Modified poly(Β-amino ester) nanoparticle for highly efficient and specific delivery of nucleic acids to the lung capillary endothelium

Abstract

Polymers based on a poly(β-amino) ester (PBAE) backbone are provided, with improved targeting of the lung endothelium following parenteral delivery. The disclosed polymers are polymers of a primary amine (A2), a secondary amine (A1), a diacrylate (D) and at least one end-capping hydrophilic compound (C) such as amine. The polymer is halogenated (hereinafter, halogenated PBAE polymer), preferably, fluorinated. The hydrophobic PBAE backbone is end capped with a hydrophilic compound by covalent attachment of these capping agents to the PBAE backbones via Michael Addition to form a halogenated PBAE. The disclosed polymers can be used to encapsulate / incorporate an active agent for targeted delivery to lung endothelial cells.

Description

[0001] MODIFIED POLY(P-AMINO ESTER) NANOPARTICLE FOR HIGHLY EFFICIENT AND SPECIFIC DELIVERY OF NUCLEIC ACIDS TO THE LUNG CAPILLARY ENDOTHELIUM

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims benefit of U.S. Provisional Application No. 63 / 515,241 filed July 24, 2023. Application No. 63 / 515,241, filed July 24, 2023, is hereby incorporated herein by reference in its entirety.

[0004] FIELD OF THE INVENTION

[0005] This invention is generally in the field of poly(P-amino ester) nanoparticle delivery.

[0006] BACKGROUND OF THE INVENTION

[0007] Endothelial dysfunction contributes to a wide range of severe pulmonary vascular diseases such as Pulmonary hypertension, pneumonia, idiopathic pulmonary fibrosis, cystic obstructive pulmonary disease, bronchopulmonary dysplasia (BPD) and alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV). The transcription factor FOXF1 plays a key role in pulmonary vascular development and endothelial repair after lung injury. Mutations in the FOXF1 gene has been associated with lung ACDMPV, a fatal congenital lung disorder, hypoplasia, and paucity of alveolar capillaries in newborns and infants1-5. Nanoparticle delivery of FOXF1 has promise for ACDMPV treatment.

[0008] Recent studies have developed innovative drugs for treatment of chronic obstructive pulmonary disease (COPD)6, however, there is still a need for versatile platforms that can deliver gene pay loads and drugs to pulmonary endothelium with high efficiency and precision. Although viral vectors have been widely used for gene delivery in preclinical studies, there are still major concerns with the use of viral vectors such as high biosafety risks, adverse immune responses, low payload capacity, and limited clinical translation due to difficulties in vector production7-10. In recent years, non-viral gene delivery systems were developed to address these limitations for gene therapy.

[0009] Nanoparticle delivery systems have been shown to serve as effective non-viral gene delivery vehicles with great potential in biomedical applications, including cancer gene therapy and the development of SARS-CoV-2 vaccines11-15. Nanoparticle-based gene therapies for pulmonary diseases can usually be divided by their administration routes, including, pulmonary administration through airways and systemic administration through intravascular route. Pulmonary administration has been widely used because it delivers the therapeutics directly to the lung via the trachea, which can increase the local accumulation of nanoparticles. But this route is affected by mucous and epithelial barriers, therefore diverting the nanoparticles to the epithelium. For endothelial targeting, the nanoparticles are delivered via systemic administrations to avoid epithelial barrier. However, the first pass metabolism may cause high transfection efficiencies in other organs resulting in low nanoparticle delivery to the lung endothelium16. To treat pulmonary vascular disorders, it is necessary to develop a nanoparticle delivery system that can efficiently deliver the cargo to the lung while targeting only endothelial cells. A polymeric -based gene delivery system based on biological fatty acids and modified hyperbranched polyethylenimine (PEI), which is capable of delivering non-integrating plasmids to the lung endothelium without targeting other cell types in the adult lung was developed17 18. However, this nanoparticle system also targets endothelial cells of other organs, such as heart, liver and kidney4, limiting potential clinical applications of the nanoparticles in lung diseases. The nanoparticle delivery system for specific delivery to the lung endothelium without targeting is needed.

[0010] Poly([3-amino) esters (PB AEs) refers to a wide range of polymers that are synthesized using the Michael Addition of amine groups to diacrylates. Thus, PBAE refers to a polymer synthesized from an acrylate and an amine by Michael addition. PBAEs have been used in biomedical applications owing to their excellent biocompatibility and biodegradability19'22, including for gene delivery23'25. However, the PBAE nanoparticles used in these applications have shown limited route of delivery and / or requirement for co-delivery with other molecules such as lipid molecules. DNA and mRNAs and DNA delivery through pulmonary or systemic delivery using the polymer-lipid nanoparticles combined with PBAEs and lipid molecules via microfluidic devices have been disclosed26 29. Other applications have included PBAE polyplexes, which do not require extra lipid molecules or devices for their straightforward synthesis. However, these PBAE polyplexes required pulmonary administration to achieve high pulmonary accumulation2030. These PBAE polyplexes are inefficient in targeting endothelial cells after pulmonary (intra-tracheal) administration.

[0011] There is a need for nanoparticulate compositions and formulations that are specific for lung endothelial cells and can be delivered intravenously.

[0012] There is also a need for methods of treating subjects to specifically target lung endothelial cells.

[0013] BRIEF SUMMARY OF THE INVENTION

[0014] Polymers based on a poly(0-amino) ester (PBAE) backbone are provided, with improved targeting of the lung endothelium following parenteral delivery. The disclosed polymers are polymers of a primary amine (A2), a secondary amine (Al), a diacrylate (D) and at least one end-capping hydrophilic compound (C) such as amine. The polymer is halogenated (hereinafter, halogenated PB AE polymer), preferably, fluorinated. The disclosed polymer backbone can be represented by the general formula I:

[0015] (D -A2 -D-Al)n-; where D is a monomeric unit derived from an di acrylate, preferably, bisphenol A ethoxylate (EO / phenol) di acrylate.

[0016] Al is a monomeric unit derived from a secondary amine, preferably, 4,4’- Trimethylenedipiperidine and

[0017] A2 is a monomeric unit derived from a primary amine, preferably, 6-amino-l-hexanol; and n is an integer ranging 2 to about 10 in some forms, for example, n is 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0018] In some forms, the hydrophobic PBAE backbone is end capped with capping agen such (for example, hydrophilic compounds) by covalent attachment of the capping agents to the PBAE backbones via Michael Addition to form a halogenated PBAE, which can be presented by the general formula II:

[0019] Ha C (D A2 D-Al)n; where D, A and n are as presented above, C is derived from a hydrophilic compound such as an amine, for example polyethylenimine (PEI) or poly(ethylene glycol) amine (PEG amine) and Ha is derived from a halogenated compound.

[0020] The disclosed polymers can be used to encapsulate / incorporate an active agent for targeted delivery to the lung endothelium. In one form, the active agent is a nucleic acid active agent.

[0021] Methods for treating subjects in need thereof, are disclosed. The methods include administering to a subject in need thereof, a therapeutically effective amount of the disclosed polymers encapsulating / incorporating an active agent. In some forms, the subject is diagnosed with or is suspected of suffering from lung endothelial cell disfunction.

[0022] BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG. 1A-C. Overview of the PBAE nanoparticle synthesis and gene delivery to the pulmonary endothelium. (FIG. 1A) Schematic of the PBAE backbone synthesis with hydrophobic monomers (red) and the hydrophilic capping agents polyethylenimine (cyan), polyethylene glycol (blue) and fluorous ligands (purple); (FIG. IB) Schematic of the PBAEs nanoparticle structure. Amphiphilic PBAEs polymers are binding the nucleic acid to form nanoparticles with hydrophilic surfaces and hydrophobic cores. (FIG. 1C) The PBAE nanoparticles were synthesized to deliver DNA plasmid to pulmonary endothelial cells after intravenous injection. FIG. ID. shows an exemplary halogenated PBAE polymer, in which the halogen is fluorine. The halogenated polymer is shown as three fragments, with lines used to indicate where the fragments are connected.

[0024] FIG. 2A-2F. The effect of fluorination of PBAE polymer on the size, charge and transfection efficiency of the nanoparticles. (FIG. 2A) In vitro transfection efficiency of PBAE nanoparticles with different formulations and polymer to DNA mass ratios. (FIG. 2B) The influence of fluorous ligands on the in vitro transfection efficiency of the PBAE P13. (FIG. 2C) The influence of fluorous ligands on the in vitro transfection efficiency of the PBAE P22. (FIG. 2D) Gel electrophoresis analysis of plasmid DNA bound to the PBAE Pl, P21, P22 and P22-F1 at different polymer to DNA mass ratios. (FIG. 2E) The hydrodynamic diameter of the PBAE P22-F1 nanoparticles at different polymer to DNA mass ratios. (FIG. 2F) The zeta potential of the PBAE P22-F1 nanoparticles at different polymer to DNA mass ratios.

[0025] FIG. 3A-D. The P22-F1 PBAE nanoparticles exhibit the highest transfection efficiency in vitro. FIG. 3A. Flow cytometry was used to analyze HEK-293T cells 48 hours after transfection with PBAE-GFP plasmid complexes at nanoparticle to DNA mass ratio of 10:1. FIG. 3B The quantification of percentages of GFP-positive (transfected) cells was performed based on flow cytometry measurements. FIG. 3CThe quantification of GFP mean fluorescence intensity (MFI) of transfected cells was performed based on flow cytometry measurements. FIG. 3D.Cell viability profiles of transfected cells after treatment with different concentrations of nanoparticles.

[0026] FIG. 4A-D. The P22-F1 PBAE nanoparticles exhibit improved endothelial targeting in the lung tissue. FIG. 4A. Fluorescent dye labeling of PBAE nanoparticles. FIG. 4B. Flow cytometry analysis of the PBAE nanoparticle distribution (blue curve) in different respiratory cell types compared to the control (red curve). FIG. 4C.Juxtaposition of cell targeting efficiency from P21, P21-F1 and P22-F1 PBAE nanoparticles (n= 3 mice were used for each group), P < 0.0001 is ****, P < 0.01 is **.

[0027] FIG. 5. Juxtaposition of MFI from respiratory cells targeted with P21, P21-F1 or P22-F1 PBAE nanoparticles (n= 3 mice were used for each group), P < 0.0001 is ****, P < 0.01 is **. Abbreviations: Endo: Endothelium; Epi, Epithelium; Fibre, Fibroblast; Hema, Hematopoietic; Neg, Lineage negative.

[0028] FIG. 6A-6C. Flow cytometry analysis of nanoparticle targeting CAP1 and CAP2 lung capillary endothelial cells. (FIG. 6A) Gating strategy to identity CAP1 and CAP2 lung capillary endothelial cells. (FIG. 6B) Histogram analysis of the P22-F1 PBAE nanoparticle targeting CAP1 and CAP2 endothelial cells (green curve) against the uninjected control (red curve). (FIG. 6C) Comparison of DyLight 650 fluorescence intensity using histograms cell uptake of the P22- F1 PBAE nanoparticles compared in CAP1 (green curve) and CAP2 (blue curve) lung capillary endothelial cells. (FIG. 6D) Juxtaposition of the targeting efficiency as percentages of cells targeted by the nanoparticles. (FIG. 6E) Juxtaposition of MFI in CAP1 and CAP2 cells.

