WO2025199294A1 - Method and apparatus for sonication-assisted intracellular mrna therapeutic delivery and transient opening of airway epithelial tight junctions - Google Patents

Abstract

Methods and devices for enhancing delivery of a biologically active molecule into a cell and/or increasing endosomal escape of mRNA within a cell. Methods and devices for improving delivery of a biologically active molecule across a cellular barrier or paracellularly.

Description

METHOD AND APPARATUS FOR SONICATION-ASSISTED INTRACELLULAR MRNA THERAPEUTIC DELIVERY AND TRANSIENT OPENING OF AIRWAY EPITHELIAL TIGHT JUNCTIONS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63/568,051, filed March 21, 2024, the content of which is hereby incorporated by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under EB027062 awarded by the National Institutes of Health and 2143620 awarded by the National Science Foundation. The government has certain rights in the invention.

TECHNICAL FIELD

[0003] The present disclosure relates to apparatuses and methods for delivering cargo to cells. For example, the disclosed apparatuses and methods may be utilized to deliver lipid nanoparticles (LNPs) carrying exogenous mRNA to a cell. Additionally, the disclosed apparatuses and methods may employ a combination of hyperosmotic treatment and mechanical vibration to deliver a drug to a cell.

BACKGROUND OF THE INVENTION

[0004] Ionizable lipid nanoparticles (LNPs) have been pivotal in combating COVID- 19, and numerous preclinical and clinical studies have highlighted their potential in nucleic acid-based therapies and vaccines. However, the effectiveness of endosomal escape for the nucleic acid cargos encapsulated in LNPs is still low, leading to suboptimal treatment outcomes and side effects. Hence, improving endosomal escape is crucial for enhancing the efficacy of nucleic acid delivery using LNPs.

[0005] In consideration of other delivery approaches and their shortcomings, it should be noted that pulmonary drug delivery offers significant advantages for local and systemic therapy by directly targeting the lungs, minimizing off-target effects, and avoiding first-pass metabolism. However, the airway epithelium, which serves as a protective barrier, presents significant challenges for effective drug delivery. Tight junctions (TJs) between epithelial cells and mucociliary clearance hinder drug absorption, especially for high molecular weight drugs. To address this, various TJ modulators, such as chelators and surfactants, have been explored, but their safety concerns limit clinical application. In clinical settings, hyperosmotic sodium chloride (NaCl) and mechanical vibration are used for mucus clearance, but their effects on TJ permeability remain underexplored.

BRIEF SUMMARY OF THE INVENTION

[0006] Methods and deices for improving mRNA delivery, or small molecule or other biologically active molecules, into cells and/or endosomal escape thereof within cells. Methods and devices for improving paracellular delivery of therapeutic molecules are provided.

[0007] A method of enhancing delivery of a biologically active molecule into a cell and/or increasing endosomal escape of mRNA within a cell, comprising applying to the cell, in the presence of the biologically active molecule or the mRNA within endosomes of the cell, an amount of sonication or mechanical vibration at 100Hz or less effective to enhance delivery of a biologically active molecule into a cell and/or increase endosomal escape of mRNA within a cell.

[0008] A method of improving delivery of a biologically active molecule across a cellular barrier or paracellularly, comprising applying to the cellular barrier (a) in the presence of the biologically active molecule, or (b) prior to administering the biologically active molecule, an amount of (i) sonication or mechanical vibration at 100Hz or less and (ii) a hypertonic solution, wherein the solution is hypertonic to the fluid in which the cell is located, effective to improve delivery of a biologically active molecule across a cellular barrier or paracellularly.

[0009] A device for effecting a method disclosed herein in a human, animal or cells, comprising (i) a sonication apparatus which sonication apparatus comprises one or more acoustic actuators or a mechanical vibration apparatus comprising one or more motorized electric actuators and (ii) one or more accelerometers, wherein the device is configured for attachment to, or communication with an amplifier, and wherein the device is optionally controlled, and/or from which signals are received wirelessly or via wire, by a computer. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] For a more complete understanding of the present disclosure and the associated features and advantages, reference is now made to the following description, taken in conjunction with the accompanying drawings, which are not to scale, and in which show:

[0011] Figures 1A-1B. Overview of oscillation-enhanced endosomal escape of mRNA. (Figure 1A) Schematic of the setup for oscillation generation and the suggested mechanism for the endosomal escape process of mRNA under oscillation. (Figure IB) The custom-built system to generate oscillation.

[0012] Figures 2A-2C. Fusion of LNPs with opposite charges induced by mechanical oscillation. (Figure 2A) Schematic diagram showing the LNP fusion between the ionizable MC3 LNPs possessing positive surface charge in acidic environment and the negatively charged 18: 1 PA LNPs induced by a mechanical oscillation. Negative staining TEM images (Figure 2B) and the size distribution obtained from DLS measurements (Figure 2C) of MC3 LNPs, mixture of MC3 LNPs and 18: 1 PA LNPs and mixture of MC3 LNPs and 18: 1 PA LNPs after oscillation at pH 6.

[0013] Figures 3A-3D. Cellular internalization of Dil-LNPs with time. (Figure 3A) Confocal laser scanning microscopy (CLSM) observations of FNE cells after incubation with Dil- LNP for 4 h and 8 h. (Figure 3B) Average fluorescent intensity of Dil-LNPs per FNE cell at 4 h and 8 h calculated from the CLSM results. (Figure 3C) Flow cytometer profile of FNE cells incubated with Dil-LNPs for 0 h, 2 h, 4 h, 8 h, 12 h and 24 h. (Figure 3D) Quantitative cellular uptake of Dil-LNPs based on the flow cytometer profile in (C). Data presented as mean ± standard deviation (SD), n=3.

[0014] Figures 4A-4B. Endosomal escape of Dil-LNPs improved by mechanical oscillation. (Figure 4A) A confocal image and a cropped region of FNE cells incubated with Dil- LNPs for 4 h and treated with the lysosome dye Lysotracker deep red (LT deep red) to show colocalization of LT deep red (red) with Dil-labeled LNPs (green). (Figure 4B) A confocal image and a cropped region of FNE cells incubated with Dil-LNPs for 4 h and treated with LT deep red as well as oscillation of 65 Hz for 5 min to show colocalization of LT deep red with Dil-labeled LNPs. Scale bars: 10 pm. [0015] Figures 5A-5B. Calcein release from endosomes prompted by mechanical oscillation. (Figure 5A) After incubation with LNPs for 4 h, a similar punctate pattern of calcein fluorescence (green) being observed within FNE cells despite the exposure of oscillation (65 Hz, 5 min). However, after incubation with LNPs for 8 h, the cells treated by oscillation exhibiting a more diffused pattern compared to the cells without being exposed to oscillation. (Figure 5B) Average cytosolic calcein fluorescence in FNE cells calculated from confocal laser scanning microscopy (CLSM) results in (A) for different groups. Data presented as mean ± SD, n=5, P- values are calculated using one-way ANOVA, ns: not significant, *P<0.05.

[0016] Figures 6A-6E. Safety test of the synergistic effect of LNPs and oscillation on FNE cells. (Figure 6A) Viability of FNE cells after different treatments being monitored by live/dead assay. Green channel: live cells, red channel: comprised/dead cells. (Figure 6B) Percentage of viability of FNE cells calculated according to live/dead assay results in (Figure 6A). (Figure 6C) Mitochondrial membrane potential of FNE cells after incubation with LNPs and subsequently exposure of oscillation (65 Hz, 5 min) monitored by JC-1 assay, with untreated FNE cells as control and carbonyl cyanide m-chlorophenyl hydrazone (CCCP)-treated cells as positive control. Green channel: JC-1 monomers, red channel: JC-1 aggregates. (Figure 6D) Quantified JC-1 aggregate/monomer ratios. (Figure 6E) Confocal images demonstrating structure of Golgi apparatus within FNE cells after different treatments (LNPs only, LNP w oscillation) with untreated cells as control. Data presented as mean ± SD, n=3, P-values are calculated using oneway ANOVA, ns: not significant, *P<0.05.

[0017] Figures 7A-7D. In vitro mRNA expression enhanced by mechanical oscillation. (Figure 7A) Expression of green fluorescent protein (GFP) within FNE cells in different groups (untreated as control, treated by LNPs for 4 h, treated by oscillation of 65 Hz for 5 min post incubation with LNPs for 4 h) observed by confocal laser scanning microscopy (CLSM). Green channel: GFP, blue channel: nuclei stained by DAPI. (Figure 7B) GFP expression within FNE cells treated under the same conditions as (A) analyzed by flow cytometry. (Figure 7C) Expression of GFP within FNE cells in different groups (untreated as control, treated by LNPs for 12 h, treated by oscillation of 65 Hz for 5 min post incubation with LNPs for 12 h) observed by CLSM. (Figure 7D) GFP expression within FNE cells treated under the same conditions as (C) analyzed by flow cytometry. [0018] Figures 8A-8B. In vitro transfection of a human lung cell line (A549). (Figure 8A) Expression of green fluorescent protein (GFP) within A549 cells in different groups (untreated as control, treated by LNPs for 12 h, treated by oscillation of 65 Hz for 5 min post incubation with LNPs for 12 h) observed by confocal laser scanning microscopy (CLSM). Green channel: GFP, blue channel: nuclei stained by DAPI. (Figure 8B) GFP expression within A549 cells treated under the same conditions as (A) analyzed by flow cytometry.

[0019] Figure 9. Output acceleration measured by accelerometer against time.

[0020] Figures 10A-10B. The TEM images (Figure 10A) and DLS measurements (Figure 10B) of MC3 LNPs at pH 6 and pH 7.

[0021] Figures 11A-11B. The TEM images (Figure 11A) and DLS measurements (Figure 11B) of 18:1 PA LNPs at pH 6 and pH 7.

[0022] Figure 12. Zeta potential of MC3 LNPs and 18: 1 PA LNPs at pH 6 and 7.

[0023] Figures 13A-13B. Scatterplots of green (Dil-LNPs) and red (Lysotracker deep red) pixel intensities of the images in Figures 4A-4B, with (Figure 13A) corresponding to Figure 4A and (Figure 13B) corresponding to Figure 4B.

[0024] Figure 14. Semi-quantification of average GFP fluorescence intensity per FNE cell calculated from confocal laser scanning microscopy (CLSM) results in Figure 7 for different groups. Data presented as mean ± SD, n=5, P-values are calculated using one-way ANOVA, *P<0.05, **P<0.01.

[0025] Figure 15. Semi-quantification of average GFP fluorescence intensity per A549 cell calculated from confocal laser scanning microscopy (CLSM) results in Figure 8. Data presented as mean ± SD, n=3, P-values are calculated using one-way ANOVA, **P<0.01.

[0026] Figures 16A-16B. Schematics of TJ structure and NaCl with mechanical stress working mechanism. (Figure 16A) Healthy epithelial layer of airway with intact TJ making it difficult for intracellular drug delivery. (Figure 16B) After treatment of hyperosmotic pressure and vibrational force, the TJ are disrupted promoting intracellular drug delivery. CC: Ciliated Cells, TJ: Tight Junction, BC: Basal Cells, mol.: molecules.

[0027] Figures 17A-17D. Experimental set-up for disrupting TJ. (Figure 17A) Schematics of the method with combined treatment of hyperosmotic pressure and mechanical stress. (Figure 17B) Photograph of the experimental set-up. (Figure 17C) Quantifying input and output acceleration Vs time for the vibrational force. (Figure 17D) Bar graph showing the input and output acceleration of the vibrational force.

[0028] Figures 18A-18B: Effect of hypertonic solution and vibration on ZO-1 localization in rat trachea. (Figure 18A) Representative fluorescent images of rat tracheal whole-mount stain for tight junction protein Zonula occludens-1 (ZO-1) following exposure to hypertonic saline (HTS) 1.8% NaCl only and combined with 70 Hz vibration for 30 minutes. (Figure 18B) Quantification of ZO-1 localization based on the Area Fraction Index (AFI) derived from the fluorescent images. AFI represents the proportion of image pixels positive for ZO-1 staining. Vibration significantly enhanced the disruptive effects of HTS on ZO-1 localization. Vib.: vibration. **P < 0.01. *P<0.05.

