TW202539696A - Composition and method for preventing or treating lung diseases - Google Patents

Abstract

Provided is a composition including an extracellular vesicle engineered to carry a payload. Also provided is a method for preventing or treating a lung disease by administering the composition.

Description

Components and methods for the prevention or treatment of lung diseases

This patent application claims the benefit of U.S. Provisional Patent Application No. 63/557,089, filed on February 23, 2024, the entire contents of which are incorporated herein by reference.

This disclosure provides pharmaceutical compositions and methods for treating lung diseases, such as acute respiratory distress syndrome (ARDS) and acute lung injury. The pharmaceutical composition comprises an extracellular vesicle (EV) carrying at least one microRNA (miRNA) and a pharmaceutically acceptable excipient thereof.

Acute respiratory distress syndrome (ARDS) is an acute, diffuse, inflammatory form of lung injury that is often fatal. ARDS is associated with microvascular endothelial damage and diffuse alveolar damage, characterized by poor oxygenation, pulmonary infiltration, and acute onset. Therefore, patients present with dyspnea and hypoxemia, which gradually worsens over 6 to 72 hours after the precipitating event, and usually require mechanical ventilation and intensive care unit level treatment.

Acute respiratory distress syndrome (ARDS) has a high mortality rate, and currently only a few effective treatments are available. The primary treatment strategy is supportive care, focusing on reducing shunt rate, increasing oxygen supply, minimizing oxygen consumption, and preventing further damage. Patients typically receive mechanical ventilation and are given diuretics to prevent fluid overload; nutritional support is also provided until their condition improves.

Therefore, the need for drugs or methods that can effectively and efficiently treat acute respiratory distress syndrome (ARDS) remains unmet.

This disclosure provides a pharmaceutical composition comprising an extracellular vesicle of let-7i-5p miRNA engineered to carry lethal 7 gene, such as human ( Homo sapiens , hsa) let-7i-5p miRNA, and a pharmaceutically acceptable excipient thereof.

In at least one embodiment disclosed herein, the extracellular vesicle is loaded with the miRNA via electroporation, lipid transfection, ultrasound treatment, or contact with calcium chloride.

In at least one embodiment of this disclosure, the extracellular vesicle is derived from an animal cell. In at least one embodiment of this disclosure, the animal cell is a mammalian cell. In at least one embodiment of this disclosure, the mammalian cell is a non-stem cell, a mesenchymal stem cell, or an immune cell. In at least one embodiment of this disclosure, the immune cell is a macrophage.

In at least one embodiment of this disclosure, the extracellular vesicle is derived from cells cultured in vitro or from the body fluids of a subject. In at least one embodiment of this disclosure, the extracellular vesicle is an exosome.

This disclosure also provides a method for the prevention or treatment of lung diseases in subjects with a need. In at least one embodiment, the method of this disclosure includes administering the above-described pharmaceutical composition to a subject with a need.

In at least one embodiment of this disclosure, a method is provided for the prevention or treatment of acute respiratory distress syndrome in subjects with a need for it. In at least one embodiment, the method of this disclosure includes administering an effective dose of the aforementioned pharmaceutical composition to the subject. In at least one embodiment of this disclosure, the acute respiratory distress syndrome is induced by sepsis. In another embodiment of this disclosure, the acute respiratory distress syndrome is induced by aspiration pneumonia.

In at least one embodiment of this disclosure, the application reduces lung damage. In at least one embodiment of this disclosure, the application improves lung function by increasing lung inspiratory volume, increasing lung dynamic compliance, reducing airway resistance, and reducing airway elasticity.

The following embodiments are used to illustrate this disclosure. Those skilled in the art can easily conceive of other effects of this disclosure based on its content. Obviously, one or more embodiments can be implemented without providing specific details. This disclosure can also be implemented or applied according to different embodiments. The following embodiments can be modified or changed to implement this disclosure in different applications without departing from its scope. This disclosure may use titles or subtitles for ease of reading, but these do not affect the scope of this disclosure.

Unless otherwise stated, the embodiments disclosed herein employ techniques such as molecular biology (including recombination techniques), microbiology, cell biology, biochemistry, immunohistochemistry, and immunology, all of which fall within the capabilities of a person of ordinary skill in their respective fields. These techniques are detailed in relevant literature, such as: *Molecular Cloning: A Laboratory Manual*, 2nd edition (Sambrook et al., 1989), Cold Spring Harbor Press; *Oligonucleotide Synthesis* (M. J. Gait, 1984); *Methods in Molecular Biology*, Humana Press; *Cell Biology: A Laboratory Notebook* (edited by J. E. Cellis, 1998), Academic Press; *Animal Cell Culture* (edited by R. I. Freshney, 1987); *Handbook of Experimental Immunology* (Weir, 1996); *Introduction to Cell and Tissue Culture* (J. P. Mather and P. E. Roberts, 1998); *Cell and Tissue Culture: Laboratory Procedures* (edited by A. Doyle, J. B. Griffiths, and D. G. Newell, 1993-8); *Methods in...* Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (edited by D. M. Weir and C. C. Blackwell); Gene Transfer Vectors for Mammalian Cells (edited by J. M. Miller and M. P. Calos, 1987); Current Protocols in Molecular Biology (edited by F. M. Ausubel et al., 1987); PCR: The Polymerase Chain Reaction (edited by Mullis et al., 1994); Current Protocols in Immunology (edited by J. E. Coligan et al., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (D. Catty (ed.), IRL Press, 1988-1989; *Monoclonal Antibodies: A Practical Approach* (P. Shepherd and C. Dean, eds.), Oxford University Press, 2000; *Using Antibodies: A Laboratory Manual* (E. Harlow and D. Lane, eds.), Cold Spring Harbor Laboratory Press, 1999; *The Antibodies* (M. Zanetti and J. D. Capra, eds.), Harwood Academic Publishers, 1995. The techniques for specific uses in particular embodiments will be further described in the following sections. Even without further detail, those skilled in the art can fully utilize this disclosure based on its description. All publications cited in this disclosure are incorporated herein by reference, and their scope is determined by their intended purpose or subject matter.

In this disclosure, all terms (including descriptive or technical terms) should be understood to have the meaning readily apparent to a person skilled in the art. However, the meaning of such terms may vary depending on the intent of a person skilled in the art, precedent, or the emergence of new technology. Furthermore, some terms may be arbitrarily chosen by the applicant; in such cases, the meaning of the chosen terms will be described in detail in the description of this disclosure. Therefore, the terms used in this disclosure are defined based on their meanings and in conjunction with the content of this specification.

In this disclosure, the singular forms “a,” “an,” and “the” include plural indicators unless otherwise expressly and unambiguously defined as a single indicator. Unless the context clearly indicates otherwise, the term “or” and the term “and/or” are used interchangeably.

Furthermore, when a part "includes" or "comprises" a set of components or steps, unless otherwise specifically stated to the contrary, that part may further include other sets of components or steps without excluding other content. Therefore, the terms "comprise," "comprising," "including," "containing," and their grammatical equivalents should be interpreted in an inclusive and open sense, meaning that they may include unexpressed additional requirements, rather than being narrowly interpreted as "consists of only."

In this disclosure, the phrase "at least one" when referring to a list of one or more elements should be understood as selecting at least one element from that list. This selection does not necessarily include at least one of every element listed, nor does it exclude any combination of elements in the list. This definition also allows for the selective presence of other elements besides those in the list referred to by the phrase "at least one," regardless of whether such elements are related to the elements in the list. Therefore, as a non-limiting example, "at least one A and B" (or equivalently, "at least one A or B", or equivalently, "at least one A and/or B") in one embodiment may refer to at least one A and may optionally include multiple A's, while no B exists (and may optionally include other elements besides B); in another embodiment, it may refer to at least one B and may optionally include multiple B's, while no A exists (and may optionally include other elements besides A); in yet another embodiment, it may refer to at least one A and may optionally include multiple A's, and at least one B and may optionally include multiple B's (and may optionally include other elements).

The term "effective amount" refers to the amount of active ingredient required to reduce, inhibit, or prevent a disease or condition, or to alleviate one or more symptoms of such a disease or condition, in a subject. As will be understood by those skilled in the art, the effective amount can vary depending on factors such as the route of administration, the use of the excipient, and the possibility of concomitant use with other therapeutic treatments.

In this disclosure, the terms “treat,” “treating,” or “treatment” refer to the administration or application of one or more active agents to a subject suffering from a disease, its symptoms or condition, or the course of the disease, in order to cure, heal, relieve, reduce, alter, correct, improve, modify, or affect the disease, its symptoms or condition, the functional impairment caused by the disease, or the course of the disease.

In this disclosure, the terms "subject" or "patient" refer to an animal that has or may develop a disease or condition in the near future. Examples of subjects include, but are not limited to, mammals, such as humans, apes, monkeys, dogs, cattle, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and genetically modified non-human animals. In at least one embodiment, the subject is a human, such as a human who has a disease or condition, is at risk of developing a disease or condition, or is potentially likely to develop a disease or condition.

In this disclosure, the terms " in vitro " and " ex vivo " refer to laboratory studies that are not conducted in humans or animals.

In this disclosure, the term " in vivo " refers to research, testing, or treatment conducted within an organism, such as a human or animal (e.g., a human).