[0029] FIG. 7A-7C. Toxicity studies of the P22-F1 PBAE nanoparticles. The peripheral blood was collected from mice 5 days after a single intravenous injection of the P22-F1 PBAE nanoparticles. (FIG. 7A) The peripheral blood was assayed for major parameters in clinical chemistry, including those related to the liver and kidney metabolic panels, n= 5 mice per group. (FIG. 7B) Major parameters in hematology after analysis of peripheral blood. (FIG. 7C) Body weight changes after nanoparticle treatment.

[0030] FIG. 8A-8C. In vivo transfection studies of P22-F1 PBAE nanoparticles. (FIG. 8A) Quantification of luminescence from different organs (n =3 mice per group (FIG. 8B) Flow cytometry shows mCherry reporter activity in endothelial cells isolated from different murine organs. Blue curve: P22-F1 PBAE nanoparticles; Red curve: untreated control. (FIG. 48C) The bar graph shows the mCherry transfection efficiency in endothelial cells from different organs (n =3 mice per group).

[0031] FIG. 9A-9C. Fluorination influences in vivo biodistribution of the PBAE nanoparticles. (FIG. 9A) The hydrodynamic diameter of D1A2C1(P1), D1A1A2C1(P21), D1A1A2C1C2(P22), and DI A1A2C1C2F1 (P22-F1) nanoparticles at different NP / DNA mass ratios. (FIG. 9B) The zeta potential of D1A2C1(P1), D1A1A2C1(P21), D1A1A2C1C2(P22), and D1A1A2C1C2F1 (P22-F1) nanoparticles at different NP / DNA mass ratios. (FIG. 9C) Bar graph shows the quantification of luminescence in the lung and spleen, (n =3 mice per group, ns is not significant, * is P < 0.05, ** is P < 0.01, *** is P < 0.001, **** is P < 0.0001).

[0032] FIG. 10A-10D. Synthesis of the PBAE polymers (FIG. 10A) A library of materials used for PBAEs synthesis; (FIG. 10B) Michael addition reactions between the diacrylate and amine groups. (FIG. 10C) Fluorescent dye labeling through the reaction between the NHS ester group and amine group. (FIG. 10D) Fluorination reaction between the amine group and fluorous ligands.

[0033] FIG. 11A-11E show!H NMR spectrums of (FIG. 11A) Bisphenol A ethoxylate diacrylate; (FIG. 11B) 4,4’ -Trimethylenedipiperidine; (FIG. 11C) 6-amino-l -hexanol; (FIG. 11D) the PBAE backbone; (FIG. HE) Fluorinated PER PEG capped PBAEs (P22-F1). FIG. 12 shows Gating strategy for the identification of cell lineage populations (CD31+ CD45- as endothelial, CD45+ as hematopoietic, CD326+ CD31- CD45- as epithelium, CD140a+ CD31- CD45- as fibroblast) from whole-lung single cell suspension.

[0034] FIG. 13. The average number of particles per cell for D1A2C1(P1), D1A1A2C1(P21), D1A1A2C1C2(P22), and D1A1A2C1C2F1 (P22-F1) nanoparticles from the lung, spleen, liver, kidney, and heart.

[0035] FIG. 14 is a Histogram analysis of PB AE nanoparticle targeted (blue curve) to different cell types compared to the uninjected control (red curve) at different NP / DNA ratios.

[0036] FIG. 15 is a Histogram analysis of mCherry transfection in endothelial cells of internal organs (lung, heart, liver, spleen, and kidney) (top panels). The DyLight 650 distribution in endothelial cells of internal organs (lung, heart, liver, spleen, and kidney) (bottom panels). Blue curve shows the nanoparticle-associated fluorescence. Red curve shows the control.

[0037] FIG. 16. mCherry transfection and DyLight 650 distribution in endothelial cells of internal organs (lung, heart, liver, spleen, kidney). Blue, nanoparticle injected mice; Red, control mice.

[0038] FIG. 17A-17E. Targeted delivery of mRNA by using the PBAE nanoparticles to pulmonary endothelial cells. (FIG. 17A) Luminescent image shows the biodistribution of luciferase expression in dissected mouse organs, the organs were harvest 4 hours after mRNA- PBAE nanoparticle injection for ex-vivo IVIS. (FIG. 17B) Quantification of luminescence from different organs. (FIG. 17C) Flow cytometry analysis of the fluorescent dye labeled mRNA- PBAE nanoparticle distribution (blue curve) in different respiratory cell types compared to the control (red curve). (FIG. 17D-17E) Bar graph of cell targeting efficiency and mean fluorescence intensity (MFI) of the fluorescent dye labeled mRNA-PBAE nanoparticle. Overall, FIG. 17A-17E shows that the nanoparticles can specifically deliver mRNAs to endothelial cells of the lung in vivo.

[0039] FIG. 18A-18C. TANFE small molecule compound encapsulated in PBAE nanoparticles exhibits endothelial cell (EC) targeting in mouse lungs. 400 ng or 1 pg of TANFE compound labelled with fluorescent Cyanine5 dye was encapsulated in nanoparticles synthesized using poly (p-amino) esters (PBAEs) backbone and capped with polyethylene glycol amine. Nanoparticle encapsulated drug was delivered in the WT C57BL6 mice lungs using retro-orbital injection. (FIG. 18A) Flow cytometry analysis of drug distribution in different cell types in lung tissue of mice injected with nanoparticle drug combination (blue curve) or empty nanoparticles (red curve). (FIG. 18B-18C) Mean fluorescent intensity (MFI) and percentage of cells receiving the nanoparticle-drug combination. Data indicate 90% of lung endothelial cells receiving nanoparticle encapsulated TANFE compound at both the doses. This was supported by higher MFI in endothelial cells as compared to Immune cells, Fibroblast, Pericytes, and Epithelial cells. Data indicated by Mean ± SEM n= 3 / group. Overall, FIG. 18A-18C shows that the nanoparticles can specifically deliver small molecule compounds to endothelial cells of the lung in vivo.

[0040] DETAILED DESCRIPTION OF THE INVENTION

[0041] The disclosed compositions and methods are based on the discovery of a facile synthesis of a polymer with hydrophobic PBAE backbones, which is capped with the hydrophilic low molecular polyethylenimine (PEI) and poly(ethylene glycol) (PEG). This amphiphilic structure provides excellent nanoparticle colloidal stability and cationic density for gene encapsulation and delivery. Application of fluorous ligands to the nanoparticles improved the performance of the PBAE nanoparticles and achieve lung-specific delivery via the systemic intravenous route. IVIS whole body imaging of experimental mice and flow cytometry analysis of multiple organs demonstrated specific gene delivery to lung microvascular endothelial cells without targeting other organs. Fluorination of PBAE was required for lung-specific endothelial delivery since the nanoparticles without fluorination did not exhibit the lung specificity. Altogether, fluorinated PBAE nanoparticles have promise for gene therapying pulmonary diseases associated with endothelial disfunction.

[0042] I. DEFINITIONS

[0043] "About" as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, in certain embodiments ±5%, in certain embodiments ±1%, in certain embodiments ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0044] The terms “bioactive agent” and “active agent”, as used interchangeably herein, include, without limitation, physiologically or pharmacologically active substances that act locally or systemically in the body. A bioactive agent is a substance used for the treatment (e.g., therapeutic agent), prevention (e.g., prophylactic agent), diagnosis (e.g., diagnostic agent), cure or mitigation of disease or illness, a substance which affects the structure or function of the body, or pro-drugs, which become biologically active or more active after they localize in a predetermined physiological environment.

[0045] As used herein, “controlled release” refers to a release profile of an agent for which the agent release characteristics of time course and / or location are chosen to accomplish therapeutic or convenience objectives not offered by conventional topical formulations. “Hydrophobic,” as used herein, refers to the property of lacking affinity for, or even repelling water. For example, the more hydrophobic a polymer (or polymer segment), the more that polymer (or polymer segment) tends to not dissolve in, not mix with, or not be wetted by water.

[0046] The terms "incorporated" and "encapsulated" refers to incorporating, formulating, or otherwise including an active agent into and / or onto a composition that allows for release, such as sustained release, of such agent in the desired application. The terms contemplate any manner by which a therapeutic agent or other material is incorporated into a polymer matrix, including for example: The terms "incorporated" and "encapsulated" refers to incorporating, formulating, or otherwise including an active agent into and / or onto a composition that allows for release, such as sustained release, of such agent in the desired application. The terms contemplate any manner by which a therapeutic agent or other material is incorporated into a polymer matrix, including for example:

[0047] The phrases "parenteral administration" and "administered parenterally" are art- recognized terms, and include modes of administration other than enteral and topical administration, typically by injection, and can include intravenous, intramuscular, intrapleural, intravascular, intradermal, intraperitoneal, transtracheal, and subcutaneous injection and infusion.

[0048] The phrase “pharmaceutically acceptable” refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues, organs, and / or bodily fluids of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio.

[0049] The phrase "pharmaceutically acceptable carrier" refers to pharmaceutically acceptable materials, compositions or vehicles, such as a liquid or solid filler, diluent, solvent or encapsulating material involved in carrying or transporting any subject composition, from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of a subject composition and not injurious to the patient.

[0050] The phrase “sustained release" refers to release of a substance over an extended period of time, in contrast to a bolus type administration in which the majority of the substance is made biologically available at one time.

[0051] The term “treating” preventing a disease, disorder or condition from occurring in an animal which may be predisposed to the disease, disorder and / or condition but has not yet been diagnosed as having it; inhibiting the disease, disorder or condition, e.g., impeding its progress; and relieving the disease, disorder, or condition, e.g., causing regression of the disease, disorder and / or condition. Treating the disease or condition includes ameliorating at least one symptom of the particular disease or condition, even if the underlying pathophysiology is not affected, such as treating the pain of a subject by administration of an analgesic agent even though such agent does not treat the cause of the pain.

[0052] The term "therapeutically effective amount" refers to an amount of the therapeutic agent that, when incorporated into and / or onto particles described herein, produces some desired effect at a reasonable benefit / risk ratio applicable to any medical treatment. The effective amount may vary depending on such factors as the disease or condition being treated, the particular targeted constructs being administered, the size of the subject, or the severity of the disease or condition. One of ordinary skill in the art may empirically determine the effective amount of a particular compound without necessitating undue experimentation. In some embodiments, the term “effective amount” refers to an amount of a therapeutic agent or prophylactic agent to reduce or diminish the symptoms of one or more diseases or disorders resulting from lung endothelial dysfunction.

[0053] IL COMPOSITIONS

[0054] A. Polymers

[0055] The disclosed polymers are based on a poly(P-amino) ester (PBAE) back, are polymers of a primary amine (A2), a secondary amine (Al), a diacrylate (D) and at least one end-capping hydrophilic compound (C) such as amine. The polymer is halogenated (hereinafter, halogenated PBAE polymer), preferably, fluorinated. The disclosed polymer backbone can be represented by the general formula I:

[0056] -(D-A2-D-A1),,-;

[0057] Where D is a monomeric unit derived from an acrylate, preferably, bisphenol A ethoxylate (EO / phenol) diacrylate.

[0058] Al is a monomeric unit derived from a secondary amine, preferably, 4,4’- Trimethylenedipiperidine and

[0059] A2 is a monomeric unit derived from a primary amine, preferably, 6-amino-l -hexanol; and n is an integer ranging 2 to about 10 in some forms, for example, n is 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some forms, n is preferably 2 or 3, where the backbone chain contains 6 diacrylate and 5 amine monomers.

[0060] An exemplary PBAE backbone is shown below.

