Plasma polymer nanoparticles carrying agents

Abstract

To provide pharmaceutical compositions comprising nanoparticles or conjugates thereof for use in treating or preventing vascular injury or vascular disease.SOLUTION: A pharmaceutical composition for use in treating or preventing vascular injury or vascular disease, comprising a conjugate, wherein the conjugate is provided for delivery to a region of a blood vessel in a patient in need thereof, and wherein the conjugate comprises: a) a nanoparticulate polymer having a mean diameter of about 1 nm to about 50 nm and formed from a plasma comprising at least one monomer selected from an alkene, an alkyne, a cycloalkene, a cycloalkyne, or a mixture thereof; or an aggregate comprising two or more of the nanoparticulate polymers, the aggregate having a mean diameter of about 5 nm to about 500 nm; and b) a biologically active agent selected from the group consisting of an anti-inflammatory cytokine, an anti-inflammatory drug, a statin drug, and an anti-proliferative drug.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to Australian Provisional Patent Application No. 2019900427, filed February 11, 2019, the contents of which are incorporated herein by reference.

[0002] FIELD OF THE DISCLOSURE The present disclosure relates to the field of nanoparticles, their conjugates, and their use in methods for treating or preventing vascular inflammation. [Background technology]

[0003] Coronary atherosclerosis is a leading cause of death and disability in Western societies. Blockage of coronary vessels leads to reduced blood flow to the myocardium, myocardial tissue damage, and ultimately myocardial infarction. However, the long-term performance of medical devices used to treat atherosclerosis, such as drug-eluting stents and angioplasty balloons, is limited by chronic inflammation at the site of the injury.

[0004] The long-term success of surgical and vascular interventions is limited by neointimal hyperplasia (NIH). In arteries, NIH is the thickening of the arterial intima after injury, such as angioplasty, stenting, or surgical repair. NIH is also used to describe thickening of vein grafts and artificial bypass grafts, resulting in reduced luminal diameter and flow, ultimately leading to graft occlusion and thrombosis. NIH can occur in all forms of vascular grafts, including both venous conduits and artificial conduits used in coronary and peripheral arterial bypasses, as well as autologous arteriovenous fistulas (AVFs) created for hemodialysis access.

[0005] Restenosis is a common adverse event following endovascular procedures such as stent insertion, balloon angioplasty, or vascular surgery. One of the contributing factors to restenosis is the inflammatory immune response elicited in response to the endovascular procedure. Restenosis is the recurrence of stenosis, a narrowing of the blood vessel, leading to reduced blood flow.

[0006] Thus, there remains a need for methods for vascular delivery of biologically active agents and drugs, such as anti-inflammatory agents. Summary of the Invention

[0007] The present disclosure describes novel therapeutic methods for localizing biologically active agents or drugs to certain regions of a blood vessel, for example, to modulate inflammation or promote healing or treatment or prevention of diseases in the blood vessel. In particular, the inventors have developed nanoparticles or nanoPs. 3 have identified that they are capable of binding agents, such as biologically active agents, drugs, and imaging agents, while retaining the biological activity of those agents both in vitro and in vivo.

[0008] Thus, in one aspect, the present disclosure provides a method of delivering an agent to a region of a blood vessel in a patient, the method comprising: a) conjugating the agent with nanoparticles to produce a conjugate; b) delivering said complex to said region of said blood vessel; The method includes:

[0009] In another aspect, the present disclosure provides a method of modulating inflammation or promoting healing in an area of a blood vessel in a patient, the method comprising: a) conjugating a biologically active agent with nanoparticles to produce a conjugate; b) delivering said complex to said region of said blood vessel; The method includes:

[0010] In another aspect, the present disclosure provides a method for maintaining a biologically active agent in an area of a blood vessel of a patient for a period of at least 14 days, the method comprising: a) conjugating the biologically active agent with nanoparticles to produce a conjugate; b) delivering said complex to said blood vessel; The method includes:

[0011] In another aspect, the present disclosure provides a nanoparticulate polymer formed from a plasma having an average diameter of about 1 nm to about 50 nm and comprising at least one monomer selected from an alkene, an alkyne, a cycloalkene, a cycloalkyne, or a mixture thereof; or an aggregate comprising two or more of the above nanoparticulate polymers and having an average diameter of about 5 nm to about 500 nm; and a biologically active agent selected from the group consisting of an anti-inflammatory cytokine, an anti-inflammatory drug, a statin drug, and an anti-proliferative drug. The present invention provides a composite comprising:

[0012] In another aspect, the present disclosure provides a nanoparticulate polymer formed from a plasma having an average diameter of about 1 nm to about 50 nm and comprising at least one monomer selected from an alkene, an alkyne, a cycloalkene, a cycloalkyne, or a mixture thereof; or an aggregate comprising two or more of the nanoparticulate polymers described above and having an average diameter of about 100 nm to about 200 nm; Interleukin-10 and The present invention provides a composite comprising:

[0013] In another aspect, the present disclosure provides a nanoparticulate polymer formed from a plasma having an average diameter of about 1 nm to about 50 nm and comprising at least one monomer selected from an alkene, an alkyne, a cycloalkene, a cycloalkyne, or a mixture thereof; or an aggregate comprising two or more of the nanoparticulate polymers described above and having an average diameter of about 100 nm to about 200 nm; Sirolimus and The present invention provides a composite comprising:

[0014] In another aspect, the present disclosure provides a nanoparticulate polymer formed from a plasma having an average diameter of about 1 nm to about 50 nm and comprising at least one monomer selected from an alkene, an alkyne, a cycloalkene, a cycloalkyne, or a mixture thereof; or an aggregate comprising two or more of the nanoparticulate polymers described above and having an average diameter of about 100 nm to about 200 nm; Sulindac and The present invention provides a composite comprising:

[0015] In another aspect, the present disclosure provides a method for treating or preventing vascular injury or disease, comprising delivering a conjugate disclosed herein to an area of a blood vessel in a patient in need thereof.

[0016] In another aspect, the present disclosure provides the use of a conjugate disclosed herein in the manufacture of a medicament for the treatment or prevention of vascular injury or vascular disease in a patient in need thereof.

[0017] The following figures form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The present disclosure may be better understood by reference to one or more of these figures in combination with the detailed description of specific embodiments presented herein. [Brief explanation of the drawings]

[0018] [Figure 1] Figure 1 shows the evaluation of IL-4 complexed with NP3 (NP3 + IL-4) as a therapeutic agent for alleviating cardiovascular disease. Direct delivery of therapeutic cargo to sites of reduced vasculature was investigated. A) The unique properties of the NP3 platform allow for rapid complexation of therapeutic compounds and improved bioavailability of these compounds upon in vivo delivery. B) M1 macrophages, which drive vascular injury / cardiovascular disease pathology, are pro-inflammatory. Inducing the phenotype of these macrophages toward an M2 anti-inflammatory state may mitigate further progression of vascular injury and promote disease regression. Various cytokines of the interleukin family, including IL-4 and IL-10, promote this M1-to-M2 phenotypic shift. C) A rat carotid artery model of blood vessels was used to determine the efficacy of NP3-conjugated interleukins for the treatment of cardiovascular pathology. [Figure 2]Figure 1 shows the loading capacity of IL-4 (blue) and IL-10 (red) onto NP3. IL-4 and IL-10 were labeled using Cy5 labels and conjugated onto NP3. Fluorescence readings of the Cy5 labels in the wash solution were quantified to measure the cargo remaining in solution (not bound to NP3), thereby determining the amount of loading onto NP3. A) Total amount of cargo bound as a function of cargo in solution. B) Binding efficiency of IL-4 and IL-10 to NP3 as a function of loading capacity. C) Emission spectra of bound IL-4 and IL-10 to confirm IL-4 and IL-10 binding. [Figure 3] Figure 1 shows validation of NP3 + IL-4 on M2 macrophage polarization in vitro. A) Scanning electron microscopy (SEM) of RAW264.76 murine macrophages showed that NP3 + IL-4 treatment increased spreading (top row) and surface roughness (bottom row) compared with untreated controls and NP3 alone, consistent with M2 activation. B) Using confocal microscopy, immunostaining for arginase-1 (ARG-1) (green), a highly expressed M2 enzyme, showed that NP3 + IL-4 significantly upregulated ARG-1 expression, indicating robust M2 activation compared with controls and NP3 alone. [Figure 4] Figure 1 shows an in vivo model of vascular injury and NP3 retention. A) Workflow of the vascular injury procedure: 1. Insert a microforceps into a ligated / isolated rat carotid artery segment. 2. Expand the forceps and rotate 360 degrees to simulate balloon injury, overexpansion injury, or denudation. 3. Remove the forceps and insert a small-diameter catheter through the same incision. 4. Deliver NP3 + IL-4 solution through the catheter and incubate in the isolated vessel for 2 minutes. 5. Suture the incision and restore blood flow. B) Tracking IL-4 retention in the isolated vessels using Cy5 labeling reveals that free IL-4 is washed out of the vessel wall immediately after restoration of blood flow. However, binding IL-4 to NP3 (NP3 + IL-4) results in significant retention in the vessel, which persists at substantial levels even after 5 days. [Figure 5]Figure 1 shows the mechanism of inhibition of neointima formation. A) Immunostaining for the presence of M2 macrophages (yellow / green) in treated carotid artery segments shows a significant increase in the NP3+IL-10 group compared with denuded, NP3+IL-4, and free IL-10. B) Immunostaining assessment of repair of injured endothelium shows that both NP3+IL-4 and NP3+IL-10 restore full endothelial integrity by 14 days after injury, but not with free IL-10 treatment. [Figure 6A] Figure 1 shows an analysis of neointima formation 2 weeks after therapeutic NP3 delivery.Representative histological images showing the extent of neointima formation in each treatment group. [Figure 6B] Figure 1 shows an analysis of neointima formation 2 weeks after therapeutic NP3 delivery. The neointima formation rate was quantified in three sections along the length of the treated carotid artery segment. The designations "proximal" and "distal" refer to the location of the vascular anastomosis in proximity to the heart. Denudation lesions lead to approximately 60% vascular occlusion after 2 weeks. This is significantly reduced to approximately 35% and 20% in the NP3 + IL-4 and NP3 + IL10 groups, respectively. Free IL-10 and NP3 alone had no significant effect on vascular occlusion, suggesting that the NP3 platform enhances the therapeutic effect of IL-10. [Figure 7] Figure 1 shows an in vivo analysis of neointimal hyperplasia in a rat carotid artery injury model using hematoxylin and eosin (H&E) staining. Test compounds included the anti-inflammatory cytokine interleukin-10 (IL-10), the antiproliferative drug sirolimus, and the nonsteroidal anti-inflammatory drug sulindac, delivered free or complexed with 200 nm NP3. When delivered on 200 nm NP3, all treatments showed inhibition of neointimal hyperplasia compared to their respective free controls. [Figure 8]Figure 1 shows an in vivo analysis of vascular re-endothelialization in a rat carotid artery injury model using von Willebrand factor (vwf) staining. Test compounds include the anti-inflammatory cytokine interleukin-10 (IL-10), the antiproliferative drug sirolimus, and the nonsteroidal anti-inflammatory drug (sulindac), delivered free or complexed with 200 nm NP3. IL-10 delivered with NP3 stimulates vascular healing (endothelialization). Surprisingly, sirolimus delivered with NP3 also stimulates vascular healing. [Figure 9] Figure 1 shows an in vitro analysis of vascular inflammation / macrophage polarization in a rat carotid artery injury model using co-staining of the mannose receptor (CD206) and CD68 cell surface receptors. Analyte compounds include the anti-inflammatory cytokine interleukin-10 (IL-10) and the nonsteroidal anti-inflammatory drug (sulindac), delivered free or complexed with 200 nm NP3. NP3-IL-10 stimulates an anti-inflammatory response by promoting M2 macrophage polarization. [Figure 10] Figure 1 shows the performance evaluation of 200 nm NP3 complexed with IL-10 in vivo in a rabbit model of iliac artery injury. A) Rabbit model of iliac artery injury. B) H&E staining of neointimal hyperplasia. C) CD31 staining of thrombosis (white dotted line). D) CD68 staining of inflammatory macrophage infiltration (white stain). [Figure 11] Figure 1 shows the in vivo performance evaluation of 200 nm NP3 complexed with IL-10 in a rabbit model of iliac artery injury. A) H&E staining analysis shows reduced hyperplasia in vessels treated with IL-10 + NP3 over a 7-day period compared to untreated controls. B) CD31 staining analysis reveals increased thrombosis over a 7-day period, which is significantly reduced in vessels treated with IL-10 + NP3. C) CD68 staining of vascular inflammation shows that IL-10 + NP3 reduces proinflammatory macrophage infiltration and inflammation over a 7-day period compared to untreated controls. [Figure 12] Both 100 nm and 200 nm nanoP3 were retained after 7 days, and 100 nm nanoP3 was also retained 2 weeks after delivery. DETAILED DESCRIPTION OF THE INVENTION

[0019] Those skilled in the art will recognize that numerous variations and / or modifications may be made to the above-described embodiments without departing from the broad general scope of the present disclosure, and the present embodiments are therefore to be considered in all respects as illustrative and not restrictive.