Extracellular vesicle is a broad term used to describe all secretory membrane vesicles. In this disclosure, the term "exosome" includes exosomes, microvesicles (also known as microparticles), ectosomes, matrix vesicles, calcifying vesicles, prostasomes, oncosomes, retrovirus-like particles, bacterial extracellular vesicles, intraluminal vesicles, and apoptotic bodies. Extracellular vesicles typically have a diameter ranging from 10 nm to 5,000 nm. In this disclosure, the term "exosome" refers to a small vesicle derived from a cell, with a diameter between 20 nm and 300 nm (e.g., 40 nm to 200 nm). This vesicle includes a membrane enclosing its internal space and is generated by budding from the cell's direct plasma membrane or by the fusion of a late endosome with the plasma membrane. Exosomes comprise lipids or fatty acids and polypeptides, and further include miRNAs as effective payloads as described in this disclosure. Exosomes may be derived from production cells and may be isolated from production cells according to their size, density, biochemical properties, or combinations thereof. Exosomes may be directly loaded with exogenous nucleic acids via electroporation, lipofection, sonication, or contact with calcium chloride. Furthermore, purified exosomes can be loaded in an ex vivo environment, for example, via electroporation.

In this disclosure, the terms "microRNA" or "miRNA" are used interchangeably to refer to unprocessed or processed RNA transcripts derived from miRNA genes. MicroRNAs (miRNAs) are non-coding RNAs, typically 19 to 25 nucleotides in length, that regulate gene expression by pairing with partially or completely complementary base sites on their target mRNAs to induce transcriptional repression or cleavage of the target mRNA.

The exosomes disclosed herein can be derived from in vitro cultured cells or from the body fluids of subjects. When exosomes are produced by autologous in vitro cell culture, various production cells can be used, such as immune cells (e.g., macrophage lines, such as RAW264.7 cells and THP-1 cells), HEK293 cells, Chinese hamster ovary (CHO) cells, or mesenchymal stem cells (MSCs).

The pharmaceutical compositions described in this disclosure can be administered at doses sufficient to treat the diseases of subjects who require them. The dosage of a pharmaceutical composition administered to a particular subject will depend on a variety of factors, such as the route of administration and the subject's physiological characteristics (including health status). For example, the appropriate dosage of a pharmaceutical composition including the miRNAs described in this disclosure may depend on a variety of factors, including but not limited to the subject's physiological characteristics (e.g., age, weight, and sex), disease progression (i.e., pathological state), and other factors well known to those skilled in the art. Various general considerations for determining appropriate dosages can be found, for example, in *Remington’s Pharmaceutical Sciences* (1990), edited by Gennaro et al., Mack Publishing Co., Easton, Pennsylvania, USA, and *Goodman and Gilman’s: The Pharmacological Bases of Therapeutics* (1990), edited by Gilman et al., Pergamon Press. Those skilled in the art can determine, through routine testing, the effective and non-toxic dosage of the pharmaceutical composition described herein, whether as a single dose or in successive doses, to achieve the desired therapeutic effect.

In therapeutic applications, the treatment will continue until the disease state or condition is reached. Furthermore, as will be understood by those skilled in the art, the optimal dosage and intervals for individual doses will depend on the nature and severity of the disease state or condition being treated, the dosage form, route of administration, and site of application, as well as the characteristics of the specific individual being treated. Such optimal conditions can also be determined using conventional techniques.

In many cases, it is expected that the pharmaceutical composition described herein will require multiple administrations. For example, it may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times. The interval between administrations may be approximately one week to approximately twelve weeks, for example, approximately one week to approximately four weeks. It will also be understood by those skilled in the art that the optimal treatment regimen can be determined through decision trials of routine treatment procedures.

The pharmaceutical composition disclosed herein can be administered by any suitable manner, such as intravenous, buccal, parenteral, intranasal, oral, sublingual, or topical administration. Therefore, the administration methods can be local, pulmonary (e.g., via nebulizer inhalation or inhalation of aerosol or powder), intranasal, intratracheal, epidermal, transdermal, oral, or parenteral administration. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion, or intracranial (e.g., intraparenchymal, intraspinal, or intraventricular) administration. In at least one embodiment, the pharmaceutical composition is suitable for intranasal administration. In at least one embodiment, the pharmaceutical composition disclosed herein is formulated as a direct-acting nasal spray. In at least one embodiment, the nasal spray disclosed herein may be self-administered by the subject at a clinical care setting.

Example

The exemplary embodiments disclosed herein will be further illustrated in the following examples, but such examples should not be construed as limiting the scope of this disclosure.

Preparation Example 1: Preparation of engineered exosomes carrying hsa-let-7i-5p miRNA by electroporation

The mouse macrophage cell line RAW264.7 was used for exosome preparation. The mouse macrophage cell line RAW264.7 (mouse macrophage-like cells; ATCC, USA) was cultured in Dulbecco modified Eagle medium (DMEM; Life Technologies) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin/streptomycin; Life Technologies). Cell culture dishes were incubated at 37°C in a 5% CO₂ incubator, with the medium changed every four days and passaged regularly. Cell culture was controlled within 8 to 10 passages for exosome production. The RAW264.7 cell line expressing hsa-let-7i-5p was established according to the method described by Madhyastha et al. (Madhyastha R. et al., Inflammation. 2021 Aug; 44(4): 1274-1287). In short, hsa-let-7i-5p plasso DNA (SC400011) was purchased from OriGene Technologies, Inc. 1 μg of hsa-let-7i-5p plasso DNA was diluted in OptiMEM (Invitrogen, New York, USA) and transfected into RAW264.7 cells by conditional electroporation at 250 V, 5 pulses, 100 ms each. The electroporated cell suspension was transferred to a 6 cm culture dish containing 2 mL of DMEM medium and incubated at 37°C for 24 hours in a CO₂ incubator. Subsequently, the cells were observed under a 200× fluorescent microscope and further quantitative culture was performed by replacing the DMEM medium with G418 antibiotic. Exosomes were isolated from the mouse macrophage RAW264.7 cell line according to the literature procedure (McDonald et al., Pain. 2014 Aug; 155(8): 1527-1539). After culturing in RAW264.7 medium for 48 hours, the medium was collected and centrifuged at low temperature. The supernatant was filtered through a 0.22 µm membrane (Merck Millipore) and then centrifuged at 100,000 g at 4°C for 90 minutes to allow the exosomes to settle. After carefully removing the supernatant, the precipitate containing exosomes was resuspended in 100 µL of PBS (phosphate buffered solution) and stored at -80°C.

To characterize the engineered exosome particles, the morphology of the exosomes was evaluated using a transmission electron microscope (Hitachi HT-7700; Hitachi, Tokyo, Japan), and their particle size was calculated using a NanoSight NS300 particle size analyzer (NTA, Malvern Panalytical, Malvern, UK) according to the manufacturer's instructions. Next, exosome markers, including CD63 and CD9, were detected using conventional immunoblotting. For example, an equal amount of protein (100 µg) was separated by electrophoresis and transferred to a nitrocellulose membrane (Bio-Rad Laboratories, Hercules, California, USA). The membrane was then reacted with primary antibodies, including anti-CD63 antibodies (Proteintech, USA, 25682-1-AP) and anti-CD9 antibodies (Proteintech, USA, 20597-1-AP). The bound antibodies were detected by chemiluminescence (ECL Plus kit; Amersham, Buckinghamshire, UK), and protein band density was measured using a densitometric assay (ImageJ).

For the engineered exosomes prepared in this example, transmission electron microscopy confirmed that they have an elliptical bilayer structure (Fig. 1A), and nanoparticle tracking assay confirmed that their particle size is approximately 100 nanometers (Fig. 1B). In addition, immunoblotting assay confirmed the presence of surface markers CD63 and CD9 (Fig. 1C).

Furthermore, the abundance of hsa-let-7i-5p miRNA in engineered exosomes was analyzed by next-generation sequencing. In short, 100 ng of total RNA was used as the starting material for small RNA sample preparation. Following the manufacturer's recommendations, the sequencing library was constructed using the QIAseq miRNA Library Kit (QIAGEN, Germany). Specifically, 3' and 5' adapters were specifically ligated to the 3' and 5' ends of the small RNA, respectively, and the first cDNA strand was synthesized using QIAseq miRNA NGS reverse transcriptase and reverse transcription primers. After PCR amplification, sequencing libraries of 170 to 200 bp fragments were selected using QIAseq magnetic beads. The quality and concentration of the purified sequencing library were evaluated using a Qsep400 system (Bioptic Inc., Taiwan) and a Qubit 2.0 fluorescent meter (Thermo Scientific, Waltham, Massachusetts, USA). Qualified libraries were then sequenced using an Illumina NovaSeq 6000 platform, and trimmed 75 bp single-end reads were generated by Genomics, BioSci & Tech Co. (New Taipei City, Taiwan). For the raw sequencing data, header sequences were removed using Trim Galore! (v0.6.6), and mature and hairpin miRNAs (miRBase v.22.1) were aligned to a reference genome using Bowtie (v1.3.0) to obtain appropriate miRNA reads. After the alignment steps were completed, the bam files were processed using Samtools (v1.12), and the miRNA expression profiles were calculated and standardized using edgeR (v3.26.5). All differentially expressed miRNAs (DEmiRNAs) were identified using DEGSeq (v1.48.0). As shown in Figure 1D, the abundance of hsa-let-7i-5p miRNA in engineered exosomes was confirmed.

Preparation Example 2: Preparation of engineered exosomes carrying hsa-let-7i-5p miRNA via transfection

RAW264.7 cells and human placental mesenchymal stem cells (hpMSCs) were used for the preparation of exosomes for comparison. RAW264.7 cells were purchased from the Bioresource Collection and Research Center, Taiwan (Hsinchu, Taiwan). Human placental mesenchymal stem cells (hpMSCs) were provided by Professor Yen-Hua Huang of Taipei Medical University (N202101014: This study on the acquisition of human placenta and separation of hpMSCs in humans has been approved by the Joint Institutional Review Board of Taipei Medical University). The culture conditions for RAW264.7 cells and hpMSCs were performed as previously reported (Chiang, M. D. et al., Antioxidants. 2022, 11, 615; Chang, C. Y. et al., Pharmaceuticals. 2021, 15, 36; Lin, C. Y. et al., Br. J. Anaesth. 2010, 104, 44-51).

