Drug delivery compositions for ocular administration of therapeutics and methods of use thereof

Abstract

To provide a composition, a device, and a method comprising one or more multilayer drug delivery capsules for delivery of a therapeutic agent to the eye.SOLUTION: A drug delivery composition comprises: one or more capsules each having a tubular shape with two closed ends, wherein each of the one or more capsules independently comprises a bi-layered wall and at least one luminal compartment; and one or more therapeutic agents each present within one or more of the at least one luminal compartment; wherein each bi-layered wall comprises an inner layer and an outer layer; wherein the inner layer comprises a first polymer having a net positive charge under physiological conditions; and wherein the outer layer independently comprises a second polymer that differs from the first polymer.SELECTED DRAWING: Figure 10

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of priority of U.S. Provisional Patent Application No. 62 / 803,388, filed on February 8, 2019, the disclosure of which is hereby incorporated by reference in its entirety.

[0002] This disclosure relates to drug delivery compositions, and more specifically, to compositions containing one or more multilayered drug delivery capsules for the delivery of therapeutic agents to the eye.

Background Art

[0003] Age - related macular degeneration (AMD) is the fourth most common cause of blindness worldwide, following cataracts, premature birth, and glaucoma. In the United States, more than 11 million people are diagnosed with exudative AMD. This number is estimated to double in 30 years. Thus, much research has been conducted to understand the disease etiology and develop treatment methods. It is widely known that the overexpression of vascular endothelial growth factor (VEGF) associated with aging stimulates angiogenesis in the choroid, which leads to irreversible damage to the retina during bleeding and scarring of the newly formed blood vessels. The current gold - standard treatment for exudative AMD is monthly intravitreal injection of anti - VEGF (such as bevacizumab or ranibizumab) to inhibit VEGF and prevent angiogenesis. However, frequent injections often lead to infections, increased intraocular pressure, and rhegmatogenous retinal detachment, as well as patient compliance problems.

[0004] Recently, there have been reports of implants and novel devices such as microparticles / nanoparticles for long-term drug delivery in the eye. Unfortunately, such implants require surgical procedures for implantation and removal. Furthermore, currently known implant devices are off-target and tend to reduce drug efficacy. Microparticles or nanoparticles have a relatively small size suitable for injection into the eye with a 30-gauge needle, but currently described microparticles or nanoparticles release therapeutic agents (such as anti-VEGF therapeutics) over a rapid release window due to biodegradation of the known particle composition in the first three months.

[0005] Accordingly, despite significant efforts directed towards the treatment of AMD or other ophthalmic disorders, there remains a lack of methods and compositions that minimize the adverse side effects of currently available treatment regimens. Furthermore, there is a need for drug delivery systems and compositions that are biodegradable and can control drug release for nine months or more after intravitreal injection. There remains a need for improved treatment approaches for the treatment of AMD and other eye diseases that require direct delivery of therapeutic agents to the eye. These and other needs are met by the present disclosure. SUMMARY OF THE INVENTION

[0006] According to an aspect (s) of the present disclosure, as implemented and broadly described herein, the present disclosure relates, in one aspect, to compositions, devices, and processes for the delivery of protein therapeutic substances (e.g., intravitreal delivery of protein therapeutic substances to the eye). The disclosed drug delivery compositions include capsules having a bilayer wall and a therapeutic agent contained therein. In a further aspect, the present disclosure relates to methods of treating ophthalmic diseases or disorders.

[0007] Accordingly, in one aspect, one or more capsules each having a tube shape with two closed ends, each of the one or more capsules independently including a multi-layer wall and at least one lumen compartment; and One or more therapeutic agents, each initially present within one or more of at least one lumen compartment comprising; each multi-layer wall independently comprising at least an inner layer and an outer layer; each inner layer comprising a first polymer having a net positive charge under physiological conditions; each outer layer independently comprising a second polymer different from the first polymer, a drug delivery composition is provided.

[0008] In some embodiments, the drug delivery composition can include two or more capsules. In some embodiments, different therapeutic agents are initially present within each of the two or more capsules. In other embodiments, the same therapeutic agent is initially present within each of the two or more capsules.

[0009] In some embodiments, at least one or one or more capsules include two or more lumen compartments. In some embodiments, different therapeutic agents are initially present within each of the two or more lumen compartments. In other embodiments, the same therapeutic agent is initially present within each of the two or more lumen compartments.

[0010] In some aspects, the first polymer can include chitosan, polyethyleneimine, protamine, polypropylimine, poly-L-lysine, poly-L-arginine, poly-D-lysine, poly-D-arginine, cellulose, dextran, poly(amidoamine), poly(2-(dimethylamino)ethyl methacrylate), derivatives thereof, or combinations thereof. In some embodiments, the first polymer can include chitosan or a derivative thereof. In some embodiments, the first polymer includes fibers having an average diameter of about 50 nm to about 1000 nm.

[0011] In some embodiments, the second polymer may include a biodegradable polymer. In some embodiments, the second polymer includes poly(ε-caprolactone) (PCL), poly-lactic acid (PLA), poly-glycolic acid (PGA), poly-lactide-co-glycolide (PLGA), polyester, poly(orthoester), poly(phosphazene), poly(phosphoric acid ester), gelatin, collagen, polyethylene glycol (PEG), its derivatives, or a combination thereof. In some embodiments, the second polymer includes PCL. In other embodiments, the second polymer includes PLA. In some embodiments, the second polymer includes fibers having an average diameter of about 100 nm to about 2000 nm.

[0012] In some embodiments, each of the one or more capsules independently has a length of about 0.1 cm to about 5 cm. In some embodiments, each of the one or more capsules independently has an inner diameter of about 100 μm to about 2000 μm. In some embodiments, each of the one or more capsules independently has an outer diameter that is about 50 μm to about 300 μm larger than the inner diameter of the same capsule. In some embodiments, each multilayer wall has a wall thickness of about 25 μm to about 150 μm. In some embodiments, each outer layer may further include pores having an average pore diameter of about 100 nm to about 10000 nm.

[0013] In some embodiments, each of the one or more drug delivery capsules has a surface charge measured as a zeta potential at pH 7.4 of about -25 mV to about 25 mV. In some embodiments, each of the one or more therapeutic agents has a net negative charge within a pH range of about 6.0 to about 7.4.

[0014] In some embodiments, at least one of the one or more therapeutic agents is an anti-VEGF agent. In some embodiments, the anti-VEGF agent is a therapeutic antibody (e.g., bevacizumab, ranibizumab, IBI305, combinations thereof). In some embodiments, the anti-VEGF agent is a VEGF decoy receptor (e.g., aflibercept). In some embodiments, the anti-VEGF agent is a tyrosine kinase inhibitor (e.g., lapatinib, sunitinib, axitinib, pazopanib, or combinations thereof).

[0015] In some embodiments, the one or more therapeutic agents can include an anti-inflammatory agent (such as cyclosporine, a steroid or non-steroidal anti-inflammatory drug), an anti-microbial agent, an immunomodulatory agent, an ocular hypotensive agent, a neuroprotective agent, gene therapy, viral vector therapy, an α-adrenergic agonist, a β-adrenergic agonist, or combinations thereof.

[0016] In another aspect, there is provided a method of treating an ophthalmic disorder in a subject, comprising injecting a therapeutically effective amount of the drug delivery composition described herein into the eye of a subject in need thereof. In some embodiments, the ophthalmic disorder is acute macular neuroretinopathy; Behcet's disease; angiogenesis (including choroidal angiogenesis); diabetic uveitis; histoplasmosis; infection (such as infection caused by fungi or viruses); macular degeneration (such as acute macular degeneration (AMD), including exudative AMD, non-exudative AMD, and exudative AMD); edema (such as macular edema, cystoid macular edema, and diabetic macular edema); multifocal choroiditis; ocular trauma affecting posterior eye sites or locations; eye tumors; retinal disorders (such as central retinal vein occlusion, diabetic retinopathy (including proliferative diabetic retinopathy), proliferative vitreoretinopathy (PVR), retinal arterial occlusive diseases, retinal detachment, uveitis retinal diseases, etc.); sympathetic ophthalmia; Vogt Koyanagi-Harada (VKH) syndrome; choroidal exudation; posterior eye pathologies caused by or affected by eye laser treatment; posterior eye pathologies that may be caused by or affected by photodynamic therapy, photocoagulation, radiation retinopathy, epiretinal membrane disorders, retinal vein branch occlusion, anterior ischemic optic neuropathy, non-retinopathy diabetic retinal dysfunction, retinitis pigmentosa, cancer, and glaucoma. In some embodiments, the ophthalmic disorder includes exudative age-related macular degeneration (exudative AMD), angiogenesis, or macular edema. In some embodiments, injecting the described composition into the eye of the subject includes injecting into the vitreous humor of the eye. In other embodiments, injecting the described composition into the eye of the subject includes intravitreal injection, subconjunctival injection, sub-Tenon's injection, retrobulbar injection, or suprachoroidal injection.

[0017] A method of producing one or more capsules for use in the drug delivery composition described herein, comprising forming a first layer of a first polymer on a conductive rod, the formation of the first layer including electrospinning using a first solution containing the first polymer in at least one organic solvent, the electrospinning being performed using a voltage difference of about 10 kV to about 30 kV; and Form a second layer of the second polymer on the first layer, wherein forming the second layer includes electrospinning a second solution comprising the first polymer and optionally a porogen onto the formed first layer, and the electrospinning is performed using a voltage difference of about 10 kV to about 30 kV. Also provided is said method including the above.

