Pullulan: A novel molecule for biomedical applications

Accepted Manuscript Title: Pullulan: A novel molecule for biomedical applications Authors: Ram Sarup Singh, Navpreet Kaur, Vikas Rana, John F. Kennedy PII: DOI: Reference:

S0144-8617(17)30488-5 http://dx.doi.org/doi:10.1016/j.carbpol.2017.04.089 CARP 12277

To appear in: Received date: Revised date: Accepted date:

12-1-2017 26-4-2017 26-4-2017

Please cite this article as: Singh, Ram Sarup., Kaur, Navpreet., Rana, Vikas., & Kennedy, John F., Pullulan: A novel molecule for biomedical applications.Carbohydrate Polymers http://dx.doi.org/10.1016/j.carbpol.2017.04.089 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Pullulan: A novel molecule for biomedical applications

Ram Sarup Singha,* [email protected] , Navpreet Kaura, Vikas Ranab, John F. Kennedyc

a

Carbohydrate and Protein Biotechnology Laboratory, Department of Biotechnology, Punjabi University, Patiala, 147 002, Punjab, India b Department of Pharmaceutical Sciences and Drug Research, Punjabi University, Patiala,147 002, Punjab, India c Chembiotech Laboratories, Advanced Science and Technology Institute, 5 The Croft, Buntsford Drive, Stoke Heath, Bromsgrove, Worcs, B60 4JE, UK *

Corresponding author: Tel./Fax: +91 175 3046262/2283073

Highlights 

Biomedical applications of pullulan and its derivatives have been discussed.

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Imperative role of pullulan in drug and gene targeting has been elaborated.

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Described pullulan as blood plasma substitute and scaffold for tissue engineering.

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Pullulan as molecular chaperone and vaccination have been discussed.

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Highlighted pullulan in film forming, insulinotropic activity and medical imaging.

Abstract Pullulan is an imperative natural polymer produced commercially by yeast like fungus Aureobasidium pullulans. It is non-toxic, non-immunogenic, non-carcinogenic and non-mutagenic in nature. The structure of pullulan consist unique linkage pattern with two α-(1→4) and one α(1→6) glycosidic bonds in maltotriose repeating units (G3). Pullulan endows distinctive physical traits due to the presence of nine hydroxyl groups on glucopyranose rings of G3 units. It can be derivatized in various forms by substituting these hydroxyl groups to enhance its utility in biomedical applications. Pullulan and its derivatives are completely explored for their applications 1

in food and pharmaceutical industries. Owing to these special properties, native pullulan and its derivatives possess potential application in multiple diagnostics. This review presents elaborated discussion on role of pullulan and its derivatives in various biomedical applications e.g. drug delivery, gene targeting, tissue engineering, vaccination, plasma substitution, chaperone-like activity, medical imaging, insulinotropic activity, pharmaceutical dosages formation, coating, etc.

Keywords: Pullulan; drug delivery; tissue engineering; vaccination; plasma expander; molecular chaperons. 1. Introduction Biopolymers are the gums produced by microbial systems, plants or can be chemically synthesized from various biological building blocks (Kaplan, 1993). The microorganisms can synthesize three types of biopolymers with unique physical and chemical characteristics i.e. polysaccharides, polyesters and proteins (Singh, Saini, & Kennedy, 2008). The microbially produced polysaccharides can be intracellular, structural or extracellular based on their morphological localization (Chawla, Bajaj, Survase, & Singhal, 2009; Vijayendra & Shamala, 2013). Some of the microbially produced biopolymers are bacterial cellulose, levan, pullulan, kefiran, xanthan, gellan, haloferax exopolysaccharides, polyhydroxyalkanoates, etc. (Singh & Kaur, 2015). Pullulan is one of these commercially emerging biopolymers mainly produced by yeast like fungus Aureobasidium pullulans. Bernier (1958) was the first to isolate pullulan from A. pullulans and its structure was also resolved by him. Later on name “pullulan” was given by Bender and coworkers (1959). It is an exopolysaccharide produced in the form of amorphous slime on the surface of microbial cells (Singh & Saini, 2008a, b; Singh, Saini, & Kennedy, 2009; Sutherland, 1998). The formation of glycolipids in fungal mycelial cells leads to accumulation of pullulan on outer surface of cells (Ono, Kawahara, & Ueda, 1977). Other pullulan producing 2

microorganisms are Tremella mesenterica (Fraser & Jennings, 1971), Cytaria harioti (Waksman, Lederkremer, & Cerezo, 1977), Cytaria darwinii (Oliva, Cirelli, & De Lederkremer, 1986), Teloschistes flavicans (Reis, Tischer, Gorrin, & Iacomini, 2002), Rhodotorula bacarum (Chi & Zhao, 2003) and Cryphonectria parasitica (Forabosco et al., 2006). Various carbon sources have been used to produce pullulan from these microbial cultures e.g. jaggery, de-oiled jatropha seed cake and corn steep liquor (Mehta, Prasad, & Choudhury, 2014), Asian palm kernel (Sugumaran, Shobana, Balaji, Ponnusam, & Gowdhaman, 2014), cassava baggase (Sugumaran, Jothi, & Ponnusami, 2014), jack fruit seed (Sugumaran, Sindhu, Sukanya, Aiswarya, & Ponnusami, 2013), glucose, sucrose, maltose, lactose (Singh et al., 2008, Singh, Singh, & Saini, 2009), peat hydrolysate (LeDuy & Boa, 1983), rice hull, (Wang, Ju, Zhou, & Wei, 2014), etc. The biochemical mechanism of fermentative synthesis of pullulan from A. pullulans is very little understood. The biosynthetic mechanism of fermentative production of pullulan from A. pullulans using sucrose as a carbon source have been investigated extensively (Taguchi, Kikuchi, Sakano, & Kobayashi, 1973). Pullulan synthesis involves two steps, first acetone drying of microbial cells followed by initiation of pullulan chain formation by uridine 5’-diphosphate glucose (UDPG) in the presence of adenosine 5’-triphosphate. UDPG is the initiator of pullulan chain as it cannot be replaced by adenosine diphosphate glucose. The first stage of the biosynthetic pathway involves UDPGmediated attachment of a D-glucose residue to lipid molecule via phosphoester bridge and at second stage D-glucose residues form lipid-linked structures. These lipid-linked glucose molecules then react with isomaltosyl to yield isopanosyl or pyranosyl residue which are polymerized into pullulan chain. Isopanose, the precursor of pullulan chain can be synthesized with the aid of phospholipid intermediate and its glucose conjugates (Catley & McDowell, 1982).

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Pullulan is a linear and unbranched polymer with maltotriose repeating units connected by α-(1→6) glycosidic bonds. The characteristic dimeric segments of pullulan are [→x)-α-Dglucopyranosyl-(1→4)-α-D-glucopyranosyl-(1→] and [→4)-α-D-glucopyranosyl-(1→6)-α-Dglucopyranosyl-(1→], where x may be either 4 or 6 for (1→4) linked segment (Singh & Saini, 2012). The co-existence of α-(1→6) and α-(1→4) glycosidic linkages in the pullulan structure can be often seen as an intermediate between structure of amylose and dextran (Petrov, Shingel, Scripko, & Tsarenkov, 2002). Pullulan has molecular formula (C6H10O5)n and molecular weight range of 4.5×104 to 6×105 Da which is greatly affected by cultivation parameters (Lee & Yoo, 1993). It is white non-hygroscopic powder, which dissolves readily in water (both hot and cold) and in dilute alkali. It is insoluble in alcohol and other organic solvents except for dimethylsulfoxide and formamide (Kimoto, Shibuya, & Shiobara, 1997). The flexibility in the conformation of pullulan chain is responsible for the hydrodynamic properties of pullulan against action of any solvent (Singh et al., 2008). The melting temperature of pullulan is 250°C and it starts to degrade by charing above 250°C. It is non-toxic, non-mutagenic, edible, odorless and tasteless (Okada et al., 1990). It has inherent physiological activity due to high concentration of hydroxyl groups in the main chain. Owing to these unique chemical and physical properties, pullulan and its derivatives hold a vital role in numerous food, pharmaceutical and biomedical applications (Cheng, Demirci, & Catchmark, 2011). The biomedical applications of pullulan include targeted drug delivery, gene delivery (Singh, Kaur, & Kennedy, 2015), tissue engineering (Singh, Kaur, Rana, & Kennedy, 2016), vaccination, capsule coating, etc. (Akiyoshi et al., 1998; Kimoto et al., 1997; Na & Bae, 2002). It can be used to prepare capsules suitable for cultural and dietary requirements of vegetarians, diabetic persons and patients with restricted diet (Anonymous, 2012; Singh & Kaur,

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2015). Apart from biomedical applications pullulan also possesses several food applications. It has GRAS (Generally Regarded As Safe) status and can be used for the production of low-calorie foods (Yatmaz & Turhan, 2012). It can function as prebiotic (Mitsuhashi, Yoneyama, & Sakai, 1990), starch replacement in liquid as well as solid foods (Hiji, 1986; Hijiya & Shiosaka, 1975a), low viscosity filler in beverages and sauces, adhesive material to bind nuts to the cookies, etc. (Hijiya & Shiosaka, 1975b). Pullulan films are clear, highly oxygen-impermeable and also have excellent mechanical properties (Yuen, 1974; Singh & Kaur, 2015). These films can be used to preserve the moisture content in food and help in reduction of microbial growth by suppressing respiration of microbial flora (Debeaufort, Quezada-Gallo, & Voilley, 1998). A film based oral care product containing pullulan has been commercialized in many countries under the brand name ‘Listerine’ (Tsujisaka & Mitsuhashi, 1993). Pullulan has been used for preparation of maltotriose syrup using pullulanase (Singh, Saini, & Kennedy, 2010a, b; Singh, Saini, & Kennedy, 2011). This review attempts to critically appraise the literature on pullulan applications in biomedical field including tissue engineering, vaccination, drug targeting, gene targeting, pharmaceutical dosage coating, plasma substitution, insulinotropic activity, medical imaging, enzyme and protein engineering, etc.