[0061] The polymer is end capped with a hydrophilic compound (C1 / C2) such as amine, for example polyethylenimine (PEI; Cl) or poly(ethylene glycol) amine (PEG amine;C2), is halogenated, preferably, fluorinated. Examples of suitable compounds for fluorination include, but are not limited to Fl (Pentadecafluorooctanoyl chloride); Pentafluoropropionic anhydride (F2), 1H,1H,2H,2H-Perfluorodecyl acrylate (F3) Pentafluoropropionic anhydride (F4), and heptafluorobutyric anhydride.

[0062] In some forms, the hydrophobic PBAE backbone is end capped with a hydrophilic compound by covalent attachment of these capping agents to the PBAE backbones via Michael Addition to form a halogenated PBAE, which can be presented by the general formula II:

[0063] Ha C-(D A2 D-Al)n-;

[0064] Where D, A and n are as presented above, C is derived from a hydrophilic compound such as an amine, for example polyethylenimine (PEI) or poly(ethylene glycol) amine (PEG amine) and Ha is derived from a halogenated compound.

[0065] An exemplary halogenated PBAE polymer is shown in FIG. ID, in which the halogen is fluorine.

[0066] In some forms, the PBAE backbone is not made from the combination of hexadecylamine, 1,4-Butanediol diacrylate and 4-amino butanol. i. Components

[0067] Acrylate (D)

[0068] Suitable acrylates that can be used in the PBAE backbone include, but at not limited to, bisphenol A ethoxylate diacrylate, and 1 ,4-butanediol diacrylate. In some forms, D is not bisphenol A glycerolate diacrylate.

[0069] Secondary amine (Al )

[0070] Suitable secondary amines that can be used in the PBAE backbone include, but are not limited to ,4’-Trimethylenedipiperidine, and hexadecylamine.

[0071] Primary Amine (A2)

[0072] Suitable primary amines that can be used in the PBAE backbone include, but are not limited to is 6-amino-l -hexanol, 5-amino-l pentanol, 4 amino-1 butanol, 1 -dodecylamine, 4-(2- amino methyl) morpholine, and 3 amino-1 propanol. Capping agent

[0073] The capping agent is a hydrophilic compound (C1 / C2) such as amine, for example polyethylenimine (PEI;), for example, Polyethyleneimine, branched Mw 800 LS / 600 GPC; In some forms the capping agent includes poly(ethylene glycol) amine (PEG amine), for example, poly(ethylene glycol) amine (PEG, Mn -2000), and Methoxypolyethylene glycol amine Mw 1000. Other suitable agents include, but are not limited to 1,3 -diaminopropane, poly-ethylene oxide)4-bis-amine, and diethylenetriamine.

[0074] In some forms, the disclosed polymers include one capping agent. In a preferred agent the disclosed polymers include one more than one capping agent. In some forms, PEG amine is incorporated into the polymer synthesis composition at about 0.5 to about 10%, preferably, at about 2 to about 8% and more preferably at about 3 to about 6%. Other capping agents that can be used include, but are not limited to Polyethyleneimine, branched Mw 1800; Polyethyleneimine, branched Mw 25000; Polyethylenimine, linear Mw 10000;Polyethylenimine, linear Mw2100; Polyethylenimine, linear Mw 5000; Methoxypolyethylene glycol amine Mw 1000; Methoxypolyethylene glycol amine, Mw 20000; Methoxypolyethylene glycol amine, Mw 750; Methoxypolyethylene glycol propyl amine, MwlOOO; and Poly(ethylene glycol) methyl ether amine, Mw 500.

[0075] Halogen Ligands

[0076] The disclosed polymers include a halogen ligand, preferably, fluoride Exemplary compounds that can be used to provide the fluoride ligan include, but at not limited to Pentadecafluorooctanoyl chloride, Pentafluoropropionic anhydride, 1H,1H,2H,2H- Perfluorodecyl acrylate and Pentafluoropropionic anhydride. ii. Method of making

[0077] The PBAE polymer backbones were synthesized using a step-growth polymerization via Michael addition between the diacrylates and amines. The hydrophobic PBAE backbones were further capped with hydrophilic capping agents to form amphiphilic PBAE polymer chains. FIG. 1A shows a schematic of the PBAE backbone synthesis with hydrophobic monomers (red) and the hydrophilic capping agents polyethylenimine (cyan), polyethylene glycol (blue) and fluorous ligands (purple).

[0078] In some forms, the ratio of A1:A2 <1, i.e., the amount of Al should not be more than the amount of A2 in the composition used to synthesize the PBAE backbone. iii. Nanoparticles

[0079] The disclosed halogenated PBAE polymers can self-assemble with the agent to be delivered (payload) such as a nucleic acid and form stable nanoparticles in aqueous medium. A schematic of the PBAEs nanoparticle structure is shown in FIG. 1C. Amphiphilic PBAEs polymers are binding the nucleic acid to form nanoparticles with hydrophilic surfaces and hydrophobic cores.

[0080] In some forms, the nanoparticle to payload ratio>10, greater than 20, greater than 30, greater than 30, greater than 40 and preferably, at least 50 or more.

[0081] In some forms, the hydrodynamic size of the nanoparticles is < 100 nM. The nanoparticle preferably does not include a lipid as part of the nanoparticle structure, i.e., the nanoparticle is not formed of polymer and lipid. iv. Active Agents

[0082] One or more active agents may be formulated alone or with excipients or encapsulated on, in or incorporated into the particles. Active agents include therapeutic, prophylactic, nutraceutical and diagnostic agents. Any suitable agent may be used. These include organic compounds, inorganic compounds, proteins, polysaccharides, nucleic acids or other materials that can be incorporated using standard techniques.

[0083] Agents to be delivered include therapeutic, nutritional, diagnostic, and prophylactic compounds. Proteins, peptides, carbohydrates, polysaccharides, nucleic acid molecules, and organic molecules, as well as diagnostic agents, can be delivered. The preferred materials to be incorporated are oligonucleotide active agents (including DNA, RNAs including mRNAs, antisense, siRNA, miRNA, anti-miRNA, piRNA, aptamers, ribozymes, external guide sequences for ribonuclease P, and triplex forming agents such as tcPNAs). In some embodiments, the active agent is a vector, plasmid, or other polynucleotide encoding an oligonucleotide such as those discussed above.

[0084] Therapeutic agents include antibiotics, antivirals, anti-parasites (helminths, protozoans), anti-cancer (referred to herein as "chemotherapeutics", including cytotoxic drugs such as doxorubicin, cyclosporine, mitomycin C, cisplatin and carboplatin, BCNU, 5FU, methotrexate, adriamycin, camptothecin, epothilones A-F, and taxol), antibodies and bioactive fragments thereof (including humanized, single chain, and chimeric antibodies), antigen and vaccine formulations, peptide drugs, anti-inflammatories, and nutraceuticals such as vitamins.

[0085] Representative classes of diagnostic materials include paramagnetic molecules, fluorescent compounds, magnetic molecules, and radionuclides. Exemplary materials include, but are not limited to, metal oxides, such as iron oxide, metallic particles, such as gold particles, etc. Biomarkers can also be conjugated to the surface for diagnostic applications.

[0086] Active agents include synthetic and natural proteins (including enzymes, peptidehormones, receptors, growth factors, antibodies, signaling molecules), and synthetic and natural nucleic acids (including RNA, DNA, anti-sense RNA, triplex DNA, inhibitory RNA (RNAi), and oligonucleotides), and biologically active portions thereof. Suitable active agents have a size greater than about 1,000 Da for small peptides and polypeptides, more typically at least about 5,000 Da and often 10,000 Da or more for proteins. Nucleic acids are more typically listed in terms of base pairs or bases (collectively "bp"). Nucleic acids with lengths above about 10 bp are typically used in the present method. More typically, useful lengths of nucleic acids for probing or therapeutic use will be in the range from about 20 bp (probes; inhibitory RNAs, etc.) to tens of thousands of bp for genes and vectors. The active agents may also be hydrophilic molecules, preferably having a low molecular weight.

[0087] B. Formulations

[0088] The compositions described herein can be formulated for enteral, parenteral, topical, or pulmonary administration. The compositions can be combined with one or more pharmaceutically acceptable carriers and / or excipients that are considered safe and effective and may 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 or ingredients.

[0089] Parenteral Formulations

[0090] For example, parenteral administration may include administration to a patient intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intravitreally, intratumorally, intramuscularly, subcutaneously, subconjunctivally, intravesicularly, intrapericardially, intraumbilically, by injection, and by infusion.

[0091] Parenteral formulations can be prepared as aqueous compositions using techniques is known in the art. Typically, such compositions can be prepared as injectable formulations, for example, solutions or suspensions; solid forms suitable for using to prepare solutions or suspensions upon the addition of a reconstitution medium prior to injection; emulsions, such as water-in-oil (w / o) emulsions, oil-in-water (o / w) emulsions, and microemulsions thereof, liposomes, or emulsomes.

[0092] The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, one or more polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), oils, such as vegetable oils (e.g., peanut oil, corn oil, sesame oil, etc.), and combinations thereof. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and / or by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride.

[0093] Solutions and dispersions of the active compounds as the free acid or base or pharmacologically acceptable salts thereof can be prepared in water or another solvent or dispersing medium suitably mixed with one or more pharmaceutically acceptable excipients including, but not limited to, surfactants, dispersants, emulsifiers, pH modifying agents, viscosity modifying agents, and combination thereof.

[0094] Suitable surfactants may be anionic, cationic, amphoteric or nonionic surface-active agents. Suitable anionic surfactants include, but are not limited to, those containing carboxylate, sulfonate and sulfate ions. Examples of anionic surfactants include sodium, potassium, ammonium of long chain alkyl sulfonates and alkyl aryl sulfonates such as sodium dodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodium dodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodium bis-(2-ethylthioxyl)-sulfosuccinate; and alkyl sulfates such as sodium lauryl sulfate. Cationic surfactants include, but are not limited to, quaternary ammonium compounds such as benzalkonium chloride, benzethonium chloride, cetrimonium bromide, stearyl dimethylbenzyl ammonium chloride, polyoxyethylene and coconut amine. Examples of nonionic surfactants include ethylene glycol monostearate, propylene glycol myristate, glyceryl monostearate, glyceryl stearate, polyglyceryl-4-oleate, sorbitan acylate, sucrose acylate, PEG- 150 laurate, PEG-400 monolaurate, polyoxyethylene monolaurate, polysorbates, polyoxyethylene octylphenylether, PEG- 1000 cetyl ether, polyoxyethylene tridecyl ether, polypropylene glycol butyl ether, Poloxamer® 401, stearoyl monoisopropanolamide, and polyoxyethylene hydrogenated tallow amide. Examples of amphoteric surfactants include sodium N-dodecyl-.beta.-alanine, sodium N-lauryl-.beta.- iminodipropionate, myristoamphoacetate, lauryl betaine and lauryl sulfobetaine.

[0095] The formulation can contain a preservative to prevent the growth of microorganisms. Suitable preservatives include, but are not limited to, parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. The formulation may also contain an antioxidant to prevent degradation of the active agent(s).

[0096] The formulation is typically buffered to a pH of 3-8 for parenteral administration upon reconstitution. Suitable buffers include, but are not limited to, phosphate buffers, acetate buffers, and citrate buffers. Water-soluble polymers are often used in formulations for parenteral administration. Suitable water-soluble polymers include, but are not limited to, polyvinylpyrrolidone, dextran, carboxymethylcellulose, and polyethylene glycol.