[0018] Also disclosed is a kit comprising (a) the disclosed drug delivery composition; (b) the disclosed drug delivery composition in a sterilized package; or (c) one of a pre-filled syringe or needle containing the disclosed drug delivery composition, and instructions for administering the drug delivery composition for treating an ophthalmic disease or disorder described herein.

[0019] Other systems, methods, characteristics, and advantages of the present disclosure will be apparent or will become apparent to those skilled in the art upon examination of the following drawings and detailed description of the invention. All such additional systems, methods, characteristics, and advantages are intended to be included within the detailed description of the invention, within the scope of the present disclosure, and protected by the appended claims. In addition, all optional preferred characteristics and modifications of the described embodiments are usable in all aspects of the present disclosure taught herein. Further, like the individual characteristics of the dependent claims, all optional preferred characteristics and modifications of the described embodiments are combinable with and interchangeable with each other.

[0020] Many aspects of the present disclosure can be better understood with reference to the following drawings. The components in the drawings are not necessarily to scale, and instead emphasis is placed on clearly illustrating the principles of the present disclosure. Further, like reference numerals designate corresponding parts throughout the several views. BRIEF DESCRIPTION OF THE DRAWINGS

[0021]

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[0022] Additional advantages of the present disclosure will be partly described in the following description, become partly apparent from the description, or can be learned by the practice of the present disclosure. The advantages of the present disclosure will be realized and achieved using the elements and combinations particularly pointed out in the appended claims. It should be understood that both the foregoing general description and the following detailed description are for illustrative and explanatory purposes only and do not limit the claimed present disclosure.

Mode for Carrying Out the Invention

[0023] Many of the modifications and other embodiments disclosed herein relate to the disclosed compositions and methods and will occur to those of ordinary skill in the art who benefit from the teachings presented in the foregoing description and the accompanying drawings. Accordingly, it is to be understood that this disclosure is not limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Those of ordinary skill in the art will recognize many variations and adaptations of the aspects described herein. These variations and adaptations are intended to be included within the teachings of this disclosure and are intended to be covered by the claims of this specification.

[0024] Certain terms are used herein, but they are used only in a general and descriptive sense and not for purposes of limitation.

[0025] As will be apparent to those of ordinary skill in the art upon reading this disclosure, each individual embodiment described and illustrated herein has discrete components and characteristics that can be readily separated from or combined with the characteristics of any of the other several embodiments without departing from the scope or spirit of this disclosure.

[0026] Any recited method can be performed in the order of recited events or in any other order that is logically possible. That is, unless otherwise expressly stated, no method or aspect described herein is intended to be construed as requiring that its steps be performed in a particular order. Accordingly, a method claim should not be construed as implying any order in the absence of specific explicit indication in the claim or description that the steps are to be limited to a particular order. This applies to any possible ambiguity in interpretation (including logical issues regarding the arrangement of steps or operation flow, plain meaning derived from grammatical construction or punctuation, or the number or type of aspects described in the specification).

[0027] All publications mentioned in this specification are hereby incorporated by reference herein for the purpose of disclosing and describing the methods and / or materials by which those publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publications by virtue of prior invention. Further, the publication dates provided herein may be different from the actual publication dates and may require independent verification.

[0028] Aspects of the present disclosure may be described and claimed in a particular statutory classification (such as a system statutory classification), but this is for convenience only, and those skilled in the art will understand that aspects of the present disclosure may be described and claimed in any statutory classification.

[0029] It should also be understood that the terms used herein are for the purpose of describing particular aspects only and are not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed compositions and methods belong. Terms (such as those defined in commonly used dictionaries) should be interpreted as having a meaning that conforms to their meaning in the context of the specification and the relevant art, and it will be further understood that they should not be interpreted in an idealized or overly formal sense unless clearly defined herein.

[0030] Prior to the description of the various aspects of the present disclosure, the following definitions are provided and should be used unless otherwise indicated. Additional terms may be defined elsewhere in the present disclosure.

[0031] Definitions As used herein, "comprising" is to be interpreted as specifying the presence of the stated feature, integer, step, or component as referred to, but not precluding the presence or addition of one or more features, integers, steps, or components, or groups thereof. Further, each of the terms "by", "comprising", "comprises", "comprised of", "including", "includes", "included", "involving", "involves", "involved", and "such as" is used in its open and non-limiting sense and may be used interchangeably. Further, the term "comprising" is intended to include examples and aspects subsumed by the terms "consisting essentially of" and "consisting of". Similarly, the term "consisting essentially of" is intended to include examples subsumed by the term "consisting of".

[0032] As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, references to "drug delivery compositions", "therapeutic agents", or "clinical conditions" include, but are not limited to, two or more such drug delivery compositions, therapeutic agents, or clinical conditions, and the like.

[0033] Note that ratios, concentrations, amounts, and other numerical data may be expressed in a range format herein. It will be further understood that each endpoint of a range is significant both in relation to and independent of the other endpoint. There are a number of values disclosed herein, and it is understood that each value is disclosed herein as "about" that particular value in addition to the value itself. For example, if the value "10" is disclosed, then "about 10" is also disclosed at that time. A range may be expressed herein as from "about" a particular value and / or to "about" another particular value. Similarly, it will be understood that when a value is expressed as an approximation by use of the antecedent "about", the particular value forms a further aspect. For example, if the value "about 10" is disclosed, then "10" is also disclosed at that time.

[0034] When a range is expressed, a further aspect includes from one particular value and / or to the other particular value. For example, when an expressly stated range includes one or both of the limits, ranges excluding either or both of those included limits are also encompassed in the disclosure (e.g., the phrase "x to y" includes ranges greater than "x" and less than "y" as well as the range from "x" to "y"). A range may also be expressed as an upper limit (e.g., "about x, y, z, or less"), and should be interpreted to include ranges of "less than x", "less than y", and "less than z" as well as the particular ranges of "about x", "about y", and "about z". Similarly, the phrase "about x, y, z, or more" should be interpreted to include ranges of "more than x", "more than y", and "more than z" as well as the particular ranges of "about x", "about y", and "about z". In addition, the phrase "from 'x' to 'y'" includes "from about 'x' to about 'y'" when "x" and "y" are numerical values.

[0035] Such a range format is used for convenience and brevity and should be understood to be interpreted in a flexible manner such that it includes not only the explicitly recited numerical values as the limits of the range, but also all individual numerical values or sub-ranges subsumed within that range as if each such numerical value and sub-range were explicitly recited. By way of illustration, a numerical range of "about 0.1% to 5%" includes not only the explicitly recited values of about 0.1% to about 5%, but also individual values within the indicated range (e.g., about 1%, about 2%, about 3%, about 4%) and sub-ranges (e.g., about 0.5% to about 1.1%; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible sub-ranges).

[0036] As used herein, the terms "about," "approximately," "is or is about," and "substantially" mean that the quantity or value in question can be the exact value, or a value that provides the same result or effect as that recited in the claims or taught herein. That is, quantities, sizes, formulations, parameters, and other quantities and characteristics are not exact and need not be exact, but can be approximate and / or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding, measurement error, and the like, as well as other factors known to those of ordinary skill in the art such that the same result or effect is obtained. In some situations, the value providing the same result or effect may not be logically determinable. In such cases, generally, when used herein and unless otherwise indicated or implied, "about" and "is or is about" are understood to mean a variation of ±10% from the stated nominal value. Generally, a quantity, size, formulation, parameter, or other quantity or characteristic is such whether or not it is expressly stated to be "about," "approximately," or "is or is about." When "about," "approximately," or "is or is about" is used before a quantitative value, the parameter is understood to include the specific quantitative value itself, unless specifically stated otherwise.

[0037] As used herein, "effective amount" can refer to an amount of a disclosed compound or pharmaceutical composition provided herein that is sufficient to achieve a beneficial or desired biological, emotional, medical, or clinical response in a cell, tissue, system, animal, or human. An effective amount can be administered in one or more administrations, applications, or dosages. The term can also encompass within its scope an amount effective to promote or restore substantially normal physiological function.

[0038] As used herein, the term "therapeutically effective amount" refers to an amount that is sufficient to achieve a desired therapeutic result or to have an effect against an undesired symptom, but generally insufficient to cause harmful side effects. The specific therapeutically effective dosage level for any particular patient will depend on a variety of factors including the disorder being treated and the severity of the disorder; the specific composition being used; the age, weight, general health, sex, and diet of the patient; the time of administration; the route of administration; the excretion rate of the specific compound being used; the duration of the treatment; drugs used in combination with or concurrently with the specific compound being used, as well as similar factors within the knowledge and expertise of the medical practitioner, and factors that may be well known in the medical arts. In the case of the treatment of a particular disease or condition, in some instances, the desired response may be to inhibit the progression of the disease or condition. This may only include temporarily delaying the progression of the disease. However, in other instances, it may be desired to permanently interrupt the progression of the disease. This can be monitored by routine diagnostic methods known to those of ordinary skill in the art for any particular disease. The desired response to the treatment of a disease or condition may be to delay or in some cases prevent the onset of the disease or condition.

