemistry, Faculty of Medicine, Zonguldak Karaelmas University, 67100 Zonguldak, Turkey3Department of Microbiology, Faculty of Medicine, Zonguldak Karaelmas University, 67100 Zonguldak, Turkey

Received 11 August 2006; accepted 18 February 2007DOI 10.1002/app.26397Published online 30 May 2007 in Wiley InterScience (www.interscience.wiley.com).

ABSTRACT: Synthesis of Poly(methyl methacrylate), PMMA-multigraft copolymers derived from linseed oil, soybean oil,and linoleic acid PMMA-g-polymeric oil/oily acid-g-poly(3-hydroxy alkanoate) (PHA), and their protein adsorption andbacterial adherence have been described. Polymeric oil/oilyacid peroxides [polymeric soybean oil peroxide (PSB), poly-meric linseed oil peroxide (PLO), and polymeric linoleic acidperoxide (PLina)] initiated the copolymerization of MMA andunsaturated PHA-soya to yield PMMA–PLO–PHA, PMMA–PSB–PHA, and PMMA–PLina–PHA multigraft copolymers.PMMA–PLina–PHA multigraft copolymers were completelysoluble while PMMA–PSB–PHA and PMMA–PLO–PHAmultigraft copolymers were partially crosslinked. Crosslinkedparts of the PLO- and PSB-multigraft copolymers were iso-lated by the sol gel analysis and characterized by swellingmeasurements in CHCl3. Soluble part of the PLO- and PSB-multigraft copolymers and completely soluble PLina-multi-graft copolymers were obtained and characterized by spectro-

scopic, thermal, gel permeation chromatography (GPC), andscanning electron microscopy (SEM) techniques. In the me-chanical properties of the PHA–PLina–PMMA, the elonga-tion at break is reduced up to � 9%, more or less preserv-ing the high stress values at its break point (48%) whencompared to PLina-g-PMMA. The solvent casting film sur-faces were studied by means of adsorption of blood pro-teins and bacterial adhesion. Insertion of the PHA intothe multigraft copolymers caused the dramatic increase inbacterial adhesion on the polymer surfaces. PHA insertioninto the graft copolymers also increased the protein ad-sorption. � 2007 Wiley Periodicals, Inc. J Appl Polym Sci 105:3448–3457, 2007

Key words: poly (3-hydroxyalkanoate)s; PHA-soya; Pseudo-monas oleovorans; autooxidation; polymeric linseed oil; poly-meric soy bean oil; polymeric linoleic acid; stress–strainbehavior; blood compatibility; bacterial adherence

INTRODUCTION

Many microorganisms produce polyesters as energyreserve material from a wide variety of carbon sub-strates,1–3 including alkanes, alcohols, alkenes, alka-noic acids, and their derivatives.4–11 Unsaturatedhydrophobic PHAs are produced by Pseudomonas oleo-vorans from edible oils such as soya bean and linseedoil,12–16 which are sticky, waxy, and soft materials.Chemical modification technique is used to improvethe mechanical and viscoelastic properties of thePHAs.17–19 Unsaturated PHA obtained from soy beanoil (PHA-soya) was grafted with poly(ethylene glycol)(PEG)20 and PMMA21,22 in our laboratories. Recently,considerable research has been concentrated on the

development of bio-based polymers using natural oilsor their derivatives as the main comonomer. Woolet al. and Aranguren et al. have, respectively, devel-oped a series of polymer resins using multifunctionalsoybean and linseed oil derivatives as a main compo-nent and styrene as a comonomer.23 In addition, per-oxidized linoleic acid polymer is a member of poly-meric peroxides24,25 and therefore can lead to multi-block copolymer.26

On the other hand, we have recently reported theautooxidation of the unsaturated edible aliphatic oilssuch as linseed oil (LO), soy bean oil (SB), and linoleicacid (Lina) to prepare polymeric oil peroxy initiators(PLO, PSB, and PLina)27–29 for free radical polymeriza-tion.

Tissue engineering uses polymer materials, includ-ing pure polymers such as PMMA, polymer blends,and copolymers as biomedical materials.30 The bio-compatibility of a polymer material can be inferred bystudying the protein adsorption on this polymer.When a polymer is implanted, the first body reactionis protein adsorption. The adsorbed proteins deter-mine later body reactions and finally determine

Correspondence to: B. Cakmaklı ([email protected]).Contract grant sponsor: Zonguldak Karaelmas University

Research Fund; contract grant number: 2004-13-02-01.Contract grant sponsor: TUB_ITAK research project; con-

tract grant number: 104M128.

