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  • 7/30/2019 Characterisation of Poly Vinyl Alcohol

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    International Journal of Emerging Technology and Advanced Engineering

    Website: www.ijetae.com (ISSN 2250-2459, Volume 2, Issue 11, November 2012)

    176

    Characterisation of Poly vinyl alcohol (PVA) / Silver nitratenanomembranes for their suitability in wound dressing

    applicationsDr. K. P. Chellamani1, P. Sundaramoorthy2, T. Sureshram3

    1,2,3The South India Textile Research Association (SITRA), Coimbatore641 014,

    Tamil Nadu, India

    Abstract - Special wound care product was developed using

    Poly vinyl alcohol (PVA) blended with Silver nitrate.

    Electrospinning technique was adopted to produce

    PVA/Silver nitrate nanomembranes, which was used as

    wound dressings. PVA/Silver nitrate wound dressings(PSNWD) have high moisture vapour transmission property

    and good antimicrobial activity. PSNWD substrates have mild

    cytotoxicity reactivity and have excellent odour absorbing

    capability. PVA/Silver nitrate wound dressing do not cause

    skin irritation even after 72 hours of contact with the wound

    and the time taken for wound healing while using PSNWD is

    just 50% of that in the case of an open wound.

    Keywords- Cytotoxicity, Electrospinning, nanomembranes,

    Poly vinyl alcohol (PVA), Silver nitrate and wound dressing.

    I. INTRODUCTION Electrospinning [1] is a technique used to process

    polymer fiber diameter in the range of micrometers tonanometers. Electrospinning process has increased in

    recent years, and this technology has been exploited for a

    wide range of applications. Among various nanomembrane

    processing techniques like drawing, template synthesis,

    phase separation and self assembly, electrospinning is the

    only method capable of producing continuous polymer

    nanofibres. Electrospun polymer nanofibers have very large

    surface area to volume ratio and flexibility in surface; these

    properties make the polymer nanofibers use in many

    important applications. Electrospun nanofibers achieve

    good result on properties required for wound dressings,

    because of its nano-size structure.

    Choice of wound dressings [2] is dependent upon the

    severity and type of injury, as well as the stage of healing

    and the presence or absence of infection. The essential

    parameters require for wound dressing to heal wounds are

    Absorptivity [3] and oxygen permeability [3]. Additionally,

    ability to conform to the wound bed, provide appropriate

    adherence/non-adherence to the healing tissue [4], provide

    a barrier to bacteria, enhanced moisture management5,

    bioactivity, resorbability [5] and occlusivity are highly

    desirable and correlated with better outcomes.

    Poly (vinyl alcohol) (PVA) has excellent chemical

    resistance, physical properties, biodegradability and good

    fiber forming capability in electrospinning techniques [6].

    PVA polymer is suitable for blending various polymers and

    additives in electrospinning process to achieve uniformnanofibers [7].

    *Correspondence to the author:

    P. Sundaramoorthy

    E-Mail ID: [email protected]

    Silver nitrate is a salt used to produce Silver

    nanoparticles by insitu polymerisation with PVA polymer.

    Silver nanoparticles have been widely used in various

    biomedical fields like wound dressing materials, body wall

    repairs, augmentation devices, tissue scaffolds,

    antimicrobial filters, and so forth. In the present study, the

    PVA polymer is blend with Silver nitrate in different blend

    proportions and optimising the electrospinning parameters

    using scanning electron microscope (SEM) analysis and

    antimicrobial activity [8]. The optimised parameter is used

    for further trials and the produced nanomembrane were

    characterised for Fourier transform infrared spectroscopy

    (FTIR) and transmission electron microscope (TEM)

    analysis. The PSNWD is test for wound dressing properties

    like Moisture vapour transmission rate (MVTR) [9],

    cytotoxicity [10], odour control capability [9], Skin

    irritation and wound healing ability

    II. MATERIALS AND METHODSA. Materials

    The polymers, chemicals and solvents used for this study

    are given below

    i. Poly Vinyl Alcohol (PVA) with a molecular weightof 1,24,000 g/mol

    ii. Silver nitrate saltiii. Nitric acid andiv. Distilled water.