RAW264.7 cells were then genetically modified to overexpress hsa-let-7i-5p. In short, confluent RAW264.7 cells were cultured in Dulbecco’s modified Eagle 126 medium (DMEM; Life Technologies) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin (both purchased from Life Technologies, USA) and maintained in a humidified incubator containing 5% CO₂ air. After attachment, RAW264.7 cells were cultured in serum-free medium (Life Technologies) for 30 minutes, followed by transfection with 2 µg of plasmids expressing hsa-let-7i-5p (pCMV-let-7i-5p, SC400011; OriGene Technologies, USA). After a 5-minute reaction, 205 µL of serum-free medium containing 5 µL of Lipofectamine 3000 (both purchased from Thermo Fisher, USA) was added to the mixture. Subsequently, a stable hsa-let-7i-5p overexpressing cell line was established using neomycin screening.

To separate engineered exosomes from RAW264.7 cells (engineered exosomes) overexpressing hsa-let-7i-5p and from hpMSCs (hpMSC exosomes), culture media were collected and centrifuged (Beckman Coulter Allegra X-15R centrifuge, 300 g, 4°C, 10 min). The supernatant was collected and filtered (0.22 mm filter, Millipore, USA), followed by ultracentrifugation (Beckman Coulter Optima L-80XP ultracentrifuge, 100,000 g, 4°C, 90 min, using Type 50.2 Ti rotors, k-factor: 157.7) to precipitate the exosomes. The precipitate was resuspended, combined, and subjected to ultracentrifugation, resuspending, and purification, followed by another ultracentrifugation. The upper fraction of the gradient was then collected, diluted, and centrifuged. The resulting crude exosome precipitate was resuspended in 1 mL of ice-cold PBS and combined. A second round of ultracentrifugation was then performed, and the resulting exosome precipitate was again resuspended in 500 µL of phosphate buffer (PBS, Life Technologies) and stored at -80°C.

The obtained exosomes were then characterized and analyzed by observing their morphology, measuring their particle size and markers, and analyzing the content of hsa-let-7i-5p within the exosomes. The morphology of the separated engineered exosomes and hpMSC exosomes was confirmed by transmission electron microscopy (TEM). In TEM analysis, the exosome suspension (3 µL) was fixed with 2% paraformaldehyde (50 µL, Sigma-Aldrich), then transferred to two Formvar carbon-coated electron microscope screens, and observed using a transmission electron microscope (JEM 1400 series, JEOL, USA). Exosome particle size analysis was performed using a NanoSight NS300 particle size analyzer (Nanosight, Malvern Panalytical, UK) according to the manufacturer's instructions. Analysis of exosome markers ALIX and CD9 was performed using the Simple Western blotting method on a WES system (ProteinSimple, Santa Clara, CA, USA) (Rumbaugh, G, et al., Methods Mol. Biol. 2011, 670, 263-74). Protein samples were diluted and prepared according to the manufacturer's instructions, denatured (95°C, 5 min), loaded into analytical plates, and subjected to electrophoretic separation, antibody reaction, and chemiluminescence detection under preset conditions in the WES system.

Primary antibodies were used to detect ALIX (anti-ALIX antibody, ab235377; Abcam, Cambridge, UK) and CD9 (anti-CD9 antibody, IR300-981; iReal Technology, Hsinchu, Taiwan). Digital images were analyzed using Compass software (ProteinSimple), and the detected protein quantification data were presented as molecular weight and signal/peak intensity.

To analyze the expression level of miRNA hsa-let-7i-5p in exosomes, droplet digital PCR (ddPCR) was used. Total RNA was extracted from hpMSC exosomes and engineered exosomes using the miRNeasy Serum/Plasma Kit (Qiagen, Hilden, Germany) according to the manufacturer's recommended procedures. Reverse transcription was performed using the TaqMan MicroRNA Assay (Thermo Fisher, Waltham, Massachusetts, USA) according to the manufacturer's instructions. Subsequently, miRNA copy number was analyzed using the QX200 ddPCR system (Bio-Rad, Hercules, California, USA). Following the manufacturer's operating procedures, reverse transcription products, primers, master mixture, and mineral oil were added to the droplet generator to produce thousands of droplets. Next, PCR amplification was performed using the TaqMan MicroRNA Assay (Thermo Fisher Scientific, Waltham, Massachusetts, USA). Microdroplets were then aspirated and the results were read using a Droplet Reader (Bio-Rad Scientific, Hercules, California, USA). Data analysis was performed using QUANTASOFT analysis software (Bio-Rad Scientific, Hercules, California, USA). Technical support for this study was provided by the National Genome Center, National Yang-Ming University (Taipei, Taiwan).

Figures 2A to 2D show the analytical results above. Observation using transmission electron microscopy (TEM) revealed that both hpMSC exosomes and engineered exosomes exhibited a typical bilayered cup morphology, as shown in Figure 2A. Immunoblotting assay confirmed the presence of the positive markers ALIX and CD9 (Figure 2B). Nanoparticle tracking analysis showed that the particle size of both types of exosomes was approximately 50 to 200 nm (Figure 2C). Furthermore, as shown in Figure 2D, the concentrations of hsa-let-7i-5p miRNA in hpMSC exosomes and engineered exosomes were 4,610 ± 310 copies and 4,207 ± 1,207 copies, respectively, both of which were significantly higher than those of exosomes isolated from unmodified RAW264.7 cells (259 ± 14 copies, both p < 0.001).

Example 1: Engineered exosomes possess biodistribution and pharmacokinetic properties suitable for treating lung diseases.

A group of mice was used to monitor the biodistribution of exosomes. To facilitate imaging, exosomes were reacted with 5 µM Cy7 mono-N-hydroxysuccinimide (NHS) ester (Amersham Biosciences, Little Chalfont Bucks, UK) at 37°C for 30 min to prepare Cy7-labeled exosomes (1 × 10⁸).

Mice were administered Cy7-labeled exosomes intraperitoneally and intratracheally. At 2, 24, and 48 hours post-injection, blood was collected via cardiac puncture, and the mice were euthanized. The heart, lungs, liver, kidneys, spleen, and bladder were then harvested. The distribution of the engineered exosomes in different organs was measured and recorded. Bioluminescent imaging was performed using an in vivo imaging system (IVIS Lumina XRMS; PerkinElmer, Waltham, MA, USA), and the resulting images were analyzed using Living Image software (PerkinElmer).

In vivo imaging results showed that the engineered exosomes had significant biodistribution in lung tissue, while pharmacokinetic assays showed that the in vivo half-life of the engineered exosomes (1 × 10⁹ particles per mouse) after intraperitoneal administration was approximately 48 hours (Figures 3A to 3C).

Similar results were observed after intratracheal administration of engineered exosomes. In vivo imaging showed that the engineered exosomes had significant biodistribution in lung tissue, and pharmacokinetic assays also showed that the in vivo half-life of engineered exosomes (1 × 10⁹ particles per mouse) after intratracheal administration was approximately 48 hours (Figures 4A to 4C).

Example 2: Engineered exosomes and hpMSC exosomes have comparable biodistribution and pharmacokinetic properties.

A separate population of 24 mice was used in this assay. In short, the mice were divided into two groups (n = 12 per group). The first group received an intraperitoneal injection of hpMSC exosomes labeled with Cy7 monoNHS ester (Amersham Biosciences, UK) at a dose of 1 × 10⁸ particles per mouse. The second group received engineered exosomes labeled with Cy7 monoNHS ester (Amersham Biosciences, UK) at a dose of 1 × 10⁹ particles per mouse. At 0, 2, 24, and 48 hours, three mice in each group were euthanized by decapitation, and all major organs were collected. Cy7 signals were detected using bioluminescent imaging assays (IVIS Lumina XRMS and Living Image software; PerkinElmer, USA).

For pharmacokinetic assays, a separate group of six mice was used, which was then divided into two groups (n = 3 per group). In short, the first group of mice received an intraperitoneal injection of hpMSC exosomes labeled with Cy7 monoNHS ester (Amersham) at a dose of 1 × 10⁸ particles per mouse; the second group of mice received engineered exosomes labeled with Cy7 monoNHS ester (Amersham) at a dose of 1 × 10⁹ particles per mouse. Continuous blood sampling was performed from three mice in each group via submandibular vein puncture before administration (0 hours) and at 2, 4, 24, and 48 hours after administration. The fluorescence of each sample was measured using a SpectraMax M5 microplate reader (Molecular Devices, USA) with an excitation wavelength of 756 nm and an emission peak of 779 nm, in order to perform pharmacokinetic analysis of hpMSC exosomes and engineered exosomes, respectively.