[0097] Sterile injectable solutions can be prepared by incorporating the active compounds in the required amount in the appropriate solvent or dispersion medium with one or more of the excipients listed above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The powders can be prepared in such a manner that the particles are porous in nature, which can increase dissolution of the particles. Methods for making porous particles are well known in the art.

[0098] The parenteral formulations described herein can be formulated for controlled release including immediate release, delayed release, extended release, pulsatile release, and combinations thereof.

[0099] Pulmonary Formulations

[0100] In one embodiment, the compounds are formulated for pulmonary delivery, such as intranasal administration or oral inhalation.

[0101] The respiratory tract is the structure involved in the exchange of gases between the atmosphere and the blood stream. The lungs are branching structures ultimately ending with the alveoli where the exchange of gases occurs. The alveolar surface area is the largest in the respiratory system and is where drug absorption occurs. The alveoli are covered by a thin epithelium without cilia or a mucus blanket and secrete surfactant phospholipids. The respiratory tract encompasses the upper airways, including the oropharynx and larynx, followed by the lower airways, which include the trachea followed by bifurcations into the bronchi and bronchioli. The upper and lower airways are called the conducting airways. The terminal bronchioli then divide into respiratory bronchiole, which then lead to the ultimate respiratory zone, the alveoli, or deep lung. The deep lung, or alveoli, is the primary target of inhaled therapeutic aerosols for systemic drug delivery.

[0102] Pulmonary administration of therapeutic compositions including low molecular weight drugs has been observed, for example, beta- androgenic antagonists to treat asthma. Other therapeutic agents that are active in the lungs have been administered systemically and targeted via pulmonary absorption. Nasal delivery is considered to be a promising technique for administration of therapeutics for the following reasons: the nose has a large surface area available for drug absorption due to the coverage of the epithelial surface by numerous microvilli, the sub epithelial layer is highly vascularized, the venous blood from the nose passes directly into the systemic circulation and therefore avoids the loss of drug by first-pass metabolism in the liver, it offers lower doses, more rapid attainment of therapeutic blood levels, quicker onset of pharmacological activity, fewer side effects, high total blood flow per cm3, porous endothelial basement membrane, and it is easily accessible.

[0103] Carriers for pulmonary formulations can be divided into those for dry powder formulations and for administration as solutions. Aerosols for the delivery of therapeutic agents to the respiratory tract are known in the art. Aerosols can be produced using standard techniques, such as ultrasonication or high-pressure treatment. For administration via the upper respiratory tract, the formulation can be formulated into a solution, e.g., water or isotonic saline, buffered or un-buffered, or as a suspension, for intranasal administration as drops or as a spray. Preferably, such solutions or suspensions are isotonic relative to nasal secretions and of about the same pH, ranging e.g., from about pH 4.0 to about pH 7.4 or, from pH 6.0 to pH 7.0. Buffers should be physiologically compatible and include, simply by way of example, phosphate buffers. For example, a representative nasal decongestant is described as being buffered to a pH of about 6.2. One skilled in the art can readily determine a suitable saline content and pH for an innocuous aqueous solution for nasal and / or upper respiratory administration.

[0104] Preferably, the aqueous solution is water, physiologically acceptable aqueous solutions containing salts and / or buffers, such as phosphate buffered saline (PBS), or any other aqueous solution acceptable for administration to an animal or human. Such solutions are well known to a person skilled in the art and include, but are not limited to, distilled water, de-ionized water, pure or ultrapure water, saline, phosphate-buffered saline (PBS). Other suitable aqueous vehicles include, but are not limited to, Ringer's solution and isotonic sodium chloride. Aqueous suspensions may include suspending agents such as cellulose derivatives, sodium alginate, polyvinyl-pyrrolidone and gum tragacanth, and a wetting agent such as lecithin. Suitable preservatives for aqueous suspensions include ethyl and n-propyl p-hydroxybenzoate.

[0105] Solvents that are low toxicity organic (i.e. nonaqueous) class 3 residual solvents, such as ethanol, acetone, ethyl acetate, tetrahydofuran, ethyl ether, and propanol may be used for the formulations. The solvent is selected based on its ability to readily aerosolize the formulation. The solvent should not detrimentally react with the compounds. An appropriate solvent should be used that dissolves the compounds or forms a suspension of the compounds. The solvent should be sufficiently volatile to enable formation of an aerosol of the solution or suspension. Additional solvents or aerosolizing agents, such as freons, can be added as desired to increase the volatility of the solution or suspension.

[0106] In one embodiment, compositions may contain minor amounts of polymers, surfactants, or other excipients well known to those of the art. In this context, "minor amounts" means no excipients are present that might affect or mediate uptake of the compounds in the lungs and that the excipients that are present are present in amount that do not adversely affect uptake of compounds in the lungs.

[0107] Dry lipid powders can be directly dispersed in ethanol because of their hydrophobic character. For lipids stored in organic solvents such as chloroform, the desired quantity of solution is placed in a vial, and the chloroform is evaporated under a stream of nitrogen to form a dry thin film on the surface of a glass vial. The film swells easily when reconstituted with ethanol. To fully disperse the lipid molecules in the organic solvent, the suspension is sonicated. Nonaqueous suspensions of lipids can also be prepared in absolute ethanol using a reusable PARI LC Iet+ nebulizer (PARI Respiratory Equipment, Monterey, CA).

[0108] Dry powder formulations ("DPFs") with large particle size have improved flowability characteristics, such as less aggregation, easier aerosolization, and potentially less phagocytosis. Dry powder aerosols for inhalation therapy are generally produced with mean diameters primarily in the range of less than 5 microns, although a preferred range is between one and ten microns in aerodynamic diameter. Large "carrier" particles (containing no drug) have been codelivered with therapeutic aerosols to aid in achieving efficient aerosolization among other possible benefits.

[0109] Polymeric particles may be prepared using single and double emulsion solvent evaporation, spray drying, solvent extraction, solvent evaporation, phase separation, simple and complex coacervation, interfacial polymerization, and other methods well known to those of ordinary skill in the art. Particles may be made using methods for making microspheres or microcapsules known in the art. The preferred methods of manufacture are by spray drying and freeze drying, which entails using a solution containing the surfactant, spraying to form droplets of the desired size, and removing the solvent.

[0110] The particles may be fabricated with the appropriate material, surface roughness, diameter and tap density for localized delivery to selected regions of the respiratory tract such as the deep lung or upper airways. For example, higher density or larger particles may be used for upper airway delivery. Similarly, a mixture of different sized particles, provided with the same or different EGS may be administered to target different regions of the lung in one administration.

[0111] Formulations for pulmonary delivery include unilamellar phospholipid vesicles, liposomes, or lipoprotein particles. Formulations and methods of making such formulations containing nucleic acid are well known to one of ordinary skill in the art. Liposomes are formed from commercially available phospholipids supplied by a variety of vendors including Avanti Polar Lipids, Inc. (Birmingham, Ala.). In one embodiment, the liposome can include a ligand molecule specific for a receptor on the surface of the target cell to direct the liposome to the target cell.

[0112] Enteral Formulation

[0113] Various particular formulation strategies have been developed for enteral delivery of active agents including nucleic acids. Reviewed in O’ Driscoll, et al., European Journal of Pharmaceutical Sciences, 133:190-204 (2019). In order to permeate the mucus gel layer in high quantities the drug delivery system has to be small enough - preferably <100 nm - and has to be mucoinert. Nanoparticles formed of nucleic acid-based therapeutics and cationic polymers exhibiting in combination a negative zeta potential seem to fulfill these demands. For small nucleic acid-based drugs that are supposed to reach the systemic circulation, permeation enhancers either making the cell membrane leakier or opening up tight junctions have already shown potential in various studies (Reviewed in O’Driscoll, et al., European Journal of Pharmaceutical Sciences, 133:190-204 (2019)).

[0114] Suitable oral dosage forms include tablets, capsules, solutions, suspensions, syrups, and lozenges. Tablets can be made using compression or molding techniques well known in the art. Gelatin or non-gelatin capsules can prepared as hard or soft capsule shells, which can encapsulate liquid, solid, and semi-solid fill materials, using techniques well known in the art. Busignies, et al., Compression of Vectors for Small Interfering RNAs Delivery: Toward Oral Administration of siRNA Lipoplexes in Tablet Forms. Mol Pharm. 2020;17(4):1159-1169 explored successful tableting of compositions containing nucleic acids.

[0115] Formulations may be prepared using a pharmaceutically acceptable carrier. As generally used herein “carrier” includes, but is not limited to, diluents, preservatives, binders, lubricants, disintegrators, swelling agents, fillers, stabilizers, and combinations thereof.

[0116] Carrier also includes all components of the coating composition, which may include plasticizers, pigments, colorants, stabilizing agents, and glidants.

[0117] Examples of suitable coating materials include, but are not limited to, cellulose polymers such as cellulose acetate phthalate, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate and hydroxypropyl methylcellulose acetate succinate; polyvinyl acetate phthalate, acrylic acid polymers and copolymers, and methacrylic resins that are commercially available under the trade name EUDRAGIT® (Roth Pharma, Westerstadt, Germany), zein, shellac, and polysaccharides.

[0118] Additionally, the coating material may contain conventional carriers such as plasticizers, pigments, colorants, glidants, stabilization agents, pore formers and surfactants.

[0119] “Diluents”, also referred to as "fillers," are typically necessary to increase the bulk of a solid dosage form so that a practical size is provided for compression of tablets or formation of beads and granules. Suitable diluents include, but are not limited to, dicalcium phosphate dihydrate, calcium sulfate, lactose, sucrose, mannitol, sorbitol, cellulose, microcrystalline cellulose, kaolin, sodium chloride, dry starch, hydrolyzed starches, pregelatinized starch, silicone dioxide, titanium oxide, magnesium aluminum silicate and powdered sugar.

[0120] “Binders” are used to impart cohesive qualities to a solid dosage formulation, and thus ensure that a tablet or bead or granule remains intact after the formation of the dosage forms. Suitable binder materials include, but are not limited to, starch, pregelatinized starch, gelatin, sugars (including sucrose, glucose, dextrose, lactose and sorbitol), polyethylene glycol, waxes, natural and synthetic gums such as acacia, tragacanth, sodium alginate, cellulose, including hydroxypropylmethylcellulose, hydroxypropylcellulose, ethylcellulose, and veegum, and synthetic polymers such as acrylic acid and methacrylic acid copolymers, methacrylic acid copolymers, methyl methacrylate copolymers, aminoalkyl methacrylate copolymers, polyacrylic acid / polymethacrylic acid and polyvinylpyrrolidone.

[0121] “Lubricants” are used to facilitate tablet manufacture. Examples of suitable lubricants include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, glycerol behenate, polyethylene glycol, talc, and mineral oil.

[0122] “Disintegrants” are used to facilitate dosage form disintegration or "breakup" after administration, and generally include, but are not limited to, starch, sodium starch glycolate, sodium carboxymethyl starch, sodium carboxymethylcellulose, hydroxypropyl cellulose, pregelatinized starch, clays, cellulose, alginine, gums or cross linked polymers, such as crosslinked PVP (Polyplasdone® XL from GAF Chemical Corp).