Journal of Applied Polymer Science, Vol. 105, 3448–3457 (2007)VVC 2007 Wiley Periodicals, Inc.

whether the material is accepted or rejected by thebody. Surface chemical structures as well as surfacemorphology can mediate protein adsorption behav-ior.31 Protein adsorption is a complex process involv-ing van der Waals, hydrophobic and electrostaticinteractions, and hydrogen bonding. The purificationof albumin is generally required for the treatment ofhypoproteinemia. Hydrophobic interaction separationhas become a popular technique for purifying proteinsand enzymes.32,33

Synthetic materials used in blood-contacting devi-ces suffer from poor hemocompatibility. To solve thisproblem, better knowledge of the mechanisms ofblood interaction with materials is necessary. A princi-pal mechanism is the adsorption of plasma proteins.When blood first comes into contact with foreignmaterials, plasma proteins are adsorbed onto the sur-faces in less than a second. The adsorption of proteinsaffects subsequent platelet adhesion, which plays amajor role in thrombegenesis on foreign surfaces.34

Human albumin, g-globulin, and fibrinogen wereused as model proteins to study the surface adsorp-tion of proteins. Protein adsorption onto polymer sur-faces is important because of its possible involvementat the initial stage of blood coagulation. Albumin isthe major constituent of blood plasma (representingabout 60% of plasma proteins) and is also one of thesmallest proteins in the plasma. As a fibrinogen mole-cule adsorbs on a polymer surface, it undergoes struc-tural, conformational, or orientational changes. Suchchanges greatly affect the binding capability of fibri-nogen molecules to platelets. The surface-bound fibri-nogen has an important role in thrombus formation.Therefore, understanding the molecular structures offibrinogen molecules adsorbed on different surfacesshould contribute to the understanding of platelet ad-hesion on such surfaces and thus the blood compati-bility of these material.31–35

Bacteria and other microorganisms have a naturaltendency to adhere to surfaces as a survival mecha-nism. This can occur in many environments includingthe living host, industrial systems, and natural waters.The general outcome of bacterial colonization of surfa-ces is the formation of an adherent layer (biofilm)composed of bacteria embedded in an organic ma-trix.36 In many biomedical applications the adhesionof bacteria to biomaterials causes undesirable inflam-mation or infection. In recent years, various groupshave therefore focused on the development of bioi-nert, biocompatible coatings that can be used to mini-mize protein adsorption and bacterial adhesion whilemaintaining the mechanical and physical properties ofthe underlying substrate.37

Polymeric materials, including pure polymers andcopolymers, are extensively applied as biomedicalmaterials. The biocompatibility of a polymeric mate-rial is important in biomedical applications such as

blood-contacting devices. Our recent studies havebeen focused on the diversification of the biomedicalpolymers. In this manner, this work refers to the syn-thesis of some new types of biodegradable polymermaterials-graft copolymers containing PHA-soya, pol-ymeric oil/oily acid, and PMMA, at a low tempera-ture with tetraethylene pentamine as a catalyst.

EXPERIMENTAL SECTION

Materials

Linseed oil was supplied from Aldrich (St. Louis,MO), soybean oil was locally supplied and both wereused as received. Linseed oil and soybean oil are tri-glycerides and they contain linoleic acid of 15.3 and 51mol %, respectively.38

Linoleic acid (cis-cis-9-12-octadecadienoic acid) wassupplied by Fluka (Steinheim, Germany), tetraethy-lene pentamine was obtained from Merck (Darmstadt,Germany) and both were used as received. MMA wassupplied by Aldrich and freed from inhibitor by vac-uum distillation over CaH2.

Human albumin and g-globulin were supplied bySigma and fibrinogen was from Fluka.

All other chemicals were reagent grade and used asreceived.

Substrates and PHA biosynthesis

Biosynthesis of the PHA-soya

Soybean oil was extracted from the related productsgrown in Turkey, and hydrolyzed13 into its fatty acidsto make them soluble in water. The acids obtainedfrom the hydrolysis of soybean oil included both satu-rated (palmitic and stearic: 18 mol %) and unsaturatedacids (oleic: 18–26 mol %, linoleic: 50–55 mol %, andlinolenic: 7–10 mol %).38,39 Stock cultures of P. oleovor-ans (ATCC 29347) were used in all growth and poly-mer production experiments. P. oleovorans was grownon soybean oily acid substrate and the resulting poly-mer was extracted by using methods in Refs. 4–6,13.

