HIV invades the CNS much sooner in the course of the infection, and establishes latent reserves within microglial cells and perivascular macrophages (15). The virus can cause HIV-associated neurocognitive disorders (HAND), characterized by memory loss, motor impairment, and behavioral changes (16). These effects generally emerge from chronic neuroinflammation, viral neurotoxiins, and even neuronal damage following immune activation-not simple infection of neurons (17).

Herpes simplex virus (HSV) is sometimes associated with herpes simplex encephalitis (HSE), which can be serious and potentially life threatening without treatment (18). After the first infection, the virus may remain dormant in the trigeminal or sacral ganglia which may reactivate causing additional episodes and potentially spreading into the brain. While either the HSV-1 or HSV-2 subtypes can cause HSE, HSV-1 is the most common cause of sporadic viral encephalitis in adults. Its rapid timing, propensity for causing temporal lobe necrosis, and long-term cognitive impairment are notable (19, 20).

This encapsulated fungal infection is one of the leading causes of fungal meningitis, especially in people with weak immune systems, like those with AIDS or who have had a transplant (21). Cryptococcus spreads through the blood and gets into the CNS. It then uses “Trojan horse” mechanisms (within infected phagocytes) to get over the BBB. It can live and reproduce in the subarachnoid space. Cryptococcal meningitis, which is what happens when this happens, is marked by increased intracranial pressure, changes in mental status, and a high death rate (22).

T. gondii is a common protozoan that lives inside cells and causes toxoplasmic encephalitis (TE), especially in people with weak immune systems (23). The parasite causes a long-lasting, hidden infection in the central nervous system (CNS), mainly in neurons and astrocytes. When latent tissue cysts in the brain become active again, they can cause seizures, localised neurological impairments, and encephalopathy (24, 25).

The BBB is a very selective and well-controlled barrier that keeps most drugs from getting into the brain. Many therapeutic compounds, including big hydrophilic medicines, cannot get through the BBB in sufficient concentrations, leading to subtherapeutic levels at the site of infection (26).

Drugs often need to be given at large systemic doses to penetrate the CNS effectively, which raises the risk of off-target effects and toxicity. This is especially bad with antifungals like amphotericin B or antiretroviral medicines, which can hurt the kidneys or liver (8).

CNS pathogens often evade immune surveillance and persist in latent or quiescent forms within protected niches such as microglia (HIV), neuronal ganglia (HSV), or neurons (T. gondii) (27). These reservoirs are inaccessible primarily to conventional drugs and can serve as sources of reactivation, contributing to recurrent or chronic disease (28).

In many CNS infections, neuronal damage results from direct pathogen activity and an exaggerated immune response. This neuroinflammatory milieu makes treatment much harder by damaging the BBB, changing how drugs are transported, and making tissue damage worse. Because of these problems, we need new, focused delivery methods to cross the BBB quickly, target just sick cells, and deliver therapeutic payloads with as few side effects as possible. Exosome-based drug delivery systems have much potential to get around these problems (28).

Importantly, the biologic behavior of each CNS pathogen will greatly impact the nature of exosome-based therapies. For example, HIV establishes latency in microglia and macrophages, and designed exosomes with ligands (e.g., RVG peptide or anti-gp120 antibodies) to deliver siRNA or CRISPR cargo can be used to target these reservoirs. HSV, on the other hand, persists in neuronal ganglia and reactivates in episodic fashion; exosomes containing anti-ICP0 siRNA or decorated with gD-binding peptides can provide pathogen-specific suppression (29). Cryptococcus crosses the BBB by “Trojan horse” mechanisms and exists in the meninges, which endorses the use of mannose- or dectin-1-modified exosomes packaged with amphotericin B or fluconazole. Finally, Toxoplasma gondii forms latent cysts in neurons and astrocytes; thus, exosomes that contain aptamers to SAG1 or CD36 in combination with a CRISPR or siRNA cargo has an added therapeutic value. By connecting pathogen biology with the design of exosomes, we can rationally dictate how to optimize therapies to manage barriers associated with latency, intracellular sanctuary and immune evasion (30).

