A therapeutic nucleic acid-encased nanoworm for gene delivery and endosomal escape

Abstract

The present invention relates to effective and safe compositions comprising anionic gold-polydopamine core-shell nanoworms as alternative gene vectors to achieve endosome escape bottlenecks. The nanoworms are useful in methods of delivering therapeutic oligonucleotides to a subject. The invention discloses a polydopamine shell supporting surface adsorption of nucleic acid. The anionic nucleic acid entrapped nanoinsect can then enter the cell without transfection agent and activate the ClC3 H + / Cl-ion exchanger in the late endosome to mediate accumulation in the vesicles of H + and Cl-, resulting in membrane rupture, ultimately escaping into the cytosolute without cell penetrating peptide or mechanical stimulation. The nanoworms can also be used to program cells (i.e., primary macrophage polarization and stem cell differentiation) and to treat diseases (such as renal fibrosis and acute liver injury).

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202480065753.2 (22) Application Date 2024.10.12 (30) Priority Data 63 / 590,241 2023.10.13 US (85) PCT International Application Entering National Phase Date 2026.04.13 (86) PCT International Application Application Data PCT / CN2024 / 124523 2024.10.12 (87) PCT International Application Publication Data WO2025 / 077898 EN 2025.04.17 (71) Applicant The Chinese University of Hong Kong Address Sha Tin, New Territories, Hong Kong, China (72) Inventors Cai Zongheng Xiao Yu (74) Patent Agency Beijing Gaowo Law Firm 11569 Patent Attorney Li Qiao (51) Int.Cl. A61K 47 / 69 (2017.01) A61K 31 / 7088 (2006.01) A61K 48 / 00 (2006.01) C12N 15 / 113 (2010.01) A61P 1 / 16 (2006.01) A61P 13 / 12 (2006.01) A61K 9 / 51 (2006.01) A61K 47 / 24 (2006.01) (54) Invention Title A Nanoworm Encapsulated with Therapeutic Nucleic Acids for Gene Delivery and Endosome Escape (57) Abstract This invention relates to effective and safe compositions comprising anionic gold-polydopamine core-shell nanoworms as alternative gene carriers to overcome endosome escape bottlenecks. The nanoworms can be used in a method for delivering therapeutic oligonucleotides to a subject. A polydopamine shell supporting surface-adsorbed nucleic acids. Nanoworms encapsulating anionic nucleic acids can then enter cells without transfection and activate ClC3 H+ / Cl- ion exchangers in late endosomes to mediate the accumulation of H+ and Cl- within vesicles, leading to membrane rupture and ultimately escape into the cytosol without cell-penetrating peptides or mechanical stimulation. Nanoworms can also be used for cell programming (i.e., primary macrophage polarization and stem cell differentiation) and for treating diseases (such as renal fibrosis and acute liver injury). Claims 2 pages Description 47 pages Sequence Listing (electronic publication) Figures 80 pages CN 122341398 A 2026.07.03 CN 1 22 34 13 98 A 1. A nanoworm composition comprising: at least two metal nanoparticle cores or nanorod cores; a polymer coating layer located on said at least two metal nanoparticle cores or nanorod cores; a therapeutic nucleic acid; and optionally, a lipid coating layer located on the polymer coating layer.2. The nanoworm composition of claim 1, wherein the at least two metal nanoparticle cores or nanorod cores are at least two gold nanoparticle cores. 3. The nanoworm composition of claim 1, wherein each of the at least two metal nanoparticle or nanorod cores has a diameter of about 40 nm. 4. The nanoworm composition of claim 1, comprising 4 or 5 metal nanoparticle cores or nanorod cores. 5. The nanoworm composition of claim 1, wherein the thickness of the polymer coating layer is from about 7 nm to about 35 nm. 6. The nanoworm composition of claim 1, wherein the polymer coating layer comprises polydopamine (PDA), polyethylene glycol (PEG), polyethyleneimine (PEI), or silica. 7. The nanoworm composition of claim 1, wherein the nanoworm composition carries an anionic charge. 8. The nanoworm composition of claim 1, wherein the therapeutic nucleic acid is an oligonucleotide or mRNA. 9. The nanoworm composition of claim 1, wherein the length of the therapeutic nucleic acid is from about 15 to about 5000 nucleotides. 10. The nanoworm composition of claim 1, wherein the therapeutic nucleic acid is antisense DNA, antisense oligonucleotide (ASO), short interfering RNA (siRNA), microRNA (miRNA), messenger RNA (mRNA), or any combination thereof. 11. The nanoworm composition of claim 8, wherein the therapeutic oligonucleotide nucleic acid is adsorbed onto the polymer coating layer. 12. The nanoworm composition of claim 10, wherein the mRNA encodes hepatocyte growth factor (HGF), chemokine receptor type 4 (CXCR4), bone morphogenetic protein 7 (BMP-7), enhanced green fluorescent protein (EGFP), red fluorescent protein (RFP), or any combination thereof; the miRNA is miR223; or the siRNA is siNog. 13. The nanoworm composition of claim 1, further comprising a lipid coating layer on the polymer coating layer, wherein the therapeutic nucleic acid is bound to the lipid coating layer. 14. The nanoworm composition according to claim 13, wherein the lipid coating layer comprises 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), cholesterol, 1,2-distearate-sn-glycerol-3-phosphocholine (DOPE), 1,1′-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azadiyl)bis(dodecane-2-ol)) (C12-200), (6Z,9Z,28Z,31Z)-heptadecane-6,9,28,31-tetraene-19-yl-4-(dimethylamino)butyrate (DLin-MC3-DMA), ((4-hydroxybutyl)azadiyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), dimethyl bis(octadecyl)ammonium bromide (DDA), 1,2-distearyl-sn-glycerol-3-phosphocholine (DSPC), 1,2-dioleoyl-3-dimethylammonium propane (DODAP), 1,2-dioleoyl-3-dimethylaminopropane (DODMA), dipalmitoylphosphatidylcholine (DPPC), or any combination thereof. 15. The nanoworm composition of claim 1, wherein the nanoworm comprises citrate-terminated gold nanoparticles (Cit-Au NP) with a diameter ranging from about 20 to about 60 nm. 16. The nanoworm composition of claim 15, wherein the nanoworm comprises Aux@PDA NW, wherein the diameter of x is about 20, about 40, or about 60 nm. Claims 1 / 2 Page 2 CN 122341398 A 17. The nanoworm composition of claim 1, wherein the nanoworm comprises Au@PDA NR (nanoran), wherein the NR has a size of about 45 nm × about 180 nm. 18. A method of nucleic acid delivery, comprising administering the nanoworm composition of claim 1 to a subject. 19. The method of claim 18, wherein the therapeutic nucleic acid modulates gene expression in the subject. 20. The method of claim 18, wherein the therapeutic nucleic acid escapes endosome and / or lysosomal degradation. 21. The method of claim 18, wherein the therapeutic nucleic acid enhances stem cell differentiation. 22. The method of claim 19, wherein the modulation of gene expression in the subject treats acute liver injury, chronic kidney disease, or a combination thereof. 23. The method of claim 18, wherein the nanoworm enters epithelial cells, hepatocytes, kidney cells, endothelial cells, primary macrophages, mesenchymal stem cells, nerve cells, or any combination thereof in the subject. 24. The composition of claim 18, wherein the nanoworm is administered via intra-articular injection, intrathecal injection, or retrobulbar injection. Claims 2 / 2 Page 3 CN 122341398 A Nanoworm Encapsulated with Therapeutic Nucleic Acid for Gene Delivery and Endosome Escape Cross-Reference to Related Applications

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 590,241, filed October 13, 2023, the entire contents of which (including any forms, figures, or drawings) are incorporated herein by reference. Background Art

[0002] In recent decades, interest in gene therapy has been rekindled. Currently, several approved and marketed gene therapies exist, such as Givlaari® (AlnylamPharmaceuticals (Cambridge, Massachusetts), Waylivra® (Akcea Therapeutics, Carlsbad, California), and Tegesedi® (Akcea Therapeutics).

[0003] Efficient intracellular delivery of nucleic acids is fundamental to important biomedical applications such as gene regulation, expression, and editing. To date, various gene delivery methods have been developed, including viral transduction, electroporation, and nanoparticle-based transfection. Viral transduction has high transduction efficiency but raises safety concerns regarding genotoxicity and insertional mutations. Electroporation is cytotoxic due to high voltage in vivo and limited penetration depth. A major advance in nucleic acid delivery has been the use of nanoparticles (NPs). NPs are among the most promising non-viral, non-physical methods in gene therapy. Although nucleic acid-based NPs have been clinically applied, there are still obstacles hindering their efficient intracellular delivery because nucleic acid-based NPs are readily degraded by nucleases and have unfavorable physicochemical properties that prevent them from being taken up by cells. Unfortunately, upon initial entry into cells, transfected genes often face significant endolysosomal encapsulation problems, leading to intralysosomal degradation and thus inefficient gene release into the cytosol. Therefore, their subsequent escape from acidic organelles is crucial for gene therapy applications.

[0004] With the global deployment of mRNA-based COVID-19 vaccines, the field of nanoparticle-based (NP) gene delivery has flourished, but since 2019, only 11 out of more than 33,000 publications have reported correlation coefficients ≤0.2 between nanoparticles and endosomes (indicating little or no spatial correlation) (Table 1). Instead, most studies focus on material design and downstream transfection efficiency, often lacking detailed information on endosome escape efficiency or the biological mechanisms of endosome escape (Table 2).

[0005] Nanoparticles provide a customized gene delivery vector with tunable size and shape and diverse functions. Many nanocarriers enable efficient cellular uptake and packaging of target genes, but endosome / lysosomal encapsulation can hinder efficacy by causing degradation, preventing gene release into the cytosol. Currently, several commonly used carriers aim to overcome the barriers to endosome escape. These include (1) cationic polymer-based nanoparticles or pH-responsive polymer-based nanoparticles (which function through the proton sponge effect), such as polyethyleneimine (PEI), polylysine, and polyamidoamine (PAMAM); (2) lipid-based nanoparticles (e.g., liposomes, lipid nanoparticles) that function through membrane fusion or the proton sponge effect (ionizable lipids); and (3) cell-penetrating peptides that function through membrane rupture. However, the probability of endosome escape for the above-mentioned carriers remains very low; it has been reported that only about 1% of ionizable lipid NPs have successfully achieved endosome escape.[1]. In addition, positively charged cationic NPs are often associated with high cytotoxicity [2].

[0006] Cationic lipids [3] and polymers [4] are classic gene carriers. Cationic lipid transfectants bind to therapeutic genes via electrostatic binding to form near-neutral nanoparticles, making them easier to enter cells. They aggregate nucleic acids into nanoparticle-based (NP) complexes to overcome the electrostatic repulsion of anionic cell membranes and promote cellular uptake. In addition, based on the well-known "proton sponge effect", cationic carriers have amine groups and sufficient pH buffering capacity to trigger the internal accumulation of protons via the endosome proton pump (V-ATPase), balancing chloride ions and ultimately increasing osmotic pressure leading to membrane rupture [5]. However, cationic carriers induce cytotoxicity or inflammatory responses [6,7] and have low endosome escape efficiency [1]. Recently, some anionic bionanomaterials have emerged as alternative gene carriers because they enter cells in large quantities via cell surface receptors [8] without causing cytotoxicity. Nevertheless, anion carriers are still subject to endosome retention [9] and typically require pH buffer groups

[10] , cell-penetrating peptides

[11] , or mechanical stimulation

[12] to escape. Gene carriers combining the advantages of cationic and anion carriers (biocompatibility, strong cellular uptake, and endosome escape driven by their physicochemical nanostructures) are rarely reported. However, efficient gene delivery remains crucial for effective gene regulation. Therefore, there is an urgent need for a safe and reliable gene delivery nanocarrier to more effectively regulate genes and thus enhance cell therapy. Summary of the Invention

[0007] The present invention relates to a novel polymer-coated nanoworm (NW). In some embodiments, the polymer is polydopamine (PDA), polyethylene glycol (PEG), polyethyleneimine (PEI), or silica. More specifically, the nanoworm is an integrated core-shell nanoparticle with metal nanochains serving as a backbone and a polymer shell for adsorbing molecules, such as oligonucleotides or an additional lipid coating layer complexed with mRNA. In some embodiments, the metal is gold (Au), copper, silver, iron oxide, cerium dioxide, zinc oxide, titanium oxide, rhodium, platinum, and iron oxide. In a preferred embodiment, the nanoparticles comprise citrate-terminated gold nanoparticles (Cit-Au NPs). In some embodiments, the diameter of the Cit-Au NPs ranges from about 20 to about 60 nm. In a preferred embodiment, the diameter of the Cit-Au NPs is about 40 nm. In some embodiments, the nanoparticles of the present invention comprise Aux@PDA NW, wherein the diameter of x is about 20, about 40, or about 60 nm. In some embodiments, the nanoparticles of the present invention comprise Au@PDA NR (nanorobars) with a size of about 45 nm × about 180 nm.

[0008] In some embodiments, the polymer nanoworms of the present invention can enter cells and escape from endosomes and / or lysosomes, effectively modulating genes. In some embodiments, the nanoworms can leave late endosomes by increasing chloride ion accumulation in intracellular vesicles, leading to vesicle swelling and subsequent membrane rupture. In some embodiments, the platform of the present invention has more effective gene regulation in vitro and in vivo compared to the widely used transfection agent Lipofectamine 3000, thereby enhancing cell therapy through genetic engineering.

[0009] In some embodiments, the nanoworms of the present invention encapsulating therapeutic nucleic acids are effective in cellular delivery with limited localization within acidic organelles such as late endosomes and lysosomes. In some embodiments, the nanoworms of the present invention maintain an overall anionic charge. In some embodiments, nanoworms can be loaded with different types of therapeutic nucleic acids, such as antisense DNA, antisense oligonucleotides (ASO), short interfering RNA (siRNA), microRNA (miRNA), and messenger RNA (mRNA); enter various cell types in vitro, such as epithelial cells, endothelial cells, primary macrophages, and human mesenchymal stromal cells, and in vivo, such as hepatocytes; and have limited co-localization with endosomes and / or lysosomes, with co-localization coefficients ranging from about 0.1 to about 0.2.

[0010] The patent or application documents contain at least one color drawing. Upon request and payment of the necessary fees, the Patent Office will provide a color drawing copy of the publication of this patent or patent application.

[0011] Figures 1A-1E show gold@polydopamine nanoworms (Au@PDA@oligonucleotide NW) encapsulated with oligonucleotides for intracellular delivery and endosome escape. Figure 1A shows the preparation route of nanoworms encapsulated with oligonucleotides for delivering various types of oligonucleotides. Figure 1B shows a representative TEM image of Au@PDA@T21NW, illustrating the nucleoshell Au@PDA NW structure. Scale bar = 50 nm. Figure 1C shows confocal images of four different cell types, illustrating the limited colocalization of Cy5-labeled Au@PDA@T21NW with the intracellular acidic compartment after 24 h of incubation. Blue = nucleus. White numbers represent the Pearson colocalization coefficient (PCC) between Cy5-labeled Au@PDA@T21NW (red) and Lysotracker (green). Figure 1D shows representative TEM images of four different cell types, illustrating the cytoplasmic accumulation of Au@PDA@T21NW after 24 h of incubation. Nu = nucleus; Cy = cytosol. Scale bar = 500 nm. Figure 1E shows the confocal images of Cy5-labeled Au@PDA@T21NW (red) in the intracellular acidic compartment.Trajectory changes in A549 cells over incubation time. EEA1, Rab9, and LAMP1 are markers of early endosomes, late endosomes, and lysosomes, respectively. Blue = cell nucleus. White numbers represent Pearson colocalization coefficients between Cy5-labeled Au@PDA@T21NW (red) and different organelle markers (green). Scale bar = 20 μm.

[0012] Figures 2A-2L show the ClC3-mediated endosome escape of Au@PDA@T21NW in A549 cells. Figure 2A shows that pharmacological inhibition of the endosome proton pump by bafloxacin A1 or chloroquine does not affect the endosome escape of Au@PDA@T21NW. White numbers represent Pearson colocalization coefficients between Cy5-labeled Au@PDA@T21NW (red) and Lysotracker (green). Scale bar = 20 μm. Figure 2B shows the titration curve of Au@PDA@T21NW. Brine and branched-chain polyethyleneimine (bPEI) were used as controls. Figures 2C and 2D show the key enrichment bioprocesses (Q<0.05) identified by comparing (c) the “Au@PDA@T21NW; 24 h” group with the “Au@PDA@T21NP 24 h” group and (d) the “Au@PDA@T21NW; 24 h” group with the “Au@PDA@T21NW; 8 h” group. All groups n=3. Figures 2E and 2F show volcano plots illustrating the distribution of DET identified by comparing (e) the “Au@PDA@T21NW; 24 h” group with the “Au@PDA@T21NP; 24 h” group and (f) the “Au@PDA@T21NW; 24 h” group with the “Au@PDA@T21NW; 8 h” group. All groups n=3. Figure 2G shows Western blot analysis, illustrating the upregulation of ClC3 in the “Au@PDA@T21NW; 24 h” group relative to the “Au@PDA@T21NP; 24 h” group (top row) and the “Au@PDA@T21NW; 8 h” group (bottom row). n=3 in one experiment. Figure 2H shows the subcellular localization of ClC3 (green) relative to intracellular vesicles (red). Scale bar = 10 μm. Figure 2I shows that RNAi-mediated ClC3 knockout significantly interferes with the escape of Au@PDA@T21NW (red) from acidic vesicles (green). Figure 2J shows that pharmacological inhibition of general chloride channels by nifluonic acid (NFA) impairs the escape of Au@PDA@T21NW (red) from acidic vesicles (green). Figure 2K shows that ClC3 activation accelerated endosome escape from Au@PDA@T21NW 8 hours after incubation. Blue = cell nucleus. Figures 2A, 2I, 2J, and 2K show white numbers, which represent Cy5-labeled Au@PDA@T21NW.Pearson colocalization coefficients (red) and Lysotracker (green). Blue = cell nucleus. Figure 2L shows a summary of PCC values ​​under different treatment conditions. Data are expressed as mean ± SEM, with n = 10 fields of view per group (approximately 150 cells were calculated, and one mean was calculated for each field of view).

[0013] Figures 3A-3H show the ion concentrations and membrane rupture of endosomes in A549 cells. Figure 3A shows the Cl- concentration of intracellular vesicles in Cy5-labeled Au@PDA@T21NW cells (top row) without siClC3 treatment or in siClC3-treated cells (bottom row). MQAE staining signal is negatively correlated with Cl- concentration. White arrows indicate that intracellular vesicles in Cy5-labeled Au@PDA@T21NW cells have relatively higher Cl- concentrations in WT A549 cells compared to A549 cells treated with siClC3 (indicated by magenta arrows). Figure 3B shows the pH determination of intracellular vesicles after 8 hours of co-incubation with Cy5-labeled Au@PDA@T21NW (magenta), dextran 10K pHrodo (green), and dextran 10K AF594 (red), with or without siClC3 treatment (top row) or with siClC3 treatment (bottom row). White arrows indicate intracellular vesicles containing Cy5-labeled Au@PDA@T21NW, while yellow arrows indicate intracellular vesicles without Cy5-labeled Au@PDA@T21NW. The R / G ratio is the ratio of the fluorescence intensity of dextran 10K pHrodo green to the fluorescence intensity of dextran 10K AF594, as shown in the heatmap. Scale bar = 10 μm. Figure 3C shows the quantification of MQAE staining fluorescence intensity based on (a). Figure 3D shows the quantification of pH of individual vesicles in WT cells and siClC3-treated cells by measuring the R / G ratio of intracellular vesicles containing Cy5-labeled Au@PDA@T21NW versus those without Cy5-labeled Au@PDA@T21NW. n = 300 intracellular vesicles. Figure 3E shows the assessment of endosome proton pump activity before and after Au@PDA@T21NW incubation. Endosomal proton pump activity was determined by the co-localization of subunits V0 and V1. White numbers indicate PCC between anti-V1 (red) and anti-V0 (green). Blue = cell nucleus. Figure 3F shows the proposed mechanism of endosome escape from Au@PDA@T21NW. Figure 3G shows green clusters, indicating that Gal8-GFP was recruited to ruptured organelle membranes after 24 h of incubation with Au@PDA@T21NW, but this phenomenon was not observed when incubated with Au@PDA@T21NP (page 3 / 47, CN 122341398 A). Blue = cell nucleus. Figure 3H shows a representative TEM image, which shows Au@PDA@T21NW (orange arrow) originates from a ruptured intracellular vesicle membrane. Cy = cytosol, ex = extracellular space.