Autooxidation of the LO, SB, and Lina

For the formation of PLO,27 PSB,28 and PLina,29 5.0 gof the oil or the oily acid was spread out in a Petri dish(f ¼ 16 cm) and exposed to sunlight in the air at roomtemperature. The upper layer gel film of the PLO,PSB, and PLina were removed by peeling off the gelfilm layers on these glass plates at the end of specifiedtime intervals (60 days). The lower parts of the gellayers were soluble polymeric oil peroxides: sPLO andsPSB, which are pink-yellow colored viscous liquids.Unlike PLO and PSB, there was no gel layer for PLina,totally pink viscous soluble liquid was observed. Thepolymeric oil peroxides were characterized by means

PROTEIN ADSORPTION AND BACTERIAL ADHERENCE 3449

Journal of Applied Polymer Science DOI 10.1002/app

of gel permeation chromatography (GPC) and peroxy-gen analysis and then used in the polymerizationof MMA with the presence of PHA-soya. Molecularweights of the polymeric oils/oily acid are listed inTable I.

Peroxygen analysis

Peroxide analysis of polymeric oil/oily acid peroxideswas carried out by refluxing a mixture of 2-propanol(50 mL), acetic acid (10 mL), saturated aqueous solu-tion of KI (1 mL), and 0.1 g of the polymeric samplefor 10 min and titrating the released iodine against thi-osulfate solution according to the literature.24 Peroxy-gen contents of the samples varied from 0.66 to 1.32wt %. Peroxide contents of the polymeric oil/oilyacids are listed in Table I.

Grafting reactions

For grafting reaction, a given amount of a polymericoil/oily acid, PHA, MMA, and tetraethylene pentam-ine catalyst were charged separately into a pyrex tube.Argon was introduced through a needle into the tubefor about 3 min to expel the air. The tightly cappedtube was then put in a water bath at 268C. After therequired time period, the contents of the tube werecoagulated in methanol. The graft copolymer sampleswere dried overnight under vacuum at 308C. Theresults and conditions of the grafting reactions arelisted in Table II.

Purification of the graft copolymers

In a typical purification procedure via fractional pre-cipitation,40,41 0.5 g of polymer sample was dissolved

in 10 mL of CHCl3. Methanol was used as a nonsol-vent and kept in a 50-mL buret. Afterwards, methanolwas added to the polymer solution with continuousstirring, until the polymer began to precipitate. At thispoint, g-value was calculated by taking the volume ra-tio of the consumed nonsolvent (methanol) to CHCl3solution of the graft copolymer. The fractionated poly-mer was dried under vacuum at room temperature.

Polymer characterization

1H NMR were recorded in CDCl3 at 178C with tetra-methylsilane as internal standard, using a 400 MHzNMR AC 400 L. A typical 1H NMR spectra of a multi-graft copolymer and its diblock/graft copolymer aregiven in Figure 1.

Molecular weights of the polymeric samples aredetermined using GPC with a Waters model 6000Asolvent delivery system with a model 401 refractiveindex detector, a mode 730 data module and twoUltrastyragel linear columns in series. Tetrahydrofu-ran was used in the elution at a flow rate of 1.0 mLmin�1. A calibration curve was generated with sixpolystyrene standards. Figure 2 shows GPC curves ofthe multigraft copolymers. GPC results of the multi-graft copolymers are listed in Table III.

Differential scanning calorimetry (DSC) was per-formed on a Netzsch DSC 204 with CC 200 liquidnitrogen cooling system to determine the glass transi-tion temperatures (Tg), and thermogravimetric analy-sis (TGA) of the obtained polymers were performedon a PL TGA 1500 instrument to determine thermaldegradation. For DSC analysis, samples were heatedfrom �50 to þ 3008C in a nitrogen environment at arate of 108C min�1. Figure 3 shows the thermogravi-metric traces of the multigraft copolymers. Thermalanalysis results are given in Table IV.

Swelling degrees of polymers obtained at equili-brium were determined by gravimetry at roomtemperature in CHCl3. Swelling ratios, qv, werecalculated using the volume ratio of swollen polymer(vswollen polymer) to dry polymer (vdry polymer).

