**3. Applications in drug delivery**

558 The Complex World of Polysaccharides

**Figure 3.** Spherical gels of curdlan and DNA observed under visible light (top and middle) and

**Figure 4.** TEM images of micro- and nano-structures of curdlan and DNA. White scale bars are 500 nm

and black scale bars are 2000 nm. Figure reproduced from [27].

polarized light (bottom). Scale bars are 1 mm each. Figures obtained from [27].

The ability to control the structure of 1,3-β-glucan based carriers has encouraged the application of these glucans in drug delivery. The glucans can be used as gels, nanoparticles, microparticles or complexes.

## **3.1. Encapsulation within gels**

Curdlan is the commonly used 1,3-β-glucan for formation of gels. Curdlan gels can be prepared by heating the suspension of curdlan in aqueous solution and then cooling it down. If the suspension is heated to 60 °C, a low-set thermally reversible gel is formed whereas if the temperature is above 80 °C, a high-set thermally irreversible gel is formed. Drugs such as indomethacin, salbutamol sulfate and prednisolone have been encapsulated in curdlan gels. The gels are prepared by mixing curdlan in drug solutions at 5-10% curdlan concentration, adding the suspension to a glass test tube, heating the test tube in water bath at the desired temperature for 10 minutes and then unmolding the gel. The experiments conducted in [32] have demonstrated that a high-set gel can lower the rate of drug release. The curdlan based gels have been able to provide a sustained release of drugs for compared to commercially available formulations. These gels can be used as drug delivery suppositories for rectal administration, which bypasses hepatic first pass clearance [32].

Curdlan gels have also been used for developing protein delivery devices. Since proteins can denature at high temperatures, the temperature required for forming curdlan gels needs to be lowered. Curdlan can form aqueous gels in the presence of hydrogen bond disrupting agents such as dimethyl sulfoxide, urea and thiocyanates [33]. This property encourages the use of chaotropes for lowering the gelling temperature. It has been demonstrated that the presence of 8 M urea can decrease the gelling temperature of curdlan from 55 °C to 37 °C. This has been used for encapsulating bovine serum albumin (BSA) as a model protein. Although gels synthesized using urea are able to demonstrate sustained release of BSA over 100 hours, the toxicity of urea is a concern. Urea also has the possibility of disrupting the hydrogen bonds of BSA and hence denaturing the protein. Thus, an alternative method of reducing the gelling temperature has been developed by modifying the backbone of curdlan to form a hydroxyethyl derivative. This system was also able to form gels at 37 °C but the BSA release was sustained for a shorter time period of 75 hours [34].

#### **3.2. Microparticles and nanoparticles**

Nanoparticles are typically used in drug delivery applications because of high drug encapsulation efficiency [35], controlled drug release and incorporation of diagnostic agents [36,37]. One of the major challenges with drug delivery is achieving specificity with cellular uptake. One method of overcoming this hurdle is by attaching targeting ligands on the surface of nanoparticles to allow enhanced uptake by specific cells [38]. Another strategy is the encapsulation of these drug loaded nanoparticle in glucan based microspheres. Glucan microspheres are derived from the cell walls of *Sacchamoryces cerevisiae* (Baker's yeast) and are 2-4 µm in size. These are porous and hollow microparticles composed mainly of 1,3-β-D-glucan and small quantities of chitin [39]. The β-glucan on the microspheres serves as a specific target for uptake by immune cells such as macrophages and dendritic cells [40]. Glucan microspheres can be used for delivering various payloads such as proteins [19], DNA [40], siRNA [16,41] and small molecules [39]. Typically small molecules are not easily entrapped within these glucan microspheres and hence the use of nanoparticles is necessary. These nanoparticles can then be loaded in glucan microspheres for enhanced uptake. Recently, two types of nanoparticles have been encapsulated within glucan microspheres: fluorescent polystyrene nanoparticles and doxorubicin loaded mesoporous silica nanoparticles [39]. The polystyrene particles are encapsulated using capillary forces from the pores of microspheres, whereas the silica nanoparticles are loaded using electrostatic interactions. These particles are then used *in vitro* to demonstrate enhanced uptake by dectin-1 expressing fibroblast cells (NIH3T3-D1 cell line). It has been observed that doxorubicin loaded silica nanoparticles were more effective when encapsulated within glucan microspheres because of enhanced uptake. These results were consistent with fluorescently tagged polystyrene nanoparticle uptake as well [39].

