Poly(amine-co-ester) polymer particles for selective lung delivery

Abstract

The present invention provides a delivery formulation for selective delivery to lung immune cells such as macrophages and monocytes. [Solution] Provided are poly(amine-co-ester) polymers, polyplexes loaded with activators, methods for forming the particles thereof, and methods for using them to deliver nucleic acid drugs with optimal uptake. Examples demonstrate key molecular weights combined with exposed carboxylic acids and / or hydroxyl groups, and methods for producing them. Typically, the compositions have lower toxicity and are more efficient in drug delivery compared to other transfection reagents of the control, or combinations thereof. In some embodiments, the compositions are suitable for in vivo delivery and can be systemically administered to a subject to treat a disease or condition.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit and priority of U.S. Patent Application No. 63 / 057,626, filed Jul. 28, 2020, and U.S. Patent Application No. 17 / 332,175, filed May 27, 2021, which are hereby incorporated by reference in their entirety.

[0002] Description of Federally Sponsored Research or Development This invention was made with government support under HL142674, HL133016, and HL150766 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] Field of the Invention The field of the invention generally relates to the selective delivery of agents to pulmonary immune cells, particularly macrophages and monocytes, and their uptake thereby, and to polymeric compositions and methods for improved pulmonary delivery of diagnostic, prophylactic, and / or therapeutic agents.

Background Art

[0004] Background of the Invention Cardiovascular diseases, such as pulmonary hypertension (PH), mainly have a harmful impact on human health. In fact, PH is defined by a mean pulmonary artery pressure higher than 20 mmHg and is the cause of more than 20,000 deaths per year in the United States alone (Simonneau G, et al. Eur Respir J. 2019;53(1); George Chest. 2014;146(2):476 - 95). PH is a heterogeneous group of clinical conditions that are classified into five groups by the World Health Organization (WHO) based on clinical symptoms, hemodynamics , pathological findings, and treatment (Simonneau).

[0005] WHO Group 1, or pulmonary arterial hypertension (PAH), includes idiopathic PAH (IPAH; previously classified as primary PH), while Group 3, caused by lung disease and / or hypoxia, is representative. Approximately half of PAH cases are IPAH, hereditary, or drug-induced. Another important subgroup is associated PAH conditions, whose primary cause is connective tissue disease, mainly systemic sclerosis (SSc; also known as scleroderma) (Hoeper MM, et al. Lancet Respir Med. 2016;4(4):306-22; Galie N, et al. Eur Heart J. 2016;37(1):67-119).

[0006] Unfortunately, PAH is highly pathological and fatal, with 50% of patients dying within 7 years of initial diagnosis (Benza Chest. 2012;142(2):448-56). Furthermore, SSc-PAH The prognosis is dramatically worse than that of IPAH (Fisher MR, et al. Arthritis Rheum. 2006;54(9):3043-50). Despite the existence of several available medications for PAH, Furthermore, there is no treatment that can induce a reversal of the disease or prevent its progression. Similarly, in patients in group 3, PH is a harbinger of a substantially poor prognosis for the underlying lung disease (Hoeper 2016).

[0007] Many cardiovascular diseases, such as atherosclerosis and arterial restenosis, are characterized by excessive and abnormal smooth muscle cells (SMCs), and similarly, coating of the distal pulmonary arterioles, which are not normally muscularized, with SMCs is an important pathological feature in pulmonary hypermuscularization (IPAH). This hypermuscularization reduces pulmonary artery compliance, which is a strong independent predictor of mortality in IPAH (Mahapatra, et al. J Am Coll Cardiol. 2006;47(4):799-803.). Current treatment for PAH mainly involves inducing vasodilation. However, these treatments do not reduce excessive muscular arterialization. The gap in treatment is mainly our Reflecting the limitations of our understanding of the etiology, further research into the pathobiology of PH is therefore of paramount importance.

[0008] Specialized pulmonary arterioles expressing platelet-derived growth factor receptor (PDGFR)-β (SMCs) clonally enlarge and give rise to pathological distal arterioles (SMCs) during hypoxia-induced hyperplasia (PH), but the regulation of this typical process is not well understood (Sheikh Cell Rep. 2014;6(5):809-17; Sheikh Sci Transl Med. 2015;7(308):308ra159). Upregulation of hypoxia-inducible factor (HIF)1-α in SMCs plays a crucial role in distal muscular arterialization, and in addition to such pathways in the SMCs themselves, non-cellular autonomous regulation is extremely important (Ball, et al. Am J Respir Crit Care Med. 2014;189(3):314-24.; Sheikh Cell Rep. 2018;23(4):1152-65). In this context, endothelial cells (ECs) are the most understudied. This is a cell type that is being treated. For example, the PDGF pathway is essential for the development and disease of vascular SMCs (Andrae et al. Genes Dev. 2008;22(10):1276-312; Seidelmann Cell Mol Life Sci. 2014;71(11):1977-99), deletion of ligand PDGF-β in EC attenuates hypoxia-induced distal pulmonary arteriole muscular arterialization, PH, and right ventricular hypertrophy (RVH) (Sheikh 2018).

[0009] Experimental hypoxia in rodents causes distal pulmonary arteriole muscular arterialization, PH, and right ventricular hypertrophy. The signaling pathway regulated by platelet-derived growth factor, abbreviated as PDGF, is essential in the pathobiology of SMCs in PH. Indeed, levels of the receptor PDGFR-β are increased in pathological SMCs, and deletion of the ligand PDGF-β in endothelial cells attenuates PH. Over the past decade, novel findings that the immune system is involved in several diseases have motivated scientists to further investigate the role of macrophages in lung pathogenesis. Hypoxia increases macrophage recruitment in the lungs, and pharmacological inhibition of selective receptors or agonists expressed by macrophages (e.g., CX3CR1, leukotriene B4) has been shown to alleviate PH; however, these products are also produced by other cell types, raising cell specificity issues.

[0010] In addition to vascular cells, immune cells including monocytes / macrophages have recently been attracting increasing attention in the context of PH (Florentin et al. Cytokine. 2017;100:11-5; Nicolls et al Am J Respir Crit Care Med. 2017;195(10):1292-95). Upon exposure to oxygen, monocytes migrate to the perivascular spaces of the lungs and differentiate into interstitial macrophages (Florentin et al. Cytokine. 2017;100:11-5; Nicolls et al. Am J Respir Crit Care Med. 2017;195(10):1292-9). Bronchoalveolar lavage in these mice demonstrates an increase in macrophages in aspirated bronchoalveolar lavage fluid (BALF) and in the remaining lung tissue (Amsellem V, et al. Am J Respir Cell Mol Biol. 2017;56(5):597-608). Similarly, cells expressing the macrophage marker CD68 are enriched proximally of occlusive lesions in the lungs of human PAH patients (Tuder et al. Am J Pathol. 1994;144(2):275-85). In rodent PH models, it has been shown that PH can be alleviated by general genetic or pharmacological inhibition of selective receptors or agonists expressed by macrophages (e.g., CX3CR1, leukotriene B4) (Amsellem, et al. Sci Transl Med. 2013;5(200):200ra117); however, these products are, Other cell types also produce macrophages in a similar manner, leading to macrophage specificity issues.

[0011] Monocytes / macrophages undoubtedly play a crucial role in the pathogenesis of PH and other vascular diseases, but their role in regulating the biology of SMCs in these situations is not fully established. It was recently demonstrated that the rare clonal increase of SMCs during atherosclerotic plaque formation is regulated by bone marrow-derived cells (most likely macrophages) (Misra A, et al. Nat Commun. 2018;9(1):2073). Furthermore, culture media conditioned with activated macrophages from atherosclerotic mice showed a large It induces the migration and proliferation of arterial SMCs (Misra 2018). In relation to PH, interlo Hypoxia exposure of macrophages pre-activated by Ikin-4 generates a conditioned medium that induces proliferation of pulmonary artery pulmonary microclasts (PASMCs) (Vergadi E, et al. Circulation). (2011;123(18):1986-95). In addition, dual inhibition of CC motif chemokine receptors 2 and 5 attenuates the induction of PASMC proliferation and migration by macrophage-conditioned medium (Abid, et al. Eur Respir J. 2019;54(4)).

[0012] It was recently found that downregulating PDGF-B in monocytes / macrophages with inefficient Csf1r-Cre-Mer-Cre moderately inhibits hypoxia-induced pulmonary vascular remodeling, but the hemodynamics and underlying pathways were not evaluated (Sheikh, Cell Reports, 2018. 23:1152; Epelman S, et al. Immunity. 2014;40(1):91-104).

[0013] Even if these cells are crucial for preventing or treating PH, there are no selective delivery methods to these cells to treat the disease or alleviate its symptoms.

[0014] Therefore, an object of the present invention is to provide an improved polymer that can selectively and effectively deliver therapeutic agents, diagnostic agents, and / or prophylactic agents to lung immune cells, particularly lung macrophages and monocytes. [Prior art documents] [Non-patent literature]

[0015] [Non-Patent Document 1] Simonneau G, et al. Eur Respir J. 2019;53(1) [Non-Patent Document 2] George Chest. 2014;146(2):476-95 [Non-Patent Document 3] Hoeper MM, et al. Lancet Respir Med. 2016;4(4):306-22

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[0016] Summary of the Invention Lung macrophage-derived PDGF-B plays a crucial role in the enlargement of pathological smegma cells (SMCs) and can be used as a therapeutic target to treat or mitigate diseases such as pulmonary hypertension (PH). Studies were conducted using mouse models, cell type-specific deletions of multiple genes, human macrophages from IPAH and SSc-PAH patients, and in vivo nanoparticle delivery of siRNA against PDGF-β. Lung macrophage depletion or PDGF-β deletion in myeloid cells attenuates hypoxia-induced distal muscular arterialization, PH, and alveolar myofibroblast accumulation. The results establish that monocytes / macrophages play a vital role in pulmonary hypertension (PH).

[0017] Using a mouse hypoxia model and human monocyte-derived macrophages, it was demonstrated that platelet-derived growth factor (PDGF)-B from macrophages is upregulated in the lungs of pulmonary hypertension (PH) patients and experimental PH mice. Macrophage-derived PDGF-B induces increased migration and proliferation of human pulmonary artery smooth muscle cells, a key component of PH pathogenesis. Furthermore, there are findings that genetic deletion of PDGF-β in myeloid cells prevents hypoxia-induced PH. The results indicate that HIF1-α and HIF2-α are upstream of PDGF-B in macrophages, and that LysM in mice exposed to hypoxia... + We have demonstrated that deletion of the Hifa gene in cells has a similar effect to PDGF-β deletion. As a complementary approach, under normoxic pressure conditions, gain-of-function mutations in HIFα in myeloid cells induce pulmonary macrophage accumulation, PDGF-β expression, distal muscular arterialization, PH, and RVH. Culture media adapted with macrophages from IPAH and SSc-PAH patients induce human PASMC (hPASMC) proliferation and migration in a PDGF-B-dependent manner. The results show that oral intratracheal administration of nanoparticles loaded with PDGF-β siRNA significantly attenuates hypoxia-induced pulmonary macrophage PDGF-β expression, distal muscular arterialization, PH, RVH, and alveolar myofibroblast accumulation. All of these demonstrate the targeting of pulmonary macrophage-derived PDGF-B as a therapeutic strategy for PH.

[0018] While several nanoparticle-based technologies are currently approved by the FDA, they are primarily administered intravenously to reach target organs via the circulation. It has been discovered that particles formed from poly(amine-co-ester) polymers can be used to selectively deliver therapeutic, prophylactic, or diagnostic agents to immune cells lining the respiratory tract, such as macrophages, so that they are taken up by them. Examples demonstrate that, when administered into the respiratory tract, particles exhibit high loading and selective uptake even without a targeted portion.

[0019] In addition to lung disorders such as PH, the diseases or disorders treated include infectious diseases, cancer, metabolic disorders, autoimmune diseases, inflammatory disorders, and age-related disorders. The particles can be administered by aerosol, inhaler, dry powder, intubation, and intravenous infusion.

[0020] The example demonstrates oral intratracheal administration of large nanoparticles (400 nm in diameter) loaded with silencing (SiO) RNA against PDGF-β to mice. These nanoparticles are preferentially taken up by pulmonary macrophages (of the total cells that take up nanoparticles, the percentage of cells that are macrophages is approximately 95% in bronchoalveolar lavage fluid and approximately 85% in residual lung after bronchoalveolar lavage). With oral intratracheal administration, the efficiency of PDGF-β silencing is high in pulmonary macrophages (>85% knockdown), and it can effectively prevent / suppress hypoxia-induced pathological distal arteriole muscular arterialization, pulmonary artery pressure, and right ventricular hypertrophy. [Brief explanation of the drawing]