42,43

Swelling ratios of the crosslinked multigraft copoly-mers are listed in Table II.

TABLE ICharacterization of the Polymeric Oil Peroxides

Polymericoil peroxide

Peroxygenanalysis (wt %)

GPC analysis

Mw MWD

sPLO 1.3 2100 1.92sPSB 1.1 4590 1.52PLina 0.7 1684 1.22

TABLE IIResults and Conditions of the Synthesis of the Multigraft Copolymers at 268C

Multigraft copolymerPLOs

(g)PSBs

(g)PLinas(g)

PHA-soya (g)

MMA(g)

Polym.time (h)

Polymer yield

Total(g)

Crosslinkeda

(wt %) qvb

57-1 (PHA–PLO–PMMA) 1.0 – – 0.5 3.0 24 1.71 75.1 24.358-1 (PHA–PSB–PMMA) – 1.0 – 0.5 3.0 24 1.54 48.0 36.059-1 (PHA–PLina–PMMA) – – 1.0 0.5 3.0 24 1.71 – –

a The rest of the percentage is soluble polymer.b Swelling ratio of the cross-linked polymer (in CHCl3).

3450 CAKMAKLI ET AL.


For the protein adsorption tests, a Shimadzu UV1601 model UV spectrophotometer was used.

Tensile test

For the polymer samples, the tensile tests are per-formed on AG-I 5 kN Shimadzu Autograph testmachine with a constant tensile speed of 0.5 mm/min.The dimensions of the test specimens are given inTable V. In this table, the stress and strain values atbreak point of the three polymer specimens are pre-sented. Figure 4 shows the stress–strain behavior ofthe specimens which are subjected to the above tensiletest.

Scanning electron microscope

Scanning electron micrographs were taken on a JEOLJXA-840A electron microscope. The specimens werefrozen under liquid nitrogen, then fractured, mounted,and coated with gold (300 A) on an Edwards S 150 Bsputter coater. The SEM was operated at 15 kV, andthe electron images were recorded directly from thecathode ray tube on a Polaroid film. The magnificationemployed was varied up to � 15,000; however, 3000,5000, 10,000, and 1000 magnifications were useful.SEM micrographs of the multigraft copolymers areshown in Figure 5.

Human blood protein adsorption test

Human albumin, g-globulin, and fibrinogen were usedto study the adsorption behavior of proteins on surfa-ces of polymer samples. Small disks (15 mm in diame-ter) of the polymer films were prepared using a punchand immersed in protein solutions containing 1 mg/mL of phosphate buffer solution (PBS) (pH 7.3–7.4) at

Figure 1 1H NMR spectra of (a) the PLina-g-PMMA29 and(b) PHA–PLina–PMMA copolymer samples (59-1).

Figure 2 GPC choromotograms of the fractional precipi-tated multigraft copolymers (57-1, 58-1, and 59-1).

TABLE IIICopolymer Content and GPC Analysis of the Multigraft

Copolymers

Run no

Molecularweight

Copolymeranalysis(mol %)

Mw (�104) MWD PHAa PMMAa

PHA–PLO–PMMA (57-1) 42.8 2.9 11 78

PHA–PSB–PMMA (58-1) 57.8 5.3 15 80

PHA–PLina–PMMA (59-1) 57.2 3.3 5 75

a From the TGA traces.

Figure 3 Thermogravimetric traces of the PLina and multi-graft copolymers/PLina: PHA–PLO–PMMA (soluble) (57-1), PHA–PLO–PMMA (crosslinked) (57-1), PHA–PSB–PMMA(soluble) (58-1), PHA–PSB–PMMA (crosslinked) (58-1), andPHA–PLina–PMMA (soluble) (59-1), multigraft copolymers.



378C for 1 h. The discs were then recovered andchanges in the protein concentrations of the solutionborne proteins were determined using an UV spectro-photometer.35,44 The amount of adsorbed protein onthe copolymer surface was calculated as follows:

AAPðmg=mLÞ ¼ 1:55�APS ðl ¼ 280 nmÞ� 0:76�APS ðl ¼ 260 nmÞ ð1Þ

where AAP is the amount of adsorbed protein andAPS is the absorbance of protein solution.