Aside from being used as a targeting moiety, 1,3-β-glucans have also been used as structural units for encapsulating insoluble drugs. One example is the use of short chain curdlan with a molecular weight of 990 Da, derived from Vietnam medicinal mushroom *Hericium erinaceum.* This curdlan has been used to synthesize a nanoparticle formulation for encapsulating curcumin, which is a water-insoluble compound with promising anti-cancer activity. Curcumin is derived from the rhizomes of the herb *Curcuma longa* and has demonstrated cancer prevention and suppression through various signaling pathways [42]. The delivery of curcumin has been enhanced by encapsulating it within curdlan to form 50 nm nanoparticles. These nanoparticles are prepared by adding curcumin dissolved in ethanol to a solution of curdlan in water and stirring at room temperature. The solvents are then evaporated and nanoparticles are purified by centrifugation to remove excess curdlan and larger aggregates. These curcumin loaded curdlan nanoparticles have been able to inhibit tumor growth in Hep-G2 cell line *in vitro* [10].

Amphiphilic derivatives of 1,3-β-glucans have also been extensively studied for formation of micellar nanoparticles with drug encapsulating capabilities. One example of an amphiphilic system is based on cholesterol-carboxymethylcurdlan. The hydrophilicity of curdlan is increased by modifying the backbone with carboxymethyl groups. The loading of hydrophobic drugs is increased by inclusion of cholesterol moieties in the curdlan backbone [17]. This system uses a remote loading method for encapsulating epirubicin, where a pH gradient between the interior and exterior of the nanoparticle is utilized to achieve high ratio of drug to carrier [43]. Remote loading method is implemented by preparing blank cholesterol-carboxymethylcurdlan nanoparticles using probe sonication, resuspending dried nanoparticles in ammonium sulfate, performing buffer exchange in sodium chloride and finally adding the desired amount of epirubicin to the solution. This method is able to achieve up to 39.6% drug loading, which is remarkable for polymeric systems. This curdlan based delivery device has been able to enhance the cytotoxicity of epirubicin *in vitro* when assessed using human cervical carcinoma (HeLa) cell lines as compared to free epirubicin. The delivery vehicle also increases the circulation half-life *in vivo* by 4.31 times as compared to free epirubicin when tested in Wistar rats. These results provide promising opportunities for utilizing a curdlan based drug delivery vehicle for enhanced efficacy of encapsulated drugs.

560 The Complex World of Polysaccharides

**3.2. Microparticles and nanoparticles** 

of 75 hours [34].

as well [39].

also able to form gels at 37 °C but the BSA release was sustained for a shorter time period

Nanoparticles are typically used in drug delivery applications because of high drug encapsulation efficiency [35], controlled drug release and incorporation of diagnostic agents [36,37]. One of the major challenges with drug delivery is achieving specificity with cellular uptake. One method of overcoming this hurdle is by attaching targeting ligands on the surface of nanoparticles to allow enhanced uptake by specific cells [38]. Another strategy is the encapsulation of these drug loaded nanoparticle in glucan based microspheres. Glucan microspheres are derived from the cell walls of *Sacchamoryces cerevisiae* (Baker's yeast) and are 2-4 µm in size. These are porous and hollow microparticles composed mainly of 1,3-β-D-glucan and small quantities of chitin [39]. The β-glucan on the microspheres serves as a specific target for uptake by immune cells such as macrophages and dendritic cells [40]. Glucan microspheres can be used for delivering various payloads such as proteins [19], DNA [40], siRNA [16,41] and small molecules [39]. Typically small molecules are not easily entrapped within these glucan microspheres and hence the use of nanoparticles is necessary. These nanoparticles can then be loaded in glucan microspheres for enhanced uptake. Recently, two types of nanoparticles have been encapsulated within glucan microspheres: fluorescent polystyrene nanoparticles and doxorubicin loaded mesoporous silica nanoparticles [39]. The polystyrene particles are encapsulated using capillary forces from the pores of microspheres, whereas the silica nanoparticles are loaded using electrostatic interactions. These particles are then used *in vitro* to demonstrate enhanced uptake by dectin-1 expressing fibroblast cells (NIH3T3-D1 cell line). It has been observed that doxorubicin loaded silica nanoparticles were more effective when encapsulated within glucan microspheres because of enhanced uptake. These results were consistent with fluorescently tagged polystyrene nanoparticle uptake

