Development of PVC membranes with clove oil as plasticizer for blood bag applications

Article history: Received on: 25/04/2016 Revised on: 15/05/2016 Accepted on: 30/06/2016 Available online: 28/07/2016 In the present study, Antimicrobial PVC films containing different amounts of clove oil as a plasticizer were prepared using traditional casting method. The physical and mechanical properties of the plasticized PVC membranes e.g. surface wettability were investigated. The increase of clove oil content demonstrated an increase in surface hydrophilicity and elongation to break the film. The thermogravimetric analysis revealed a decrease of polymer thermal stability by increasing clove oil concentration. The antibacterial activities against four different bacterial strains (two-gram positive: Staphylococcus aureus and Bacillus cereus & two-gram negative: Pseudomonas aeruginosa and Escherichia coli) were promoted by addition of clove oil. Although the natural source of clove oil, the bio-evaluation of plasticized membranes showed an increase in hemolysis percent (%) and thrombus weight. It can be concluded that the addition of clove to PVC need to further studies for applying in blood bags.


INTRODUCTION
Starting from the 17th century, transfers of blood from healthy volunteer to patients was established as a medical treatment for some diseases and during a surgical operation (Greening et al., 2010).Until 1950, glass bottles were the most standard containers for blood transfusions and storage.Several problems were associated with using these old fashion vessels related to sterilizations, breakability, and the presence of air bubbles surround valves and during a blood transfusion (Ramsey and Schmidt, 2009;Duran, 1939).All of these reasons driving scientists to develop a new generation of flexible bags based on polymeric materials.Polyvinylchloride (PVC) plays an essential DEHP can easily elute from PVC products into solutions that contact with the plastic and the migrated DEHP is directly and/or indirectly introduced into the human body (Allwod, 1986;Loff et al., 2000;Tickner et al., 2001).By 1967, Guess reported that DEHP leached from the plastic blood bags into plasma (Guess et al., 1967).Later, Jaeger and Rubin detected DEHP that present at levels of 50-70 mg/l (Jaeger and Rubin, 1970).The release of DEHP depends on some factors, covering the lipids content in the blood, the storage time and the temperature.DEHP is ranked as a reproductive toxin.
Self-disinfected PVC membranes to be used in biomedical take attention of scientists in last decay.Several approaches were done to achieve this goal.Including grafting of PVC surfaces with biocidal molecules to prevent bacterial growth on the surface.Indeed, antibacterial PVC surfaces were successfully prepared by grafting of Heparin (Zhou and Meyerhoff, 2005), Chitosan (Asadinezhad et al., 2010;Mao et al., 2004;Mohyeldin et al., 2015a), PEG (Balakrishnana et al., 2005), silver nitrate (Balazs et al., 2004), aromatic thiol compounds (Herrero et al., 2006).
Today the modern studies awarded to increase antimicrobial and haemocompatibility of the native polymer besides improving mechanical and physical properties of the polymer itself without adding any hazard materials (Omer et al., 2016).
Natural products such as plant essential oils (EOs) have used in human health such as functional food, food additives, medicine, nutritional supplements and cosmetic manufacturing.Essential oils also called volatile oils, are aromatic oily liquids obtained from plant materials such as flowers, buds, seeds, leaves, twigs, bark, herbs, wood, fruits, and roots.
Plant EOs have antimicrobial, antioxidant and antimutagenic activities, and potentially beneficial effects on certain health conditions (BURT 2004).The antimicrobial activity of essential oils is due to some small terpenoids and phenol compounds (Oussalah et al., 2006).Several of these are classified as generally recognized as safe (Sharififar et al., 2007).Essential oils such as tea tree oil, lavender oil, thyme oil, peppermint oil and eugenol oil have been traditionally used by people for various purposes in different parts of the world.
The aim of the work is to plasticize PVC with eugenol that considered the main component of clove oil instead of synthetic plasticizer DEHP to be used in blood bag applications.

Bacteria
Four bacterial strains were used for evaluating the antibacterial activity of plasticized PVC membranes.These were included two Gram-negative strains (E. coli and P. aeruginosa) and two Gram-positive (S. aureus and B. cereus).All bacteria were provided from Genetic Engineering and Biotechnology Research Institute (GEBRI), City of Scientific Research and Technological Applications (SRTA-City).The strains were refreshed through inoculating in LB broth (peptone 1%, yeast extract 0.5%, NaCl 1%, and pH 7±0.2) and they were incubated overnight at 37°C and 150 rpm in a rotary shaker.

