Glucan Particles as Carriers of Nanoparticles for Macrophage

Chapter 3

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Glucan Particles as Carriers of Nanoparticles for Macrophage-Targeted Delivery Ernesto Soto and Gary Ostroff* Program in Molecular Medicine, University of Massachusetts Medical School, 373 Plantation Street, Worcester, Massachusetts 01605, USA *E-mail: [email protected]

Glucan particles (GP) are 2-4 μm hollow and porous microspheres derived from Saccharomyces cerevisiae (Baker’s yeast) that provide an efficient system for encapsulation, protection, and oral or systemic macrophage-targeted delivery of macromolecules, such as DNA, siRNA and proteins using either a polyplex or layer-by-layer (LbL) synthesis methods. We have recently extended the application of GPs to the delivery of nanoparticles (NP) that are encapsulated within the hollow cavity of GPs or bound to the outer surface of chemically derivatized GPs (Soto, et al. J. Drug Delivery, 2012, Article ID 143524). GP mediated delivery of nanoparticles provides the advantages of (1) encapsulation of materials that are difficult to prepare in situ within the glucan particles, such as nanomaterials composed of water-insoluble components, (2) β1,3-D-glucan receptor-targeted delivery of nanoparticles to phagocytic innate immune cells, (3) small molecule loaded nanoparticles for small drug molecule delivery, and (4) targeted delivery of nanoparticles with an intrinsic property, such as magnetic or gold nanoparticles, thus increasing the versatility of the GPs for theranostic applications. Examples of nanoparticles that have been formulated with GPs include magnetic iron oxide nanoparticles, gold nanoparticles, quantum dots and adeno-associated virus (AAV).

© 2012 American Chemical Society In Nanomaterials for Biomedicine; Nagarajan, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Introduction Advances in rational drug design and rapid screening (1), combined with the discovery of promising new approaches to treat diseases such as gene (2, 3) and siRNA therapies (4, 5), have spurred the development of an increasing number of novel drug delivery technologies. An effective drug delivery system must address several hurdles including issues of drug solubility, in vivo stability, efficient targeting, poor pharmacokinetics, toxicity and adverse side effects. Nanotechnology-based drug delivery systems represent a promising approach to fulfill the need for new delivery techniques. The field of nanoparticle based drug delivery has its origins in the discovery of liposomes (6) and polymer-drug conjugates (7). The development of methods for the synthesis, characterization and precise control over size and shape design (i.e. Particle Replication in Non-wetting Templates (PRINT) technology (8, 9)) of nanoparticles has made it possible over the past 20 years to explore a variety of materials for nanoparticle based drug delivery (10, 11), including natural and synthetic polymers, metal nanoparticles (gold, magnetic iron oxide), quantum dots, silica, dendrimers, and virus-like particles. The successful development of nanoparticle based delivery systems is exemplified by the use of nanomaterials for approved anticancer drug formulations (12, 13), and it is anticipated that the number of applications using nanotechnology for drug delivery will continue to expand in the near future. The interest in using nanoparticles for drug delivery is driven by high nanoparticle drug loading capacity, improved drug solubility and bioavailability, improved pharmacokinetics and therapeutic index, reduced immunogenicity, and the potential for targeted and controlled release (10, 11, 14). Careful synthetic design also allows for the synthesis of nanoparticles with precise size and shape and texture, key factors affecting penetration across biological barriers and cellular uptake mechanisms. More recently, research on nanoparticles has also focused on the design of nanoparticulate theranostic agents (15). The term theranostic, a term derived from therapeutic and diagnostic, was initially proposed by Funkhouser (16) in 2002, and it refers to materials that combine therapeutic drug delivery (i.e. gene therapy, chemotherapy, radiation, photodynamic therapy) and diagnostic imaging (i.e. magnetic resonance imaging (MRI), positron emission tomography/single-photon emission computed tomography (PET/SPECT), near-infrared (NIR) fluorescence functionality). These properties allow for the design of multifunctional nanoparticles that can be loaded with a therapeutic drug (i.e. siRNA) and an imaging agent (i.e. Gd(III) for MRI imaging), which can be delivered at the same time within the same dose. In the case of some nanoparticles (i.e. magnetic iron oxide, quantum dots, gold nanoparticles) the imaging application is an intrinsic property of the nanoparticle. Although the theranostics field is relatively new, significant advances have been made in the past decade to design promising nanoparticulate materials. The combination of therapeutic and diagnostic capabilities has the promise to drive the application of nanoparticle based materials in the biomedical field.

