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Design of macroscopically ordered liquid crystalline hydrogel columns knitted with nanosilver for topical applications Sudha Janardhanan Devaki, Reshma Lali Raveendran, and Nishanth Kumar Sasidharan Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00706 • Publication Date (Web): 21 Feb 2017 Downloaded from http://pubs.acs.org on February 22, 2017

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Bioconjugate Chemistry

macroscopically ordered liquid crystalline hydrogel columns knitted with nanosilver for topical applications 171x159mm (96 x 96 DPI)

ACS Paragon Plus Environment


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Design of macroscopically ordered liquid crystalline hydrogel columns knitted with nanosilver for topical applications Reshma Lali Raveendrana, Nishanth Kumar Sasidharanb, Sudha J. Devakia,*

a Chemical Sciences and Technology Division, CSIR-National Institute for Interdisciplinary Science and Technology (CSIR-NIIST), Thiruvananthapuram, 695019, India. b Agro-Processing and Technology Division, CSIR-National Institute for Interdisciplinary Science and Technology (CSIR-NIIST), Thiruvananthapuram, 695019, India.

ABSTRACT: Design of liquid crystalline hydrogels knitted with silver nanoparticles in macroscopic ordering are becoming subject of research interest due to their promising multifunctional applications in biomedical and opto-electronic applications. The present work describes the development of liquid crystalline Schiff-based hydrogel decorated with silver nanoparticles and demonstration of its antifungal applications. Schiff base was prepared from polyglucanaldehyde and chitosan and the former was prepared by the oxidation of amylose (polyglucopyranose) isolated from abundantly available unutilised jackfruit seed starch. Selfassembled silver columns decorated macroscopically ordered networks were prepared in a single step of in-situ condensation and reduction/complexation process. The various noncovalent interactions among the –OH, –C=O , and –NH imparts rigidity and ordering for the formation of macroscopically ordered liquid crystalline hydrogel and the Ag(I) complexation evidenced from the studies made by FT-IR spectroscopy in combination with rheology and microscopic techniques such as SEM, TEM, AFM, XRD and PLM. The antifungal studies were screened using species of Candida by disc diffusion method. The MIC and MFC values, in vitro antifungal studies, reactive oxygen species (ROS) production and propidium iodide (PI) uptake results suggests that the present macroscopically ordered liquid crystalline hydrogel system can be considered as an excellent candidate for topical applications. All these results suggests that this design strategy can be exploited for the incorporation of biologically relevant metal nanoparticles for developing unique robust hydrogels for multifunctional applications. KEYWORDS: Polyglucanaldehyde, Schiff base, liquid crystalline hydrogel, nanosilver, antifungal.


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INTRODUCTION Recently, there have been overwhelming research interest among the research community for the development of macroscopically aligned liquid crystalline hydrogels especially decorated with noble metal nanoparticles for potential applications in the areas such as biomedical, antimicrobial, optoelectronic or catalytic materials, as well as the surface enhanced Raman scattering

substrates1- 6. Metal nanoparticles patterned macroscopically ordered soft hydrogel

can result in materials with synergistic properties of both metal nanoparticles and also ordered soft hydrogels with improved multifunctional properties.7-9 Hydrogels are three dimensional polymer structures that can absorb profuse amount of water without losing their structural integrity. They have received special attention due to its special properties such as softness, stimuli responsiveness, water permeability, swelling, self -healing and biocompatibility and are held together by either physical interactions (chain entanglements, van der Waals forces, hydrogen bonds, crystallite associations, and/or ionic interactions) or chemical cross-links (covalent bonding). They find applications in various fields such as in agriculture, hygiene, biomedical, wound dressing, scaffolds/implants, tissue engineering, pollutant absorbents and biosensors, synthetic extracellular matrix, sensors, actuators, enzyme immobilization, smart hydrogels, etc. They are made from natural polymers and their salts such as starch, protein, gelatin, sodium alginate, hyaluronate, hemicellulose, lignin, cellulose, chitin, and their derivatives16-18. In biomedical applications natural hydrogels made from biopolymers are using extensively due to their high biocompatibility, biodegradability, tissue mimicking properties etc. The three-dimensional network structures of hydrogels helps them to incorporate various nanoparticles (metallic, inorganic, bioactive, etc.) leads to hybrid hydrogels with enhanced mechanical properties and unique optical, electric, magnetic, or biological properties19-21. These hydrogels are formed by molecular self-assembly through non-covalent interactions such as hydrogen bonding, van der Waals forces and π-π and electrostatic interactions. These interactions helps in reversible sol−gel or gel− sol transitions, the prepared hydrogels to be processable and also facilitate the stimuli-responsive behaviors of the hydrogels. The combination of metal nanoparticle and soft hydrogel can results in materials with synergistic properties of both and found numerous applications in catalysis, electronics, design of sensors, and luminescence devices, in photonics, biotechnology, and medicine 22-26. Also aligned liquid crystalline gels have well-defined network, a large interfacial area, and the possibility to entrap solutes within the gel