2. Pullulan derivatives Pullulan is an as excellent carrier for therapeutic molecules to directly target various body organs like liver, lungs, brain, spleen, etc. (Prajapati, Jani, & Khanda, 2013; Singh & Saini, 2014) and it releases specific cytotoxic molecules to the particular infected site (Gupta & Gupta, 2004; Na & Bae, 2002). Although pullulan itself have numerous potential applications, further chemical modifications increase its utility in biomedical field by lowering the enzymatic degradation of

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pullulan. Pullulan consists of nine hydroxyl groups on its pyranose rings per maltotriose unit. The modifications of pullulan can be done via various chemical reactions to substitute the hydroxyl groups with desired chemical moieties (Shingel, 2004; Singh et al., 2015). The chemical reactions involve esterification, etherification, sulfation, oxidation, co-polymerization, amidification, etc. (Bataille, Meddahi-Pelle, Visage, Letourneur, & Chaubet, 2011). Various pullulan derivatives with potential application in the biomedical field and their structures are given in Table 1. Different derivatizations improve pullulan in a variable manner; hence every derivative possesses unique physico-chemical properties. The modifications due to derivatization of pullulan for each derivative are explained briefly. The hydroxyl groups of pullulan cross-link with carboxylate groups via ester linkages to form carboxymethyl pullulan (CMP) which results final negative charge on the derivative (Dulong, Mocanu, & LeCerf, 2007) and the derivative has high molecular weight than native pullulan (Nogusa, Yano, Okuno, Hamana, & Inoue, 1995). The sensitivity of CMP towards pH and ionic strength is very high and it possesses enhanced vascular permeability (Takakura & Hashida, 1996). Pullulan can be tailored hydrophobically by binding cholesterol group to its main chain. Cholesterol bearing pullulan (CHP) self-aggregates in aqueous solution to form spherical hydrogel nanoparticles (Akiyoshi & Sunamoto, 1996; Akiyoshi, Deguchi, Mariguchi, Yamaguchi, & Sunamoto, 1993; Akiyoshi, Deguchi, Tajma, Nishikawa, & Sunamoto, 1997). Another derivative, pullulan acetate (PA) can be prepared by acetylation of pullulan. PA has the property to self-aggregate in aqueous solution forming thermodynamically stable micelles with a hydrophobic core and hydrophilic outer cover (Yokoyama, Kwon, Okano, Sakurai, & Kataoka, 1994). The succinylation process form a reactive pullulan derivative which can be further modified in pH stable polymeric drug conjugate (Bruneel & Schacht, 1994). Pullulan-polyetheramine derivative can be synthesized by terminating one end

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of pullulan chain via chloroformate activation (Bruneel & Schacht, 1993). This derivative can easily conjugate with various thermo-sensitive and pH-sensitive blocks by reductive amination (Belbekhouche, Ali, Dulong, Picton, & LeCerf, 2011). Polyethyleneimine (PEI) pullulan is a derivative with quite high cationic charge density, blood compatibility and stability in the presence of plasma (Kim & Rossi, 2008; Rekha & Sharma, 2011a; Zhang, Tan, et al., 2009). The cationized form of pullulan can be obtained by addition of diethylaminoethylamine (DEAE) to pullulan backbone (Juan, Hlawaty, Chaubet, Letourneur, & Feldman, 2007) or by insertion of the thiol group to pullulan with the help of protamine (Priya, Rekha, & Sharma, 2014). The conjugation of pullulan with a chelating agent, diethylenetriamine pentaacetic acid, helps in maintaining the biological activity of attached molecules (Suginoshita, Tabata, Moriyasu, Ikada, & Chiba, 2001). Another derivative pullulan folate can be synthesized by folic acid treatment which has higher efficiency to internalize the cancer cells and sustained release of drugs (Scomparin, Salmaso, Bersani, Satchi-Fainaro, & Caliceti, 2011). Further addition of maleic anhydride to pullulan-folate enhances its pH sensitivity (Zhang et al., 2011). Hydrophobically modified pullulan derivatives can be formed by addition of L-lactide. Poly(L-lactide) and poly(DL-lactide-co-glycolide) grafting helps in sustained drug release via diffusion method (Cho, Park, & Na, 2009; Jeong et al., 2006). The combination of pullulan-folate and poly(DL-lactideco-glycolide) improves the carrier efficiency of pullulan nanoparticles for anticancer agents (Lee et al., 2015). All trans retinoic acid when combined with pullulan nanogels, enhances the anticancer effect of conjugated drug (Lee, Jeong, Seo, & Na, 2013; Lotan, 1995). Deoxycholic acid acts as a hydrophobic moiety and when conjugate with pullulan, it increases its affinity towards cancer cells (Na, Park, Jo, & Lee, 2006). Various chemical and physical stimuli have been applied on pullulan to have reversible light-responsive nanogels (Wang, Chen, Yang, Yang, & Liu,

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2014). The light responsive nanogels prepared from amphiphilic spiropyrane modified pullulan (SpP) can be controlled by photostimulation. Pullulan-g-poly(L-lysine) nanoparticles have been considered a potential non-viral carrier for safe gene delivery with negligible cytotoxicity (Park et al., 2012).

3. Biomedical applications 3.1 Drug delivery Pullulan nanogels, nanoparticles and microspheres can act as efficient drug delivery systems with enhanced permeability and retention effect. The drugs conjugated with these forms tend to accumulate in tumor/diseased tissue or cells much more than they do in normal tissues and reduce the toxicity of drug towards normal cells. These forms can undergo cell internalization with the help of cell adhesion receptor integrin (Absolom, Zingg, & Neumann, 1987; Haas & Plow, 1994) e.g. lectin-like receptors on liver cells result in higher biological affinity of pullulan towards liver (Seymour et al., 1991; Toth, Thomas, Broitman, & Zamcheck, 1985). The high molecular weight pullulan has short half-life period in blood circulation and it accumulates in the liver (Kaneo, Tanaka, Nakano, & Yamaguchi, 2001; Yamaoka, Tabata, & Ikada, 1993). Pullulan can conjugate interferon and this complex helps in controlling hepatitis C virus disease by directly targeting the liver without any side effects (Davis et al., 1989; Xi et al., 1996). It can also control the dose requirement for liver targeting, when injected intravenously. The addition of cyclodextrin to pullulan microspheres improves the stability of drug (diclofenac), its dissolution rate and bioavailability (Fundueanu et al., 2003; Horiuchi, Hirayama, & Uekama, 1991; Irie & Uekama, 1997; Loftsson & Brewster, 1996). Various pH sensitive and temperature dependent pullulan drug delivery systems can be prepared by addition of required oligomers pendants to neutral pullulan.

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The pullulan microspheres with semitelechelic poly(N-isopropyl acrylamide-co-acrylamide) oligomer pendants act as temperature dependent non-toxic drug delivery systems (Fundueanu et al., 2010). These pendants strictly show on-off mechanism with respect to the lower critical solution temperature (LCST), i.e. below LCST pendants remain in extended form to restrict the drug release and above LCST pendants shrink to allow drug delivery (Chu, Park, Yamaguchi, & Nakao, 2001; Ding, Fong, Long, Stayton, & Hoffman, 2001; Fundueanu, Constantin, & Ascenzi, 2008). Similarly, pH sensitive pullulan drug delivery systems work on the basis of a pH difference in normal cells and cancer cells. The pH sensitive pullulan-doxorubicin conjugate remains stable at neutral pH. However, the tumor cells have lower pH which leads drug release in tumor cells (Maeda, Wu, Sawa, Matsumura, & Hori, 2000; Ulbrich & Subr, 2004). This delivery system is safe and also has higher drug retention in tumor cells with low cardiotoxicity (Lu, Liang, Fan, Gu, & Zhang, 2010). A lot of research has been done on preparation and application of pullulan derivatives for targeted drug delivery to the desired organ or cells. Evaluation of various pullulan derivatives formulations as potential carriers for drugs is depicted in Table 2. The hydrophobically modified CHP can be used as a replacement of antibody immunotherapy for the treatment of Alzheimer’s disease which is a neurological disorder (Boridy, Takahashi, Akiyoshi, & Maysinger, 2009). The neutral CHP nanogels can reduce the toxicity in central nervous system for both primary cortical and microglial cells. CHP nanoparticles help in curing diseased neuron cells via blood-brain barrier (Vinogradov, Batrakova, & Kabanov, 2004). It also helps in lowering excessive blood glucose level in diabetic patients by acting as insulin carrier (Akiyoshi et al., 1998). The insulin conjugated with CHP shows long-term stability and higher biological activity than native insulin. It can reduce the adverse effect of continuous injections and frequency of drug injections; only a small drug

