Research Article New Biofunctional Loading of Natural ...downloads.hindawi.com/journals/ijbm/2016/6964938.pdf · New Biofunctional Loading of Natural Antimicrobial Agent in Biodegradable

Research ArticleNew Biofunctional Loading of Natural Antimicrobial Agent inBiodegradable Polymeric Films for Biomedical Applications

Bakhtawar Ghafoor Murtaza Najabat Ali Umar Ansari Muhammad Faraz BhattiMariam Mir Hafsah Akhtar and Fatima Darakhshan

Biomedical Engineering and Sciences Department School of Mechanical and Manufacturing Engineering (SMME)National University of Sciences and Technology (NUST) Islamabad Pakistan

Correspondence should be addressed to Murtaza Najabat Ali murtaza bmehotmailcom

Received 9 July 2016 Revised 5 September 2016 Accepted 9 October 2016

Academic Editor Rosalind Labow

Copyright copy 2016 Bakhtawar Ghafoor et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The study focuses on the development of novel Aloe vera based polymeric composite films and antimicrobial suture coatingsPolyvinyl alcohol (PVA) a synthetic biocompatible and biodegradable polymer was combined with Aloe vera a natural herbused for soothing burning effects and cosmetic purposes The properties of these two materials were combined together to getadditional benefits such as wound healing and prevention of surgical site infections PVA and Aloe vera were mixed in a fixedquantity to produce polymer based films The films were screened for antibacterial and antifungal activity against bacterial (Ecoli P aeruginosa) and fungal strains (Aspergillus flavus and Aspergillus tubingensis) screened Aloe vera based PVA films showedantimicrobial activity against all the strains the lowest Aloe vera concentration (5) showed the highest activity against all thestrains In vitro degradation and release profile of these films was also evaluated The coating for sutures was prepared in vitroantibacterial tests of these coated sutures were carried out and later on in vivo studies of these coated sutures were also performedThe results showed that sutures coated with Aloe veraPVA coating solution have antibacterial effects and thus have the potential tobe used in the prevention of surgical site infections and Aloe veraPVA based films have the potential to be used for wound healingpurposes

1 Introduction

Nosocomial infections are hospital-acquired infections(HAI) that usually develop in patients during their hospitalstay affecting the health expenditure of the patient [1]The main factors that make patients prone to nosocomialinfection include concurrent infections medical devicessurgery immunosuppressive agents and emergence ofmulti-drug resistant pathogens Pathogens are responsible for suchinfections known as nosocomial pathogens Among them90 bacterial pathogens are involved however mycobacte-rial viral fungal or protozoal agents are less commonlyinvolved [2] According to the data Escherichia coli Staphylo-coccus aureus enterococci and Pseudomonas aeruginosaare the most common nosocomial pathogens [3] Amongthe fungal pathogens Candida albicans [4] Aspergillus sppand especially Aspergillus fumigatus A flavus and A terreus

have also been reported as the common cause of nosocomialinfection in highly immunocompromised patients Thesepathogens can be transmitted through either inhalation ordirect contact with occlusive materials [5 6]

One of the reasons of nosocomial infections is surgicalsite infections (SSIs) mainly caused due to infected suturematerials used in surgery and medical implants [7] Theseinfections are usually difficult to resolve and may causecomplications in extreme cases In order to prevent surgicalsite infections scientists have been using several natural andsynthetic materials like plant extracts and polymers whichmay be used as coating materials on surface of medicaldevices such as surgical implants or sutures [8] The additionof antibiotics to these coating biomaterials can providethe local delivery of antibiotic directly at implantation orsuture site thereby decreasing the onset of infection [9]Synthetic and natural biomaterials have also been used in

Hindawi Publishing CorporationInternational Journal of BiomaterialsVolume 2016 Article ID 6964938 9 pageshttpdxdoiorg10115520166964938

2 International Journal of Biomaterials

other biomedical applications such as drug delivery systemswound infections and antitumor and anti-inflammatoryagents [10]

Among synthetic biomaterials one of the extensivelyused polymers is poly(vinyl alcohol) (PVA) Due to itssuitable chemical and physical properties biocompatibilitybiodegradability easy preparation with excellent film form-ing properties and nontoxic nature PVA has been studiedintensively in different biomedical applications includingwound dressings contact lenses coatings for sutures andcatheters [11 12]

Aloe vera as a natural source of bioactive compounds iswidely studied for biomedical applications Aloe vera belongsto the Liliaceae family and is known as the oldest therapeuticherb It has the ability to promote wound healing as well astreat burnt areas on the skin [13 14] Due to its propertiesmany researchers have shown the antibacterial antiviralantitumor and anti-inflammatory activity of different partsof Aloe vera such as its stem root and leaf extracts [15ndash17]The chemical composition of Aloe vera has also proved itspotential use in cosmetic formulations food supplementsand medical devices [15 18 19]

The inner part of Aloe vera contains a clear mucilaginoustissue commonly known as Aloe gel The major portion ofAloe gel contains water while almost 1 contains bioac-tive compounds such asaloin emodin (anthraquinones)flavonoids saponin and Aloe-mannan along with many dif-ferent amino acids and vitaminsThese bioactive compoundsplay a major role for antibacterial activity of Aloe gel [15 2021]

The present work focuses on the antibacterial and anti-fungal activity of Aloe veraPVA composite membranesand the application of these blends in the prevention ofnosocomial infections for the specific purpose of investingthis sutures coated with the PVAAloe gel blend have beenused for both in vitro and in vivo analysisAloe veraPVAfilmshave been characterized through SEM and FTIR analysisThe results of in vitro and in vivo analysis proposed that thiscomposition can be used as a coating for the prevention ofsurgical site infections that are caused by infections throughsutures To our knowledge such biofunctional Aloe loadedPVA coatings have not been investigated against surgicalinfection causing bacteria in recent studiesThe results of ourstudy indicate the potential of such coatings as part of largerpreventive measures against surgical infections

