Pharmaceutical applications of natural polysaccharides

CHAPTER 2

Pharmaceutical applications of natural polysaccharides Zahra Shariatinia Department of Chemistry, Amirkabir University of Technology (Tehran Polytechnic), Tehran, Iran

Chapter Outline List 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

of abbreviations 15 Introduction 16 Application of natural polysaccharides Application of natural polysaccharides Application of natural polysaccharides Application of natural polysaccharides Application of natural polysaccharides Application of natural polysaccharides Application of natural polysaccharides Application of natural polysaccharides Application of natural polysaccharides Application of natural polysaccharides Application of natural polysaccharides additives/packaging materials 48 13. Conclusion 50 Acknowledgment 50 References 50

in in in in in in in in in in in

cell encapsulation 19 pharmaceutics/drug delivery 29 gene delivery 33 protein binding 36 wound healing 38 tissue engineering 41 bioimaging 42 preparation of contact lenses 45 preparation of implants 46 preparation of antibacterial textiles/papers preparation of antimicrobial food

List of abbreviations AgNPs ALG-PAAm AO/PI BADSCs BSA CMCel CMT CMV

Silver nanoparticles Alginate-polyacrylamide Acridine orange/propidium iodide Brown adipose-derived stem cells Bovine serum albumin carboxymethyl cellulose Carboxymethyl tamarind Cytomegalovirus

Natural Polysaccharides in Drug Delivery and Biomedical Applications. https://doi.org/10.1016/B978-0-12-817055-7.00002-9 Copyright © 2019 Elsevier Inc. All rights reserved.

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16 Chapter 2 CO CS CS CS-NS Cur CVB3 DOX E. coli GAGs GEM GK GT HA HASCs HIV HMSN HSV IOLs MNPs MPECHs MR n-HAp/CS-TSP NS NT2 PGA PVP QDs QPG S. aureus SCL SiRNA SO SPI TCL TML

Clove oil Chitosan St-AgNPs: Chitosan:starch-silver nanoparticles Chitosaneneem seed Curcumin Coxsackievirus B3 Doxorubicin hydrochloride Escherichia coli Glycosaminoglycans Gemcitabine hydrochloride Gum karaya Gum tragacanth Hyaluronic acid Human adiposeederived stem cells Human immunodeficiency virus Hollow mesoporous silica nanoparticles Herpes simplex virus Intraocular lens Magnetic nanoparticles Magnetic responsive polyelectrolyte complex hydrogels Mauran Nano-hydroxyapatite/chitosan-tamarind seed polysaccharide Nanostarch NTera2 Propylene glycol alginate Poly(vinyl pyrrolidone) Quantum dots Quaternized analogs of pectic galactan Staphylococcus aureus Soft contact lens Small interference RNA Sandalwood oil Soybean protein isolate Therapeutic contact lens Timolol maleate

1. Introduction Natural polymers are more preferred compared to the synthetic ones because they are easily accessible, prone to chemical modification, renewable, economic, nontoxic, stable, hydrophilic, biocompatible, and biodegradable compared to expensive synthetic polymers that have shown environmental and toxicity problems along with long-time synthetic methods [1e4]. Polysaccharides are the most plentiful natural biopolymers which are of growing attraction as effective materials in various biomedical fields, and this can be due to their inherent exceptional valuable features [5e9]. Polysaccharides possess several functional groups and display variable physicochemical properties and essential biological activities that make them suitable materials in numerous pharmaceutical areas such as drug

Pharmaceutical applications of natural polysaccharides 17 delivery and tissue engineering [10e13]. Thus, the number of natural carbohydrates used to deliver desired materials for particular pharmaceutical applications is increasing [14e16]. Encapsulation of living cells into polymeric materials is usually done with the aim of protecting the cells against destruction by the immune system. This method was first introduced in 1933 when Bisceglie et al. investigated the encapsulation influence on the survival of tumor cells in the abdominal cavities of pigs and found that long cell survival was attained through enveloping cells by immunoprotective membranes [17]. Hence, Bisceglie used amnion tissue membrane; however, he did not distinguish the ability of such method to treat diseases. In 1950, Algire and coworkers presented the idea of the “diffusion chamber” in order to graft therapeutic cells [18]. Also, they emphasized on the significance of using biocompatible polymers having sustained and favored properties which are a requirement for therapeutic purposes [18]. Since then, numerous people employed the encapsulation route to treat diverse kinds of diseases [19]. Numerous diseases can be overcome using this technology including anemia [20], hemophilia B [21], dwarfism [22], pituitary disorders [23], liver [24] and kidney failure [25], diabetes mellitus [26], and central nervous system insufficiency [27]. Today, encapsulation in polymers is one of the most frequently utilized cell immobilization technologies because of its simple and mild preparation procedures [28]. As an example, alginate is widely used in cell encapsulation [29]. Polysaccharides are also attractive candidates for gene delivery. Moreover, the modification of nonionic and hydrophilic polysaccharides improves the half-life of polyplexes in blood circulation through avoiding their unwanted interactions with serum proteins thus preventing their clearance with the reticuloendothelial cells [30,31]. Recently, the interactions that occur between biodegradable natural polysaccharides and therapeutic proteins have broadly been examined [32e36]. Such interactions are very important to assess the macroscopic characteristics of processed foods like texture, flow, and stability [37]. Polysaccharides can be processed as ingredients of functional foods and serve as edible films [38], interfacial stabilizers [39,40], microcapsules [41,42], electrostatic gels [43,44], and alternatives for meat or fat [45]. Furthermore, the interactions that occur between polysaccharides and proteins are essential to determine the physical and structural properties of formulated foods [46,47]. It is known that limited drugs are accessible in modern medicine to stimulate wound healing process. Besides, despite the considerable progress in this area, highly safe and effective wound healing treatments are not available. Consequently, advanced investigations have been done to find innovative natural medicines with potential activities to hasten and increase wound healing effect during the healing progression [48]. Currently, polysaccharide biopolymers have been found as promising agents in different forms [49].

18 Chapter 2 For instance, it was exhibited that plant polysaccharides accelerated healing, modulated the inflammatory stage [50e52], and stimulated proliferation of dermal keratinocytes and fibroblasts [53]. Natural biomaterials are commonly exploited as scaffolds in tissue engineering applications in various forms such as bioceramics, hydrogels, and composites (nanocomposites/ biocomposites) [54e56]. The increased attention to the natural polymers is mostly related to the growing concerns and awareness to the environmental problems of plastics subsequent to their being discarded in the environment post usage. Indeed, natural and biobased polymers can decrease plastic waste production and CO2 emission [57]. Moreover, natural biodegradable polymers such as proteins and polysaccharides are significantly similar to the extracellular matrix, chemically adaptable, biodegradable, biocompatible, and reveal required biological potential [58,59] for making biobased polymers. After the pioneer application of chitosan (CS) and its derivatives in the aqueous colloidal synthesis of CdS quantum dots (QDs) [60], QDs capped with carbohydrate polymers were increasingly used in chemical analysis and biomedical applications [61e65]. For instance, carbon quantum dots possessing tunable and very strong fluorescence properties were employed in optronics, sensors, and biomedicines [66]. The systemic administration of medicines in ocular diseases is usually unsuccessful, primarily due to the bloodeocular barrier. The ocular barriers are blooderetinal and bloodeaqueous barriers that mainly prevent drug absorption from the blood [67,68]. Recently, drug-laden contact lenses are widely used to treat ocular illnesses. Contact lenses are curved and thin plastic lenses that are worn on the cornea for protection of the eye and/or to correct vision [69]. Currently, contact lenses can serve as drug carriers to control anterior segment diseases. Contact lenses are able to enhance the drugs’ residence time period on the eye to >30 min, and this is longer compared to 2 min when eye drops are used, confirming they can enhance bioavailability of drug on the cornea [70]. Additionally, the exposure of drug in the systemic circulation along with its side effects will be decreased. The drug-laden contact lenses could be utilized for longer times by patients to decrease required administration rate. Titanium and its alloys are commonly applied as implants in the dental and orthodontic areas because they have shown exceptional mechanical properties, osteoconductivity, and biocompatibility [71,72]. Nevertheless, infection of implants is a serious clinical problem which has led to implant failure, long hospitalization, and also death [73,74]. The bioinert characteristic is the inherent shortcoming of titanium-based implants [75]. As a result, scientists tried to modify the titanium surface by coatings and/or nanostructures in order to increase osteogenesis inducing capability and inhibit bacterial growth [76e78]. For example, coating the titanium surface by antibacterial materials such as chitosan is of great interest [79].

Pharmaceutical applications of natural polysaccharides 19 Production of antibacterial textiles is an imperative part in the textile manufacturing. Although several chemicals and approaches are existing to produce antibacterial textiles, some of them are toxic to humans [80,81]. Thus, in order to overcome this drawback, natural polymers as ecofriendly and biocompatible materials are employed in fabrication of antibacterial textiles. It is well known that papers are used for diverse applications including in paper indicators; sensors; filters; printing/writing, packaging, and household products [82e84]. Paper has unique features such as low weight, low cost, low environmental impact, and suitable mechanical properties [85]. However, paper has poor barrier properties along with poor grease/oil resistance [86]. They are susceptible to react with fat molecules because they have lipophilic properties that lead to damaging the printed papers. Several polymers like chitosan were examined to alter the surface of papers with the purpose of enhancing their characteristics [87,88]. Because application of synthetic polymers results in environmental problems and their recycling is hard, ecofriendly preparation methods are broadly explored to achieve antimicrobial papers [89,90]. The frequent usage of plastic packaging materials in the food industry has brought about pollution problems. Accordingly, application of biodegradable packaging biopolymers has been proposed in order to decrease the environmental contamination [91]. An antimicrobial packaging can interact with products or headspace in order to decrease, delay, and/or inhibit the development of microorganisms which can exist on food surfaces [92]. This strategy can tackle the contamination of food, reduce danger of pathogen growing, and extend the food shelf life. Also, the increasing demand for using natural preservatives and additive-free food motivate the introduction of natural antimicrobial agents to the packaging materials that can gradually migrate to the food medium and eliminate the requirement for consuming extra amounts of preservatives that are directly introduced into the food products [93,94]. In this chapter, the most recent research results on pharmaceutical applications of natural polysaccharides in miscellaneous biomedical areas including cell encapsulation, drug/gene delivery, wound healing, protein binding, tissue engineering, bioimaging, preparation of contact lenses and implants, antibacterial textiles/papers, and antibacterial food additives/ packaging materials (Scheme 2.1) will be presented.

2. Application of natural polysaccharides in cell encapsulation Cell-based therapy includes implantation or delivery of living cells or their sustained growth in patients to treat certain diseases [95]. On contrary to small drug molecules and biologicals like engineered antibodies and proteins that are the principal therapeutic materials for various diseases, the cell-based therapy transports complex living compounds

20 Chapter 2

Cell encapsulation ation

Drug delivery

Gene delivery

Wound healing g

P Protein binding

Bioimaging

Natural Polysaccharides Tissue engineering ing Implants

Antibacterial textiles/papers p p

Contact lens Food packaging

Food additives F

Scheme 2.1 The different pharmaceutical applications of natural polysaccharides.

to modulate their function, sensing, and responding to the environment. One of the foremost advantages of employing cell encapsulation is its potential to use allogenic cells for transplantation as it can protect encapsulated cells from being destroyed by the immune system [96]. Also, it can decrease usage of antiimmunoresponse therapeutics and subsequent influences of traditional transplants through avoiding the body immunoresponse [97]. Cell encapsulation leads to the sustained drug release from encapsulated cells in comparison to the common drug administration that caused underdosing or overdosing effects [96]. Besides, cell encapsulation is able to resolve the challenge of drug localization in a place because encapsulated cells are transplanted to desired site(s) with superior efficacy and lesser whole dosage than traditional approaches [98]. Some therapeutic applications for encapsulated cells are treatments of diabetes, degenerative bone/joint disease, hepatic and central nervous system diseases. It is known that brown adipose derived stem cells (BADSCs) are favorite stem cells for the treatment of myocardial infarction because they can effectively and spontaneously differentiate to cardiomyocytes [99]. In this context, a neotype three-dimensional cell expansion method was developed for BADSCs. For this purpose, “clickable” zwitterionic starch hydrogels were prepared from starch derived from methacrylate modified sulfobetaine using dithiol-functionalized poly(ethylene glycol) cross-linker through the “thiol-ene” Michael addition reaction. Furthermore, CGRGDS peptide was immobilized onto the hydrogels by a comparable “clickable” method. The Young’s moduli were