[0021] [Figure 1-1]Figures 1A-1F are graphs showing that lung macrophages accumulate under hypoxia and are crucial for hypoxia-induced pulmonary vascular remodeling and PH. Wild-type mice were exposed to hypoxia (10% FiO2) for up to 21 days as indicated, or maintained under normal oxygen conditions. BALF and residual lung tissue were collected, and single-cell suspensions were subjected to flow cytometry analysis. The percentage of total cells in designated compartments that were CD64+Ly6G- macrophages was determined. n=3 mice per time point. Figures 1A and 1B are graphs of CD64+Ly6G- cells (%) in BALF (Figure 1A) and residual lung (Figure 1B) against the number of days of hypoxia. Figures 1C-1D are graphs of RVSP (mmHg) (Figure 1C) and RV / (LV+S) (Figure 1D, Fulton index (F; weight ratio of right ventricle [RV] to the sum of left ventricle [LV] and septum [S]) for normal oxygen and hypoxia, n=3 mice). Liposomes containing PBS (vehicle) or clodronate were administered orally intratracheally at the start of hypoxia (or normal oxygen as control) and every 3 days during the subsequent 21 days of treatment. Figures 1E and 1F show the percentage of CD64+Ly6G-macrophages in total cells of the BALF (Figure 1G) and residual lung (Figure 1H), n=3 mice. [Figure 1-2] Same as above. [Figure 2-1]Figures 2A-2F are graphs showing that PDGF-β levels in lung macrophages increase with hypoxia, and that PDGF-β deficiency in LysM+ cells attenuates distal muscular arterialization and PH. CD64+Ly6G- cells from BALF (Figure 2A) and residual lung (Figure 2B) were isolated by FACS from wild-type mice exposed to hypoxia (10% FiO2) for up to 21 days as indicated, or under normal oxygen conditions. PDGF-β mRNA levels were measured by qRT-PCR (see Table 1). n=3 mice per time point, and qRT-PCR was performed in triplicate. Figures 2C-2F show PDGF-β (flox / flox) mice lacking Cre or possessing both Cre and LysM-Cre, which were exposed to hypoxia for 21 days or maintained under normal oxygen conditions. Figure 2C, RVSP; Figure 2D, RV / (LV+S); Figure 2E, changes in RV / LV+S; and Figure 2F, myofibroblasts / 100 alveoli. Figure 2C shows the difference in Fulton indices between hypoxic and normal oxygen levels, stratified by genotype. One-way ANOVA with Tukey's multiple comparison test (compared to normal oxygen levels, *, **, ***, #, p<0.05, <0.01, <0.001, <0.0001, respectively) was used in Figures 2C-2D, and Student's t-test was used in Figure 2E. [Figure 2-2] Same as above. [Figure 3] Figures 3A-3D. Vhl deletion in LysM+ cells induces distal muscular arterialization and PH under normal oxygen conditions. Vhl(flox / flox) mice lacking Cre or possessing both LysM-Cre were maintained under normal oxygen conditions for 49 days postnatally. Figure 3A shows isolated BALF cells and PDGF-β transcript levels measured by the Fulton index (Figure 3C, BALF; 3B, lung). The number of macrophages (stars) per 100 alveoli was quantified (Figure 3D). More than 500 alveoli were quantified per mouse. n=3 mice. Student's t-test was used. [Figure 4-1]Figures 4A-4F. Hif1a deletion in myeloid cells attenuates hypoxia-induced PDGF-β expression, distal muscular arterialization, and PH. BALF cells were isolated from wild-type mice in normal oxygen or hypoxic conditions (10% FiO2, up to 21 days). HIF1-α and β-actin proteins were evaluated by Western blotting with densitometry of HIF1-α compared to β-actin. n=3 mice per time point. One-way ANOVA with Tukey's multiple comparison test. Hif1a(flox / flox) mice lacking Cre or also possessing LysM-Cre were exposed to hypoxia for 3 or 21 days. On day 3 of hypoxia, PDGF-β transcript levels in BALF cells were determined by qRT-PCR (Figures 4A, 4B). Lung vibratome sections were stained for SMA, macrophage marker CD64, and nucleus (DAPI). The number of macrophages and alveolar myofibroblasts per 100 alveoli was quantified (Figures 4C, 4D). qRT-PCR was performed in triplicate on 3-5 mice. More than 700 alveoli were quantified per mouse. On day 21 of hypoxia, vibratome sections with distal arterioles in the L, L1, A1, and L1 regions were stained for SMA, CD31, and RVSP, and the Fulton index was measured as shown in Figures 4E, 4F. (3 mice). [Figure 4-2] Same as above. [Figure 5-1]Figures 5A–5F. Hif2a deletion in LysM+ cells attenuates hypoxia-induced PDGF-β expression, distal muscular arterialization, and PH. BALF cells were isolated from wild-type mice exposed to normal oxygen or hypoxia (10% FiO2) for up to 21 days. HIF2-α and β-actin protein levels were assessed by densitometry of HIF2-α compared to β-actin using Western blotting. n=3 mice for each time point. Figure 5A. One-way ANOVA with Tukey's multiple comparison test. Figures 5B–5F. Hif2a(flox / flox) mice lacking Cre or also possessing LysM-Cre were exposed to hypoxia for 3 or 21 days. On day 3 of hypoxia, BALF cells were isolated, and PDGF-β mRNA levels were determined by qRT-PCR (Figure 5B). Vibratome sections of the lung were stained for SMA, CD64, and the nucleus (DAPI). The number of macrophages and alveolar myofibroblasts per 100 alveoli was quantified (Figures 5C-5D). n=3-5 mice, qRT-PCR was performed in triplicate. More than 700 alveoli were quantified per mouse. On day 21 of hypoxia, vibratome sections with distal arterioles in the L, L1, A1, and L1 regions were stained for SMA, MECA-32, and RVSP, and the Fulton index was measured (Figures 5E, 5F). n=3 mice. Student's t-test. [Figure 5-2] Same as above. [Figure 6-1]Figures 6A-6E. PDGF-B secreted by macrophages from PAH patients promotes the proliferation and migration of hPASMCs. Monocytes were isolated from peripheral blood mononuclear cells of human controls and IPAH or SSc-PAH patients and differentiated into macrophages in culture. Figure 6A, macrophages derived from human control monocytes were cultured for 12 hours under normal oxygen pressure or hypoxic (3% O2) conditions, and PDGF-β mRNA levels were measured by qRT-PCR. n=3 humans (2 females and 1 male, 30-60 years old), qRT-PCR was performed in triplicate. Figure 6B, PDGF-β mRNA levels of macrophages from controls and PAH patients were assayed using qRT-PCR. n=5 humans and n=9 controls per PAH diagnostic class (see Table S2), qRT-PCR was performed in triplicate. Figure 6C shows that hPASMCs were cultured for 24 hours in medium pre-conditioned with control and patient macrophages. BrdU was added during the last 10 hours of this incubation. Cells were then stained for BrdU and the nucleus (propidium iodide [PI]). In Figure 6C, the percentage of total cells (PI+ nuclei) expressing BrdU for control humans and patients was normalized to this percentage for controls. In Figure 6D, anti-PDGF-B blocking antibody or control IgG was added to the conditioned medium 1 hour before incubation with hPASMCs. The results are the ratio of the percentage of total (PI+) cells that are BrdU+ compared to anti-PDGF-B treatment, stratified by patient diagnostic class. n=3 humans and n=6 controls per PAH diagnostic class (see Table S3), 10 microscopic fields per human, 30–60 cells per field. Culture media pre-conditioned with control or patient macrophages were treated with anti-PDGF-B blocking antibody or control IgG antibody for 1 hour and then placed in the lower chamber of a Boyden apparatus. hPASMC was added to the upper chamber, and migration toward the conditioned medium was evaluated for 8 hours. Migratory cells (i.e., cells on the underside of the membrane) were stained with crystal violet. Figure 6E shows the quantification of migratory cells and IgG treatment compared to control patients.n=4 humans and n=3 controls per PAH class (see Table 4), 5 microscopic fields per human, 8–90 cells per field. One-way ANOVA and Student's t-test with Tukey's multiple comparison test were used. Compared to IPAH, the results were #, ##, p<0.05, <0.01, and compared to the corresponding IgG controls, the results were *, **, ***, ns, p<0.05, <0.01, <0.0001, respectively, not significant. [Figure 6-2] Same as above. [Figure 7-1]Figures 7A-7F. Nanoparticle-mediated knockdown of PDGF-β attenuates distal arteriole muscular arterialization, myofibroblast accumulation, and pH. Nanoparticles (400 nm in diameter) loaded with the dye DiD were administered orally and intratracheally to normoxic mice. After 12 hours, cells from BALF and residual lung were stained for CD64 and subjected to flow cytometry analysis. Figure 7A shows the percentage of BALF or residual lung (RL) cells containing DiD+ nanoparticles (400 or 200 nm in diameter as indicated) expressing CD64. n=3 mice per treatment. BALF cells were collected from normoxic mice, cultured with 400 nm nanoparticles loaded with DiD for 6 hours, and then stained for the nucleus (DAPI). Figures 7B-7F show 400 nm nanoparticles loaded with PDGF-β-targeted siRNA or scrambled (Scr)RNA, administered to mice at the onset of hypoxia, and then administered twice weekly thereafter. Lungs were isolated from hypoxic mice on day 3, stained for Ly6G and CD64, and subjected to flow cytometry. The percentage of CD64+Ly6G-macrophages was quantified in Figure 7B. n=3 mice per treatment. In Figure 7C, PDGF-β RNA levels of CD64+Ly6G-macrophages were quantified by qRT-PCR. n=3 mice per treatment, qRT-PCR was performed in triplicate. In Figures 7D-7F, mice were treated with hypoxia for 21 days or maintained in a normal oxygen state. In hypoxic mice, sections containing distal arterioles in the L.L1.A1 region or alveolar region were stained for CD31 and SMA. RVSP (Figure 7D), Fulton index (Figure 7E), and myofibroblast count per 100 alveoli were measured (Figure 7F). More than 500 alveoli per mouse were quantified. One-way ANOVA with Tukey's multiple comparison test and Student's t-test were used. Compared to normal oxygen levels*, p<0.05.ns, not significant. Scale bars, 10 μm (D) and 25 μm (I, L). [Figure 7-2] Same as above. [Figure 8] Figure 8 is a schematic diagram of the methods used for animal and human studies. [Modes for carrying out the invention]

[0022] Detailed description of the invention I. Definition As used herein, the term "polyplex" typically refers to polymeric microparticles and / or nanoparticles, or micelles, in which one or more polynucleotides are encapsulated, dispersed, and / or associated with the surface thereof.

[0023] The term “microparticles” includes objects having an average diameter of approximately 1 micron or larger, up to approximately 1000 microns. The term “microparticles” also includes microspheres and microcapsules, as well as structures that may not readily fit into either of the above two categories. Microparticles can be spherical or non-spherical and may have any regular or irregular shape. Structures with an average diameter of less than approximately 1 micron (1000 nm) are called “nanoparticles” and include “nanospheres” and “nanocapsules.” The term “diameter” is used to refer to either physical diameter or hydrodynamic diameter. The diameter of an essentially spherical particle may refer to either physical or hydrodynamic diameter. The diameter of a nanospherical particle may refer to hydrodynamic diameter. As used herein, the diameter of a non-spherical particle may refer to the maximum straight-line distance between two points on the surface of the particle. When referring to multiple particles, the particle diameter typically refers to the average diameter of the particles. Particle diameter can be measured using a variety of techniques in the art, including, but not limited to, dynamic light scattering and confocal microscopy.

[0024] Compositions containing microparticles or nanoparticles may contain particles of a certain range of particle sizes. In certain embodiments, the particle size distribution may be uniform, for example, within less than about 20% standard deviation of the mean volume diameter, and in other embodiments, it may be even more uniform, for example, within about 10%, 8%, 5%, 3%, or 2% of the median volume diameter.

[0025] As used herein, “pharmaceutically acceptable” means a compound, material, composition, and / or dosage form that is suitable for use in contact with human and animal tissues, organs, and / or bodily fluids, within the bounds of sound medical judgment, without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio.

[0026] As used herein, the term "biocompatible" means one or more materials that are neither toxic to a host (e.g., an animal or a human) nor decompose in the host at a rate that produces monomeric or oligomeric subunits or other byproducts at toxic concentrations (if the material decomposes).

[0027] As used herein, the term "biodegradable" means that a material typically breaks down or disintegrates into its constituent subunits through hydrolysis or enzymatic action.

[0028] As used herein, the term "surfactant" refers to a substance that reduces the surface tension of a liquid.

[0029] As used herein, "sustained release" refers to the release of a substance over a long period of time, as opposed to a bolus dose in which the entire amount of the substance is made biologically available at once.

[0030] The terms "parenteral administration" and "administered parenterally" are recognized terms in the art and include, but are not limited to, modes of administration other than enteral and local administration, such as injection, including intravenous, intramuscular, intrapleural, intravascular, intrapericardial, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, and intrasternal injections and infusions.

[0031] The term “targeted portion,” as used herein, refers to a portion localized at or away from a specific location. The portion may be, for example, a protein, nucleic acid, nucleic acid analog, carbohydrate, or small molecule. The entity may be, for example, a therapeutic compound such as a small molecule, or a diagnostic entity such as a detectable label.

[0032] The term "alkyl" refers to a radical of a saturated aliphatic group and includes linear alkyl groups, branched alkyl groups, cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups.

[0033] In preferred embodiments, linear or branched alkyl groups have 30 or fewer carbon atoms in their skeleton (for example, C1-C1 for a linear group). 30 Regarding the branched chain, C3~C 30 ), preferably having 20 or fewer carbon atoms, more preferably 15 or fewer, and most preferably 10 or fewer. All integer values ​​for the number of skeletal carbon atoms between 1 and 30 are intended and disclosed for linear or branched alkyls. Similarly, preferred cycloalkyls have 3 to 10 carbon atoms in their ring structure, more preferably 5, 6, or 7 carbon atoms in their ring structure. All integer values ​​for the number of ring carbon atoms between 3 and 10 are intended and disclosed for cycloalkyls.

[0034] When used throughout this specification, the examples, and the claims, the term “alkyl” (or “lower alkyl”) is intended to include both “unsubstituted alkyl” and “substituted alkyl,” the latter referring to an alkyl moiety having one or more substituents that replace hydrogens on one or more carbons of a hydrocarbon backbone. Such substituents include, but are not limited to, halogens, hydroxyls, carbonyls (e.g., carboxyls, alkoxycarbonyls, formyls, or acyls), thiocarbonyls (e.g., thioesters, thioacetates, or thioformates), alkoxyls, phosphoryls, phosphates, phosphonates, phosphinates, aminos, amides, amidines, imines, cyanos, nitros, azides, sulfhydryls, alkylthios, sulfates, sulfonates, sulfamoyls, sulfonamides, sulfonyls, heterocyclyls, aralkyls, or aromatic or heteroaromatic moieties.

[0035] Unless otherwise specified, “lower alkyl” as used herein means an alkyl group as defined above, but having 1 to 10 carbon atoms, more preferably 1 to 6 carbon atoms, in its skeletal structure. Similarly, “lower alkenyl” and “lower alkynyl” have similar chain lengths. Throughout this application, preferred alkyl groups are lower alkyl groups. In preferred embodiments, substituents designated as alkyl in this specification are lower alkyl groups.

[0036] Those skilled in the art will understand that, where appropriate, the substituted portions on the hydrocarbon chain may themselves be substituted. For example, substituents on substituted alkyl groups include halogens, hydroxyl, nitro, thiol, amino, azide, imino, amide, phosphoryl (including phosphonates and phosphinates), sulfonyl (including sulfates, sulfonamides, sulfamoyl and sulfonate), and silyl groups, as well as ethers, alkylthio, carbonyl (including ketones, aldehydes, carboxylates, and esters), -CF3, -CN, etc. Cycloalkyls can be substituted in the same manner.

[0037] When used herein, "aryl" means C5-C 10 This refers to a ring system consisting of one aromatic, heterocyclic, fused aromatic, fused heterocyclic, diaromatic, or bihetereocyclic rings. In some forms, the ring system has 3 to 50 carbon atoms. In a broad sense, "aryl" as used herein refers to 5, 6, 7, 8, 9, 10 and 24-membered monocyclic aromatic groups that may contain 0 to 4 heteroatoms, such as benzene, naphthalene, anthracene, phenanthrene, chrysene, pyrene, corannulene, coronene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine. These aryl groups having heteroatoms in the ring structure may also be called "aryl heterocyclic" or "heteroaromatic." The aromatic ring may be substituted at one or more ring positions with one or more substituents, but are not limited to, halogens, azides, alkyls, aralkyls, alkenyls, alkynyls, cycloalkyls, hydroxyls, alkoxyls, aminos (or quaternized aminos), nitros, sulfhydryls, iminos, amides, phosphonates, phosphinates, carbonyls, carboxyls, silyls, ethers, alkylthios, sulfonyls, sulfonamides, ketones, aldehydes, esters, heterocyclyls, aromatic or heteroaromatic moieties, -CF3, -CN; and combinations thereof.

[0038] The term "aryl" also includes polycyclic ring systems (i.e., "fused rings") having two or more cyclic rings in which two or more carbon atoms are common to two adjacent rings, where at least one of the rings is aromatic, and the other cyclic rings or rings may be, for example, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, and / or heterocyclic rings. Examples of heterocyclic rings include, but are not limited to, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, Benzoxazolinil, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinil, carbazolyl, 4aH-carbazolyl, carborinil, chromanil, clomenil, sinnolinil, decahydroquinolinil, 2H,6H-1,5,2-dithiadinil, dihydrofloflo[2,3-b]tetrahydrofuran, furanil, flazanil, imidazolidinil, imidazolinil, imidazolyl, 1H-indazolyl, indolenil, indolinil, Indolidinyl, indolyl, 3H-indolyl, isatinoyl, isobenzofuranil, isochromanil, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholinyl, naphthilidinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl, oxazolyl, oxin Drill, pyrimidinil, phenanthrolinil, phenanthrolinil, phenazinil, phenothiazinil, phenoxatinil, phenoxadinil, phthalazinil, piperadinil, piperidinil, piperidonil, 4-piperidonil, piperonil, pteridinil, prinil, pyranil, pyrazinil, pyrazolidinil, pyrazolinil, pyrazolyl, pyridazinil, pyridoxazole, pyridoimidazole, pyridothiazole, pyridinil, pyridyl, pyrimidinil, pyrrolidinil, pyrrolinil, 2H-pyrrolyl, pyro Examples include lyl, quinazolinyl, quinolinyl, 4H-quinolidinyl, quinoxalinyl, quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, tetrazolyl, 6H-1,2,5-thiadiadinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienoxazolyl, thienoimidazolyl, thiophenyl, and xanthenyl. One or more rings may be substituted as defined above for "aryl".

[0039] "Alkoxy" refers to the alkyl group defined above, having the indicated number of carbon atoms attached via oxygen crosslinking. Examples of alkoxys include, but are not limited to, methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, s-butoxy, n-pentoxy, s-pentoxy, and their derivatives.

[0040] Primary amines are formed when one of the three hydrogen atoms in ammonia is replaced by a substituted or unsubstituted alkyl group or a substituted or unsubstituted aryl group. Secondary amines have one hydrogen atom and two organic substituents (substituted or unsubstituted alkyl, substituted or unsubstituted aryl, or a combination thereof) bonded to the nitrogen. In tertiary amines, the nitrogen has three organic substituents.

[0041] As used herein, "substituted" means that one or more atoms or groups of atoms on a monomer are replaced by one or more atoms or groups of atoms different from the atoms or groups of atoms being replaced. In some embodiments, one or more hydrogens on a monomer are replaced by one or more atoms or groups of atoms. Examples of functional groups that can replace hydrogens are listed above in the definition. In some embodiments, one or more functional groups can be added that alter the chemical and / or physical properties of the resulting monomer / polymer, such as charge or hydrophilicity / hydrophobicity. Examples of substituents include, but are not limited to, halogens, hydroxyls, carbonyls (e.g., carboxyls, alkoxycarbonyls, formyls, or acyls), thiocarbonyls (e.g., thioesters, thioacetates, or thioformates), alkoxyls, phosphoryls, phosphates, phosphonates, phosphinates, aminos, amides, amidines, imines, cyanos, nitros, azides, sulfhydryls, alkylthios, sulfates, sulfonates, sulfamoyls, sulfonamides, sulfonyls, nitros, heterocyclyls, aralkyls, or aromatic or heteroaromatic moieties.

[0042] Unless otherwise indicated, this disclosure encompasses the prior art of molecular biology, microbiology, cell biology, and recombinant DNA, which are within the scope of the art. For example, Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3rd edition (2001); Current Protocols In Molecular Biology [(Ausubel, et al. eds., (1987)]; Coligan, Dunn, Ploegh, Speicher and Wingfeld, eds. (1995) Current Protocols in Protein Science (John Wiley & Sons, Inc.); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (See MJ MacPherson, BD Hames and GR Taylor eds. (1995)) I want to be treated that way.

