Cholesterol-mediated anchoring of enzyme-loaded liposomes within disulfide-stabilized polymer carrier capsules

Abstract

Polymer capsules containing multiple liposomes, termed capsosomes, are a promising new concept toward the design of artificial cells. Herein, we report on the fundamental aspects underpinning the assembly of capsosomes. A stable and high loading of intact liposomal cargo into a polymer film was achieved by non-covalently sandwiching the liposomes between a tailor-made cholesterol-modified poly(l-lysine) (PLL_c) precursor layer and a poly(methacrylic acid)-co-(cholesteryl methacrylate) (PMA_c) capping layer. The film assembly, optimized on planar surfaces, was successfully transferred onto colloidal substrates, and a polymer membrane was subsequently assembled by the alternating adsorption of poly(N-vinyl pyrrolidone) (PVP) and thiol-modified poly(methacrylic acid) (PMA_SH) onto the pre-adsorbed layer of liposomes. Upon removal of the silica template, stable capsosomes encapsulating the enzyme luciferase or β-lactamase within their liposomal sub-compartments were obtained at both assembly (pH 4) and physiological conditions (pH 7.4). Excellent retention of the liposomes and the enzymatic cargo within the polymer carrier capsules was observed for up to 14 days. These engineered capsosomes are particularly attractive as autonomous microreactors, which can be utilized to repetitively add smaller reactants to cause successive distinct reactions within the capsosomes and simultaneously release the products to the surrounding environment, bringing these systems one step closer toward constructing artificial cells.

Introduction

Biological cells, which separate their interior from the exterior media by a lipid bilayer, operate by performing multiple enzymatic cascade reactions within predefined sub-compartments, the cell organelles. The lipid barrier is equipped with various biomolecules, including ion channels and transmembrane proteins, which serve as specific gates. Artificial cells [1], [2], on the other hand, do not require the complex multifunctionality of their biological counterparts, but rather can be more simply designed to perform a specific activity. However, certain prerequisites have to be fulfilled. Among them is the need of a micron-sized vessel with specific permeability that provides the structural scaffold and the encapsulated machinery that enables confined specific reactions to be conducted. Polymer [3], [4], [5] or lipid [6], [7] vesicles and polymer capsules [8], [9], [10], [11], [12], [13] are suitable platforms that are being explored as microreactors to conduct encapsulated reactions and as advanced therapeutic delivery vehicles.

Recently, we reported the construction of a new class of colloidal carriers/microreactors, termed capsosomes [14]. These are obtained by incorporating intact liposomes into polymer capsules, assembled by the layer-by-layer (LbL) technique [15], [16]. The first generation of capsosomes we reported consist of intact, unsaturated, zwitterionic 50 nm liposomes incorporated in a non-biodegradable polyelectrolyte film assembled from poly(styrene sulfonate) (PSS) and poly(allylamine hydrochloride) (PAH) [14]. We confirmed the functionality of the capsosomes by performing a triggered quantitative enzymatic reaction using β-lactamase encapsulated within the liposomal sub-compartments [17]. These capsosomes are of interest because the polymer capsule provides a structural scaffold with controllable permeability and the liposomes divide the interior into sub-compartments, thus potentially allowing parallel encapsulated enzymatic cascade reactions within confined systems. The liposomes are well-suited to encapsulate small hydrophobic and hydrophilic drugs or fragile biomolecules. Furthermore, these loaded liposomes can be incorporated in the polymer film at different regions, giving access to multi-strata films that are expected to be useful for the co-administration of complementary drugs.

In the current study, we examine two main fundamental aspects regarding capsosome assembly and performance that strongly affect their functionality: (i) the loading efficiency and stability of the liposomal cargo into the polymer film; and (ii) the structural properties of the capsosomes containing enzyme-loaded liposomes. To optimize the loading of intact liposomes into polymer multilayer films, we introduce a non-covalent linkage concept for liposomes based on cholesterol-functionalized polymers. Further, the choice of the building blocks of the polymer carrier capsules governs the structural properties, including the long-term stability of the capsosomes, and thus the encapsulated species. PSS and PAH, for instance, provide a non-degradable carrier capsule that is attractive if repetitive function over an extended period of time is required. On the other hand, (bio)degradable polymer membranes enable the body to deconstruct the carrier vehicles into their building blocks after their task is fulfilled. Among a variety of polymer systems that are suitable for the assembly of (bio)degradable membranes [18], [19], [20], [21], [22], poly(N-vinyl pyrrolidone) (PVP) and thiol-modified poly(methacrylic acid) (PMA_SH) were chosen. Cross-linking the thiols of the PMA_SH and the release of PVP at physiological conditions yield colloidally stable, (bio)degradable, single-component PMA capsules [19]. We have demonstrated that these PMA capsules can be used for the encapsulation of DNA [23], [24], drug-containing oil-droplets [25] and peptides [26], and to eradicate colon cancer cells in vitro [25] and stimulate T-cells for vaccination [26]. Equally important as the choice of the individual building blocks is an understanding of the structural characteristics of the polymer carrier capsule containing liposomal cargo. In particular, the polymer membrane assembly and stability in physiological media, as well as the selective diffusion of biomolecules across the polymer membrane are key factors to be investigated.

Capsosome engineering can be achieved by individually addressing the challenges in each assembly step. In this study, we synthesize cholesterol-modified poly(l-lysine) (PLL_c) and poly(methacrylic acid)-co-(cholesteryl methacrylate) (PMA_c) (Scheme 1). We characterize suitable polymer precursor layers to maximize liposome adsorption depending on the charge and phase transition temperature of the liposomes and examine various capping layers to stably anchor liposomes to the surface and to enable the subsequent multilayer film assembly (Scheme 2). We provide details on the optimal film assembly conditions on planar surfaces and subsequently transfer the assembly of these films onto colloidal substrates to form the capsosomes. With the goal to control the properties and the function of the capsosomes, we present data on the structural integrity of the capsosomes, the cross-linking of the thiols in the film, the encapsulation efficiency of the enzymatic cargo within the polymer carrier capsules under various conditions, and the long-term stability of the capsosomes.