Pharmaceutical applications of natural polysaccharides 21 dependent on the concentration of precursor solutions and changed from 22.28 to 74.81 kPa. Outstanding antifouling property was observed upon the incorporation of zwitterionic fragments. The BADSCs were evenly encapsulated into the hydrogels and they were cultured for 10 days. High cell proliferation was seen and its extent was increased by decreasing the mechanical strength and addition of the CGRGDS. Interestingly, the cell “stemness” was well preserved during the encapsulation/culture and the released cells from the hydrogels excellently kept the capability of efficient spontaneous cardiomyogenic differentiation. Thus, it was found that zwitterionic starch hydrogel was suitable for growth and “stemness” preservation of BADSCs. The cellular viability and morphology within the 10-day culture were followed through acridine orange-propidium iodide (AO-PI) staining. Also, Fig. 2.1 reveals that cells preserve spherical shape in this period, as they are surrounded by hydrogel and cannot be stretched by growing over the TCP’s surface. Nevertheless, size of cells noticeably increases with time illustrating the cells are not restrained in hydrogel and they remold their neighboring environment by growing. Moreover, dramatic continuous cell proliferations happened so that little dead cells are observed in all hydrogels. Thus, hydrogels are very biocompatible and advantageous for the growth of cells. Fig. 2.2 exhibits that the BADSCs constantly express GATA-4 during the entire culture time that is increased in all hydrogels which confirms the cells expanded in the hydrogels outstandingly conserve the cardiac-differentiate potentiality. Moreover, introducing CGRGDS leads to GATA-4 expression by slightly more amount of cells when compared with that without CGRGDS. This designates that the CGRGDS causes cellular function preservation for adherent stem cells [99]. The cell encapsulation in alginate was carried out to study the differentiation process of embryonic cancer stem NTera2 (NT2) cells to dopamine generating cells [100]. Encapsulation of cells into polymer beads led to their isolation by immune system and made them suitable for transplantation and achieving an auspicious tool to deliver bioactive agents to the brain. The alginate polysaccharide was employed in this process because it is one of the most commonly used materials, which is well adopted by several tissues such as the brain. Also, two diverse initial cell concentrations (0.5
106 and 1.0
106/mL) were examined to recognize which one would more homogeneously distribute in the alginate beads and reveal superior cell viability at various culture stages. Two diverse CaCl2 concentrations of the gelling bath (1.0 and 0.1 M) were used to acquire beads with altered permeability including LP (low permeability) and HP (high permeability) beads for CaCl2 concentrations of 1.0 and 0.1 M, respectively. Then, the encapsulated cells were preserved inside a humid incubator with 95% humidity and 5% CO2 for 30 days and the cell solution was renewed every 2e3 days. Pure alginate beads were obtained as reference by the same method. The designations of the various samples are given in Table 2.1.

22 Chapter 2 Figure 2.1 AO/PI staining fluorescence images of BADSCs cultured in different S/P hydrogels with/without CGRGDS and ST/P-7.5 hydrogel for 1, 4, 7, and 10 days [99].

Pharmaceutical applications of natural polysaccharides 23

Figure 2.2 GATA-4 immunofluorescence staining of BADSCs cultured in different S/P hydrogels with/without CGRGDS and ST/P-7.5 hydrogel for 1, 4, 7, and 10 days [99].

24 Chapter 2 Table 2.1: Designation of the systems based on NT2 cells encapsulated within alginate beads in different conditions [100]. Sample 0.5NT2-HP 0.5NT2-LP 1.0NT2-HP 1.0NT2-LP

[NT2 cell] (106/mL) 0.5 0.5 1.0 1.0

[CaCl2] gelling bath (M) 0.1 1.0 0.1 1.0

It was found that higher number of cells stimulated clusters formation and caused better interactions of encapsulated cells and subsequently promoted mitotic effect. The live/dead cells distribution within the alginate polymeric beads was confirmed by the fluorescein diacetate/propidium iodide staining using fluorescence microscopy imaging which proved the existence of living neuronal positive cells. The activities of the encapsulated NT2 cells were established through dopamine formation. Hence, the NT2-alginate system was a valuable active platform that could produce/release dopamine and could be used for the treatment of Parkinson disease. One day after culture, all cells were evenly well encapsulated into the alginate beads (Fig. 2.3A). Greater mitotic effect was observed 8 days after culture (Fig. 2.3B) in capsules prepared using the maximum cell concentration (1.0
106/mL). Large amount of cells supported cluster creation and caused more efficient interactions among encapsulated cells and promoted mitotic effect. Cells in the LP capsules showed incomplete localized growth possibly because of uneven distribution of cells when the encapsulation is initiated. The fluorescence images of 1.0NT2-HP subsequent to diverse culture durations (7, 9, 11, 16, and 21 days, Fig. 2.4) reveal that the encapsulated cells are increased due to their strong mitotic activities. Nevertheless, a progressive rise in the mortality of cells was seen (nuclei were stained in red color) mainly apparent for the separated cells that could not be organized as clusters. In all cases, cell viability was preeminent relative to the moderate or little cell death [100]. The ever-growing clinical usage of cell-based therapy leads to development of systems in order to store and distribute cell therapy materials that are suitable for the clinical applications [101]. Recently, encapsulation to alginate was performed to increase the maintenance of human adiposeederived stem cells (hASCs) in the course of hypothermic storing and to find a large-scale production process. A dropwise technique was adopted to create scalable alginate beads using the calcium cross-linker which produced 3500 jelly beads in 1 minute. Also, the influence of alginate amount was assessed on viscosity properties of nonjelly sodium alginates, mechanical characteristics, and structures of Ca cross-linked alginate beads using diverse alginate and calcium quantities. The mechanical strength mainly depended on alginate quantity and 1.2% alginate cross-linked using 100 mM CaCl2 revealed a tensile stress of 35 kPa. Using the optimum parameters for

Pharmaceutical applications of natural polysaccharides 25

Figure 2.3 Optical microscopy images of encapsulated cells for different samples: (A) 0.5NT2-HP, 1.0NT2-HP, and 1.0NT2-LP, after 1 day of culture (bar ¼ 500 mm (left) and 200 mm (right)); (B) 0.5NT2-HP, 1.0NT2-HP, and 1.0NT2-LP, after 8 days of culture (bar ¼ 500 mm (left), 200 mm (middle), and 100 mm (right)) [100].

26 Chapter 2

Figure 2.4 Fluorescence microscopy images of cells encapsulated within 1.0NT2-HP beads, at different days of culture (bar ¼ 200 mm). Living cells are stained green with fluorescein diacetate, whereas dead cells are stained red using propidium iodide (for interpretation of the references to color in this figure legend, the reader is referred to the web version of this article) [100].

Pharmaceutical applications of natural polysaccharides 27 the beads, the human stem cells were immobilized and the encapsulated hASCs did not exhibit defeat in cell viability, and they also showed a homogeneous distribution after large-scale manufacture. After storing, the released cells were attached and revealed a normal shape once they were returned into the culture medium. Consequently, a scalable process was developed for encapsulation/storage of stem cells that was appropriate for the cell treatment supply chain. To explore the spatial cells distribution after encapsulation and their viability, some imaging techniques were carried out 48 h following the bead preparation. Beads displayed a spherical morphology along with a small tail (Fig. 2.5A) and such appearance was

Figure 2.5 The spatial distribution of cells through alginate beads. About 48 h after encapsulation, hASCs in beads were examined by phase microscopy (A), infrared imaging (B), and confocal microscopy (CeE). (CeE) hASCs were stained with calcein-AM (live indicator; green) and ethidium homodimer-1 (dead indicator: red) and maximal projection (C), Orthogonal projection (D) and 3D projection (E) images were captured. Arrowheads indicate the position of dead cells (for interpretation of the references to color in this figure legend, the reader is referred to the web version of this article) [101].

28 Chapter 2 preserved subsequent to the creation of most beads (Fig. 2.5B). Also, uniform cell distribution was confirmed through infrared images of encapsulated cells which evidently represented bead shapes with a greater intensity at overlap points (see Fig. 2.5B). Confocal images of live-dead stained encapsulated cells indicated great viabilities at earlier times so that cells exhibited appropriate distributions from projected Z-stacks (Fig. 2.5C) but little dead cells were observed (specified with arrow-heads). It was obvious in orthogonal XZ and YZ planes (Fig. 2.5D) with all dead and live cells dispersed in bead depth (the dead cells are shown by arrow-heads). Such cellular dispersion was revealed by volume analysis (see Fig. 2.5E). The uniform dispersal of deceased cells without viability loss approves that cell death was not stimulated by possible external stresses which cells may experience in the course of processing. The hASCs binding was examined using normal tissue culture for 24 h after hypothermic storing for 1 or 7 days. The hASCs cultured for 24 h one day after encapsulation revealed a regular spindle-shaped fibroblast resembling morphology which demonstrated suitable attachment capability (see Fig. 2.6). There were not any noticeable differences between nonencapsulated (control) and encapsulated hASCs stored in identical conditions. However, subsequent to 7-days storage, the alginate encapsulation preserved attachment (Fig. 2.6) but encapsulated cells demonstrated a practical attachment although with

Figure 2.6 Morphology and attachment of encapsulated ASCs following storage. hASCs were stored for either 1 or 7 days before plating at 5000 cells/cm2and returning to normal tissue culture conditions. Scale bars ¼ 200 mm [101].

Pharmaceutical applications of natural polysaccharides 29 slightly lower ability compared to 1 day; the control samples revealed significantly decreased attached cells along with more rounded dead cells [101].

3. Application of natural polysaccharides in pharmaceutics/drug delivery Natural polysaccharides (NP) are broadly used in drug delivery applications. Mauran (MR) is a polysaccharide macromolecule that is extracted from Halomonas maura halophilic bacterium. The antioxidant properties of MR-chitosan (CS) nanoparticles were examined to determine their usefulness for biomedical applications, and it was found that they could effectively defend against oxidative stress [102]. The ability of extremophilic mauran-based nanoparticles was investigated for in vitro and ex vivo scavenging of reactive oxygen species. The 5-fluorouracil incorporated MR-CS nanoparticles were assessed for cancer inhibition and their healing efficacy was compared using glioma and breast adenocarcinoma cells confirming they could specifically affect the cancer cells. The fluorescent labeled nanoparticles were utilized to evaluate the cell internalization by the nanocarriers by means of flow cytometry and confocal microscopic imaging indicating effective cellular uptake and absorption by such nanoparticles which were suitable for bioimaging as well as recognizing the cell binding by such nanoparticles. Moreover, the internalized fluorescent nanocarriers revealed a minor effect on the normal cells’ integrity. The cellular recognition and absorption of the MR-CS NPs were evaluated by fluorescent images of FITC-tagged L929 cells treated by MR-CS NPs. The FITC tagged CS was employed to create MR-CS NPs through polyelectrolyte complexation. The dye tagged NPs were introduced into the cells and their images were obtained by means of confocal microscope using a 488 nm laser. The fluorescent images of MR-CS-FITC NPs absorbed by the L929, GI-1, and MCF7 cells are indicated in Fig. 2.7A, E, and I show bright field imaginings and Fig. 2.7B, F, and J exhibit the DAPI stained nuclei. Fig. 2.7C, G, and K reveal green fluorescing MR-CS NPs and Fig. 2.7D, H, and L depict merged images of L929, GI-1, and MCF7 cells, respectively. The absorption tests in 24 h proved that MR-CS NPs had desirable cell acceptance without oxidative destruction and cell membrane interruption by the MR existence in L929 cells. Consequently, fluorescent images of normal cells supported the results achieved by antioxidant tests confirming MR was a useful biomaterial to promote the antioxidant mechanism up to the 500 g/mL concentration. Likewise, confocal microscopy images of cancer cells supported the flow cytometry data. Fig. 2.7 approved that the number of fluorescent NPs that was absorbed via GI-1 and MCF7 cells were rather lower compared to that of L929 cells after incubation for 24 h. Comparable results were observed for the SR-MR-CS NPs. Fig. 2.8 shows fluorescent images for the SR-MR/CS NPs absorbed by the L929, GI-1, and MCF7 cells. Fig. 2.8A, E, and I reveal bright field images, Fig. 2.8B, F, and J demonstrate the DAPI stained nuclei. Fig. 2.8C, G, and K illustrate the green fluorescing MR-CS NPs

30 Chapter 2

Figure 2.7 FITC tagged MR/CS nanoparticle absorption studies using confocal microscopy; (A), (E), and (I) bright field images of L929, MCF7, & G1 cells; (B), (F), and (J) DAPI stained nucleus; (C), (G), and (K) FITC tagged MR/CS nanoparticles accumulated in cells; (D), (H), and (L) merged images [102].

and Fig. 2.8D, H, and L depict the merged images of L929, GI-1, and MCF7 cells, respectively. The confocal and flow cytometry tests proved that the MR-CS was an effective carrier for drug molecules to release them at the target sites. The biocompatibility and antioxidant characteristics of such particles enable their applications as drug carriers for cancer therapy, and MR-CS NPs could be utilized as an effective targeting material [102]. The gum karaya (GK) was employed to synthesize gold nanoparticles (GNP) which were applied to deliver anticancer drug [103]. The GK nanoparticles displayed extraordinary biocompatibility throughout cell survival test using both normal Chinese hamster ovary

Pharmaceutical applications of natural polysaccharides 31

Figure 2.8 Sypro-ruby tagged MR/CS nanoparticle absorption studies using confocal microscopy; (A), (E), and (I), bright field images of L929, MCF7, & G1 cells; (B), (F), and (J), DAPI stained nucleus; (C), (G), and (K), Sypro-ruby tagged MR/CS nanoparticles accumulated in cells; (D), (H), and (L) merged images [102].

cells plus A549 human nonsmall cell lung cancer cells as well as in the course of hemolytic toxicity assessments. The anticancer drug gemcitabine hydrochloride (GEM) was incorporated into the nanoparticles indicating a drug loading efficiency of 19.2%. The GEM loaded nanoparticles presented superior inhibition against cancer cells growth in clonogenic and antiproliferation tests relative to the native GEM. This result was associated to greater generation of reactive oxygen species using the GEM containing nanoparticles in A549 cells compared to the native GEM confirming the GK had a noteworthy capacity to synthesize biocompatible gold nanoparticles, and it could be applied as an anticancer drug delivery vehicle. The influence of formulations on the A549

32 Chapter 2

Figure 2.9 Effect on the morphology of A549 human lung cancer cells incubated with gemcitabine hydrochloride (GEM) and GEM loaded gum karaya stabilized gold nanoparticles (GEM-GNP). Untreated cells are shown as control [103].