[0014] Figures 4A-4H show that in vitro nanoworm transfection with oligonucleotides can induce polarization of BMDM and osteogenic differentiation of hMSCs. Figure 4A shows that Cy5-labeled Au@PDA@miR-223 NW (red) did not accumulate in the acidic organelles (green) of BMDM after 24 h of incubation. Figure 4B shows a representative TEM image of endosome escape from BMDM after 24 h of incubation with Au@PDA@miR-223 NW. Figure 4C shows the qRT-PCR results of the M2 phenotypic marker in BMDM. n=6 in each group in 2 experiments. Figure 4D shows a confocal image showing the repolarization of BMDM from M1 to M2, which has been demonstrated by the stronger M2 marker (CD206; green) and the weaker M1 marker (CD80; red). Figure 4E shows that Cy5-labeled Au@PDA@siNog NW (red) did not accumulate in acidic organelles (green) in hMSCs after 24 h of incubation. White numbers represent the Pearson colocalization coefficients of Cy5-labeled Au@PDA@siNog NW (red) and Lysotracker (green). Blue in Figures 4A, 4D, and 4E represents cell nuclei. Figure 4F shows a representative TEM image of endosome escape from Au@PDA@siNog NW in hMSCs after 24 h of incubation. Figure 4G shows the results of qRT-PCR assays of osteogenic markers in hMSCs 14 days after induced differentiation. n=6 per group in two experiments. Figure 4H shows calcium deposition in hMSCs stained with Alizarin Red 14 days after osteogenic differentiation. Scale bar = 100 μm. n=3 per group in one experiment. Figures 4C and 4G show data expressed as mean ± SEM. Statistical significance was calculated post-hoc by one-way ANOVA and Tukey's test. ns = no significant difference (P>0.05).

[0015] Figures 5A-5K show the transfection of mRNA in hMSCs by lipid nanoworms in vitro for cell therapy and to alleviate renal fibrosis. Figure 5A shows a schematic diagram of the synthesis of Au@PDA NWs coated with mRNA and lipids. Figure 5B shows that Cy5-labeled Au@PDA@mCXCR4 / BMP7 NWs did not accumulate in acidic organelles (green) after 24 h of incubation. White numbers represent the Pearson colocalization coefficients of Cy5-labeled Au@PDA@mCXCR4 / mBMP7 NWs (red) and Lysotracker (green). Blue = cell nucleus. Figure 5C shows a confocal image showing Au@PDA@lipid NWs incubated for 24 h with CXCR4-GFP-expressing cells.hMSCs co-transfected with mRNA (green) and mRNA expressing BMP7-OFP (red). Blue = cell nucleus. Figure 5D shows a schematic diagram of hMSC-based treatment for renal fibrosis in mice. After the left ureter was ligated twice with sutures, UUO mice received a single intravenous injection of saline or hMSCs under different treatments on day 3 and were sacrificed on day 14 post-UUO surgery. Figure 5E shows the quantification of CXCR4 and BMP7 expression in vitro. The therapeutic protein levels of hMSCs were assessed by ELISA 24 h post-transfection. Figure 5F shows in vitro fluorescence images of UUO kidneys, where the strongest fluorescence is shown in the group of hMSCs transfected with DiR-labeled Au@PDA@mCXCR4 / mBMP7 (NWs-hMSCCXCR4 / BMP7) 24 h post-injection. Lipo-hMSCCXCR4 / BMP7: hMSCs transfected with Lipo+mCXCR4 / mBMP7. Figure 5G shows IHC images illustrating the expression of Col-I and α-SMA in UUO kidney sections. Figures 5H and 5I show quantification of Col-I-positive and α-SMA-positive regions in UUO kidney sections, based on an average of 6 images per mouse. n=7 per group in both experiments. Figures 5J and 5K show the mRNA expression of Col-I and α-SMA quantified by qRT-PCR. Figure 5L shows the quantification of BMP7 levels in UUO kidneys by ELISA on day 14. Data are presented as mean ± SEM. Statistical significance was calculated using two-way ANOVA with Tukey's multiple comparison test. ns = no significant difference (P>0.05). n=7 per group in both experiments.

[0016] Figures 6A-6I show the transfection of hepatocytes with nanoworms in vivo to reduce acute liver injury (ALI). Figure 6A shows the disease progression and treatment plan for acute liver injury. Following intraperitoneal injection of APAP to induce acute liver injury, mice received a single intravenous injection of saline, Au@PDA@mHGF NW, or a mixture of Lipofectamine and mHGF on day 1 and were sacrificed on day 2. Figure 6B shows the cellular distribution of Cy5-labeled Au@PDA@mHGF NW (red) in the liver of mice with acute liver injury 24 h after injection. Blue = cell nuclei. Green = hepatocytes (labeled with anti-HNF4α antibody). Figure 6C shows a TEM image of the liver in acute liver injury, showing the accumulation of Au@PDA@mHGF NW in intracellular vesicles at 5 h post-injection (green arrows), but they remain in the cytosol (Cy) at 10 h post-injection (blue arrows). Orange arrows indicate Au@PDA@mHGF NW from the cytosol. (Instructions for use 4 / 47, page 7, CN 122341398 A)Escape from the ruptured membrane. Nu = nucleus. Cy = cytosol. Figure 6D shows a confocal image of the liver in acute liver injury, illustrating limited colocalization of Cy5-labeled Au@PDA@mHGF NW and organelles (late endosomes labeled with anti-Rab9 antibody and lysosomes labeled with anti-LAMP1 antibody, respectively) 10 hours post-injection. White numbers represent Pearson colocalization coefficients of Cy5-labeled Au@PDA@mHGF NW (red) and organelles (green). Blue = nucleus. Figures 6E, 6F, and 6I show apoptotic cells (TUNEL positive; green) in liver sections of acute liver injury by histological staining and serum ALT levels 24 h post-treatment, demonstrating that Au@PDA@mHGF NW most effectively reduced the necrotic area (yellow dashed line). Blue = nucleus. Figures 6G and 6H show quantification of necrotic and TUNEL positive areas. n=7 per group in 2 experiments. Data are expressed as mean ± SEM. All statistical data were analyzed using two-way ANOVA and Tukey's multiple comparison test was used. ns = no significant difference (P>0.05).

[0017] Figures 7A-7G show the improvement of streptozotocin (STZ)-induced diabetic nephropathy by mRNA-transfected MSCs. Figure 7A shows a schematic diagram of disease progression and treatment. Figure 7B shows the HGF secretion concentrations measured by ELISA in conditioned medium of Lipo+mHGF-transfected MSCs, Au@PDA@mHGF NW-transfected hMSCs, and initial hMSCs. Data are expressed as mean ± SEM. All statistical data were analyzed using two-way ANOVA and Tukey's multiple comparison test was used. ns = no significant difference (P>0.05). Figure 7C shows an ex vivo image of the biodistribution of injected hMSCs in the kidneys 24 h after NIRF imaging. Figure 7D shows the organ-level distribution of DiR-labeled initial hMSCs, Lipo-hMSCHGF, and LNW-hMSCHGF in STZ-induced diabetic mice 24 h after injection. Representative ex vivo NIRF images of STZ-induced diabetic mice after intravenous injection of free DiR dye molecules, DiR-labeled initial hMSCs, Lipo-hMSCHGF, and LNW-hMSCHGF. Figure 7E shows a typical IHC image, which shows the expression of type I collagen and α-SMA in UUO kidneys. Figure 7F shows the quantification of Col-I positive regions in IHC stained sections, and Figure 7G shows the quantification of α-SMA positive regions in IHC stained sections. For Figures 7E and 7F, kidney section data were obtained from an average of 6 images per mouse, with n=7 mice per group (spanning two independent experiments). Data are expressed as mean ± SEM.

[0018] Figures 8A-8C show citrate-capped 40nm Au NP (Figure 8A), Au40@PDARepresentative TEM images of NP (Fig. 8B) and Au40@PDA NW (Fig. 8C).

[0019] Figs. 9A-9F show representative TEM images of citrate-capped 60nm Au NP (Fig. 9A), Au60@PDA NW (Fig. 9B), citrate-capped 20nm Au NP (Fig. 9C), Au20@PDA NW (Fig. 9D), citrate-capped Au45NR (45×200 nm) (Fig. 9E), and Au45@PDA NR (Fig. 9F).

[0020] Figs. 10A-10D show the cytotoxicity of oligonucleotide nanoworms in vitro. (Figure 10A) shows Au40@PDA@T21NW, (Figure 10B) shows Au40@PDA@asEGFP NW, (Figure 10C) shows Au40@PDA@miR223 NW, and (Figure 10D) shows Au40@PDA@siNog-NW, which were incubated with A549 cells, bEnd.3 cells, BMDM, and hMSC for 24 h, respectively. Cells remained largely viable after incubation with all concentrations of oligonucleotide-encapsulated nanoworms as per the AlamarBlue assay. Data are presented as mean ± SEM. Statistical significance was determined post-hoc by one-way ANOVA and Tukey's test. ns: no significant difference (P>0.05). n=3 per group in one experiment.

[0021] Figures 11A-11D show the cytotoxicity of the pharmacological inhibitors. A549 cells (Fig. 11A), bEnd.3 cells (Fig. 11B), BMDM (Fig. 11C), and hMSCs (Fig. 11D) were incubated for 4 hours with various inhibitors of cellular uptake pathways, including 250 μg / mL amiloride, 25 μg / mL pembrolizumin III, 50 mM sodium azide, and 50 μg / mL fucoidan. Cells remained largely viable after incubation with all tested inhibitors as determined by the AlamarBlue assay. Data are presented as mean ± SEM. Statistical significance was assessed post-hoc by one-way ANOVA and Tukey's test. ns: no significant difference (P>0.05). In one experiment, n=6 per group.

[0022] Figs. 12A-12D illustrate the cellular uptake pathway of oligonucleotide nanoworms. A549 cells (Fig. 12A), bEnd.3 cells (Fig. 12B), BMDM (Fig. 12C), and hMSCs (Fig. 12D) were pre-incubated for 1 hour with various cellular uptake pathway inhibitors, and then respectively with nanoworms coated with T21, nanoworms coated with asEGFP, nanoworms coated with miR223, and nanoworms coated with siNog. (Instruction manual, page 5 / 47, 8 CN 122341398 A)Nanoworms were co-incubated for 4 hours. Cell uptake levels were measured by ICP-MS. Data are expressed as mean ± SEM. Post-hoc analysis was performed using the Tukey test, and statistical significance was calculated by one-way ANOVA. ns: no significant difference (P>0.05). In one experiment, n=6 per group.

[0023] Figures 13A-13B show representative confocal and transmission electron microscopy images of Au40@PDA@T21NP in A549 cells. Figure 13A shows that Au40@PDA@T21NP was highly co-localized with Lysotracker after 24 h of incubation. Blue = DAPI (nucleus). White numbers represent the Pearson colocalization coefficient (PCC) of Cy5-labeled Au40@PDA@T21NP (red) and Lysotracker (green). Figure 13B shows TEM images confirming that Au40@PDA@T21NP was encapsulated in intracellular vesicles. Nu = nucleus. Cy = Cytosol. Ex = Extracellular space. Yellow asterisks indicate vesicles. The two smaller images on the right show magnification of the boxed area of ​​the larger image on the left.

[0024] Figures 14A-14B show representative TEM images of Au40@PDA@T21NW in A549 cells. Most Au40@PDA@T21NW were encapsulated in late endosomes 8 hours after incubation (Figure 14A) and freed and accumulated in the cytosol (or extravesicles) after 24 h of incubation (Figure 14B). Nu = Nucleus. Cy = Cytosol. Ex = Extracellular space. Yellow asterisks indicate vesicles, and blue arrows indicate free c in the cytosol. In Figures 14A and 14B, the two smaller images on the right show magnification of the boxed area (blue or yellow) of the larger image on the left.

[0025] Figures 15A-15B show that Au20@PDA@T21NW (Figure 15A) and Au60@PDA@T21NW (red) (Figure 15B) were trapped in lysosomes (green) after 24 h of incubation, unlike Au40@PDA@T21NW which escaped the endosomes. Blue = cell nucleus. White numbers represent the Pearson colocalization coefficients between Cy5-labeled NW (red) and Lysotracker (green).

[0026] Figures 16A-16B show endosome escape and cellular uptake of Au45@PDA@T21NR in A549 cells. Figure 16A shows the colocalization of Au45@PDA@T21NR in A549 cells after 24 h of culture. Blue = cell nucleus. White numbers represent the Pearson colocalization coefficients between Cy5-labeled Au45@PDA@T21NR (red) and Lysotracker (green). Figure 16B shows the ICP-MS results, indicating that the intake of Au45@PDA@T21NR was approximately 65% ​​lower than that of the Au40@PDA@T21NW group. Data are presented as mean ± SEM.Figure 17 shows the zeta potential of Au40@PDA@T21NW in assay buffer. The data show that Au@PDA@T21NW remains negatively charged in the pH range of 4.5 to 7.5. Data are expressed as mean ± SEM. In one experiment, n=3 per group. Figure 18 shows the effect of Na+, K+ and Ca2+ channels on endosome escape of T21NW in A549 cells. For all pharmacological inhibitors tested, there was no significant escape of Au40@PDA@T21NW (red) from lysosomes (green) after 24 h of incubation. White numbers represent the Pearson colocalization coefficients of Au@PDA@T21NW (red) and Lysotracker (green). Blue = cell nucleus.

[0029] Figure 19 shows the effects of Na+, K+, Ca2+, Cl- channels and endosome proton pumps on Au40@PDA@T21NW endosome escape in bEnd.3 cells, BMDM, and hMSCs. White numbers represent Pearson colocalization coefficients for Au@PDA@T21NW (red) and Lysotracker (green). For bEnd.3 cells, NFA (a typical universal Cl- inhibitor) had little effect on endosome escape, and limited inhibition of Ca2+ channels and endosome proton pumps. For BMDM and hMSCs, all tested inhibitors had little or no significant effect on Au@PDA@T21NW endosome escape.

[0030] Figures 20A-20B show the quantification of ClC3 expression protein blot data under different comparisons, including (Figure 20A) time-dependent comparisons and (Figure 20B) shape-dependent comparisons. Data are expressed as mean ± SEM. Statistical significance was calculated using Student's t-test. In one experiment, n=3 per group.

[0031] Figure 21 shows the subcellular localization of ClC3 relative to intracellular vesicles in bEnd.3 cells, BMDM, and hMSCs. ClC3 strongly colocalizes with lysosomes (LAMP1) and less so with late endosomes (Rab9). White numbers represent the Pearson colocalization coefficients between ClC3 (green) and intracellular vesicles (red). Scale bar = 10 μm.

[0032] Figure 22 shows the validation of siClC3 knockout in A549 cells. Cells were incubated with two different siClC3 sequences at three different concentrations using Lipofectamine 3000, and their relative mRNA expression of ClC3 was assessed 48 hours after incubation. Randomized siRNA sequences (siNC) served as negative controls. Data are presented as mean ± SEM. Statistical significance was assessed by one-way analysis.Post-hoc analysis was performed using ANOVA and Tukey's test. ns = no significant difference (P>0.05). n=3 per group in one experiment.

[0033] Figures 23A-23B show the validation of bufotoxin activating ClC3 in A549 cells. Figure 23A shows the relative mRNA expression of ClC3, and Figure 23B shows the cell viability after 8 hours of incubation with different concentrations of bufotoxin. We chose 0.5 μM bufotoxin for subsequent studies because it effectively upregulated ClC3 with low cytotoxicity. Data are expressed as mean ± SEM. Statistical significance was post-hoc analysis using one-way ANOVA and Dunnett's test. ns = no significant difference (P>0.05). n=3 per group in one experiment.

[0034] Figure 24 shows the validation of siClC5 knockout in A549 cells. Cells were incubated with three different concentrations of siClC5 sequences using Lipofectamine 3000, and the relative mRNA expression of ClC3 was assessed 48 hours after incubation. Data are presented as mean ± SEM. Statistical significance was assessed post-hoc by one-way ANOVA and Tukey's test. ns = no significant difference (P>0.05). n=3 per group in one experiment.

[0035] Figure 25 shows the experimental design for intracellular vesicle pH assay. A549 cells were incubated for 8 hours with a mixture of dextran 10K pHrodo green, dextran 10K AF594, and Cy5-labeled Au@PDA@T21NW. Some vesicles contained only dextran 10K pHrodo green, only dextran 10K AF594, both dyes, only Cy5-labeled Au@PDA@T21NW, dextran 10K pHrodo green and Cy5-labeled Au@PDA@T21NW, Cy5-labeled dextran 10K AF594 and Au@PDA@T21NW. Only intracellular vesicles that were positive for both dextran 10K AF594 and dextran 10K pHrodo green were considered for pH determination.

[0036] Figures 26A-26D show endosome rupture due to treatment with Au@PDA@T21NW. Other representative confocal images of Gal8-GFP transfected A549 cells, untreated (Fig. 26A), (Fig. 26B) treated with Au@PDA@T21NW for 24 h, and (Fig. 26C) treated with Au@PDA@T21NP for 24 h. Green clusters indicate Gal8-GFP recruitment to ruptured organelle membranes after 24 h of incubation with T21NW. Blue = cell nuclei. Scale bar = 10 μm. Fig. 26D shows the quantification of Gal8 clusters. Data are presented as mean ± SEM. n = 20 per group in 3 experiments. Post-hoc analysis was performed using the Tukey test, with one-way analysis.Analysis of variance was used to calculate statistical significance. ns: no significant difference (P>0.05).

[0037] Figures 27A-27B show the rupture of intracellular vesicle membranes. Representative TEM images show the vesicle membrane rupture of A549 cells (Figure 27A) and hMSCs (Figure 27B) after 8 hours of incubation with Au@PDA@T21NW and Au@PDA@siNog NW, respectively. Orange arrows indicate that nanoworms are escaping from the ruptured vesicle membranes. Ex = extracellular space. Cy = cytosol.

[0038] Figures 28A-28D show the knockout of EGFP mediated by Au@PDA@asEGFP NW in bEnd.3 cells. Figure 28A shows a confocal image of bEnd.3 cells expressing EGFP, showing limited colocalization of Cy5-labeled Au@PDA@asEGFP-NW with the intracellular acidic compartment after 24 h of incubation. Blue = cell nucleus. White numbers represent the Pearson colocalization coefficient (PCC) between Cy5-labeled Au@PDA@EGFP-NW (red) and Lysotracker (green). Figure 28B shows representative confocal images of bEnd.3 cells expressing EGFP after various treatments. Green = EGFP; Blue = cell nucleus. Figure 28C shows the percentage of EGFP-positive cells, and Figure 28D shows the mean fluorescence intensity (MFI) of EGFP as shown by flow cytometry. Data are presented as mean ± SEM, with 3 cells per group for 1 experiment. Post-hoc analysis was performed using the Tukey test, and statistical significance was calculated by one-way ANOVA. ns: no significant difference (P>0.05).

[0039] Figure 29 shows representative TEM images of bEnd.3 cells, showing cytoplasmic accumulation of Au@PDA@asEGFP-NW (blue arrow) 24 h after incubation. Nu = cell nucleus; Cy = cytosol; Ex = extracellular space. The two smaller images on the right show magnification of the boxed area (blue or yellow) of the larger image on the left.

[0040] Figure 30 shows a representative TEM image of BMDM, showing cytoplasmic accumulation of Au@PDA@miR-223 NW (blue arrow, page 7 / 47, manual 10 CN 122341398 A) 24 h after incubation. Nu = nucleus; Cy = cytosol; Ex = extracellular space. The two smaller images on the right show magnification of the boxed area (blue or yellow) of the larger image on the left.

[0041] Figure 31 shows the in vitro transfection of miR-223 by nanoworms to induce M2 polarization of BMDM. qRT-PCR assay of M1 phenotypic markers. Data are expressed as mean ± SEM. Statistical significance was analyzed post-hoc by one-way ANOVA and Tukey's test. ns = no significant difference (P>0.05). n=6 per group in 2 experiments.