[0029] Figures 19A-19C: Impact of hypertonic Saline and vibration on ciliary structure in rat trachea. (Figure 19A) Fluorescent images of rat tracheal whole mounts stained for acetylated a-tubulin to visualize cilia without any treatment and following exposure to hypertonic saline (HTS) 1.8% NaCl only or in combination with 70 Hz vibration for 30 minutes. (Figure 19B) Quantification of ciliary density based on the Area Fraction Index (AFI) derived from the fluorescent images. AFI represents the percentage of pixels within the image that exhibit ciliary staining above a defined threshold. (Figure 19C) Scanning electron microscopy (SEM) images depicting the luminal surface of rat tracheas exposed to HTS 1.8% NaCl only or in combination with 70 Hz vibration for 30 minutes. Vib.: vibration. ***P < 0.001.

[0030] Figures 20A-20D: Bioimpedance Measurement for Assessing Tight Junction Disruption in Rat Trachea. (Figure 20A) Schematic representation of electrical current flow through intact and disrupted airway tissue. (Figure 20B) Cross-sectional view of the rat trachea with the four-probe bioimpedance measurement system. (Figure 20C) Experimental setup for measuring bioimpedance of airway tissue, including electrodes, impedance meter, and computer. (Figure 20D) Bar graph illustrating changes in bioimpedance of trachea tissue at 120 kHz alternating current frequency for control, HTS only, and HTS combined with 70 Hz vibration experimental groups. Vib.: vibration. **P < 0.01. *P<0.05.

[0031] Figures 21A-21C: Dextran incorporation in airway tissue. (Figure 21A) Schematics of mechanism of dextran penetration through the open tight junctions in airway. (Figure 21B) XZ plane view of the dextran penetration in hypertonic solution (HTS) and HTS with vibration; green fluorescence is dextran. (Figure 21C) Graphical representation of the average distance penetrated by dextran in different experimental groups. Vib.: vibration ****P<0.0001. ***P < 0.001. *P<0.05.

DETAILED DESCRIPTION OF THE INVENTION

[0032] In the following description, details are set forth by way of example to facilitate discussion of the disclosed subject matter. It should be apparent to a person of ordinary skill in the art, however, that the disclosed implementations are exemplary and not exhaustive of all possible implementations.

[0033] A method of enhancing delivery of a biologically active molecule into a cell and/or increasing endosomal escape of mRNA within a cell, comprising applying to the cell, in the presence of the biologically active molecule or the mRNA within endosomes of the cell, an amount of sonication or mechanical vibration at 100Hz or less effective to enhance delivery of a biologically active molecule into a cell and/or increase endosomal escape of mRNA within a cell.

[0034] In embodiments the method further comprises administering, or having administered, the mRNA-containing composition or biologically active molecule to the cell.

[0035] In embodiments, the biologically active molecule comprises a nucleic acid, optionally mRNA.

[0036] In embodiments, the biologically active molecule is encapsulated within a liposomal nanoparticle or ionizable lipid nanoparticle (LNP).

[0037] In embodiments, sonication or mechanical vibration is applied at 60Hz to 70Hz.

[0038] In embodiments, the sonication or mechanical vibration is applied at 10Hz to 20Hz. In embodiments, the sonication or mechanical vibration is applied at 20Hz to 30Hz. In embodiments, the sonication or mechanical vibration is applied at 30Hz to 40Hz. In embodiments, the sonication or mechanical vibration is applied at 40Hz to 50Hz. In embodiments, the sonication or mechanical vibration is applied at 50Hz to 60Hz. In embodiments, the sonication or mechanical vibration is applied at 60Hz to 70Hz. In embodiments, the sonication or mechanical vibration is applied at 70Hz to 80Hz. In embodiments, the sonication or mechanical vibration is applied at 80Hz to 90Hz. In embodiments, the sonication or mechanical vibration is applied at 90Hz to 100Hz. [0039] In embodiments, the method effects increased endosomal escape for a mRNA cargo of a LNP over the level of endosomal escape in the absence of sonication or mechanical vibration.

[0040] In embodiments, the method effects increased mRNA transfection over the level of mRNA transfection in the absence of sonication or mechanical vibration.

[0041] In embodiments, the sonication or mechanical vibration is applied from an electromagnetic device.

[0042] In embodiments, the sonication or mechanical vibration is applied at up to 0.5 to 0.7 g.

[0043] In embodiments, the sonication or mechanical vibration does not compromise cell viability or does not induce damage on mitochondrial membrane potential and/or on Golgi apparatus structure.

[0044] In embodiments, the cell is within a mammalian subject.

[0045] In embodiments, the cell is a cell of an organ. In embodiments, the cell is in a tissue.

In embodiments, the tissue or organ is a porous tissue or organ.

[0046] In embodiments, the cell is a cell of a lung.

[0047] In embodiments, the sonication or mechanical vibration is applied for up to 5 minutes, 10 minutes, 15 minutes, 20 minutes or 30 minutes.

[0048] In embodiments, the sonication or mechanical vibration is applied to a subject at 10 to 26 hours after the biologically active molecule or mRNA-containing composition has been administered to the subject.

[0049] In embodiments, the method does not deform the plasma membrane of the cell.

[0050] In embodiments, the mRNA encodes a protein or peptide of a virus, bacteria or tumor antigen.

[0051] In embodiments, the cell is in vitro.

[0052] In embodiments, the biologically active molecule is a small molecule, peptide, or protein.

[0053] In embodiments, a hypertonic or hyperosmotic solution is not applied to the cell.

[0054] In embodiments, the cell is an epithelial cell or is a basal cell. [0055] In embodiments, the liposomal nanoparticle comprises one or more of 3060il0, tetrakis(8-methylnonyl) 3,3',3",3'"-(((methylazanediyl) bis(propane-3,l diyl))bis (azanetriyl))tetrapropionate; 9A1P9, decyl (2-(dioctylammonio)ethyl) phosphate; A2-Iso5- 2DC18, ethyl 5,5-di((Z)-heptadec-8-en-l-yl)-l-(3-(pyrrolidin-l-yl)propyl)-2,5-dihydro-lH- imidazole-2-carboxylate; ALC-0315, ((4-hydroxybutyl)azanediyl)bis(hexane-6, l-diyl)bis(2- hexyldecanoate); ALC-0159, 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide; 0- sitosterol, (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylheptan-2-yl)-10,13- dimethyl-2,3,4,7,8,9,10,1 l,12,13,14,15,16,17-tetradecahydro-lH-cyclopenta[a]phenanthren-3-ol; B ME-016B, bis(2-(dodecyldisulfanyl)ethyl) 3 ,3 '-((3 -methyl-9-oxo- 10-oxa- 13,14-dithi a-3 ,6- diazahexacosyl)azanediyl)dipropionate; BHEM-Cholesterol, 2-(((((3S,8S,9S,10R,13R,14S,17R)- 10,13 -dimethyl- 17-((R)-6-methy lheptan-2-y l)-2, 3 ,4, 7,8,9,10,11,12,13,14, 15,16,17- tetradecahydro-lH-cyclopenta[a]phenanthren-3-yl)oxy)carbonyl)amino)-N,N-bis(2- hydroxyethyl)-N-methylethan-l-aminium bromide; C12-200, l,l'-((2-(4-(2-((2-(bis(2- hydroxydodecyl)amino)ethyl) (2-hydroxydodecyl)amino)ethyl) piperazin-l-yl)ethyl)azanediyl) bis(dodecan-2-ol); cKK-E12, 3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5- dione; DC-Cholesterol, 3p-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol; DLin-MC3- DMA, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino) butanoate; DOPE, l,2-dioleoyl-sn-glycero-3 -phosphoethanolamine; DOSPA, 2,3-dioleyloxy-N-[2- (sperminecarboxamido)ethyl]-N,N-dimethyl-l-propanaminium trifluoroacetate; DOTAP, 1,2- dioleoyl-3-trimethylammonium-propane; DOTMA, l,2-di-O-octadecenyl-3- trimethylammonium-propane; DSPC, l,2-distearoyl-sn-glycero-3-phosphocholine; ePC, ethylphosphatidylcholine; FTT5, hexa(octan-3-yl) 9, 9', 9", 9"', 9"", 9"'"- ((((benzene-1,3,5- tricarbonyl)yris(azanediyl)) tris (propane-3,1 -diyl)) tris(azanetriyl))hexanonanoate; Lipid H (SM- 102), heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6- (undecyloxy)hexyl)amino) octanoate; OF- Deg-Lin, (((3,6-dioxopiperazine-2,5-diyl)bis(butane-4, l-diyl))bis(azanetriyl))tetrakis(ethane- 2,1-diyl) (9Z,9'Z,9"Z,9"'Z,12Z,12'Z,12''Z,12"'Z)-tetrakis (octadeca-9,12-dienoate); PEG2000- DMG, l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000; TT3, Nl,N3,N5-tris(3- (didodecylamino)propyl)benzene-l,3,5-tricarboxamide, and ionizable amphiphilic Janus dendrimers (lAJDs). In embodiments, the composition comprises hybrid lipid-polymer nanoparticles comprising a PLGA core and a dipalmitoylphosphatidylcholine (DPPC) shell. [0056] In embodiments, the mRNA is for a human pathogen antigen or a human tumor antigen.

Non-limiting examples of mRNAs are set forth in Table 1.

[0057] Table 1 - Exemplary mRNAs

[0058] In embodiments, the biologically active molecule is for a lung therapy. In embodiments, examples of the biologically active molecule are set forth in Table 2.

[0059] Table 2: Exemplary lung diseases and biologically active molecules that can be used in the methods:

[0060] In embodiments, the biologically active molecule comprises inhaled mRNA to enhance cystic fibrosis transmembrane receptor (CFTR) expression in cystic fibrosis. In embodiments, the biologically active molecule comprises a nucleic acid. In embodiments, the biologically active molecule comprises RNAi, siRNA, antisense oligonucleotide (ASO). In embodiments, the biologically active molecule comprises sgRNA, together with the Cas enzyme, delivered as nucleic acids via a lipid nanoparticle.

[0061] A method is also provided herein for non-porous tissue or organ such as a liver, a method of enhancing delivery of a biologically active molecule into a cell of the non-porous tissue or organ and/or increasing endosomal escape of mRNA within a cell of said tissue or organ, comprising applying to the cell, in the presence of the biologically active molecule or the mRNA within endosomes of the cell, an amount of sonication or mechanical vibration at over 100Hz up to 1,000Hz effective to enhance delivery of a biologically active molecule into a cell and/or increase endosomal escape of mRNA within a cell.

[0062] A method of improving delivery of a biologically active molecule across a cellular barrier or paracellularly, comprising applying to the cellular barrier (a) in the presence of the biologically active molecule, or (b) prior to administering the biologically active molecule, an amount of (i) sonication or mechanical vibration at 100Hz or less and (ii) a hypertonic solution, wherein the solution is hypertonic to the fluid in which the cell is located, effective to improve delivery of a biologically active molecule across a cellular barrier or paracellularly.

[0063] In embodiments, said cell barrier includes tight junction connections between two or more adjacent cells, and method effects an increase in permeability of the cellular barrier.

[0064] In embodiments, the cellular barrier comprises epithelia.

[0065] In embodiments, the cellular barrier comprises lung or tracheal epithelia.

[0066] In embodiments, the sonication or mechanical vibration is applied at 65Hz to 75Hz. [0067] In embodiments, the sonication or mechanical vibration is applied at 10Hz to 20Hz. In embodiments, the sonication or mechanical vibration is applied at 20Hz to 30Hz. In embodiments, the sonication or mechanical vibration is applied at 30Hz to 40Hz. In embodiments, the sonication or mechanical vibration is applied at 40Hz to 50Hz. In embodiments, the sonication or mechanical vibration is applied at 50Hz to 60Hz. In embodiments, the sonication or mechanical vibration is applied at 60Hz to 70Hz. In embodiments, the sonication or mechanical vibration is applied at 70Hz to 80Hz. In embodiments, the sonication or mechanical vibration is applied at 80Hz to 90Hz. In embodiments, the sonication or mechanical vibration is applied at 90Hz to 100Hz.

[0068] In embodiments, the sonication or mechanical vibration is applied from an electromagnetic device.

[0069] In embodiments, the sonication or mechanical vibration is applied at up to 0.5 to 0.7 g.

[0070] In embodiments, the hypertonic solution comprises NaCl.

[0071] In embodiments, the hypertonic solution comprises 1.8% NaCl.

[0072] In embodiments, the combination of hypertonic solution and sonication or mechanical vibration at 100Hz or less effects an increase in cellular barrier permeability more quickly than hypertonic solution alone and permits withdrawal of the hypertonic solution from being in the presence of the cell in a shorter time while still effecting the same or greater increased cellular barrier permeability.