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    B. Methods

    The electrospinning technique was used to produce thePVA/Silver nitrate nanomembranes. In electrospinning

    technique, there are two methods for the preparation of

    polymer solution.

    1. Method I:In this method, the polymer is dissolved in

    the solvent to form polymer solution.

    2. Method II:In this method, the polymer is heated upto

    its melting point and thereby the polymer solution is

    formed.

    In the present study, Method I was adopted to prepare

    the polymer solution.

    C. Solution Preparation

    The n gram of polymer is weighed with the help of

    weighing balance and m milliliter of solvent is measured

    with the help of micropipette.

    The concentration of the polymer solution is determined

    by the formula,

    Concentration =n gram of polymer

    X 100 (%)m milliliter of solvent

    D. PVA/Silver Nitrate Blend Solution Preparation

    The n gram of PVA and y gram of Silver nitrate salt

    were dissolved in m millilitre of distilled water (pH of

    distilled water - 4).

    PVA/Silver nitrate blend solution of 0.5* %, 1 % & 2 %were prepared at 80 C.

    * 0.5 % by weight of Silver nitrate in the blend solution of

    PVA/Silver nitrate.

    E. Electrospinning of Nanomembrane

    The term nano represents the size of the material in

    10-9

    meter. The Polymeric nanofibers can be processed by a

    number of techniques like Drawing, Template Synthesis,

    Phase Separation, Self-Assembly and Electrospinning.

    Among the various processing techniques, electrospinning

    is the only method capable of producing continuous

    polymer nanofibres. Electrospinning is a unique technology

    that can produce non-woven fibrous articles with fiberdiameters ranging from tens of nanometers to microns, a

    size range that is otherwise difficult to achieve by

    conventional techniques.

    A schematic representation of Electrospinning set up is

    given in Figure 1. The basic setup for electrospinning

    consists of three major components: a high-voltage power

    supply, a spinneret (a metallic needle), and a collector (agrounded conductor).

    A high electrical supply, typically 130 kV, is applied to

    a polymer solution contained in a syringe.

    Fig 1 Schematic Diagram of Electrospinning Setup

    The electrospinning process is governed by manyparameters, classified broadly into solution parameters,

    process parameters, and ambient parameters. Solution

    parameters include viscosity, conductivity, molecular

    weight, and surface tension. Process parameters include

    applied electric field, tip to collector distance and feeding

    or flow rate. Ambient parameters include temperature &

    humidity maintained during electrospinning process. These

    parameters significantly affect the fiber morphology

    obtained. By proper manipulation of these parameters,

    nanofibers of desired morphology and linear density can be

    obtained.

    III. PREPARATION AND OPTIMISATION OF PVA/SILVERNITRATE NANOMEMBRANE

    PVA/Silver nitrate solution was prepared by In-situ

    polymerisation method. In this method, the Silver nitrate

    and PVA were mixed in distilled water with pH value at

    4.0 (pH value of distilled water maintained by adding

    Nitric acid with the same). Maintaining pH value of the

    distilled water at 4.0 is mainly to control the ionisation

    reaction of Silver ions. To produce PVA/Silver nitrate

    nanomembrane, 4 different combinations of PVA/Silver

    nitrate were taken. They are,

    - 10% PVA/ 0.5*% Silver nitrate- 10% PVA/ 1.0% Silver nitrate- 10% PVA/ 1.5% Silver nitrate and- 10% PVA/ 2.0% Silver nitrate

    * 0.5% by weight of Silver nitrate on the weight of PVA

    solution with which Silver nitrate is added.

    These 4 combinations of PVA/Silver nitrate were

    electrospun to form nanomembranes.

