Nanoparticles derived from herbal preparations May represent a novel nucleic acid therapy

A significant number of nucleic acid therapies have been approved, highlighting the vast potential of nucleic acid therapeutics. Although numerous delivery strategies have been investigated, and progress has been made in nucleic acid delivery systems, the oral delivery route still presents a significant obstacle. Recently, a novel and promising force in oral nucleic acid delivery has emerged, namely Decoctosomes and Bencaosomes, obtained from the decoction of natural herbs, which refers to boiled Chinese herbal medicines. The active components of Decoctosomes include small molecules, peptides, and small RNAs (sRNAs). sRNAs were the first targets of extensive research. Additionally, Bencaosomes have been proven effective in several disease mouse models as artificially enhanced decoctosome products. This perspective summarizes the development and possible future directions of Decoctosomes and Bencaosomes, two varieties of nanoparticles that may open the door to new possibilities in nucleic acid therapy.


| INTRODUCTION
The development of nucleic acid therapy dates back almost 50 years.This therapeutic approach uses nucleic acid molecules (i.e., microRNA (miRNA), short interfering RNA (siRNA), antisense oligonucleotides (ASOs), messenger RNA (mRNA), plasmid DNA) to modulate gene expression to treat or prevent various diseases, such as cancer, virus infection, lung disorders, liver diseases and nervous system diseases. 1,22][3] Although it holds promising prospects in medicine development, use of nucleic acids as therapeutic agents faces significant challenges.For example, nucleases quickly degrade them, activate the immune system, or cannot be easily shifted toward cells to function. 1,2Excitingly, with advancements in chemical synthesis and delivery systems, the field has witnessed rapid development, leading to the approval of 18 nucleic acidbased therapies to date. 4 For instance, ONPATTRO® and GIVLAARI™, two kinds of siRNA drugs based on the mechanism of RNA interference (RNAi), have been approved for commercial application. 5Additionally, mRNA technology is also a promising research field.mRNA vaccines (i.e., BNT 162b2 and mRNA-1273) have successfully combated the COVID-19 pandemic and attracted global interest. 6,7umerous techniques of nucleic acid administration are known to exist, including subcutaneous, intramuscular, intravenous, and local injections, such as intravitreal injections. 1 However, the most straightforward, familiar and convenient drug administration, the oral nucleic acid delivery method continues to pose significant challenges, encountering intrinsic barriers such as the gastrointestinal acidic environment, mucus clearance and enzymatic degradation. 8,9Additionally, when oral nucleic acid drugs are absorbed through the gastrointestinal tract's epithelial cells, they must cope with the obstacles of Intracellular barriers such as the cell membrane, end lysosomal entrapment, and intracellular localization. 9Therefore, developing nucleic acid delivery systems with enhanced efficiency is vital to achieving optimal outcomes for treatment. 10So far, numerous nanoparticle-based systems such as polymeric nanoparticles, lipid-based nanoparticles (LNPs) and extracellular vesicles (EVs) have been developed for oral nucleic acid delivery.Nucleic acids are encapsulated and protected in nanoparticles, which frees them from degradation in physiological environments. 8,11he increasing global attention on nucleic acid delivery systems has led to innovations in overcoming various biological barriers. 12Current nucleic acid therapeutic delivery systems can be broadly divided into virus-based carriers and non-viral carriers. 1The ladder includes adeno-associated virus (AAV), adenoviruses and lentiviruses, and the former involves polymers, cationic liposomes, NPS, inorganic nanoparticles and EVs. 13 Recently, a nanovesicular substance nominated Decoctosomes was discovered in herbal preparations after boiling (i.e., the decoction). 14These vesicles contain small molecules, peptides, and sRNAs, displaying more effective anti-fibrotic and anti-inflammatory actions than external decoction treatment. 145][16] Interestingly, acquiring Bencaosomes requires heating a mixture of nucleic acid and lipids to promote the embedding of nucleic acid in the lipid membrane to increase the stability of the complex. 14Nevertheless, based on the fact that Decoctosomes and Bencaosomes are the latest novel discoveries, their firm similarities and differences in structure and function still need further investigation to verify.
Altogether, the main emphasis of the current study was on the developmental process and prospective trends of Decoctosomes and Bencaosomes, which may promote the development of oral nucleic acid treatments and be therapeutically used in the future.

| Controversy regarding nucleic acid molecules originating from herbal medicine
Herbal decoctions, referred to as "decoction" in China, are rich in a variety of substances, many kinds of which remain unexplained. 17,18Recent research highlighted that high-temperature boiling forms supramolecular structures, acting as drug carriers to promote biological activity due to complex interactions. 19However, the study of supramolecules in decoctions is still in progress with limited research methodologies.][22][23][24][25] However, scholars have debated the extent to which microRNAs (miRNAs) derived from exogenous plants and decoctions can penetrate mammalian organisms and produce significant biological effects. 26,27The debate concerning cross-kingdom regulation by plant-derived miRNAs can be categorized into the following points.Firstly, reproducibility and Contamination.Some researchers question the validity of the findings because they have not been able to reproduce them.Concerns are raised about the possible contamination of samples or biases introduced during sequencing. 28Besides, Zhang et al. state that those questioning their work might use a sequencing approach biased against detecting plant miRNAs. 25Also, alternative explanations argue that the observed effects could be artefacts of experimental design, such as cross-contamination during library preparation and low-biomass samples. 29n response to Professor Jiang's group's findings, Lu argued that the highly conserved animal miRNA sequences found in plant sRNA indicate a non-plant origin, which infers that animal sRNA contamination in decoctions. 302][33] It's worth mentioning that traditional Chinese medicine is effective in treating patients with COVID-19 in China, but the specific mechanism is complex and challenging to clarify. 34Interestingly, Jiang's group first reported that in the peripheral blood mononuclear cells (PBMCs) samples of COVID-19 patients treated with traditional Chinese medicine, many sRNAs derived from herbal sources were identified.The predicted target genes of these sRNAs showed approximately 80% overlap with the differentially expressed genes (DEGs) in COVID-19 patients, suggesting a potential interplay between sRNAs and the regulation of DEGs during treatment. 33However, because the disease progression itself might influence DEGs, the underlying molecular mechanisms still need validation and clarification.