[0039] For example, it is well within the skill of those of ordinary skill in the art to start the dosage of a compound at a level lower than that required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dosage can be divided into multiple dosages for the purpose of administration. Therefore, a single-dose composition can contain such an amount or an approximation thereof to constitute a daily dosage. The dosage can be adjusted by an individual physician in any event of contraindication. It is generally preferred that the maximum dosage (alone or in combination with other therapeutic agents) of the pharmacological agents of the present invention, i.e., the highest safe amount in accordance with sound medical judgment, be used. However, it will be understood by those of ordinary skill in the art that a patient may claim a lower dosage or tolerance for medical, psychological, or practical other reasons.

[0040] The response to a therapeutically effective dose of the disclosed drug delivery composition can be measured by determining the therapeutic or physiological action of the treatment or medicament (such as a decrease or absence of disease symptoms following administration of the treatment or pharmacological agent). Other assays are known to those of skill in the art and can be used for measuring the level of response. The amount of treatment can be varied, for example, by increasing or decreasing the amount of the disclosed compound and / or pharmaceutical composition, by varying the disclosed compound and / or pharmaceutical composition administered, by changing the route of administration, by changing the dosing timing, etc. The dosage can vary and can be administered daily for one or more days in one or more doses. Guidance can be found in the literature for appropriate dosages for a given class of pharmaceutical products.

[0041] As used herein, the term "prophylactically effective amount" refers to an amount effective for preventing the onset or development of a disease or condition.

[0042] As used herein, the terms "prevent" or "preventing" refer to precluding, avoiding, averting, forestalling, stopping, or hindering something from occurring, particularly by prior action. It is understood that the use of the other two terms is also expressly disclosed when reducing, inhibiting, or preventing is used herein, unless specifically indicated otherwise.

[0043] As used herein, the terms "optional" or "optionally" mean that the subsequent described event or circumstance may or may not occur and that the description includes examples where the event or circumstance occurs and examples where it does not.

[0044] As used herein, "therapeutic agent" can refer to any substance, compound, molecule, and the like that can be biologically active or, otherwise, can induce pharmacological, immunogenic, biological, and / or physiological effects in a subject administered by local and / or systemic action. A therapeutic agent can be a primary active agent, or, in other words, a component(s) of a composition to which the overall or partial effect of the composition is attributed. A therapeutic agent can be a secondary therapeutic agent, or, in other words, an additional part of the composition and / or a component(s) of the composition to which other effects are attributed. Thus, the term encompasses compounds or chemical substances that are traditionally regarded as being drugs, vaccines, and biopharmaceuticals (including molecules such as proteins, peptides, hormones, nucleic acids, gene constructs, and the like). Examples of therapeutic agents are described in well-known literature references such as the Merck Index (14th edition), the Physicians’ Desk Reference (64th edition), and The Pharmacological Basis of Therapeutics (12th edition), and include, without limitation, pharmaceuticals; vitamins; mineral supplements; substances used for the treatment, prevention, diagnosis, cure, or alleviation of a disease or illness; substances that affect the structure or function of the body, or prodrugs that may become or become more biologically active after being placed in a physiological environment.For example, the term "therapeutic agent" includes adjuvants; anti-infective substances (such as antibiotics and antiviral agents); analgesic substances and combinations of analgesic substances, anorectic substances, anti-inflammatory agents, antiepileptic substances, local and general anesthetic substances, hypnotic substances, sedative substances, antipsychotic agents, neuroleptic agents, antidepressant substances, anxiolytic substances, antagonists, neuronal blockers, anticholinergic agents and cholinergic-like agents, antimuscarinic agents and muscarinic agents, antiadrenergic substances, antiarrhythmic substances, antihypertensive agents, hormones, and nutrients, anti-arthritis substances, anti-asthma agents, antispasmodic substances, antihistamine substances, antiemetic substances, antineoplastic substances, anti-itch substances, antipyretic substances; antiepileptic substances, cardiovascular drugs (including calcium channel blockers, β-blockers, β-agonists, and antiarrhythmic substances), antihypertensive substances, diuretic substances, vasodilator substances; central nervous system stimulants; cold remedies; decongestant substances; diagnostic substances; hormones; bone growth stimulating substances and bone resorption inhibiting substances; immunosuppressive substances; muscle relaxants; psychostimulants; sedative substances; tranquilizers; proteins, peptides, and fragments thereof (whether naturally occurring, chemically synthesized, or produced recombinantly); and nucleic acid molecules (in the polymeric form of two or more nucleotides of either ribonucleotide (RNA) or deoxyribonucleotide (DNA), including double-stranded and single-stranded molecules, gene constructs, expression vectors, antisense molecules, and the like), small molecules (such as doxorubicin), and other biologically active macromolecules (such as proteins and enzymes), and is not limited thereto, and includes compounds or compositions for use in all major therapeutic areas. The agent can be a biologically active agent used in medical applications including veterinary medicine, agriculture (such as by plants), and other areas. The term "therapeutic agent" includes pharmaceuticals; vitamins; mineral supplements; substances used for the treatment, prevention, diagnosis, cure, or alleviation of a disease or illness; or substances that affect the structure or function of the body; or prodrugs that may become biologically active or more active after being placed in a given physiological environment, without limitation.

[0045] It is understood that the disclosure of the therapeutic agent in the present specification also discloses pharmaceutically acceptable salts, pharmaceutically acceptable esters, pharmaceutically acceptable amides, prodrug forms, and derivatives of the therapeutic agent.

[0046] The term "pharmaceutically acceptable salt", as used herein, means a salt of an active ingredient prepared with an acid or a base that is tolerated by a biological system, or tolerated by a subject, or tolerated by a biological system and tolerated by a subject when administered in a therapeutically effective amount. When the compounds of the present disclosure contain relatively acidic functional groups, the base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either without adding a liquid or in a suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include, but are not limited to, sodium salts, potassium salts, calcium salts, ammonium salts, organic amino salts, magnesium salts, lithium salts, strontium salts, or similar salts. When the compounds of the present disclosure contain relatively basic functional groups, the acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either without adding a liquid or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, monohydrogencarbonic acid, phosphoric acid, monohydrogenphosphoric acid, dihydrogenphosphoric acid, sulfuric acid, monohydrogensulfuric acid, hydroiodic acid, or phosphorous acid, and the like, and salts derived from relatively non-toxic organic acids such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, glutaric acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, methanesulfonic acid, and the like. Also included are salts of amino acids such as arginine salts and the like, and salts of organic acids such as glucuronic acid or galactunoric acid, and the like.

[0047] The term "pharmaceutically acceptable ester" refers to esters of the compounds of the present disclosure that hydrolyze in vivo and are readily degradable in the human body to leave the parent compound or its salt. Examples of pharmaceutically acceptable non-toxic esters of the present disclosure include C1-C6 alkyl esters and C5-C7 cycloalkyl esters, with C1-C4 alkyl esters being preferred. The esters of the disclosed compounds can be prepared according to conventional methods. Pharmaceutically acceptable esters can be added to hydroxy group-containing compounds by reacting the compound with an acid and an alkyl carboxylic acid (such as acetic acid), or an acid and an aryl carboxylic acid (such as benzoic acid). In the case of compounds containing a carboxylic acid group, pharmaceutically acceptable esters are prepared from the carboxylic acid group-containing compound by reacting the compound with a base such as triethylamine and an alkyl halide (such as methyl iodide, benzyl iodide, cyclopentyl iodide, or an alkyl triflate). Esters can also be prepared by reacting the compound with an acid (such as hydrochloric acid) and an alcohol (such as ethanol or methanol).

[0048] The term "pharmaceutically acceptable amide" refers to the non-toxic amides of the present disclosure derived from ammonia (primary C1-C6 alkylamines and secondary C1-C6 dialkylamines). In the case of secondary amines, the amine can also be in the form of a 5- or 6-membered heterocycle containing one nitrogen atom. Amides derived from ammonia (C1-C3 alkyl primary amides and C1-C2 dialkyl secondary amides) are preferred. The amides of the disclosed compounds can be prepared according to conventional methods. Pharmaceutically acceptable amides can be prepared from compounds containing a primary or secondary amine group by reacting the compound with an alkyl anhydride, aryl anhydride, halogenated acyl, or halogenated aroyl. In the case of a compound containing a carboxylic acid group, a pharmaceutically acceptable amide is prepared from the compound containing a carboxylic acid group by reacting the compound with a base (such as triethylamine), a dehydrating agent (such as dicyclohexylcarbodiimide or carbonyldiimidazole), and an alkylamine (dialkylamine) (such as methylamine, diethylamine, and piperidine). Amides can also be prepared by reacting the compound with an acid (such as sulfuric acid) and an alkylcarboxylic acid (such as acetic acid), or an acid and an arylcarboxylic acid (such as benzoic acid under dehydrating conditions such as adding molecular sieves). The composition can contain the compounds of the present disclosure in the form of a pharmaceutically acceptable prodrug.