2 Materials and Methods

21 Collection of Plant Material Fresh Aloe vera plants werecollected from local nurseries and the leaves washed wellwith distilled water to remove all contaminants presentat the surface The gel was harvested from the leaves inan autoclaved container and kept at room temperature forfurther use the storage time of the gels at room temperaturewas one minute During this time period any residual solidleaf particles were mechanically separated from the gel

22 Test Organisms for In Vitro Studies In order to investi-gate antimicrobial and antifungal activity (in vitro studies)

pure cultures of bacterial and fungal strains including Pseu-domonas aeruginosa (P aeruginosa) Escherichia coli (E coli)Aspergillus tubingensis and Aspergillus flavus were obtainedfrom Mycovirus Research Lab National University of Sci-ences and Technology (NUST) H-12 Islamabad The purebacterial and fungal cultures were stored in nutrient agar at4∘C

23 Suture Materials Commercially available silk braidedblack surgical sutures nonabsorbable (15 metric size 4-0)supplied by Foosin Medical supplies Inc Ltd ShandongChina and manufactured by WEGOSUTURES were used tocarry out in vitro and in vivo studies The suture material wasdelivered in sterile single peelable foil packages and storedat room temperature For investigation the sutures were cutinto defined lengths (1 cm) under aseptic conditions

24 Preparation of Aloe vera Based PVA Films Polyvinylalcohol (PVA) supplied by AppliChem Germany a biocom-patible polymer was used for the formation of polymerAloevera films [22] Dimethyl Formamide (DMF) manufacturedby TEDIA Company Inc USA was selected as solvent for theformation of PVA-Aloe vera films due to its high volatility

Solvent-casting method was used for the fabrication ofAloe vera gelPVA films 1 g of PVA was dissolved in 40mL ofDMFThe solution was stirred with a constant RPM of 580 at60∘Cuntil PVAwas completely dissolved and a clear solutionwas obtained This was followed by the addition of differentamounts of Aloe gel Aloe gel was added in the amounts of5 10 15 and 20 respectively for the fabrication ofAloe veraPVA films with varying Aloe gel compositions Theheating was turned off while constant magnetic stirring wascontinued to obtain a homogenized mixture of Aloe vera geland PVA in DMF The mixture was poured into Petri dishesand placed in oven at 37∘C for 20 h to evaporate the solventcompletely and dry films were harvested for further testingA solution of PVA in DMF was also prepared by the sameprocedure to obtain PVA films that were used as control forantimicrobial activity

25 Antimicrobial Testing of Aloe vera Based PVA FilmsThe antifungal and antibacterial activities of films wereevaluated using standard procedure of disc diffusion [23]For antibacterial activity sterile nutrient agar (pH 74) wasprepared using Tryptone 10 g supplied by BioWorld USAyeast extract 5 g supplied by MERCK Germany SodiumChloride 10 g supplied by AnalaR England and nutrient agar12 g supplied by MERCK Germany dissolved in 1000mL ofdistilledwater After autoclaving the nutrient agarwas pouredin Petri dishes which were inoculated with the 01mL ofbacterial inoculum from preculture of test bacterial strains

For antifungal investigation sterile potato dextrose agarwas prepared and poured onto the Petri plates and purefungal cultures were obtained from test fungal strains

For disc diffusion test films were cut into discs of about7mm in diameter and placed on the bacterial and fungalinoculated plates with certain distances Each Petri platecontained six discs one of which included the control sterileWhatman filter paper number 1 PVA film and other four

International Journal of Biomaterials 3

Aloe verapolymer based films with varying concentrationsof Aloe vera (5 10 15 and 20) Antimicrobial activityof pure Aloe vera gel was also recorded using well diffusionmethod 10mm of diameter of well was made in solid agarmedium in which 01mL of pure Aloe vera gel was deliveredinto the well after incubating the plate with the bacterialstrains

For antibacterial testing a positive control (Tetracyclinedisc) was used All plates were incubated at 37∘C for 24 hThe zone of inhibition diameter in millimeter (mm) wasmeasured The study was performed in triplicate and meanwas calculated

26 Characterization of Aloe vera Based PVA Films

261 Fourier Transform Infrared (FTIR) Analysis Fouriertransform infrared (FTIR) spectroscopy (Perkin Elmer spec-trum 100 FTIR spectrophotometer) of Aloe veraPVA filmswas carried out (at 256 scans 8 cmminus1 resolution) to investigatethe presence of functional groups and types of interactionbetween the Aloe vera and PVA components

262 Morphological Analysis SEM Scanning ElectronMicroscopy (SEM) was performed to find out the surfacemorphology of the casted filmsThe assessment of the surfacemorphology of the Aloe veraPVA based films was doneusing JSM-6490A Analytical scanning electron microscope(JEOL Tokyo Japan) SEM images were collected at anactivation voltage of 20 kV

27 In Vitro Degradation and Aloe Release Profile Testingof Aloe vera Based PVA Film The degradation profile wasassessed by recording weight differences after regular timeintervals while Aloe release profile of Aloe veraPVA filmswas assessed through UV-Vis spectrophotometry A portionof Aloe veraPVA films with measurable size (110158401015840 by 110158401015840)were cut and placed in 3mL of PBS (pH 74) at 37∘C Theremaining PBS was removed after every 10-minute intervaland replaced with fresh 3mL of PBSThe films were weighedbefore addition of PBS and afterwards they were taken out ofthe PBS solution inwet state theweightswere subtracted andrecordedMoreover the drained PBS solutionswere evaluatedfor Aloe release profile by UV-VIS spectrophotometer (Sys-tronics 2202) absorbance at 120582max = 301 nm The degradationand release tests were carried out in triplicate and an averagevalue was calculated

28 Coating for the Sutures Dip coating method was used tocoat the sutures For dip coating the solutionwas prepared bymixing 2 g of Aloe vera and 1 g of PVA in 40mL of DMF Thesutures (30 cm length) were first sterilized and then dippedin the dip coating solution (for 60 minutes) followed byremoval and air drying of suture for 24 h The confirmationof coating of the suture was done by measuring the weightbefore coating and after coating

