Macro and nano shaped polysaccharide hydrogels as drug delivery sytems
Giorgia D’Arrigo
XXV cycle
Department of Drug Chemistry and Technologies, “Sapienza” University of Rome, p.le Aldo Moro 5, 00185 Rome, Italy.
Center for Pharmaceutical Biotechnology & Nanomedicine, Northeastern University, Boston, MA 02115, USA.
Prof. Franco Alhaique Prof. Pietro Matricardi
It is not the strongest of the species that survives, nor the most intelligent that survives. It is the one that is the most adaptable to change.
Charles Darwin
Table of contents:
Chapter 1: Polysaccharide hydrogels in drug/protein delivery: from macro- to nano-systems……………………………..………...... ……12
Chapter 2: Aim and outline of the thesis…………..……………………………………….235
Chapter 3: Hyaluronic acid methacrylate derivatives and calcium alginate interpenetrated hydrogel networks for biomedical applications: physico-chemical characterization and protein release. ……...…………………………………………………………………….....278
Chapter 4: Calcium alginate/dextran methacrylate IPN beads as protecting carriers for protein delivery. ……………………………………………………………….……...……..453
Chapter 5: Self-assembled gellan-based nanohydrogels as a tool for prednisolone delivery……………………………………………………………………………..………..653
Chapter 6: Gellan gum nanohydrogel: a multi-drug delivery system for combination therapy. ……………………………………………………………………………….……..853
Chapter 7: Summary and Perspectives……………………………………………..……. 1095
Acknowledgements………………………………………………………………………..114
1
Polysaccharide hydrogels in drug/protein delivery: from macro- to nano- systems
Chapter 1
Polysaccharide hydrogels in drug/protein delivery: from macro- to nano- systems
1.1 Polysaccharide hydrogels.
Hydrogels are three-dimensional polymeric networks capable of imbibing large amounts of water or biological fluids. Their ability to absorb water is attributed to the presence of hydrophilic moieties such as hydroxyl and carboxyl groups as well as ethers, amines and sulfates in the polymers forming the hydrogel structure.
Among the numerous polymers used to obtain hydrogels, polysaccharides show several advantages over other macromolecules: they are abundant, available from renewable sources such as algae, plants, cultures of microbial selected strains and can be also obtained by recombinant DNA techniques. Furthermore, polysaccharides have structures and properties that cannot be easily mimicked by synthetic compounds and are also generally cheaper than synthetic polymers [1].
Owing to their high water content, their soft and rubbery consistency and low interfacial tension with water or biological fluids, polysaccharidic hydrogels resemble natural soft tissue more than any other type of polymeric biomaterials. Their physico-chemical similarity to the native extracellular matrix, both from the point of view of the composition and of the mechanical properties, makes them highly biocompatible and permeable for oxygen, nutrients and other metabolites. Furthermore, they are relatively deformable and can be formulated in a variety of physical forms, including macroscopic networks such as films or slabs, and smaller networks such as microparticles and nanoparticles. As a result, polysaccharidic hydrogels are used in clinical practice and experimental medicine for a wide range of applications, including tissue engineering, cellular immobilization, diagnostics and drug delivery [2].
Among the polysaccharides that have been proposed for hydrogel preparation, gellan gum, hyaluronic acid, alginate and dextran can be considered as the most extensively used.
Gellan gum is an anionic polysaccharide produced by Sphingomonas elodea with a complex tetrasaccharide repeating unit of β-D-glucose, β-D-glucuronic acid, β-D-glucose, and α-L-rhamnose, with a free carboxyl group. Gellan gum is largely used in the food industry and biotechnology because it forms transparent hydrogels that are more resistant to heat and acidic medium as compared to other polysaccharide hydrogels. Recently, gellan gum hydrogels showed potential applications in the engineering of cartilaginous tissues due to their visco-elastic properties and lack of cytotoxicity. The polymer solution gelation is due to the thermally reversible ordered helix-coil transition of the polymer chains and the formation of junction zones by the stacking of the macromolecules in the double helix form. The helix aggregation and gel formation are enhanced by cations, with divalent cations being stronger gelation promoters than monovalent cations [3-6].
