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DNA tetrahedron nanoparticles service as a help carrier and adjvant of mRNA vaccine

Journal of Translational Medicine volume 22, Article number: 1024 (2024) Cite this article

Abstract

Aim of the study

To investigate the potential of DNA nanoparticles (DNPs) as carriers and adjuvants for mRNA vaccines.

Materials and methods

Customized oligonucleotides were assembled into DNA tetrahedra (DNA-TH), which were subsequently complexed with streptavidin and mRNA encoding green fluorescent protein (GFP). Various assays were conducted to evaluat the stability of the DNPs, their cellular uptake, immune activation potential, and GFP mRNA transcription efficiency. P53-mutant HSC-3 cells were used to establish a subcutaneous xenograft tumor model to explore the effects of DNPs as carriers and adjuvants in a disease model.

Results

The DNPs were remained stable extracellularly and rapidly taken up by antigen-presenting cells. Compared to naked GFP mRNA, DNPs statistically significantly activated immune responses and facilitated GFP mRNA transcription and protein expression both in vitro and in vivo. Immunization with DNP-GFP mRNA complexes induced higher antibody titers compared to naked mRNA. The DNPs demonstrated good biocompatibility. DNP-p53 inhibited the growth of subcutaneous xenograft tumors in mice with p53-mutant HSC-3 cells, outperforming both the naked p53 mRNA and blank control groups, with a statistically significant difference (P < 0.05).

Conclusion

DNA nanoparticles show promise for improving mRNA vaccine delivery and efficacy. Further optimization of these nanoparticles could lead to highly effective mRNA vaccine carriers with broad applications.

Introdution

The global COVID-19 pandemic of 2019 presented significant challenges to public health. Vaccines have emerged as crucial tools in combating viral infections, with mRNA vaccines, in particular, demonstrating exceptional effectiveness. They have demonstrated remarkable potential and promising prospects in the development of COVID-19 vaccines [1], placing the advancement of mRNA technology at the forefront of current vaccine research.

Although mRNA vaccines have proven their ability to confer rapid and safe protection against infectious diseases, further investigation is required to enhance mRNA design, delivery, and explore applications beyond prophylaxis [2]. The large size and negative charge of mRNA molecules (ranging from 104 to 106 Da) hinder their cellular uptake. Additionally, naked mRNA is prone to rapid degradation by extracellular RNA enzymes, resulting in poor stability. In vivo, mRNA is engulfed by cells of the innate immune system and degraded by nucleases. Therefore, developing carrier systems that can shield mRNA from degradation, facilitate its delivery to target cells, and enable the transcription of target proteins without inducing toxicity or unwanted immunogenicity is crucial for advancing next-generation mRNA vaccines [3].

Current research on mRNA carrier systems explores a diverse array of materials, including lipids, viruses, polymers, peptides, small molecules, metals, and more [4]. These materials exhibit varying physicochemical properties, such as nanoparticles, microparticles, conjugate structures, solid states, hydrogels, hyaluronic acid nanohydrogel [5], and others. Among these, nanoparticles (NPs) have gained prominence due to their ability to traverse physiological barriers and capillaries, enabling targeted delivery, which marks a significant advancement in recent years [6]. NPs have shown notable potential across multiple domains, including vaccine development [7], drug targeting [8], and cancer therapy [5, 9].

Within the realm of non-viral nanoplatforms for mRNA delivery, common options include lipid nanoparticles (LNPs), lipoplexes (LPX), polymer-based polyplexes, lipid shell-coated lipopolyplexes (LPPs), cationic lipid-assisted nanoparticles (CLANs), inorganic nanoparticles, and cationic nanoemulsions [10]. Each platform has its own set of advantages and limitations [11]. LNPs, extensively utilized in commercial applications, are noted for their delivery efficiency and availability. However, they also pose risks of toxicity, apoptosis, and inflammatory responses [12]. Thus, there remains a pressing need for the development of mRNA delivery strategies that are both safe and efficient, with low-toxicity.

Recently, DNA nanotechnology has emerged as a promising avenue for enhancing mRNA transfection [13], leveraging the concept of designing specific sequences to create stable geometric linkage structures within nucleotide chains [14]. Li et al. developed a thermo-responsive dynamic nanocomplex integrated with biodegradable shell polysaccharides to enhance mRNA stability, facilitating the release and translation of mRNA via endocytosis within the cytoplasm [15]. Similarly, Lv et al. designed a smart DNA-based nanosystem comprising four monomers, forming a nanoframework that efficiently promotes mRNA transfection in macrophages [16]. Despite these advances, research into DNA-based nanomaterials for mRNA delivery remains in its early stages [17].

