In vivo Delivery Processes and Development Strategies of Lipid Nanoparticles.

Lipid nanoparticles (LNPs) represent an advanced and highly efficient delivery system for RNA molecules, demonstrating exceptional biocompatibility and remarkable delivery efficiency. This is evidenced by the clinical authorization of three LNP formulations: Patisiran, BNT162b2, and mRNA-1273. To further maximize the efficacy of RNA-based therapy, it is imperative to develop more potent LNP delivery systems that can effectively protect inherently unstable and negatively charged RNA molecules from degradation by nucleases, while facilitating their cellular uptake into target cells. Therefore, this review presents feasible strategies commonly employed for the development of efficient LNP delivery systems. The strategies encompass combinatorial chemistry, a rational design approach, the derivatization strategy of functional molecules, the optimization of LNP formulations, and adjustment of particle size and charge property of LNPs. Prior to introducing these developing strategies, in vivo delivery processes of LNPs, a crucial determinant influencing the clinical translation of LNP formulations, is described to better understand how to develop LNP delivery systems.

Design, synthesis, and in vitro gene transfer efficacy of novel ionizable cholesterol derivatives.

ABSTACTThe medicinal properties of genetic drugs are highly dependent on the design of delivery systems. Ionizable cationic lipids are considered core materials in delivery systems. However, there has not yet been a widespread consensus on the relationship between the wide diversity of lipid structure design and gene delivery efficiency. The aims of the research work were to synthesize ionizable cholesterol derivatives (iChol-lipids) and to evaluate their potential applications as gene delivery vector. A series of iChol-lipids with different head groups were synthesized with carbamate bond spacer. The chemical structures were characterized by 1H NMR, MS, melting range, and pKa. The interactions between iChol-lipids and MALAT1-siRNA were studied by molecular dynamics simulations and compared with market available DC-Chol, which revealed that hydrogen bonds, salt-bridge, and electrostatic interaction were probably involved. The self-assemble behaviors of these lipids were intensively investigated and evaluated by dynamic laser scattering in the presence of different helper lipids and PEGylated lipids. Their plasmid binding ability, transfection efficiency, hemolytic toxicity, and cytotoxicity were fully studied. IZ-Chol-LNPs was proved to be highly potential to effectively complex with DNA, and endosome escape mechanisms mediated by proton sponge effect was verified by pH-sensitive fluorescence probe BCFL.

A Cu+ /Thiourea Dendrimer Achieves Excellent Cytosolic Protein Delivery via Enhanced Cell Uptake and Endosome Escape.

Intracellular protein delivery has attracted considerable attention in the development of protein-based therapeutics, however, the design of highly efficient materials for robust delivery of native proteins remains challenging. This study proposes a Cu+ -based coordination polymer for cytosolic protein delivery with high efficacy and robustness. The phenylthiourea grafted dendrimer is coordinated with cuprous ions to prepare the polymeric carrier, which efficiently bind cargo proteins via a combination of coordination, ionic and hydrophobic interactions. The incorporation of Cu+ ions in the polymer greatly improves its cellular uptake and endosomal escape. The cuprous-based coordination polymer successfully delivered a variety of structurally diverse proteins into various cell lines with reserved bioactivities. This study provides a new type of coordination polymers for cytosolic delivery of biomacromolecules.

Structural analysis of polysaccharide/antisense DNA complexes during cytoplasmic target mRNA hybridization.

We previously demonstrated antisense oligonucleotides (AS-ODNs) delivery system based on the complex formed with poly (dA) and schizophyllan, a type of ß-1,3-glucan. This complex enables efficient intracellular delivery of AS-ODNs. In this communication, we investigated the cytoplasmic translocation of the complex itself and its mechanism of action on mRNA. As a result, we found that the complex moved into the cytoplasm while keeping its structure, and AS-ODN hybridized with the target mRNA. This result encourages pharmaceutical applications of the complex.

Discovery of endosomalytic cell-penetrating peptides based on bacterial membrane-targeting sequences.