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matrix, along with long-term stability, makes them valuable for a variety of applications. Hence different therapeutic compounds such as analgesics, antibiotics, antifungal, anticancer, vitamins, antiasthmatic, immunosuppressive etc. have been either incorporated or itself used for the formation of the liquid crystals with encouraging results in medical field.27-29 This aligned system will also help for the easy release of silver ions which will give better antifungal property. Among the metal nanoparticles, silver nanoparticle have gained special interest due to its antibacterial and antifungal activities. It was later found that out of all the metals with antimicrobial properties, silver has the most effective antimicrobial action and the least toxicity to animal cells30. Colloidal silver was already known to be a better antibacterial and antifungal agent. It can be used to cure most of the topical fungal infections affected on eyes, ears, mouth and skin etc31. Athlete’s foot and stubborn toe nail fungal infections can also be wiped out by using colloidal silver instead of potentially harmful antifungal drugs that have been known to harm the liver. Silver nanoparticle as antimicrobial agents has been applied in different medical fields such as for ocular applications, sustained drug delivery, surgical catheters, and dressing of infected wounds. The antimicrobial activity of silver nanoparticle is proposed to arise from the release of silver ions and will cause damage to the bacterial cell wall and destroys the metabolic responses32-33. Various mechanisms have been suggested for the antibacterial activity of AgNPs34-35. Compared with other metals, silver exhibits higher toxicity to microorganisms while it exhibits lower toxicity to mammalian cells36. Thus hydrogels are expected to act as a better scaffold for silver nanoparticles and it can release silver ions in a controlled manner. Starch is a natural semicrystalline biopolymer of D-glucose. It is a natural polysaccharide distributed in various plant species and is abundant in cereal grains, legumes, tubers and immature fruits37. Jackfruit (Artocarpus heterophyllus Lam.) is important natural plant of Southeast Asia and the endocarp of jackfruit seed contains 80 % starch which is presently low cost and unutilized. Starch consists of two macromolecules, α- 1, 4- D-glucose (amylose) and α1, 6- D-glucose (amylopectin). Amylose is linear and amylopectin is branched and owing to its complete biodegradability, low cost and renewability, starch is considered as a promising candidate for developing sustainable biomaterials. Further starch-based hydrogel nanocomposites are very important in both academic and industrial fields owing to their abundance, availability and potential applications in several technologies.


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Albeit their potential applications, the structural inhomogeneity and lack of effective energy dissipation mechanism lower the mechanical strength of hydrogels. The mechanical and physical properties of starch based hydrogels can be improved by crosslinking with natural or synthetic polysaccharide or by chemical modification38-39. Chitosan is another important natural polysaccharide composed of β - (1–4) linked D-glucosamine and N-acetyl D-glucosamine residues. It is nontoxic, biocompatible, and biodegradable pH-dependent cationic polymer that has recently found many applications in the biomedical field. It also has important biological properties, such as antimicrobial activity and ionic binding affinity with DNA. In addition to good film and gel forming properties, one of chitosan’s most promising feature is its excellent ability to produce three-dimensional scaffolds. Research in the development of novel sustainable antifungal agents for topical applications is receiving interest. Fungi produces a wide spectrum of infections especially in the tropical regions. Candida species such as Candida albicans, Candida tropicalis, Candida glabrata and Candida parapsilosis are most widespread among them. Hence it is an urgent need for developing new antifungal agent against these Candida species. In this work we have prepared nanocomposites hydrogel by in-situ condensation and reduction method. Amylose was extracted from starch and oxidized to polyglucanaldehyde. Then it is coupled with amino containing natural polymer chitosan through Schiff base formation. Silver nanoparticles were incorporated to the Schiff base by insitu reduction method. The resulting hydrogels were characterized by 1H NMR, FT-IR, and UV -Visible spectroscopy. Swelling properties were also evaluated. Morphology was studied by using SEM, TEM, AFM and PLM. The viscoelastic properties were studied by rheological measurements. The in vitro antifungal activities were tested against four wound infectious fungi Candida albicans, Candida tropicalis, Candida parapsilosis and Candida glabrata. The minimum inhibitory concentration and minimum fungicidal concentration were found out. The capacity of the prepared nanocomposites hydrogel in intracellular Reactive Oxygen Species (ROS) production and PI uptake was also studied.