Figure 2.10 Clonogenic assay: GEM loaded gum karaya stabilized gold nanoparticles (GEM-GNP) showed greater inhibition of colony formation activity of A549 human lung cancer cells as compared to native GEM and untreated cells (control) [103].

cells morphologies are displayed in Fig. 2.9. Furthermore, colony formation inhibition test was accomplished to confirm the extended antiproliferation influence of GEM-GNP. Less colonies were detected for the cells treated by GEM-GNP sample compared to control and GEM-treated cells (Fig. 2.10). In another study, Cassia obtusifolia seed mucilage was extracted and used in drug delivery [104]. The seed mucilage was assessed for the occurrence of polysaccharide and a mucilage-based biodegradable film was achieved using seeds of C. obtusifolia. The in vitro oral acute toxicity assay and degradation in simulated body fluids revealed a great LD50 > 2 g/(kg of body weight) which established the benign nature of this excipient. The diclofenac incorporated film illustrated a continuous drug release that was attributed to the swelling of film and dug diffusion. Thus, it was validated that the C. obtusifolia seed mucilage could be used as a film-forming excipient showing improved features for drug delivery purposes. Phytochemical tests for the identification of seed mucilage proved the occurrence of carbohydrate and mucilage (Table 2.2). Nonetheless, the tests did not show existence of starches, alkaloids, glycosides, tannins, and steroids. The carbohydrate

Pharmaceutical applications of natural polysaccharides 33 Table 2.2: Phytochemical identification tests of isolated mucilage from Cassia obtusifolia seed [104]. Identification tests Test for carbohydrates Test for starches Test for proteins and amino acids Test for mucilage Test for glycosides Test for alkaloids Test for steroids and sterols Test for tannins

Name of tests

Observation

Molisch test Iodine test Ninhydrin test

þ  

Ruthenium red test Legal, Keller-Killiani, Borntrager tests Mayer, Dragendroff tests Libermann-Burchard test Ferric chloride, lead acetate tests

þ    

þ, Present; , Absent.

Table 2.3: Physical properties of Cassia obtusifolia seed mucilageebased film [104]. Physical property

Results

Film thickness (mm) Weight measurement (mg) Moisture content (%) pH Folding endurance Mechanical strength (kg/mm2) Mucoadhesive force (dynes/cm2)

0.14  0.01 0.92  0.02 0.92  0.02 6.96  0.05 >500 0.2 2400.60  89.45

chemical analysis exhibited that polysaccharides were present in the sample. Table 2.3 represents the physical properties of C. obtusifolia seed mucilage film.

4. Application of natural polysaccharides in gene delivery Genes are inherited units and their defects or malfunctioning results in several conditions and diseases including cystic fibrosis and cancer. Most of the traditional treatments involve drug-based methods focusing on symptoms/signs instead of the fundamental root reason of the disease which is the faulty genes. Therefore, in order to effectively treat genetic disorders, defective genes must be corrected or augmented at molecular level through the gene therapy approach [105,106]. Although clinical trials were started in early 1990s, this method is yet at the infancy step as there is trouble in development of a perfect vector to protect/deliver nucleic acids to the desired target sites without any negative influence [107e109]. Sugar-containing cationic polymers show high potential to deliver genes such as therapeutic RNA interference (RNAi).

34 Chapter 2 Table 2.4: Particle size and zeta potential of siRNA-loaded 6AC-100 nanoparticlesa [110]. Weight ratio (siRNA/6AC-100) 1:10 1:5

Charge ratio (¡/þ) 1:14 1:7

Particle size (nm) 92.9  3.3 182.7  5.1

Polydispersity index ([(m2)/G2]) 0.267 0.099

Zeta potential (mV) 21.7  0.1 17.5  1.1

a Particle preparation conditions: 6AC-100 concentration: 1.0 mg/mL (PBS pH7.4), siRNA concentration: for weight ratio 1:10, 0.1 mg/mL (7.0 mM) and for weight ratio 1:5,0.2 mg/mL (14 mM), T: 25  2 C. Data shown are the mean  standard deviation (n ¼ 3).

The RNAi can downregulate the gene expression as posttranscriptionally and this is an important therapeutic process which can highly decrease the amount of disease causing proteins which are not treated using common small drug molecules. Nonetheless, clinical usage of small interference RNA (siRNA) needs designing effective siRNA sequences and developing benign and proficient delivery formulations. In order to achieve a siRNA delivery system which is also biocompatible, natural polysaccharide curdlan was modified chemically by a regioselective method so that amine groups were introduced on the glucose groups [110]. The resultant 6-amino-curdlan was watersoluble that produced NPs after its complexation with siRNAs. The zeta potentials and particle sizes of siRNA-loaded 6AC-100 nanoparticles are provided in Table 2.4. The curdlan-based NPs powerfully delivered siRNAs to mouse primary cells and human cancer cells and decreased 70%e90% of the target mRNA amount. Furthermore, 6-amino-curdlan nanoparticle could deliver the siRNA targeting eGFP to the mouse embryonic stem cell that soundly expressed eGFP and substantially reduced the GFP protein concentration. Hence, the curdlan-based nanoparticle was proposed as an auspicious vehicle to deliver short RNAs in order to diminish endogenous mRNAs. It was investigated to find if the 6AC polymer could deliver siRNA to cells using the curdlan-based nanoparticles. For this purpose, A549 cells were treated with Cy3-labeled RNA (Cy3-siRNA) that was complexed to 6AC-100 NPs; then the siRNA cell internalization was tracked using fluorescence microscope. For most cells treated by 6AC-100 NPs, after 4 h transfection, strong fluorescence signals were seen signifying 6AC100 could effectively deliver siRNA into cytoplasm (see Fig. 2.11). In order to evaluate the transfection efficacy of 6AC-100 on stem cells, a complex of siRNA and 6AC-100 was used to target eGFP into mouse embryonic stem cells (mES cells) which continuously expressed eGFP. Fig. 2.12 exhibits that the GFP signal was greatly decreased 24 h after transfection using 25 nM siRNA complexed with 6AC-100 confirming 6AC-100 powerfully delivered siRNA into stem cells and reduced gene expression at the protein level [110]. Glycosaminoglycans (GAGs) are known as naturally occurring polymers that are generally employed in gene delivery to enhance stability and reduce toxicity and nonspecific interactions, thus improving transfection efficacy. The sorbitan esterebased lipid NPs functionalized by hyaluronic acid and GAGs chondroitin sulfate were used as gene

Pharmaceutical applications of natural polysaccharides 35

Figure 2.11 Internalization of siRNA in cancer cells by 6AC-100. A549 cells were transfected with Cy3-labeled siRNA complexed with 6AC-100 nanoparticles. Localization of dye-labeled siRNA after 4 h was monitored by fluorescence microscopy [110].

Figure 2.12 In vitro silencing of endogenous mRNA by 6AC-100 nanoparticles in mouse embryonic stem cells. Fluorescence microscope image of mES cells stably expressing eGFP treated with nontargeting siRNA complexed with 6AC-100 nanoparticles, or siRNA targeting GFP complexed with 6AC-100 nanoparticles. mRNA levels are expressed as percent of control [110].

delivery vehicles [111]. Such nanoplatforms incorporated with plasmid DNA were evaluated for the physicochemical properties and stability, protection capacity, and proficient transfection of cells using the improved green fluorescence of plasmid protein in vitro along with the in vivo and in vitro biocompatibility. It was established that compounds with extraordinary biological significance and targeting capacity (like hyaluronic acid and chondroitin sulfate) could fruitfully be introduced in the sorbitan esterebased nanoparticles to stabilize both nanosystems and protect the plasmid DNA. It was found that adding the hyaluronic acid and chondroitin sulfate caused long-standing stability of the nanoplatforms in both lyophilized and liquid states confirming they could be used in industry. Such functionalized nanosystems could transfect A549 cells without

36 Chapter 2 affecting the cells’ viability and revealed innocent safety profiles in vivo approving the ability of these nanoparticles as gene delivery platforms.

5. Application of natural polysaccharides in protein binding Proteins are natural polyelectrolytes having exceptional functional characteristics like the capacity to produce emulsions and gels. Thus, they can be used in combination with natural polysaccharides for biomedical applications like the encapsulation of bioactive materials [112]. Also, it is well known that functionality and stability of protein can be enhanced under unfavorable conditions through complex creation between protein and polysaccharide. The nature of interactions among polysaccharides and proteins can be electrostatic and/or covalent [113]. In a recent work, polysaccharide-protein-surfactant complexes were achieved through coprecipitation method using propylene glycol alginate (PGA), zein, as well as lecithin or rhamnolipid. Such ternary complexes were used as delivery platforms to increase the curcumin (Cur) bioavailability and stability [114]. The curcumin-containing zein-PGA, zein, zein-PGA-lecithin, and zein-PGA-rhamnolipid complexes were denoted as Z-P-Cur, Z-Cur, Z-P-L-Cur, and Z-P-R-Cur, respectively. Encapsulation efficiency of the curcumin was increased due to the existence of surfactants and polysaccharides in the complexes compared to the pure zein NPs as Z-P-Cur (67%), Z-Cur (21%), Z-P-R-Cur (92%), as well as Z-P-L-Cur (94%). Incorporating the surfactants to the complexes considerably enhanced the bioaccessibility and photostability of curcumin. Hence, the developed ternary complexes were promising means for encapsulation, protection, and delivery of hydrophobic nutraceuticals for usage in pharmaceuticals, supplements, and foods. The carrageenan and xanthan gum, respectively, indicated high and medium negatively charged polysaccharides. Diverse proportions of these biopolymers were heated along with soybean protein isolate (SPI) [115]. After mixing by simulated stomach juice, both carrageenan-SPI and xanthan-SPI immediately underwent self-assembled gelation using the biopolymer ratios greater than 0.01. At upper biopolymer ratios, a stronger gel was formed. Also, highly negatively charged carrageenan produced a stronger gel compared to that combined with xanthan gum. It was found that the SPI digestibility was postponed after mixing with the polysaccharides, and it is improved by increasing the biopolymer ratio. Fig. 2.13 exhibits the SEM images of xanthan-SPI (A and B) as well as carrageenanSPI (C and D) (polysaccharides/SPI ratio ¼ 0.1) which immediately were mixed with SGF (A and C) after 1 h digestion (B and D). Polysaccharides having higher negative charge could more strongly delay the SPI digestion. Besides, the microstructures of both the carrageenan-SPI and xanthan-SPI gels were observed by scanning electron microscopy before and after simulated stomach digestion which confirmed the gels delayed the SPI digestion [115].

Pharmaceutical applications of natural polysaccharides 37

Figure 2.13 SEM images of xanthan-SPI (A and B) and carrageenan-SPI (C and D) (polysaccharides: SPI ratio of 0.1) immediately mixed with SGF (A and C) and after 1 h digestion (B and D) [115].