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    During electrospinning, process parameters like i)

    Applied voltage, ii) Flow rate of solution and iii) Tip tocollector distance were optimised by adopting the same

    method followed for optimisation of process parameters for

    spinning PVA/Chitosan nanomembrane. A total numbers of

    27 trials were conducted in the optimisation process.

    Optimum values of electrospinning process parameters for

    the 4 combinations of PVA/Silver nitrate are given in

    Table 1.

    Table 1

    Optimum Values of Process Parameters* for the PVA / Silver NitrateNanomembranes

    S.No.

    Combinations

    Process parameters

    10%

    PVA/

    0.5%Silver

    nitrate

    10%

    PVA/

    1.0%Silver

    nitrate

    10%

    PVA/

    1.5%Silver

    nitrate

    10%

    PVA/

    2.0%Silver

    nitrate

    1.Applied voltage

    (kV)18 15 18 18

    2.Flow rate

    (milliliter/hr)0.5 0.5 1.0 0.5

    3.Tip to collector

    distance (cm)13 8 10 8

    *Optimum process parameter in electrospinning is one

    where uniform fibres without any bead could be produced.

    Figure 2, shows the electrospun PVA/Silver nitrate

    nanomembrane obtained after 5 hours of process (21 cm

    length).

    Fig 2 Electrospun PVA/Silver Nitrate Nanomembrane (after 5 hours

    of processing)

    SEM picture of PVA/Silver nitrate nanomembrane

    produced using different combinations of PVA/Silvernitrate are shown in Appendix I. PVA/Silver nitrate

    nanomembrane produced using different blend proportions

    were also tested for their antibacterial activity.

    A. Antimicrobial Activity of PVA/Silver NitrateNanomembranes

    The antimicrobial activity of the nanomembrane is very

    important for wound dressing applications. Antimicrobial

    test was conducted based on quantitative analysis by JIS L

    1902 method. Bacteria used in this study were

    staphylococcus aureus (ATCC 6538)*.

    *S. Aureus is a Gram positive micro-organism.

    Antimicrobial activity of the nanomembranes developedin various percentages of PVA/ silver nitrate viz 10/0.5,

    10/1, 10/1.5 and 10/2 was tested as per the standard JIS L

    1902. Briefly, 0.2 ml of the Staphylococcus aureus has

    been inoculated on 0.5 g of each sterile test material and

    incubated for 18 h at 37 C. Following incubation, ratio of

    microbiostasis was calculated using the formula F=

    Mb -Maand the result was expressed in log reduction. Here

    F is growth value, Mb is average value of common

    logarithm of number of living bacteria after 18 hr andMais

    average of value of common logarithm of number of living

    bacteria at 0th

    hr. Antimicrobial activity of the PVA/ silver

    nitrate nanomembranes and 100% PVA nanomembranes

    are given in Table 2.The results confirm that amongvarious combinations of PVA/ silver nitrate, the

    nanomembranes developed using 10% PVA/ 2% Silver

    nitrate, completely arrested the bacterial growth (100 %)

    when compared to control (0 %)*.Hence this combinationhas been chosen for further studies.

    Table 2

    Antimicrobial Activity of PVA/silver Nitrate Nanomembrane

    S.No.

    PVA/Silver nitrate

    nanomembrane

    combinations

    % Bacteria reduction

    after 18 h of treatment Remarks

    Control Treated

    1 10% / 0.5% 0 54Moderate

    activity

    2 10% / 1.0% 0 75Good

    activity

    3 10% / 1.5% 0 82Goodactivity

    4 10% / 2.0% 0 100Very Good

    activity

    *Control refers to nanomembranes made out of 100% PVA.