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RESULTS AND DISCUSSION Formation of Schiff based hydrogel Amylose i.e. poly (1-4)-α-D-glucopyranose was isolated and purified from Jackfruit seed starch as per reported procedure40 and the details are given in the experimental section and structure. Further poly(1-4)-α-D-glucopyranose was oxidized to poly(1-4)-α-D-glucanadehyde using sodium periodate as the oxidizing agent and the formation of aldehyde was confirmed by FTIR and 1H NMR spectroscopy. FTIR spectra of poly (1-4)-α-D-glucopyranose and poly(1-4)-α-Dglucanaldehyde are shown in Figure 1a and 1b respectively. Poly(1-4)-α-D-glucopyranose exhibited characteristic absorption bands of O–H stretching at 3290 cm−1, and of C–H stretching at 2931cm−1. FTIR spectra of poly(1-4)-α-D-glucanadehyde exhibited the characteristic peak carbonyl peak of aldehyde functional group is at 1739 cm-1 which is absent in the native gluocpyranose ring. Further confirmed by 1H NMR and details are depicted in the experimental section. Schiff base was prepared by the condensation of polyglucanaldehyde and chitosan i.e, poly-(D) glucosamine as per the procedure given in the experimental section39. Chitosan was selected as the amino containing cross-linker because of its biocompatibility. Aldehyde group of glucanaldehyde react with the amino group of chitosan and forms Schiff base. Similar procedure was used for the preparation silver nanoparticles entrapped hydrogel by conducting the preparation of Schiff base in presence of silver ions. The secondary hydroxyl groups and also the amino group can reduce silver ions to form silver nanoclusters stabilized inside the gel. Apart from stabilization the silver clusters by the hydroxyl group, the lone pair of electrons present in the carbonyl group and the imino group can from coordination with Ag (1). Formation of the silver hydrogen interaction and various noncovalent interactions present in the hydrogel which is leading to the formation of macroscopically aligned liquid crystalline hydrogel was further strengthened by the studies made by FTIR spectroscopy, XRD and microscopic analysis. The robust nature of the hydrogel observed by rheological analysis and the presence of silver nanoparticles observed by studying the surface plasmon resonance spectra by UV-Vis spectroscopy. The schematic representation of the formation of hydrogel is shown in Scheme 1.


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. Scheme 1. Schematic representation of the formation of silver nanoparticle entrapped Schiff base (SCS) hydrogel The FTIR spectra of Schiff base showed characteristic band of -C=N- at 1640 cm−1 and disappearance of band at 1383 cm−1 −NH2 characteristic of amino group present in chitosan. The changes in the band position of the hydroxyl group polyglucopyranose (~3290 cm−1), polyglucanaldehyde ( ~3280 cm−1) and the -C=N- group of Schiff base ( 3250 cm−1) and also the nature of the band transformed from broad to narrow suggests the establishment of large extend of hydrogen bond interaction present in Schiff base. The band at 2800 cm−1 in the Schiff



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base spectra suggests the presence of OH…N intramolecular hydrogen bond interaction. The formation of silver- Schiff base co-ordination bond Ag (1) results an increase in C=0 stretching frequency from 1739 to 1750 cm-1 and C=N frequency also shifted to 1615 cm-1. Further formation of complex structure and crystallization formation of silver inside the liquid crystalline hydrogel supported the studies made from XRD, UV-Vis spectroscopy and various microscopic analysis. The presence silver nanoparticles present in the SCS hydrogels were studied by UV-Visible spectroscopy which are expected to exhibit optical spectra related to the surface plasmon resonance energy of the silver nanoparticles present in the hydrogel. The UV-Vis spectra of SCS1, SCS2, SCS3 and SCS4 gels with concentration of silver ions 0.06 M, 0.08 M, 0.1 M and 0.15 M respectively are shown in Figure 1b. UV-Vis spectra showed absorption maxima corresponding to the surface plasmon resonance energy of the silver nanoparticles and it is varied with the amount of silver present in the nanocomposites. SCSs exhibited two peaks at 290 nm and 400-417 nm. The peak at 290 nm corresponds to the –C=N- group and the second peak corresponds to the surface plasmon resonance peak of silver nanoparticle. From SCS1 to SCS4 the SPR peak get shifted from 401 nm to 417 nm. As the concentration of silver ions increased from 0.06 M to 0.15 M in SCS1 to SCS4, spectra showed more intense and sharper bands. The nature and position of the absorption maxima changes with increase with the concentration of silver nanoparticles. This attributed to the formation of more uniform silver nanoparticles. The UV-Vis spectra of the films were measured for 30 days and observed UV-maxima at 330-450 nm confirms the presence and stability of silver nanoparticles in the hydrogel matrix for 30days. Corresponding spectra is incorporated in the supporting information as Figure S1.


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(a)

(b)

III II

1750 cm-1

I

Figure 1. (a) FT-IR spectra of I) amylose II) polyglucanaldehyde and III) SCS4 hydrogel (Schiff base hydrogel) (b) UV-Visible absorption spectra of SCSs

Swelling studies The swelling behavior of the hydrogels was studied by varying the concentration of silver nitrate in phosphate buffer solution at pH of 7 and is shown in Figure S2. As moving from SC to SCS4 the swelling density gets decreased. Increase in concentration of silver ions, a decrease in swelling ratio observed from 136 % to 75%. This may be due to the formation of more entanglement and crosslinks due to the presence of silver nanocrystallites (columnar liquid crystalline junctions formed along the macroscopically ordered hydrogels). These observations were supported by the studies made under polarized light microscopy. This is because of the enhancement in the crosslinking density and thereby decrease in the swelling ratio of SCs. The pores inside the composite hydrogels were easily blocked by knots of self-assembled liquid crystalline columns of silver – Schiff base complexes. This may block the penetration of water molecule into the interior of the gels. Rheology The viscoelastic property of the prepared hydrogel was studied using rheology. Viscoelastic properties were conducted on oscillatory mode rheometer under varying strain for the observation of the linear viscoelastic region. Rheological studies give evidence for the storage modulus (G') which corresponds to the solid like behavior and loss modulus (G") stands for viscous like behavior. The amplitude sweep measurement of SC & SCS were performed to determine the linear visco-elastic region and it is given in Figure S3 A & B. Rheogram exhibited