[0043] II. Particles Particles for efficient and selective delivery to the lungs are typically formed from biodegradable, biocompatible polymers. These are typically nanoparticles with a size of less than 1000 nm, more preferably less than 500 nm, and most preferably at least 100 nm. An example demonstrates that nanoparticles between 200 and 400 nm selectively target lung immune cells such as monocytes and macrophages.

[0044] polymer Polymers comprising poly(amine-co-esters), poly(amine-co-amides), or combinations thereof, as well as polyplexes and solid core particles formed therefrom. Poly(amine-co-esters) are discussed in WO2013 / 082529, WO2017 / 151623, WO2017 / 197128, U.S. Publication No. 2016 / 0251477, U.S. Publication No. 2015 / 0073041, and U.S. Patent No. 9,272,043.

[0045] Substituting diester monomers in a polymer with diacids, such as sebacic acid, yields polymers having a mixture of hydroxyl and carboxyl end groups. Both of these end groups can be activated with 1,1'-carbodimidazole. The activated product can react with amine-containing molecules to produce polymers with novel end groups.

[0046] The polymer can be further hydrolyzed to release more active end groups, such as -OH and -COOH, both of which typically originate from the hydrolysis of ester bonds in the polymer by incubating the polymer for several days or weeks at a controlled temperature (e.g., 37°C or 100°C) (referred to herein as "actuation"). (It is hydrolyzed). In some embodiments, the polymer is not hydrolyzed and may therefore be called "not activated".

[0047] In some embodiments, when used to form polyplexes, the content of hydrophobic monomers in the polymer is increased compared to the content of the same hydrophobic monomers. Increasing the content of hydrophobic monomers in the polymer forms a polymer that can form solid core nanoparticles in the presence of nucleic acids, including RNA.

[0048] Unlike polyplexes, these particles are stable for extended periods during incubation in buffer water or serum, or upon administration to animals (e.g., injection). They also provide sustained release of nucleic acids (e.g., siRNA) resulting in long-lasting activity (e.g., siRNA-mediated knockdown).

[0049] A. Polymer structure Poly(amine-co-esters) or poly(amine-co-amides) are described herein. In some forms, the polymer has the structure shown in formula I: [ka] [In the formula, n is an integer from 1 to 30, m, o, and p are independent integers between 1 and 20. x, y, and q are independent integers between 1 and 1000. R x is hydrogen, a substituted or unsubstituted alkyl, a substituted or unsubstituted aryl, or a substituted or unsubstituted alkoxy. Z and Z' are independently O or NR' (wherein R' is hydrogen, a substituted or unsubstituted alkyl, or a substituted or unsubstituted aryl), R1 and R2 are chemical entities containing a hydroxyl group, a primary amine group, a secondary amine group, a tertiary amine group, or a combination thereof. It has.

[0050] R x Examples of the R' group include, but are not limited to, hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, and their homologs and isomers, such as n-pentyl, n-hexyl, n-heptyl, n-octyl, phenyl, naphthalyl, anthracenyl, phenanthryl, crisenyl, pyrenyl, tolyl, xylyl, and others.

[0051] In certain embodiments, the values ​​of x, y, and / or q are such that the weight-average molecular weight of the polymer is greater than 20,000 daltons, greater than 15,000 daltons, greater than 10,000 daltons, greater than 5,000 daltons, and greater than 2,000 daltons. In some forms, the weight-average molecular weight of the polymer is between about 2,000 daltons and about 20,000 daltons, more preferably between about 5,000 daltons and about 10,000 daltons.

[0052] The polymer can be prepared from one or more lactones, one or more amine-diols (Z and Z'=O), triamines (Z and Z'=NR'), or hydroxy-diamines (Z=O and Z'=NR', or Z=NR' and Z'=O) and one or more diacids or diesters. In those embodiments in which two or more different lactones, diacids or diesters and / or triamines, amine-diols, or hydroxy-diamine monomers are used, the values ​​of n, o, p, and / or m may be the same or different.

[0053] In some forms, the percentage composition of lactone units is between approximately 10% and 100%, and is calculated as lactone unit-to-(lactone unit + diester / diacid). Expressed in terms of molar ratio, the lactone unit-to-(lactone unit + diester / diacid) content is between approximately 0.1 and 1, i.e., x / (x+q) is between approximately 0.1 and 1. Preferably, the number of carbon atoms in the lactone unit is between approximately 10 and 24, more preferably between approximately 12 and 16. Most preferably, the number of carbon atoms in the lactone unit is 12 (dodecalactone), 15 (pentadecalactone), or 16 (hexadecalactone).

[0054] In some forms, Z is the same as Z'.

[0055] In some forms, Z is O and Z' is O. In some forms, Z is NR' and Z' is NR'. In some forms, Z is O and Z' is NR'. In some forms, Z is NR' and Z' is O.

[0056] In some forms, Z' is O, and n is an integer between 1 and 24, for example, 4, 10, 13, or 14. In some forms, Z is also O.

[0057] In some forms, Z' is O, n is an integer between 1 and 24, e.g., 4, 10, 13, or 14, and m is an integer between 1 and 10, e.g., 4, 5, 6, 7, or 8. In some forms, Z is also O.

[0058] In some forms, Z' is O, n is an integer between 1 and 24, e.g., 4, 10, 13, or 14, m is an integer between 1 and 10, e.g., 4, 5, 6, 7, or 8, as are o and p, the same integers between 1 and 6, e.g., 2, 3, or 4. In some forms, Z is also O.

[0059] In some embodiments, Z' is O, n is an integer from 1 to 24, e.g., 4, 10, 13, or 14, m is an integer from 1 to 10, e.g., 4, 5, 6, 7, or 8, and R is an alkyl, e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, and their homologs and isomers, e.g., n-pentyl, n-hexyl, n-heptyl, and n-octyl, or an aryl, e.g., phenyl, naphthalyl, anthracenyl, phenanthryl, crisenyl, pyrenyl, tolyl, or xylyl. In some forms, Z is also O.

[0060] In some forms, n is 14 (e.g., pentadecalactone, PDL), m is 7 (e.g., sebacic acid), and o and p are 2 (e.g., N-methyldiethanolamine, MDEA).

[0061] In some embodiments, the polyplex or particles are formed from polymers R1 and / or R2 that are not associated with the corresponding polyplex, where R1 and / or R2 are: [ka] Consists of, or includes.

[0062] In some embodiments, the polyplex or particles formed from the polymer have R1 and / or R2: [ka] Compared to the corresponding polyplex consisting of or containing the same, it exhibits improved loading of nucleic acid cargo, such as RNA, more specifically mRNA, improved cellular transfection, improved intracellular endosomal release, or a combination thereof.

[0063] In some forms, the polymer has the structure of formula II. [ka] [In the formula, J1 and J2 are either independently connected or absent.] R3 and R4 are substituted alkyl groups independently containing a hydroxyl group, a primary amine group, a secondary amine group, a tertiary amine group, or a combination thereof. In some forms, the molecular weight of R3, R4, or both is 500 daltons or less, 200 daltons or less, or 100 daltons or less.

[0064] In some forms, J1 is -O- or -NH-.

[0065] In some forms, J2 is -C(O)NH- or -C(O)O-.

[0066] In some configurations, R3 is identical to R4.

[0067] Preferably, R3 and / or R4 are linear.

[0068] In some forms, R3, R4, or both contain a primary amine group. In some forms, R3, R4, or both contain a primary amine group and one or more secondary or tertiary amine groups.

[0069] In some forms, R3, R4, or both contain a hydroxyl group. In some forms, R3, R4, or both contain a hydroxyl group and one or more amine groups, preferably secondary or tertiary amine groups. In some forms, R3, R4, or both contain a hydroxyl group and no amine group.

[0070] In some forms, at least one of R3 and R4 does not contain a hydroxyl group.

[0071] In some forms, R3, R4, or both are - unsubstituted C1 - C 10 alkylene - Aq - unsubstituted C1 - C 10 alkylene - Bq, - unsubstituted C1 - C 10 alkylene - Aq - substituted C1 - C 10 alkylene - Bq, - substituted C1 - C 10 alkylene - Aq - unsubstituted C1 - C 10 alkylene - Bq, or - substituted C1 - C 10 alkylene - Aq - substituted C1 - C 10 alkylene - Bq (where Aq is absent or - NR5 - and Bq is hydroxyl, primary amine, secondary amine, or tertiary amine, and R5 is hydrogen, substituted or unsubstituted alkyl, or substituted or unsubstituted aryl).

[0072] In some forms, R3, R4, or both are selected from the groups shown in Figure 1.

[0073] In some forms, the polymer has the structure of Formula III. [ka]

[0074] The monomer unit may be substituted with one or more substituents at one or more positions. Exemplary substituents include, but are not limited to, alkyl groups, cyclic alkyl groups, alkene groups, cyclic alkene groups, alkynes, halogens, hydroxyls, carbonyls (e.g., carboxyls, alkoxycarbonyls, formyls, or acyls), thiocarbonyls (e.g., thioesters, thioacetates, or thioformates), alkoxyls, phosphoryls, phosphates, phosphonates, phosphinates, aminos, amides, amidines, imines, cyanos, nitros, azides, sulfhydryls, alkylthios, sulfates, sulfonates, sulfamoyls, sulfonamides, sulfonyls, nitros, heterocyclyls, aralkyls, or aromatic or heteroaromatic moieties.

[0075] The polymer is preferably biocompatible. Lactones readily available in various ring sizes are known to have low toxicity: for example, polyesters prepared from small lactones such as poly(caprolactone) and poly(p-dioxanone) are commercially available biomaterials used in clinical applications. 16 ~C 24 Lactones and their polyester derivatives are natural products identified in organisms such as bees. Lactones containing between 16 and 24 ring carbon atoms are specifically intended and disclosed.

[0076] In some forms, the polymer can be further activated via temperature-controlled hydrolysis, thereby exposing one or more activated end groups. These one or more activated end groups may be, for example, hydroxyl or carboxylic acid end groups, both of which can arise from the hydrolysis of ester bonds within the polymer. The activated polymer may have a weight-average molecular weight of about 5–25 kDa, preferably about 5–10 kDa. As used herein, the term “about” means a small variation within the acceptable parameters. For clarity, “about” refers to ±10% of a given value. In some forms, the activated polymer contains R1 or R2 at one end and a hydroxyl or carboxylic acid end group at the other, arising from hydrolysis.

[0077] In some forms, the polymer has the structure of formula IV. [ka]

[0078] In some forms, the polymer has the structure of formula V. [ka]

[0079] In some forms, the polymer has the structure of formula VI. [ka] [In the formula, X' is either -OH or -NHR']

[0080] Formulas VI, V, and VI represent the structures of intermediate products. They can be used to synthesize a wide variety of polymers having the structures of formulas I, II, or III.

[0081] B.PEG blocking polymer The polymer can be used to deliver drugs in formulations of particles, such as microparticles or nanoparticles, or micelles, which can release, for example, one or more therapeutic, prophylactic, and / or diagnostic agents in a controlled release manner over a desired period of time.

[0082] pH-responsive micelle nanosupports are often formed via the self-assembly of amphiphilic block copolymers, consisting of a hydrophilic (e.g., PEG) outer shell and a hydrophobic inner core capable of responding to the medium pH. Typically, when the medium pH is changed from neutral or slightly basic to weakly acidic, the micelle core undergoes accelerated degradation, becoming completely soluble in water or substantially expanding in an aqueous medium. As a result, micelles encapsulating drugs with slow drug release rates at physiological pH can be triggered by acidic pH to rapidly release (unload) the drug molecules. Polymer segments constituting the micelle core in previous reports include poly(orthoesters), poly(β-aminoesters), poly(L-histidine), and others. The main drawbacks of most previous micelle systems are the need for multiple steps to prepare the copolymers, the difficulty in controlling the polymer molecular weight, and the difficulty in adjusting the polymer composition during copolymer synthesis.

[0083] The copolymers exhibited release rate variations as a function of pH. The in vitro drug release behavior of DTX-encapsulated micelles of PEG2K-PPMS copolymer samples (PEG2K-PPMS-11%PDL, PEG2K-PPMS-30%PDL, and PEG2K-PPMS-51%PDL) was studied in PBS solution at both a physiological pH of 7.4 and an acidic pH of 5.0. Generally, DTX release from all micelle samples followed a two-phase release kinetics and exhibited significant pH dependence. DTX-loaded PEG2K-PPMS copolymer micelles rapidly released 25–45% of the drug during the first 12 hours, followed by a gradual release of another 25–40% over the next 132 hours. The effect of medium pH on drug release rate was substantial. For example, at the end of the incubation period (144 hours), the accumulated DTX values ​​released from the micelles of PEG2K-PPMS-11%PDL, PEG2K-PPMS-30%PDL, and PEG2K-PPMS-51%PDL copolymers were 66%, 60%, and 55% at a physiological pH of 7.4, respectively, and correspondingly at an acidic pH of 5.0. The release rates increase to 85%, 81%, and 75%. The pH-triggered acceleration observed for DTX release from PEG2K-PPMS copolymer micelles is consistent with previous observations that a change in medium pH from 7.4 to 5.0 causes significant swelling of the micelles due to protonation and size increase of the micelle PPMS core. This pH-triggered increase in micelle size clearly facilitates the diffusion and release of captured DTX from the micelle core into the aqueous medium. At a given pH, the DTX release rate is likely controlled by the interaction between the drug and the PPMS matrix in the micelle core. Since PDL-rich PEG2K-PPMS copolymers are expected to form strong hydrophobic domains in their micelle cores to better capture and retain hydrophobic DTX molecules, drug release from such copolymer micelles should be more gradual and sustained. This hypothesis is supported by experimental results showing that at both pH 7.4 and 5.0, the rate of DTX release from PEG2K-PPMS copolymer micelles decreases as the PDL content in the PPMS chain segment of the copolymer increases.

[0084] Upon micelle uptake by tumor cells, the micelle particles are subjected to endosomal capture at a pH range of 5.5–6.0 and to lysosome capture at a pH range of 4.5–5.0. As the results clearly indicate, these acidic environments inevitably trigger rapid DTX release from PEG2K-PPMS copolymer micelles, thereby enhancing the cytotoxicity of drug-loaded micelles. The amino groups in the copolymer act as proton sponges to facilitate endosomal escape. Therefore, the pH-responsive properties exhibited by PEG2K-PPMS copolymer micelles are highly desirable, making them excellent carriers for the delivery of anticancer drugs.

[0085] C. Method for preparing polymers Polymers are generally modified from synthetic polymers. Exemplary synthetic polymers include poly(amine-co-esters) formed from lactones, dialkyl acids, and dialkylamines. Methods for synthesizing poly(amine-co-esters) from lactones, dialkyl acids, and dialkylamines using enzyme catalysts, such as lipase, are also provided. Exemplary lactones are disclosed in U.S. Patent Application Publication No. 20170121454.

[0086] D. Particles formed from polymers Using polymers, microparticles and / or nanoparticles can be prepared in which one or more therapeutic, diagnostic, or preventive agents are encapsulated therein. The agents can be encapsulated within the particles, dispersed within the polymer matrix forming the particles, covalently or acovalently associated on the surface of the particles, or a combination of these methods.

[0087] The release rate can be controlled by altering the monomer composition and / or molecular weight of the polymer, thereby altering the degradation rate. For example, if simple hydrolysis is the primary degradation mechanism, increasing the hydrophobicity of the polymer can slow the degradation rate and therefore increase the release period. In all cases, the polymer composition is selected so that an effective amount of nucleic acid is released to achieve the desired purpose / result.

[0088] E. polyplex and micelles The gene delivery ability of polycationic polymers has been found to depend on several factors, including the polymer's molecular weight, hydrophobicity, and charge density. Many synthetic polycationic materials have been tested as vectors for nonviral gene delivery, but almost all are ineffective due to their low efficiency or high toxicity. Most previously described polycationic vectors exhibit high charge density, which is considered a key requirement for effective DNA condensation. As a result, they deliver genes with high efficiency in vitro. While this is possible, its in vivo applications are limited due to toxicity associated with excessive charge density.

[0089] High molecular weight polymers, particularly terpolymers, have low charge densities. In addition, their hydrophobicity can be altered by selecting lactone comonomers with specific ring sizes and by adjusting the lactone content in the polymer. The high molecular weight and increased hydrophobicity of lactone-diester-aminodiol terpolymers compensate for the low charge density, providing efficient gene delivery with minimal toxicity.

[0090] In preferred embodiments, the terpolymer exhibits efficient gene delivery with reduced toxicity. The terpolymer may be significantly more efficient than commercially available nonviral vectors. For example, based on luciferase expression assays, the terpolymer may be more than 100 times more efficient than commercially available nonviral vectors, such as PEI and LIPOFECTAMINE® 2000, while exhibiting minimal toxicity at toxic doses up to 0.5 mg / ml compared to these commercially available nonviral vectors. Preferably, the terpolymer is in nucleic acid It is nontoxic at concentrations suitable for both in vitro and in vivo transfection. For example, in some embodiments, the terpolymer causes less nonspecific cell death compared to other approaches to cell transfection. A preferred terpolymer is ω-pentadecalactone-diethyl sebacate-N-methyldiethanolamine terpolymer (also known as terpolymer III-20%PDL) containing 20% ​​PDL.

