Bioengineered nanotechnology for nucleic acid delivery

Abstract

Nucleic acid-based therapy has emerged as a promising therapeutic approach for treating various diseases, such as genetic disorders, cancers, and viral infections. Diverse nucleic acid delivery systems have been reported, and some, including lipid nanoparticles, have exhibited clinical success. In parallel, bioengineered nucleic acid delivery nanocarriers have also gained significant attention due to their flexible functional design and excellent biocompatibility. In this review, we summarize recent advances in bioengineered nucleic acid delivery nanocarriers, focusing on exosomes, cell membrane-derived nanovesicles, protein nanocages, and virus-like particles. We highlight their unique features, advantages for nucleic acid delivery, and biomedical applications. Furthermore, we discuss the challenges that bioengineered nanocarriers face towards clinical translation and the possible avenues for their further development. This review ultimately underlines the potential of bioengineered nanotechnology for the advancement of nucleic acid therapy.

1. Introduction

Gene therapy is based on the use of nucleic acids, including DNA, small RNAs (e.g., siRNA and miRNA), mRNA, CRISPR gene editing tools, antisense oligonucleotides (ASO), among others [1–5]. The structures and properties of the nucleic acids applied for the upregulation, disruption, or editing of genes typically differ. For example, gene upregulation often uses exogenous mRNA or DNA, while disruption typically employs RNA interference with siRNAs or shRNAs. For gene editing, tools like CRISPR-Cas systems rely on guide RNAs tailored to specific genomic sequences [6,7]. Gene therapy thus holds immense promise for the treatment of a multitude of diseases, such as genetic disorders, metabolic diseases, infection diseases, and cancers [8–10], by introducing, replacing, correcting, or silencing specific genes within an individual’s cells. This therapy is centered around altering the physiological processes by directly manipulating genes, rather than by intervening in biochemical reactions to induce pathological changes as many traditional drugs do.

The success of gene therapy largely hinges on the effective delivery of therapeutic nucleic acids to target cells, a task that is fraught with numerous challenges [11–14]. The obstacles to nucleic acid delivery in vivo include: 1) extracellular barriers such as extracellular matrix adsorption, clearance by mononuclear phagocyte system and renal system, and nuclease degradation; 2) difficulties in cellular uptake; and 3) collection of the genes by endosomes/lysosomes upon entering the cell. Furthermore, maintaining the integrity of the genetic material during transit, targeting specific cells or tissues, circumventing immune responses, and ensuring safety and minimal side effects are also barriers for efficient gene therapy [15–17]. Therefore, finding a suitable vehicle to overcome the in vivo barriers of nucleic acid delivery is a pressing task that must be accomplished to achieve gene safety, high efficiency, and controllable expression.

The gene delivery vehicles currently used mainly include viral vectors and non-viral vectors. Viral vectors, such as retrovirus [18], lenti-virus [19], and adenovirus [20,21], have high transfection efficiency, and have shown promise in nucleic acid drug delivery. For example, Gendicine was successfully approved in 2003 to treat certain types of squamous cell carcinoma of the head and neck [22]. Subsequently, the US Food and Drug Administration (FDA) has approved quite a few viral gene therapy drugs, such as Glybera for lipoprotein lipase deficiency, Strimvelis for severe combined immunodeficiency, and more recently Roctavian and Hemgenix for hemophilia [23–25]. However, viral gene therapy may potentially cause adverse reactions such as immune response and potential genome mutagenesis [26]. Additionally, the amount of transferred genes may be limited, up to now, as only a maximum of 40 kb gene fragments can be delivered [27]. In parallel, non-viral vectors, particularly nanoparticles, have been widely used in the field of nucleic acid delivery due to their relatively safe, easily adjustable physical and chemical properties, mass production, low cost, and large load capacity. The first clinically approved siRNA nanomedicine (Onpattro) uses lipid nanoparticles to efficiently deliver siRNA to the liver. Lipid nanoparticle-based mRNA vaccines [28,29] have also been clinically approved for COVID-19 (Comirnaty and Spikevax) [30,31], and are in clinical trials for other infection diseases, cancer, genetic disorders, and others.

Bioengineered nanotechnology has recently emerged for promising non-viral delivery of nucleic acids. Bioengineered nanocarriers, such as exosomes [32,33], cell membrane-derived nanovesicles [34], protein nanocages [35], and virus-like particles [36], emulate nature’s strategies for packaging and delivering genetic materials. By harnessing the intrinsic design principles of biological entities, these bioengineered nanocarriers aim to provide efficient, targeted, and safe delivery of therapeutic genes. In addition, these nanocarriers span a diverse range of structures and mechanisms [37,38], taking inspiration from biological entities, such as viruses, bacteria, and cells themselves. Exosomes, a kind of extracellular vesicles, capitalize on natural intercellular communication mechanisms to deliver genetic materials [39]. Currently, four exosomes products have entered the clinical stage [40]. For example, in a clinical trial (NCT03608631), mesenchymal stem cell exosomes containing siRNA targeting the KrasG12D mutant gene are being used to treat pancreatic cancer [41]. Another clinical research (NCT03384433) is exploring the therapeutic effect of mesenchymal stem cell exosomes carrying miR-124 on ischemic heart disease [42]. Cell membrane-derived nanovesicles are nanocarriers composed only of cell membrane components, without any cell contents [43,44]. Compared to exosomes, they are easier to obtain and feature favorable biocompatibility, controllable size, and high drug-loading capacity. Currently, by using genetic engineering techniques to express functional ligands on the cell membrane, such as fusogen, multifunctional cell membrane nanovesicles can be prepared to achieve efficient RNA delivery [45]. Protein nanocages and virus-like particles simulate their design principles from viral capsids, providing a protective shell for genetic material and facilitating efficient entry into target cells. For instance, the Selective Endogenous eNcapsidation for cellular Delivery (SEND) system, which was inspired by the mammalian retrovirus-like protein PEG10, has been developed to deliver mRNA and the CRISPR-Cas9 gene editing system [46]. Each of these systems offers unique advantages and presents its own set of challenges, providing a rich landscape for exploration and innovation. Specifically, bioengineered nanotechnology can further endow these biomimetic delivery nanocarriers with multifunctionality [47–49], enhancing their nucleic acid loading rate, lesion targeting specificity, and controllability. For instance, the efficiency of targeting liver cancer greatly improves when the NGR peptide is displayed on the surface of red blood cell membranes [50]; similarly, the expression of positively charged peptides onto the interior surface of ferritin nanocage increases the nucleic acid loading rate, resulting in more effective immune activation [51]; moreover, through genetic engineering modifications, Photorhabdus asymbiotica-derived contractile injection systems can achieve highly efficient loading of various nucleic acids [52]. Therefore, bioengineered nanotechnology can effectively improve nanocarriers, transforming them into novel nucleic acid delivery systems with high efficiency and biosafety.