The formation of exosomes is a tightly regulated process involving both ESCRT-dependent and ESCRT-independent ways that exosomes develop, and the process is strictly controlled (33). The Endosomal Sorting Complex Required for Transport (ESCRT) machinery helps the endosomal membrane bud inward to make intraluminal vesicles (ILVs) inside MVBs in the ESCRT-dependent pathway. ESCRT-0, -I, -II, and -III are important parts of ESCRT that help choose cargo, change the shape of membranes, and split vesicles (34). Proteins like tetraspanins (CD63 and CD81) and lipids like ceramides help make ILVs by bending membranes and sorting specific cargo into exosomes. These pathways do not depend on ESCRT Figure 1. When MVBs are fully grown or mature, they can either fuse with lysosomes for degradation or with the plasma membrane to release their ILVs as exosomes into the extracellular space (35).

Exosomes carry unique surface markers, including CD9, CD63, CD81, ALIX, and TSG101. They also have cargo important for their function, such as miRNAs controlling gene expression in cells that take them in (36). Proteins and enzymes change how the immune system works or assist cells in metabolising, as well as lipids that help keep membranes stable and make it easier for vesicles to fuse. These molecular contents are carefully packaged, often showing the health or illness condition of the parent cell. This makes exosomes possible indicators for CNS ailments (37). Comparison with other delivery systems, exosomes have advantages as CNS delivery vehicles including but not limited to their ability to cross the blood–brain barrier (BBB), low immunogenicity, and inherent biocompatibility (38). Conversely, liposomes are established nanocarriers with sufficient drug loading capabilities and scalable manufacture; however, they are often labile to rapid clearance and have limited BBB penetrability. Polymeric nanoparticles have tunable physicochemical properties and controlled release, but may present cytotoxicity and an immune response, which negatively impacts long-term safety (39). Viral vectors are highly efficacious in gene delivery but raise fears surrounding immunogenicity, mutagenesis, and communication among regulatory hurdles. Exosomes, in comparison, afford a natural targeting mechanism and decreased toxicity (40). Nevertheless, challenges remain for exosomes in the form of scalability, heterogeneity, and reliable data for standardization. Hence, there is no single system which is the universally optimal solution, but exosomes can balance efficacy with safety for therapies in CNS infections (as long as current limitations are solved by future investigations).

Neurones, astrocytes, oligodendrocytes, and microglia are all neural cells that release exosomes in the CNS. Moving bioactive molecules between cells helps with important bodily functions such as synaptic plasticity, neuronal survival, myelination, and neuroinflammation (41). Their intrinsic capacity to breach the BBB and tendency to go towards inflamed or infected neural regions make them suitable delivery vehicles for therapeutic interventions in CNS diseases. Recent studies have shown that exosomes can be modified to carry therapeutic substances (including antiretrovirals, antifungal medicines, and CRISPR/siRNA constructs) to parts of the brain that are infected or injured (9). Their low immunogenicity, long circulation half-life, and capacity to avoid phagocytic clearance make them even better candidates for brain-targeted therapy, especially in CNS infections when standard treatments do not reach therapeutic levels (9).

Surface engineering is a central strategy in exosome modification, enabling specific interaction with infected cells or facilitating BBB penetration. Two primary approaches are employed: ligand display and membrane anchoring of targeting moieties (45). One of the most important parts of exosome-based targeted therapy is ensuring that the therapeutic vesicles are sent directly to the cells in the CNS infected with pathogens. Pathogen-specific ligand display is a method that makes this level of accuracy possible. It involves targeting moieties (ligands) on the surface of exosomes so that they can find and bind to pathogen-related indicators or host cell receptors that have been changed by infection. This method makes exosomes more selective, easier to take up, and more effective as treatments, especially when it comes to diseases that infect the brain, such as Mycobacterium tuberculosis, HIV, Cryptococcus neoformans, Herpes simplex virus (HSV), and Toxoplasma gondii (44) Table 1. The principles of pathogen-specific ligand display involves engineering the exosome membrane to present molecular recognition elements that bind selectively to: Pathogen-specific antigens (e.g., viral glycoproteins, fungal capsular components). Host cell receptors are regulated during infection (e.g., mannose receptor CD206, ICAM-1) and Cellular stress or inflammatory markers in infected brain tissue. These ligands may include: Peptides, Antibodies or antibody fragments, Aptamers, Lectins or sugar moieties and single-chain variable fragments (scFvs) (51) Table 1.

In addition to surface targeting, exosomes can be genetically engineered to encapsulate specific nucleic acid-based therapeutics that modulate gene expression in target cells.