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dose is efficient for proper drug delivery (Degim & Celebi, 2007; Shi & Li, 2005). The modification of CHP nanoparticles with amine leads to higher efficiency in vitro and in vivo as a carrier of anticancer drug (docetaxel) to target lung cancer cells (Satoh, Chen, Aoyama, Date, & Akiyoshi, 2008). CHP coated liposomes also have excellent liver targeting ability both in vivo and in vitro (Taniguchi et al., 1999). The hydrophobically modified CMP nanoparticles form hydrazone bonds with drugs and help in their safe delivery to tumor cells. CMP has antioxidant properties and shows a higher affinity towards lymph nodes and spleen (Horie et al., 1999). CMP nanoparticles are pH sensitive, have liver targeting property and work well in pH range of 4.5-6.5 (Li et al., 2014; Lu et al., 2009). The conjugation of drugs with CMP leads to longer circulation time of drug in blood, higher retention in tumor cells with no hemolysis and lesser cardiotoxicity (Lu et al., 2010). CMP can conjugate with immunosuppressants for the treatment of autoimmune diseases (Masuda et al., 2001). Thermosensitive CMP nanoparticles are formed by addition of jeffamines (Mocanu, Mihai, Dulong, Picton, & LeCerf, 2011). These nanoparticles are amphiphilic in nature and can retain acidic, hydrophobic and basic drugs for controlled drug delivery applications (Mocanu, Nichifor, Picton, About-Jaudet, & Le Cerf, 2014). Various human diseases (atherosclerosis, graft rejection, ischemia, asthma, etc.) can be treated with CMP-Sialyl Lewis X complex. It can easily bind at inflammatory sites with the expression of cell binding molecule E-selectin which helps in receptor mediated cell specific drug delivery (Bevilacqua, Nelson, Mannori, & Cecconi, 1994; Rohde et al., 1992). The small-sized (<100 nm) thermodynamically stable PA micelles can be used for longer circulation of drugs in blood and their delivery to the targeted tumor cells/tissues (Yokoyama et al., 1994). The nanoparticle form of PA encapsulates hydrophobic drugs in its core and diseases like panic disorder, tumor, etc. can be treated easliy (Jeong, Nah, Na, Cho, & Kim, 1999; Jung,

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Jeong, & Kim, 2003; Kumar, Kumar, Suguna, Sastry, & Mandal, 2012). These nanoparticles can also attain the property of being pH sensitive by the addition of sulfadimethoxine or oligosulfadimethoxine to enhance drug delivery (Na, Lee, & Bae, 2004). It can be used for the treatment of diseased cells/organs with lower pH than normal cells and blood, e.g. ischemia, tumor, cancer, etc. Addition of carboxymethylated poly(ethylene glycol) to PA improves hydrophobic drug release (Jung et al., 2003), also helps in avoidance of macrophages uptake and protein absorption (Jung, Jeong, Kim, & Kim, 2004). PA nanoparticles with ionic strength sensitivity can aggregate in the human body with higher ionic strength (0.15) and accumulate in tumor cells for targeted delivery of radioisotopes (Park et al., 2007). Folate modification of PA nanoparticles improves its drug entrapment and release efficiency. Folate modified PA shows higher cytotoxicity towards cancer cells of human nasopharyngeal epidermal and increases the cellular uptake of drug (Zhang et al., 2010). Succinylated pullulan (SP) are functionally appropriate derivative form for proper drug attachment and targeting specific sites (Barker, Tun, Doss, Gray, & Kennedy, 1971; Doane, Shasha, Stout, Rusell, & Rist, 1968). The SP microspheres modified with poly(vinyl alcohol) encapsulated in cellulose acetate butyrate shell shows stability at acidic pH and helps in drug delivery to intestine (Constantin et al., 2007). Grafting of pullulan with poly(N-isopropyl acrylamide-co-acrylamide) followed by treatment with succinic anhydride results sensitivity of SP microspheres towards temperature and pH (Constantin et al., 2011; Fundueanu et al., 2008). Various never-ending liver diseases can be cured by using diethylenetriamine penta acetic acid (DTPA) pullulan as a carrier of interferon (Suginoshita et al., 2002). DTPA reduces the loss of interferon biological activity by acting as a chelaing agent (Suginoshita et al., 2001). It have higher cellular uptake of drug and more cytotoxic for acidic cancer cells. Pullulan-g-poly(Llactide) and pullulan-g-poly(DL-lactide-co-glycolide) help in degradation of cancer cells via

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controlled and long term drug delivery (Cho et al., 2009). L-Lactide acts as hydrophobic moiety and can carry water insoluble anticancer drugs. Temperature triggers the internalization of thermosensitive pullulan-g-poly(L-lactide)-drug nanogels in cancer cells. These nanogels further show stronger cytotoxic effect towards the cancer cells and help in treatment of the chronic disease (Seo, Lee, Jung, & Na, 2012). Pullulan-deoxycholic acid nanogels can be used for efficient drug carrier as deoxycholic acid has hydrophobic moiety and the final complex has low critical association concentration (Na et al., 2006). Folate receptors are present on the apical membrane of polarized epithelia, so they have very limited access to normal cells than diseased cells. The density of folate receptors increases with the increase in cancer cells. Folate decorated pullulan has a high therapeutic index of drug and can easily target brain, kidney, ovary, lung or breast cancer (Lu & Low, 2002). The pH sensitive folate-decorated maleilated pullulan (FA-MP) enhances the cellular uptake of drug, increases cytotoxicity and have higher antitumor effects (Li et al., 2013). These nanoparticles can lower the problem of multidrug resistance, but they also have few side effects. The stimulus-sensitive SpPs nanogels exhibit light controlled release of drugs and also has applications in tissue engineering (Wang, Ju, et al., 2014). The colorless Sps only absorb ultraviolet light and are light-switchable with reversible photochemical process. UV light causes SpPs to merocyanine conversion and visible light grounds reversion of merocyanine to SpP with no free radical generation. These nanogels help in delivery of accurate drug dose to the specific patient via wavelength adjustments (Agasti et al., 2009; Uda, Hiraishi, Ohnishi, Nakahara, & Kimura, 2010). Recently, a hybrid pullulan hydrogel was obtained by a two-step process with pH- and temperature-sensitive properties (Asmarandei, Fundueanu, Cristea, Harabagiu, & Constantin, 2013). The chemical cross-linking of NIPAAm in the presence of CMP and secondary reticulation of the pullulan leads to the formation of porous hydrogels which increase the swelling/deswelling

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properties and enhance the loading capacity. The novel pH-responsive nanoparticle system containing charge reversible pullulan-based shell, poly(β-amino ester) and poly(lactic-co-glycolic acid) core is a promising candidate as a carrier for drugs against hepatocellular carcinoma (Zhang et al., 2016). Hepatocellular carcinoma is the third most common cause of cancer death in humans, particularly in Southeast Asia and Africa (Blechacz & Mishra, 2013).

3.2 Gene delivery Pullulan and pullulan derivatives can act as potential carriers of genes or protein (Grenha & Rodrigues, 2013). The nanoparticles of pullulan with hydrophilic core help in targeted gene delivery without exhibiting any cytotoxicity to the normal cells (Gupta & Gupta, 2004). Pullulan nanoparticles can bind the desired ligands with their surface and protect them from DNase degradation. Evaluation of pullulan derivative formulations as a carrier of genes or DNA to target various organs or cells is depicted in Table 3. Pullulan hydrogels encapsulate pBUDLacZ with high loading efficiency and possess sustained release of DNA. The cationized pullulan has high affinity with DNA and genes as compared to neutral pullulan. Tubular DEAE pullulan hydrogels can be used for gene transfer to vascular smooth muscle cells or local arteries. These hydrogels have good cationic properties which ease the gene targeting to the desired site (Juan, Ducrocq, et al., 2007). These tubular forms can be molded into 3D matrices for protection of genes (SEAP gene) from DNase I degradation and for significant gene transfer (Juan, Hlawaty, et al., 2007). DEAE pullulan can be used for gene therapy by delivery of small interfering RNA (siRNA) to arteries and also facilitates gene silencing in vascular cells (Juan et al., 2009). PEI-pullulan with high cationic charge density helps in liver targeted gene delivery. It shows cellular uptake of drugs and transfection to Hep G2 cells (Rekha & Sharma, 2011a). PEI-pullulan helps in treatment of

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diseases (tumors, viral infections, etc.) by conjugating with siRNA (Kim & Rossi, 2008; Zhang, Tan, et al., 2009). siRNA-PEI pullulan systemic injections results an increase in mortality rate (Kang et al., 2010). Further, folate modification of PEI pullulan leads to higher gene transfection and enhances the gene silencing effect (Wang, Dou, & Bao, 2014). The metal coordination (Zn2+) helps in enhanced gene expression; hence the metal ions can be used for gene or DNA delivery to liver (Hosseinkhani, Aoyama, Ogawa, & Tabata, 2002). Pullulan-g-poly(Llysine) nanoparticles are non-viral gene carrier with negligible cytotoxicity and safe gene delivery (Park et al., 2012). Pullulan-spermine conjugate can act as a delivery system for easy transfection of DNA to brain endothelial cells with higher cellular uptake and acceptable cytotoxicity (Thomsen, Lichota, Kim, & Moos, 2011). This conjugate transports notch intracellular domain (NICD) gene to the stromal cells of bone marrow. NICD gene secrets dopamine via reverse transfection technique and dopamine helps in treatment of Parkinson’s disease (Nagane, Kitada, Wakao, Dezawa, & Tabata, 2009). The complex can also be used for targeting human bladder cancer (Kanatani et al., 2006) and neuronal gene delivery (Thakor, Teng, & Tabata, 2009). Pullulan modified with allergens helps in immunotherapy for effective treatment of Japanese cedar pollinosis (Pawankar et al., 2001). The derivative CHP consists of hydrophobic cholesterol group and hydrophilic pullulan chain (Lee & Akiyoshi, 2004). The nanogels of CHP form hydrophobic cholesterol moieties by self-aggregation which helps in loading various soluble proteins (Akiyoshi, Nishikawa, Shichibe, & Sunamoto, 1995; Nishikawa, Akiyoshi, & Sunamoto, 1994). CHP can be used for immune cell therapy by incorporation of protein (truncated HER2-147) to pullulan and then loading on dendritic cells (Ikuta et al., 2002). CHP can also be used for thermal stabilization and refolding of proteins and enzymes for their delivery to desired sites.