In this review, we focus on this unique type of bioengineered nanocarriers for nucleic acid delivery, which mainly includes exosomes, cell membrane-derived nanovesicles, protein nanocages, and virus-like particles. We present a thorough overview of their unique features, loading methods, advantages for nucleic acid delivery, and biomedical applications with various nucleic acids (DNA, mRNA, miRNA, siRNA, ASO, and CRISPR tools) (Fig. 1). We further discuss the challenges associated with these bioengineered nanocarriers regarding clinical translation and our perspectives for their further development. We expect that this comprehensive review article, the first of its kind, will provide new insights into the development of a new generation of nucleic acid therapy for more effective and safer disease treatment and prevention.

Fig. 1.

Schematic representation of bioengineered nanotechnology for nucleic acid delivery. Natural nanocarriers, such as exosomes, cell membrane-derived nanovesicles, protein nanocages, and virus-like particles, could be engineered into multifunctional vehicles capable of efficiently encapsulating nucleic acids and protecting them from degradation, while also exhibiting specific targeting ability, high biocompatibility, prolonged circulation, good biosafety, and scalability. Created with BioRender.

2. Exosomes for nucleic acid delivery

Exosomes are a specific type of extracellular vesicles, with diameters ranging from 30 to 150 nm, released from cells after the fusion of multivesicular bodies with the cellular membrane [53]. They possess a bilayer lipid structure and have been demonstrated to contain a variety of different RNA molecules, including miRNA, sncRNA, and mRNA. This implicates their pivotal roles in numerous physiological processes, including immune responses, cell proliferation and differentiation, and tissue repair. Moreover, exosomes have been found to contribute to the progression of several diseases [54,55], such as cancer, neurodegenerative diseases, and cardiovascular diseases. Virtually all types of cells, including immune cells (dendritic cells, T cells, B cells), platelets, muscle cells, neurons, tumor cells, as well as various types of epithelial and endothelial cells, can generate exosomes. Thus, exosomes possess the potential for RNA drug delivery and, due to the specific functions of exosomes derived from different cells, can be developed into personalized nucleic acid delivery vehicles.

Compared to other nucleic acid delivery platforms, exosomes present several distinct advantages [47,56–58]: 1) Specific proteins on the membrane surface bestow exosomes with homing characteristics and easy-to-modify features, enabling targeted delivery, crossing of the blood-brain barrier, and reaching poorly perfused tissues or regions; 2) Exosomes can be extracted from the patient’s cells to minimize cytotoxicity and immunogenicity; 3) They can evade phagocytosis by the mononuclear phagocyte system, allowing for prolonged in vivo retention for better therapeutic effects; 4) Functional exosomes from certain sources (with regenerative and anti-inflammatory properties) can enhance the effect of gene therapy; 5) They can form hybrid nanocarriers, such as with liposomes, for encapsulating and delivering large gene molecules such as DNA and plasmids, with low toxicity.

The objective of nucleic acid delivery is to ensure the effective transfer of genetic materials into cells and its subsequent expression, leading to the desired production of proteins or therapeutic outcomes. Ongoing efforts are dedicated to developing and refining nucleic acid delivery techniques to enhance efficiency, specificity, and safety [59,60]. The selection of delivery vectors, modifications for targeted cell recognition, and optimization of intracellular trafficking are crucial considerations in achieving successful nucleic acid delivery. Progress in nucleic acid delivery holds significant potential for diverse applications, such as gene therapy for genetic disorders [61], genetic manipulation of cells or organisms [62], and biomedical research [53]. Given these benefits, the exploration of bioengineered exosomes that carry specific genes for precise therapeutic effects through genetic engineering represents a promising avenue of investigation.

2.1. Nucleic acid loading methods for exosomes

The efficient loading of nucleic acid molecules is a fundamental requirement for the implementation of exosome-based gene therapy. However, the lipid bilayer structure of exosomes poses challenges for the loading of hydrophilic nucleic acid molecules [63]. Currently, several methods are employed for the loading of exogenous nucleic acid molecules into exosomes, including incubation, electroporation, sonication, and repeated freeze-thaw cycles.

Incubation represents a widely utilized approach for the preparation of exosome-based nucleic acid drugs [64]. During this process, purified exosomes and nucleic acid molecules are co-incubated in an appropriate buffer, typically at a temperature of 37°C. This facilitates the absorption and transportation of nucleic acid molecules by the exosomes. One notable advantage of this method is its ability to maintain the integrity of the exosomes. Moreover, certain studies have employed cholesterol modification of siRNA to enhance their efficiency in entering exosomes [65]. Another effective method involves the use of cationic liposomes for gene loading, followed by encapsulation of these liposomes into exosomes through incubation [66]. Notably, this approach has successfully enabled the loading of mRNA encoding the spike and nucleocapsid antigens of the novel coronavirus into exosomes, resulting in an antiviral immune response that persisted for at least 84 days.

Electroporation is a technique that employs transient, high-voltage pulses to disrupt the bilayer membrane structure of exosomes, creating small gaps that facilitate the entry of nucleic acid molecules [67]. Upon completion of the pulse, these gaps are automatically repaired, thereby stably encapsulating the nucleic acid molecules within the exosomes. Electroporation offers several advantages, including simplicity, speed, cost-effectiveness, and ease of control. In studies, the silencing of the mutated KRAS gene was successfully achieved by encapsulating siRNA into exosomes derived from fibroblasts, showcasing their therapeutic potential for pancreatic cancer treatment [68]. Furthermore, engineered exosomes developed using this method, adhering to Good Manufacturing Practice (GMP) specifications, have progressed to Phase 1 clinical trials (NCT03608631) [69]. However, electroporation has certain limitations. For instance, the pulses can induce nucleic acid molecule aggregation and may also compromise exosome stability [70]. Therefore, further optimization is necessary to improve the technology of electroporation for nucleic acid molecule loading.

Sonication shares similarities with electroporation as it utilizes ultrasonic waves’ mechanical and thermal effects to temporarily create micropores in the exosome membrane [71], allowing the entry of nucleic acid molecules. Research reports indicate that by optimizing sonication parameters (500 V, 2 kHz, 20% power, 6 cycles, each cycle comprising a 4-second pulse and a 2-second pause), micropores can be formed in exosomes, facilitating the entry of nucleic acid molecules [72]. Additionally, compared to electroporation, sonication can enhance the encapsulation rate of exosomal genes from 0.16% to 2.96% [73]. However, high-frequency ultrasound can damage large molecular weight nucleic acids like mRNA and DNA, reducing their activity. Consequently, further exploration is required to optimize sonication for suitable loading of nucleic acids into exosomes.

The repeated freeze-thaw method involves a rapid freezing and thawing of the mixture of exosomes and genes, disrupting the bilayer without the use of an electric field or ultrasonic wave. Through three freeze-thaw cycles, ice crystals formed during the freezing process transiently disrupt the phospholipid bilayer, allowing the introduction of genes into exosomes [74]. It has been reported that miR-140 was successfully encapsulated into exosomes using the repeated freeze-thaw method. This nucleic acid delivery system promotes chondrogenic differentiation by positively regulating the expression of chondrogenesis-related genes. Although the repeated freeze-thaw method maintains the exosome membrane structure more effectively compared to other techniques [75], the gene drugs’ activity may undergo changes during the repeated freeze-thaw process.