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linear viscoelastic region ~ 5%. Further , amplitude sweep experiment showed G' and G" cross over point at a strain value of 167% for SC and for SCS4 strain value increased to 185% which supports our earlier observation that on incorporation of silver nanoparticle enhances the strength of the hydrogels. Further, frequency sweep measurements of the hydrogels, SC and SCS were studied at room temperature and are shown in Figure 2a. Rheogram exhibited characteristic profile of G' and G" in which both the values independent of the frequency applied. Moreover, these gels exhibited storage modulus higher than loss modulus characteristic of elastic and robust behavior of gels. SC exhibited storage modulus value of 3000 Pa and loss modulus value of 548 Pa. Also storage modulus and loss modulus value is independent of angular frequency suggests typical gel formation. Storage and loss modulus values of SCS enhanced with increasing concentration of silver ions. It revealed that while incorporating silver nanoparticles in the gel matrix, strength of the hydrogel get enhanced. This is a clear evidence for the significant role of silver nanoparticles in the cross-linking of hydrogel. The viscosity- shear rate relationship of the SC & SCSs were determined from flow test by performing rheological measurement under rotatory mode which is given in Figure 2b. SC and all the formulations of SCS showed non- Newtonian flow behavior with shear thinning property at a shear rate ranges from 0.1 to 100 s-1. The viscosity of SCS enhanced with increase in concentration of silver ions. The increase in viscosity of hydrogels from SC to SCS4 is due to the enhancement in the cross-linking density of the hydrogels.

(a)

(b)

Figure 2. a) Frequency sweep measurement of SC and SCS hydrogels b) Shear rate against viscosity plot of SC and SCS hydrogels.


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Liquid crystalline mesophase formation Morphology and the formation of supramolecular columnar liquid crystalline silver Schiff base complexes decorated with silver nanocrystallites confirmed by studies made through various microscopic techniques such as PLM, SEM, TEM and AFM in combination with XRD. The PLM images of Schiff-base (SC) and silver – Schiff base (SCS) are shown in Figure 3a, 3b and 3c. The SC exhibited liquid crystalline mesophase due to the presence of the mesogen C=N group in the Schiff base and also presence of rigidity established through various hydrogen bonding interaction. PLM image of SC observed as macroscopically aligned well-ordered fibers. (Fig 3a). However, morphology of SCS exhibited necklace patterns of macroscopic chains decorated with tetragonal columnar phase. These columnar liquid crystalline junctions can be attributing from the supramolecular mesophase formation of self-assembled silver-Schiff base complex. Silver nanoparticles are entrapping inside the columns during reduction in presence of amine and hydroxyl groups present in the system. These loosely bound silver nanocrystallites can be exploited for various applications since it can be made available through controlled release as demonstrated for the antifungal properties as illustrated in this paper. It has also been observed the morphological changes on increasing the amount of silver ions in the systems. The PLM images of SCS2 and SCS4 are shown in Figure 3b and 3c, respectively. The PLM image of SCS4 exhibited more centres of well-defined tetragonal columnar junctions compared with the PLM image of SCS2 due to the formation of more number of silver- Schiff- base complex centres in SCS4 which forms self-assembled tetragonal columnar features along the macroscopically aligned fibres. This design strategy will help for the easy release of silver ions.



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Figure 3. PLM images of (a) SC (b) SCS2 (c) SCS4, SEM images of d) SC e) SCS2 f) & g) global macroscopic alignment of SCS4 h) Hydrogel column with nanosilver i) EDAX spectra of SCS4

Further morphology of the Schiff base and complex of silver Schiff base was studied by SEM in combination with EDS and is shown in Fig 3. SEM image of SC observed as macroscopically aligned fibrillar morphology as observed under PLM. SEM images of SCS showed bunches of macroscopically oriented fibres having junctions of columnar features as shown in Figure 3d, 3e, 3f. SEM images taken from focusing the centre of junctions given in Figure 3g and 3h. Presence of silver nanoparticles confirmed by EDAX spectra of SCS4 is shown in Figure 3i. It was found that 3.15 weight % of Ag+ ions were present in the SCS4 hydrogel.