58 In Nanomaterials for Biomedicine; Nagarajan, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Nanoparticle Design for Drug Delivery Three primary parameters need to be considered for the successful design of nanoparticles as drug carriers: (1) a method to load and deliver a drug, (2) the mechanism of cell uptake, and (3) payload release. A payload can be loaded by multiple general strategies including: physical entrapment or payload absorption onto the NP using non-covalent interactions, or covalent binding to the NP using degradable or non-degradable bonds. Targeting of NPs can be achieved by three approaches: (a) passive accumulation of NPs by the enhanced permeability and retention (EPR) effect, such as the case of NP accumulation through the leaky vasculature of tumors (13), (b) NPs can be partially targeted by attaching ligands with specificity to receptors that are over-expressed in certain cells (i.e. folate and transferrin receptors in cancer cells (17–21)). However, this mechanism has the disadvantage of off-target effects as the receptors can be expressed on other types of non-target cells, and (c) targeting of cell populations with high selectivity by grafting specific targeting moieties (i.e. antibodies, aptamers, peptides, oligonucleotides) to nanoparticle surfaces recognized by cell surface receptors known to be expressed only on target cells (i.e. antibodies to target prostate-specific membrane antigen (PSMA) (22), or galactose to target asialoglycoprotein receptors on hepatocyte cells (23)). Finally, efficient payload release from NPs is accomplished by using stimuli-responsive components. Nanoparticles offer the advantage of selective chemical modification to incorporate components that will respond to an internal (reducing nature of cellular environment, change in pH) or external stimulus (applied magnetic field, radiation exposure). These stimuli activate a chemical mechanism to degrade or destabilize a carrier and release physically entrapped payloads (24, 25), or to cleave a degradable bond and release molecules that are covalently bound to the NP (26, 27). The most critical challenge of current nanoparticle based drug delivery systems is the lack of optimal strategies for selective and efficient targeted delivery. Several undesired processes such as off-target accumulation in other organs tissues and cells, rapid particle clearance from in vivo circulation (especially particles less than 5 nm) (28, 29), opsonization and macrophage clearance (30, 31), and complement activation by proteins that causes hypersensitivity reactions (32) reduce the number of nanoparticles reaching target cells, thus resulting in using high nanoparticle concentrations to achieve a therapeutic response. Synthetic design of nanoparticles allows for specific control of nanoparticle functionalization to introduce chemical groups that reduce undesired processes. For example multifunctional NPs coated with cell surface receptor-targeting ligands combined with PEG polymer brush coats inhibit protein adsorption reducing macrophage uptake and rapid clearance by the RES; and control of particle size, shape and texture alters cellular internalization and trafficking (32–35). We have developed a drug delivery system based on 1,3-β-D-glucan (β-glucan) microparticles (GP) that allows for receptor-targeted delivery to macrophage and dendritic cells that express β-glucan receptors. The combined use of GPs for binding/encapsulation of nanoparticles containing a payload drug 59 In Nanomaterials for Biomedicine; Nagarajan, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