Hyaluronic acid (or hyaluronan, HA) is a linear, not sulfated glycosamino-glycan composed of β-1,4-linked disaccharide units of β-1,3-linked glucuronic acid and N-acetyl-D-glucosamine. HA is one of the components of the extracellular matrix (ECM) and is present at high concentrations in all connective tissues (cartilage, vitreous humour and synovial fluids). Moreover, due to its capacity to interact with some cell receptors, HA plays an important role in several processes such as cell proliferation, migration and differentiation [7]. It is isolated either from animal sources or from bacteria through a process of fermentation or direct isolation; more recently it is obtained from recombinant Bacillus Subtilis sp. that is recognized as a GRAS microorganism [8]. The properties of HA can be modified and tailored in many ways in order to obtain materials with new physico-chemical and biological characteristics, as hydrophobicity, amphiphilicity and specific biological activities. In particular, carboxylic groups are frequently modified by esterification and amidation reactions mostly carried out using carbodiimide-assisted coupling reactions. Because of its excellent biocompatibility and biodegradability, HA is one of the biopolymers most frequently used in the biomedical field and in several industrial applications. Actually, numerous HA linear or cross-linked derivatives that have been synthesized are employed for tissue repair, wound healing, treatment of joint diseases, anticancer drug delivery, and as scaffolds for tissue engineering [9].
Dextran is a bacterial polysaccharide consisting of consecutive α-1,6 linked D-glucopyranose units in their main chains (more than 50% of the total linkages) with some side chains stemming from α-1,2, α-1,3 or α-1,4 branch linkages. Dextran is used in medicine as an antithrombotic agent to reduce blood viscosity and as a plasma expander [10]. Dextran can be modified in several ways to obtain chemically cross-linkable polymers. The functionalization of the dextran chains with methacrylate moieties (dex-MA) [11] allows hydrogel formation by radical polymerization, initiated chemically or by UV radiation. The derivatization of dextran chains with hydroxyethyl-methacrylate (dex-HEMA) [12], thus containing hydrolysable esters,allows biodegradability of the hydrogel network.
Alginate (Alg) is a well known polysaccharide produced by bacteria or extracted from marine brown algae. It is composed of a polymer backbone of 1,4 linked β-D mannuronic acid (M) and α-L-guluronic acid (G) residues. Alg contains homopolymeric M- and G-blocks, that are joined by regions of alternating structures. The physico-chemical properties of Alg strongly depend on the M/G ratio as well as on the structure of the alternating zones. Also, the gelling ability of this polysaccharide in aqueous solutions, arising from the interactions between the carboxylic acid moieties and divalent counter ions (i.e. calcium, lead, and copper), is highly dependent on the M/G ratio. It has been found that the G-blocks are responsible for the typical “egg-box” structure [7]. The Alg gel formation driven by divalent ions is generally very fast and the resulting hydrogels have been investigated for many industrial and biomedical applications.
1.2 Hydrogel nomenclature.
Hydrogels are broadly classified into two categories: chemical and physical hydrogels.
Chemical hydrogels are covalently cross-linked networks obtained by radical polymerization, enzyme-mediated polymerization, click-chemistry, Michael addition, etc... [13]. Physical hydrogels are held together by molecular entanglements, hydrogen bondsand/or ionicand hydrophobic interactions [14]. All these interactions are reversible and can be disrupted by changes in physical conditions or stress application.Usually, chemical cross-linking leads to mechanically strong hydrogels, while the physically cross-linked ones are generally weaker. Simultaneous physical and chemical cross-linking methods have also been applied leading to hydrogel networks that combine their specific properties. These heterogeneous polymer systems are generally composed by combinations of different polymers (graft polymers, block copolymers, interpenetrating polymers and AB cross-linked copolymers).
In recent years particular attention has been focused on networks obtained by interpenetration of different macromolecules, better known with the acronyms IPN and semi-IPN. The IUPAC definition of IPN is as follows: “a polymer comprising two or more networks which are at least partially interlaced on a molecular scale but not covalently bonded to each other and cannot be separated unless chemical bonds are broken” [15].The IUPAC definition of semi-IPN is the following one: “a polymer comprising one or more networks and one or more linear or branched polymer(s) characterized by the penetration on a molecular scale of at least one of the networks by at least some of the linear or branched macromolecules” [16].Polymer interpenetration leads to new systems with improved properties, which quite often are substantially different from those of the individual polymers. In several systems synergism of properties is also observed. The combination and synergism of properties can be exploited to modify and tailor the characteristics of the resulting material to meet specific needs. Because of these features, IPN hydrogels appear to be suitable for various applications, particularly in the field of drug delivery.Therapeutic agents can either be physically entrapped into the interpenetrated polymeric matrix or covalently bound to the polymer backbone. For instance, hydroxyl groups of polysaccharides allow direct reaction with drugs containing carboxylic groups; as in the case of methacrylic-functionalized non-steroidal anti-inflammatory drugs,conjugated to a methacrylic-functionalized dextran via UV irradiation [17].