[0049] The terms "pharmaceutically acceptable prodrug" or "prodrug" refer to prodrugs of the compounds of the present disclosure that are suitable for use in contact with the tissues of humans and lower animals within the scope of sound medical judgment, without undue toxicity, irritation, allergic response, and the like, are balanced in a reasonable benefit / risk ratio, and are effective for their intended uses. The prodrugs of the present disclosure can be rapidly transformed in vivo, for example, by hydrolysis in the blood, into the parent compounds having the structures of the disclosed compounds. Detailed discussions are provided in T. Higuchi and V. Stella, Pro-drugs as Novel Delivery Systems, V. 14 of the A.C.S. Symposium Series, and Edward B. Roche, ed., Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press (1987).

[0050] As used herein, "kit" means a collection of at least two components that make up the kit. The components together constitute a functional unit for a given purpose. The individual member components can be physically packaged together or separately. For example, a kit that includes instructions for using the kit may or may not physically include the instructions with the other individual member components. Instead, the instructions can be provided in paper form, or on a computer-readable memory device, or downloaded from an Internet website, or provided as a separate member component in any of electronic forms as a recorded format.

[0051] As used herein, "instructions (s)" means a document that describes related materials or methodologies related to the kit. These materials may include any combination of the following: background information, a list of components and information on obtaining them (such as purchasing information), a concise or detailed protocol for using the kit, troubleshooting, references, technical support, and other related documents. The instructions may be provided in paper form, or on a computer-readable memory device, or downloaded from an Internet website, or in any electronic form as a recorded format, either with the kit or as a separate member component. The instructions can include one or more documents and are intended to include future revised versions.

[0052] As used interchangeably herein, "subject", "individual", or "patient" can refer to a vertebrate organism such as a mammal (e.g., human). "Subject" can also refer to cells, cell populations, tissues, organs, or organisms, preferably humans and their components.

[0053] As used herein, the terms "treating" and "treatment" can generally refer to obtaining a desired pharmacological and / or physiological effect. The effect can be prophylactic (but not necessarily) with respect to preventing or partially preventing a disease, symptom, or condition thereof (such as an ophthalmic disorder). The effect can be therapeutic with respect to the partial or complete cure of a disease, condition, symptom, or adverse effect resulting from a disease, disorder, or condition. The term "treatment" as used herein can include any treatment of an ophthalmic disorder in a subject (particularly a human), and includes the following: (a) preventing a disease from occurring in a subject who may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., preventing its onset; and (c) reducing the disease, i.e., alleviating or remitting the disease and / or its symptoms or condition, and can include any one or more of the foregoing. The term "treatment" as used herein can refer to treatment alone, prophylaxis alone, or both treatment and prophylaxis. Those in need of treatment (the subjects in need thereof) can include those already having a disorder and / or those in whom the disorder should be prevented. As used herein, the term "treating" can include inhibiting a disease, disorder, or condition (e.g., preventing its progression); and reducing a disease, disorder, or condition (e.g., causing regression of the disease, disorder, and / or condition). Treatment of a disease, disorder, or condition can include alleviating at least one symptom of a particular disease, disorder, or condition (e.g., treating a subject's pain by administration of an analgesic even if such agent does not treat the cause of the pain) even if the underlying pathophysiology is not affected.

[0054] As used herein, "dosage", "unit dosage", or "dose" refers to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of the disclosed compound and / or its pharmaceutical composition calculated to produce the desired response(s), if any, attendant upon administration thereof.

[0055] As used herein, "therapeutic" can refer to treating, curing, and / or alleviating a disease, disorder, condition, or side effect, or reducing the rate of progression of a disease, disorder, condition, or side effect.

[0056] As used herein, nomenclature for compounds (including organic compounds) can be given using common names, IUPAC, IUBMB, or CAS recommendations for nomenclature. When one or more stereochemical features are present, the Cahn-Ingold-Prelog rules for stereochemistry can be used to denote stereochemical priorities, E / Z designations, and the like. One of ordinary skill in the art can readily confirm the structure of a compound given its name by systematic reduction of the compound structure using naming rules or by commercially available software such as CHEMDRAW™ (Cambridgesoft Corporation, U.S.A.).

[0057] Unless otherwise specified, temperatures referred to herein are based on atmospheric pressure (i.e., 1 atmosphere).

[0058] Drug delivery compositions having therapeutic or clinical utility are described herein. Methods of preparing or making the disclosed drug delivery compositions are also described herein. Methods of administering the disclosed drug delivery compositions to a subject in need thereof are also described herein. In some embodiments, the subject can have a clinical condition or lesion (such as an ophthalmic disorder). Other compositions, compounds, methods, features, and advantages of the present disclosure will be apparent or will become apparent to those skilled in the art upon examination of the following drawings, detailed description, and examples. All such additional compositions, compounds, methods, features, and advantages are intended to be included within the detailed description and within the scope of the present disclosure.

[0059] Drug delivery composition Vascular endothelial growth factor (VEGF) is an essential regulatory factor involved in abnormal angiogenesis, which supports rapid tumor growth and the formation of exudative age-related macular degeneration (AMD) (see Holmes, D.I.R. and I. Zachary, The vascular endothelial growth factor (VEGF) family: angiogenic factors in health and disease. Genome biology, 2005. 6(2): p. 209-209; Shibuya, M., Vascular Endothelial Growth Factor (VEGF) and Its Receptor (VEGFR) Signaling in Angiogenesis: A Crucial Target for Anti- and Pro-Angiogenic Therapies. Genes & cancer, 2011. 2(12): p. 1097-1105; and Ferrara, N., Role of vascular endothelial growth factor in the regulation of angiogenesis. Kidney International, 1999. 56(3): p. 794-814). Anti-angiogenic strategies have been proposed to delay exudative AMD (see Ferrara, N., et al., Discovery and development of bevacizumab, an anti-VEGF antibody for treating cancer. 2004. 3(5): p. 391; and Niu, G. and X. Chen, Vascular endothelial growth factor as an anti-angiogenic target for cancer therapy. Current drug targets, 2010. 11(8): p. 1000-1017).Humanized monoclonal antibodies (anti-VEGF) are used in ophthalmology for the treatment of off-label use of exudative AMD (see Ferrara, N., et al., Discovery and development of bevacizumab, an anti-VEGF antibody for treating cancer. 2004. 3(5): p. 391; and Presta, L.G., et al., Humanization of an anti-vascular endothelial growth factor monoclonal antibody for the therapy of solid tumors and other disorders. 1997. 57(20): p. 4593-4599).

[0060] The treatment of this age-related retinal disease currently relies on the use of anti-angiogenic agents to delay or interrupt its progression. Intravitreal injection of anti-VEGF therapeutic substances (such as bevacizumab and ranibizumab) constitutes the current gold-standard treatment for exudative AMD and prevents irreversible retinal damage caused by VEGF-induced initiation of subretinal choroidal neovascularization (CNV) and bleeding and scarring of newly formed blood vessels (see Delplace, V., S. Payne, and M. J. J. o. C. R. Shoichet, Delivery strategies for treatment of age-related ocular diseases: From a biological understanding to biomaterial solutions. 2015. 219: p. 652-668; and Ohr, M. and P. K. J. E. o. o. p. K. Kaiser, Intravitreal aflibercept injection for neovascular (wet) age-related macular degeneration. 2012. 13(4): p. 585-591). Bevacizumab is widely used, for example, in the treatment of exudative AMD because of its relatively low cost.However, because these protein therapeutics have a short half-life in the vitreous humor, they often require frequent (up to monthly) intravitreal injections to maintain efficacy in the eye (see Hard, A.L. and A.J.A.p.Hellstrom, On safety, pharmacokinetics and dosage of bevacizumab in ROP treatment - a review. 2011. 100(12): p. 1523 - 1527; and Stewart, M.W., et al., Pharmacokinetic rationale for dosing every 2 weeks versus 4 weeks with intravitreal ranibizumab, bevacizumab, and aflibercept (vascular endothelial growth factor Trap-eye). Retina, 2012. 32(3): p. 434 - 457). Unfortunately, this regimen results in side effects that frequently include pain, infection, endophthalmitis, increased intraocular pressure, inflammation, retinal detachment, and cataract formation (see Sampat, K.M. and S.J.J.C.o.i.o.Garg, Complications of intravitreal injections. 2010. 21(3): p. 178 - 183). A major barrier to treatment is the high cost associated with each injection, imposing the burden of monthly treatment on patients and their families (see Heimes, B., et al., Compliance von Patienten mit altersabhängiger Makuladegeneration unter Anti-VEGF-Therapie. Der Ophthalmologe, 2016. 113(11): p. 925 - 932). Thus, there is a clear need for easier and more efficient treatments for exudative AMD.

