Liquid–liquid phase separation (LLPS) induces the formation of membrane-less droplet-like compartments through the segregation of biomolecules into distinct liquid phases within a solution, without the requirement for a surrounding lipid bilayer1. These membrane-less droplet-like compartments, which arise from LLPS, possess unique properties and functions essential for cellular organization, biomolecular condensation, and cellular signaling2. Recent advancements have increasingly enabled the utilization of these membrane―less compartments as delivery platforms3. However, LLPS assemblies often fail to meet long-term storage and application requirements due to challenges such as coalescence and disassembly4.
Recently, a pioneering study published in Nature Chemistry by Gu's group5 introduced an innovative coacervate vesicle delivery system leveraging liquid–liquid phase separation to enhance the efficient and targeted delivery of biopharmaceuticals (Fig. 1). Under an appropriate ratio of components, cholesterol-modified single-strand DNA (Chol-ssDNA) and histone molecules could self-assemble to generate highly stable liquid–liquid phase separation (LLPS)-assembled coacervate vesicles (CVs) through a simple mixing process. Unlike phospholipid-based membrane-bound vesicles, CVs lacked a surface membrane but featured a dense liquid layer surrounding a water-filled core. This unique configuration overcomes the limitations commonly associated with phospholipid membranes, such as low permeability and physical barriers, thereby enabling the effective encapsulation of viral particles, mRNA, cytokines, peptides, and various therapeutic agents while maintaining their biological activity.
Figure 1.
A schematic illustration of coacervate vesicle for oncolytic virus packing and intracellular delivery (copyright © 2025 Springer Nature).
Researchers employed oncolytic virus, specifically oncolytic adenovirus serotype 11 (ad11), as model drugs to explore the potential of CVs for drug delivery. In vitro experiments demonstrated that CVs effectively improved oncolytic virus infection in tumor cells, regardless of whether these cells expressed the requisite receptor for viral infections. The results highlighted that CVs notably enhanced the uptake of oncolytic virus in various tumor cell lines by up to 48-fold. Mechanistically, by evaluating the uptake of oncolytic virus by tumor cells in the presence of multiple inhibitors, Gu's group5 identified distinct uptake pathways facilitated by CVs and elucidated the roles of macropinocytosis or phagocytosis in CV-cell interactions.
In a mouse model, the enhanced tumor infection facilitated by oncolytic viruses delivered via CVs not only extended tumor residence but also intensified their anti-tumor efficacy. Notably, the augmented anti-tumor effect induced by CVs was noticeable even when tumor volumes reached 500 mm³. Additionally, CVs demonstrated favorable biosafety profiles. Throughout the treatment period, no significant organ damage or alterations in body weight were noted. Transient fluctuations in transaminase levels during treatment were observed in blood biochemical indexes, gradually normalizing over time. The pivotal aspect of oncolytic virus therapy lies in the induction of the anti-tumor immune response. CVs significantly facilitated the infiltration of CD8+ T cells into tumors during oncolytic virus therapy. Furthermore, compared to direct intratumoral injection of oncolytic virus, the delivery of oncolytic virus via CVs effectively increased the presence of M1 macrophages within and decreased the abundance of M2 macrophages in tumor tissues, thus successfully modulating the tumor microenvironment.
LLPS carriers encounter significant limitations in drug delivery, including the potential instability of phase-separated structures and challenges in regulating drug release kinetics4. The shape and topological characteristics of these phase-separated carriers play a crucial role in influencing drug delivery efficiency and specificity, underscoring the necessity for a deeper comprehension of how these aspects impact drug transport within such systems6. Gu's group5 have effectively optimized the morphology of condensate droplets by adjusting the ratio of Chol-ssDNA to histones, thereby enhancing the efficacy of oncolytic virus against tumors with low infectious receptor expression and inducing significant changes in the tumor microenvironment. Extensive research has delved into exploring the topology, physicochemical attributes, and intracellular delivery pathways associated with LLPS to delve further into the underlying mechanisms. This study has revealed that hydrophobicity, charge-based interactions, and electrostatic effects of multivalent interactions are pivotal determinants of the phase state and act as essential driving forces for condensation. This research, centered on the development of CVs based on LLPS and assembly mechanisms, introduces an innovative delivery platform for the efficient transportation of biotechnological drugs and cancer therapies. With its uncomplicated preparation process and outstanding biological safety profile, this carrier showcases significant promise as a versatile drug delivery system.
In contrast to the intricate extraction procedures associated with natural condensates and the instability often observed in membraneless compartments, the simplicity of operation and robust stability of CVs significantly enhance their potential for clinical translation. Moving forward, comprehensive preclinical studies are imperative to thoroughly evaluate the in vivo stability and safety of the vector before clinical implementation. Given the remarkable efficiency of CVs in delivering a wide range of biological drugs, its application is expected to extend beyond cancer treatment to include the delivery of proteins, mRNAs, and other therapeutic agents across various disease indications. The advancement of this technology holds the promise of providing substantial benefits to patients grappling with tumors or some refractory diseases.
Author contributions
Zao Ji wrote the manuscript. Jianbin Bi, Hong-Xu Liu and Heran Li revised and supervised the manuscript.
Conflicts of interest
The authors declare no conflict of interest.
Acknowledgments
This work was supported by the National Nature Science Foundation of China (No. 82073286), and State Key Laboratory of Neurology and Oncology Drug Development (No. SKLSIM-F-202418, China).
Footnotes
Peer review under the responsibility of Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical Sciences.
Contributor Information
Jianbin Bi, Email: bijianbin_cmu@163.com.
Hong-Xu Liu, Email: hongxuliu@qq.com.
Heran Li, Email: liheranmm@163.com.
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