[0020] General Techniques and Definitions Unless otherwise defined, all technical and scientific terms used herein should be understood to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., of immunology, molecular biology, immunohistochemistry, biochemistry, oncology, and pharmacology).

[0021] The present disclosure will be carried out without undue experimentation using, unless otherwise indicated, conventional techniques of molecular biology, recombinant DNA technology, immunology and pharmacology. Such procedures are described, for example, in Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, Fourth Edition (2012), volumes I, II, and III, all of which are incorporated herein by reference; DNA Cloning: A Practical Approach, volumes I and II (D.N. Glover, Second Edition, 1995), IRL Press, Oxford, the entire text; Oligonucleotide Synthesis: A Practical Approach (M.J. Gait, ed., 1984), IRL Press, Oxford, the entire text, and in particular the articles by Gait, p. 22; Atkinson et al., pp. 35-81; Sproat et al., pp. 83-115; and Wu et al., pp. 135-151, therein; Nucleic Acid Hybridization: A Practical Approach (B.D. Hames & S.J. Higgins, eds., 1985), IRL Press, Oxford, the entire text. Press, Oxford, entire text; Immobilized Cells and Enzymes: A Practical Approach (1986) IRL Press, Oxford, entire text; Perbal, B., A Practical Guide to Molecular Cloning (1984), and Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.), entire series.

[0022] Those skilled in the art will appreciate that the present disclosure is susceptible to variations and modifications other than those specifically described. The present disclosure is to be understood to include all such variations and modifications. The present disclosure also includes all steps, features, compositions, and compounds referred to or indicated herein, individually or collectively, and any and all combinations of any two or more of the steps or features.

[0023] The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for purposes of example only. Functionally equivalent products, compositions, and methods are clearly within the scope of the present disclosure as described herein.

[0024] Each feature of any particular aspect or embodiment or embodiment of the present disclosure may be applied, mutatis mutandis, to any other aspect or embodiment or embodiment of the present disclosure.

[0025] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, references to a single step, composition, group of steps, or group of compositions should be interpreted as encompassing one and more (i.e., one or more) of that step, composition, group of steps, or group of compositions.

[0026] As used herein, the singular forms "a," "and," and "the" include the plurals of these words unless the context clearly dictates otherwise. For example, reference to "a bacterium" includes a plurality of such bacterium, and reference to "an allergen" is a reference to one or more allergens.

[0027] As used herein, the term "about" encompasses a tolerance of 10% in any value(s) to which it is attached. For the avoidance of doubt, the term "about" should be understood to encompass an explicitly qualified reference to the integer in question (e.g., "about 10" should be understood to encompass an explicit reference to 10).

[0028] The term "and / or", e.g., "X and / or Y", shall be construed to mean either "X and Y" or "X or Y", and shall be construed as explicitly endorsing both meanings or either meaning.

[0029] Throughout this specification, the word "comprise" or variations such as "comprises" or "comprising" will be understood to mean the inclusion of a specified element, integer, or step, or group of elements, integers, or steps, but not the exclusion of any other element, integer, or step, or group of elements, integers, or steps.

[0030] The present inventors have disclosed in WO2018 / 112543 (the contents of which are incorporated herein by reference in their entirety) that 3 ", nanoP3, " NanoP 3 ", nanoP 3 Materials”, “NanoP 3 The authors demonstrated the production of nanoparticulate materials, described as "nanoparticle materials" or "NP3." 3 The materials can serve as a versatile and multifunctional class of nanocarriers that can be easily functionalized. 3 The material is nanoP 3 embedded within the material, nanoP 3 by reaction with radicals diffusing to the surface of the material and / or nanoP 3 A wide range of biomolecules and drugs can be conjugated by reaction with moieties / functional groups formed on the surface of the material or its composite.

[0031] The inventors have found that delivery of biologically active agents and / or imaging agents complexed to nanoparticles can increase the bioavailability and / or persistence of the delivered agents and drugs in vivo in a region of a blood vessel, for example at the site of injury to a blood vessel, compared to the agent when injected free into the region of the blood vessel (i.e., the agent is delivered in a form that is not complexed to the nanoparticles described herein).

[0032] Thus, in some embodiments, the complex is retained in the region of the blood vessel for a longer period of time than the uncomplexed biologically active agent is retained in the region of the blood vessel.

[0033] The present disclosure provides methods for localizing biologically active agents and modulating inflammation in blood vessels in response to vascular intervention. The present disclosure also provides methods for treating or preventing vascular disease or injury.

[0034] Inflammation, restenosis, and neointimal hyperplasia The disclosed methods can be used to deliver and / or localize an agent, such as a biologically active agent or an imaging agent, to a region of a blood vessel in a patient. Alternatively or additionally, the disclosed methods can be used to modulate inflammation or promote healing in a region of a blood vessel in a patient.

[0035] In one aspect, the present disclosure provides a method for delivering an agent to a region of a blood vessel in a patient, the method comprising: a) conjugating the agent with nanoparticles to produce a conjugate; b) delivering said complex to said region of said blood vessel; The method includes:

[0036] In one embodiment, the nanoparticles are nanoparticulate polymers formed from a plasma containing at least one monomer selected from an alkene, an alkyne, a cycloalkene, a cycloalkyne, or a mixture thereof, having an average diameter of about 1 nm to about 50 nm; or aggregates containing two or more of the nanoparticulate polymers described above and having an average diameter of about 5 nm to about 500 nm.

[0037] In one embodiment, the agent is a biologically active agent.

[0038] In another embodiment, the agent is an imaging agent.

[0039] In one aspect, the present disclosure provides a method of modulating inflammation in an area of a blood vessel in a patient, comprising: a) conjugating a biologically active agent with nanoparticles to produce a conjugate; b) delivering said complex to said region of said blood vessel; The method includes:

[0040] In one example, regulation of inflammation is mediated by macrophage polarization.

[0041] In one aspect, the present disclosure provides a method of promoting healing in a region of a blood vessel in a patient, comprising: a) conjugating a biologically active agent with nanoparticles to produce a conjugate; b) delivering said complex to said region of said blood vessel; The method includes:

[0042] Inflammation is triggered when tissues are subjected to one or more of a variety of insults. This response consists of a cascade of events, including the release of various chemical mediators and the recruitment of circulating blood cells (platelets and leukocytes) to the site of injury and their subsequent activation.

[0043] Without wishing to be bound by theory, restenosis is believed to be a natural healing process in response to arterial injury that occurs during any form of angioplasty. This highly complex healing process results in intimal hyperplasia, more specifically, the migration and proliferation of medial smooth muscle cells (SMCs). The problem with this arterial healing process is that it may not be stopped. The artery continues to "heal" until it becomes blocked. It is important to note that restenosis is not the redeposition of plaque-like cholesterol material that originally blocked the artery.

[0044] Without wishing to be bound by theory, it is believed that successful angioplasty of a stenotic lesion results in plaque disruption, dissection into the media, denudation and destruction of endothelial cells, exposure of thrombogenic collagen, release of tissue thromboplastin, and an increased decrease in prostacyclin production leading to aggregation of activated platelets.

[0045] Activated platelets release several mitogenic factors, including platelet-derived growth factor (PDGF), epidermal growth factor, and transforming growth factor. PDGF has both mitogenic and chemotactic properties and can therefore induce both SMC migration from the media to the intima and proliferation (intimal hyperplasia). PDGF induces SMC proliferation by binding to specific PDGF receptors. PDGF binding to these receptors results in deoxyribonucleic acid (DNA) synthesis and new cell replication. Mild endothelial injury can lead to platelet adhesion and subsequent activation by PDGF. Therefore, even the deposition of a monolayer of platelets may well induce SMC proliferation.

[0046] Deeper arterial injury, which may be associated with complex stenotic lesions, may result in more widespread platelet deposition and activation, leading to even greater availability of mitogens and, in turn, increased SMC proliferation and intimal hyperplasia. Arterial injury from angioplasty may result in the release of PDGF-like compounds not only from platelets, but also from macrophages, monocytes, endothelial cells, or SMCs themselves.

[0047] Activated SMCs from human atheromas or experimental arterial injury (described below) secrete PDGF-like molecules, which appear to perpetuate SMC proliferation by their own release of PDGF-like substances. Thus, any or all of the cells capable of secreting PDGF-related substances (platelets, macrophages, monocytes, endothelial cells, and smooth muscle cells) may contribute to the cascade of restenosis after angioplasty.

[0048] One way to prevent restenosis is to stop the proliferation of smooth muscle cells. Therefore, without wishing to be bound by theory, some possible ways to stop restenosis include: Reducing platelet adhesion and aggregation at the site of arterial injury; Blocking the expression of growth factors and their receptors, developing competitive antagonists of the above growth factors; Interfering with receptor signaling in responsive cells, or Inhibiting smooth muscle proliferation There is.

[0049] nanoparticles The term "nanoparticle" means "nanoP 3 " or "NP3." The terms "nanoparticle" or "nanoP" can be used interchangeably. 3 " or "NP3" refers to nanoparticulate material having a particle size of less than 100 microns, unless otherwise specified or clear from the context in which the term is used. 3has a particle size of about 50 to 500, 100 to 500, 200 to 500, 5 to 200, 5 to 100, 5 to 50, 5 to 20, 20 to 100, 100 to 300, or 200 to 400 nm, for example, about 5, 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 nm, or about 5 to about 400 nm, or about 5 to about 300 nm, or about 5 to about 200 nm, or about 5 to about 100 nm, or about 50 to about 100 nm, or about 100 to about 500 nm, or about 150 to about 500 nm, Alternatively, the nanoP3 may be in the range of about 180 nm to about 500 nm, or about 100 to about 400 nm, or about 150 to about 400 nm, or about 180 to about 400 nm, or about 100 to about 300 nm, or about 150 to about 300 nm, or about 180 to about 300 nm, or about 100 to about 200 nm, or about 150 to about 200 nm, or about 180 to about 200 nm, or about 150 to about 250 nm, or about 180 to about 250 nm, or about 200 to about 400 nm, or about 200 nm to about 300 nm, or a mixture thereof. In one embodiment, the nanoP3 is about 200 nm. In another embodiment, the nanoP3 is about 50 to about 100 nm. It will be understood that particle size, as used herein, refers to the diameter or average diameter of the nanoparticulate material. The term "nanoP3" may be used interchangeably with "nanoP3" to refer to the nanoparticle size or average diameter of the nanoparticle material. 3 " or "NP3" encompasses both "nanoparticulate polymers" and "aggregates," as defined herein, unless otherwise specified or apparent from the context in which the term is used. Thus, for example, the particle sizes described above apply equally to nanoparticles or aggregates of nanoparticles. In one preferred embodiment, the nanoparticulate material comprises a plasma polymer. The plasma polymer can be formed by condensation of fragments in a plasma, and the material can be covalently bonded to one or more compounds, e.g., one or more reactants, including organic or organometallic species.