Amount of adsorbed protein on the copolymer sur-face was calculated from the difference between UVabsorbency of a standard solution and that of the solu-tion after the polymer disc was removed (each experi-ment was repeated seven times).29 Obtained valuesfor AAP were divided by disk area (mg/cm2). Resultsof adsorption measurements of albumin, g-globulin,and fibrinogen on the prepared PMMA and copoly-mer disc surfaces are listed in Table VI.

Bacterial adherence

One Staphylococcus epidermidis and one Escherichia coliisolates obtained from two different patients who hadinfections related to intravascular and urinary cathe-ters were used for the adherence tests. The bacteriawere kept frozen at �808C in skim milk. Ten microli-ters of the bacterium culture was inoculated onto ablood agar plates (Oxoid, UK); tryptone (14.0 g/L),peptone (4.5 g/L), yeast extract (4.5 g/L), sodiumchloride (5.0 g/L), agar (12.5 g/L), and sheep blood(7 wt %), and kept overnight at 378C. Bacterial suspen-sions of 108 colony forming unit (CFU) per mL wereprepared for each bacterium for the adherence testsaccording to the method in cited Refs. 45–47. For this

purpose, polymer disc (thickness �1 mm, ø ¼ 6 mm)was placed under sterile conditions in 1 mL of bacte-rial suspension and incubated at 378C for 30 min.Polymer disc was removed and rinsed with 2 mL ster-ile PBS three times for 60 s to eliminate nonadheringbacteria. The polymer disc was then transferred into1 mL of PBS in glass tube and agitated for 3 min viavortex at 2400 rpm/min. A 10 mL sample of PBS con-taining dislodged bacteria was seeded on blood agarplates and spread to facilitate subsequent colonycounting. Tenfold dilutions were made to calculate anaccurate count of bacteria adhered to the polymer discsurfaces. Tenfold-dilutions colonies were counted bythe naked eye after 24 h of incubation at 378C. The bac-terial density per polymer type (CFU/mL/mm2) wascalculated by dividing the colony number mean bythe total surface area (mm2) of the polymer disc.Results of the bacterial adhesion on the multigraftcopolymers by direct counting of viable adherent bac-teria are released by vortex agitation. The bacterialdensity (CFU/mL/mm2) was calculated by dividingthe colony number mean by the total surface area ofthe polymer disc.

RESULTS ANDDISCUSSION

Medium chain length (mcl) PHAs may be elastomericbut have very low mechanical strength. Therefore, forpackaging materials, biomedical applications, tissueengineering, and other specific applications, the phys-ical and mechanical properties of microbial polyestersneed to be diversified and improved.19 PMMA is anacrylic hydrophobic biostable polymer that is widelyused in the biomedical field as bone cement in ortho-pedics and traumatology and as implant carrier forsustained local delivery of antiinflammatory or antibi-

TABLE IVThermal Analysis of the Multigraft Copolymers

Polymer

DSC TGA

Tg1 (8C) Tg2 (8C) Td (8C) Td1 (8C) Td2 (8C) Td3 (8C)

PMMA–PLO–PHA (soluble) – – 194 293 396PMMA–PLO–PHA (crosslinked) 52 120 186 284 434PMMA–PSB–PHA (soluble) 115 174 266 398PMMA–PSB–PHA (crosslinked) 50 – – 183 262 417PMMA–PLina–PHA (soluble) 82 – 170 305 401

TABLE VDimensions of the Tensile Test Specimens and Test Data

Thickness(mm)

Width(mm)

Gaugelength(mm)

Stress at break(N/mm2)

Strain at break(%)

PMMA 0,2150 5 15 12.5 34PLina-g-PMMA (67-2) 0.05 5 15 55 30PHA–PLina–PMMA (59-1) 0.05 5 15 48 9



otics drugs.48 The presence of oil/fatty acid chain inthe polymer structure improves some physical prop-erties of polymer in terms of flexibility, adhesion,resistances of water and chemicals. Because of theirsource and structural nature, triglyceride oils canwidely be used as themselves. In bioapplications theirbiocompatibility and/or biodegradability play an im-portant role.49

