Toxoplasma gondii-derived nanocarriers: leveraging protozoan membrane biology for scalable immune modulation and therapeutic delivery

Abstract

Cell membrane-derived nanovesicles (CMNVs) are nanoscale lipid bilayer structures obtained from cellular membranes that serve as biomimetic drug delivery platforms, offering immune evasion, targeting, and surface functionalization capabilities. While most CMNVs originate from mammalian cells, Toxoplasma gondii (T. gondii), a genetically tractable protozoan with a structurally distinct membrane, offers a high-yield and underexplored source for producing T. gondii-derived CMNVs (TgCMNVs). These vesicles are obtained from the parasite's plasma membrane and inner membrane complex and retain unique features including abundant GPI-anchored SRS proteins, phosphatidylthreonine-rich lipids, and an editable genome, enabling versatile engineering via genetic and chemical strategies. We review methods for TgCMNV fabrication, purification, and functionalization, and evaluate their potential in immunomodulation, attenuation of tissue injury, cancer immunotherapy, and self-adjuvanting vaccine design. By combining intrinsic immune engagement with programmable surface architecture, TgCMNVs could serve as a complementary and adaptable platform alongside established CMNV systems. Finally, we discuss key translational considerations, including scalable production, immunogenicity control, regulatory compliance, and stability testing, which will be essential for assessing the feasibility of TgCMNVs in clinical applications.

Graphical abstract

Highlights

• •TgCMNV emerge as a transformative, robust, and versatile platform for next-generation drug delivery
• •TgCMNV’s lipids and proteins (e.g., GPI, SAG1) drive immune modulation, circulation, and functional design.
• •TgCMNV hold promise in controlling inflammation, reprogramming cell death, immunotherapy, and vaccines.
• •This review maps TgCMNV membrane composition, engineering strategies, and therapeutic opportunities.

1. Introduction

Cell membrane-derived nanovesicles (CMNVs) are a potent new class of drug delivery systems, offering biomimetic phospholipid bilayers that encapsulate therapeutic cargos while presenting natural cellular surfaces [1,2]. These features enable prolonged circulation, immune modulation, and receptor-mediated targeting, often surpassing synthetic carriers in biocompatibility and functional versatility [3,4]. CMNV-based approaches have been investigated for tumor therapy, barrier penetration, and tissue regeneration [3,5]. Their versatility stems from the diversity of membrane sources, including bacterial outer membranes, eukaryotic cells, and secreted vesicles, which preserve native biomolecules important for disease-site homing and vesicle stability [4,[6], [7], [8]]. Genetic engineering and chemical modification strategies have further expanded their diagnostic and therapeutic utility [9].

Nanomedicine has progressed from first-generation liposomes that rely on the enhanced permeability and retention (EPR) effect, to PEGylated second-generation systems with improved pharmacokinetics, and more recently, to third-generation CMNVs that incorporate whole-cell membranes from red blood cells, immune cells, tumor cells, and bacteria [1,[10], [11], [12]]. These advanced vesicles inherit cell-specific traits such as homotypic targeting and barrier translocation [13]. Despite these advances, mammalian-derived CMNVs face important challenges, including limited scalability, slow genetic engineering processes, and membrane fragility, which often lead to the use of hybridization strategies. Most platforms remain restricted to mammalian sources.

Expanding into non-mammalian systems may help overcome these limitations. Toxoplasma gondii (T. gondii), a widely distributed intracellular protozoan, offers an attractive complementary source. It combines rapid proliferation with advanced genetic tools and a distinctive membrane architecture, including the inner membrane complex (IMC), phosphatidylthreonine-rich lipids, and abundant, engineerable GPI-anchored proteins [[14], [15], [16]]. These features confer structural stability, prolonged circulation potential, and flexible functionalization options. Compared with mammalian cells, T. gondii cultures yield larger amounts of membrane material in shorter timeframes, supporting scalable production and efficient engineering [15,17,18].

These emerging findings suggest that the biomedical community should invest effort into developing T. gondii-derived CMNVs (TgCMNVs), as their unique biological and structural properties offer capabilities not readily achievable with existing nanocarrier systems. In this review, we explore the potential of TgCMNVs as next-generation nanocarriers. We describe their membrane composition and engineering strategies, evaluate their applications in immune modulation, tissue protection, cancer immunotherapy, and vaccine development, and outline the key challenges and opportunities for advancing TgCMNVs toward clinical translation.

2. T. gondii membrane lipids, surface proteins, and nanocarrier construction strategies

2.1. Membrane architecture of T. gondii: a structural basis for nanocarrier engineering

Toxoplasma gondii (T. gondii) is a unicellular protozoan of the phylum Apicomplexa and the sole species of its genus [18]. It exists in three major genotypes (types I, II, and III) that differ in virulence. Type I strains, such as RH and GT1, replicate rapidly and are highly lethal in mice, whereas types II (e.g., ME49, PRU) and III (e.g., VEG, CTG) are less virulent and typically associated with chronic infection [19]. Notably, differences in virulence arise primarily from soluble effector proteins secreted by rhoptries, micronemes, and dense granules, rather than from surface membrane proteins [20]. These effectors modulate host immune responses and account for strain-specific pathogenicity [21].