29 In Vitro Evaluation of Coated and Uncoated SuturesThe silk sutures (with and without Aloe veraPVA coating)were evaluated in vitro for antibacterial activity against two

bacterial strains that is E coli and P aeruginosa Nutrientagar media (pH 74) plates were prepared and the coatedsuture of the size 4 cm was placed over agar The plateswere then inoculated with bacterial strains (E coli and Paeruginosa) and antibacterial activity was recorded

210 InVivo Evaluation of Coated Sutures BALBcmicewerepurchased from National Institute of Health (NIH) for the invivo analysis of coated sutures To check the antimicrobialactivity of the coated sutures in vivo mice were given anincision of about 2 cm on both sides of the spineThe incisionwas inoculated with E coli 30 times 106 colony forming unit(CFU) of 100 120583L with the help of a syringe A coated suturewas then placed in one incision whereas an uncoated suturewas placed in the other incision A discontinuous suturingwas done to close the incision site The same procedure wascarried with the mice using P aeruginosa (50 times 106 CFU) of100 120583L for inoculationThe entire experiment was performedin triplicate using sterilized instrumentsThe sutured incisionsites were covered with surgical tape for two days After twodays sutures from both sides of mice were taken out andplaced in separate 15mL centrifuge tubes containing 100120583LPBS solution the sutures were placed on the Petri dishescontaining nutrient agar and placed in an incubator at 37∘Covernight

211 Statistical Analysis All the quantitative data wereexpressed as mean value with standard deviation The statis-tical analyses of the results were done by using 119905-test in GraphPad Prism 60 software The values that were 119901 lt 005 wereconsidered statistically significant value

3 Results and Discussion

31 Scanning Electron Microscopy (SEM) The surface mor-phology of different films was assessed by SEM which hasbeen demonstrated in Figure 1 The SEM images showed theaggregates ofAloe veradispersed on the surface of filmswhichcontributed to the film surface roughness Similar results havebeen reported by Pereira et al while studying the propertiesof alginate based Aloe vera films [8]

32 Fourier Transform Infrared (FTIR) FTIR analysis wasperformed to identify the nature of linkages betweenPVAandAloe vera The FTIR spectra of pureAloe vera PVA Aloe verainDMF andAloe veraPVAfilmswith varying concentrationshave been shown in Figure 2The peak that appeared between3500 cmminus1 and 3200 cmminus1 in all films indicates the presenceof hydroxyl group (OH) [24] The absorption band between3000 cmminus1 and 2800 cmminus1 centered at 293268 cmminus1 in 5Aloe veraPVA and 2926 cmminus1 in 20 Aloe veraPVA Bothpeaks had shifted from 2922 cmminus1 this was a characteristic ofasymmetric stretching of CH

2groups [25]The shift indicated

the intermolecular interactions at these functional groupsin Aloe vera and PVA The peaks obtained at the range of1720 cmminus1 to 1710 cmminus1 correspond to the stretching of C=Ogroup which indicated the presence of carbonyl compoundsinAloe veraThe presence of C-O-C (phenol ether) groupwas


(a) (b)

(c) (d)

Figure 1 SEM images of Aloe verapolymer films (a) Film with 5 Aloe vera concentration at 20 kV and times100 magnification (b) Film with10 concentration at 10 kV and times100 magnification (c) Film with 15 at 20 kV and times100 magnification and (d) with 20 concentration at20 kV and times100 magnification

FTIR spectra of Aloe verapolymer with varying concentration

51520

Pure PVAPVA in DMF

15

5

20

PVA

PVA in DMF

80

130

180

230

280

330

380

430

480

530

T

3000 2000 1000 04000(cmminus1)

(a)

FTIR spectra of Aloe verapolymer with varying

Aloe in DMFAloe

62

63

64

65

66

67

68

69

70

T

3000 2000 1000 04000(cmminus1)

concentration

(b)

Figure 2 FTIR results of (a) PVA film and Aloe verapolymer films with 5 15 and 20 and (b) results of pure Aloe and Aloe incubatedin DMF


control Aloe vera

Antibacterial activity of Aloe verapolymer filmswith different concentration

E coliP aeruginosa

0

5

10

15

20

25

Zone

of i

nhib

ition

(mm

)

5 10 15 20 Positive PurePVA

Concentration of Aloe verapolymer filmsBacterial strains

(a)

films control Aloe

Antifungal activity of Aloe vera based polymer filmswith varying concentration

Aspergillus flavusAspergillus tubingensis

0

5

10

15

20

25

30

Zone

of i

nhib

ition

(mm

)


Concentration of Aloe verapolymer films ()

(b)

Figure 3 Graphical representation of antibacterial (a) and antifungal (b) activity of different concentrations of Aloe verapolymer films119910-axis shows zones in mm while 119909-axis shows varying concentration of Aloe verapolymer films

indicated by the bands located at 1036 cmminus1 in films having20 Aloe veraPVA concentration The peak in pure Aloevera at 1075 cmminus1 [25] was shifted to 1036 cmminus1 indicatingthe presence of C-N functional groups in the films theshift observed in the peak can be attributed to interactionsbetween amine groups and hydroxyl groups of Aloe vera andPVA respectively [26] The absorption band 1460 cmminus1 to1410 cmminus1 appeared in all concentrations of Aloe veraPVAfilms hence representing symmetric stretching vibrations ofCOOH groups in films [26] The broad peak at 1150 cmminus1 to1130 cmminus1 could indicate either (C-O) stretching vibrationsin films with concentrations of 5 and 15 The absorptionpeaks obtained at 860 cmminus1 to 840 cmminus1 correspond torocking vibrations of CH