Cell-penetrating peptides (CPPs) are prominent scaffolds for drug developments and related research, particularly the endocytic delivery of biomacromolecules. Effective cargo release from endosomes prior to lysosomal degradation is a crucial step, where the rational design and selection of CPPs remains a challenge and calls for deeper mechanistic understandings. Here, we have investigated a strategy of designing CPPs that selectively disrupt endosomal membranes based on bacterial membrane targeting sequences (MTSs). Six synthesized MTS peptides all exhibit cell-penetrating abilities, among which two d-peptides (d-EcMTS and d-TpMTS) are able to escape from endosomes and localize at ER after entering the cell. The utility of this strategy has been demonstrated by the intracellular delivery of green fluorescent protein (GFP). Together, these results suggest that the large pool of bacterial MTSs may be a rich source for the development of novel CPPs.

Strategy for Cytoplasmic Delivery Using Inorganic Particles.

Endosome escape is a key process for intracellular uptake of intact biomolecules and therapeutics, such as nucleic acids. Lysosome escape is a more common pathway during endocytosis, while some biomolecular, organic and inorganic materials are found to enhance the endosome escape, and several mechanisms have been proposed accordingly. Specifically, some inorganic nanomaterials show their unique mechanisms of action for enhanced endosome escape, including salt osmotic effect and gas blast effect. These inorganic nanomaterials are basically weakly alkaline and are naturally featured with the anti-acidification capacity, with limited solubility in neutral solutions. This review paper has briefly presented the strategies in the design of inorganic nanoparticle-based cellular delivery vehicles with endosome escapability and discussed a few typical inorganic nanomaterials that are currently widely examined for delivery purpose. A brief summary and prospect for this kind of inorganic nanomaterials are provided.

Mannosylation of pH-sensitive liposomes promoted cytoplasmic delivery of protein to macrophages: green fluorescent protein (GFP) performed as an endosomal escape tracer.

Conventional non-pH-sensitive liposomes for cytoplasmic delivery of protein suffer from poor efficiency. Here we investigated mannosylated pH-sensitive liposomes (MAN-PSL) for cytoplasmic delivery of protein to macrophages RAW 264.7 using PSL and non-pH-sensitive liposomes for comparison. We characterised the pH-dependent fluorescence of green fluorescent protein (GFP) and encapsulated it in liposomes as an intracellular trafficking tracer. GFP showed a reversed 'S'-shaped pH-fluorescence curve with a dramatic signal loss at acidic pH. GFP stored at 4 °C with light protection showed a half-life of 10 days (pH 5-8). The entrapment efficiency of GFP was dominated by the volume ratio of intraliposomal core to external medium for thin-film hydration. Mannosylation did not affect the pH-responsiveness of PSL. Confocal microscopy elucidated that mannosylation promoted the cellular uptake of PSL. For both these liposomes, the strongest, homogeneously distributed GFP fluorescence in the cytoplasm was found at 3 h, confirming efficient endosomal escape of GFP. Conversely, internalisation of non-pH-sensitive liposomes was slow (peaked at 12 h) and both Nile Red and GFP signals remained weak and punctuated in the cytosol. In conclusion, GFP performed as a probe for endosome escape of liposomal cargo. Mannosylation facilitated the internalisation of PSL without compromising their endosomal escape ability.

Efficient Cytosolic Delivery Using Crystalline Nanoflowers Assembled from Fluorinated Peptoids.

The design and synthesis of biocompatible nanomaterials as cargoes for the intracellular delivery of therapeutic proteins or genes have attracted intense attention because of their potential for use in therapeutics. Despite the advances in this area, very few nanomaterials can be efficiently delivered to the cytosol. To address these challenges, crystalline nanoflower-like particles are designed and synthesized from fluorinated sequence-defined peptoids; the crystallinity and fluorination of these particles enable highly efficient cytosolic delivery with minimal cytotoxicity. A cytosol delivery rate of 80% has been achieved for the fluorinated peptoid nanoflowers. Furthermore, these nanocrystals can carry therapeutic genes, such as mRNA and effectively deliver the payload into the cytosol, demonstrating the universal delivery capability of the nanocrystals. The results indicate that self-assembly of crystalline nanomaterials from fluorinated peptoids paves a new way toward development of nanocargoes with efficient cytosolic gene delivery capability.

Light-Triggered Genome Editing: Cre Recombinase Mediated Gene Editing with Near-Infrared Light.

A light-activated genome editing platform based on the release of enzymes from a plasmonic nanoparticle carrier when exposed to biocompatible near-infrared light pulses is described. The platform relies on the robust affinity of polyhistidine tags to nitrilotriacetic acid in the presence of copper which is attached to double-stranded nucleic acids self-assembled on the gold nanoparticle surface. A protein fusion of the Cre recombinase containing a TAT internalization peptide sequence to achieve endosomal localization is also employed. High-resolution gene knock-in of a red fluorescent reporter is observed using a commercial two-photon microscope. High-throughput irradiation is described to generate useful quantities of edited cells.