2.2. Exosomes based nucleic acid delivery nanocarriers

Various types of RNA, including miRNA and siRNA, as well as DNA, have been confirmed to be present in biologically derived exosomes. These exosomes can originate from various cellular sources, with nearly all cells capable of producing them. Research has revealed that exosomes derived from different cell types exhibit distinct biological functions. For example, exosomes derived from pancreatic cancer cells have been shown to prepare distant organs for metastasis by creating a pre-metastatic niche [76]. Dendritic cell-derived exosomes can carry MHC-peptide complexes and co-stimulatory molecules, stimulating T cell responses, making them potential candidates for cancer and infectious disease vaccines [77]. Additionally, exosomes derived from mesenchymal stem cells can enhance tissue repair processes and have shown promise in treating conditions like myocardial infarction and kidney injury [78]. Thus, exosomes not only serve as excellent nucleic acid delivery vehicles but can also possess specific functionalities based on their cellular origin, paving the way for their development as novel therapeutic agents.

Small RNAs are a broad class of short non-coding RNAs that are typically 20–30 nucleotides in length and play crucial roles in gene regulation at both the transcriptional and post-transcriptional levels [78]. These include microRNAs (miRNAs) and small interfering RNAs (siRNAs). They participate in diverse biological processes, including development, cell differentiation, apoptosis, and immune response, and their dysregulation is often linked to various diseases, particularly cancers. For better application, exosomes can encapsulate small RNAs, protecting them from enzymatic degradation in the extracellular environment, thereby enhancing their stability while the innate functions of exosomes could enhance delivery efficacy. Recently, researchers engineered exosomes for loading RNA by constructing fusion proteins in which the exosomal membrane protein CD9 is fused to an RNA-binding protein, and thus could bind the RNA [79]. They fused CD9 to human antigen R (HuR), an RNA-binding protein that interacts with miR-155 with relatively high affinity. In exosome-packaging cells, when miR-155 was overexpressed, the fused CD9-HuR successfully enriched miR-155 into exosomes. Furthermore, miR-155 encapsulated in exosomes could in turn be efficiently delivered into recipient cells and recognize endogenous targets. Based on this strategy, the delivery of arbitrary RNA can be achieved (Fig. 2A). The exosomes functionalized with the CD9-HuR fusion protein were characterized by the presence of inclusive markers TSG101 and CD63, and the absence of marker GM130. Morphologically, these functionalized exosomes are similar in size and shape to empty exosomes. Furthermore, the CD9-HuR functionalized exosomes effectively delivered miR-155 to the liver and downregulated the expression of miR-155 targeted SOCS1 (Fig. 2B). Therefore, the use of exosomes for small RNA delivery presents a promising avenue in the field of RNA therapeutics.

Fig. 2.

(A) Schematic showing that in packaging cells, CD9-HuR fusion protein recruits target miRNA or mRNA into exosomes through RNA-HuR recognition; released to act as functional miRNA or mRNA. (B) The CD9-HuR fusion protein functionalized exosomes were identified by the inclusive markers of TSG101 and CD63, and exclusive marker GM130. The size and shape of the functionalized exosomes were similar to that of empty exosomes. The CD9-HuR functionalized exosomes could deliver miR-155 in the liver efficiently, and down-regulate miR-155 targeting SOCS1. Reproduced with the permission from Ref. [79]. Copyright © 2019 American Chemical Society.

Therapeutic mRNA holds considerable potential for the treatment of a diverse range of diseases, encompassing genetic disorders, cancers, and infectious diseases. mRNA-based therapeutics [80], particularly mRNA vaccines developed for diseases such as COVID-19, have drawn extensive attention in recent years, signifying their immense potential for clinical applications [81]. Exosomes can encapsulate mRNA, shielding it from extracellular RNases, thereby enhancing its stability and half-life within the bloodstream and imparting specific targeting capabilities.

For instance, researchers have engineered interleukin-10 (IL-10) mRNA to respond to miR-155 through a modified Hepatitis C Virus Internal Ribosome Entry Site (HCV-IRES). Encapsulation of this engineered IL-10 mRNA within exosomes enabled efficient delivery to macrophages within atherosclerotic plaques of ApoE-deficient mice. IRES-IL-10 expression plasmids were transfected into HEK293T cells, allowing the transcribed IRES-IL-10 mRNA to be passively loaded into secreted exosomes [82]. The subsequent creation of a miR-155-activated IL-10 mRNA expression system confirmed in an ApoE-deficient mouse model that exosome-encapsulated IRES-IL-10 mRNA (Exo IRES-IL-10) can be delivered to plaques for precise control of inflammation, thereby mitigating atherosclerosis (Fig. 3). In another study [83], researchers generated exosomes rich in engineered Bone Morphogenetic Protein 2 (Bmp2) mRNA by co-transfecting 293T cells with the non-annotated P-body dissociating polypeptide (NoBody) and a Bmp2 artificial plasmid. Loading GelMA with an appropriate size and continuous gaps encouraged sustained exosome release, which significantly augmented bone repair in a rat cranial defect model treated with ExoBMP2+NoBody.

Fig. 3.

Schematic representation of the exosome preparation and isolation process, and representative transmission electron microscope images of Exo^None, Exo^Empty, and Exo^IRES-IL−10; qPCR analysis of IL-10 mRNA in the isolated exosomes showed that exosomes have great mRNA encapsulation efficacy; two weeks injection of Exo^IRES-IL−10 increased the IL-10 mRNA while decreasing the expression of IL-1β, TNF-α and IL-6 in the plaque. Exo^IRES-IL−10 could deliver functional IRES-IL-10 mRNA into the plaque for precise control of inflammation. Reproduced with the permission from Ref. [82]. Copyright © 2021 Ivyspring International Publisher.

Exosomes are being explored for their potential to serve as vehicles for delivering DNA, effectively enhancing cancer treatment strategies. For instance, by genetically modifying exosomes derived from tumor cells with immunostimulatory CpG DNA, it’s possible to deliver tumor antigens and adjuvants simultaneously to the same antigen presenting cells (APCs), thus inducing potent antitumor immunity [84]. This methodology using CpG-SAV-exo effectively stimulates tumor antigen-specific immune responses and hinders B16BL6 tumor growth in mice, thereby providing a valuable approach for exosome-based cancer immunotherapy (Fig. 4A). The process of tethering DNA to exosomes, alongside functional motifs, nucleic acids, and bioactive proteins, has been streamlined to be both rapid and efficient. For example, exosomes isolated from THP1 cells were tethered with 18-mer DNA possessing a 5’ tetraethylene glycol (TEG) spacer and cholesterol modification (Chol-DNA) [85]. This was achieved through vortexing the exosomes with Chol-DNA in a buffer solution for 5 minutes at room temperature. FasL, useful in immunotherapy but unsuitable for systemic therapy, causes spatially restricted apoptosis of PCI-13 cells when presented in the Exoss-DNA-SA-FasL pattern, which remains biologically active in the solid phase (Fig. 4B).

Fig. 4.