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3.3 Tissue engineering Tissue engineering is a process to enhance self-healing potential of the damaged tissues or organs by creating a cell suitable environment using an appropriate artificial 3-dimensional (3D) scaffold (Tabata, 2003; Singh et al., 2016). 3D scaffold structure varies with type of collagen fibers in extracellular matrix (ECM) of injured tissues (Perumal, Antipova, & Orgel, 2008). A number of biopolymers which have properties same as natural tissues can be molded into various forms like scaffolds, hydrogels, micro-molded matrices, micro-beads and nanoparticles for tissue engineering application (Mallick & Cox, 2013). The application of these biopolymers in tissue engineering mainly involves surface modification practices. The surface properties of pullulan can be easily enhanced by substitution of desired chemical moieties on its hydroxyl groups (Kumar, Saini, Pandit, & Ali, 2012). Pullulan has excellent mechanical properties, high hydration capacity and excellent biocompatible (Chaouat, Le Visage, Autissier, Chaubet, & Letourneur, 2006; Lack et al., 2004; Shingel, 2004). Owing to these properties, pullulan based scaffolds have a promising role in facilitating cell-based dermal replacement, tissue engineering of vascular cells and bone regeneration (Fig. 1). Pullulan hydrogel scaffolds helps in building cell-laden micro-tissue complex to encapsulate injured cells for their regeneration and proliferation (Bae et al., 2011). The hydrogels have hydrated atmosphere, interconnected pore structure and macro-porosity. They assist controlled release of metabolites and nutrients to the targeted sites (Anseth, Bowman, & BrannonPeppas, 1996). The hydrogels of pullulan/dextran composite have application in maxillofacial and orthopedic surgeries. They help in stimulating bone cell differentiation of host mesenchymal stem cells (MSCs). Further, nanocrystalline hydroxyapatite particles (nHAP) coating enhances the mechanical properties of these 3D hydrogels and also improves the attachment ability of cells to

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these constructs. The coating helps in higher osteoconductivity and improves the compressive modulus of these 3D pullulan structures. The nHAP modified pullulan/dextran scaffolds can activate early calcification which leads osteoid tissue formation in osseous and non-osseous sites (Fricain et al., 2013). Amrita and co-workers (2015) revealed the potential of pullulan hydrogels in non-load bearing bone tissue engineering. Pullulan hydrogels allow smooth muscle cells (SMCs) adhesion, spreading and proliferation for vascular tissue engineering. They have antiadhesion properties and help in reducing various problems like infertility, postoperative pain, intestinal obstruction, etc. (Bang, Lee, Ko, Kim, & Kwon, 2016). Novel pullulan bioconjugate can be used for selective breast cancer bone metastases treatment (Bonzi, Salmaso, Scomparin, EldarBoock, Satchi-Fainaro, & Caliceti, 2015). Heparin conjugated carboxylated pullulan have suitability as a substrate for vascular EC growth (Na, Shin, Yun, Park, & Lee, 2003). CMPchondroitin sulfate hydrogels act as a scaffold for cartilage tissue regeneration (Chen et al., 2016). The cholesteryl and acryloyl group-bearing pullulan hydrogel system induces osteoprogenitor cell infiltration both inside and outside the hydrogels which futher helps in delivery of multiple proteins to skull bone defects (Fujioka-Kobayashi et al., 2012). Hydrogels have acquired considerable attention as wound dressings as well. They have similar physical characteristics to natural soft tissues and can absorb a large quantity of aqueous liquid. Hydrogel wound dressings can provide a moist environment to the wound and protect it from bacterial infection (Barnett & Irving, 1991; Lay-Flurrie, 2004; Loke, Lau, Yong, Khor, & Sum, 2000). CMP-cystamine hydrogels possess all these properties along with excellent mechanical strength and can be used potentially as an ideal wound dressing for clinical applications (Li et al., 2011). These hydrogels also act as carriers for antimicrobial agents without any loss in their bioactivity. Pullulan-collagen composite hydrogels have consistent porosity and

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can replicate dermal architecture and successfully incorporate stem cells for early wound healing (Galvez et al., 2009). These hydrogels can shield MSCs from oxidative damage due to their antioxidant properties (Wong, Rustad, Glotzbach, et al., 2011). They can be modified with saltinduced phase inversion technique which improves cell recruitment and activation to accerelate wound healing (Wong, Rustad, Galvez, et al., 2011). The addition of adipose-derived mesenchymal stem cells (ASCs) to pullulan/collagen hydrogels enhances the expression of monocyte chemoattractant protein-1 on both transcriptional and protein levels (Garg et al., 2012). ASCs seeded pullulan-collagen hydrogels have potential for angiogenesis and sustained cell recruitment. Phosphorylated pullulan (PPL) possesses good adhesion property with hard tissue via ionic bonding and helps in bone defect repair (Shiozaki et al., 2011). PPL can act as carrier for antibacterial agents and fasten the regeneration of femur bone defects. Further, the addition of βtricalcium phosphate enhances PPL’s water solubility, biocompatibility and mechanical properties for bone tissue engineering (Takahata et al., 2015). The carboxylated pullulan in conjugation with human-like collagen and 1,4-butanediol diglycidyl ether can act as a promising soft filler for tissue engineering (Li et al., 2015). The 3D pullulan-cellulose acetate scaffolds possess cytocompatibility to ease cell attachment, spreading and proliferation for skin tissue engineering (Atila, Keskin, & Tezcaner, 2015). The nanogels of CHP can entrap hydrophobic drugs for tissue engineering. CHP nanogels conjugated with prostaglandin E1 generate a moist environment in full thickness wounds which promotes neovascularization, neoepithelialization and controlled release of prostaglandin E1 for regeneration of injury (Kobayashi, Katakura, Morimoto, Akiyoshi, & Kasugai, 2009). The novel amphiphilic pullulan nanogel-crosslinked hydrogels possess strong mechanical properties, higher biodegradability and can act as universal scaffolds for tissue engineering (Hashimoto, Mukai, Sawada, Sasaki, & Akiyoshi, 2015). The hydrogels can work properly even

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without any growth factors to improve cell infiltration and regenration of damaged tissues. The microbeads of pullulan/dextran-nHAP with few hundred millimeter size can act as potential scaffolds for repairing bone defect of variable size (Schlaubitz et al., 2014). These microbeads can augment osteoid tissue formation by enhancing the interconnectivity of cells and maintenance of their trabecular-like structural organization. Pullulan microspheres are another convenient way to repair bone defects (Aydogdu, Keskina, Baran, & Tezcaner, 2016). These microspheres are surface modified to have biocompatible spherical microcarriers of an injectable size which can be applied to small incisions. The stability and compatibility of pullulan microspheres can be enhanced by the addition of trisodium trimetaphosphate, silk fibroin coating and simulated body fluid incubation. Pullulan can be casted in films which possess excellent biomedical application i.e. skin tissue engineering. The pullulan films are biodegradable, biocompatible, have good mechanical properties and can augment wound healing (Suguna, 2014). They can be used as a dressing for incision and excision injuries by increasing the rate of contraction and re-epithelialization. Films prepared from a blend of pullulan, pyruvate, antioxidant, unsaturated and saturated fatty acids can act as a bioadhesive wound dressing (Leung, Martin, & Leone, 2001). They can also be improvised with additional medicinal agents like bacitracin zinc, neomycin sulfate, polymyxin B sulfate, etc. (Leung, Martin, & Leone, 2005). Porous nanofibrous pullulan/poly(vinyl acetate) membranes significantly enhance cell proliferation rate and can be used for growth of fibroblasts (Jeong, Kim, & Kim, 2013). The crosslinked nanofibrous membranes of pullulan/tannic acid/chitosan composite are water stable and allow cells to pass through their fibrous structure for interlayer regeneration of deep and intricate wounds (Xu, Weng, Gilkerson, Materon, & Lozano, 2015).