Autooxidation of the oils and linoleic acid caused theformation of polymeric oil/oily acid peroxides (sPLO,sPSB, and PLina), which were characterized by meansof molecular weight measurements and the peroxygenanalysis (see Table I). Molecular weights of the poly-meric peroxides were changing between from 1684 to4690 Da. Peroxygen content of the polymeric peroxideswere found between from 0.66 to 1.32. So, these macro-peroxy initiators were successfully used to initiate freeradical polymerization of methyl methacrylate. Sch-eme 1 explains the simple design of the graft copoly-merization mechanism. In this manner, polymeric oil/oily acid peroxide produces polymeric oil/oily acidradicals, which can both initiate the polymerization ofMMA and attack to the double bonds of PHA.22

In this work, free radical polymerization of MMAwas initiated by each polymeric oil/oily acid peroxideinitiator in the presence of PHA to give partly cross-linked PMMA–PLO–PHA and PMMA–PSB–PHAmulti-graft copolymers, and completely soluble PMMA–PLina–PHA multigraft copolymers. Results and con-ditions of the graft copolymerization are listed inTable II.

Crosslinked and soluble copolymer fractions wereisolated by means of chloroform extraction. Swellingdegrees of the crosslinked multigraft copolymers atequilibrium indicated that the crosslinking density islow (e.g., qv ¼ 24.3 and 36.0).

Soluble fractions of the multigraft copolymers werefractionally precipitated to determine the g values ofthe graft copolymers. Homo-PHA and homo-PMMAprecipitated in the g ranges of 1.4–2.2 and 2.8–4.1respectively, while homo-PLO, homo-PSB, and homo-PLina were soluble in chloroform–methanol mixture

Figure 4 Stress–strain curves for tested polymers.

Figure 5 SEM micrographs of the multigraft copolymers:(a) PHA–PLO–PMMA (57-1) (left: �5000 and right:�10,000), (b) PHA–PSB–PMMA (58-1) (left: �3000 and right:�15,000), (c) PHA–PLina–PMMA (59-1) (left: �3000 andright: �15,000).



(g > 10). However, PLO–PHA–PMMA, PSB–PHA–PMMA, and PLina–PHA–PMMA multigraft copoly-mer fractions precipitated in g values of 0.4–2.0 and2.0–4.0, respectively. Because g values of the multi-graft copolymers with PMMA and PHA-homopoly-mers were almost superimposed, fractional precipita-tion was useful only in determining the g values of themultigraft copolymers instead of isolating pure graftcopolymers from the related homopolymers exceptunreacted polymeric oil/oily acid. Homo-PLO, -PSB,and -PLina, pale yellow liquids, were already elimi-nated by staying in the solution during the precipita-tion procedure. As we will discuss later, unimodalGPC curves can be attributed to the pure graft copoly-mers freed from the related homopolymers.

1H NMR spectra of the soluble multigraft copoly-mer samples contained the characteristic peaks ofPMMA ��COOCH3 at 3.7 ppm, PHA ��CH��O�� at5.2–5.4 ppm, and the peaks of the triglyceride��CH��O��, CH2��O�� at 4.1–4.4 ppm. Typical 1 HNMR spectra of the PLina multigraft copolymer andPLina-g-PMMA diblock/graft copolymer are shownin Figure 1. The characteristic signals of the additionalPHA blocks were observed in d: 1.0–2.0 and 5.0–5.4ppm.

GPC was used to determine the molecular weightsand polydispersity of the fractionated multigraftcopolymers (see Table III). GPC chromatograms of thegraft copolymers were all unimodal as shown in Fig-ure 2, which can be attributed to the pure multigraftcopolymer samples.

Thermal analysis of the graft copolymers was per-formed by DSC and TGA. Table IV lists the glass tran-sition (Tg) and decomposition temperature (Td). PHAand polymeric oils caused the plasticizer effect and itis observed that Tg of multigraft polymer decreased to508C where Tg of homo PMMA is � 1058C. We havealso observed peroxide decompositions in the samesample at around 1158C. In TGA curves, the graftcopolymers have three decomposition steps: decom-position at 170–1908C may come from the peroxidedecomposition of the undecomposed peroxide groupsof polymeric oil peroxides. 285–3008C belongs to thedecomposition of the PHA blocks and Td around4008C belongs to the PMMA and polymeric oil blocks.

Copolymer analysis was performed using the TGAcurves. Thus, PHA content in the graft copolymer wascalculated in a range from 5 to15 mol % (see Table III).