(A) Schematic diagram of the preparation of CpG DNA-modified exosomes. Reproduced with the permission from Ref. [84]. Copyright © 2016 Elsevier. (B) Schematic diagram of the tethering method of cholesterol-oligonucleotides on exosomal membranes. Reproduced with the permission from Ref. [85]. Copyright © 2019 American Chemical Society.

Moreover, exosomes play a significant role in intercellular communication and are emerging as a promising platform for nucleic acid delivery due to their inherent characteristics and capabilities. Considerable interest has been directed toward developing exosome-based gene therapies, with several clinical trials currently investigating the safety, efficacy, and applicability of these therapies across a range of diseases. For instance, ExoCarta (NCT03608631) is conducting a Phase I clinical trial on engineered exosomes as delivery vehicles for siRNA against the KRASG12D mutation in patients with metastatic pancreatic cancer [41]; while miR-124 loaded exosomes are being studied for their potential to ameliorate brain injury by promoting neurogenesis for stroke recovery (NCT03384433) [42].

In summary, while exosomes hold great promise as platforms for nucleic acid delivery, reaching their full potential requires surmounting numerous technical and regulatory challenges. For example, the lipid bilayer structure of exosomes makes it impossible for hydrophilic nucleic acid molecules to be simply loaded, and methods such as electroporation need to be utilized in order to increase the solubility of the drug and to improve the efficiency of drug utilization. Secondly, exosomes, because they contain a variety of heterogeneous compositions with different cellular functions, will exhibit immunogenic effects when an inappropriate source of exosomes is not selected. Lastly, exosomes have a similar morphology, an overlapping range of sizes, and a very small size compared to that of other cellular constituents, which makes it difficult to isolate large-scale and high-purity exosomes [86]. Nevertheless, with the swift progress being made in the field, exosomes continue to show promise as nucleic acid delivery vectors for therapeutic applications.

3. Cell membrane-derived nanovesicles for nucleic acid delivery

Cell membrane-derived nanovesicles are formed by directly protruding from and subsequently detaching from the cell membrane. These vesicles retain only the membrane components, such as proteins and lipids, from the source cell and do not contain internal cellular contents. These naturally occurring structures are collected directly from cells, inherently possessing the capacity to interact with and fuse to cellular membranes, enabling them to deliver genetic materials into cells with high efficiency. A major advantage of cell membrane-derived nanovesicles is their biocompatibility and low immunogenicity, resulting from their natural origins [87]. These traits allow them to evade the immune system, prolong their circulation time within the body, and thus, enhance their nucleic acid delivery efficiency. Furthermore, genetically engineered methods could endow specific targeting ability to nanovesicles, which could display the targeting molecules onto the cell membrane surfaces [88,89]. Therefore, these nanovesicles can be designed to carry a variety of genetic materials, including DNA, mRNA, and siRNA. The encapsulation of these substances can protect them from degradation, increase their stability, and thereby improve delivery efficiency.

Researchers have currently prepared various types of biomimetic nanovesicles that replicate the unique structure, properties, and biosynthetic pathways of natural cell membranes. The most widely studied cell types include erythrocytes [90], leukocytes [91], platelets [92], cancer cells [93], bacterial membranes [94], along with the integration of two different biological membranes to combine the functionalities of multiple cell types into a singular nanovesicle, maximizing functionality and enhancing delivery to target tissues.

3.1. Erythrocyte derived nanovesicle for nucleic acid delivery

Erythrocytes, as delivery systems capable of transporting nucleic acids, represent the most extensively studied cell type [95]. The ability of erythrocytes to fundamentally alter drug pharmacokinetics is attributed to their long circulatory lifespan in the body (PK), biocompatibility, and reduced immunogenicity [96]. Utilizing electroporation, a therapeutic RNA delivery system incorporating human erythrocytes can be constructed, carrying antisense oligonucleotides, Cas9 mRNA, and guide RNA (Fig. 5) [97]. Furthermore, a strategy for particle surface modification has been developed through the extrusion method of coating biodegradable polylactic acidhydroxyacetic acid (PLGA) particles on natural erythrocyte membranes to evade macrophage uptake and systemic clearance. While preserving the function of the polymer, this method disguises the nanoparticle surface with the outer surface of the erythrocyte for extended circulation [90]. Additionally, it has been discovered that vesicles derived from cholesterol-rich erythrocyte membranes can enhance their drug encapsulation potential [98]. These vesicles have been shown to increase the stability and improve the loading efficiency of adriamycin, offering superior antitumor efficacy in comparison to the free drug.

Fig. 5.

RNA drug delivery using erythrocyte extracellular vesicles. Reproduced with the permission from Ref. [97]. Copyright © 2018 Nature Publishing Group.

A strong targeting ability is an essential characteristic of a superior transport carrier [99]. However, the targeting ability of erythrocytes as a standalone delivery vehicle is limited. Precise control over the cellular uptake and the drug release profile of the delivery system can further improve therapeutic efficacy and selectivity. The capability of erythrocytes to accumulate at the target site can be significantly augmented by coating the erythrocyte membrane with antibodies, harnessing antigen-antibody interactions [100].

3.2. Platelet derived nanovesicle for nucleic acid delivery

Platelets possess known circulatory roles, undergoing interactions with vascular endothelium, cancer cells, and immune system constituents. These attributes are passed on to platelet-derived nanovesicles [101]. This enables efficient distribution within the body and supports their prolonged circulation, a key necessity for delivering RNA and maintaining sustained therapeutic effects. Platelet-derived nanovesicles, being autologous biological entities, exhibit excellent tolerance and low immunogenicity, reducing the chances of adverse immune responses often associated with synthetic RNA delivery vectors. Additionally, the natural propensity of platelets to accumulate at sites of injury and disease, including tumors, might be shared by platelet-derived nanovesicles, enabling specific and efficient RNA delivery [102]. Furthermore, the surface of these nanovesicles can be modified with specific ligands to enhance the targeting of designated cell types or tissues [103,104]. As such, platelet-derived nanovesicles are an emerging promising nucleic acid delivery system capable of encapsulating various biomolecules, including different types of RNAs, such as siRNA, miRNA, and mRNA.

Successful siRNA therapy relies on the effective delivery of therapeutic siRNAs to target cells without degradation. Nanovesicles not only bolster the stability and binding affinity of siRNAs but also shield them from nuclease action, enhancing RNA transfection efficiency. A novel bioengineered platform was developed for targeted siRNA delivery where synthetic siRNAs were loaded within porous metal-organic framework (MOF) nanoparticles that were subsequently cloaked with platelet membranes (Fig. 6). This MOF core enables high siRNA loading capacity, and the platelet membranes interact proficiently with various disease-related matrices, exhibiting excellent tumor targeting capabilities. This platform provides an effective means for achieving gene silencing in vivo, potentially broadening the applicability of siRNA in various diseases [105].

Fig. 6.

