Erythrocyte-based drug delivery

Erythrocyte-based drug delivery systems (EBDDS) use red blood cells, their membranes, or their components to release and deliver pharmaceutical agents throughout the body in a controlled manner.^[1] Because red blood cells circulate for long periods of time and are potentially non-immunogenic, they are an attractive vector for drug delivery via the circulatory system.^[1][2][3] Erythrocytes can be used intact as carriers,^[3] or alternately empty erythrocyte membranes or nanoscale vesicles derived from erythrocytes may be used.

As early as 1973, it was recognized that red blood cells can be loaded with therapeutic enzymes, in order to avoid a possible immune reaction to the enzyme or rapid degradation of the enzyme if injected directly into blood.^[4] In humans, red blood cells have a half-life in circulation of 10-15 days before clearance by the lymphoreticular system and exhibit a natural tendency to absorb a variety of drugs.^[5] Together, these properties allow for controlled release of pharmaceutical products over an extended time, and/or targeting of erythrocyte-destroying cells such as macrophages in the spleen or liver. One of the earliest proposed applications of EBDDS was in enzyme replacement therapy for Gaucher disease, in which lipids accumulate in spleen macrophages; the enzymes not present in this disease could thus be delivered directly to macrophages by red blood cells.^[4]

Furthermore, red blood cells are not immunogenic as long as the blood type is compatible with the recipient. This property allows potentially immunogenic substances such as proteins to be safely carried through the bloodstream.

The success of EBDDSs depends heavily on the method that is used to load therapeutic agents onto or into the cells. There are many strategies that have been developed to optimize the drug stability, controlled release, and encapsulation, while also preserving the functionality and biocompatibility.^[6] The methods range from passive loading, such as dialysis based encapsulation of compounds like chlorpromazine,^[7] to complicated bioengineering approaches, such as electroporation and microfluidics.^[8] Other innovations include using natural cell membranes to camouflage nanoparticles to enhance circulation and immune evasion,^[9] as well as internal versus surface drug attachment strategies that are able to influence delivery efficiency and cellular behavior. Controlled release systems are further expanding the utility of erythrocyte carriers by allowing site-specific or stimulus-triggered drug release.^[6] This section will explore these drug loading methods in detail, highlighting their principles, efficience, and potential clinical applications.

One of the simplest and most widely used strategies for drug encapsulation is passive loading, which is typically reliant on diffusion-based mechanisms.^[7] For example, hypotonic dialysis is a method that leverages osmotic pressure differences to temporarily permeabilize the red blood cell membrane, allowing for the passive diffusion of drugs into the cells.^[7] In the process, the drug diffuses across a semipermeable membrane into the lipid vesicles or cells, which are driven by concentration gradients.^[7] While this is straightforward and it avoids the need for complex equipment, it often results in lower loading efficiencies and is limited to drugs that are sufficiently amphiphilic or hydrophobic to cross membranes.^[7] Nevertheless, passive loading remains like hypotonic dialysis remains a valuable approach due to its relative simplicity and compatibility with the delicate structure of erythrocytes.^[7]

Biomimetic approaches to deliver drugs have gained prominence with the use of erythrocyte membranes to camouflage nanoparticles. This method involves coating polymeric nanoparticles with natural erythrocyte membranes, creating a delivery system that is able to mimic the properties of native erythrocytes and prolong the circulation time . Further research includes the development of erythrocyte membrane-coated poly(lactic-co-glycolic acid) nanoparticles for combined chemotherapy and hypoxia-activated therapy.^[9] These systems not only enhance tumor targeting , but this approach leverages the inherent properties of erythrocyte membranes, which contribute to extended half-life and biocompatibility, making these carriers versatile in applications for inflammatory diseases and cancer.^[1]

There are 2 different strategies for drug incorporation into erythrocytes, internal encapsulation or surface absorption, which impact safety and therapeutic effectiveness. Internal loading protects the drugs from premature degradation, compared to surface loading which facilitates rapid delivery at the cost of potential immune recognition. Erythrocyte hitchhiking strategies, where nanoparticles are tethered to the cell surface non-covalently, have shown encouraging outcomes in boosting organ-specific delivery.^[10] It is also researched that the choice of the loading route should also account for the erythrocyte deformability and the integrity of the membrane to ensure that the carrier is compatible with tissue targeting and blood flow.