3.4 Vaccination

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The human body consists of different cell types such as blood cells, brain cells and skin cells, with specific functional genes in them. These genes signal the cells to make proteins for their healthy functioning and formation of tissues. Cancer begins with the mutation in specific genes of healthy cells which lead to change the functionality of proteins and also cause abnormal growth of cells. Cancer has the ability to invade surrounding tissues, metastasize and may be fatal if left untreated. The treatment of cancer involves proper immune response of antigen peptides against the cancer cells. The development of an efficient system for delivery of these peptides to major histocompatibility complex (MHC) molecules is the major issue. The exogenous soluble proteins cannot induce CD8+ cytotoxic T lymphocytes (CTLs) as they can rarely enter the MHC class I pathway and the endosome (Apostolopoulos, Pietersz, Loveland, Sandrin, & McKenzie, 1995; Fenton, Taub, Kwak, Smith, & Longo, 1993; Malik, Gross, Ulrich, & Hoffman, 1993). Various adjuvants can be used in vaccination to modify the immune response (Noguchi et al., 1991; Schirmbeck, Bohm, & Reimann, 1994). However, novel peptide and protein vaccines can enhance the cellular immunity with or without adjuvants (Disis et al., 1996; Raychaudhuri, John, & Morrow, 1993). Cholesterol bearing pullulan (CHP) and cholesterol bearing mannan (CHM) complexed with truncated protein encoded by human epidermal growth factor receptor 2 (HER2) proto-oncogene can act as a protein delivery system for cancer immunotherapy (Akiyoshi et al., 1993). HER2 has the ability to over express in breast, stomach, ovarian and bladder cancers with high frequency (Slamon et al., 1989). The hydrophobized CHP and CHM can enhance the cellular and humoral immunity of soluble proteins (Gu et al., 1998). These novel vaccines have potential role in cancer prevention and cancer therapy (Shiku et al., 2000). CHP-HER2 vaccine can be used to cure cancer in bone marrow derived dendritic cells. This vaccine can safely induce HER2 specific CD8+ and CD4+ T cell immune responses (Kageyama et al., 2008; Kitano et al., 2006). It

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can also induce HER2 specific humoral responses in HER2 expressing tumor patients and further granulocyte-macrophage colony-stimulating factor accelerates their responses. Hence CHP-HER2 vaccine can be considered efficient for application in various categories of tumor antigens. Shiku and Kageyama (2008) patented the method for preparation of cancer vaccine by combining cancer antigen (HER2, NY-ESO-l or MAGE A4) and hydrophobized polymer (CHP or CHM) with an agent extracted from hemolytic Streptococcus. The agent can bind with a tolllike receptor to stimulate and activate the entire antigen presenting immune cells i.e. killer T cells and helper T cells. The CHP or CHM vaccine helps in production of antibodies against the cancer antigens and intensify the cellular immunity to damage cancer cells. NY-ESO-1 is the most immunogenic tumor antigen with expression range of 20 to 80% depending on the stage of tumor (Gnjatic et al., 2006). It can be expressed in cancer cells of breast, bladder, prostate, melanoma, sarcoma and ovaries. NY-ESO-1 protein helps in treatment of existing cancer and prevents further development of NY-ESO-1 positive cancers (Karbach et al., 2010). CHP-NY-ESO-1 vaccine dose (200 µg) can induce efficient immune response and have better survival benefits (Kageyama et al., 2013). NY-ESO-1 antigen can also be expressed in approximately 20-30% of esophageal cancers (Fujita et al., 2004). The combined vaccine of CHP-NY-ESO-1 and CHP-HER2 is safe for esophageal cancer patients with mild adverse effects. OK-432 stimulates toll like receptor 4 and activates antigen-presenting cells (Okamoto et al., 2004); but it has no effect on immunogenicity of both NY-ESO-1 and HER2 antigens. Hence, targeting multiple tumor antigens is feasible even without antigenic interactions between the antigens (Aoki et al., 2009). Pneumococcal disease is an infection caused by bacterium Streptococcus pneumonia. The primary source for spread of pneumococcal infection among humans is nasal carriage of pneumococci (Leiberman, Dagan, Leibovitz, Yagupsky, & Fliss, 1999). An optimal vaccine

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strategy can induce protective immunity against both colonization and invasive disease. A nanogel based delivery system consisting of cationic CHP (cCHP) and pneumococcal surface protein A can act as an adjuvant free mucosal vaccine against the respiratory infection of Pneumococcus (Kong et al., 2013; Yuki et al., 2012). Nanogel-based nasal vaccination can induce high levels of antigen-specific serum IgG, nasal and bronchial secretory IgA antibodies. The intranasal coadministration of Clostridium botulinum type-A neurotoxin (BoHc/A) and cCHP nanogel can act as a vaccine (Nochi et al., 2010). It can adhere with nasal epithelium and then transferred to mucosal dendritic cells. The vaccine helps in inducing antibody responses without addition of any mucosal adjuvant. The cCHP-BoHc/A vaccine does not accumulate in olfactory bulbs or brain. Additionally tetanus toxoid can be conjugated with cCHP nanogels to induce strong tetanustoxoid-specific systemic and mucosal immune responses. Mitsuhasi and Koyama (1987) patented the process for formation of virus vaccine with higher antibody production ability. The vaccine can be prepared by covalently conjugating virus to the saccharide (pullulan, elsinan, etc., and their hydrolysates). The covalent bonding between virus and saccharide enhances the production of IgG and IgM which further helps in treatment of viral diseases and diminishing IgE production.

3.5 Film forming agent The pharmaceutical dosage forms are the means by which drug molecules are delivered to the site of action within the body. These dosage forms should be of good quality so as reduce the environmental effect on drug. In 1970's, rapidly dissolving dosage forms were introduced as an alternative to the conventional tablets. These novel dosage forms are lyophilized wafers, thin strips and films, prepared by freeze drying, vacuum drying and spray drying. Film coating can be prepared by compression, extrusion and evaporation of mixture containing various polymers e.g.

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gelatin, pullulan, pullulan ester, polyvinyl alcohol, amylase, etc. (Hijiya & Shiosaka, 1975b, c). Coating helps in ingestion ease of capsule, covers unpleasant taste of drug and improves patient compliance. Coating improves tablet’s appearance and chipping resistance, along with targeted drug release to the particular site and helps in reducing tablet processing time (Childers, Oren, & Seidler, 1991). The shelf-life and strength of capsule can be increased by coating with sugar solution containing pullulan derivatives i.e. pullulan esters and/or pullulan ether produced by substitution reactions using radicals (Miyamoto, Goto, Sato, Okano, & IIjima, 1986). Soft capsules can be film coated with blend of pullulan and other binding substrates e.g. gelatin, agar, etc. These pullulan coated capsules exhibit good strength and higher solubility (Hayakawa, Maruyama, & Fukasawa, 2011). Capsules coating can be done with water-soluble neutral pullulan which forms solid low oxygen permeable film on drying. The mixture consists of pullulan solution, surfactants and setting systems (Scott, Cade, & He, 2005). These coatings facilitate targeted delivery of encapsulated drug to the desired sites. Childers et al. (1991) patented the process for coating tablets via aqueous diffusion method using a mixture of hydrophilic pullulan and insoluble ethylacrylatemethylacrylate complex. Pullulan coating on tablet surface along with some lubricants (polyethylene glycol), suspending agent (Talc) and colorants can reduce the brownish change in color of tablet with time (Izutsu, Sogo, Okamoto, & Tanaka, 1987). Coating reduces the disintegration of tablet and improves the appearance of tablet to total white color or desired color. Addition of pullulan improves the stability and bioavailability of drugs (nisoldipine) by enhancing their solubility (Chatap, Maurya, Deshmukh, & Zawar, 2013). Further involvement of chitosan reduces the flushing action of saliva over drugs and together these polymers can lower the wetting time and disintegration time of tablets. The pullulan coated tablets facilitate easy execution of bound molecules to remote sites by getting immediately dissolved in water. These tablets remain

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stable at room temperature for long period and help in easy shipping and storage of bioassay molecules (Jahanshahi-Anbuhi et al., 2014). The hardness of tablets augments with increase in concentration of pullulan (Patel, Chauhan, Patel, & Patel, 2012). Pullulan and its derivatives can be molded into physiologically acceptable and orally consumable films (Nagar, Chauhan, & Mohd Yasir, 2011). The oral films consist of saliva stimulating agents, which help in disintegration of pullulan on tongue or in buccal cavity (Dixit & Puthi, 2009). Various drugs can bind with these oral films and released to the systemic circulation. These pullulan films can be modified by addition of antimicrobial agents e.g. essential oils thymol, eucalyptol, methanol, methyl salicylate, etc. (Leung, Loene, Kumar, Kulkarni, & Sorg, 2006), salivary stimulants and deodorizing agents to cure moth problems like dental plaque, gingivitis and bad breath (Leung, Loene, Kumar, Kulkarni, & Sorg, 2008). Taste masking agents (amberlite) can be added to pullulan edible films to increase their compliance with patients (Bess, Kulkarni, Ambike, & Rampay, 2010). The rapidly dissolving dosage form containing cetirizine hydrochloride coated with pullulan possesses stability for six months at room temperature and has disintegration time of 30 sec (Mishra & Amin, 2011). Various other factors can be added to have acceptable mechanical properties e.g. casting surfaces (teflon), plasticizers (polyethylene glycol 400) and taste masking components (sweeteners, flavors, citric acid, etc.). New pullulan-tailormade derivatives prepared by matrix-assisted pulsed laser evaporation (MAPLE) processing possess applications as cell growth substrate, artificial organ lining and immunology testing agents. MAPLE processing is a deposition technique which modulates drug release by varying the thickness of pullulan in multilayer implementations (Cristescu et al., 2006; Jelinek et al., 2007). High quality pullulan films can be prepared by using MAPLE processing e.g. triacetate-pullulan (Cristescu et al., 2006), cinnamate-pullulan and tosylate-pullulan (Jelinek et al., 2007). The

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production cost of pullulan based films can be reduced by blending pullulan with different compatible polymers like carboxymethyl cellulose and sodium alginate (Tong, Xiao, & Lim, 2008).