[0061] Conventional delivery systems in the form of implants and particles have been developed to achieve controlled release for possible AMD treatment (see Delplace, V., S. Payne, and M. J. J. o. C. R. Shoichet, Delivery strategies for treatment of age-related ocular diseases: From a biological understanding to biomaterial solutions. 2015. 219: p. 652-668; Radhakrishnan, K., et al., Protein delivery to the back of the eye: barriers, carriers and stability of anti-VEGF proteins. 2017. 22(2): p. 416-423; Imperiale, J. C., G. B. Acosta, and A. J. J. o. C. R. Sosnik, Polymer-based carriers for ophthalmic drug delivery. 2018; and Lee, S. S., et al., Biodegradable implants for sustained drug release in the eye. 2010. 27(10): p. 2043-2053). Compared to micro / nanoparticle-based systems, implants have higher stability and drug payload due to their larger size (see Kim, Y. C., et al., Ocular delivery of macromolecules. Journal of Controlled Release, 2014. 190: p. 172-181). However, most implant-based treatments are associated with difficulties regarding injection, and for non-biodegradable intraocular lenses, additional surgical procedures for implantation and removal are required (see Silva, G. R. d., et al., Implants as drug delivery devices for the treatment of eye diseases. Brazilian Journal of Pharmaceutical Sciences, 2010. 46: p. 585-595).These are accompanied by postoperative complications and increased costs. Additionally, long-term sustained release from either particles or implants has been difficult due to inadequate physical and chemical drug retention. For example, the (lactic acid-glycolic acid) copolymer (PLGA), one of the most commonly used polymers for drug delivery, is characterized by rapid hydrolysis and often leads to therapeutic release for up to 90 days (see Li, F., et al., Controlled release of bevacizumab through nanospheres for extended treatment of age-related macular degeneration. 2012.6: p. 54; and Sousa, F., et al., A new paradigm for antiangiogenic therapy through controlled release of bevacizumab from PLGA nanoparticles. 2017.7(1): p. 3736). Additional drawbacks include the formation of acidic by-products that can induce inflammation and exacerbate the foreign body reaction (see Lu, L., M. J. Yaszemski, and A. G. J. B. Mikos, Retinal pigment epithelium engineering using synthetic biodegradable polymers. 2001.22(24): p. 3345-3355).

[0062] Disclosed herein is a drug delivery composition comprising injectable and biodegradable multi-layer capsules loaded with a therapeutic agent (e.g., bevacizumab) for achieving a higher drug loading rate and a longer drug release duration compared to conventionally available injectable drug delivery devices. To achieve highly sustainable and controllable drug release, drug delivery compositions are disclosed herein that include, for example, a nanoporous PCL outer shell and a chitosan inner layer to achieve physical trapping and electrostatic-based chemisorption, respectively. A hollow structure encapsulated by a two-layer hybrid shell was utilized to load sufficient therapeutic substance for long-term drug release of at least one year. More specifically, the overall drug delivery composition is prepared by combining materials processing techniques including electrospinning, sintering, and salt leaching. The disclosed method provides a centrally hollow cylindrical microrod with a high aspect ratio to enable injection feasibility via a 21-gauge or smaller needle for intravitreal implantation delivery. By optimizing the chemical and physical structure of the capsules using the disclosed method, a stable and controlled release of protein therapeutic substances can be obtained for more than 10 months using the disclosed drug delivery composition. By reducing the frequency of injections via a small-gauge needle, the disclosed drug delivery composition may be able to improve the quality of life of patients with exudative AMD.

[0063] Thus, in one aspect, one or more capsules, each having a tube shape with two closed ends, wherein each of the one or more capsules independently includes a multi-layer wall and at least one lumen compartment; and One or more therapeutic agents, each initially present within one or more of the at least one lumen compartment, Comprising, Each multi-layer wall independently includes at least an inner layer and an outer layer; Each inner layer independently includes a first polymer having a net positive charge under physiological conditions; Each outer layer independently includes a second polymer different from the first polymer, A drug delivery composition is provided.

[0064] In some embodiments, the drug delivery composition can include two or more capsules (e.g., two capsules, three capsules, four capsules, five capsules, six capsules, seven capsules, eight capsules, nine capsules, ten capsules, or more). In such embodiments, the two or more capsules can each include the same composition for the multilayer walls of each capsule, or they can be different in their compositions. In some embodiments, the same therapeutic agent or different therapeutic agents can initially be present within each of the two or more capsules.

[0065] In some embodiments, each capsule in the drug delivery composition can independently include two or more luminal compartments (e.g., two luminal compartments, three luminal compartments, four luminal compartments, or more). In some embodiments, the same therapeutic agent or different therapeutic agents can initially be present within each of the two or more luminal compartments within a single capsule.

[0066] In some embodiments, each capsule independently has a length of from about 0.1 cm to about 5 cm (e.g., from 0.5 cm to about 3 cm, or from 1 cm to about 3 cm). In some embodiments, each capsule independently has a length of from about 0.1 cm to 5 cm, 0.5 cm to 5 cm, 1 cm to 5 cm, 2 cm to 5 cm, 3 cm to 5 cm, 4 cm to 5 cm, 0.1 cm to 4 cm, 0.5 to 4 cm, 1 cm to 4 cm, 2 cm to 4 cm, 3 cm to 4 cm, 0.1 cm to 3 cm, 0.5 cm to 3 cm, 1 cm to 3 cm, 2 cm to 3 cm, 0.1 cm to 2 cm, 0.5 cm to 2 cm, 1 cm to 2 cm, 0.1 cm to 1 cm, 0.5 to 1 cm, or from 0.1 to 0.5 cm.

[0067] In some embodiments, the multilayer wall has a wall thickness of about 25 μm to about 150 μm (e.g., about 70 μm to about 100 μm, about 75 μm to about 95 μm, or about 80 μm to about 90 μm). In some embodiments, the multilayer wall is about 50 μm to 150 μm, about 55 μm to 150 μm, about 60 μm to about 150 μm, about 65 μm to about 150 μm, about 70 μm to about 150 μm, about 75 μm to about 150 μm, about 80 μm to about 150 μm, about 90 μm to about 150 μm, about 95 μm to about 150 μm, about 100 μm to about 150 μm, about 110 μm to about 150 μm, about 125 μm to about 150 μm, about 140 μm to about 150 μm, about 50 μm to 140 μm, about 55 μm to 140 μm, about 60 μm to about 140 μm, about 65 μm to about 140 μm, about 70 μm to about 140 μm, about 75 μm to about 140 μm, about 80 μm to about 140 μm, about 90 μm to about 140 μm, about 95 μm to about 140 μm, about 100 μm to about 140 μm, about 110 μm to about 140 μm, about 125 μm to about 140 μm, about 50 μm to 125 μm, about 55 μm to 125 μm, about 60 μm to about 125 μm, about 65 μm to about 125 μm, about 70 μm to about 125 μm, about 75 μm to about 125 μm, about 80 μm to about 125 μm, about 90 μm to about 125 μm, about 95 μm to about 125 μm, about 100 μm to about 125 μm, about 110 μm to about 125 μm, about 50 μm to 110 μm, about 55 μm to 110 μm, about 60 μm to about 110 μm, about 65 μm to about 110 μm, about 70 μm to about 110 μm, about 75 μm to about 110 μm, about 80 μm to about 110 μm, about 90 μm to about 110 μm, about 95 μm to about 110 μm, about 100 μm to about 110 μm, about 50 μm to 100 μm, about 55 μm to 100 μm, about 60 μm to about 100 μm, about 65 μm to about 100 μm, about 70 μm to about 100 μm, about 75 μm to about 100 μm, about 80 μm to about 100 μm, about 90 μm to about 100 μm, about 95 μm to about 100 μm, about 50 μm to 95 μm, about 55 μm to 95 μm, about 60 μm to about 95 μm, about 65 μm to about 95 μm, about 70 μm to about 95 μm, about 75 μm to about 95 μm, about 80 μm to about 95 μm, about 90 μm to about 95 μm, about 50 μm to 90 μm, about 55 μm to 90 μm, about 60 μm to about 90 μm, about 65 μm to about 90 μm, about 70 μm to about 90 μm, about 75 μm to about 90 μm, about 80 μm to about 90 μm,It has a wall thickness of about 50 μm to 80 μm, about 55 μm to 80 μm, about 60 μm to about 80 μm, about 65 μm to about 80 μm, about 70 μm to about 80 μm, about 75 μm to about 80 μm, about 50 μm to 75 μm, about 55 μm to 75 μm, about 60 μm to about 75 μm, about 65 μm to about 75 μm, about 70 μm to about 75 μm, about 50 μm to 70 μm, about 55 μm to 70 μm, about 60 μm to about 70 μm, about 65 μm to about 70 μm, about 50 μm to 65 μm, about 55 μm to 65 μm, about 60 μm to about 65 μm, about 50 μm to 60 μm, about 55 μm to 60 μm, and about 50 μm to about 55 μm.

[0068] In some embodiments, the thickness of the inner layer can range from about 1 μm to about 100 μm. In some embodiments, the thickness of the inner layer can range from about 100 nm to about 990 nm (e.g., about 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, or 990 nm).

[0069] In some embodiments, the thickness of the outer layer can range from about 1 μm to about 100 μm. In some embodiments, the thickness of the outer layer can range from about 100 nm to about 990 nm (e.g., about 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, or 990 nm).