[0091] Micelles can be prepared using polymers, such as PEG-block-containing polymers. The average micelle size is typically in the range of about 100 to about 500 nm, preferably about 100 to about 400 nm, more preferably about 100 to about 300 nm, more preferably about 150 to about 200 nm, and most preferably about 160 to about 190 nm, which were stable at a physiological pH of 7.4 in the presence of serum proteins. The copolymer has high blood compatibility and exhibits minimal activity that induces hemolysis and agglutination.

[0092] The size and zeta potential of the micelles were found to change significantly when the pH of the aqueous medium containing the micelles was altered. For example, the trends of the size-pH and zeta-pH curves are remarkably similar for micelles of three PEG2K-PPMS copolymers with different PDL content (11%, 30%, and 51%). It is evident that the average size of the micelle samples gradually increased as the medium pH was decreased from 7.4 to 5.0, and then remained almost constant when the pH value was below 5.0. This pH-response behavior observed for the micelles is expected when the pH is decreased from 7.4 to 5.0, as the PPMS core of the micelle becomes protonated and more hydrophilic, thus absorbing more water molecules from the aqueous medium and causing the micelle to swell. The micelle core is already sufficiently protonated at pH 5.0, and as a result, the micelle size remains fairly constant even when the pH is further decreased from 5.0. The effect of the PDL content in the PEG2K-PPMS copolymer on the degree of change in micelle size between pH values ​​of 7.4 and 5.0 is also significant. It is expected that reducing the PDL content in the copolymer and increasing the tertiary amino group content will increase the ability of the micelle core to absorb protons and water molecules. Thus, when the pH was reduced from 7.4 to 5.0, the change in average micelle size was more significant for PEG2K-PPMS-11%PDL (200nm to 234nm) compared with PEG2K-PPMS-30%PDL (184nm to 214nm) and PEG2K-PPMS-51%PDL (163nm to 182nm) (Figure 5A).

[0093] The zeta potential of micelles in aqueous media also exhibits substantial pH dependence. At physiological and alkaline pH (7.4-8.5), the zeta potential of blank PEG2K-PPMS copolymer micelles is also pH-dependent. The surface charge was negative, but changed to positive when the medium pH was reduced to the acidic range (4.0–6.0). For example, micelles of PEG2K-PPMS-11%PDL, PEG2K-PPMS-30%PDL, and PEG2K-PPMS-51%PDL had zeta potential values ​​of -5.8, -7.1, and -5.1 mV at pH 7.4, and correspondingly +7.6, +5.8, and +4.0 mV at the lower pH 5.0. Based on the above considerations, this dependence of surface charge on pH is due to the protonation or deprotonation of the PPMS core of the micelles at different medium pHs. At alkaline pH (7.4–8.5), most of the amino groups in the micelles are probably not protonated, and the micelle particles remain negatively charged due to the absorption of HPO42- and / or H2PO4- anions in PBS by the micelles. In particular, at pH 8.5, the zeta potential values ​​were -8.1mV, -7.9mV, and -9.0mV for PEG2K-PPMS-11%PDL, PEG2K-PPMS-30%PDL, and PEG2K-PPMS-51%PDL, respectively. Decreasing the pH from 7.4 to 5.0 resulted in the near-protonation of the tertiary amino portion in the micelle PPMS core, making the micelles positively charged particles. Consistently, among the three micelle samples, the PEG2K-PPMS-11%PDL micelles, possessing the greatest protonation capacity, exhibited the highest zeta potential values ​​at pH 4.0–5.0, while the PEG2K-PPMS-51%PDL micelles, with the least protonation capacity, showed the lowest zeta potential. The observed micelle surface charge response to medium pH suggests that the negative surface charge of micelles at physiological pH is highly desirable because it can reduce the interaction between micelles and serum proteins in the blood, thereby extending their in vivo circulation time. On the other hand, reversing the surface charge to a positive value at an extracellular pH of approximately 6.5 may enhance the uptake of these micelles by target tumor cells.

[0094] The surface charge of particles / micelles is slightly negative in PBS solution (0.01 M, pH=7.4), which is beneficial for the in vivo drug delivery application of micelles. It is known that nanoparticles with a nearly neutral surface charge (zeta potential between -10 and +10 mV) can reduce their uptake by the reticuloendothelial system (RES) and increase their circulation time in the blood. The negative surface charge of micelles allows for hydrogen bonding interactions between the anion and the ether group of the PEG shell or the amino group of the PPMS core, enabling the micelle particles to deliver HPO4 in PBS. 2- and / or H2PO4 - This may be due to anion absorption. In the case of amphiphilic block copolymer micelles, hydrophilic chain segments (e.g., PEG) in the outer shell of the micelle can shield the charge in the micelle core, and long-chain blocks are expected to be more effective than short-chain blocks in reducing the zeta potential. Therefore, significantly lower zeta potential values ​​were observed for PEG5K-PPMS copolymer micelles compared to PEG2K-PPMS copolymer micelles.

[0095] Copolymer micelles are pH-responsive: when the medium pH was lowered from 7.4 to 5.0, micelle size significantly increased, but the surface charge of the micelles reversed from negative to positive. Correspondingly, DTX-encapsulated copolymer micelles showed a gradual and sustained drug release at pH 7.4, but a significantly accelerated DTX release at the acidic pH 5.0. Since it is known that the tumor microenvironment is typically slightly acidic (e.g., 5.7–7.0) as a result of lactate accumulation due to poor oxygen perfusion, this phenomenon can be utilized to improve drug release at the tumor site. In contrast, the extracellular pH of normal tissues and blood is slightly basic (pH 7.2–7.4). Thus, enhanced drug delivery efficiency is predicted for anticancer drug-loaded micelles that are pH-responsive, with acidic pH triggering accelerated drug release. Furthermore, after micelles are taken up by tumor cells via the endocytosis pathway, they encounter even more acidic conditions (pH = 4.0-6.0) in endosomes and lysosomes, which can further increase the cytotoxicity of drug-encapsulated micelles.

[0096] F. Therapeutic agents, prophylactic agents, and diagnostic agents Polymers can be used to encapsulate, mix with, or ionically or covalently couple various therapeutic, prophylactic, or diagnostic agents. A wide variety of bioactive materials can be encapsulated or incorporated.

[0097] Compounds with a wide range of molecular weights (e.g., 100 to 500,000 grams or more per mole) can be encapsulated. In some forms, the encapsulated and delivered drug may be a small molecule drug (i.e., a non-polymeric drug with a molecular weight of less than 2,000 daltons, less than 1,500 daltons, less than 1,000 daltons, less than 750 daltons, or less than 500 daltons) or a polymer (e.g., oligomers or polymers), such as proteins, peptides, nucleic acids, etc. Suitable small molecule active agents include organic, inorganic, and / or organometallic compounds.

[0098] Examples of suitable therapeutic and prophylactic agents include synthetic inorganic and organic compounds, proteins and peptides, polysaccharides and other sugars, lipids, and DNA and RNA nucleic acid sequences having therapeutic, prophylactic, or diagnostic activity. Nucleic acid sequences include genes, antisense molecules that bind to complementary DNA and inhibit transcription, and ribozymes. Examples of suitable materials include proteins, e.g., antibodies, receptor ligands, and enzymes, peptides, e.g., adhesion peptides, sugars and polysaccharides, synthetic organic or inorganic drugs, and nucleic acids. Drugs preferred for delivery are those specific to the treatment of lung diseases or disorders, particularly pulmonary hypertension (PH). In the case of PH, most drugs are vasodilators (e.g., endothelin antagonists, prostacyclin analogs, phosphodiesterase inhibitors), which result in relaxation of smooth muscle cells, and in our view, it is not meaningful to use them in strategies targeting lung macrophages. Drugs targeting the immune system in PH appear to be underutilized. In the case of COPD, similarly inhaled bronchodilators cause relaxation of the airway smear chain (SMC), but appropriate corticosteroids can be used.

[0099] The results demonstrate remarkable selectivity for delivery to and uptake by lung immune cells, making this delivery system particularly well-suited for local delivery to the lungs of antiviral agents, such as those involved in the treatment of viral diseases like COVID-19, diseases like pulmonary fibrosis, and lung cancer. Similarly, it has clear advantages with respect to the delivery of immunomodulatory agents for the treatment of chronic obstructive pulmonary disease (COPD).

[0100] Examples of therapeutic agents that can be incorporated into particles include, but are not limited to, immunomodulators, anti-infective agents (including antivirals or antibiotics), chemotherapeutic agents, monoclonal antibodies or fragments thereof or humanized versions thereof, enzymes, growth factors, growth inhibitors, hormones, hormone antagonists, and nucleic acid molecules.

[0101] Immunomodulators include anti-inflammatory agents, ligands that bind to Toll-like receptors and activate the innate immune system, molecules that mobilize and optimize the adaptive immune system, molecules that activate or upmodulate the action of cytotoxic T lymphocytes, natural killer cells and helper T cells, as well as molecules that deactivate or downmodulate suppressor or regulatory T cells, and agents that promote the uptake of particles into cells, including dendritic cells and other antigen-presenting cells. Exemplary immunomodulators include cytokines, xanthines, interleukins, interferons, oligodeoxynucleotides, glucans, growth factors (e.g., TNF, CSF, GM-CSF, and G-CSF), hormones (e.g., estrogens (diethylstilbestrol, estradiol), androgens (testosterone, HALOTESTIN® (fluoxymesterone)), progestins (MEGACE® (megestrol acetate), PROVERA® (medroxyprogesterone acetate)), and cosine. Examples include luticosteroids (prednisone, dexamethasone, hydrocortisone).

[0102] Oligonucleotide drugs include DNA, RNA, antisense, aptamers, small interfering RNAs, ribozymes, external guide sequences for ribonuclease P, and triple-stranding agents.

[0103] Typical chemotherapy agents include alkylating agents (e.g., cisplatin, carboplatin, oxaliplatin, mechloretamine, cyclophosphamide, chlorambucil, dacarbazine, lomustine, carmustine, procarbazine, chlorambucil, and ifosfamide), antimetabolites (e.g., fluorouracil (5-FU), gemcitabine, methotrexate, cytosine arabinoside, fludarabine, and phloxuridine), mitotic inhibitors (taxanes, e.g., paclitaxel and docetaxel), and Vinca alkaloids (including vincristine, vinblastine, vinorelbine, and vindesine), anthracyclines (including doxorubicin, daunorubicin, barurubicin, idarubicin, and epirubicin, and actinomycins, including actinomycin D), cytotoxic antibiotics (including mitomycin, plicamycin, and bleomycin), topoisomerase inhibitors (including camptothecin, including camptothecin, irinotecan, and topotecan, and derivatives of epipodophyllotoxin, including amsacrin, etoposide, phosphate etoposide, and teniposide), antibodies against vascular endothelial growth factor (VEGF), such as bevacizumab (AVASTIN®), Other anti-VEGF compounds include thalidomide (THALOMID®) and its derivatives, e.g., lenalidomide (REVLIMID®); endostatins; angiostatins; receptor tyrosine kinase (RTK) inhibitors, e.g., sunitinib (SUTENT®); tyrosine kinase inhibitors, e.g., sorafenib (Nexavar®), erlotinib (Tarceva®), pazopanib, axitinib, and lapatinib; transforming growth factor-α or transforming growth factor-β inhibitors, as well as antibodies against epidermal growth factor receptors, e.g., panitumumab (VECTIBIX®) and cetuximab (ERBITUX®).

[0104] Examples of immunological adjuvants that can associate with particles include, but are not limited to, TLR ligands, type C lectin receptor ligands, NOD-like receptor ligands, RLR ligands, and RAGE ligands. Examples of TLR ligands include lipopolysaccharides (LPS) and their derivatives, as well as lipid A and its derivatives, such as, but are not limited to, monophosphoryl lipid A (MPL), glycopyranosyl lipid A, PET-lipid A, and 3-O-desacyl-4'-monophosphoryl lipid A.

[0105] The particles may also contain antigens and / or adjuvants (i.e., molecules that enhance the immune response). Peptide, protein, and DNA-based vaccines may be used to induce immunity against a variety of diseases or conditions. Cell-mediated immunity is necessary to detect and destroy virus-infected cells. Most conventional vaccines (e.g., protein-based vaccines) can only induce humoral immunity. DNA-based vaccines represent a unique means of vaccination against viruses or parasites, as they can induce both humoral and cell-mediated immunity. DNA vaccines consist of two main components: a DNA carrier (or delivery vehicle) and DNA encoding an antigen. The DNA carrier can protect the DNA from degradation and facilitate the entry of the DNA into specific tissues or cells and its expression at efficient levels.

[0106] Typical diagnostic methods include X-ray, fluorescence, magnetic resonance imaging, radioactivity, ultrasound, and computer Examples of drugs detectable by CT and positron emission tomography (PET) include ultrasound contrast agents, which are typically gases such as air, oxygen, or perfluorocarbons. Exemplary diagnostic agents include paramagnetic molecules, fluorescent compounds, magnetic molecules, and radionuclides, as well as X-ray contrast agents.

[0107] In some embodiments, particles produced using the methods described herein contain less than 80%, less than 75%, less than 70%, less than 60%, less than 50% by weight, less than 40% by weight, less than 30% by weight, less than 20% by weight, less than 15% by weight, less than 10% by weight, less than 5% by weight, less than 1% by weight, less than 0.5% by weight, or less than 0.1% by weight of the agent. In some embodiments, the agent may be a mixture of pharmaceutically active agents. The loading percentage depends on a variety of factors, including the agent to be encapsulated, the polymer used to prepare the particles, and the method used to prepare the particles.

[0108] Polynucleotides Polymer particles can be used to transfect cells with nucleic acids. Polynucleotides can encode one or more proteins, functional nucleic acids, or combinations thereof. Polynucleotides can be monocistronic or polycistronic. In some embodiments, polynucleotides are polygenic.

[0109] In some embodiments, the polynucleotides are transfected into cells and remain extrachromosomal. In some embodiments, the polynucleotides are introduced into host cells and integrated into the host cell's genome.

[0110] In some embodiments, polynucleotides are incorporated into or as part of a vector. Methods for constructing expression vectors containing gene sequences and appropriate transcription and translation regulatory elements are well known in the art. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Expression vectors generally contain regulatory sequences and elements necessary for the translation and / or transcription of an inserted coding sequence, which may be, for example, a polynucleotide of interest. The coding sequence may be operably linked to promoters and / or enhancers that help control the expression of the desired gene product. Promoters used in biotechnology are of different types depending on the type of gene expression control intended. They can generally be divided into constitutive promoters, tissue-specific or developmental stage-specific promoters, inductive promoters, and synthetic promoters.

[0111] Several virus-based expression systems can be used; for example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, and simian virus 40 (SV40). Early and late promoters of SV40 virus are useful because they can be readily obtained from the virus as fragments that also contain the SV40 virus origin of replication. Smaller or larger SV40 fragments can also be used, provided they contain a sequence of approximately 250 bp extending from the HindIII site toward the BglI site located at the viral origin of replication.

[0112] Specific start signals may also be necessary for the efficient translation of the composition. These signals include the ATG start codon and adjacent sequences. Further exogenous translational regulatory signals, including the ATG start codon, may be required. In eukaryotic expression, it would typically be desirable to incorporate a suitable polyadenylation site into the transcription unit if the signal was not contained within the original cloned segment. Typically, the poly-A addition site is located approximately 30–2000 nucleotides "downstream" of the protein's termination site at a pre-transcriptional position.

[0113] For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, a cell line that stably expresses the protein-coding construct can be engineered. Rather than using an expression vector containing a viral replication origin, host cells can be transformed with a vector controlled by appropriate expression regulatory elements (e.g., promoter, enhancer, sequence, transcription terminator, polyadenylation site, etc.) and a selection marker. After introducing exogenous DNA, the engineered cells may be grown in enriched medium for 1-2 days, and then switched to a selection medium. The selection marker in the recombinant plasmid confers resistance to selection, allowing cells to stably incorporate the plasmid into their chromosomes, grow, and form a focus, which can then be cloned and expanded into a cell line. ru.

[0114] In a preferred embodiment, the polynucleotide cargo is RNA, such as mRNA. The mRNA can encode the polypeptide of interest.

[0115] In some embodiments, the mRNA has a cap and / or a 3' poly(A) tail at its 5' end, which can modulate ribosome binding, translation initiation, and mRNA stability in cells.

[0116] A polynucleotide can encode one or more polynucleotides of interest.

[0117] In some embodiments, polynucleotides replenish or replace defective polynucleotides in organisms.

[0118] In some embodiments, the polynucleotide includes a selection marker, such as a selection marker effective in eukaryotic cells, such as a drug resistance selection marker. In some embodiments, the polynucleotide includes a reporter gene.