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TEM images were studied for strengthening the macroscopic aligned fiber formation and also presence of silver in the hydrogel and are shown in Figure 4. The size of the silver nanoparticles measured as 20 nm in Figure 4 a & b. The selected-area electron diffraction study (SAED) pattern was observed by directing the electron beam perpendicular to the silver nanoparticles. The diffraction spots pattern shown in the Figure 4c shows that the silver nanoparticles in the hydrogel are well crystalline in nature. In order to confirm the crystalline nature of silver nanoparticles X-ray diffraction study was carried out. The XRD spectra of SC and SCS4 hydrogels are shown in Figure 4d. The XRD pattern of SC exhibited broad signals in the diffractogram revealing the absence of highly ordered crystalline phase. However it showed maxima at 2θ values of 31.70, 42.00, 49.180 corresponding to interlayer ordering present in the Schiff base. Moreover, XRD pattern of SCS4 showed Bragg diffraction peaks at 2θ values of 38.28°, 44.04°, 64.34° corresponding to (111), (200) and (220) Bragg reflections indicating the face centered cubic crystalline nature of silver. The AFM images of SC and SCS4 were shown in Figure 5. The porous and macroscopically ordered columnar phase of the liquid crystalline hydrogel is well shown in Fig. 5a and its 3D image in Fig. 5c.In SCS4 hydrogel the silver nanoparticles were distributed in the porous gel structure which is shown in Figure 5c. 3D image of SCS4 is also shown in Figure 5f. The height profile of SC showed 170 nm fibres and SCS4 were measured as 25-35 nm corresponding to the size of the columnar features present in the SCS4 silver- Schiff base hydrogel. corresponding figures are shown 5b and 5e respectively.


And the


(b)

(a)

(d)

(c)

SC SCS4

Intensity (Counts)

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(200) (111) (222)

20

30

40

50

60

70

Diffraction Angle (2θ)

Figure 4. HR-TEM images of a) SCS2 b) SCS4 c) SAED pattern of SCS hydrogel SCS4 hydrogel D) XRD spectra of SC and SCS

(a)

(b)

(c)

2.0µm

(d)

(e)

(f)

2.5µm

Figure 5. a) & b) AFM images and height profile of SC c) 3 D image of SC d) & e) AFM image and height profile of SCS4 f) 3D image of SCS4


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In vitro antifungal susceptibility assay of SCS Due to the mammoth emergence of the fungal diseases, researchers are intensively directed towards the discovery and development of novel antifungal agents. Detection of the resistance of these agents in a microbe are the same for bacteria, fungi and large parasitic organisms. Normally the detection strategy is to measure the reduction in the microbial growth resulting from exposure to an inhibitor. Presently several technologically sophisticated instruments are reported and available for the evaluation of the efficacy of these antimicrobial agents. The simplest approach is to measure the scrutiny of zones of growth inhibition in the territory of microbial cultures around the discs impregnated with an antimicrobial substance. The most widely used quantitative measurement is the study of minimum inhibitory concentration (MIC) of an antimicrobial. Cultures containing serial dilutions of an agent are used to establish the lowest concentration that has prevented development of detectable microbial growth. Among the fungal species, Candida species are responsible for the pathogenic infection in humans. The antifungal assay was done against four Candida species viz. Candida albicans, Candida tropicalis, Candida parapsilosis and Candida glabrata through disk diffusion assay. The results are given in Figure 6. All the formulations exhibited better antifungal activity. Comparative value of zone of inhibition for all the batches of SCS are depicted in Table 1. A standard drug Amphotericin B and SC hydrogel were used as the control. It can be observed that SCS4 showed highest zone of inhibition. So SCS4 was selected to determine the MIC and MFC. The minimum inhibitory concentrations (MIC) and minimum fungicidal concentrations (MFC) were determined. The MIC were determined as the lowest concentrations of antifungal agent that significantly inhibit target cell growth and MFC were determined as the lowest concentrations that show either no growth or fewer than three colonies to obtain a 99–99.5% killing activity. The MIC values for Candida albicans, Candida tropicalis, Candida glabrata and Candida parapsilosis were 16 µg/mL, 32 µg/mL, 32 µg/mL and 64 µg/mL respectively. The corresponding MFC values were 32 µg/mL, 64 µg/mL, 34 µg/mL and 128 µg/mL.



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Table 1. Zone of inhibition values of control (drug & SC) and SCSs against fungus Candida albicans, Candida tropicalis, Candida parapsilosis and Candida glabrata.

Fungi

Candida albicans Candida tropicalis Candida parapsilosis Candida glabrata -

SC

Zone of inhibition (Dia. in mm) SCS1 SCS2 SCS3

SCS4

-

11±0.52

14±0

20±2.52

29±0.52

Amphotericin B (Standard drug) 17±1.57

-

12±1.77

20±1

30±1.77

31±1.52

15±0.52

-

-

11±0.77

11±0.52

12±0.77

12±0.77

14±1.77

13±1.77

19±1.52

15±1

15±1

16±1.72

Indicate no activity

Intracellular Reactive oxygen species production Metal nanoparticle can interact with bacterial cell and produces reactive oxygen species such as hydroxyl radicals OH•, superoxide ions O2− •, H2O2, and hydroperoxyl radicals, which produce oxidative stress in the cells and will damage proteins and nucleic acids