Biodistribution and pharmacokinetic analysis results are shown in Figures 5A to 5C. In mice receiving Cy7-conjugated hpMSC exosomes, bioluminescence imaging showed significantly higher Cy7 signal in the lungs, liver, kidneys, and spleen at 2 hours post-administration compared to baseline (all p < 0.05) (Figure 5A). However, at 24 and 48 hours post-administration, there were no significant differences in Cy7 signal in these organs compared to baseline. Furthermore, there were no significant differences in Cy7 signal in the heart and bladder at any time point. These results indicate that hpMSC exosomes are significantly distributed in the lungs, liver, kidneys, and spleen shortly after administration, but the duration is less than 24 hours. In mice receiving Cy7-conjugated engineered exosomes, bioluminescence imaging showed significantly higher Cy7 signal in the heart, lungs, kidneys, and spleen at 2 hours post-administration compared to baseline (all p < 0.05) (Figure 5B). However, at 24 and 48 hours post-administration, there were no significant differences in Cy7 signal in the heart, lungs, and spleen compared to baseline, while the Cy7 signal in the liver and kidneys remained significantly elevated (all p < 0.05). Similar to hpMSC exosomes, there were no significant differences in Cy7 signal in the bladder at any time point. These data show that engineered exosomes are significantly distributed in the heart, lungs, liver, kidneys and spleen after administration.

Pharmacokinetic analysis showed that the plasma concentrations of both hpMSC exosomes and engineered exosomes peaked 4 hours after administration. The half-life of hpMSC exosomes was approximately 16 hours, while that of engineered exosomes was approximately 48 hours (Figure 5C).

Example 3: Engineered exosomes can alleviate sepsis-induced lung damage.

In a study on sepsis in mice, sepsis was induced by intraperitoneal injection of 25 mg/kg lipopolysaccharide (LPS, E. coli 0127:B8 endotoxin; Sigma-Aldrich, St. Louis, MO, USA). In short, adult male C57BL/6 mice were randomly assigned to two groups: one group received intraperitoneal administration of LPS (LPS group); the other group received intraperitoneal administration of engineered exosomes carrying hsa-let-7i-5p miRNA concurrently with LPS administration (1 × 10⁹ particles/mouse; LPSEExo group). The engineered exosomes were administered 2 hours and 26 hours after LPS administration. In addition, control groups were set up, including a mouse group that received physiological saline solution (Sham group) and a mouse group that received only intraperitoneal administration of engineered exosomes (1 × 10⁹ particles/mouse) carrying hsa-let-7i-5p miRNA (EExo group).

After 48 hours of close observation, the 48-hour survival rate was assessed. The results showed that the 48-hour survival rate of the LPS group was significantly higher than that of the LPS group (p = 0.0204), as shown in Figure 6A.

The surviving mice were then euthanized to assess and compare the degree of lung damage in each group.

First, a portion of each group of mice was instilled with 10% formalin solution (Sigma-Aldrich) via a tracheostomy tube, followed by the harvesting of lung tissue for histological analysis. The formalin-instilled lung tissue was placed in paraffin wax, then serially sectioned and stained with hematoxylin and eosin (HE). Lung injury was assessed based on histological features, and alveolar wall edema, hemorrhage, vascular congestion, and polymorphonuclear leukocyte (PMN) infiltration were observed under a light microscope. Each histological feature was scored on a scale of 0 to 5 (normal to severe), and a total score (i.e., lung injury score) was calculated to determine the degree of lung injury. Based on histological analysis and lung injury scoring results using hematoxylin-eosin (HE) staining, the degree of lung injury in the LPS group was significantly higher than that in the LPSExo group (p = 0.0012; Figure 6B).

In addition, mice in each group underwent midline abdominal laparotomy and sternotomy to expose the abdominal aorta and lungs. After euthanasia, the trachea was ligated, and the left and right lungs were freshly harvested. The freshly harvested lung tissue was then divided and collected. Half of the lung tissue samples were rapidly frozen in liquid nitrogen and stored at -80°C for subsequent analysis, while the other half of the lung tissue samples were used to determine the wet/dry weight (W/D) ratio of lung water content (an indicator of lung damage). In short, fresh lung tissue samples were weighed, dried in an 80°C oven for 24 hours, and weighed again to calculate the W/D ratio of the lung tissue samples. The results showed that the wet weight/dry weight ratio of the LPS group was significantly higher than that of the LPSExo group (p < 0.0001; Figure 6C).

To assess lung function, anesthetized mice underwent tracheostomy, and a 20G tube (B. Braun, Melsungen, Germany) was inserted as the tracheostomy tube. This tube was then connected to a computerized small animal ventilator (flexiVent FX; SCIREQ Inc., Montreal, Canada). Mechanical ventilation was set to a ventilation rate of 150 breaths per minute and a tidal volume of 0.2 mL. Airway resistance and dynamic compliance were recorded using the fliiWare 8 system (SCIREQ Inc., Montreal, Canada). In addition, inspiratory capacity (IC), resistance (Rrs), dynamic compliance (Crs), elastance (Ers), forced expiratory volume in 0.1 sec (FEV0.1), and forced expiratory volume (FEV) were measured. As shown in Figures 6D to 6I, the results of pulmonary function tests showed that, compared with the control groups (Sham group and EExo group), the LPS group had significantly reduced inspiratory volume, dynamic compliance, and forced expiratory volume, while resistance and elasticity were significantly increased. However, in the LPSExo group mice, after administration of engineered exosomes carrying hsa-let-7i-5p miRNA, the lung damage was reversed, with increased inspiratory volume, dynamic compliance, and forced expiratory volume, while resistance and elasticity decreased.

To assess lung tissue inflammation, bronchoalveolar lavage fluid (BALF) was collected and analyzed. Specifically, one group of anesthetized mice from each group underwent five lavages via a tracheostomy tube with 1 mL of sterile saline solution to collect BALF samples. BALF samples were analyzed using the IDEXX ProCyte Dx Automated Hematology Analyzer (software version 00-33_51). ProCyte generated an automated feline white blood cell differential count covering five categories: total neutrophils, lymphocytes, monocytes, eosinophils, and basophils. The results showed that the total cell count and the cell count of each type in the bronchoalveolar lavage fluid of the LPS group were significantly higher than those of the control groups (Sham group and EExo group); however, after administering engineered exosomes carrying hsa-let-7i-5p miRNA to mice in the LPS group, the inflammatory response was reversed and the cell count was significantly reduced (p < 0.0001, p = 0.0044, p < 0.0001, p = 0.0002; Figures 6J to 6M).

Example 4: Engineered exosomes alleviate lung damage induced by aspiration pneumonia

In a study of aspiration pneumonia in mice, aspiration pneumonia was induced by intratracheal administration of gastric contents. To simulate gastric contents, a mixture containing xanthan gum-based thickener (12 mg/mL), pepsin (2 mg/mL), and lipopolysaccharide (2.5 mg/mL) was prepared, and the pH was adjusted to 1.6. Adult male C57BL/6 mice were randomly assigned to two groups: Group 1 received intratracheal administration of the simulated gastric contents mixture (AP group); Group 2 received intratracheal administration of the simulated gastric contents mixture in combination with administration of engineered exosomes carrying hsa-let-7i-5p miRNA (1 × 10⁹ particles/mouse; APEExo group). The engineered exosomes were administered intratracheally 2 hours and 26 hours after the mice inhaled the simulated gastric contents mixture. In addition, control groups were also set up, including a mouse group that received physiological saline solution (Sham group) and a mouse group that received only intratracheal administration of engineered exosomes carrying hsa-let-7i-5p miRNA (1 × 10⁹ particles/mouse) (EExo group).

After 48 hours of close observation, all mice were euthanized, and the degree of lung damage was assessed and compared among the groups using the same methods described above. Histological analysis and lung damage scoring results showed that the degree of lung damage in the AP group was significantly higher than that in the APEExo group (p < 0.001; Figure 7A). In addition, the wet/dry weight ratio results showed that the wet/dry weight ratio in the AP group was also significantly higher than that in the APEExo group (p < 0.001; Figure 7B). Further pulmonary function tests showed that, compared with the APEExo group, the AP group had significantly lower inspiratory volume and dynamic compliance, while the resistance was significantly higher (p = 0.006, p < 0.001, p < 0.001; Figures 7C to 7E).

In addition, the results of the total cell count and differential cell count of bronchoalveolar lavage fluid showed that the cell count in the AP group was significantly higher than that in the APEExo group (both p < 0.001; Figures 7F to 7I).

The data from the above examples confirm that lipopolysaccharide-induced sepsis and aspiration pneumonia induced by gastric contents can lead to acute respiratory distress syndrome (ARDS) and lung damage. Furthermore, this disclosure provides clear evidence that engineered exosomes carrying hsa-let-7i-5p miRNA have potent therapeutic potential, as administration of these engineered exosomes can alleviate ARDS and lung damage induced by sepsis and aspiration pneumonia.

Example 5: Engineered exosome hpMSC stem cells show similar therapeutic effects on lung diseases.

This study employed a widely used LPS-induced single-pathogen sepsis model to analyze the therapeutic effects of exosomes on lung disease. In short, following previously reported methods, the experiment was conducted via intraperitoneal injection of a Gram-negative endotoxin (25 mg/kg, LPS, *E. coli* O127:B8, Sigma-Aldrich, USA) (Chang, C. Y. et al., Pharmaceuticals. 2020, 13, 280). Seven- to eight-week-old adult male wild-type C57BL/6 mice (purchased from the National Laboratory Animal Center, Taipei, Taiwan) were used. All mice had free access to standard experimental feed and water and were subjected to a 12:12 hour light-dark cycle. The raising and handling of mice followed the guidelines of the National Institutes of Health (NIH).

In this experiment, mice were randomly assigned to different groups and received different treatments: intraperitoneal injection of saline solution (0.5 mL, Sham group); saline solution supplemented with hpMSC exosomes (MExo group); saline solution supplemented with engineered exosomes (EExo group); LPS treatment only (LPS group); LPS plus hpMSC exosomes (LMExo group); LPS plus engineered exosomes (LEExo group); LPS plus inhibitor-treated hpMSC exosomes (LMExoi group); or LPS plus inhibitor-treated engineered exosomes (LEExoi group).