3.6 Molecular chaperons Molecular chaperons are unique supramolecular assemblies between self-aggregated hydrophobic polymer and soluble protein. The chaperons can enhance the thermal stability of protein structure and its enzymatic activity. The self-assembling method is an efficient and versatile technique using associating polymers as building blocks for preparing functional different nanobiomaterials and nanogels. The nanoparticles of hydrophobized cholesterol bearing pullulan (CHP) can act as molecular chaperones for application in enzyme engineering (Nishikawa et al., 1994). CHP increases thermal stability of protein by entrapping the denatured form of αchymotrypsin inside CHP self-aggregate and releasing the refolded protein (Fig. 2). The refolded form of chymotrypsin remain stable even after heating and have 74% of original enzyme activity with no thermal unfolding in protein structure. The hydrogel nanoparticles of hydrophobized CHP also enhance the thermal stability and refolding of heat denatured carbonic anhydrase B (Akiyoshi, Sasaki, & Sunamoto, 1999). CHP can completely prevent the irreversible aggregation of CAB on heating. β-cyclodextrin (CD) also plays important role in dissociation of self-aggregated CHP (Nomura et al., 2005) and release refolded form of enzyme with almost 100% recovery of activity (Akiyoshi et al., 1999). CD controls binding ability of host nanogels to proteins. CHP nanogels can prevent the aggregation of GdmCl denatured citrate synthase and carbonic anhydrase by trapping the refolded intermediate proteins (Nomura et al., 2003). CHP nanogels help in stability of lipase both thermally and colloidally (Sawada & Akiyoshi, 2010). The

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lyophilization-induced lipase aggregation and denaturation on heating of lipase is inhibited by complexing it with CHP nanogels. Along with thermostability, CHP improves lipase activity also. The thermal stabilization and refolding of denatured horseradish peroxidase (HRP) can be enhanced by aggregation with CHP nanogels (Sawada, Sasaki, Nomura, & Akiyoshi, 2011). Addition of CD to the complex increases the recovery of native HRP enzyme activity up to 80%. The hybrid hydrogels of CHP-hyaluronan are used for sustained delivery of protein without any denaturation (Hirakura et al., 2010). These hydrogels are degradable at cross-linking sites and HA backbone helps in biocompatible and safe release of proteins. Various stimulus-sensitive (heat and light) and surface modified nanogels can be designed for recovery of proteins and enzymes. The photoresponsive nanogels of spiropyrane-bearing pullulan can act as molecular chaperones via stimulus sensitive activity (Hirakura, Nomura, Aoyama, & Akiyoshi, 2004). These nanogels are switched on by photostimulation and they significantly enhance the activity of enzyme citrate synthase. The polymerization of CHP with 2methacryloyloxyethyl phosphorylcholine by radical polymerization in a semi-dilute aqueous solution enhances the properties of CHP nanogels (Morimoto, Endo, Ohtomi, Iwasaki, & Akiyoshi, 2005). The complex shows high chaperone-like activity via host-guest interaction by trapping denatured insulin and releasing in refolded form. CHP nanogels have comparable properties with natural chaperone i.e. GroEL-GroES system (Asayama, Swada, Taguchi, & Akiyoshi, 2008). These CHP nanogels can be immobilized on various surfaces e.g. beads, for batch wise renaturation and refolding chromatography of denatured proteins (Nomura et al., 2003).

3.7 Plasma expander

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The plasma expander/blood plasma substitute is a colloidal component which has similar properties to the blood plasma, such as proper colloidal osmotic effect, osmotic pressure and viscosity. The plasma expander used should be non-pyrogenic, non-toxic and compatible with the human body (Igarashi, Nomura, Naito, & Yoshida, 1983). A colloidal component of modified pullulan has great therapeutic potential and can be used as blood plasma substitute (Kulicke & Heinze, 2006; Shingel & Petrov, 2002). As illustrated in Fig. 3, when polytrauma occurs due to a severe accident or other circumstances, there occurs very high amount of blood loss which causes decrase in osmotic pressure. The blood plasma volume expanders have to be used in order to maintain the blood circulation and maintain the osmotic pressure of blood vessels. The adequate concentration of derivatized pullulan colloid, with enhanced biocompatibility, can simply metabolize and helps in balancing the blood loss. It can be easily removed after the desired therapeutic effect (Akiyoshi et al., 1999; Donabedian & McCarthy, 1998; Igarashi et al., 1983; Petrov & Shtykova, 1988). γ-irradiated pullulan with low molecular weight and viscosity have potential application as a blood plasma substitute (Gapanovich et al., 1992; Phillips, 1972). Various surfactants (cationic and anionic) can conjugate with γ-irradiated pullulan to give fractions with a narrower molecular weight distribution range (Catley, Ramsay, & Servis, 1986; Kikuchi, Taguchi, Sakano, & Kobayashi, 1973; Tsianou & Alexandridis, 1999). These fractions of modified pullulan can be used for production of blood plasma substitute (Shingel & Petrov, 2002). Due to different chemical modifications, pullulan becomes resistance to amylase action which suppresses the rate of pullulan deterioration in blood vessels (Shingel & Petrov, 2001). Various hemodynamic constraints can be controlled by isovolumetric blood replacement with modified pullulan, such as blood circulation index, contraction rate of cardiac, volumetric cardiac output and normalization of blood microcirculation. The fractionalized pullulan with molecular weight distribution range of

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30,000 to 90,000 helps in treatment and sometimes prevention of hemorrhage (Igarashi et al., 1983). Fractionation can be done with water miscible organic solvent such as methanol, ethanol, acetone and isopropanol. Various isotonic agents used for dilution of fractionated pullulan are sodium chloride, glucose, sorbitol, xylitol, sodium chloride, glucose mixture, etc.

3.8 Insulinotropic activity Insulinotropic activity is a process to stimulate or affect the production of insulin in diabetic patients and to control the further activity of insulin for treatment of diabetes. Diabetes is a chronic condition which affects the ability of body to break down sugars of food into simpler forms, which leads to accumulation of unutilized sugar in blood. High blood glucose level can damage the blood vessels of kidneys, heart, eyes, etc. and if left untreated can eventually cause kidney disease, heart disease, blindness and nerve damages in feet. Research has been done to produce artificial pancreas by combining insulin-secreting cells with biomaterials for the treatment of diabetes mellitus (Colton & Avgoustiniatos, 1991; Hou & Bae, 1999; Lanza, Sullivan, & Chick, 1992; Mikos, Papadaki, Kouvroukoglou, Ishaug, & Thomson, 1994). The problem is immunomodulating membranes and extracellular matrix has impact on physiological functioning of these artificial structures. Along with, a large number of pancreatic islets and proper oxygen supply are crucial requirements (Colton, 1995; Keymeulen, Teng, Vetri, Gorus, & Pipeleers, 1992; Suzuki et al., 1998; Warnock et al., 1990). These issues can be resolved by using pullulansulfonylurea (SUP) conjugate (Kim, Su, Kun, Sung, & You, 2003). SUP has stimulatory effect on insulinotropic activity of pancreatic islets. SUP helps in regaining the insulin secretion pattern of islets microcapsule. SUP conjugated islets microcapsule can sustain their morphology in an intact manner and show higher insulin secretion at both low and high glucose concentrations as compared

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to islets without SUP. These pullulan conjugate microcapsules have better ability in responding to any change in glucose levels than other microcapsules.

3.9 Medical imaging Medical imaging is a technique used for visual representation of body interior by labeling the inner body cells with fluorescent probes. Quantum dots (QDs) can be used as fluorescent probes for live cell imaging to track whole cells or intracellular biomolecules. These QDs are semiconductor nanocrystals with excellent properties such as broad excitation, bright fluorescence, high photo-stability and narrow emission spectra (Alivisatos, 1996; Arya et al., 2005; Chan et al., 2002; Michalet et al., 2005; Mitchell, 2001). However, the delivery of QDs into living cells is a major challenge as uptake efficiency of cells for QDs is generally low. Along with a high amount of QDs is required for proper cell imaging due to their distinct surface and size properties. The cationic liposome can act as carrier for QDs to overcome the above issues (Derfus, Chan, & Bhatia, 2004; Voura, Jaiswal, Mattoussi, & Simon, 2004). Cationic liposomes can interact with negatively charged cell membrane and promote endocytosis. Instead of their high efficiency for QDs delivery, liposomes form aggregates of several hundred nanometers in cytoplasm, which limits their use in bioimaging. The amine modified cholesterol bearing pullulan (CHPNH2) as a carrier for QDs can surmount all these difficulties and allow proper intracellular labeling (Hasegawa, Nomura, Kaul, Hirano, & Akiyoshi, 2005). The cellular uptake efficiency is much higher for CHP conjugated QDs as compared to conventional carriers. The monodisperse hybrid QDs-CHPNH2 nanoparticles possess promising application as fluorescent probe for medical imaging of various human cells. Infrared dye 900 conjugated CHP nanoparticles, i.e. near-infrared polymer nanogels (NIR-PNG),

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show least dispersion and longer retention in sentinel nodes (Kong et al., 2015). These NIR-PNG tracers have good sensitivity and specificity for sentinel nodes and can be potentially used as optimal tracers in gastric cancer for sentinel node navigation surgery. DTPA can be used for magnetic resonance imaging of the heptocytes for cancer treatment (Yim et al., 2011).