Bencaosomes
Professor Jiang's group first proposed Decoctosomes and Bencaosomes. 14In China, most herbal medicines are traditionally boiled before use.To explain how sRNA in decoctions remain intact and are absorbed across species into humans, Jiang's group first demonstrated the existence of phosphocholines in the boiled Chinese herbal medicine Hong Jing Tian (HJT) that promoted sRNA entry into mouse and human cells to exert antipulmonary fibrosis effects. 35Subsequently, transmission electron microscopy (TEM) and Zetasizer characterized nano-vesicular structures in HJT-decoction and PGYdecoction (i.e., a decoction of Chinese herbal medicine Pu Gong Ying), hence named Decoctosomes.Nevertheless, these Decoctosomes exhibited superior anti-fibrotic and anti-inflammatory effects compared to decoction alone. 14dditionally, after identifying Decoctosomes, they synthesized single sphinganine (d22: 0) in vitro, followed by mixing and heating it at 90°C with specific sRNA sequenced and synthesized in vitro from Decoctosomes, and finally created thermally stable vesicular lipidnucleic acid complexes with excellent oral therapeutic effects, named Bencaosomes. 14,35Notably, through a series of physical chemistry experiments, 90°C heating of the mixture of nucleic acid and lipid promoted the insertion of nucleic acid into the lipid layer to increase the stability of the complex. 14Recent studies have validated more about Bencaosomes in mouse models of acute lung injury and angiotensin II-induced hypertension. 15,16his suggests that the utilization of these artificially stabilized nano-vesicular particles has the potential to serve as a novel form of oral nucleic acid therapy while also explaining the efficacy of Chinese herbal decoctions.

| CONCLUSION AND OUTLOOK
This perspective provides a concise overview of the progress made in creating Decoctosomes and Bencaosomes, nanoparticle-like materials obtained from herbal medicines.It's worth mentioning that, recently plantderived exosome-like nanoparticles (PELNs) have been extensively studied to prevent and treat various diseases. 36,37As Professor Qiao's group said, the development and update of PELNs separation and characterization methods will improve the quality control system of PELNs, and the application of PELNs in disease prevention and treatment will enrich the evidence of crosskingdom regulation in the field of plants.However, up to now there have been some controversies in the biochemical characterization or marker identification of PELNs.The approaches include comprehensive omics analysis of lipids, proteins and nucleic acids.Syntaxin PENETRATION 1 (PEN1) and TETRASPANIN 8 (TET8) were used for specific biomarker identification, but this still lacks consensus.Thin layer chromatography (TLC) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) techniques were employed for sample identification by comparison with known reference substances. 38The perspective also highlights that there is still more to be explored regarding the biochemical characterization of Decoctosomes.On the contrary, as a simplified version of Decoctosomes, the basic structure of Bencaosomes is clear (i.e., a single lipid layer such as sphinganine (d22: 0) added a specific type of sRNA), and it also has a similar therapeutic effect compared with Decoctosomes. 14hese two novel herbal nano-items, Decoctosomes and Bencaosomes, focus on their potential utilization in oral nucleic acid treatments. 8,9,1433,35 However, the field is in its early stages, with fundamental concerns regarding their detailed mechanisms, interactions, and downstream molecular effects still unresolved.Further research may clarify the specific mechanisms and optimized delivery methods of Decoctosomes and Bencaosomes.In the future, experts may look at a wider range of herbs, making this new treatment method more practical.Continued research will yield more profound knowledge of these nanoparticles and, potentially, novel nucleic acid treatments, opening a new chapter in the effort to overcome biological barriers (Figure 1).

CONFLICT OF INTEREST STATEMENT
The authors declare no conflict of interests.
Remarkably, these nano-items can be isolated by ultracentrifugation of decoction and found intact through transmission electron microscopy (TEM).Their heat stability may explain the traditional preparation of most Chinese herbal medicines by boiling.Moreover, the sRNAs and lipids obtained through techniques such as sequencing using a sequencer and liquid chromatography-mass spectrometry (LC-MS) can be synthesized separately in vitro and then used to co-assemble a novel, simplified version of Decoctosomes, nominated as Bencaosomes.Additionally, heating the mixture of sRNA and lipid molecules at 90°C alters their physical and chemical characteristics, leading to the insertion of sRNAs into the lipid layers, which is helpful for the encapsulation of sRNAs to conduct Bencaosomes in a firm state.More importantly, Decoctosomes and Bencaosomes' good efficacy validation in mice disease models opened up a new therapeutic channel for oral nucleic acid therapies, and it is essential to explore and develop their potential more thoroughly.
This work was partially sponsored by the National Key R&D Program of China (2022YFA1104900 and 2022YFA 1104904), the Innovation Team Project (2023KCXTD007) and the Special Project in Key Areas (2021ZDZX2011) of the Department of Education of Guangdong Province, the Natural Science Foundation of Guangdong Province (2021A1515010013), the Guangzhou Municipal Science and Technology Project (202201011760), and the President Foundation of the Integrated Hospital of Traditional Chinese Medicine of Southern Medical University (1202101003).