[0042] Figures 32A-32C show the in vitro transfection of miR-223 by nanoworms to induce BMDM depolarization. Figure 32A shows a confocal image illustrating M1 depolarization of BMDM, demonstrated by a stronger M2 marker (CD206; green) and a weaker M1 marker (CD80; red). Blue = nucleus. Scale bar = 20 μm. qRT-PCR assay of M1 phenotypic markers (Figure 32B) and M2 phenotypic markers (Figure 32C). Data are expressed as mean ± SEM. Statistical significance was post-hocly analyzed by one-way ANOVA and Tukey's test. ns = no significant difference (P>0.05). n = 6 per group in both experiments.

[0043] Figure 33 shows a representative TEM image of hMSCs, showing cytoplasmic accumulation of Au@PDA@siNog NW (blue arrow) 24 h after incubation. Nu = nucleus; Cy = cytosol; Ex = extracellular space. The two smaller images on the right show magnification of the boxed area (blue or yellow) of the larger image on the left.

[0044] Figures 34A-34C show the in vitro siNog transfection-induced osteogenic differentiation of hMSCs by nanoworms. Figure 34A shows the qRT-PCR results confirming the knockout of Noggin 7 days after incubation. Calcium deposits (red) stained with Alizarin Red (Figure 34C) were quantified 7 days after osteogenic differentiation (Figure 34B). Scale bar = 100 μm. Data are expressed as mean ± SEM. Statistical significance was analyzed post-hoc by one-way ANOVA and Tukey's test. ns = no significant difference (P>0.05). n = 3 or 6 per group in 1-2 experiments. OM: osteogenic mediator; original NW: Au@PDA NW without siNog.

[0045] Figure 35 shows the native agarose gel electrophoresis of mRNA synthesized by in vitro transfection (IVT). From left to right, the samples are RNA gradient standards (500-9000 nt), mEGFP, mBMP-7-OFP, mHGF, and mCXCR4-GFP. The expected sizes of mEGFP, mBMP-7-OFP, mHGF, and mCXCR4-GFP are ~1100 nt, 2200 nt, 2500 nt, and 2100 nt, respectively.

[0046] Figure 36 shows the gel retardation analysis of mRNA loaded on Au@PDA@lipid NW. Model mRNA (mEGFP) was compounded with Au@PDA@lipid NW at different weight ratios from 1:20 to 1:100 (mEGFP: Au@PDA@lipid). The mRNA loading was saturated at a ratio of 1:50.

[0047] Figure 37: Trajectory of Cy5-labeled nanoworms (red) encapsulating mRNA in hMSCs versus incubation time.Time-lapse confocal images showed that the mRNA-encapsulated nanoworms were highly colocalized with acidic organelles (stained with Lysotracker) 5 hours after incubation and escaped from these vesicles after 10 hours of incubation. Blue = cell nucleus. White numbers represent the Pearson colocalization coefficient between Cy5-labeled mRNA-encapsulated nanoworms (red) and Lysotracker (green).

[0048] Figures 38A-38B show the accumulation of Cl- in hMSCs. Figure 38A shows the Cl- accumulation in intracellular vesicles containing mRNA-encapsulated nanoworms in hMSCs that were untreated with siClC3 (top row) or treated with siClC3 (bottom row). The MQAE staining signal was negatively correlated with the Cl- concentration. White arrows indicate that the vesicles containing Cy5-labeled mRNA-encapsulated nanoworms in WT hMSCs had a relatively high Cl- concentration compared to the vesicles in siClC3-treated hMSCs. Figure 38B shows the quantification of MQAE staining fluorescence intensity based on Figure 38A. Data are expressed as mean ± SEM. Statistical significance was calculated using Student's t-test. n = 300 intracellular vesicles per group. ns = no significant difference (P > 0.05).

[0049] Figures 39A-39C show the pH measurements of hMSC intracellular vesicles. Figure 39A shows the pH measurements of intracellular vesicles untreated (top row) or treated with siClC3 (bottom row) after 5 hours of co-incubation with mRNA-encapsulated nanoworms (magneta), dextran 10K pHrodo (green), and dextran 10K AF594 (red). White arrows indicate intracellular vesicles containing Cy5-labeled mRNA-encapsulated nanoworms. Scale bar = 10 µm. Figure 39B shows the change in endosomal proton pump activity over time after incubation of hMSCs with mRNA-encapsulated nanoworms. After incubation for 5 hours, the activity of the endosperm pump was enhanced. Blue = cell nucleus. White numbers represent the Pearson colocalization coefficients between V1 (green) and V0 (red). Figure 39C shows the quantification of pH of individual vesicles in WT hMSCs and siClC3-treated hMSCs by measuring the R / G ratio of intracellular vesicles of Cy5-labeled and non-Cy5-labeled nanoworms containing mRNA. Data are expressed as mean ± SEM. Statistical significance was calculated by Student's t-test. n = 300 intracellular vesicles per group. ns = no significant difference (P>0.05).

[0050] Figures 40A-40C show that lipid-coated nanoworms (Au@PDA@lipid NW) can transfect mRNA in vitro.EGFP was expressed in hMSCs. Figure 40A shows representative fluorescence images confirming the expression of EGFP (green) due to transfection with Lipofectamine 3000 and LNW at 24 h and 48 h after incubation. Scale bar = 50 μm. Figure 40B shows the transfection efficiency of mEGFP (as determined by flow cytometry). Figure 40C shows the cell viability at 24 h and 48 h after incubation. Data are expressed as mean ± SEM. Statistical significance was determined post-hoc by one-way ANOVA and Tukey's test. ns = no significant difference (P>0.05). n=6 per group in both experiments.

[0051] Figures 41A-41C show the transfection of CXCR4 and BMP7 mRNA in hMSCs by lipid nanoworms in vitro. Figure 41A shows the in vitro transfection efficiency of mCXCR4-GFP and mBMP-7-OFP at 24 h after transfection with hMSCs, as assessed by flow cytometry. Figures 41B-41C show the relative expression of CXCR4 (Figure 41B) and BMP-7 (Figure 41C) in hMSCs under different treatments. Data obtained by ELIZA assays showed that LNW resulted in the highest expression of both mRNAs compared to initial hMSCs and liposome transfection. Data are presented as mean ± SEM. Statistical significance was assessed post-hoc by one-way ANOVA and Tukey's test. ns = no significant difference (P>0.05). n=3 per group in one experiment.

[0052] Figure 42 shows the establishment of renal fibrosis 3 days post-UUO surgery. Kidneys from UUO mice were collected 3 days post-surgery and stained with IHC to assess the expression of Col-I and α-SMA in the UUO kidney and the contralateral kidney (CL kidney).

[0053] Figure 43 shows the accumulation of injected hMSCs in major visceral organs. Figure 43 shows representative in vitro near-infrared fluorescence imaging (NIRF) of UUO mice 24 h after intravenous injection of DiR-labeled hMSCs. The stronger fluorescence in the kidneys of the Au@PDA@mCXCR4 / BMP7 NW-transfected hMSCs (NWs-hMSCCXCR4 / BMP7) group compared to the initial hMSCs and liposome-transfected hMSCs (Lipo-hMSCCXR4 / BPM7) group indicates enhanced homing ability of hMSCs to UUO kidneys due to strong mCXCR4 transfection mediated by Au@PDA@mCXCR4 / mBMP7 NW.

[0054] Figures 44A-44B show the quantification of Col-I positive regions (Figure 44A) and α-SMA positive regions (Figure 44B) in UUO kidney slices based on an average of 6 images per mouse. Data are expressed as mean ± SEM. Statistical significance was determined post-hoc by one-way ANOVA and Tukey's test. n=7 in each of the two experiments.

[0055] Figures 45A-45B show the in vivo toxicity of NW-hMSCmCXCR4 / BMP7 in UUO mice 7 days after intravenous injection (or 14 days after UUO surgery). No changes were observed in blood chemistry, cell counts, and liver function (Figure 45A) after injection of Au@PDA@mCXCR4 / BMP7 NW transfected hMSCs (Figure 45A). Gray shaded areas represent the normal range for mouse markers or cell counts. WBC: White blood cells. RBC: Red blood cells. Absolute neutrophils, lymphocytes, or monocytes (10³ / μL): Number of cells per unit volume of blood. Neutrophils %, Lymphocytes %, Monocytes %: Percentage of cells in total white blood cells. RBC (10⁶ / μL): Number of cells per unit volume of blood. AST: Aspartate aminotransferase. ALT: Alanine aminotransferase. Data from one experiment, n=3.

[0056] Figure 46 shows the toxicity of NW-hMSCCXCR4 / BMP7 in UUO mice. Histological examination of the heart, liver, lungs, and spleen showed that NW-hMSCCXCR4 / BMP7 did not cause significant changes in tissue morphology 11 days after injection. Representative images were selected from n=3 mice in each group in one experiment.

[0057] Figures 47A-47C show the distribution of Au@PDA@mHGF NW in mice with acute liver injury. Gold content was determined by ICP-MS. Figure 47A shows the hematologic pharmacokinetics. Figure 47B shows the organ-level distribution data, which shows that Au@PDA@mHGF NW accumulated most abundantly in the liver and spleen (>60% ID within 24 h after injection). Figure 47C shows the amount of HGF in liver tissue 24 h after injection as measured by ELISA. Data are expressed as mean ± SEM. Statistical significance was determined post-hoc by one-way ANOVA and Tukey's test. ns = no significant difference (P>0.05). n = 3 or 6 per group in one experiment.

[0058] Figure 48 shows the in vivo toxicity of Au@PDA@mHGF NW in mice with acute liver injury. Histological examination of the heart, kidneys, lungs, and spleen showed no significant changes in tissue morphology 24 h after Au@PDA@mHGF NW injection. Representative images were selected from n = 3 mice per group in one experiment. Scale bar = 100 µm.

[0059] Figure 49 shows the in vivo toxicity 24 h after intravenous injection of Au@PDA@mHGF NW in mice with acute liver injury (or 48 h after APAP disease induction). Au@PDA@mHGF-NW did not alter blood chemistry or cell counts. Gray shaded areas represent the normal range for mouse markers or cell counts. WBC: white blood cells. RBC: red blood cells. Absolute neutrophils, lymphocytes.Or monocytes (10³ / μL): Number of cells per unit volume of blood. Neutrophils%, Lymphocytes%, Monocytes%,: Percentage of cells in total white blood cells. RBCs (10⁶ / μL): Number of cells per unit volume of blood. Data from one experiment with n=3. Table 1. In vivo escape of nanoparticles for gene delivery with colocalization coefficient (<0.5) in literature from 2019 to 2024. Instruction manual 10 / 47 pages 13 CN 122341398 A Instruction manual 11 / 47 pages 14 CN 122341398 A Instruction manual 12 / 47 pages 15 CN 122341398 A Table 2. In vivo escape of NPs for gene delivery in literature from 2019 to 2024, but colocalization coefficients are not reported. Specification page 13 / 47, page 16, CN 122341398 A; Specification page 14 / 47, page 17, CN 122341398 A, Table 3. Primer sequences used in this invention. Specification page 15 / 47, page 18, CN 122341398 A; Specification page 16 / 47, page 19, CN 122341398 A, Table 4. Hydrated particle size and zeta potential determined in water by dynamic light scattering (DLS) at room temperature (RT). For serum stability testing, different types of nanoworms were incubated in DMEM containing 10% FBS at 37°C for 24 h. Specification page 17 / 47, page 20, CN 122341398 A, Table 5. Oligonucleotide loading on Au40@PDA NW determined by fluorescence analysis. Table 6. Top-enriched GO entries identified from differentially expressed genes (DEG) in A549 cells based on paired comparisons of the “Au@PDA@T21NW 24h” group versus the “Au@PDA@T21NP 24h” group. Data from one experiment, n=3. Q<0.05. Bold GO entries are associated with intracellular vesicles and ion homeostasis. Table 7. Top-enriched GO entries identified from differentially expressed genes (DEG) in A549 cells based on paired comparisons of the “Au@PDA@T21NW 24h” group versus the “Au@PDA@T21NW 8h” group. Data from one experiment, n=3. Q<0.05. Bold GO entries are associated with intracellular vesicles and ion homeostasis. (Instruction manual 18 / 47 pages 21 CN 122341398 A) Table 8. Physicochemical characterization of different types of mRNA-encapsulated nanoworms by DLS. PDI = Polydispersity Index. Instructions 19 / 47, page 22, CN 122341398 A, Supplementary Table 9. mRNA loading on Au@PDA@lipid NW. Sequence Description

[0060] SEQ IDNO:1: miR223

[0061] SEQ ID NO:2: siNog

[0062] SEQ ID NO:3: Antisense DNA against enhanced green fluorescent protein (asEGFP)

[0063] SEQ ID NO:4: Mouse GAPDH forward sequence 5' to 3'

[0064] SEQ ID NO:5: Mouse GAPDH reverse sequence 3' to 5'

[0065] SEQ ID NO:6: Human GADPH forward sequence 5' to 3'

[0066] SEQ ID NO:7: Human GADPH reverse sequence 3' to 5'

[0067] SEQ ID NO:8: Mouse IL-12 forward sequence 5' to 3'

[0068] SEQ ID NO:9: Mouse IL-12 reverse sequence 3' to 5'

[0069] SEQ ID NO:10: Mouse IL-10 forward sequence 5' to 3'

[0070] SEQ ID NO:11: Mouse IL-10 reverse sequence 3' to 5'

[0071] SEQ ID NO:12: Mouse CD80 forward sequence 5' to 3'

[0072] SEQ ID NO:13: Mouse CD80 reverse sequence 3' to 5'

[0073] SEQ ID NO:14: Mouse CD206 forward sequence 5' to 3'

[0074] SEQ ID NO:15: Mouse CD206 reverse sequence 3' to 5'

[0075] SEQ ID NO:16: Mouse TNF-α forward sequence 5' to 3'

[0076] SEQ ID NO:17: Mouse TNF-α reverse sequence 3' to 5'

[0077] SEQ ID NO:18: Mouse TGF-β forward sequence 5' to 3'

[0078] SEQ ID NO:19: Mouse TGF-β reverse sequence 3' to 5'

[0079] SEQ ID NO:20: Mouse INF-γ Forward sequences 5' to 3' Specification 20 / 47 pages 23 CN 122341398 A

[0080] SEQ ID NO:21: Mouse INF-γ reverse sequence 3' to 5'

[0081] SEQ ID NO:22: Mouse Arg1 forward sequence 5' to 3'

[0082] SEQ ID NO:23: Mouse Arg1 reverse sequence 3' to 5'

[0083] SEQ ID NO:24: Human Noggin forward sequence 5' to 3'

[0084] SEQ ID NO:25: Human Noggin reverse sequence 3' to 5'

[0085] SEQ ID NO:26: Human BMP-2 forward sequence 5' to 3'

[0086] SEQ ID NO:27: Human BMP-2 reverse sequence 3' to 5'

[0087] SEQ ID NO:28: Human RUNX2 forward sequence 5' to 3'

[0088] SEQ ID NO:29: Human RUNX2 reverse sequence 3' to 5'

[0089] SEQ ID NO:30: Human C1C3 forward sequence 5' to 3'

[0090] SEQ ID NO:31: Human C1C3 reverse sequence 3' to 5'

[0091] SEQ ID NO:32: Human Col-1 forward sequence 5' to 3'

[0092] SEQ ID NO:33: Human Col-1 reverse sequence 3' to 5'

[0093] SEQ ID NO:34: Mouse α-SMA forward sequence 5' to 3'

[0094] SEQ ID NO:35: Mouse α-SMA reverse sequence 3' to 5' Detailed Description

[0095] The present invention provides an effective and safe composition comprising nanoworms, which can be used for a method of delivering nucleic acids to a subject. More specifically, the present invention provides a polymer-coated nanoworm (NW). Furthermore, the present invention provides a nanoworm encapsulated with therapeutic nucleic acid for efficient cellular delivery with limited localization within acidic organelles.

[0096] Selected Definitions

[0097] The singular forms “a,” “an,” and “the” used herein also include the plural forms unless the context clearly indicates otherwise. Furthermore, if “including,” “includes,” “having,” “has,” “with,” or variations thereof are used in the detailed description and / or claims, these terms are intended to encompass in a manner similar to “comprising.” Transitional terms / phrases (and any grammatical variations thereof) “comprising,” “comprises,” “comprise,” “consisting essentially of,” “consisting,” and “consistits” are used interchangeably.

[0098] The phrase "consisting essentially of" or "consists essentially of" indicates that the claims cover embodiments comprising the specified materials or steps, as well as embodiments that do not substantially affect the nature and novelty of the claims.

[0099] The term "about" refers to a specific value within an acceptable margin of error as determined by a person skilled in the art, depending in part on how that value is determined, i.e., the limitations of the measuring system. The term "about" is used in compositions containing a certain amount of an ingredient.At that time, these compositions contain a specified amount of the ingredient, with a variation (error range) of 0-10% around the value (X±10%). In other cases, the term "about" means providing a variation (error range) of 0-10% around a given value (X±10%). Obviously, such variation represents a range that is 10% higher or lower than the given value, for example, X±1%, X±2%, X±3%, X±4%, X±5%, X±6%, X±7%, X±8%, X±9%, or X±10%.

[0100] In this disclosure, ranges are expressed in abbreviated form to avoid having to list and describe every value in the range in detail. Specification 21 / 47 pages 24 CN 122341398 A Where appropriate, any suitable value in the range may be selected as the upper limit, lower limit, or end point of the range. For example, the range 0.1–1.0 represents final values ​​of 0.1 and 1.0, and intermediate values ​​of 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and all intermediate ranges included in the range 0.1–1.0, such as 0.2–0.5, 0.2–0.8, 0.7–1.0, etc. Values ​​having at least two significant figures within a range are conceivable; for example, the range 5–10 represents all values ​​between 5.0 and 10.0 and between 5.00 and 10.00, including final values. When ranges are used herein, combinations and sub-combinations of ranges (e.g., sub-ranges within the disclosed ranges) and specific embodiments thereof are explicitly included.

[0101] As used herein, the terms “effective therapeutic amount,” “effective therapeutic dose,” “effective amount,” and “effective dose” refer to the amount or dose of a compound or composition that, when administered to a subject, is capable of treating, preventing, or improving a subject’s condition, disease, or disorder. In other words, when administered to a subject, the dose is “effectively therapeutic.” The actual dose will vary depending on many factors, including, but not limited to, the specific condition, disease, or disorder being treated, prevented, or improved; the severity of the condition; the patient’s weight, height, age, and health status; and the route of administration.

[0102] As used herein, the term “treatment” means eradication; reduction; improvement; mitigation; relief; reduction or delay of symptom onset; slowing of the rate of degeneration or decline; reduction of the degree of weakness at the end of degeneration; and / or improvement of the physical or mental health of the subject, or reversal of the signs or symptoms of a health condition, disease, or disorder to any extent, including but not requiring a complete cure of the condition, disease, or disorder. Treatment may cure, improve, or partially improve a disease. “Treatment” may also include improving or enhancing a condition or characteristic, for example, bringing the function of a specific system in the body to a higher state of health or homeostasis.

[0103] As used herein, “prevention” of a health condition, disease, or disorder means avoiding, delaying, preventing, or minimizing.The onset of specific signs or symptoms of the condition, disease, or disorder. Prevention may be absolute or complete, but is not necessary; this means that the signs or symptoms may still develop later. Prevention may include reducing the severity of the onset of the condition, disease, or disorder, and / or inhibiting the progression of the condition, disease, or disorder to a more severe condition, disease, or disorder.

[0104] In some embodiments of the invention, the method includes administering multiple doses of the nanoworms of the invention. The method may include administering 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, or more therapeutically effective doses of a composition comprising the compounds of the invention described herein. In some embodiments, the dosage is administered over a period of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 14 days, 21 days, 30 days, 2 months, 3 months, 6 months, 9 months, 1 year, 1.5 years, 2 years, 2.5 years, 5 years, or more than 10 years. The frequency and duration of administration of the multi-dose composition are intended to prevent or treat diseases, such as kidney disease. Furthermore, treating a subject with an effective therapeutic amount of the compound of the present invention may include a single treatment or may include a series of treatments. It should also be understood that the effective dose of the compound used for treatment may be increased or decreased during a particular treatment. Changes in dosage may cause and become apparent in the results of tests for kidney disease, such as glomerular filtration rate. In some embodiments of the invention, the method includes administering the compound multiple times a day, including but not limited to twice a day, three times a day, and four times a day.

