In situ monitoring of the formation of lipidic non-lamellar liquid crystalline depot formulations in synovial fluid

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Abstract

Administration of parenteral liquid crystalline phases, forming in-vivo with tunable nanostructural features and sustained release properties, offers an attractive approach for treatment of infections and local drug delivery. It has also a potential use for postoperative pain management after arthroscopic knee surgery. However, the optimal use of this drug delivery principle requires an improved understanding of the involved dynamic structural transitions after administration of low-viscous stimulus-responsive lipid precursors and their fate after direct contact with the biological environment. These precursors (preformulations) are typically based on a single biologically relevant lipid (or a lipid combination) with non-lamellar liquid crystalline phase forming propensity. In relation to liquid crystalline depot design for intra-articular drug delivery, it was our interest in the present study to shed light on such dynamic structural transitions by combining synchrotron SAXS with a remote controlled addition of synovial fluid (or buffer containing 2% (w/v) albumin). This combination allowed for monitoring in real-time the hydration-triggered dynamic structural events on exposure of the lipid precursor (organic stock solution consisting of the binary lipid mixture of monoolein and castor oil) to excess synovial fluid (or excess buffer). The synchrotron SAXS findings indicate a fast generation of inverse bicontinuous cubic phases within few seconds. The effects of (i) the organic solvent N-methyl-2-pyrolidone (NMP), (ii) the lipid composition, and (iii) the albumin content on modulating the structures of the self-assembled lipid aggregates and the implications of the experimental findings in the design of liquid crystalline depots for intra-articular drug delivery are discussed.

Introduction

The development of biodegradable parenteral sustained release formulations has gained increasing research interest in the last two decades [1], [2], [3], [4], [5], [6], [7], [8]. Among the various approaches in designing parenteral drug delivery systems, in situ forming implants are attractive candidates as efficient sustained release matrices [1], [2], [5], [6], [9]. In addition to the attractiveness of these stimuli-triggered in situ forming systems in sustaining drug release, they could minimize undesirable side effects and improve patient compliance by avoiding repeated drug administration [1], [4]. The advantages of their utilization include among others, ease of administration and use of less invasive small needles [2], [4], [10]. The parenteral depots are typically used for local chemotherapy, local ocular drug delivery, and infection treatments [2], [6].

Following sol–gel approach in the formation process [1], [2], [4], various parenteral dosage forms with sustained release properties are reported in the literature. The applied process is based on the injection of a low viscous solution that is converted at the administration site in the body to a viscous gel-like matrix or a solid depot [1], [2], [4]. These depots are formed in response to a change in temperature, hydration level or pH [2], [4], [11]. They can also be formed in the presence of an external trigger, e.g., a heat or a light source [4]. Among these depots, the in situ forming drug delivery systems based on inverted-type lyotropic non-lamellar liquid crystalline phases can offer an interesting approach to the design of parenteral formulations with tunable nanostructures and sustained release properties [1], [2], [6], [7], [10], [12], [13], [14], [15]. These lyotropic non-lamellar self-assemblies are tolerable and capable of solubilizing hydrophilic, amphiphilic as well as hydrophobic drugs, and are stable against dilution in excess water [3], [4], [6], [9], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25]. Of most interest is the in situ formation of inverse cubic and hexagonal liquid crystalline delivery systems at the administration site [5], [6], [10], [19], [26]. This in situ generation of non-lamellar self-assemblies, also known in the literature as the in situ formation of solidifying organogels, occurs in response of injectable low-viscous stimulus-responsive precursors (drug preformulations) to the biological environmental stimulus [1], [3], [4], [7], [10], [13], [26], [27]. The hydration-induced transition from a low water-containing micellar solution or a water-free organic solution consisting of single surfactant-like lipid (or also binary or ternary lipid combinations) to highly viscous inverse cubic or hexagonal liquid crystalline systems is attractive method in the design of injectable formulations. Here, the gelation occurs at physiological conditions without the need for an external trigger or the addition of a copolymerization agent [4], [10]. In spite of the potential of the non-lamellar liquid crystalline phases as parenteral depots for loading and delivering various drugs, the number of studies on the mechanism of their formation is still limited [3], [10]. It is vital to understand the transition pathways under non-equilibrium conditions and the possible formation of intermediate nanostructures [3], [28]. This is endeavored by mimicking the direct exposure of the surfactant-like lipids to the biological environment upon the injection of the drug precursors [3]. The fast in situ formation of these highly viscous cubic or hexagonal phases calls for applying synchrotron time-resolved small angle X-ray scattering (TR-SAXS) for real-time monitoring of the structural dynamic processes [3], [28], [29], [30].