Building upon previous work on DNA tetrahedral nanostructure [18], this study introduces a DNA nanoparticle incorporating a poly-T sequence that binds to the 3’ polyadenosine (poly(A)) tail of GFP-mRNA through complementary base pairing, termed DNP-Gm. Given the exclusive use of DNA and straptavidin, the resulting structure exhibits excellent biocompatibility. The aim of this study is to investigate whether this DNA nanoparticle can serve as a vaccine carrier/adjuvant structure, enhancing the expression of GFP-mRNA and eliciting an immune antibody response in mice.

Materials and methods

Synthesis of the double Helix DNA tetrahedron (DNA-TH)

The basic structure of the DNA tetrahedron (DNA-TH) was self-assembled following a protocol established by Yung Chung’s research group [18]. Four customized oligonucleotide strands were mixed at a molar ratio of 1:3:3:3 (L: S:M: LT) in a Tris-acetic acid-EDTA-Mg2+ (TAE/Mg2+) buffer (Table s3) to prepare stock solutions with a final concentration of 2.5 µmol/L (Table s4). The mixture was heated to 95 °C for 2 min, then gradually cooled to room temperature over 1 h, and finally stored at 4 °C. All DNA strands were procured from General Biol. The sequences of the DNA strands are provided in Table s2.

Synthesis of DNA-TH with streptavidin/GFP-encoding mRNA complex

The S strand of DNA-TH was labeled with biotin, allowing it to bind with streptavidin (SA) non-covalently. DNA-TH and SA (Thermo Scientific, 434301) were mixed at a final concentration ratio of 1:4 and incubated at room temperature for one hour to form complete DNA tetrahedron nanoparticles, referred to as DNP (Figure s1). DNP was then incubated with GFP mRNA (Gm, Thermo Scientific, 88880) at 40 °C for 10 min, followed by storage at 4 °C overnight. The final concentration of GFP mRNA after incubation with DNP is 625 nM (10×).

Gel electrophoresis

The assembled DNA structure was characterized using 3.5% non-denaturing polyacrylamide gel electrophoresis (PAGE) at 37 °C. After electrophoresis for two hours, the gel was stained with ethidium bromide (EB) solution, and images were captured using a BIO-RAD gel imager.

Dynamic Light Scattering (DLS)

The hydrodynamic diameter of the samples was measured using a Zetasizer Nano ZS90 unit (Malvern Instruments, Malvern, UK).

Fluorescence Resonance Energy Transfer (FRET) assay

YOYO-1 (Invitrogen, Y3601) and Alexa Fluor 546 (AF546) were selected as a FRET-pair. YOYO-1, capable of binding to DNA double strands, served as the donor moiety, while AF546-labeled SA (Life Technologies, S11225) functioned as the acceptor. The emission spectrum of YOYO-1 overlaps with the excitation spectrum of AF546, as shown in Figure s2A. The excitation efficiency of YOYO-1 for AF546 is highest at 547 nm, fulfilling the fundamental conditions for FRET. DNase-1 at a final concentration of 2 U/mL was used for treatment. Changes in fluorescence spectra were recorded every 5 min at 37 °C.

In vitro, FRET efficiency was quantified by defining the ratio of the fluorescence intensity of AF546 at 570 nm to the fluorescence intensity of the donor group YOYO-1 at 510 nm (Acceptor/Donor ratio). The FRET efficiency at each time point was normalized by the calculated efficiency value when the DNP was intact (Figure s2B).

In vivo FRET efficiency was measured using flow cytometry. The FRET efficiency was expressed as the difference between the fluorescence intensity of F-DNP at FL-2 and the background value, which was the fluorescence intensity of AF546-DNP at FL-2. The FRET efficiency at each assay time point was normalized by the starting time point value of one efficiency.

Cell culture

RAW264.7 cells were obtained from the American Type Culture Collection (ATCC), while DC2.4 cells were obtained from Applied Biosystems. Dulbecco’s Modified Eagle’s Medium (DMEM, ATCC), Roswell Park Memorial Institute Medium (RPMI, ATCC), Fetal Bovine Serum (FBS, Gibco), 0.25% Trypsin-EDTA (Gibco, catalog number: 25200-056), and Penicillin-Streptomycin Solution, 100x (Thermo-Fisher Scientific) were used for cell culture.

RAW264.7 cells were maintained in DMEM supplemented with 10% FBS and 1% Penicillin-Streptomycin Solution (P/S). DC2.4 cells were cultured in RPMI supplemented with 10% FBS and 1% P/S. Cells were grown in complete growth medium using 10 cm cell culture dishes or cell culture flasks within a humidified CO2 incubator (95% air, 5% CO2, 37 °C).