1.3 Physical forms of polysaccharide hydrogels.
Polysaccharide hydrogels can be formulated in the form of macro- and nano- systems. Macroscopic networks include films, slabs and beads and one of their most promising applications is related to tissue engineering. Recently, nano- sized hydrogel networks attracted an ever increasing attention because of the relevant variations of their physical (mechanical, electrical, optical, etc.) properties when compared to macroscopic systems. For example, the increase in area/volume ratio significantly modifies mechanical, thermal and catalytic properties, leading to new biomaterials suitable for a wide variety of applications. In 2007 the micro-hydrogel was defined by IUPAC as a “particle of hydrogel of any shape with an equivalent diameter of approximately 0.1 to 100 µm” [18]. A nano-hydrogel was then defined as a: “particle of hydrogel of any shape with an equivalent diameter of approximately 1 to 100 nm”. Micro-and nano-hydrogels can be produced by a large number of procedures such as self-assembling, suspension, emulsion, precipitation or dispersion polymerization, micro-molding, droplet generation, micro-fluidics, etc.The mean size measurements of water-swollen nano-hydrogels and micro-hydrogels may be challenging. Transmission electron microscopy and scanning electron microscopy, suitable for dried samples, cannot be applied for swollen hydrogels. Polymer chains collapse during drying leading to structures that are not present in the wet state. It is thus advisable to supplement studies in the dry state (electron microscopy, mercury intrusion porosimetry, etc.) with scanning confocal microscopy in the swollen state. Dynamic light scattering (DLS) is also useful to obtain not only an average hydrodynamic radius of microparticles and nanoparticles but also a size distribution [19].
1.4. Nanohydrogels and solid polymeric nanoparticles.
Because of their soft and rubbery consistency, nanohydrogels show a variety of advantagesoversolid polymeric nanoparticles. Such peculiar consistency minimizes frictional irritation of surrounding cells and tissues and allows specific deformations of the material that is capable to pass through pores without losing its original form and shape. Furthermore, nanohydrogels can often behave as “smart” materials, capable to respond to external physical/chemical/mechanical stimuli.According to their starting materials, nanohydrogels usually have good compatibility with aqueous fluids without dissolving and are consequently useful as biomaterials, as controlled release devices and electrophoresis gels. On the other side, polymeric nanoparticles, such as nanoparticles of PLGA or PCL, are usually obtained from synthetic polymers and do not show good compatibility with water, their shape cannot be modulated and their consistency does not allow deformations. Furthermore, it was recently reported that polysaccharidic nanohydrogels can evade the clearance by mononuclear phagocyte system (MPS). While the neutral and hydrophilic nature of polysaccharidic nanohydrogels avoids the macrophage uptake, reduces opsonization in the bloodstream and hinders immunogenic responses; solid polymeric nanoparticles are rapidly cleared from systemic circulation by the MPS and are sequestered in the liver, the kidneys or the spleen. To avoid the MPS, thereby increasing circulation life times, PEG-coating of the polymeric nanoparticles is required [20, 21]. Inspired by the polysaccharidic nanohydrogel ability to minimize premature clearance by macrophages, Alhareth et al. [22] decorated the nanoparticles surface with dextran chains in order to prolong the biodistribution of the system.
2.1. Polysaccharide hydrogels as drug delivery systems.
The unique physical properties of polysaccharidic hydrogels sparked particular interest in their use in drug delivery applications. Their highly porous structure allows drug loading into the hydrogels matrix followed by drug release through the gel network. Indeed, a depot formulation is created from which drugs slowly elute, maintaining high local concentration in the surrounding tissues over an extended period of time. Drug release mechanisms from hydrogels can be classified as: a) diffusion-controlled, b) swelling-controlled, and c) chemically-controlled. The drug diffusion out of a hydrogel matrix is primarily dependent on mesh sizes (ξ) within the hydrogel matrix which, in turn, is affected by several parameters, including the degree of cross-linking and the chemical structure of the macromolecular components. In the case of the swelling-controlled mechanism, swelling rate becomes the rate limiting factor. Chemically-controlled release is determined by chemical reactions occurring within the hydrogel matrix. These reactions include polymeric chain cleavage via hydrolytic or enzymatic degradation, or reversible/irreversible reactions occurring between the polymer network and the loaded drug [2]. For pratical applications, drug delivery vehicles and their degradation products must have good biocompatibility properties and should not show toxic, allergic or inflammatory effects. Thus, polysaccharidic hydrogels appear to be particularly suitable as drug delivery systems, since they are usually not toxic and biocompatible, may protect drug activity and improve drug transport through the biological barriers.