Controlled release technologies in EBDDSs offer site-specific and temporal precision in drug delivery. There are outlined mechanisms such as pH-sensitive and enzyme-responsive release, which ensure that the drug payload is discharged only in the targeted pathological environments, such as inflammatory sites or tumor tissues.^[6] These designs aim to reduce the systemic toxicity and improve therapeutic indices.^[6] This research has been expanded on by discussing erythrocyte derived carriers that are able to respond to external stimuli like light and temperature, while it has also been demonstrated that refunctionalized erythrocyte derived carriers can be engineered to increase tumor specificity and immune evasion. Such advancements underscore the adaptability of erythrocytes in complex therapeutic scenarios.

Bioengineering advances have improved the efficiency and the precision of drug loading into erythrocytes. Research as highlighted techniques such as hypotonic dialysis, microinjection, chemical coupling, and electroporation, which each enable different drug types to be loaded without compromising the cell viability.^[8] Electroporation is particularly important for delivering nucleic acids, while hypotonic methods are better suited for small molecules.^[7][11] Research has also shown that the use of erythrocyte derived extracellular vesicles for RNA delivery, which is able to combine the natural biocompatibility of erythrocytes with the therapeutic potential of genetic medicine.^[11] These methods show potential in the development of personalized and multifunctional carriers for the treatment of chronic diseases.

Erythrocyte-based drug delivery systems are a versatile approach that has been explored for the delivery of various types of therapeutic agents, including steroids, peptides/proteins (antigens), nucleic acids (RNA drugs), and chemotherapeutic agents.^[9][12][11] The application of EBDDS spans a diverse range of diseases from neurodegenerative disorders to cancers and inflammatory conditions.

Glucocorticoids, particularly dexamethasone sodium phosphate (DSP), have been successfully encapsulated into autologous erythrocytes to create long-circulating, controlled-release formulations.^[12] This technique has allowed for the gradual release of dexamethasone over a period of several weeks. The choice of DSP stems from its high anti-inflammatory potency and similar structure to betamethasone. Encapsulation of these compounds into erythrocytes enables a repeated dosing without requiring high systemic concentrations or frequent administration.^[12]

Erythrocytes can be chemically engineered to carry a wide variety of peptides and proteins, particularly through covalent attachment. An example of a widely used method involves a sortase A-mediated conjugation, in which peptide ligands are covalently bound to the surface of erythrocytes without disrupting the membrane's integrity. This platform supports the stable presentation of both natural and synthetic peptides, while enabling the transport of complex protein fragments or immunogenic sequences. The conjugation process maintains the erythrocyte morphology and circulation time, even when decorated with multiple surface-bound proteins.

Red blood cell extracellular vesicles (RBCEVs) have also emerged as a delivery system for various RNA-based therapeutics. These vesicles are devoid of nuclear material and capable of loading and transporting antisense oligonucleotides (ASOs), mRNA, and CRISPR-Cas9 components including guide RNAs (gRNAs). The RNA payloads can be introduced via electroporation which allows for efficient encapsulation as well as preserving vesicle stability and function. Studies have confirmed high delivery efficiency, minimal aggregation, and protection of nucleic acids from degradation during circulation.^[11]

Encapsulation of chemotherapeutic agents in erythrocyte-membrane-coated nanoparticles has been explored in order to enhance drug delivery profiles and reduce systemic degradation. For example, dual loading of curcumin ( a polyphenolic cytotoxic compound) and tirapazamine (a hypoxia-activated prodrug) into poly(lactic-co-glycolic acid) (PLGA) nanoparticles, followed by erythrocyte membrane coating, has been demonstrated.^[9] These formulations retain structural integrity and exhibit consistent physicochemical characteristics such as size and charge.^[9] The erythrocyte coating also improves colloidal stability and supports effective incorporation of hydrophobic agents.

Hydrophilic compounds, including bisphosphonates such as clodronate, can be efficiently loaded into RBC-derived nanovesicles (RDNVs). These carriers maintain structural compatibility with aqueous environments and allow for a stable encapsulation of water-soluble molecules. Surface markers like CD47 play a role in modulating vesicle uptake. However, clodronate-loaded RDNVs (whether derived from wild-type or CD47-knockout erythrocytes) exhibit sufficient encapsulation efficiency and vesicle integrity in storage and circulation.