[0073] In embodiments, the method further comprises administering a biologically active molecule into a lung of the subject via aerosol inhalation or bronchoscopic liquid instillation.

[0074] In embodiments, the biologically active molecule is administered to a subject within 1 hour of the subject’s lungs being subjected to (i) and (ii).

[0075] In embodiments, the sonication or vibration applies low-intensity acoustic oscillation and/or does not elicit acoustic cavitation.

[0076] In embodiments, the vibration energy is elicited for 15-30 minutes on the subject. In embodiments, the vibration energy is applied from 12-24 hours after the subject has been administered the biologically active molecule. In embodiments, the biologically active molecule is administered by inhalation to the lung of the subject. [0077] A device for effecting a method as described herein in a human, animal or cells, comprising (i) a sonication apparatus which sonication apparatus comprises one or more acoustic actuators or a mechanical vibration apparatus comprising one or more motorized electric actuators and (ii) one or more accelerometers, wherein the device is configured for attachment to, or communication with an amplifier,

[0078] and wherein the device is optionally controlled, and/or from which signals are received wirelessly or via wire, by a computer.

[0079] In embodiments, the device applies sonication or mechanical vibration at 60Hz to 75Hz.

[0080] In embodiments, the device applies sonication or mechanical vibration via electromagnetic means.

[0081] In embodiments, the sonication or mechanical vibration is applied at up to 0.5 to 0.75

8-

[0082] In embodiments, the device comprises an acoustic actuator that can generate sonication-based vibration of from 10-100 Hz.

[0083] In embodiments, the device comprises a motorized electric actuator that can generate mechanical vibration of from 10-100 Hz.

[0084] In embodiments, the device further comprises a computer-based controller that feeds a specific waveform to the one or more acoustic actuators.

[0085] In embodiments, the device comprises an acoustic amplifier that modulates intensity of the sonication.

[0086] In embodiments, the one or more accelerometers can monitor the vibration response of the human, animal or cells being treated.

[0087] In embodiments, the device is wearable by a human.

[0088] In embodiments, the device is shaped as a vest for a human.

[0089] In embodiments, the device is shaped as a vest for a mammalian animal. [0090] In embodiments, the device comprises a horizontal platform on which a cell-containing receptable or small animal is placed, and wherein the horizontal platform transmits the sonication or mechanical vibration to the cells or small animal. In embodiments, the small animal is a rodent.

[0091] Wearable devices can be fabricated for typical human dimensions such as adult human dimensions, or pediatric dimensions, and can be fabricated from, e.g., typical synthetic clothes fabrics (e.g., derived from petroleum-based chemicals) with, for example, head holes, fixabe or tighten-able straps for securing the device etc.

[0092] Also provided is a non-transitory computer readable storage medium comprising instructions for performing a method as described herein via control of mechanical or acoustic actuators in a device of the invention and, optionally, adjusting the strength or frequency of the resultant vibration through feedback via one or more accelerometers. Also provided are the methods herein as implemented by a computer.

[0084] Now that embodiments of the present invention have been shown and described in detail, various modifications and improvements thereon can become readily apparent to those skilled in the art. Accordingly, the exemplary embodiments of the present invention, as set forth above, are intended to be illustrative, not limiting. The spirit and scope of the present invention is to be construed broadly. Those of ordinary skill in the art will recognize that the method and apparatus of the present invention described herein and others implied have many applications; therefore, the present invention which is the subject of this application is not limited by or to the representative examples and/or methods disclosed herein, nor limited by or to the preferred embodiments described herein. Moreover, various other embodiments and modifications to these exemplary embodiments may be made by those skilled in the relevant art without departing from the scope or spirit of these inventions. Accordingly, the inventions are not to be limited by the foregoing specification, except as by the appended claims.

[0085] In this respect, various inventive concepts may be embodied as a non-transitory computer readable storage medium (or multiple non-transitory computer readable storage media) (e.g., a computer memory of any suitable type including transitory or non-transitory digital storage units, circuit configurations in Field Programmable Gate Arrays or other semiconductor devices, or other tangible computer storage medium) encoded with one or more programs that, when executed on one or more computers or other processors, perform methods that implement one or more of the various embodiments described above. When implemented in software (e.g., as an app), the software code may be executed on any suitable processor or collection of processors, whether provided in a single computer or distributed among multiple computers.

[0086] Further, it should be appreciated that a computer may be embodied in any of a number of forms, such as a rack-mounted computer, a desktop computer, a laptop computer, or a tablet computer, as non-limiting examples. Additionally, a computer may be embedded in a device not generally regarded as a computer but with suitable processing capabilities, including a Personal Digital Assistant (PDA), a smartphone or any other suitable portable or fixed electronic device.

[0087] Such computers, such as the computers of the HPC, may be capable of performing the methods and algorithms in this disclosure. In embodiments, such methods and algorithms may be incapable of being performed by hand - e.g., with pen and paper.

[0088] Also, a computer may have one or more communication devices, which may be used to interconnect the computer to one or more other devices and/or systems, such as, for example, one or more networks in any suitable form, including a local area network or a wide area network, such as an enterprise network, and intelligent network (IN) or the Internet. Such networks may be based on any suitable technology and may operate according to any suitable protocol and may include wireless networks or wired networks.

[0089] Also, a computer may have one or more input devices and/or one or more output devices. These devices can be used, among other things, to present a user interface. Examples of output devices that may be used to provide a user interface include printers or display screens for visual presentation of output and speakers or other sound generating devices for audible presentation of output. Examples of input devices that may be used for a user interface include keyboards, and pointing devices, such as mice, touch pads, and digitizing tablets. As another example, a computer may receive input information through speech recognition or in other audible formats.

[0090] The non-transitory computer readable medium or media may be transportable, such that the program or programs stored thereon may be loaded onto one or more different computers or other processors to implement various one or more of the embodiments described above. In embodiments, computer readable media may be non- transitory media.

[0091] The terms “program,” “app,” and “software” are used herein in a generic sense to refer to any type of computer code or set of computer-executable instructions that may be employed to program a computer or other processor to implement various embodiments as described above. Additionally, it should be appreciated that, according to one aspect, one or more computer programs that when executed perform methods of this application need not reside on a single computer or processor but may be distributed in a modular fashion among a number of different computers or processors to implement various embodiments of this application.

[0092] Computer-executable instructions may be in many forms, such as program modules, executed by one or more computers or other devices. Generally, program modules include routines, programs, objects, components, data structures, etc. that perform particular tasks or implement particular abstract data types. The functionality of the program modules may be combined or distributed as desired in various embodiments.

[0093] Databases may include computer readable memory (also referred to as ‘memory’). For example, data storage space 3memlN may be and/or include computer readable memory, used to store data as described in the disclosure. Memory may be embodied by suitable hardware, including but not limited to the following: hard disk drives, serial advanced technology attachment (SATA) hard drives, SATA solid state drives (SSDs), non-volatile memory express (NVMe) SSDs, tape drives.

[0094] Also, data in databases may be stored in computer-readable media in any suitable form. For simplicity of illustration, databases may be shown to have fields that are related through location in the data structure. Such relationships may likewise be achieved by assigning storage for the fields with locations in a computer-readable medium that convey relationship between the fields. However, any suitable mechanism may be used to establish a relationship between information in fields of a data structure, including through the use of pointers, tags or other mechanisms that establish relationship between data elements.

[0093] Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

[0094] In the discussion unless otherwise stated, adjectives such as “substantially” and “about” modifying a condition or relationship characteristic of a feature or features of an embodiment of the invention, are understood to mean that the condition or characteristic is defined to within tolerances that are acceptable for operation of the embodiment for an application for which it is intended. In embodiments, about means within a standard deviation using measurements generally acceptable in the art. In embodiments, about means a range extending to +/- 10% of the specified value. In embodiments, about includes the specified value. Unless otherwise indicated, the word “or” in the specification and claims is considered to be the inclusive “or” rather than the exclusive or, and indicates at least one of and any combination of items it conjoins.

[0095] It should be understood that the terms “a” and “an” as used above and elsewhere herein refer to “one or more” of the enumerated components. It will be clear to one of ordinary skill in the art that the use of the singular includes the plural unless specifically stated otherwise. Therefore, the terms “a,” “an” and “at least one” are used interchangeably in this application.

[0096] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains.

[0097] For purposes of better understanding the present teachings and in no way limiting the scope of the teachings, unless otherwise indicated, all numbers expressing quantities, percentages or proportions, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0098] In the description and claims of the present application, each of the verbs, “comprise,” “include” and “have” and conjugates thereof, are used to indicate that the object or objects of the verb are not necessarily a complete listing of components, elements or parts of the subject or subjects of the verb. Other terms as used herein are meant to be defined by their well-known meanings in the art.

General

[0099] For the foregoing embodiments, each embodiment disclosed herein is contemplated as being applicable to each of the other disclosed embodiment. [0100] As used herein, all headings are simply for organization and are not intended to limit the disclosure in any manner. The content of any individual section may be equally applicable to all sections. All combinations of the various elements disclosed herein are within the scope of the invention.

[0101] Additional obj ects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.

[0102] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

[0103] Examples are provided below to facilitate a more complete understanding of the invention. The following examples illustrate the exemplary modes of making and practicing the invention. However, the scope of the invention is not limited to specific embodiments disclosed in these Examples, which are for purposes of illustration only. Specifically, the DN proteins, methods, compositions, and polynucleotides herein disclosed are further illustrated in the following examples, which are provided by way of illustration and are not intended to be limiting.

EXAMPLES

Example 1 - Enhancing Cytoplasmic Expression of Exogenous mRNA through Dynamic Stimulation

Brief Overview

[0104] Here, a mechanical oscillation (frequency: 65 Hz) is utilized to prompt the LNP- mediated endosomal escape. The results reveal this mechanical oscillation can induce the combination and fusion between LNPs with opposite surface charges, enhance endosomal escape of mRNA, and increase the transfection efficiency of mRNA. Additionally, cell viability remains high at 99.3% after treatment with oscillation, which is comparable to that of untreated cells. Furthermore, there is no obvious damage to mitochondrial membrane potential and Golgi apparatus integrity. Thus, this work presents a user-friendly and safe approach to enhancing endosomal escape of mRNA and boosting gene expression. As a result, our work can be utilized in both research and clinical fields to facilitate LNP -based delivery by enabling more effective release of LNP-encapsulated cargos from endosomes.

1. Introduction

[0105] In vitro transcribed (IVT) mRNA has recently come into focus as a new drug class^[11 due to several unique advantages compared with other drugs for therapeutically manipulating protein levels in tissues, including DNA, small molecules^[21 and proteins. First, IVT mRNA-based drugs exhibit a relatively high transfection efficiency by functioning directly within cytoplasm, eliminating the necessity to enter the nucleus to be functional which is required by DNA therapeutics. Additionally, mRNA doesn’t integrate into the genome, mitigating the risk of insertional mutagenesis, a concern commonly associated with DNA therapeutics. Furthermore, mRNA is transiently active and can be efficiently degraded in the body via ribonucleases, contributing to low toxicity. ^[3] Moreover, the production of mRNA is relatively simple and cost- effective compared with small molecule drugs, making it an appealing option for treating diseases induced by mutant proteins. ^[3b’ ^4] Additionally, mRNA therapeutics leverage human cells to synthesize proteins, overcoming the challenge of obtaining fully human post-translational modifications during protein drug development and potentially improving therapeutic efficacy. ^|S| However, there are few barriers to impede the development of mRNA therapeutics. mRNA is a negatively charged macromolecule with short half-life, owing to susceptibility to RNases. ^{[3c> 61} Hence, it is difficult for mRNA to pass through the anionic cell membrane to access cytoplasm for functionality. In addition, mRNA can elicit an innate immune response, greatly restricting its translatability to clinical use.¹⁷¹.

[0106] To overcome cellular barriers, improve instability and immunogenicity, various materials have been developed for mRNA delivery, such as lipid and lipid-like materials,^¹ polymers, ^[91 and protein derivatives. ^[10] Among them, ionizable lipid nanoparticles (LNPs) have attracted increasing attention in recent years, ^[11] due to their high encapsulation efficiency, structural stability, and simple preparation. ^[12] Furthermore, LNPs can swap their electrostatic charges from neutral charge in physiological pH to positive charge in acidic environments, ^[12al which improves their in vivo circulation, reduces their biotoxicity and enhances endosomal escape by inducing membrane fusion and subsequently endosomal rupture. ^{| l 2a}‘ ^13J Thus, LNPs have undergone extensive research and been clinically deployed for mRNA delivery to prevent and treat disease, ^[14] which paves the way for the successful development of mRNA vaccines for COVID- 19.

