Enhancing Transduction and Immune Resilience in Viral Gene Therapy Through Erythrocyte-Derived Membrane Encapsulation

Abstract

Viral vectors such as adenovirus (Ad) and lentivirus (LV) are central to gene therapy owing to their transduction efficiency and broad applicability; however, their clinical translation is often limited by immunogenicity, rapid clearance, and reduced bioavailability. Non-enveloped Ad vectors are highly susceptible to neutralization by pre-existing antibodies, while enveloped LVs remain vulnerable to immune surveillance and off-target clearance. In this study, a biomimetic encapsulation strategy using erythrocyte-derived membranes (EDMs) is reported to enhance viral immune resilience and functional gene delivery. Ad-GFP and LV-mCherry were successfully encapsulated within EDM using an extrusion-based assembly approach, resulting in uniform membrane-coated particles with physicochemical properties characteristic of erythrocyte membranes. EDM encapsulation significantly enhanced in vitro transduction efficiency of both viral platforms across multiple cancer cell lines without compromising viral activity. Notably, EDM-Ad-GFP demonstrated robust protection against neutralizing antibodies, achieving significantly higher transduction of HEK293 cells in the presence of diluted human serum compared to unencapsulated Ad. These findings indicate that EDM encapsulation can effectively shield viral vectors from immune recognition while improving cellular uptake and transduction performance. Collectively, this work establishes EDM encapsulation as a versatile and scalable platform to enhance the efficacy, durability, and translational potential of viral gene delivery systems.

1. Introduction

Viral vectors have been widely established as essential tools in gene therapy due to their intrinsic ability to efficiently deliver genetic material into host cells. Their use has enabled significant advances in oncology, genetic medicine, infectious diseases, and regenerative medicine [1]. Among the most utilized platforms, adenovirus type 5 (Ad) and lentiviral vectors (LVs) have been extensively characterized and deployed in both preclinical and clinical settings [2,3,4,5]. Ads are valued for their high transduction efficiency, episomal gene expression, and cargo capacity, whereas LVs enable stable genomic integration and sustained transgene expression in dividing and non-dividing cells [6,7,8]. Despite these advantages, the clinical translation and repeated administration of viral vectors remain constrained by immunogenicity, rapid clearance, and reduced bioavailability [9].

Immune recognition represents a major limitation for viral gene delivery, particularly for the non-enveloped Ad. Pre-existing neutralizing antibodies against Ad are highly prevalent in the human population and significantly reduce vector efficacy by blocking cellular entry and accelerating systemic clearance [9]. This immune barrier not only limits therapeutic potency but also restricts vector re-dosing and long-term treatment strategies. Although enveloped LVs are and comparatively less susceptible to immediate antibody neutralization, they remain vulnerable to opsonization, immune surveillance, and off-target clearance, which can adversely impact transduction efficiency and biodistribution [10].

To address these challenges, biomimetic approaches have been increasingly investigated to enhance viral vector stability, immune evasion, and functional delivery [11,12]. Cell membrane-based encapsulation strategies have emerged as a promising solution by leveraging naturally occurring biological interfaces to mask foreign payloads from immune detection. Among these, erythrocyte-derived membranes (EDMs) offer distinct advantages due to their abundance, biocompatibility, long circulation lifespan, and minimal immunogenicity [13,14,15]. Erythrocyte membranes possess native surface proteins associated with immune tolerance and self-recognition, making them particularly attractive as protective coatings for therapeutic agents [13,14,15].

In this study, Ads and LVs are encapsulated within erythrocyte-derived membranes using an extrusion-based assembly approach. This encapsulation strategy is designed to create an EDM shield around viral vectors, enhancing viral immune resilience and improving blood viral concentration. EDM encapsulation provides physical and immunological protection for Ad vectors against neutralizing antibodies, enabling preserved infectivity and improved functional delivery. It also enhances transduction performance of LVs without compromising biological activity. Collectively, this work presents a versatile and scalable EDM-based encapsulation platform that improves viral gene delivery and offers a broadly applicable strategy for enhancing the efficacy of viral gene therapy systems.