Beyond biological modification, chemical strategies have been widely applied to improve exosome robustness, payload stability, and cellular targeting (57). These includes;

Click chemistry enhances the therapeutic utility of exosomes in several pathogen-targeted applications HIV-Associated Neuroinfections: Enables display of gp120-binding aptamers on exosomes for targeting infected microglia (106). Herpes Simplex Virus (HSV): Facilitates loading of anti-HSV siRNA inside exosomes with modified membrane proteins for enhanced neuronal uptake (57).Toxoplasmosis: Allows tethering of parasite-targeting antibodies on exosomal surfaces to direct them toward infected astrocytes (107). Tuberculous Meningitis (TBM): Supports BBB-targeted delivery of anti-TNF-α siRNA using click-functionalised exosomes (104, 105). The challenges for consideration include toxicity. A study showed copper toxicity (CuAAC) reaction that requires careful post-reaction purification or copper-free variants (93, 94). Ensuring batch-to-batch consistency in functionalisation is necessary for clinical-grade applications for scalability (108). Modifications must preserve exosome structure and prevent immune clearance (109), thereby enhancing the biocompatibility of ligands. Surface modifications may not always guarantee internalisation into the intended subcellular compartment (102), improving intracellular targeting. However, prospects could involve developing multifunctional click-compatible ligands (e.g., dual BBB-targeting and infection-targeting)—integration with bioorthogonal imaging for real-time tracking of therapeutic delivery (108). Scalable GMP-compliant click chemistry platforms for clinical-grade exosome production—application of click-to-release systems where cargo is activated only in the infected microenvironment (110). The click chemistry gives us a strong and flexible set of tools for changing the surfaces and interiors of exosomes. This makes it possible to deliver drugs to infected areas in the brain very selectively, efficiently, and stably. Exosomes are a critical molecular method for developing precision treatment for brain-infecting diseases because they work well with biological systems and have natural targeting and transport capabilities (110).

Aptamers can be conjugated to exosomes using several chemical and genetic methods:

Aptamer conjugation has become a flexible and valuable way to make exosomes functional for targeted delivery, notably in the problematic area of central nervous system (CNS) infections (112). The Systematic Evolution of Ligands by Exponential Enrichment (SELEX) technique chooses aptamers, which are short, single-stranded DNA or RNA oligonucleotides, to bind strongly and selectively to a wide range of targets, such as proteins, cells, and tiny molecules (45, 113). The rationale for Aptamer Use in brain-targeted exosome therapy is that aptamers can resemble antibodies in recognising targets because of their unique structural folding. They also have benefits such as being less immunogenic and poisonous. Easier to make and change, better stability in various physiological circumstances and a high binding selectivity with an affinity of nanomolar to picomolar (114). In the context of CNS-targeted therapies, aptamers can be selected to bind to: Blood-brain barrier (BBB) transporters (e.g., transferrin receptor, insulin receptor), Pathogen-specific antigens (e.g., surface proteins of Neisseria meningitidis, Cryptococcus neoformans, or viral envelope proteins), infected cells or inflammatory microenvironment markers (e.g., ICAM-1, VCAM-1, and CD44) (114, 115).

Several methods have been developed to alter or enhance the lipid composition of exosomes either pre-isolation (during biogenesis) or post-isolation (after purification). The post-isolation lipid insertion strategy involves passively inserting or incorporating modified lipids into the exosome membrane (45). For instances, cholesterol-conjugated ligands such as molecules like miRNA, aptamers, or peptides can be attached to cholesterol and inserted into exosome membranes via hydrophobic interactions (73, 74). Also, PEGylated lipids (e.g., DSPE-PEG), here the insertion of polyethene glycol (PEG)-linked lipids can prolong circulation time, reduce opsonisation and clearance by macrophages, and provide anchor points for further ligand attachment (e.g., via NHS ester or maleimide groups) (122). Lastly is targeting ligand-lipid conjugates such as RVG (rabies virus glycoprotein peptide), transferrin, or folate can be conjugated to lipids like DSPE and inserted to enhance brain or pathogen-specific targeting (123). On the other hand, is the pre-isolation lipid engineering in donor cells that involves lipid supplementation in culture, were the parent cells are cultured with exogenous modified lipids, which get incorporated into budding exosomes (124). Also, genetic modification of donor cells that involves overexpression of enzymes like ceramide synthase or phospholipid remodelling enzymes changes the lipid profile of secreted exosomes, improving membrane fluidity or targeting properties (125).