2bonds in PVA [27] The bending

of C-H alkyl groups present in Aloe vera and PVA at a peakrange of 950 cmminus1 to 940 cmminus1 can easily be seen in FTIRresults A new peak at 217118 cmminus1 in 5 216769 cmminus1 in15 and 2168 cmminus1 in 20 Aloe veraPVA film indicates theoccurrence of interactions between CH group of PVA withCH group of Aloe vera The band at 1660 cmminus1 and 1264 cmminus1in 20 Aloe veraPVA film demonstrated the interactionbetween hydrogen groups and C-O-C of PVA and C=O andC-O-C groups of Aloe vera [25 28 29]

The occurrence of peaks of COOH C-H C-O-C NH2

and OH shows that the pharmacologically active compoundsof Aloe vera such as anthraquinones saponins and polysac-charides are still in their active form This can be furthercorrelated with antibacterial activity which confirmed that

the active components are still intact and are not affectedthrough interactions with PVA Thus the stability of phar-macologically active moiety ofAloe after preparation in DMFand loading in PVA films has been confirmed

33 Antimicrobial Testing Results of Films TheAloe veraPVAfilms when positioned on bacterial and fungal inoculatedplates gave zones of inhibition which were recorded after 24 hof positioning the films (Figure 3) All films demonstratedthe antimicrobial activity due to the release of Aloe vera fromthe surface of the films The maximum activity was indicatedby 5 Aloe veraPVA combination The potential reasoncould be the presence of a lower number of interactionsbetween Aloe vera and PVA because of lower concentrationsof Aloe gel they were not chemically bound to each otherthus keeping the components and their respective functionalgroups of Aloe vera chemically active against microbialactivity as shown by FTIR results Increased levels (1015 and 20) of Aloe vera in the PVA blend lead tothe increased interactions between pharmacologically activecomponents of Aloe vera and PVA which causes the shiftin FTIR peak (Figure 2) thus such interactions of AloeveraPVAmay influence antimicrobial activity Renisheya JoyJeba Malar et al demonstrated the antimicrobial activity ofDMSO extracts of Aloe vera gel against human pathogensand highest zone of inhibition (13mm) against E coli wasrecorded [30] In another study the zone of inhibition againstE coli P aeruginosa and Aspergillus flavus was recorded


510

1520

10 20 30 40 50 60 700Time (minutes)

65

70

75

80

85

90

95

100

105

Chan

ge in

wei

ght (

)

Concentration

Figure 4 Degradation profile of Aloe verapolymer films withvarying concentration Time in minutes is shown on 119909-axis and change in weight is shown on 119910-axis

510

1520

0

1

2

3

4

5

6

7

8

Rele

ase o

f Alo

e ver

a (c

once

ntra

tion

())

10 20 30 40 50 600Time (minutes)

Figure 5 Aloe vera release profile of Aloe verapolymer films withdifferent concentrations 119909-axis shows time in minutes and 119910-axisshows release of Aloe vera concentration in

as 15mm 20mm and 15mm respectively [31] In currentresearch the mean zone of inhibition is 15mm for both Ecoli and P aeruginosa and 16mm for Aspergillus tubingensis(Figures 4 and 5) while for pure Aloe vera the zone was19mm and no zone was recorded against PVA films Thusit can be concluded that blend of Aloe vera and PVA hasnot much affected the antimicrobial activity of Aloe vera andantimicrobial activity of Aloe vera was maintained in blendform

34 Degradation of Aloe vera Release Profile Test ResultsThe degradation profile of Aloe veraPVA composite was

evaluated by recording weight loss at predetermined timepoints (Figure 4) The degradation profile was divided intothree stages during first 10 minutes a sudden loss of weightwas observed due to the initial burst release of Aloe verafollowed by sudden increase in the weight of the films(Figure 4) because of the absorption of buffer solution bythe PVA PVA when exposed to aqueous media absorbs theliquid and swells resulting in an increase in weight laterit becomes solvated and starts losing mass [32] Howeverafter 30 minutes the weight loss by the films became linearThe rate of swelling of the PVA films after the initial burstdecreasedwith the increase in the ratio of theAloe componentof the filmsThis was due to the fact that with the increase inthe Aloe vera concentration of PVA based films absorptionof the liquid medium by the PVA decreased [33]

The initial burst release followed by slow surface releaseof Aloe vera from the polymer based Aloe vera films wasobserved (Figure 5) An initial burst release of Aloe verafrom the surface of the films was detected during the first 10minutes This may be attributed to the presence of aggregatesof Aloe vera components on the film surface (verified laterby SEM images of the films) The aggregates of Aloe veraover the surface of the films were observed causing the initialburst release Later the amount of Aloe vera released fromthe surface decreased because of entrapment of Aloe vera inPVA mass During first 10 minutes Aloe vera was releasedonly by diffusion from the surface while after 20 minutesthe degradation of Aloe verapolymer film also contributesto the release of Aloe vera [34] The release profile of allthe concentrations that is 5 10 15 and 20 showedthe same behavior but with the increase in concentrationfrom 5 to 20 greater initial burst release was observedwhich is due to the increased amount of Aloe vera Moreoverincreased concentration resulted in decreased Aloe releasefrom the surface in the later stages because increasing theAloeamount lowers the rate of diffusion of Aloe from the surface[35]

An accelerated degradation and release study is per-formed for short period of time because keeping the applica-tion in mind the chance of infections occurring is greater atinitial stagesTheAloe verapolymer composite film is flexibleand can easily be placed on body surfaces hence makingit an ideal candidate for wound healing devices The initialAloe vera release from the surface is intended to be used asan antimicrobial so as to prevent the entry and proliferationof the microbes into the wound area [36] Also slow releasemarks the potential for an ideal microbial-free environmentfor wound healing [36]

35 Coating of the Sutures The dry weight of the suturesbefore dipping into the coating solution was 0045 g and afterdipping into the coating solution was increased to 0075 gThis increase inweight demonstrated the coating of the suturewith the coating material

36 In Vitro Testing of Coated Suture The zones of inhi-bitions against both the bacterial strains (E coli and Paeruginosa) were evaluated with coated and uncoated sutures(Figure 6) The results were compared with uncoated sutures