[0050] The nanoP3 material may be a homopolymer or a copolymer. Examples of suitable nanoP3 materials and methods for deriving suitable nanoP3 materials are described in PCT Publication No. WO2018 / 112543, page 21, line 2 to page 28, line 12, which is incorporated herein by reference.

[0051] Nanoparticulate polymers can be produced in the presence of a gas from Groups 15, 16, or 17 of the periodic table, such as nitrogen. Fragments of this gas may be incorporated into the nanoparticulate polymer. For example, the presence of nitrogen can enhance the nanoparticulate polymer or nanoP 3 Amine, imine, or nitrile groups, or mixtures thereof, may be present in the material, and therefore the nanoparticulate polymers disclosed herein may contain nitrogen.

[0052] Nitrogen has been found to be suitable not only as a carrier but also as a reactive, non-polymerizing gas. This means that nitrogen can be incorporated into the nanoparticulate material to impart specific physicochemical properties to the resulting functionalized nanoparticulate material. Furthermore, nitrogen may enable the production of nanoparticles with different morphologies that would not be possible without the use of nitrogen. The inclusion of other gases, such as gases from the same family as nitrogen, is also expected to provide additional flexibility in tuning the nanoparticle production mechanism and physicochemical properties.

[0053] In one embodiment, the nanoP 3 The material is obtained from a plasma containing at least one monomer as described herein. 3 The material is produced in the presence of a gas, such as nitrogen, and fragments of the gas are introduced into the nanoparticulate polymer.

[0054] The above nanoP 3The nitrogen:carbon atomic ratio of the material may be about 0.01:1 to about 2:3. For example, the nitrogen:carbon atomic ratio of the nanoparticulate polymer may be about 0.05 to about 1, or about 0.1 to about 1, or about 0.15 to about 1, or about 0.2 to about 1, or about 0.25 to about 1, or about 0.3 to about 1, or about 0.35 to about 1, or about 0.4 to about 1, or about 0.45 to about 1, or about 0.5 to about 1, or about 0.55 to about 1, or about 0.6 to about 1, or about 0.65 to about 1. Alternatively, the nitrogen:carbon atomic ratio of the nanoparticulate polymer may be about 0.1 to about 1:2. In one example, the nitrogen:carbon atomic ratio of the nanoparticulate polymer may be about 0.35 to about 0.5 or about 0.35 to about 1. In another example, the nanoparticulate polymer may have a nitrogen:carbon elemental ratio of about 0.38.

[0055] The nanoP described herein 3 The material preferably comprises at least one binding site capable of binding one or more compounds, such as organic or organometallic compounds, or a second species as defined herein.

[0056] In one embodiment, the nanoP 3 The material comprises at least a binding site capable of binding one or more compounds, the binding site comprising an unpaired electron capable of binding an organic or organometallic compound, or a second species as defined herein.

[0057] The above nanoP 3The material may contain unpaired electrons in the polymer. These unpaired electrons may be present on or near the surface of the nanoparticle. The unpaired electrons may be present at a depth of 40 nm or less within the nanoparticle material particle, or within about 30, 20, or 10 nm of the surface, or about 10 to about 40 nm from the surface, or about 10 to 30 nm, 20 to 40 nm, or 20 to 30 nm from the surface, or about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, or 40 nm from the surface. The unpaired electrons may be present at various depths, from about 0 to about 40 nm. In some cases, the unpaired electrons may be present at depths greater than 40 nm. The unpaired electrons may be present throughout the volume of the nanoparticle material. This allows the material to react with a second species, such as an organic or organometallic species, covalently bonding the species to the nanoparticle polymer to form a complex.

[0058] In one embodiment, nanoP 3 The nanoP material has an average particle size of about 5 nm to about 500 nm. 3 The material includes an organic plasma polymer, nanoP 3 (nanoparticle polymer or aggregate thereof) contains unpaired electrons, thereby enabling covalent bonding with organic or organometallic species. 3 Materials are provided.

[0059] In another embodiment, the nanoparticulate material or nanoP 3 The material includes at least one functional moiety that is capable of chemically or physically bonding with a second species.

[0060] The above nanoP 3 The particle size of the material can be measured by scanning electron microscopy, transmission electron microscopy, small angle laser light scattering, photon correlation spectroscopy, differential mobility spectroscopy, or some other suitable technique. 3The particle size distribution of the material may be narrow or broad, and the standard deviation of the particle size distribution may be about 1% to about 500%, or about 1-200, 1-100, 1-50, 1-20, 1-10, 1-5, 1-2, 10-500, 20-500, 50-500, 100-500, 200-500, 10-100, 10-50, or 50-100%, for example, about 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500%, of the average particle size. In some cases, the particles may be substantially monodisperse, that is, all particles may have substantially the same diameter (eg, within about 10%, or about 5%, or about 2% of the same diameter).

[0061] The above nanoP 3 The material may include an organic plasma polymer. Plasma polymers are characterized by a heterogeneous, dense, highly crosslinked network. The plasma polymer may be amorphous. The plasma polymer may be generated in the plasma by reaction (e.g., ionization and fragmentation) of reactive species generated from organic gases and other reactive gases in the gas mixture, or in the plasma / gas mixture by reactive species resulting from ionization and fragmentation of gases in the gas mixture.

[0062] The above nanoP 3 Materials can be characterized by many methods, including, but not limited to, electron paramagnetic resonance (EPR) spectroscopy, infrared spectroscopy (such as Fourier transform infrared spectroscopy), Raman spectroscopy, UV-VIS spectroscopy, elemental analysis (e.g., X-ray photoelectron spectroscopy), soft X-ray spectroscopy, measurement of zeta potential, nuclear magnetic resonance (NMR) spectroscopy, mass spectroscopy, gel permeation chromatography, scanning electron microscopy (SEM), transmission electron microscopy (TEM), small-angle laser light scattering, photon correlation spectroscopy, differential mobility spectroscopy, elastic recoil detection analysis (ERDA), or neutron scattering.

[0063] The above nanoP 3The materials (e.g., the nanoparticulate polymers or aggregates) can be characterized by one or more of the following features: a broad electron paramagnetic resonance peak centered in the range of about 3470 G to about 3520 G and / or corresponding to a g-factor in the range of about 2.001 to about 2.005; Approximately 10 19 ~about 10 15 spins / cm 3 Spin densities in the range of Approximately 10 measured by electron paramagnetic resonance within approximately 0 to 240 hours after synthesis 17 ~about 10 15 spins / cm 3 Spin densities in the range of One or more absorption bands in the infrared spectrum centered on: *Approx. 3680~2700cm -1 range, *About 1800~1200cm -1 range, *Approx. 2330~2020cm -1 range, *About 1200~1010cm -1 range, and / or *About 1010~700cm -1 range, One or more absorption bands in the infrared spectrum centered on: *About 3600~3100cm -1 range, and / or *About 3100~2700cm -1 range, Zeta potential in the range of approximately -100mV to approximately +100mV, a zeta potential in the range of about -80 mV to about +80 mV measured in a solution within the pH range of about 2 to about 10, or Nitrogen:carbon elemental ratio of about 0.1:1 to about 2:3.

[0064] In one embodiment, the nanoP 3The material, nanoparticulate polymer, or aggregate is characterized using EPR spectroscopy, and the nanoparticulate polymer or aggregate may exhibit a broad electron paramagnetic resonance peak centered in the range of about 3470 G to about 3520 G and / or corresponding to a g-factor in the range of about 2.001 to about 2.005.

[0065] In one embodiment, the nanoP 3 The materials, nanoparticulate polymers, or aggregates exhibited a solubility of about 10 mol / L as measured by EPR spectroscopy within about 0 to about 2 hours after synthesis. 19 ~about 10 15 spins / cm 3 The measurements may be performed at about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 65 minutes, about 70 minutes, about 75 minutes, about 80 minutes, about 85 minutes, about 90 minutes, about 95 minutes, about 100 minutes, about 105 minutes, about 110 minutes, about 115 minutes, or about 120 minutes after synthesis.

[0066] In one embodiment, the nanoP 3 The material, nanoparticulate polymer, or aggregate has a molecular weight of about 10, as measured by electron paramagnetic resonance within about 0 to about 240 hours after synthesis. 17 ~about 10 15 spins / cm 3 The measurements may be performed at about 0.5 hours, about 1 hour, about 2 hours, about 4 hours, about 5 hours, about 6 hours, about 8 hours, about 10 hours, about 20 hours, about 30 hours, about 40 hours, about 50 hours, about 60 hours, about 70 hours, about 80 hours, about 90 hours, about 100 hours, about 110 hours, about 120 hours, about 130 hours, about 140 hours, about 150 hours, about 160 hours, about 170 hours, about 180 hours, about 190 hours, about 200 hours, about 210 hours, about 220 hours, about 230 hours, or about 240 hours after synthesis.

[0067] In one embodiment, the nanoP 3The material, nanoparticulate polymer, or aggregate may be characterized using infrared spectroscopy (such as Fourier transform infrared spectroscopy). For example, the nanoparticulate polymer or aggregate may exhibit one or more absorption bands in the infrared spectrum centered around: ·Approx. 3680~2700cm -1 range, ·About 1800~1200cm -1 range, ·About 2330~2020cm -1 range, ·About 1200~1010cm -1 range, ·About 1010~700cm -1 range, ·About 3600~3100cm -1 range, ·About 3100~2700cm -1 range, and / or · A mixture of them.

[0068] In another embodiment, the nanoP 3 The material, nanoparticulate polymer, or aggregate is characterized using the zeta potential of the nanoparticulate polymer or aggregate. 3 The zeta potential of the material, nanoparticulate polymer, or aggregate may be from about −100 mV to about +100 mV. For example, the zeta potential may be in the range of from about −50 mV to about 60 mV.

[0069] In a further embodiment, the nanoP 3 The zeta potential of the material, nanoparticulate polymer, or aggregate, when measured in a solution within a pH range of about 2 to about 10, ranges from about -80 mV to about +80 mV.

[0070] The above nanoP 3 The surface morphology of the material may be rough and cauliflower-like. 3 The formation of the material may be due to the aggregation of the nanoparticulate polymers.3 The material may be an aggregate of the nanoparticulate polymer. 3 The materials and aggregates may be spherical or approximately spherical. The particle size of the nanoparticulate polymer may be about 1 to about 50 nm, or about 5 to 10, 5 to 10, 10 to 50, 20 to 50, or 10 to 30 nm, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nm. The aggregates (and associated nanoparticulate polymers) may contain embedded highly reactive radicals whose amount decreases over time. The nanoP 3 Materials may also contain long-lived, stable radicals embedded in the delocalized orbitals of carbon clusters. These stable radicals (secondary radicals) may result from reactions involving highly reactive radicals (primary radicals). 3 The surface of the material may be hydrophilic. 3 The material may be easily dispersed in water, which may allow the bioactivity of the bioactive molecules immobilized on the surface to be maintained. The surface may become hydrophilic as a result of the oxidation of radicals during or after generation by exposing the surface to air. After complexation with one or more second species, the nanoP 3 The material composite may be hydrophilic or hydrophobic. 3 The material complex may exhibit amphiphilic properties.

[0071] Depending on the monomers used or the conditions applied during polymerization, the above nanoP 3 The material may be crosslinked. As used herein, the term "crosslinked" refers to a polymer composition containing intramolecular and / or intermolecular bonds. These crosslinks may be covalent or non-covalent in nature. Non-covalent bonds include hydrogen bonds, electrostatic bonds, and ionic bonds.