2. Materials and Methods

2.1. Reagents and Cell Lines

Green Fluorescent Protein (GFP) expressing replication-deficient Ad (Ad-GFP) was purchased from Baylor College of Medicine, Houston, TX, USA (Vector: Ad5-CMV-eGFP). LV expressing mCherry (LV-mCherry) was purchased from GenScript, Piscataway, NJ, USA (Vector: GLV3-CMV-EGFP-Puro-EF1a-mCherry). 4T1 (mouse breast cancer cells), MDA-MB-231 (human breast cancer cells), HEK293 (human embryonic kidney cells) and CT26 (mouse colon cancer cells) cell lines were purchased from American Type Culture Collection (ATCC). Dulbecco’s Modified Eagle Medium (DMEM) with high glucose (Cytiva, Marlborough, MA, USA Catalog #SH30081.01) was supplemented with 10% of Fetal Bovine Serum (FBS, Corning, Glendale, AZ, USA, Catalog #35-011-CV) and 1% of Pen Strep Glutamine (PSG, Thermo Fisher Scientific, Waltham, MA, USA, Catalog #10378016) to prepare the complete media for HEK293 cell culturing. Roswell Park Memorial Institute (RPMI) 1640 (Thermo Fisher Scientific, Waltham, MA, USA, Catalog #11875093) and RPMI 1640 medium no folic acid (Thermo Fisher Scientific, Waltham, MA, USA, Catalog #27016021) were supplemented with 10% FBS and 1% PSG to prepare the complete RPMI (RP-10) for 4T1 and CT26 cell culturing. A 50:50 mixture of complete RP-10 and complete DMEM was used for culturing MDA-MB-231-GFP and MDA-MB-231 cells. All cells were cultured at 37 °C and 5% CO₂ in the complete media.

2.2. Derivation of EDM from Human Whole Blood and Encapsulation Procedure

All in vitro experiments were performed using EDM prepared from human donor red blood cells. EDM was produced using an established protocol for enrichment of cellular membranes, as previously described [16]. Briefly, Type O Rh-negative whole blood from a single donor was obtained from BioIVT (Westbury, NY, USA). Red blood cells were isolated and subjected to osmotic lysis to release intracellular contents, after which the membrane fraction was collected and purified using tangential flow filtration. The resulting EDM preparation was stored at −20 °C until further use. Reproducibility of the EDM preparation and encapsulation procedures has been previously established through process development, optimization, and quality control measures.

Virus encapsulation was performed using fine membrane extrusion as previously described [11,17]. Briefly, an Avanti mini extruder (Avanti Polar Lipids, Inc., Alabaster, AL, USA, Catalog #610000-1EA) was used, and a 1.0 µm pore-size polytetrafluoroethylene (PTFE) membrane (Sterlitech, Auburn, WA, USA, Catalog #PTFE1025100) was cut to size and sanitized by soaking all components in 70% isopropanol, followed by rinsing with sterile deionized water. The pre-extrusion mixture contained either Ad-GFP or LV-mCherry combined with EDM to yield working concentrations of 10¹¹ Transducing Units (TU)/mL EDM and 10⁸ TU/mL EDM, respectively, based on a 1 mg/mL concentration of EDM as measured through total protein content. The EDM–virus suspension was processed by repeated back-and-forth extrusion through the PTFE membrane using syringe pumps. Figure 1A illustrates EDM-encapsulated Ad and EDM-encapsulated LV. The encapsulation process is schematically illustrated in Figure 1B–D. The resulting EDM-encapsulated viruses (EDM-Ad-GFP or EDM-LV-mCherry) were collected in sterile 1.5 mL microcentrifuge tubes and used immediately for subsequent experiments.

For the Ad-GFP and LV-mCherry comparison groups, the viral vectors were used as received from the vendors and were not subjected to any additional processing or extrusion steps.

2.3. Dynamic Light Scattering (DLS) and Zeta Potential Measurements

Particle size measurements were performed using a Malvern Zetasizer Nano ZS instrument (Malvern Panalytical, Malvern, UK). Prior to analysis, samples were diluted 1:10 in phosphate-buffered saline (PBS). Dynamic light scattering (DLS) measurements were conducted using the instrument’s automatic measurement mode, with five runs of 10 s each recorded per sample. The average hydrodynamic diameter and polydispersity index (PDI) were calculated using Zetasizer software (version 8.02, Malvern Panalytical, Malvern, UK).

For zeta potential analysis, measurements were also performed in automatic mode. A minimum of five and up to fifteen measurements were acquired for each sample, with a 45 s interval between acquisitions. The average zeta potential values were calculated using the same Zetasizer software. All measurements were performed in triplicate (n = 3).