In the MExo and LMExo groups, two doses of hpMSC exosomes (1 × 10⁸ particles per mouse) were administered intraperitoneally at 2 hours and 26 hours after physiological saline or LPS injection, respectively. In the EExo and LEExo groups, two doses of engineered exosomes (1 × 10⁹ particles per mouse) were administered intraperitoneally at 2 hours and 26 hours after physiological saline or LPS injection, respectively. Similarly, in the LMExoi and LEExoi groups, two doses of inhibitor-treated hpMSC exosomes (1 × 10⁸ particles per mouse) or inhibitor-treated engineered exosomes (1 × 10⁹ particles per mouse) were administered intraperitoneally at 2 hours and 26 hours after LPS injection, respectively. The inhibitor-treated exosomes were used to further clarify the mechanism of action of hsa-let-7i-5p miRNA. Specifically, the miRNA inhibitor is an oligonucleotide with the sequence 5’-AACAGCACAAACUACUACCUCA-3’ (SEQ ID NO. 1). The miRNA inhibitor was introduced into engineered exosomes and MSC exosomes via precipitation, resuspending, transfer, electroporation (150 V/100 µF), removal of free miRNA, and ultracentrifugation. Subsequently, the precipitates of engineered exosomes and MSC exosomes treated with the miRNA inhibitor were resuspended and stored at -80°C.

The dosages of MSC exosomes and inhibitor-treated MSC exosomes were determined based on previous data obtained in obese mice with sepsis (Chiang, M. D. et al., Antioxidants. 2022, 11, 615; Chang, C. Y. et al., Pharmaceuticals. 2021, 15, 36). Furthermore, the dosage of engineered exosomes was determined based on preliminary data (as shown in Figure 8), which indicated that in LPS-treated mice, administration of 1 × 10⁹ particles per mouse (instead of 1 × 10⁸ particles) significantly improved survival. Therefore, the dosage of inhibitor-treated engineered exosomes was also determined accordingly.

To analyze the survival rate and plasma cytokines of LPS-induced single-pathogen sepsis animal models, this study used an independent group of 78 mice. Eighteen mice were assigned to the Sham, MExo, and EExo groups (n = 6 per group), and the remaining 60 mice were assigned to the LPS, LMExo, LMExoi, LEExo, and LEExoi groups (n = 12 per group). Each of the Sham, MExo, and EExo groups had 6 mice, and each of the LPS, LMExo, LMExoi, LEExo, and LEExoi groups had 12 mice. All mice were closely monitored for 48 hours to determine the 48-hour (48-h) survival rate of each group, with the disappearance of blood pressure pulsation used as the mortality criterion. Survival differences among groups were compared using Kaplan-Meier survival analysis curves, as shown in Figure 9A. The results showed that the 48-hour survival rates of the Sham, MExo, and EExo groups were all 100%; in contrast, the 48-hour survival rate of the LPS group was significantly lower than that of the Sham group (50% vs. 100%, p = 0.016), indicating that lipopolysaccharide (LPS) can induce significant mortality. Furthermore, the 48-hour survival rate of the LExo group was significantly higher than that of the LPS group (92% vs. 50%, p = 0.020), demonstrating that engineered exosomes can mitigate the harmful effects of LPS and improve mouse survival. Conversely, the 48-hour survival rate in the LEExoi group was significantly lower than that in the LEExo group (42% vs. 92%, p = 0.008), indicating that hsa-let-7i-5p inhibition can significantly reduce the therapeutic effect of engineered exosomes. A similar trend was also observed in the LPS, LMExo, and LMExoi groups. Furthermore, the difference in survival rate between the LMExo and LEExo groups was not statistically significant (75% vs. 92%, p > 0.05).

After survival determination, five mice from each group were randomly selected, anesthetized, and blood samples were collected via cardiac puncture to assess the degree of systemic inflammation. This assessment was performed by measuring the concentration of cytokines in the plasma samples. The obtained blood samples were placed in heparin anticoagulant tubes (Venosafe, Terumo Europe) and centrifuged at 2,000 g for 10 minutes. The supernatant plasma sample was then collected and stored at -20°C for subsequent analysis. Plasma cytokine concentrations were measured using enzyme-linked immunosorbent assay (ELISA) within 7 days of sample collection. The concentrations of cytokines (including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-6) in plasma were measured using an ELISA kit for TNF-α, IL-1β, and IL-6 (Enzo Life Science, Farmingdale, NY, USA) and performed according to the manufacturer’s instructions.

The results of plasma cytokine analysis are shown in Figure 9B. TNF-α concentrations were low in the Sham, MExo, and EExo groups, while the TNF-α concentration in the LPS group was significantly higher than that in the Sham group (p < 0.001). TNF-α concentrations were comparable in the LMExo and LEExo groups, and both groups had significantly lower TNF-α concentrations than the LPS group (both p < 0.001). Conversely, TNF-α concentrations in the LMExoi and LEExoi groups were significantly higher than their corresponding exosome treatment groups (both p < 0.001). Similar trends were observed in the determination of IL-1β and IL-6. Overall, these results show that hpMSC exosomes and engineered exosomes have similar effects in alleviating lipopolysaccharide-induced systemic inflammation. Furthermore, inhibition of hsa-let-7i-5p weakens the therapeutic efficacy of both exosomes in this regard.

Next, LPS-induced lung damage was assessed. Lung tissue and bronchoalveolar lavage fluid (BALF) were collected and analyzed from an independent population of 240 mice. The Sham, MExo, and EExo groups each had 54 mice (n = 18 per group); the LPS, LMExoi, and LEExoi groups each had 126 mice (n = 42 per group); and the LMExo and LEExo groups each had 60 mice (n = 30 per group). The sample size for each group was determined based on 48-hour survival data to ensure that at least 18 mice from each group were surviving and available for analysis in subsequent measurements.

Forty-eight hours after administration of LPS or saline solution, the lung tissue of six surviving mice in each group (Group 1) was removed via decapitation and rapidly frozen in liquid nitrogen, then stored at -80°C for subsequent analysis. For the six surviving mice in Group 2, after decapitation, tracheostomy was performed and a tracheostomy tube was inserted, followed by ligation of the left main bronchus. The left lung lobe was then freshly harvested for wet/dry weight (W/D) ratio determination to assess the degree of pulmonary edema. Subsequently, the right lung was perfused with 10% formalin (Sigma-Aldrich) at constant pressure for tissue staining. Lung tissues fixed in formalin were embedded in paraffin, sectioned serially, and stained with hematoxylin and eosin (HE). Under a light microscope, the morphological features of lung injury were assessed based on alveolar wall edema, vascular congestion, hemorrhage, and polymorphonuclear leukocyte (PMN) infiltration. Each histological feature was scored according to a lung injury scoring system (0: normal, 5: severe), and a total score was calculated to determine the degree of lung injury. As for the determination of the wet/dry weight (W/D) ratio (i.e., the index of tissue moisture content), fresh left lung tissue was sampled from 6 surviving mice in the second group of each group. The samples were weighed first, then dried in an 80°C oven for 24 hours, and then weighed again to calculate the wet/dry weight ratio.

Figure 10A shows the histological analysis of lung tissue and its corresponding lung injury score. Histological examination revealed normal lung tissue features in the Sham, MExo, and EExo groups. In contrast, the LPS, LMExo, LMExoi, LEExo, and LEExoi groups showed lung injury features, including increased polymorphonuclear leukocyte (PMN) infiltration, focal necrosis, and hemorrhage/congestion. The lung injury scores of the Sham, MExo, and EExo groups were lower, while the lung injury score of the LPS group was significantly higher than that of the Sham group (p < 0.001). Furthermore, the lung injury scores of the LMExo and LEExo groups were comparable, and both were significantly lower than those of the LPS group (p < 0.001). The study found that the lung injury scores of the LMExoi and LEExoi groups were significantly higher than those of their respective exosome treatment groups (p < 0.001 and p = 0.001, respectively). The wet/dry weight (W/D) ratio results are shown in Figure 10B.

In addition, six surviving mice from the third group of each group underwent five bronchoalveolar lavages, each with 1 mL of sterile saline solution, and bronchoalveolar lavage fluid (BALF) was collected. Total cell count and differential cell count were then determined. Bronchoalveolar lavage fluid data are shown in Figure 10C.

The wet/dry weight ratio of lung tissue (Figure 10B) and the cell count data from bronchoalveolar lavage fluid (Figure 10C) were consistent with the lung injury scoring trend shown in Figure 10A. These findings collectively highlight the considerable efficacy of hpMSC exosomes and engineered exosomes in reducing lipopolysaccharide-induced lung injury. Furthermore, inhibition of hsa-let-7i-5p attenuated the therapeutic effects of both exosomes in this context.

To assess lung function, an independent population of 80 mice was used, with 18 mice assigned to the Sham, MExo, and EExo groups (n = 6 each), 42 mice assigned to the LPS, LMExoi, and LEExoi groups (n = 14 each), and 20 mice assigned to the LMExo and LEExo groups (n = 10 each). The sample size for each group was determined based on 48-hour survival data to ensure at least 6 surviving mice from each group were available for the assay. Subsequently, 6 surviving mice were independently selected from each group for lung function testing. 48 hours after administration of LPS or saline solution, all 6 surviving mice were anesthetized (Zoletil/Xylazine, 40/10 mg/kg, intraperitoneal injection). Next, tracheostomy was performed on the mice, and a tracheostomy tube (22-gauge intravenous catheter, Terumo Corp., Tokyo, Japan) was inserted to facilitate pulmonary function testing using a computerized small animal ventilator (flexiVent FX; SCIREQ Inc., Montreal, Canada). Mechanical ventilation was set to a ventilation rate of 150 breaths per minute and a tidal volume of 0.2 mL. Parameters including inspiratory volume, airway resistance, and dynamic compliance were recorded using the fliiWare 8 system (SCIREQ).