In another research, CS and bovine serum albumin (BSA) were employed to fabricate BSA-CS nanogels through a green facile self-assembly procedure [116]. The nanogels were utilized to encapsulate doxorubicin hydrochloride (DOX) with 46.3% entrapment efficiency in order to understand both less cytotoxicity and gradual release of DOX. Also, the pure DOX and DOX-containing BSA-CS (DOX-BSA-CS) revealed IC50 amounts equal to 0.05 and 0.22 mg/mL, respectively, against growth of SGC7901 cells. The DOX cytotoxicity significantly declined in 24 h after its encapsulation into the nanogels demonstrating the loaded drug was slowly released during 24 h indicating BSA-CS was an appropriate sustained release platform. The cell uptake tests pointed out that the DOXBSA-CS was more rapidly diffused to the cancer cells compared to the pure drug. Also, it was evidenced that the DOX-BSA-CS induced apoptosis of gastric cancer cells 7901 was superior to the plain drug confirming it was favorable for the gastric cancer treatment.

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6. Application of natural polysaccharides in wound healing Cutaneous wound healing is one of the most important processes in numerous pathologies, which is required in postsurgery scars and burns. It is well recognized that the wound healing occurs as a dynamic interactive phenomenon that can substitute missing and devitalized tissue layers and cells through interacting processes classified as hemostasis, inflammation, proliferation, and matrix remodeling [117]. It is essential that an injured/ wound site immediately be covered with a dressing that is able to prevent microbial invasion, preserve wet medium for efficient skin regeneration, let gaseous passage, and adsorb exudates [118]. Nevertheless, commercially existing wound dressings only meet some of these standards. Consequently, the development of novel nanodressings using nanocomposites is of growing importance [119]. Recently, polymeric nanocomposites have been developed as wound dressing materials. Among such materials, polysaccharide nanocomposites are proficient candidates because of their exceptional tissue mimicking and biocompatibility characteristics [120]. They can mimic full-thickness skin wounds because these kinds of dressing have morphologies reminiscent of the native skin and appropriate features for a good wound healing process [120]. It is known that starch is a plentiful, relatively low-cost, and ecological material which can be prepared as nanoparticles and fillers to fabricate bionanocomposite wound dressing materials. In a recent effort, symmetric and asymmetric porous films were fabricated using chitosan (CS) and poly(vinyl pyrrolidone) (PVP) along with nanostarch (NS) as filler through salt leaching process for wound dressing usage [121]. Symmetric CS-PVPnanostarch (CSPNS) films containing 3 and 1%wt of nanostarch were achieved without their coating by stearic acid but the CS-PVP-nanostarch-stearic acid (CSPNSeS) asymmetric film was obtained through coating with stearic acid. It was found that the stearic acid covered surface had microporous, hydrophobic, bacterial antiadhesion characteristics whereas the hydrophilic stearic acid uncoated surface displayed higher bactericidal and noncytotoxic properties with a very porous structure. All of the asymmetric and symmetric films demonstrated nearly identical barrier; mechanical, hemolytic, and swelling features indicating the stearic acid did not influence the hemolytic and physical properties while the nanostarch amount significantly affected these features. The CSPNS1%-S film showed outstanding Staphylococcus aureus antiadhesion capacity. Moreover, excision wound healing in vivo verified that the CSPNS1%-S film illustrated improved healing process in addition to enhanced collagen development and reepithelialization. In vivo wound healing experiment was accomplished using adult albino rats; images indicating wound healing are displayed in Fig. 2.14. In the day wound was created, wound areas were identical in all groups. Granulation tissues were detected on day 7 in wounds covered by CPNS1%-S so that the healed rates were 32% and 20% for the CP-S dressing

Pharmaceutical applications of natural polysaccharides 39

Figure 2.14 In vivo wound healing analysis of control, CP-S, and CPNS1%-S dressing material [121].

and control, respectively. Wound areas were considerably decreased on day 14 in group dressed with CPNS1%-S and wound bleeding was stopped so that healing rates were improved to 81%, 75%, and 60% for the CPNS1%-S, CP-S, and control dressing, respectively. All wounds were entirely cured but the healing rate was very greater for the CPNS1%-S compared to those of the CP-S dressing and control. It is noteworthy that high rate of wound closure using CPNS1%-S was attributable to synergistic influence of CS, PVP, stearic acid, and nanostarch due to their mechanical, barrier, antibacterial, anticell adhesion, cytotoxicity, and hemolytic properties. The healing efficacy of the wound dressing was studied by histological analysis. Fig. 2.15 illustrates the histological analysis results for the CPNS1%-S dressing, CP-S, and control. Inflammatory cells seen on day 7 were greater in CP-S and control but fresh blood vessels were observed and inflammatory cells were highly reduced for the CPNS1%-S dressed sample. Collagen fiber was created on day 14 in CPNS1%-S that supported generation of additional granulation tissues. Hair follicle cells were seen on day 21 using the CPNS1%-S

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Figure 2.15 Histological Images of control, CP-S, and CPNS1%-S dressing material [121].

and CP-S materials. Such findings confirmed the powerful influence of CPNS1%-S as an asymmetric wound dressing to healing wounds [121]. In another study, the antioxidant, hemolytic activity and in vivo wound healing effect of FWEP polysaccharide which was extracted from fenugreek (Trigonella foenum-graecum) seeds were assessed [122]. The in vivo and in vitro antioxidant activities were estimated by different tests, and it was revealed that the FWEP had strong antioxidant potency whereas hemolytic activity was not witnessed to bovine erythrocytes. Also, the FWEP hydrogel was applied on a wound position in a rat model which substantially improved wound healing, as well as expedited wound closure, 14 days after the wound generation. The histological analysis validated formation of entirely reepithelialized wound and whole epidermal renewal. Overall, it was established that FWEP exhibited high wound healing capacity that was probably related to its antioxidant activity. Several dextran-based bionanocomposite films incorporated with sandalwood oil (SO) and clove oil (CO) were prepared which could prevent infection as a result of their intrinsic antibacterial potency and modify the wound healing process to accelerate scarfree healing [123]. The dextran-nanosoy-glycerol-chitosan (DNG-CS) nanocomposites were fabricated followed by adding SO and CO to acquire herbal DNG-CS-CO and DNG-CS-SO nanodressing materials which demonstrated >98% bactericidal effects against both Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) microorganisms only

Pharmaceutical applications of natural polysaccharides 41 using very low loading amounts of 10% and 5% for SO and CO, respectively. Such encapsulation approach led to controlled EO diffusion during 72 h measured for drug effectiveness by means of bacterial reduction counting and serial disk diffusion assays. The bacterial adherence test established the proficiency of these dressings to stop microbial invasion. The in vivo wound healing test by means of DNG-CS-CO dressings on male Swiss albino mice (BALB/c strain) revealed full healing in 14 days along with extraordinary efficiency in scar inhibition. Also, the histological analysis proved that CO and SO treatments brought about ordered collagen deposition together with fibroblast migration.

7. Application of natural polysaccharides in tissue engineering The structural and biological functions of polysaccharides have made them appropriate compounds to be exploited in tissue engineering. Such biomaterials exhibit appropriate biochemical and mechanical properties for tissue engineering applications [124]. The polysaccharides reveal numerous features as eligible biomaterials for tissue engineering including biodegradability, biocompatibility, and the cell delivery capacity [125]. An ideal biomaterial not only has a suitable chemical structure but also it shows favorable macroscopic structural properties, that is, the biomaterial scaffold has a porous structure to allow mass transportation (diffusion as well as permeability) [126]. Further, the biomaterial should reproduce the mechanical, elastic, and organizational characteristics of native tissues, and this is especially imperative for vital and very specific tissues like the cardiac tissues [127,128]. The best biomaterial should have a structure to stimulate cell attachment/growth once enabling its organization and perhaps differentiation to a very well-ordered biomimetic structure [129]. Also, it should be prone to resist high and permanent mechanical stresses. Another foremost role is related to the integration with host tissue and final substitution by the extracellular matrix of the host [130]. The biomaterials should have biological activities to accelerate the tissue repair. For instance, cell recruitment, angiogenesis, and cardiomyocyte protection properties are beneficial for the treatment of heart diseases [131]. Also, the tissue engineering products must be effective and economical considering their functionality and production simplicity [132]. Polysaccharides are auspicious materials meeting most of these criteria that can be used as eligible biomaterials for tissue engineering. Nanocomposites of nanohydroxyapatite, chitosan, and tamarind seed polysaccharide (n-HAp/CS-TSP) were fabricated in weight ratios equal to 70:10:10, 70:15:15, and 70:20:20, respectively [133]. The n-HAp/CS-TSP (70:10:20) exhibited the most rough and porous surface, improved thermal stability in addition to maximum compressive modulus, and strength. Also, the n-HAp/CS-TSP (70:10:20) showed greater swelling, satisfactory degradation, as well as enhanced biomineralization within simulated body fluid in

42 Chapter 2 comparison to the nanocomposites n-HAp/CS and n-HAp/CS-TSP (70:15:15 and 70:20:10). The n-HAp/CS-TSP (70:15:15) indicated greater nontoxic activity to MG-63 cells plus superior hemocompatibility. Thus, the n-HAp/CS-TSP nanocomposites were considered as more suitable biomaterials for bone tissue engineering relative to the n-HAp/ CS nanobiocomposite. Recently, a series of hydrogels were synthesized through incorporating the synthetic hydroxyethyl methacryate monomer along with a semisynthetic polymer backbone (carboxymethyl tamarind, CMT) in diverse molar ratios [134]. Such materials were denoted as CMT:HEMA hydrogels. The biocompatibility tests using NIH-3T3, HaCaT, in addition to mouse dermal fibroblast cells established that these materials are biocompatible and they were not cytotoxic. The mitochondrial functionality assays proved that they were safe and noncytotoxic. The hemolytic assessment using red blood cells plus acute skin irritation experiment on SD rats verified that they were appropriate for the ex vivo applications. Finally, it was suggested that these hydrogels were favorable for in vivo commercial and clinical applications to treat skin disorders. Skin edema and erythema were not detected during the experiment (Fig. 2.16A). Microscopic assessment of various skin sections which were stained using eosin and hematoxylin did not display morphological variations in dermal or epidermal skin layers in tested animals relative to the control (Fig. 2.16B). Weight gain and feed consumption were similar in both control and test groups. Based on gross, systemic notes and skin microscopic analysis, it was found that the CMT:HEMA was well accepted by all animals upon topical skin usage [134]. Xanthan gum and CS were blended using Fe3O4 magnetic nanoparticles (MNPs) to create magnetic responsive polyelectrolyte complex hydrogels (MPECHs) through in situ ionic complexation by means of D-(þ)-glucuronic acid d-lactone to acidifying the medium [135]. It was shown that adding Fe3O4 MNPs to the PECH highly enhanced mechanical characteristics as well as storage modulus. The in vitro cell culture using NIH3T3 fibroblast cells on MPECHs displayed increased growth and adhesion of cells under an external magnetic field compared to the native PECH confirming the MPECH can be utilized as a magnetic stimulated material in tissue regeneration.

8. Application of natural polysaccharides in bioimaging Nowadays, nanocomposites of natural polysaccharides and quantum dots (QDs) are widely used in bioimaging applications [136]. Recently, sodium carboxymethyl cellulose (CMCel) was used as a biocompatible and multifunctional polysaccharide to synthesize fluorescent ZnCdS alloyed quantum dot nanostructures at room temperature by an aqueous green method [137]. The core-shell nanoconjugates were comprised of the ZnCdS semiconductor

Pharmaceutical applications of natural polysaccharides 43

Figure 2.16 Ex-vivo corrosion/irritation test for CMT:HEMA hydrogel. (A) Pictographic representation of the area of the dorsal surface of SD rats where the control and hydrogel solution was topically applied. Images were taken at 24, 48, and 72 h after the application of hydrogel. (B) Histopathology of skin is represented through H & E staining of skin tissue of three independent animals after 15 days of application of hydrogel and control (top panel). Images were acquired by 40X objective. Lower panel represents enlarged view of the epidermis of the same tissue sections [134].

QD core along with the CMCel shell. Besides, CMCel functional groups controlled the diameters of colloidal wateresoluble nanocrystals and their hydrodynamic sizes. Also, the nanoconjugates were cytocompatible and luminescent which were used in bioimaging of human osteosarcoma cancer cells, approving that such polysaccharide-based fluorescent conjugates were auspicious nanoformulations for diagnosis and bioimaging of cancer cells. Quaternized analogs of pectic galactan (QPG) were prepared through the reaction of 3chloro-2-hydroxypropyl trimethyl ammonium chloride and pectic galactan in aqueous sodium hydroxide environment [138]. The QPG had electrostatic interactions with plasmid DNA in aqueous medium and formed complexes with spherical reduced shape showing nanometer sizes ranging from of 60e160 nm. These complexes were fluorescently labeled by 5-DTAF and added into the C6 rat glioma cells. It was indicated that they could eternalize the cells and reach close to the nucleus in 24 h justifying this modified natural polysaccharide could also be used as a biodegradable and biocompatible gene delivery and cell specific carrier.