Laboratory-adapted RH strains, which proliferate exclusively in the tachyzoite stage and lack the ability to form tissue cysts, are widely used in research [22]. Their rapid replication, short life cycle, and scalability make them an efficient source of membrane material for CMNV production [23]. In nature, T. gondii completes its sexual cycle only within the feline intestinal epithelium, while its asexual cycle, comprising tachyzoites and tissue cysts, occurs in virtually all warm-blooded animals [24]. In vitro, tachyzoites are the dominant form, exhibiting robust intracellular replication and high membrane yield, which supports large-scale nanovesicle production. As a unicellular eukaryote (4–7 μm × 2–4 μm), T. gondii has a complex architecture with specialized apical organelles (micronemes, rhoptries, and dense granules) that facilitate host-cell invasion and immune modulation [20]. The secretory proteins from these organelles, often characterized by short transmembrane domains, are largely removed during membrane isolation, which markedly reduces the potential immunogenicity of derived vesicles [21,25]. In contrast, the extracted plasma membrane (PM) retains structural lipids and surface proteins, providing a stable, engineerable, and comparatively safer foundation for CMNV design (Table 1).

Table 1.

Transition of functional mechanism: From active invasion to passive targeting.

Characteristics	Live T. gondii	TgCMNVs
Interaction Mode	Active invasion (secretion of ROP/GRA effector proteins)	Passive targeting (membrane protein-receptor binding)
Motility Mechanism	Actin-driven gliding motility mediated by apical complex	Receptor-mediated endocytosis
Pathogenicity	Replication-dependent tissue damage; actively disrupts host cell cycle/death pathways	Replication-incompetent; utilizes recognition signals only
Immune Response	Potent proinflammatory cascades and tissue damage	Altered immunogenicity (intracellular pathogenic components removed); activates memory cells (vaccine potential)

Abbreviations:TgCMNV: T. gondii-derived CMNVs; GRAs: Dense Granule Proteins; ROPs: Rhoptry Proteins.

A hallmark of T. gondii is its pellicle, a tripartite membrane system composed of the outer plasma membrane (PM) and the underlying inner membrane complex (IMC), which consists of two closely apposed lipid bilayers supported by subpellicular microtubules (Fig. 1) [26]. The PM contains key structural elements, including cholesterol, channel proteins, and GPI-anchored ligands, while the IMC provides mechanical strength and contributes to curvature control. The actin-myosin gliding apparatus spans both membrane systems, enabling parasite motility. This three-layered pellicle imparts compositional versatility and resistance to mechanical stress, properties that can be inherited by TgCMNVs. Vesicles can be derived solely from the PM, as in most mammalian CMNV systems, or from PM-IMC composites, which may alter vesicle rigidity, curvature, and protein distribution. This architectural diversity offers opportunities to fine-tune the biophysical and functional characteristics of TgCMNVs for specific biomedical applications.

Fig. 1.

Schematic of T. gondii membrane structure and nanovesicle derivation. The pellicle of T. gondii consists of an outer plasma membrane (PM) and a two-layered inner membrane complex (IMC), supported by actin-myosin motors and microtubules. Structural and functional elements such as GPI-anchored SRS proteins, cholesterol, channel proteins, and intramembranous particles are localized across different membrane domains. Nanovesicles may be derived from the PM alone, the IMC, or a PM-IMC hybrid, resulting in distinct vesicle types with tunable properties.

2.2. Lipid composition and chemical programmability of T. gondii membranes

T. gondii membranes contain a diverse mix of neutral and polar lipids that influence curvature, fluidity, and host interactions [27]. Because the parasite cannot synthesize sufficient lipids de novo, it acquires them via three key routes: (i) direct acquisition of host-derived molecules such as low-density lipoproteins and fatty acid precursors [28,29]; (ii) metabolic remodeling, often via apicoplast-dependent pathways [28]; and (iii) biosynthesis of core phospholipids, including phosphatidylcholine (PtdCho), phosphatidylethanolamine (PtdEtn), phosphatidylserine (PtdSer), and phosphatidylinositol (PtdIns) [30,31]. A notable feature is the synthesis of phosphatidylthreonine (PtdThr), an anionic lipid largely unique to apicomplexan parasites [32,33]. PtdThr supports calcium homeostasis and membrane integrity and may be regulated by the phosphatidylserine synthase (PTS) gene [34]. Chemical inhibition of PtdCho or PtdEtn biosynthesis severely compromises parasite viability, underscoring the potential of lipid pathways as drug targets [30,35]. Importantly, T. gondii exhibits lipid plasticity, readily incorporating exogenous analogs, such as BODIPY-tagged fatty acids, into its membrane, providing a route for chemically programmable vesicle production [36].

Click chemistry provides an efficient means to functionalize these membranes. Copper-catalyzed azide-alkyne cycloaddition (CuAAC) enables high-density, site-specific attachment of ligands or probes under mild conditions, preserving vesicle integrity [37]. This method is well-suited to post-extraction surface functionalization, particularly given T. gondii's capacity to retain reactive lipid anchors (Fig. 2). Additionally, GPI-anchored proteins which is abundant on the T. gondii surface, can be selectively modified via metabolic glycan labeling using non-natural sugars, such as azido-mannose or alkyne-sialic acid [38]. These orthogonal strategies allow fine control over vesicle surface chemistry, enabling dual-function designs for targeted delivery and immune modulation.

Fig. 2.

Targeted optimization strategies for TgCMNVs Four complementary approaches enable precise engineering of T. gondii-derived CMNVs: (a) hybridizing membranes with tumor or immune cell membranes to inherit their native targeting and tissue affinity; (b) genetically engineering the parasite to express functional surface proteins via CRISPR/Cas9 or plasmid-based systems; (c) metabolically incorporating modified lipid precursors or analogs through endogenous biosynthetic or scavenging pathways; and (d) modifying lipids post-extraction using site-specific click chemistry or direct lipid insertion. Together, these strategies allow construction of nanovesicles with tunable surface properties for therapeutic, diagnostic, and vaccine applications.