[0107] However, despite the Food and Drug Administration (FDA) approval, Emergency Use Authorization (EUA), and successful applications in clinics, only 1-4% of nucleic acids encapsulated inside LNPs can successfully escape from the endosome and reach the cytoplasm)^{13, 151} limiting efficient intracellular expression of mRNA drugs. To overcome this barrier, different strategies have been developed, mainly including: ^[12a] (1) designing new ionizable lipids by adding unsaturation)¹⁶¹ adding degradable groups such as ester and disulfide bonds, ¹¹⁷¹ and introducing bioactive molecules such as aminoglycosides and spermine ¹⁸¹ (2) optimizing other lipid molecule components including cholesterol and phospholipid, ^[19] and (3) adding auxiliary materials, such as cell-penetrating peptide and polyhistidine)²⁰¹ Although these strategies have achieved higher mRNA expression efficiency, they are generally involved in design, synthesis, and introduction of new molecules, which increases the development cost and raises potential safety issues. Thus, proposing a strategy to address inefficient endosomal escape, while maintaining low cost and high clinical translatability, is of significant importance.

[0108] Mechanical oscillation can increase the motion speeds of particles in a medium, thereby increasing the kinetic energy. And the increased kinetic energy would assist particles in overcoming resistance to movement, leading to elevated Brownian motion of particles and thus, more rapid mixing of fluid elements. Consequently, mechanical oscillation has been widely employed in mixing viscous flows)²¹¹ As reported, it's essential for LNPs to fuse with the endosomal membrane to achieve endosomal escape. This process necessitates the diffusion of LNPs to reach the endosomal membrane and subsequently contact and fuse with it, requiring specific energy to overcome the energy barrier^[22( Thus, we hypothesized that mechanical oscillation could increase the kinetic energy of LNPs, facilitating their diffusion inside endosomes and leading to a higher likelihood of contact with the endosomal membrane. This elevated energy would also assist LNPs in overcoming the energy barrier and forming a nonbilayer hexagonal (HII) structure, ¹²³¹ thereby facilitating better fusion of lipids between LNPs and the endosomal membrane. Additionally, exposure to physical stress may facilitate the disruption of the fusion structure, further enhancing endosomal escape efficiency (Figure 1A).

[0109] To test our hypothesis, we designed an electromagnetic shaker to generate mechanical oscillations and assess their potential to facilitate endosomal escape (Figure IB). Notably, our initial results demonstrated that the mechanical oscillation of 65 Hz with acceleration around 0.6 g (Figure 9) could improve efficiency of endosomal escape and, importantly, the efficacy of mRNA transfection. To the best of our knowledge, the use of oscillation to facilitate endosomal escape has not been previously reported. Furthermore, the mechanical oscillation employed in our experiments was within the clinically accepted range and did not comprise cell viability or induce obvious damage on mitochondrial membrane potential and Golgi apparatus structure, affirming the biosafety of our strategy. Collectively, these findings demonstrate the potential of oscillation to enhance LNP -based mRNA delivery, offering promising possibilities for a wide range of applications, such as cancer immunotherapy, ^[24] protein-replacement therapies^[3b- ²⁵¹ and regenerative medicine. ^[26]

2, Results and Discussion

2.1 - Characterization of Lipid Nanoparticles

[0110] Initially, we studied the interaction of a “benchmark” LNPs (MC3 LNPs) with an anionic LNPs (18: 1 PA LNPs) to investigate the ability of mechanical oscillation to induce fusion between MC3 LNPs and negatively charged lipid structure, which plays a pivotal role in LNP- facilitated endosomal escape (Figure 2A). The MC3 LNPs are composed of DLin-MC3-DMA : DSPC : cholesterol : DMG-PEG2000 fixed at a molar ratio of 50 : 10 : 38.5 : 1.5 that has been used in the US Food and Drug Administration (FDA) approved Onpattro (Patisiran) formulation.^{111 1} The obtained MC3 LNPs display spherical structure with diameter around 132 nm in negative staining TEM images in both acidic (pH 6) pH and neutral (pH 7) pH (Figure 2B, Figure 10A), smaller than the corresponding hydrodynamic diameter of about 190 nm determined by Dynamic Light Scattering (DLS) (Figure 2C, Figure 10B). Compared with TME results, the DLS measurements showed larger mean diameters and wider size distributions due to the contribution of solvation shell on the measured hydrodynamic diameter^[27] and higher emphasis on larger LNPs.^[28] Furthermore, the LNP sample was subjected to high vacuum conditions during TEM imaging, which would lead to particle collapse, making them appear smaller than the hydrodynamic sizes measured by DLS.^[29] Once 18: 1 PA LNPs with TEM-measured diameter around 70 nm (Figure 11A), hydrodynamic diameter of about 122 nm (Figure 11B) and zeta potential of -27.9 mV (Figure 12) were added when pH was 6, TEM images demonstrated an interesting phenomenon where a few smaller nanoparticles attached to the surface of a larger nanoparticle (Figure 2B). Furthermore, the main peak in DLS results shifted to 295 nm and a new peak appeared in 2670 nm (Figure 2C), which could be attributed to the electrostatic attraction between these two oppositely charged LNPs, leading to the attachment, and even aggregation between them, and thus, the increased measured size. We then want to know whether application of external oscillation would prompt the fusion between these two LNPs. Thus, an oscillation with frequency of 65 Hz that is within the range of clinical usage^[30] was applied to the LNPs mixture. Excitingly, we observed the combination between two LNPs in TEM images, where their boundaries were not discernible, indicating a successful fusion between them (Figure 2B). We also noticed that a transition from two distinct peaks to a single broad peak centered between the original peaks in the DLS results (Figure 2C). The transition observed could be attributed to oscillation, which facilitated both contact and fusion between LNPs of opposite charges, thereby increasing the measured size of the LNP mixture. Simultaneously, it led to the disassembly of large LNP aggregates, reducing the measured size of them. Ultimately, this resulted in a peak centered between the original two peaks. Based on the discovery, we hypothesized the mechanical oscillation could also prompt the fusion between ionizable LNPs with endosomal membrane that also has negative charges, leading to enhanced endosomal escape as well as improved efficacy of mRNA expression.

2.2 - Cellular Uptake ofMC3 LNPs

[0111] Prior to evaluating the impact of mechanical oscillation on endosomal escape, we examined the cellular uptake of MC3 LNPs in fallopian tube nonepithelial (FNE) cells using confocal microscopy. In this study, MC3 LNPs were labeled by non-exchangeable lipid tracer (Dil) with excitation and emission peaks at 550 and 564 nm, respectively. As shown in Figure 3A and Figure 3B, the diffused fluorescence signal of labeled LNPs was visualized in the cells after 4 h of incubation with intensity of 3.63 x 10⁴ a.u. per cell, and the intensity was obviously increased to 13.1 x io⁴ a.u. per cell after 8 h of incubation. Flow cytometry results also demonstrated that the LNPs can be efficiently internalized into FNE cells in a time-dependent manner (Figure 3C and Figure 3D).

2.3 Synergistic Effect of LNPs and Mechanical Oscillation on Endosomal Escape

[0112] To assess the ability of mechanical oscillation to enhance endosomal escape of MC3 LNPs, we incubated FNE cells with Dil-LNPs for 4 h before oscillation. After oscillation (65 Hz, 5 min), the cells were stained with Ly sotracker deep red (LT deep red) and then imaged by confocal microscopy. The confocal images show higher colocalization of Dil-LNPs with LT deep red within FNE cells without exposure of the oscillation relative to the cells treated by oscillation (colocalization appears yellow color in Figure 4). To more clearly demonstrate the colocalization, the boxed regions in Figure 4A and Figure 4B were enlarged for clarity. The colocalization was further evaluated by colocalization scatter plot where colocalized signal falls on the y = x line, free LNPs falls on the y-axis, and endosomes/lysosomes not containing LNPs fall on the x-axis. Compared with the scattergram of the group without oscillation in Figure 13A, the scattergram of the group with oscillation in Figure 13B shows a more obvious “two-tailed” split, reflecting a more dissociation between LNPs and endosomes/lysosomes. The Pearson’s correlation coefficient (PCC) was also calculated in order to assess the endosomal escape efficiency of each treatment. In the case of without oscillation, the PCC approached 0.60 ± 0.02, while that of the group with oscillation was 0.46 ± 0.12. These combined data demonstrate the potency of mechanical oscillation to promote endosomal escape of LNPs. We reasoned that the observed improvement in endosomal escape of LNPs could be attributed to an increased likelihood of fusion between LNPs and the endosomal membrane, followed by disruption of the fusion structure due to the oscillation, which facilitates the release of LNPs from the endosomes. However, we did not observe the fusion structure or structural changes in the LNPs after oscillation under confocal microscopy, due to its maximum resolution of approximately 200 nm, which is insufficient to resolve the changes in LNPs’ structure.

[0113] The ability of oscillation to enhance endosomal escape was also evaluated by calcein assay, a widely used method for studying endosomal escape^[31] (Figure 5), due to calcein's stability, cost-effectiveness, and ease of use. When FNE cells were exposed to the MC3 LNPs and calcein, a cell impermeable dye, both (MC3 LNPs and calcein) would get entrapped into endosomes, leading to punctate fluorescence in cell cytoplasm. However, when the calcein released from endosomes into cytoplasm, a diffused calcein signal would be visualized in cytoplasm.¹³²¹ In the oscillation-treated group, the results showed a significant increase in cytosolic calcein fluorescence after an 8-h incubation with LNPs. This suggested that oscillation could effectively enhance the release of calcein from endosome (Figure 5A and Figure 5B). All these data collectively confirm the synergistic effect of LNPs-oscillation combination in facilitating endosomal escape, which could be attributed to the heightened kinetic energy of LNPs induced by oscillation that increased their likelihood of close contact with endosomal membrane. Furthermore, the heightened kinetic energy could further aid LNPs in surmounting the energy barrier for fusion with the endosomal membrane, thereby resulting in more efficient membrane fusion, prompting endosomal escape, and potentially enhancing the efficacy of gene delivery.

2.4 - The Combination Effect of LNPs and Mechanical Oscillation on Cell Viability

[0114] Before conducting mRNA delivery, the combination effect of LNPs and oscillation on cell viability was investigated using live/dead assay on FNE cells. After incubation with MC3 LNPs for 12 h, FNE cells were subsequently exposed to vibration at 65 Hz for 5 min. Then, we assessed the viability of FNE cells and observed that 99.3% FNE cells still remained viable, comparable to the viability of untreated cells (Figure 6A and Figure 6B). We also investigated cell viability after treatment with either LNPs or oscillation alone. The results show that cell viability in both groups is comparable to that of the untreated group (99.4% for only LNPs group and 99.5% for only oscillation group), suggesting that neither LNPs nor oscillation alone adversely affect cell viability. All results confirm that both MC3 LNPs and oscillation won’t harm FNE cells, indicating the safety of employing the oscillation strategy to improve endosomal escape of LNPs.

[0115] Additionally, we also evaluated the safety of our strategy by examining its influence on mitochondrial membrane potential (A\|/m) and the integrity of Golgi apparatus, which couldn’t be reflected by live/dead assay. Mitochondrial membrane potential has been served as an indicator of cells’ health and functional status)³³¹ Therefore, the JC-1 probe, capable of monitoring A\|/m, was utilized in our study. JC-1 tends to aggregate in the mitochondrial matrix and exhibits red fluorescence when A\|/_m is high. Conversely, JC-1 resides in cytoplasm in a monomeric formulation and displays green fluorescence when A\|/m is lost. Thus, the ratio of red fluorescence (J-aggregate) to green fluorescence (J-monomer) serves as a critical parameter for characterizing mitochondrial state.¹³⁴¹ As shown by confocal microscopy results in Figure 6C, the green fluorescence of JC-1 monomers within FNE cells incubated with MC3 LNPs and subsequently subjected to oscillation was similar to that within untreated cells, while it was much weaker than that within the FNE cells treated by carbonyl cyanide 3 -chlorophenylhydrazone (CCCP), a mitochondrial membrane potential disrupter. In contrast, the red fluorescence of JC-1 aggregates was stronger than that within the CCCP -treated cells. The ratios of the red to green fluorescence intensity in the cells receiving LNPs and subsequently exposed to oscillation (98.5%) were found to be comparable to the ratio in the untreated cells (85.1%), yet much higher than the ratios in the CCCP-treated cells (36.2%), based on the flow cytometry results (Figure 6D). This observation indicated that the combination of LNPs with oscillation of 65 Hz for 5 min wouldn’t damage mitochondrial membrane potential. Then, the immunofluorescence staining was performed to investigate the combination influence of LNPs and oscillation on the structure of Golgi apparatus. And the results showed that no obvious alteration in the structure of Golgi apparatus was caused by LNPs or the oscillation (65 Hz, 5 min) (Figure 6E). All results together implied that our strategy is less likely to cause obvious damage to mitochondria and Golgi apparatus, further confirming the safety of our strategy. Thus, we moved forward to test the synergistic effect of LNPs and oscillation on mRNA delivery in the next stage.