(A)Platelet membrane-coated siRNA-loaded MOFs (P-MOF-siRNA) for gene silencing. (B) Relative survivin mRNA expression in SK-BR-3 cells after incubation with siRNA^Sur, P-MOF, P-MOF-siRNA^NC, P-MOF-siRNA^Sur, or R-MOF-siRNA^Sur, and quantification of nanoparticle localization within the tumor after intravenous administration with fluorescently labeled P-MOF-siRNA^Sur or R-MOF-siRNA^Sur. Reproduced with the permission from Ref. [105]. Copyright © 2020 American Association for the Advancement of Science.

3.3. Bacteria membrane-derived nanovesicle for nucleic acid delivery

Bacterial membrane-derived nanovesicles (BMNs) offer an intriguing platform for nucleic acid delivery, thanks to their intrinsic biological properties [106,107]. Their natural ability to encapsulate biological cargo, in conjunction with their biocompatibility, earmarks them as potential alternatives or adjuncts to traditional synthetic vectors used in gene therapy.

BMNs possess inherent biocompatibility, which may help reduce systemic toxicity and immune responses frequently seen with synthetic vectors. BMNs also provide a versatile platform for functionalization, allowing the potential to load a variety of biological cargoes, such as nucleic acids, proteins, and therapeutic agents [108,109]. This functionalization can be further optimized for targeted delivery, enhancing treatment effectiveness. The biological origin and diminutive size of BMNs contribute to their ability to evade the host immune system, potentially leading to extended circulation times and enhanced delivery efficiency of therapeutic genes. Imperatively, genetic engineering techniques can be utilized to modify the bacterial host, changing the surface proteins or ligands of BMNs to improve their affinity for specific cell types, tissues, or organs, thus increasing their specificity and reducing off-target effects. As reported, researchers utilized an RNA-binding protein to decorate outer membrane vesicles (OMVs) to rapidly display mRNA antigens via surface adsorption [110]. This OMV-based mRNA delivery system, primarily located in the paracortical region of draining lymph nodes, induces effective T cell activation and efficiently delivers mRNA antigens to DCs for effective translation, triggering immune system activation and inhibiting tumor progression (Fig. 7).

Fig. 7.

Bacterial outer membrane vesicles rapidly display mRNA antigen surfaces for tumor vaccine production. Reproduced with the permission from Ref. [110]. Copyright © 2022 John Wily and Sons.

Nevertheless, the effective use of BMNs in gene therapy also introduces several obstacles. The natural variability of BMNs in dimensions, surface protein constituents, and lipid structure can lead to noticeable differences between batches, potentially influencing their therapeutic performance and the repeatability of findings [111]. The maintenance of BMNs’ stability under physiological conditions for extended durations is paramount for efficient nucleic acid delivery. Challenges related to upholding structural integrity, averting aggregation, and conserving the bioactivity of the encased cargoes must be resolved [112]. While BMNs are typically seen as safe, there’s a persisting possibility of bacterial contamination during the production phase and the risk of unintentional immune responses that cannot be fully ruled out. Rigorous production and purification protocols are required to guarantee safety.

In conclusion, though BMNs offer potential as nucleic acid delivery vectors, their successful transition into clinical settings requires a comprehensive understanding of their properties, as well as meticulous optimization and standardization of production and application procedures. It’s anticipated that ongoing research will further the development of BMNs as an effective instrument in gene therapy applications.

3.4. Tumor cell membrane nanovesicles for nucleic acid delivery

Cancer cell membrane-derived nanovesicles (CCM-NVs) represent a novel addition to the nucleic acid delivery field, providing several distinctive benefits. These vesicles, due to their origin, inherently possess homing capabilities to their source cancer cells, along with the identical surface proteins [113,114]. This homotypic targeting property permits CCM-NVs to bind specifically to, and be internalized by cancer cells, rendering them into a highly selective delivery system for therapeutic agents. Moreover, the surface of CCM-NVs can undergo further modification with targeting ligands to improve their affinity towards specific receptors on cancer cells, thereby boosting nucleic acid delivery efficiency [115]. Leveraging these advantages, the interior of CCM-NVs can be configured to house different therapeutic agents, such as genes, siRNA, or drugs. This adaptability renders them versatile vehicles, customizable for various therapeutic applications.

As demonstrated in a recent report, researchers utilized bladder cancer cell membrane-derived nanovesicles as drug carriers to achieve cancer-specific delivery, circumventing the permeation barrier and off-target effects on normal urothelial cells in bladder cancer treatment [116]. Furthermore, given the unique functionalities of different cell membrane types, researchers have explored the concept of chimeric cell membrane nanovesicles to create multifunctional nanocarriers [117]. For instance, a type of macrophage-tumor chimeric cell membrane was employed to encase HIF-1 siRNA and magnetic nanoparticles, harnessing the stealth properties of macrophages while targeting tumor cells. This enabled extended circulation, magnetic resonance imaging (MRI) guidance for penetration into hypoxic sites, and tumor accumulation [118]. Therefore, the thoughtful design and application of various cell membranes to produce nanocarriers that cater to different disease treatments represents a highly promising strategy.

4. Protein nanocages for nucleic acid delivery

Protein nanocages are comprised of multiple protein subunits, arranging themselves into typically hollow, cage-like structures. These nanocages self-assemble into highly symmetric nanostructures with diverse shapes, such as polyhedral, spherical, or tubular forms [119]. Using genetic engineering techniques, protein nanocages are designed to possess specific properties and functionalities [48,120–122]. Their well-defined sizes and structures allow for precise control over their assembly and characteristics, marking them as an intriguing class of nanomaterials that amalgamate the versatility and unique self-assembly features of proteins with the advantages of nanotechnology. Furthermore, the interior and exterior surfaces of protein nanocages can be modified to encapsulate or bind specific molecules, enabling their wide applicability across diverse fields such as targeted drug delivery, molecular imaging, biomaterials, and nanoscale engineering.

Among the variety of nucleic acid delivery carriers, protein nanocage-based genes exhibit considerable efficacy against various infectious diseases. Nucleic acid delivery using protein nanocages leverages the nanocage structure to efficiently transport and deliver genetic materials, like DNA or RNA, into target cells [123,124]. Protein nanocages bring multiple advantages to nucleic acid delivery, rendering them desirable vectors for introducing genetic material into cells. First, protein nanocages, being derived from natural proteins, possess inherent biocompatibility and are less likely to stimulate an immune response or induce toxicity. This reduces the likelihood of adverse effects in nucleic acid delivery applications. Second, protein nanocages provide a protective environment for the enclosed genetic material, insulating it from degradation by nucleases and other cellular enzymes. The stable structure of the nanocage helps maintain the cargo’s integrity during transportation and delivery. Third, protein nanocages can undergo modification with targeting ligands or peptides on their surfaces to amplify their specificity and selectivity for specific cell types or tissues, improving nucleic acid delivery efficiency and minimizing off-target effects. Moreover, protein nanocages can be designed to release the enclosed genetic material in a controlled manner, with stimuli-responsive elements such as pH- or enzyme-sensitive linkages triggering cargo release at specific cellular or tissue environments. This enhances the efficiency and specificity of nucleic acid delivery. Finally, protein nanocages can be genetically engineered and modified to fine-tune their properties, including stability, cargo-loading capacity, and surface functionality, allowing for their optimization for specific nucleic acid delivery applications.