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Figure 2.17 Representative confocal image of C6 cellular path of QPG/pGFP complexes 24 h after exposure to the complexes. Bars represent 10 mm [138].

QPG-pGFP labeled complexes were administered to C6 rat glioma cells. Fig. 2.17 indicates the confocal image. Cells were observed after 24 h contact with the complexes. The labeled QPG (QPG-5-DTAF) was used to obtain images of the complexes (green). The fluorescent labeled membrane was yellow but the nucleus had a blue color. Thus, after 24 h, the complexes were entered inside the cells so that they were adjacent to the nucleus [138]. An enzyme and redox dual-stimuli responsive carrier called HMSN-SS-CDPEI@HA was achieved using carbon dots coated hollow mesoporous silica nanoparticles (HMSN) to be used in targeted drug delivery and cell bioimaging [139]. The positively charged CDPEI NPs were obtained using polyethylenimine (PEI) and grafted onto the HMSN pore openings via disulfide bonds which were employed as gatekeepers in order to entrap drugs inside the hollow cavities. Natural polysaccharide hyaluronic acid (HA) was also grafted onto the HMSN surfaces in order to examine targeted drug delivery, enhanced stability, as well as controlled drug release. Doxorubicin was used as an anticancer drug in the experiments because of its extensive clinical applications. The in vitro drug release test established that doxorubicin-containing HMSN-SS-CDPEI@HA had enzyme and redox dual-responsive drug release characteristics. Besides, the HMSN-SS-CDPEI@HA illustrated exceptional biocompatibility and fluorescent properties. The confocal laser scanning microscopy and flow cytometry established that the HMSN-SS-CDPEI@HA had a greater cell uptake through the CD44-receptor mediated endocytosis using CD44-receptor

Pharmaceutical applications of natural polysaccharides 45 overexpressed A549 cells compared to the NIH-3T3 (receptor-negative) cells which led to superior cytotoxicity effect against A549 cells relative to the NIH 3T3 cells. Hence, the HMSN-SS-CDPEI@HA was prepared to be a dual-stimuli responsive, real-time imaging, and targeted drug delivery platform which could be a promising system for the cancer treatment.

9. Application of natural polysaccharides in preparation of contact lenses Therapeutic ophthalmic lenses with prolonged drug release have been developed with the aim of circumventing tedious and fruitless eye drop administration. Hence, coating the contact lenses with natural polymers containing desired drugs is usually carried out [140]. Recently, the layer-by-layer deposition was applied using alginate and CS natural polymers in order to regulate releasing diverse ophthalmic drugs using three kinds of lenses including one silicone-based hydrogel used as a soft contact lens (SCL) with the drug releasing ability plus two commercially existing lenses called Definitive 50 for SCLs and CI26Y for intraocular lens (IOLs) [141]. The optimized coating was composed of a (alginateeCaCl2)/(CS þ glyoxal) double layer on top and an alginate-CaCl2 final layer to stop the CS degradation with tear fluid proteins. It was found that this coating had outstanding properties in controlling the release of diclofenac antiinflammatory drug once maintaining or increasing the physical features of the lenses. It was found that the coating could control the diclofenac release from IOL and SCL lenses at least for 1 week. Also, it was very hydrophilic (water contact angle z 0) as well as biocompatible; thus, it could avoid using additional surface treatments to improve the user relief. A therapeutic contact lens (TCL) with extended wear was prepared to sustainably deliver timolol maleate (TML) through molecular imprinting method [142]. The TCL contained a TML imprinted in a copolymer made up of carboxymethyl chitosan-g-hydroxy ethyl methacrylate-g-polyacrylamide (CmCS-g-HEMA-g-pAAm) introduced into polyHEMA framework. The TML was reloaded into the lens with an exceptional reloading capacity (6.53 mg of TML/TCL). Furthermore, the drug release accomplished in lacrimal fluid well obeyed the Higuchi model, which revealed the diffusion mechanism happened without polymer degradation. The TML drug release kinetics showed a continuous release which was suitable to achieve TML therapeutic index; besides, it could provide an appropriate medicine for glaucoma. The hydrogel contact lenses have attracted great interest as carriers in oculopathy treatment but traditional hydrogels do not demonstrate outstanding drug loading and controlled release effects which is due to lack of extra interactions of simple hydrophilic polymeric chains with drug molecules. To overcome these problems, some functional hydrogels were synthesized for the delivery of ophthalmic drug in oculopathy treatment [143]. Thus, mono-GMA-b-CD monomer and MA-b-CD cross-linker were added into the

46 Chapter 2 hydrogel via copolymerization reaction. The equilibrium swelling ratios and contact angles of hydrogels were changed with variations in ratios of mono-GMA-b-CD and MA-b-CD. The hydrogels revealed similar water loss, suitable transparency, and elastomer rheological characteristics. The functional hydrogels containing b-CD presented improved protein resistance as well as noticeably higher drug encapsulation compared to traditional hydrogels. Also, the drug release from the hydrogels was changed depending on the MA-b-CD and mono-GMA-b-CD ratios. The in vivo tests proved that hydrogel contact lenses had higher efficiency in decreasing intraocular tension compared with commercial eye drops confirming they are favorable for application in oculopathy therapy.

10. Application of natural polysaccharides in preparation of implants Polysaccharide hydrogels form three-dimensional matrixes of hydrophilic polymeric chains which are able to preserve great amount of water within the macromolecular structure; thus, they are interesting materials for preparation of implants [144,145]. Also, they have suitable features like adjustable mechanical and antibacterial properties, enhanced fluid film lubrication, in addition to friction decrease which lead to their application as injectable materials to fill out defects with any shapes [146]. So far, metal covered implants are widely employed against dental pathogens that cause biofilm creation as well as dental implants failure. Such nanoparticles can be applied together with natural polysaccharide biomacromolecules to improve the potential of these biologically active compounds. Alginate-polyacrylamide (ALG-PAAm) hydrogels were fabricated as orthopedic prosthesis materials and indentation experiments were performed to evaluate their mechanical features [147]. Also, their tribological responses were assessed by means of reciprocating sliding movement alongside alumina ceramic ball. The hydrogels were prepared by means of two diverse amounts of cross-linker in order to investigate the cross-linker influence on their wear resistances. Different loads and sliding speeds were used in absence and presence of bovine serum as a lubricant to mimic human gait/running cycle. The mass loss of each dried sample was measured using thermogravimetric analysis before and after every experiment and the wear volume was examined by profilometry. Increasing the cross-linker amount improved the elastic modulus up to 21% and the hardness to 32%. The mass loss was enhanced using a greater loading irrespective of cross-linker ratio. Nevertheless, using a higher sliding speed, a smaller amount of material was removed as a result of wear. Upon lubrication using the utmost load, the lowest average friction coefficient was attained for hydrogels containing 0.06% cross-linking agent, which is favorable in comparison to that measured for the articular cartilage. The observation of worn surfaces by means of electron microscopy indicated that adhesion was the principal wear mechanism. Also, hydrogels having superior cross-linking densities exhibited greater tribological performance and stiffness under lubrication.

Pharmaceutical applications of natural polysaccharides 47 In another work, silver (Ag) conjugated CS nanoparticles were prepared to be used as an effective coating compound for the titanium dental implants [148]. The bioactive CS was prepared using Aspergillus flavus Af09 and then conjugated to Ag NPs. The Ag-CS nanoparticles exhibited satisfactory growth inhibition capacity against the two main dental pathogens, namely, Streptococcus mutans and Porphyromonas gingivalis. The Ag-CS NPs inhibited the bacterial adhesion and decreased biofilm creation. Moreover, the nanoparticles inhibited the quorum sensing generation in both microorganisms. The nanoparticles were biocompatible, as they did not display any cell cytotoxicity approving that they could be used as suitable coatings for the titanium dental implants that can result in corrosion resistant dental implants with greater passivating properties. Some CS-based hydrogel implants were fabricated and employed for regeneration of peripheral nervous tissue by electrodeposition technique by means of a solution containing CS and an organic acid [149]. To enhance mechanical strengths of implants, hydroxyapatite was introduced into the solution which was also utilized as a source for calcium ions. Effects of additive and polymer concentrations were evaluated on chemical, mechanical, and biological features of the implants. It was found that the physicochemical characteristics of the resulting structures were extremely related to the original composition of solution. The in vitro proinflammatory as well as cytotoxic assessments exhibited biocompatibility of developed implants which proposed that the animal tests could be carried out.

11. Application of natural polysaccharides in preparation of antibacterial textiles/papers Production of antibacterial textiles is significant in the textile industry. Although a lot of chemicals and methods exist to manufacture antibacterial textiles, all of them are not benign to humans [150e152]. In order to overcome these shortcomings, biocompatible and ecofriendly materials should be developed and used for the fabrication of antibacterial textiles. The natural biopolymeric polysaccharides which are commonly utilized in biomedical application are suitable candidates for the preparation of antibacterial textiles/papers [81]. An ecological green synthesis of chitosan/neem seed (CS/NS) composite was carried out using aqueous neem seed extract through coprecipitation technique [153]. Cotton fabrics were modified with glutaraldehyde and citric acid as cross-linkers and then the composite was coated on the cotton fabric through chemical linkages formed between cellulose and composite. The bactericidal potency of the CS/NS composite covered cotton fabric was assessed in absence and presence of cross-linkers using both Gram-negative and Gram-positive bacteria by means of agar well diffusion technique. It was validated that the CS/NS composite coated on the cotton fabric showed a higher bactericidal effect

48 Chapter 2 compared to the cotton fabric lacking cross-linkers. Accordingly, the CS-neem seed composite could be utilized to achieve medicinal textiles. A facile and economical method was developed to achieve antibacterial cotton fabric using biodegradable gum tragacanth (GT) and silver nanoparticles (AgNPs) [154]. Diverse GT concentrations (2, 4, and 6 g/L) and an Ag amount of 5% relative to weight of dry GT were employed to examine their influences on the physical, mechanical, biological properties and antibacterial efficacy (against E. coli and S. aureus) of cotton fabric. Adding low amount of Ag NPs in the composite enhanced the antibacterial effect of fabric relative to fabric only treated with GT. Additionally, the cotton treated with 4% GT-Ag showed appropriate stiffness and tensile strength in comparison to the fabric treated with 6% GT-Ag composite. It was found that the GT as well as GT-Ag treated fabrics were biocompatible to fibroblast cells. In another effort, papers coated with chitosan:starch-silver nanoparticles (CS:St-AgNPs) were prepared to be applied in antimicrobial packages [155]. The St-AgNPs had a spherical morphology and a mean diameter of 7 nm. CS was added to the St-AgNPs mixture in diverse ratios (9:1, 8:2, 7:3, and 5:5) and the effect of various CS:St-AgNPs ratios were assessed on the papers characteristics including oil and water resistance, mechanical properties, and antimicrobial capacities. The characteristics of the papers coated by nanoparticles were highly related to the CS:St-AgNPs ratio. The CS:St-AgNPs coated paper fabricated using the 9:1 ratio exhibited exceptional mechanical characteristics and suitable resistance to oil and water. The CS:St-AgNPs coated papers presented significant improvement in oil and water resistance, mechanical strength, antifungal and bactericidal efficacy confirming they were potential candidates for preparation of functional antimicrobial textiles/papers.