(a)

E coli5mm

(b)

Figure 6 In vitro testing against E coli of (a) uncoated suture and (b) coated suture

Table 1 In vitro testing of coated and uncoated sutures against Ecoli and P aeruginosa

Bacteria Zone of inhibition (mm)Coated suture Uncoated suture

E coli 46 plusmn 0577 0 plusmn 0

P aeruginosa 316 plusmn 0288 0 plusmn 0

which demonstrated no zone of inhibition using a paired 119905-test (Table 1)

The zone of inhibition with E coli was 46 plusmn 0577mm(mean of three triplicates) 119901 value = 00051 while with Paeruginosa it was 316 plusmn 028mm (mean of three triplicates)119901 value lt 00028

37 In Vivo Testing of Coated Sutures The silk sutures coatedwith Aloe verapolymer coatings showed significant reduc-tion in microbial colonization by E coli and P aeruginosain mice models (Table 2) The coated sutures demonstratedreduction in E coli to about 97 (119901 lt 00001) and 80 withP aeruginosa (119901 lt 00001) (Figure 7)

In this present study silk sutures coated with Aloeverapolymer coating exhibited substantial zone of inhibi-tions against E coli and P aeruginosa in vitro because ofthe pharmacologically active components present in Aloevera such as anthraquinones which remain active after beingblended with PVA Coated sutures showed results againstbacterial strains while no inhibition zones were observedwith uncoated sutures For in vivo studies mice models wereused in which control and test sutures were used in the sameanimal and the incision site was inoculated with a knownnumber of bacteria to evaluate the effectiveness of the coatedsutures The results of both in vivo and in vitro studies alongwith FTIR confirmed that the active components that areresponsible for antimicrobial activity present inAloe vera stillremain in their active form

SutureswithAloe verapolymer coating illustrated notice-able reduction in the growth of the P aeruginosa and evengreater reduction against E coli The test results of the invivo and in vitro investigations suggested that sutures with

Table 2 In vivo bacterial colonization of suture with coatedmaterial

Bacterial strains Log CFUexplanteda kill bacteriarelative toinoculumintroduced

119901 valueb

E coliWith coatedmaterial 03 97

lt00001Without coatedmaterial gt300 NA

P aeruginosaWith coatedmaterial 11 80

lt00001Without coatedmaterial gt300 NAaAverage of three animalsbPaired 119905-testNA not applicable

Aloe verapolymer coating are bactericidal It was verified bycalculating the bacterial colony count at incision site whichwas reduced in case of coated sutures this shows that theAloe veraPVA compositemay be used as a suture coating thathas the potential to prevent the spread of infections duringsurgical procedures

4 Conclusion

The biocompatibility and biodegradative properties of PVAhave been combinedwith the intrinsic bactericidal propertiesof Aloe vera The composition was screened for antimicrobialactivity against bacterial and fungal strains that is E coliP aeruginosa Aspergillus flavus and Aspergillus tubingensisrespectively The polymeric films with lowest concentration(5) of Aloe vera illustrated the best results with regard toantimicrobial activity against all the strains Commerciallyavailable sutures were coated with Aloe veraPVA solutionand tested for antimicrobial activity in in vitro and invivo systems These coated sutures illustrated a potential


(a) (b)

Figure 7 In vivo colonization of E coli with coated and uncoated suture (a) shows the results of in vivo antibacterial activity with coatedsuture while (b) shows the in vivo antibacterial results with uncoated sutures

for antibacterialantifungal coatings in commercial surgicalsutures that can play a role in preventing infections at surgicalsites Biocompatibility tests and clinical trials need to be con-ducted to better ascertain the potential of this Aloepolymercomposite as an option for use in surgical procedures as asuture coating as part of prophylactic measures to preventsurgical infections

Competing Interests

The authors declare that they have no competing interests

References

[1] A Nautiyal N V Satheesh Madhav et al ldquoReview on nosoco-mial infectionsrdquo Caribbean Journal of Science and Technologyvol 3 pp 781ndash788 2015

[2] G D Taylor M Buchanan-Chell T Kirkland M McKenzieand R Wiens ldquoNosocomial gram-negative bacteremiardquo Inter-national Journal of Infectious Diseases vol 1 no 4 pp 202ndash2051997

[3] T C Horan M Andrus and M A Dudeck ldquoCDCNHSNsurveillance definition of health carendashassociated infection andcriteria for specific types of infections in the acute care settingrdquoAmerican Journal of InfectionControl vol 36 no 5 pp 309ndash3322008

[4] S N Banerjee T G Emori D H Culver et al ldquoSecular trendsin nosocomial primary bloodstream infections in the UnitedStates 1980ndash1989 National Nosocomial Infections SurveillanceSystemrdquo The American Journal of Medicine vol 91 no 3 pp86Sndash89S 1991

[5] G P Bodey ldquoThe emergence of fungi as major hospitalpathogensrdquo Journal of Hospital Infection vol 11 pp 411ndash4261988

[6] S K Fridkin and W R Jarvis ldquoEpidemiology of nosocomialfungal infectionsrdquo Clinical Microbiology Reviews vol 9 no 4pp 499ndash511 1996

[7] C D Owens and K Stoessel ldquoSurgical site infections epi-demiology microbiology and preventionrdquo Journal of HospitalInfection vol 70 supplement 2 pp 3ndash10 2008

[8] R Pereira A Tojeira D C Vaz A Mendes and P BartololdquoPreparation and characterization of films based on alginateand aloe verardquo International Journal of Polymer Analysis andCharacterization vol 16 no 7 pp 449ndash464 2011

[9] S Goldstein R J Levy V Labhasetwar and J F BonadioldquoCompositions and methods for coating medical devicesrdquo USPatent 6143037 A 1996

[10] M G Cascone B Sim and D Sandra ldquoBlends of synthetic andnatural polymers as drug delivery systems for growth hormonerdquoBiomaterials vol 16 no 7 pp 569ndash574 1995