Aside from being used as a targeting moiety, 1,3-β-glucans have also been used as structural units for encapsulating insoluble drugs. One example is the use of short chain curdlan with a molecular weight of 990 Da, derived from Vietnam medicinal mushroom *Hericium erinaceum.* This curdlan has been used to synthesize a nanoparticle formulation for encapsulating curcumin, which is a water-insoluble compound with promising anti-cancer activity. Curcumin is derived from the rhizomes of the herb *Curcuma longa* and has demonstrated cancer prevention and suppression through various signaling pathways [42]. The delivery of curcumin has been enhanced by encapsulating it within curdlan to form 50 nm nanoparticles. These nanoparticles are prepared by adding curcumin dissolved in ethanol to a solution of curdlan in water and stirring at room temperature. The solvents are then evaporated and nanoparticles are purified by centrifugation to remove excess curdlan and larger aggregates. These curcumin loaded curdlan nanoparticles have been able to

inhibit tumor growth in Hep-G2 cell line *in vitro* [10].

Alternatively, curdlan can also be used as the hydrophobic component in an amphiphilic drug delivery vehicle. To achieve this a graft copolymer of curdlan and poly(ethylene glycol) (PEG) has been synthesized [5]. PEG is a commonly used polymer for enhanced biocompatibility [44-48]. Doxorubicin can be incorporated in graft copolymer nanoparticle by nanoprecipitation method. In this technique, the copolymer and drug are dissolved in a common water miscible solvent and then added to magnetically stirred water in a drop-wise manner. The mixing causes self-assembly of polymers where hydrophobic components are at the core of the nanoparticle and hydrophilic components are at the surface. In the case of doxorubicin and curdlan-graft-PEG, dimethyl sulfoxide is used as the common solvent [5]. A schematic of the nanoparticle is presented in Figure 5.

The structure of curdlan-graft-PEG nanoparticles has been confirmed using TEM. The samples were stained using phosphotungstic acid, which acts as a negative stain. This makes the background appear dark and the samples of interest appear bright. Additionally, since phosophotungstic acid is hydrophilic, hydrophobic components will be excluded from the acid and hence appear brighter than hydrophilic components. As observed in Figure 6, the nanoparticles are about 109 nm in size. Doxorubicin is visible as the bright center of the nanoparticles. Figure 6 (B) shows the three distinct layers of the nanoparticle, which correspond well with the schematic presented in Figure 5 [5].

The doxorubicin formulation with curdlan-graft-PEG was tested *in vitro* to determine its drug release profile and it demonstrated sustained release over 24 hours following a Fickian diffusion release model. Since PEG graft is supposed to improve the biocompatibility of the nanoparticles, a hemolysis assay was implemented to assess this property. Sheep red blood cells (RBCs) were used for this assay and nanoparticles of interest were incubated with the

RBCs for one hour at 37 °C. The amount of lysis was compared to a negative control of veronal buffered saline and positive control of deionized water. Curdlan-graft-PEG nanoparticles showed hemolysis below 5% at clinically relevant concentrations [5], which is considered biocompatible [35].

**Figure 5.** Schematic of nanoparticle synthesized using curdlan-graft-poly(ethylene glycol) and doxorubicin. Doxorubicin is at the core, surrounded by curdlan and PEG forms the shell. Figure adapted from [5].

**Figure 6.** TEM images of doxorubicin encapsulated within curdlan-graft-PEG nanoparticles. (A) Bright center indicates doxorubicin and surroundings are darker because of higher electron density; (B) Three layers of the nanoparticle can be seen: bright doxorubicin core, dense curdlan layer in the middle and sparse PEG layer on the shell. Scale bars are 200 nm. Figure adapted from [5].

Therefore, nanoparticles and microparticles based on 1,3-β-glucans provide promising opportunities for drug delivery as structural units and as targeting ligands. The combination of these properties can be exploited for developing a drug delivery vehicle with enhanced potency.