aims to integrate the structural advantages of nanoparticles with the targeting capabilities of glucan microparticles to selectively target nanoparticles to macrophages via β-glucan mediated phagocytosis of the GP carriers. A challenge with nanoparticle delivery is unintended partial uptake and clearance by phagocytic cells, such as macrophages. In contrast, glucan particles are selectively targeted to phagocytic macrophages by both a size (2-4 micron) and a receptor mediated uptake mechanism. GPs remain in macrophage endosomes for a longer time compared to nanoparticle uptake by pinocytosis or other non-receptor mediated mechanisms. We previous reported the kinetics of glucan particle intracellular metabolism showing that GPs remain intact in macrophages for several days and are slowly degraded into soluble β-glucan fragments (36). The ability of targeting GPs into macrophages and their slow degradation makes GPs an ideal drug carrier to target diseases or ameliorate symptoms in diseases in which macrophages play a significant role. Specific examples of diseases in which GP drug targeting to macrophages could play a significant role include: (1) targeting of antibiotics into alveolar macrophages to inhibit replication of Mycobacterium tuberculosis, (2) anti-inflammatory drug delivery to macrophages that promote pathogenic responses in inflammatory diseases such as diabetes, rheumatoid arthritis, Chrohn’s disease and psoriasis, (3) use of GPs loaded with antigens for vaccine delivery (37, 38).

β-Glucan Based Drug Delivery 1,3-β-D-glucans are a class of polysaccharides consisting of D-glucose monomers naturally found in fungi, algae, plants and some bacteria. Their most studied application is based on their ability to activate the immune system. Two receptors, dectin-1 (D1) and the Complement Receptor 3 (CR3 or CD11b/CD18), present on the surface of innate immune cells have been found to be primarily responsible for binding to 1,3-β-D-glucans (39) and the signaling mediated by both receptors has been characterized at the molecular level (40–42). In addition to immuno-stimulatory properties and their use as adjuvants in pharmaceutical formulations, glucans have been used in drug delivery systems as actual drug carriers, or in combination with other materials to form suitable drug delivery systems. The most significant applications of 1,3-β-D-glucan for drug delivery can be classified in four categories: (1) the use of 1,3-β-D-glucan (primarily curdlan, scleroglucan and their synthetic derivatives) as carriers in drug formulations consisting of hydrogels, tablets and ingestible films (43–46), (2) the preparation of particles coated with 1,3-β-D-glucan (i.e. use of curdlan for stabilization of liposomes (47, 48), lipid nanoparticles of glyceryl caprate for entrapment of doxorubicin (49), superparamagnetic iron oxide nanoparticles coated with β-glucan for MRI diagnosing of liver metastasis (50)); (3) the use of soluble 1,3-β-D-glucan for encapsulation and delivery of macromolecules (i.e. DNA delivery with schizophyllan complexes (51–53)); and (4) the development of the yeast 1,3-β-D-glucan particle drug delivery technology.