[0119] Polynucleotides can be or encode functional nucleic acids. Functional nucleic acids are nucleic acid molecules that have a specific function, such as binding to a target molecule or catalyzing a particular reaction. Functional nucleic acid molecules can be divided into the following non-restrictive categories: antisense molecules, siRNA, miRNA, aptamers, ribozymes, triple-stranding molecules, RNAi, and external guide sequences. Functional nucleic acid molecules can act as effectors, inhibitors, modulators, and stimulants of specific activities possessed by target molecules, or functional nucleic acid molecules may possess de novo activity independently of any other molecule.

[0120] Functional nucleic acid molecules can interact with any macromolecule, such as DNA, RNA, polypeptides, or carbohydrate chains. Therefore, functional nucleic acids can interact with the mRNA or genomic DNA of a target polypeptide, or they can interact with the polypeptide itself. In many cases, functional nucleic acids are designed to interact with other nucleic acids based on sequence homology between the target molecule and the functional nucleic acid molecule. In other situations, specific recognition between a functional nucleic acid molecule and a target molecule is not based on sequence homology between the two molecules, but rather on the formation of a tertiary structure that allows specific recognition to occur.

[0121] Antisense molecules are designed to interact with target nucleic acid molecules through either standard or atypical base pairing. The interaction between the antisense molecule and the target molecule is designed to promote the destruction of the target molecule, for example, by RNAseH-mediated RNA-DNA hybrid degradation. Alternatively, the antisense molecule may interact with the target molecule through transcription or replication. Antisense molecules are designed to interrupt processing functions that would normally occur. Antisense molecules can be designed based on the sequence of the target molecule. There are many methods for optimizing antisense efficiency by finding the most accessible region of the target molecule. Exemplary methods include in vitro selection experiments, as well as DNA modification studies using DMS and DEPC. Antisense molecules are designed to interrupt processing functions that would normally occur. -6 , 10 -8 , 10 -10 , or 10 -12 Dissociation constant (K) less than or equal to (K) d It is preferable that the target molecule is bound to the target molecule.

[0122] Aptamers are molecules that interact with target molecules, preferably in a specific manner. Typically, aptamers are small nucleic acids ranging from 15 to 50 nucleotides in length that fold into defined secondary and tertiary structures such as stem-loops or G quartets. Aptamers can bind to small molecules such as ATP and theophylline, as well as larger molecules such as reverse transcriptase and thrombin. -12 K from target molecules with a M or less d It can bond very strongly. The aptamer is 10 -6 , 10 -8 , 10 -10 , or 10 -12 K less than d It is preferable for the aptamer to bind to the target molecule. The aptamer can bind to the target molecule with very high specificity. For example, aptamers have been isolated that have a difference of more than 10,000 times in binding affinity between the target molecule and another molecule that differs only at a single position on the molecule. The aptamer binds to the background binding molecule via K d The K in the target molecule is at most 1 / 10, 1 / 100, 1 / 10000, or 1 / 1000000. d It is preferable that the background molecules are different polypeptides. When comparing molecules such as polypeptides, it is preferable that the background molecules are different polypeptides.

[0123] Ribozymes are nucleic acid molecules that can catalyze either intramolecular or intermolecular chemical reactions. Ribozymes are preferably those that catalyze intermolecular reactions. There are several different types of ribozymes that catalyze nuclease or nucleic acid polymerase-type reactions based on ribozymes found in nature, such as hammerhead ribozymes. There are also some ribozymes that are not found in nature but have been engineered to catalyze specific reactions de novo. Preferred ribozymes cleave RNA or DNA substrates, and more preferably RNA substrates. Ribozymes typically cleave nucleic acid substrates by recognition and binding to a target substrate and subsequent cleavage. This recognition is often primarily based on the interaction of standard or non-standard base pairs. Because target substrate recognition is based on the target substrate sequence, this property makes ribozymes particularly good candidates for target-specific cleavage of nucleic acids.

[0124] Triple-helix-forming functional nucleic acid molecules are molecules that can interact with either double-stranded or single-stranded nucleic acids. When a triple-helix molecule interacts with a target region, a structure called a triple helix is ​​formed, which contains three strands of DNA that form a complex dependent on both Watson-Crick and Hoogsteen base pairing. Triple-helix molecules are preferred because they can bind to target regions with high affinity and specificity. -6 , 10 -8 , 10 -10 , or 10 -12 K less than d It is preferable that it binds to the target molecule.

[0125] An external guide sequence (EGS) is a molecule that binds to a target nucleic acid molecule to form a complex, which is then recognized by RNase P and subsequently cleaves the target molecule. EGS can be designed to specifically target a selected RNA molecule. RNAse P assists in the processing of transfer RNA (tRNA) within cells. Bacterial RNAse P can be recruited to cleave virtually any RNA sequence by using an EGS that mimics a native tRNA substrate in the target RNA:EGS complex. Similarly, the targeted cleavage of RNA by eukaryotic EGS / RNAse P can be used to cleave desired targets within eukaryotic cells. EGS molecules facilitate the cleavage of various different target molecules. Typical examples of methods for producing and using are known in the art.

[0126] Gene expression can also be effectively silenced in a highly specific manner by RNA interference (RNAi). This silencing was originally observed by adding double-stranded RNA (dsRNA) (Fire, et al. (1998) Nature, 391:806-11; Napoli, et al. (1990) Plant Cell 2:279-89; Hannon, (2002) Nature, 418:244-51). When dsRNA enters a cell, it is cleaved by the RNase III-like enzyme Dicer into 21-23 nucleotide long double-stranded small interfering RNA (siRNA) containing two nucleotide overhangs at its 3' end (Elbashir, et al. (2001) Genes Dev., 15:188-200; Bernstein, et al. (2001) Nature, 409:363-6; Hammond, et al. (2000) Nature, 404:293-6). In the ATP-dependent step, the siRNA is incorporated into a multi-subunit protein complex commonly known as the RNAi-induced silencing complex (RISC), which guides the siRNA to the target RNA sequence (Nykanen, et al. (2001) Cell, 107:309-21). At some point, the siRNA double helix unwinds, and the antisense strand remains bound to RISC, which is thought to direct the degradation of the complementary mRNA sequence by a combination of endonucleases and exonucleases (Martinez, et al. (2002) Cell, 110:563-74). However, iRNA or siRNA The effects or their uses are not limited to any type of mechanism.

[0127] Short interfering RNAs (siRNAs) are double-stranded RNAs that can induce sequence-specific post-transcriptional gene silencing, thereby reducing or further inhibiting gene expression. For example, an siRNA can trigger the specific degradation of homologous RNA molecules, such as mRNA, within a region of sequence identity between the siRNA and the target RNA. For instance, WO02 / 44321 discloses siRNAs capable of sequence-specific degradation of target mRNA upon base pairing with the 3' overhang end, and methods for constructing these siRNAs are incorporated herein by reference. Sequence-specific gene silencing can be achieved in mammalian cells using synthetic short double-stranded RNAs that mimic siRNA produced by enzyme dicers (Elbashir, et al. (2001) Nature, 411:494-498) (Ui-Tei, et al. (2000) FEBS Lett 479:79-82). siRNA can be synthesized chemically or in vitro, or may result from short double-stranded hairpin-like RNA (shRNA) that is processed into siRNA inside cells. Synthetic siRNA is generally designed using algorithms and conventional DNA / RNA synthesizers. Suppliers include Ambion (Austin, Texas), ChemGenes (Ashland, Massachusetts), Dharmacon (Lafayette, Colorado), Glen Research (Sterling, Virginia), MWB Biotech (Esbersberg, Germany), Proligo (Boulder, Colorado), and Qiagen (Vento, The Netherlands). siRNA can be synthesized in vitro using kits such as Ambion's SILENCER® siRNA construction kit. It can also be synthesized at a microscope.

[0128] siRNA production from vectors is more commonly carried out by the transcription of short hairpin RNAse (shRNA). Kits for generating shRNA-containing vectors are available, such as Imgenex's GENESUPPRESSOR™ construction kit and Invitrogen's BLOCK-IT™ inducible RNAi plasmid and lentiviral vector.

[0129] Polynucleotides are typically DNA or RNA nucleotides that consist of a heterocyclic base (nucleic acid base), a sugar moiety attached to the heterocyclic base, and a phosphate moiety that esterifies the hydroxyl function of the sugar moiety. The major naturally occurring nucleotides are heterocyclic salts. The material contains uracil, thymine, cytosine, adenine, and guanine as bases, as well as ribose or deoxyribose sugars linked by phosphodiester bonds.

[0130] Polynucleotides may consist of nucleotide analogs that have been chemically modified to improve stability, half-life, or specificity or affinity to a target sequence compared to their DNA or RNA counterparts. Chemical modifications include modifications to nucleic acid bases, sugar moieties, nucleotide linkages, or combinations thereof. As used herein, “modified nucleotide” or “chemically modified nucleotide” defines a nucleotide having one or more chemical modifications to heterocyclic bases, sugar moieties, or phosphate moiety components. In some embodiments, the charge of the modified nucleotide is reduced compared to DNA or RNA oligonucleotides of the same nucleic acid base sequence. For example, oligonucleotides may have a low negative charge, no charge, or a positive charge. Modifications should not impede, but preferably enhance, the oligonucleotide’s ability to enter cells and perform functions such as inhibiting gene expression as discussed above.

[0131] Typically, nucleoside analogs support bases capable of hydrogen bonding via Watson-Crick base pairing to standard polynucleotide bases, where the analog's backbone presents the bases in a sequence-specific manner, enabling such hydrogen bonding between the oligonucleotide analog molecule and the bases in a standard polynucleotide (e.g., single-stranded RNA or single-stranded DNA). Preferred analogs are those having a substantially uncharged, phosphorus-containing backbone.

[0132] The efficiency of polynucleotide delivery using polymers can be affected by the positive charge on the polyplex surface. For example, a polyplex zeta potential of +8.9 mV may attract and bind to negatively charged plasma proteins in the blood during circulation, leading to rapid clearance by the reticuloendothelial system (RES). Efficiency can also be affected by the instability of polyplex nanoparticles. For example, as discussed in the examples below, polyplex particles incubated in NaAc buffer containing 10% serum nearly doubled in size within 15 minutes and increased more than tenfold after 75 minutes. As a result of this size increase, the enlarged polyplex may be removed from circulation by uptake in the liver. Therefore, in some embodiments, polyplexes are treated or coated to improve polynucleotide delivery efficiency. In some embodiments, coating improves the cell-specific targeting of the polyplex, improves stability (i.e., stabilizes the size of the polyplex in vivo), increases the half-life of the polyplex in vivo (i.e., in systemic circulation), or a combination thereof, compared to a control. In some embodiments, the control is uncoated polyplex.

[0133] An exemplary polyplex coating for targeting tumor cells is polyE-mRGD. As used herein, polyE-mRGD refers to a synthetic peptide containing three segments: a first segment containing a polyglutamic acid (polyE) stretch that is negatively charged at physiological pH and therefore can electrostatically bond to the positively charged surface of a polyplex; a second segment containing a neutral polyglycine stretch that functions as a neutral linker; and RGD and α v β3 and α v A third segment containing an RGD sequence that binds to tumor endothelium through interaction with β5.

[0134] The polynucleotide delivery efficiency of polyplexes can be improved by coating the particles with a drug that is negatively charged at physiological pH. Preferably, the negatively charged drug can electrostatically bond to the positively charged surface of the polyplex. The negatively charged drug neutralizes the charge of the polyplex or reverses the charge of the polyplex. This is possible. Therefore, in some embodiments, a negatively charged agent imparts a net negative charge to the polyplex.

[0135] In some embodiments, the negatively charged agent is a negatively charged polypeptide. For example, the polypeptide may include aspartic acid, glutamic acid, or a combination thereof, such that the total charge of the polypeptide is negative at neutral pH. Increasing the negative charge on the particle surface can reduce or prevent the negative interactions described above, and more positively charged particles attract and bind to negatively charged plasma proteins in the circulating blood, resulting in rapid clearance by the reticuloendothelial system (RES). In some embodiments, the zeta potential of the particles is about -15 mV to about 10 mV, preferably about -15 mV to about 8 mV, more preferably about -10 mV to about 8 mV, and more preferably about -8 mV to about 8 mV. The zeta potential may be even more negative or even more positive than the above range under conditions where the particles are stable (i.e., do not aggregate, etc.) and are not easily removed from the bloodstream. The zeta potential can be manipulated by coating or functionalizing the particle surface with one or more parts that alter the surface charge. Alternatively, the monomers themselves can be functionalized, and / or additional monomers that alter the surface charge can be introduced into the polymer.

[0136] Resistance to aggregation can be important because maintaining a small particle size limits clearance by the liver and maintains the transfection ability of polyplex particles to target cells. Therefore, in preferred embodiments, the polyplex is resistant to aggregation. Preferably, the polyplex, with or without coating, has a radius of about 1 nm to 1000 nm, more preferably about 1 nm to about 500 nm, and most preferably about 15 nm to about 250 nm. For example, in some embodiments, polynucleotide-loaded coated polyplex has a radius of about 150 nm to 275 nm.

[0137] The size of the polyplex can be adjusted by using the ratio of polynucleotide weight to polymer weight (polynucleotide:polymer), the content and amount of polyplex coating, or a combination thereof.

[0138] G. Preparations The formulation is prepared using a pharmaceutically acceptable “carrier” composed of materials considered safe and effective, and can be administered to an individual without causing undesirable biological side effects or unwanted interactions. The “carrier” is all components present in the pharmaceutical formulation other than the active ingredient. The term “carrier” includes, but is not limited to, diluents, binders, lubricants, disintegrants, fillers, and coating compositions. For detailed information on materials, equipment, and processes for preparing tablets and delayed-release dosage forms, see Pharmaceutical Dosage Forms: Tablets, eds. Lieberman et al. (New York: Marcel Dekker, Inc., 1989) and Ansel et al., Pharmaceutical See Dosage Forms and Drug Delivery Systems, 6th sup.th Ed. (Media, PA: Williams & Wilkins, 1995).

[0139] Preferred formulations for pulmonary delivery are aerosols, inhalers, dry powders, pharmaceutically acceptable carriers for administration by intubation and intravenous infusion.

[0140] III. Method for preparing particles or polyplexes The particles can be prepared using a variety of techniques known in the art. The techniques used may depend on a variety of factors, including the polymer used to form the nanoparticles, the desired size range of the resulting particles, and the suitability of the material to be encapsulated.

[0141] Methods known in the art that can be used to prepare nanoparticles include, but are not limited to, polyelectrolyte condensation (see Suk et al., Biomaterials, 27, 5143-5150 (2006)); single and double emulsions; nanoparticle molding; and electrostatic self-assembly (e.g., polyethyleneimine-DNA or liposomes).

[0142] In one embodiment, the loaded particles are typically prepared by mixing a solution of the polymer in an organic solvent with the polynucleotide of the choice. The polymer solution is prepared by dissolving or suspending the polymer in a solvent. The solvent must be selected so as not to adversely affect the nucleic acid being encapsulated (e.g., not destabilizing or degrading it). Suitable solvents include, but are not limited to, DMSO and methylene chloride. The concentration of the polymer in the solvent can be varied as needed. In some embodiments, the concentration is, for example, 25 mg / ml. The polymer solution can also be diluted in a buffer, such as sodium acetate buffer.

[0143] Next, the polymer solution is mixed with the encapsulation agent, such as a polynucleotide. The agent can be dissolved in a solvent to form a solution before being mixed with the polymer solution. In some embodiments, the agent is dissolved in a physiological buffer before being mixed with the polymer solution. The ratio of the polymer solution volume to the agent solution volume may be 1:1. The polymer and agent combination is typically incubated for several minutes before the solution is used for its desired purpose, such as transfection, to form particles. For example, a polymer / polynucleotide solution may be incubated for 2, 5, 10, or longer than 10 minutes before the solution is used for transfection. Incubation may be at room temperature.

[0144] In some embodiments, the particles are also incubated with a solution containing a coating agent before use. The particle solution can be incubated with the coating agent for 2, 5, 10, or longer than 10 minutes before using Polyplex for transfection. Incubation may be at room temperature.

[0145] In some embodiments, if the drug is a polynucleotide, the polynucleotide is first complexed with a polycation before being mixed with the polymer. Complexation can be achieved by mixing the polynucleotide and polycation in an appropriate molar ratio. When a polyamine is used as the polycation species, it is useful to determine the molar ratio (N / P ratio) of polyamine nitrogen to polynucleotide phosphate. In preferred embodiments, the inhibitory RNA and polyamine are mixed together in an N / P ratio of approximately 1:1 to 1:25, preferably about 8:1 to 15:1, to form a complex. The volume of polyamine solution required to achieve a particular molar ratio is given by the following formula:

number

[0146] The term "polycation" refers to a positively charged compound at a selected pH, preferably physiological pH. A polycation preferably refers to a compound having at least two positive charges. The polycationic moiety has a positive charge of about 2 to about 15, preferably about 2 to about 12, and more preferably about 2 to about 8, at a selected pH value. Many polycations are known in the art. Suitable components of polycations include basic amino acids and their derivatives, e.g., arginine, asparagine, glutamine, lysine, and histidine; cationic dendrimers; and aminopolysaccharides. Suitable polycations may have linear structures, e.g., linear tetralysine, branched, or dendrimer structures.