Targeting strategies play a crucial role in enhancing the efficacy, safety, and specificity of EBDDSs. Using the intrinsic properties of erythrocytes - like immune evasion, long circulation half-life, and biocompatibility - researchers have been able to develop better methods to more accurately direct payloads to specific tissues, organs, or diseases microenvironments.^[1] These methods fall into various categories, which include passive targeting via natural biodistribution, active targeting through surface functionalization, cell-specific delivery through hitchhiking strategies, and stimuli-responsive targeting mechanisms.^{[1][9][10][6]} When they are appropriately designed, these strategies can significantly reduce the off-target effects, which improve pharmacokinetics, and expand the therapeutic potential of the red blood cell-based carriers across various clinical contexts.^[10]

Passive targeting is able to utilize the RBC's inherent long circulation time and biocompatibility. Because they are already naturally present in the bloodstream, they are able to persist for weeks, nd can act as stealth carriers that passively accumulate in certain organs or tissues through natural vascular filtration mechanisms.^[1] For example, the liver and spleen are known to clear modified or aged erythrocytes, which has been exploited for targeted drug delivery in inflammatory or autoimmune disorders.^[1] It can also be noted that such biodistribution patterns can fe fine-tuned depending on the surface modifications or the degree of drug encapsulation, which optimizes the passive accumulation in target tissues. Although it is less precise than active targeting, passive approaches are still important in RBC-mediated delivery because of their minimal immune activation and simplicity^[6]

To improve the targeting specificity, erythrocytes can be actively modified with peptides, antibodies, ligands, or aptamers that bind to specific cellular receptors. This approach allows for selective delivery to diseased tissue or cells, while making sure their healthy counterparts are not affected. There are strategies where erythrocytes' surfaces are engineered to carry targeting ligands for inflammatory endothelial markers or tumor vasculature.^[1] It has also been demonstrated that conjugating hypoxia-responsive elements or tumor-targeting peptides to erythrocyte membranes significantly enhances the uptake by the cancer cells while reducing the off-target effects.^[9] Furthermore, it has been shown that nanocarriers that are hitchhiking on RBCs and tagged with endothelial-specific antibodies are able to drastically increase delivery to target sites by several orders of magnitude.^[10] Such surface functionalization is able to open the door to personalized drug delivery and precision medicine by enabling the targeting of specific disease markers.^[1]

One strategy involves the "hitchhiking" of therapeutic nanoparticles on the surface of erythrocytes, which allows them to utilize the cell's natural trafficking routes to reach the target tissues. In this approach, nanocarriers or drugs are attached non-covalently to the erythrocyte's membrane before injection. Once they are injected and in circulation, they detach at specific sites, such as the liver, lungs, or spleen, depending on the molecular and hemodynamic cues.^[10] Research has shown the effectiveness of erythrocyte hitchhiking in delivering drugs to vascular endothelia and reducing systemic exposure. This approach has also been extended by creating erythrocyte-derived nanovesicles that preferentially home in on macrophages, which is a key strategy in immunomodulatory therapy. This method allows for a spatial control of drug distribution and can be combined with both active and passive targeting strategies to optimize the therapeutic outcomes.^[10]

Another emerging area involves engineering erythrocyte carriers that respond to specific disease-related stimuli - such as enzymes, oxidative stress, or pH - to release their payload only under pathological conditions. Research has shown that pH-sensitive carriers that remain intact under normal physiological conditions, but rapidly release drugs in acidic tumor microenvironments.^[6] Similarly, there have been carriers that have been developed to respond to tumor-associated enzymes or inflammatory cytokines, ensuring that there is localized delivery. These approaches not only enhance the therapeutic specificity but they also minimize the side effects associated with systemic drug exposure.^[6] When integrated with active targeting methods, the stimuli-responsive delivery significantly enhances the therapeutic index of erythrocyte-based platforms.

EBDDS have been increasingly recognized for their ability to improve performance of therapeutic agents across a broad range of clinical contexts. These systems have demonstrated utility in improving drug stability, prolonging circulation, reducing systemic toxicity, and enabling targeted delivery. As a result, they have been explored for use in cancer therapy, enzyme replacement therapy, anticoagulation, and immune modulation.

EBDDSs have been studied in oncology for their possible role in optimizing chemotherapeutic delivery and adjusting the therapeutic index of enzyme- and gene-based therapies. Encapsulation of L-asparaginase (L-ASNase) within erythrocytes (ASNase-ERY) has been one of the most advanced applications in this area. This formulation allows sustained depletion of circulating asparagine -a critical nutrient for certain leukemia cells- while shielding the enzyme from immune recognition and degradation. Clinical trials of ASNase-ERY in patients with acute lymphoblastic leukemia (ALL) and pancreatic ductal adenocarcinoma (PDAC) have demonstrated extended drug half-life, reduced hypersensitivity reactions, and improved overall survival outcomes.^[13] Chemotherapeutic agents such as daunorubicin, doxorubicin, and cytosine arabinoside have also been delivered using erythrocytes. Early studies utilized whole red blood cells for drug encapsulation, as opposed to more recent developments that have focused on nanoerythrosomes and RBC membrane-coated particles. These approaches enable controlled drug release, enhanced tumor accumulation, and reduced cardiotoxicity, especially for agents like doxorubicin known to cause cumulative cardiac damage.^[13]