60 In Nanomaterials for Biomedicine; Nagarajan, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Glucan Particles (GPs) are highly purified, biodegradable, porous and hollow 2-4 μm microspherical particles prepared from Baker’s yeast. Upon systemic administration, the GPs are internalized through a receptor-mediated process by phagocytic cells, such as monocytes, macrophages, neutrophils and dendritic cells (36, 37). The use of glucan particles for macromolecule (DNA, siRNA, protein, vaccine) delivery has been demonstrated both in vitro and in vivo (37, 38, 54–56). Macromolecules such as nucleic acids can be efficiently loaded into GPs using either polyplex core or layer-by-layer synthesis approaches to produce glucan particle encapsulated DNA or siRNA polyplexes (54, 55). The GPs offer protection and macrophage targeted delivery allowing a significant reduction in the amount of DNA or siRNA required for efficient transfection or gene silencing compared to the corresponding control polyplex samples prepared in the absence of GPs. A significant advance in the use of GPs for macrophage targeted delivery was the development of Glucan encapsulated siRNA particles (GeRPs) for oral and parenteral delivery of Map4k4-siRNA (55). These GeRPs consisted of glucan particle encapsulated tRNA/polyethylenimine cores onto which siRNA and other components were adsorbed by LbL synthesis. The GeRPs effectively suppressed lipopolysaccharide induced systemic inflammation protecting septic mice at siRNA doses 10-fold lower than required in in vivo studies reported using other available siRNA delivery methods (55). More recently, we engineered a simpler formulation method than the original multi-layered GeRPs by directly forming amphiphatic peptide (Endo-Porter (EP))/siRNA nanocomplexes in empty glucan shells. These formulations efficiently induced gene silencing in macrophages that internalized the glucan particles/siRNA-EP complexes, but not other cell types (56). The advantages of GP delivery have been recognized by others for diverse applications. In independent work, Zhu (57) reported the use of GeRPs containing antiviral vp28-siRNA for delivery into Marsupenaeus japonicus shrimp to inhibit replication of white spot syndrome virus (WSSV), which is a major shrimp viral pathogen responsible for worldwide economic losses to shrimp aquaculture. Figueiredo (58) used glucan particles for delivery of an amphiphatic/lipophilic gadolinium MRI imaging agent (Gd-DOTAMA(C18)2; DOTAMA = 1,4,7,10-tetraazacyclododecane-N,N′,N′′,N′′′-tetraacetic acid monoamide). Particles containing an oil/water microemulsion to trap the amphiphatic MRI agent were used for MRI imaging of mice grafted subcutaneously with B16 melanoma cells. Following 48 h post-injection of the paramagnetic GPs the particles were observed in lymph nodes near the tumor as a result of rapid macrophage uptake of GP Gd and subsequent delivery to the lymph node by migrating macrophages. We have also used GPs for the encapsulation of small drug molecules, such as the antibiotic rifampicin (rif) (59). Rifampicin was loaded in soluble form into glucan particles, precipitated by a change in pH and subsequently trapped by loading of alginate or chitosan to form a hydrogel matrix to slow the release of rifampicin from the particles. These GP rif particles enhanced in vitro macrophage killing of TB. However, the use of glucan particles for 61 In Nanomaterials for Biomedicine; Nagarajan, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


small drug molecule delivery has been generally limited by the lack of efficient methods to load and retain small drug molecules inside GPs. The combined use of nanoparticles for drug binding and GP encapsulation of nanoparticles for targeted delivery represents a promising approach to expand the applications for GP delivery. The use of glucan particles for macrophage-targeted delivery of nanoparticles was initially reported using fluorescent polystyrene nanoparticles and mesoporous-silica nanoparticles containing the chemotherapeutic drug doxorubicin (Dox) (60). Nanoparticles containing Dox were loaded inside the hollow cavity of the glucan particles or non-covalently attached to the outer surface of chemically-derivatized GPs. GPs containing doxorubicin-mesoporous silica nanoparticles efficiently delivered the drug into GP phagocytic cell line 3T3-D1 expressing the dectin-1 receptor, resulting in enhanced Dox-mediated growth arrest. The advantages of combining glucan particle and existing nanoparticle drug delivery methods include: (1) 1,3-β-D-glucan receptor-targeted delivery to macrophage and dendritic cells. Although, others have reported the use of different nanoparticles coated with β-glucan for targeted delivery to cells expressing β-glucan receptors (49, 50, 61), these systems present the disadvantage of each uptake event delivering just one nanoparticle as opposed to the possibility of delivering hundreds to thousands of nanoparticles in one glucan particle uptake event due to the high loading capacity of GPs, (2) the encapsulation of payload complexes that cannot be prepared in situ as the synthetic conditions are not compatible with GPs, (3) the loading of nanoparticles with small neutral and hydrophobic drug molecules into GPs that is not possible to load by polyplex trapping or LbL methods, and (4) the incorporation of nanoparticles with an intrinsic property, such as magnetic nanoparticles, thus increasing the versatility of the particles and creating potential theranostic agents. A current disadvantage with theranostics is the mismatch in dose required for imaging and for therapeutic use, the dosages required for nuclear imaging and anticancer therapeutics are several orders of magnitude different. Other MRI agents, such as those based on gadolinium require considerably higher doses than the typical therapeutic drug due to the low sensitivity of MRI (62). A potential advantage of GPs for delivery of nanoparticles for theranostic applications is that NPs for imaging and NPs containing the therapeutic drug can be independently co-loaded in the same GP at the optimal concentration required for each function. Here we report on the different strategies developed to efficiently create Glucan Particle – Nanoparticle (GP/NP) hybrid systems and present examples of potential applications using quantum dots, gold nanoparticles, magnetic iron oxide nanoparticles and adeno-associated virus.