[0072] One potential benefit of cross-linking is that the resulting nanoP 3The stability of the material and, potentially, the composites formed from such materials. For example, crosslinking can improve the stability of the nanoP polymer compared to a similar composition that is not crosslinked. 3 The solubility of the material (or the resulting composite) may be reduced. 3 The crosslinked nature of the material (or composite formed therefrom) may improve the chemical resistance of the nanoparticulate material or the resulting composite.

[0073] The above nanoP 3 can be further doped with inorganic elements or compounds, or organometallic compounds, which act as image-enhancing contrast agents in medical imaging diagnostic techniques. 3 can be doped with magnetic resonance imaging (MRI) contrast agents, such as iron oxide or gadolinium compounds. Examples of image-enhancing contrast agents include fluorescent dyes (e.g., Alexa 680, indocyanine green, and Cy5.5); 11 C. 13 N, 15 O. 18 F, 32 P, 51 Mn, 52m Mn, 52 Fe, 55 Co, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 72 As, 73 Se, 75 Br, 76 Br, 82m Rb, 83 Sr, 86 Y, 90 Y, 89 Zr, 94m Tc, 94 Tc, 99m Tc, 110 In, 111 In, 120 I, 123 I, 124 I, 125 I, 131 I, 154 Gd,155 Gd, 156 Gd, 157 Gd, 158 Gd, 177 Lu, 186 Re, 188 Re, and 223 isotopes and radionuclides such as Ra; paramagnetic ions such as chromium(III), manganese(II), iron(III), iron(II), cobalt(II), nickel(II), copper(II), neodymium(III), samarium(III), ytterbium(III), gadolinium(III), vanadium(II), terbium(III), dysprosium(III), holmium(III), or erbium(III); metals such as lanthanum(III), gold(III), lead(II), and bismuth(III); chromium(III), manganese(II), iron(III ), iron(II), cobalt(II), nickel(II), copper(II), neodymium(III), samarium(III), ytterbium(III), gadolinium(III), vanadium(II), terbium(III), dysprosium(III), holmium(III), or erbium(III) oxides; metals such as lanthanum(III), gold(III), lead(II), and bismuth(III), including iron oxide and gadolinium oxide; ultrasound contrast agents such as liposomes; and radiopaque agents such as barium, gallium, and thallium compounds. The image-enhancing contrast agent may be any of the nanoP 3 It may be introduced directly onto the material or indirectly through the use of an intermediate functional group such as a chelating agent.

[0074] In an exemplary process, the nanoP3 material can be prepared by plasma polymerization via activation of a gaseous mixture of N2 / C2H2 / Ar at 150 mTorr and application of 50 W of radio frequency power.

[0075] monomer The nanoP described herein 3 The material is derived from one or more monomers.

[0076] In one embodiment, the one or more monomers are used in gaseous form, and the nanoP 3 Generate materials.

[0077] The monomer may be a hydrocarbon. Examples of hydrocarbons include alkenes, alkynes, cycloalkenes, and cycloalkynes.

[0078] Examples of suitable alkene monomers include, but are not limited to, ethylene, propene, 1-butene, 1-pentene, 1-hexene, 1-heptene, 1-octene, 1-nonene, 1-decene, isomers thereof, or mixtures thereof.

[0079] Examples of suitable alkyne monomers include, but are not limited to, ethyne (acetylene), propyne, 1-butyne, 1-pentyne, 1-hexyne, 1-heptyne, 1-octyne, 1-nonyne, 1-decyne, isomers thereof, or mixtures thereof.

[0080] Examples of suitable cycloalkene monomers include, but are not limited to, cyclopropene, cyclobutene, cyclopentene, cyclohexene, cycloheptene, cyclooctene, 1,3-cyclohexadiene, 1,4-cyclohexadiene, 1,5-cyclooctadiene, isomers thereof, or mixtures thereof.

[0081] Examples of suitable cycloalkyne monomers include, but are not limited to, cycloheptyne, cyclooctyne, cyclononyne, isomers thereof, or mixtures thereof.

[0082] In one embodiment, an alkene is used as a monomer, which may be the only monomer utilized to form the nanoparticulate polymer, or the alkene may be used in the presence of at least one other monomer, such as another alkene and / or an alkyne, cycloalkene, or cycloalkyne, to form a copolymer.

[0083] In one embodiment, an alkyne is used as a monomer. The alkyne may be the only monomer utilized to form the nanoparticulate polymer, or the alkene may be used in the presence of at least one other monomer, such as another alkyne and / or alkene, cycloalkene, or cycloalkyne, to form a copolymer. In a further embodiment, acetylene is used as a monomer, either alone or in the presence of at least one other monomer.

[0084] In another embodiment, acetylene is used as a monomer in combination with at least one other monomer, for example, at least one other monomer that is an alkene, alkyne, cycloalkene, or cycloalkyne.

[0085] In one embodiment, a cycloalkene is used as a monomer, which may be the only monomer utilized to form the nanoparticulate polymer, or the cycloalkene may be used in the presence of at least one other monomer, such as another cycloalkene and / or an alkene, alkyne, or cycloalkyne, to form a copolymer.

[0086] In one embodiment, a cycloalkyne is used as a monomer, which may be the only monomer utilized to form the nanoparticulate polymer, or the cycloalkyne may be used in the presence of at least one other monomer, such as another cycloalkyne and / or an alkene, alkyne, or cycloalkene, to form a copolymer.

[0087] The above nanoP 3 Other monomers that can be used to form include perfluorocarbons, ethers, esters, amines, alcohols, or carboxylic acids.

[0088] Examples of suitable perfluorocarbons include, but are not limited to, perfluoroallylbenzene.

[0089] Examples of suitable ethers include, but are not limited to, diethylene glycol vinyl ether, diethylene glycol divinyl ether, diethylene glycol monoallyl ether, or mixtures thereof.

[0090] Examples of suitable amines include, but are not limited to, allylamine, cyclopropylamine, poly(vinylamine), or mixtures thereof.

[0091] Examples of suitable alcohols include, but are not limited to, poly(vinyl alcohol), allyl alcohol, ethanol, or mixtures thereof.

[0092] Examples of suitable carboxylic acids include, but are not limited to, acrylic acid.

[0093] biologically active agents As used herein, the term "biologically active agent" refers to any agent (e.g., a peptide, polypeptide, nucleic acid, or small molecule drug) that has biological and / or pharmacological activity in vivo. One of ordinary skill in the art will appreciate that biologically active agents or drugs known to prevent restenosis or prevent inflammation, or agents that treat cardiovascular disease, may be suitable agents for complexation with the nanoparticles described herein. Examples of suitable biologically active agents include, but are not limited to, antiplatelet and anticoagulant agents, antithrombotic and fibrinolytic agents, antireplicative and antiproliferative agents, anti-inflammatory agents, cardiovascular drugs, proteins, peptides, and nucleotides.

[0094] As used herein, the term "peptide" is intended to refer to any polymer comprising amino acids linked by peptide bonds. The term "peptide" is intended to encompass not only polymers assembled using ribosomes, but also polymers assembled enzymatically (i.e., non-ribosomal peptides) and synthetically assembled polymers. In various embodiments, the term "peptide" may be considered synonymous with "protein" or "polypeptide." In various embodiments, the term "peptide" may be limited to polymers of more than 50 amino acids, or alternatively, to polymers of 50 or fewer amino acids. While in various embodiments, the term "peptide" is intended to include only amino acids as monomer units of the polymer, in various embodiments, the term "peptide" includes additional components and / or modifications to the amino acid backbone. For example, in various embodiments, the term "peptide" may apply not only to core polymers of amino acids but also to derivatives of such core polymers, such as core polymers having pendant polyethylene glycol groups or amide groups at the amino or carboxy termini of the amino acid chain.

[0095] A "peptidomimetic" can be a molecule, such as a peptide, modified peptide, or any other molecule, that biologically mimics an active ligand or a biomolecule, such as an enzyme substrate or cytokine. For example, a peptidomimetic may antagonize, stimulate, or otherwise modulate the physiological activity of a cytokine involved in the inflammatory process. Alternatively, the peptidomimetic may mimic the activity of the native protein in treating cardiovascular disease.

[0096] As used herein, the term "protein" refers to a sequence of amino acids whose chain length is sufficient to form tertiary and / or quaternary structures at higher levels of the sequence of amino acids. The molecular weight of the protein may range from about 300 Da to about 150 kDa. The molecular weight of the protein may be greater than 150 kDa or less than 300 Da.

[0097] Antiplatelet and anticoagulant agents Adhesion and platelet aggregation can be prevented by antiplatelet and anticoagulant agents. In one example, the biologically active agent is an antiplatelet agent. In another example, the biologically active agent is an anticoagulant agent.

[0098] Examples of suitable antiplatelet agents include, but are not limited to, aspirin and dipyridamole. Aspirin is classified as an analgesic, antipyretic, anti-inflammatory, and antiplatelet agent. Clinical trials have shown that aspirin reduces the risk of sudden death and / or nonfatal reinfarction in patients after a myocardial infarction (heart attack). The proposed mechanism of action of aspirin is directly related to platelets. Aspirin somehow blocks platelets, limiting blood clotting. This prevents the cascade of platelet aggregation seen in thrombus and subsequent restenosis. Therefore, aspirin is a candidate for a restenosis inhibitor. Dipyridamole is a drug similar to aspirin in that it possesses antiplatelet properties. Dipyridamole is also classified as a coronary vasodilator. Dipyridamole increases coronary blood flow by primary selective dilation of the coronary arteries without altering systemic blood pressure or peripheral arterial blood flow. These vasodilatory properties are thought to be potentially beneficial in preventing restenosis.

[0099] Examples of suitable anticoagulants include, but are not limited to, heparin, coumadin, protamine, and hirudin. These drugs function as anticoagulants by preventing the production of thrombin, a binding agent that causes blood to clot. This may also reduce the cascade effect of platelet aggregation at the lesion site, thereby reducing restenosis. When protamine is used in the presence of heparin, it acts as a heparin antagonist, blocking the effects of heparin. However, protamine used alone acts as an anticoagulant. Hirudin was chosen because it is not normally present in the human body. Hirudin is a drug found in the salivary glands of leeches. Hirudin is a highly concentrated anticoagulant that acts in a manner similar to heparin, coumadin, and protamine.

[0100] Antithrombotic and fibrinolytic agents In one example, the biologically active agent is an antithrombotic agent, hi another example, the biologically active agent is a fibrinolytic agent.

[0101] Examples of antithrombotic and fibrinolytic agents include, but are not limited to, glycoprotein IIb / IIIa inhibitors, direct thrombin inhibitors, heparin, low molecular weight heparin, platelet adenosine diphosphate (ADP) receptor inhibitors, fibrinolytic agents (including streptokinase, urokinase, recombinant tissue plasminogen activator, reteplase, and tenecteplase), enzymes (including streptokinase, urokinase, tissue plasminogen activator (tPA), and plasmin), or mixtures thereof.

[0102] Anti-Replication and Anti-Proliferation Agents There are several types of drugs that interfere with cell replication. Without wishing to be bound by theory, antimitotic agents (cytotoxic agents) act directly to prevent cells from mitosis (replication), while antimetabolites prevent deoxyribonucleic acid (DNA) synthesis and thus replication. In one example, the biologically active agent is an anti-replicative agent. In one example, the biologically active agent is an anti-proliferative agent.

[0103] Examples of suitable anti-replicative agents include, but are not limited to, methotrexate, colchicine, azathioprine, vincristine, vinblastine, fluorouracil, adriamycin, and mutamycin.

[0104] Examples of suitable antiproliferative agents include, but are not limited to, target of rapamycin (mTOR) inhibitors (including sirolimus, everolimus, and ABT-578), paclitaxel, and antitumor agents (including alkylating agents such as cyclophosphamide, mechlorethamine, chlorambucil, melphalan, carmustine, lomustine, ifosfamide, procarbazine, dacarbazine, temozolomide, altretamine, cisplatin, carboplatin, and oxaliplatin). In one embodiment, the biologically active agent is paclitaxel.