2.4. In Vitro Transduction

Cells were plated in 96-well plates at a density of 3 × 10⁴ cells per well and incubated overnight in complete culture medium at 37 °C in a humidified incubator with 5% CO₂ to allow attachment. The next day (day 1), cells were exposed to either Ad-GFP or EDM-encapsulated Ad-GFP at a multiplicity of infection (MOI) of 50 plaque-forming units (PFU) per cell and maintained under standard culture conditions (37 °C, 5% CO₂). In parallel experiments, LV-mCherry or EDM-LV-mCherry was added to the cells at an MOI of 1 PFU per cell on day 1 and incubated under the same conditions. All treatment formulations were administered in complete culture medium. No transduction enhancers were used, the culture medium was not replaced during the experiment, and cells were not washed during or after the transduction period.

2.5. Fluorescence Microscopy

Forty-eight hours after transduction, cells treated with Ad-GFP or EDM–Ad-GFP were imaged using a Keyence BZ-X710 fluorescence microscope (Keyence Corporation of America, Itasca, IL, USA) equipped with a GFP filter set (excitation 470/40 nm, emission 525/50 nm, dichroic mirror 495 nm). Cells exposed to LV-mCherry or EDM-LV-mCherry were visualized using a TexasRed filter set (excitation 560/40 nm, emission 630/75 nm, dichroic mirror 585 nm). Representative fluorescence images were captured with a 4× objective lens to enable comparative evaluation of transduction across experimental groups.

2.6. In Vitro Neutralizing Antibody Protection Assay

HEK293 cells were seeded into 96-well plates at a density of 1 × 10⁴ cells per well and maintained in complete culture medium at 37 °C with 5% CO₂. After allowing the cells to adhere for 24 h, a neutralization assay was conducted. The culture medium was carefully removed without disturbing the cell monolayer. EDM-Ad-GFP or Ad-GFP was then combined with human serum diluted to 1:10 and applied to the cells at an MOI of 100 in a volume sufficient to cover the cells (day 1). The cells were incubated under standard culture conditions (37 °C, 5% CO₂) for 1 h. Following this incubation, the treatment mixture was removed and replaced with fresh complete medium while minimizing disturbance to the cells. The cells were further incubated for an additional 24 h [9]. On day 2, fluorescence microscopy was performed, and GFP signal intensity was quantified using a Tecan F PLEX Infinite 200 Pro microplate reader (Tecan Group Ltd., Männedorf, Switzerland) [9].

2.7. Statistical Analysis

Data analysis was performed using Prism version 10.4.1 (GraphPad Software LLC, Boston, MA, USA). Differences between two groups were evaluated using a two-tailed unpaired t-test, and p values less than 0.05 were considered statistically significant.

3. Results

3.1. Characterization of EDM-Ad-GFP and EDM-LV-mCherry

Hydrodynamic size (z-average) and zeta potentials were measured for empty EDM, Ad-GFP, EDM-Ad-GFP, LV-mCherry, and EDM-LV-mCherry (

Table 1. Particle size (z-average) using DLS, and zeta potential of EDM-Ad-GFP and EDM-LV-mCherry (n = 3).

Formulation	z-Average (nm)	Polydispersity Index (PDI)	Zeta Potential (mV)
EDM	686 ± 42	0.30 ± 0.07	−12.7 ± 0.3
Ad-GFP	96 ± 2	0.27 ± 0.06	−5.1 ± 0.1
EDM-Ad-GFP	743 ± 37	0.50 ± 0.03	−11.1 ± 0.7
LV-mCherry	135 ± 7	0.26 ± 0.04	−4.8 ± 0.8
EDM-LV-mCherry	732 ± 14	0.46 ± 0.01	−13.1 ± 0.4

). The size distribution by intensity was recorded (Figures S1 and S2). As shown in Table 1, encapsulation of Ad-GFP and LV-mCherry within EDM results in a marked increase in hydrodynamic diameter—from native viral sizes of approximately 96–135 nm to ~730–740 nm for the EDM-encapsulated formulations—along with a shift in zeta potential toward values characteristic of the EDM, consistent with successful incorporation of the viruses within the EDM.