[0123] “Stabilizers” are used to inhibit or retard drug decomposition reactions, which include, by way of example, oxidative reactions. Suitable stabilizers include, but are not limited to, antioxidants, butylated hydroxytoluene (BHT); ascorbic acid, its salts and esters; Vitamin E, tocopherol and its salts; sulfites such as sodium metabisulphite; cysteine and its derivatives; citric acid; propyl gallate, and butylated hydroxyanisole (BHA). • Controlled Release Enteral Formulations

[0124] Oral dosage forms, such as capsules, tablets, solutions, and suspensions, can for formulated for controlled release. For example, the one or more compounds and optional one or more additional active agents can be formulated into nanoparticles, microparticles, and combinations thereof, and encapsulated in a soft or hard gelatin or non-gelatin capsule or dispersed in a dispersing medium to form an oral suspension or syrup. The particles can be formed of the drug and a controlled release polymer or matrix. Alternatively, the drug particles can be coated with one or more controlled release coatings prior to incorporation in to the finished dosage form.

[0125] In another embodiment, the one or more compounds and optional one or more additional active agents are dispersed in a matrix material, which gels or emulsifies upon contact with an aqueous medium, such as physiological fluids. In the case of gels, the matrix swells entrapping the active agents, which are released slowly over time by diffusion and / or degradation of the matrix material. Such matrices can be formulated as tablets or as fill materials for hard and soft capsules.

[0126] In still another embodiment, the one or more compounds, and optional one or more additional active agents are formulated into a sold oral dosage form, such as a tablet or capsule, and the solid dosage form is coated with one or more controlled release coatings, such as a delayed release coatings or extended release coatings. The coating or coatings may also contain the compounds and / or additional active agents.

[0127] (1 ) Extended release dosage forms

[0128] The extended release formulations are generally prepared as diffusion or osmotic systems, which are known in the art. A diffusion system typically consists of two types of devices, a reservoir and a matrix, and is well known and described in the art. The matrix devices are generally prepared by compressing the drug with a slowly dissolving polymer carrier into a tablet form. The three major types of materials used in the preparation of matrix devices are insoluble plastics, hydrophilic polymers, and fatty compounds. Plastic matrices include, but are not limited to, methyl acrylate-methyl methacrylate, polyvinyl chloride, and polyethylene. Hydrophilic polymers include, but are not limited to, cellulosic polymers such as methyl and ethyl cellulose, hydroxyalkylcelluloses such as hydroxypropyl-cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and Carbopol® 934, polyethylene oxides and mixtures thereof. Fatty compounds include, but are not limited to, various waxes such as carnauba wax and glyceryl tristearate and wax-type substances including hydrogenated castor oil or hydrogenated vegetable oil, or mixtures thereof. In certain preferred embodiments, the plastic material is a pharmaceutically acceptable acrylic polymer, including but not limited to, acrylic acid and methacrylic acid copolymers, methyl methacrylate, methyl methacrylate copolymers, ethoxyethyl methacrylates, cyanoethyl methacrylate, aminoalkyl methacrylate copolymer, poly (aery lie acid), poly (methacrylic acid), methacrylic acid alkylamine copolymer poly(methyl methacrylate), poly(methacrylic acid)(anhydride), polymethacrylate, polyacrylamide, poly(methacrylic acid anhydride), and glycidyl methacrylate copolymers.

[0129] In certain preferred embodiments, the acrylic polymer is comprised of one or more ammonio methacrylate copolymers. Ammonio methacrylate copolymers are well known in the art, and are described in NF XVII as fully polymerized copolymers of acrylic and methacrylic acid esters with a low content of quaternary ammonium groups.

[0130] In one preferred embodiment, the acrylic polymer is an acrylic resin lacquer such as that which is commercially available from Rohm Pharma under the tradename EUDRAGIT t®. In further preferred embodiments, the acrylic polymer comprises a mixture of two acrylic resin lacquers commercially available from Rohm Pharma under the tradenames EUDRAGIT® RL30D and EUDRAGIT ® RS30D, respectively. EUDRAGIT® RL30D and EUDRAGIT ® RS30D are copolymers of acrylic and methacrylic esters with a low content of quaternary ammonium groups, the molar ratio of ammonium groups to the remaining neutral (meth (acrylic esters being 1:20 in EUDRAGIT ® RL30D and 1 :40 in EUDRAGIT® RS30D. The mean molecular weight is about 150,000. EUDRAGIT ® S-100 and EUDRAGIT ® L-100 are also preferred. The code designations RL (high permeability) and RS (low permeability) refer to the permeability properties of these agents. EUDRAGIT ® RL / RS mixtures are insoluble in water and in digestive fluids. However, multiparticulate systems formed to include the same are swellable and permeable in aqueous solutions and digestive fluids.

[0131] The polymers described above such as EUDRAGIT ® RL / RS may be mixed together in any desired ratio in order to ultimately obtain a sustained-release formulation having a desirable dissolution profile. Desirable sustained-release multiparticulate systems may be obtained, for instance, from 100% EUDRAGIT® RL, 50% EUDRAGIT® RL and 50% EUDRAGIT t® RS, and 10% EUDRAGIT® RL and 90% EUDRAGIT® RS. One skilled in the art will recognize that other acrylic polymers may also be used, such as, for example, EUDRAGIT® L.

[0132] Alternatively, extended release formulations can be prepared using osmotic systems or by applying a semi-permeable coating to the dosage form. In the latter case, the desired drug release profile can be achieved by combining low permeable and high permeable coating materials in suitable proportion. The devices with different drug release mechanisms described above can be combined in a final dosage form comprising single or multiple units. Examples of multiple units include, but are not limited to, multilayer tablets and capsules containing tablets, beads, or granules An immediate release portion can be added to the extended release system by means of either applying an immediate release layer on top of the extended release core using a coating or compression process or in a multiple unit system such as a capsule containing extended and immediate release beads.

[0133] Extended release tablets containing hydrophilic polymers are prepared by techniques commonly known in the art such as direct compression, wet granulation, or dry granulation. Their formulations usually incorporate polymers, diluents, binders, and lubricants as well as the active pharmaceutical ingredient. The usual diluents include inert powdered substances such as starches, powdered cellulose, especially crystalline and microcrystalline cellulose, sugars such as fructose, mannitol and sucrose, grain flours and similar edible powders. Typical diluents include, for example, various types of starch, lactose, mannitol, kaolin, calcium phosphate or sulfate, inorganic salts such as sodium chloride and powdered sugar. Powdered cellulose derivatives are also useful. Typical tablet binders include substances such as starch, gelatin and sugars such as lactose, fructose, and glucose. Natural and synthetic gums, including acacia, alginates, methylcellulose, and polyvinylpyrrolidone can also be used. Polyethylene glycol, hydrophilic polymers, ethylcellulose and waxes can also serve as binders. A lubricant is necessary in a tablet formulation to prevent the tablet and punches from sticking in the die. The lubricant is chosen from such slippery solids as talc, magnesium and calcium stearate, stearic acid and hydrogenated vegetable oils.

[0134] Extended release tablets containing wax materials are generally prepared using methods known in the art such as a direct blend method, a congealing method, and an aqueous dispersion method. In the congealing method, the drug is mixed with a wax material and either spray - congealed or congealed and screened and processed.

[0135] (2) Delayed release dosage forms

[0136] Delayed release formulations can be created by coating a solid dosage form with a polymer film, which is insoluble in the acidic environment of the stomach, and soluble in the neutral environment of the small intestine.

[0137] The delayed release dosage units can be prepared, for example, by coating a drug or a drug-containing composition with a selected coating material. The drug-containing composition may be, e.g., a tablet for incorporation into a capsule, a tablet for use as an inner core in a "coated core" dosage form, or a plurality of drug-containing beads, particles or granules, for incorporation into either a tablet or capsule. Preferred coating materials include bioerodible, gradually hydrolyzable, gradually water-soluble, and / or enzymatically degradable polymers, and may be conventional "enteric" polymers. Enteric polymers, as will be appreciated by those skilled in the art, become soluble in the higher pH environment of the lower gastrointestinal tract or slowly erode as the dosage form passes through the gastrointestinal tract, while enzymatically degradable polymers are degraded by bacterial enzymes present in the lower gastrointestinal tract, particularly in the colon. Suitable coating materials for effecting delayed release include, but are not limited to, cellulosic polymers such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxymethyl cellulose, hydroxypropyl methyl cellulose, hydroxypropyl methyl cellulose acetate succinate, hydroxypropylmethyl cellulose phthalate, methylcellulose, ethyl cellulose, cellulose acetate, cellulose acetate phthalate, cellulose acetate trimellitate and carboxymethylcellulose sodium; acrylic acid polymers and copolymers, preferably formed from acrylic acid, methacrylic acid, methyl acrylate, ethyl acrylate, methyl methacrylate and / or ethyl methacrylate, and other methacrylic resins that are commercially available under the tradename Eudragit® (Rohm Pharma; Westerstadt, Germany), including EUDRAGIT® L30D-55 and L100-55 (soluble at pH 5.5 and above), EUDRAGIT® L-100 (soluble at pH 6.0 and above), EUDRAGIT® S (soluble at pH 7.0 and above, as a result of a higher degree of esterification), and EUDRAGITS® NE, RL and RS (water-insoluble polymers having different degrees of permeability and expandability); vinyl polymers and copolymers such as polyvinyl pyrrolidone, vinyl acetate, vinylacetate phthalate, vinylacetate crotonic acid copolymer, and ethylene- vinyl acetate copolymer; enzymatically degradable polymers such as azo polymers, pectin, chitosan, amylose and guar gum; zein and shellac. Combinations of different coating materials may also be used. Multi-layer coatings using different polymers may also be applied.

[0138] The preferred coating weights for particular coating materials may be readily determined by those skilled in the art by evaluating individual release profiles for tablets, beads and granules prepared with different quantities of various coating materials. It is the combination of materials, method and form of application that produce the desired release characteristics, which one can determine only from the clinical studies.

[0139] The coating composition may include conventional additives, such as plasticizers, pigments, colorants, stabilizing agents, glidants, etc. A plasticizer is normally present to reduce the fragility of the coating, and will generally represent about 10 wt. % to 50 wt. % relative to the dry weight of the polymer. Examples of typical plasticizers include polyethylene glycol, propylene glycol, triacetin, dimethyl phthalate, diethyl phthalate, dibutyl phthalate, dibutyl sebacate, triethyl citrate, tributyl citrate, triethyl acetyl citrate, castor oil and acetylated monoglycerides. A stabilizing agent is preferably used to stabilize particles in the dispersion. Typical stabilizing agents are nonionic emulsifiers such as sorbitan esters, polysorbates and polyvinylpyrrolidone. Glidants are recommended to reduce sticking effects during film formation and drying, and will generally represent approximately 25 wt. % to 100 wt. % of the polymer weight in the coating solution. One effective glidant is talc. Other glidants such as magnesium stearate and glycerol monostearates may also be used. Pigments such as titanium dioxide may also be used. Small quantities of an anti-foaming agent, such as a silicone (e.g., simethicone), may also be added to the coating composition.

[0140] III. METHODS OF USE

[0141] The disclosed compositions are used in subjects in need thereof, to delivery an active agent to lung endothelial cells.

[0142] In some forms subject suffers from or is suspected of suffering from an endothelial cell dysfunction. Endothelial cell dysfunction occurs in a variety of acute and chronic pulmonary diseases including pulmonary hypertension, viral and bacterial pneumonia, bronchopulmonary dysplasia, cystic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), lung cancer, asthma, and congenital lung diseases such as alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) and cystic fibrosis (CF) among others.