[11] J Walker G Young C Hunt and T Henderson ldquoMulti-centreevaluation of two daily disposable contact lensesrdquo Contact Lensand Anterior Eye vol 30 no 2 pp 125ndash133 2007

[12] S-H Yang Y-S J Lee F-H Lin J-M Yang and K-SChen ldquoChitosanpoly(vinyl alcohol) blending hydrogel coatingimproves the surface characteristics of segmented polyurethaneurethral cathetersrdquo Journal of Biomedical Materials ResearchmdashPart B Applied Biomaterials vol 83 no 2 pp 304ndash313 2007

[13] J M Schmidt and J S Greenspoon ldquoAloe vera dermal woundgel is associated with a delay in wound healingrdquo Obstetrics ampGynecology vol 78 no 1 pp 115ndash117 1991

[14] M Y Wani N Hasan and M A Malik ldquoChitosan and Aloevera two gifts of naturerdquo Journal of Dispersion Science andTechnology vol 31 no 6 pp 799ndash811 2010

[15] J H Hamman ldquoComposition and applications of Aloe vera leafgelrdquoMolecules vol 13 no 8 pp 1599ndash1616 2008

[16] R Pandey and A Mishra ldquoAntibacterial activities of crudeextract of Aloe barbadensis to clinically isolated bacterialpathogensrdquo Applied Biochemistry and Biotechnology vol 160no 5 pp 1356ndash1361 2010

[17] T Reynolds and A C Dweck ldquoAloe vera leaf gel a reviewupdaterdquo Journal of Ethnopharmacology vol 68 no 1ndash3 pp 3ndash371999

[18] M H Radha and N P Laxmipriya ldquoEvaluation of biologicalproperties and clinical effectiveness of Aloe vera a systematic


reviewrdquo Journal of Traditional and Complementary Medicinevol 5 no 1 pp 21ndash26 2015

[19] S S Silva S G Caridade J F Mano and R L Reis ldquoEffectof crosslinking in chitosanaloe vera-based membranes forbiomedical applicationsrdquo Carbohydrate Polymers vol 98 no 1pp 581ndash588 2013

[20] M Fani and J Kohanteb ldquoInhibitory activity of Aloe vera gelon some clinically isolated cariogenic and periodontopathicbacteriardquo Journal of Oral Science vol 54 no 1 pp 15ndash21 2012

[21] A Surjushe R Vasani and D G Saple ldquoAloe vera a shortreviewrdquo Indian Journal of Dermatology vol 53 no 4 pp 163ndash166 2008

[22] A Aytimur S Kocyigit and I Uslu ldquoSynthesis and charac-terization of poly(vinyl alcohol)poly(vinyl pyrrolidone)-iodinenanofibers with poloxamer 188 and chitosanrdquo Polymer-PlasticsTechnology and Engineering vol 52 no 7 pp 661ndash666 2013

[23] AW BauerWMKirby J C Sherris andM Turck ldquoAntibioticsusceptibility testing by a standardized single disk methodrdquoAmerican Journal of Clinical Pathology vol 45 no 4 pp 493ndash496 1966

[24] R A Kumar M Gayathiri S Ravi P Kabilar and S Velmuru-gan ldquoSpectroscopy studies on the status of aloin in Aloe veraand commercial samplesrdquo Journal of Experimental Sciences vol2 no 8 pp 10ndash13 2011

[25] Z X Lim and K Y Cheong ldquoEffects of drying temperature andethanol concentration on bipolar switching characteristics ofnatural Aloe vera-based memory devicesrdquo Physical ChemistryChemical Physics vol 17 no 40 pp 26833ndash26853 2015

[26] K S Venkatesh S R Krishnamoorthi N S Palani et al ldquoFacileone step synthesis of novel TiO

2nanocoral by solndashgel method

using Aloe vera plant extractrdquo Indian Journal of Physics vol 89no 5 pp 445ndash452 2015

[27] G-M Kim Fabrication of Bio-Nanocomposite Nanofibers Mim-icking the Mineralized Hard Tissues via Electrospinning ProcessINTECH Open Access Publisher 2010

[28] G-M Kim ldquoFabrication of bio-nanocomposite nanofibersmimicking the mineralized hard tissues via electrospinningprocessrdquo in Nanofibers A Kumar Ed chapter 4 pp 69ndash88InTech Rijeka Croatia 2010

[29] N A Abdullah Shukry K Ahmad Sekak M R Ahmad andT J Bustami Effendi ldquoCharacteristics of electrospun PVA-Aloevera nanofibres produced via electrospinningrdquo in Proceedingsof the International Colloquium in Textile Engineering FashionApparel and Design 2014 (ICTEFAD 2014) pp 7ndash10 Springer2014

[30] T Renisheya Joy JebaMalarM Johnson SNancyBeaulah R SLaju G Anupriya and T Renola Joy Jeba Ethal ldquoAnti-bacterialand antifungal activity of Aloe vera gel extractrdquo InternationalJournal of Biomedical and Advance Research vol 3 no 3 pp184ndash187 2012

[31] S Arunkumar and M Muthuselvam ldquoAnalysis of phytochem-ical constituents and antimicrobial activities of Aloe vera Lagainst clinical pathogensrdquo World Journal of Agricultural Sci-ences vol 5 no 5 pp 572ndash576 2009

[32] E-R Kenawy F I Abdel-Hay M H El-Newehy and GE Wnek ldquoControlled release of ketoprofen from electrospunpoly(vinyl alcohol) nanofibersrdquoMaterials Science and Engineer-ing A vol 459 no 1-2 pp 390ndash396 2007

[33] M Jannesari J VarshosazMMorshed andM Zamani ldquoCom-posite poly (vinyl alcohol)poly (vinyl acetate) electrospun

nanofibrous mats as a novel wound dressing matrix for con-trolled release of drugsrdquo International Journal of Nanomedicinevol 6 pp 993ndash1003 2011