Therefore, protein nanocages’ benefits in nucleic acid delivery position them as valuable assets in the field of gene therapy. Their nonviral nature, enhanced stability, targeted delivery capabilities, ample cargo capacity, controlled release, biocompatibility, and engineering versatility contribute to their potential in progressing gene therapy, genome editing, and fundamental molecular biology research [125]. The following discussion summarizes the advancements and prospects with regard to protein nanocages for nucleic acid delivery.

4.1. The nucleic acid delivery process of protein nanocage

The principal procedure of employing protein nanocages for nucleic acid delivery encompasses: 1) The design and engineering of protein nanocages to possess specific attributes favorable for gene loading and release. This could involve modifying the interior surface of the nanocage with a positive charge peptide [126]; 2) Further modification of protein nanocages with targeting ligands on their surfaces enables specific cell recognition and binding [127]. These targeting ligands can be designed to interact with receptors or markers existing on the surface of target cells, aiding their internalization; 3) The protein nanocages, encapsulating the genetic materials, are absorbed by the target cells via different mechanisms, such as receptor-mediated endocytosis. This process of cellular internalization ensures the delivery of the genetic material into the cytoplasm of the target cells; 4) Once within the target cells, the protein nanocages release the encapsulated nucleic acid into the cytoplasm. Depending on the nucleic acid type and its desired action, additional mechanisms might be employed to facilitate entry into the cell nucleus where gene expression can occur; 5) The delivered nucleic acid can then undergo gene expression within the target cells. In the case of DNA, it might undergo transcription and translation to produce the desired protein. Conversely, RNA-based genetic material, like mRNA, can be directly translated into proteins within the cytoplasm.

The process of nucleic acid delivery using protein nanocages is a vibrant research area, and specific strategies and modifications might vary depending on the unique nanocage design and target cells. This approach carries promise for various applications, including gene therapy, genetic engineering, and regenerative medicine.

4.2. Applications of protein nanocages for nucleic acid delivery

Protein nanocages embody considerable potential for nucleic acid delivery, boasting numerous advantages and applications in this domain. These entities serve as conduits for delivering therapeutic genes to target cells, aiding in the treatment of genetic anomalies, cancers, and other conditions [128]. The nanocages shield the genetic material from degradation, streamline its cellular uptake, and manage its release, thereby augmenting the effectiveness and efficiency of gene therapy. Protein nanocages can also carry gene editing tools, such as CRISPR-Cas9 complexes, facilitating precise genome alterations in target cells. Additionally, protein nanocages prove useful in the investigation of gene expression, cellular operations, and regulatory mechanisms.

Protein nanocages have demonstrated remarkable potential as nucleic acid delivery vehicles for addressing various diseases. Numerous studies have examined the use of protein nanocages in nucleic acid delivery for specific diseases like genetic disorders, cancer, neurodegenerative diseases, cardiovascular diseases, and inherited metabolic disorders [123]. In this context, we emphasize the recently developed protein nanocage-based nucleic acid delivery techniques that enhance therapeutic efficacy by leveraging the intrinsic properties of protein nanocages.

4.2.1. Ferritin-based nanocages for nucleic acid delivery

Ferritins, ubiquitous non-viral protein cages found in animals, plants, and bacteria, perform critical roles in iron storage, detoxification, and protection against oxidants. As a naturally occurring protein, ferritin forms a hollow nanocage structure and has been widely investigated and engineered for diverse applications. It is well recognized that ferritin nanocages can function as carriers for drug delivery, with their hollow interior cavity capable of housing a variety of therapeutic molecules such as drugs, peptides, or nucleic acids [129].

Based on the structural analysis of ferritin, researchers selectively mutated the negatively charged amino acids on ferritin’s inner surface to engineer a ferritin carrier with a positively charged inner cavity suitable for nucleic acid complexation (Fig. 8). Following this, a ferritin mutant (E61K/E64R/E140K/E147K) boasting high stability and nucleic acid loading ability was selected as a nucleic acid carrier after systematically evaluating nucleic acid loading capacity. The experimental results indicate that the ferritin carrier exhibits universal and efficient loading capacity for different types of TLR (TLR3, TLR7/8, and TLR9) nucleic acid ligands. This significantly improves the in vivo and in vitro delivery efficiency of TLR nucleic acid ligands, enabling efficient cellular uptake, specific intracellular localization, and enhanced immune activation of TLR nucleic acid ligands [51].

Fig. 8.

Using bioengineering technology to replace the negative amino acid residues in the cavity of wild-type HFn (WT HFn) with positive amino acid residues, a series of HFn(+) mutants with different degrees of positive mutations were constructed; Optimal HFn(+) nanocarriers that efficiently and universally load different TLR-activating nucleic acids to realize nanocage carrier-mediated enhancement of anti-tumor immunotherapy. Reproduced with the permission from Ref. [51]. Copyright © 2022 Elsevier.

Recently, growing endeavors have been directed toward establishing potent gene carriers for the treatment of brain-related diseases. An example of such advancements is the self-assembled heavy chain ferritin (HFn) nanoparticle (NP). A study revealed that HFn+ NPs, derived through genetic engineering methods, offer a glimpse into the systematic de novo design of versatile protein cages for Blood-Brain Barrier (BBB) traversal and effective siRNA delivery [130]. This research offers crucial insights for the future development of nucleic acid delivery carriers with BBB-crossing capabilities. Nonetheless, further exploration is warranted to assess the expansion of the inner cavity and the modification of the charge environment to effectively evaluate the loading potential of ferritin.

It is crucial, however, to underscore that research in the field of ferritin-based nucleic acid delivery is still nascent. While ferritin-based nanocages provide numerous advantages for nucleic acid delivery, they are also confronted with several challenges that need to be resolved. These include constraints related to cargo size, intracellular release, transfection efficiency, targeting specificity, and immune response [131]. The overcoming of these challenges demands sustained research and development initiatives. Key areas of focus to surmount these hurdles include optimization of the ferritin nanocage design, improvement of cargo-loading methods, enhancement of targeting strategies, and refinement of delivery protocols, all of which are instrumental in further bolstering the potential of ferritin-based nanocages for nucleic acid delivery.

4.2.2. Heat shock proteins for nucleic acid delivery

Heat shock proteins (HSPs) comprise a family of proteins that play integral roles in cellular stress responses, and are essential in protein folding, stabilization, and intracellular transportation [132]. Although HSPs are not typically employed for nucleic acid delivery, certain research investigations have examined their potential as carriers for delivering genetic material.

Scientific studies have investigated the use of HSPs as vehicles for nucleic acid delivery. These proteins can be modified or genetically engineered to interact with nucleic acids, such as plasmid DNA or RNA molecules, protecting them from degradation, aiding cellular uptake, and assisting in intracellular transportation [133,134]. Furthermore, HSPs can form complexes with DNA molecules, thereby shielding them from nucleolytic degradation, which increases the stability of the genetic material and promotes its delivery into cells. Additionally, the DNA-HSP interaction may promote cellular uptake via receptor-mediated endocytosis [135]. Furthermore, HSPs can be employed to bolster the efficiency of conventional nucleic acid delivery methods, such as lipofection or viral vectors [133]. Co-transfecting cells with HSPs and the target genetic material can enhance the intracellular transport and expression of the transgene.