Beyond molecular composition, the biophysical properties of T. gondii membranes support engineering flexibility. A low cholesterol-to-phospholipid ratio enhances fluidity and facilitates spontaneous nanoparticle wrapping [39,40]. Combined with short replication cycles and tolerance to chemical perturbation, this enables rapid recovery after modification. PtdThr adds further utility by supporting calcium buffering and membrane stabilization—traits rarely observed in mammalian systems [32,33,41]. The integration of functional components, including small, functionalized amines and nanoparticles such as gold, expands the utility of TgCMNV to diagnostic imaging and therapeutic delivery [28,29].

Multiple functionalization strategies can be applied to TgCMNVs (Fig. 2). Membrane hybridization with immune or tumor cell membranes can introduce homotypic targeting ligands. Genetic engineering allows stable expression of fusion proteins on scaffolds such as SAG1, while metabolic incorporation of modified lipid precursors during parasite growth provides programmable surface chemistry. Post-synthetic modifications, including click chemistry and metabolic glycan labeling, add an additional layer of functional tuning (Fig. 2). Together, these modular approaches may provide a flexible basis for future therapeutic, diagnostic, and vaccine applications.

2.3. GPI-anchored SRS proteins and genetic engineering strategies

The plasma membrane of T. gondii is densely populated with glycosylphosphatidylinositol (GPI)-anchored surface proteins belonging to the SAG1-related sequence (SRS) family [42,43]. These proteins mediate host-cell adhesion and shape host-pathogen interactions across different stages of the parasite life cycle (Table 2) [44]. Despite their sequence diversity, most GPI-anchored SRS proteins share a conserved domain architecture: an N-terminal signal peptide, a C-terminal hydrophobic anchor, and a GPI-modified linker that facilitates membrane tethering [45]. Among these, SAG1 (P30) is the most abundant and extensively studied, representing up to 5 % of the total protein content and accounting for 20–25 % of the total membrane protein content in tachyzoites [46]. SAG1 binds lysine-rich coiled-coil structures or sulfated proteoglycans on host cells [46,47]. Blocking SAG1 can reduce adhesion by over 70 %, underscoring its functional centrality [48,49]. Importantly, while SAG1 is not a determinant of virulence, its high-level and genotype-consistent expression makes it a practical and well-characterized scaffold for targeted vesicle engineering [18,50]. In contrast, stage-specific or polymorphic antigens such as SAG2 are less suitable for consistent surface display (Table 2) [51].

Table 2.

Expression stage, abundance, and host cell targeting characteristics of Key T. gondii Membrane Proteins.

Membrane Protein	Gene ID	Expression Stage	Expression abundance in EES (TPM)	Host Cellular Targets	References
SAG1/P30	TGGT1_233460	Tachyzoites only		Lysine-rich coiled-coil proteins or Sulfated proteoglycans	[45,52]
	TGME49_233460
SAG2A/P22	TGGT1_271050	Tachyzoites only		Zinc finger proteins	[45,53]
	TGME49_271050
SAG3/P43	TGGT1_308020	Both tachyzoites and bradyzoites (low abundance)		Same as SAG1	[[54], [55], [56]]
	TGME49_308020
SABP1	TGME49_225940	Both tachyzoites and bradyzoites (low abundance)		Sialic acid on host cell surface	[57,58]
SAG2C/D	TGGT1_207160	Predominantly bradyzoites		Not determined	[51,59]
	TGME49_207160
SRS9	TGME49_320190	Predominantly bradyzoites		Not determined	[[60], [61], [62]]
BSR4	TGGT1_320180	Predominantly bradyzoites		Not determined	[63]
	TGME49_320180

Abundance is expressed in transcripts per million (TPM) based on available transcriptomic datasets and the data of expression abundance in EES was obtained from the ToxoDB database (https://toxodb.org/toxo/app). Horizontal bars represent normalized TPM values on a uniform scale across all listed proteins, allowing visual comparison of relative expression levels. Host-cell targeting characteristics are derived from published functional studies.

Abbreviations: EES: Enteroepithelial Stages; SABP1, sialic acid binding protein-1; SAG: surface antigen; SRS: SAG1-related sequence; TPM: Transcripts Per Million.

GPI anchoring: GPI anchors, highly conserved across eukaryotic parasites, are synthesized in the endoplasmic reticulum and covalently linked to the C-terminus of SRS proteins (Fig. 3a) [[64], [65], [66]]. This lipid-glycan conjugation stabilizes membrane localization and facilitates dynamic organization of surface proteins into functional clusters [66]. For TgCMNV applications, GPI anchoring offers a dual advantage: it ensures robust surface expression of engineered proteins and allows modular modifications without compromising membrane integrity. Because the anchoring region is structurally conserved and biochemically tractable, it provides a stable platform for recombinant fusion constructs designed to display targeting ligands, immune epitopes, or imaging handles.

Fig. 3.

Genetic engineering of GPI-anchored SRS proteins in T. gondii (exemplified by SAG1) (a) Native SAG1 biosynthesis involves transcription, signal peptide cleavage in the ER, and GPI anchoring at the C-terminus for stable membrane insertion. (b) Recombinant SAG1-tag fusions can be introduced via CRISPR-Cas9 or plasmid-based methods, provided that key domains (signal peptide, folding structure, and GPI anchor) are retained. Fusion inserts (e.g., targeting peptides) must preserve proper topology to ensure surface exposure.

Surface reprogramming via genetic engineering: The availability of precise gene-editing tools in T. gondii, including CRISPR-Cas9 and site-specific plasmid integration, enables programmable modification of its membrane protein landscape [45]. Engineered fusion proteins incorporating functional domains, such as tumor-targeting peptides, nanobodies, or cytokine mimetics, can be stably expressed on the parasite surface via insertion into SAG1 or SAG3 backbones (Fig. 3b). Designing such constructs requires preservation of essential trafficking motifs, including the signal peptide for ER targeting and the GPI anchoring sequence for membrane insertion. Linkers rich in glycine and serine may be introduced to maintain independent folding of native and inserted domains. Structural modeling, informed by known disulfide patterns and cysteine frameworks, facilitates rational design of constructs that preserve both function and stability.