[0105] As used herein, “subject” means an animal, such as a mammal, such as a human. The methods described herein may be used in humans and non-human animals. In some embodiments, the subject is a mammal (such as an animal model of a disease), and in some embodiments, the subject is a human. The terms “subject” and “patient” are used interchangeably. Animals may be, for example, humans, pigs, horses, goats, cats, mice, rats, dogs, apes, fish, chimpanzees, orangutans, guinea pigs, hamsters, cows, sheep, birds, chickens, and any other vertebrates or invertebrates. In some embodiments, the subject is a cell line, such as A549 human lung cells, bEnd.3 endothelial cells, bone marrow-derived macrophages (BMDM), and human mesenchymal stem cells (hMSC).

[0106] As used herein, the “isolated” or “purified” compound is substantially free of other compounds. In some embodiments, the purified compound is at least 60 wt.% (dry weight) of the target compound. Preferably, the formulation is at least 75 wt.% of the target compound, more preferably at least 90 wt.%, and most preferably at least 99 wt.%. For example, the purified compound is the desired compound. (See page 22 / 47 of CN 122341398 A for the specification of the desired compound.)The compound is present in at least 90 wt.%, 91 wt.%, 92 wt.%, 93 wt.%, 94 wt.%, 95 wt.%, 98 wt.%, 99 wt.%, or 100 wt.% (w / w). Purity can be determined by any suitable standard method, such as column chromatography, thin-layer chromatography, or high-performance liquid chromatography (HPLC).

[0107] In this disclosure, the term "isolated nucleic acid" molecule refers to a nucleic acid molecule isolated from other nucleic acid molecules typically associated with isolated nucleic acid molecules. Thus, "isolated nucleic acid molecule" includes, but is not limited to, nucleic acid molecules that do not contain the nucleotide sequence at one or both ends of a natural flanking nucleic acid in the genome of the organism from which the isolated nucleic acid is derived (e.g., cDNA or genomic DNA fragments produced by PCR or restriction endonuclease digestion). Such isolated nucleic acid molecules are typically introduced into vectors (e.g., cloning vectors or expression vectors) for ease of manipulation or production of fusion nucleic acid molecules. Furthermore, isolated nucleic acid molecules may include engineered nucleic acid molecules, such as recombinant or synthetic nucleic acid molecules. Nucleic acid molecules present in hundreds of millions to millions of other nucleic acid molecules, such as in nucleic acid libraries (e.g., cDNA or genomic libraries) or gels (e.g., agarose or polyacrylamide) containing restriction-digested genomic DNA, are not “isolated nucleic acids.”

[0108] The terms “nucleic acid” or “polynucleotide” refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in single-stranded or double-stranded form and polymers thereof. Unless specifically limited, the term covers nucleic acids containing known natural nucleotide analogs that have similar binding properties to a reference nucleic acid and are metabolized in a manner similar to that of natural nucleotides. Unless otherwise stated, a particular nucleic acid sequence also implicitly includes variants of its conserved modifications (e.g., degenerate codon substitutions), alleles, orthologs, single nucleotide polymorphisms (SNPs), and complementary sequences, as well as explicitly indicated sequences. Specifically, degenerate codon substitution can be achieved by generating a sequence in which the third position of one or more selected (or all) codons is replaced by a mixture of bases and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The term nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene.

[0109] As used herein, the terms “oligonucleotide” and “oligonucleotide” are used interchangeably to describe short single strands of synthetic DNA or RNA, such as sequences of about 5 nucleic acid bases to about 500 nucleic acid bases.The terms “oligonucleotide delivery” and “oligomeric nucleotide delivery” may be used interchangeably to describe methods of delivering oligonucleotides into cells or targeting specific cell types, tissues, or organs.

[0110] The term “gene” refers to a segment of DNA involved in the production of a polypeptide chain; it includes regions before and after coding regions (leading and trailing) involved in the transcription / translation and transcription / translation regulation of the gene product, as well as insertion sequences (introns) between individual coding segments (exons).

[0111] In some embodiments, a subject is treated with a “significant reduction or elimination of gene expression or function of the protein encoded by the gene.” Compared to an untreated subject, this phrase means a reduction in gene expression of at least (or at least about) 30%. Therefore, the treated subjects exhibited a significant reduction or elimination of gene expression, or a reduction in the expression of a gene or active protein, ranging from about 30% to about 99.99%, about 40% to about 99.99%, about 50% to about 99.99%, about 60% to about 99.99%, about 70% to about 99.99%, about 80% to about 99.99%, about 90% to about 99.99%, or no expression of the gene or the active protein it encodes (expression was completely suppressed).

[0112] “Reduction” means a negative change of at least 1%, 5%, 10%, 25%, 50%, 75%, or 100%.

[0113] “Increase” means a positive change of at least 1%, 5%, 10%, 25%, 50%, 75%, or 100%.

[0114] As used herein, “pharmacokinetics” refers to the branch of pharmacology that specifically studies the movement of drugs in the body.

[0115] As used herein, the terms “determine,” “assay,” “assess,” and “analyze” are used interchangeably, including quantitative and qualitative assays.

[0116] As used herein, “stromal cell differentiation” refers to the process by which stromal cells transform into differentiated cells. Stromal cell transplantation occurs when stromal cells replace cells damaged by chemotherapy or disease.

[0117] As used herein, “macrophage polarization” refers to the process by which macrophages produce different functional phenotypes in response to specific microenvironmental stimuli and signals. Nanoworm Composition

[0118] The present invention provides a polymer-coated nanoworm, such as a polydopamine-coated nanoworm (PDA NW), having a one-dimensional assembly that can reduce the endosome and / or lysosomal encapsulation of nucleic acids when administered to a subject. In some embodiments, nanoworms are integrated core-shell nanoparticles having a backbone of metal nanochains (e.g., gold (Au) nanochains) and a polymer shell (e.g., a polydopamine shell) for adsorbing nucleic acids (e.g., oligonucleotides), or an additional lipid coating layer for complexing with nucleic acid molecules (e.g., mRNA). In some embodiments, the length of the oligonucleotide can be from about 15 to about 50 bases.In some embodiments, the length of the mRNA can be from about 700 to about 5000 bases. In some embodiments, the metal nanochains are gold (Au), copper, silver, iron oxide, cerium dioxide, zinc oxide, titanium oxide, rhodium, platinum, iron oxide, or any combination thereof. In some embodiments, the metal nanoparticles can be capped with citrate.

[0119] In some embodiments, the present invention includes anionic gold polydopamine core-shell nanoworms (Au@PDA NW), which serve as a bifunctional agent for gene delivery and activation of endosome escape. In some embodiments, the gold core is capable of tracking endosome escape by transmission electron microscopy (TEM), and the PDA shell supports the surface adsorption of mRNAs such as DNA, siRNA, miRNA, and additional lipids. In some embodiments, the nanoworms encapsulating nucleic acids are generally anionic and can enter cells without transfection agents, including but not limited to A549, bEnd3 endothelial cells, primary bone marrow-derived macrophages (BMDM), and human mesenchymal stromal cells (hMSC). Unbound by any theory, the nucleic acid-coated nanoworms naturally enter the cell, activate the ClC3 H+ / Cl- ion exchanger, mediate endosome escape, accumulate H+ and Cl-, leading to membrane rupture, and robustly escape from late endosomes with PCC ≤ 0.2 without cell-penetrating peptides or mechanical stimulation. In a preferred embodiment, the endosome escape mechanism differs from the proton sponge effect due to the involvement of ClC3. In a more preferred embodiment, the anionic gold polydopamine core-shell nanoworms of the present invention can be used to program cellular responses, such as in primary macrophage polarization and stem cell differentiation, and for the treatment of diseases, including but not limited to renal fibrosis and acute liver injury.

[0120] In some embodiments, to prepare polymer-coated nanoworms, metal nanoparticles can be sonicated with a polymer to produce core-shell nanochains. In some embodiments, sonication is performed at a frequency of about 40 kHz for a duration of about 1 hour. In some embodiments, the diameter of each metal nanoparticle is about 10 nm to about 100 nm or about 40 nm. In some embodiments, the nanoworms have 2, 3, 4, 5, 6, or 7 metal nanoparticle cores in the chain. In a preferred embodiment, each chain of nanoworms has 4 or 5 metal nanoparticle cores. In some embodiments, the thickness of the polymer shell is about 7 nm to about 35 nm or about 20 nm. In some embodiments, the polymer includes polydopamine, polyethylene glycol, polyethylene oxide, polylactic acid, polyglycolic acid, polyethylene oxide-polylactic acid, polyethylene oxide-polyglycolic acid, polyglycolic acid / polylactic acid, sodium alginate, dextran, polyethyleneimine (PEI), silica, or any combination thereof.

[0121] In some embodiments, oligonucleotides can be adsorbed onto the polymer under acidic conditions via electrostatic interactions.On the coated nanoworms, for example under conditions of pH less than or equal to about 3.0 or about 2.5. In some embodiments, the oligonucleotide can be labeled with a dye, such as anthocyanin (Cy). In some embodiments, to produce oligonucleotide-containing nanoworms, the negatively charged oligonucleotide Cy5-labeled 21-thymine (Cy5-T21) can be adsorbed onto the polymer-coated nanoworms by electrostatic interaction under acidic conditions. In some embodiments, the oligonucleotide is about 15 to about 50 bases in length. In some embodiments, the oligonucleotide can be a single-stranded oligonucleotide, such as microRNA, and / or a double-stranded oligonucleotide, such as siRNA. In some embodiments, to determine the oligonucleotide loading on the nanoworms, nucleic acids, such as Cy5-labeled oligonucleotides or mRNA, can be labeled, and then the concentration of oligonucleotides in the supernatant can be subtracted after purification. In some embodiments, for adsorbing oligonucleotides, the oligonucleotides can be mixed with nanoworms in an acidic buffer, such as an acidic citrate buffer with a pH less than or about 3. In some embodiments, for adsorbing mRNA, the mRNA can be mixed with nanoworms in nuclease-free water with a pH of about 7. In some embodiments, exemplary oligonucleotides include miR223 (3'-CCCAUAAACUGUUUGACUG-5'; SEQ ID NO:1) and siNog (5'-AAC ACU UAC ACU CGG AAA UGA UGG G-3'; SEQ ID ID:2). In some implementations, the mRNA may encode hepatocyte growth factor (HGF), chemokine receptor type 4 (CXCR4), bone morphogenetic protein 7 (BMP-7), and enhanced green fluorescent protein (EGFP); SEQ ID NO: 3:UAC CAC UCG UUC CCG CUC CUC GAC AAG UGG CCC CAC CAC GGG UAG_ GAC CAG CUC GAC CUG CCG CUG CAU UUG CCG GUG UUC AAG UCG CAC AGG CCG CUC CCG CUC CCG CUA CGG UGG AUG CCG UUC GAC UGG GAC UUC AAG UAG_ ACG UGG UGG CCG UUC GAC GGG CAC GGG ACC GGG UGG GAG CAC UGG UGG GAC UGG AUG CCG CAC GUC ACG AAG UCG GCG AUG GGG CUG GUG UAC UUC GUC GUGCUG AAG AAG UUC AGG CGG UAC GGG CUU CCG AUG CAG GUC CUC GCG UGG UAG_ AAG AAG UUC CUG CUG CCG UUG AUG UUC UGG GCG CGG CUC CAC UUC AAG CUC CCG CUG UGG GAC CAC UUG GCG UAG_ CUC GAC UUC CCG UAG_ CUG AAG UUC CUC CUG CCG UUG UAG_ GAC CCC GUG UUC GAC CUC AUG UUG AUG UUG UCG GUG UUG CAG AUA UAG_ UAC CGG CUG UUC GUC UUC UUG CCG UAG_ UUC CAC UUG AAG UUC UAG_ GCG GUG UUG UAG_ CUC CUG CCG UCG CAC GUC GAG CGG CUG GUG AUG GUC GUC UUG UGG GGG UAG_ CCG CUG CCG GGG CAC GAC GAC GGG CUG UUG GUG AUG GAC UCG UGG GUC AGG CGG GAC UCG UUU CUG GGG UUG CUC UUC GCG CUA GUG UAC CAG GAC GAC CUC AAG CAC UGG CGG CGG CCC UAG_ UGA_ GAG CCG UAC CUG CUC GAC AUG UUC AUU), red fluorescent protein (RFP), or any combination thereof, the miRNA being miR223, or the siRNA being siNog.

[0122] In some embodiments, the polymer-coated nanoworms may also include a lipid coating layer. In some embodiments, the lipid coating layer comprises lipids such as 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), cholesterol, 1,2-distearate-sn-glycerol-3-phosphocholine (DOPE), 1,1′-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azadiyl)bis(dodecyl-2-ol)) (C12-200), and (6Z,9Z,28Z,31Z)-heptadecano-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate (DLin-MC3-The lipids are: DMA, ((4-hydroxybutyl)azadiyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), dimethyl bis(octadecyl)ammonium bromide (DDA), 1,2-distearyl-sn-glycerol-3-phosphocholine (DSPC), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), 1,2-dioleoyl-3-dimethylaminopropane (DODMA), dipalmitoylphosphatidylcholine (DPPC), or any combination thereof. In some embodiments, the concentration of DOTAP is from about 33 mol% to about 37 mol% or about 35 mol%. In some embodiments, the concentration of cholesterol is from about 47 mol% to about 51 mol% or about 49 mol%. In some embodiments, the concentration of DOPE is from about 14 mol% to about 18 mol% or about 16 mol%. In some embodiments, mol% of each lipid refers to the entire lipid mixture (e.g., cholesterol, DOTAP, and DOPE) initially added to the polymer-coated nanoworms. The percentage of the mixture. In some embodiments, the average hydrodynamic size of the lipid and polymer-coated nanoworms is about 220 nm to about 275 nm or about 252 nm, and the zeta potential is about +30 mV to about +45 mV or about +40 mV. In some embodiments, the lipid and polymer-coated nanoworms can be formed by mixing lipids with polymer-coated nanoworms.

[0123] In some embodiments, the lipid and polymer-coated nanoworms may also contain oligonucleotides, such as mRNA, which can be prepared by mixing lipid and polymer-coated nanoworms with oligonucleotides. In some embodiments, oligonucleotides can be adsorbed onto the lipid and polymer-coated nanoworms by electrostatic interaction under acidic conditions, for example, at a pH less than or equal to about 3.0 or about 2.5. In some embodiments, the positive charge of the lipid coating layer on the nanoworms allows macromolecular nucleic acids (e.g., mRNA) to complex with the lipid nanoworms in nuclease-free water. In some embodiments, For nanoworms coated with oligonucleotides, shorter oligonucleotides can be adsorbed onto the polydopamine shell of the nanoworms via electrostatic interactions in an acidic citrate buffer, thereby protonating and acidifying the polymer shell. In some embodiments, the average hydrodynamic size of the lipid and polymer-coated nanoworms with therapeutic mRNA is about 225 nm to about 280 nm or about 258 nm, and the total zeta potential is about -30 mV to about -50 mV to about -32 mV.

[0124] In some embodiments, the nanoworms may have a total anionic charge and can be readily loaded with different types ofThe therapeutic nucleic acids include, for example, antisense oligonucleotides (ASO), short interfering RNA (siRNA), microRNA (miRNA), and messenger RNA (mRNA). In some embodiments, the zeta potential of the nanoworms is less than about -10 mV (e.g., -20 mV). In some embodiments, the negative charge on the surface of the nanoworms is uniformly dispersed. In some embodiments, the nanoworms have limited co-localization with endosomes and / or lysosomes, with a co-localization coefficient of about 0.1 to about 0.2. Compared with widely used transfection agents (e.g., Lipofectamine 3000), the nanoworms of the present invention exhibit more effective gene regulation to support in vitro stem cell differentiation and macrophage polarization, more effective in vitro gene generation expression to support in vivo stem cell-based treatment of renal fibrosis, and finally, in vivo gene generation expression to salvage liver injury. Method of administration of the nanoworm composition

[0125] In some embodiments, the nanoworms of the present invention can deliver various types of therapeutic nucleic acids, such as ASO, siRNA, miRNA, and mRNA, in vitro and in vivo, with low cytotoxicity and high endosome escape efficiency. In some embodiments, escape efficiency can be quantified using confocal imaging of nanoworms such as intracellularly labeled nucleic acids of nanoworms. In some embodiments, acidic organelles, such as late endosomes and lysosomes, can be stained with dyes such as LysoTracker. In some embodiments, a Pearson correlation coefficient (PCC) of colocalization of two signals <0.4, as determined by ImageJ, indicates less overlap between the stained organelles and the labeled nanoworms, indicating escape has occurred. In some embodiments, a PCC >0.6 indicates strong overlap, indicating that the nanoworms are trapped in the acidic compartment. In a preferred embodiment, PCC ≤ 0.2.

[0126] In some embodiments, the therapeutic nucleic acid can be used to treat a variety of diseases and conditions, such as chronic kidney disease, acute liver disease, cancer, cell-based immunotherapy, or any combination thereof. In some embodiments, the nanoworms enter epithelial cells, hepatocytes, kidney cells, endothelial cells, primary macrophages, mesenchymal stem cells, nerve cells, or any combination thereof in the body of a subject.

[0127] Several applications of the compositions of the invention are described in specific embodiments, administered at specific time intervals. In some embodiments, the test nanoworms can be used for in vitro transfection, ex vivo transfection, and then injected into a subject as a cell therapy, or for in vivo transfection directly by injecting nanoworms into a subject.

[0128] In some embodiments, for in vitro transfection, cells can be administered once with specific nucleic acid-coated nanoworms for approximately 2 to 3 days to induce cellular processes (e.g., macrophage polarization, stem cell differentiation, gene knockout). In some embodiments, for ex vivo transfection, stem cells can be incubated with lipid nanoworms coated with therapeutic nucleic acids for one...The transfected stem cells can be injected into a subject, such as a subject with kidney disease, after approximately 12 hours. In some embodiments, the time between approximately 1 day and approximately 6 months, or approximately 11 days and approximately 4 weeks, may be before efficacy evaluation. In some embodiments, for direct in vivo transfection, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, approximately 20, approximately 30, approximately 40, approximately 50, approximately 100 or more doses of lipid nanoworms encapsulated with therapeutic nucleic acids may be administered to a subject, such as a subject with acute liver injury. In some embodiments, the time between approximately 6 hours and approximately 168 hours or approximately 48 hours may be before efficacy evaluation.

[0129] Those skilled in the art will understand that the dosage of the compositions of the present invention varies depending on, for example, the route of administration, the specific nanoworms used in the composition (pages 26 / 47 of CN 122341398 A), other drugs administered, and the age, condition, sex, and severity of disease of the subject, as described above. The effective dose of the nanoworm composition described in this invention is typically between about 1 μg / kg body weight and 100 mg / kg body weight. Examples of such dosage ranges include, but are not limited to, about 1.5 μg / kg to about 90 mg / kg, about 2 μg / kg to about 80 mg / kg, about 5 μg / kg to about 70 mg / kg, about 7.5 μg / kg to about 65 mg / kg, about 10 μg / kg to about 60 mg / kg, about 12.5 μg / kg to about 55 mg / kg, about 15 μg / kg to about 50 mg / kg, about 17.5 μg / kg to about 45 mg / kg, about 20 μg / kg to about 40 mg / kg, about 22.5 μg / kg to about 35 mg / kg, about 25 μg / kg to about 30 mg / kg, about 27.5 μg / kg to about 25 mg / kg, about 30 μg / kg to about 20 mg / kg, about 32.5 μg / kg to about 18 mg / kg, about 35 μg / kg to about 17 mg / kg, and about 37.5 μg / kg to about 16 mg / kg. Approximately 40 μg / kg to approximately 15 mg / kg, approximately 42.5 μg / kg to approximately 14 mg / kg, approximately 45 μg / kg to approximately 13 mg / kg, approximately 47.5 μg / kg to approximately 12 mg / kg, approximately 50 μg / kg to approximately 11 mg / kg, approximately 52.5 μg / kg to approximately 10 mg / kg, approximately 55 μg / kg to approximately 9 mg / kg, approximately 57.5 μg / kg to approximately 8 mg / kg, approximately 60 μg / kg to approximately 7 mg / kg, approximately 62.5 μg / kg to approximately 6 mg / kg, approximately 65 μg / kg to approximately 5 mg / kg, approximately 67.5 μg / kg to approximately 4 mg / kg, approximately 70 μg / kg to approximately 3 mg / kg, approximately 72.5 μg / kg to approximately 2 mg / kg, approximately 75 μg / kg to approximately 3 mg / kg, approximately 72.5 μg / kg to approximately 2 mg / kg, approximately 75 μg / kg to approximately 10 mg / kg, approximately 55 μg / kg to approximately 9 mg / kg, approximately 57.5 μg / kg to approximately 8 mg / kg, approximately 60 μg / kg to approximately 7 mg / kg, approximately 62.5 μg / kg to approximately 6 mg / kg, approximately 65 μg / kg to approximately 5 mg / kg, approximately 67.5 μg / kg to approximately 4 mg / kg, approximately 70 μg / kg to approximately 3 mg / kg, approximately 72.5 μg / kg to approximately 2 mg / kg, approximately 75 μg / kg to approximately 10 mg / kg, approximately 75 μg / kg to approximately 10 mg / kg, approximately 55 μg / kg to approximately 10 mg / kg, approximately 55 μg / kg to approximately 10 mg / kg, approximately 65 μg / kg

[0130] In some embodiments, the nanoworms of the present invention are administered at doses of about 50 µg / kg to about 200 µg / kg, preferably about 75 µg / kg to 150 µg / kg, or most preferably about 100 µg / kg to 120 µg / kg.