#### **3.3. Glucan complexes with polynucleotides**

562 The Complex World of Polysaccharides

considered biocompatible [35].

adapted from [5].

potency.

RBCs for one hour at 37 °C. The amount of lysis was compared to a negative control of veronal buffered saline and positive control of deionized water. Curdlan-graft-PEG nanoparticles showed hemolysis below 5% at clinically relevant concentrations [5], which is

**Figure 5.** Schematic of nanoparticle synthesized using curdlan-graft-poly(ethylene glycol) and doxorubicin. Doxorubicin is at the core, surrounded by curdlan and PEG forms the shell. Figure

**Figure 6.** TEM images of doxorubicin encapsulated within curdlan-graft-PEG nanoparticles.

middle and sparse PEG layer on the shell. Scale bars are 200 nm. Figure adapted from [5].

(A) Bright center indicates doxorubicin and surroundings are darker because of higher electron density; (B) Three layers of the nanoparticle can be seen: bright doxorubicin core, dense curdlan layer in the

Therefore, nanoparticles and microparticles based on 1,3-β-glucans provide promising opportunities for drug delivery as structural units and as targeting ligands. The combination of these properties can be exploited for developing a drug delivery vehicle with enhanced Polynucleotides have found several applications in drug delivery as active agents due to their therapeutic effects [16,41,49]. One of the challenges faced by polynucleotide delivery is their rapid degradation *in vivo*. Encapsulation of polynucleotides thus becomes necessary for maintaining their function. 1,3-β-glucans are a preferred choice for forming these complexes because of their helix forming capabilities. In the past, several polysaccharides have been tested for their ability to form complexes with the polynucleotide poly(C) and the complexation was assessed using circular dichorism (CD) where a change in the spectrum indicates complex formation. Only schizophyllan (1,3-β-glucan with one 1,6 branch every three units) and lentinan (1,3-β-glucan with two 1,6 branch every five units) have demonstrated changes in CD spectra, whereas curdlan, amylose (1,4-α-glucan), dextran (1,6-αglucan) and pullulan (1,4-α-1,6-α-glucan) did not show any changes. This implies that only soluble 1,3-β-glucans are able to form complexes with poly(C). Commercially available curdlan is unable to form a complex because of its low solubility in water, which leads to precipitation [50]. The solubility of curdlan can be improved by reducing the molecular weight of the polymer. At lower molecular weight, curdlan is able to form complexes with poly(C) but these complexes are not as stable as the ones formed with schizophyllan [51]. Complexes have also been formed with poly(A) using schizophyllan but poly(U) showed no complexation, which suggests that polysaccharides can bind to polynucleotides in a specific manner [52]. Curdlan backbone has also been modified with carbohydrate molecules using click chemistry to improve the solubility and hence induce complex formation with poly(C) [53].

Hitherto, only homo-sequence polynucleotides have been discussed. Often therapeutic polynucleotides are composed of heterogeneous base pairs. Drug delivery vehicles for hetero-sequence oligonucleotides have also been developed by synthesizing cationic curdlan chains. CpG DNA has demonstrated immune stimulating effects but it needs to be preserved from degradation [54]. Cationic curdlan, synthesized using click chemistry, has been able to form complexes with CpG DNA and increase the cellular uptake in macrophage-like cell line J774.A1. The complex also induces an increase in cytokine (IL-12) secretion, which suggests activation of the macrophage cells [54]. Another strategy of binding hetero-sequence oligonucleotides is by modification of one terminal to attach a homo-sequence such as poly(A). A schizophyllan derivative has been utilized for binding modified antisense oligonucleotides. Schizophyllan has been modified with galactose and PEG units to enhance cellular uptake. It has been observed that the antisense effect was maximized with the use of schizophyllan derivative when the complex is administered to hepatoblastoma HepG2 and melanoma A375 cell lines [55]. Thus, 1,3-β-glucan based drug delivery devices serve as biocompatible carriers for a variety of nucleotides.

### **4. Applications in immunotherapy**

1,3-β-glucans have been known to generate an immune response including stimulation of cytokine production, oxidative burst, increased phagocyte and lymphocyte proliferation as well as phagocytosis of opsonized tissues. Various mechanisms are responsible for this activation and this response has been utilized for varied applications including cancer resistance, disease immunity and wound healing [56].
