The combination of liposomes and protein delivery represents a novel biotechnological approach with the objective of enhancing the stability and delivery efficiency of protein drugs. As a synthetic membrane-like structure, liposomes exhibit excellent biocompatibility and controllability, enabling close binding with proteins for targeted delivery and sustained release of proteins. The ways in which liposomes enter cells mainly include direct fusion with the plasma membrane, entry through endocytosis, and lipid exchange with the plasma membrane. These methods all rely on the interaction and charge matching between liposomes and the cell membrane [26, 27]. The binding modes of liposomes and proteins can be broadly classified into two categories: encapsulation and modification. In the context of encapsulation strategies, liposomes utilize their double-layer membrane structure to encapsulate proteins, thereby forming stable liposome-protein complexes. This strategy is particularly well-suited for the delivery of therapeutic proteins, including glucose oxidase, catalase, horseradish peroxidase, and superoxide dismutase [28,29,30,31]. Proteins such as enzymes are susceptible to various factors in the in vitro environment, leading to structural damage and loss of activity. As a protective barrier, liposomes can isolate the external environment from interfering with proteins, maintaining their stability and activity. At the same time, liposomes can also provide a suitable microenvironment that helps proteins release slowly in the body, extending their action time [32]. Liposomes can also be combined with polypeptide proteins through modification strategies. This strategy has wide applications in fields such as vaccine adjuvants [33,34,35]. Through specific chemical modifications, liposomes can form stable covalent or non-covalent bonds with polypeptide proteins to achieve efficient loading of proteins. As a carrier, liposomes can also target the delivery of polypeptide proteins to specific tissues and organs, improving the immune effect and safety of vaccine adjuvants [36].

Therapeutic enzymes have now become effective therapeutic drugs for many major diseases and play an important role in the treatment of congenital enzyme deficiency. For example, infantile neurofibromatosis, a lysosomal storage disorder characterized by the accumulation of metabolites in lysosomes due to the lack of ppt1, can lead to the formation of inclusions known as granular halophilic deposits [37, 38]. Enzyme replacement therapy (ERT) helps restore the blocked function of tissues or cells by supplementing these missing enzymes, thereby alleviating the disease. However, despite the potential efficacy of ERT, its translation into clinical applications has indeed been hindered by several factors. These obstacles may include enzyme source, stability, and immunogenicity [39].

Encapsulating enzyme-based proteins within liposomes addresses specific challenges, including enhancing their stability and mitigating immunogenicity. Santi et al. described the use of liposomal delivery of ppt1 enzyme for the treatment of infantile neurofibromatosis [37]. These liposomes containing enzymes can restore stable levels of enzyme activity in fibroblasts of CLN1 patients, promote the delivery of proteins to the central nervous system, and affect intracellular biological pathways. In addition, the delivery of catalytic enzymes, such as glucose oxidase, can help kill tumor cells (Fig. 2). In previous research, glucose oxidase was encapsulated and delivered into tumor cells by liposomes [32]. The manganese-based nanoprobes NanoMn-GOx-PTX are mainly composed of a manganese core and a phospholipid bilayer shell, which together carry glucose oxidase, paclitaxel, and fluorescent dye. The platform is capable of releasing manganese ions and payloads in a pH-dependent manner within tumor cells. Subsequently, glucose oxidase catalyzes the production of hydrogen peroxide from glucose, which is further catalyzed by manganese ions to produce reactive oxygen species. Combined with the antitumor effect of paclitaxel, it shows strong anti-tumor effects. In addition, recent research reports that Wang et al. developed a liposome-based enzyme nanoreactor, which cleverly encapsulates glucose oxidase GOx and horseradish peroxidase HRP together in the aqueous core of liposomes. Through encapsulation of liposomes, the two enzymes can work together in the same space, significantly improving the efficiency of the entire tandem reaction. GOx can effectively consume glucose in tumor cells and produce gluconic acid and hydrogen peroxide. The production of gluconic acid can reduce the pH value of the local environment and increase the concentration of hydrogen peroxide, which together promote the catalytic efficiency of HRP, resulting in the production of highly cytotoxic hydroxyl radicals ·OH, ultimately achieving the goal of killing tumor cells [17] .