[34] R Rosenberg W Devenney S Siegel and N Dan ldquoAnomalousrelease of hydrophilic drugs from poly(120576-caprolactone) matri-cesrdquoMolecular Pharmaceutics vol 4 no 6 pp 943ndash948 2007

[35] S G Kumbar and T M Aminabhavi ldquoSynthesis and char-acterization of modified chitosan microspheres effect of thegrafting ratio on the controlled release of nifedipine throughmicrospheresrdquo Journal of Applied Polymer Science vol 89 no11 pp 2940ndash2949 2003

[36] S-J Park and K-S Kim ldquoInfluence of hydrophobe on therelease behavior of vinyl acetate miniemulsion polymerizationrdquoColloids and Surfaces B Biointerfaces vol 46 no 1 pp 52ndash562005

Submit your manuscripts athttpwwwhindawicom

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CorrosionInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Polymer ScienceInternational Journal of



CeramicsJournal of


CompositesJournal of

NanoparticlesJournal of



International Journal of

Biomaterials


NanoscienceJournal of

TextilesHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Journal of

NanotechnologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

CrystallographyJournal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


CoatingsJournal of

Advances in

Materials Science and EngineeringHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Smart Materials Research



MetallurgyJournal of


BioMed Research International

MaterialsJournal of


Nano

materials


Journal ofNanomaterials


other biomedical applications such as drug delivery systemswound infections and antitumor and anti-inflammatoryagents [10]

Among synthetic biomaterials one of the extensivelyused polymers is poly(vinyl alcohol) (PVA) Due to itssuitable chemical and physical properties biocompatibilitybiodegradability easy preparation with excellent film form-ing properties and nontoxic nature PVA has been studiedintensively in different biomedical applications includingwound dressings contact lenses coatings for sutures andcatheters [11 12]

Aloe vera as a natural source of bioactive compounds iswidely studied for biomedical applications Aloe vera belongsto the Liliaceae family and is known as the oldest therapeuticherb It has the ability to promote wound healing as well astreat burnt areas on the skin [13 14] Due to its propertiesmany researchers have shown the antibacterial antiviralantitumor and anti-inflammatory activity of different partsof Aloe vera such as its stem root and leaf extracts [15ndash17]The chemical composition of Aloe vera has also proved itspotential use in cosmetic formulations food supplementsand medical devices [15 18 19]

The inner part of Aloe vera contains a clear mucilaginoustissue commonly known as Aloe gel The major portion ofAloe gel contains water while almost 1 contains bioac-tive compounds such asaloin emodin (anthraquinones)flavonoids saponin and Aloe-mannan along with many dif-ferent amino acids and vitaminsThese bioactive compoundsplay a major role for antibacterial activity of Aloe gel [15 2021]

The present work focuses on the antibacterial and anti-fungal activity of Aloe veraPVA composite membranesand the application of these blends in the prevention ofnosocomial infections for the specific purpose of investingthis sutures coated with the PVAAloe gel blend have beenused for both in vitro and in vivo analysisAloe veraPVAfilmshave been characterized through SEM and FTIR analysisThe results of in vitro and in vivo analysis proposed that thiscomposition can be used as a coating for the prevention ofsurgical site infections that are caused by infections throughsutures To our knowledge such biofunctional Aloe loadedPVA coatings have not been investigated against surgicalinfection causing bacteria in recent studiesThe results of ourstudy indicate the potential of such coatings as part of largerpreventive measures against surgical infections

2 Materials and Methods

21 Collection of Plant Material Fresh Aloe vera plants werecollected from local nurseries and the leaves washed wellwith distilled water to remove all contaminants presentat the surface The gel was harvested from the leaves inan autoclaved container and kept at room temperature forfurther use the storage time of the gels at room temperaturewas one minute During this time period any residual solidleaf particles were mechanically separated from the gel

22 Test Organisms for In Vitro Studies In order to investi-gate antimicrobial and antifungal activity (in vitro studies)

pure cultures of bacterial and fungal strains including Pseu-domonas aeruginosa (P aeruginosa) Escherichia coli (E coli)Aspergillus tubingensis and Aspergillus flavus were obtainedfrom Mycovirus Research Lab National University of Sci-ences and Technology (NUST) H-12 Islamabad The purebacterial and fungal cultures were stored in nutrient agar at4∘C

23 Suture Materials Commercially available silk braidedblack surgical sutures nonabsorbable (15 metric size 4-0)supplied by Foosin Medical supplies Inc Ltd ShandongChina and manufactured by WEGOSUTURES were used tocarry out in vitro and in vivo studies The suture material wasdelivered in sterile single peelable foil packages and storedat room temperature For investigation the sutures were cutinto defined lengths (1 cm) under aseptic conditions

24 Preparation of Aloe vera Based PVA Films Polyvinylalcohol (PVA) supplied by AppliChem Germany a biocom-patible polymer was used for the formation of polymerAloevera films [22] Dimethyl Formamide (DMF) manufacturedby TEDIA Company Inc USA was selected as solvent for theformation of PVA-Aloe vera films due to its high volatility

Solvent-casting method was used for the fabrication ofAloe vera gelPVA films 1 g of PVA was dissolved in 40mL ofDMFThe solution was stirred with a constant RPM of 580 at60∘Cuntil PVAwas completely dissolved and a clear solutionwas obtained This was followed by the addition of differentamounts of Aloe gel Aloe gel was added in the amounts of5 10 15 and 20 respectively for the fabrication ofAloe veraPVA films with varying Aloe gel compositions Theheating was turned off while constant magnetic stirring wascontinued to obtain a homogenized mixture of Aloe vera geland PVA in DMF The mixture was poured into Petri dishesand placed in oven at 37∘C for 20 h to evaporate the solventcompletely and dry films were harvested for further testingA solution of PVA in DMF was also prepared by the sameprocedure to obtain PVA films that were used as control forantimicrobial activity