[0105] In another embodiment, the biologically active agent is sirolimus ((1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28E,30S,32S,35R)-1,18-dihydroxy-12-[(2R)-1-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]propan-2-yl]-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-11,36-dioxa-4-azatricyclo[30.3.1.0] 4,9 ]Hexatriaconta-16,24,26,28-tetraene-2,3,10,14,20-pentone; CAS No. 53123-88-9). Sirolimus, also known as rapamycin, is a macrolide compound believed to inhibit T and B cell activation by reducing their sensitivity to IL-2 through mTOR inhibition. Sirolimus is an immunosuppressant and has previously been reported to be contraindicated for wound healing. Impaired wound healing at surgical sites is observed in many patients receiving this drug. Areas of blood vessels treated with sirolimus are known to experience delayed reendothelialization due to the drug's antiproliferative effects. Sirolimus is represented by Formula I. Formula I:

[0106] [ka]

[0107] anti-inflammatory agents Anti-inflammatory biologically active agents or drugs may also be useful locally to suppress inflammation caused by injury to the luminal tissue during angioplasty.

[0108] In one example, the biologically active agent is an anti-inflammatory agent. For example, the anti-inflammatory agent can be an anti-inflammatory drug or a biomolecule.

[0109] Suitable anti-inflammatory drugs include, but are not limited to, corticosteroids such as dexamethasone, betamethasone, and prednisone, and widely used immunosuppressants such as sulindac (2-[(3Z)-6-fluoro-2-methyl-3-[(4-methylsulfinylphenyl)methylidene]inden-1-yl]acetic acid; CAS No. 38194-50-2), naproxen (CAS No. 22204-53-1), and aspirin (CAS No. 50-78-2). In one embodiment, the biologically active agent is sulindac (2-[(3Z)-6-fluoro-2-methyl-3-[(4-methylsulfinylphenyl)methylidene]inden-1-yl]acetic acid; CAS No. 38194-50-2). Sulindac, shown as Formula II, is a nonsteroidal anti-inflammatory drug. Formula II:

[0110] [ka]

[0111] Suitable anti-inflammatory agents include cytokines or fragments thereof. Cytokines are small proteins (approximately 5-20 kDa) that are important in cell signaling. Inflammation is characterized by the interaction between pro-inflammatory and anti-inflammatory cytokines. Cytokines include chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors. Suitable examples include, but are not limited to, anti-inflammatory cytokines such as IL4, IL-10, IL-13, IFN-α, and transforming growth factor-β.

[0112] It is known in the art that macrophage polarization can be alternatively activated by interleukins. The M1 phenotype is pro-inflammatory, while the M2 phenotype is anti-inflammatory. Thus, one skilled in the art will recognize that interleukins known to activate the M2 anti-inflammatory phenotype are suitable biologically active agents for conjugation to the nanoparticles described herein. In one example, the biologically active agent is a cytokine or a fragment thereof. In one example, the biologically active agent is interleukin-4 (IL-4). In another example, the biologically active agent is interleukin-10 (IL-10).

[0113] Cardiovascular drugs In one example, the biologically active agent may be a drug for treating cardiovascular disease. Suitable drugs include, but are not limited to, ACE inhibitors, anti-gotensin receptor blockers, calcium channel blockers, vasodilators, and statins (also known as HMG-CoA reductase inhibitors). For example, the biologically active agent is a statin, such as simvastatin, pitavastatin, lovastatin, fluvastatin, or a mixture thereof. In one embodiment, the biologically active agent is simvastatin.

[0114] It is also contemplated that nitric oxide-releasing agents may be suitable agents for conjugation to the nanoparticles described herein.

[0115] antibody In one embodiment, the biologically active agent is an antibody. The antibody may be an antibody that can target the biologically active agent to the correct location in the blood vessel. Additionally or alternatively, the antibody may be an antagonist, such as a cytokine inhibitor.

[0116] As used herein, the term "antibody" or "antibodies" refers to an antibody that binds to one or more immunoglobulin chains, e.g., V L and a polypeptide comprising V H The term "antibody" is intended to encompass proteins comprising a variable region composed of a polypeptide comprising: V, Vc, Vdc, Vs ... H and V L interact to form an Fv containing an antigen-binding region capable of specifically binding one or a few closely related antigens. Generally, light chains from mammals are either kappa or lambda light chains, and heavy chains from mammals are alpha, delta, epsilon, gamma, or mu. The antibody may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. The term "antibody" also encompasses humanized antibodies, deimmunized antibodies, non-depleting antibodies, non-activating antibodies, primatized antibodies, human antibodies, and chimeric antibodies. As used herein, the term "antibody" is also intended to include forms other than full-length, intact, or whole antibody molecules, such as Fab, F(ab'), and Fv, which are capable of binding an epitopic determinant. These formats are sometimes referred to as "fragments" of antibodies. These antibody formats retain some ability to selectively bind to the target protein, and examples of these antibody formats include: (1) Fab: The fragment that contains a monovalent binding fragment of an antibody molecule and can be produced by digesting whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain; (2) Fab': The fragment of an antibody molecule that can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of one heavy chain; two Fab' fragments are obtained per antibody molecule; (3) (Fab')2: The fragment of an antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab)2 is a dimer of two Fab' fragments linked by two disulfide bonds; (4) Fv: defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; (5) Single-chain antibody ("SCA"): defined as a genetically engineered molecule containing a light chain variable region and a heavy chain variable region linked by an appropriate polypeptide linker as a genetically fused single-chain molecule; such single-chain antibodies may be in the form of multimers such as diabodies, triabodies, and tetrabodies, which may or may not be multispecific; and (6) Single-domain antibodies, typically variable heavy domains without light chains These include, but are not limited to:

[0117] Thus, the antibodies described herein may comprise separate heavy chains, light chains, Fab, Fab', F(ab'), Fc, variable light chain domains without any heavy chains, variable heavy chain domains without light chains, and Fv. Such fragments may be produced by recombinant DNA techniques or by enzymatic or chemical separation of intact immunoglobulins. Any of the antibodies or fragments thereof described herein and others known in the art may be used in conjunction with the nanoP antibodies disclosed herein. 3 It can be complexed with particles.

[0118] Thus, the biologically active agent may be an antibody or a protein or peptide that targets a factor involved in the inflammatory process.

[0119] As used herein, the term "targeting ligand" refers to a molecule that binds to or interacts with a target molecule. Generally, the nature of the interaction or binding is non-covalent, e.g., by hydrogen, electrostatic, or van der Waals interactions, but may also be covalent.

[0120] As used herein, the term "ligand" refers to a compound that targets a biological marker. Examples of ligands include, but are not limited to, proteins, peptides, antibodies, antibody fragments, saccharides, carbohydrates, glycans, cytokines, chemokines, nucleotides, lectins, lipids, receptors, steroids, neurotransmitters, Cluster of Designation / Differentiation (CD) markers, imprinted polymers, and the like.

[0121] Examples of targeting ligands include, but are not limited to, nuclear localization signals (e.g., KR[PAATKKAGQA]KKKK), RGD, NGR, folate, transferrin, GM-CSF, galactosamine, anti-VEGFR, anti-ERBB2, anti-CD20, anti-CD22, anti-CD19, anti-CD33, anti-CD25, anti-tenascin, anti-CEA, anti-MUC1, anti-TAG72, anti-HLA-DR10, or mixtures thereof.

[0122] Polynucleotides The biologically active agent may be a polynucleotide that can regulate the inflammatory process. As used herein, the term "polynucleotide" is intended to encompass DNA, RNA, antisense polynucleotides, ribozymes, interfering RNA, siRNA, microRNA, and any other polynucleotides known in the art. The polynucleotide may encode a protein or functional RNA (such as interfering RNA) that can disrupt inflammation. Thus, the polynucleotide may be a polynucleotide vector or a plasmid.

[0123] Examples of gene targeting agents include, but are not limited to, DNA (gDNA, cDNA), RNA (sense RNA, antisense RNA, mRNA, tRNA, rRNA, small interfering RNA (siRNA), small hairpin RNA (ShRNA), microRNA (miRNA), small nucleolar RNA (SnoRNA, small nuclear RNA (snRNA))), ribozymes, aptamers, DNAzymes, antisense oligonucleotides, vectors, plasmids, other ribonuclease-type complexes, and mixtures thereof. For example, the biologically active agent may be an siRNA directed against the p65 subunit of NF-κB, thereby altering NF-κB-mediated inflammation, or a gene targeting agent directed against IkB kinase or AP-1, encoding cytokine expression and / or cytokine receptor expression.

[0124] stem cells The biologically active agent may be a stem cell, such as a skeletal myoblast, a bone marrow-derived stem cell, a bone marrow-derived mononuclear cell, a bone marrow-derived hematopoietic stem cell and endothelial progenitor cell, a mesenchymal stromal / stem cell, a cardiac muscle stem cell and progenitor cell, an induced pluripotent stem cell, or a mixture thereof. For example, the stem cell may be a mesenchymal stromal / stem cell that adopts an immunosuppressive phenotype in the presence of inflammatory cytokines.

[0125] contrast agents In one embodiment, the agent is a contrast agent. The contrast agent can be used in vivo to examine vascular structure, function, and angiogenesis. Examples of suitable contrast agents include, but are not limited to, luciferase; fluorescently labeled dyes and antibodies, contrast agents (including iopamidol, iohexol, and ioxilan); barium sulfate; indocyanine green (ICG), and mixtures thereof.

[0126] The imaging agent may be an image-enhancing imaging agent. Examples of suitable image-enhancing imaging agents include fluorescent dyes (e.g., Alexa 680, indocyanine green, and Cy5.5); fluorescent dyes (e.g., Alexa 680, indocyanine green, and Cy5.5); 11 C. 13 N, 15 O. 18 F, 32 P, 51 Mn, 52m Mn, 52 Fe, 55 Co, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 72 As, 73 Se, 75 Br, 76 Br, 82m Rb, 83 Sr, 86 Y, 90 Y, 89 Zr, 94m Tc, 94 Tc, 99m Tc, 110 In, 111 In, 120 I, 123 I, 124 I, 125 I, 131 I, 154 Gd, 155 Gd, 156 Gd, 157 Gd, 158 Gd, 177 Lu, 186 Re, 188 Re, and 223isotopes and radionuclides such as Ra; paramagnetic ions such as chromium(III), manganese(II), iron(III), iron(II), cobalt(II), nickel(II), copper(II), neodymium(III), samarium(III), ytterbium(III), gadolinium(III), vanadium(II), terbium(III), dysprosium(III), holmium(III), or erbium(III); metals such as lanthanum(III), gold(III), lead(II), and bismuth(III); chromium(III), manganese(II), iron(III), iron(II) (II), cobalt(II), nickel(II), copper(II), neodymium(III), samarium(III), ytterbium(III), gadolinium(III), vanadium(II), terbium(III), dysprosium(III), holmium(III), or erbium(III) oxides; metals such as lanthanum(III), gold(III), lead(II), and bismuth(III), including iron oxide and gadolinium oxide; ultrasound contrast agents such as liposomes; and radiopaque agents such as barium, gallium, and thallium compounds. The image-enhancing contrast agent may be any of the nanoP 3 It may be directly conjugated onto the material or indirectly conjugated through the use of an intermediate functional group such as a chelating agent.

[0127] Complex As used herein, the term "conjugate" refers to a molecule formed by binding one or more compounds to a nanoparticulate polymer or an aggregate comprising a nanoparticulate polymer. The "one or more compounds" may be a biologically active agent, as defined herein. The binding may be via a covalent bond or electrostatic interaction.