Flow cytometry analysis of antigen-presenting cells (APCs) cellular uptake and intracellular stability

1 × 105 cells were seeded in triplicate in a 24-well plate in 500 µl of complete growth medium. Cells were allowed to adhere and rest overnight. The next morning, the medium was removed and cells were washed with 1×PBS. Cells were then stimulated with different samples in an incubator at 37 °C and 5% CO2. After stimulation at designated time points, the medium was aspirated, And cells were washed twice with 1 ml of 1×PBS. Cells were harvested by scraping and centrifuge for 3 min. The cell pellet was washed with 1×PBS twice, resuspended in 500 µl cold 1×PBS, and kept on ice. Fluorescence of cells was analyzed using flow cytometry. Histograms and corresponding mean fluorescence intensities (MFI) were compared to assess cellular uptake and intracellular stability.

Fluorescence microscopy to detect uptake of APCs

DC2.4 cells were harvested during the logarithmic growth phase and washed with 1×PBS. The cell density was then was adjusted to 1 × 105 cells/mL to create a cell suspension. 500 µL of this suspension was added to each well of a 24-well plate. The cells were cultured until adherence. A blank control group and an experimental group were set up, with three replicates in each group. Experimental reagents were added to the cell culture medium containing magnesium ions (final concentration: 12.5 mM). Cells were incubated for different time points, and images were captured using a fluorescence microscope to visualize the uptake of experimental reagents. The extent of uptake by the APCs was analyzed.

Flow cytometry analysis of APCs activation

5 × 105 cells were seeded in triplicate in a 24-well plate in 500 µl of complete growth medium. Cells were allowed to adhere and rest overnight. The next morning, the medium was removed, and cells were washed with 1×PBS. 500 µl of complete growth medium containing Mg2+ was added to the wells. Different stimulation buffers were added to achieve a final concentration of SA of 50 nM in each group. An equal volume of 1×TAE/Mg2+ buffer was added as a negative control, while lipopolysaccharide (LPS) at a final concentration of 1 µg/ml was used as a positive control. After stimulation, DC2.4 cells underwent cell surface cluster of differentiation (CD) 40 staining using PE-anti CD40 antibody. Fluorescence intensity was detected using flow cytometry, and the fluorescence signal intensity of the FL-2 channel was recorded. The negative control group’s fluorescence intensity was set to one, and the fold increase of the other groups relative to the negative control was calculated.

Small animal imaging

Eight weeks old male BALB/c JGpt mice were used in the experiment. The experimental group received injections of DNP-Gm, while the control group received injections of naked GFP mRNA (naked-Gm). Each mouse was injected with 20 µl of sample to ensure a consistent dose of GFP mRNA at a final concentration of 62.5 nmol/L. The blank control group received buffer-only injections. Each group consisted of one mouse. The IVIS In Vivo Imaging System (PerkinElmer, IVIS Lumina III) was used to detect GFP mRNA transcription in vivo. Following tail vein injection, mice were anesthetized before imaging. Images were captured at different time points. Successful transcription of GFP mRNA was indicated by detectable green fluorescence. The IVIS In Vivo Imaging System software recorded the fluorescence intensity at each time point. Results were adjusted to ensure that the fluorescence of the blank group was set to zero, minimizing interference from intrinsic fluorescence. The experiment was conducted three times.

Immunization protocol

Eight-week-old male BALB/c JGpt mice were used in this study. The experimental group received injections of DNP-Gm, the control group received injections of naked-Gm, and the blank control group received buffer-only injections. Each group consisted of three mice, and the experiment was replicated three times. Each mouse received a 20 µl injection to ensure uniform dosing of GFP mRNA at a final concentration of 62.5 nmol/L. Injections were administered via the tail vein on days 1, 27, and 42.

Starting from the first immunization, 200 µl of blood was collected from the cheek of each mouse every seven days. A portion of the blood was used for blood marker testing, while the remaining portion was centrifuged to isolate serum for subsequent experiments, as shown in Fig. 4Ca. These subsequent experiments included the detection of serum-related indices and the assessment of serum GFP antibody levels.

Enzyme-linked immuno sorbent assay (ELISA) to detect mouse serum anti-GFP antibody titers

Serum GFP antibodies in immunized mice were detected using the Mouse GFP ELISA KIT (96 Test, ZC-38490). Experimental procedures followed the manufacturer’s instructions (Method s4).