Glucan Particle-Nanoparticle Loading Strategies (GP/NP Delivery) As previously reported (60), it is possible to use glucan particles for nanoparticle delivery by either encapsulation of nanoparticles inside glucan particles (GP-NP) or by binding of nanoparticles to the outer surface of chemically-modified glucan particles (NP-GP). The nanoparticle location is 62 In Nanomaterials for Biomedicine; Nagarajan, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


dependent on nanoparticle size. NPs of average diameter less than 30 nm can diffuse through the GP shell matrix and be trapped inside the cavity by physical or chemical methods. Larger nanoparticles, which are unable to penetrate into GPs must be loaded by binding to the outer surface of chemically-derivatized glucan particles. The NP-GP system offers the advantage of more rapid release due to less nanoparticle aggregation and overcomes the possible slow diffusion of a payload from the glucan particles. In contrast, the GP-NP system, encapsulating nanoparticles inside GPs is a more advantageous strategy due to (1) higher nanoparticle loading capacity, and (2) protection of NPs by the glucan shell in transit to target cells. An alternative GP nanoparticle loading strategy to overcome NP size limitation is the in situ synthesis of nanoparticles inside GPs for those nanoparticles that can be synthesized under conditions that are compatible with GPs. The mechanisms by which pre-formed nanoparticles remain incorporated inside or on the surface of glucan particles include physical aggregation, physical embedment of nanoparticles in a polymer gel, covalent crosslinking of nanoparticles, non-covalent electrostatic interactions, or affinity binding (i.e. streptavidin-biotin) of nanoparticles to either surface derivatized GPs or polymers loaded inside the hollow GP cavity. Using either commercially available nanoparticles of narrow size distribution or nanoparticles synthesized in our laboratory we developed different loading strategies to incorporate NPs inside or on the surface of GPs. Glucan Particle Encapsulated Nanoparticles (GP-NP) Pre-formed nanoparticles of less than 30 nm in diameter can be loaded through the pores of glucan particles (Figure 1). Ideally, the nanoparticles are in a stable suspension to prevent aggregation outside the GPs. Nanoparticle loading is accomplished by wetting the glucan particles in a sub-hydrodynamic volume of the nanoparticle suspension to ensure complete absorption of the NP suspension into GPs by capillary action. Subsequent solvent hydration/lyophilization steps hydraulically push the nanoparticles into GPs by capillary action. In most formulations the solvent is water, although depending on the nanoparticles solvents such as 70% ethanol are more suitable to foster NP aggregation inside GPs. The nanoparticles are retained inside GPs by either particle aggregation, physical or chemical trapping. Spontaneous nanoparticle aggregation occurs with some nanoparticles during the lyophilization steps involved in GP-NP synthesis. However NPs less prone to aggregation quickly diffuse out of the GPs upon resuspension of the GP-NP sample in a volume higher than the hydrodynamic volume. To ensure that the majority of nanoparticles (either single particle or large aggregates) are effectively retained inside the GPs we have investigated the use of cationic or anionic polymers to non-covalently bind nanoparticles functionalized with corresponding anionic or cationic groups. Additionally, more stable samples can be prepared by covalent chemical crosslinking of nanoparticles with a polymer (i.e. EDC crosslinking of carboxylated labeled nanoparticles with a cationic polymer like chitosan or PEI). Other strategies include the non-covalent affinity binding of biotin-surface-modified nanoparticles interacting 63 In Nanomaterials for Biomedicine; Nagarajan, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