Lipid-modified exosomes have been explored in multiple therapeutic contexts, such as:

Conventional antiretroviral therapy (ART) is often ineffective in eliminating HIV reservoirs in the brain due to limited drug penetration and persistence of the virus in microglial cells. Engineered exosomes have shown promise in overcoming these barriers (134).

Current antifungal drugs have toxic profiles and don't get into the brain very well, which makes it very hard to treat cryptococcal meningitis. Researchers are working on exosome-based delivery systems to make them more effective and less harmful (136).

Toxoplasmic encephalitis remains a significant cause of mortality in immunocompromised populations, and new molecular approaches are needed to overcome the parasite's ability to form latent cysts in the brain (140).

Infections in the CNS can cause neuroinflammation, oxidative stress, and damage to neurons, long after the pathogens are gone. Exosomes can also transport neurotropic and anti-inflammatory drugs to help rehabilitate and stop long-term neurological problems (142).

Several administration routes have been explored to maximise exosome delivery to the brain. The choice of route influences distribution, onset of action, and clinical feasibility (148).

Exosomes must be tracked in vivo to evaluate distribution, clearance, and CNS specificity to assess their therapeutic relevance. Multiple labelling and imaging techniques have been developed, including;

Several preclinical studies have confirmed the ability of engineered exosomes to cross the BBB and selectively target CNS pathogens. RVG-modified exosomes delivering anti-HIV siRNA showed specific uptake by HIV-infected microglia and reduced viral replication in the CNS. In Cryptococcus infection models, mannose-modified exosomes loaded with amphotericin B localised preferentially to infected meninges and reduced fungal burden significantly. Exosomes tagged with T. gondii-specific aptamers demonstrated high specificity for parasitic cysts in the brain and suppressed cystogenesis when carrying gene-silencing cargo (156). This review again shows that optimising delivery routes and surface engineering are important for getting targeted therapy, reducing off-target effects, and increasing the therapeutic index in CNS infections. By carefully choosing the right ways to give exosomes and doing precise molecular engineering, we may get them to the CNS areas more effectively. Modern tracking technology gives us much information about how exosomes move through the body and where they go. These new ideas make using exosome-based medicines in clinical settings possible to treat hard-to-treat brain infections (157).

Exosome production remains difficult to scale reproducibly, with yields varying by donor cell type, culture conditions, and passage number. Although bioreactors and tangential flow filtration (TFF) improve yield and purity compared to ultracentrifugation or precipitation, batch-to-batch reproducibility and GMP-compliant release criteria (identity, purity, potency) are not yet standardized (180).

While generally considered biocompatible, engineered or allogeneic exosomes may carry PAMPs or inflammatory molecules that raise immunogenicity concerns (181). PEGylation and membrane engineering (e.g., CD47 expression) can mitigate risk, but regulators require extensive long-term toxicology, biodistribution, and off-target data. Preclinical reports of low toxicity for exosome-based siRNA, CRISPR, or antifungal delivery (158) remain too limited in scale to satisfy clinical standards (159).

Exosome therapies fall into a gray zone between biologics, cell-derived products, and nanomedicine, complicating approval pathways. Regulatory agencies (FDA, EMA, CDSCO) demand source traceability, sterility, reproducible characterization, and validated potency assays. Although early-phase trials in cancer and neurodegeneration demonstrate safety and CNS targeting, no product has yet been approved for CNS infections, reflecting the regulatory bottlenecks (160).

Currently, no harmonized GMP standards exist for exosome therapeutics. Key analytical markers—particle size (30–150 nm), zeta potential, protein cargo (CD9, CD63, CD81), and RNA content—are inconsistently applied, and robust potency assays are undeveloped. Lack of clear release specifications and validated QC assays for clinical-grade exosomes remains a critical barrier to regulatory approval (161). Also, the challenge of batch-to-batch consistency requires stringent protocols for donor cell characterisation, culture conditions, and post-processing storage (e.g., lyophilisation, cryopreservation). Like Quality Control Technologies are Nanoparticle tracking analysis (NTA), Western blotting and flow cytometry for surface markers (CD63, CD81, CD9) and High-throughput RNA sequencing for transcript cargo profiling (162).