[0070] In some embodiments, the tube shape of the drug delivery capsule has an inner diameter of from about 100 μm to about 1000 μm (such as from about 100 μm to about 1000 μm, from about 100 μm to about 500 μm, or from 100 μm to about 300 μm).In some embodiments, the inner diameter of the tube-shaped drug delivery capsule is from about 100 μm to about 2000 μm, from 200 μm to about 2000 μm, from about 300 μm to about 2000 μm, from about 400 μm to about 2000 μm, from about 500 μm to about 2000 μm, from about 600 μm to about 2000 μm, from about 700 μm to about 2000 μm, from about 800 μm to about 2000 μm, from about 900 μm to about 2000 μm, from about 1000 μm to about 2000 μm, from about 1500 μm to about 2000 μm, from about 100 μm to about 1500 μm, from 200 μm to about 1500 μm, from about 300 μm to about 1500 μm, from about 400 μm to about 1500 μm, from about 500 μm to about 1500 μm, from about 600 μm to about 1500 μm, from about 700 μm to about 1500 μm, from about 800 μm to about 1500 μm, from about 900 μm to about 1500 μm, from about 1000 μm to about 1500 μm, from about 100 μm to about 1000 μm, from 200 μm to about 1000 μm, from about 300 μm to about 1000 μm, from about 400 μm to about 1000 μm, from about 500 μm to about 1000 μm, from about 600 μm to about 1000 μm, from about 700 μm to about 1000 μm, from about 800 μm to about 1000 μm, from about 900 μm to about 1000 μm, from about 100 μm to about 900 μm, from 200 μm to about 900 μm, from about 300 μm to about 900 μm, from about 400 μm to about 900 μm, from about 500 μm to about 900 μm, from about 600 μm to about 900 μm, from about 700 μm to about 900 μm, from about 800 μm to about 900 μm, from about 100 μm to about 800 μm, from 200 μm to about 800 μm, from about 300 μm to about 800 μm, from about 400 μm to about 800 μm, from about 500 μm to about 800 μm, from about 600 μm to about 800 μm, from about 700 μm to about 800 μm, from about 100 μm to about 700 μm, from 200 μm to about 700 μm, from about 300 μm to about 700 μm, from about 400 μm to about 700 μm, from about 500 μm to about 700 μm, from about 600 μm to about 700 μm, from about 100 μm to about 600 μm, from 200 μm to about 600 μm, from about 300 μm to about 600 μm, from about 400 μm to about 600 μm, from about 500 μm to about 600 μm, from about 100 μm to about 500 μm, from 200 μm to about 500 μm, from about 300 μm to about 500 μm, from about 400 μm to about 500 μm, from about 100 μm to about 400 μm, from 200 μm to about 400 μm, from about 300 μm to about 400 μm, from about 100 μm to about 300 μm, from 200 μm to about 300 μm, and from about 100 μm to about 200 μm.In some embodiments, the tube shape has an outer diameter that is about 100 μm to about 300 μm larger than the inner diameter (e.g., about 100 μm to about 300 μm, 150 μm to about 300 μm, 200 μm to about 300 μm, about 250 μm to about 300 μm, about 100 μm to about 250 μm, about 150 μm to about 250 μm, about 200 μm to about 250 μm, about 100 μm to about 200 μm, about 150 μm to about 200 μm, or about 100 μm to about 150 μm larger than the inner diameter).

[0071] In some embodiments, the first polymer can include chitosan, polyethyleneimine, protamine, polypropylenimine, poly-L-lysine, poly-L-arginine, poly-D-lysine, poly-D-arginine, cellulose, dextran, poly(amidoamine), poly(2-(dimethylamino)ethyl methacrylate), derivatives thereof, or combinations thereof.

[0072] In some embodiments, the first polymer includes chitosan or a derivative thereof. Chitosan can have a degree of deacetylation of about 60% to about 90%; at least about 70%, at least about 75%, at least about 80% degree of deacetylation.

[0073] In some embodiments, the first polymer has a molecular weight of about 50 kDa to about 500 kDa (e.g., about 100 kDa to about 500 kDa, about 100 kDa to about 400 kDa, about 200 kDa to about 400 kDa, about 300 kDa to about 400 kDa, or about 310 kDa to about 375 kDa). In some embodiments, the first polymer has a molecular weight of about 10 kDa or more (e.g., about 15 kDa or more, about 20 kDa or more, about 30 kDa or more, about 40 kDa or more, about 50 kDa or more, about 60 kDa or more, about 70 kDa or more, about 90 kDa or more, about 90 kDa or more, or about 100 kDa or more).

[0074] In some embodiments, the first polymer used in the inner layer contains fibers. In some embodiments, the fibers can have a diameter of about 50 nm to about 1000 nm (e.g., about 100 nm to about 400 nm). In some embodiments, the fibers can have a diameter of about 50 nm to about 1000 nm, about 100 nm to about 1000 nm, about 200 nm to about 1000 nm, about 400 nm to about 1000 nm, about 600 nm to about 1000 nm, about 800 nm to about 1000 nm, about 50 nm to about 800 nm, about 100 nm to about 800 nm, about 200 nm to about 800 nm, about 400 nm to about 800 nm, about 600 nm to about 800 nm, about 50 nm to about 600 nm, about 100 nm to about 600 nm, about 200 nm to about 600 nm, about 400 nm to about 600 nm, about 50 nm to about 400 nm, about 100 nm to about 400 nm, about 200 nm to about 400 nm, about 50 nm to about 200 nm, about 100 nm to about 200 nm, or about 50 nm to about 100 nm.

[0075] In some embodiments, the second polymer can include poly(ε-caprolactone) (PCL), poly-lactic acid (PLA), poly-glycolic acid (PGA), poly-lactide-co-glycolide (PLGA), polyester, poly(ot her) ester, poly(phosphazene), poly(phosphate ester), gelatin, collagen, polyethylene glycol (PEG), its derivatives, and combinations thereof. In other embodiments, the second polymer can include PLGA, PCL, PLA, PGA, PEG, polysorbate, poly(ε-caprolactone-thioethyl ethylene phosphate) (PCLEEP), polyvinyl alcohol (PVA), or combinations thereof. In some embodiments, the second polymer includes PLGA, PCL, PLA, PGA, or combinations thereof. In some embodiments, the second polymer can include PLGA, PCK, PLA, or combinations thereof. In some embodiments, the second polymer includes PLGA. In some embodiments, the second polymer includes PCL. In some embodiments, the second polymer includes PLA.

[0076] In some embodiments, the second polymer has a molecular weight of from about 50 kDa to about 500 kDa (e.g., from about 100 kDa to about 500 kDa, from about 100 kDa to about 400 kDa, from about 200 kDa to about 400 kDa, from about 300 kDa to about 400 kDa, or from about 310 kDa to about 375 kDa). In some embodiments, the second polymer has a molecular weight of about 10 kDa or greater (e.g., about 15 kDa or greater, about 20 kDa or greater, about 30 kDa or greater, about 40 kDa or greater, about 50 kDa or greater, about 60 kDa or greater, about 70 kDa or greater, about 90 kDa or greater, about 90 kDa or greater, or about 100 kDa or greater).

[0077] In some embodiments, the second polymer is biodegradable in vivo and exhibits good tolerance throughout the duration of the presence and degradation of the composition. In some embodiments, under physiological conditions, the second polymer degrades by random scission of the chains, which results in biphasic degradation. First, even as the molecular weight decreases, the physical structure is not significantly affected. Degradation occurs throughout the polymer material and proceeds until a critical molecular weight is reached at which the degradation products are small enough to solubilize. At this point, the structure begins to become significantly porous and hydrated. In some embodiments, the second polymer has a molecular weight of about 90 kDa or greater and does not degrade until after 6 months or more in the eye of the subject. In some embodiments, the molecular weight of the biodegradable polymer is selected to adjust the degradation time of the material in vivo.

[0078] In some embodiments, the second polymer may include a blend of a high molecular weight polymer and a low molecular weight polymer. In some embodiments, the high molecular weight polymer may be about 25 kDa or greater (e.g., about 30 kDa or greater, 40 kDa or greater, 50 kDa or greater, 60 kDa or greater, 70 kDa or greater, 80 kDa or greater, 90 kDa or greater, or 100 kDa or greater), and the low molecular weight polymer may be about 20 kDa or less (e.g., 15 kDa or less, 10 kDa or less, 8 kDa or less, 6 kDa or less, or 4 kDa or less). In some embodiments, the ratio of the high molecular weight polymer to the lower molecular weight polymer is between about 1:9 and about 9:1 (e.g., between about 2:8 and about 8:2, between about 2:8 and about 6:4, or between about 2:8 and about 1:1).

[0079] In some embodiments, the outer layer of the second polymer used in the outer layer contains fibers. In some embodiments, the fibers can have a diameter of about 100 nm to about 2000 nm (e.g., about 500 nm to about 1000 nm). In some embodiments, the fibers can have a diameter of about 100 nm to about 2000 nm, about 250 nm to about 2000 nm, about 500 nm to about 2000 nm, about 750 nm to about 2000 nm, about 1000 nm to about 2000 nm, about 1500 nm to about 2000 nm, about 100 nm to about 1500 nm, about 250 nm to about 1500 nm, about 500 nm to about 1500 nm, about 750 nm to about 1500 nm, about 1000 nm to about 1500 nm, about 100 nm to about 1000 nm, about 250 nm to about 1000 nm, about 500 nm to about 1000 nm, about 750 nm to about 1000 nm, about 100 nm to about 750 nm, about 250 nm to about 750 nm, about 500 nm to about 750 nm, about 100 nm to about 500 nm, about 250 nm to about 500 nm, or about 100 nm to about 250 nm.