2.5 Synergistic Effect of LNPs and Mechanical Oscillation on mRNA Expression

[0116] The last objective was to investigate the ability of mechanical oscillation to enhance mRNA delivery. FNE cells were incubated with the commercialized LNPs carrying the enhanced green fluorescence protein (eGFP) mRNA for 4 h (Figure 7A and Figure 7B) and 12 h incubation (Figure 7C and Figure 7D). Then, the eGFP fluorescence intensity was analyzed after 4 h post removing LNPs for eGFP expression. As shown by results of confocal microscopy (Figure 7A and Figure 7C) and flow cytometry (Figure 7B and Figure 7D), the green fluorescence intensity of eGFP was stronger in the groups with oscillation treatment, compared with the corresponding groups without oscillation exposure. The fluorescence intensity of GFP in FNE cells shows a significant rise, with a 43.2% increase during the 4-h incubation and a 67.9% increase during the 12-h incubation (Figure 14). We also observed that although the number of cells expressing eGFP mRNA increased with time, the rate of the increase caused by oscillation declined over time from 1.67 times (4-h incubation) (Figure 7B) to 1.07 times (12-h incubation) (Figure 7D). We speculate that this phenomenon is likely because the contribution of oscillation to endosomal escape was overshadowed by the impact of ionizable lipids in LNPs, as more LNPs entered the cells over time. [0117] Encouraged by the increased gene expression achieved by oscillation, we further assessed this strategy in human lung cancer cells, A549. In this experiment, A549 cells were incubated with the LNPs for 12 h before removing the LNPs. After 4-h period for gene expression without the LNPs, we used confocal microscopy (Figure 8A) and flow cytometry (Figure 8B) to evaluate the eGFP expression and found 243% increase in GFP fluorescence intensity (Figure 15) and 1.15-fold increase in cell number induced by the oscillation (65 Hz, 5min). This experiment further evidence that the combination of LNPs and oscillation enable the slightly enhanced mRNA expression.

[0118] Although previous studies have reported mRNA and LNPs are fragile and can be disrupted by external physical force, ^{|3? l} but no reduction in mRNA delivery was observed in our study. By contrast, we observed an increase induced by 65 Hz oscillation that facilitates endosomal escape likely by enhancing fusion between LNPs and the endosomal membrane. Furthermore, while external oscillation can induce rupture of LNPs,¹³⁵¹ in this context, this feature could be utilized to potentially enhance mRNA release from the LNPs, leading to more efficient delivery of mRNA into the cytoplasm. On the other hand, we didn’t observe any side effects from our strategy on the cells in this study, including cell viability, mitochondrial membrane potential and Golgi apparatus integrity. Although further investigation is needed to fully understand the safety of our oscillation strategy, such as TEM analysis and functional assessments of various organelles, the subsequent mRNA expression results also suggest that our oscillation approach does not significantly impair cellular function. Additionally, mechanical oscillation has presented wide proposed applications, including assisting in clearing airway secretions for cystic fibrosis patients, enhancing muscle strength for athletes¹³⁶¹, and improving physical performance in elderly individuals. ^[30] Moreover, studies have reported that vibrations within the frequency range of 30- 100 Hz and with an intensity lower than 1 g are generally deemed safe for the human body.^[30] The oscillation we utilized in this study falls within these parameters, further confirming the safety of our strategy.

[0119] While various mechanical force-based techniques have been developed, such as microinj ection, ¹³⁷¹ microfluidics-based technologies,¹³⁸¹ and acoustic shock waves, ¹³⁹¹ aiming to improve the intracellular delivery of foreign nucleic acids by deforming or opening plasma membrane, the utilization of mechanical oscillation to enhance mRNA delivery efficiency is the first reported instance, to the best of our knowledge. In contrast to the mechanical techniques, the oscillation doesn’t involve the deformation of plasma membrane, which may induce unexpected damage to the plasma membrane, leading to premature senescence or reduced cellular viability.¹⁴⁰¹ Moreover, mechanical oscillation could be easily applied in in vivo, which presents a challenge for techniques such as microinjection and microfluidics-based technologies.

[0120] In light of the broad applications of mRNA therapeutics, our strategy could be utilized in facilitating various disease treatment. For example, our strategy could complement LNP -based delivery of mRNA for treating diseases stemming from the lack of specific functional proteins, such as cystic fibrosis, classic galactosemia,¹⁴¹¹ arginase deficiency.¹⁴²¹ It could also be used to enhance the immune response to mRNA vaccines through applying local oscillation directly to the injection site, potentially enabling the vaccines to more effectively prevent the spread of infectious diseases. Moreover, it could improve in vitro transfection efficiency in various cell types for diverse applications, such as transfecting dendritic cells (DCs) for cancer immunotherapy and guiding stem cells to differentiate into target cells for regenerative medicine. In addition, our approach, while valuable for mRNA therapeutics, could potentially improve the delivery efficiency of other drugs. This includes therapeutic small molecules,¹⁴³¹ peptides¹⁴⁴¹ and proteins¹⁴⁵¹ that can be incorporated into drug carriers that achieve endosomal escape through membrane fusion, such as solid LNPs modified with fusogenic peptides.¹⁸⁶’ ^45-461 As a result, this approach could be used to facilitate the delivery of a variety of drugs for the treatment of different diseases.

[0121 ] Accordingly, this disclosure teaches a novel mechanical oscillation strategy to enhance the endosomal escape and expression of mRNA in ionizable lipid nanoparticles (LNPs). Moreover, the safety of this method is confirmed, showing no significant impact on cellular membranous organelles or viability. Thus, this approach advances mRNA therapeutics and LNP -based drug delivery.

3, Experimental Section

3.1 - Materials

[0122] DLin-MC3-DMA (Cat. No. 555308) was purchased from MedKoo Biosciences. 1,2- Distearoyl-sn-glycero-3-phosphocholine (DSPC) (Cat. No. 850365P), DMG-PEG (MW 2000) (DMG-PEG2000) (Cat. No. 880151P) and 18: 1 PA (Cat. No. 840875P) were purchased from Avanti Polar Lipids. Cholesterol (Cat. No. C3045), methanol (Cat. No. 34860-2L-R) and calcein (Cat. No. C0875) were purchased from Sigma-Aldrich. Ethanol (Cat. No. AC615090010) was purchased from Fisher Scientific. Citric acid monohydrate (Cat. No. 97062-512) and tri-sodium citrate dihydrate (Cat. No. BDH9288) were purchased from VWR. l,l'-dioctadecyl-3,3,3',3'- tetramethylindocarbocyanine perchlorate (Dil), LysoTracker deep red, live/dead viability assay, GM 130 antibody (Cat. No. MA5-35107), JC-1 assay kit (Cat. No. M34152) and Dulbecco's modified eagle medium (DMEM) (Cat. No. 11965084) were purchased from ThermoFisher Scientific. F-12K medium (Cat. No. 30-2004), fetal bovine serum (Cat. No. 30-2020) and penicillin-stretomycin solution (Cat. No. 30-2300) were purchased from ATCC. 4,6-diamidino-2- phenylindole (DAPI) (Cat. No. ab228549) was purchased from Abeam. EGFP mRNA-lipid nanoparticle (LNP) (Cat. No. PM-LNP-0021) was purchased from ProMab Biotechnologies. Formvar/carbon supported copper grids (Cat. No. FCF200-Cu-50) and UranyLess EM Stain (Cat. No. 22409) were purchased from Electron Microscopy Sciences.

3.2 Preparation of Lipid Nanoparticles

[0123] Empty ionizable DLin-MC3-DMA lipid nanoparticles (MC3 LNPs) were formed using the modified ethanol dilution method. Briefly, DLin-MC3-DMA, DSPC, cholesterol and DMG- PEG2000 were dissolved in ethanol at molar ratios of 50: 10:38.5: 1.5. Then, the lipid mixture was dropwisely added to 10 mM citrate buffer (pH 4) under rigorous stirring at an aqueous to ethanol ratio of 3/1 by volume (3/1, aq./ethanol, vol. /vol.). After that, the mixture was stirred for 2 hours at room temperature and then, dialyzed against deionized water (DI water) overnight. After dialysis, the hydrodynamic diameter, the PDI and (^-potential of the synthesized LNPs were measured by using a Zetasizer Nano-S (Malvern) at 25°C. For cellular uptake experiment, nonexchangeable lipid tracer (Dil) was added to lipid mixtures at a concentration of 0.2 mol% to synthesize LNP -Dil. To prepare the anionic 18: 1 PA LNPs, 18: 1 lipid was initially dissolved in methanol, then mixed with DSPC and cholesterol in ethanol at molar ratios of 54:22:24, and finally added into DI water. After the formation of 18: 1 PA LNPs, the fresh LNPs were dialyzed against deionized water (DI water) overnight.

3.3 Negative Strain Transmission Electron Microscopy (TEM)

[0124] 5 pL LNP suspension was added to a glow-discharged formvar/carbon supported copper grid. After 30 seconds, the LNP solution was removed from the grid and then, 5 pL UranyLess staining solution was added for negative staining. After 5 seconds, the staining solution was removed. Once the grid became dry, TEM (JEOL 2100Plus) operating at 120 kV was used to image all samples.

3.4 In Vitro Cellular Uptake Experiments

[0125] Fallopian tube non-ciliated epithelial (FNE) cells (kind gift from Dr. Marcin Iwanicki’s group, Stevens Institute of Technology) and A549 (CCL-185) purchased from ATCC were used to investigate the uptake of the home-made MC3 LNPs. 1 m cell suspension was seeded on sterilized poly-D-lysine (PDL)-coated glass coverslips (13 mm diameter) placed in 12-well plates at a density of 5 * 10⁴/mL. The cells were allowed to adhere overnight at 37 °C in a humidified 5% CO2 atmosphere before being incubated with LNP-Dil (30 pg/mL of lipids). The cells were incubated with the LNP-Dil for different times (37 °C, 5% CO2). After cell/particle incubation, the cells were washed with phosphate-based buffer saine (PBS) and then fixed in 4% paraformaldehyde (PF A) for 10 min at room temperature. Then, the fixed cells were rinsed again with PBS and stained with 5 mM DAPI solution. After mounting, microscopy was carried out on a confocal microscope (LSM 880, Zeiss). Fluorescent images were collected and analyzed using Fiji/ImageJ Software for visualizing intracellular internalization. We also employed a flow cytometer (ThermoFisher) to further quantitatively analyze the time-dependent internalization of LNPs.

3.5 In Vitro Endosomal Escape Analysis

[0126] The influence of oscillation on the endosomal escape of LNPs was first assessed by colocalization of LysoTracker deep red and Dil-labeled LNPs. Briefly, FNE cells were seeded on the sterilized poly-D-lysine (PDL)-coated glass coverslips placed in 12-well plates and incubated at 37 °C, 5% CO2 overnight. The LNP-Dil (30 pg/mL of lipids) were added and cells were incubated with the LNPs at 37 °C, 5% CO2 for 4 h. After incubation, cells were washed 3 times with PBS and then, exposed to 1 mL of PBS containing 100 nM Lysotracker deep red for 2 min at room temperature. After being rinsed 3 times with PBS, the cells were exposed to 1 mL fresh DMEM, and then the oscillation with frequency of 65 Hz would be applied for 5 min in the group: LNP-Dil + oscillation. After oscillation, the cells in all groups were placed at 37 °C, 5% CO2 for 2 h before imaging to allow the fusion between the LNPs and endosomal membrane. 40x waterimmersion objective of the confocal microscope (LSM 880, Zeiss) and excitation/emission wavelengths of 647 nm/668 nm (red channel) and 549 nm/565 nm (green channel) were used for detection of lysosomes and LNPs, respectively. Colocalization over space and Pearson’s coefficient were analyzed using the plugin Coloc 2 in the Fiji software by simultaneously looking at the green and red channels.