HSC-3 xenograft mouse model

BALB/c mice were randomly assigned to groups via a block randomization method, and the dorsal skin was prepared. A total of 1 × 106 p53-mutated HSC-3 cells (human oral squamous cell carcinoma cell lines) were resuspended in 0.1 ml of serum-free culture medium and injected subcutaneously into the backs of the mice. Wild-type p53 mRNA served as the target mRNA, and DNP-p53 particles (DNP conjugated to p53 mRNA) were constructed following the previously described methodology for DNP-Gm. The experimental groups included a DNP-p53 group and a Naked-p53 mRNA (Naked-p53mR) group, with each mouse receiving an equal dose of p53 mRNA via intravenous injection into the tail vein. The control group received 0.1 ml of serum-free culture medium. Tumor size was measured every three days, with tumor volume calculated as length × width × height (mm³). On day 36 post-tumor cell injection, mice were euthanized using carbon dioxide, and dorsal tumor tissue was isolated.

Immunohistochemistry staining (IHC)

Tumor tissue was fixed in paraffin and sectioned to a thickness of 5 μm. The sections were deparaffinized and rehydrated, and antigen retrieval was conducted using pH 6.0 citrate buffer. Sections were incubated with anti-proliferating cell nuclear antigen (PCNA) antibody (Abcam, ab92552), diluted 1:1000, at 4 °C for 24 h, followed by incubation with a secondary antibody at room temperature for 1 h. Visualization was achieved using DAB substrate solution. Positive cells were counted microscopically, recording the number of PCNA-positive cells per hundred cells.

Results

Synthesis and in vitro stability of DNP

The study synthesized a DNA nanostructure (DNP) consisting of DNA and streptavidin (SA) protein. Initially, DNA tetrahedron (DNA-TH) was synthesized (Table s2) and then combined with SA via biotin to form DNP (Figs. 1A, s1). Continuous electrophoresis at 37 °C indicated successful linkage of SA to the DNA scaffold, as evidenced by the thickening band (between 800 and 900 bp) on the PAGE gel (Fig. 1B). Dynamic Light Scattering (DLS) revealed an average DNP size of 20–30 nm (Fig. 1C).

FRET assay monitored DNP integrity, using YOYO-1 and AF546 as donor and acceptor, respectively (Figure s2A). The fully synthesized fluorescence-labeled DNP, denoted as YO&AF-DNP, was subjected to analysis. Fluorescence intensity changes upon DNAase-1 treatment indicated DNP disintegration (Figs. 1D, s2B).

Magnesium ion concentration was crucial for DNP stability, with 12.5 mM Mg2 + required for maintaining structure (Fig. 1E). Treatment with splenocyte lysate, saliva, and peritoneal extracts led to gradual DNP disintegration (Fig. 1F). Methods for preparing splenocyte lysate, saliva, and peritoneal extracts are detailed in the Supplemental Material Method S1-3.

Fig. 1
figure 1

Synthesis and in vitro characterization of DNP. (A) Structural diagram of DNP. (B) PAGE gel of DNA-TH and DNP. (C) DLS size determination of DNP. SA was a control. (D) FRET assay showing DNP stability. Fluorescence intensity of AF546 at 570 nm gradually decreased, indicating the decomposition of DNA structure. (E) Impact of magnesium ion concentration on DNP stability. (F) Stability in body fluids and lysates

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APCs uptake and intracellular stability of DNP

The uptake of AF488-labeled DNP (AF488SA-DNP) by DC2.4 cells was rapid, with significant uptake observed within five minutes, compared to minimal uptake of free AF488-SA and partial uptake of AF488SA-CpG (Fig. 2A). After one hour, free SA was not internalized, while AF488SA-CpG uptake increased but remained lower than DNP uptake (Fig. 2B).

Intracellular DNP stability was assessed using FRET, showing degradation within approximately 1.5 h post-internalization, with a 50% decrease in FRET efficiency in RAW246.7 and DC2.4 cells (Fig. 2C and E).

DNP activation of immune co-stimulatory molecule CD40 expression on the surface of APCs

After stimulating DC2.4 cells with various DNA structures, CD40 expression on the cell surface was evaluated using PE-anti-CD40 antibody staining, and fluorescence intensity was measured by flow cytometry. The fluorescence intensity of the negative control group was set as a baseline value of one. DNP significantly increased CD40 expression on DC2.4 cells, indicating enhanced activation compared to both SA and SA-CpG (Fig. 2F).