PEG-Benzaldehyde-Hydrazone-Lipid Based PEG-Sheddable pH-Sensitive Liposomes: Abilities for Endosomal Escape and Long Circulation.

PURPOSE: To fabricate an acid-cleavable PEG polymer for the development of PEG-cleavable pH-sensitive liposomes (CL-pPSL), and to investigate their ability for endosomal escape and long circulation. METHODS: PEG-benzaldehyde-hydrazone-cholesteryl hemisuccinate (PEGB-Hz-CHEMS) containing hydrazone and ester bonds was synthesised and used to fabricate a dual pH-sensitive CL-pPSL. Non-cleavable PEGylated pH-sensitive liposome (pPSL) was used as a reference and gemcitabine as a model drug. The cell uptake and endosomal escape were investigated in pancreatic cancer Mia PaCa-2 cells and pharmacokinetics were studied in rats. RESULTS: The CL-pPSL showed accelerated drug release at endosomal pH 5.0 compared to pPSL. Compared to pPSL, CL-pPSL released their fluorescent payload to cytosol more efficiently and showed a 1.4-fold increase in intracellular gemcitabine concentration and higher cytotoxicity. In rats, injection of gemcitabine loaded CL-pPSL resulted in a slightly smaller Vd (149 ± 27 ml/kg; 170 ± 30 ml/kg) and shorter terminal T1/2 (5.4 ± 0.3 h; 5.8 ± 0.6 h) (both p > 0.05) but a significantly lower AUC (p < 0.01), than pPSL, due to the lower PEGylation degree (1.7 mol%) which means a 'mushroom' configuration of PEG. A five-time increase in the dose with CL-pPSL resulted in a 11-fold increase in AUC and a longer T1/2 (8.2 ± 0.5 h). CONCLUSION: The PEG-detachment from the CL-pPSL enhanced endosome escape efficiency compared with pPSL, without significantly compromising their stealth abilities.

Boosting Intracellular Delivery of Lipid Nanoparticle-Encapsulated mRNA.

Intracellular delivery of mRNA holds great potential for vaccine1-3 and therapeutic4 discovery and development. Despite increasing recognition of the utility of lipid-based nanoparticles (LNPs) for intracellular delivery of mRNA, particle engineering is hindered by insufficient understanding of endosomal escape, which is believed to be a main limiter of cytosolic availability and activity of the nucleic acid inside the cell. Using a series of CRISPR-based genetic perturbations of the lysosomal pathway, we have identified that late endosome/lysosome (LE/Ly) formation is essential for functional delivery of exogenously presented mRNA. Lysosomes provide a spatiotemporal hub to orchestrate mTOR signaling and are known to control cell proliferation, nutrient sensing, ribosomal biogenesis, and mRNA translation. Through modulation of the mTOR pathway we were able to enhance or inhibit LNP-mediated mRNA delivery. To further boost intracellular delivery of mRNA, we screened 212 bioactive lipid-like molecules that are either enriched in vesicular compartments or modulate cell signaling. Surprisingly, we have discovered that leukotriene-antagonists, clinically approved for treatment of asthma and other lung diseases, enhance intracellular mRNA delivery in vitro (over 3-fold, p < 0.005) and in vivo (over 2-fold, p < 0.005). Understanding LNP-mediated intracellular delivery will inspire the next generation of RNA therapeutics that have high potency and limited toxicity.

A Light Responsive Nanoparticle-Based Delivery System Using Pheophorbide A Graft Polyethylenimine for Dendritic Cell-Based Cancer Immunotherapy.