While GPI-anchored SRS proteins are optimal scaffolds, other candidates such as microneme-derived adhesins offer additional functional versatility, though their transient expression and proteolytic shedding complicate stable display [[67], [68], [69]]. Taken together, T. gondii's genetic plasticity supports the construction of customizable TgCMNVs equipped for applications ranging from receptor-targeted drug delivery to antigen-specific immunotherapy.

2.4. Preparation and evaluation of T. gondii membrane-derived nanovesicles

Preparation workflow: TgCMNVs are produced using protocols adapted from established eukaryotic cell membrane-derived nanocarrier methods, originally developed for mammalian cells (Fig. 4), underscoring the broad applicability of these fabrication strategies. Production begins with isolating plasma membranes from wild-type or genetically engineered T. gondii tachyzoites. Lysis methods, which typically a combination of non-denaturing detergents, mechanical shear, or freeze-thaw cycles, are selected to preserve membrane-associated proteins and lipids [10,70]. The enriched membrane fractions are then converted into nanoscale vesicles through physical fragmentation and extrusion [3], yielding unilamellar structures with controlled size, uniform surface properties, and preserved bioactivity. Membrane fragments can be reconstituted directly into vesicles or combined with nanoparticle cores to improve stability or enable imaging. Formation conditions, such as temperature and electrostatic compatibility, are optimized to maintain lipid bilayer integrity and fluidity, while post-processing steps (e.g., solvent exchange or filtration) enhance homogeneity and concentration [71].

Fig. 4.

Schematic workflow for the preparation of T. gondii cell membrane-derived nanovesicles (TgCMNVs). Wild-type or genetically engineered T. gondii tachyzoites are subjected to lysis buffer treatment (non-denaturing buffer), followed by homogenization (30 % power, 3 s on/2 s off cycles) to disrupt cells. Membrane fractions are collected through sequential centrifugation at 5000×g for 10 min at 4 °C, 20,000×g for 20 min at 4 °C, and 200,000×g for 45–60 min at 4 °C. The purified membrane vesicles are fused with nanoparticle cores using sonication (20 Hz, 130 W, 20 min). Vesicle size is then refined through serial extrusion using polycarbonate membrane filters (800 nm, 400 nm, 220 nm, and 100 nm; 10 passes per filter). Transmission electron microscopy (TEM) images confirm vesicle morphology at key steps.

Customization strategies: TgCMNVs support both biosynthetic and post-synthetic modifications. During parasite cultivation, genetic engineering can be used to incorporate functional ligands such as tumor-homing peptides or immunomodulatory proteins. After fabrication, chemical approaches, such as click chemistry or lipid insertion, enable precise surface tuning to adjust circulation time, targeting specificity, and immune visibility. This two-tiered strategy, combining biosynthetic programming with synthetic editing, allows membrane composition to be matched to specific therapeutic or diagnostic goals (Table 3).

Table 3.

Characteristics of nanovesicles derived from bacterial, mammalian cell, and T. gondii membranes.

Characteristics	Bacterial-derived	Mammalian Cell-derived	T. gondii-derived	TgCMNVs Advantages
Scalable culture method	Nitrogen- and carbon-containing medium	Serum-containing media	HFF/HeLa cells or murine hosts	Flexible in vivo/in vitro expansion
Sterility requirements	Strict	Strict	Aseptic for cells; non-sterile for mice	Reduced contamination risk in vivo
Immunogenicity	Strong (TLR4 activation)	Moderate or Low (cell-type dependent)	Moderate (Virulence factors removed)	Safer for systemic delivery
Mechanical stability	Low (Lacks cholesterol and cytoskeleton)	Moderate	High (IMC reinforcement)	The IMC enhances the stability of the submembrane network, providing resistance to shear stress
Genetic stability	Mutation-prone (plasmid loss)	Stable	Stable across passages	Consistent quality
Inherent targeting capability	None	Homing to immune/tumor cells	Neural affinity	SAGs enable host cell recognition and enhance adhesion
Tissue tropism	None	Tissue-specific (e.g., liver or tumor)	CNS-specific targeting	Ideal for neuro applications
Genetic editing difficulty	Limited PTM capacity	Low efficiency (polyploidy)	Moderate (haploid)	Balanced flexibility

Abbreviations: CNS: Central Nervous System, IMC: inner membrane complex; TLR: Toll-like Receptor, SAGs: surface antigens, PTM: Post-Translational Modification. CNS targeting listed here is based on live-parasite tropism; applicability to TgCMNVs remains to be validated.

Evaluation and safety validation: Comprehensive characterization is critical for translational readiness. TEM verifies vesicle morphology and lamellarity, while DLS and zeta potential measurements define size, colloidal stability, and surface charge, key parameters for biodistribution and uptake. Proteomic profiling confirms retention of signature membrane proteins and lipids, ensuring biological fidelity. Functional assays assess stability under physiological and storage conditions, as well as cargo encapsulation and release. To strengthen biosafety assurance, additional quality control tests such as cytokine induction in macrophages and residual ROP kinase activity assays can confirm the absence of active virulence factors.

Host membrane separation and pathogenicity control: Because T. gondii is cultured in mammalian host cells, purification must completely remove host-derived membranes. This often requires multi-step centrifugation, membrane density profiling, or immunoaffinity depletion. Batch validation should include host-cell marker exclusion assays and functional testing under immunologically relevant conditions. To confirm complete removal of pathogenic components, proteomic mass spectrometry can be used to detect residual virulence effectors; a reference list of these proteins is provided in Table 4.

