Cholesterol-mediated anchoring of enzyme-loaded liposomes within disulfide-stabilized polymer carrier capsules
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) (PMASH) were chosen. Cross-linking the thiols of the PMASH 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) (PLLc) and poly(methacrylic acid)-co-(cholesteryl methacrylate) (PMAc) (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.
Section snippets
Materials
Poly(l-lysine) (PLL, 40,000–60,000 Da), poly(N-vinyl pyrrolidone) (PVP, 10,000 Da), methacrylic acid, 4-(2-hydroxyethyl)piperazine-1-ethane-sulfonic acid (HEPES), sodium chloride (NaCl), sodium acetate (NaOAc), chloroform, cholesterol, triethylamine (TEA, 99%), dichloromethane (DCM), hydroquinone, methacryloyl chloride, hydrochloric acid (HCl), sodium bicarbonate (NaHCO3), magnesium sulphate (MgSO4), methanol, 4-nitrophenol chloroformate (NPC, 95%), 2,2′-azoisobutyronitrile (AIBN), dioxane,
Results and discussion
To optimize and control the formation of (bio)degradable and selectively permeable capsosomes, we exploit a new approach of sandwiching liposomes between two polymer layers by using cholesterol as a non-covalent linker (Scheme 2). PLLc was synthesized by attaching cholesterol to the amines of the polymer via NPC, while controlled radical copolymerization via RAFT was chosen to synthesize cholesterol-modified PMA due to its monomer compatibility, good end-group purity, and control afforded over
Conclusions
We have demonstrated the successful synthesis of cholesterol-modified PLL and PMA, and that different types of liposomes can be stably anchored non-covalently to the polymer films by sandwiching them between a tailor-made PLLc precursor layer and a PMAc capping layer. Experiments on planar and colloidal substrates showed that PMASH/PVP multilayer films can be subsequently assembled on top of the anchored liposomes. We examined two approaches to achieve thiol-to-disulfide conversion with the use
Acknowledgements
This work was supported by the Australian Research Council under the Federation Fellowship and Discovery Project schemes and the Swiss National Science Foundation (SNF, PBEZB-118906). We thank Kerry Breheney for providing the β-lactamase.
References (45)
- et al.
Artificial cells: prospects for biotechnology
Trends Biotechnol
(2002) - et al.
Artificial cells: building bioinspired systems using small-scale biology
Trends Biotechnol
(2008) - et al.
Layer-by-layer engineered capsules and their applications
Curr Opin Colloid Interface Sci
(2006) - et al.
Construction and enzymatic degradation of multilayered poly-l-lysine/DNA films
Biomaterials
(2006) - et al.
Advances in RAFT polymerization: the synthesis of polymers with defined end-groups
Polymer
(2005) - et al.
Arrays of lipid bilayers and liposomes on patterned polyelectrolyte templates
J Colloid Interface Sci
(2006) - et al.
Cholesterol interactions with phospholipids in membranes
Prog Lipid Res
(2002) - et al.
Complementary liposomes based on phosphatidylcholine: interaction effectiveness vs protective coating
J Colloid Interface Sci
(2002) - et al.
Positional assembly of enzymes in polymersome nanoreactors for cascade reactions
Angew Chem Int Ed
(2007) - et al.
Polymer vesicles containing small vesicles within interior aqueous compartments and pH-responsive transmembrane channels
Angew Chem Int Ed
(2008)
Enzymes containing porous polymersomes as nano reaction vessels for cascade reactions
Org Biomol Chem
Recent advances with liposomes as pharmaceutical carriers
Nat Rev Drug Discov
Enzymatic reaction in a vesicular microreactor: peptaibol-facilitated substrate transport
Chem Biodivers
Nanoengineering of inorganic and hybrid hollow spheres by colloidal templating
Science
Novel hollow polymer shells by colloid-templated assembly of polyelectrolytes
Angew Chem Int Ed
Nanoengineered polymer capsules: tools for detection, controlled delivery, site-specific manipulation
Small
Polymeric microcapsules for synthetic applications
Macromol Biosci
Polyelectrolyte microcapsules for biomedical applications
Soft Matter
Capsosomes: sub-compartmentalizing polyelectrolyte capsules using liposomes
Langmuir
Buildup of ultrathin multilayer films by a self-assembly process: II. Consecutive adsorption of anionic and cationic bipolar amphiphiles and polyelectrolytes on charged surfaces
Ber Bunsenges Phys Chem
Fuzzy nanoassemblies: toward layered polymeric multicomposites
Science
A micro-reactor with thousands of sub-compartments: enzyme-loaded liposomes within polymer capsules
Angew Chem Int Ed
Cited by (100)
Multicompartment polymer capsules
2022, Supramolecular MaterialsBiocatalytic self-assembled synthetic vesicles and coacervates: From single compartment to artificial cells
2022, Advances in Colloid and Interface ScienceCitation Excerpt :For example, Kataoka and coworkers developed injectable enzyme-loaded PICsomes (100 nm) as nanoreactors based on PEG-based block aniomers (PEG-b-PAsp), catiomers (Homo-P(Asp-AP)), and enzyme β-galactosidase (β-gal) or lysozyme for selective in vivo EPT to tumor cells [52]. Similarly, using alternate layers of polyelectrolytes and liposomes/ enzymes, e.g., GOx/PEI/CAT [63], GOx/CAT [64], PSS/PAH [65], PLLc/liposomes/PMAc/PVP/PMASH [66,67], PS-PIAT [43,45,58,59] layer-by-layer (LbL) capsules have been formed (Fig. 2B(c)). Such arrangement of polyelectrolyte provides sufficient movement of substrate and product across the capsule membrane, beneficial in enzyme nanoreactor development [104,137,138].
Enzyme-responsive polymer composites and their applications
2020, Smart Polymer Nanocomposites: Biomedical and Environmental ApplicationsEnzymes as key features in therapeutic cell mimicry
2017, Advanced Drug Delivery ReviewsRecent advances in compartmentalized synthetic architectures as drug carriers, cell mimics and artificial organelles
2017, Colloids and Surfaces B: BiointerfacesCitation Excerpt :In particular, cholesterol-modified poly(L-Lysine), poly(methacrylic acid)-co-(cholesteryl methacrylate), poly(N-vinyl pyrrolidone)-block-(cholesteryl acrylate) and oleic acid-modified poly(methacrylic acid)-co-(oleyl methacrylate) have been successfully employed as precursor, separation and capping layers [84–88]. This strategy enabled for the incorporation of zwitterionic or negatively charged saturated or unsaturated liposomes without rupturing or displacement from polymer surfaces [84,86–88]. Non-degradable capsosomes have been assembled by employing the PSS and PAH polymer pair, since they afford structurally stable, non-aggregating capsules over an extended period of time [89].
Synthetic Cells Revisited: Artificial Cells Construction Using Polymeric Building Blocks
2024, Advanced Science