In this study, the photochemical internalization (PCI) technique was adopted in a nanoparticle-based antigen delivery system to enhance antigen-specific CD8+ T cell immune response for cancer immunotherapy. Pheophorbide A, a hydrophobic photosensitizer, grafted with polyethylenimine (PheoA-PEI) with endosome escape activity and near-infrared imaging capability was prepared. A model antigen ovalbumin (OVA) was then complexed with PheoA-PEI to form PheoA-PEI/OVA nanoparticles (PheoA-PEI/OVA NPs) that are responsive to light. Flow cytometry analysis revealed increased endocytosis in a murine dendritic cell line (DC2.4) that was treated with PheoA-PEI/OVA NPs compared to free OVA. Generation of reactive oxygen species (ROS) in DC2.4 cells was also confirmed quantitatively and qualitatively using 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). Confocal laser scanning microscopy (CLSM) further demonstrated that the PheoA-PEI/OVA NPs enhanced cytosolic antigen release after light stimulation. Moreover, PheoA-PEI/OVA NP treated DC2.4 cells exhibited enhanced cross-presentation to B3Z T cell hybridoma in vitro after light irradiation, substantially increased compared to those treated with free OVA. Consistently, in vivo results revealed upregulation of CD3+CD8+T lymphocytes in tumors of mice treated with dendritic cells plus PheoA-PEI/OVA NPs and light irradiation. The activated T cell response is partly responsible for the inhibitory effect on E.G7 tumor growth in mice immunized with dendritic cells plus PheoA-PEI/OVA NPs and light irradiation. Our results demonstrate the feasibility to enhance antigen-specific CD8+ T cell immune response by light-responsive nanoparticle-based vaccine delivery for cancer immunotherapy.

Curcumin-loaded nanoliposomes linked to homing peptides for integrin targeting and neuropilin-1-mediated internalization.

CONTEXT: Curcumin, a naturally occurring polyphenol, has been extensively studied for its broad-spectrum anticancer effects. The potential benefits are, however, limited due to its poor water solubility and rapid degradation which result in low bioavailability on administration. OBJECTIVES: This study encapsulates curcumin in nanoliposomes including an integrin-homing peptide combined with a C end R neuropilin-1 targeting motif for targeted delivery and receptor-mediated internalization, respectively. MATERIALS AND METHODS: The linear GHHNGR (Glycine-Histidine-Histidine-Asparagine-Glycine-Arginine) was synthesized through F-moc chemistry on 2-chlorotrityl chloride resin and conjugated to oleic acid. The lipoyl-peptide units were then co-assembled with lecithin and 0-75 mole % Tween-80 into liposomes. Curcumin was passively entrapped using a film hydration technique and its degradation profile was examined within seven consecutive days. The cytotoxic effects of the curcumin-loaded liposomes were studied on MCF-7 and MDA-MB-468, during 24 h exposure in MTT assay. RESULTS: The maximum curcumin entrapment (15.5% W/W) and minimum degradation (< 23%) were obtained in a pH switch loading method from 5.7 to 8, in nanoliposomes (< 50 nm) containing oleyl-peptide, lecithin and Tween-80 (1:1:0.75 mole ratio). The oleyl-peptide did not prove any haemolytic activity (< 1.5%) up to 10-fold of its experimental concentration. The curcumin-loaded liposomes displayed significant reduction in the viabilities of MCF-7 (IC50 3.8 µM) and MDA-MB-468 (IC50 5.4 µM). DISCUSSION AND CONCLUSION: This study indicated potential advantages of the peptide-conjugated liposomes in drug transport to the cancer cells. This feature might be an outcome of probable interactions between the targeted nanoliposomes with the integrin and neuropilin-1 receptors.

ChAcNLS, a Novel Modification to Antibody-Conjugates Permitting Target Cell-Specific Endosomal Escape, Localization to the Nucleus, and Enhanced Total Intracellular Accumulation.