3.2. Comparative In Vitro Transduction of Ad-GFP and EDM-Ad-GFP

Comparative in vitro transduction of Ad-GFP and EDM-Ad-GFP was studied on 4T1 mouse breast cancer cells, MDA-MB-231 human breast cancer cells, and CT26 mouse colon cancer cells at MOI 50 (Figure 2 and Figure 3). The results demonstrate that in vitro transduction performance of EDM-Ad-GFP is significantly higher than that of Ad-GFP in all cell lines.

3.3. Comparative In Vitro Transduction of LV-mCherry and EDM- LV-mCherry

Comparative in vitro transduction of LV-mCherry and EDM-LV-mCherry was studied on 4T1 mouse breast cancer cells, MDA-MB-231 human breast cancer cells, and CT26 mouse colon cancer cells at MOI 1 (Figure 4 and Figure 5). The results demonstrate that in vitro transduction performance of EDM-Ad-GFP is significantly higher than that of Ad-GFP in all cell lines.

3.4. Comparative In Vitro Protection Against Neutralizing Antibodies Ad-GFP and EDM-Ad-GFP

Enveloped viruses such as LV are generally less susceptible to direct neutralization by antibodies targeting capsid proteins, as their lipid envelope can shield viral structural components. In contrast, non-enveloped viruses like Ad are more readily neutralized by pre-existing antibodies that directly recognize and bind exposed capsid proteins. Comparative in vitro transduction studies were performed to evaluate the impact of neutralizing antibodies on the non-enveloped Ad-GFP and to assess the protective effect of EDM encapsulation. The results demonstrated that EDM-Ad-GFP achieved significantly higher transduction of HEK293 cells (p ≤ 0.0001) in the presence of 1:10 diluted neutralizing antibody serum (Figure 6).

4. Discussion

The physicochemical characterization confirmed successful encapsulation of both Ad-GFP and LV-mCherry within EDM, as evidenced by the substantial increase in hydrodynamic diameter from native virus particles (~96–135 nm) to ~730–740 nm for EDM-encapsulated formulations, with concomitant shifts in zeta potential toward values characteristic of the EDM vesicles. These findings indicate effective encapsulation of viral particles. Importantly, EDM encapsulation did not compromise viral functionality; instead, it significantly enhanced in vitro transduction across multiple cancer cell lines. EDM-Ad-GFP consistently outperformed unencapsulated Ad-GFP in 4T1, MDA-MB-231, and CT26 cells at equivalent MOI, suggesting that EDM encapsulation may improve cellular interaction, uptake, or intracellular trafficking, potentially through altered surface charge, receptor engagement, or endocytic pathways.

A similar enhancement was observed for EDM-LV-mCherry, despite lentiviruses already being enveloped, indicating that EDM encapsulation provides additional benefits beyond native viral envelopes, possibly by stabilizing particles or promoting more efficient cell–virus interactions. Recent advances in cell membrane-based nanocarriers have demonstrated that biomimetic membranes derived from erythrocytes, platelets, or cancer cells can enhance nanoparticle stability, prolong circulation time, and facilitate biological interactions through the presence of native membrane proteins and lipids. Such biomimetic platforms have increasingly been explored as delivery vehicles for drugs, nanoparticles, and biologics due to their ability to mimic natural cellular interfaces and improve biological compatibility.

EDM encapsulation also conferred significant protection against neutralizing antibodies for the non-enveloped adenovirus, as demonstrated by robust transduction of HEK293 cells in the presence of diluted neutralizing serum. This result supports the hypothesis that EDM encapsulation can shield exposed viral capsid epitopes from antibody recognition, partially mimicking immune-evasive features of enveloped viruses. Immune masking strategies are increasingly recognized as an important approach in viral vector engineering, where surface shielding—through polymers, lipid coatings, or membrane cloaking—can reduce antibody recognition and complement activation while preserving infectivity. Incorporating EDM encapsulation may provide a biologically derived stealth layer that reduces immune detection while maintaining viral functionality.

In addition to immune shielding, several strategies have been explored to enhance viral transduction efficiency in gene therapy platforms, including chemical or lipid-based coating of viral particles and nanoparticle-assisted delivery systems that promote cellular uptake or protect vectors from extracellular degradation. The enhanced transduction observed with EDM-encapsulated viruses in this study suggests that membrane-based encapsulation may represent a complementary strategy for improving viral delivery efficiency.

From a translational perspective, while the current study demonstrates improved viral transduction in vitro, further investigation will be required to evaluate immunogenicity, biodistribution, pharmacokinetics, and bioavailability of EDM-coated viral particles following systemic or local administration.