41-42

. The intracellular

ROS production was measured by H2-DCFDA assay. In the presence of ROS, H2-DCFDA is oxidized and form green fluorescent dichlorofluoroscein upon excitation. SCS hydrogel containing with ordered columnar silver can release silver ions which can interact with fungus and produces reactive oxygen species. The Ag (1) in the SCS hydrogel is weakly bound and so can easily released in a controllable way 43. On interaction with Candida species SCS will induce the ROS production which will cause oxidative stress 44 in the cells. It can results in the damage of the cell membrane and subsequently lead to the death of the cell. The percentage of reactive oxygen species production was also calculated. The fluorescence microscope images and % ROS production is given in Figure 6 b. Studies on the interaction of SCS with fungal cells The interaction of fungal cells with silver nanoparticle entrapped hydrogels were studied by propidium iodide assay. SCS4 was taken as the typical hydrogel for studying the interaction with four fungal species Candida albicans, Candida tropicalis, Candida parapsilosis and Candida glabrata. The percentage of PI uptake is also given in Figure 6c. The PI uptake result is associated with the occurrence of substantial damage to the membrane, indicating alteration of


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cell membrane potential, which finally causes cell death45. PI could enter the cell and bind to DNA, showing red fluorescence. SCS4 hydrogel shows higher propidium iodide uptake compared to the control SC hydrogel.



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Figure 6. a)Disk diffusion assay of SCS1, SCS2, SCS3 & SCS4 against Candida albicans, Candida tropicalis, Candida parapsilosis & Candida glabrata.b) Fluorescence microscope images of fungal species treated with H2- DCFDA i) Candida albicans ii) Candida tropicalis iii) Candida parapsilosis iv) Candida glabrata and v) Representation of ROS production with SC hydrogel as the control. c) Fluorescence microscope images of i) Candida albicans ii) Candida tropicalis iii) Candida parapsilosis iv) Candida glabrata treated with SCS4 by propidium iodide assay and v) Representation of PI uptake of Candida cells with SC hydrogel as the control.

There is hydrogen bond interaction between the neighbouring - N-H and carboxylate oxygen atoms. These intermolecular hydrogen bonds involved by the nitrogen and oxygen atoms from weak Ag-O and Ag-N bonding 46. The coordination donor atoms to the silver (1) center and ease of scission of the coordination bond attributed to the crucial roles in leading to the increased antifungal activities. The mechanism of antimicrobial action of silver nanoparticles is still a debated topic. However various mechanism have been suggested. We have studied the interaction of SCS with fungal cells by intracellular ROS production and PI uptake assay. Both assay helps to know the mechanism of action of SCS with fungal cells. Upon interaction with fungal cells SCS produce reactive oxygen species production and it induces oxidative stress in the cell. That will cause damage to the cell membrane and finally leading to cell death. The interaction of AgNPs with the cells was investigated by PI uptake assay. PI can enter into the cell through membrane and bind to DNA which will cause the death of the bacteria and it exhibits strong red fluorescence. CONCLUSIONS In conclusion, we have successfully prepared and characterized novel macroscopically aligned liquid crystalline complex of silver Schiff base antifungal hydrogel from low cost starch and chitosan by simple facile strategy. The robust hydrogels showed storage modulus value of 2×104 Pa. The focal conic columnar phase of crosslinking junctions along the macroscopically aligned fibres confirmed by various microscopic techniques, PLM, SEM, TEM and AFM. The size of the silver nanoparticle was found to be ~ 20 nm. The antifungal applications of the hydrogels were studied by disc diffusion assay and it shows better zone of inhibition, MIC and MFC values and it suggests it as very efficient antifungal agent for topical applications. Apart from its antifungal application, the design strategy can be exploited for the development of macroscopically aligned liquid crystalline metallogels for its interdisciplinary application in electronic and tissue engineering.


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EXPERIMENTAL SECTION Materials Amylose extracted from jackfruit seed starch40, chitosan (Sigma Aldrich), glacial acetic acid – (99%, Fisher scientific); sodium periodate (99.8%, Sigma Aldrich,), silver nitrate (99.5%, Emerck Limited, Mumbai, India). Candida albicans MTCC 277, Candida tropicalis MTCC 230, Candida glabrata MTCC 3019 and Candida parapsilosis MTCC 6510 were used for antifungal studies. All the test microorganisms were procured from Microbial Type Culture Collection Centre (MTCC), IMTECH, Chandigarh, India. The test fungi were maintained on potato dextrose agar (PDA) slants. The fungal cells were cultured in potato dextrose broth (PDB) with aeration at 37 ºC. The cell growth was determined by measuring optical density at 600 nm with a microliter ELISA plate reader. Isolation of amylose (polyglucopyranose) from starch Amylose was extracted from starch by an already reported procedure which is describes as follows40. Amylose was extracted at 2% concentration by adding a starch slurry to water at 98 0C and maintaining this temperature for 11-15 minutes while stirring the solution. pH of the solution was held at 6.0 with phosphate buffer. The extraction was done in a three necked flask with volume three times higher than the starch volume. The solution was stirred with a mechanical stirrer regulated to a speed of 200 r.p.m. Solutions were cooled rapidly to room temperature by immersion in a bath at 0-2 0C. The granule residues, which consisted mainly of amylopectin, were separated from the dissolved amylose by centrifugation. A compact gel layer, occupying 25 to 27% of the volume of the initial dispersion, formed after sedimentation for half an hour. Separations were also made by centrifugation for 1 hour in an ultracentrifuge operating at 20,000 r.p.m. Supernatants were less turbid and contained slightly lower concentrations of polysaccharide. From 75 to 85% of the amylose in a starch appeared in the supernatant layer after centrifugation. The gel layer was extracted by redispersion at 98 0C by gentle stirring for 15 minutes and was separated as a supernatant solution by centrifuging and cooled solution as before. Procedure of the same repeated twice to extract all the amylose.



