Biopolymeric Coacervate Microvectors for the Delivery of Functional Proteins to Cells

Abstract

Although protein therapeutics have exceptional potential for nextgeneration therapies, delivery of these macromolecules is hindered due to their vulnerability to biological deactivation, [6-9] poor native membrane permeability, and short circulation time if utilized intravenously. [10-12] For these reasons, the development of protein delivery technologies is a vital endeavor that promises to unlock the potential of this significant class of bioactive macromolecules. [13,14] An emerging strategy to address the challenge of protein delivery is via the use of coacervate particles, which is appealing due to their ability to spontaneously sequester various macromolecular cargoes, [15,16] and to undergo direct cellular interactions. [17] In these terms, coacervates have been described as biomimetic microcontainers due to their physicochemical semblance to the cell cytoplasm, albeit without an external membrane. [18,19] Although being a relative newcomer to the field of therapeutic protein delivery, coacervate-based materials have received significant attention in biomedical research and have already been used for drug delivery. [20,21] For example, coacervate-based materials have shown great promise for controlled growth factor delivery in vivo for attenuation of disc degeneration, persistent angiogenesis, preservation of The extent to which biologic payloads can be effectively delivered to cells is a limiting factor in the development of new therapies. Limitations arise from the lack of pharmacokinetic stability of biologics in vivo. Encapsulating biologics in a protective delivery vector has the potential to improve delivery profile and enhance performance. Coacervate microdroplets are developed as cell-mimetic materials with established potential for the stabilization of biological molecules, such as proteins and nucleic acids. Here, the development of biodegradable coacervate microvectors (comprising synthetically modified amylose polymers) is presented, for the delivery of biologic payloads to cells. Amylose-based coacervate microdroplets are stable under physiological conditions (e.g., temperature and ionic strength), are noncytotoxic owing to their biopolymeric structure, spontaneously interacted with the cell membrane, and are able to deliver and release proteinaceous payloads beyond the plasma membrane. In particular, myoglobin, an oxygen storage and antioxidant protein, is successfully delivered into human mesenchymal stem cells (hMSCs) within 24 h. Furthermore, coacervate microvectors are implemented for the delivery of human bone morphogenetic protein 2 growth factor, inducing differentiation of hMSCs into osteoprogenitor cells. This study demonstrates the potential of coacervate microdroplets as delivery microvectors for biomedical research and the development of new therapies.

Interfacial Structure and Tension of Polyelectrolyte Complex Coacervates

“…Polyelectrolyte complex coacervation refers to an associative liquid–liquid phase separation in solutions of oppositely charged polyelectrolytes into a polymer-rich liquid coacervate phase and an equilibrium polymer-deficient supernatant phase. − Due to its fundamental role in a variety of fields such as hydrogel fabrication, − drug delivery, , and formation of membrane-less organelles in cells, − polyelectrolyte complex coacervation has received a great deal of attention in recent years from both the polymer physics and biophysics communities, and significant progresses have been made in understanding the bulk phase behaviors. ,− For example, it is now well accepted that the addition of monovalent salt can weaken the degree of phase separation, and the miscibility gap vanishes above a critical salt concentration. , Likewise, the effects of other parameters such as temperature, charge fraction, chain length, polymer concentration, sequence, and stoichiometry on the bulk properties of complex coacervation have been extensively examined in the literature; we refer to several recent excellent reviews ,− for these progresses.…”

Biomolecular Liquid–Liquid Phase Separation for Biotechnology

“…On the same note, currently, we have seen a substantial increase in therapeutic protein therapies with LLPS. One such demonstration even delivered functional myoglobin to human stem cells using amylose-based coacervates [ 186 ]. When more effective protein therapies are developed, the usage of phase-separated protein complexes may become even more prevalent [ 187 ].…”

Connecting primitive phase separation to biotechnology, synthetic biology, and engineering

“…Development of such technologies that form phase-separated droplets composed of multiple proteins, which themselves do not phase separate naturally, could help in the design of LLPS systems capable of transport of active protein cascades to specific locations in human patients. Therapeutic protein therapies have grown significantly in the recent few years; one such demonstration even utilized amylose-based coacervates to deliver functional myoglobin to human stem cells (figure 3) (Xiao et al 2020). As such, the use of phase-separated protein complexes may become even more widespread as more efficient protein therapies are discovered (Lagassé et al 2017), suggesting that application of LLPS to therapeutic protein delivery could be an important avenue of investigation going forward.…”