[0127] Calcein release assay was also performed for studying endosomal escape. Briefly, FNE cells at a density of 5 x 10⁴/mL were seeded on the sterilized PDL-coated glass coverslips placed in 12-well plates and cultured overnight in a CO2 incubator at 37 °C. Next day, the cells were incubated with media-containing calcein (0.5 pM) and the LNPs (30 pg/mL of lipids) for 4 h or 8 h. Then, the cells were washed with PBS three times. Then, in the group of LNPs + oscillation, the cells would be vibrated at 65 Hz for 5 min. After oscillation, the cells in all groups were kept in fresh DMEM medium for 2 h, and then observed under the confocal microscope with 40x waterimmersion objective using a FITC filter. Images were pseudocolored by Fiji software. The fluorescent intensities of cytosolic calcein in 5 images per sample were semi-quantified using Fiji software to evaluate the efficiency of endosomal escape, following the established protocol .^{|3 ld|}

3.6 Cell Viability Assay

[0128] Live/dead viability assay was performed to evaluate cell viability after various treatments: only oscillation (65 Hz, 5 min), MC3 LNPs (30 pg/mL of lipids), MC3 LNPs (30 pg/mL of lipids) + oscillation (65 Hz, 5 min). Briefly, for MC3 LNPs and MC3 LNPs + oscillation groups, FNE cells would be treated with MC3 LNPs for 12 h. After treatments, the FNE cells were rinsed with PBS and then stained in PBS containing 4 pM calcein AM (stained live cells) and 8 pM EthD-1 (stained dead cells) for 15 min at room temperature. Then, the FNE cells were washed three times with PBS. After mounting, the stained cells were observed using a fluorescent microscope (Olympus BX53). The live and dead cells exhibited green and red fluorescence, respectively. The viability of the fallopian cells was analyzed by calculating the percentage of dead cells. Untreated FNE cells were used as a control group in this experiment.

3.7 JC-1 Assay

[0129] FNE cells were treated for 12 h with MC3 LNPs (30 pg/mL of lipids) in 12-well plates and were then washed three times with PBS. For MC3 LNPs + oscillation group, oscillation with frequency of 65 Hz would be applied for 5 min. Then, the cells would be placed at 37 °C, 5% CO2 for 2 h. After that, the cells were rinsed with PBS again and then were incubated with 1 pg/mL of 5,5^Z ,6,6' -tetrachloro-l,!⁷ ,3,3' -tetraethylbenzimidazolcarbocyanine iodide (JC-1) in DMEM culture medium at 37°C for 20 min. The cells were rinsed with PBS before being analyzed by flow cytometer and imaged by confocal microscope. The ratio of red/green fluorescence intensity was analyzed by the FlowJo software.

3.8 Immunofluor e scent Staining

[0130] FNE cells were attached to the sterilized PDL-coated glass coverslips placed in 12-well plates and incubated at 37 °C, 5% CO2 overnight. Then, cells were incubated with MC3 LNPs (30 pg/mL of lipids) at 37 °C, 5% CO2 for 12 h. After incubation, cells were washed 3 times with PBS and then exposed to 1 mL fresh DMEM. Then, the cells in MC3 LNPs + oscillation group were vibrated at frequency of 65 Hz for 5 min, and then placed at 37 °C, 5% CO2 for 2 h before staining. For immunofluorescent staining, cells were washed with PBS, and then fixed in 4% PFA solution for 1 h at room temperature. After fixation, cells were permeabilized and rinsed with 0. 1% Triton- X 100 in PBS (PBST) three times with 5 minutes for each time. Then, cells were blocked with 0.5% bovine serum protein (BSA) in PBST for 30 minutes at room temperature. After blocking, the fixed cells were incubated with rabbit monoclonal anti-Golgi matrix protein 130 (GM130) antibodies (1:200) which were diluted in the blocking buffer overnight in refrigerator. Next day, the cells on the slides were incubated with secondary antibodies (donkey anti-rabbit, 1 :500) (Jackson ImmunoResearch, Cat. No. 711-605-152) conjugated with Cy5 for 2 h at room temperature, and then stained with 5 pM DAPI in PBS. Then, the cells were washed with PBS three times before being mounted on glass slides with mounting media. After immunostaining, images were taken with a Zeiss LSM 880 confocal microscope.

3.9 In Vitro EGFP-mRNA Expression

[0131] In vitro transcription was performed to investigate the influence of oscillation on expression of EGFP-mRNA encapsulated in commercialized LNPs, where two cell strains (FNE and A549 cells) were employed to test our approach. In detail, cells were seeded in 12-well plates at 5 x 10⁴ cells/well and incubated at 37 °C, 5% CO2 overnight. The cells were then transfected with EGFP mRNA-LNPs according to the manufacturer’s instructions. After incubation of 4 or 12 h, the LNPs were removed, and the cells were rinsed three times with PBS. Then, the cells in EGFP mRNA-LNPs + oscillation group were vibrated at 65 Hz for 5 min in fresh DMEM and cultured in 5% CO2 incubator at 37 °C for another 4 h, allowing more expression of EGFP-mRNA. Four hours later, the cells were collected, and resuspended in FACS buffer before running on a flow cytometer. Untreated blank FNE cells were served as a negative control for GFP+ to determine the percentage of positive cells in different groups (LNPs, LNPs + oscillation). In each group, 20,000 cells were counted for EGFP-mRNA expression analysis. The confocal microscope was also utilized to assess EGFP-mRNA expression in different groups. Images were analyzed using Fiji software. We first subtracted background fluorescence from each image. Cell counts were obtained from the DAPI channel, and GFP fluorescence was then normalized to these counts. A minimum of three images was quantified for each group.

3.10 - Statistical Analysis

[0132] Statistical analysis was performed using GraphPad Prism (GraphPad Software, Inc., La Jolla, CA, USA). All data were presented as the mean ± standard deviation (n>3). Statistical differences were assessed by one-way ANOVA with Tukey’s multiple comparison post-hoc test. Differences were considered to be statistically significant at p < 0.05.

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Brief Overview

[0179] In this study, we investigate the synergistic effects of hypertonic saline (HTS) solution (1.8% NaCl) and mechanical vibration (70 Hz) on the modulation of TJs in the rat tracheal epithelium. Our results show that this combination effectively increases epithelial permeability, offering a novel and safe strategy for enhancing pulmonary drug delivery. This work provides insights into utilizing established clinical techniques to overcome barriers in pulmonary drug administration, paving the way for more effective treatments.

1. Introduction

[0180] Drug delivery across pulmonary epithelia is a promising route for local and systemic therapy¹' ². Pulmonary drug delivery allows for the direct delivery of high concentrations of drugs to the site of action, reducing potential off-target effects in other organs, avoiding first-pass metabolism, and eventually resulting in a rapid response with the reduced risk of systemic side effects³'⁸. Unlike systemic administration, the pulmonary route offers the advantages of being minimally invasive and suitable for delivery of a wide range of molecules, such as proteins and nucleic acids, due to the absence of digestive enzymes found in oral administration⁹'¹² However, the airway epithelium lining the respiratory tract serves as a protective barrier for lungs, creating challenges for effective pulmonary administration. For example, the mucociliary flow, a crucial barrier functions of the airway epithelium, can trap and expel drugs from the respiratory tract¹³'¹⁵. Additionally, tight junctions (TJ) formed between epithelial cells (Figure 16A) form the defensive barrier, further limiting drug absorption^{16, 17}. To achieve effective pulmonary drug delivery, new safe approaches are needed to overcome these challenges.

[0181] To overcome TJ barrier and increase drug absorption, TJ modulators, such as chelators, surfactants, fatty acids and TJ toxins, have been used to modulate TJ permeability¹⁸'²². For example, McCray’s group has shown that the combination of calcium chelator: ethylenegly-col- bis-(2-aminoethylether)-N,N,N’,N’-tetra acetic acid (EGTA) and hypotonic buffer could modulate the permeability of airway epithelial TJ and enhanced gene transfer¹⁸. While these modulators have demonstrated promise in overcoming the challenges posed by TJs for drug delivery, their applications also raise significant safety concerns. Prolonged exposure or high doses of TJ modulators may cause irreversible damage to TJs, hindering their clinical translation²¹.

[0182] In clinics, hyperosmotic sodium chloride (NaCl) and mechanical vibration are used to remove mucus from airways²³. Additionally, hypertonic solutions can open TJs between airway epithelial cells^{19, 20, 24, 25}. The mechanism behind this effect may involve cell shrinkage induced by osmotic stress, which alters organization of TJ proteins, leading to TJ disruption. Simultaneously, vibration can also destabilize the TJ proteins, leading to increased paracellular permeability. This is because the vibration can induce mechanical stress on cells, causing alterations in cellular actin cytoskeleton that interacts with TJ proteins to maintain their structure and function, leading to the modulation of TJs (Figure 16B).

[0183] Although the combination of hyperosmotic NaCl and mechanical vibration have been deployed for clearing mucus in clinics, the influence of this approach on permeability of TJs hasn’t been investigated. Thus, in this study, we explored the synergistic effects of hypertonic saline (HTS) solution (1.8% NaCl) and mechanical vibration (70 Hz) on modulation of TJs for enhancement of permeability of the airway epithelium. We assessed one of TJ proteins (zonula occludens-1), cilia, electrical impedance, and paracellular permeability of the rat tracheal epithelium after our osmo-mechanical treatment. Excitingly, the results demonstrate that this combination can effectively modulate TJs and increase the permeability of epithelial cells. To the best of our knowledge, this is the first time to apply this clinical airway clearance therapy to increase epithelial junction permeability. Meanwhile, our work also offers a safe and feasible strategy for overcoming the challenges associated with pulmonary drug delivery.

2, Methods

2.1 - Isolation of rat trachea

[0184] This study utilized Sprague-Dawley rats (SAS SD Rats; Charles River Laboratories; weight: 200-250 g). All animal procedures were conducted in accordance with an animal protocol approved by the Institute for Animal Care and Use Committee (IACUC) at Stevens Institute of Technology. Freshly isolated rat tracheas were used for the experiments. Rats were euthanized with a 5% isoflurane overdose for 20 mins, followed by the removal of connective tissues and gentle washing of the tracheas with 1 x phosphate-buffered saline (PB S) (Cat. No. 10-0044, Sakura Genemed) to remove blood. The washed tracheas were then stored in PBS until they were used for the subsequent experiments using 1.8% sodium chloride (NaCl) (Cat. No. 7647-14-15, ThermoFisher scientific) and 70 Hz Vibrational force for 30 mins.

2.2 Custom designed electromagnetic shaker

[0185] The experimental setup utilized an electromagnetic shaker built on a translational stage (TBB1212, ThorLabs). The actuator (TT25-8 PUCK, Dayton audio) was secured between two acrylic plastic sheets, which were supported by holders and posts. An accelerometer (WT61C, WitMotion) was positioned on top of the stage to measure vibrations. The actuator of the shaker was connected to an amplifier (K3118, Kinter), which in turn was linked to a computer for control and data acquisition.

2.3 HTS with vibration treatment

[0186] After the rat tracheal tissue was isolated, it was placed in a petri dish and exposed to 1.8% Hypertonic NaCl diluted in DI water. The dish was then placed on an electromagnetic shaker for 30-minute periods and exposed to a computer-generated sinusoidal wave with a frequency of 70 Hz. The frequency and amplitude of the actuator were adjusted using the computer and amplifier. An accelerometer was employed to continuously monitor the response of the actuator, ensuring accurate delivery of the vibrational force.

2.4 Immunofluore scent staining

[0187] Rat tracheas were isolated, sectioned, and subjected to experimental treatments. Tissue sections were fixed in 4% paraformaldehyde (Cat. No. 30525-89-4, Thermofisher Scientific) overnight to preserve tissue architecture. Non-specific binding sites were blocked with 0.5% BSA (Cat. No. 9048-46-8, VWR) and 0.5% Triton X-100 (Cat. No. 9002-93-01, VWR) in PBS for 1 hour at room temperature. Antibodies for ZO-l(Cat. No. sc-33725, Santa Cruz Biotechnology) and for cilia (Cat. No. SAB5600134, Sigma-Aldrich) were then applied to the sections and incubated overnight at 4°C. Following three washes with 0.5% Triton X-100 in PBS. After additional washes, nuclear staining was performed using DAPI (4’,6-diamidino-2-phenylindole) (Cat. No. ab228549, Abeam) for 15 minutes, followed by final washes. Slides were mounted with an anti-fade mounting medium (Cat. No. H-1700-10, Vector Laboratories) for subsequent confocal microscopy analysis using LSM 880, Zeiss. This protocol enabled the visualization and localization of target proteins within the tracheal tissue. 2.5 - Quantification ofZO-1 and cilia

[0188] Initially, the image was converted to grayscale and subjected to appropriate thresholding (for this experiment; min value: 39 intensity and max value: 255 intensity) to differentiate the stained region from the background. Subsequently, the parameter counting tool was employed to measure the total area of the stained region. The area fraction index for threshold image was calculated by dividing the area of positive staining by the total area of the image, providing a quantitative assessment of protein expression and distribution within the tissue. This analysis was performed on multiple images per experimental condition to ensure statistical reliability.