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Oxidation of amylose (polyglucopyranose) Amylose (polyglucopyranose) was oxidized by an already reported method47. Amylose and sodium periodate (1:0.5 molar ratio) were dissolved in distilled water with vigorous mechanical stirring. The pH of the solution was kept at 7 reaction temperature was set at 37 °C. After 12 h, the slurry was filtered and the product was washed with deionized water for several times, and then lyophilized. A white powder was obtained with an yield of 85%. Formation of aldehyde confirmed by FTIR and 1H NMR. The 1H NMR spectrum of the polyglucanaldehyde shows hydroxyl protons at 4.68–5.6 ppm, and peaks at 5.10 ppm (C2–OH), 5.52 and 5.41 ppm (C3– OH), and 4.68 ppm (C6–OH). Peak at 9.36 ppm obtained correponds to the dialdehyde groups. 1

H NMR spectrum is given in supporting information as Figure S4.

Preparation of Schiff base hydrogel from polyglucanaldehyde and chitosan (SC) 1 mL of 5% polyglucanaldehyde solution was taken into a vial. To that 1 mL of 2% chitosan in acetic acid solution was added and mixed it to get homogenous mixture by constant stirring. The pH of the Schiff base solution was adjusted to 7 by the addition of alkali. Gel formation was observed on keeping the solution for 12 hours at room temperature. Schiff base was isolated by centrifugation and formation was confirmed by FTIR and 1H NMR. The 1H NMR spectra of Schiff base shows peak at 7.50 ppm which corresponds to the -CH=N- proton. The peak for hydroxyl protons obtained as 3.65 ppm (-CH2-OH), 3.58 ppm, 4.02 ppm and 4.00 ppm (CH-OH). Preparation of silver nanoparticle entrapped Schiff base hydrogel (SCS) 1 mL of 5% polyglucanaldehyde solution, 1 mL of 2% chitosan were taken in different vials and mixed to get homogenous solution by constant stirring. Then add 700 μL of different molar concentrations (0.06 M, 0.08 M, 0.1 M and 0.15 M) of silver nitrate solution. During the addition of AgNO3, immediate gel formation was observed. The colour of the SCS hydrogel observed to vary from light yellow to brown on increasing concentration of silver nitrate. The different batches of the gels were designated as SCS1, SCS2, SCS3 and SCS4. Swelling studies The weight of the dried hydrogel was noted and then it was immersed directly in buffer solution of pH 7 at room temperature for 24 hrs. The weight of the swollen hydrogel was noted. The equilibrium percentage of the product was then calculated as:

% swelling =

x 100


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Where Wh is the weight of the product after hydration for 24 hours while Wd is the weight of the dried product. Antifungal susceptibility test Agar plate disc diffusion assay The SCS formulations were screened for their antifungal activity against four fungi by disc diffusion method. Four Candida species Candida albicans, Candida tropicalis, Candida parapsilosis and Candida glabrata were used for the assay. 100 μL of overnight candidal cultures were spread over PDA agar plates. 0.6 cm filter paper discs containing 50 μL of SCSs (50 μg/disc) were placed on the agar. Following which, the plates were incubated overnight at 37˚C in an upright position. Clear, the distinct zone of inhibition was envisaged surrounding the discs containing antimicrobial substances. Amphotericin B (100 μg/disc) was used as the positive drug control. The antimicrobial activity of the test agents was determined by measuring the zone of inhibition expressed in mm. Minimum inhibitory and minimum fungicidal concentration Minimum inhibitory concentrations (MICs) and minimum fungicidal concentration (MFCs) were determined by the broth microdilution method according to the recommendation of Clinical and Laboratory Standards Institute (Clinical and Laboratory Standards Institute, 2008) using 96-well microliter plates with minor modification. Briefly, ten twofold serial dilutions of SCS hydrogel particle were prepared in the appropriate broth concentrations ranging from 2 to 1000 μg/mL. Each well was inocubated with 5 μL of fungal suspension at a density of 107 CFU/mL, while microliter plates were incubated at 37°C for 48 h, and the fungal growth was determined by measuring the OD value at 600 nm. Minimum inhibitory concentrations were determined as the lowest concentrations that inhibited the growth of the test fungi by ≥90% compared with that of the agent-free growth control. Minimum inhibitory concentrations were obtained from three independent experiments that performed in triplicate. After the appropriate incubation time, the presence (or absence) of growth was observed visually. The formation of cell clusters or “buttons” in the plate wells was considered. The MIC is defined as the lowest concentration of the antifungal agent that inhibits the growth of the organism. From the corresponding concentrations to MIC and higher values of this SCS concentration, we took an aliquot of 20 𝜇L for each of these fungal cultures grown in media with SCS and reACS Paragon Plus Environment