Fig. 2
figure 2

DNP uptake by antigen-presenting cells, intracellular stability of DNP, and stimulatory effect on DC2.4 cells. (A) Flow cytometry analysis of SA, SA-CpG, and DNP uptake by DC2.4 cells. The DNP (red line) shows rapid uptake within 5 min, with higher uptake levels compared to other structures. (B) Mean fluorescence intensity (MFI) and percentage of positive cells (per 10,000 cells) in the flow assay. Both the MFI and the percentage of positive cells for DNP are significantly higher than those observed for other groups (***: P < 0.01). (C and D) Flow-validated FRET to evaluate the stability of DNP within cells. The FL-1 channel detects fluorescent signals in the range of 515 to 545 nm, while FL-2 detects signals in the range of 564 to 606 nm. FRET occurs when both fluorophores are present, resulting in increased fluorescence intensity in FL-2. (E) Decay of FRET efficiency over time when in DC2.4 and RAW264.7 cells after DNP uptake. FRET efficiency decreases to 50% of its initial value at approximately 50 min in RAW264.7 cells and around 70 min in DC2.4 cells. (F) Activation of DC2.4 cells following stimulation with different DNA structures. Compared to the SA and SA-CpG groups, DNP significantly enhances the activation of DC2.4 cells (***: P < 0.01)

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Intracellular transcription of GFP mRNA and expression of GFP protein

The poly-T sequence of the LT strand was utilized to bind with the 3’ Poly (A) tail of the mRNA, forming DNP-Gm (Fig. 3A). DC2.4 cells were co-cultured with either DNP-Gm or naked-Gm (control group). GFP fluorescence was detected after 30 min, indicating successful mRNA transcription and subsequent protein synthesis. The fluorescence intensity continued to increase over 12 h, with DNP-Gm showing higher GFP fluorescence compared to naked-Gm, as confirmed by ImageJ analysis and flow cytometry (Fig. 3B and D).

Fig. 3
figure 3

(A) Schematic illustration showing the synthesis of DNP-Gm, which combines DNP and GFP mRNA. (B) After 12 h of co-incubation with DC2.4 cells, DNP-Gm demonstrated stronger GFP fluorescence intensity compared to control naked-Gm group. (C) ImageJ analysis quantified the fluorescence intensity in images, revealing a statistically significant difference (***: p < 0.01). (D) Flow assay results depicting the percentage of GFP positive cells (per 10,000 cells) also showed a statistically significant difference(***: p < 0.01)

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In vivo transcription of GFP mRNA

To investigate the transcription of GFP mRNA in vivo, Cy-7 labeled SA was used to synthesize DNP, serving as a trace marker. Following the injection of samples into the tail vein, GFP fluorescence intensity was monitored using a small animal imaging system. Fluorescence data from the blank group was set to zero to minimize interference from the animals’ intrinsic fluorescence (Fig. 4A). This standardization allowed for more accurate measurement and comparison of the fluorescence signals among experimental groups.

Two hours post-injection, GFP fluorescence was detected in the DNP-Gm group, indicating successful in vivo transcription of GFP protein. In contrast, no GFP fluorescence was observed in the naked-Gm group at this time point. Over time, GFP fluorescence intensity gradually increased in both groups, with the DNP-Gm group showing significantly higher intensity compared to both the naked-Gm and blank groups (Fig. 4A). The difference in GFP fluorescence intensity between the DNP-Gm group and the other groups was statistically significant (Fig. 4B).

After 12 h, the mice were euthanized, and lymph nodes (LNs) were extracted for further analysis. LNs from the DNP-Gm group showed higher GFP fluorescence intensity, indicative of greater GFP transcription, compared to the naked-Gm group (Fig. 4D). GFP fluorescence intensity was quantified using ImageJ software on five 20-fold magnified images of lymph nodes from each group (Figure s3A). The difference in fluorescence intensity was found to be statistically significant (Figure s3B).

Inmunization efficiency

To evaluate the immunization efficacy of DNP-Gm in stimulating GFP antibodies production, mice were immunized on days 1, 27, and 42. Blood samples were collected every 7 days starting from the first immunization, and serum was isolated for analysis (Fig. 4Ca). ELISA analysis of the serum revealed that on day 21, the serum GFP antibody titer in the DNP-Gm group reached its peaked, showing a statistically significant difference compared to the control group receiving naked-Gm (P < 0.01). By day 28, the GFP antibody titer began to decline, although it remained significantly higher than that of the control group (P < 0.05). Over time, the serum GFP antibody titer in the DNP-Gm group continued to decrease, with no statistically significant difference compared to the naked-Gm group by the end of the study (Fig. 4Cb).