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    IV. CHARACTERISATION OF PVA/SILVER NITRATENANOMEMBRANE

    A. Moisture Vapour Transmission Rate (MVTR) of PVA/Silver Nitrate Nanomembrane

    The MVTR is an important criteria for an wound

    dressing material. The liquid formed inside the wound layer

    first changes to vapour state and then transported to

    atmosphere. This moisture vapour transmission helps to

    heal the wound; otherwise will be wound infection. The

    PVA/Silver nitrate nanomembranes were tested for MVTR

    as per BS EN 137262: 2002 [9].

    A test sample of 40 mm diameter is taken and fixed over

    a container of 35.7 mm inner diameter, containing 20 ml of

    distilled water. The test sample container is weighed (W 1)

    before the start of the test. Then the container is kept insidean incubator for 24 hours (conditions maintained inside the

    incubator i) Temperature: 37 1 C and ii) RH 20%). After

    24 hours, the container is taken out and again weighed

    (W2).MVTR is calculated based on the formula,

    X = (W1W2) 1000 x 24/T

    Where,

    X is MVTR (g/m2/24 hours)

    W1 is the mass of the container, sample and liquid in

    grams

    W2 is the mass of the container, sample and liquid ingrams after the test duration

    and

    T is the test period in hours

    MVTR values of PVA/ Silver nitrate nanomembrane

    samples provided in this study are given in Table 3.

    Table 3

    MVTR Values of PVA/Silver Nitrate Nanomembranes

    S. No.PVA/Silver nitrate

    nanomembrane combinations

    MVTR (g/m2/24

    hours)

    1. 10 %/2 % 3060

    2. 10%/1.5 % 3070

    3. 10%/1.0 % 31204. 10%/0.5 % 3180

    With increase in the concentration of Silver nitrate in

    PVA, MVTR values tend to increase. This is attribute to

    the reduction in the diameter of the corresponding

    nanomembrane substrates. For an infected skin, MVTR

    value of 2000 to 2500 in good [11]. Hence, all the

    PVA/Silver nitrate nanomembrane substrates made in this

    study can be considered as suitable for infected skins.

    B. Fourier-Transform Infrared Spectroscopy (FT-IR) forPVA/ Silver Nitrate Nanomembrane

    Functional groups present in PVA/Silver nitrate

    electrospun nanomembranes were studied using FTIR

    spectroscopy.

    The FTIR spectra of electrospun PVA/ Silver nitrate

    nanomembrane (Combination of 10% / 2%) are shown in

    Figure 3.

    Fig 3 FT-IR Spectra of PVA/Silver Nitrate Nanomembrane

    In FTIR spectra, X-axis represents wavenumbers and Y-

    axis, the transmittance. It is discernable from the FTIRspectra, that these are peaks at i) Wavenumbers of 3336

    cm-1

    (peak 1) and ii) wavenumber of range below 500 cm-1

    (peak 2). Peak 1 indicates the presence ofOH groups and

    peak 2, the presence of silver groups. This inturn confirms

    the presence of PVA polymer and nano Silver

    materials in the substrates produced.

    C. Cytotoxicity Studies on PVA/Silver NitrateNanomembrane

    An in vitro cytotoxicity test was conducted for the

    PVA/Silver nitrate substrates using Direct contact method

    was performed using test sample PVA/Silver nitrate

    nanomembrane as per ISO 10993-5 [10].

    In this method, a monolayer culture of L-929 is used as

    the medium. Test samples, negative and positive controls

    were placed on the medium. After incubation at 37 1 C

    for 24 hours, the monolayer medium is examined

    microscopically for the response around the test specimen.

    The reactivity levels observed are graded as 0,1,2,3 & 4

    based on zone of lysis, vacuolization, detachment and

    membrane disintegration as explained in Table 4.

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    180

    Table 4

    Grading of Cytotoxicity

    Reactivity Description of reactivity zone Grade

    NoneNo detectable zone around or under

    sample0

    SlightlySome malformed or degenerated cells

    under specimen1

    Mild Zone limited to area under specimen 2

    ModerateZone extending specimen size up to 0.33

    cm3

    SevereZone extended farther than 0.33 cm

    beyond specimen4

    As per ISO 10993 5, reactivity grade higher than 2 is

    considered as cytotoxicity. The PVA/Silver nitratenanomembrane substrates produced in this study (10%/2%)

    were found to have Mild reactivity and hence graded as

    2 as far as cytotoxicity is concerned.