The many advantages of HSPs for nucleic acid delivery render them promising candidates for such applications. Leveraging HSPs for nucleic acid delivery yields efficient cellular uptake, protection of genetic material, increased stability and expression, immune response activation, chaperone-mediated intracellular transport, and enhanced biocompatibility. These beneficial attributes, combined with their potential for targeted delivery and versatility, lend HSPs significant promise in achieving effective nucleic acid delivery. Nevertheless, more comprehensive research and development are required to optimize and improve the application of HSPs for nucleic acid delivery across various therapeutic scenarios.

5. Virus-like nanoparticle for nucleic acid delivery

Virus-like particles (VLPs) are multi-protein structures that closely resemble the configuration and conformation of actual viruses but, as they lack the viral genome, they can be safely employed [136]. Derived from both enveloped and non-enveloped viruses, these VLPs self-assemble from one or more viral structural proteins, and can spontaneously form particles that approximate the size and shape of the original virus. These VLP nanocages can be engineered to encapsulate and convey genetic material such as DNA or RNA to target cells [137,138]. By protecting the payload from degradation and facilitating efficient delivery, they demonstrate potential as vehicles for gene therapy and gene editing.

Among the advantages of utilizing VLP nanocages for nucleic acid delivery are their inherent biocompatibility, their capacity to safeguard genetic materials from degradation, and their potential for targeted delivery [139]. Additionally, the protein structure of the VLP can be genetically modified to introduce functional elements such as targeting ligands to improve specificity and efficiency [138,140,141]. The promise of nucleic acid delivery using VLP nanocages extends across various fields, including gene therapy, regenerative medicine, and biomedical research. Ongoing improvements in VLP engineering and a deeper understanding of cellular uptake mechanisms will further increase the efficacy and safety of VLP-based nucleic acid delivery systems.

Moreover, a retrovirus-like protein is a biomolecular structure that mimics the properties and morphology of a virus particle but lacks the viral genome [142]. Such structures self-assemble from the virus’s structural proteins and bear striking resemblance in size and shape to the original virus, but are incapable of replication and causing infection as they are devoid of the virus’s genetic material. Intriguingly, researchers have found that a retrovirus-like protein expressed by a gene that was integrated into the human genome following infection by an ancestral virus, PEG10, can safeguard its genetic material in a protective shell and transport mRNA between cells [46]. This suggests that PEG10 holds the potential to be developed into an effective RNA delivery vehicle. Based on PEG10, researchers fused and expressed the fusion protein, thereby enhancing the cell fusion capability of PEG10 (Fig. 9). The system is referred to as Selective Endogenous eNcapsidation for cellular Delivery (SEND). When the team tested SEND’s capacity to edit specific genes using the CRISPR-Cas9 system, the results indicated that co-packaging of Cas9 with sgRNA using the SEND system facilitated about 40% editing at specific chromosome locations. Notably, as the SEND system comprises proteins naturally produced in the body, it is unlikely to provoke an immune response. This suggests that SEND holds significant potential for clinical translation.

Fig. 9.

The construction of the SEND system and the verification of its delivery efficiency of mRNA and CRISPR gene editing tools. Reproduced with the permission from Ref. [46]. Copyright © 2021 American Association for the Advancement of Science.

Further, this research team developed a new delivery system, which is expected to accurately deliver any nucleic acids to any target human cells. Contractile injection systems (CIS) are syringe-like macromolecular complexes abundantly present in bacteria and archaea, which can drive spikes through the cell membrane to inject intraluminal protein loads into its recognized targets in eukaryotic cells [143]. Inspired by this, the researchers focused on the tail fiber region of the CIS system, because this region allows the CIS system to specifically target various cell types. Using AlphaFold, an artificial intelligence program for protein structure prediction, the research team modified the tail fibers to precisely target different proteins on the surface of human cells (Fig. 10) [52]. The results showed that the nucleic acids delivery achieved by the CIS system was highly specific in vitro cell culture experiments. Efficient delivery is only possible with the correct epitope-antibody pairing. In live mouse experiments, the CIS system successfully completed intracranial nucleic acid delivery without causing any activation of immune cells or production of inflammatory cytokines.

Fig. 10.

CIS-based delivery system for specific and efficient nucleic acid delivery. Reproduced with the permission from Ref. [52]. Copyright © 2023 Nature Publishing Group.

VLPs are particularly advantageous in their ability to breach the BBB, facilitating treatment for neurological diseases [144,145]. The BBB, constituted by brain microvasculature, restricts the entry of certain substances from the blood into brain tissue. As a result, the transit of therapeutic drugs for brain diseases through the BBB can be limited, with only some lipid-soluble drugs, small molecules, and drugs transported by carrier proteins able to access brain tissue. However, research has shown that the accurate design and construction of shape and size of VLPs can enhance the precision of targeting ability and biocompatibility of the drug released, improving its effectiveness in crossing the BBB. Researchers have successfully constructed an endogenous virus-like system to load and deliver mRNA in vivo [146]. By incorporating an endogenous retrovirus-like Arc protein coat into leukocyte-derived extracellular vesicles and stabilizing it using Arc 5’UTR RNA, mRNA was efficiently delivered across the BBB. The study also constructed a new AAV variant capable of retrograde transport in the CNS. These virus-like nanoparticles may become the principal nucleic acid delivery vectors in the future for targeted delivery at systemic target sites.

In summary, the VLP-based nucleic acid delivery system has several benefits such as high biological safety, delivery efficiency, and versatility, and is anticipated to provide a new alternative to viral vectors and LNP vectors.

6. Conclusions and perspectives

Bioengineered nanocarriers, such as exosomes, cell membrane-derived nanovesicles, protein nanocages, and VLPs, present exceptional advantages for nucleic acid delivery, demonstrating promising potential for transformative biomedical applications. As outlined in Table 1, we have summarized the main research reports regarding these bioengineered nucleic acid delivery nanocarriers and their applications in biomedicine. To date, nucleic acid drugs delivered by exosomes derived from mesenchymal stem cells have entered clinical trials, leveraging the unique advantages of exosomes. This includes mesenchymal stem cell exosomes containing siRNA targeting the KrasG12D mutant gene being used to treat pancreatic cancer (NCT03608631) [41], as well as mesenchymal stem cell exosomes carrying miR-124 for the treatment of ischemic heart disease (NCT03384433) [42]. Despite their growing popularity in research and the advancement of some products into clinical trial phases, these nanocarriers also encounter numerous challenges such as complex production processes, potential immune responses, and stability issues [147,148]. Based on these findings, we summarized the advantages and disadvantages of the four types of bioengineered nanocarriers in Table 2, and expect that future research efforts should focus on nanocarrier optimization for clinical use, including refinement of targeting strategies, reduction of immunogenicity, and stability enhancements.

Table 1.

Bioengineered nanocarriers for nucleic acid delivery.