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suspended in 900 𝜇L PDB culture medium in order to re-activate viable remnant cells and further

they were incubated at 35°C for 24 h. Then, aliquots from each well were withdrawn and Petri dish with PDA medium were inoculated for checking absence of fungal growth. These cultured microplates were incubated for 48 h at 35°C. The MFC was defined as the lowest product concentration that inhibited growth of the yeast or permitted less than three CFUs to occur, resulting thus in 99.9% fungicidal activity. Biological activity assays were performed in duplicate, and the results were expressed as the arithmetic mean of the MIC and MFC. Intracellular Reactive Oxygen Species (ROS) measurement The amount of ROS produced by the target cells was measured by fluorometric assay using 2′, 7′-dichlorodihydrofluorescein diacetate (H2-DCFDA), as per the protocol of Kobayashi et al48. For this purpose, overnight-grown Candida cells were harvested and washed with PBS and adjusted to an OD600 of 0.5 in 10 mL of PBS. Five hundred microliters of cell suspension (2×106/mL) was taken in 24-wells plate and treated with MIC concentration of gel at 30 0C for 2

h. After incubation, 10 mM H2-DCFDA prepared in DMSO was added and plate was incubated at 50 0C. The untreated cells stained with H2-DCFDA without gel served as control. The fluorescence emitted by the cells was also visualized microscopically under fluorescence microscope (BD Bioscience). SC hydrogel without silver nanoparticle was used as the control for this study. Propidium iodide influx assay to detect membrane damage Candida cells in the log phase (2×106/mL PDB), resuspended in PBS, were treated with MIC concentration of the gel and incubated for 2 h at 30°C. Cells were then harvested by centrifugation and suspended in PBS. Subsequently, the cells were treated with 9 μm propidium iodide and incubated for 5 min at room temperature49. The cells were analyzed using fluorescence microscopy (BD Bioscience). SC hydrogel without silver nanoparticle was used as the control for this study. Characterization FT-IR spectroscopic measurements were made with a fully computerized Nicolet Impact 400D FT-IR spectrophotometer. IR spectra were recorded in the range of 4000 cm-1 to 400 cm-1. All spectra were corrected for the presence of moisture and carbon dioxide in the optical path. The experiments were performed for a scan of 45 times and with a resolution of 4 cm−1.

1

H NMR

spectra was obtained on a 500 MHz Bruker Advance DPX NMR spectrometer using TMS as


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internal standard. Chemical shifts were given in parts per million relative to TMS in CDCl3. Optical properties of the prepared hydrogels were studied by dispersing the sample in distilled water and recording the spectra using UV-Visible spectrophotometer (Shimadzu model 2100) in the range of 200-700 nm. For SEM measurements, samples were subjected for thin gold coating using a JEOL JFC-1200 fine coater and the probing side was inserted into JEOL JSM-5600 LV scanning electron microscope. TEM measurements were carried out using FEI (TEC-NAI G2 30 S-TWIN) with an accelerating voltage of 100 kV. For TEM measurements, the samples were casted on a carbon-coated copper grid and dried in vacuum at room temperature before observation. Rheological properties of the silver hydrogel hybrid composites were measured using Anton Paar Modulated Compact Rheometre-150 Physica (Germany). Parallel plate sensor with a diameter of 50 mm and a gap size of 1mm were used. The frequency sweep was performed from 0.01-100 rad s-1 to determine the storage modulus and loss modulus at a constant strain of 5% which is determined from LVR from the amplitude sweep measurement. The viscosity of the hydrogel with shear rate was measured by rotational mode with a shear rate of 0.1 to 100 s-1. Atomic force microscopy (AFM) images were recorded under ambient conditions using a Ntegra multimode Nanoscope IV operating in the tapping mode regime. Microfabricated silicon cantilever tips (MPP-11100-10) with a resonance frequency of 284-299 kHz and a spring constant of 20-80 N m1- were used. The scan rate varied from 0.5 to 1.5 Hz. X-ray diffraction studies were done using X-ray diffractometer (Philips X’pert Pro, Netherlands) with CuKα radiation (λ~0.154 nm) employing X’ celarator detector and monochromator at the diffraction beam side. Polarized light micrographs (PLM) were taken in an Olympus BX 51 microscope after drop casting the solution of the sample in a clean dry glass plate.

Supporting information UV-Visible absorption studies of SCS4 for 30 days, Swelling of SC and SCSs hydrogels at pH 7, Angular sweep measurement of SC and SCS4,

1

H NMR spectra of poly(1-4)-α-D-

glucanaldehyde AUTHOR INFORMATION *E-mail: [email protected].



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ACKNOWLEDGEMENTS We thank KSCSTE (GAP 134739) for the financial support. The authors are grateful to Dr. A. Ajayaghosh, Director, CSIR-NIIST, TVM, for his constant encouragement and support. We thank Mr. Kiran Mohan, Ms. Soumya, Mr. Prithviraj, Mr. Aswin and Mr. Vishnu for TEM, SEM, XRD and AFM. Nishanth Kumar S thank DST-SERB for providing financial support from young scientist programme. REFERENCES (1)

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Design of Macroscopically Ordered Liquid ... - ACS Publications

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