    D. Odour Control Ability of PVA/Silver NitrateNanomembrane

    The objective of the test is to assess the resistance of

    wound dressing to penetration by odour. The sample is

    tested as per BS EN 13726 6: 2003 method [9]. A

    stainless steel container of 35.7mm inner diameter and

    40mm height is used for carrying out the test. The test

    consists of two parts.

    1. Part I: Here, the container is heated for 1 hour at 105 C.Then, Nitrogen gas is purged inside the container. After

    this, 0.5 l of pure Diethylamine is also passed inside the

    container and the container is kept inside anincubator at 37C for 20 minutes. After 20 minutes, 250 l sample of

    Nitrogen gas is removed from the container and injected

    into GC (Gas Chromatography) instrument. The GC

    instrument estimates the intensity ofDiethylamine presentin Nitrogen gas and provides the information in the form of

    a graph (Figure 4 a). In the graph, the intensity of

    diethylamine present in Nitrogen gas is plotted against

    time.

    2. Part II: The container is heated for 1 hour at 105 C.

    After this, a 1.3% w/v solution of Diethylamine (0.26 gramof Diethylamine dissolved in 20 ml of distilled water) is

    added into the container. Then, the test specimen is kept

    inside the container, Nitrogen gas is purged inside the

    container and the container is kept inside an incubator at 37

    C for 30 minutes. After 30 minutes, 250 l sample of

    Nitrogen gas is removed from the container and injected

    into GC (Gas Chromatography) instrument for the analysis

    of intensity of Diethylamine in Nitrogen gas. The

    information is given in Figure 4 b.

    Fig 4 a) Diethylamine Vs time in Control specimen

    Fig 4 b) Diethylamine Vs time in PVA/Silver nitrate nanomembrane

    In the Graph, the peak shown at 25 27 minutesrepresents the Diethylamine. The Diethylamine present in

    PVA/Silver nitrate nanomembrane is just 5% of that

    exhibited by control specimen. Hence, PVA/Silver nitrate

    nanomembrane has excellent odour absorbing capability.

    E. Skin Irritation Potential of PVA/Silver NitrateNanomembrane

    PVA/Silver nitrate nanomembrane samples were

    evaluated for potential skin irritation, when they are used

    for covering the wound. The evaluation was as per ASTM

    F 719-81 standards. The test method is explained in

    Appendix II. Six healthy rabbit were selected for the study

    and PVA/Silver nitrate wound dressings were applied on

    the 6 rabbits. The study has shown that PVA/Silver nitrate

    wound dressing do not causes any skin irritation even after

    72 hours of contact with the wound.

    F. Wound Healing Ability of PVA/Silver NitrateNanomembrane

    The extent of wound healing provided by a given wound

    dressing was evaluated using the method proposed by

    Morton & Malone.

    Time (minutes)

    IntensityofDiethylamin

    e

    I

    i

    i

    l

    i

    Time (minutes)

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    As per this method, twelve healthy rats were employed

    for the experimentation and they were separated into 2groups (Group I and Group II) each with 6 rats. Excision of

    wounds was made on the rats as per the method suggested

    by Morton & Malone [12].

    The rats were anaesthetized with an aesthetic ether and

    placed in operation table in their natural position. A squarewound of about 1.5 cm (width) x 0.2 cm (depth) was made

    on depilated ethanol-sterilized dorsal thoracic region of

    rats. Infection was made on wounds by staphylococci

    aureus.