Fig. 4
figure 4

(A) In vivo transcription of GFP mRNA: GFP fluorescence was detected two hours after injection of DNP-Gm into the tail vein of mice. No GFP fluorescence was observed in the control group injected with naked-Gm. The fluorescence intensity of the blank group was adjusted to zero to minimize interference from intrinsic animal fluorescence. (B) Fluorescence intensity was recorded at each time point using small animal imaging system software. The GFP fluorescence intensity in the DNP-Gm group was significantly higher than that in the control group, with a statistically significant difference (***: p < 0.01). (C) LNs were extracted from mice after 12 h. Cy7-SA fluorescence representing DNP. GFP fluorescence intensity were statistically significantly higher in the DNP-Gm group compared to the naked-Gm group. (D) (a) Immunization strategy. (b) Serum GFP antibody titer: On day 21, the serum GFP antibody titer in the DNP-Gm group reached its peak, significantly higher than in the control naked-Gm group (***: p < 0.01). The difference remained statistically significant on day 28 (*: p < 0.05). Subsequently, the serum GFP antibody titer in the DNP-Gm group gradually decreased and was not statistically different from the control group

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Safety evaluation

We conducted a safety assessment to evaluate the potential adverse effects of DNP-Gm. Serum biochemical analysis revealed no significant differences in key indicators, including blood glucose level, serum alanine aminotransferase (ALT), serum aspartate aminotransferase (AST), creatinine (Cr), and blood urea nitrogen (BUN), between the DNP-Gm treated group and the control group. Statistical analysis indicated no significant disparity in the test results (Table s5).

Furthermore, we performed a morphological analysis of major organs (lung, liver, brain, spleen, kidney, and heart) using hematoxylin and eosin (H&E) staining (Fig. 5A). The analysis showed normal cell morphology and intact cell nuclei in these organs. In comparison to the control group, there was no significant difference in tissue necrosis and morphology in the DNP-Gm treated mice, demonstrating the biocompatibility of DNP with major peripheral organs.

DNP-p53 inhibits growth of subcutaneous xenograft tumors in mice with p53-mutant HSC-3 cells

To evaluate the efficacy of DNPs as mRNA carriers and adjuvants, we targeted p53 mRNA and established a subcutaneous xenograft tumor model in mice. Isolated tumor tissues are shown in Fig. 5B. Following the injection of DNP-p53, the inhibitory effect on tumor growth was greater than that observed with naked p53 mRNA, leading to a significant reduction in tumor volume compared to the control group (P < 0.05) (Fig. 5C). In the analyzed tumor tissues, the expression rate of PCNA-positive cells in the DNP-p53 group was lower than in both the naked p53 mRNA and blank control groups (Fig. 5D), with this difference also being statistically significant (P < 0.05).

Fig. 5
figure 5

(A) Hematoxylin and eosin (H&E) staining for morphological analysis of major organs, including the lung, liver, brain, spleen, kidney, and heart. (B) DNP-p53 inhibits the growth of subcutaneous xenograft tumors derived from mutant-p53 HSC-3 cells, resulting in a significant reduction in tumor size compared to both naked p53 mRNA and blank control groups. The separated tumor tissues were subjected to PCNA staining, where yellow coloration indicates the presence of PCNA-positive cells. (C) Tumor volume was measured every three days, demonstrating that tumors in the DNP-p53 group were smaller than those in the naked mRNA and blank control groups, with the difference being statistically significant (*: P < 0.05). (D) The expression rate of PCNA-positive cells in the tumor tissues of the DNP-p53 group was lower than that in the naked mRNA and blank control groups, with this difference also being statistically significant (*: P < 0.05)

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Discussion

With the promising potential of mRNA vaccines, researchers has shifted from merely synthesizing targeted mRNA to developing specific packaging and delivery systems. Key factors in this development include the choice of carrier materials, their chemical structure, physicochemical propertie, and application methods [2].

Choice of carrier material

In this study, DNA was selected as the primary biological material for constructing carriers/adjuvants for mRNA vaccines. Although DNA is a fundamental component of the human body, it was previously overlooked as a vaccine carrier due to its susceptibility to degradation by endogenous nucleases and limited cellular uptake. Recent advancements in nucleotide chain design have led to the creation of various DNA nanostructures with controllable size, shape, and spatial addressing capabilities, including DNA origami [14]. These advancements offer a functional platform for its biological applications [19].

DNA possesses inherent advantages as a synthetic vaccine carrier, including excellent biocompatibility, small size, simple structure, modifiability, and programmable design. Various DNA nanoparticles have been explored in preclinical research as delivery systems for chemotherapy drugs and nucleic acid drugs [20]. Leveraging DNA nanotechnology, we introduced sequences complementary to the mRNA 3’ poly(A) tail and successfully synthesized DNPs for constructing mRNA vaccines targeting GFP as the reporting protein.