As shown in Figure 10D, the inspiratory volume and dynamic compliance of the LPS group were significantly lower than those of the Sham group (both p < 0.001), while airway resistance was significantly higher (p < 0.001). In the LMExo group, compared with the LPS group, the inspiratory volume was significantly higher (p = 0.001), while the airway resistance was significantly lower (p = 0.005), but the dynamic compliance was not significantly different from that of the LPS group. Similarly, in the LEExo group, compared with the LPS group, the airway resistance was significantly lower (p = 0.001), while the dynamic compliance was significantly higher (p = 0.005), but the inspiratory volume was not significantly different. When comparing the LMExoi and LMExo groups, the LMExoi group had a significantly lower inspiratory volume (p = 0.002) and a significantly higher drag (p = 0.004). Similarly, when comparing the LEExoi and LEExo groups, the LEExoi group had significantly lower inspiratory volume and dynamic compliance (p = 0.010 and p = 0.018, respectively), while its drag was significantly higher (p = 0.008). Furthermore, the differences in inspiratory volume, dynamic compliance, and airway drag between the LMExo and LEExo groups were not statistically significant (all p > 0.05). These results further emphasize that hpMSC exosomes and engineered exosomes have comparable therapeutic effects in preventing or reversing LPS-induced lung function loss. Furthermore, inhibition of hsa-let-7i-5p weakens the therapeutic effects of both exosomes in this regard.

In addition, the degree of lung inflammation was assessed in an LPS-induced single-pathogen sepsis model, including analysis of the activation of the upstream regulator nuclear factor-κB (NF-κB) and lung cytokine levels. Five mice were randomly selected from six surviving mice in the first group, and their rapidly frozen lung tissue was used for this assay. The activation of the upstream regulator NF-κB in the lung tissue was determined using the Simple Western blotting method. The Simple Western blotting was performed according to the aforementioned method, using primary antibodies against phosphorylated NF-κB (3033L, Cell Signaling Technology, Danvers, MA, USA) and actin (A2228, Sigma-Aldrich). Furthermore, lung cytokine concentrations were also analyzed using ELISA. Lung tissue processing methods are described in Chiang, M. D. et al., Antioxidants. 2022, 11, 615. The levels of cytokines such as TNF-α, IL-1β, and IL-6 were measured using an ELISA kit (Enzo Life Science, Farmingdale, NY, USA).

Figure 11A shows the expression levels of the upstream regulator NF-κB in lung tissue. Phosphorylated NF-κB (p-NF-κB) expression was extremely low in the Sham, MExo, and EExo groups. Conversely, p-NF-κB expression was significantly higher in the LPS group than in the Sham group (p < 0.001), confirming that lipopolysaccharide (LPS) significantly upregulates NF-κB expression. The results showed that p-NF-κB expression levels were similar in the LMExo and LEExo groups, and both groups showed significantly lower p-NF-κB expression levels than the LPS group (p < 0.001 and p = 0.006, respectively). Furthermore, the p-NF-κB expression levels in both the LMExoi and LEExoi groups were significantly higher than those in their respective exosome treatment groups (p < 0.001). Figure 11B shows the expression levels of TNF-α, IL-1β, and IL-6 in lung tissue, with trends consistent with p-NF-κB expression. These results further confirm that hpMSC exosomes and engineered exosomes have considerable efficacy in reducing LPS-induced lung inflammation. Moreover, these data clearly demonstrate the role of hsa-let-7i-5p in the therapeutic effects of hpMSC exosomes and engineered exosomes.

Furthermore, this study aimed to further investigate the polarization of macrophages in lung tissue in a LPS-induced single-pathogen sepsis model. Five mice were randomly selected from six surviving mice in the first group, and their rapidly frozen lung tissue was used for this assay. The activation of hypoxia-inducible factor-1α (HIF-1α, a macrophage M1 phase polarization promoting factor) and inducible nitric oxide synthase (iNOS, a macrophage M1 phase polarization marker) in lung tissue was measured using the Simple Western blotting method. The Simple Western blotting was performed according to the methods described above. The primary antibodies used included anti-HIF-1α antibody (IR113-466, iReal Technology), anti-iNOS antibody (IR231-856, iReal Technology), and anti-actin antibody (A2228, Sigma-Aldrich). In addition, iNOS expression was analyzed by immunohistochemical staining of paraffin sections of lung tissue. Five mice were randomly selected from six surviving mice in the second group, and their left lung tissue perfused with formalin was used for this assay. After processing, the tissue sections were reacted with anti-iNOS antibody (IR231-856, iReal Technology), and then imaged using a scanning system (TissueGnostics Axio Observer Z1 microscope; TissueGnostics GmbH, Austria) and analyzed using image analysis software (Image J, free software provided by the US NIH).

Figure 12A shows the expression level of HIF-1α (M1 phase polarization promoting factor) in lung tissue. HIF-1α expression was extremely low in the Sham, MExo, and EExo groups. In contrast, the HIF-1α expression level in the LPS group was significantly higher than that in the Sham group (p = 0.001), confirming that lipopolysaccharide can upregulate HIF-1α expression in mouse lung tissue. The results showed that the HIF-1α expression levels in the LMExo and LEExo groups were similar, and both groups showed significantly lower HIF-1α expression levels than the LPS group (p = 0.003 and p = 0.001, respectively). Furthermore, the HIF-1α expression levels in the LMExoi and LEExoi groups were significantly higher than those in their corresponding exosome treatment groups (p = 0.049 and p = 0.032, respectively).

Figure 12A shows data on the expression of iNOS, a marker of macrophage M1 phase polarization, derived from rapidly frozen lung tissue from five randomly selected mice out of six surviving mice in each group, measured as described previously. Figure 12B shows iNOS expression data from the left lung tissue of five randomly selected mice out of six surviving mice in each group, perfused with formalin. The iNOS expression levels in lung tissue (Figures 12A and 12B) show a similar trend to HIF-1α expression levels (Figure 12A). These results further highlight the considerable efficacy of hpMSC exosomes and engineered exosomes in reducing lipopolysaccharide-induced macrophage polarization in mouse lung tissue. Furthermore, these results also highlight the role of hsa-let-7i-5p in mediating the therapeutic effects of hpMSC exosomes and engineered exosomes.

In addition, lung oxidation assays were performed, including endogenous antioxidant enzyme assays and lipid peroxidation assays. Briefly, this assay used rapidly frozen lung tissue from five mice randomly selected from the first batch of six surviving mice in each group. The expression of the oxidation regulator superoxide dismutase 2 (SOD-2) was also measured using the Simple Western blotting method. The Simple Western blotting method was performed as described above, using primary antibodies against SOD-2 (ab68155, Abcam, Cambridge, MA, USA) and β-actin (actin, A2228, Sigma-Aldrich).

The expression of oxidative stress markers was determined by immunohistochemistry (IHC) staining, which involved staining paraffin sections of lung tissue with an antibody targeting malondialdehyde (MDA, ab27642, Abcam, Cambridge, MA, USA), a protein associated with lipid peroxidation. Five mice were randomly selected from six surviving mice in a second group, and their left lung tissue, perfused with formalin, was used for this assay. After processing and reaction with the MDA antibody, all tissue sections were observed under a microscope (TissueGnostics Axio Observer Z1 Microscope, TissueGnostics, Vienna, Austria) and analyzed using image analysis software (Image J).

Figure 13A shows the SOD2 expression level in lung tissue. Extremely low SOD2 expression was observed in the Sham, MExo, and EExo groups. The SOD2 expression level was significantly higher in the LPS group compared to the Sham group (p = 0.015). The results showed that the SOD2 expression levels in the LMExo and LEExo groups were similar, and both groups had significantly lower SOD2 expression levels than the LPS group (p = 0.007 and p = 0.043, respectively). Furthermore, the SOD2 expression level in the LMExoi group was significantly higher than that in the LMExo group (p = 0.001); similarly, the SOD2 expression level in the LEExoi group was also higher than that in the LEExo group (p = 0.005).

Figure 13B shows the lipid peroxidation status in lung tissue, assessed by the amount of MDA (lipid peroxidation marker). The Sham, MExo, and EExo groups had lower MDA levels. However, the LPS group had significantly higher MDA levels than the Sham group (p < 0.001). The results showed that the LMExo and LEExo groups had similar MDA levels, and both groups had significantly lower MDA levels than the LPS group (both p < 0.001). Conversely, the LMExoi group had significantly higher MDA levels than the LMExo group (p < 0.001); similarly, the LEExoi group also had higher MDA levels than the LEExo group (p < 0.001). These results generally indicate that hpMSC exosomes and engineered exosomes are equally effective in reducing lipopolysaccharide-induced oxidative damage in the lungs. Furthermore, these results further highlight the role of hsa-let-7i-5p in modulating the therapeutic effects of both exosomes.