Preparation of PVC membranes
The plasticized PVC membranes were prepared by using traditional casting method.PVC (1 g) was dissolved in 25 ml THF at room temperature.A predetermined amount of clove oil was added to the solution under stirring to obtain a homogenous solution.On a clean glass petri dish, the solution was cast at room temperature for 48 hrs to ensure complete solvent evaporation.Once the membrane was dried and separated from the Petri dish, it was rinsed with 50 ml of distilled water.The wet membranes were spread out and allowed to dry under vacuum for 24 hrs at room temperature.Six different plasticized PVC membranes with clove oil were studied and coded as sample PVC-0, PVC-C 0.1, PVC-C 0.2, PVC-C 0.3, PVC-C 0.4 and PVC-C 0.5 containing different amounts of clove oil 0, 0.1, 0.2, 0.3, 0.4 and 0.5 Wt % respectively.

UV-Vis Spectroscopic analysis
The electronic absorbance of plasticized PVC membranes was done using spectrophotometer (Model Ultrospec 2000 made in England) scanned from 190 -1000 nm.

Infrared spectroscopic analysis (FT-IR)
The structure of plasticized PVC membranes was investigated by Fourier Transform Infrared Spectrophotometer (Shimadzu FTIR -8400 S, Japan).

Thermogravimetric analysis (TGA)
Thermal analysis of plasticized PVC membranes was carried out using Thermal Gravimetric Analyzer (Shimadzu TGA -50, Japan).The thermograms were performed from ambient temperature to 600 ºC at heating rate 10 ºC/min under Nitrogen gas with flow rate 20 ml/min.

Scanning electron microscope (SEM)
Scanning of plasticized PVC membranes were carried out using Scanning Electron Microscope (Joel Jsm 6360LA, Japan).

Tensile testing
Universal tensile test were performed in an AG-1S, SHIMADZU universal testing machine, according to ASTM D-882 standards for testing tensile properties of paper and paper broad using a constant rate of elongation apparatus.Membrane thickness measurements were determined with an electronic digital micrometer.

Contact angle measurements
Static water contact angle measurements were performed at room temperature using (advanced Gonimeter model 500-F1) in a sessile drop configuration (using ultrapure water as the liquid), coupled with a video camera and image analysis software.At least, ten droplet images were obtained for each film (Mohyeldin et al., 2015b).

Surface roughness
The surface roughness of the substrate used for blood contact materials is critical.The average roughness was measured using surface roughness tester SJ-201P, Japan.Samples were mounted on a glass slide with a double-sided tap.Minimum sample dimensions were 25 mm x 25 mm.All results are the average of triplicate measurements (Mohy Eldin et al., 2008).

Optical properties
Film color was measured by an X-Rite Model Sp64 Made in USA.The colorimeter was calibrated with white and black plates.A white standard color plate for the instrument calibration was used as a background for color measurements of the films.The system provides the values of three color

Antibacterial assay Broth evaluation method
Antimicrobial activity of plasticized PVC membranes was measured according to the reported method previously (Skyttä and Mattila., 1991).Briefly, the bacteria were inoculated in a Luria-Bertani medium (LB medium) containing (1% peptone, 0.5% yeast extract, and 1% NaCl).The test tubes were incubated at 37ºC for 24 hrs with shaking.The obtained bacterial suspension was diluted with the same medium (100 fold).Then, 0.1 ml of diluted bacteria suspension was cultured in a ten ml of LB medium, which contains a piece of membranes (5 cm x 1 cm).The inoculated medium was maintained at 37ºC and 150 rpm for 24 hrs.The turbidity of bacteria was measured using UV-visible spectrophotometer at 620 nm.

Agar diffusion method
Agar-well diffusion method was applied for screening the antibacterial activities of plasticized PVC membranes against E. coli, P. aeruginosa, Salmonella sp., S. aureus and B. cereus as described by the reported method (Castro et al., 2011).Briefly, 50 µl overnight cultures of the indicator microorganisms were swabbed on LB agar medium.Small discs of PVC membranes were placed on the agar surface.The plates were left in the refrigerator at 4°C for 2 hrs to allow diffusion of materials into the agar.The plates were incubated at 37°C for 24 hrs then, they were investigated, and the bacterial inhibitions were photographed using gel documentation system.