2.6 - Scanning electron microscopy (SEM) of airway lumen

[0189] The luminal surface of the tracheal tissue were treated and visualized via scanning electron microscope (SEM). The tissues were cut into small tissue samples and fixed using 2.5% Glutaraldehyde (Cat. No. 16520, Electron Microscopy Science) at pH 7.0 for overnight at 4 deg Celsius. The fixed tissue were then washed three times for 5 mins each with IX PBS and dehydrated with graded series of aqueous ethanol solution concentration: 25%, 50%, 70%, 85%, 95%, and 100% (v/v). The tissues were kept in hexamethyldisilane solvent overnight in fume hood. The tissues were airdried and then coated with gold using a sputter coater. The samples were now ready to be imaged using scanning electron microscope (SEM) with an accelerated voltage of 2kV.

2.7 - Epithelial barrier function test

[0190] The permeability assay was performed on the native and on the treated tissues. We used 2 mg/ml Fluorescein isothiocyanate-dextran (3-5 kDa, Cat. No. 60842-46-8, Sigma-Aldrich). After the treatment, the tissue were exposed to diluted dextran molecules in DI water for 15 mins and was washed with IX PBS. The samples were then fluorescently labelled with DAPI and ZO- 1 antibody to localize the penetration depth of the dextran in the epithelial layer of airway tissue. The stained sampled were visualized in the confocal microscopy.

2.8 - Bioimpedance measurement system

[0191 ] Electrical properties of the rat trachea were assessed using a precision LCR meter (E4980AL, Keysight). To enhance measurement accuracy, we implemented a four-probe technique. During measurements, electrodes make direct contact with the tissue to assess its electrical characteristics. An alternating electrical current (AC) with varying frequencies (250 Hz to 135 kHz) and a maximum amplitude of 100 pA was supplied into the tissue using two currentcarrying electrodes. Two voltage pick-up electrodes simultaneously measured the potential distribution at the measurement site. We quantified the obtained electrical resistance (R) and reactance (X) across the entire frequency range, but our analysis focused on data collected at 120 kHz. The acquired data was then used to calculate bioimpedance (Z) using the²³:

[0192] Z = R + jX where |Z| = fR² + X²

[0193] Four tungsten electrodes (SE-T, Lucas Signatone; length: 30 cm; tip diameter: 5 pm) were strategically positioned using micro positioners (S-725SLM or S-725SRM, Lucas Signatone), ensuring precise placement. The electrode tip positions were monitored in real-time using a custom-built microscope equipped with a 10x LWD objective lens (375-039, Mitutoyo), a tube lens (AC254-100-A-ML, Thorlabs), and a high-resolution scientific CMOS camera (pco. panda 4.2, PCO). The output of the microscope was displayed on a computer screen for visual verification.

2.9 - Statistical analysis

[0194] Statistical analysis was performed using GraphPad Prism (GraphPad Software, Inc., La Jolla, CA, USA). All data were presented as the mean ± standard deviation (n>3). Statistical differences were assessed by one-way ANOVA with Tukey’s multiple comparison post-hoc test. Differences were considered to be statistically significant at p < 0.05.

3, Results

3.1 - Characterization of the mechanical vibrational force

[0195] For the experiment, we utilized isolated rat tracheal tissue exposed to HTS in a petri dish (Figure 17A). To induce vibration, we employed a custom designed shaker controlled by a computer-generated sinusoidal waveform whose output was continuously monitored by accelerometer for accuracy (Figure 17B). We used the accelerometer to measure the acceleration of the vibrational force overtime both at the actuator and the tissue. The acceleration of the sinusoidal wave demonstrated a minor 9% reduction in the vibrational force as it propagates from the actuator to the tissue through a sample stage (Figure 17C). The amplitude of the wave indicates the maximum acceleration, which was recorded to be approximately 0.69 gravity force (F_g) at the actuator to 0.62 F_g at the tissue. [0196] Therefore, the mechanical oscillation of 70 Hz with maximum acceleration of around 0.6 F_g could transiently permeabilize the airway epithelial TJ (Figure 17D). Notably, this vibration frequency used in the experiment is approximately in a clinically accepted range of 20 - 65 Hz used in mechano-acoustic device for airway clearance techniques^{26, 27}. Overall, we will study the synergistic effect of osmosis and vibration in creating a temporary pathway for enhanced drug delivery to the airway epithelium.

3.2 Synergistic effect of HTS and Oscillation on ZO-1

[0197] To assess the impact of combined osmo-mechanical treatment visually and quantitatively on TJ integrity in airway tissue, Zonula occludens-1 (ZO-1) was immunofluorescently stained. Figure 18A presents fluorescent images of rat trachea wholemounts stained for ZO-1 and the images compare the localization of ZO-1 in tissues unexposed, exposed to HTS (1.8% NaCl) alone, and HTS in combination with 70 Hz vibration for 30 minutes. The airway tissue (control) with no treatment shows intact distribution of ZO-1 along the epithelial cells, indicating a preserved TJ structure. However, in the tissue with HTS treatment only, the distribution and localization of ZO-1 protein showed empty spaces surrounded by disrupted pattern of ZO-1 distribution. The group with HTS in combination with 70 Hz vibration further showed decreased in staining intensity and potential redistribution of the protein. This suggests that the combined treatment has had a more significant impact on compromising TJ integrity.

[0198] To quantitatively assess the visual changes in ZO-1 localization, we calculated the area fraction index (AFI) of ZO-1 signals in the fluorescent images (Figure 18B). AFI is the ratio of the total number of pixels above a specified threshold (min value: 39 intensity and max value: 255 intensity) to the total number of pixels in the image²⁸. This metric provides a numerical measure of the relative abundance of ZO-1 within the tissue. The AFI of ZO-1 in native untreated tissue samples was approximately 32%, indicating a substantial presence of intact ZO-1 protein. Exposure to HTS alone significantly decreased AFI to around 20%, likely due to ZO-1 disruption and loss of fluorescence signal. Notably, the combination of HTS and vibration further reduced AFI to nearly 11%. This suggests that vibration exacerbated the disruptive effects of HTS on TJC, potentially leading to both disruption of ZO-1 localization and a decrease in its abundance. 3.3 The combination effects of HTS and vibration on cilia structure

[0199] To study the effect of the treatment on cilia structure, we performed immunofluorescent staining of rat trachea for acetylated a-tubulin, a protein marker for cilia. The confocal microscopy images were acquired and analyzed to compare for the distribution of cilia in tissues that were exposed to HTS alone and in combination with 70 Hz vibration for 30 minutes (Figure 19A). In untreated control samples, the cilia were observed to be evenly distributed along the apical surface of epithelial cells. However, both treatment groups exhibited notable alterations in ciliary morphology, orientation, and density. While HTS treatment alone did not significantly affect ciliary density, it did induce ciliary disorganization and detachment. In contrast, the combination of HTS and vibration resulted in a marked decrease in ciliary density, indicating that the combined treatment has disrupted the ciliary structure.

[0200] To quantitatively assess changes in ciliary density, we calculated the AH of ciliary staining in the fluorescent images (Figure 19B). AFI provides a numerical measure of the relative abundance of cilia within the tissue. In native tissue, the AFI of cilia was approximately 41% indicating relatively high density and well-organized structure. This value was reduced to 35% in samples treated with HTS alone likely due to ciliary disorganization. However, when vibration was applied in combination with HTS, the ciliary AFI dropped to approximately 10%. This reduction reflects substantial decrease in the ciliary density and changes in ciliary morphology.

[0201] To visually confirm the changes observed in immunofluorescence, we employed scanning electron microscopy (SEM) to examine the luminal surface of the rat trachea (Figure 19C). This will allow a detailed examination of the ciliary architecture. SEM images revealed a similar trend to that observed in the immunofluorescent images, revealing variations in ciliary density. The native rat trachea exhibited a dense ciliary structure. While the treated group with HTS and vibration revealed morphological abnormalities such as shortened or damaged cilia along with ciliary detachment. These changes would suggest the effect of the treatment on the mucociliary function.

3.4 Quantification of the effects of tissue permeability on tissue bioimpedance

[0202] We investigated whether the permeability of the tissues altered via the treatments can be evaluated by measuring the electrical properties, in particular bioelectrical impedance (bioimpedance), of the airway epithelium. Based on the mechanism of current flow through tissue, that at low frequencies, current primarily flow through the extracellular fluid, bypassing cell membranes, while at high frequencies, capacitive pathways dominate, enabling current to pass through cell membranes²⁹. Therefore, we hypothesized that at when high frequency current flows is supplied, tissue with intact intracellular junctions would impede the flow of electrical current, leading to higher resistance, while disrupted tissue with compromised intracellular junctions would allow for easier current flow, resulting in decreased resistance³⁰ (Figure 20A).

[0203] To accomplish this, we constructed a bioelectrical measurement platform that employs a four-point electrode configuration³⁰ (Figure 20B). An alternating current (AC) of varying frequencies (250 Hz to 135 kHz) was applied, and the resulting potential distribution was measured on native tissue and treated tissue samples^{29, 30} (Figure 20C). Bioimpedance of the samples were calculated across the frequency range, while the bioimpedance values measured from different tissue samples were analyzed at 120 kHz.

[0204] The results demonstrated a significant decrease in bioimpedance of about 6.0 x 10² Q in the HTS alone group compared to the 9.0 x 10² in the control group (Figure 20D). This suggests that the HTS alone disrupts the tissue’s electrical properties by the permeabilizing the intracellular junctions. Furthermore, the HTS combined with vibration group exhibited an even lower bioimpedance of nearly 4.5 x 10² compares to the 6.0 x 10² of HTS alone group, indicating that the addition of vibration further enhances alteration in the electrical properties. This suggests that the combined effect of HTS and vibration is more potent in altering tissue permeability.

3.5 Assessing the treatment-induced change in tissue permeability

[0205] A dextran penetration assay was conducted to quantify the increase in permeability caused by TJ disruption³¹. Fluorescein isothiocyanate - dextran (FITC-Dextran), a versatile fluorescent tracer, was applied to the apical surface of the airway epithelium. Its penetration depth was measured using z-stack confocal imaging over 15 minutes (Figure 21A). The localized concentration gradient drove dextran diffusion through the tissue, facilitated by open TJ³². The distance traveled by dextran molecules was used to estimate epithelial barrier permeability and the effectiveness of TJ disruption treatments. Figure 21B presents XZ plane views of dextran penetration in the control, HTS, and HTS with vibration groups, with green fluorescence indicating the presence of dextran. The results demonstrate a significant enhancement of dextran penetration in both treated groups compared to the control. This suggests that the treatments effectively disrupt TJ, creating a more permeable pathway for drug molecules to reach the underlying tissues.

[0206] To obtain a comprehensive understanding of dextran penetration, the average penetration distance was calculated from multiple experiments and graphically represented in Figure 21C. The average distance covered by the dextran in HTS with vibration group was approximately 139.4 ± 35.5 pm, which was much higher than the depth travelled by dextran in control (47.4 + 11.0 pm. Expectedly, dextran penetration was slightly lower in the HTS-only group (109.0 ± 12.2 pm) than in the HTS with vibration group. These data further confirm the increased permeability induced by the combined HTS and vibration treatment to significantly enhance drug delivery.

4. Discussion

[0207] In this study, we investigated the combined effects of HTS and vibration as a potential strategy to modulate the airway epithelial permeability that can promote drug or gene delivery to the cells in the sub epithelial regions. A significant challenge is the location of the basal cells, which are anchored to the basal membrane beneath the pseudostratified epithelial cells of the airway. The epithelial cells form a semi-permeable barrier due to the presence of TJ between them, creating a formidable obstacle for drug delivery^{33, 34}. Disrupting the integrity of TJ using the combined application of HTS and vibration can increase permeability, potentially facilitating drug delivery to underlying tissues (Figures 16A-16B and Figures 17A-17D). We used confocal microscopy and electron beam microscopy to visualize and quantify the structural changes in the airway epithelium caused by this osmo-mechanical treatment. Furthermore, we characterized the electrical properties of the tissue using bioimpedance measurements and the permeability characteristics using dextran incorporation to further understand the impact of this treatment on drug delivery.