In addition, the process of lung cell death and apoptosis in a single-pathogen sepsis model induced by LPS was evaluated. Five mice were randomly selected from six surviving mice in the first group, and their rapidly frozen lung tissue was used for this assay. The performance of the pro-apoptotic protein cleaved caspase-3 was also measured using the Simple Western blotting method. The Simple Western blotting was performed according to the above method, using primary antibodies against cleaved caspase-3 (IR96-401, iReal Technology) and actin (A2228, Sigma-Aldrich). On the other hand, apoptosis was detected in lung tissue using a terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay (TUNEL assay), performed using an in situ cell death detection kit (Roche, USA). Five mice were randomly selected from six surviving mice in a second group, and their formalin-perfused left lung tissue was used for this assay. Tissue sections were stained with 4',6-dimidinyl-2-phenylindole (DAPI, Pierce) to mark total cell nuclei, and the sections were observed using a conjugated focal microscope (LMS-780). The TUNEL positivity rate was calculated according to the method described by Chiang, M. D. et al. (Antioxidants. 2022, 11, 615).

Figure 14A shows the expression levels of the pro-apoptotic proteins cleavage caspase 3 (17 kDa and 19 kDa) in lung tissue. Caspase 3 (17 kDa) expression was extremely low in the Sham, MExo, and EExo groups. Conversely, the LPS group showed significantly higher caspase 3 (17 kDa) expression than the Sham group (p < 0.001). The results showed that the LMExo and LEExo groups had similar caspase 3 (17 kDa) expression levels, and both groups showed significantly lower levels than the LPS group (p < 0.001). In comparison, the levels of cleavage caspase 3 (17 kDa) in the LMExoi and LEExoi groups were significantly higher than those in their respective exosome treatment groups (p = 0.004 and p < 0.001, respectively). Furthermore, the results for cleavage caspase 3 (19 kDa) were similar to those for cleavage caspase 3 (17 kDa), with the only exceptions being that the differences between the LEExo and LPS groups and between the LEExoi and LEExo groups did not reach statistical significance.

In Figure 14B, TUNEL assays and DNA fragmentation data from TUNEL-positive cell counts reveal the apoptotic state of lung tissue. These results are consistent with the trend observed in cleavage caspase 3 (17 kDa) expression (Figure 14A). Overall, these findings demonstrate that hpMSC exosomes and engineered exosomes are equally effective in reducing lipopolysaccharide-induced apoptosis in mouse lung cells. Furthermore, these results further highlight the role of hsa-let-7i-5p in modulating the therapeutic effects of both exosomes.

Although some embodiments of this disclosure have been described in detail above, those skilled in the art can make various modifications and variations to the illustrated embodiments without substantially departing from the teachings of this disclosure. All such modifications and variations are covered within the scope of this disclosure, as defined in the appended patent applications.

This disclosure can be further understood by reading the following description of the embodiments and referring to one or more of the accompanying figures.

Figures 1A through 1D show the characteristics of engineered exosomes and their carriers. Figure 1A shows an image depicting the morphology of engineered exosomes. Figure 1B shows the particle size and number of engineered exosomes. Figure 1C shows the results of Western blot analysis, demonstrating the expression of CD63 and CD9 markers on the surface of engineered exosomes. Figure 1D shows the results of next-generation sequencing (NGS) analysis, showing the content of hsa-let-7i-5p miRNA within exosomes. Scale bar: 200 nm. Vector: Exosomes isolated from RAW264.7 cells. EExo: Engineered exosomes isolated from RAW264.7 cells overexpressing hsa-let-7i-5p miRNA via plasmid-mediated synthesis. PBS: Phosphate buffer.

Figures 2A through 2D show the characteristics of exosomes isolated from genetically modified RAW264.7 cells and human placental mesenchymal stem cells (hpMSCs). Figure 2A shows representative transmission electron microscopy images of the isolated exosomes. Figure 2B shows representative colloidal imaging results of ALIX and CD9 markers in the isolated exosomes analyzed using the Simple Western blotting method. Figure 2C shows the particle size analysis results of the isolated exosomes. Figure 2D shows the microRNA concentration of hsa-let-7i-5p in the isolated exosomes. Data are from three batches of exosomes per group and are presented as mean ± standard deviation. *p < 0.05 compared to the REXO group. EExo: an engineered exosome obtained from HSA-let-7i-5p overexpressed RAW264.7 cells; MExo: an exosome obtained from human placental-derived mesenchymal stem cells; RExo: an exosome obtained from unmodified RAW264.7 cells.

Figures 3A to 3C show the biodistribution assay and pharmacokinetic analysis results of engineered exosomes carrying hsa-let-7i-5p miRNA administered intraperitoneally. Figures 3A and 3B show the in vivo biodistribution of exosomes conjugated with Cy7 monoNHS ester (1 × 10⁹ particles/mouse) in the heart, lungs, liver, kidneys, spleen, and bladder of mice, measured by in vitro bioluminescence imaging analysis at 0, 2, 24, and 48 hours after intraperitoneal administration. Figure 3C shows the pharmacokinetic analysis results of EExo, where the concentration of engineered exosomes in plasma was measured by the ExoCounter assay.

Figures 4A to 4C show the biodistribution and pharmacokinetic analysis results of engineered exosomes carrying hsa-let-7i-5p miRNA administered intratracheally. Figures 4A and 4B show the in vivo biodistribution of exosomes conjugated with Cy7 monoNHS ester (1 × 10⁹ particles/mouse) in the heart, lungs, liver, kidneys, spleen, and bladder of mice, measured by in vitro bioluminescence imaging analysis at 0, 2, 24, and 48 hours after intratracheal administration. Figure 4C shows the pharmacokinetic analysis results of EExo, where the concentration of engineered exosomes in plasma was measured by the ExoCounter assay.

Figures 5A to 5C show the biodistribution assays and pharmacokinetic analyses of exosomes isolated from genetically modified RAW264.7 cells and human placental mesenchymal stem cells (hpMSCs). Figures 5A and 5B show the biodistribution of Cy7 monoNHS ester-conjugated hpMSC exosomes (MExo, 1 × 10⁸ particles/mouse) and engineered exosomes (EExo, 1 × 10⁹ particles/mouse) in the heart, lungs, liver, kidneys, spleen, and bladder of mice, respectively. These biodistributions were measured at 0, 2, 24, and 48 hours after intraperitoneal administration using in vitro bioluminescence imaging analysis. At each time point, three mice from each group were sacrificed to obtain data. Figure 5C shows the pharmacokinetic analysis of MExo and EExo. Plasma concentrations of MExo and EExo were measured using Cy7 single NHS ester signal intensity analysis. The concentrations of MExo and EExo were measured 0.5 hours after intraperitoneal administration and used as baseline data. Data were obtained from 3 mice per group. hpMSC exosomes (MExo): exosomes obtained from human placental-derived mesenchymal stem cells. Engineered exosomes (EExo): exosomes obtained from RAW264.7 cells overexpressing hsa-let-7i-5p.

Figures 6A through 6M show the effect of intraperitoneal administration of engineered exosomes carrying hsa-let-7i-5p miRNA on reducing sepsis-induced lung damage. Figure 6A shows the 48-hour survival rate of each mouse group, where * indicates p < 0.05 compared to the Sham group; # indicates p < 0.05 compared to the Sham/EExo group; and p < 0.05 compared to the LPS/LPSEExo group. Figure 6B shows the histological feature assessment and lung damage scoring performed using hematoxylin-eosin (HE) staining. Figure 6C shows the wet/dry weight (W/D) ratio. Figures 6D to 6I show the results of lung function assessments, including inspiratory volume, dynamic compliance, airway resistance, airway elasticity (Ers), and forced expiratory volume. Figures 6J to 6M show the cellular composition of bronchoalveolar fluid (BALF). Sham: Sham surgery group, in which mice were given only physiological saline. EExo: Engineered exosome group (1 × 10⁹ particles per mouse), in which mice were given engineered exosomes. LPS: Sepsis model group, in which mice were induced to have sepsis by lipopolysaccharide (25 mg/kg, intraperitoneal administration). LPSEExo: LPS+EExo group, in which mice induced to have sepsis were given engineered exosomes.

Figures 7A to 7I show the effect of intratracheal administration of engineered exosomes carrying hsa-let-7i-5p miRNA on reducing lung damage induced by aspiration pneumonia. Figure 7A shows histological characterization by hematoxylin-eosin (HE) staining. Figure 7B shows the wet/dry weight (W/D) ratio. Figures 7C to 7E show the results of pulmonary function assessments, including inspiratory volume, dynamic compliance, and airway resistance. Figures 7F to 7I show the cellular composition of bronchoalveolar fluid (BALF). Sham: sham surgery group, in which mice were given only physiological saline. EExo: Engineered exosome group (1 × 10⁹ particles per mouse), in which mice were administered engineered exosomes. AP: Aspiration pneumonia model group, in which mice developed aspiration pneumonia via gastric contents. APEExo: AP+EExo group, in which mice induced with aspiration pneumonia were administered engineered exosomes.

Figure 8 shows the 48-hour (48-h) survival rate of the exosome dosage required for survival testing of LPS-treated mice in the preliminary experiment.

Figures 9A and 9B show the survival rate and plasma cytokines in LPS-induced single-pathogen sepsis animal models. Figure 9A shows the 48-hour (48-h) survival rate, determined by calculating the number of mice surviving during the 48-hour observation period after administration of physiological saline or lipopolysaccharide. Data were obtained from 6 mice in the Sham, MExo, and EExo groups, and 12 mice in the LPS, LMExo, LMExoi, LEExo, and LEExoi groups. * p < 0.05 compared to the Sham group in the LPS group. # p < 0.05 compared to the LPS group in the LEExo group. ˄ p < 0.05 compared to the LEExoi group in the LEExo group. Figure 9B shows the concentrations of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-6 in plasma, obtained using enzyme-linked immunosorbent assay (ELISA). Data for each group were obtained from five mice, and all measurements were taken 48 hours after administration of saline or lipopolysaccharide. Data are expressed as mean ± standard deviation. * p < 0.05 compared to the Sham group. # p < 0.05 compared to the LPS group.† p < 0.05 compared to the LMExoi group. ˄ p < 0.05 compared to the LEExoi group. Sham: saline group. MExo: saline plus hpMSC exosome group. EExo: Physiological saline solution plus engineered exosomes. LPS: Lipopolysaccharide (LPS) group. LMExo: LPS plus hpMSC exosomes group. LMExoi: LPS plus hpMSC exosomes treated with inhibitor. LEExo: LPS plus engineered exosomes group. LEExoi: LPS plus engineered exosomes treated with inhibitor.