[0131] In some embodiments, the effective therapeutic amount of the nanoworm composition of the present invention can be administered intravenously, orally, rectally, bronchially, nasally, topically, orally, sublingually, transdermally, vaginally, intramuscularly, intraperitoneally, intraarterially, intracerebrally, or intraocularly, or in a form suitable for inhalation or inhalation, including powder and liquid aerosol administration, or via a sustained-release system, such as a semi-permeable matrix of a solid hydrophobic polymer containing the nanoworms of the present invention. Administration can also be via other carriers or solvents, such as patches, micelles, liposomes, vesicles, implants (e.g., microimplants), synthetic polymers, microspheres, nanoparticles, etc.

[0132] In some embodiments, the nanoworm composition of the present invention can be formulated for parenteral administration, for example by injection, such as bolus or continuous infusion. Furthermore, the nanoparticle composition can be present in unit dose form in ampoules, pre-filled syringes, and small-volume infusions, or in multi-dose containers with or without preservatives. The nanoparticle composition can be in the form of a suspension, solution, or emulsion in an oil or aqueous carrier. The composition may also contain formulation agents such as suspending agents, stabilizers, and / or dispersants. In a further embodiment, the active ingredient of the composition of the present invention may be in powder form, prepared by aseptic separation of sterile solids or by lyophilization in solution with a suitable carrier (e.g., sterile pyrogen-free water) prior to use.

[0133] Advantageously, our nanoworms do not have significant concerns about genotoxicity. The nanoworms of the present invention do not apply any voltage to cells and can overcome delivery barriers to deep tissues via various routes of administration (e.g., intra-articular injection, intrathecal injection, and retrobulbar injection).

[0134] In some embodiments, nanoworms encapsulating nucleic acids effectively modulate genes by efficiently entering multiple cell lines and escaping endosomes and / or lysosomes. In some embodiments, nanoworms can increase or decrease transcription and / or translation rates by at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, or100%. This invention provides a nanoworm for efficient cell delivery with limited localization within acidic organelles such as late endosomes and lysosomes. In some embodiments, the nanoworm can leave late endosomes by increasing chloride ion accumulation in intracellular vesicles, leading to vesicle swelling and subsequent membrane rupture. Materials and Methods Lysotracker Staining Specification 27 / 47 pages 30 CN 122341398 A

[0135] Lysotracker staining uses Lysotraker probes to label lysosomes in living cells. Lysotracker staining is performed on mammalian cells and yeast with fluorescent dyes, which can be blue, green, visible red, and far-infrared fluorescent dyes. In some embodiments, lysotracker staining can be performed under confocal imaging. In one embodiment, the invention found that the colocalization of Cy5-T21@NW and Lysotracker signals was consistently low across all tested cell lines, with Pearson correlation coefficients (PCC) of 0.1–0.2 (Figure 1D). This was achieved by lysotracker staining.

[0136] In some embodiments, confocal images of Lysotracker staining can show the effect of treatment on organelle colocalization. For example, the method of the present invention found that bafloxacin A1 treatment did not alter the colocalization of acidic organelles and nanoworms, indicating that endosome escape of nanoworms is not based on the proton sponge effect (Figure 2A).

[0137] In other embodiments, Lysotracker staining can be used to test the endosome escape efficiency of nanoworms. For example, the endosome escape efficiency of nanoworms coated with asEGFP was tested by Lysotracker staining. This showed that the PCC was 0.134 after 24 h of culture (Figure 28A). Immunofluorescence (IF) Staining

[0138] IF staining is a method that allows visualization of multiple components in a given tissue or cell type. IF staining is performed by a combination of specific antibodies labeled with fluorophores, which are fluorescent compounds that re-emit light upon photoexcitation.

[0139] Figures 4A and 4D show the results of IF staining using red, green, and blue dyes. In some embodiments, IF staining can be used to assess polarization. As shown in Figure 4D, strong CD80-positive (M1-specific marker) signals were found in the PBS, raw nanoworm, and free miR223 groups, while cells pretreated with Au@PDA@miR-223 NW showed the weakest CD80 signal but a stronger signal for CD206 (M2-specific marker). qRT-PCR analysis also confirmed that M1-related genes (such as IL-12, CD80, TNF-α, and IFN-γ) were present in Au@PDA@miR-223.The NW group showed significant inhibition of expression (downregulated by at least 50%), while the Lipo+miR-223 group showed moderate inhibition of expression (downregulated by about 25%).

[0140] Intracellular trajectory of A549 cells

[0141] Cells were seeded in 35 mm confocal culture dishes and incubated with 0.2 nM Cy5-labeled Au@PDA@T21NW in 1 mL of complete DMEM at different time points. After two PBS washes, the cells were fixed with cold methanol for 10 minutes and with ice-cold acetone for 1 minute. The cells were then blocked with 2% BSA at room temperature for 1 hour and incubated overnight at 4°C with primary antibodies of EEA1 (1:100; Abcam, ab2900), Rab 9 (1:100 dilution; Abcam; ab179815), or LAMP1 (1:150 dilution; Abcam, ab24170) in 2% BSA. After two PBS washes, cells were stained with 2 μg / mL Alexa Fluor 488-labeled goat anti-rabbit secondary antibody (Invitrogen, A-11008) in 2% BSA for 1 hour at room temperature, followed by staining with 1 μg / mL DAPI (Invitrogen, D1306) in PBS for 10 minutes, and then washed twice with PBS. Cells were mounted with fluorescence-attenuating mounting media (Thermo Scientific; P36980) for confocal laser scanning microscopy. The excitation wavelengths for DAPI, Alexa Fluor 488, and Cy5 were 405 nm, 488 nm, and 650 nm, respectively. The emission wavelengths for DAPI, Alexa Fluor 488, and Cy5 were 410–470 nm, 495–600 nm, and 655–750 nm, respectively.

[0142] CCl3 colocalization staining of A549 cells

[0143] After fixation and BSA blocking, cells were stained with CCl3 primary antibody (1:100; Invitrogen, #MA5-45554). After washing twice with PBS, cells were stained with 2 μg / mL Alexa Fluor 488-labeled goat rabbit secondary antibody and Cy5-labeled goat mouse secondary antibody (Invitrogen, A10524) dissolved in 2% BSA for 1 hour at room temperature, then stained with 1 μg / mL DAPI dissolved in PBS for 10 minutes at room temperature, and then washed twice with PBS.

[0144] BMDM Polarization

[0145] After fixation and BSA blocking, polarized BMDM cells were stained overnight at 4°C with anti-CD206 (1:150; Abcam, ab64693) and CD80 antibody (1:100; Abcam, ab254579) primary antibodies dissolved in 2.5% BSA. After three washes with PBS, the cells were injected with 1 μg / mL...Alexa Fluor 488-labeled goat anti-rabbit secondary antibody was stained at room temperature for 1 hour, stained with 1 μg / mL DAPI dissolved in PBS for 10 minutes, and then rinsed twice with PBS.

[0146] Tissue Sections

[0147] Fresh tissue was embedded in OCT and stored at -80°C. 8 μm thick tissue cryosections were dried for 20 minutes and fixed with 4% PFA at room temperature for 15 minutes. Next, the cryosections were blocked with 2.5% horse serum (Vector Laboratories, S-2012-50) at room temperature for 1 hour and stained overnight at 4°C with HNF4α primary antibody dissolved in 2.5% horse serum (1:100; Thermo Scientific, MA1199). After rinsing with PBS, frozen sections were stained at room temperature with Alexa Fluor 532-labeled goat anti-mouse secondary antibody (1:1000; Invitrogen, A11002) for 1 hour, stained with 1 μg / mL DAPI dissolved in PBS for 10 minutes, washed, and mounted with antifade mounters for confocal laser scanning microscopy. The excitation and emission wavelengths of Alexa Fluor 532 are 532 nm and 542–640 nm, respectively. Western Blotting

[0148] Western blotting is a laboratory technique used to detect specific proteins in blood or tissue samples. This method uses gel electrophoresis to separate proteins from the sample. Western blotting begins by placing the antigen sample into a separation gel. The separated proteins are then transferred to a nitrocellulose sheet (blot) in a blot chamber. After the blot chamber, antibodies are labeled, and the blot is immunostained. The sample is then subjected to autoradiography to form a fixed autoradiographic pattern, where the antigen bands can be observed.

[0149] In some embodiments, RNA sequencing results can be validated by Western blotting. Figure 2G shows Western blotting analysis of ClC3 expression in A549 cells treated with nanoworms and Au@PDA NP, and in A549 cells treated with nanoworms for “8 h” and “24 h”. Oligonucleotide Delivery

[0150] Oligonucleotide delivery, or oligonucleotide delivery, describes a method of delivering oligonucleotides into cells or targeting specific cell types, tissues, or organs. In some embodiments, this method can be used to silence gene expression. Oligonucleotides are short single strands of DNA or RNA and are the starting point for many biological methods. Oligonucleotides can be used for gene detection, forensic research, and sequencing. In some embodiments, oligonucleotides can be administered via parenteral routes, including intravenous infusion or subcutaneous injection.

[0151] In one embodiment, to produce nanoworms containing oligonucleotides (oligonucleotides), we use acidic conditions...Under conditions where negatively charged oligonucleotides, anthocyanin (Cy)5-labeled 21-thymine (Cy5-T21), were adsorbed onto nanoworms via electrostatic interactions to verify the hypothesis.

[0152] The method of the present invention further provides applications for nanoworms to deliver oligomers into cells. Synthesis of Au@PDA NW and Au@PDA NR

[0153] Citrate-terminated Au NPs (Cit-Au NPs) with a diameter of approximately 40 nm were synthesized according to established seed-mediated growth methods. Ci-Au NPs and Cit-Au NRs (45 nm × 180 nm) with diameters of approximately 20 nm and 60 nm were purchased from Nanopartz. Au@PDA NW and Au@PDA NR were prepared using our published method. In short, dilute 1 mL of 0.1 nM Au NP (x=20, 40, or 60 nM) and Au NR by adding 1 mL of Tris buffer (10 mM, pH=8.5). Then, under sonication, rapidly inject 2 mL of freshly prepared dopamine (DA) Tris buffer solution (1 mg / mL) into the diluted Au NP solution. After 60 minutes, collect the resulting NW and NR and purify them twice by centrifugation at 5000 rpm (Au40@PDA NW, Au60@PDA NW, and Au40@PDA NR) or 10000 rpm (for Au20@PDA NW) for 15 minutes. Resuspend the purified nanoparticles in Nanopure water and store at 4°C for future use. Instructions for Use, Pages 29 / 47, 32, CN 122341398 A

[0154] Synthesis of Unassembled Au@PDA NP

[0155] Briefly, 1 mL of Cit-Au NP in a 40 nm stock solution (0.1 nm) was injected into 1 mL of HS-PEG5000-COOH (Jenkem; 0.1 mg / mL) dissolved in Nanopure water. After sonication for 1 hour, Au@PEG NP was collected twice by centrifugation at 10000 rpm for 15 minutes. Next, 1 mL of Au@PEG NP was diluted to 0.05 nM by adding 1 mL of Tris buffer (5 mM, pH = 8.5), and then 2 mL of freshly prepared 0.5 mg / mL DA was rapidly injected into the Tris buffer. After sonication for 1 hour, the unassembled product Au@PDA NP was collected and purified by repeated centrifugation at 5000 rpm for 15 minutes twice. The purified Au@PDA NP was resuspended in Nanopure water and stored at 4°C. Synthesis of oligonucleotide-encapsulated nanoparticles (Au@PDA@oligo NW)

[0156] Unless otherwise stated, oligonucleotide-coated nanoworms were produced using Au@PDA NWs with a 40 nm Au core as the starting material. Typically, concentrated Au@PDA NWs were resuspended in 5 mL of citrate buffer (20 mM, pH 3, prepared with nuclease-free water) to a final concentration of 1 nM. After 30 minutes of sonication to adjust the negatively charged PDA shell of the nanoworms to a positive surface charge, an equal volume of oligonucleotide solution (500 nM, nuclease-free water) was added to the nanoworms; the oligonucleotides could be T21 DNA, ASO targeting enhanced green fluorescent protein (asEGFP), miR-223, or siRNA targeting Noggin (Idobio). After 1 hour of incubation, the resulting oligonucleotide-coated Au@PDA NWs were collected twice by centrifugation at 10,000 rpm for 15 minutes at 4 °C, and then resuspended in nuclease-free water and stored at 4 °C. Loading oligonucleotides onto Au@PDA NPs will follow the same method as Au@PDA NWs, keeping the total concentration of the gold core constant. Preparation of Au@PDA NWs coated with mRNA and lipids

[0157] Step 1. Synthesis of lipid-coated nanoworms

[0158] DOTAP (1,2-dioleoyl-3-trimethylpropane ammonium; Cayman, 15110), cholesterol (Sigma, C8667) and DOPE (dioleoylphosphatidylethanolamine; Cayman (15091)) were added to chloroform (≥99.8%, Fisher Chemical) at a molar ratio of 35:49:16 to maintain the total lipid concentration at 1 mg / mL. After rotary evaporation of the lipid mixture at 32°C to remove chloroform, 10 mL of Au@PDA NW (0.5 mg / mL Nanopure water) was added dropwise to the dried lipid membrane. After sonicating the mixture for 1 hour, the purified LNW was dispersed in Nanopure water and stored at 4°C by removing excess lipids twice by centrifugation at 5000 rpm for 15 minutes.

[0159] Step 2. Synthesis of mRNA by in vitro transcription (IVT)

[0160] The circular plasmid DNA template of pEGFP (Addgene, BPK1098) was linearized in 16 µL of nuclease-free water by adding 1 µL of FastDigest BshTI (Thermo Scientific, FD1464) and 2 µL of digestion buffer per µg of DNA for 1 hour at 37°C. Similarly, pHGF and pBMP7 were linearized by adding FastDigest XbaI (Thermo Science, FD0684).The circular plasmid DNA templates containing OFP and pCXCR4-GFP (Sino-Biological) were linearized. After purification using a Monarch® PCR & DNA Cleanup Kit (NEB, T1030S), the linearized DNA template was transcribed in vitro using the T7 HiScribe ARCA mRNA Transcription Kit (with tailing modification) (NEB, E2060S). Briefly, 2 µL (0.5 µg / µL) of linearized DNA was mixed with 10 µL of 2X ARCA / NTP Mix and 2 µL of T7 RNA polymerase Mix, diluted to 20 µL with nuclease-free water, and incubated at 37°C for 30 min. After removing unreacted DNA template by adding 2 µL of DNase I and incubating at 37°C for 15 min, the mRNA was tailed with poly(A) polymerase at 37°C for 30 min. After purification using the Monarch® RNA Cleanup Kit (NEB, T2040L), the concentration of the mRNA product was characterized using Nanodrop (Thermo Scientific), and the molecular weight was characterized by gel electrophoresis. A 1.2% non-denaturing agarose gel was prepared in diethyl pyrocarbonate (DEPC; Macklin, D6079 X) treated TBE (ethylenediaminetetraacetic acid triborate) buffer with 1X SYBR gold nucleic acid gel staining (1:10000 dilution; Thermo Scientific, S11494). For each gel lane, 2 µL of single-stranded RNA gradient standards (NEB, N0362S) or mRNA product (200 ng) was mixed with 8 µL of RNA loading dye, heated at 90°C for 2 minutes, cooled on ice for 2 minutes, and then loaded. After electrophoresis at 80 mV for 1 hour, the gel was imaged using a ChemiDoc™ Touch imaging system (Bio-Rad). The mRNA was a product stored at -70°C.

[0161] Step 3. Preparation of Au@PDA NW coated with mRNA and lipids

[0162] Typically, 1 mL of mRNA was added to 1 mL of lipid-coated Au@PDA NW (in nuclease-free water) at a mass ratio of 1:100. After incubation for 30 minutes, the mixture was centrifuged twice at 5000 rpm for 15 minutes at 4°C to remove excess mRNA. Characterization of Au@PDA NW and Au@PDA NP

[0163] The nanoworms and Au@PDA were determined by inductively coupled plasma mass spectrometry (ICP-MS) (Agilent 7900) with reference to a standard curve (parts per billion (ppb)) of known gold concentration (Au 197 isotope).NP concentration. To quantify the gold content in nanoworm solutions, Au@PDA NP solutions, tissues, and cells used for efficacy studies, we first converted the raw ICP-MS data from ppb to μg / L using the equation 1ppb = 1 μg / L, and then multiplied it by the volume of 2% dilute nitric acid containing the digested sample.

[0164] The hydrodynamic diameter and zeta potential of the nanoworms and nanoparticles were determined using a DelsaMax PRO dynamic light scattering (DLS) analyzer (Beckman Coulter). The reported DLS data represent values ​​from three independent determinations. The quality of the DLS data was explained by analyzing the autocorrelation function and its fit, with the sum of squares (SOS) threshold set to less than 100. (Only determinations with SOS values ​​<100 were counted). Slight batch-to-batch variability was observed as the independent samples were used for different determinations or experiments. To test colloidal stability, the nanoworm solution was dispersed in DMEM containing 10% fetal bovine serum (FBS; Gibco, 10270106). After incubation at 37°C for 24 h, the nanoworm solution was analyzed using UV-Vis spectroscopy and DLS. The reported DLS values ​​represent the mean ± standard deviation (SD) of three independent determinations.

[0165] Following prior literature, the loading of each nucleic acid on the nanoworms was calculated based on fluorescence assays. The concentrations of Cy5-labeled oligonucleotides before and after adsorption onto nanoworms or Au@PDA NPs were determined using a microplate reader (MULTISKAN GO, Thermo Scientific), and the nucleic acid concentration difference was then divided by the concentrations of the nanoworms and nanoparticles to obtain the nucleic acid loading. Cell Culture

[0166] All cell types were maintained at 37°C and 5% CO2. A549 lung epithelial cells (ATCC) and bEnd.3 brain endothelial cells (ATCC) were cultured in Dulbecco modified Eagle medium (DMEM; Gibco, 12100046) supplemented with 10% feBAsade and 1% penicillin-streptomycin (P / s; Gibco, 15140122). Human mesenchymal stromal cells (hMSCs, Lonza) were cultured in α-minimum essential medium (α-MEM; Gibco, 11900073) supplemented with 16.7% FBS, 1% P / s, and 1% L-glutamine (Gibco, 25030081). Bone marrow-derived macrophages (BMDM) were isolated from 9–11-week-old Balb / c mice. Briefly, the hind legs were dislocated from the hip bone and disinfected by washing three times with 75% ethanol for 1 minute. After removing the bone below the knee joint by cutting the ligaments, the separated femur and tibia were rinsed with pre-cooled phosphate-buffered saline (PBS; Gibco, 21600010). The ends of the femur and tibia were cut and rinsed with 5 mL of PBS containing 10% FBS.Each bone marrow cavity was rinsed with 1640 medium (Gibco, 11875093). Cells were pelleted by centrifugation at 1500 rpm for 5 min, treated with frozen red blood cell lysis buffer (BioLegend, 420301) for 2 min, pelleted again, and cultured for 7 days in DMEM containing 10% FBS, 1% P / S and 50 ng / mL colony-stimulating factor-1 (CSF-1; SinoBiological, 51112-MNAH). Cytotoxicity

[0167] Cells were seeded in 96-well plates and cultured to approximately 80% confluence. Cells were then incubated for 24 h with Au@PDA@oligo NW (0.2 nM) in 0.1 mL of complete medium or mRNA LNW (10 μg) in 0.1 mL of complete α-MEM. Cell viability was determined by the alamarBlue assay (Invitrogen, DAL1025) according to the manufacturer's protocol after two PBS washes. Intracellular chloride ion staining instructions, pages 31 / 47, CN 122341398 A

[0168] Cells were seeded in 35 mm confocal culture dishes (SPL Life Sciences) and cultured to 80-90% confluence. Next, 1 mL of 10 mM MQAE (N-(ethoxycarbonylmethyl)-6-methoxyquinolineonium bromide; Invitrogen, E3101) prepared in OptiMEM (Gibco, 31985070) was added. After incubation at 37°C for 30 minutes, the staining solution was removed, cells were washed twice with PBS, and phenol red-free DMEM (Gibco, 31053028) was added. Live cell images were acquired using a confocal laser scanning microscope (TCS SP8, Leica) under the same imaging settings. The excitation and emission wavelengths of MQAE were 405 nm and 410-480 nm, respectively.