[0128] The nanoparticulate polymers, aggregates, or nanoPs described herein 3The material is conjugated with a drug. The drug can be a biologically active drug or an imaging agent. In one embodiment, the drug is a biologically active drug. In another embodiment, the drug is an imaging agent. The binding of the drug can be controlled by changing the pH of the reaction conditions during the conjugation process. The pH of the solution influences the binding of nanoP through the protonation or deprotonation of surface functional groups such as amine and carboxylic acid groups. 3 For example, the positively charged complex nanoP 3 Binding to the material is 3 This may be improved by increasing the pH of the solution containing the material and the agent. 3 The negatively charged nanoparticles are formed by deprotonation of the carboxylic acid surface groups, which also stabilizes the nanoparticles due to repulsion between the negatively charged particles. 3 Binding to the material is 3 This may be improved by lowering the pH of the solution containing the material and the agent.

[0129] Herein, nanoP in the formation of a complex 3 The use of materials (eg, nanoparticle polymers, aggregates, or mixtures thereof) is disclosed.

[0130] The complex may include only a single agent, or the complex may include two or more different agents, for example, two, three, or four second agents.

[0131] The above nanoP 3 Materials can generally be directly bonded to drugs, such as biologically active drugs or imaging agents, by, for example, covalent or ionic bonds. When the nanoparticle polymer or aggregate is a plasma polymer, the bonding process is generally rapid and can proceed under mild conditions. The bonding can occur via unpaired electrons (i.e., radical sites) in the polymer structure or via the nanoP 3By adding a suitable biologically active agent or by forming a nanoP 3 This is due to functional groups introduced onto the resulting composite by reaction with air (or another gas) or some other fluid to which the material is exposed. 3 The material may include monomer units that contain functional groups to which drugs can be chemically attached. 3 Conjugation with materials may introduce functional groups onto or into the resulting conjugate, which can be used for further chemical reactions or as binding sites for processes such as biochemical / biological processes carried out under in vitro or in vivo conditions.

[0132] As used herein, functional moieties (or "functional groups") refer to the monomers, nanoP 3 Materials, or nanoP 3 A group of atoms present on a composite containing a material that can react with another complementary functional group, such as another functional group present on a drug. Functional groups include, but are not limited to, the following groups: carboxylic acid (-(C=O)OH), carbonyl, primary or secondary amine (-NH, -NH-), nitric oxide, maleimide, thiol (-SH), sulfonic acid (-(O=S=O)OH), carbonate, carbamate (-O(C=O)N<), hydroxy (-OH), aldehyde (-(C=O)H), ketone (-(C=O)-), hydrazine (>NN<), isocyanate, isothiocyanate, phosphate (-O(P=O)OHOH), phosphonate (-O(P=O)OHH), haloacetyl, alkyl halide, acryloyl, aryl fluoride, hydroxylamine, disulfide, vinyl sulfone, vinyl ketone, diazoalkane, oxirane, and aziridine, or mixtures thereof.

[0133] Thus, in one aspect, the present disclosure provides: a nanoparticulate polymer formed from a plasma having an average diameter of about 1 nm to about 50 nm and comprising at least one monomer selected from an alkene, an alkyne, a cycloalkene, a cycloalkyne, or a mixture thereof; or an aggregate comprising two or more of the nanoparticulate polymers described above and having an average diameter of about 5 nm to about 500 nm; a biologically active agent selected from the group consisting of anti-inflammatory cytokines, anti-inflammatory drugs, statins, and anti-proliferative drugs; The present invention provides a composite comprising:

[0134] In one embodiment, the average diameter of the aggregates is about 5 to 500 nm, or about 5 to about 400 nm, or about 5 to about 300 nm, or about 5 to about 200 nm, or about 5 to about 100 nm, or about 50 to about 100 nm, or about 100 to about 500 nm, or about 150 to about 500 nm, or about 180 nm to about 500 nm, or about 100 to about 400 nm, or about 150 to about 400 nm, or about The average diameter of the aggregates is in the range of 180 to about 400 nm, or about 100 to about 300 nm, or about 150 to 300 nm, or about 180 to 300 nm, or about 100 to about 200 nm, or about 150 to about 200 nm, or about 180 to about 200 nm, or about 150 to about 250 nm, or about 180 to about 250 nm, or about 200 to about 400 nm, or about 200 nm to about 300 nm, or a mixture thereof. In one embodiment, the average diameter of the aggregates is about 200 nm. In one embodiment, the average diameter of the aggregates is about 100 nm. In one embodiment, the average diameter of the aggregates is about 100 nm to about 200 nm. In another embodiment, the average diameter of the aggregates is about 50 to about 100 nm.

[0135] In one embodiment, the anti-inflammatory cytokine is IL-4. The IL-4 is about 0.5 μg, about 0.6 μg, about 0.7 μg, about 0.8 μg, about 0.9 μg, about 1.0 μg, or about 1.1 μg IL-4 / 10 9 - may be bound to nanoP3 at an equivalent concentration at the loading capacity of nanoP3 or alternatively at an equivalent amount of nanoP3.

[0136] In one embodiment, the anti-inflammatory cytokine is IL-10. The IL-10 is about 0.5 μg, about 0.6 μg, about 0.7 μg, about 0.8 μg, about 0.9 μg, about 1 μg, about 1.1 μg, about 1.2 μg, about 1.3 μg, or about 1.4 μg IL-10 / 10 9 The IL-10 may be bound to nanoP3 at an equivalent concentration in the loading capacity of nanoP3, or alternatively, in an amount of nanoP3. In one example, the IL-10 is about 1.3 μg IL-10 / 10 9 - may be bound to nanoP3 at an equivalent concentration at the loading capacity of nanoP3 or alternatively at an equivalent amount of nanoP3.

[0137] Thus, in another aspect, the present disclosure provides a method for producing a pharmaceutical composition comprising: a nanoparticulate polymer formed from a plasma having an average diameter of about 1 nm to about 50 nm and comprising at least one monomer selected from an alkene, an alkyne, a cycloalkene, a cycloalkyne, or a mixture thereof; or an aggregate comprising two or more of the nanoparticulate polymers described above and having an average diameter of about 100 nm to about 200 nm; IL-10 and Alternatively, the mean diameters described herein may be used.

[0138] In one embodiment, the antiproliferative drug is sirolimus. The sirolimus is administered at a dose of about 1.5 μg to about 3 μg, or about 1.5 μg to about 2.5 μg, or about 2 μg to about 2.5 μg, or about 2 μg to about 3 μg. 9 The sirolimus may be bound to nanoP3 at a concentration equivalent to the loading capacity of nanoP3, or alternatively, at an equivalent concentration in nanoP3. In one example, the sirolimus is about 2.50 μg sirolimus / 10 9 - may be bound to nanoP3 at an equivalent concentration at the loading capacity of nanoP3 or alternatively at an equivalent amount of nanoP3.

[0139] Surprisingly, the present inventors found that the sirolimus bound to nanoP3 increased reendothelialization and thus promoted vascular healing, a finding contrary to the effects of free sirolimus.

[0140] Thus, in another aspect, the present disclosure provides a method for producing a pharmaceutical composition comprising: a nanoparticulate polymer formed from a plasma having an average diameter of about 1 nm to about 50 nm and comprising at least one monomer selected from an alkene, an alkyne, a cycloalkene, a cycloalkyne, or a mixture thereof; or an aggregate comprising two or more of the nanoparticulate polymers described above and having an average diameter of about 100 nm to about 200 nm; Sirolimus and Alternatively, the mean diameters described herein may be used.

[0141] In one embodiment, the anti-inflammatory drug is sulindac. The sulindac is administered at a dose of about 2 μg to about 3.5 μg, or about 2.5 μg to about 3.5 μg, or about 2.5 μg to about 3.1 μg. 9 - The loading capacity of nanoP3, or alternatively, the nanoP3 may be bound to the nanoP3 at an equivalent concentration in an amount of nanoP3. In one example, the sulindac is about 3.05 μg sulindac / 10 9 - may be bound to nanoP3 at an equivalent concentration at the loading capacity of nanoP3 or alternatively at an equivalent amount of nanoP3.

[0142] Thus, in another aspect, the present disclosure provides a method for producing a pharmaceutical composition comprising: a nanoparticulate polymer formed from a plasma having an average diameter of about 1 nm to about 50 nm and comprising at least one monomer selected from an alkene, an alkyne, a cycloalkene, a cycloalkyne, or a mixture thereof; or an aggregate comprising two or more of the nanoparticulate polymers described above and having an average diameter of about 100 nm to about 200 nm; Sulindac and Alternatively, the mean diameters described herein may be used.

[0143] It is contemplated that the nanoparticulate polymers, aggregates thereof, or composites thereof can be incorporated into scaffolds suitable for treating incisions / wounds or vascular injuries. For example, the nanoparticulate polymers, aggregates thereof, or composites thereof can be used on or in implants such as cardiac patches, vascular grafts, and stents.

[0144] Manufacture of the complex Details of the preparation of the nanoP3 material and nanoP3 composites are described in PCT Publication No. WO2018 / 112543, page 49, line 18 to page 71, line 7, which is incorporated herein by reference. An exemplary process for preparing the composite is provided below.

[0145] NanoP3 material can be prepared by plasma polymerization via activation of a gaseous mixture of N2 / C2H2 / Ar at 150 mTorr and application of 50 W of radio frequency power. Using fluorescently labeled molecules such as Cy5, the loading capacity and binding efficiency of nanoP3 for biologically active agents such as IL-4 and IL-10 can be measured to establish optimal incubation parameters for both in vitro and in vivo processing.

[0146] The biologically active agent is mixed with the nanoP3 in ultrapure water in a total reaction volume of 1 ml and incubated statically at room temperature for 1 hour. After 1 hour of incubation, the remaining wash solution can be used to analyze the binding kinetics on a Clariostar monochromator microplate reader (BMG Labtech, Germany).

[0147] Pharmaceutical Composition The complex may be present in a pharmaceutical composition. Details of suitable pharmaceutical compositions and methods for preparing such compositions are described in PCT Publication No. WO2018 / 112543, page 40, line 7 to page 44, line 26, which is incorporated herein by reference.

[0148] Delivery of the complex to the blood vessels Disclosed herein are methods for localizing a biologically active agent to a region of a blood vessel, modulating inflammation in a region of a blood vessel, and retaining a biologically active agent at a delivery site in a blood vessel by complexing the biologically active agent with a nanoparticle, thereby producing a complex, and delivering the complex to the region of the blood vessel.

[0149] The complex can be delivered to the blood vessel by any suitable delivery method known in the art, including, but not limited to, catheters, stents, stented vascular grafts, grafts, and valves, or by direct injection into the blood vessel.

[0150] In one example, the catheter is an occlusion perfusion catheter, hi another example, the catheter is a sweating balloon catheter.

[0151] After delivery, the complex is retained at the delivery site in the blood vessel. In one embodiment, the complex is retained in the region of the blood vessel for a longer period of time than the uncomplexed biologically active agent is retained in the region of the blood vessel. For example, the complex may be retained in the region of the blood vessel for at least 1.2, 1.2, 1.5, 1.7, or 2 times the period of time the uncomplexed biologically active agent is retained in the region of the blood vessel, or the complex is retained at the delivery site for at least 1 day, or at least 2 days, or at least 3 days, or at least 4 days, or at least 5 days, or at least 6 days, or at least 7 days, or at least 8 days, or at least 9 days, or at least 10 days, or at least 11 days, or at least 12 days, or at least 13 days, or at least 14 days. In one example, the complex is retained at the delivery site for at least 1 day. In another example, the complex is retained at the delivery site for at least 5 days. In another example, the complex remains at the delivery site for at least 14 days.