[0143] Examples

[0144] Materials and Methods

[0145] Materials and Plasmids

[0146] All materials for PBAEs synthesis were purchased from Sigma-Aldrich (St. Louis, MO, USA) including Bisphenol A ethoxylate (EO / phenol) diacrylate (DI), 4,4’- Trimethylenedipiperidine (Al), 6-amino-l-hexanol (A2), polyethylenimine (PEI, Mn -600) (Cl), poly(ethylene glycol) amine (PEG, Mn -2000) (C2), Pentadecafluorooctanoyl chloride (Fl), Pentafluoropropionic anhydride (F2), 1H,1H,2H,2H-Perfluorodecyl acrylate (F3) Pentafluoropropionic anhydride (F4); the structures of F1-F4 are shown below.

[0147] Fl (Pentadecafluorooctanoyl chloride)

[0148] F2 (Pentafluoropropionic anhydride);

[0149] F3 (1H,1H,2H,2H-Perfluorodecyl acrylate)

[0150] F4 (Pentafluoropropionic anhydride)

[0151] D-Luciferin, Potassium Salt was purchased from Gold Biotechnology (St. Louis, MO, USA). mCherry was cloned into the Multiple Cloning Site (MCS) of the pMC.EFla-MCS- SV40polyA Parental Plasmid (PP) to produce mCherry-PP (Minicircle DNA technology, System Biosciences). Briefly, mCherry-PPs were transfected into ZYCY10P3S2T E. coli Minicircle producer competent cells (MPCs) which harbored an arabinose-inducible system to express <bC31 integrase and the LScel endonuclease simultaneously. Following propagation of PPs in MPCs, arabinose was added to the media to induce <t>C3 I integrase and the I-Scel endonuclease expression. In this process, <t>C31 integrase mediated an intramolecular recombination between cis-positioned attP and attB sites on PPs, creating the MC and a bacterial backbone. LScel endonuclease digested the bacterial backbone through the 32 copies of I-Scel restriction sites, incorporated in the DNA, yielding clean MC DNA. DyLight 650 NHS ester, SnakeSkin™ Dialysis Tubing, 3.5K MWCO were obtained through ThermoFisher Scientific (Waltham, MA, USA). Cell culture Cell Counting Kit-8 (CCK-8) was purchased from Apexbio (Houston, TX, USA). CMV-turboGFP plasmid was purchased from Evrogen. gWiz luciferase-encoding DNA plasmid was purchased from Aldevron (Fargo, ND, USA). All antibodies used for flow cytometry and immunostaining were purchased from BioLegend (San Diego, CA, USA), BD Bioscience (Franklin Lakes, NJ, USA), MilliporeSigma (Burlington, MA, USA) and ThermoFisher (eBioscience) and listed in Table 1. Table 1 Antibodies used for flow cytometry and immunostaining.

[0152] PBAE nanoparticle synthesis and characterization

[0153] The PBAE polymer backbone was synthesized and capped in anhydrous dimethylsulfoxide (DMSO) at various temperatures with different monomer ratios. Briefly, the A2 monomer (primary amine) and DI monomer (diacrylates) were mixed at 90 °C for 24 hours. The Al monomer (secondary amine) was added to the mixture at 50 °C for another 24 hours. The backbone polymers were diluted in DMSO to a concentration of 200 mg / ml. Following the backbone synthesis, an excess of end-capping amine, including PEG and PEI, was added and reacted at 40 °C for an additional 24 hours. For the fluorinated polymers, the fluorination agent was added into the capping amine at a fluorination agent with the PEI mole ratio of 1:4. The PBAE polymers were stored at -20 °C.

[0154] Before assembling with plasmid, PBAE polymers were dialyzed against deionized H2O using a 3.5 kDa membrane to remove the excess of capping agents and DMSO. The product polymers were dissolved in a 25 mM HEPES buffer (pH=7.4) at a concentration of 10 mg / ml. To prepare DNA- encapsulated polyplex nanoparticle, DNA and PBAEs were simply mixed by pipette thoroughly. For the fluorescent nanoparticles, polymers were fluorescently tagged by mixing the NHS -functionalized fluorophores with polymers at room temperature using a vortex. The nanoparticles were allowed to rest at room temperature for at least 30 min and used no later than 4 h following formulation.

[0155] The hydrodynamic diameter and surface potential of the PBAE nanoparticles were determined by dynamic light scattering (DLS) using a Zetasizer Nano-ZS (Malvern, Malvern, UK). The 1H- NMR (nuclear magnetic resonance) was taken in deuterated chloroform on a Bruker AV 400 MHz spectrometer (Bruker, MA, USA). Gel electrophoresis was performed using Tris / borate / ethylenediaminetetraacetic acid (TBE)-based agarose gels (0.8% w / v, 0.5x TBE) at 120 V and imaged using a Bio-Rad Gel Doc. In vitro transfections and cytotoxicity assay

[0156] The HEK-293T cell line was cultured in Dulbecco’s Modified Eagle Medium (DMEM, Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco) and 1% v / v Penicillin Streptomycin (Invitrogen) in a controlled environment at 37 °C and 5% CO2. For green fluorescent protein (GFP) transfection, the cells were seeded onto a 96-well polystyrene tissue culture plate 24 hours before transfection. Typically, the GFP plasmid and polymer were diluted into 100 ng / l and 1 pg / pl, separately, following by mixing in the OPTI-MEM (Gibco) at certain ratios. Nanoparticles encapsulated with GFP plasmid were added to the cells at a concentration of 100 ng DNA / well. After 4 h incubation, the medium was replaced with fresh DMEM medium with 10% FBS, and the plate was incubated for an additional 48 hours. The GFP intensity was registered by a Flexstation 3 Multi-Mode Microplate Reader (Molecular Devices, CA, USA). The microscopic images of the GFP expression with different nanoparticle formulation were taken by EVOS M7000 fluorescence microscope. For confocal microscopy, 8 chamber-slides were used for cell culture and immunostaining. The cells were fixed using 4% PFA (Paraformaldehyde, Electron Microscopy Sciences, Hatfield, PA, USA), and the cell nuclei were counterstained with DAPI (4’, 6-diamidino-2-phenylindole, blue). The images were captured using a Nikon AIR GaAsP inverted confocal microscope. The in vitro cell viability was assessed using the Cell Counting Kit. The nanoparticles were diluted to different concentrations and added to cells for 24 hours. The assay absorbance was measured at a wavelength of 450 nm using a Microplate Reader.

[0157] Animal studies

[0158] All animal experiments were approved by the Institutional Animal Care and Use Committee. NIH guidelines for laboratory animal care and safety were strictly followed. Adult CD1 and SKH-1 mice (Charles River Laboratories, 8-12 weeks) were used for animal studies. For the lung targeting and nanoparticle localization experiments, CD1 mice were intravenously injected with DyLight 650 labeled nanoparticles and the cells from dissected lung tissue were harvested 24 hours after the nanoparticle injection. Luciferase reporter activity was measured by using the IVIS Lumina III Imaging system and the IVIS Spectrum CT In Vivo Imaging System (PerkinElmer, MA, USA). For ex- vivo IVIS imaging, CD1 mice were injected intraperitoneally with 200 pL D-luciferin (30 mg / mL) 48 hours after the nanoparticle administration. The mice were sacrificed 10 mins after the luciferin injection and internal organs (heart, liver, spleen, lung, and kidney) were collected for luminescence imaging. The SKH-1 hairless mice were used for in-vivo IVIS image. The mice were injected intraperitoneally with D-luciferin as described

[0028] , and the whole-body imaging was performed 10 min following the D-luciferin injection. The luminescence intensity was quantified using Living Image software.

[0159] Flow Cytometry

[0160] Lungs from CD1 mice were digested in DMEM medium supplemented with 0.5 mg mL- 1 and DNase, 100 pg mL-1 liberase. The red blood cells were removed using ACK lysis buffer for 5 min on ice. Non-specific background was blocked in autoMACS® Running Buffer with TruStain FcX™ (anti-mouse CD16 / 32) antibody (BioLegend, 101320). Cells were then stained with CD31 Ab labeled with eF450, CD45 Ab labeled with AF700, CD140a Ab labeled with PE Cy7, CD326 Ab labeled with PerCP-Cy5.5 (Supplemental table si). Dead cells were stained with 7-AAD Viability Dye (BioLegend, 420404). CD31+ CD45- CD326- CD140a- cell population was identified as endothelial cells. CD45+ CD31- CD326- CD140a- cell population was identified as hematopoietic cells. CD326+ CD31- CD45- CD140a- cells were identified as epithelial cells, CD140a+ CD31- CD45- CD326- cells as fibroblasts, and CD140a- CD31- CD45- CD326- as lineage-negative cells, which include pericytes, smooth muscle and other respiratory cell types

[0017] . FACS analysis was performed using a BD Biosciences LSR II cytometry and data were analyzed by FlowJo software.

[0161] Immunofluorescence

[0162] Lungs from CD 1 mice were collected and cryosections (7 pm) were prepared for immunofluorescence as described previously

[0017] , The rat anti-CD31 and mouse anti-aSMA antibodies were used to identify endothelial and smooth muscle cells, respectively. After primary antibodies, cells were incubated with the donkey anti-mouse antibodies labeled with AF594 and donkey anti-mouse antibodies labeled with AF488. Fluorescence images were captured by a Zeiss Axioplan2 microscope and Nikon AIR confocal microscope. Analysis was performed using Imaris software, and the quantification of nanoparticles in the images was performed using imageJ software as reported before

[0036] .

[0163] Clinical toxicity evaluation

[0164] Blood and serum were collected from the mice 5 days after the nanoparticle injection. Ten CD1 mice were used for the toxicology study (5 for the experimental group treated with nanoparticle at NP:DNA=50:l ratio, 5 for the control group treated with glucose injection solution). The peripheral blood was collected in MINICOLLECT® K2E K2EDTA blood sample tubes. MiniCollect® TUBE 0.8 ml CAT Serum Separator (Greiner Bio-One, Kremsmunster, Austria) was used to prepare the blood serum. Collected samples were analyzed by IDEXX BioAnalytics (North Grafton, MA, USA) for clinical chemistry and hematology of the peripheral blood. Statistics

[0165] Unpaired, two-tailed student’s t-test in Graphpad Prism was used to determine statistical significance. P values <0.05 were considered significant. All data were shown as mean ± SD for groups of at least three replicates.

[0166] Results

[0167] PBAEs Polymer Synthesis, Optimization and Characterization

[0168] A library of monomers was used for PBAE polymer synthesis and fluorination (Fig.10). The PBAE polymer backbones were synthesized using a step-growth polymerization via Michael addition between the diacrylates and amines. The hydrophobic PBAE backbones were further capped with hydrophilic capping agents to form amphiphilic PBAE polymer chains (Fig. 1A). The characterization data on the PBAEs are shown in the Supplemental materials including the typical chemical structures and 1H-NMR spectrum of the starting materials (Fig. 11 A-C), and the synthesized polymers (Fig. 11 D-E). Resonance signals from terminal acrylate groups on the DI (Bisphenol A ethoxylate diacrylate) and uncapped PBAE backbones were observed at 5.8 ppm - 6.5 ppm (Fig. 11 A and Fig. HD). These resonance peaks disappeared upon capping with Cl (PEI) and C2 (PEG), indicating the successful covalent attachment of these capping agents to the PBAE backbones via Michael Addition (Fig. HE). Furthermore, the characteristic peaks from Al (4,4’ -Trimethylenedipiperidine, Fig. 11B) and A2 (6-amino-l -hexanol, Fig. 11C) are present in the products after polymerization (Fig. HD and Fig. HE), indicating successful synthesis of PBAE polymers. The cationic amphiphilic PBAEs can self-assemble with the nucleic acid and form stable nanoparticles in aqueous medium (Fig.1 B).