Evaluation of haemocompatibility
The hemolysis tests were performed as described in American Society for Testing and Materials (ASTM) (ASTM F 756-00, 2000) (US Pharmacopeia XXIII., 1994;Hassan et al., 2010).Anticoagulated blood was used for this purpose.The tested samples were prepared by adding 1 ml of anticoagulant acid citrate dextrose solution (ACD) to 9 ml of fresh blood.Before performing the tests, Samples (1 cm 2 ) were placed in polypropylene test tubes, and 7 ml of phosphate buffer solution pH 7.0 (PBS) were added.After 72 hrs of incubation at 37°C, the PBS was removed, and 1ml ACD blood (9.02 mg/ml) was added to each sample and maintained at 37°C for 3 hrs.Positive and negative controls were prepared by adding the same amount of ACD blood to 7 ml of water and PBS, respectively.Each tube was gently inverted twice each 30 min to maintain contact with the blood with the material.After incubation, each fluid was transferred to a suitable tube and centrifuged at 2000 rpm for 15 min.The hemoglobin released by hemolysis was measured by the absorbance of the supernatants at 540 nm using a spectrophotometer (Model Ultrospec 2000).The percentage of hemolysis was calculated from the absorbance (A) as follows equation: of According to ASTM F 756-00 (2000) materials can be classified into three different categories according to their haemolytic index (haemolysis %): materials with percentages of haemolysis over 5% are considered haemolytic; while the ones with haemolytic index between 5% and 2% are classified as slightly haemolytic.Finally, when the material presents a hemolysis percentage below 2%, it is considered as a non-haemolytic material.

Thrombogenicity
Evaluation of thrombus formation on polymeric surfaces was carried out using a gravimetric method (Imai and Nose., 1972).Anticoagulated rabbit blood was used for this purpose.Anticoagulated was prepared by adding 1 ml of anticoagulant acid citrate dextrose solution (ACD) and 9 mL of fresh rabbit blood.
Before performing the tests, samples were immersed in PBS at a constant temperature of 37ºC.After 48 hrs of incubation, the PBS was removed, and the ACD blood was put in contact with the surface of the membranes and also to an empty Petri dish, which acted as a positive control.Blood clotting tests were initiated by adding 0.02 ml of a 10 M calcium chloride solution and were stopped after 45 min by the addition of 5 ml of water.Resultant clots were fixed with 5 ml of a 36% formaldehyde solution and were then dried with tissue paper and finally weighed.At least three measurements were performed for each sample, and the mean values are reported (US Pharmacopeia XXIII., 1994).

RESULTS AND DISCUSSION
In the current research, PVC membranes were improved by utilized clove oil as a plasticizer.Changes of Physical and mechanical properties of membranes were studied beside its biological activates.

Physical analysis Water uptake
Figure 1 represents the effect of clove oil content on the membranes water uptake.Obtained results show a neglected increase in water uptake.Hydrophobic character of clove oil prevents the adoption of water molecules to plasticized PVC membranes.

Roughness
It is well-known that some surface characteristics affect the extent of adhesion between two adjacent materials.One of such parameters is the surface roughness as surface asperities at the nanoscale level govern the overall adhesive forces.Figure 2 demonstrates an increase surface roughness by increased content of clove oil.That may be explained by the effect of plasticizer molecules in disruption of polymer chains during membranes casting (Chae et al., 2008;Brewer., 1984;Chapman et al., 2010).