12. Application of natural polysaccharides in preparation of antimicrobial food additives/packaging materials Food safety is a major challenge; especially, it is vital due to the growing consumer demand for fast foods. Consequently, significance of food storage as well as preservation is enhanced. In fact, there is an increasing attention for natural and higher quality foods prepared without using chemical preservers. Application of natural antimicrobial agents to produce safe and healthy foods is a favorable outlook [156]. Recently, corn starch was employed as a polymeric material to develop antimicrobial packaging materials using pediocin or nisin as food preservatives [157]. Halloysite clay was utilized as nanofiller to reinforce the films and bacteriocins were adsorbed on nanoclay before its addition to the film forming solutions. The films were active against

Pharmaceutical applications of natural polysaccharides 49 Clostridium perfringens and Listeria monocytogenes and halloysite preserved antimicrobial efficacy compared to the films lacking nanofiller. It was found that the adsorption method was promising to retain a suitable crystallinity and uniform shape for the films. For the nanocomposite containing nisin, adsorption enhanced the water barrier property. The mechanical resistances of films loaded by the halloysite were increased. Also, the elongation at break was considerably improved for films containing pediocin or nisin in addition to those incorporated with nisin and halloysite. Thus, these antimicrobial nanocomposite films were suitable food packaging materials. Several antimicrobial films were achieved using calcium alginate and lysozyme [158]. In order to optimize the antimicrobial activity, ultrasonic irradiation was used to enhance the immobilization efficacy. Diverse ultrasonic duration and power were applied. It was found that sonication speeded up the lysozyme immobilization rate and increased the lysozyme amount immobilized on the supports. The catalytic performance of the microbicidal film was assessed by the turbidimetric test and showed the greatest value using 147.8 W. Also, the antimicrobial effect was enhanced by sonication and determined through the inhibition zone method. To discover the mechanism of ultrasonic influence on lysozyme immobilization, the changes in lysozyme structures were studied before and after the ultrasonic irradiation. The fluorescence and circular dichroism spectra demonstrated that sonication influenced both the secondary and tertiary structures of lysozyme. Also, structural variations in the enzyme improved the enzymatic activity. Sonication impacts on the films’ microstructures were followed by the scanning electron microscopy and it was observed that the film surface had several cracks after sonication. Nisin is a famous bacteriocin which is approved to be used as a food additive in food preservation. Some nisin-incorporated pectin-inulin particles were achieved in order to avoid interaction of this bacteriocin with food components [159]. To prepare particles, pectins with diverse esterification degrees were utilized. Combining pectin and inulin improved the effectiveness of nisin addition compared to nisin-pectin samples. For all pectins examined using nisin amounts of 0.1e1.0 mg/mL in pH values of 4.0e7.0, the loading efficiency was 100%. The inulin and pectin combination for particle preparation slightly enhanced the microbicidal potency of nisin compared to nisin-pectin particles. Also, the antimicrobial effects of nisin-containing pectin-inulin particles depended on the pectin esterification degree. All particles having low pectin esterification degree or no esterified pectic acid revealed greater activity than the particles with high pectin esterification degree. High nisin-loading and comparable antimicrobial potency of inulinpectin particles compared to those of the nisin-pectin particles proved that combining inulin with pectin was very effective to produce antimicrobial particles for application in food industry.

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13. Conclusion In this work, the pharmaceutical applications of natural polysaccharides were reviewed in numerous biomedical fields such as cell encapsulation, drug/gene delivery, protein binding, wound healing, tissue engineering, bioimaging, preparation of contact lenses and implants, antibacterial textiles/papers, and antibacterial food additives/packaging materials. In case of cell encapsulation, alginate was used to encapsulate the human adiposeederived stem cells in order to increase their maintenance during hypothermic storage and produce them in large scale. The gold nanoparticles were synthesized using gum karaya and applied to deliver the gemcitabine hydrochloride anticancer drug. For gene delivery application, 6-amino-curdlan was used to achieve nanoparticles through its complexation with siRNAs which delivered siRNAs to mouse primary cells and human cancer cells and decreased 70%e90% of the target mRNA level. As an example of protein binding, the polysaccharide-protein-surfactant complexes were obtained using propylene glycol alginate, zein along with lecithin or rhamnolipid and applied as curcumin delivery platforms. Also, the FWEP polysaccharide isolated from fenugreek (Trigonella foenumgraecum) seeds revealed antioxidant, hemolytic, and in vivo wound healing features. The blends of xanthan gum and CS using Fe3O4magnetic nanoparticles yielded magnetic responsive polyelectrolyte complex hydrogels which were appropriate materials for tissue engineering. The nanocomposites fabricated using natural polysaccharides and quantum dots were applied in bioimaging applications. Moreover, natural polysaccharides containing various drugs were employed to coat contact lenses in order to release the desired drug in a controlled rate. As implant materials, the alginate/polyacrylamide hybrid hydrogels were fabricated as orthopedic prosthesis compounds. Also, the natural polysaccharides were utilized to prepare antibacterial textiles and papers. The corn starch was applied to obtain antimicrobial packaging materials using nisin or pediocin for food preservation. Finally, it can be stated that natural polysaccharides are very valuable materials that have found significant pharmaceutical applications.

Acknowledgment The authors appreciatively acknowledge all financial supports of this research by the Research Office of Amirkabir University of Technology (Tehran Polytechnic).

References [1] Seidi F, Jenjob R, Phakkeeree T, Crespy D. Saccharides, oligosaccharides, and polysaccharides nanoparticles for biomedical applications. J Control Release 2018;284:188e212. [2] Shariatinia Z, Zahraee Z. Controlled release of metformin from chitosanebased nanocomposite films containing mesoporous MCM-41 nanoparticles as novel drug delivery systems. J Colloid Interface Sci 2017;501:60e76.

Pharmaceutical applications of natural polysaccharides 51 [3] Kohsari I, Shariatinia Z, Pourmortazavi SM. Antibacterial electrospun chitosan-polyethylene oxide nanocomposite mats containing ZIF-8 nanoparticles. Int J Biol Macromol 2016;91:778e88. [4] Simo´a G, Ferna´ndez-Ferna´ndeza E, Vila-Crespob J, Ruipe´rezb V, Rodrı´guez-Nogales JM. Research progress in coating techniques of alginate gel polymer forcell encapsulation. Carbohydr Polym 2017;170:1e14. [5] Kohsari I, Shariatinia Z, Pourmortazavi SM. Antibacterial electrospun chitosanepolyethylene oxidenanocomposite mats containing bioactive silver nanoparticles. Carbohydr Polym 2016;140:287e98. [6] Fazli Y, Shariatinia Z, Kohsari I, Azadmehr A, Pourmortazavi SM. A novel chitosan-polyethylene oxide nanofibrous mat designed for controlled co-release of hydrocortisone and imipenem/cilastatin drugs. Int J Pharm 2016;513:636e47. [7] Germershaus O, Lu¨hmann T, Rybak J-C, Ritzer J, Meinel L. Application of natural and semi-synthetic polymers for the delivery of sensitive drugs. Int Mater Rev 2015;60:101e30. [8] Shariatinia Z, Nikfar Z. Synthesis and antibacterial activities of novel nanocomposite films of chitosan/ phosphoramide/Fe3O4 NPs. Int J Biol Macromol 2013;60:226e34. [9] Shariatinia Z, Nikfar Z, Gholivand K, Abolghasemi Tarei S. Antibacterial activities of novel nanocomposite biofilms of chitosan/phosphoramide/Ag NPs. Polym Compos 2015;36:454e66. [10] Ngwuluka NC, Ochekpe NA, Aruoma OI. Naturapolyceutics: the science of utilizing natural polymers for drug delivery. Polymers 2014;6:1312e32. [11] Cordeiro AS, Jose´ Alonso M, de la Fuente M. Nanoengineering of vaccines using natural polysaccharides. Biotechnol Adv 2015;33:1279e93. [12] Shariatinia Z, Mazloom Jalali A. Chitosan-based hydrogels: preparation, properties and applications. Int J Biol Macromol 2018;115:194e220. [13] Shariatinia Z, Fazli M. Mechanical properties and antibacterial activities of novel nanobiocomposite films of chitosan and starch. Food Hydrocolloids 2015;46:112e24. [14] Fazli Y, Shariatinia Z. Controlled release of cefazolin sodium antibiotic drug from electrospun chitosan-polyethylene oxide nanofibrous mats. Mater Sci Eng C 2017;71:641e52. [15] Shelke NB, James R, Laurencin CT, Kumbar SG. Polysaccharide biomaterials for drug delivery and regenerative engineering. Polym Adv Technol 2014;25:448e60. [16] Ali A, Ahmed S. A review on chitosan and its nanocomposites in drug delivery. Int J Biol Macromol 2018;109:273e86. [17] Zamboni F, Collins MN. Cell based therapeutics in type 1 diabetes mellitus. Int J Pharm 2017;521:346e56. [18] Algire GH, Weaver JM, Prehn RT. Growth of cells in vivo in diffusion chambers. I. Survival of homografts in mice. J Natl Cancer Inst 1954;15:493e507. [19] Rokstad AMA, Lacı´k I, de Vos P, Strand BL. Advances in biocompatibility and physico-chemical characterization of microspheres for cell encapsulation. Adv Drug Deliv Rev 2014;67e68:111e30. [20] Koo J, Chang TSM. Secretion of erythropoietin from microencapsulated rat kidney cells. Int J Artif Organs 1993;16:557e60. [21] Liu HW, Ofosu FA, Chang PL. Expression of human factor IX by microencapsulated recombinant fibroblasts. Hum Gene Ther 1993;4:291e301. [22] Chang PL, Shen N, Westcott AJ. Delivery of recombinant gene products with microencapsulated cells in vivo. Hum Gene Ther 1993;4:433e40. [23] Colton CK. Implantable biohybrid artificial organs. Cell Transplant 1995;4:415e36. [24] Uludag H, Sefton MV. Microencapsulated human hepatoma (HepG2) cells: in vitro growth and protein release. J Biomed Mater Res 1993;27:1213e24. [25] Cieslinski DA, David Humes H. Tissue engineering of a bioartificial kidney. Biotechnol Bioeng 1994;43:678e81. [26] Lim F, Sun AM. Microencapsulated islets as bioartificial endocrine pancreas. Science 1980;210:908e10. [27] Aebischer P, Goddard M, Signore AP, Timpson RL. Functional recovery in hemiparkinsonian primates transplanted with polymer-encapsulated PC12 cells. Exp Neurol 1994;126:151e8.

52 Chapter 2 [28] Dulieu C, Poncelet D, Neufeld RJ. Encapsulation and immobilization techniques. In: Ku¨htreiber WM, Lanza RP, Chick WL, editors. Cell encapsulation technology and therapeutics. Bosto: Birkha¨user; 1999. p. 3e17. [29] De Vos P, Lazarjani HA, Poncelet D, Faas MM. Polymers in cell encapsulation from an enveloped cell perspective. Adv Drug Deliv Rev 2014;67:15e34. [30] Doh K-O, Yeo Y. Application of polysaccharides for surface modification of nanomedicines. Ther Deliv 2012;3:1447e56. [31] Passirani C, Barratt G, Devissaguet JP, Labarre D. Interactions of nanoparticles bearing heparin or dextran covalently bound to poly(methylmethacrylate) with the complement system. Life Sci 1998;62:775e85. [32] Shariatinia Z. Pharmaceutical applications of chitosan. Adv Colloid Interface Sci 2019;263:131e94. [33] Costa JR, Silva NC, Sarmento B, Pintado M. Delivery systems for antimicrobial peptides and proteins: towards optimization of bioavailability and targeting. Curr Pharmaceut Biotechnol 2017;18:108e20. [34] Gaber M, Medhat W, Hany M, Saher N, Fang J-Y, Elzoghby A. Protein lipid nanohybrids as emerging platforms for drug and gene delivery: challenges and outcomes. J Control Release 2017;254:75e91. [35] Shariatinia Z. Carboxymethyl chitosan: properties and biomedical applications. Int J Biol Macromol 2018;120:1406e19. [36] Song M, Li L, Zhang Y, Chen K, Wang H, Gong R. Carboxymethyl-b-cyclodextrin grafted chitosan nanoparticles as oral delivery carrier of protein drugs. React Funct Polym 2017;117:10e5. [37] Tolstoguzov V. Some thermodynamic considerations in food formulation. Food Hydrocolloids 2003;17:1e23. [38] Ferreira CO, Nunes CA, Delgadillo I, Lopes-Da-Silva JA. Characterization of chitosan-whey protein films at acid pH. Food Res Int 2009;42:807e13. [39] Dickinson E. Interfacial structure and stability of food emulsions as affected by protein-polysaccharide interactions. Soft Matter 2008;4:932e42. [40] Gu YS, Decker EA, Mcclements DJ. Application of multi-component biopolymer layers to improve the freeze-thaw stability of oil-in-water emulsions: b-Lactoglobulin-i-carrageenan-gelatin. J Food Eng 2007;80:1246e54. [41] Nori MP, Favaro-Trindade CS, Alencar SMD, Thomazini M, de Camargo Balieiro JC, Contreras Castillo CJ. Microencapsulation of propolis extract by complex coacervation. LWT - Food Sci Technol 2011;44:429e35. [42] Xiao JX, Huang GQ, Wang SQ, Sun YT. Microencapsulation of capsanthin by soybean protein isolatechitosan coacervation and microcapsule stability evaluation. J Appl Polym Sci 2014;131:257e65. [43] Laneuville SI, Turgeon SL, Sanchez C, Paquin P. Gelation of native b-lactoglobulin induced by electrostatic attractive interaction with xanthan gum. Langmuir 2006;22:7351. [44] Le XT, Turgeon SL. Rheological and structural study of electrostatic crosslinked xanthan gum hydrogels induced by b-lactoglobulin. Soft Matter 2013;9:3063e73. [45] Chen WS, Soucie WG. Edible fibrous serum milk protein/xanthan gum complexes. 1985. United States Patent, 4559233. [46] Schmitt C, Sanchez C, Desobrybanon S, Hardy J. Structure and technofunctional properties of proteinpolysaccharide complexes: a review. Crit Rev Food Sci Nutr 1998;38:689e753. [47] Strauss G, Gibson SM. Plant phenolics as cross-linkers of gelatin gels and gelatin-based coacervates for use as food ingredients. Food Hydrocolloids 2004;18:81e9. [48] Abdel-Azim NS, Shams KA, Shahat AA, El Missiry MM, Ismail SI, Hammouda FM. Egyptian herbal drug industry: challenges and future prospects. Res J Med Plant 2011;5:136e44. [49] Kulkarni AD, Joshi AA, Patil CL, Amale PD, Patel HM, Surana SJ, Belgamwar VS, Chaudhari KS, Pardeshi CV. Xyloglucan: a functional biomacromolecule for drug delivery applications. Int J Biol Macromol 2017;104:799e812. [50] Pereira LDP, Mota MRL, Brizeno LAC, Nogueira FC, Ferreira EGM, Pereira MG, Assreuy AMS. Modulator effect of a polysaccharide-rich extract from Caesalpinia ferrea stem barks in rat cutaneous wound healing: role of TNF-a IL-1b, NO, TGF-b. J Ethnopharmacol 2016;187:213e23.