[0169] Ratio imaging for intracellular pH determination

[0170] Cells were seeded in 35 mm confocal culture dishes and cultured to 80-90% confluence. The pH of intracellular acidic vesicles was analyzed by ratio analysis of the fluorescence intensity of the pH-sensitive dye pHrodo green (10 kDa dextran, Invitrogen, P10361) and the pH-insensitive dye Alexa Fluor 594 (10 kDa dextran; Invitrogen, D22913) on the same day. After co-incubation with both dyes at a concentration of 100 μg / mL for 8 hours, cells were fixed with 4% formaldehyde for 10 minutes and then incubated for 2 hours in calibration buffer (Invitrogen, P35379) at pH ranges of 4.5, 5.5, 6.5, and 7.5 before confocal imaging.Hours. In each image, the individual integrated intensities of the two dyes at different pH values ​​were calculated in each acidic vesicle in three independent experiments, with three replicates per experiment (n=300 vesicles). Generally, the fluorescence intensity of pHrodo green increased with decreasing pH, but the fluorescence intensity of Alexa 594 remained consistent. The R / G fluorescence ratio of Alexa 594 to pHrodo green was then calculated and plotted. To determine the pH of intracellular vesicles containing nanoworms, cells were co-incubated for 8 hours with Cy5-labeled Au@PDAT21NW (0.2 nM), pHrodo green (100 μg / mL), and Alexa Fluor 594 (100 μg / mL) and observed in real time under a confocal microscope. Approximately 300 vesicles were counted per group. Gal8 recruitment assay

[0171] Cells were seeded on 35 mm confocal culture dishes and cultured to approximately 80% confluence. Next, 500 ng of Gal8-GFP pDNA (addgene, 127191) and 1.5 μL of Lipofectamine 3000 (Invitrogen, L3000008) were added to 50 μL of OptiMEM; after 10 minutes, the mixture was diluted with 100 μL of complete culture medium. Cells were incubated with transfection medium for 6 hours, then replenished with fresh complete culture medium and incubated for another 36 hours. Next, cells were incubated for 24 hours in DMEM (supplemented with 1% FBS) with 0.2 nM Au@PDAT21NW or Au@PDAT21NP. Cells were fixed with cold methanol for 10 minutes and stained with DAPI (4',6-diamidinyl-2-phenylindole; Thermo Scientific, D1306) for confocal microscopy observation. The excitation wavelengths of DAPI and Gal8-GFP were 405 nm and 488 nm, respectively, and the emission wavelengths were 410-480 nm and 495-600 nm, respectively. Pharmacological blocking

[0172] Cells were seeded in 24-well plates and cultured to approximately 80% confluence. Before incubation with nanoworms, cells were pretreated with 0.3 mL of complete DMEM containing chemical inhibitors for 1 hour. These inhibitors included phenytoin III (2.5 μg / mL; MCE, HY-N6718), dynasore (120 μM; Cayman Chemical, #HY-N6717), methyl-β-cyclodextrin (MβCD) (10 mM; Tokyo Chemical Industry, M1356), sodium azide (0.1%; Sigma, S2002), and amiloride (0.5 mg / mL; Sigma, BP008). Next, the medium containing the inhibitors was removed, and 0.4 mL of fresh complete DMEM was added to the cells.DMEM containing the original concentration and 0.2 nM of the same inhibitor T21@NW. After 2 hours of incubation, cells were washed twice with PBS and digested with trypsin (0.25% trypsin-EDTA, Gibco), and cell counts were performed using a hemocytometer. Cells were further centrifuged at 1500 rpm for 5 minutes to form a pellet. Cell pellets were digested overnight in 0.25 mL aqua regia (3:1 v / v ratio of 38% HCl and 68% HNO3) and diluted to 5 mL with matrix solution (2% HCl, 2% HNO3) for ICP-MS determination of cell-associated Au content (Agilent 7900). Data are presented as mean ± standard deviation for four biological replicates (i.e., 3 wells per treatment group). Instructions for Use, Pages 32 / 47, No. 35, CN 122341398 A

[0173] Live-cell imaging of intracellular transport

[0174] After two PBS washes, the nanoworm-treated cells were stained with 100 nM LysoTracker Green DND-26 (Thermo Scientific, L7526) in 1 mL of phenol red-free complete DMEM for 30 minutes. Next, the cells were washed, stained with Hoechst 33342 (Thermo Sciences; #62249) for 10 minutes, washed again, and 1 mL of phenol red-free complete DMEM was added for confocal microscopy observation. The excitation wavelengths of Au@PDA@T21NW labeled with Hoechst 33342, LysoTracker Green DND-26, and Cy5 were 405, 488, and 650 nm, respectively, and their corresponding emission wavelengths were 410-480 nm, 495-580 nm, and 655-750 nm, respectively. Bio-transmission electron microscopy (Bio-TEM) imaging

[0175] Freshly harvested cell particles or tissues were fixed with glutaraldehyde (2.5% in PB; EMS, 16000) for 2 hours and stained with osmium tetroxide (1% dissolved in PB; EM, 19100) for 2 hours. The samples were dehydrated in an increasing ethanol gradient (30%, 50%, 70%, 80%, 90%, 100%) and propylene oxide, embedded in Epon 812 resin (EMS), and polymerized at 55°C for 48 hours. Ultrathin sections with a thickness of approximately 70 nm were deposited on a 200-mesh copper grid (Electron Microscopy Sciences, G200 Cu), stained with 4% uranyl acetate in 50% methanol / water (EMS, 541-09-3) and Reynolds lead citrate, and observed under TEM at a beam voltage of 100 kV (Hitachi H7700). siRNA transfection

[0176] Cells were seeded in 24-well plates and cultured to approximately 80% confluence. A transfection solution prepared with 50 µL OptiMEM contained 1.2 µL Lipo and 40 pmole of small interfering RNA (siRNA) specifically targeting human or mouse ClC3 (Dharmacon SMARTpool; Santa Cruz, sc-42381), human ClC5 (Santa Cruz) (sc-42385), or a non-targeting siRNA control (siCtrl) (Idobio). After incubation for 10 minutes and dilution with 350 µL of complete DMEM, the transfection mixture was added to each well of the 24-well plate. Cells were incubated for 48 hours prior to harvesting RNA for q-PCR analysis or confocal imaging. Immunoblotting

[0177] Cells were lysed with 0.2 mL of ice-cold RIPA buffer (Themo Scientific, 89900) and Pierce protease inhibitor (Thermo Scientific, A32963). After gently rotating on ice for 5 minutes, the lysate was centrifuged at 14000×g at 4°C to remove precipitated cell debris. The supernatant was subjected to quinoline carboxylic acid (BCA) determination (Thermo Scientific, #23225) to quantify protein concentration. The sample was then loaded onto a 10% gel [with sodium dodecyl sulfate (SDS)] for reducing polyacrylamide gel electrophoresis (PAGE). After transferring the gel protein onto a polyvinylidene fluoride (PVDF) membrane using Power Blotter Station (Invitrogen), the membrane was blocked for 1 hour at room temperature with 3% bovine serum albumin (BSA; Rocland Immunochemicals, BSA-50) in TBST (Tris-buffered saline containing Tween-20). The membrane was then incubated overnight at 4°C with primary antibodies against ClC3 (1:1000 in 3% BSA / TBST; Thermo Scientific, MA5-27709) and anti-β-tubulin (1:1000 in 3% BSA / TBST; Abcam, ab108342), and then soaked for 1 hour in 5% milk (Bio-Rad) / TBST with secondary antibodies conjugated to horseradish peroxidase (HRP) (1:1000 anti-rabbit, Bio-Rad 1706515; 1:3000 anti-mouse, Invitrogen 31430). After washing five times with TBST, proteins on the blot were detected using the ChemiDoc™ Touch imaging system with Clarity Max ECL protein blot substrate (Bio-Rad1705062). BMDM polarization

[0178] For M1 depolarization, BMDM from day 7 was mixed with 1 mL of 0.2Au@PDA@miR-223 NW cells were incubated in 1 mL of complete DMEM for 12 h. After two PBS washes, cells were stimulated with 0.4 mL of complete medium containing 100 ng / mL LPS (lipopolysaccharide; Sigma, L7895). After 24 h, the LPS-containing medium was removed, and cells were cultured in fresh complete medium or lysed with RNAiso Plus (Takara, #9108) to isolate RNA. For M2 repolarization, BMDM cells were stimulated on day 7 with 1 mL of complete DMEM containing 100 ng / mL LPS and 25 ng / mL IFN-γ (Sinobiological, 11725-H08H). Instructions 33 / 47, page 36, CN 122341398 A. After 24 h, remove the medium containing LPS / IFN-γ, wash twice with PBS, and repolarize the cells by incubating them in 1 mL of complete DMEM with 0.2 nM Au@PDA@miR-223 NW for 48 hours. hMSC osteogenic differentiation

[0179] hMSCs were seeded in 24-well plates until approximately 90% confluence. Next, hMSCs were treated with 0.4 mL of osteogenic medium (OM) containing 0.2 nM (0.05 mM ascorbic acid, 10 mM glycerophosphate, and 0.1 μM dexamethasone in complete α-MEM) for 7 out of 14 days, with the medium being changed every 3 days. After fixing with 95% ethanol for 10 minutes at room temperature and rinsing three times with PBS, cells were stained with 400 μL Alizarin Red S staining solution (Abcam, ab146374) at 37°C for 45 minutes, washed three times with Nanopure water, and observed under an optical microscope (10x objective) to confirm stem cell differentiation. To quantify calcium deposition, cells were treated with 200 μL 10% acetic acid for 30 minutes at room temperature, agitated to separate the cell monolayer, transferred to microcentrifuge tubes, heated at 85°C for 10 minutes, and cooled on ice for 5 minutes. After centrifugation at 10,000 rpm for 15 minutes and removal of the supernatant, 75 μL 10% ammonium hydroxide was added to dissolve the precipitate. Finally, the absorbance of the particulate sample at 405 nm, measured by a microplate reader, indicated the level of Alizarin Red (or calcium deposition). TUNEL staining

[0180] The Click iTTMPlus in situ apoptosis detection TUNEL assay (Invitrogen, C10617; Alexa Fluor 488) was used to visualize and quantify apoptotic and necrotic cells. 5 μm thick paraffin-embedded liver sections were stained according to the manufacturer's instructions, co-stained with DAPI for 10 minutes at room temperature, and mounted with Antifade Mountant for confocal laser scanning.Microscope. The excitation wavelengths of DAPI and Alexa Fluor 488 were 405 nm and 488 nm, respectively. The emission wavelength ranges of DAPI and Alexa Fluor 488 were 415–480 nm and 495–650 nm, respectively. Whole transcriptome analysis was performed using total RNA sequencing (RNA-Seq)

[0181] Cells with 80–90% confluence were seeded in 6-well plates and incubated overnight with complete DMEM. The cells were then divided into 4 groups (3 wells per group) to investigate the effects of nanoparticle shape and incubation time. After incubation, Au@PDA@T21NW cells were sent to the Beijing Genomics Institute (BGI) for cDNA preparation and whole transcriptome sequencing. The process followed standardized procedures monitored by the BGI quality control system. mRNA was isolated from total RNA using oligonucleotide (dT) magnetic beads according to the manufacturer’s cDNA library construction instructions. Double-stranded cDNA was sequenced using the DNBseq platform. On the DNBseq platform, at least 20 million high-quality reads were generated for each sample for data analysis. BGI performed differentially expressed gene (DEG) detection, DEG gene ontology (GO) analysis, and other gene expression-based analyses. GO items with a corrected p-value (Q value) of 0.05 or lower were considered significantly enriched in DEG. RNA isolation and qRT-PCR analysis

[0182] RNA was isolated using RNAiso Plus and reverse transcribed to produce cDNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, K1622). qRT-PCR was performed on a StepOnePlus real-time PCR system (Applied Biosystems) using the TB SYBR Green Premix Ex Taq Kit (Takara, RR82WR) according to the manufacturer's instructions. Transcriptional levels were analyzed using the ΔΔCT method and normalized relative to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Gene expression was quantified using pre-designed primers purchased from Shanghai Rui Mian Bio-Tech (see sequences in Table 3). Animals

[0183] All procedures followed the guidelines set forth by the Animal Experimentation Ethics Committee of the Chinese University of Hong Kong (CUHK). Male Balb / c and C57 mice aged 9 to 11 weeks were used and randomly assigned to different treatment groups. All mice were housed in a temperature- and humidity-controlled environment with a 12-hour light / dark cycle. For all biodistribution and efficacy studies, sample size (n) represents biological replicates. Unilateral ureteral obstruction (UUO) mouse model instruction manual 34 / 47 pages 37 CN 122341398 A

[0184] Male Balb / c mice aged 9–11 weeks were anesthetized by intraperitoneal injection of 0.2 mL of saline containing ketamine (100 mg / kg; Alfasan International BV) and toluidine (10 mg / kg; Alfasa International). The peritoneum was incised along the midline, the left ureter was dissected, and ligated twice with 5-0 sutures (Ningbo Cheng-He Microsurgical Instruments). After repositioning the intestines, the peritoneum was sutured. The mice were placed under a heat lamp to maintain body temperature until recovery from anesthesia. For analgesia, the mice were subcutaneously injected three times every 12 hours postoperatively with 0.1 mL of saline containing buprenorphine (Temgesic, 0.05 mg / kg). To verify the development of renal tubulointerstitial fibrosis, the mice were sacrificed 7 days postoperatively, and the kidneys were weighed and processed for histological analysis. Acute liver injury (ALI) mouse model induced by acetaminophen (APAP)

[0185] Male C57BL / 6 mice aged 10-12 weeks were fasted for 12 hours before injection of APAP (Solarbio, IA0030) to induce acute liver injury. After a single intraperitoneal injection of 0.3 mL 500 mg / kg APAP (by body weight) in saline, the mice were allowed free access to food. To verify the establishment of the acute liver injury model, the mice were sacrificed 24 h after APAP injection, serum was collected to determine alanine aminotransferase (ALT) levels (an indicator of liver function), and livers were collected for histological analysis. In vitro fluorescence imaging of hMSC biodistribution in UUO mice

[0186] Three days after ligation, UUO mice received a single intravenous injection of free DiR dye (10 mg / kg body weight; Invitrogen, D12731) or DiR-labeled hMSCs at 5 × 10⁵ cells / kg body weight (initial hMSCs, LipomCXCR4 / BMP7-treated hMSCs, and LNWmCXCR4 / BMP7-treated hMSCs). To label hMSCs with DiR, 10 μM DiR staining solution prepared in complete α-MEM was added to the cells and incubated at 37°C for 30 min. The staining solution was washed off, and the hMSCs were treated with trypsin for cell counting using a hematology analyzer. The hMSCs were further centrifuged at 1500 rpm for 5 min and resuspended in 200 μL PBS for injection. The injected mice were sacrificed 24 h after injection and perfused with PBS before imaging. In vitro fluorescence imaging of excised visceral organs (liver, lung, heart, spleen, and kidney) was performed using an Odyssey infrared imaging system (excitation wavelength: 700 nm, emission wavelength: ≥700 nm). Blood pharmacokinetics

[0187] Mice were intravenously injected via the tail vein with 0.1 mL containing 10 μg...LNW of mHGF. At different time points, 0.5 mL of blood was aspirated by terminal endocardial puncture (using a 25-gauge needle) and stored in EDTA-coated tubes (Becton Dickinson). After centrifugation at 1500×g for 10 minutes, 0.2 mL of plasma from the supernatant was collected, dissolved in 0.5 mL of aqua regia, and further diluted to 5 mL with matrix solution (2% HCl, 2% HNO3) for ICP-MS determination of Au content. Data were fitted to a single exponential decay model using Prism Graphpad software. In vivo toxicity of Au@PDA@mHGF NW

[0188] Mice with acute liver injury were given a single intravenous injection of 0.15 mL of 0.5 mg Au@PDA@mHGF NW dissolved in PBS containing 10 μg mHGF. Twenty-four hours later, 0.5 mL of whole blood was collected from the heart via intracardiac puncture under anesthesia. The blood was allowed to stand at room temperature for 30 minutes and then centrifuged at 2000 × g for 10 minutes at 4°C. Approximately 200 μL of serum was collected from the supernatant and sent to PathLab Medical Laboratories (Hong Kong, China) on the same day for blood biochemical analysis. Heart, liver, spleen, kidney, and lung sections were trimmed and fixed in 10% neutral buffered formalin for 48 hours, encased in paraffin, and cut into 5 μm thick sections. Paraffin sections were dewaxed in xylene (5 min × 3 times), hydrated with a series of ethanol solutions (100%, 95%, 70%) and deionized water, and counterstained with hematoxylin and eosin (H&E; Sigma) for 2 min and 1 min, respectively. The stained sections were dehydrated in ethanol, clarified in xylene, and mounted with DPX patches (Sigma) for visualization in bright-field mode on a Ti-E motorized inverted fluorescence microscope (Nikon). Protein purification for proteomics analysis

[0189] The desorbed protein was mixed with 5 times loading buffer (Beyotime) and boiled for 5 minutes. The prepared sample and protein marker (page 35 / 47, CN 122341398 A) were loaded into the wells of a 12% separation gel. The bands were separated by PAGE at 80 kV for 15 minutes, and then separated again at 120 kV for 15 minutes. The gel was stained with Coomassie Brilliant Blue (Beyotime). Three gel slices of each gel were cut into 1 mm3 cubes, transferred to 1.5 mL microcentrifuge tubes, and washed with 500 μL of 50 mM ammonium bicarbonate / acetonitrile (ACN) (1:1, v / v) solution until Coomassie Brilliant Blue disappeared. After discarding the supernatant, 500 μL of ACN was added, and the mixture was incubated for 10 minutes until the gel blocks became opaque and stuck together. After removing ACN, the gel sections were prepared in 10 mM dithiothreitol (DTT) / 50 mL solution.Rehydrate the gel in mM ammonium bicarbonate and incubate at 56°C for 1 hour, discarding the supernatant. During the rinsing step, add 500 μL of ACN and incubate the mixture for another 10 minutes, then remove the ACN. Next, incubate the gel sections in the dark at room temperature in 50 mM iodoacetamide (IAA) / 50 mM ammonium bicarbonate for 1 hour, discarding the IAA / ammonium bicarbonate. After repeating the rinsing step, incubate the gel blocks in proteomics-grade trypsin (Promega) digestion solution on ice for 45 minutes. Add 5–20 μL of enzyme digestion solution and digest overnight at 37°C. Add 100 μL of extraction solution (5% trifluoroacetic acid (TFA) – 50% ACN – 45% ddH2O) to each sample, incubate the mixture in a 37°C water bath for 1 hour, then sonicate for 5 minutes. Transfer the extract to fresh microcentrifuge tubes and repeat the extraction. LC-MS / MS Proteomics Analysis

[0190] All reagents were chromatographic grade and purchased from Thermo Fisher Scientific. Extracted peptides were lyophilized and resuspended in 10 μL of 0.1% formic acid and separated using an Orbitrap Eclipse mass spectrometer (Thermo Fisher Scientific) equipped with a 150 μm × 15 cm self-made column packed with Acclaim PepMap RPLC C18 (1.9 μm, 100 Å, Dr. Maisch GmbH). An organic gradient was driven by a Nanoflow UPLC system at a flow rate of 600 nL / min over 120 minutes using buffer A (0.1% formic acid aqueous solution) and buffer B (20% 0.1% formic acid aqueous solution – 80% acetonitrile). The gradient was maintained for 3 minutes at 4% to 8% B, 86 minutes at 8% to 28% B, 20 minutes at 28% to 40% B, 1 minute at 40% to 95% B, and 10 minutes at 95% to 95% B. The eluted peptides were directly sprayed into the mass spectrometer. Ten MS / MS data-dependent scans and one high-resolution (60,000 m / z at 400 m / z) full-scan mass spectra were acquired simultaneously to provide the amino acid sequence and mass-to-charge ratio of the selected peptide ions. Data processing and statistical analysis

[0191] Data analysis and graph construction were performed using Prism (GraphPad) and Excel software. Unpaired two-tailed t-tests were performed to determine the statistical significance of two group comparisons. Unpaired one-way ANOVA was performed post-hocly using the Tukey test to determine the statistical significance of multiple group comparisons. The normality of the mean sampling distribution was verified by the Shapiro-Wilk test. The homogeneity of variance was verified by the Bartlett test. Results were considered significant at P < 0.05.