2.2. Switching from macro- to nano- hydrogel systems in drug delivery applications.
Polysaccharidic hydrogels, despite of their properties, when formulated in macroscopic networks show several limitations. In recent years, significant efforts have been devoted to overcome such limitations. For this purpose, micro- and nano-scale systems were studied and developed. Hydrogels nanoparticles (nanohydrogels) possess, at the same time, features and characteristics of hydrogels and nanosystems. Therefore, they benefit from both the hydrophilicity, flexibility, versatility, high water absorptivity and biocompatibility of hydrogels and all the advantages of nanoparticles.Nanometer scale is found frequently in biological systems, consequently nanohydrogels offer a number of treatment strategies unachievable with conventional hydrogels [23, 24].
Drug uptake and target accumulation: challenge of hydrogels formulated in macroscopic networks VS advantages of nanohydrogels.
Many conventional hydrogels formulated in macroscopic networks show a low tensile strength [2] that can cause a premature dissolution or a removal of the macroscopic systems from a specific target, thus limiting their use in load-bearing applications. Hydrogel slabs and films can swell and subsequently dissolve in aqueous environment, sometimes within a few hours for highly hydrophilic polymers. This hampers the drug accumulation in the target and reduces the cellular drug uptake.
A variety of strategies have been attempted to prolong the residence time of hydrogels in the site of action thus favoring drug accumulation and uptake. For example, a simple approach was the use of muco-adhesive polysaccharides such as chitosan, hyaluronic acid or gellan gum:muco-adhesive polysaccharidic macro-hydrogels are able to interact with the mucus leading to a prolonged permanence of the device in the body. Recently, Varshosaz et al. [25] underlined the advantages of the use of muco-adhesive polysaccharidic hydrogels in the nasal release of insulin. They obtained a good control of drug release and the muco-adhesive propertiesincreased insulin absorption. The hydrogel ability to stick to the targetwas also improved by combining two or more polymers with muco-adhesive properties.Uccello-Barretta et al [26], for example, investigated the muco-adhesive properties of tamarind seed polysaccharide and hyaluronic acid mixtures by NMR spectroscopy. They found that the muco-adhesivity of the polymeric mixtures increased with respect to that of the single polysaccharides and was strongly dependent on polysaccharideratios.
Even though macroscopic hydrogel networks can take advantage of the muco-adhesive property that prolongs the residence time in the specific site, only by formulating hydrogels in the form of nanoparticles it is possible to manipulate the molecule structures and obtain an efficient drug uptake and selective drug accumulation [2].Nanohydrogels allow to obtain passive or active targeting and offer unique advantages in drug delivery, unachievable with macroscopic hydrogels networks. Passive delivery refers to the transport through compromised endothelial barriers. Sinus endothelium, tumor neo-vasculature and inflammatory endothelium have an increased capillary permeability, which allows a high rate of nanoparticles accumulation, based on the “enhanced permeability and retention” (EPR) effect. Hydrogels formulated in slabs or films cannot take advantage of this effect. Nanohydrogels, because of their small size, spontaneously accumulate in pathological areas, leading to higher drug accumulation in the target site and enhanced therapeutic benefits. Yong Woo Cho [27] investigated the tumoral distribution of self-assembled nanoparticles, based on EPR effect, using fluorescein and radio-labeled nanoparticles. Self-assembled nanoparticles were prepared from amphiphilic chitosan derivatives, and their tissue distribution was examined in tumor-bearing mice. The nanoparticles were sustained at a high level throughout the 14 day experimental period, indicating their long systemic retention in the blood circulation. The ɣ-images provided clear evidence of selective tumor localization and confocal microscopy revealed the nanoparticles to be preferentially localized in the perivascular regions, confirming their extravasation to the tumors through the hyperpermeable angiogenic tumor vasculature.