[0147] Examples of polycations include, but are not limited to, synthetic polycations based on acrylamide and 2-acrylamide-2-methylpropanetrimethylamine, poly(N-ethyl-4-vinylpyridine) or similar quaternized polypyridines, diethylaminoethyl polymers and dextran conjugates, polymyxin B sulfate, lipopolyamines, poly(allylamines), for example, the strong polycation poly(dimethyldiallylammonium chloride), polyethyleneimines, polyblens, and polypeptides, for example, protamines, histone polypeptides, polylysine, polyarginine, and polyornithine.

[0148] In some embodiments, the polycation is a polyamine. A polyamine is a compound having two or more primary amine groups. Suitable naturally occurring polyamines include, but are not limited to, spermine, spermidine, cadaverine, and putrescine. In preferred embodiments, the polyamine is spermidine.

[0149] In another embodiment, the polycation is a cyclic polyamine. Cyclic polyamines are known in the art and are described, for example, in U.S. Patent No. 5,698,546, WO1993 / 012096, and WO2002 / 010142. Examples of cyclic polyamines include, but are not limited to, cyclenes.

[0150] Spermine and spermidine are derivatives of putrescine (1,4-diaminobutane), produced from L-ornithine by the action of ODC (ornithine decarboxylase). L-ornithine is the product of L-arginine degradation by arginase. Spermidine is a triamine structure produced by spermidine synthase (SpdS), which catalyzes the monoalkylation of putrescine (1,4-diaminobutane) with the decarboxylation S-adenosylmethionine (dcAdoMet) 3-aminopropyl donor. Formal alkylation of both amino groups of putrescine with the 3-aminopropyl donor yields symmetrical tetraaminespermine. Spermine biosynthesis proceeds to spermidine by the action of spermine synthase (SpmS) in the presence of dcAdoMet. The 3-aminopropyl donor (dcAdoMet) is derived from S-adenosylmethionine by the sequential conversion of L-methionine by methionine adenosyltransferase, followed by decarboxylation by AdoMetDC (S-adenosylmethionine decarboxylase). Therefore, putrescine, spermidine, and spermine are metabolites derived from the amino acids L-arginine (L-ornithine, putrescine) and L-methionine (dcAdoMet, aminopropyl donor).

[0151] IV. Methods using particles / micelles Using particles, an effective amount of one or more therapeutic, diagnostic, and / or prophylactic agents can be delivered to a patient requiring such treatment. The amount of drug administered can be easily determined by the prescribing physician and depends on the patient's age and weight, as well as the disease or disorder being treated.

[0152] The composition is administered to the target lung in a therapeutically effective dose. When used herein, "effective dose" refers to a therapeutically effective dose. The term "therapeutic dose" or "therapeutic dose" means a dose sufficient to treat, inhibit, or alleviate one or more symptoms of the disorder being treated, or otherwise sufficient to provide the desired pharmacological and / or physiological effect. The exact dose varies depending on various factors, including subject-dependent variables (e.g., age, immune system health), the disease, and the treatment being administered. [Examples]

[0153] The present invention can be further understood by referring to the following non-limiting examples demonstrating a method for selectively treating one or more symptoms of pulmonary hypertension by selectively targeting platelet-derived growth factor inhibitors using PACE nanoparticles.

[0154] Pathological characteristics of pulmonary hypertension ("PH"): Distal pulmonary arteriole muscular arterialization elevated pulmonary artery blood pressure Right ventricular hypertrophy (RVH)

[0155] Platelet-derived growth factor (PDGF)-B from endothelial cells is important for the pathogenesis of pulmonary hypertension (PH), but the role of lung macrophages and macrophage-derived PDGF-B in PH is not fully understood.

[0156] The following study demonstrates that lung macrophage-derived PDGF-β plays a crucial role in pathological SMC enlargement in PH, and that PDGF-β inhibitors can be selectively delivered to lung macrophages and monocytes for treatment.

[0157] (Example 1) Alveolar and parenchymal macrophages accumulate in hypoxic conditions, and their depletion leads to distal muscular arterialization and reduced pH. A pH model was created by exposing wild-type or transgenic mice to hypoxia for up to 21 days. Measurements and analyses were performed on lung tissue, BALF cells, and the heart. Furthermore, fresh whole blood from human patients was studied, primary monocytes were isolated and differentiated into macrophages, and the RNA content in these cells was analyzed, as well as the effect of acclimatization media from such cultures on SMC migration and proliferation.

[0158] Methods and materials animal research Mice were obtained from Jackson Laboratory. C57BL / 6 mice were used for wild-type studies, and the mice were LysM-Cre (Clausen Transgenic Res. 1999;8(4):265-77; Cowburn. Proc Natl Acad Sci US A. 2016;113(31):8801-6), ROSA26R (mTmG / mTmG) (Muzumdar MD, Tasic B, Miyamichi K, Li L, and Luo L. A global double-fluorescent Cre reporter mouse. Genesis. 2007;45(9):593-605), PDGF-β (flox / flox) (Enge M, et al. EMBO J. 2002;21(16):4307-16), Vhl (flox / flox) (Haase Proc Natl Acad Sci US A. 2001;98(4):1583-8), Hif1a (flox / flo x) (Ryan. Cancer Res. 2000;60(15):4010-5), or Hif2a (flox / f lox) (Gruber, Proc Natl Acad Sci US A. 2007;104(7):2301-6) was used. Male and female mice aged 10-16 weeks, as well as sex and age-matched controls, were used.

[0159] Hypoxia exposure and hemodynamic measurements Mice were subjected to hypoxic (10% FiO2) conditions with a controller and oxygen sensor. Mice were placed in a BioSpherix® chamber for up to 21 days. After hypoxia treatment, RVSP was measured. Next, the mice were euthanized by isoflurane inhalation, and in addition to lung collection, the heart was collected to determine the Fulton index, which is the weight ratio of RV to the sum of LV and septum (S) (Sheikh Cell Rep. 2014;6(5):809-17). Technicians performing hemodynamic measurements were blinded to the treatment group and the genotype of the mice.

[0160] Bronchoalveolar lavage fluid and lung tissue collection After euthanasia, PBS was perfused into the lungs via RV. For analysis of the entire lung, both the left and right lungs were collected directly after perfusion. For BALF collection, 1 ml of PBS was injected into the alveoli through the trachea, and then aspirated from the trachea. This procedure was repeated once, and the collected BALF was pooled. The BALF was centrifuged at 830 g (GS-6R centrifuge, Beckman Coulter) at 4°C for 10 minutes, and the cell pellet was collected. For FACS experiments on the remaining lung after BALF removal, the right main bronchus was ligated, and the right lung was excised. For immunohistochemistry, the left lung was inflated with 2% low-melting-point agarose and placed in ice-cold PBS. Once the agarose solidified, the left lung was immersed overnight in Dent fixative (4:1 methanol:DMSO) at 4°C, washed the following day, and stored in 100% methanol at -80°C.

[0161] Nanoparticle formulations and administration Nanoparticles were administered orally and intratracheally to wild-type mice. Clodronate or PDGF-β siRNA-loaded nanoparticles were administered at the start of hypoxia and every three days thereafter for up to 21 days of hypoxia. Mice administered with dye-loaded nanoparticles were maintained under normal oxygen conditions for 6 hours and then euthanized. To deplete phagocytic cells, 0.25 mg clodronate or PBS was loaded, and 50 μL of liposomes dissolved in PBS (Liposoma Research) was injected. For the evaluation of nanoparticle uptake or PDGF-β knockdown, PACE nanoparticles composed of poly(pentadecalactone-co-n-methyldiethanolamine co-sebacate) with an acid terminus along with 50% lactone (PPMS-50COOH) were formulated using modified single emulsion or double emulsion solvent evaporation techniques (Kauffman Biomacromolecules. 2018;19(9):3861-73). Briefly, in dye-loaded nanoparticle formulations (approximately 200 or 400 nm in diameter), 0.2 wt% DiD (ThermoFisher) relative to the polymer was used. DMSO (10 μL of 10 mg / mL solution) was dissolved in 50 mg of the polymer immediately before single emulsion formulation. PDGF-β For nanoparticles loaded with siRNA and scrambled (Scr)RNA, the nucleic acid cargo (Dharmacon, 50 nM) was dissolved in sodium acetate buffer (25 mM, pH 5.8) and then subjected to a double emulsion assay. Nanoparticle parameters (stratified by siPDGF-β or Scr loading) were assayed, including hydrodynamic diameter (404 ± 8 or 386 ± 7 nm), size distribution (PDI; 0.218 ± 0.004 or 0.238 ± 0.007) and zeta potential (9.4 ± 0.3 or 10.8 ± 0.5 mV) using dynamic light scattering (Zetasizer Pro, Malvern Panalytical), and siRNA loading efficiency (69.6 ± 1.2 or 64.3 ± 0.5%) using the QuantIT RiboGreen assay (ThermoFisher). Nanoparticles (0.2 mg) were suspended in 50 μL of PBS and administered to mice.To confirm the uptake of nanoparticles by macrophages in the culture, BALF cell pellets were resuspended in mouse cell culture medium (RPMI [Thermo Scientific], 10% fetal bovine serum [FBS; Invitrogen], 5% penicillin / streptomycin [Life Technologies]) and incubated with nanoparticles loaded with 0.25 mg / ml DiD at 37°C for 6 hours.

[0162] immunohistochemistry Regarding immunohistochemical analysis, the left lung, stored in 100% methanol, was subjected to 5% H2O2 / After peroxidase deactivation by incubation in methanol at RT for 15 minutes, the lungs were sequentially re-humidified in PBS with 75%, 50%, 25%, and 0% methanol. The re-humidified lungs were sectioned into 150 μm thick sections using a vibratome, incubated overnight at 4°C in IHC blocking buffer (5% goat serum in 0.5% Triton® X-100 / PBS [PBS-T]), and then stained with primary antibody in IHC blocking buffer for 3 days at 4°C. The sections were then washed three times in PBS-T, incubated overnight at 4°C with secondary antibody in IHC blocking buffer, washed five times in PBS-T, mounted on glass slides using Dako mounting medium, and stored at 4°C. The primary antibodies used were rat anti-MECA-32 (1:15, Developmental Studies Hybridoma Bank [DSHB]), rat anti-CD31-FITC (1:250, BD Biosciences), mouse anti-CD64-APC (1:250, Biolegend), rat anti-CD68-APC (1:50, Miltenyi Biotec), and mouse anti-SMA Cy3 clone 1A4 (1:250, Sigma). The secondary antibody used was Alexa488 anti-rat (1:250, Invitrogen). Nuclei were stained with DAPI (1:500).

[0163] imaging Images of stained sections were scanned using a confocal microscope (PerkinElmer UltraView). Images were acquired using a VOX spin disk or Leica SP8 point scan. Images were processed using Adobe Photoshop®. To analyze distal muscular arterialization, the inventors previously described and indicated two types in the left lung as L.L1.A1.L1 and L.L1.A1.M1 (Sheikh 2014; Sheikh 2015). We focused on two specific arteriole beds. Their nomenclature is derived from the nearest airway with a typical branching pattern in adult mice (Sheikh et al Cell Rep. 2014;6(5):809-17, Metzger. Nature. 2008;453(7196):745-50). Their diameter and branching pattern. Based on this, pulmonary arterioles are classified as proximal (P; diameter > 75 mm), intermediate (M; 25-75 mm), and distal (D; < 25 mm), as well as nominally L, left main bronchus; L1, L2, L3, lateral branch; M1, M2, medial branch; A1, A2, anterior branch.

[0164] Human research All procedures involving human subjects were approved by the Yale University Institutional Ethics Board (IRB #1307012431 and #1005006865), and the inventors followed all applicable ethical rules. Written informed consent was obtained from all participants prior to enrollment in the study.

[0165] Isolation of human monocytes and differentiation into macrophages Fresh whole blood from IPAH and SSc-PAH patients and healthy controls at the Department of Pulmonary and Vascular Diseases, Yale University School of Medicine, was collected as an unspecified sample using the Greif method. The monocytes were provided to the lab. Based on the previously described method (Bennett J Exp Med. 1966;123(1):145-60; Karlsson, et al. Exp Hematol. 2008;36(9):1167-75), the monocytes were subjected to sterilization. The cells were separated and differentiated into macrophages. Briefly, fresh whole blood was 3-fold diluted in HBSS, loaded onto a Ficoll-Histopaque column (Fisher Scientific), and centrifuged at 830g for 30 minutes. The peripheral blood mononuclear cell phase was aspirated, 3-fold diluted in HBSS, and centrifuged at 830g for 10 minutes. To ensure platelet removal, the pellet was resuspended in 3 ml of HBSS and centrifuged again at 830g for 10 minutes. Next, the pellet was resuspended in RPMI containing 10% FBS, and the cells were adhered to a plastic cell culture dish at 37°C for 1 hour. Monocytes preferentially adhered to the plastic (37). Adhesion was performed (Figure S6A, B). Suspended cells were discarded, adherent cells were washed with PBS, incubated with 5 mM EDTA in PBS for 10 minutes, and collected for staining and flow cytometry, or cultured in macrophage differentiation medium (ImmunoCult®-SF Macrophage Medium and 1 ng / ml Macrophage Colony Stimulating Factor [both from StemCell Technologies]). The medium was replaced with fresh macrophage differentiation medium on day 4. On day 6, the medium was changed to ImmunoCult®-SF Macrophage Medium, and after 12 hours, the conditioned medium was collected and cells were harvested. For hypoxic studies, macrophages derived from healthy donor monocytes were exposed for 12 hours in either normal oxygen or 3% O2 ​​in RPMI supplemented with 1% FBS and 5% penicillin-streptomycin.

[0166] hPASMC culture and proliferation assay hPASMC (American Type Culture Collection) cells were cultured for up to six passages in M199 medium supplemented with 10% FBS, 1% penicillin / streptomycin, 2 ng / ml fibroblast growth factor (Promega), and 3 ng / ml epidermal growth factor (Promega). Growth was evaluated as previously described with minor modifications (Dave J. Dev Cell. 2018;44(6):665-78 e6). MCs were cultured overnight on culture slides (BD Falcon) pre-coated with trypsin and fibronectin (10 μg / mL in PBS). The following day, the cells were washed with PBS and serum depleted overnight in M199 supplemented with 0.5% FBS. The cells were then washed with PBS and cultured for 24 hours in medium acclimatized with human controls or patient-derived macrophages pre-treated or untreated with 20 μg / ml IgG control or anti-PDGF-B blocking antibody (R&D Systems) at 37°C for 1 hour. During the last 10 hours of this incubation, 10 μg / ml BrdU(Sigma) was added to the cells. The slides were fixed in 4% paraformaldehyde for 30 minutes, rinsed in 0.3% Tris and 1.5% glycine water for 15 minutes, incubated in 2N HCl at 37°C for 30 minutes, washed with 0.1M boric acid, and incubated in PBS-T with 1% FBS for 1 hour. hPASMCs were stained with rat anti-BrdU primary antibody (1:100, BioRad) in 1% FBS in PBS-T for 1 hour, washed three times with 0.5% Tween® 20 in PBS, and then incubated for 1 hour with goat anti-rat secondary antibody conjugated to Alexa488 (1:500, Molecular Probes) and PI (1:500, Sigma) in 1% FBS in PBS-T. Finally, the slides were washed three times with 0.5% Tween 20 in PBS and mounted on fluorescent mounting medium (Dako). Growth was performed using BrdU + The total PI was + The percentage was calculated as hPASMC. At least 10 fields were scored for each control or patient.

[0167] SMC migration assay Cell migration was evaluated using the method described in Dave Dev Cell. 2018;44(6):665-78 e6. Briefly, hPASMCs were trypsinized and immediately added to the upper part of a polycarbonate membrane (Corning Costar, 8 μm pore size) in a Boyden chamber. The lower compartment of the Boyden chamber contained culture medium conditioned with human control and patient-derived macrophages, either pre-treated with 20 μg / ml anti-PDGF-B blocking antibody or IgG control at 37°C for 1 hour or untreated. The hPASMCs were allowed to migrate toward the lower chamber for 8 hours, at which point the membrane was fixed in 4% paraformaldehyde for 30 minutes, stained with 0.1% crystal violet, and washed with water. The upper surface of the membrane was scraped with a cotton swab to remove non-migrating cells, and cells on the lower surface (i.e., migrating cells) were imaged and counted.

[0168] statistics All data are expressed as mean ± standard deviation. Student's t-test (independent, two-tailed) and one-way ANOVA were used to compare the means of two groups and multiple groups, respectively (GraphPad Prism software). The statistical significance threshold was set at p ≤ 0.05. All studies assumed a normal distribution.

[0169] result Figure 1A-1 of the results shows alveolar and residual lung parenchymal macrophages, CD64 + Ly6G - This indicates that cells accumulate under hypoxic conditions.