25 Antimicrobial Testing of Aloe vera Based PVA FilmsThe antifungal and antibacterial activities of films wereevaluated using standard procedure of disc diffusion [23]For antibacterial activity sterile nutrient agar (pH 74) wasprepared using Tryptone 10 g supplied by BioWorld USAyeast extract 5 g supplied by MERCK Germany SodiumChloride 10 g supplied by AnalaR England and nutrient agar12 g supplied by MERCK Germany dissolved in 1000mL ofdistilledwater After autoclaving the nutrient agarwas pouredin Petri dishes which were inoculated with the 01mL ofbacterial inoculum from preculture of test bacterial strains

For antifungal investigation sterile potato dextrose agarwas prepared and poured onto the Petri plates and purefungal cultures were obtained from test fungal strains

For disc diffusion test films were cut into discs of about7mm in diameter and placed on the bacterial and fungalinoculated plates with certain distances Each Petri platecontained six discs one of which included the control sterileWhatman filter paper number 1 PVA film and other four



















(a) (b)

(c) (d)



51520

Pure PVAPVA in DMF

15

5

20

PVA

PVA in DMF

80

130

180

230

280

330

380

430

480

530

T

3000 2000 1000 04000(cmminus1)

(a)


Aloe in DMFAloe

62

63

64

65

66

67

68

69

70

T

3000 2000 1000 04000(cmminus1)

concentration

(b)



control Aloe vera


E coliP aeruginosa

0

5

10

15

20

25

Zone

of i

nhib

ition

(mm

)



(a)

films control Aloe



0

5

10

15

20

25

30

Zone

of i

nhib

ition

(mm

)



(b)










510

1520

10 20 30 40 50 60 700Time (minutes)

65

70

75

80

85

90

95

100

105

Chan

ge in

wei

ght (

)

Concentration


510

1520

0

1

2

3

4

5

6

7

8

Rele

ase o

f Alo

e ver

a (c

once

ntra

tion

())

10 20 30 40 50 600Time (minutes)










(a)

E coli5mm

(b)













119901 valueb






4 Conclusion



(a) (b)



Competing Interests


References

















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials





















(a) (b)

(c) (d)



51520

Pure PVAPVA in DMF

15

5

20

PVA

PVA in DMF

80

130

180

230

280

330

380

430

480

530

T

3000 2000 1000 04000(cmminus1)

(a)


Aloe in DMFAloe

62

63

64

65

66

67

68

69

70

T

3000 2000 1000 04000(cmminus1)

concentration

(b)



control Aloe vera


E coliP aeruginosa

0

5

10

15

20

25

Zone

of i

nhib

ition

(mm

)



(a)

films control Aloe



0

5

10

15

20

25

30

Zone

of i

nhib

ition

(mm

)



(b)










510

1520

10 20 30 40 50 60 700Time (minutes)

65

70

75

80

85

90

95

100

105

Chan

ge in

wei

ght (

)

Concentration


510

1520

0

1

2

3

4

5

6

7

8

Rele

ase o

f Alo

e ver

a (c

once

ntra

tion

())

10 20 30 40 50 600Time (minutes)










(a)

E coli5mm

(b)













119901 valueb






4 Conclusion



(a) (b)



Competing Interests


References

















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




(a) (b)

(c) (d)



51520

Pure PVAPVA in DMF

15

5

20

PVA

PVA in DMF

80

130

180

230

280

330

380

430

480

530

T

3000 2000 1000 04000(cmminus1)

(a)


Aloe in DMFAloe

62

63

64

65

66

67

68

69

70

T

3000 2000 1000 04000(cmminus1)

concentration

(b)



control Aloe vera


E coliP aeruginosa

0

5

10

15

20

25

Zone

of i

nhib

ition

(mm

)



(a)

films control Aloe



0

5

10

15

20

25

30

Zone

of i

nhib

ition

(mm

)



(b)










510

1520

10 20 30 40 50 60 700Time (minutes)

65

70

75

80

85

90

95

100

105

Chan

ge in

wei

ght (

)

Concentration


510

1520

0

1

2

3

4

5

6

7

8

Rele

ase o

f Alo

e ver

a (c

once

ntra

tion

())

10 20 30 40 50 600Time (minutes)










(a)

E coli5mm

(b)













119901 valueb






4 Conclusion



(a) (b)



Competing Interests


References

















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




control Aloe vera


E coliP aeruginosa

0

5

10

15

20

25

Zone

of i

nhib

ition

(mm

)



(a)

films control Aloe



0

5

10

15

20

25

30

Zone

of i

nhib

ition

(mm

)



(b)










510

1520

10 20 30 40 50 60 700Time (minutes)

65

70

75

80

85

90

95

100

105

Chan

ge in

wei

ght (

)

Concentration


510

1520

0

1

2

3

4

5

6

7

8

Rele

ase o

f Alo

e ver

a (c

once

ntra

tion

())

10 20 30 40 50 600Time (minutes)










(a)

E coli5mm

(b)













119901 valueb






4 Conclusion



(a) (b)



Competing Interests


References

















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




510

1520

10 20 30 40 50 60 700Time (minutes)

65

70

75

80

85

90

95

100

105

Chan

ge in

wei

ght (

)

Concentration


510

1520

0

1

2

3

4

5

6

7

8

Rele

ase o

f Alo

e ver

a (c

once

ntra

tion

())

10 20 30 40 50 600Time (minutes)










(a)

E coli5mm

(b)













119901 valueb






4 Conclusion



(a) (b)



Competing Interests


References

















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




(a)

E coli5mm

(b)













119901 valueb






4 Conclusion



(a) (b)



Competing Interests


References

















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




(a) (b)



Competing Interests


References

















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials

































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials










CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials



Documents

Research Article New Biofunctional Loading of Natural ...downloads.hindawi.com/journals/ijbm/2016/6964938.pdf · New Biofunctional Loading of Natural Antimicrobial Agent in Biodegradable