    Table 5

    Wound Healing in Open Wound and PVA/Silver Nitrate Nanomembrane Treated Wound

    Type of woundsExtent of Wound healing

    Day 0 Day 7 Day 14 Day 21 Day 28 Day 35

    Open wound

    PVA / Silver

    nitrate

    nanomembrane

    Group I rats were left as they were withoutapplication of any wound dressing (open wound)

    Group II rats were treated using PVA/Silver nitratenanomembrane wound dressing

    The dressings were applied on the wounds of the rats

    every day till the epithelialisation was complete. The extent

    of wound contraction was studied by tracing the raw wound

    area in a tracing paper on 0th

    day, 7th

    day, 14th

    day, 21th

    day,

    28th

    day and 35th

    day.

    The weights of the traced portions of the wounded area

    of rats subjected to different treatments were measured

    using electronic balance. Based on the difference in weight,

    the superiority or otherwise of a particular wound dressing

    is determined. Table 5 shows, the extent of wound healing

    noticed while using different wound dressings on differentdays.

    The weights of the traced portions of open wound and

    PVA/ Silver nitrate nanomembrane treated wound ondifferent days are shown in Table It is clear from Table 6,

    that there is a decrease in wound area with the application

    of PVA/ Silver nitrate nanomembrane wound dressings.

    Table 6

    Weight Of The Traced Portions Of Wounds On Different Days

    Type of

    wound

    Weight of traced portions of the wound (grams)

    Day 0 Day 7Day

    14

    Day

    21

    Day

    28

    Day

    35

    Open

    wound100 154.89

    110.7

    882.44 40.72

    11.2

    3

    PSNWDtreatedwound

    100 61.74 20.87 0

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    G. Transmission Electron Microscope (TEM) Analysis ofPVA/Silver Nitrate Nanomembrane

    The Transmission electron microscope (TEM) analysis

    was used to study the size and distribution of Silver

    nanoparticles inside the single PVA/Silver nitrate nanofiber

    (Figure 5).

    Fig 5 TEM Image of PVA/Silver Nitrate Nanofiber

    In the Figure 5, the black dots represent the Silver

    nanoparticles. It is clear that the distribution of Silver

    nanoparticles on the nanofibre is quite uniform. The

    average size of the Silver nanoparticles was found to

    be 3.7 nm.

    V. CONCLUSIONS SUMMARISING THE ACHIEVEMENTSAND INDICATION OF SCOPE FOR FURTHER WORK

    SITRA has developed special type of wound dressingviz) PVA/Silver nitrate nanomembrane wound

    dressing (PSNWD).

    PSNWD are found to be ideally suited for infectedskins in view of their high moisture vapour

    transmission property (MVTR).

    PSNWD Substrates have good antimicrobial activity. PSNWD substrates have mild cytotoxicity reactivity

    and exhibited excellent odour absorbing capability.

    PVA/Silver nitrate substrates do not cause any skinirritation even after 72 hours of contact with thewound.

    The time taken for wound healing while usingPSNWD is around 50% lower as compared to that in

    the case of an open wound.

    REFERENCES

    [1] Zheng-Ming Huang et al., 2003. A review on polymer nanofibers byelectrospinning and their applications in nanocomposites. Compos.

    Sci. Technol. 63: 2223-2253.

    [2] Marian McCord, 2009. Challenges in advanced nanofiber wounddressings (Annual report, National Textile Center). Retrived from

    http://ebookbrowse.com/f09-ns06-a9-pdf-d55286555.

    [3] Joshua S. et al., 2008. Wound healing dressing and drug deliverysystems: A review. J. Pharm. Sci. 97 (8): 2892-2922.

    [4] Zhong S. P. et al., 2010. Tissue scaffolds for skin wound healing anddermal reconstruction. WIREs Nanomedicine and Nanotechnology.

    2:510-525.

    [5] Mehrdad Kokabi et al., 2007. PVA-Clay nanocomposite hydrogelsfor wound dressing. Eur. Polym. J. 43: 773-781.