[0170] As shown in Figures 2A-2B, there is a similar increase in PDGF-β mRNA, peaking at levels of approximately 6-fold and 9-fold increases in alveolar and residual lung macrophages, respectively.

[0171] Figures 2C-2F demonstrate that macrophage depletion reduces muscular arterialization, right ventricular systolic pressure, and right ventricular hypertrophy in animals under hypoxic conditions.

[0172] Using LysM-Cre mice carrying the loxP transallele, specific genes were deleted in myeloid cells. After 21 days of hypoxia, mice with myeloid cells deficient in PDGF-β or hypoxia-inducible factor 2a were protected from distal arteriole muscular arterialization and PH. As shown in Figures 3A-3D, von Hippel-Lindau plays a crucial role in the degradation of hypoxia-inducible factors, and the results indicate that deletion of the von Hippel-Lindau gene in myeloid cells leads to distal arteriole muscular arterialization and PH under normal oxygen conditions.

[0173] As shown in Figures 4A-4F, 5A-5F, and 7A-7F, the pharmacological downmodulation effect of delivering PDGF-β siRNA-loaded nanoparticles in lung macrophages was evaluated. In bronchoalveolar lavage fluid, PDGF-β siRNA reduced PDGF-β levels by 90%. These siPDGF-β nanoparticles attenuated hypoxia-induced distal pulmonary arteriole muscular arterialization, PH, and right ventricular hypertrophy.

[0174] Finally, in Figures 6A–6E, human macrophages and SMCs were studied to assess the clinical relevance of this research. First, in macrophages from healthy donors, exposure to hypoxia increased PDGF-β transcript levels 2.5-fold (Figures 6A–6B). In addition, PDGF-β levels in macrophages from PH patients were enhanced 5-fold and 10-fold, respectively, by idiopathic etiology or scleroderma. See Figures 6C–6D. Similarly, medium conditioned with patient macrophages increased SMC proliferation by approximately 6-fold. Furthermore, pretreatment of PH patient-conditioned medium with an anti-PDGF-B blocking antibody inhibited this SMC proliferation. See Figure 6E. Similarly, PH patient-conditioned medium induced SMC migration by approximately 4-fold, and anti-PDGF-B pretreatment reduced this effect by approximately 50%.

[0175] Figure 8 is a schematic diagram illustrating the methods used herein for mouse and human studies.

[0176] Considering these factors together, studies using experimental models and cells isolated from human pulmonary hypertension patients demonstrate that macrophage hypoxia-inducible factor and PDGF-B play major roles in SMC and right ventricular remodeling and PH. Furthermore, nanoparticle-mediated silencing of PDGF-β in lung macrophages is therapeutic. Immunohistochemical analysis of distal muscular arterialization in this study of the foregoing identified airway branches, left bronchus- We focused on specific pulmonary arteriole beds adjacent to the first collateral branch secondary branch - first anterior branch - first collateral branch or first medial branch (L.L1.A1.L1 or L.L1.A1.M1). Under normal oxygen pressure conditions, the distal arterioles in these beds did not undergo muscular arterialization, but underwent a typical process of muscular arterialization upon hypoxic exposure (Sheikh Cell Rep. 2014;6(5):809-17; Sheikh Sci Transl Med. 2015;7(308):308ra159; Sheikh Cell Rep. 2018;23(4):1152-65).

[0177] In addition to developing distal arteriole muscular arterialization and PH, the lungs of mice exposed to hypoxia accumulate excessive macrophages (Amsellem Am J Respir Cell Mol Biol.). (2017;56(5):597-60818, Stenmark Circ Res. 2006;99(7):675-91; Rabinovitch Annu Rev Pathol. 2007;2:369-99) (Figure 1A~C). Maintenance under hypoxia (10% FiO2) The time course of lung macrophage accumulation during PH in wild-type mice was determined for up to 21 days. The pulmonary vascular system was washed, and CD64 was analyzed using flow cytometry. + Ly6G -Macrophages were isolated from bronchoalveolar lavage fluid (BALF) and residual lung tissue after BALF. The percentage of macrophages in BALF gradually increased, reaching statistical significance at day 21 of hypoxia compared to normal oxygen conditions. In contrast, macrophages from residual lung tissue increased 2.9 ± 0.5 times at day 3 of hypoxia and up to 10.8 ± 1.1 times at day 21 of hypoxia.

[0178] The effects of clodronate on hypoxia-induced distal muscular arterialization and pulmonary hypertension (PH) by depleting alveolar and residual macrophages were evaluated. Wild-type mice were orally administered intratracheally twice weekly at the start of hypoxia and during the following 21 days of hypoxia to deplete phagocytic cells, using either clodronate-loaded liposomes or phosphate-buffered saline (PBS)-loaded liposomes as a control. Clodronate-treated mice showed reduced hypoxia-induced distal muscular arterialization, right ventricular systolic pressure (RVSP; equal to pulmonary artery systolic pressure), and RVH as measured by the Fulton index (i.e., the weight ratio of the right ventricle [RV] to the sum of the left ventricle [LV] and septum [S]). Compared to control liposomes, treatment with clodronate-loaded liposomes reduced macrophages in the BALF by approximately 50% and macrophages in the residual lung by approximately 65% ​​(Figure 1E, 1F). Under basal conditions, the presence of myofibroblasts in adult lungs is extremely rare, but hypoxia has been shown to induce a significant increase in the number of these cells (Sheikh Cell Rep. 2014;6(5):809-17, Chen J Appl Physiol (1985). 2006;100(2):564-71). Depletion of myeloid cells significantly inhibits hypoxia-induced accumulation of alveolar myofibroblasts.

[0179] Lung macrophage PDGF-β is upregulated by hypoxia, and PDGF-β deficiency in the LysM-Cre lineage attenuates PH. Exposure of mice to hypoxia increases PDGF-B levels throughout the lungs and especially in the pulmonary EC (Sheikh 2015; Sheikh 2018); however, not all pulmonary PDGF-B levels are necessarily elevated. It does not originate from EC. Therefore, CD64 isolated by FACS from the BALF and residual lung of mice exposed to hypoxia for up to 21 days. + Ly6G - The time course of PDGF-β expression in macrophages was calculated. PDGF-β mRNA levels were measured by qRT-PCR and compared to normal oxygen levels. They increased within 1 day of hypoxia, peaking on day 3 at levels 5.6±0.2 and 9.3±0.2 times higher for BALF and residual lung, respectively (Figure 2A, B). To further confirm the upregulation of PDGF-β in monocytes / macrophages, LysM-Cre was used to label this population. LysM-Cre, ROSA26R (mTmG / mTmG) Mice were exposed to hypoxia for 21 days or maintained under normal oxygen conditions, after which GFP was produced. + Cells were isolated from the entire lung by FACS. PDGF-β mRNA levels increased 2.1 ± 0.4 times in cells isolated from hypoxic mice. Similarly, GFP isolated from BALF of normoxic mice. + When cells are cultured under hypoxic conditions (3% O2) in contrast to normal oxygen pressure conditions, the same They had increased PDGF-β mRNA levels.

[0180] Next, we evaluated whether monocyte / macrophage-derived PDGF-β contributes to hypoxia-induced pH. Previously, we investigated Csf1r-Mer-iCre-Mer and PDGF-β. (flox / flox) Tamoxifen treatment in mice was found to weakly reduce pathological distal pulmonary arteriole muscular arterialization (Sheik 2018), but it did not affect PH, RVH, and myofibroblast accumulation. The effect was not studied. Inducible Csf1r-Cre is extremely inefficient in inducing recombination (Qian Nature. 2011;475(7355):222-5; Epelman Immunity. 2014;40(1):91-104), and in this specification, in order to avoid this inefficiency, constitutive LysM-Cre PDGF-β was deleted using [a specific method] (Figure S3A). PDGF-β (flox / flox)In the background, mice possessing LysM Cre showed reduced distal muscular arterialization and PH after 21 days of hypoxic exposure compared to mice lacking Cre (Figure 2C, D). LysM-Cre, PDGF-β (flox / flox) The Fulton index of PDGF-β (flox / flox) Compared to the Fulton index in mice, there was a tendency for it to decrease in hypoxia and increase in normal oxygen conditions, but these differences did not reach statistical significance (Figure 2E). However, when the difference in the Fulton index between hypoxic and normal oxygen conditions was stratified by genotype, LysM-Cre and PDGF-β showed a tendency to increase. (flox / flox) In mice, a significant reduction of 46±7% was observed in this difference (Figure 2F). Finally, PDGF-β deficiency in myeloid cells reduced myofibroblasts by approximately 60% in both 3-day and 21-day hypoxic exposure (Figure 2G, H, S4A, B). Thus, myeloid-derived PDGF-B plays a crucial role in hypoxia-induced pulmonary vascular remodeling and PH.

[0181] LysM-Cre-mediated deletion in von Hippel-Lindau induces PDGF-β expression and pulmonary vascular remodeling under normal oxygen conditions. Considering the crucial role of myeloid cell-derived PDGF-B in the pathogenesis of PH, we evaluated the underlying mechanisms of hypoxia-induced PDGF-β expression by this cell type. Hypoxia-inducible factor (HIF) is a heterodimer of HIF1-β and the HIFα isoform, i.e., either HIF1-α or HIF2-α. In mice exposed to hypoxia, EC HIF regulates autonomous PDGF-β expression in cells, as well as distal muscular arterialization and PH. Using oxygen as a substrate, HIFα undergoes proline hydroxylation, a modification that facilitates binding to von Hipperlindau (VHL)-E3 ubiquitin ligase and ultimately facilitates proteasome-mediated degradation. Thus, HIFα accumulates when oxygen is scarce or when the associated ubiquitination degradation pathway is inhibited, for example, by Vhl deletion. Under normal oxygen pressure conditions, Vhl (flox / flox) Compared to a mouse, LysM-Cre, Vhl(flox / flox) Mice exhibit reduced Vhl in BALF cells and increased Hif1a, Hif2a, and PDGF-β levels (Figure 3A, S3D-F). Furthermore, Vhl deficiency in myeloid cells induces distal muscular arterialization, PH, and RVH under normal oxygen conditions (Figure 3B-C), as well as pulmonary macrophage accumulation (Figure 3D).

[0182] Next, we evaluated whether Vhl deficiency enhances the effect of relatively short (7-day) exposure to hypoxia. At this point, Vhl with LysM-Cre... (flox / flox) The mice have BALF cell PDGF-β mRNA levels that are significantly increased by 7.6 ± 1.2 times compared to mice lacking Cre. Furthermore, LysM + Vhl deletion in cells induced a marked enhancement of distal muscular arterialization, as well as an increase in RVSP and RVH, after short-term hypoxic exposure.

[0183] Myeloid cell HIFα regulates PDGF-β expression and hypoxia-induced distal muscular arterialization, RVH, and PH. To complement experiments in which the HIF pathway is induced in this way by deleting Vhl, LysM + We conducted studies in which Hif1a or Hif2a was deleted in cells. First, from the time course of hypoxia exposure in wild-type mice, it was revealed that HIF1-α and HIF2-α were upregulated in BALF cells by hypoxia on day 3 (Figure 4A, 5A). At this point, Hif1a (flox / flox) or Hif2a (flox / flox) Background mice, also possessing LysM-Cre, had reduced levels of PDGF-β and either Hif1a or Hif2a in BALF cells compared to mice lacking Cre (Figures 4B, 5B). In addition, the accumulation of cells expressing the macrophage marker CD64 and myofibroblasts in the lungs was substantially reduced by Hif1a or Hif2a deletion (Figures 4C-D, 5C-D). Furthermore, analysis at 21 days of hypoxia showed that Hif1a (flox / flox) or Hif2a (flox / flox)We demonstrated that LysM-Cre mice possessing [specific trait] exhibited reduced distal pulmonary arteriole muscular arterialization, RVSP, and Fulton index (Figures 4E-F, 5E-F). Thus, considering the PDGF-β, Vhl, Hif1a, and Hif2a deletion experiments together, the results suggest that PDGF-B expression by myeloid cells is cell-autonomously modulated by both HIFα isoforms, and is an important factor in regulating pulmonary vascular remodeling and PH.

[0184] Macrophage-derived PDGF-B increases in PAH patients and induces SMC proliferation and migration. Given the prominent role of macrophages and myeloid-derived PDGF-B in pathological pulmonary muscular arterialization in mice, we then attempted to extrapolate these findings to human PAH patients. First, we analyzed PDGF-β levels from human macrophages. Peripheral blood mononuclear cell fractions were isolated from fresh whole blood of control humans by Ficoll column centrifugation and enriched with respect to monocytes by adhesion to plastic. Adherent cells were incubated with macrophage colony-stimulating factor to differentiate them into macrophages, and exposure of macrophages to hypoxia (3% O2) for 12 hours induced a 2.6 ± 0.6-fold increase in PDGF-β transcript levels in contrast to normal oxygen conditions (Figure 6A). As strong clinically relevant evidence of this study, PDGF-β levels in macrophages differentiated from circulating monocytes in IPAH and SSc-PAH patients were enhanced by 5.1 ± 1.8 and 10.7 ± 4.8-fold, respectively, compared to those of control humans (Figure 6B).

[0185] The effect of macrophage-conditioned medium from PAH patients on hPASMC proliferation and the role of PDGF-B in this medium were evaluated. hPASMCs were cultured for 24 hours in newly differentiated macrophage-conditioned medium, and BrdU was added during the last 10 hours of incubation. Proliferative activity was observed compared to the control (i.e., BrdU was added). + ) Cells (Propidium iodide [PI] +The percentage of nuclei was determined (Figure 6C). Relative increases of 4.6±0.3 and 7.0±1.9-fold, respectively, were observed in media conditioned with macrophages derived from IPAH and SSc-PAH patients, respectively. To assess the contribution of PDGF-B to these effects, macrophage-conditioned media were incubated for 1 hour with an anti-PDGF-B blocking antibody or IgG control before adding to hPASMCs. In macrophages derived from control patients, hPASMC proliferation was not altered by anti-PDGF-B pretreatment, but this pretreatment significantly inhibited the proliferation of hPASMCs induced by media conditioned with IPAH or SSc-PAH macrophages (Figure 6D).

[0186] Next, a similar approach was used to investigate the effect of macrophage-conditioned medium and PDGF-B within it on hPASMC migration. hPASMC migration from the upper chamber to the lower chamber containing the pre-treated conditioned medium was evaluated using anti-PDGF-B or IgG control antibodies, similar to the proliferation studies. Regarding IgG control pretreatment... Furthermore, conditioned media from IPAH or SSc-PAH macrophages induced migration 3.0±0.8 times and 4.2±0.8 times, respectively, compared to conditioned media from control macrophages. In addition, compared to IgG pretreatment, anti-PDGF-B pretreatment reduced hPASMC migration by approximately 40-50% using IPAH or SSc-PAH macrophage-conditioned media. In contrast, PDGF-B pretreatment of conditioned media from human controls did not affect hPASMC migration.

[0187] Nanoparticle delivery of siPDGF-β attenuates hypoxia-induced pH. After demonstrating the importance of myeloid-derived PDGF-B in experimental PH and the induction effect of PDGF-B from macrophages of PAH patients on hPASMCs, we pharmacologically downregulated this ligand in lung macrophages by delivering nanoparticles formed from poly(amine-co-ester)[PACE] polymer and PDGF-β siRNA. Previous studies have shown that similar nanoparticles can enable sustained silencing of protein expression in cells that internalize the particles. First, 400 or 200 nm diameter nanoparticles composed of (poly(pentadecalactone-co-n-methyldiethanolamine co-sebacate)) (PPMS-50COOH) with an acid terminus containing 50% lactone loaded with the dye DiD were orally administered intratracheally to wild-type mice, and uptake by lung cells expressing the macrophage marker CD64 was evaluated 12 hours later using flow cytometry analysis (Figure 7A). For both 400 and 200 nm diameter nanoparticles, CD64 + The majority of cells were DiD-labeled (>99% in BALF and approximately 92% in residual lung). Similarly, CD64 + The percentage of DiD-labeled cells was high and comparable for 400 nm and 200 nm diameter particles in BALF (95±1% and 93±3%, respectively); however, in residual lung, these percentages decreased to 86±1% for 400 nm particles and 62±1% for 200 nm particles (Figure 7C). Therefore, all further experiments were performed with 400 nm diameter nanoparticles. To confirm uptake, isolated BALF cells were cultured with DiD-labeled nanoparticles for 6 hours, and these cells exhibited perinuclear fluorescence.