Carrier	Nucleic acid	Size	Targeted gene(s)	Loading method	Efficiency	Indication	Ref.
Exosome	mRNA	~100 nm	HIV-1 LTR	Transfection	Significant reduction in viral expression	Epigenetic suppression of HIV-1	[158]
Exosome	miRNA	NA	EGFR	Liposome transfection	Tripling	Breast cancer	[159]
Exosome	miRNA	63.2±1.77 nm	NF-κB and TNF-α signaling molecules	Co-incubation	NA	Atherosclerosis	[160]
Exosome	miRNA	75 nm	NA	Lentiviral transfection	Significant increase	Bone regeneration	[161]
Exosome	Cas9 & gRNA	50–150 nm	PARP-1	Electroporation	Complete inhibition of PARP-3 expression	Ovarian cancer	[162]
Exosome	CRISPR/Cas9	50–200 nm	KrasG12D	Transfection	NA	Pancreatic cancer	[163]
Exosome	CRISPR/Cas9	NA	p53	Electroporation	NA	Liver diseases	[61]
Exosome-AAV	DNA	NA	Cochlear hair cells	Genetic cloning, Transfection	Superior to conventional AAV vectors	Hearing impairment	[164]
Hybridized exosome	CRISPR/Cas9	0.1–1.0 μm	Mesenchymal stem cells (MSCs)	Co-incubation	NA	Encapsulation of large plasmids	[165]
Cell membrane vesicle	siRNA	175 nm	NA	Membrane extrusion	Effective promotion	Breast cancer	[105]
Cell membrane vesicle	siRNA	~60 nm	HIF-1	Membrane extrusion	Significant increase	Blocking HIF-1 function	[118]
Cell membrane vesicle	RNA	~140 nm	miR-125b	Electroporation	~80%	Leukemia and breast cancer	[97]
Ferritin	mRNA	30 nm	SARS-CoV-2	Transfection	Produced broad-spectrum neutralizing antibodies	mRNA vaccines targeting SARS-CoV-2	[166]
Ferritin	siRNA & miRNA	8 nm	InsR	Co-incubation	Significant gene silencing	Colon adenocarcinoma liver carcinoma cell	[167]
Ferritin	Cas9 and gRNA	26 nm	EGFP	Disassembly/assembly	Satisfied genome editing efficiency	Hela cells	[168]
Heat shock protein	DNA	NA	NF-κB	Co-incubation	Successfully intranuclear delivery	Pre-B 70Z/3 cells	[169]
Heat shock protein	siRNA & miRNA	55.8 nm	TERT	Co-incubation	Significant gene silencing	Colorectal cancer	[134]
Virus-like particle	DNA	NA	GFP	Electroporation	Successfully over-expression	HepG2	[170]
Virus-like particle	siRNA	~27 nm	GFP/Akt	Encapsulation	Significant gene silencing	Breast cancer	[171]
Virus-like particle	miRNA	25 nm	miR-146a	Transformation	Successfully over-expression	HeLa, HepG2, Huh-7, andPBMCs	[172]
Virus-like particle	shRNA	NA	Bcl-2	Urea dissociation/association	Significant gene silencing	Cervical cancer cells	[173]
Virus-like particle	Cas9 and gRNA	NA	Brachyury gene	Cloning	Significant gene silencing	Chordoma	[174]
Virus-like particle	mRNA and Cas9	~50 nm	Cre-mRNA	Packaging	~60%	N2a cell lines	[46]
Virus-like particle	mRNA and Cas9	~116 nm	GFP	Novel payload	~100%	loxP-tdTomato mice	[52]

Table 2.

Pros and cons of bioengineered nanocarriers for nucleic acid delivery.

Bioengineered nanocarriers	Pros	Cons
Exosomes	Easy modification Low immunogenicity Long circulation Natural targeting properties	Limited loading efficiency Undefined composition Difficulty in isolating exosomes on a large scale and with high purity Difficulty in storage and stability
Cell membrane-derived nanovesicles	High biocompatibility and low immunogenicity High engineering potential Natural targeting abilities Size-controllable	Limited loading efficiency Difficulty in storage and stability
Protein nanocage	High biocompatibility Defined size and shape Excellent engineering potential	Complex production Drug loading limitation Potential immunogenicity
Virus-like nanoparticle	Attractive self-assembly properties Personalized design for targeted delivery	Particle instability Intrinsic immunogenicity Limitation in antigen fusions

The importance of drug formulation safety cannot be overstated. Preliminary research indicates that these bioengineered nucleic acid delivery nanocarriers offer superior biocompatibility and immune adaptability when compared to chemically synthesized nanoparticles, but these results require further substantiation through larger studies and extended investigation periods [149]. Moreover, production costs could inhibit the progression of drug formulations [150]. The production of bio-origin materials generally incurs higher expenses than that of chemical drugs. One specific challenge is the efficient isolation and purification of exosomes from biological samples, as the yield and purity can drastically impact their therapeutic potential [151,152]. Techniques for loading therapeutic genes into exosomes effectively while preserving their integrity are still being developed. The need for upscaling exosome production to meet clinical needs while ensuring consistency across batches presents a significant challenge. Therefore, there is a critical need for the development of standardized and simplified production methods for bioengineered nucleic acid delivery nanocarriers. This may require the development of innovative bioengineering technologies to boost production efficiency and scalability without compromising the biocompatibility and delivery efficiency of nanoparticles. Furthermore, ensuring the consistent quality of bioengineered nanoparticles throughout production is vital.

Moreover, bioengineered nanocarriers must be capable of transporting adequate quantities of nucleic acid to achieve the intended therapeutic effect. Sometimes, the loading efficiency of nucleic acid into the nanocarriers might be low, necessitating higher dosages and potentially increasing side effect risks. For instance, nanocarriers derived from cell membranes, including exosomes, have faced this challenge, with loading capacities of less than 10% [73,153,154]. Therefore, enhancing the gene payload capacity of bioengineered nucleic acid delivery nanocarriers is a vital research direction. Furthermore, precise control over the release of loaded gene therapeutics is critical [155,156]. Once inside the target cells, the genetic material must be efficiently released from the nanocarriers and reach the appropriate cellular compartments for expression. Although these major nanocarriers can deliver genes, the mechanisms facilitating efficient gene release within cells are not fully understood. Therefore, further studies on the intracellular behavior of these nanocarriers are required to aid the design of new nanoparticle structures and gene encapsulation strategies.

Lastly, the stability of bioengineered nanocarriers is a major constraint in their development and cost reduction efforts. For example, protein or gene drugs, such as the COVID-19 vaccine, necessitate cold-chain transportation [157]. Therefore, enhancing the long-term stability of bio-origin nanoparticles is a substantial technical challenge.

Addressing these challenges will require ongoing research and development efforts, along with the collaborative input of researchers from diverse fields, encompassing biology, materials science, and engineering. Despite these obstacles, the prospective benefits of bio-engineered nanotechnology make it a captivating research focus with the potential to significantly impact nucleic acid therapy and other medical areas.

Data availability

No data was used for the research described in the article.