Table 4.

Gene IDs, protein functions, and post-secretion localization of key virulence effectors in T. gondii tachyzoites.

Gene ID	Protein Name	Protein Function	Post-Secretion Localization
TGME49_309590	ROP1	Knockout does not affect growth/invasion/virulence but causes abnormal rhoptry morphology	PVM
TGME49_308090	ROP5	Modulates host immune response	PVM
TGME49_312270	ROP13	Knockout does not affect virulence	Host cytoplasm
TGME49_262730	ROP16	Modulates host immune response	PVM
TGME49_205250	ROP18	Modulates host immune response; knockout reduces virulence	PVM
TGME49_242100	ROP38	Modulates host immune response	PVM
TGME49_210370	ROP54	Modifies GBP2 to inhibit host immune response	PVM
TGME49_314500	SUB2	Involved in rhoptry protein maturation; knockout causes abnormal rhoptry morphology	–
TGME49_269885	TLN1	Protease	PV membrane
TGME49_214080	Toxoflin	Disrupts host actin cytoskeleton to promote invasion	Host cytoplasm
TGME49_299060	NHE2	Knockout causes defective egress under calcium induction	–
TGME49_270250	GRA1	Calcium-binding	Soluble within PV
TGME49_227620	GRA2	Forms IVN; knockout attenuates murine virulence	IVN membrane
TGME49_203310	GRA3	Knockout reduces proliferation rate and virulence	PV/IVN membrane
TGME49_286450	GRA5	Modulates host immune response	PV/IVN membrane
TGME49_275440	GRA6	Forms IVN; knockout attenuates murine virulence	IVN membrane
TGME49_203310	GRA7	Integrates into ROP18/17/5 complex; disrupts host IRGs	PV/IVN membrane
TGME49_239740	GRA14	Knockout reduces proliferation rate	PV/IVN membrane
TGME49_275470	GRA15	Modulates host immune response (activates NF-κB pathway; regulates IL-12 production)	PVM
TGME49_208830	GRA16	Modulates host immune response (controls host p53 transcription)	Host nucleus
TGME49_222170	GRA17	Transports small molecules between PV and host cytosol	PVM
TGME49_288840	GRA18	Modulates host immune response	Host cytoplasm
TGME49_297880	GRA23	Transports small molecules between PV and host cytosol	PVM
TGME49_230180	GRA24	Modulates host immune response (induces sustained p38α autophosphorylation)	Host nucleus
TGME49_290700	GRA25	Modulates host immune response (induces macrophage CCL2/CXCL1 secretion; knockout reduces virulence)	PV
TGME49_226380	GRA35	Activates host macrophage pyroptosis pathway	PV/IVN membrane
TGME49_289380	GRA39	Knockout reduces proliferation rate	PV/IVN membrane
TGGT1_236870	GRA42	Activates host macrophage pyroptosis pathway	PVM
TGGT1_237015	GRA43	Activates host macrophage pyroptosis pathway	PVM
TGME49_228170	GRA44	Knockout reduces proliferation rate	PV/IVN membrane
TGME49_240060	TgIST	Modulates host immune response (suppresses host IFN pathway)	Host nucleus
TGME49_291890	MIC1	Adhesin binding sialylated oligosaccharides; forms complex with MIC4/MIC6	Extracellular (host interaction)
TGME49_201780	MIC2	Adhesin binding M2AP; critical for motility/invasion	Extracellular (host interaction)
TGME49_319560	MIC3	Adhesin forming complex with MIC8; knockout reduces virulence	Extracellular (host interaction)
TGME49_208030	MIC4	Adhesin binding galactose; forms complex with MIC2/MIC6	Extracellular (host interaction)
TGME49_277080	MIC5	Inhibits SUB1 protease; knockout increases SUB1 activity	Extracellular (host interaction)
TGME49_218520	MIC6	Facilitates complex trafficking/positioning with MIC2/MIC4	Extracellular (host interaction)
TGME49_260190	MIC13	Adhesin binding sialylated oligosaccharides	Extracellular (host interaction)
TGME49_218240	MIC25	Knockout does not affect proliferation/invasion	Extracellular (host interaction)

Abbreviations: GRA: Dense Granule Protein; IST: Inhibitor of STAT Transcription; IVN: Intravacuolar Network; MIC: Microneme Protein; NHE: Sodium-Hydrogen Exchanger; PV: Parasitophorous Vacuole; ROP: Rhoptry Protein; SUB: Rhoptry Neck Secretory Vesicle; TLN1: Toxolylin1.

3. Potential applications of TgCMNVs

3.1. Modulating inflammation and tissue repair via host-TgCMNV interactions

As an obligate intracellular protozoan that has co-evolved with its host, T. gondii has developed sophisticated strategies to reprogram host cell signaling in ways that promote its persistence while modulating inflammation and tissue remodeling [72,73]. These include temporal regulation of programmed cell death (apoptosis, autophagy, and pyroptosis), remodeling of the extracellular matrix (ECM), and modulation of innate immune effectors such as platelets and complement pathways. In particular, the parasite's ability to balance immune activation with tissue repair offers a conceptual blueprint for designing membrane-derived nanovesicles capable of fine-tuning inflammatory responses. Recent studies reveal that T. gondii-derived effector molecules such as macrophage migration inhibitory factor and dense granule protein GRA15 contribute to anti-fibrotic reprogramming by recruiting monocyte-derived macrophages, enhancing MCP-1 expression, and facilitating the resolution of fibrosis in hepatic injury models [[74], [75], [76]]. These context-specific immunomodulatory effects suggest that TgCMNVs, if properly engineered, could inherit and amplify these beneficial traits for applications in regenerative medicine and fibrotic disease.