Tensile test

In Table V and Figure 4, the stress and strain values atbreak point of the three polymer specimens are pre-sented. When the graphs are observed, PMMA has �

TABLE VIProtein Adsorption Results on the Multigraft Copolymers

PolymerAlbumin(mg/cm2)

g-Globulin(mg/cm2)

Fibrinogen(mg/cm2) Reference

PMMA 7.1 3.1 20.7 16PLO-g-PMMA (run no: 39-6) 2.7 2.3 19.4 16PHA–PLO–PMMA (run no: 57-1) 6.4 3.3 19.6 This workPSB-g-PMMA (run no: 56-5) 4.6 3.1 20.1 16PHA–PSB–PMMA (run no: 58-1) 4.4 3.5 19.8 This workPLina-g-PMMA (run no: 67-1) 0.0 0.0 0.0 16PHA–PLina–PMMA (run no: 59-1) 3.0 3.7 19.6 This work

Scheme 1 Formation of the multigraft copolymers.



12.5 MPa stress at break point and 34% strain at breakpoint. It is worth noting that the data for PMMA mayshow discrepancies with the data given in the litera-ture because these properties are highly dependent onthe tacticity of PMMA. Containing PLina in its struc-ture, PLina-g-PMMA copolymer has increased valuesfor the stress at its break point (55 MPa), more or lesspreserving the strain value at its break point (30%)when compared to PMMA. However, for the PHA–PLina–PMMA copolymer, which contains both PLinaand PHA, the strain at break point is reduced up to�9%, more or less preserving the high stress values atits break point (48%) when compared to PLina-g-PMMA.

SEM analysis

SEM analysis showed the microstructure of the frac-tured surface of the copolymers obtained. Figure 5indicates the SEM pictures of the copolymer samplesof PHA–PLO–PMMA, PHA–PSB–PMMA, and PHA–PLina–PMMA. Homogeneous structure was observedfor PHA–PLO–PMMA (sample 57-1), PHA–PSB–PMMA (sample 58-1), and PHA–PLina–PMMA (sample59-1) copolymers where tiny holes are present within[see Fig. 5(a–c)].

Human blood protein adsorption test

Polymeric materials, including pure polymers andcopolymers, are extensively applied as biomedicalmaterials. The biocompatibility of a polymeric mate-rial can be inferred by studying the protein adsorptionon the polymer. After a polymeric material is im-planted, the first body reaction is protein adsorptionwhich is an undesired effect. However, the treatmentof hypoproteinemia requires the purification of albu-min. Because of aforementioned reasons, it is crucialto know the protein adsorption behavior of the poly-meric materials used as biomaterials. Surface mor-phology and surface chemical structures can alsomediate protein adsorption behavior. Results of pro-tein adsorption measurements with albumin, g-globu-lin, and fibrinogen onto the prepared samples areshown in Table VI.

Less amount of albumin, g globulin, and fibrinogenadsorptions are observed for PLO-g-PMMA, PSB-g-PMMA, PLina-g-PMMA when compared to PMMA.However, the albumin adsorption is reduced for thePHA containing multiblock copolymers when com-pared to PMMA while it is increased when comparedto PLO-g-PMMA, PSB-g-PMMA, PLina-g-PMMA. Onthe other hand, g-globulin adsorption of the PHA con-taining multiblock copolymers is increased whencompared with PMMA and PLO-g-PMMA, PSB-g-PMMA, PLina-g-PMMA. For fibrinogen adsorption,the values for PHA–PLO–PMMA and PHA–PLina–

PMMA are reduced compared with PMMA whilethey are increased when compared to PLO-g-PMMAand PLina-g-PMMA, but values for PHA–PSB–PMMAare lower than those which are measured for bothPMMA and PSB-g-PMMA.

These results showed that PLO, PSB, PLina, andPHA blocks affect the mechanical strength and ductil-ity of copolymers and play an important role indecreasing the adsorption of protein.

Bacterial adherence

Bacterial adherence to polymer surfaces varied signifi-cantly depending on the polymer type as well as thestrain of the bacteria.37 There are many studiesinvolved in bacterial adhesion on different polymersurfaces.50–52 The factors involved in the initial adhe-sion of bacteria to a substrate can be explained interms of nonspecific interactions (electrostatic forces,hydrogen bonds, and Van der Waals forces) andhydrophobic interactions. Van der Waals forces whichare usually attractive, come in to play at a separationdistance (between bacteria the repellent electrostaticforces increase due to an overlap of the electron cloudsof both bacteria and surface) of ‡ 50 nm and hold thebacteria relatively weakly to the surface.53–55 At a sep-aration distance of about 10–20 nm, the bacterial cell,although weakly held, is kept away from the substratesurface by electrostatic repulsion forces. At � 2 nm,water adsorbed to bacteria or substrate surfaces canact as a barrier to bacterial attachment. The exclusionof water to enable attachment is not kinetically favor-able; hydrophobic interactions, however, if present(usually within 2 nm of the surface) can help excludewater through nonpolar regions on both surfaces.Once a separation of � 1 nm is reached, other adhe-sion forces such as ionic bridging, hydrogen bonding,and ligand–receptor interactions occur.56,57

