Alginate-Based Delivery Systems for Bevacizumab Local Therapy: In Vitro Structural Features and Release Properties

Abstract

Alginate-based polyelectrolyte complexes (PECs) and hydrogel were engineered as platforms for local bevacizumab (BVZ) therapy. This study provides deep comprehension on the microstructures of such systems, and their correlation with drug-release patterns. PECs and hydrogel were characterized using Fourier transform infrared spectroscopy, small-angle X-ray scattering, scanning electron microscopy, atomic force microscopy, and porosimetry. Structural investigations indicated that PECs are formed by supramolecular interactions, resulting in physically cross-linked polymer networks, whereas the BVZ-loaded hydrogel has a more compact and rigid structure, promoting better entrapment of BVZ. PECs and hydrogel were able to control the BVZ release for 4 and 8 days, respectively. Their release profiles correlated best with the Higuchi and Korsmeyer-Peppas models, respectively, indicating drug diffusion as the limiting step for drug release. Furthermore, BVZ remained biologically active in vitro after its incorporation into the hydrogel system. Together, these studies confirm that PECs and hydrogel exhibit different porous structures and physicochemical properties, making them promising platforms that allow the modulation of BVZ release meeting different requirements.

Introduction

In the field of polymeric materials, innovative release systems with a wide range of physicochemical and structural properties have been developed, thanks to advancement of pharmaceutical technology and material science.

Natural polymers used as drug delivery platforms have drawn increasing attention because of their wide variety of structures. Some of these polymers are composed, for example, by hydrophilic groups and surface charges, which allow them to be chemically or physically modified. Thus, new, biocompatible, and biodegradable materials with specific properties can be produced.1, 2, 3, 4, 5, 6 The understanding and ability to control the parameters involved in the production of natural polymer–based systems allow structural properties to be manipulated and acquire specific biological performance.7, 8

Alginates are natural polymers isolated mainly from brown algae such as Laminaria hyperborea and Lessonia nigrescens, both found in coastal regions. Chemically, they are composed of a linear unbranched structure of 1,4-linked α-l-guluronic (G) and β-d-mannuronic acid (M), copolymers connected by glycosidic bonds with variable molecular weight, containing sequences of same (MMM or GGG) or alternating (MGMG) units.9, 10, 11, 12 The ratio and distribution of each monomer in the composition are closely related to the nature of the alginate source and its extraction process. Alginate polymer chains exhibit pK_a values of 3.4 and 3.6 for mannuronic and guluronic acid, respectively.¹³ At physiological pH, they compose a pattern surrounded by negative charges which interact with positively charged molecules by supramolecular interactions and spontaneously self-assemble into polyelectrolyte complexes (PECs) of oppositely charged polyelectrolytes, which represents a great promise as a tool for the development of drug delivery systems.⁶ Furthermore, in the presence of divalent ions, they form 3-dimensional (3D) networks by sol-gel transition, creating a stiff egg-box structure.14, 15, 16 The hydrogel matrix can be structured through cross-linking provided by covalent or secondary bonds, originating chemical or physical hydrogels, respectively.¹⁷

Physical hydrogels are widely applied for biomedical purposes because of their flexible and dynamic structures, formed by supramolecular interactions, including hydrogen bonds, hydrophobic interaction, as well as ionic and electrostatic bonds.18, 19 These hydrogels are very useful for drug delivery in specific target areas because of their capacity to protect the drug from hostile environments; to control the release rates by changing the structure in response to environmental stimuli; and to degrade with time.20, 21

Alginate polysaccharides cross-linked with calcium must be highlighted because of their low interfacial tension with the surrounding biological environment and high capacity to absorb biological fluids, which renders mechanical and compositional similarity to the natural extracellular matrix.²² In addition, their 3D porous structures, obtained in mild conditions, feature a distinctive drug delivery profile, which can be modulated through drug-matrix associations.²³

The encapsulation and release of protein-based drugs, such as monoclonal antibodies (mAbs), using alginate hydrogel or alginate PECs can significantly enhance their efficiency and targeting.14, 24, 25, 26, 27 This engineering design may become especially attractive in high-cost therapies, because they enable the release of reduced doses of drugs.

Bevacizumab (BVZ) is a recombinant humanized MAb which acts against human VEGF-A in all isoforms and in bioactive proteolytic fragments.²⁸ This therapeutic agent was approved by the U.S. Food and Drug Administration (FDA) in 2004 and is widely used for cancer treatment.²⁹ Furthermore, BVZ is widely used as an off-label therapy for diseases associated with neovascularization, which provides even more reasons to consider it.³⁰ Although therapies with BVZ still require intravenous administration, as protein macromolecules, BVZ exhibits a complex 3D structure, physicochemical instability, and susceptibility to environmental factors.31, 32 An alternative approach to its systemic administration may be localized therapy using polymer systems administered at the targeted site. Injectable platforms might promote the mAb retention because tumor microenvironment poses barrier to the distribution and retention of mAb molecules.³³ This promising strategy may enhance BVZ therapeutic effect and decrease its considerable side effects besides offering the possibility of drug maintenance at the site of action.24, 34 In addition, due to high polymer biocompatibility, it may provide drug-controlled release in a minimally invasive manner. Moreover, BVZ-release patterns from the alginate hydrogel or PECs can be modulated by alginate-protein interactions.14, 26, 32

Previously, we exploited BVZ-loaded calcium alginate hydrogels as anti-angiogenic delivery systems for cancer therapy. They were produced by the previous formation of PECs between the alginate polymer and the BVZ drug, followed by a subsequent cross-linking process using calcium chloride. The developed hydrogel worked effectively as a delivery platform for the resumption of anti-VEGF local cancer therapy. In addition, it improved BVZ antiangiogenic and antineoplastic activity.²⁴ Nonetheless, considering that these polymeric matrices might behave differently as release platforms, the PEC structure produced to engineer these systems, as well as the release patterns of BVZ from hydrogel or PECs, has not yet been addressed. The supramolecular associations involved in the PECs formation should provide a release system driven particularly by drug-polymer interactions. On the other hand, for hydrogel systems, different mechanisms may be involved, in addition to drug diffusion throughout the polymer network.³⁵ Therefore, precisely controlling the mechanical properties of those systems is critical for the desired application.²²

Thus, the scope of the present work is to deepen our investigation into the microstructures of alginate-BVZ PECs and BVZ-loaded hydrogels, in an attempt to understand some of the structural particularities related to the system’s release profile. Small-angle X-ray scattering (SAXS), atomic force microscopy (AFM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM) analyses were extensively reported to provide insights about PECs and hydrogel microstructures, as well as about the rules of protein macromolecule and polymer chain interactions for release purposes.36, 37, 38, 39, 40, 41 Morphological and structural properties were discussed in relation to their release kinetics and mechanisms. BVZ bioactivity into developed systems was also investigated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell toxicity assays. Our study proves that PECs and alginate hydrogel exhibit differentiated microstructural compositions and physicochemical properties which can be engineered and modulated making them promising platforms for BVZ release.