4. Future perspectives The native pullulan and its derivatives possess numerous biomedicinal and pharmaceutical applications due to their unique structural properties. These modified pullulan derivatives have been explored for in vitro studies only. The major focus of research should be on investigation of in vivo efficiency of pullulan and its derivatives. The derivative like cationized pullulan has been successfully developed as proficient gene carrier for in vitro studies (Priya et al., 2014; Rekha & Sharma, 2011a). Additional efforts should be done for evaluation of their in vivo gene transfection efficacy. Further, cationized pullulan coated stents can be used for treatment of cardiovascular diseases. The folate conjugated pullulan acetate nanoparticles are successfully used as drug carriers in vitro (Zhang et al., 2011). In vivo examination of these nanoparticles for treatment of cancer cells is yet to be done. Pullulan-spermine conjugate has been explored for in vitro DNA release studies to treat the neuron related diseases and also approved for gene transfer to human stem cells (Thakor et al., 2009). Further, in vivo experimentation for targeted delivery of genes/DNA to various stromal cells is under consideration. The hydrogels of pullulan have non-fouling surface properties and are less explored in bone tissue engineering applications (Amrita et al., 2015). In future, clinical trials can be done to investigate their role in promoting the growth of bone tissues which leads to the bone formation in large bone defect. The tubular molds of pullulan-based hydrogels can be studied for in vivo

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implantation of vascular cells which may lead to augmentation of cellular growth and further tissue formation (Autissier, Letourneur, & Le Visage, 2007). The cholesterol bearing pullulan hydrogels may be examined as a tool for improving the protein-dependent bone healing efficiency. Their conjugation with proteins may open the possibility of vaccination and also help in immunotherapy to control tumor expansion in high-risk stage patients (Karbach et al., 2010). The surface modified cholesterol bearing pullulan hydrogels may have potential application as artificial chaperone for protein refolding in post-genome era (Morimoto, Endo, Iwasaki, & Akiyoshi, 2005; Nomura et al., 2003). The sulfonylurea-pullulan conjugate enhances insulin secretion in islet microcapsules at different glucose concentrations (Kim et al., 2003). However, biohybrid artificial pancreas can be developed in future for long term maintenance of insulin secretion. Pullulan can be casted in microporous beads which may be applicable in mineralization of bone defect and its repair (Schlaubitz et al., 2014). The future use of these microporous beads may be generation of a novel fill-up material for repair of variable sized bone defects. These findings suggest that emerging innovations can improve the properties of pullulan, thereby opening new avenues for applications of pullulan in biomedical field.

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Fig. 1: Role of pullulan based scaffolds in tissue engineering-based regeneration therapy. SMCs: Smooth Muscle Cells; BMP2: Bone Morphogenetic Protein 2; HFGF18: Human Fibroblast Growth Factor 18; MSCs: Mesenchymal Stem Cells; HUVECs: Human Umbilical Vein Endothelial Cells; MEFCL: Mouse Embryonic Fibroblast Cell Line; HOCL: Human Osteoprogenitor Cell Line; PE1: Prostaglandin E1.

Fig. 2: Schematic representation of pullulan as an artificial molecular chaperone.

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Fig. 3: Functionality of pullulan as a blood plasma expander.

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70

Table 1 Biomedical applications of various pullulan derivatives. Pullulan derivative

Structure

Formulation

Biomedical application(s)

Reference

Cholesterol bearing pullulan

Immunoliposomes

Human lungs, human Sunamoto stomach & mouse lungs (1987) targeting

Pullulan acetate

Nanoparticles

Human throat epidermal Zhang, Gao, et al. carcinoma cell line (2009) treatment

Carboxymethyl pullullan

Nanoparticles

Mouse breast treatment

Succinylated pullulan

Microspheres

Intestine targeting

Constantin et al. (2007)

Pullulan-b-polyetheramine

Films

N.S.

Belbekhouche et al. (2011)

et

al.

cancer Lu et al. (2009)

Contd…. 71

Table 1 Contd…. Pullulan folate

Nanogels

Photodynamic therapy

Bae & Na (2010)

Maleilated pullulan

Conjugate

Tumor cells treatment

Zhang et al. (2011)

Diethylene triamine penta acetic acid pullulan

Magnetic Liver targeting resonance functionalities

Yim et al. (2011)

All trans retinoic bearing pullulan

Nanogels

Anticancer therapy

Lee et al. (2013)

Nanospheres

Cancer cells treatment

Jeong et al. (2006)

acid

Poly(DL-Lactide-coglycolide)-grafted pullulan

Contd…. 72

Table 1 Contd…. Pullulan-g-poly(L-lactide)

Nanoparticles

Cancer cells treatment

Cho et al. (2009)

Pullulan/deoxycholic acid

Nanogels

Cancer cells treatment

Na et al. (2006)

Diethylaminoethylamine pullulan

3 D matrices

Gene transfer

Juan, Hlawaty, et al. (2007)

Polyethylenimine pullulan

Conjugate

Liver cell gene delivery

Rekha & Sharma (2011a)

Pullulan-g-poly(L-lysine)

Nanoparticles

Gene carrier

Park et al. (2012)

73

Table 2 Evaluation of targeted drug delivery formulations prepared from various pullulan derivatives. Pullulan Formulation(s) Drug(s) Performance derivative In vitro In vivo CHP

Reference(s)

Nanoparticles

Doxorubicin

After 24 h at 37°C and pH=7.4, 93.57% Showed antitumor effect on broad Li, Yawata, et al. drug released range of dose (40 mg/Kg) in Balb/c (2014) nude mice and CHP coating reduced adverse effects of drug

Nanoparticles

Epirubicin

N.S.

BCHP

Nanoparticles

Mitoxantrone

At 37°C pH=3.5 (tumor cells), >90% N.S. drug released

Yang, Wang, et al. (2014)

CMP

Nanoparticles

Doxorubicin

At pH=5.0 (tumor cells), 67% drug N.S. released due to equilibrium shift and hydrazone bond of free drug-polymer cleaved

Lu et al. (2008)

Conjugate

Doxorubicin

N.S.

Nogusa, Yamamoto, et al. (2000); Nogusa, Yano, et al. (2000)

Nanoplexes

Doxorubicin

At pH=5.0 (tumor cells), 85% drug N.S. released and polymer reduced off target side effects of drug

Improved pharmacokinetic parameters Shen et of drug in female Wistar rats: Half-life (2014) time (19.33 h); Maximum plasma concentration (10.45 mg/L); Area under curve from time 0 to time infinity (48.36 h.mg/L); Mean residence time (25.76 h); Clearance (0.23 L/h/Kg)

Rapidly cleaved even with a low enzyme activity and contributed to loss of the preferential drug release with high enzyme activity

Vora et (2014)

al.

al.

Contd…. 74

Table 2 Contd…. OCMPJe

Nanoparticles

Diclofenac At 40°C, ~70% drug released for both N.S. (acidic); α- drugs (acidic and hydrophobic) tocopherol (hydrophobic)

Mocanu et al. (2014)

ACMP

Nanoparticles

Doxorubicin

At pH=5.0 (tumor cells), >85% drug N.S. released

Li, Bian, et al. (2014)

PA

Microspheres

Naproxen

After 13 h at pH=7.4, ionization of N.S. carboxylic group occured which led in swelling of polymer and 90% drug released

Bishwambhar & Suneetha (2012)

Nanospheres

Clonazepam

N.S.

Jeong et al. (1999); Jung et al. (2003)

Nanoparticles

Silymarin

1:4 ratio of drug and polymer showed N.S. proper drug release at 24 th h

Kumar (2012)

Microsphere

Indomethacin

At pH=7.4, ~90% drug diffused out

Na et al. (1997)

Nanoparticles

Adriamycin

At pH 6.8, cytotoxicity towards breast N.S. tumor cell line was enhanced

Nanoparticles

Lopinavir

Drug followed biphasic release pattern via reciprocal time release model and after 6.96 h 50% drug released with mean dissolution time of 40.71 h

Nanoparticles

Epirubicin

After 72 h at pH=7.4, 61.4% drug N.S. released

Zhang, Gao, et al. (2009)

Microspheres

Diclofenac

Within 1 h at pH 7.4, polymer swollen N.S. and 82% drug released

Fundueanu et al. (2003)

PCyD

N.S.

N.S.

et

al.

Na, Lee, et al. (2003)

Improved pharmacokinetic parameters Ravi et of drug in male Wistar rats: Area under (2014) curve (3782.5 h.ng/mL); Maximum plasma concentration (1060.34 ng/mL); Mean residence time (6.75 h)

al.

Contd…. 75

Table 2 Contd…. PS

Microspheres

Diclofenac

At pH=7.4, swollen up to 20-times in N.S. intestinal fluid causing rupture of CAB and higher drug release

Conjugate

Cisplatin

After 4 h at pH=7.6, drug release rate After 24 h post administration, drug Wang improved distributed in tumor-bearing cells of (2015) nude mice and inhibited tumor growth at doses of 3.5 and 7.0 mmol/kg

Microspheres

Lysozyme

At pH 7.4, polymer showed phase N.S. transition and swollen up to 28-times for efficient drug release

Fundueanu et al. (2008)

Poly(NIPAA Microspheres m-co-AAm) PS

Lysozyme

At 37°C, polymer showed phase N.S. transition and swollen up to 28-times for efficient drug release

Fundueanu et al. (2008)

Microspheres

Fluorescein isothiocyanatedextran 4000

At 37°C, pendant units collapsed N.S. without any steric interaction and >75% drug released

Fundueanu et al. (2010)

Nanogels

Doxorubicin

69% drug released with first order N.S. kinetics

Cho et al. (2009)

Nanogels

Doxorubicin

After 50 h at 42°C, polymer became N.S. more compact causing 100% drug release by squeezing effect

Seo et al. (2012)

Nanogels

Doxorubicin

After 30 h at pH=5.0 (tumor cells), 53% N.S. drug released

Li et al. (2013)

Conjugate

Doxorubicin

After 30 h at pH=7.4, 68.71% drug N.S. released

Zhang (2011)

Nanospheres

Clonazepam

Induced higher hydrophilic N.S. environment which lowered hydrophobic drug-polymer bond and drug diffused out easily

Jung et (2004)

CPS

PUPL

FA-MP

PEP

76

Constantin et al. (2007) et

et

Contd….

al.

al. al.