Pharmaceutical applications of natural polysaccharides 53 [51] Trombetta D, Puglia C, Perri D, Licata A, Pergolizzi S, Lauriano ER. Effect of polysaccharides from Opuntia ficus-indica (L.) cladodes on the healing of dermal wounds in the rat. Phytomedicine 2006;13:352e8. [52] Bae JS, Jang KH, Park SC, Jin HK. Promotion of dermal wound healing by polysaccharides isolated from Phellinus gilvus in rats. J Vet Med Sci 2005;67:111e4. [53] Tabandeh MR, Oryon A, Mohammadalipour A. Polysaccharides of Aloe vera induce MMP-3 and TIMP-2 gene expression during the skin wound repair of rat. Int J Biol Macromol 2014;65:424e30. [54] Kumar A, Madhusudana Rao K, Han SS. Application of xanthan gum as polysaccharide in tissue engineering: a review. Carbohydr Polym 2018;180:128e44. [55] Ulery BD, Nair LS, Laurencin CT. Biomedical applications of biodegradable polymers. J Polym Sci B Polym Phys 2011;49:832e64. [56] Zohuri GH, Sandaroos R, Ahmadjo S, Damavandi S, Rabiee A, Shamekhi MA. Tissue engineering: biomaterial application, Encycl. Biomed. Polym. Polym. Biomater. CRC Press; 2015. p. 7901e32. [57] Salerno A, Pascual CD. Bio-based polymers, supercritical fluids and tissue engineering. Process Biochem 2015;50:826e38. [58] Silva AK, Juenet M, Meddahi-Pelle´ A, Letourneur D. Polysaccharide-based strategies for heart tissue engineering. Carbohydr Polym 2015;116:267e77. [59] Pina S, Oliveira JM, Reis RL. Natural-based nanocomposites for bone tissue engineering and regenerative medicine: a review. Adv Mater 2015;27:1143e69. [60] Mansur HS, Mansur AAP, Curti E, de Almeida MV. Bioconjugation of quantum-dots with chitosan and N,N,N-trimethyl chitosan. Carbohydr Polym 2012;90:189e96. [61] Luna-Martı´nez JF, Herna´ndez-Uresti DB, Reyes-Melo ME, Guerrero-Salazar CA, Gonza´lezGonza´lez VA, Sepu´lveda-Guzma´n S. Synthesis and optical characterization of ZnSesodium carboxymethyl cellulose nanocomposite films. Carbohydr Polym 2011;84:566e70. [62] Mansur AAP, Ramanery FP, Oliveira LC, Mansur HS. Carboxymethylchitosan functionalization of Bi2S3 quantum dots: towards eco-friendly fluorescent core-shell nanoprobes. Carbohydr Polym 2016;146:455e66. [63] Tang CR, Su ZH, Lin BG, Huang HW, Zeng YL, Li S, Huang H, Wang YJ, Li CX, Shen GL, Yu RQ. A novel method for iodate determination using cadmium sulfide quantum dots as fluorescence probes. Anal Chim Acta 2010;678:203e7. [64] Chen L, Lai C, Marchewka R, Berry RM, Tam KC. Use of CdS quantum dot-functionalized cellulose nanocrystal films for anti-counterfeiting applications. Nanoscale 2016;8:13288e96. [65] Chan WCW, Nie SM. Quantum dot bioconjugates for ultrasensitive nonisotopic detection. Science 1998;281:2016e8. [66] Das R, Bandyopadhyay R, Pramanik P. Carbon quantum dots from natural resource: a review. Materials Today Chemistry 2018;8:96e109. [67] Toda R, Kawazu K, Oyabu M, Miyazaki T, Kiuchi Y. Comparison of drug permeabilities across the blooderetinal barrier, bloodeaqueous humor barrier, and bloodebrain barrier. J Pharm Sci 2011;100:3904e11. [68] Raviola G. The structural basis of the blood-ocular barriers. Exp Eye Res 1977;25:27e63. [69] Garnett BD. The contact lens manual: a practical fitting guide. Hum Immunol 1993;63:S78. [70] Creech JL, Chauhan A, Radke CJ. Dispersive mixing in the posterior tear film under a soft contact lens. Ind Eng Chem Res 2001;40(14):3015e26. [71] Helsen JA, Breme HJ. Metals as biomaterials. 1998. p. 30e40. Chichester, New York. [72] Neoh KG, Hu X, Zheng D, Kang ET. Balancing osteoblast functions andbacterial adhesion on functionalized titanium surfaces. Biomaterials 2012;33:2813e22. [73] Li M, Liu X, Xu Z, Yeung KWK, Wu S. Dopamine modified organic-inorganic hybrid coating for antimicrobial and osteogenesis. ACS Appl Mater Interfaces 2016;8(49):33972e81. [74] Darouiche RO. Treatment of infections associated with surgical implants. N Engl J Med 2004;350:1422e9.

54 Chapter 2 [75] Liu X, Li M, Zhu Y, Yeung KWK, Chu PK, Wu S. The modulation of stem cell behaviors by functionalized nanoceramic coatings on Ti-based implants. Bioact Mater 2016;1:65e76. [76] Wu S, Liu X, Yeung KWK, Liu C, Yang X. Biomimetic porous scaffolds for bone tissue engineering. Mater Sci Eng R 2014;80:1e36. [77] Xu Z, Man L, Xia L, Liu X, Fei M, Wu S, Yeung KWK, Han Y, Chu PK. Antibacterial activity of silver doped titanate nanowires on Ti implants. ACS Appl Mater Interfaces 2016;8:16584e94. [78] Zhou T, Zhu Y, Li X, Liu X, Yeung KWK, Wu S, Wang X, Cui Z, Yang X, Chu PK. Surface functionalization of biomaterials by radical polymerization. Prog Mater Sci 2016;83:191e235. [79] Rinaudo M. Chitin and chitosan: properties and applications. Prog Polym Sci 2006;31:603e32. [80] Rajendran R, Radhai R, Kotresh TM, Csiszar E. Development of antimicrobial cotton fabrics using herb loaded nanoparticles. Carbohydr Polym 2013;91. 613-317. [81] Ul-Ialam S, Shahid M, Mohammad F. Green chemistry approaches to develop antimicrobial textiles based on sustainable biopolymers-a review. Ind Eng Chem Res 2013;52:5245e60. [82] Mahato K, Srivastava A, Chandra P. Paper based diagnostics for personalized health care: emerging technologies and commercial aspects. Biosens Bioelectron 2017;15:246e59. [83] Reshetnyak EA, Ostrovskaya VM, Goloviznina KV, Kamneva NN. Influence of tetraalkylammonium halides on analytical properties of universal acid-base indicator paper. J Mol Liq 2017;248:610e5. [84] Schoonover DV, Gibson HW. Facile removal of tosyl chloride from tosylates using cellulosic materials, e.g., filter paper. Tetrahedron Lett 2017;58:242e4. [85] Rastogi VK, Samyn P. Bio-based coatings for paper applications. Coatings 2015;5:887e930. [86] Piselli A, Garbagnoli P, Alfieri I, Lorenzi A, Curto BD. Natural-based coatings for food paper packaging. Int J Des Sci Technol 2014;20:55e78. [87] Khwaldia K, Arab-Tehrany E, Desobry S. Biopolymer coatings on paper packaging materials. Compr Rev Food Sci F 2010;9:82e91. [88] Ma Y, Liu P, Si C, Liu Z. Chitosan nanoparticles: preparation and application in antibacterial paper. J Macromol Sci B 2010;49:994e1001. [89] Gottesman R, Shukla S, Perkas N, Solovyov LA, Nitzan Y, Gedanken A. Sonochemical coating of paper by microbiocidal silver nanoparticles. Langmuir 2011;27:720e6. [90] Brobbey KJ, Haapanen J, Gunell M, Ma¨kela¨ JM, Eerola E, Toivakka M, Saarinen JJ. One-step flame synthesis of silver nanoparticles for roll-to-roll production of antibacterial paper. Appl Surf Sci 2017;420:558e65. [91] Mlalila N, Hilonga A, Swai H, Devlieghere F, Ragaert P. Antimicrobial packaging based on starch, poly(3-hydroxybutyrate) and poly(lactic-co-glycolide) materials and application challenges. Trends Food Sci Technol 2018;74:1e11. [92] Appendini P, Hotchkiss JH. Review of antimicrobial food packaging. Innov Food Sci Emerg Technol 2002;3:113e26. [93] Quintavalla S, Vicini L. Antimicrobial food packaging in meat industry. Meat Sci 2002;62:373e80. [94] Sung S-Y, Sin LT, Tee T-T, Bee S-T, Rahmat AR, Rahman WAWA, Tan AC, Vikhraman M. Antimicrobial agents for food packaging applications. Trends Food Sci Technol 2013;33:110e23. [95] Wang J-Z, Ding Z-Q, Zhang F, Ye W-B. Recent development in cell encapsulations and their therapeutic applications. Mater Sci Eng C 2017;77:1247e60. [96] Orive G, Santos E, Pedraz J, Herna´ndez R. Application of cell encapsulation for controlled delivery of biological therapeutics. Adv Drug Deliv Rev 2014;67:3e14. ˚ , Blennow K, [97] Ferreira D, Westman E, Eyjolfsdottir H, Almqvist P, Lind G, Linderoth B, Seiger A Karami A, Darreh-Shori T. Brain changes in Alzheimer’s disease patients with implanted encapsulated cells releasing nerve growth factor. J Alzheimers Dis 2015;43:1059e72. [98] Vegas AJ, Veiseh O, Gu¨rtler M, Millman JR, Pagliuca FW, Bader AR, Doloff JC, Li J, Chen M, Olejnik K. Long-term glycemic control using polymer-encapsulated human stem cell-derived beta cells in immune-competent mice. Nat Med 2016;22(3):306e11.