The development of in situ forming liquid crystalline systems is particularly attractive in the treatment of arthritis diseases via the intra-articular administration route [5], [9], [31]. The design of these in situ forming liquid crystalline formulations with sustained release properties and the optimal use, require among others: (i) in vitro and in vivo evaluation studies, and (ii) understanding of the dynamics of the structural transitions and specifically, the time required for the gelation process [10] (liquid crystalline formation). In this work, we exclusively focus on the structural events that take place on exposure of a low viscous lipid precursor (preformulation) to the biological environment. In the preparation of this lipidic precursor, the water-soluble organic solvent N-methyl-2-pyrolidone (NMP) was selected due to its biocompatibility [32]. Once the precursor is exposed to excess buffer (or biological medium), NMP starts to diffuse into the excess of buffer, and at one point, its concentration drops below the solubility limit for both lipids (monoolein and castor oil). It induces, therefore, the self-assembly into inverse non-lamellar liquid crystalline phases in the new aqueous environment. A combination of synchrotron small-angle X-ray scattering (SAXS) with remote controlled addition of synovial fluid was applied to mimic the self-assembly behavior under physiological conditions. The implication of these detected dynamic structural changes in relation to drug delivery applications is discussed in the light of our experimental findings.

Section snippets

Materials

Monoolein (MO, product name: RIKEMAL-XO-100) with a purity ≥90% was obtained from Riken Vitamin Co. (Tokyo, Japan). Castor oil (CO), human serum albumin (HSA), and the organic solvent N-methyl-2-pyrolidone (NMP) were purchased from Sigma-Aldrich (St. Louis, USA). In the experiments, a 67 mM phosphate buffer solution (PBS) at pH 7.4 was used. The albumin solution composed of 2.0% (w/v) HSA in PBS and prepared at pH 7.4.

Synovial fluid (SF) from arthritis patients was kindly donated by the Parker

Effects of lipid composition, NMP content, and albumin on the structural features of MO- and MO/CO-based systems

Fig. 1 shows the molecular structures of MO and glyceryl triricinoleate, which is the main constituent of CO. Before conducting the dynamic investigations on the potential generation of inverse non-lamellar liquid crystalline phases on exposure of a lipid precursor to excess SF, one should bear in mind the high sensitivity of such phases to variations in lipid composition, inclusion of an organic solvent such as NMP, and aqueous (or biological) medium composition [7], [9], [13], [14], [16], [34]

Conclusions

There is a growing interest in the development of injectable low-viscous stimulus-responsive drug precursors that convert in situ to inverse non-lamellar liquid crystalline phases at the administration site in the body. The attractiveness of the in situ formation of these inverse self-assemblies relies on their unique structural features, biological relevance, and sustained drug release properties [16], [34], [23], [24], [25]. However, the optimal use requires extensive characterization as the

CRediT authorship contribution statement

Anan Yaghmur: Conceptualization, Validation, Supervision, Investigation, Formal analysis, Data curation, Writing - original draft, Writing - review & editing. Michael Rappolt: Conceptualization, Validation, Investigation, Methodology, Writing - review & editing. Anne Louise Uldall Jonassen: Investigation, Formal analysis. Mechthild Schmitt: Investigation. Susan Weng Larsen: Conceptualization, Validation, Supervision, Methodology, Writing - review & editing.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

AY acknowledges financial support from the Danish Natural Sciences Research Council (DanScatt) for SAXS experiments.

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