[0192] All patents, patent applications, provisional applications and publications mentioned or cited herein, including all figures and tables, are incorporated herein by reference in their entirety, provided that they do not contradict the express teachings of this specification.

[0193] The following examples illustrate specific steps for carrying out the invention. These examples should not be construed as limiting. Unless otherwise stated, all percentages are by weight and all solvent mixtures are by volume. Example 1 - Preparation of nanomaterials encapsulated with oligonucleotides

[0194] We prepared Au40@PDA NW (Figure 1A) by sonicating citrate-terminated 40 nm gold NPs with monomeric dopamine at alkaline pH for 1 hour, following previous work.

[20] Au40@PDA NW had 4-5 gold cores, a PDA shell approximately 20 nm thick, approximately 224 nm long, and a zeta potential of -25.7 mV (Figures 1B and 8C). We then constructed oligonucleotide-encapsulated nanoworms (Au40@PDA@T21NW) by flipping their surface charge to positive at pH 3 and electrostatically adsorbing polythymidine (a pattern non-coding DNA) using 21T (T21). Each Au40@PDA@T21NW contained approximately 1024 strands, was stable for 24 h in 10% fetal bovine serum (FBS) at 37°C (Tables 4 and 5), and was non-toxic to A549 lung epithelial cells, bEnd.3 brain endothelial cells, primary bone marrow-derived macrophages (BMDM), or human mesenchymal stem cells (hMSC) 24 h after incubation (Figures 10A-10D). Amiloride treatment reduced the association of T21-coated nanoworms with all four cell types, most significantly among all the pharmacologically inhibitory agents tested, suggesting that macropinocytosis is a key uptake pathway (similar to PDA-coated NPs)

[21] . Treatment with fucoidan also weakened cell binding, suggesting that scavenger receptors play a role in uptake (similar to oligonucleotide-coupled NPs) [8] (Figs. 11A-12D). Example 2 - Endosomal escape of oligonucleotide-coated NWs

[0195] We treated A549 cells with anthocyanin (Cy)5-labeled Au40@PDA@T21NWs and monitored their cellular transport by confocal imaging. Lysotracker staining under confocal imaging confirmed that the colocalization of Cy5-T21@NWs with Lysotracker signals was consistently low in all cell lines tested, with Pearson correlation coefficients (PCC) of 0.1–0.2 (Fig. 1C). In addition to fluorescence imaging, we also used bioTEM imaging to locate the position of T21@NW after entry into cells. Consistent with the imaging data, the bioTEM images showed that after 24 h of incubation, most of the T21@NW was exposed in the cytosol, indicating endosome escape.The ability to colocalize vesicles is universal across different cell types (Fig. 1D). We then specifically labeled intracellular vesicles with anti-Rab 9 (late endosome) and anti-LAPM1 (lysosome) antibodies and tracked their distribution using time-lapse confocal imaging. A large number of Cy5-T21@NWs entered cells after 4 hours of incubation and significantly accumulated in late endosomes at 8 hours, with a PCC value of 0.781. We found that the PCC of Rab 9 and Cy5-T21@NWs decreased to 0.407 at 12 hours, indicating that more than half of the Cy5-T21@NWs escaped from late endosomes. Most T21@NWs escaped from late endosomes at 24 hours, with a PPC value of 0.305. In contrast, lysosome colocalization remained at ~0.3 at all time points tested (Fig. 1E). Therefore, nanoworms escape from late endosomes before reaching lysosomes.

[0196] To elucidate the effect of nanostructure shape on endosome escape, we prepared a control nanoparticle, “Au40@PDA@T21NP” (Fig. 8B), by adsorbing chains of the same density (approximately 380 chains per NP) onto unassembled Au@PDA NPs. The gold core size and PDA shell thickness were the same as those of Au40@PDA@T21NW. After incubation for 24 h, Au40@PDA-T21NPs were mainly located within acidic organelles (PCC ~0.755; Fig. 13), matching the data we reported for PDA NPs

[21] and DNA-encapsulated NPs [8]. We prepared elongated Au@PDA NP nanostructures of different sizes and shapes while maintaining similar concentrations added to cells. We fabricated Au20@PDA@T21 and Au60@PDA@T21NW nanorods using 20 and 60 nm gold cores (Figs. 9A–9D and Table 4); the nanoworms composed of larger cores contained fewer cores (approximately 6 cores for Au20@PDA@T21NW and approximately 2 cores for Au60@PDA@T21NW). Both nanoworms were highly localized to acidic organelles (PCC > 0.7) (Figs. 15A and 15B). However, when we prepared unassembled Au@PDA nanorods (core size: 45 × 200 nm) encapsulated with T21 with similar size, PDA thickness, and aspect ratio to Au40@PDA@T21NW (Figs. 9E–9F), the nanorods showed almost no co-localization with acidic organelles (PCC = 0.137), but exhibited reduced cellular uptake compared to Au40@PDA@T21NW (Figs. 16A and 16B). These data indicate that an aspect ratio of approximately 3.5 and a length of approximately 240 nm are suitable parameters for endosome escape. Example 3 – The role of CLC3 chloride ion exchanger in endosome escape of nanoworms

[0197] The proton sponge effect is the mainstream mechanism for endosome escape. Here, bafloxacin A1 or chloroquine (two endosomes) are used.Pretreatment of A549 cells with an acidification inhibitor did not impair Au40@PDA@T21NW endosome escape (Fig. 2A). Furthermore, the nanoworms did not exhibit pH buffering capacity between 4.5 (lysosomes) and 7.5 (extracellular space) (Fig. 2B), with a zeta potential below -25 mV (Fig. 17). We investigated other possible mechanisms of endosome escape beyond the proton sponge effect. Pretreatment with nitrofluoric acid (a general pharmacological blocker of Cl- channels) disrupted NW endosome escape, but pretreatment with lidocaine (a Na+ channel blocker), amiodarone (a K+ channel blocker), or nifedipine (a Ca2+ channel blocker) did not (Figs. 18 and 19). Therefore, we hypothesize that Cl- plays a role in endosome escape, but it remains unclear which specific Cl- channel is involved.

[0198] To investigate the mechanism of endosome escape, we used unbiased RNA sequencing (RNA-seq) to analyze changes in gene expression after incubation of nanoworms with A549 cells, screening for differentially expressed mRNA transcripts (DETs), with a statistical significance set at Q < 0.05. Initially, we explored the shape effect by comparing gene changes after 24 h of continuous treatment with Au40@PDA@T21NW and Au40@PDA@T21NP. This comparison showed enrichment of Gene Ontology (GO) entries related to late endosomes, cytoplasm, membrane, and cytosol (Figure 2C and Table 6). Then, we explored the time-dependent effect by comparing gene changes after 24 h of continuous incubation of Au40@PDA@T21NW (when NW was in cytosol) and 8 h (when NW was in late endosomes). This comparison showed enrichment of GO entries associated with transport vesicles, as well as late endosomes and endosome membranes (Fig. 2D and Table 7). Nevertheless, it remains unclear which DETs are critical.

[0199] Based on our RNA-seq data, we screened for Cl--related DETs with a log2 fold change >2 (Q < 0.05) and detected a sharp upregulation of ClC3 (H+ / 2Cl- exchanger) in two comparisons (Fig. 2E and 2F), with a log2 fold change of 20.12 (Q = 5.69 × 10⁻⁵) for “Au40@PDA@T21NW 24 h vs. Au40@PDA@T21NP 24 h” and a log2 fold change of 9.93 (Q = 0.029) for “Au40@PDA@T21NW 24 h vs. Au40@PDA@T21NW 8 h”. There are nine ClC subtypes in mammals. Among them, ClC1, ClC2, ClC-Ka, and ClC-Kb are plasma membrane Cl- channels, while ClC3 to ClC7 are H+ / 2Cl- exchangers in endosomes or lysosomes, used to regulate luminal acidification and Cl- accumulation.21. In DET analysis, we found that ClC5, TTYH1 (Tweety family member 1, a Cl- channel), and TMEM206 (a proton-activated Cl- channel) were also upregulated. Western blot analysis showed that ClC3 expression in A549 cells of the “NW 24 h” group was significantly higher than that of the “NP 24 h” or “NW 8 h” groups (Figs. 2G, 20A, and 20B). Confocal immunofluorescence confirmed that ClC3 was mainly expressed in lysosomes (PCC ~0.662) and moderately expressed in late endosomes (PCC=0.530) (Figs. 2H and 21). Therefore, we hypothesize that ClC3 mediates endosome escape in NW.

[0200] As supporting evidence, gene knockout via RNA interference using two different siRNA sequences targeting ClC3 (Fig. 22) increased nanoworm accumulation in acidic organelles of A549 cells at 24 h post-culture (PCC > 0.7; Fig. 2I), but not with the non-specific control siRNA sequence. Activation of ClC3 with bufotoxin

[22] [verified by quantitative reverse transcription polymerase chain reaction (qRT-PCR); Figs. 23A and 23B] showed limited overlap between nanoworms and acidic organelles at 8 h post-incubation (PCC = 0.249; Fig. 2K), while nanoworms were trapped in late endosomes without bufotoxin treatment (Fig. 1E). These data highlight the crucial role of CCl3 in endosome escape. Meanwhile, siRNA knockout of ClC5 resulted in modest accumulation in acidic organelles (PCC = 0.442; Figs. 2L and 24), suggesting that ClC5 plays a minor role in endosome escape. Example 4—Ion Accumulation and Endosomal Membrane Rupture

[0201] Since ClC3 is an ion exchanger, we tracked changes in H+ and Cl- concentrations in acidic organelles induced by nanoworm treatment. We used a Cl- indicator fluorescent dye called MQAE (N-(ethoxycarbonylmethyl)-6-methoxyquinolineonium bromide) to qualitatively assess Cl- content in wild-type A549 cells; MQAE fluorescence decreased with increasing Cl- concentration. Since MQAE is not a Cl-specific dye for late endosomes or lysosomes, we co-incubated cells with Cy5-labeled nanoworms and TRITC-labeled dextran, a tracer nanoparticle that enters cells via macropinocytosis, the same pathway used by nanoworms

[21] ; interestingly, after 8 hours of incubation, the colocalization of MQAE and Cy5 fluorescence was limited, indicating that the Cl- concentration in vesicles containing nanoworms was higher than in vesicles without nanoworms. However, in ClC3-silenced A549 cells, there was stronger overlap between MQAE and Cy5 fluorescence, indicating that Cl- in nanoworm organelles was significantly reduced compared to wild-type cells.The concentration decreased (Figs. 3A and 3C). Next, we used ratiometric assays to detect the pH of nanoworm-containing vesicles

[24] based on the fluorescence ratio of the pH-insensitive dye Alexa Fluor 594 coupled with dextran (red) and the pH-sensitive dye Rodo Green coupled with dextran (green) (Fig. 25); the fluorescence of Rodo Green decreased with increasing pH. When both dyes colocalized with Cy5-labeled nanoworms, the red-to-green ratio (R / G) fluorescence should indicate the pH of nanoworm-containing vesicles. After incubating wild-type A549 cells with two pH dyes without nanoworms for 8 hours, the mean pH of the organelles was 5.44, which is characteristic of late endosomes. In contrast, treatment of wild-type cells with nanoworms reduced the mean pH to 5.20, which means that the organelles were acidified. In ClC3-silenced cells, the organelle pH values ​​before and after nanoworm treatment were 5.70 and 5.55, respectively (Fig. 3B and 3D), both lower than those in wild-type cells. To investigate the function of the endosome proton pump, we assessed its activity by analyzing the interaction of the major subunits V0 and V1. Confocal images showed that A549 cells treated with nanoworms for 8 hours exhibited significantly enhanced colocalization of the two major subunits of the endosome proton pump compared to untreated cells (Fig. 3E), indicating stronger endosome proton pump activity after nanoworm treatment. These data suggest that nanoworm treatment increased the concentrations of H+ and Cl- in organelles, a cellular event regulated by ClC3. Therefore, we hypothesize that internalized nanoworms are transported to late endosomes, where they upregulate ClC3, leading to the accumulation of H+ and Cl-, ultimately resulting in nanoworm membrane rupture and endosome escape (Fig. 3F).

[0202] To verify this claim, we prepared A549 cells expressing galectin-8 (Gal8) fused with green fluorescent protein (GFP). Gal8-GFP is a cytoplasmic protein that aggregates in the inner lobules of the ruptured endosome membrane, producing strong GFP fluorescence [25,26]. Treatment of Gal8-GFP-expressing cells with Au@PDA@T21NW produced punctate spots (approximately 15 spots per cell), demonstrating endosome rupture (Figs. 3G and 26). No obvious Gal8 spots were observed in untreated or Au@PDA@T21NP-treated cells. Transmission electron microscopy imaging also captured the damaged endosome membrane and Au@PDA@T21NW escaping into the cytosol, with rupture sites on membrane segments near the nanoworms rather than throughout the entire membrane (Figs. 3H and 27). Example 5 – In Vitro Delivery of Multiple Oligonucleotide Types to Multiple Cell Types

[0203] We used Au40@PDA NW to deliver antisense DNA, miRNA, or siRNA to different cell types. These oligonucleotides…Nucleotide-loaded nanoworms were derived from the same method used to link T21 to Au40@PDA NWs. They were similar in size, surface charge, and oligonucleotide load to Au40@PDA@T21NWs, stable in FBS (Tables 4 and 5), entered cells without transfection agents, were non-cytotoxic (Figs. 10A–10D), and escaped endosomes 24 h after incubation (Fig. 28A; Figs. 4A, 4B, 4E, and 4F; Figs. 29, 30, and 33). In each application, we added the same amount of oligonucleotides to cells in all treatment groups.

[0204] Our first application was antisense gene regulation, where we delivered antisense DNA targeting enhanced GFP (asEGFP) to bEnd.3 cells expressing EGFP. In addition to the asEGFP-loaded nanoworms, cells were also treated with free asEGFP, Lipofectamine 3000 (a commercial transfection agent), and a mixture of Lipofectamine 3000 and asEGFP (Lipo+ asEGFP). Confocal images showed that the EGFP-encapsulated nanoworms reduced EGFP fluorescence, and flow cytometry data showed that after 72 hours of incubation, the number of EGFP-positive cells decreased by 60% compared to other groups (Figs. 28B and 28C). Liposome-assisted delivery showed a reduction of approximately 34% in EGFP-positive cells (Fig. 28D).

[0205] Our second application was macrophage polarization, where we delivered miR-223 (an M2 macrophage stimulating factor

[27] ) to primary BMDMs pre-induced to the M1 phenotype. M1 BMDMs were M2 repolarized by treatment with miR-223-encapsulated nanoworms, free miR-223, pristine Au@PDA, or Lipo+miR-223 for 48 hours. Nanoworms coated with miR-223 most effectively activated M2-related genes (IL-10, CD206, TGF-β, and arginase-I) and inhibited M1-related genes (IL-12, CD80, TNF-α, and IFN-γ) (Fig. 4C and 31). Nanoworms coated with miR-223 produced elongated cell shapes (M2 phenotype), but other treatments resulted in flattened, round cell shapes (M1 phenotype). Nanoworms coated with miR-223 produced strong C206 signaling but weak CD80 signaling, but pristine Au@PDA NW or free miR-223 produced strong CD206 signaling but weak CD80 signaling. Lipofectamine-assisted delivery produced fewer M2-like and CD206-positive cells (Fig. 4D).

[0206] Our third application was stem cell differentiation, where we delivered siRNA targeting Noggin (a negative regulator of osteogenic differentiation

[17] ) to hMSCs. The treatment lasted 14 days and included nanobots encapsulated with siNog, free siNog, and pristine Au@PDA.NW and Lipo+siNog. Nanoworms coated with siNog most effectively inhibited Noggin and activated osteogenic-related genes [bone morphogenetic protein (BMP)-2 and Runt-related transcription factor 2], and produced the most calcium deposition (Fig. 4G, 34A, 34B, and 34C). Lipofectamine-assisted delivery resulted in moderate inhibition of Noggin and increased calcium deposition. Specification 39 / 47 pages 42 CN 122341398 A Example 6—Preparation of mRNA-coated nanoworms

[0207] We further utilized Au@PDA NW to deliver mRNA, which is a larger target gene. Unlike oligonucleotides, direct adsorption of EGFP-encoding mRNA (mEGFP) onto Au@PDA NWs did not generate sufficient mRNA loading or protein expression. Therefore, we coated Au@PDA NWs with cationic lipids [1,2-dioleoyl-3-trimethylammonium propane (DOTAP), 1,2-distearate-sn-glycerol-3-phosphocholine (DOPE)] and cholesterol for mRNA adsorption (Fig. 5A). Au@PDA@lipid NWs were larger than Au@PDA NWs (278.5 nm) and positively charged (+30.5 mV), but when adsorbed with mEGFP at a w / w ratio of 1:50 (Figs. 35 and 36), the resulting Au@PDA@mEGFP-NWs were larger (280.3 nm), negatively charged (-35.2 mV), and contained approximately 68 mEGFP molecules (Tables 8 and 9). Confocal imaging confirmed that NWs escaped from late endosomes 10 hours after incubation (Fig. 37). Similar to Au@PDA@T21NW, Au@PDA@mEGFP-NW also enhanced ClC3 expression, endosome proton pump activity, and Cl- and H+ concentrations in vesicles (Fig. 38A-39C). Au@PDA@mEGFP-NW-transfected hMSCs were more effective than Lipofectamine, as evidenced by the increased proportion of EGFP-positive cells and cell viability 48 hours after incubation (Fig. 40A-40C). Example 7 - mRNA delivery to hMSCs as ex vivo cell therapy for renal fibrosis

[0208] We used nanoworms to deliver mRNA to hMSCs for cell programming, and then injected the genetically engineered hMSCs into mice with unilateral urethral obstruction (UUO; a well-established model of renal fibrosis) for ex vivo cell therapy. On the lipid-coated nanoworms, we adsorbed equal amounts of mRNA encoding BMP-7 (an anti-fibrotic protein) fused to orange fluorescent protein (OFP)

[28] and mRNA encoding C-X-C chemokine receptor type 4 (CXCR4; used for homing fibrosis sites) fused to green fluorescent protein

[29] .The Au@PDA@mCXCR4 / mBMP7 NW had similar physicochemical parameters and serum stability to the mEGFP-coated LNW (288.1 nm, -33.7 mV), entered hMSCs without inducing toxicity, and showed almost no colocalization with acidic organelles (Pearson coefficient ~0.091) (Fig. 5B). 85% of the transfected hMSCs supported the expression of both fluorescently labeled therapeutic proteins (Fig. 5C), and were more effective than Lipofectamine when the same amount of mRNA was added (Figs. 41A-41C).