with biotin-modified polymers through avidin or streptavidin bridges between the nanoparticles and polymers. A click chemistry reaction is another possibility to generate stable polymer/nanoparticle aggregates inside GPs. Figure 1 shows microscopic images of GP-NP samples prepared with fluorescently labeled 20 nm carboxylated nanoparticles. The carboxylate groups on the nanoparticle surface allow for electrostatic crosslinking with polyethylenimine (PEI) or other cationic polymers forming nanoparticle aggregates or cores inside the GPs. The use of a cationic polymer to bind nanoparticles also offers the possibility of using the cationic nanoparticle core for binding of an anionic payload molecule (i.e. DNA or siRNA).

Figure 1. (A) Schematic representation of nanoparticle loading inside GPs, (B) Microscopic image of GPs containing a core of fluorescently labeled polystyrene (20 nm) nanoparticles crosslinked with PEI (fluorescent image on the left, overlay of fluorescent and brightfield image on the right). (see color insert)

In Situ Synthesis of GP-NP As indicated above, nanoparticle size is the key limiting factor to the preparation of encapsulated GP-NP samples. An alternative strategy to load larger nanoparticles is the in situ synthesis of NPs inside GPs for those NPs that can be synthesized under conditions compatible with GPs (Figure 2A). Two key parameters were identified for successful in situ synthesis of nanoparticles: (1) efficient loading of nanoparticle starting materials and their stability prior to (2) generation of optimal nanoparticle formation reaction conditions within glucan particles. For example, although we can efficiently load starting materials for in situ synthesis of mesoporous silica nanoparticles (MSN) or poly(lactic-co-glycolic acid) (PLGA) nanoparticles, it is difficult to generate exact reaction conditions (i.e. stable surfactant micelle for MSN synthesis or water/oil emulsion for synthesis of PLGA) resulting in GPs containing mixture of MSN or PLGA particles and side products (i.e. silicon dioxide from decomposition of tetraethylorthosilicate used for MSN synthesis). 64 In Nanomaterials for Biomedicine; Nagarajan, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Figure 2. (A) Schematic representation of in situ synthesis of NPs inside GPs, (B) Bright field microscopic images of empty GPs and magnetic GP FexOy NPs. We have successfully synthesized in situ GP-NPs containing iron oxide nanoparticles. Colloidal iron oxide nanoparticles are of interest for their magnetic properties as MRI contrast agents. Iron oxide nanoparticle cores were prepared inside GPs by co-precipitation of acidic aqueous solutions of iron chloride (III) and iron chloride (II) in the presence of tetramethylammonium hydroxide (TMAOH). Optimal iron salts and TMAOH concentrations, as well as temperature were based on the work by Babes (63), to generate paramagnetic particle aggregates inside GPs (GP FexOy NPs). The brightfield microscopic images in Figure 2B show the difference between empty GPs and GPs containing magnetic iron oxide nanoparticles. Nanoparticle Binding to the Outer GP Surface (NP-GP) Nanoparticles of average diameter greater than 30 nm will not efficiently penetrate through the GP shell and can be chemically bound to the surface of derivatized GPs, as previously demonstrated for anionic polystyrene nanoparticles or anionic mesoporous silica nanoparticles electrostatically bound to GPs derivatized with cationic PEI (60). GPs are essentially neutral with exception of a small amount of positively charged chitosan. However, the chitosan is largely buried in the glucan matrix and not readily available for electrostatic binding of anionic nanoparticles. Cationic GPs can be synthesized by covalent modification of the GP surface with cationic polymers (i.e. PEI, chitosan, PLL) via a reductive amination synthetic approach. This synthetic strategy allows functionalization of the reducing terminal glucose monomers (

Glucan Particles as Carriers of Nanoparticles for Macrophage

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