The DNPs were synthesized using only DNA and streptavidin protein, demonstrating good biocompatibility and safety without the need for additional synthetic materials. For safety considerations, we aimed for the nanostructure to degrade after entering the body. Stability studies revealed that DNPs can degrade within a limited time after cellular entry, similar to mRNA, which undergoes transient expression and enzyme-mediated degradation. This property ensures the safety profile of mRNA for therapeutic use. The combination of these advantages enhances the biocompatibility and safety of synthetic vaccines. Compared to the issues associated with prolonged circulation and accumulation of lipid nanoparticles (LNPs) materials [6], DNA materials present a promising alternative for mRNA vaccine carriers.

Structural design and physicochemical properties of carriers

Previous strategies for carrier design focused on encapsulating mRNA molecules within nanoparticles to protect them from degradation by RNA enzymes [21]. For example, LNPs have been used to achieve GFP mRNA transcription and GFP protein expression in mice, including in skin, spleen, and other organs after subcutaneous injection [22], as well as in retinal tissue after intravitreal injection [23]. While effective, this approach presents the challenge of releasing mRNA molecules from the nanoparticles [24]. In our study, the DNPs were designed to target the 3’ poly(A) tail of mRNA at the structural level rather than by encapsulating the mRNA. The Yang group has reported similar designs, where nanoparticles capture mRNA through base pairing of poly-T sequences with poly(A) tails below the lower critical solution temperature (LCST) of 4 °C [16]. This approach may represent a new direction for developing mRNA carriers.

The length of the 3’ poly(A) tail indirectly regulates mRNA translation and half-life. Previous research has shown that modifications to the poly(A) tail [25], such as direct nucleotide modification or incorporation of non-A residues, can enhance mRNA stability and translation [26]. In our study, DNPs binds to the 3’ poly(A) tail via poly-T sequences, and the resulting three-dimensional structure may mimic the poly(A) binding protein (PABP) mechanism, affecting mRNA stability [27].

The DNPs are simple and small, with sizes ranging from 20 to 30 nm. This makes them significantly smaller than LNP-based carrier structures [28] and smaller than DNA nanoparticles with additional components [29, 30]. The small diameter of the DNPs also provide good dispersibility [18], potentially influencing the uniformity and stability of mRNA vaccine synthesis [31]. Further studies should explore the stability of mRNA after binding with DNPs.

The nanoparticle structure must exhibit in vitro stability to maintain its integrity before being taken up by APCs. Our results indicate that DNPs remain stable for at least two hours under 37 °C electrophoretic conditions, reflecting their relative in vitro stability. This stability is closely correlated with magnesium ions concentration [29]. Our research findings also confirm that, compared to naked-Gm, DNP-Gm offers better mRNA transfection efficiency and higher GFP expression in DC2.4 cells. However, further comparative studies between DNP and LNP structures in terms of kinetics and metabolism are needed to explore their relative effectiveness and design strategies.

Effects of immune response

The DNPs structure integrate CpG oligo-deoxynucleotides (ODN) sequences (mouse-specific ODN-1826) with adjuvant effects, representing a CpG motif common in bacteria or viruses [32]. These motifs are recognized by toll-like receptors (TLRs) 9, initiating intracellular signal cascades that upregulate pro-inflammatory cytokines, chemokines, and co-stimulatory molecules [33]. TLR9 is abundantly expressed in various human immune cells, including dendritic cells (DCs), monocytes, and B cells [34]. The incorporation of CpG adjuvants with nucleic acid nanodevices enhances CpG stability in endolysosomes and improves immunological performance [35].

Our results indicate that mice in the DNP-Gm group exhibit favorable short-term immune responses, with higher titers during early stages of immunity. However, this effect was not sustained long-term, with no increase in antibody titers following re-immunization. Several mechanisms may contribute to this observation. First, the natural decline of the immune system results in a transient peak in antibody titers post-immunization due to effector B cell activation and proliferation, followed by a decrease as effector B cells diminish [36]. Second, antigen clearance may be a contributing factor. As the GFP protein is eliminated, immune stimulation decreases, leading to reduced antibody production. The maintenance of memory B cells generally necessitates either continuous antigen presence or periodic antigen exposure. Therefore, rapid antigen clearance could potentially affect the stability and longevity of memory B cells [37]. Third, antibody-dependent negative feedback could inhibit further B cell activation and antibody production to prevent excessive immune responses [36]. Further studies are needed to optimize mRNA vaccine design and introducing auxiliary ligands to promote memory T/B cells formation.