The design of antibody-conjugates (ACs) for delivering molecules for targeted applications in humans has sufficiently progressed to demonstrate clinical efficacy in certain malignancies and reduced systemic toxicity that occurs with standard nontargeted therapies. One area that can advance clinical success for ACs will be to increase their intracellular accumulation. However, entrapment and degradation in the endosomal-lysosomal pathway, on which ACs are reliant for the depositing of their molecular payload inside target cells, leads to reduced intracellular accumulation. Innovative approaches that can manipulate this pathway may provide a strategy for increasing accumulation. We hypothesized that escape from entrapment inside the endosomal-lysosomal pathway and redirected trafficking to the nucleus could be an effective approach to increase intracellular AC accumulation in target cells. Cholic acid (ChAc) was coupled to the peptide CGYGPKKKRKVGG containing the nuclear localization sequence (NLS) from SV-40 large T-antigen, which is termed ChAcNLS. ChAcNLS was conjugated to the mAb 7G3 (7G3-ChAcNLS), which has nanomolar affinity for the cell-surface leukemic antigen interleukin-3 receptor-α (IL-3Rα). Our aim was to determine whether 7G3-ChAcNLS increased intracellular accumulation while retaining nanomolar affinity and IL-3Rα-positive cell selectivity. Competition ELISA and cell treatment assays were performed. Cell fractionation, confocal microscopy, flow cytometry, and Western blot techniques were used to determine the level of antibody accumulation inside cells and in corresponding nuclei. In addition, the radioisotope copper-64 ((64)Cu) was also utilized as a surrogate molecular cargo to evaluate nuclear and intracellular accumulation by radioactivity counting. 7G3-ChAcNLS effectively escaped endosome entrapment and degradation resulting in a unique intracellular distribution pattern. mAb modification with ChAcNLS maintained 7G3 nM affinity and produced high selectivity for IL-3Rα-positive cells. In contrast, 7G3 ACs with the ability to either escape endosome entrapment or traffic to the nucleus was not superior to 7G3-ChAcNLS for increasing intracellular accumulation. Transportation of (64)Cu when complexed to 7G3-ChAcNLS also resulted in increased nuclear and intracellular radioactivity accumulation. Thus, ChAcNLS is a novel mAb functionalizing technology that demonstrates its ability to increase AC intracellular accumulation in target cells through escaping endosome entrapment coupled to nuclear trafficking.

Intracellular Availability of pDNA and mRNA after Transfection: A Comparative Study among Polyplexes, Lipoplexes, and Lipopolyplexes.

Intracellular availability of nucleic acids from synthetic vectors is critical and directly influences the transfection efficiency (TE). Herein, we evaluated the TE of polymer- and lipid-based nanoplexes (polyplexes, lipoplexes and lipopolyplexes) of EGFP-encoding mRNA and pDNA. To determine the translation and transcription efficiency of each nucleic acid nanoplex, in vitro expression was measured in HEK293T7 cells that permit gene expression in the cytoplasmic region. Globally, mRNA transfection profile was well corroborative with cytoplasmic transfection of pT7-pDNA as well as with nuclear transfection of pCMV-DNA. Irrespective of the nucleic acid, high TE was observed with histidinylated l-polyethylenimine (His-lPEI) polyplexes and dioleyl succinyl paromomycin/O,O-dioleyl-N-histamine phosphoramidate (DOPS/MM27) lipoplexes. Moreover, His-lPEI polyplexes yielded higher in vitro expression of EGFP for pDNA than for mRNA. Furthermore, a significant enhancement in the TE in the presence of an excess of His-lPEI was observed indicating that this polymer promotes cytosolic delivery. Compared to other nanoplexes, His-lPEI polyplex showed high intracellular availability of DNA and mRNA along with low cytotoxicity, owing to its rapid (complete or partial) unpacking in the cytosol and/or endosomes. This study gives an insight that, whether with mRNA or pDNA, enhancing nanoplex unpacking in the endosomes and cytosol would improve the delivery of nucleic acid in the cytosol and particularly in the case of pDNA where a sufficient available amount of pDNA in the cytoplasm would definitely improve its transport toward the nucleus.

Development of Long-Circulating pH-Sensitive Liposomes to Circumvent Gemcitabine Resistance in Pancreatic Cancer Cells.

PURPOSES: To develop pH-sensitive liposomes (PSL) containing a high content of gemcitabine; and to investigate whether drug loading (DL) would alter the in vitro and pharmacokinetic properties. METHODS: PSL with a high DL were obtained using a modified small-volume incubation method. The DL effects on drug release rate and in vitro cytotoxicity of PSL were evaluated using MIA PaCa-2 pancreatic cancer cells and their pharmacokinetics investigated in rats. RESULTS: The highest DL of 4.5 ± 0.1% was achieved for gemcitabine in PSL with 145 ± 5 nm diameter. DL did not alter the in vitro release rate from PSL. The IC50 (48 h) of PSL (DL 0.5 and 4.5%) and non pH-sensitive liposomes (NPSL, DL 4.2%) were 1.1 ± 0.1, 0.7 ± 0.1 and 37.0 ± 7.5 µM, respectively. The PSL resulted in a 4.2-fold increase in its elimination half-life (6.2 h) compared to gemcitabine solution (1.4 h) in rats. No significant difference in pharmacokinetic parameters was observed between the two PSL (DL 0.5 and 4.5%). CONCLUSION: The PSL offered advantages over NPSL in restoring the sensitivity of pancreatic cancer cells to gemcitabine without requiring a high DL. DL in the PSL did not alter release rate, cytotoxicity or their long-circulating properties. Graphical Abstract ᅟ.