Wettability
When the polymer materials come in contact with blood they can cause different undesired host responses like thrombosis, inflammatory reactions, and infections.The first event, which occurs, after exposure of biomaterials to blood, is the adsorption of blood proteins.Surface physicochemical properties of the materials as wettability greatly influence the amount and conformational changes of adsorbed proteins.
Figure 3 illustrates that increase of contact angle of PVC membranes plasticized with clove oil.These results suggest that clove oil enhancement hydrophobic nature and decrease wettability of PVC membranes.Wettability is believed to play an important role for the amount and the conformational changes of adsorbed proteins  , 1969;Norde and Lyklema, 1991).The hydrophobic interactions seem to be the dominant force driving protein adsorption/ unfolding on the surface (Norde and Lyklema., 1991).The hydrophobic interactions and their importance in protein adsorption were firstly indicated by studies showing that the protein adsorption increased with the decreasing wettability of the surface (Brash and Horbett, 1995)so called "hydrophobic rule."The water structure is that which makes differences between hydrophobic and hydrophilic surfaces (Vogler, 1998).The interaction between a hydrophobic surface and a protein originates mainly from an entropy gain due to water desorption from the solid surface and from the protein molecule (Norde, 1986).In contrast water molecules near to a hydrophilic surface exhibit relatively more-dense water structure in an extended 3D network of self-associating molecules.This type of water structure is less reactive and.Therefore, it's hard to be removed (Norde and Lyklema, 1991).To hydrophilic surfaces, the proteins are adsorbed weakly with a conformation near to their native state.As a result the protein adsorption to hydrophilic substrata is generally reversible, whereas to hydrophobic one's it is not.Denaturation of the adsorbed protein by hydrophobic-hydrophobic interactions with the substrate can also contribute to an irreversible adsorption (Chinn et al., 1992).As a result, the biological function of a given protein could be changed and altered, when it is adsorbed to a hydrophobic surface.

Color measurement
The membranes color is necessary for consumer acceptance and general performance.Film color dimension (L*, a*, and b*) and total color (∆E) were measured using color hunter (X-Rite Model SP64).(See Table 1), Data obtained show a slight decrease in membrane brightness (∆L*), and total color difference (∆E*).In the other hand, it demonstrates an increase in yellowish color of membranes.

UV-Vis Spectra
Figure 4 shows the electronic absorption of membranes from 200 to 400 nm.Collected charts demonstrate characteristic absorption peaks from 260-290 nm that may be attributed to the presence of the phenolic components in clove oil (i.e.Eugenol, βcaryophyllene).The increase of absorption bands and its shift to higher wavelength were attributed to increasing of clove oil content in membranes.

FT-IR analysis
Figure 5 illustrates FT-IR absorption of neat PVC and plasticized PVC membranes.Beside main characteristic peak of neat PVC membranes (C-H stretching mode observed at 2947 cm −1 , CH 2 deformation mode at 1340 cm −1 , C-H rocking mode at 1247 cm −1 , trans C-H wagging mode at 958 cm −1 , C-Cl stretching mode at 856 cm −1 , and cis C-H wagging mode at 619 cm −1 ), plasticized membranes exhibit a characteristic peaks phenolic components of clove oil (OH phenolic at 3520 cm -1 , =C-H stretching at 3060 cm -1 and aromatic -C=C stretching at 1514 and 1616 cm -1 ).It was clearly seen that the intensity of these peaks with increase clove oil content.

Mechanical characterization
Mechanical properties of plasticized PVC membranes with different amounts of eugenol were determined from critical breaking point of stretching.Maximum stress σ max (Nm −2 ) was evaluated as the ratio of the stretching force divided by the crosssectional area of broken membrane piece.The maximum strain λ max was measured as the elongation ratio of the initial length of the test piece.The result showed that the maximum stress decreases with increasing clove oil content (figure 6) While, the elongation percent was increased.This may be explained as a result of lubricant action of clove oil between PVC chains.

Thermogravimetric analysis (TGA)
Figure 7 illustrates thermograms of neat and plasticized PVC.As shown in figure 7, the nest PVC exhibits two distinct stages.The first decomposition starts from 247°C to 305°C with a maximum decomposition temperature rate at 280°C.This stage of degradation is attributed to autocatalytic dehydrochlorination reaction (zipper elimination) with the subsequent formation of conjugated double bonds (Simon, 1990;1992;Bacaloglu and Fisch, 1994;McNeill et al., 1995).After the loss of the first HCl molecule, the subsequent unsaturated structure formed in a PVC chain is an allylic chlorine structure.However, this allylic chlorine stimulates the next loss of an HCl molecule, and the repeated process leads to the chain or zip dehydrochlorination (Folarin and Sadiku, 2011)