Table 2 Contd…. FP

Nanogels

Pheophorbide-A Showed >80% fluorescence intensity due to pullulan-Pheophorbide A ester bond cleavage by enzymatic reaction (8.5 U/mL esterase and 0.1% tween80)

Shown maximum fluorescence at 12 h Bae and in Balb/c nude mice having HeLa cells (2010) and retained singlet oxygen quantum yield (0.43) for ~30 days

Conjugate

Doxorubicin

After 12 h pH=5.5 (cancer cells), 50% Improved pharmacokinetic parameters Scomparin et al. drug released and complete release of drug in Balb/c mice: Central (2011) taken after 40 h compartment volume (1.01 mL); Area under the curve (1.247 h.mg/mL); Half-life (α=0.2 h, β=15.7 h); Steady state distribution volume (1.7 mL); Clearance (0.08 mL/h); Elimination rate constant (0.078); Central to peripheral compartment rate constant (2.71); Peripheral to central compartment rate constant (3.86)

Nanoparticles

Epirubicin

After 72 h pH=7.4, ~90% drug released N.S.

Zhang (2010)

et

Na

al.

PEu

Microparticles/ Risedronate tablets

Followed Korsmeyer-Peppas model N.S. with complete drug release at 450 min

Velasquez et al. (2014)

PUL/DOCA

Nanogels

Doxorubicin

After 24 h pH=7.5, 61% drug released N.S. following first order kinetics

Na et al. (2006)

PulG

Nanospheres

Adriamycin

Following diffusion mechanism >80% N.S. drug released in 10 days

Jeong (2006)

PAuNPs

Nanoparticles

5-Fluorouracil

After 48 h pH=7.4, 45.88% drug After 2 h of administration ~55% drug Ganeshkumar et released accumulated in male Wistar rat liver al. (2014) Table 2 Contd…. and ~50% cancer growth inhibited

CAP

Conjugate

Interferon

N.S.

et

al.

Improved accumulation of drug in Xi et al. (1996) Balb/c mice liver with higher induction of 2’,5’-oligoadenylate synthetase 77

Contd….

PP

Conjugate

Doxorubicin

N.S.

Improved antitumor efficacy and drug Lu et al. (2010) circulation time in blood along with reducing toxicity of drug and hemolysis

NURPA

Nanoparticles

Methotrexate & combretastatin A4

After 24 h pH=5.0 (tumor cells), 85.3% Enhanced antitumor and Wang drug released antiangiogenic effects with prolonged (2013) circulation time in Balb/c nude mice containing human heptoma PLC/PRF/5 cells

OURPA

Nanoparticles

Adriamycin

After 24 h pH=5.8 (tumor cells), 72.1% N.S. drug released

Guo et al. (2014)

MoP

Particles

Salbutamol sulfate

Showed a predominant deposition at the N.S. lower stages in lung cells and fine particle fractions (FPF, particle size 4.46 μm) was as high as 59.7%

Xu, Jiang, et al. (2015)

Abbreviations: N.S.: Not specified; CHP: Cholesterol bearing pullulan; BCHP: Biotin modified cholesterol bearing pullulan; CMP: Carboxymethyl pullulan; OCMPJe: Oxidized carboxymethyl pullulan jeffamines; ACMP: Aminated carboxymethyl pullulan; PA: Pullulan acetate; PCyD: Pullulan cyclodextrin; PS: Pullulan succinylate; CAB: Cellulose acetate butyrate; CPS: Carboxylated pullulan succinylate; Poly(NIPAAm-co-AAm)PS: Poly(N-isopropylacrylamide-co-acrylamide) pullulan succinylate; PUPL: Pullulan-g-poly(L-lactide); FAMP: Folate-decorated maleilated pullulan; PEP: Poly(ethylene glycol) grafted pullulan; FP: Folate modified pullulan; PEu: PullulanEudragit® S100; PUL/DOCA: Pullulan/deoxycholic acid; PulG: Poly(DL-lactide-co-glycolide) grafted pullulan; PAuNPs: Pullulan stabilized gold nanoparticles; CAP: Cyanuric chloride; PP: pH sensitive pullulan; NURPA: N-Urocanyl pullulan; OURPA: O-Urocanyl pullulan; MoP: Modified pullulan

78

et

al.

Table 3 Evaluation of targeted gene delivery formulations prepared from various pullulan derivatives. Pullulan Formulation Gene(s) Performance derivative In vitro In vivo DEAEP

Transfection of DNA in vascular N.S. smooth muscle cells

Juan, Ducrocq, et al. (2007)

3 dimensional pSEAP matrices

Transfection of vector expressing N.S. alkaline phosphatase and improved activity by 150-fold

Juan, Hlawaty, et al. (2007)

Nanoparticles

siRNA

N.S.

Nanoparticles

pGFP & pGL3

Expression of pGFP in HepG2 N.S. cells by pGFP:polymer (1:4) and pGL3 expressed higher in the presence of serum

Nanoparticles

pGFP

pGFP:polymer (1:5 ratio) showed After 3 h, 46.7% gene Rekha & Sharma gene transfection of pGFP transfection and liver binding (2011b) in mice

PPB

Nanoplexes

p53

PEI (10 kD) showed good N.S. transfection efficacy and 90% death in C6 cells

Ambattu & Rekha (2015a, b)

PPF

Complex

siRNA and Polymer:DNA (6.25) showed N.S. plasmid DNA higher gene transfection in presence of serum. Polymer:siRNA (12.5) showed highly efficient gene silencing in presence and absence of serum in HeLa cells

Wang, Dou, et al. (2014)

PEIP

Tubular hydrogels

Plasmid DNA

Reference

Reduced toxicity of gene and Kang mortality of Balb/c mice at (2010) 22°C in 12 h light/dark cycle

et

al.

Rekha & Sharma (2011a)

Contd… 79

Table 3 Contd…. PS

Nanoparticles

Plasmid EGFP- Showed an initial release within N.S. p53 first 5 days and continued during 9 days of incubation

Eslaminejad et al. (2016)

Conjugate

RFP-cDNA

After 48 h showed good N.S. transfection efficacy in RBEC and HBMEC with 60-80% confluency and less cytotoxicity

Thomsen et al. (2011)

PULPLL

Nanoparticles

pEGFP-N1 plasmid

Showed successful gene N.S. transfection in HEK293, HepG2 and KB cell lines

Park et al. (2012)

SP

Microspheres

Lysozyme

Improved stability of enzyme, N.S. suppressed denaturation of enzyme and showed long term protein delivery at slow rate (<80% in 27 days)

Kim and (2010)

HP

Nanoparticles

pBUDLacZ plasmid

Showed gene transfection in COS- N.S. 7 cells at 0.25 mg/mL of pullulan

Gupta and Gupta (2004)

PPA

Conjugate

p53

Polymer:gene (25:1) acted as N.S. vector for gene transfection in C 6 cells with >80% cell death after 48 h

Priya (2014)

PAEP

Conjugate

Methotrexate and GFP

After 24 h complete distribution of N.S. gene in tumor cells occured In 2 h, polymer showed an initial N.S. burst (~30%) of the encapsulated protein Exhibited much higher gene N.S. transfection efficiencies and cellular uptake rates in HepG2 cell lines than in Hella cell lines

Liu et al. (2014)

PSu & AP Nanoparticles

Transmucosal protein

PBGV

pRL-CMV

N.S.

80

et

Na

al.

Dionisio et al. (2013, 2016) Yang, Niu, et al. (2014)

Contd…

Abbreviations: N.S.: Not specified; DEAEP: Diethylaminoethylamine pullulan; pSEAP: Secreted embryonic alkaline phosphatase plasmid; PEIP: Polyethylenimine pullulan; siRNA: Small interfering RNA; pGFP: Green fluorescent protein plasmid; pGL3: Plasmid backbone vector; HepG2: Hepatocellular carcinoma; PPB: Polyethyleneimine pullulan betaine; PPF: Polyethyleneimine pullulan folate; PS: Pullulanspermine; RFP-cDNA: Red fluorescent protein Hc red containing cDNA; RBEC: Rat brain endothelial cells; HBMEC: Human brain microvascular endothelial cells; PULPLL: Pullulan-g-poly(L-lysine); pEGFP-N1: Enhanced Green fluorescent protein plasmid with N terminal fusion; KB: Keratin by immunoperoxidase staining; SP: Succinylated pullulan; HP: Hydrogel pullulan; PPA: Pullulan-protamine; PAEP: Poly(β-amino) ester pullulan; PSu: Pullulan succinylate; AP: aminated pullulan; PBGV: Pullulan based gene vector; pRL: Renilla luciferase control vector

81