3.2. Reprogramming cell death pathways: immunological mimicry and therapeutic leverage

T. gondii has been shown to suppress apoptosis in immune cells by blocking caspase activation, preserving its intracellular niche and limiting immunopathology [77,78]. Notably, the parasite's outer membrane exposes phosphatidylserine (PS), a hallmark of apoptotic cells, which may facilitate its uptake by macrophages through apoptotic mimicry [79,80]. This observation raises the intriguing possibility that TgCMNVs, by retaining PS residues on their surface, could co-opt endogenous apoptotic clearance mechanisms mediated by TIM-family receptors (e.g., TIM-1, TIM-4) [81,82]. These pathways are known to promote anti-inflammatory reprogramming and cross-presentation. Although TIM-4 is prominently expressed in peritoneal resident macrophages, TIM-3 is distributed more broadly and is also modulated by T. gondii during pregnancy and infection-associated immunosuppression [83]. Whether membrane-derived vesicles can differentially engage these receptors to direct macrophage fate remains an open question, a fertile area for translational exploration. Interestingly, not all T. gondii strains or contexts lead to suppression of cell death. In some settings, the parasite promotes apoptosis via MST2 activation and Hippo signaling, particularly in epithelial or pulmonary cell models [84]. This dual capacity, either promoting or suppressing host cell death, likely reflects the parasite's stage-specific, cell type-specific, and protein-dependent interactions [72,84,85]. For TgCMNV applications, this complexity suggests that cargo selection and vesicle engineering will need to be context-specific, particularly when balancing inflammation resolution against tissue regeneration.

Beyond apoptosis, the parasite also modulates pyroptosis, a caspase-1-dependent inflammatory cell death pathway [72,85]. While dense granule and rhoptry proteins are implicated in inflammasome activation, the precise contribution of membrane constituents remains unresolved [72,85]. Notably, GPI anchors appear not to influence host cell viability directly, offering a potential advantage for constructing non-cytotoxic yet immunologically active vesicles [86].

3.3. Hijacking host motility and matrix remodeling for vesicle targeting

The dissemination of T. gondii from the intestinal epithelium to distal tissues relies on its ability to reprogram host immune cells and remodel the extracellular matrix (ECM) [[87], [88], [89]]. This is driven by a network of surface and secreted effectors, including SRS-family membrane proteins and the plasma membrane-anchored phosphatase PPM5C, which together promote stable adhesion to polarized epithelial and endothelial surfaces [90]. Following invasion, the effector TgWIP induces a mesenchymal-to-amoeboid transition in dendritic cells, enhancing integrin-dependent motility and enabling swift tissue traversal [[91], [92], [93], [94]]. Notably, naive T cells infected with T. gondii exhibit impaired migration compared to memory T cells, suggesting antigen-experience-dependent motility programs [95]. Hyperdissemination observed in Type I strains underscores a genetic basis for migratory behavior, hinting at engineering opportunities for tailoring vesicle tropism [96]. In deeper tissues, infected macrophages show reduced adhesion and migratory capacity, enabling passive transport via circulation and contributing to systemic spread [94]. These shifts illustrate how T. gondii exploits host cell plasticity for immune evasion and barrier penetration, the mechanisms that TgCMNVs could inherit or reprogram for therapeutic purposes.

Beyond immune cells, T. gondii exerts multifaceted control over the ECM microenvironment. It reprograms host receptors such as integrins and hyaluronan receptors and induces matrix-degrading enzymes like gelatinases [88,89,97]. GPI-anchored SRS proteins upregulate MMP-9, facilitating ECM breakdown, while MIC2, a microneme protein essential for gliding motility, binds fibrin and laminin and mediates directional migration via actomyosin contractility [88,98,99]. MIC2 also interacts with host proteins including LAMTOR1 and RNaseH2B, underscoring its multifunctionality at the immune-matrix interface [100,101]. TgCMNVs that retain or present engineered MIC2 or GPI components could potentially be directed to inflamed or fibrotic tissues by engaging integrin β1 or enhancing MMP-9-mediated remodeling, an approach relevant to diseases involving pathological ECM accumulation, such as tumor invasion or organ fibrosis.

To fully harness these capabilities, however, several mechanistic aspects still need to be clarified. For instance, what molecular determinants underlie the hypermotility of Type I T. gondii strains, and can these features be stably encoded in vesicle membranes? Disentangling the respective contributions of membrane-bound versus secreted effectors to tissue dissemination may unlock new strategies for engineering TgCMNVs with programmable migratory behavior and tissue-specific tropism.

3.4. Attenuating complement activation and NET-associated damage

T. gondii actively modulates the complement system, recruiting host regulators like factor H (FH) to inhibit C3b deposition and membrane attack complex (MAC) formation via the alternative pathway [102,103]. It also co-opts C4-binding protein (C4BP) and cloaks surface glycan motifs to attenuate the classical and lectin pathways [102]. Notably, Type I and II strains adopt distinct strategies—Type II enhances lectin pathway activation to support dissemination, while Type I favors immune stealth [102]. These mechanisms provide a design template for TgCMNVs. By inheriting complement-regulatory features such as FH-binding GPI-anchored SRS proteins or surface sialylation, TgCMNVs may be tailored to suppress complement-mediated inflammation in autoimmune and transplant-related pathologies [102]. The possibility of tuning vesicle composition to selectively modulate distinct complement arms (classical vs. alternative) presents an underexplored but translatable direction.