Targeted intracellular delivery of proteins with spatial and temporal control.

While a host of methods exist to deliver genetic materials or small molecules to cells, very few are available for protein delivery to the cytosol. We describe a modular, light-activated nanocarrier that transports proteins into cells by receptor-mediated endocytosis and delivers the cargo to the cytosol by light triggered endosomal escape. The platform is based on hollow gold nanoshells (HGN) with polyhistidine tagged proteins attached through an avidity-enhanced, nickel chelation linking layer; here, we used green fluorescent protein (GFP) as a model deliverable cargo. Endosomal uptake of the GFP loaded nanocarrier was mediated by a C-end Rule (CendR) internalizing peptide fused to the GFP. Focused femtosecond pulsed-laser excitation triggered protein release from the nanocarrier and endosome disruption, and the released protein was capable of targeting the nucleoli, a model intracellular organelle. We further demonstrate the generality of the approach by loading and releasing Sox2 and p53. This method for targeting of individual cells, with resolution similar to microinjection, provides spatial and temporal control over protein delivery.

Improved nuclear delivery of antisense 2'-Ome RNA by conjugation with the histidine-rich peptide H5WYG.

BACKGROUND: Antisense oligonucleotides are promising medicines for treating various diseases, although their efficiency still requires high doses. Their delivery in the cytosol and nucleus to reach their mRNA targets would increase their efficiency at the same time as reducing the dose. METHODS: We conjugated the histidine-rich peptide H5WYG (GLFHAIAHFIHGGWHGLIHGWYG) at the 5'-end of the RNase H-incompetent antisense 2'-O-methyl-phosphodiester oligonucleotide (2'-Ome RNA705) targeting aberrant splicing of luciferase pre-mRNA in HeLa pLuc705 cells. H5WYG was also conjugated with 2'-Ome-RNA705 labelled by fluorescein at the 3'-end. Then, H5WYG-2'-Ome-RNA705 conjugate and 2'-Ome-RNA705 were formulated with lipofectamine to favor their uptake in HeLa pLuc705 cells. RESULTS: Confocal microscopy showed that, after 4 h and overnight incubation, the presence of fluorescein-labelled 2'-Ome-RNA705 in the cytosol and nucleus was enhanced when the oligonucleotide was conjugated with H5WYG. We found that H5WYG-2'-Ome-RNA705 increased the splicing redirection and restoration of a functional luciferase mRNA. Luciferase activity and luciferase mRNA levels in these cells were 6.6- and two-fold higher, respectively, with H5WYG-2'-Ome-RNA705 than with 2'-Ome-RNA705. CONCLUSIONS: The results of the present study show that the conjugation of 2'-Ome antisense RNA to peptide H5WYG is a good strategy for improving its cytosol delivery, accumulation in the nucleus and antisense activity.

Effects of Histidine Oligomers in Lipid Nanoparticles on siRNA Delivery.

In this study, histidine oligomer (oHis; 10mer)-incorporating LNPs (H10LNPs) are developed as a novel carrier for efficient siRNA delivery. Notably, the unmodified oHis (10mer) is greatly incorporated within LNPs through ionic interaction with siRNAs, which serves as an endosome escape enhancer. H10LNPs with a size of ≈65 nm demonstrate a significantly enhanced extent of endosomal escape, as evidenced by calcein assay and confocal microscopy images of intracellular fluorescence, surpassing conventional LNPs. Furthermore, the half inhibitory concentration (IC50) of the human endogenous globotriaosylceramide synthase (Gb3 synthase) gene in H10LNPs-treated cells exhibits a significant threefold decrease, compared to that in LNP-treated cells. Notably, H10LNPs maintain comparable biocompatibility and biodistribution both in vitro and in vivo. Considering that the fabricated siRNA H10LNPs exhibit excellent biocompatibility and superior gene silencing activity over conventional LNPs, these particles can be harnessed for the safe delivery of therapeutic siRNAs. Additionally, this study introduces promising, feasible, simple, and alternative formulation processes for integrating unmodified functional cationic peptides into LNPs to enhance the delivery efficiency of a wide range of nucleic acid-based drugs.