[0209] We intravenously (iv) injected a single dose of saline, initial hMSCs, hMSCs transfected with mCXCR4 / mBMP7 nanoworms, or hMSCs transfected with both mRNAs using Lipofectamine into UUO mice. Three days after UUO surgery, the right UUO kidney showed fibrosis; the left contralateral kidney remained healthy (Fig. 42). In vitro validation showed that Au@PDA@mCXCR4 / mBMP7 NW more effectively transfected both mRNAs into hMSCs than Lipofectamine (Fig. 5E). In vitro fluorescence imaging showed that 24 h post-injection, nanoworm-transfected hMSCs accumulated the most in the kidneys of patients with renal fibrosis (UUO) and the least in other key organs (liver, spleen, and lungs), demonstrating their superior homing ability (Figs. 5F and 43). At sacrifice (14 days post-UUO surgery or 11 days post-treatment), the nanoworm-transfected hMSC group exhibited the strongest inhibitory effect on type I collagen and α-smooth muscle actin (both markers of renal fibrosis) by immunohistochemistry (IHC; Figs. 5G-5I, 44A, and 44B) and qRT-PCR (Figs. 5J and 5K), and showed the highest expression of BMP7 by enzyme-linked immunosorbent assay (ELISA; Fig. 5L), demonstrating that nanoworm-promoted genetic engineering enhanced the therapeutic effect of hMSCs in UUO kidneys. Blood tests and histological examination of major organs showed no in vivo toxicity after treatment with nanoworms coated with mCXCR4 / mBMP7 (Figs. 45A, 45B, and 46). Example 8 - In vivo delivery of mRNA to hepatocytes for the treatment of acute liver injury

[0210] We used nanoworms to deliver mRNA to hepatocytes in vivo to reduce acetaminophen (APAP)-induced acute liver injury (ALI) in mice (Fig. 6A). We adsorbed mRNA encoding hepatocyte growth factor (mHGF) onto lipid-coated nanoworms to accelerate hepatocyte recovery and improve liver function.30 Nanoworms coated with mHGFP had similar properties to those coated with mEGFP (Tables 8 and 9), with a blood half-life of approximately 1 hour, as determined by ICP-MS, 24 h post-injection.Accumulation in the liver (30% of the injected dose) (Figs. 47A and 47B). Cy5-labeled nanoworms coated with mHGFP entered hepatocytes (Fig. 6B) without overlapping with late endosomes (PCC ~ 0.202) or lysosomes (PCC ~ 0.153) (Fig. 6D). Five hours after injection, TEM imaging of the liver captured the accumulation of nanoworms in organelles, and 10 hours after injection, nanoworms were located in the cytosol (with vesicle membrane rupture) (Fig. 6C), demonstrating endosome escape.

[0211] After animal recovery (24 h after APAP induction), we intravenously injected acute liver injury mice with saline, raw Au@PDA@lipid NW, Au@PDA@mHGF NW, or Lipo+mHGF, and then sacrificed them 24 h after treatment. ELISA showed that nanoworms coated with mHGFP were most effective in increasing HGF expression in the liver (Fig. 47C), reducing necrotic area (Fig. 6E and 6G) ​​and apoptotic cells (Fig. 6F and 6H), and decreasing serum alanine aminotransferase (ALT; a liver function indicator) by approximately 50% (Fig. 6I), demonstrating the efficacy of NW-assisted in vivo transfection and anti-ALI. At 24 h post-injection, nanoworms coated with mHGFP did not cause toxicity or accumulation in major visceral organs (Fig. 48 and 49), demonstrating in vivo safety.

[0212] It should be understood that the embodiments and implementations described herein are for illustrative purposes only, and various modifications or changes will be suggested to those skilled in the art, which will be included within the spirit and scope of this application. Furthermore, any element or limitation of any invention or implementation thereof disclosed herein may be combined with any and / or all other elements or limitations disclosed herein (alone or in any combination) or any other invention or implementation thereof, and all such combinations are considered to be within the scope of the invention, but not limited thereto. Example Embodiments

[0213] Embodiment 1. A nanoworm composition comprising: at least two metal nanoparticle cores; a polymer coating layer on the at least two metal nanoparticle cores; a therapeutic nucleic acid; and optionally, a lipid coating layer on the polymer coating layer.

[0214] Embodiment 2. The nanoworm composition according to Embodiment 1, wherein the at least two metal nanoparticle cores are at least two gold nanoparticle cores.

[0215] Embodiment 3. The nanoworm composition according to any of the preceding embodiments, wherein each of the at least two metal nanoparticle cores has a diameter of about 40 nm.

[0216] Embodiment 4. The nanoworm composition according to any of the preceding embodiments, comprising 4 or 5 metal nanoparticle cores.

[0217] Embodiment 5. The nanoworm composition according to any of the preceding embodiments, wherein the polymer coating layer...The thickness is from about 7 nm to about 35 nm.

[0218] Embodiment 6. The nanoworm composition according to any of the preceding embodiments, wherein the polymer coating layer comprises polydopamine (PDA), polyethylene glycol (PEG), polyethyleneimine (PEI), or silica.

[0219] Embodiment 7. The nanoworm composition according to any of the preceding embodiments, wherein the nanoworm composition carries an anionic charge.

[0220] Embodiment 8. The nanoworm composition according to any of the preceding embodiments, wherein the therapeutic nucleic acid is an oligonucleotide or mRNA.

[0221] Embodiment 9. The nanoworm composition according to any of the preceding embodiments, wherein the length of the therapeutic nucleic acid is from about 15 to about 5000 nucleotides.

[0222] Embodiment 10. The nanoworm composition according to any of the preceding embodiments, wherein the therapeutic nucleic acid is an antisense oligonucleotide (ASO), short interfering RNA (siRNA), microRNA (miRNA), messenger RNA (mRNA), or any combination thereof.

[0223] Embodiment 11. The nanoworm composition according to Embodiment 8, wherein a therapeutic oligonucleotide nucleic acid is adsorbed onto a polymer coating layer. Specification 41 / 47 pages 44 CN 122341398 A

[0224] Embodiment 12. The nanoworm composition according to Embodiment 10, wherein the mRNA encodes hepatocyte growth factor (HGF), chemokine receptor type 4 (CXCR4), bone morphogenetic protein 7 (BMP-7), enhanced green fluorescent protein (EGFP), red fluorescent protein (RFP), or any combination thereof; the miRNA is miR223; or the siRNA is siNog.

[0225] Embodiment 13. The nanoworm composition according to any of the preceding embodiments, comprising at least two metal nanoparticle cores; a polymer coating layer on the at least two metal nanoparticle cores; and a lipid coating layer on the polymer coating layer, wherein a therapeutic nucleic acid is bound to the lipid coating layer.

[0226] Embodiment 14. The nanoworm composition according to Embodiment 13, wherein the lipid coating layer comprises 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), cholesterol, 1,2-distearate-sn-glycerol-3-phosphocholine (DOPE), 1,1′-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azadiyl)bis(dodecane-2-ol)) (C12-200), (6Z,9Z,28Z,31Z)-heptadecane-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate (DLin-MC3-DMA), ((4-hydroxybutyl)azadiyl]di(Hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), dimethyl bis(octadecyl)ammonium bromide (DDA), 1,2-distearate-sn-glycerol-3-phosphocholine (DSPC), 1,2-dioleoyl-3-dimethylammonium propane (DODAP), 1,2-dioleoyl-3-dimethylaminopropane (DODMA), dipalmitoylphosphatidylcholine (DPPC), or any combination thereof.

[0227] Embodiment 15. The nanoworm composition according to any of the preceding embodiments, wherein the nanoworm comprises citrate-terminated gold nanoparticles (Cit-Au NP) with a diameter ranging from about 20 to about 60 nm.

[0228] Embodiment 16. The nanoworm composition according to any of the preceding embodiments, wherein the nanoworm comprises Aux@PDA NW, wherein the diameter of x is about 20, about 40, or about 60 nm.

[0229] Embodiment 17. The nanoworm composition according to any of the preceding embodiments, wherein the nanoworm comprises Au@PDANR (nanoran), wherein the size of the NR is about 45 nm × about 180 nm.

[0230] Embodiment 18. A method of nucleic acid delivery comprising administering the nanoworm composition of Embodiment 1 to a subject.

[0231] Embodiment 19. The method according to Embodiment 18, wherein the therapeutic nucleic acid regulates gene expression in the subject.

[0232] Embodiment 20. The method according to Embodiment 18, wherein the therapeutic nucleic acid escapes from endosomes and / or is degraded by lysosomes.

[0233] Embodiment 21. The method according to Embodiment 18, wherein the therapeutic nucleic acid enhances stromal cell differentiation.

[0234] Embodiment 22. The method according to Embodiment 19, wherein the regulation of gene expression in the subject treats acute liver injury, chronic kidney disease, or a combination thereof.

[0235] Embodiment 23. The method according to Embodiment 18, wherein the nanoworms enter epithelial cells, hepatocytes, kidney cells, endothelial cells, primary macrophages, mesenchymal stem cells, nerve cells, or any combination thereof in the body of a subject.

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Specification 47 / 47 pages 50 CN 122341398 A Figure 1A Figure 1B Figure 1C Specification Appendix 1 / 80 pages 51 CN 122341398 A Figure 1D Figure 1E Specification Appendix 2 / 80 pages 52 CN 122341398 A Figure 2A Figure 2B Figure 2C Appendix 3 / 80 page 53 CN 122341398 A Figure 2D Figure 2E Appendix 4 / 80 page 54 CN 122341398 A Figure 2F Figure 2G Appendix 5 / 80 page 55 CN 122341398 A Figure 2H Appendix 6 / 80 page 56 CN 122341398 A Figure 2I Figure 2J Appendix 7 / 80 page 57 CN 122341398 A Figure 2K Figure 2L Appendix 8 / 80 page 58 CN 122341398 A Figure 3A Appendix 9 / 80 page 59 CN 122341398 A Figure 3B Figure 3C Appendix 10 / 80 page 60 CN 122341398 A Figure 3D Figure 3E Appendix 11 / 80 page 61 CN 122341398 A Figure 3F Figure 3G Instruction Manual Drawings 12 / 80 Page 62 CN 122341398 A Figure 3H Figure 4A Instruction Manual Drawings 13 / 80 Page 63 CN 122341398 A Figure 4B Figure 4C Instruction Manual Drawings 14 / 80 Page 64 CN 122341398 A Figure 4D Figure 4E Instruction Manual Drawings 15 / 80 Page 65 CN 122341398 A Figure 4F Figure 4G Figure 4H Instruction Manual Drawings 16 / 80 Page 66 CN122341398 A Figure 5A Figure 5B Instruction Manual Drawings 17 / 80 Page 67 CN 122341398 A Figure 5C Figure 5D Instruction Manual Drawings 18 / 80 Page 68 CN 122341398 A Figure 5E Figure 5F Instruction Manual Drawings 19 / 80 Page 69 CN 122341398 A Figure 5G Figure 5H Instruction Manual Drawings 20 / 80 Page 70 CN 122341398 A Figure 5I Figure 5J Instruction Manual Drawings 21 / 80 Page 71 CN 122341398 A Figure 5K Figure 5L Instruction Manual Drawings 22 / 80 Page 72 CN 122341398 A Figure 6A Figure 6B Instruction Manual Drawings 23 / 80 Page 73 CN 122341398 A Figure 6C Figure 6D Instruction Manual Drawings 24 / 80 Page 74 CN 122341398 A Figure 6E Figure 6F Figure 6G Instruction Manual Drawings 25 / 80 Page 75 CN 122341398 A Figure 6H Figure 6I Instruction Manual Drawings 26 / 80 Page 76 CN 122341398 A Figure 7A Figure 7B Instruction Manual Drawings 27 / 80 Page 77 CN 122341398 A Figure 7C Instruction Manual Drawings 28 / 80 Page 78 CN 122341398 A Figure 7D Instruction Manual Drawings 29 / 80 Page 79 CN 122341398 A Figure 7E Figure 7F Instruction Manual Drawings 30 / 80 Page 80 CN 122341398 A Figure 7G Figure 8A Instruction Manual Drawings 31 / 80 Page 81 CN 122341398 A Figure 8B Figure 8C Instruction Manual Drawings 32 / 80 Page 82 CN 122341398 A Figure 9A Figure 9B Instruction Manual Drawings 33 / 80 Page 83 CN 122341398 A Figure 9C Figure 9D Instruction Manual Drawings 34 / 80 Page 84 CN 122341398 A Figure 9E Figure 9F Instruction Manual Drawings 35 / 80 Page 85 CN 122341398 A Figure 10A Figure 10B Instruction Manual Drawings 36 / 80 Page 86 CN 122341398 A Figure 10C Figure 10D Instruction Manual Drawings 37 / 80 Page 87 CN 122341398 A Figure 11A Figure 11B Instruction Manual Drawings 38 / 80 Page 88 CN 122341398 A Figure 11C Figure 11D Instruction Manual DrawingsFigure 39 / 80, page 89, CN 122341398 A; Figure 12A, Figure 12B; Instruction Manual Drawings, pages 40 / 80, page 90, CN 122341398 A; Figure 12C, Figure 12D; Instruction Manual Drawings, pages 41 / 80, page 91, CN 122341398 A; Figure 13A; Instruction Manual Drawings, pages 42 / 80, page 92, CN 122341398 A; Figure 13B, Figure 14A; Instruction Manual Drawings, pages 43 / 80, page 93, CN 122341398 A; Figure 14B, Figure 15A; Instruction Manual Drawings, pages 44 / 80, page 94, CN 122341398 A; Figure 15B, Figure 16A; Instruction Manual Drawings, pages 45 / 80, page 95, CN 122341398 A; Figure 16B, Figure 17; Instruction Manual Drawings, pages 46 / 80, page 96, CN 122341398 A; Figure 18; Instruction Manual Drawings, pages 47 / 80 Page 97 CN 122341398 A Figure 19 Instruction Manual Drawings 48 / 80 Page 98 CN 122341398 A Figure 20A Figure 20B Instruction Manual Drawings 49 / 80 Page 99 CN 122341398 A Figure 21 Instruction Manual Drawings 50 / 80 Page 100 CN 122341398 A Figure 22 Figure 23A Instruction Manual Drawings 51 / 80 Page 101 CN 122341398 A Figure 23B Figure 24 Instruction Manual Drawings 52 / 80 Page 102 CN 122341398 A Figure 25 Figure 26A Instruction Manual Drawings 53 / 80 Page 103 CN 122341398 A Figure 26B Figure 26C Instruction Manual Drawings 54 / 80 Page 104 CN 122341398 A Figure 26D Figure 27A Instruction Manual Drawings 55 / 80 Page 105 CN 122341398 A Figure 27B Figure 28A Instruction Manual Drawings 56 / 80 Pages 106 CN 122341398 A Figure 28B Figure 28C Instruction Manual Drawings 57 / 80 Pages 107 CN 122341398 A Figure 28D Figure 29 Instruction Manual Drawings 58 / 80 Pages 108 CN 122341398 A Figure 30 Figure 31 Instruction Manual Drawings 59 / 80 Pages 109 CN 122341398 A Figure 32A Instruction Manual Drawings 60 / 80 Pages 110 CN 122341398 A Figure 32B Instruction Manual Drawings 61 / 80 Pages 111 CN122341398 A Figure 32C Instruction Manual Drawings 62 / 80 Pages 112 CN 122341398 A Figure 33 Figure 34A Instruction Manual Drawings 63 / 80 Pages 113 CN 122341398 A Figure 34B Figure 34C Instruction Manual Drawings 64 / 80 Pages 114 CN 122341398 A Figure 35 Instruction Manual Drawings 65 / 80 Pages 115 CN 122341398 A Figure 36 Figure 37 Instruction Manual Drawings 66 / 80 Pages 116 CN 122341398 A Figure 38A Figure 38B Instruction Manual Drawings 67 / 80 Pages 117 CN 122341398 A Figure 39A Figure 39B Instruction Manual Drawings 68 / 80 Pages 118 CN 122341398 A Figure 39C Figure 40A Instruction Manual Drawings 69 / 80 Pages 119 CN 122341398 A Figure 40B Figure 40C Instruction Manual Drawings Page 70 / 80 120 CN 122341398 A Figure 41A Figure 41B Instruction Manual Drawings Page 71 / 80 121 CN 122341398 A Figure 41C Instruction Manual Drawings Page 72 / 80 122 CN 122341398 A Figure 42 Instruction Manual Drawings Page 73 / 80 123 CN 122341398 A Figure 43 Figure 44A Instruction Manual Drawings Page 74 / 80 124 CN 122341398 A Figure 44B Instruction Manual Drawings Page 75 / 80 125 CN 122341398 A Figure 45A Instruction Manual Drawings Page 76 / 80 126 CN 122341398 A Figure 45B Figure 46 Instruction Manual Drawings Page 77 / 80 127 CN 122341398 A Figure 47A Figure 47B Instruction Manual Drawings Page 78 / 80 128 CN 122341398 A Figure 47C Figure 48 Instruction Manual Drawings Page 79 / 80 129 CN 122341398 A Figure 49 Instruction Manual Drawings Page 80 / 80 130 CN 122341398 A

Claims

1. A nanoworm composition comprising: At least two metal nanoparticle cores or nanorod cores; A polymer coating layer is located on the at least two metal nanoparticle cores or nanorod cores; Therapeutic nucleic acids; and Optionally, a lipid coating layer is applied over a polymer coating layer.

2. The nanoworm composition according to claim 1, wherein the at least two metal nanoparticle cores or nanorod cores are at least two gold nanoparticle cores.

3. The nanoworm composition according to claim 1, wherein each of the at least two metal nanoparticles or nanorod cores has a diameter of about 40 nm.

4. The nanoworm composition according to claim 1, comprising 4 or 5 metal nanoparticle cores or nanorod cores.

5. The nanoworm composition according to claim 1, wherein the thickness of the polymer coating layer is from about 7 nm to about 35 nm.

6. The nanoworm composition according to claim 1, wherein the polymer coating layer comprises polydopamine (PDA), polyethylene glycol (PEG), polyethyleneimine (PEI), or silica.

7. The nanoworm composition according to claim 1, wherein the nanoworm composition carries an anionic charge.

8. The nanoworm composition according to claim 1, wherein the therapeutic nucleic acid is an oligonucleotide or mRNA.

9. The nanoworm composition according to claim 1, wherein the therapeutic nucleic acid is about 15 to about 5000 nucleotides in length.

10. The nanoworm composition according to claim 1, wherein the therapeutic nucleic acid is antisense DNA, antisense oligonucleotide (ASO), short interfering RNA (siRNA), microRNA (miRNA), messenger RNA (mRNA), or any combination thereof.

11. The nanoworm composition of claim 8, wherein the therapeutic oligonucleotide nucleic acid is adsorbed onto the polymer coating layer.

12. The nanoworm composition of claim 10, wherein the mRNA encodes hepatocyte growth factor (HGF), chemokine receptor type 4 (CXCR4), bone morphogenetic protein 7 (BMP-7), enhanced green fluorescent protein (EGFP), red fluorescent protein (RFP), or any combination thereof; wherein the miRNA is miR223; or wherein the siRNA is siNog.

13. The nanoworm composition according to claim 1, further comprising: A lipid coating layer on a polymer coating layer, wherein the therapeutic nucleic acid is bound to the lipid coating layer.

14. The nanoworm composition according to claim 13, wherein the lipid coating layer comprises 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), cholesterol, 1,2-distearate-sn-glycerol-3-phosphocholine (DOPE), 1,1′-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azadiyl)bis(dodecane-2-ol)) (C12-200), (6Z,9Z,28Z,31Z)-heptadecane-6,9,28, 31-Tetraen-19-yl 4-(dimethylamino)butyrate (DLin-MC3-DMA), ((4-hydroxybutyl)azadiyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), dimethyl bis(octadecyl)ammonium bromide (DDA), 1,2-distearyl-sn-glycerol-3-phosphocholine (DSPC), 1,2-dioleoyl-3-dimethylammonium propane (DODAP), 1,2-dioleoyl-3-dimethylaminopropane (DODMA), dipalmitoylphosphatidylcholine (DPPC), or any combination thereof.

15. The nanoworm composition of claim 1, wherein the nanoworm comprises citrate-terminated gold nanoparticles (Cit-Au NP) with a diameter ranging from about 20 to about 60 nm.

16. The nanoworm composition of claim 15, wherein the nanoworm comprises Au x @PDA NW, where the diameter of x is approximately 20, 40, or 60 nm.

17. The nanoworm composition of claim 1, wherein the nanoworm comprises Au@PDA NR (nanorobars) having a size of about 45 nm × about 180 nm.

18. A method for nucleic acid delivery, comprising administering the nanoworm composition of claim 1 to a subject.

19. The method of claim 18, wherein the therapeutic nucleic acid regulates gene expression in the subject.

20. The method of claim 18, wherein the therapeutic nucleic acid escapes endosome and / or lysosomal degradation.

21. The method of claim 18, wherein the therapeutic nucleic acid enhances stem cell differentiation.

22. The method of claim 19, wherein the regulation of gene expression in the subject is used to treat acute liver injury, chronic kidney disease, or a combination thereof.

23. The method of claim 18, wherein the nanoworms enter the epithelial cells, hepatocytes, kidney cells, endothelial cells, primary macrophages, mesenchymal stem cells, nerve cells, or any combination thereof in the subject.

24. The composition of claim 18, wherein the nanoworm is administered via intra-articular injection, intrathecal injection, or postbulbar injection.

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