Liposome-mediated delivery of GOx for antitumor therapy. (a) A diagrammatic representation of the GISLs’ constituent parts and the anticancer mechanism exerted by GOx, dc-IR825, and sorafenib. Reproduced with permission from Ref [40]. (b) A schematic outlining the fabrication of the cell metabolism regulator ATO/GOx PLP and its utilization in cancer treatment through a PpIX translocation and cell respiration substrate redistribution mechanism. Reproduced with permission from Ref [41]. (c) A depiction of a liposomal delivery system (SN-38∩LP@Fe3O4/GOx) and a ROS field effect transistor for the enhancement of chemodynamic therapy. Reproduced with permission from Ref [42]

Due to their unique catalytic function, enzymes can not only undergo color reactions with their substrates but also serve as tools for detecting enzyme activity [43,44,45]. Therefore, they are often used as model proteins to evaluate the efficiency of delivery tools. This process is usually achieved by observing fluorescence phenomena or detecting the activity of enzyme in the cytoplasm. For example, superoxide dismutase (SOD) has a special function of catalyzing the conversion process of anionic superoxide radicals in molecular oxygen and hydrogen peroxide. In the medical field, this enzyme is widely used, especially in the treatment of diseases caused by ROS, such as rheumatoid arthritis, various inflammatory diseases, and ischemia-reperfusion injury [46]. However, it is worth noting that direct administration of SOD without the assistance of a suitable delivery system faces many limitations. Its half-life in the blood is relatively short. This makes it difficult to effectively accumulate in damaged areas and is easily filtered by the kidneys [47]. In this regard, the presence of PEG on the surface of liposomes plays a crucial role [48]. It can significantly reduce the opsonization of liposomes by the mononuclear phagocytic system, thereby effectively promoting the delivery effect of SOD-loaded liposomes.

Nevertheless, despite the considerable promise of liposomes in protein delivery, the process of encapsulating proteins is not without shortcomings. A notable disadvantage is that this process may potentially impair the functionality of the proteins in question. Such damage frequently arises from the intricate procedures involved in liposome preparation, whereby multiple steps may have a detrimental impact on the structure and functionality of proteins [49,50,51]. For instance, the pH value employed during liposome preparation represents a pivotal factor. The pH level exerts a direct influence on the self-assembly process of phospholipid molecules, which, in turn, affects the stability of the encapsulated proteins. Furthermore, the temperature of the solution is a significant factor that influences the activity of liposome-encapsulated proteins. It is imperative that the temperature of the solution be strictly controlled during the preparation of liposomes, as this will ensure that the phospholipid molecules can correctly self-assemble and encapsulate proteins.

Clustered regularly interspaced short palindromic repeats (CRISPR) associated protein 9 (Cas9) is a promising gene editing tool for treating diseases at the genetic level [52]. However, the challenge of safely and effectively delivering CRISPR/Cas9 to host cells limits its clinical application [53]. Compared to delivering genes, direct delivery of Cas9 RNP can immediately function without protein expression processes [7, 54,55,56]. The large size of Cas9 protein, approximately 160 kDa, prevents it from being directly delivered into cells. Liposome packaging technology can not only directly deliver Cas9 RNP into cells, but also partially protect it from degradation. The ability of liposomes to deliver intact Cas9 RNP represents a key advantage of this approach, ensuring effective and non-toxic gene editing.

Applications of Liposome-Facilitated Delivery of RNP in Diverse Diseases. (a) A lipid nanoparticle (LNP) encapsulated Cas9 RNP delivery system is presented. PCL nanofibrils (NFs), which mimic the bone tissue microenvironment, are coated with mesenchymal stem cell membrane (MSCM) and loaded with the CXCL12α cytokine along with LNP-encapsulated Cas9 RNP. This LNP-Cas9 RNP/MSCM-NF/CXCL12α complex can be injected into the bone marrow cavity to induce chemotaxis of leukemia blasts or leukemia stem cells (LSCs), enhancing gene editing cargo delivery efficacy. Reproduced with permission from Ref [57]. (b) CRISPR RNP delivery via LNPs enables widespread in vivo genome editing in the mouse cornea. Reproduced with permission from Ref [54]. (c) A lipid nanoparticle-mediated hit-and-run approach achieves efficient and safe in situ gene editing in human skin. Reproduced with permission from Ref [51]