Exosome platforms hold significant promise for individualised therapies, especially in settings where rapid, tailored interventions are needed:

One critical area of future research involves elucidating the molecular and cellular mechanisms underlying exosome-pathogen interactions. Exosomes can inhibit or facilitate pathogen dissemination by serving as decoys or carriers. Understanding: How viruses like HIV, SARS-CoV-2, or Zika exploit exosomal pathways, the role of exosomal cargo (miRNAs, proteins, lipids) in modulating immune responses, Host-pathogen communication through exosomal signaling, will not only improve our comprehension of disease progression but may also enable the repurposing of exosomes as anti-viral or antimicrobial delivery tools. High-resolution proteomics and single-exosome RNA sequencing technologies will be instrumental in uncovering these mechanisms (166).

Natural exosomes are safe for living things, but they have problems with scalability, heterogeneity, and loading cargo efficiently. So, people have been paying attention to the rise of synthetic exosomes (or biomimetic vesicles). These manufactured structures copy the physical and biological properties of natural exosomes, which allow changes in their composition and surface in a regulated way, encapsulate drugs more effectively and precisely, and make them more stable and last longer. Hybrid technologies, including exosome-liposome hybrids or exosome-coated nanoparticles, also have the advantage of targeting naturally and having engineered functionality. These platforms may prove especially useful in crossing the blood-brain barrier (BBB) or targeting hard-to-reach tissues (167).

The integration of computational biology and Artificial intelligence (AI) is being increasingly used in exosome engineering, including cargo loading, surface modification, targeting, and quality control. Machine learning can help predict the most efficient encapsulation of RNA or drug; while there are programs (e.g., with AlphaFold) that could assist with designing ligands or peptides for improved targeting toward the brain (168). For example, AI-guided optimization of miRNA loading for glioblastoma treatment, as well also computational docking, were used to support engineering RVG-tagged exosomes that can cross the blood–brain barrier (169). AI has also made patient-specific predictions by analyzing exosomal biomarkers and supporting personalized medicine. Next generation, AI-driven imaging is expected to improve reproducibility and the quality of evidence that goes into manufacturing. The incorporation of robotics for liquid handling and integrating AI with high-throughput screening and design may help lead to more efficient scaled-up exosome-based therapies for clinical use (170). Moreover, 3D bio printing can aid in fabricating exosome-loaded scaffolds for regenerative applications, such as neural repair or cardiac tissue engineering. Alongside this, organ-on-chip platforms (like brain-on-chip or liver-on-chip) are being developed to mimic in vivo environments, providing a more accurate preclinical assessment of exosome-based therapeutics, including toxicity and permeability studies (171).

Combinatory Medicines Combinatory medicines refer to therapies that include two or more agents that work synergistically, have better resistance profiles, or are less toxic than monotherapies. This approach is increasingly important in multifactorial diseases including cancer, infections, and neurological disorders with multiple therapeutic targets (172). For instance, chemotherapy plus immunotherapy (e.g., PD-1 inhibitor plus paclitaxel) has demonstrated improved survival rates in patients with non-small cell lung cancer (173). In infectious diseases, antiretroviral therapy (ART) involves combination therapies that target different stages of the HIV life cycle to reduce viral resistance. The use of a multiple-drug regimen to treat tuberculosis (isoniazid, rifampicin, ethambutol, pyrazinamide) is essential to ensure resistant strains do not develop and to ensure that patients ultimately receive a curative treatment (174). In the neurological context, levo-dopa with carbidopa used as a co-medication in Parkinson's disease (PD) to increase efficacy and decrease undesirable systemic side effects. There is ongoing research into co-delivery of drugs (e.g., doxorubicin with siRNA) via various delivery vehicles including nanoparticles or exosomes to improve delivery targeting and maximize therapeutic outcomes (24, 25, 175).

While the exosome engineering for targeted therapy for brain-infecting pathogens is promising, the clinical application of exosomes still requires standardised isolation, characterisation, and storage protocols (176). Therefore, future research could focus on creating potency tests and quality control measures for clinical-grade exosomes, setting up production lines that follow good manufacturing practices (GMP), and making rules for making synthetic or hybrid exosome compositions. Long-term clinical trials are also necessary to determine the long-term safety, immunogenicity, and therapeutic results of exosome-based therapeutics in different patient groups. Exosome research is at a crucial point, with progress in several fields coming together to realise their therapeutic potential fully (158). Future work should harness molecular tools, delivery approaches, and translational challenges, using synthetic biology, nanotechnology and AI to enhance targeting, scalability, and safety for exosome based therapies for brain pathogens. As the area grows, it will be important to deal with mechanistic gaps, translational obstacles, and regulatory hurdles to successfully move from bench to bedside (177, 178).