Figures 10A through 10D show lung injury and functional assessment in an animal model of LPS-induced single-pathogen sepsis. Figure 10A shows representative histological features of lung injury in lung tissue, assessed under a light microscope (200×) after hematoxylin and eosin (HE) staining, and includes data on lung injury scoring. Data for each group were obtained from 6 mice. Figure 10B shows the wet/dry weight ratio (W/D ratio) of lung tissue. Data for each group were obtained from 6 mice. Figure 10C shows the number of white blood cells (WBC), neutrophils, lymphocytes, and monocytes in collected bronchoalveolar lavage fluid (BALF). Data for each group were obtained from 6 mice. All measurements were taken 48 hours after administration of saline or lipopolysaccharide. Data are presented as mean ± standard deviation. * p < 0.05 compared to the Sham group. # p < 0.05 compared to the LPS group.† p < 0.05 compared to the LMExoi group. ˄ p < 0.05 compared to the LEExoi group. Figure 10D shows the results of lung function assessments, including inspiratory volume, airway resistance, and dynamic compliance. Data for each parameter in each group were obtained from 6 mice. Sham: Saline group. MExo: Saline plus hpMSC exosomes group. EExo: Saline plus engineered exosomes group. LPS: Lipopolysaccharide (LPS) group. LMExo: LPS plus hpMSC exosomes group. LMExoi: hpMSC exosomes treated with LPS and an inhibitor. LEExo: LPS-treated engineered exosomes. LEExoi: LPS-treated engineered exosomes.

Figures 11A and 11B show the similar efficacy of hpMSC exosomes and engineered exosomes in reducing lipopolysaccharide-induced lung inflammation in mice. Figure 11A shows representative colloidal images of phosphorylated nuclear factor-κB (p-NF-κB) and actin (internal standard), analyzed using the Simple Western blot method, and displays the relative band density of the p-NF-κB/actin ratio in lung tissue. Data for each group were obtained from 5 mice. Figure 11B shows the concentrations of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-6 in lung tissue, measured using enzyme-linked immunosorbent assay (ELISA). Data for each group were obtained from 5 mice. All measurements were taken 48 hours after administration of physiological saline or lipopolysaccharide. Data are expressed as mean ± standard deviation. * p < 0.05 compared to the Sham group. # p < 0.05 compared to the LPS group. † p < 0.05 compared to the LMExoi group. ˄ p < 0.05 compared to the LEExoi group. Sham: Physiological saline group. MExo: Physiological saline plus hpMSC exosomes group. EExo: Physiological saline plus engineered exosomes group. LPS: Lipopolysaccharide (LPS) group. LMExo: LPS plus hpMSC exosomes group. LMExoi: LPS plus hpMSC exosomes treated with an inhibitor. LEExo: LPS plus engineered exosomes group. LEExoi: an engineered exosome group treated with LPS and an inhibitor.

Figures 12A and 12B show the similar efficacy of hpMSC exosomes and engineered exosomes in reducing lipopolysaccharide-induced macrophage polarization in lung tissue of mice. Figure 12A shows representative colloidal images of hypoxia-inducible factor-1α (HIF-1α), inducible nitric oxide synthase (iNOS), and actin (internal standard). This data was analyzed using the Simple Western blot method, and the relative band densities of the HIF-1α/actin and iNOS/actin ratios in lung tissue are shown. Each set of data was obtained from 5 mice. Figure 12B shows representative microscopic images (indicated by arrows) of iNOS immunohistochemical staining analysis and the quantitative total intensity of iNOS in lung tissue. Data for each group were obtained from 5 mice. All measurements were taken 48 hours after administration of saline or lipopolysaccharide. Data are expressed as mean ± standard deviation. * p < 0.05 compared with the Sham group. # p < 0.05 compared with the LPS group.† p < 0.05 compared with the LMExoi group. ˄ p < 0.05 compared with the LEExoi group. Sham: saline group. MExo: saline plus hpMSC exosome group. EExo: saline plus engineered exosome group. LPS: lipopolysaccharide (LPS) group. LMExo: LPS plus hpMSC exosomes. LMExoi: LPS plus inhibitor-treated hpMSC exosomes. LEExo: LPS plus engineered exosomes. LEExoi: LPS plus inhibitor-treated engineered exosomes.

Figures 13A and 13B show the similar efficacy of hpMSC exosomes and engineered exosomes in reducing lipopolysaccharide-induced lung oxidative damage in mice. Figure 13A shows representative colloidal images of superoxide dismutase 2 (SOD2) and actin (internal standard), analyzed using the Simple Western blotting method, and displays the relative band density of the SOD2/actin ratio in lung tissue. Data for each group were obtained from 5 mice. Figure 13B shows representative microscopic images (indicated by arrows) of myeloperoxidase (MPO) immunohistochemical staining analysis and the quantitative total intensity of MPO in lung tissue. Data for each group were obtained from 5 mice. All measurements were taken 48 hours after administration of physiological saline or lipopolysaccharide. Data are expressed as mean ± standard deviation. * p < 0.05 compared to the Sham group. # p < 0.05 compared to the LPS group. † p < 0.05 compared to the LMExoi group. ˄ p < 0.05 compared to the LEExoi group. Sham: Physiological saline group. MExo: Physiological saline plus hpMSC exosomes group. EExo: Physiological saline plus engineered exosomes group. LPS: Lipopolysaccharide (LPS) group. LMExo: LPS plus hpMSC exosomes group. LMExoi: LPS plus hpMSC exosomes treated with an inhibitor. LEExo: LPS plus engineered exosomes group. LEExoi: an engineered exosome group treated with LPS and an inhibitor.

Figures 14A and 14B show the similar efficacy of hpMSC exosomes and engineered exosomes in reducing lipopolysaccharide-induced lung cell apoptosis in mice. Figure 14A shows representative colloidal images of pro-apoptotic cleaved caspase 3 (19 kDa and 17 kDa) and actin (internal standard), analyzed using Simple Western blotting, and shows the relative band densities of the cleaved caspase 3 (19 kDa)/actin and cleaved caspase 3 (17 kDa)/actin ratios in lung tissue. Each set of data was obtained from 5 mice. Figure 14B shows representative fluorescence micrographs of the TUNEL assay, which labels fragmented DNA (indicated by arrows), and displays the count of TUNEL-positive cells in lung tissue. Data for each group were obtained from 5 mice. All measurements were taken 48 hours after administration of physiological saline or lipopolysaccharide. Data are expressed as mean ± standard deviation. * p < 0.05 compared to the Sham group. # p < 0.05 compared to the LPS group.† p < 0.05 compared to the LMExoi group. ˄ p < 0.05 compared to the LEExoi group. Sham: physiological saline group. MExo: physiological saline plus hpMSC exosome group. EExo: physiological saline plus engineered exosome group. LPS: Lipopolysaccharide (LPS) group. LMExo: LPS plus hpMSC exosome group. LMExoi: LPS plus inhibitor-treated hpMSC exosome group. LEExo: LPS plus engineered exosome group. LEExoi: LPS plus inhibitor-treated engineered exosome group.

TW202539696A_113143017_SEQL.xml

Claims (20)

A pharmaceutical composition comprising extracellular vesicles carrying let-7i-5p miRNA and a pharmaceutically acceptable excipient thereof. The pharmaceutical composition as claimed in claim 1, wherein the extracellular vesicles are loaded with let-7i-5p miRNA via electroporation, lipid transfection, ultrasound treatment, or contact with calcium chloride. The pharmaceutical composition as described in claim 2, wherein the extracellular vesicles are derived from animal cells. The pharmaceutical composition as described in claim 3, wherein the animal cell is a mammalian cell. The pharmaceutical composition as described in claim 4, wherein the mammalian cell is a non-stem cell. The pharmaceutical composition as described in claim 4, wherein the mammalian cell is a mesenchymal stem cell. The pharmaceutical composition as described in claim 4, wherein the mammalian cell is an immune cell. The pharmaceutical composition as described in claim 7, wherein the immune cell is a macrophage. The pharmaceutical composition as described in claim 2, wherein the extracellular vesicles are derived from cells cultured in vitro or from the body fluids of a subject. The pharmaceutical composition as described in claim 1, wherein the extracellular vesicle is an exosome. A method for the prevention or treatment of lung disease in subjects in need, comprising administering to the subject an effective dose of the pharmaceutical composition as claimed in claim 1. The method described in claim 11, wherein the lung disease is acute respiratory distress syndrome. The method described in claim 12, wherein the acute respiratory distress syndrome is induced by sepsis. The method described in claim 12, wherein the acute respiratory distress syndrome is induced by aspiration pneumonia. The method described in claim 11, wherein the application reduces lung damage. The method as described in claim 11, wherein the application improves lung function. The method as described in claim 11, wherein the application increases the inspiratory volume of the lungs. The method as described in claim 11, wherein the application increases the dynamic compliance of the lungs. The method as described in claim 11, wherein the application reduces airway resistance. The method as described in claim 11, wherein the application reduces airway elasticity.