Haemocompatibility
Several essential requirements must be taken into consideration during preparation and qualification of medical devices, especially blood contact materials.Blood compatibility is recognized to play critical parameter during evaluation of wound dressing membranes.The value of haemolysis is taken as a mentor test.Figure 10 illustrates haemolysis percent of the prepared membranes.Haemolysis is regarded as an especially significant screening test.Once it provides quantification of small levels of plasma hemoglobin, which may not be measurable under in vivo conditions.As reported in the literature (ISO 10993-4(1999)), it is not possible to define a universal level of acceptable or unacceptable amounts of haemolysis.Although blood compatible materials should be nonhaemolytic, in practice several medical devices cause haemolysis.This means that when such haemolytic effect takes place, it is important to make sure that clinical benefits overcome these risks and that the values of haemolysis are within acceptable limits.According to ASTM F 756-00 (2000), materials can be classified into three different categories according to their haemolytic index (haemolysis %): materials with percentages of haemolysis over than 5% are considered haemolytic; while the ones with haemolysis index between 5% and 2% are classified as slightly haemolytic.Finally, when material presents a haemolysis percentage below 2%, it is considered as a nonhaemolytic material.Figure 11 shows a disastrous increase in haemolysis percent of blood contact at a high amount of clove oil (from 0.4-0.5 %).

Thrombogenicity
As the membrane is designed to be used topically in contact with blood, it is important to evaluate its tissue and blood compatibility.Furthermore, the thrombogenic character is a desirable property in membranes.Figure 11 shows the weights of blood clots obtained on thrombogenicity test.It was observed that clot formation is higher in the plasticized compare with neat PVC membranes than in the control so; the polymers are classified as thrombogenic.
This characteristic is directly related to the hydrophilicity of the materials.When placed in contact with a hydrophobic surface, proteins adsorb to it in a strong and irreversible way, while at hydrophilic surfaces proteins adsorb weakly and reversibly (Abdou and Hassan, 2014).This relation between hydrophilicity and thrombosis was confirmed by the higher value of thrombus weight that was formed when blood contacted with PVC membranes, and that will increase by clove oil percent as a

CONCLUSION
PVC membranes plasticized with clove oil were prepared and characterized.The Results showed improvement in physical and mechanical properties of the membranes.Additionally, Bioevaluation study showed increasing of antibacterial activities against both Gram-positive and Gram-negative bacteria with increasing of clove oil concentrations within membranes.On the other hand, there are prominently increase in blood hemolysis and thrombus weight.This study should be subjected to further analysis and investigations to obtain a good PVC membrane with potent features to apply in blood bags manufacture.

Fig. 1 :
Fig. 1: water uptake PVC membranes plasticized with different contents of clove oil.

Fig. 2 :
Fig. 2: Surface roughness of PVC membranes plasticized with different contents of clove oil.

Fig. 3 :
Fig. 3: contact angle measurements of PVC membranes plasticized with different contents of clove oil.

Fig. 4 :
Fig. 4: Electronic spectrum of PVC membranes plasticized with different contents of clove oil.

Fig. 5 :
Fig. 5: FT-IR spectrum of PVC membranes plasticized with different contents of clove oil.

Fig. 6 :
Fig.6: stress strain parameters of neat PVC and that plasticized with different amount of clove oil.
. The second decomposition started from 450°C, which presumably corresponds to the degradation of the resulting unsaturated hydrocarbons in the dehydrochlorination of PVC.The addition of clove oil generates a new weight loss in the temperature range 100-200 C due to evaporation of the volatile components of clove oil (Folarin and Sadiku, 2011).

Fig. 7 :
Fig. 7: TGA of neat PVC membranes compared with plasticized membranes with different amounts of clove oil.Membrane bio-evaluationAntibacterial evaluationInhibition activity of the neat and plasticized PVC membranes comparing to neat PVC membrane was tested against two-gram positive bacteria: S. aureus and B. cereus & two-gram negative bacteria: P. aeruginosa and E. coli.The growths inhibition of the neat and plasticized PCV membranes is shown in

Fig. 8 :
Fig. 8: Antibacterial activity of plasticized PVC membranes with different contents of clove oil against four different bacterial strains.

Fig. 10 :
Fig. 10: Hemolysis percentage of neat PVC and that plasticized ith different amounts of clove oil.
an increase in hydrophobicity.Finally, the prepared membranes should be applied on an animal model for more investigations.

Fig. 11 :
Fig. 11: Thrombogenicity of neat PVC and that plasticized ith different amount of clove oil.

Table 1 :
Color values of plasticized PVC membranes with clove oil.