[0188] Next, we evaluated whether nanoparticles loaded with siRNA targeting PDGF-β could improve the effects of hypoxia exposure on the mouse lung. The PDGF-β siRNA oligonucleotide was used because transfection into BALF cells reduced PDGF-β levels by 91 ± 1% compared to Scr RNA treatment. Nanoparticles loaded with this siPDGF-β or Scr RNA were administered intratracheally by mouth twice a week at the onset of hypoxia and during up to 21 days of hypoxia exposure. On day 3 or 21 of hypoxia, CD64 + LysG - The percentage of cells that were macrophages was not different between mice treated with the two nanoparticle types (Figures 7B–C). Next, we determined the effect of siPDGF-β-nanoparticles on macrophage PDGF-β RNA levels at day 3, the time point of maximal PDGF-β levels (see Figures 2A, B). Nanoparticles loaded with siPDGF-β reduced lung macrophage PDGF-β levels by 86 ± 11% (Figure 7C). Finally, siPDGF-β-nanoparticle treatment during 21 days of hypoxia exposure significantly attenuated distal pulmonary arteriolar muscularization, PH, RVH, and accumulation of myofibroblasts (Figures 7D–F).

[0189] Discussion The increase in the SMC lineage is becoming increasingly recognized as an important factor in diverse cardiovascular diseases; however, understanding the non-cellular autonomous regulation of SMCs by cell types other than ECs during these pathological situations as well as normal vascular development has not yet been achieved. Phagocytic cells, including macrophages, play fundamental roles in both the innate immune system and the etiology of diverse cardiovascular diseases, including PH. During embryonic development, fetal macrophage progenitor cells are mobilized to the normal lung and differentiate into macrophages, and then these resident macrophages It is maintained by local proliferation. In contrast, in PH, increased monocytes are found in the pulmonary vascular system and perivascular region, giving rise to pulmonary macrophages. Vascular SMCs and pulmonary macrophages are undoubtedly important cell types in PH, but a crucial unresolved question is whether and how pulmonary macrophages regulate SMCs in this context. In this specification, our studies using a mouse model of PH, as well as human macrophages from IPAH and SSc-PAH patients, demonstrate that macrophage-derived PDGF-B induces pathological SMC enlargement and PH, thereby establishing macrophage-derived PDGF-B as a key factor in this paradigm. Furthermore, our findings regarding nanoparticle-derived PDGF-β siRNA offer an interesting therapeutic approach.

[0190] Intratracheal administration of clodronate-containing liposomes has previously been shown to deplete alveolar macrophages and reduce hypoxia-induced PH and RVH in rats. Herein, we demonstrate that such treatment in mice reduces macrophages in the remaining lung and BALF, and also attenuates distal muscular arterialization and hemodynamic changes (Figure 1). While this approach is beneficial for chronically depleted macrophages in the short term, it is impractical given their essential role in innate immunity. Thus, a preferred strategy is to target specific macrophage-derived gene products.

[0191] Along these lines of thought, PDGF is widely involved in the etiology of PH. In human IPAH, the mRNA levels of the ligands PDGFA, PDGF-B, and the receptors PDGFRA and PDGFRB are upregulated in small pulmonary vessels, and PDGFR-β protein is increased in whole lung lysates. Mice with a knock-in mutant Pdgfrb encoding a protein defective in mediating downstream PI3K and PLC-γ signaling blunted hypoxic-induced pulmonary vascular remodeling, PH, and RVH. In a fetal lamb model of PH induced by partial ligation of the ductus arteriosus in utero, injection of an anti-PDGF-B aptamer into the pulmonary artery reduced the severity of pulmonary vascular remodeling by half and RVH by two-thirds. Moreover, overall PDGF-β (+ / -) mice lack hypoxic-induced distal pulmonary arteriolar SMCs, and EC-specific deletion of PDGF-β reduces but does not completely prevent distal muscularization. Here, the inventors show that exposure of mice to hypoxia significantly upregulates the expression of PDGF-β by alveolar and residual lung macrophages (by day 3 of hypoxia), and LysM-Cre, PDGF-β (flox / flox) mice demonstrate substantial attenuation of distal muscularization and PH. Interestingly, although there is a tendency for reduction of RVH in these hypoxic mice, the RV weight ratio under normoxia tends to increase in these mutants and thus does not reach statistical significance. Indeed, the hypoxia-induced increase in RVH stratified by genotype is reduced by approximately 50% by PDGF-β deletion. The explanation for the tendency for increased RV weight ratio under basal conditions is unclear, but the inventors suggest that bone marrow-derived PDGF-B may limit RV mass during normal development and / or maintenance.

[0192] These data indicate that lung macrophage-derived PDGF-B plays a crucial role in hypoxia; however, the regulation of PDGF-B expression in this cell type is not well understood. When mice were exposed to hypoxia, lung EC increased PDGF-β levels in a HIF1-α-dependent manner, and it was found that deletion of myeloid Hif1a or Hif2a reduced PDGF-β levels in lung macrophages compared to control mice. The data suggest that Hif1a deletion in myeloid cells protects against hypoxia-induced hypoxia. In addition, LysM-Cre, Hif2a (flox / flox) The mice were protected from schistosomiasis-induced pH, and the results show that these mice also attenuated hypoxia-induced pH. Complementary HIF gain-of-function studies (i.e., bone marrow) The Vhl deletion in the macrophage system suggests that HIF in lung macrophages is sufficient to induce autonomous PDGF-β expression, distal muscular arterialization, PH, and RVH under normal oxygen pressure conditions (Figure 3). Thus, HIF-induced PDGF-β in macrophages is considered essential for the hypoxic response and hemodynamic changes in vascular remodeling.

[0193] These findings demonstrate that, similar to distal arteriole muscular arterialization, pulmonary macrophages induce alveolar myofibroblast accumulation in hypoxic lungs (Figure 1), and that myeloid-derived PDGF-β, Hif1a, and Hif2a are crucial for this process (Figures 2, 4, and 5). Pulmonary myofibroblasts play a vital role in alveolar septal formation during normal alveolar formation in early postnatal mice, but these cells are then very rare in adult lungs. In fibrotic diseases, myofibroblasts are involved in the production of a large amount of excess extracellular matrix, and macrophages secrete pro-fibrosis factors that recruit and activate myofibroblasts. In contrast, the role of monocytes / macrophages in regulating hypoxia-induced alveolar myofibroblasts has not been previously reported. PDGFR-β + The cells produce more than 40% of hypoxia-induced myofibroblasts in the lungs (R. Chandran, I. Kabir, A. Sheikh, ELH and DMG, undisclosed data), SMA + Cells account for only about 20% of the supply. The result was PDGFR-β + SMA - This is consistent with other studies suggesting that pulmonary pericytes are an important cell type in PH.

[0194] Approximately 10–15% of SSc patients develop PAH, which is the leading cause of death in these patients. In fact, the 3-year survival rate is estimated to be only 49% in SSc-PAH patients compared to 84% in IPAH patients. One factor contributing to this higher mortality rate is the mutational response to standard anti-PAH treatment in SSc-PAH patients compared to IPAH patients. In addition, anti-PDGFR-β immunohistochemical staining is enhanced in small blood vessels in SSc-PAH patients compared to IPAH patients. The number of circulating monocytes does not differ between these PAH patient populations; however, the results show that PDGF-β levels are enhanced in macrophages derived from these monocytes in SSc-PAH patients compared to control humans. Furthermore, macrophages from these two classes of PAH patients induce SMC proliferation and migration primarily in a PDGF-B-dependent manner. A study published 25 years ago reported that PDGF-B protein levels were increased in BALF in general SSc patients (i.e., patients not evaluated for PH) compared to controls. Thus, a strategy targeting macrophage-derived PDGF-B may be effective in PAH.

[0195] Imatinib is a tyrosine kinase inhibitor that is active against BCR-ABL, c-KIT, PDGFR-α, and -β in cancer applications. Daily injections of imatinib reverse monoclotaline-induced pulmonary vascular remodeling, PH, and RVH in rats or chronic hypoxia-induced pulmonary vascular hypertension in mice. Unfortunately, these positive results were not extrapolated to PAH patients in Imatinib in Pulmonary Arterial Hypertension, a Randomized Efficacy Study (IMPRES). Overall, 94% of patients discontinued this oral imatinib study, and serious and unexpected adverse effects, including subdural hematoma, were common. However, IMPRES patients who were able to maintain long-term with imatinib showed improved functional class and 6-minute walk distance. These results further highlight the need for anti-PH therapies that target specific pathways (e.g., PDGF-B-mediated) in specific cell types of the lung (e.g., macrophages).

[0196] Oral intratracheal administration of PPMS polymer-formulated nanoparticles loaded with siRNA targeting PDGF-β substantially downregulates macrophage-derived PDGF-β and prevents hypoxia-induced distal pulmonary arteriole muscular arteriovenous hypertrichosis (PH) and RVH. These nanoparticles are pulmonary macrophages. Lung macrophages specifically and broadly phagocytose the lungs. Previous studies have shown that intratracheal or intravenous delivery of nanoparticles containing drugs effective in human PAH, including prostacyclin analogs and sildenafil, attenuates PH in experimental rodent models. The only previous report on nanoparticle-mediated RNA interference in this context demonstrated that intravenous delivery of antisense oligonucleotide microRNA (anti-miR)-145, aimed at directly targeting SMCs, alleviates hypoxia / Sugen-5416-induced PH in rats; however, in addition to the lungs, this anti-miR accumulates in the liver, spleen, and kidneys. The herein approach of orally intratracheally administering siRNA-loaded nanoparticles is advantageous because it specifically and potently targets selective gene products in lung macrophages, thereby promising to limit undesirable effects. Furthermore, PPMS polymer-formulated nanoparticles are non-toxic, biodegradable, and protect their cargo from degradation.

[0197] In summary, studies using experimental models and cells isolated from human PAH patients demonstrate that HIF-regulated expression of PDGF-B by macrophages plays a major role in SMC remodeling, PH, and RVH. Furthermore, nanoparticle-mediated silencing of PDGF-β in lung macrophages is a therapeutic strategy that warrants further intensive research.

[0198] summary: The results indicate that PACE nanoparticles provided selective uptake in lung macrophages and monocytes after oral (or pulmonary) administration.

[0199] The results also establish that this method of delivery of PDGF-β inhibitors was effective in treating PH.

[0200] Figures 1A–1F show that macrophages from BALF and residual lung tissue increase with hypoxic exposure. Mice were exposed to normal oxygen or hypoxia (10% FiO2) for 0–21 days. CD64+Ly6G-macrophages were isolated from BALF by FACS and subjected to qRT-PCR for PDGF-β. Similarly, CD64+Ly6G-macrophages were isolated from residual lung tissue and their PDGF-β mRNA levels were evaluated.

[0201] Figures 2A–2F demonstrate that macrophage depletion is protective against pulmonary hypertension. Mice were exposed to normal oxygen or hypoxia (10% FiO2) for 21 days and simultaneously administered oral intratracheal liposomes loaded with clodronate or vehicle twice weekly. Clodronate treatment reduced distal arterial muscular arterialization, as indicated by right ventricular systolic pressure (RVSP; equal to pulmonary artery systolic pressure) and Fulton index in hypoxic mice.

[0202] As shown in Figures 3A-3D, 4A-4F, and 5A-5F, hypoxia induces PDGF-β in macrophages of the BALF and residual lung tissue. In the LysM-Cre lineage, PDGF-β or Hif2a deletion attenuates hypoxia-induced distal muscular arterialization and PH, while Vhl deletion induces spontaneous PH.

[0203] PDGF-β and Hif2a deletions in myeloid cells provide protection against PH, while Vhl deletion leads to PH under normal oxygen pressure conditions. Mice were exposed to hypoxia or normal oxygen conditions as indicated. Lung sections with distal arterioles from mice lacking Cre or possessing LysM-Cre and having loxP transalleles for PDGF-β, Hif2a, or Vhl were examined for SMC (alpha-smooth muscle actin [SMA]) and endothelium. Cells (EC;MECA-32) were stained for a marker.

[0204] Myeloid cells from human PH patients have elevated PDGF-β levels, which induce SMC proliferation and migration.

[0205] Figures 6A–6E show that macrophage-derived PDGF-B promotes the proliferation and migration of pulmonary artery cells (SMCs) in patients with idiopathic and scleroderma-associated hypertension (PAH). Human macrophages were cultured for 12 hours under normal or hypoxic (3% O2) conditions, and PDGF-β mRNA in macrophages from controls and PAH patients was measured by qRT-PCR. The BrdU assay was performed on human pulmonary artery SMCs cultured with patient or control culture medium (CM). Anti-PDGF-B blocking Ab or control IgG was added to the CM. Migrated cells were quantified compared to controls using a migration assay in which SMCs were added to the upper part of a Boyden chamber and CM containing either anti-PDGF-B Ab or IgG was added to the lower part.

[0206] Figures 7A–7F show that PACE nanoparticle (NP)-mediated PDGF-β knockdown in myeloid cells attenuates pulmonary hypertension (PH). Mice were exposed to normal oxygen or hypoxia and simultaneously administered NPs loaded with scrambled (Scr)RNA or PDGF-β-targeted siRNA. CD64+Ly6G-macrophages isolated by FACS were subjected to qRT-PCR for PDGF-β mRNA. Lung sections containing distal arterioles were stained for SMA and CD31 (EC marker). RVSP and Fulton index were measured. Nanoparticle delivery of siPDGF-β to lung macrophages attenuates hypoxia-induced distal muscular arterialization, PH, and RVH.

[0207] Therefore, the results establish the following: 1. PH can be treated or prevented by administering PDGF-β to the lungs; and 2. Selective drug uptake in lung macrophages and monocytes can be achieved using nanoparticles formed from PACE polymers.

[0208] Unless otherwise defined, all scientific and technical terms used herein have the same meaning as those commonly understood by those skilled in the art to which this invention pertains. Publications cited herein and the materials they refer to are specifically incorporated herein by reference.

[0209] Those skilled in the art can recognize or confirm, by mere ordinary experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be covered by the following claims.

Claims

1. A delivery formulation for selective delivery to lung immune cells such as macrophages and monocytes, with the following formula: 【Chemistry 9】 The polymer comprises nanoparticles having an average diameter between 100 and 500 nm, preferably between 200 and 400 nm, wherein the formula includes, n is an integer from 1 to 30, m, o, and p are independently integers from 1 to 20, x, y, and q are independently integers from 1 to 1000, Rx is hydrogen, a substituted or unsubstituted alkyl, a substituted or unsubstituted aryl, or a substituted or unsubstituted alkoxy, Z and Z' are independently O or NR', where R' is hydrogen, a substituted or unsubstituted alkyl, or a substituted or unsubstituted aryl. R １ and R ２ This is a chemical entity containing a hydroxyl group, a primary amine group, a secondary amine group, a tertiary amine group, or a combination thereof. Delivery formulation.

2. R1 and / or R2 【Chemistry 10】 The formulation described in claim 1, not the formulation described in claim 1.

3. The formulation according to claim 1, wherein the polymer is in the form of a polyplex containing nucleic acids or particles thereof.

4. R1 and / or R2 are: 【Chemistry 11】 The formulation according to claim 3, comprising:

5. The aforementioned polymer is given by formula II: 【Chemistry 12】 It has the structure, in the formula, J １ and J ２ It is either an independent connecting part or does not exist. R ３ and R ４ This is a substituted alkyl group containing a hydroxyl group, a primary amine group, a secondary amine group, a tertiary amine group, or a combination thereof. The formulation according to claim 1.

6. The aforementioned polymer is formula III: 【Chemistry 13】 The formulation according to claim 1, having the structure.

7. The formulation according to claim 1, wherein the polymer has a weight-average molecular weight between about 2,000 daltons and 20,000 daltons, preferably between about 2,000 daltons and 10,000 daltons, and most preferably between about 2,000 daltons and 7,000 daltons, when measured by gel permeation chromatography using a narrow polydispersible polystyrene standard.

8. The formulation according to any one of claims 1 to 7, wherein the nanoparticles include a therapeutic agent, a preventive agent, or a diagnostic agent.

9. The formulation according to claim 8, wherein the drug is for the treatment, prevention, or diagnosis of lung injury or disease.

10. The formulation according to claim 8, wherein the drug is a PDGF-β inhibitor.

11. The formulation according to claim 8, wherein the drug is a protein or peptide, a sugar or carbohydrate, a lipid, a lipoprotein or lipopolysaccharide, a nucleic acid molecule, or a small molecule having a molecular weight of less than 2000 daltons.

12. The formulation according to claim 8, which is formulated for administration as an aerosol by intravenous infusion, nebulizer, inhaler, ventilator, or respiratory mask, or as a dry powder.

13. The formulation according to claim 8, wherein the drug is present in an amount that allows for local delivery of the drug to the respiratory system rather than to the whole body.

14. A method for treating an individual in need of treatment, comprising administering an effective amount of the formulation according to any one of claims 8 to 13.

15. The method according to claim 14, wherein a PDGF-β inhibitor is administered to an individual having pulmonary hypertension.

16. The method according to claim 14, wherein the individual has congestive heart failure.

17. The method according to claim 14, wherein the individual has pulmonary fibrosis.

18. The method according to claim 14, wherein the individual has lung cancer.

19. The method according to claim 14, wherein the individual has or is at risk of developing acute respiratory distress syndrome.

20. The method according to claim 14, wherein the individual has a viral disease such as COVID-19.

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