[0208] The confocal images revealed a significant disruption of TJ in airway epithelial tissue following treatment with the HTS and vibration (Figures 18A-3B), indicated by structural changes and decreased abundance of ZO-1. To further explore the effect of the osmo-mechanical treatment on the epithelium, we quantified changes in the cilia structure (Figures 19A-4C). High-frequency vibration, in particular, can induce mechanical stress on the cilia, leading to detachment from the apical membrane³⁵. This stress may result from the rapid oscillation of the vibrating surface, which can disrupt the adhesive forces between the cilia and the underlying cell membrane. Additionally, high-frequency vibration may generate shear forces that can physically tear the cilia away³⁶. While HTS (Sodium Chloride, NaCl) is generally considered less damaging to the airway epithelium compared to other substances, it can still cause some degree of irritation and inflammation³⁷. Prolonged exposure to high concentrations of NaCl can lead to osmotic stress, which can disrupt cellular homeostasis and potentially contribute to cilia detachment³⁷.

[0209] The confocal and SEM images provided compelling evidence of ciliary disruption in airway epithelial tissue following the treatment (Figure 19C). While these changes may temporarily impair mucociliary clearance, it is important to note that cilia are highly regenerative structures and can readily recover following tissue damage³⁶. The ability of cilia to regenerate is crucial for maintaining normal airway function and preventing the accumulation of mucus and debris. Therefore, the temporary disruption of cilia, coupled with the potential for rapid regeneration, suggests that this treatment creates a transient pathway for drug delivery.

[0210] We further showed that disruption of the TJ between the epithelial cells caused by the physical tissue damage can be quantified by measuring the electrical properties of the local airway tissue^{29, 30} (Figures 20A-20D). A significant decrease in bioimpedance was observed in both the HTS-only and HTS with vibration groups compared to the control group, indicating a disruption of tissue electrical properties. This reduction in bioimpedance is likely attributed to increased tissue permeability, which allows for easier current flow. The relationship between bioimpedance and tissue permeability can be understood in terms of the electrical properties of the tissue. In tissues with intact intracellular junctions, the electrical current encounters greater resistance due to the presence of barriers that impede its flow. These barriers include cell membranes, TJ, and other cellular structures^{30, 38}. When tissue permeability increases, these barriers are disrupted or compromised, allowing for easier current flow. This reduced resistance leads to a decrease in bioimpedance³⁰. The observed changes in bioimpedance are consistent with the findings from the confocal and SEM images, which demonstrated disruptions in TJ structure. These results collectively highlight the ability of bioimpedance measurements to serve as a non-invasive and sensitive indicator of tissue damage and alterations in permeability.

[0211] To further check the permeability of the pathway created by this osmo-mechanical treatment, we performed a dextran penetration assay (Figures 21A-21C). The dextran was chosen as a tracer molecule as it is a highly hydrophilic molecule that can readily interact with water molecules, forming a hydrated layer around its structure³⁹. This hydration layer facilitates the diffusion of dextran across biological barriers, such as epithelial tissues allowing a precise measurement of paracellular diffusion. We incorporated FITC-dextran (molecular weight: 4 kDa) into the tissues for this assay. This molecular weight falls within a range that is optimal for penetrating epithelial tissues without being excessively large or small³². Larger dextran molecules might face greater resistance in diffusing through tissue barriers, while smaller molecules could be more readily cleared before reaching the target tissues. Therefore, FITC-dextran represents a well-balanced choice for permeability assays, providing a reliable and informative measure of tissue barrier function³¹.

[0212] Based on the histological cross-section image analysis from previous literature, we calculated rat tracheal tissue width to be approximately 150-250 pm. The epithelial cell layer thickness within this measurement ranges from 30 to 50 pm⁴⁰'⁴². The basal cells are located at the base of the basal membrane. Dextran penetration depth serves as a valuable indicator of epithelial layer permeability by calculating the distance travelled^{31, 32}. The observed dextran penetration in the treated groups was beyond the epithelial layer, this highlights the potential for treatment to enhance epithelial permeability and suggest future research exploring the delivering of larger molecules. Additionally, the distribution of dextran within the tissue can be analyzed to provide insights into the heterogeneity of permeability changes. For instance, a more widespread distribution of dextran indicates a uniform disruption of TJ across the epithelial surface. This could be particularly important for applications where consistent permeability is desired.

5, Conclusion

[0213] In conclusion, our study investigated the effects of HTS and vibration on airway epithelial TJ and their subsequent impact on drug delivery and mucociliary function. We demonstrated that the combined treatment significantly disrupted TJ, leading to increased paracellular permeability and enhanced drug delivery. The confocal and SEM images showed redistribution of proteins and cilia structure. Bioimpedance measurements confirmed these findings as indicated by the decrease in the electrical resistance (i.e., bioimpedance) of the tissue following treatment. 6. Example 2 References

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[0256] The above disclosed subject matter is to be considered illustrative, and not restrictive, and the appended claims are intended to cover all such modifications, enhancements, and other implementations which fall within the true spirit and scope of the present disclosure. Thus, to the maximum extent allowed by law, the scope of the present disclosure is to be determined by the broadest permissible interpretation of the following claims and their equivalents and shall not be restricted or limited by the foregoing detailed description.

[0257] Throughout this application, various publications are referenced, including referenced in parenthesis. The disclosures of all publications mentioned in this application in their entireties are hereby incorporated by reference into this application in order to provide additional description of the art to which this invention pertains and of the features in the art which can be employed with this invention.

Claims

1. A method of enhancing delivery of a biologically active molecule into a cell and/or increasing endosomal escape of mRNA within a cell, comprising applying to the cell, in the presence of the biologically active molecule or the mRNA within endosomes of the cell, an amount of sonication or mechanical vibration at 100Hz or less effective to enhance delivery of a biologically active molecule into a cell and/or increase endosomal escape of mRNA within a cell.

2. The method of Claim 1, further comprising administering, or having administered, the mRNA-containing composition or biologically active molecule to the cell.

3. The method of Claim 1 or 2, wherein the biologically active molecule comprises a nucleic acid, optionally mRNA.

4. The method of Claim 1, 2 or 3, wherein the biologically active molecule is encapsulated within a liposomal nanoparticle or ionizable lipid nanoparticle (LNP).

5. The method of any of Claims 1-4, wherein the sonication or mechanical vibration is applied at 60Hz to 70Hz.

6. The method of any of Claims 4-5, which effects increased endosomal escape for a mRNA cargo of a LNP over the level of endosomal escape in the absence of sonication or mechanical vibration.

7. The method of any of Claims 4-6, which effects increased mRNA transfection over the level of mRNA transfection in the absence of sonication or mechanical vibration.

8. The method of any of Claims 1-7, wherein the sonication or mechanical vibration is applied from an electromagnetic device.

9. The method of any of Claims 1-7, wherein the sonication or mechanical vibration is applied at up to 0.5 to 0.7 g.

10. The method of any of Claims 1-9, wherein the sonication or mechanical vibration does not compromise cell viability or does not induce damage on mitochondrial membrane potential and/or on Golgi apparatus structure.

11. The method of any of Claims 1-10, wherein the cell is within a mammalian subject.

12. The method of any of Claims 1-11, wherein the cell is a cell of an organ.

13. The method of any of Claims 1-12, wherein the cell is a cell of a lung.

14. The method of any of Claims 1-13, wherein the sonication or mechanical vibration is applied for up to 5 minutes, 10 minutes, 15 minutes, 20 minutes or 30 minutes.

15. The method of any of Claims 1-14, wherein the sonication or mechanical vibration is applied to a subject at 10 to 26 hours after the biologically active molecule or mRNA- containing composition has been administered to the subject.

16. The method of any of Claims 1-15, which does not deform the plasma membrane of the cell.

17. The method of any of Claims 3-16, wherein the mRNA encodes a protein or peptide of a virus, bacteria or tumor antigen.

18. The method of any of Claims 1-10 or 12-17, wherein the cell is in vitro.

19. The method of any of Claims 1-2, 4-5, or 8-18, wherein the biologically active molecule is a small molecule, peptide, or protein.

20. The method of any of Claims 1-19, wherein a hypertonic or hyperosmotic solution is not applied to the cell.

21. The method of any of Claims 1-12 or 14-20, wherein the cell is an epithelial cell or is a basal cell.

22. A method of improving delivery of a biologically active molecule across a cellular barrier or paracellularly, comprising applying to the cellular barrier (a) in the presence of the biologically active molecule, or (b) prior to administering the biologically active molecule, an amount of (i) sonication or mechanical vibration at 100Hz or less and (ii) a hypertonic solution, wherein the solution is hypertonic to the fluid in which the cell is located, effective to improve delivery of a biologically active molecule across a cellular barrier or paracellularly.

23. The method of Claim 22, wherein said cell barrier includes tight junction connections between two or more adjacent cells, and method effects an increase in permeability of the cellular barrier.

24. The method of Claim 22 or 23, wherein the cellular barrier comprises epithelia.

25. The method of Claim 22 or 23, wherein the cellular barrier comprises lung or tracheal epithelia.

26. The method of any of Claims 22-25, wherein the sonication or mechanical vibration is applied at 65Hz to 75Hz.

27. The method of any of Claims 22-26, wherein the sonication or mechanical vibration is applied from an electromagnetic device.

28. The method of any of Claims 22-27, wherein the sonication or mechanical vibration is applied at up to 0.5 to 0.7 g.

29. The method of any of Claims 22-28, wherein the hypertonic solution comprises NaCl.

30. The method Claim 29, wherein the hypertonic solution comprises 1.8% NaCl.

31. The method of any of Claims 22-30, wherein the combination of hypertonic solution and sonication or mechanical vibration at 100Hz or less effects an increase in cellular barrier permeability more quickly than hypertonic solution alone and permits withdrawal of the hypertonic solution from being in the presence of the cell in a shorter time while still effecting the same or greater increased cellular barrier permeability.

32. The method of any of Claims 22-31, further comprising administering a biologically active molecule into a lung of the subject via aerosol inhalation or bronchoscopic liquid instillation.

33. The method of Claim 32, wherein the biologically active molecule is administered to a subject within 1 hour of the subject’s lungs being subjected to (i) and (ii).

34. The method of any of claims 1-33, wherein the sonication or vibration applies low- intensity acoustic oscillation and/or does not elicit acoustic cavitation.

35. A device for effecting the method of any of Claims 1-34 in a human, animal or cells, comprising (i) a sonication apparatus which sonication apparatus comprises one or more acoustic actuators or a mechanical vibration apparatus comprising one or more motorized electric actuators and (ii) one or more accelerometers, wherein the device is configured for attachment to, or communication with an amplifier, and wherein the device is optionally controlled, and/or from which signals are received wirelessly or via wire, by a computer.

36. The device of Claim 35, which applies sonication or mechanical vibration at 60Hz to 75Hz.

37. The device of Claim 35 or 36, which applies sonication or mechanical vibration via electromagnetic means.

38. The device of Claim 35, 36 or 37, wherein the sonication or mechanical vibration is applied at up to 0.5 to 0.75 g.

39. The device of any of Claims 35 - 38, wherein the device comprises an acoustic actuator that can generate sonication-based vibration of from 10-100 Hz.

40. The device of any of Claims 35 - 38, wherein the device comprises a motorized electric actuator that can generate mechanical vibration of from 10-100 Hz.

41. The device of any of Claims 35 - 40, wherein the device further comprises a computer- based controller that feeds a specific waveform to the one or more acoustic actuators.

42. The device of any of Claims 35 - 41, wherein the device comprises an acoustic amplifier that modulates intensity of the sonication.

43. The device of any of Claims 35 - 42, wherein the one or more accelerometers can monitor the vibration response of the human, animal or cells being treated.

44. The device of any of Claims 35-43, wherein the device is wearable by a human.

45. The device of any of Claims 35-43, wherein the device is shaped as a vest for a human.

46. The device of any of Claims 35-43, wherein the device is shaped as a vest for a mammalian animal.

47. The device of any of Claims 35-43, wherein the device comprises a horizontal platform on which a cell-containing receptable or small animal is placed, and wherein the horizontal platform transmits the sonication or mechanical vibration to the cells or small animal.

48. The device of Claim 47, wherein the small animal is a rodent.