DNA-based nanomaterials in vaccine design applications

Vaccines based on antigen proteins or pathogen/tumor epitopes offer advantages in immunological precision and safety [38]. In recent decades, DNA nanomaterials have achieved significant advancements in vaccine development, particularly for tumor therapy. Researchers have developed various immunotherapies targeting distinct components and processes within the cancer immune cycle, leading to transformative changes in the treatment of neoplastic diseases [39]. Tumor vaccines, as a form of immunotherapy, work by introducing specific tumor antigens, which induces the death of effector T cells and the generation of tumor-specific memory T cells. This process leverages the body’s innate immune system to suppress or eliminate tumors [40].

Encouragingly, several DNA nanomaterial-based immunotherapeutic platforms have demonstrated promising potential in preclinical studies, offering alternative approaches to enhance antitumor immune responses and sensitize tumors to immunotherapies safely and effectively [20]. A common design strategy involves using DNA nanostructures to protect tumor antigens and act as immune adjuvants. For example, Liu et al. assembled two types of molecular adjuvants and an antigen peptide within a tubular DNA origami structure. pH-responsive DNA ‘locking strands’ positioned outside the nanostructures facilitate the release of the vaccine within lysosomes of antigen-presenting cells, exposing the adjuvants and antigens to elicit a robust immune response [41]. These innovations highlight the advantages of programmable DNA design and may suggest unique developmental directions for DNA-based systems compared to other carrier/adjuvant structures.

In this study, GFP transcribed from GFP mRNA was utilized as a model antigen to evaluate the potential of DNPs as vectors and adjuvants for mRNA vaccines. However, the efficacy of DNPs may vary in actual disease contexts. To investigate the effectiveness of DNPs in tumor therapy, we established a mouse xenograft model using p53-mutated HSC3 cells. The results indicated that DNP-p53, compared to naked mRNA, effectively slowed tumor growth, suggesting potential applications of DNPs in disease models. Nonetheless, this verification is in its early stages, and further research is needed to elucidate the specific mechanisms underlying tumor growth inhibition. In a p53-null hepatocellular carcinoma model, the use of a p53 mRNA vaccine to restore p53 protein expression has been shown to improve the immune microenvironment and achieve enhanced anti-tumor effects [42].

Additionally, parallel comparisons with other widely studied carrier systems, such as lipid nanoparticles (LNPs), are essential. As the most extensively studied carriers for mRNA vaccines, LNPs have undergone various modifications [43] and structural improvements [44] to enhance delivery efficiency. Insights from LNPs development can significantly inform and refine the design of DNA-based nanomaterials.

Conclusion

DNA nanoparticles demonstrate potential for improving mRNA vaccine delivery and efficacy. Their ability to efficiently deliver mRNA, activate immune responses, and induce antigen-specific antibody production suggests their promise in the development of mRNA vaccine. Further optimization of these nanoparticles could lead to highly effective mRNA vaccine carriers with broad applications.

Data availability

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

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Acknowledgements

We express our gratitude to Dr. Xiaowei Liu for generously sharing insights into the design and assembly of tetrahedral DNA. We also thank Prof. Yung Chang from Arizona State University for providing valuable technical suggestions. Furthermore, we also acknowledge the guidance and support of Dean Qianming Chen from Zhejiang University School of Stomatology and Prof. Xin Zeng from the West China School/Hospital of Stomatology of Sichuan University for their invaluable contributions to this project.

Funding

This project supported by Sichuan Science and Technology Program, Natural Science Foundation of Sichuan Province, China (Grant No. 2024NSFSC1881) and the Talent Youth Foundation of the Sichuan Academy of Medical Sciences & Sichuan Provincial People’s Hospital (Grant No. 2021QN06, awarded to Lili Wang).

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Author notes

  1. Henglang Liu and Xianxian Li contributed equally to this work and should be considered co-first authors.

Authors and Affiliations

  1. Department of Stomatology, Sichuan Provincial People’s Hospital, University of Electronic Science and Technology of China, Chengdu, Sichuan, China

    Henglang Liu, Xianxian Li, Jing Yang, Qun Lu & Lili Wang

  2. School of Medicine, University of Electronic Science and Technology of China, Chengdu, Sichuan, China

    Ruike Yan & Lili Wang

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  1. Henglang Liu
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Contributions

HL: Writing-original draft, Project administrartion. XL: Project administration, Methodology. RY: Formal analysis, Visualization. JY: Investigation. QL: Validation, Date curation. LW: Writing-review & editing, Supervision.

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Correspondence to Lili Wang.

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Liu, H., Li, X., Yan, R. et al. DNA tetrahedron nanoparticles service as a help carrier and adjvant of mRNA vaccine. J Transl Med 22, 1024 (2024). https://doi.org/10.1186/s12967-024-05837-w

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