[0080] In some embodiments, the outer layer may further include pores. In other embodiments, the outer layer does not include pores. In some embodiments, the outer layer includes pores having an average pore diameter of about 1 nm to about 990 nm (e.g., about 1 nm to about 100 nm, about 2 nm to about 700 nm, about 3 nm to about 400 nm, about 5 nm to about 200 nm, or about 7 nm to about 50 nm). In some embodiments, the outer layer includes pores having an average pore diameter of about 100 nm to 1000 nm (e.g., 350 nm to 650 nm). In some embodiments, the outer layer is about 100 nm to about 1000 nm, 200 nm to about 1000 nm, 300 nm to about 1000 nm, about 400 nm to about 1000 nm, about 450 nm to about 1000 nm, about 500 nm to about 1000 nm, about 550 nm to about 1000 nm, about 600 nm to about 1000 nm, about 650 nm to about 1000 nm, about 700 nm to about 1000 nm, about 800 nm to about 1000 nm, about 900 nm to about 1000 nm, about 100 nm to about 900 nm, 200 nm to about 900 nm, 300 nm to about 900 nm, about 400 nm to about 900 nm, about 450 nm to about 900 nm, about 500 nm to about 900 nm, about 550 nm to about 900 nm, about 600 nm to about 900 nm, about 650 nm to about 900 nm, about 700 nm to about 900 nm, about 800 nm to about 900 nm, about 100 nm to about 800 nm, 200 nm to about 800 nm, 300 nm to about 800 nm, about 400 nm to about 800 nm, about 450 nm to about 800 nm, about 500 nm to about 800 nm, about 550 nm to about 800 nm, about 600 nm to about 800 nm, about 650 nm to about 800 nm, about 700 nm to about 800 nm, about 100 nm to about 700 nm, 200 nm to about 700 nm, 300 nm to about 700 nm, about 400 nm to about 700 nm, about 450 nm to about 700 nm, about 500 nm to about 700 nm, about 550 nm to about 700 nm, about 600 nm to about 700 nm, about 650 nm to about 700 nm, about 100 nm to about 650 nm, 200 nm to about 650 nm, 300 nm to about 650 nm, about 400 nm to about 650 nm, about 450 nm to about 650 nm, about 500 nm to about 650 nm, about 550 nm to about 650 nm, about 600 nm to about 650 nm, about 100 nm to about 600 nm, 200 nm to about 600 nm, 300 nm to about 600 nm, about 400 nm to about 600 nm,It contains pores having an average pore diameter of from about 450 nm to about 600 nm, from about 500 nm to about 600 nm, from about 550 nm to about 600 nm, from about 100 nm to about 550 nm, from 200 nm to about 550 nm, from 300 nm to about 550 nm, from about 400 nm to about 550 nm, from about 450 nm to about 550 nm, from about 500 nm to about 550 nm, from about 100 nm to about 500 nm, from 200 nm to about 500 nm, from 300 nm to about 500 nm, from about 400 nm to about 500 nm, from about 450 nm to about 500 nm, from about 100 nm to about 450 nm, from 200 nm to about 450 nm, from 300 nm to about 450 nm, from about 400 nm to about 450 nm, from about 100 nm to about 400 nm, from 200 nm to about 400 nm, from 300 nm to about 400 nm, from about 100 nm to about 300 nm, from 200 nm to about 300 nm, from about 100 nm to about 200 nm. In some embodiments, the size of the average pores is similar to the size of the therapeutic agent(s) such that one or more therapeutic agents diffuse through the nanopores via single file diffusion or facilitated diffusion. When the desired therapeutic agent is of a sufficiently small size (e.g., having a molecular weight of less than 500) and can readily diffuse through the outer layer of the capsule, the pores may not be necessary.,

[0081] In some embodiments, the composition of the first polymer or the second polymer can provide a melting temperature between about 50 °C and about 70 °C. In some embodiments, the composition of the first polymer or the second polymer is provided to provide a glass transition temperature (T g ) between about -50 °C and about -80 °C.

[0082] In some embodiments, each of the one or more capsules independently has a surface charge measurement as a zeta potential at pH 7.5 of from about -25 mV to about 25 mV (e.g., from about -20 mV to about 20 mV, from about -15 mV to about 15 mV, from about -10 mV to about 10 mV, from about -5 mV to about 5 mV, from about -1 mV to about 1 mV, from about -0.5 mV to about 0.5 mV, or from about -0.1 mV to about 0.1 mV).

[0083] In some embodiments, the composition of the first polymer and the second polymer is selected such that 50% of the mass for one or more of the layers remains after at least three months when exposed to physiological conditions. If desired, the degradation rate of one or more of the layers can be accelerated by adjustment of such aspects (such as layer thickness or porosity) in the manufacture of the capsule or by increasing the hydrophilicity of the polymer composition used to manufacture one or more of the layers.

[0084] Therapeutic agent In a further aspect, the disclosure also provides one or more therapeutic agents that can be used in the compositions disclosed herein.

[0085] In some embodiments, each of the one or more therapeutic agents has a net negative charge within a pH range of about 6.0 to about 7.4.

[0086] As used herein, "therapeutic agent" refers to one or more therapeutic agents, active ingredients, or substances that can be used to treat an ocular medical condition or cancer. Therapeutic agents are typically provided in a form that is ophthalmically acceptable and does not cause an adverse reaction when the compositions disclosed herein are placed in the eye. As discussed herein, therapeutic agents can be released from the disclosed compositions in a biologically active form. For example, when released from the system into the eye, therapeutic agents can retain their three-dimensional structure.

[0087] As used herein, the term "therapeutic agent" is further understood to include any synthetic or naturally occurring biologically active compound or composition of matter that, when administered to an organism (human or non-human animal), induces a desired pharmacological, immunogenic, and / or physiological effect by local and / or systemic action. Thus, the term encompasses compounds or chemical substances that are traditionally regarded as drugs, vaccines, and biopharmaceuticals (including molecules such as proteins, peptides, hormones, nucleic acids, gene constructs, and the like). Examples of therapeutic agents are described in well-known reference works such as the Merck Index (14th edition), the Physicians’ Desk Reference (64th edition), and The Pharmacological Basis of Therapeutics (12th edition), and include, by way of example, pharmaceuticals; vitamins; mineral supplements; substances used for the treatment, prevention, diagnosis, cure, or alleviation of a disease or disorder; substances that affect the structure or function of the body, or prodrugs that may become or more active biologically after placement in the physiological environment, without limitation.For example, the term "therapeutic agent" includes adjuvants; anti-infective substances (such as antibiotics and antiviral agents); analgesic substances and combinations of analgesic substances, anorectic substances, anti-inflammatory agents, antiepileptic substances, local and general anesthetic substances, hypnotic substances, sedative substances, antipsychotic agents, neuroleptic agents, antidepressant substances, anxiolytic substances, antagonists, neuronal blockers, anticholinergic agents and cholinergic-like agents, antimuscarinic agents and muscarinic agents, antiadrenergic substances, antiarrhythmic substances, antihypertensive agents, hormones, and nutrients, anti-arthritis substances, anti-asthma agents, antispasmodic substances, antihistamine substances, antiemetic substances, antineoplastic substances, anti-itch substances, antipyretic substances; anti-convulsant substances, cardiovascular drugs (including calcium channel blockers, β-blockers, β-agonists, and antiarrhythmic substances), antihypertensive substances, diuretic substances, vasodilator substances; central nervous system stimulants; cold remedies; decongestants; diagnostic substances; hormones; bone growth stimulants and bone resorption inhibitors; immunosuppressive substances; muscle relaxants; psychostimulants; sedative substances; tranquilizers; proteins, peptides, and fragments thereof (whether naturally occurring, chemically synthesized, or produced recombinantly); and nucleic acid molecules (in the polymeric form of two or more nucleotides of either ribonucleotide (RNA) or deoxyribonucleotide (DNA), including double-stranded and single-stranded molecules, gene constructs, expression vectors, antisense molecules, and the like), small molecules (such as doxorubicin), and other biologically active macromolecules (such as proteins and enzymes), and is not limited thereto, and includes compounds or compositions for use in all major therapeutic areas. The agent can be a biologically active agent used in medical applications including veterinary medicine, agriculture (such as those by plants), and other areas. The term "therapeutic agent" includes pharmaceuticals; vitamins; mineral supplements; substances used for the treatment, prevention, diagnosis, cure, or alleviation of diseases or illnesses; or substances that affect the structure or function of the body; or prodrugs that may become or become more biologically active after being placed in a given physiological environment, without limitation.

[0088] In some embodiments, the therapeutic agent may include a β-blocking substance (such as timolol, betaxolol, levobetaxolol, and carteolol); a miotic substance (such as pilocarpine); a carbonic anhydrase inhibitor; a serotonin agonist; a muscarinic agonist; a dopamine agonist; an adrenergic agonist (including apraclonidine and brimonidine); an anti-angiogenic agent; an anti-infective agent (such as quinolones (e.g., ciprofloxacin) and aminoglycosides (e.g., tobramycin and gentamicin)); a non-steroidal anti-inflammatory agent and a steroidal anti-inflammatory agent (such as suprofen, diclofenac, ketorolac, rimexolone, and tetrahydrocortisol); a growth factor (such as EGF); an immunosuppressant; and an anti-allergy agent (such as olopatadine); a prostaglandin (such as latanoprost); 15-ketolatanoprost; travoprost; and drugs useful for the treatment of ophthalmic disorders or eye diseases such as isopropyl unoprostone.