Liposome-delivered gene editing protein complexes have been widely used in the study of various diseases (Fig. 3), such as gene editing in ophthalmology, otology, and dermatology [51, 54,55,56, 58, 59]. In the field of corneal diseases, Mirjalili Mohanna et al. tested a new LNP platform by providing pre-complexed RNPs and template DNA to cultured mouse cortical neurons, and achieved successful in vitro genome editing. Then, the LNP-encapsulated RNP and DNA templates were directly injected into the mouse cornea to evaluate in vivo delivery. This study demonstrated extensive genome editing in the cornea using LNP-RNPs for the first time [54]. In the field of hearing disorders, Tao et al. found that the in vivo delivery of liposome-mediated CRISPR-Cas9 RNP complexes can lead to the specific editing of Obl alleles [59]. In vivo genome editing promotes the survival and restoration of function in outer hair cells, thereby restoring hearing. By lipid and AAV mediated delivery of Streptococcus pyogenes Cas9 (SpCas9) RNP complexes, researchers have improved hearing in mouse models of dominant hearing loss with hair cell origin by targeting the mutant allele of transmembrane channel-like 1 [59]. In the realm of skin diseases, Bolsoni et al. have investigated the potential of LNP to deliver gene editing tools into the living epidermis of human skin, enabling efficient in situ gene editing that could potentially cure rare monogenic skin diseases [51]. Therefore, the aforementioned studies have demonstrated the extensive applications and potential of liposome-delivered gene editing protein complexes in various disease areas.

To facilitate the clinical translation of liposome-based genome editing therapies, several novel liposome systems with responsive controlled release have been reported for the efficient delivery of CRISPR-Cas9 RNP into target cells [56, 60, 61]. Light-triggered liposome systems have been used to achieve temporal and spatial controlled release of CRISPR-Cas9 RNP [56]. By incorporating photosensitive molecules, such as Verteporfin (VP), into liposomes and exposing them to specific wavelength light, the liposomes undergo structural instability, leading to the controlled release of RNP for gene editing. For example, Aksoy et al. developed light-triggered liposomes that controllably release CRISPR-Cas9 ribonucleoprotein by incorporating the clinically used photosensitive molecule VP into the lipid bilayer and then rationally designing it. Under 690 nm wavelength light irradiation, VP reacts with available oxygen molecules and generates singlet oxygen, which rapidly oxidizes unsaturated lipid components and leads to structural instability of the liposomes and the release of ribonucleoprotein [56]. This regulatory mechanism restricts CRISPR-Cas9 activation to designated target sites, thereby achieving enhanced tissue- and cell-type specificity. Furthermore, Yan et al. reported a phosphorylated DNA-engineered liposome system capable of responding to stimuli to achieve cell-specific intracellular delivery and genome editing [61]. The liposome design mimics the viral fusion process, which can trigger membrane fusion under pH or UV stimulation to achieve cytoplasmic delivery of proteins. This strategy is highly efficient in delivering proteins of varying sizes and charges to target cells. In summary, these liposome systems offer innovative strategies for temporal and spatial control and cell-specific delivery of CRISPR-Cas9 RNP, thereby paving the way for safer and more effective genome editing therapies.

Liposome-protein complexes, as efficient and precise tools, are widely applied in cell labeling and imaging techniques [43, 62]. By combining the excellent membrane fusion properties of liposomes with the specific recognition capabilities of proteins, scientists are able to accurately label and meticulously observe cells at the microscopic level. This complex not only exhibits high targeting ability, enabling precise localization to target cells or specific intracellular regions, but also produces excellent imaging results, clearly revealing the morphology, structure, and functional status of cells.