In this study, the adherence of bacteria to copoly-mer PMMAs was compared with that of PMMA. OneS. epidermidis and one E. coli to the PMMA and graftcopolymers were used for the adherence tests. Whilethe bacterial adhesion on PMMA for S. epidermidis andE. coli were the same, with insertion of PLO and PSBto PMMA, bacterial adhesion decreased significantlyfor E. coli (100- and 300-fold respectively) and S. epider-midis (twofold for each). Insertion of PLina did notaffect E. coli adhesion when compared with PMMA,but S. epidermidis adhesion increased � 45-fold. PHAinsertion to grafts, adhesion of both bacteria was in-creased significantly but this increment was higher forE. coli than for S. epidermidis. PHA insertion to PLO–PMMA and PSB–PMMA grafts increased the bacterialadhesion for S. epidermidis � 70- and 207-fold, respec-tively, while insertion of PHA to PLina–PMMAresulted in twofold increase. With the PHA insertionto PLO–PMMA, PSB–PMMA, and PLina–PMMA



grafts, bacterial adhesion increased 257-, 1439-, and 3-,7-fold respectively, for E. coli (Table VII). Very fewmicrobes have purely hydrophobic or charged surfa-ces. Their surfaces are complex, possessing chargedresidues as well as hydrophobic residues.58 It can beconcluded adhesion of bacteria on pure linoleic acid ismuch higher than the triglycerides. As explainedabove, hydrophobic interactions contribute to the ini-tial adhesion of microorganism to surfaces and con-cordant with this finding, in our study, PHA insertionto whole binary grafts resulted in increased bacterialadhesion. Even though more hydrophobic grafts withinsertion of PLO and PSB to PMMA have beenobtained, bacterial adhesion on that grafts (especiallyfor E. coli) decreased independently from the polymerhydrophobicity.

CONCLUSIONS

As macro-peroxyinitiators, PLO, PSB, and PLina initi-ate the free radical polymerization of the unsaturatedPHA and synthetic MMA and biodegradable and bio-compatible multiblock copolymers are obtained byaltering the properties of monomers. Diversification ofthe biomaterials can be diversified by using edibleoils. This can be done in two ways, either by directsynthesis of the polymeric peroxides via autooxida-tion or by obtaining polyesters from microorganisms.It is crucial to know the protein adsorption behaviorand bacterial adherence of the polymeric materials tobe used as biomaterials. Polymeric oil peroxides haveantimicrobial properties and nonprotein adsorbabilitywhile microbial polyester, PHA, affects the bacteriaadherence and protein adsorption. Thus, insertion ofthe PHA into the multigraft copolymers caused thedramatic increase in bacterial adhesion on the poly-mer surfaces. PHA insertion into the graft copolymersdecreased the albumin and g-globulin adsorption butincreased the fibrinogen adsorption when compared

to PMMA. PLO, PSB, and PLina blocks affect the me-chanical strength and ductility of polymers and playan important role in decreasing the adsorption of pro-tein and bacterial adherence.

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TABLE VIIResults of the Bacterial Adhesion on the Multigraft

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PMMA 46,000 43,000 15PLO-g-PMMA(39-6) 27,000 428 This workPMMA–PLO–PHA (57-1) 1,900,000 110,000 This workPSB-g-PMMA (56-5) 28,000 132 15PMMA–PSB–PHA (58-1) 5,800,000 190,000 This workPLina-g-PMMA (67-2) 2,100,000 43,000 This workPMMA–PLina–PHA (59-1) 4,200,000 160,000 This work

Determined by direct counting of viable adherent bacteriareleased by vortex agitation ((CFUs/mL)/mm2). The bacte-rial density ((CFUs/mL)/mm2) was calculated by dividingthe colony number mean by the total surface area of thepolymer disk.



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