[0152] Treatment method Also disclosed herein are methods for treating or preventing vascular injury or disease, comprising delivering to a subject a conjugate as defined herein or a pharmaceutical composition comprising a conjugate as defined herein. The term "treating" is used herein to encompass both therapeutic and prophylactic treatment. Accordingly, the methods of treatment disclosed herein may encompass methods of preventing one or more symptoms of a disease, disorder, or condition. It is understood that "treating" may also be interpreted as alleviating any one or more symptoms of a disease, disorder, or condition. Thus, "treating" encompasses a reduction in vascular occlusion or neointima formation and / or an increase in the rate of reendothelialization compared to a patient not receiving a conjugate as disclosed herein or receiving only an unconjugated biologically active agent. As used herein, "promoting healing" refers to an increase in any marker of healing compared to a patient not receiving a conjugate as disclosed herein or receiving only an unconjugated biologically active agent. "Healing" may refer to the healing of a vascular wound. It is understood that the term "healing" encompasses processes such as endothelialization. Therefore, any reference herein to promoting healing should be understood to refer to promoting endothelialization.

[0153] In one aspect, the present disclosure provides a method for treating or preventing vascular injury or disease, comprising delivering a complex as defined herein to an area of a blood vessel in a patient in need thereof.

[0154] Thus, in one example, the present disclosure provides: a nanoparticulate polymer formed from a plasma having an average diameter of about 1 nm to about 50 nm and comprising at least one monomer selected from an alkene, an alkyne, a cycloalkene, a cycloalkyne, or a mixture thereof; or an aggregate comprising two or more of the nanoparticulate polymers described above and having an average diameter of about 100 nm to about 200 nm; i) interleukin-10, or ii) sulindac, or iii) Sirolimus and The present invention provides a method for treating or preventing vascular injury or disease, comprising delivering a complex comprising:

[0155] In another aspect, the present disclosure provides a method of promoting healing comprising delivering a complex as defined herein to an area of a blood vessel in a patient in need thereof.

[0156] Thus, in one example, the present disclosure provides: a nanoparticulate polymer formed from a plasma having an average diameter of about 1 nm to about 50 nm and comprising at least one monomer selected from an alkene, an alkyne, a cycloalkene, a cycloalkyne, or a mixture thereof; or an aggregate comprising two or more of the nanoparticulate polymers described above and having an average diameter of about 100 nm to about 200 nm; i) interleukin-10, or ii) Sirolimus and The present invention provides a method for promoting healing, comprising delivering a complex comprising:

[0157] Also disclosed herein is the use of a conjugate as defined herein in the manufacture of a medicament for the treatment or prevention of a disease in a patient.

[0158] In one aspect, the disclosure provides the use of a conjugate as defined herein in the manufacture of a medicament for the treatment or prevention of vascular injury or vascular disease in a patient in need thereof.

[0159] In one aspect, the disclosure provides the use of a conjugate as defined herein in the manufacture of a medicament for promoting healing in a patient in need thereof.

[0160] In one embodiment, the conjugates defined herein are used as or in the manufacture of a medicament.

[0161] In another embodiment, the conjugate defined herein is for use in the treatment or prevention of vascular disease.

[0162] In another embodiment, the complexes defined herein are for use in promoting healing.

[0163] In another aspect, the present disclosure provides a conjugate as defined herein when used in the treatment or prevention of vascular disease.

[0164] In another aspect, the present disclosure provides a complex as defined herein when used to promote healing.

[0165] The conjugates can be provided in an "effective amount," for example, when the appropriate compound is added to a pharmaceutical composition. The phrase "effective amount" is intended to mean the amount of the conjugate that will elicit the desired biological or medical response in a tissue, system, animal, or human being sought by a researcher, veterinarian, physician, or other clinician administering the compound of a composition comprising the compound.

[0166] The "effective amount" will depend on many factors, including the potency of the particular conjugate. The weight and age of the patient may also be factors for those skilled in the art in determining the concentration of compound that the patient should receive.

[0167] The phrases "administration of" a compound and / or "administering" a compound should be understood to mean providing a conjugate or a pharmaceutical composition comprising a conjugate as defined herein to a patient in need of treatment.

[0168] The recipient of the conjugates provided herein may be a human being, male or female.

[0169] Alternatively, the recipient of the nanoparticles, aggregates, or complexes; or pharmaceutical compositions comprising the nanoparticles, aggregates, or complexes, may be a non-human animal. "Non-human animals" or "non-human animals" refers to the animal kingdom excluding humans, and includes both male and female vertebrates and invertebrates, including warm-blooded animals, including mammals (including, but not limited to, primates, dogs, cats, cows, pigs, sheep, goats, rats, guinea pigs, horses, or other bovine, ovine, equine, canine, feline, rodent, or murine species), birds, insects, reptiles, fish, and amphibians.

[0170] Recipients of the conjugates and pharmaceutically acceptable compositions are referred to herein using the interchangeable terms "patient," "recipient," "individual," and "subject." These four terms are used interchangeably and refer to any human or animal as defined herein (unless otherwise indicated). The patient may be undergoing or have previously undergone an endovascular intervention. In one example, the patient is undergoing an endovascular intervention. In another example, the patient has previously undergone an endovascular intervention. The vascular injury may be due to an endovascular intervention. For example, the endovascular intervention may be stent placement or balloon angioplasty. In one example, the endovascular intervention may be stent placement. In another example, the endovascular intervention is balloon angioplasty.

[0171] Potential diseases, disorders, or conditions that may be treated using the conjugates described herein include, but are not limited to, vascular diseases such as atherosclerosis, peripheral arterial disease, acute coronary syndromes; gastrointestinal diseases, and renal diseases.

[0172] Any discussion of any document, statute, material, device, article, or the like contained in this specification should not be construed as an admission that any or all of that matter formed part of the prior art or was common general knowledge in the art relevant to the present disclosure by virtue of its existence prior to the priority date of the appended claims. [Example]

[0173] Example 1 Surface binding of interleukins to nanoP3 Carrier-free recombinant rat IL-4 and IL-10 (R&D Systems, USA) were dissolved in phosphate-buffered saline (PBS) at a stock concentration of 100 ng / μl. To confirm binding kinetics, each interleukin was tagged with a Lightning-link Cy5 antibody label (Novus Biologicals, USA). 1 × 10 IL-4 and IL-10 were dissolved in ultrapure water (Thermofisher, USA). 9 For each nanoP3 (200 nm diameter), 1.42 μg of either IL-4 or 1.32 μg of IL-10 was added. Water was added to bring the total reaction volume to 1 ml, and the plates were incubated at room temperature for 1 hour. After the 1-hour incubation, the remaining wash buffer was used to analyze binding kinetics using a Clariostar monochromator microplate reader (BMG Labtech, Germany).

[0174] The binding efficiency of nanoP3 to IL-4 in solution was 99%, which was the highest at 0.5±0.01 μg / 10 9 - corresponds to the particle loading capacity. The loading capacity is further increased to 1.1 ± 0.02 μg / 10 9 The maximum binding efficiency of nanoP3 to IL-10 was 99.9%, which was 0.5±0.02 μg / 10 9 -corresponding to the total mass loading capacity of the particles. IL-10 was 0.80±0.02μg / 10 9 - Further loading of the particles was observed, which corresponds to a binding efficiency of 40%. All experiments were carried out in ultrapure water (pH = 6.5) at room temperature. The incubation time was 30 min.

[0175] 1A is a schematic diagram of the functionalization of nanoP3. Small molecules, imaging agents, targeting ligands, or proteins are incubated with nanoP3 to functionalize the nanoP3.

[0176] Figure 1B shows the response of macrophages to IL-4 and IL-10, which can shift the phenotype of M1 macrophages (pro-inflammatory) to M2 macrophages (anti-inflammatory).

[0177] Figure 2 shows the loading capacity of nanoP3 with IL-4 and IL-10. The binding efficiency of IL-4 and IL-10 is 100%. The emission spectrum confirms that nanoP3 can bind to IL-4 and IL-10.

[0178] Example 2 IL-4 bound to nanoP3 results in M2 macrophage polarization Inducing the macrophage phenotype toward the M2 anti-inflammatory state of macrophages may mitigate further progression of vascular injury and promote disease regression. Various cytokines of the interleukin family, including IL-4 and IL-10, promote this shift from M1 to M2 phenotype.

[0179] We sought to investigate the effect of NP3+IL-4 on macrophage polarization in vitro. Untreated 246.7 mouse macrophage cells (ATCC, USA) were cultured at 5 × 10 3 IL-4-bound nanoP3 was cultured at 1 × 10 cells / well. 5 NanoP3 was added to macrophage cultures at a concentration of 1000µg / well. After 24 hours, macrophages were fixed in 4% paraformaldehyde, followed by scanning electron microscopy (SEM) and confocal microscopy imaging. Confocal staining was performed using actin cytoskeleton stain (Abcam, USA) and anti-arginase-1 antibody (Abcam, USA).

[0180] Figure 3 shows that nanoP3-IL4 activates M2 compared to untreated macrophages and macrophages treated with nanoP3 alone. Confocal staining confirmed that ARG-1, a highly expressed M2 enzyme, was significantly upregulated in macrophages treated with nanoP3-IL4, further confirming M2 activation.

[0181] Example 3 Rat Carotid Artery Injury and NanoP3 Delivery Model (I) A rat carotid artery model of vascular injury was used to determine the efficacy of NP3-conjugated interleukins for treating cardiovascular pathology in vivo (FIG. 1C).

[0182] Approval for the study was obtained from the Sydney Local Health District Animal Welfare Committee (Protocol No. 2017 / 006). Experiments were conducted in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. Male Sprague-Dawley rats (7 weeks old) were purchased from the Laboratory Animal Service (NSW, Australia). Anesthesia was induced in rats with a single intramuscular injection of ketamine (75 mg / kg) and medetomidine (0.5 mg / kg). The common carotid artery was isolated and double ligated approximately 1 cm apart. A small incision was made at the distal end through which a micro-intracranial forceps (World Precision Instruments, USA) was inserted. The forceps were extended to their full width and rotated 360 degrees to injure the entire luminal surface area of the vessel. This was repeated five times, after which the forceps were withdrawn and a 22G catheter was inserted into the same incision. Through this catheter, 2 x 10 cells were injected in RPMI medium in a total volume of approximately 80 μl. 8A solution of IL-4- or IL-10-conjugated nanoP3 was injected into the vessel at a concentration of 100 μg / ml. The nanoP3 solution was incubated for 2 minutes and then completely withdrawn from the vessel. The incision was closed with 9-0 nylon suture, and both ligatures were replaced to reestablish blood flow. The isolated vessel segments were then explanted 14 days later for pathological evaluation.

[0183] Figure 4B shows that free IL-4 was rapidly washed out of the vessel wall upon restoration of blood flow, but nanoP3-bound IL-4 was significantly retained in the vessels and persisted at significant levels even after 5 days.

[0184] Figure 5 shows that neointima formation is inhibited. Immunostaining (yellow / green) for the presence of M2 macrophages in treated carotid artery segments shows a significant increase in the NP3 + IL-10 group compared with denuded, NP3 + IL-4, and free IL-10. Assessment of repair of injured endothelium by immunostaining shows that both NP3 + IL-4 and NP3 + IL-10 restore sufficient endothelial integrity by 14 days after injury. However, this is not seen with free IL-10 treatment.

[0185] After 14 days in vivo, vascular explants were fixed overnight in 4% PFA, dehydrated with ethanol, and then embedded in paraffin. The embedded vessel segments were then cut longitudinally into 5-μm-thick sections. Staining of M2 macrophages and luminal endothelium was performed using anti-CD206 (Abcam, USA) and anti-von Willebrand factor (Sigma, USA) antibodies. Fluorescence imaging was performed using Alexa-fluor 594 secondary antibody. Neointima formation was stained using hematoxylin and eosin (H&E) staining. Normalized hyperplasia was calculated as the total area of hyperplasia divided by the area of the original vessel lumen.

