A facile one-pot synthesis of microgels and nanogels of laminarin for biomedical applications

Highlights

• Single-step p(LAM) micro/nanogel preparation was accomplished for the first time.

• P(LAM) particles were fabricated by water-in-oil microemulsion polymerization.

• Surface tunability of p(LAM) particles were demonstrated by CSA modification.

• The bare and CSA modified p(LAM) particles were found to be blood-compatible.

• P(LAM) particles hold great promise as natural/nontoxic drug delivery.

Abstract

Hypothesis

Laminarin (LAM) as a nontoxic, biodegradable, and biocompatible marine polysaccharide, has been reported for its ingenious bioactivities such as antioxidant, antitumor antiapoptotic anti-inflammatory, immunomodulatory and dietary fiber activities, and distinct physicochemical structure possess a remarkably promising potential in biomaterial science. Synthesis of LAM-based microgels and bulk hydrogels have been reported in two stages: modification of LAM polysaccharide with polymerizable functional groups and subsequent crosslinking reaction. Therefore, here an easier and more effortless methods to prepare poly(laminarin) (p(LAM)) particles were tackled.

Experimental

A direct and facile single step fabrication of micro/nanogels of p(LAM) for the first time by means of reverse micelle microemulsion system were illustrated. Preparation of p(LAM) particles were achieved by the well-known Oxa-Michael addition reaction mechanism using divinyl sulfone as the crosslinker.

Findings

P(LAM) particles in 0.3–10 µm size range in spherical morphologies were prepared with 93 ± 7% yield and functionalized with chlorosulfonic acid (CSA) demonstrating their chemical modifiability for variety of agents e.g., targeting ligands. The bare and modified p(LAM) particles showed excellent blood compatibility with hemolytic indices of <1% and blood clotting indices higher than 90%. The reported p(LAM) particles hold great promise as natural alternative surrogates in biomedical applications including drug delivery.

Introduction

Hydrogels are 3D colloidal networks of polymers formed by the physical or chemical crosslinking of the corresponding precursors. They are also known for their ability to absorb and retain high volumes of water thereby can serve as soft and flexible scaffolding platforms in tissue engineering and wound dressing applications [1], [2]. Microgels and nanogels in particular have been holding remarkable capacity as reservoirs for various drugs and bioactive agents [3], [4], [5], [6]. They can be specialized through fine-tuning of their surface chemistry and provide targeted and controllable drug release [7], [8], [9]. Recently accelerating interest has been aroused in designing bio-based micro and nanogels from renewable biocompatible and biodegradable precursors such as polysaccharides, proteins, and polyphenols [10]. Marine-derived polysaccharides are one such class of prominent biopolymers emerging with a remarkable potency to be used as natural and bioactive precursors in the fabrication of various polymeric structures [11], [12]. Besides, agar, carrageenan, ulvan, fucoidan, alginate, and laminarin (LAM) has recently been explored and attracted for its remarkable potential in biomedicine due not only to its inherently biocompatible, biodegradable, and non-toxic nature but also for its highly sophisticated physiological bioactivities [13], [14]. LAM is available in great abundance as the main storage polysaccharide of algae. It is a low molecular weight glucan comprised of β-(1-3) linked glucose chains with occasional 6-O inter-strand branching. The chemical structure of LAM was reported to exhibit various degrees of polymerization, branching, and variations in its bioactivity depending on the source organism [15], [16]. Many researchers have highlighted bio-functional potential and health-promoting effects of LAM [17] including, antioxidant, antitumor [18], [19], anti-apoptotic [20], anti-inflammatory, immunomodulatory [21], and dietary fiber activities [22]. The antioxidant property of LAM was evaluated by Choi and colleagues. In their study, a significant correlation has been made between molecular weight and antioxidant capacity of LAM whereby by gamma irradiation mediated degradation of LAM have imparted increased levels of radical scavenging ability than non-degraded polysaccharide form [23]. Although LAM is a relatively weaker antioxidant in comparison to other biomolecules such as polyphenols [24], it reduces oxidative stress and promotes stimulation of the body’s own antioxidant mechanism by increasing the activity of antioxidant and anti-inflammatory enzymes [25], hence exert beneficial effects. Besides the bioactive benevolences, intrinsically low viscosity, and the natural hydrophilic backbone of LAM can be conveniently fortified by chemical modifications for potential biomedical applications. From the biomedical standpoint, in spite of the exhaustive structural and functional characterization of LAM polysaccharide and its spectacular potential therein, practical translation of LAM into biomaterial science seemed to be somehow hindered and progressed at a slow pace. However, in recent years, LAM has recuperated the scientific interest and acquired the temptation of many researchers [13].

LAM has been used as functionalization and synthesis of micro/nano architectures, and exemplary materials include sulfated LAM preparations as anticoagulant [26], and anti-metastatic [27] agents, support for the synthesis of Ag nanoparticles [28], and protoporphyrin IX loaded hematin-conjugated (Pp IX-loaded HLDM) nanomicelles [29]. Moreover, microgels [30], and bulk hydrogels [31] of LAM were synthesized by radical photopolymerization upon chemical modification with methacrylate functional groups. Modified LAM microgels were studied for attachment of human adipose stem cells for cell encapsulation applications. Likewise, Wang and coworkers prepared LAM based micro and macro hydrogels by using the same methacrylate functionalization method and reported enhancements on their mechanical properties [32]. Recently, Castanheira and colleagues reported the preparation of LAM microparticles via Cu(I) catalyzed Huisgen cycloaddition mechanism after modification of LAM polysaccharides with alkyne and azide groups [33]. Although, preparation of LAM based micro/macro hydrogels are possible after incorporation of polymerizable functional groups, less demanding new strategies are required to further amplify the research and potential applications of LAM for pharmaceutical and biomedical implementation.

Therefore, the state-of-art and novelty engaged in this study for the fabrication of LAM micro/nanogel formulations unlike the reported counterparts are a direct synthesis of crosslinked particles without the requirement of prior modification to introduce polymerizable moieties onto LAM backbone by employing a single-step reverse microemulsion polymerization. To the best of the authors’ knowledge, this is the first report demonstrating the one-pot synthesis of crosslinked p(LAM) micro/nanogel particles via water-in-oil microemulsion (w/o) technique. Optic microscopy, scanning electron microscopy (SEM), dynamic light scattering (DLS), and Fourier transform infrared (FT-IR) spectroscopy was used to corroborate particle formation as well as for the assessment of morphology, and hydrodynamic size of the particles. Moreover, to demonstrate the suitability of the hydroxyl groups of p(LAM) for surface functionalization and to endow additional versatility, p(LAM) particles were chemically modified with chlorosulfonic acid (CSA) on the basis of earlier reports where sulfated modification enhanced antitumor activity of LAM polysaccharides [34]. Thermal decomposition profiles of bare and CSA-modified p(LAM) (p(LAM)-CSA) particles were determined by thermogravimetric (TG) analysis. Furthermore, membrane degradation and blood clotting characteristics of the p(LAM) and P(LAM)-CSA particles were evaluated through in vitro hemolysis and coagulation assays.