[0169] To exclude ineffective formulations and select the most effective nanoparticles for further in vivo testing, a set of initial in vitro transfection experiments were performed using HEK-293T cells. Variations of the polymer formulations influenced the transfection efficiency (Fig. 2A). The changes in Al to A2 ratio significantly altered the transfection efficiency of the nanoparticles. For nanoparticles with more Al than A2, the gelation of PBAE polymers caused failure in the nanoparticle synthesis. PBAEs with only A2 in the backbones yielded lower transfection efficiencies compared to PBAEs containing both Al and A2 in the backbone. Interestingly, incorporation of PEG (3 - 6%) in capping agents increased the transfection efficiency as shown in Figure 2A (Pl, PH, and P21 polymers without PEG capping; P2, P12, and P22 polymers with 3% PEG capping; and P3, P13, and P23 polymers with 6% PEG capping, Supplemental Table 4).

[0170] Table 4. Synthesis conditions for the PBAE polymer synthesis

[0171] To investigate the influence of fluorous ligands, the PBAE formulations with the highest transfection efficiency (Pl 3 and P22) were used for fluorination. The Fl fluorination increased the transfection efficiency, especially for the P22 polymer (Fig. 2 B-C). However, the transfection efficiency was lower if the length of the fluorous ligands was further increased (F3 and F4). The gel electrophoresis analysis was used to determine the w / w ratio required to stabilize DNA in the nanoparticles (Fig. 2D). PBAEs with only A2 in the backbone and capped with PEI encapsulated all the DNA at a polymer to DNA mass ratio of 2 (Pl). After using Al in the formulation (P21) and 3% of PEG in capping agents (P22), the required mass ratio for DNA full encapsulation was increased to 3. The fluorination (P22-F1) did not influence DNA encapsulation into PBAE nanoparticles (Fig. 2D). The size and surface charge of the P22-F1 PBAEs with different nanoparticle to DNA mass ratio (NP / DNA) were determined by dynamic light scattering. Before assembling with DNA, the average hydrodynamic size of polymer nanoparticles was 50 nm, however, the nanoparticle size was significantly increased after assembly with DNA (Fig. 2E). When the polymer to DNA ratio was set at 2.5, the nanoparticles were unstable and aggregated into large particles (Fig. 2F), most likely, due to the neutral surface charge, which is known to decrease electrostatic repulsive forces between the nanoparticles

[0037] , After the polymer to DNA mass ratio reached 5, the nanoparticle size was decreased to 150 nm and the surface charge was increased to 20 mV (Fig. 2 E-F). The nanoparticle size further decreased to 90 nm after increasing the polymer / DNA ratio to 50 (Fig. 2E).

[0172] Fluorescence microscopy and flow cytometry were used to measure the transfection efficiency of PBAE nanoparticles after the PEG modification and fluorination. HEK-293T cells were transfected with nanoparticles containing the CMV-GFP reporter plasmid. The nanoparticles containing both PEG and fluorous ligands exhibited brighter GFP fluorescence compared to nanoparticles containing either PEG or fluorous ligands (data not shown). Flow cytometry was used to quantify the transfection efficiency of the nanoparticles in HEK-293T cells in vitro (Fig. 3A-C). Compared to nanoparticles without PEG, the nanoparticles containing PEG increased the brightness of GFP in cells but did not change the percentage of transfected cells (Fig. 3A-C). In contrast, the fluorination of nanoparticles increased both the transfection efficiency and mean fluorescence intensity (MFI) (Fig. 3A-C). There were no significant changes in cell viability after the PEG modification and fluorination of the PBAE nanoparticles (Fig. 3D). Consistent with flow cytometry measurement, fluorescence microscopy demonstrated high efficiency of cell transfections and detected GFP in both nucleus and cytoplasm of transfected cells (data not shown).

[0173] In Vivo Targeting of the PBAE nanoparticles

[0174] To track the nanoparticles in vivo, the DyLight 650 fluorescent dye with NHS ester was used to label the PBAE polymer surface of the nanoparticles (Fig. 4A). DyLight 650-labeled nanoparticles were injected intravenously and 24 hours after the injection, mouse lungs were dissected and used for flow cytometry analysis to identify the cell types containing the nanoparticles (Fig. 1C). Immunostaining for CD31, CD45, CD326 and CD140a cell surface markers identified several cell populations in the lung tissue, including fibroblasts, endothelial, epithelial, and hematopoietic cells. Cells without these markers were identified as lineage negative cell, which contain pericytes, smooth muscle cells and other respiratory cell types (Fig. 12A-12E). The PBAE nanoparticles mainly targeted lung endothelial cells with relatively low distribution to other cell types. Compared to the nanoparticles containing only the PEI capping, the PBAE nanoparticles with PEG and Fl (P22-F1) exhibited an increased targeting efficiency of endothelial cells without significant changes in other cell types (Fig. 4B-C). Moreover, the P22-F1 nanoparticles exhibited an increased MFI (5), demonstrating that more endothelial cells were targeted with greater efficiency. Thus, the PEGylation and fluorination remarkably enhanced the efficiency of endothelial targeting in vivo.

[0175] Confocal microscopy was used to track the distribution of DyLight 650-labeled P22-F1 PBAE nanoparticles in the lung tissue. To visualize the microvasculature and large pulmonary vessels, frozen lung sections from mice treated with P22-F1 PBAE nanoparticles were stained for endothelial marker CD31 (PECAM1) and smooth muscle cell marker aSMA. DyLight 650- labeled nanoparticles were highly disseminated throughout the pulmonary microvasculature in the alveolar region (data not shown). In contrast, the nanoparticles were not found in endothelial cells of large pulmonary arteries and veins (data not shown). Based on deconvolution of 3D high magnification confocal images followed by the cell surface reconstruction, the P22-F1 PBAE nanoparticles successfully internalized in the capillary endothelial cells in the alveolar region (data not shown). Compared to Pl, P21 and P22 nanoparticle formulations, the P22-F1 PBAE nanoparticles were the most effective in targeting lung endothelial cells (Fig. 13; and data not shown), consistent with flow cytometry data (Fig. 4B-D). The P22-F1 PBAE nanoparticle targeting of the spleen was restricted to capillaries located in the red pulp (data not shown), whereas the nanoparticle targeting in the kidney was restricted to capillaries of glomeruli (data not shown). The P22-F1 PBAE nanoparticles detection in the heart (data not shown) and liver (data not shown) was low. Average number of the P22-F1 PBAE nanoparticles in the endothelial cells of the heart, liver, kidney and spleen were significantly lower compared to the lung endothelial cells (Fig. 13).

[0176] Recent single-cell RNA sequencing studies demonstrated that lung capillary endothelial cells consist of 2 cell types in the alveolar region: general capillary cells (CAP1 or gCAPs) and aerocytes (CAP2 or aCAPs)

[0038] . Therefore, flow cytometry was used with CD117, CD31 and CD45 antibodies to distinguish between CAP1 and CAP2 (Figure 6A)

[0039] . Both CAP1 and CAP2 capillary endothelial cells were targeted by the P22-F1 PBAE nanoparticles with high efficiency (Fig. 6B). However, CAP2 cells had higher MFI (Fig. 6 C-E), consistent with higher transfection rate due to larger cell surface area in CAP2 compared to CAP1 cells

[0038] . Thus, the P22-F1 PBAE nanoparticles accumulate in both CAP1 and CAP2 lung capillary endothelial cells without targeting the endothelium of other organs, arteries, and veins.

[0177] The P22-F1 PBAE nanoparticles are not toxic

[0178] Since biosafety is one of the most important considerations for nanoparticle delivery systems in biomedical and clinical applications, the safety profile was evaluated of the P22-F1 PBAE nanoparticles by measuring multiple clinical chemistry and hematology parameters in the peripheral blood of nanoparticle-injected mice.

[0179] After treatment with P22-F1 PBAE nanoparticles, mice had normal levels of alkaline phosphatase (ALP), aspartate transaminase (AST), Alanine aminotransferase (ALT) and Lactate dehydrogenase (LDH) (Fig. 7A and Table 2).

[0180] Compared to vehicle-treated mice, there were no differences in concentrations of total protein, albumin, BUN, creatinine, and other clinical chemistry parameters. ALT, ALP AST, LDH, bilirubin, albumin, and total protein are usually related to the liver function. Lack of abnormalities in these parameters indicate normal liver function and the lack of liver toxicity. Although ALT value was slightly increased after the nanoparticle injection compared to the control group (table 2), the value was within the normal range. The creatinine test is typically used as a measure of the kidney function, and no significant increase of creatinine was found in the P22-F1 PBAE nanoparticle-treated group. These results demonstrated that the P22-F1 PBAE nanoparticles are nontoxic to the liver and kidney after a single intravenous administration in mice. In the hematology tests, the nanoparticle-treated group had no changes in the levels of hematologic parameters compared to the control group (Fig. 6B and Table 3).

[0181] Measurements of white blood cells, red blood cells, and platelets in the peripheral blood used to diagnose inflammation, clotting disorders, and other blood diseases, remained within normal ranges after P22-F1 PBAE nanoparticle treatment, showing the benign nature of the nanoparticles. Thus, the PBAE nanoparticles did not alter the composition and viscosity of the peripheral blood. Furthermore, the body weight changes in mice were monitored for 5 days after the treatment. There were no body weight changes after the P22-F1 PBAE nanoparticle injection compared to the control group (Fig. 7C). Altogether, the fluorinated PBAE nanoparticles (P22- Fl) did not exhibit toxicity as demonstrated by multiple clinical chemistry and hematology parameters and the lack of changes in the body weights.

[0182] The whole-body analysis of the P22-F1 PBAE nanoparticle efficiency and biodistribution The P22-F1 PBAE nanoparticles were used for the in vivo biodistribution studies, in which the Luciferase reporter plasmid (gWiz-Luc) was encapsulated into the nanoparticles (NP) and injected intravenously to adult mice at a dose of 20 pg DNA per mouse at different NP / DNA mass ratios. Twenty-four hours after the injection, different organs were dissected and examined for bioluminescence. At NP / DNA ratios of 5:1 and 10:1, low luciferase expression was mostly observed in the spleen (data not shown). At higher NP / DNA ratios, luciferase expression in the lung tissue was dramatically increased, while the luciferase expression in the spleen remained low (data not shown). The highest luciferase expression in the lung tissue was reached at NP / DNA mass ratio of 40: 1 and 50:1 (Fig. 8 A and data not shown). Luciferase expression in the liver, kidney, and heart was undetectable (Fig. 8A and data not shown). Thus, the biodistribution and targeting efficiency of P22-F1 PBAE nanoparticles were found to be highly dependent on the NP / DNA ratio; the NP / DNA ratios of 40: 1 and 50:1 resulted in the most effective lungspecific delivery. To further confirm lung-specific delivery, the in vivo bioluminescence imaging (IVIS) and the microCT imaging of nanoparticle-treated mice were performed, demonstrating specific delivery of P22-F1 PBAE nanoparticles to the lung tissue in vivo (Fig. data not shown). Low luciferase expression in the spleen was only detected by IVIS but not by MicroCT (data not shown). At NP / DNA ratios of 40: 1 and 50:1, luciferase activities in the lung were > 10-fold higher than in spleen. (Fig. 8A).