    [6] Bin Ding, 2002. Preparation and characterisation of nanoscaledPoly(Vinyl alcohol) fibers via electrospinning. Fiber. Polym. 3(2):

    73-79.

    [7] Kim Soo-Hwan. et al., 2011. Antibacterial activity of Silver-nanoparticles against staphylococcus aureus and Escherichia coli.

    Korean J. Microbiol. Biotechnol. 39 (1):77-85.

    [8] JIS L 1902: 2008. Testing for antibacterial activity and efficacy ontextile products. Japanese Industrial Standard (JIS). ICS 07.100.99:59.080.01.

    [9] BS EN 13726: 2002. Testing methods for primary wound dressings.British Standard. ICS 11.120.20.

    [10] ISO 10993-5: 2009. Biological evaluation of medical devices Part5: Tests for in vitro cytotoxicity. International standard.

    [11] Ching-Wen Lou, Ching-Wen Lin, Yueh-Sheng Chen, Chun-HsuYao, Zen-Shoung lin, Chieh-Yu Chao and Jia-Horng Lin. 2008.

    Dressing properties evaluation of Tencel/Cotton nonwoven fabriccoated with Chitosan for wound. Text. Res. J. 78(3):248-253.

    [12] Morton J. J. P. and Malone M. H., 1972. Evaluation of vulneraryactivity by an open wound procedure in rats. Arch. Int. Pharmacod.

    T. 196:117-126.

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    Appendix I

    SEM pictures of PVA/Silver nitrate nanomembrane produced using different combinations of PVA/Silver nitrate

    S.

    No.

    PVA / Silver nitrate

    combination

    SEM pictures of PVA/ Silver nitrate

    nanomembrane

    Nanofiber

    diameter

    (nm)

    Results

    1. 10% PVA/ 0.5% Silver

    nitrate

    242 56 Uniform fibers.

    No bead

    formation.

    2. 10% PVA/ 1.0% Silver

    nitrate

    234 153 Uniform fibers.

    No bead

    formation.

    3. 10% PVA/ 1.5% Silver

    nitrate

    184 54 Uniform fibers.

    No bead

    formation.

    4. 10% PVA/ 2.0% Silver

    nitrate

    169 75 Non-uniform

    fibers.

    Bead formation

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    Appendix II

    Standard test method for assessing skin irritation

    (ASTM F 719-81 standard)

    Principle of Measurement

    Exposure of skin to the test material is accomplished by

    means of a patch test technique employing two intact sites

    on the back of each of six albino rabbits. The skin is

    clipped free of hair one day prior to testing. The test

    substance is applied using 0.5 ml for liquids, 0.5 g for

    solids or semisolids and a 2.5 by 2.5 cm square patch for

    films. After application, each test site is covered with a 2.5by 2.5 cm gauze flat and entire trunk is occluded with a

    polyethylene sleeve. After 24 hours the sleeve, flat and test

    material are removed and test sites are evaluated for

    erytheme and edema.

    Scoring Method

    Using the criteria given in Table 7, the test sites are

    scored for Erythema (ER) and Edema (ED).

    Table 7Skin Reactions Score For Erythema And Edema Formations

    Reaction Description ScoreErythema (ER) Erythema and Eschar

    No erythema

    Very slight erythema (barely perceptible)

    Well-defined erythema (pale red in colour)

    Moderate to severe erythema (red and area well defined)

    Severe erythema (beet redness to slight eschar formation)

    0

    1

    2

    3

    4

    Edema (ED) Edema formation

    No edema

    Very slight edema (barely perceptible)

    Slight edema (edges of area well defined by definite raising)

    Moderate edema (edges raising approximately 1 millimeter)

    Severe edema (raised more than 1 mm and extending beyond the area of

    exposure)

    0

    1

    2

    3

    4

    Test sites can also be scored for erythema and edema at

    48 hours as well as 72 hours after removal (as per the usage

    requirement) using the criteria given in Table 7.