The use of liposome-protein complexes for cell labeling and imaging has enabled scientists to gain deeper insights into the biological processes occurring within cells. This encompasses a multitude of processes, including cell growth, division, metabolism, and signal transduction. Such understanding not only elucidates the enigmas of life but also furnishes a crucial theoretical foundation and practical guidance for the diagnosis and treatment of diseases. For instance, Li et al. have successfully developed a multi-faceted ultrasound molecular probe known as cell-penetrating peptide-modified 10-hydroxycamptothecin-loaded phase-transformation lipid nanoparticles, or simply iRGD-ICG-10-HCPT-PFP-NPs [63]. When combined with low-intensity focused ultrasound, liposome protein probe can be used for precise diagnosis and treatment of hepatocellular carcinoma. This probe exhibits excellent targeting ability, enabling ultrasound/photoacoustic (PA) dual-mode imaging. It can penetrate deeply into the tumor, achieving a better therapeutic effect, and thus provides new ideas and methods for the diagnosis and treatment of liver cancer. Three-dimensional optical microscopy plays a crucial role in understanding and optimizing the delivery of nanomedicines [64]. However, unfortunately, the process of tissue clearing often removes liposomes, preventing the technique from achieving three-dimensional imaging of liposomes within tissues. Fortunately, Professor Warren C. W. Chan designed a protein tag named REMNANT, which can not only attach to liposomes but also crosslink with tissues while remaining stable during the clearing process, thus enabling three-dimensional imaging of liposomes in intact tissues [25]. The REMNANT tag can also monitor the release rate of liposome contents in tissues in real time. This innovative method not only helps researchers observe the behavior of degradable materials in vivo, but also provides valuable guidance for the engineering design of imaging techniques and drug delivery vehicles.

Redirecting host cell signaling pathways through the utilization of bacterial effectors via cationic lipid-mediated intracellular protein delivery. Reproduced with permission from Ref [65]

Additionally, fluorescent proteins have been successfully utilized by researchers in combination with liposome delivery technology to target proteins, enabling clear observation of the absorption efficiency and subcellular localization of nanoparticles, providing new perspectives and tools for research in nanomedicine and drug delivery [65,66,67]. Yang et al. have reported the use of a high-throughput liposome screening strategy to successfully achieve intracellular delivery of OspF mediated by cationic liposomes [65]. This strategy effectively specifically inhibits the MAPK signaling pathway and tumor growth in cancer cells, as well as specifically regulates the immune response of macrophages. To further improve the encapsulation efficiency of lipid nanoparticles during intracellular delivery, researchers genetically fused OspF with a negatively charged green fluorescent protein. This fusion promotes the self-assembly of cationic lipid nanoparticles through electrostatic interactions, and provides strong support for studying protein transport behavior at the cellular level. Overall, the application of liposome-protein complexes has not only deepened our understanding of liposome delivery mechanisms, but also opened up new prospects and potential applications for future drug delivery and disease treatment fields (Fig. 4).

In recent years, the cytosolic delivery of protein drugs through protein nanoparticles has emerged as a significant breakthrough in the field of biomedicine [68, 69]. This technology utilizes single or multiple protein molecules to form nanoparticles with nanoscale dimensions through self-assembly, enabling efficient cytosolic delivery. This section will focus on three innovative protein nanoparticle design methods. These methods not only demonstrate the potential of protein nanoparticles in the biomedical field, but also provide new perspectives and strategies for the development of cytosolic delivery technology. The following is a brief overview of these three design methods: The first is the design of protein nanoparticles based on phase-separated condensates. This method utilizes the interactions between different proteins and controls environmental conditions such as temperature, pH, or ionic strength to induce phase separation and subsequent formation of condensates [70, 71]. These condensates serve as drug carriers or imaging tools to perform specific functions within cells. A second innovative design approach is the use of computer-designed protein nanoparticles. The advancement of bioinformatics and computational biology has enabled the utilisation of computer simulations and algorithm optimisation in the design of protein nanoparticles with defined structures and functions. This method allows for precise control over the dimensions, configuration, and surface characteristics of nanoparticles, thereby enabling precise regulation of the drug delivery process. Moreover, computer-aided design can predict the interactions between nanoparticles and cells, thereby providing valuable insight for optimizing delivery efficiency and minimizing side effects. The third category of protein nanoparticles is based on cell-penetrating peptide nanocarriers. Cell-penetrating peptides are short peptides that possess the capacity to traverse cellular membranes. The combination of cell-penetrating peptides with proteins allows for the creation of peptide-modified nanocarriers, which can facilitate the transmembrane delivery of proteins in an efficient manner. These innovative design methods for protein nanoparticles possess unique characteristics, providing new perspectives and advanced tools for advancements in cytosolic protein delivery technology.