[0183] Flow cytometry was used to investigate the influence of the NP / DNA ratio on the specificity of the P22-F1 PBAE nanoparticles in the lung tissue. Increasing the NP / DNA ratio had no effect on cell specificity; The nanoparticles were mainly found in lung endothelial cells, while they did not efficiently target fibroblasts, epithelial, hematopoietic, and lineage-negative cells, the latter of which contain pericytes, smooth muscle and other respiratory cell types (Fig. 14). These results demonstrate that the P22-F1 PBAE nanoparticles can deliver functional DNA (luciferase reporter) to endothelial cells in the lung tissue at high NP / DNA ratios.

[0184] Next, it was investigated whether the P22-F1 PBAE nanoparticles can target endothelial cells in other organs. The mCherry minicircle DNA reporter plasmid was encapsulated into the P22-F1 PBAE nanoparticles at NP / DNA ratio of 50:1 and delivered intravenously. mCherry transfection efficiency in the lung endothelium was significantly higher compared to the endothelial cells isolated from the spleen (Fig. 8B-C). mCherry expression in endothelial cells of the heart, liver, and kidney was undetectable (Fig. 8B-C). Interestingly, the mCherry reporter was only detected in lung endothelial cells, whereas DyLight-650 labeled nanoparticles were present in endothelial cells of other organs (Fig. 15-16). Thus, while the P22-F1 PBAE nanoparticles entered (or attached to) endothelial cells of many organs, the plasmid functional activity was only detected in lung endothelial cells. Altogether, the in vivo mCherry transfection flow cytometry analysis showed that the P22-F1 PBAE nanoparticles efficiently transfected lung microvascular endothelial cells without targeting endothelial cells of other organs, a finding consistent with IVIS whole-body imaging and microCT.

[0185] Fluorination is required to maintain the size and charge of the P22-F1 PBAE nanoparticles for lung endothelial-specific targeting

[0186] To investigate the factors that influence the in vivo transfection efficiency and biodistribution of the P22-F1 PBAE nanoparticles, the nanoparticles with four different formulations (Pl, P21. P22, and P22-F1) were selected to perform DLS analysis and the in vivo distribution imaging. For the nanoparticles with only one amine (A2) in the backbone (Pl), the hydrodynamic diameter of the nanoparticles did not change when the NP / DNA ratio was increasing (Fig. 9A). However, for the nanoparticles with two amines (Al and A2), the size of the nanoparticles was decreased after increasing the NP / DNA ratio (Fig. 9A). It is possible that the terpolymer chains have higher flexibility, making the size of the nanoparticles more controllable during the self-assemble. Compared to nanoparticles without fluorination (P21 and P22), the fluorinated PBAE nanoparticles (P22-F1) exhibit a much smaller hydrodynamic diameter at the low NP / DNA ratio, indicating that fluorination stabilizes the polymers chains in the nanoparticle complexes. Even at high NP / DNA ratios, the fluorinated PBAE nanoparticles exhibited a smaller size compared to the nanoparticles without fluorination (Fig. 9A). Measurement of zeta potentials showed that P21, P22, and P22-F1 nanoparticles have lower zeta potentials compared to the Pl formulation. However, zeta potentials for P21, P22, and P22-F1 were similar at high NP / DNA ratios (Fig. 9B). The bioluminescence whole-body imaging showed that the in vivo transfection efficiency and biodistribution are highly dependent on the formulations of PBAE polymers. Compared to the terpolymers, the transfection only occurred in the spleen for Pl nanoparticles (Fig. 9C and data not shown). Bioluminescence distributions for the nanoparticles assembled by the terpolymers in the absence or presence of PEG (P21 vs P22) were similar and observed in both the lung and spleen (Fig. 9C and data not shown). Fluorination of P22 nanoparticles (P22-F1) significantly improved bioluminescence in the lung tissue but decreased luciferase activity in the spleen (Fig. 9C and data not shown). Thus, fluorination is required for P22-F1 nanoparticle-mediated delivery of functional DNA plasmid to lung microvascular endothelial cells in vivo.

[0187] Discussion

[0188] In this study, a fluorinated PBAE nanoparticle formulation capable of targeting pulmonary microvascular endothelium with high transfection efficiency and organ specificity has been designed. The initial studies focused on developing effective formulations for in vitro transfection and found that the alkyl chain amines in backbone synthesis can significantly enhance the transfection performance of the PBAE nanoparticles. The PBAE polymer synthesis was found to be sensitively controlled by the Al to A2 ratio for optimum transfection efficiencies. PEG has been widely used in nanoparticle synthesis to improve nanoparticle stability, increase circulation time in vivo, and reduce toxicity [30,42]. In this study, the addition of PEG at 3% - 6% in the end caps was found to increase the transfection efficiency. However, further increasing the PEG ratio in end caps reduced transfection. Therefore, the best PEGylation ratio for this amphiphilic PBAE capping was controlled in the range of 3% - 6%. Moreover, there was a large difference between the different fluorous ligands in terms of in vitro transfection efficiency. Based on the data herein, the transfection efficiency can only be increased by modification of Pentadecafluorooctanoyl chloride (Fl) and by changing the fluorous ligand type. However, increasing the length of the fluorous ligands may have negative influence on transfections. Unlike PEGylation, fluorination not only increased the percentage of transfected cells but also improved the strength of the transfection. While not being bound by theory, these results support the hypothesis that halogenated ligands such as the fluorous ligands used here make it possible for the PBAE nanoparticles to enter the cell more efficiently.

[0189] While the in vivo transfection is more complex, the data herein showed high endothelial targeting efficiency via the amphiphilic PBAE nanoparticles. PEGylation increased both the targeting percentage and strength as it, likely, delays nanoparticle clearance in circulation before reaching pulmonary endothelium, while fluorination exerts a similar effect on the nanoparticles in vivo.. The PBAE nanoparticles were primarily located in the lung microvasculature as supported by confocal microscopy and flow cytometry for CAP1 and CAP2 lung capillary endothelial cells. Moreover, the nanoparticle size and charge influence the bio-distribution of the nanoparticles and are dependent on NP / DNA ratio. Interestingly, in vivo luciferase imaging demonstrated that at low NP / DNA ratio of 10, the nanoparticles have a relatively large size (135.83+2.41 nm), and the transfection occurs mainly in the spleen. With the increase of NP / DNA ratio to 50, the size of the nanoparticles was reduced (86.00+0.32 nm) dramatically, changing bioluminescence distribution in which the lung becomes the major targeted organ. Compared to nanoparticle sizes, the changes in the surface charge were insignificant when increasing the NP / DNA ratio. Thus, it is possible that small sizes of the PBAE nanoparticles at high NP / DNA ratios can enhance the nanoparticles by avoiding the elimination through the spleen, leading to the accumulation of the nanoparticles in lungs via interaction between the nanoparticles and the microvascular bed. 5. Conclusion

[0190] In conclusion, a facile synthesis of amphiphilic PB AE nanoparticles for pulmonary endothelial specific targeting and effective delivery was developed. The transfection efficiency of PB AE nanoparticles can be optimized by changing the ratio of two alkyl chains in the backbones. The addition of PEG in the end cap and a suitable fluorination can further increase the transfection efficiency. Based on flow cytometry and confocal imaging, the PBAE nanoparticles were mainly taken up by lung microvascular endothelial cells. The optimized formulation with PEG and fluorous ligand modifications increased the endothelial targeting efficiency to above 90%. Based on IVIS whole body imaging and microCT, at low NP / DNA ratios, the transfection occurred only in the spleen, but the lung became the primary targeted organ after increasing the NP / DNA ratio. Clinical chemistry and hematology of the peripheral blood showed that the PBAE nanoparticles are nontoxic in experimental mice. The present study shows that these specifically designed PBAE nanoparticles have promise for developing important therapies to correct endothelial dysfunctions in pulmonary vascular diseases.

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Claims

CLAIMSWe claim:

1. A halogenated polymer based on a poly(P-amino) ester (PBAE) backbone having the general formula II:Ha C-(D A2 D-Al)n-; wherein:D is derived from a diacrylate;Al is derived from a secondary amine;A2 is derived from a primacy amine;C is derived from a hydrophilic compound;Ha is derived from a halogenated compound; and n is an integer ranging n is an integer ranging 2 to about 10.

2. The polymer of claim 1, wherein the diacrylate is selected from the group consisting of bisphenol A ethoxylate diacrylate, and 1 ,4-butanediol diacrylate. In some forms, D is not Bisphenol A glycerolate diacrylate.

3. The polymer of claim 1 or 2, wherein the secondary amine is 4’- Trimethylenedipiperidine and Hexadecylamine.

4. The polymer of any one of claims 1-3, wherein the primary amine is selected from the group consisting of 6-amino-l-hexanol, 5-amino-l pentanol, 4 amino- 1 butanol, and 3 amino- 1 propanol.

5. The polymer of any one of claims 1-4, wherein the capping agent is selected from the group consisting of polyethylenimine (PEI;), for example, Polyethyleneimine, branched Mw 800 LS / 600 GPC; In some forms the capping agent includes poly(ethylene glycol) amine (PEG amine), for example, poly(ethylene glycol) amine (PEG, Mn -2000), and Methoxypolyethylene glycol amine Mw 1000.

6. The polymer of any one of claims 1-5, wherein the halogenated compound is a fluorinated compound.

7. The polymer of claim 6, wherein the fluorinated compound is selected from the group consisting of:Fl (Pentadecafluorooctanoyl chloride)F2 (Pentafluoropropionic anhydride);FFgrCFpgG X OJL CFgCrpFgF3 (1H,1H,2H,2H- Perfluorodecyl acrylate); andF4 (Pentafluoropropionic anhydride)8. The polymer of any one of claims 1-7, wherein the diacrylate, is bisphenol A ethoxylate (EO / phenol) diacrylate.

9. The polymer of any one of claims 1-8, wherein the secondary amine, is 4,4’-Trimethylenedipiperidine.

10. The polymer of any one of claims 1-9, wherein the primary amine is -amino-l -hexanol.

11. The polymer of any one of claims 1-10, wherein the hydrophilic compound is polyethylenimine (PEI) or poly (ethylene glycol) amine (PEG amine).The polymer of any one of claims 1-10, wherein the halogenated compound is Fl(Pentadecafluorooctanoyl chloride)12. The polymer of claim 11, comprising about 3 to about 6% PEG amine.

13. The polymer of any one of claims 1-12, wherein the ratio of AA1:A2<1.

14. A composition comprising nanoparticles, wherein the nanoparticles comprise the polymer of any one of claims 1-13.

15. The composition of claim 14, further comprising a bioactive agent.

16. The composition of claim 15, wherein the bioactive agent is a therapeutic, prophylactic, nutraceutical and diagnostic agent.

17. The composition of claim 15 or 16, wherein the active agent is an oligonucleotide, optionally, wherein the oligonucleotide is encapsulated in the nanoparticles.

18. The composition of claim 17, wherein the oligonucleotide is a DNA of RNA molecule.

19. The composition of claim 18, wherein the RNA comprises an mRNA, siRNA, miRNA, anti-miRNA, piRNA, aptamer, ribozyme, or an external guide sequences for a nuclease enzyme.

20. The composition of claim any one of claims 17-19, wherein the polymer to nucleotide ratio is at least 50.

21. The composition of claim any one of claims 17-19, wherein the average hydrodynamic size of polymer nanoparticles is less than 100 nm22. A pharmaceutical comprising the composition of any one of claims 14-21 in a pharmaceutically acceptable excipient.

23. A method of treating a subject in need thereof, comprising administering the composition of any one of claims 17-21 or the pharmaceutical of claim 22, to the subject.

24. The method of claim 23, wherein the subject is diagnosed with or is suspected of suffering from lung endothelial cell disfunction.

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