T. gondii can be effectively captured by extracellular traps (ETs), particularly neutrophil extracellular traps (NETs), largely through TLR2/4-ERK signaling induced by its GPIs [[104], [105], [106]]. While effective against infection, excessive NETosis contributes to tissue damage in conditions like acute lung and kidney injury (ALI, AKI), lupus, and cancer metastasis [[107], [108], [109], [110], [111]]. TgCMNVs may mitigate such damage by serving as decoys that absorb ROS or deliver therapeutic agents, such as DNase I or PAD4 inhibitors, directly into NET-rich microenvironments, enhancing bioavailability and specificity [5,111,112]. Engineering TgCMNVs with controlled GPI density and surface orientation could help balance immunostimulation with tissue protection, a balance that is critical in settings such as sepsis or ischemia-reperfusion injury.

Beyond complement and NETs, T. gondii interacts extensively with the vascular system. Its ligands bind ICAM-1(intercellular cell adhesion molecule-1) [113], VCAM-1 (vascular cell adhesion molecule) [114], VWA (von Willebrand factor) [115], and activate the platelet-activating factor receptor [116], influencing coagulation and vascular tone. TgCMNVs carrying these surface elements could be adapted for vascular-targeted therapies, particularly in thromboinflammatory disorders. Whether such vesicles can be fine-tuned to reduce endothelial activation or modulate microvascular thrombosis remains an open translational question.

3.5. Applications in antitumor immunotherapy

T. gondii has demonstrated potent antitumor properties in preclinical models, largely through activation of type 1 immune responses [117,118]. Live or attenuated parasites elicit robust IL-12 and IFN-γ secretion, promoting cytotoxic T lymphocyte recruitment and remodeling the tumor microenvironment toward an immunostimulatory state [118]. While prior studies focused on secreted proteins such as ROP18, GRA15, or profilin, recent work highlights the immunogenicity of membrane-bound molecules, specifically SAG1 and SAG3, which contain MHC-I epitopes that trigger CD8⁺ T cell responses [18,119,120]. Likewise, parasite GPIs engage TLR2/4, mimicking bacterial PAMPs and amplifying innate immune signaling. These immunological features are retained in TgCMNVs [104,121]. Though vesicle immunogenicity is milder than live parasites, strategic inclusion of native surface proteins or GPI-mimetics allows for rational tuning of immune activation. In mouse models, parasite-membrane-coated nanoparticles have successfully induced tumor regression and increased intratumoral CD8⁺ T cells, underscoring their translational potential.

The nanoscale size of TgCMNVs (can be controlled around 100–200 nm, Fig. 4) supports passive accumulation in tumors via the EPR effect. Yet their real strength lies in programmable targeting [6,11]. Through genetic engineering, tumor-specific ligands, such as anti-PD-L1 nanobodies or RGD peptides, can be displayed on abundant membrane scaffolds like SAG1 [115]. This avoids the instability and inefficiency of post-synthetic conjugation seen in synthetic carriers. Additional targeting moieties like folate or aptamers may be incorporated to match tumor surface receptor profiles and reduce off-target delivery [38].

TgCMNVs inherently combine antigen delivery with immune activation. This is particularly attractive in immune-excluded tumor, where checkpoint blockade alone often fails. By co-delivering parasite antigens and immune ligands in a single construct, TgCMNVs may recruit antigen-presenting cells and prime de novo T cell responses. Such vesicles could be deployed alone or as adjuvants in therapeutic vaccines—stimulating dendritic cells in draining lymph nodes while homing to the tumor core. Collectively, TgCMNVs bridge a gap between synthetic carriers and live microbial vectors, enabling programmable, immune-active nanotherapy for solid tumors.

3.6. TgCMNVs as a vaccine platform against T. gondii

Vaccine development for T. gondii has historically centered on subunit, DNA, and live-attenuated approaches, each balancing immunogenicity with safety and scalability [122]. Nanocarriers such as PLGA and chitosan have improved antigen stability and delivery kinetics, but few platforms integrate both antigen presentation and delivery into a single system. TgCMNVs offer this dual functionality, embedding immunogenic surface proteins within a biocompatible, customizable vesicle [123,124]. Key surface antigens from the SRS family, particularly SAG1, SAG2, and SAG3, are highly conserved and abundantly expressed during the tachyzoite stage, making them ideal for durable B- and T-cell activation [46,125,126]. While GPIs alone may be insufficient for protection, likely due to glycan shielding, they act as potent PAMPs, activating TLR2/4 when co-presented with GPI-anchored SRS proteins [127]. This synergy appears critical for eliciting robust Th1-biased immune responses and long-lasting cellular immunity. TgCMNVs can also be engineered to expand antigenic coverage beyond acute infection. Incorporation of bradyzoite-stage antigens such as BAG1 [126], or epitope-optimized chimeric constructs, mimics the breadth of whole-organism vaccines without their safety risks. Immunoinformatic-guided selection of conserved epitopes from targets like SABP1 may enhance cross-stage protection and immunological memory, enabling precision design of multi-epitope vaccines [58].

These vesicles offer distinct advantages: native antigen presentation without exogenous adjuvants, stable architecture suited for prolonged circulation, and a genetically tractable platform for modular antigen insertion. Still, challenges remain [128]. Compared to live parasites, isolated membranes may require co-delivery of cytokines or additional immunostimulants to achieve comparable immunogenicity. Furthermore, clinical translation will demand scalable production, consistent antigen composition across strains, and long-term stability under cold-chain constraints. TgCMNVs thus represent a next-generation self-adjuvanting vaccine platform, offering the antigenic complexity of whole-parasite strategies with the safety and tunability of engineered nanocarriers. Whether applied alone or within heterologous prime-boost regimens, they provide a promising route toward precision immunization against T. gondii and potentially other intracellular pathogens.