[0186] Analysis of neointima formation 2 weeks after therapeutic nanoP3 delivery showed that vascular occlusion was reduced by approximately 35% and 20% of the cross-sectional lumen area in the NP3 + IL-4 and NP3 + IL-10 groups, respectively. Free IL-10 and nanoP3 alone had no significant effect on vascular occlusion, suggesting that the nanoP3 platform enhances the therapeutic effect of IL-10 (Figure 6).

[0187] Example 4 Rat Carotid Artery Injury and NanoP3 Delivery Model (II) A rat carotid artery model of vascular injury was used to determine the efficacy of NP3 plus IL-10, NP3 plus sirolimus, or NP3 plus sulindac to treat cardiovascular pathology in vivo. Carrier-free recombinant rat IL-10 (1.34 μg / 10 9 -nanoP3; R&D systems, USA), sirolimus (2.50 μg / 10 9 -nanoP3; rapamycin, Sigma-Merck, USA), sulindac (3.05 μg / 10 9 -nanoP3; Sigma-Merck, USA), or Cy7 fluorescent labeling (5.03 μg / 10 9 -nanoP3; CF750 Antibody Label, Sigma-Merck, USA) was diluted with sterile water to 2 × 10 9 It was complexed with nanoP3.

[0188] Through the catheter, 2 x 10 cells were transferred to RPMI medium in a total volume of approximately 80 μl. 8 Rats were subjected to vascular injury as described in Example 3, except that solutions of IL-10-conjugated nanoP3, sirolimus-conjugated nanoP3, or sulindac-conjugated nanoP3 were infused at concentrations of nanoP3 (IL-10, 0.268 μg; sirolimus, 0.5 μg; sulindac, 0.61 μg; Cy7, 1.01 μg).

[0189] FIG. 7 shows that delivery of NP3 plus IL-10, NP3 plus sirolimus, or NP3 plus sulindac inhibits neointimal hyperplasia compared with delivery of free drug in a rat carotid artery injury model.

[0190] Vascular re-endothelialization in rat carotid artery models treated with NP3 + IL-10, NP3 + sirolimus, or NP3 + sulindac was also examined using von Willebrand factor (vwf) staining as described in Example 3. Sirolimus is a drug known to impair wound healing. Surprisingly, we found that both NP3 + sirolimus and NP3 + IL-10 stimulated healing (endothelialization), even though sirolimus has previously been shown to impair wound healing (Figure 8).

[0191] Example 5 IL-10 and Sulindac Bound to nanoP3 Cause M2 Macrophage Polarization The present inventors sought to examine the effects of NP3 plus IL-10 and NP3 plus sulindac on macrophage polarization in vitro.

[0192] Untreated 246.7 mouse macrophage cells (ATCC, USA) were cultured at 5 × 10 3 Cells / well were cultured. IL-10 (1.34 μg / 10 9 -nanoP3) and sulindac (3.05 μg / 10 9 -nanoP3) was conjugated to nanoP3 as described in Example 4.

[0193] NP3+IL-10 or NP3+sulindac at 1×10 5 NanoP3 was added to macrophage cultures at a concentration of 1000µg / well. After 24 hours, macrophages were fixed in 4% paraformaldehyde, followed by scanning electron microscopy (SEM) and confocal microscopy imaging. Confocal staining was performed using actin cytoskeleton stain (Abcam, USA) and anti-arginase-1 antibody (Abcam, USA).

[0194] Figure 9 shows that M2 is activated by NP3 + IL-10 compared to untreated macrophages ("denuded") and macrophages treated with nanoP3 alone ("+NP3"). Confocal staining confirmed that ARG-1, a highly expressed M2 enzyme, was significantly upregulated in macrophages treated with NP3 + IL-10, further confirming M2 activation.

[0195] Example 6 Rabbit Iliac Bone Injury and NanoP3 Delivery Model The rabbit iliac bone injury model was used to evaluate the efficacy of NP3+IL-10 treatment.

[0196] Approval for the study was obtained from the University of Sydney Animal Ethics Committee (AEC) (protocol 2019-1653). Experiments were conducted in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. A central incision (approximately 1 cm long) was made in the skin of the groin, and the right femoral artery was exposed after blunt dissection of the muscle layer. The artery was ligated at its distal end, and a small incision was made above the ligature to allow insertion of a 5F sheath (Abbott, TREK coronary dilatation catheter, 3.25 mm). The sheath was secured in place with sutures (3-0, silk). A 0.014-inch guidewire was inserted through the sheath and advanced retrograde into the abdominal aorta. A 3.25 mm angioplasty balloon catheter was then inserted over the guidewire and advanced into the aorta. NP3 delivery and location in the surrounding vasculature were measured by intravenous injection of contrast fluid ISOVUE 370 or OMNIPAQUE (75.5 g / 100 ml), 25%–50% (v / v) contrast / saline, followed by angiography.

[0197] The iliac artery was denuded by inflating the balloon to a diameter of 3.1 mm at the bifurcation of the abdominal aorta and slowly withdrawing it into the femoral artery three times (1 min per denudation). The same balloon was reinserted through the main incision into the injured iliac artery up to the bifurcation and inflated to 6-8 atmospheres to occlude proximal blood flow. Immediately after blood flow was occluded, IL-10 nanoP3 solution (3 × 10 8 A total of 2 ml of saline solution was delivered to distend the artery and incubated for 2 minutes, after which the remaining solution was aspirated through the sheath. The sheath was removed, and the femoral artery was permanently ligated with 3-0 silk suture, followed by removal of the sheath. The exposed area was closed with 3-0 silk suture using individual stitches and two layers.

[0198] Figure 10 shows the in vivo performance evaluation of 200 nm NPs complexed with IL-10 in a rabbit model of iliac artery injury. A) Rabbit model of iliac artery injury. B) H&E staining of neointimal hyperplasia. C) CD31 staining of thrombosis (white dotted line). D) CD68 staining of inflammatory macrophage infiltration (white stain). Vessels treated with NP3 + IL-10 showed reduced occlusion, thrombosis, and inflammation. Figure 11 shows that hyperplasia was reduced in NP3 + IL-10-treated vessels over a 7-day period compared to untreated controls. Furthermore, the incidence of thrombosis increased over a 7-day period, which was significantly reduced in NP3 + IL-10-treated vessels. CD68 staining of vascular inflammation indicates that NP3 + IL-10 reduced infiltration of proinflammatory macrophages and attenuated inflammation over a 7-day period compared to untreated controls.

[0199] Example 7 Retention of nanoP3 in a rat carotid artery injury model In a rat carotid artery injury model, we compared the retention of 200 nm nanoP3 and 100 nm nanoP3 (NP3).

[0200] The Cy7 fluorophore was conjugated to either 200 nm or 100 nm diameter nanoP3.

[0201] Through the catheter, 2 x 10 cells were cultured in RPMI medium in a total volume of approximately 80 µl. 8 Rats were subjected to vascular injury as described in Example 3, except that a solution containing Cy7 conjugated to 100 nm nanoP3 or Cy7 conjugated to 200 nm nanoP3 was injected at a concentration of nanoP3.

[0202] FIG. 12 shows that both 100 nm and 200 nm nanoP3 were retained after 7 days, and that 100 nm nanoP3 was also retained 2 weeks after delivery.

[0203] NanoP3 was detected after 14 days regardless of the particle size used. There was some variation in the retention profile depending on the particle size of nanoP3 used. Therefore, a particularly favorable retention time can be achieved by selecting a suitable nanoP3 particle size.

Claims

1. 1. A method for delivering a drug to an area of a blood vessel in a patient, comprising: a) conjugating the drug with nanoparticles to form a conjugate; b) delivering the complex to the region of the blood vessel; The method comprising:

2. The method of claim 1 , wherein the agent is a biologically active agent or an imaging agent.

3. 1. A method of modulating inflammation or promoting healing in an area of a blood vessel in a patient, comprising: a) conjugating a biologically active agent with nanoparticles to produce a conjugate; b) delivering the complex to the region of the blood vessel; The method comprising:

4. 4. The method of any one of claims 1 to 3, wherein the complex is retained in the region of the blood vessel for a period of time that is longer than the period of time that an uncomplexed biologically active agent is retained in the region of the blood vessel.

5. 5. The method of claim 4, wherein the complex is retained at the delivery site in the blood vessel for at least 1 day.

6. 6. The method of claim 5, wherein the complex is retained at the delivery site in the blood vessel for at least 5 days.

7. 6. The method of claim 5, wherein the complex is retained at the delivery site in the blood vessel for at least 14 days.

8. The method of any one of claims 1 to 7, wherein the biologically active agent is an anti-inflammatory cytokine, an anti-inflammatory drug, a limus drug, a statin drug, or an anti-proliferative drug.

9. 9. The method of claim 8, wherein the biologically active agent is an anti-inflammatory cytokine.

10. 10. The method of claim 9, wherein the anti-inflammatory cytokine is interleukin-4 or interleukin-10.

11. the anti-inflammatory drug is sulindac, or the statin drug is simvastatin, or the antiproliferative agent is paclitaxel, sirolimus, or an mTOR inhibitor; The method of claim 8.

12. The method of any one of claims 3 to 10, wherein the modulation of inflammation is mediated by macrophage polarization.

13. 1. A method for maintaining a biologically active agent in an area of a blood vessel of a patient for a period of at least 14 days, comprising: a) conjugating the biologically active agent with nanoparticles to form a conjugate; b) delivering the complex to the blood vessel; The method comprising:

14. The method of any one of claims 1 to 13, wherein the complex is delivered to the blood vessel using a catheter.

15. 15. The method of claim 14, wherein the catheter is an occlusion perfusion catheter or a sweating balloon catheter.

16. The method of any one of claims 1 to 15, wherein the patient is undergoing or has undergone an endovascular intervention.

17. a nanoparticulate polymer formed from a plasma having an average diameter of about 1 nm to about 50 nm and comprising at least one monomer selected from an alkene, an alkyne, a cycloalkene, a cycloalkyne, or a mixture thereof; or an aggregate comprising two or more of said nanoparticulate polymers and having an average diameter of about 5 nm to about 500 nm; a biologically active agent selected from the group consisting of anti-inflammatory cytokines, anti-inflammatory drugs, statins, and anti-proliferative drugs; A complex containing

18. 18. The conjugate of claim 17, wherein the biologically active agent is an anti-inflammatory cytokine.

19. 19. The conjugate of claim 18, wherein the anti-inflammatory cytokine is interleukin-4 or interleukin-10.

20. the anti-inflammatory drug is sulindac, or the statin drug is simvastatin, or The antiproliferative agent is paclitaxel, sirolimus, or an mTOR inhibitor. The composite of claim 17.

21. a nanoparticulate polymer formed from a plasma having an average diameter of about 1 nm to about 50 nm and comprising at least one monomer selected from an alkene, an alkyne, a cycloalkene, a cycloalkyne, or a mixture thereof; or an aggregate comprising two or more of said nanoparticulate polymers and having an average diameter of about 100 nm to about 200 nm; i) interleukin-10, ii) sulindac, or iii) Sirolimus A complex containing:

22. A method for treating or preventing vascular injury or disease, comprising delivering the complex of any one of claims 17 to 21 to an area of a blood vessel in a patient in need thereof.

23. 23. The method of claim 22, wherein the complex is delivered to the region of the blood vessel using a catheter.

24. 24. The method of claim 23, wherein the catheter is an occlusion perfusion catheter or a sweating balloon catheter.

25. The method of any one of claims 22 to 24, wherein the vascular injury is the result of an intravascular intervention.

26. The method according to any one of claims 22 to 24, wherein the vascular injury is neointimal hyperplasia or restenosis.

27. The method of any one of claims 22 to 24, wherein the vascular disease is atherosclerosis.

28. Use of a conjugate according to any one of claims 17 to 21 in the manufacture of a medicament for the treatment or prevention of vascular injury or vascular disease in a patient in need thereof.

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