Additionally, the fusion of erythrocyte membranes with various nano-materials has led to the creation of camouflaged nanoparticles that are capable of delivering small molecules, photodynamic/photothermal agents, siRNAs, and cancer vaccines. These systems combine the long-circulating properties of erythrocytes with the multifunctionality of nanoparticles, making them good candidates for both treatment and imaging applications in solid tumors. [8; Rao et al. (2016)]

Erythrocyte carriers have been employed for the delivery of therapeutic enzymes that require prolonged systemic activity or are otherwise prone to rapid inactivation. For example, glutamine synthetase, adenosine deaminase, and β-galactosidase have been successfully encapsulated within erythrocytes to treat metabolic conditions such as hepatic encephalopathy and enzyme deficiencies. In these cases, erythrocytes serve as circulating bioreactors that allow continuous enzymatic conversion of target substrates while protecting the enzymes from immune clearance.

EBDDS carriers have been used to deliver anticoagulants such as low molecular weight heparin (LMWH) and thrombolytic agents like plasminogen activators (PAs). Encapsulation helps maintain therapeutic levels in circulation and prevents rapid renal clearance. Importantly, this strategy allows antithrombotic activity to be confined within the vasculature, thereby minimizing hemorrhagic risks associated with systemic administration.^[13]

Erythrocyte-based carriers have enabled targeted delivery of immunomodulatory agents with reduced immunogenicity. Peptide antigens covalently linked to erythrocyte membranes have been used to induce antigen-specific immune tolerance in preclinical models of autoimmune diseases such as multiple sclerosis and type 1 diabetes. This strategy suppresses auto-reactive T and B cell responses without broadly suppressing the immune system. In oncology, erythrocyte-encapsulated interleukin-2 and tumor-associated antigens have been used to stimulate anti-tumor immune responses and enhance vaccine efficacy.^[13]

Due to their natural clearance by the reticuloendothelial system, erythrocyte derived nano-vesicles have been leveraged for targeted drug delivery to macrophages. These carriers are especially suited for administering hydrophilic drugs like clodronate, a bisphosphonate used to deplete macrophages. Studies have shown that erythrocyte-derived nano-vesicles preferentially accumulate in macrophage-rich organs such as the liver and spleen, and can achieve more efficient cellular uptake and reduced toxicity compared to traditional liposomal formulations.

While EBDDs offer a lot of advantages - biocompatibility, prolonged circulation, and immune evasion - they are not without their limitations.^[1] These systems are able to utilize the natural properties of red blood cells to improve the therapeutic delivery, but translating them from the lab to clinical use involves overcoming biological, technical and regulatory barriers.

EBBDSs face several biological and practical challenges. Techniques like microfluidics, electroporation, and dialysis, though effective for loading the drugs, can compromise the integrity of the membrane and reduce the viability of erythrocytes.^[7][8] Achieving high loading efficiency without damaging the erythrocytes remains a important challenge, especially for commercial or large-scale applications.[3^[7][8] Controlled drug release strategies are still in development, and current methods like pH-sensitive or passive systems often lack the precision that is required for consistent outcomes.^[6] Surface modifications or drug attachments can also increase recognition by the immune system, which reduces the circulation time and overall effectiveness. In addition to these technical obstacles, logistical and regulatory issues complicate clinical translation. These challenges include maintaining sterility, sourcing erythrocytes, and standardizing preparation protocols across batches.^[1]

Despite these challenges, future research efforts are focused on addressing the current challenges and exploring clinical applications of EBDDSs. Innovations in synthetic biology and nanotechnology are enabling hybrid erythrocyte-based platforms with enhanced controlled drug release and targeting. These systems can be engineered to respond to stimuli such as enzymatic activity or pH, which improves the site-specific delivery.^[6] Erythrocyte-derived carries also show the potential for personalized medicine and gene therapy, particularly for delivering RNA-based therapies and CRISPS components.^[13][11] Additionally, integrating the diagnostic agents into these systems supports real time monitoring and theranostic applications. While further research is needed to overcome the technical and regulatory obstacles, interdisciplinary advancements continue to move EBDDSs close to a clinical reality.

• Drug Carrier
• Blood Substitute
• Drug Delivery
• Cancer Therapy
• Erythrocyte Sedimentation Rate
• Intracellular Delivery
• Immunomodulation
• Immunotherapy