Pharmaceutical applications of natural polysaccharides 55 [99] Dong D, Hao T, Wang C, Zhang Y, Qin Z, Yang B, Fang W, Ye L, Yao F, Li J. Zwitterionic starchbased hydrogel for the expansion and “stemness” maintenance of brown adipose derived stem cells. Biomaterials 2018;157:149e60. [100] Cacciotti I, Ceci C, Bianco A, Pistritto G. Neuro-differentiated Ntera2 cancer stem cells encapsulated in alginate beads: first evidence of biological functionality. Mater Sci Eng C 2017;81:32e8. [101] Swioklo S, Ding P, Pacek AW, Connon CJ. Process parameters for the high-scale production of alginateencapsulated stem cells for storage and distribution throughout the cell therapy supply chain. Process Biochem 2017;59:289e96. [102] Raveendran S, Palaninathan V, Nagaoka Y, Fukuda T, Iwai S, Higashi T, Mizuki T, Sakamoto Y, Mohanan PV, Maekawa T, Kumar DS. Extremophilic polysaccharide nanoparticles for cancer nanotherapyand evaluation of antioxidant properties. Int J Biol Macromol 2015;76:310e9. [103] Pooja D, Panyaram S, Kulhari H, Reddy B, Rachamalla SS, Sistla R. Natural polysaccharide functionalized gold nanoparticles asbiocompatible drug delivery carrier. Int J Biol Macromol 2015;80:48e56. [104] Deore UV, Mahajan HS. Isolation and characterization of natural polysaccharide from Cassia Obtustifolia seed mucilage as film forming material for drug delivery. Int J Biol Macromol 2018;115:1071e8. [105] Anderson W. Human gene therapy. Science 1992;256:808e13. [106] Oliveira AV, Rosa da Costa AM, Silva GA. Non-viral strategies for ocular gene delivery. Mater Sci Eng C 2017;77:1275e89. [107] Rosenberg SA, Aebersold P, Cornetta K, Kasid A, Morgan RA, Moen R, Karson EM, Lotze MT, Yang JC, Topalian SL, Merino MJ, Culver K, Miller AD, Blaese RM, Anderson WF. Gene transfer into humansdimmunotherapy of patients with advanced melanoma, using tumor-infiltrating lymphocytes modified by retroviral gene transduction. N Engl J Med 1990;323:570e8. [108] Leiden JM. Gene therapy-promise, pitfalls, and prognosis. N Engl J Med 1995;333:871e3. [109] Cotrim AP, Baum BJ. Gene therapy: some history, applications, problems, and prospects. Toxicol Pathol 2008;36:97e103. [110] Han J, Jia C, Borjihan W, Ganbold T, Tariq M. Rana, Huricha Baigude, Preparation of novel curdlan nanoparticles for intracellular siRNA delivery, Carbohydr. Polymers 2015;117:324e30. [111] Fernandez-Pin˜eiro I, Pensado A, Badiola I, Sanchez A. Development and characterisation of chondroitin sulfate- and hyaluronic acid-incorporated sorbitan ester nanoparticles as gene delivery systems. Eur J Pharm Biopharm 2018;125:85e94. [112] Wang R, Tian Z, Chen L. Nano-encapsulations liberated from barley protein microparticles for oral delivery of bioactive compounds. Int J Pharm 2011;406:153e62. [113] Dickinson E. Hydrocolloids as emulsifiers and emulsion stabilizers. Food Hydrocolloids 2009;23:1473e82. [114] Dai L, Wei Y, Sun C, Mao L, McClements DJ, Gao Y. Development of protein-polysaccharide-surfactant ternary complex particles as delivery vehicles for curcumin. Food Hydrocolloids 2018;85:75e85. [115] Hu B, Chen Q, Cai Q, Fan Y, Wilde PJ, Rong Z, Zeng X. Gelation of soybean protein and polysaccharides delays digestion. Food Chem 2017;221:1598e605. [116] Wang Y, Xu S, Xiong W, Pei Y, Li B, Chen Y. Nanogels fabricated from bovine serum albumin and chitosan viaself-assembly for delivery of anticancer drug. Colloids Surfaces B Biointerfaces 2016;146:107e13. [117] Zhang H, Chen J, Cen Y. Burn wound healing potential of a polysaccharide from Sanguisorba officinalis L. in mice. Int J Biol Macromol 2018;112:862e7. [118] Selig HF, Lumenta DB, Giretzlehner M, Jeschke MG, Upton D, Kamolz LP. The properties of an "ideal" burn wound dressingdwhat do we need in daily clinical practice? Results of a worldwide online survey among burn care specialists. Burns 2012;38:960e6. [119] Waghmare VS, Wadke PR, Dyawanapelly S, Deshpande A, Jain R, Dandekar P. Starch based nanofibrous scaffolds for wound healing applications. Bioactive Mater 2018;3:255e66.

56 Chapter 2 [120] Basu A, Kunduru KR, Abtew E, Domb AJ. Polysaccharide-based conjugates for biomedical applications. Bioconjug Chem 2015;26:1396e412. [121] Poonguzhali R, Khaleel Basha S, Sugantha Kumari V. Fabrication of asymmetric nanostarch reinforced Chitosan/PVP membrane and its evaluation as an antibacterial patch for in vivo wound healing application. Int J Biol Macromol 2018;114:204e13. [122] Ktari N, Trabelsi I, Bardaa S, Triki M, Bkhairia I, Slama-Ben Salem RB, Nasri M, Salah RB. Antioxidant and hemolytic activities, and effects in rat cutaneous wound healing of a novel polysaccharide from fenugreek (Trigonellafoenum-graecum) seeds. Int J Biol Macromol 2017;95:625e34. [123] Singh S, Gupta A, Sharma D, Gupta B. Dextran based herbal nanobiocomposite membranes for scar free wound healing. Int J Biol Macromol 2018;113:227e39. [124] Chi N-H, Yang M-C, Chung T-W, Chou N-K, Wang S-S. Cardiac repair using chitosan-hyaluronan/silk fibroin patches in a rat heart model with myocardial infarction. Carbohydr Polym 2013;92:591e7. [125] Silvestri A, Boffito M, Sartori S, Ciardelli G. Biomimetic materials and scaffolds for myocardial tissue regeneration. Macromol Biosci 2013;13:984e1019. [126] Hollister SJ. Porous scaffold design for tissue engineering. Nat Mater 2005;4:518e24. [127] De Mulder EL, Buma P, Hannink G. Anisotropic porous biodegradable scaffolds for musculoskeletal tissue engineering. Materials 2009;2:1674e96. [128] Saludas L, Pascual-Gil S, Pro´sper F, Garbayo E, Blanco-Prieto M. Hydrogel based approaches for cardiac tissue engineering. Int J Pharm 2017;523:454e75. [129] Sokolsky-Papkov M, Agashi K, Olaye A, Shakesheff K, Domb AJ. Polymer carriers for drug delivery in tissue engineering. Adv Drug Deliv Rev 2007;59:187e206. [130] Liu Y, Wang S, Zhang R. Composite poly(lactic acid)/chitosan nanofibrous scaffolds for cardiac tissue engineering. Int J Biol Macromol 2017;103:1130e7. [131] Nelson DM, Ma Z, Fujimoto KL, Hashizume R, Wagner WR. Intra-myocardial biomaterial injection therapy in the treatment of heart failure: materials, outcomes and challenges. Acta Biomater 2011;7:1e15. [132] Place ES, Evans ND, Stevens MM. Complexity in biomaterials fortissue engineering. Nat Mater 2009;8:457e70. [133] Shakir M, Zia I, Rehman A, Ullah R. Fabrication and characterization of nanoengineered biocompatible n-HA/chitosan-tamarind seed polysaccharide: bio-inspired nanocomposites for bone tissue engineering. Int J Biol Macromol 2018;111:903e16. [134] Choudhury P, Kumar S, Singh A, Kumar A, Kaur N, Sanyasi S, Chawla S, Goswami C, Goswami L. Hydroxyethyl methacrylate grafted carboxy methyl tamarind (CMT-g-HEMA) polysaccharide based matrix as a suitable scaffold for skin tissue engineering. Carbohydr Polym 2018;189:87e98. [135] Madhusudana Rao K, Kumar A, Han SS. Polysaccharide-based magnetically responsive polyelectrolyte hydrogels for tissue engineering applications. J Mater Sci Technol 2018;34:1371e7. [136] Alhaique F, Matricardi P, Meo CD, Coviello T, Montanari E. Polysaccharide-based self-assembling nanohydrogels: an overview on 25-years research on pullulan. J Drug Deliv Sci Technol 2015;30:300e9. [137] Mansur AAP, de Carvalho FG, Mansur RL, Carvalho SM, de Oliveira LC, Mansur HS. Carboxymethylcellulose/ZnCdS fluorescent quantum dot nanoconjugates for cancer cell bioimaging. Int J Biol Macromol 2017;96:675e86. [138] Chintakunta R, Buaron N, Kahn N, Moriah A, Lifshiz R, Goldbart R, Traitel T, Tyler B, Brem H, Kost J. Synthesis, characterization, and self-assembly with plasmid DNA of a quaternary ammonium derivative of pectic galactan and its fluorescent labeling for bioimaging applications, Carbohydr. Polymers 2016;150:308e18. [139] Zhao Q, Wang S, Yang Y, Li X, Di D, Zhang C, Jiang T, Wang S. Hyaluronic acid and carbon dotsgated hollow mesoporous silica for redox and enzyme-triggered targeted drug delivery and bioimaging. Mater Sci Eng C 2017;78:475e84. [140] Xu J, Xue Y, Hu G, Lin T, Gou J, Yin T, He H, Zhang Y, Tang X. A comprehensive review on contact lens for ophthalmic drug delivery. J Control Release 2018;281:97e118.

Pharmaceutical applications of natural polysaccharides 57 [141] Silva D, Pinto LFV, Bozukova D, Santos LF, Serro AP, Saramago B. Chitosan/alginate based multilayers to control drug release from ophthalmic lens. Colloids Surfaces B Biointerfaces 2016;147:81e9. [142] Anirudhan TS, Nair AS, Parvathy J. Extended wear therapeutic contact lens fabricated from timolol imprinted carboxymethyl chitosan-g-hydroxy ethyl methacrylate-g-polyacrylamide as a onetime medication for glaucoma. Eur J Pharm Biopharm 2016;109:61e71. [143] Hu X, Tan H, Hao L. Functional hydrogel contact lens for drug delivery in the application of oculopathy therapy. Int J Mech Behav Biomed Mater 2016;64:43e52. [144] Blum MM, Ovaert TC. Investigation of friction and surface degradation of innovative boundary lubricant functionalized hydrogel material for use as artificial articular cartilage. Wear 2013;301:201e9. [145] Kobayashi M, Chang Y-S, Oka M. A two year in vivo study of polyvinyl alcohol hydrogel (PVA-H) artificial meniscus. Biomaterials 2005;26:3243e8. [146] Lin P, Zhang R, Wang X, Cai M, Yang J, Yu B, Zhou F. Articular cartilage inspired bilayer tough hydrogel prepared by interfacial modulated polymerization showing excellent combination of high loadbearing and low friction performance. ACS Macro Lett 2016;5:1191e5. [147] Arjmandi M, Ramezani M, Nand A, Neitzert T. Experimental study on friction and wear properties of interpenetrating polymer network alginate-polyacrylamide hydrogels for use in minimally invasive joint implants. Wear 2018;406e407:194e204. [148] Divakar DD, Jastaniyah NT, Altamimi HG, Alnakhli YO, Muzaheed, Alkheraif AA, Haleem S. Enhanced antimicrobial activity of naturally derived bioactivemolecule chitosan conjugated silver nanoparticle against dental implant pathogens. Int J Biol Macromol 2018;108:790e7. [149] Nawrotek K, Tylman M, Rudnicka K, Balcerzak J, Kami nski K. Chitosan-based hydrogel implants enriched with calcium ions intended for peripheral nervous tissue regeneration. Carbohydr Polym 2016;136:764e71. [150] Maryan AS, Montazer M, Harifi T. Synthesis of nano silver on cellulosic denim fabric producing yellow colored garment with antibacterial properties, Carbohydr. Polymers 2015;115:568e74. [151] Shariatinia Z, Shekarriz S, Mirhosseini Mousavi HS, Maghsoudi N, Nikfar Z. Disperse dyeing and antibacterial properties of nylon and wool fibers using two novel nanosized copper(II) complexes bearing phosphoramide ligands. Arab J Chem 2017;10:944e55. [152] Shariatinia Z, Javeri N, Shekarriz S. Flame retardant cotton fibers produced using novel synthesized halogen-free phosphoramide nanoparticles. Carbohydr Polym 2015;118:183e98. [153] Revathi T, Thambidurai S. Synthesis of chitosan incorporated neem seed extract (Azadirachtaindica) for medical textiles. Int J Biol Macromol 2017;104:1890e6. [154] Ranjbar-Mohammadi M. Production of cotton fabrics with durable antibacterial property by using gum tragacanth and silver. Int J Biol Macromol 2018;109:476e82. [155] Jung J, Kasi G, Seo J. Development of functional antimicrobial papers using chitosan/starch-silver nanoparticles. Int J Biol Macromol 2018;112:530e6. [156] Gyawali R, Ibrahim SA. Natural products as antimicrobial agents. Food Control 2014;46:412e29. [157] Meira SMM, Zehetmeyer G, Werner JO, Brandelli A. A novel active packaging material based on starch-halloysite nanocomposites incorporating antimicrobial peptides. Food Hydrocolloids 2017;63:561e70. [158] Wang D, Lv R, Ma X, Zou M, Wang W, Yan L, Ding T, Ye X, Liu D. Lysozyme immobilization on the calcium alginate film under sonication: development of an antimicrobial film. Food Hydrocolloids 2018;83:1e8. [159] Krivorotova T, Staneviciene R, Luksa J, Serviene E, Sereikaite J. Preparation and characterization of nisin-loaded pectin-inulin particles as antimicrobials. LWT - Food Sci Technol 2016;72:518e24.