Phase-separated protein self-assembly condensates delivery is a cutting-edge biotechnology that utilizes the phase separation properties of proteins under specific conditions to form condensates through self-assembly, thereby achieving targeted delivery of proteins. Phase separation is a process where intracellular proteins or protein-RNA complexes spontaneously form distinct “phases,” involving both attractive and repulsive interactions among proteins, as well as thermodynamic driving forces [72, 73]. Condensates form within cells typically through spontaneous assembly via interactions among biomacromolecules such as proteins and RNAs. These condensates are not meant to restrict molecular diffusion but rather to provide a locally high-concentration environment to facilitate specific intermolecular interactions. Within the condensates, interactions among molecules, such as electrostatic and hydrophobic interactions, cause them to tightly cluster together. However, this clustering is not completely enclosed but rather possesses a certain degree of permeability. Therefore, molecules can still diffuse to a certain extent within the condensates and between the condensates and the surrounding cytoplasm [74].

The condensates encapsulate proteins within their interior and deliver them to specific target locations through intracellular transport mechanisms [75]. The process of intracellular transport of phase-separated condensates may involve the following mechanisms: direct membrane translocation and entry into the cell through classical endocytosis. Direct membrane translocation refers to the mode of protein delivery within the cell that avoids capture by endosomes/lysosomes, directly fusing with the membrane to transport the cargo into the cytoplasm. For example, redox-responsive peptide(HBpep) coacervates and magnetically responsive peptide (DgHBP-2) coacervates [76, 77]. Phase-separated condensates can also be internalized by cells through energy-dependent endocytosis [78, 79].The classical endocytosis pathway primarily includes the macropinocytosis pathway, the clathrin-mediated endocytosis pathway, and the caveolin-mediated endocytosis pathway [80]. Some condensates are internalized through endocytosis to deliver coupled anticancer drugs to target cells. For example, Dittrich et al. developed Flutax-2 peptide nanoparticles functionalized with transferrin, which enter cells through clathrin-mediated TfR endocytosis [79]. After entering the cytoplasm, condensates can be transported by molecular motors attached to microtubules or microfilaments [81]. Furthermore, condensates in the cytoplasm may also be encapsulated within vesicles, enabling their transport inside and outside the cell through the fusion and separation processes of the vesicles with the cell membrane. For example, phase-separated YBX1 condensates recruit miRNAs and selectively sort them into exosomes [82]. Compared to other delivery vehicles, such as liposomes and polymeric nanoparticles, protein condensates form almost instantaneously, exhibiting negligible cytotoxicity from their peptide building blocks and eliminating the need for organic solvents that may reduce the biological activity of encapsulated molecules. This ensures both efficiency and safety in the delivery process.

Phase-separated protein peptides are capable of loading macromolecules, traversing cell membranes, and delivering their payloads within cells, thereby overcoming the major limitation of the difficulty in intracellular delivery of macromolecules (Fig. 5). Yu et al. reported a study on glucose-driven droplet formation in supramolecular peptide and therapeutic protein complexes. The study revealed that under specific conditions, these complexes can respond to glucose stimulation, leading to phase separation and droplet formation. This discovery not only provides a new perspective for understanding intermolecular interactions among biomolecules but also opens up new avenues for drug delivery and biological therapy [20]. In another study, Sun et al. developed short His-rich, pH-responsive beak peptide (HBpep) coacervates that bind to disulfide bonds containing self-sacrificial fragments (HBpep-SR), which can trigger the disintegration of droplets in a reducing environment, promoting the intracellular delivery of protein drugs [76]. HBpep condensates can cross cell membranes independently of endocytosis, demonstrating their potential for intracellular delivery of therapeutic agents. At low pH, histidine-rich HBpep exists as monomers, but rapidly phase separates into condensed microdroplets at neutral pH, during which process it can absorb and integrate various macromolecules from the solution. These examples fully demonstrate the versatility and practicality of phase-separated protein self-assembly condensates in the delivery of therapeutic proteins.

Currently, the technology of phase-separated protein self-assembly condensates for protein delivery has demonstrated potential application value in multiple fields, such as drug delivery, gene therapy, and cell regeneration [83,