4. Discussion and future directions

TgCMNVs combine features of evolutionary immunobiology with modern nanotechnology. These vesicles retain the biological sophistication of their parent organism, rapid proliferation, a genetically tractable genome, and complex membrane architecture, while inheriting the functional hallmarks of cell membrane-derived nanocarriers (CMNVs), including natural surface biocompatibility, immune modulatory potential, and customizable functionalization. Their distinct membrane composition, including GPI-anchored SRS proteins and phosphatidylthreonine-enriched lipids, enables unique applications across immunotherapy, vaccine delivery, inflammation resolution, and tissue-targeted delivery.

Unlike conventional CMNV platforms derived from mammalian or bacterial cells, TgCMNVs offer both high scalability and inherent immunostimulation. As a eukaryotic parasite, T. gondii grows rapidly in vitro and tolerates a wide range of genetic and chemical modifications. Its membranes include innate immune agonists, such as GPIs that trigger TLR2/4, and conserved surface antigens like SAG1 that can directly engage adaptive immunity. This allows TgCMNVs to operate without exogenous adjuvants while offering opportunities for precision targeting via surface display of immune ligands, tumor-homing peptides, or cytokine mimetics. TgCMNVs offer dual functionalization capacity, either through genetic engineering during parasite growth or via chemical modification after membrane isolation, which broadens their potential range of applications. This two-tiered strategy enables integration of therapeutic payloads, targeting ligands, or immune-modulating factors within a single vesicle construct. Furthermore, their capacity to mimic apoptotic cell clearance and interface with ECM and complement systems makes them especially relevant for addressing fibrotic diseases, autoimmunity, and thromboinflammatory conditions.

Despite their promise, several translational barriers must be addressed for TgCMNVs to achieve clinical impact. First, scalable and reproducible manufacturing remains essential, as variations in size, protein density, or lipid composition can alter biodistribution and immune activation. This can be addressed by adapting microfluidic or continuous-flow extrusion systems to the unique membrane properties of T. gondii, paired with in-line quality analytics for batch-to-batch control. Second, biosafety and immunogenicity balance are critical. Removal of residual rhoptry and dense granule proteins must be confirmed through proteomic screening (Table 4), while rigorous in vivo pipelines, including cytokine profiling, systemic toxicity studies, and long-term biodistribution tracking, will ensure safety without loss of key functional properties. Third, targeting specificity should be optimized for each application by tuning ligand density, vesicle charge, elasticity, and cargo stability; predictive modeling combined with rational surface engineering can accelerate this process for diverse environments such as tumors, fibrotic tissues, or immune-privileged sites. Fourth, biological variability in GPI structure, glycan patterns, and SRS protein expression between strains may influence performance; standardizing on a defined production strain or replicating its membrane profile synthetically will support consistency. Finally, storage stability and clinical readiness require attention. Optimized formulations for lyophilization, cryoprotection, or matrix encapsulation, along with robust QC metrics such as morphology, zeta potential, and antigen retention, will be vital for regulatory compliance and clinical translation. These steps should align with established regulatory frameworks for cell-derived nanocarriers, including documented safety validation, pathogen inactivation verification, and lot release criteria.

To advance the translational potential of TgCMNVs, several priority areas merit strategic focus. Expanding the antigenic repertoire, particularly through incorporation of bradyzoite-stage proteins such as BAG1 or epitope-optimized constructs like SABP1, may improve immune coverage against latent infections while preserving safety. Parallel efforts to refine functionalization strategies, including the genetic fusion of immunomodulatory ligands (e.g., PD-L1 nanobodies, CD40L mimetics) and post-synthetic modifications via click chemistry or lipid anchoring, could enhance targeting fidelity across diverse disease models. The therapeutic scope of TgCMNVs also warrants broadening, given their compatibility with complement regulators, matrix remodeling enzymes, and vascular adhesion pathways—traits that position them to address thrombo-inflammation, autoimmune pathology, and tissue fibrosis. Finally, scalable manufacturing remains pivotal. Establishing GMP-compliant pipelines for membrane assembly, vesicle standardization, and in-line quality analytics will be essential for regulatory acceptance and clinical deployment.

5. Conclusions

T. gondii-derived cell membrane nanovesicles may represent a promising expansion of the biomimetic nanomedicine landscape. Harnessing evolutionary features such as abundant GPI-anchored proteins, phosphatidylthreonine-rich lipids, and a highly editable genome, TgCMNVs integrate immunological sophistication, engineering flexibility, and production scalability. Their ability to present native antigens, coupled with compatibility for both genetic and chemical functionalization, could provide a versatile platform for targeted drug delivery, cancer immunotherapy, and self-adjuvating vaccine design. These vesicles combine biological recognition with customizable surface architecture, an increasingly important combination for advanced therapeutics. Translational barriers remain, including biosafety validation, membrane consistency, and large-scale manufacturing. However, advances in microfluidic fabrication, automated quality control, and computational epitope mapping are helping to close these gaps. As the field moves beyond conventional mammalian and bacterial sources, T. gondii offers a distinctive and underexplored foundation for engineering programmable nanocarriers that can modulate immunity and meet the demands of precision medicine.

CRediT authorship contribution statement

Jiating Chen: Writing – original draft, Visualization, Validation, Resources, Investigation, Data curation, Conceptualization. Pengfei Zhang: Writing – original draft, Investigation. Hongjuan Peng: Writing – review & editing, Conceptualization. Jihong Chen: Writing – review & editing, Validation, Funding acquisition, Conceptualization.

Data availability

No datasets were generated or analysed during the current study.

Ethics approval and consent to participate

Not applicable. This study did not involve human participants, animal experiments, or personal data collection requiring ethical approval.

Declaration of competing interests

The authors declare no competing interests.