[0089] In some embodiments, the therapeutic agent is selected from the group consisting of an anti-inflammatory agent, a calcineurin inhibitor, an antibiotic, a nicotinic acetylcholine receptor agonist, and an anti-lymphangiogenesis agent. In some embodiments, the anti-inflammatory agent can be cyclosporine. In some embodiments, the calcineurin inhibitor can be voclosporin. In some embodiments, the antibiotic can be selected from the group consisting of amikacin, gentamicin, kanamycin, neomycin, netilmicin, streptomycin, tobramycin, teicoplanin, vancomycin, azithromycin, clarithromycin, dirithromycin, erythromycin, Roxithromycin, troleandomycin, amoxicillin, ampicillin, azlocillin, carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, mezlocillin, nafcillin, penicillin, piperacillin, ticarcillin, bacitracin, colistin, polymyxin B, ciprofloxacin, enoxacin, gatifloxacin, levofloxacin, lomefloxacin, moxifloxacin, norfloxacin, ofloxacin, trovafloxacin, mafenide, sulfacetamide, sulfamethizole, sulfasalazine, sulfisoxazole, trimethoprim, cotrimoxazole, demeclocycline, doxycycline, minocycline, oxytetracycline, and tetracycline. In some embodiments, the nicotinic acetylcholine receptor agonist can be any of pilocarpine, atropine, nicotine, epibatidine, lobeline, or imidacloprid. In some embodiments, the anti-lymphangiogenesis agent can be a vascular endothelial growth factor C (VEGF-C) antibody, a VEGF-D antibody, or a VEGF-3 antibody.

[0090] In some embodiments, the therapeutic agent may be selected from beta blockers (such as levobunolol (BETAGAN), timolol (BETIMOL, TIMOPTIC), betaxolol (BETOPTIC), and metipranolol (OPTIPRANOLOL)); alpha - agonists (such as apraclonidine (IOPIDINE) and brimonidine (ALPHAGAN)); carbonic anhydrase inhibitors (such as acetazolamide, methazolamide, dorzolamide (TRUSOPT), and brinzolamide (AZOPT)); prostaglandins or prostaglandin analogs (such as latanoprost (XALATAN), bimatoprost (LUMIGAN), and travoprost (TRAVATAN)); miotics or cholinergic agents (such as pilocarpine (ISOPTO CARPINE, PILOPINE) and carbachol (ISOPTO CARBACHOL)); epinephrine compounds (such as dipivefrin (PROPINE)); forskolin; or neuroprotective compounds (such as brimonidine and memantine); steroid derivatives (such as 2 - methoxyestradiol or its analogs or derivatives); or antibiotics.

[0091] The term "VEGF" refers to vascular endothelial growth factors that induce angiogenesis or the angiogenic process (including, but not limited to, increased permeability). As used herein, the term "VEGF" includes the various subtypes of VEGF (also known as vascular permeability factor (VPF) and VEGF-A) that result from alternative splicing of, for example, the VEGF-A / VPF gene, including VEGF121, VEGF165, and VEGF189. Further, as used herein, the term "VEGF" includes VEGF-related angiogenic factors (such as placental growth factor (PIGF), VEGF-B, VEGF-C, and VEGF-D, and VEGF-E, etc.) that act through cognate VEFG receptors (i.e., VEGFR) to induce angiogenesis or the angiogenic process. The term "VEGF" includes any member of the class of growth factors that bind to VEGF receptors (VEGFR-1 (Flt-1), VEGFR-2 (KDR / Flk-1), or VEGFR-3 (FLT-4)). The term "VEGF" may be used to refer to a "VEGF" polypeptide or a "VEGF" encoding gene or nucleic acid.

[0092] The term "anti-VEGF agent" refers to an agent that reduces or inhibits, either partially or completely, the activity or production of VEGF. An anti-VEGF agent can directly or indirectly reduce or inhibit the activity or production of specific VEGF (such as VEGF165). Further, the "anti-VEGF agent" includes an agent that acts on either the VEGF ligand or its cognate receptor so as to reduce or inhibit the receptor signal associated with VEGF. Non-limiting examples of "anti-VEGF agents" include antisense molecules, ribozymes, or RNAi that target VEGF nucleic acids; anti-VEGF aptamers, anti-VEGF antibodies to VEGF itself or its receptor, or soluble VEGF receptor decoys that prevent the binding of VEGF to its cognate receptor; antisense molecules, ribozymes, or RNAi that target cognate VEGF receptor (VEGFR) nucleic acids; anti-VEGFR aptamers or anti-VEGFR antibodies that bind to the cognate VEGFR receptor; and VEGFR tyrosine kinase inhibitors.

[0093] In some embodiments, the therapeutic agent may include an anti-VEGF agent. Representative examples of anti-VEGF agents include ranibizumab, bevacizumab, aflibercept, KH902 VEGF receptor-Fc, fusion protein, 2C3 antibody, ORA102, pegaptanib, bevaciranib, SIRNA-027, decursin, decursinol, picropodophyllin, guggulsterone, PLG101, eicosanoid LXA4, PTK787, pazopanib, axitinib, CDDO-Me, CDDO-Imm, silibinin, β-hydroxyisovaleryl silibinin, ganglioside GM3, DC101 antibody, Mab25 antibody, Mab73 antibody, 4A5 antibody, 4E10 antibody, 5F12 antibody, VA01 antibody, BL2 antibody, VEGF-related protein, sFLT01, sFLT02, peptide B3, TG100801, sorafenib, G6-31 antibody, fusion antibody, and an antibody that binds to an epitope of VEGF. Additional non-limiting examples of anti-VEGF agents useful in the present method include substances that specifically bind to one or more of human vascular endothelial growth factor-A (VEGF-A), human vascular endothelial growth factor-B (VEGF-B), human vascular endothelial growth factor-C (VEGF-C), human vascular endothelial growth factor-D (VEGF-D), and human vascular endothelial growth factor-E (VEGF-E), and an antibody that binds to an epitope of VEGF.

[0094] In various embodiments, the anti-VEGF agent is the antibody ranibizumab or a pharmaceutically acceptable salt thereof. Ranibizumab is commercially available under the trademark LUCENTIS. In another embodiment, the anti-VEGF agent is the antibody bevacizumab or a pharmaceutically acceptable salt thereof. Bevacizumab is commercially available under the trademark AVASTIN. In another embodiment, the anti-VEGF agent is aflibercept or a pharmaceutically acceptable salt thereof. Aflibercept is commercially available under the trademark EYLEA. In one embodiment, the anti-VEGF agent is pegaptinib or a pharmaceutically acceptable salt thereof. Pegaptinib is commercially available under the trademark MACUGEN. In another embodiment, the anti-VEGF agent is an antibody or antibody fragment that binds to an epitope of VEGF (such as an epitope of VEGF-A, VEGF-B, VEGF-C, VEGF-D, or VEGF-E). In some embodiments, the VEGF antagonist binds to an epitope of VEGF such that the binding of VEGF to VEGFR is inhibited. In one embodiment, the epitope encompasses components of the three-dimensional structure of VEGF presented such that the epitope is exposed on the surface of the folded VEGF molecule. In one embodiment, the epitope is a linear amino acid sequence from VEGF.

[0095] In various aspects, the therapeutic agent can include an agent that blocks or inhibits VEGF-mediated activity (e.g., one or more VEGF antisense nucleic acids). The present disclosure provides the therapeutic or prophylactic use of a nucleic acid comprising at least 6 nucleotides that is antisense to a gene or cDNA encoding VEGF or a portion thereof. As used herein, a VEGF “antisense” nucleic acid refers to a nucleic acid capable of hybridizing by virtue of some sequence complementarity to a portion of the RNA (preferably mRNA) encoding VEGF. The antisense nucleic acid can be complementary to the coding region and / or non-coding region of the mRNA encoding VEGF. Such antisense nucleic acids have utility as compounds that prevent VEGF expression and can be used in the treatment of diabetes. The antisense nucleic acids of the present disclosure are double-stranded or single-stranded oligonucleotides, RNA, or DNA, or modifications or derivatives thereof, and can be administered directly to cells or produced intracellularly by transcription of an exogenously introduced sequence.

[0096] The VEGF antisense nucleic acid is at least 6 nucleotides, preferably an oligonucleotide in the range of 6 to about 50 oligonucleotides. In certain embodiments, the oligonucleotide is at least 10 nucleotides, at least 15 nucleotides, at least 100 nucleotides, or at least 200 nucleotides. The oligonucleotide can be DNA or RNA or a chimeric mixture, or derivatives or modified versions thereof, and can be single-stranded or double-stranded. In addition, the antisense molecule can be a polymer that is a nucleic acid mimic (such as PNA, morpholino oligo, and LNA). Other types of antisense molecules include short double-stranded RNAs known as siRNAs, and short hairpin RNAs, and long dsRNAs (>50 bp, but usually ≧500 bp).

[0097] In various embodiments, the therapeutic agent can include one or more ribozyme molecules designed to catalytically cleave transcripts of the gene mRNA encoding VEGF and prevent translation of the target gene mRNA and thus expression of the gene product.

[0098] A ribozyme is an enzymatic RNA molecule capable of catalyzing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to a complementary target RNA, followed by an endonucleolytic cleavage event. The composition of the ribozyme molecule must contain one or more sequences complementary to the target gene mRNA and must contain a well-known catalytic sequence responsible for mRNA cleavage. For this sequence, see, for example, U.S. Patent No. 5,093,246. Ribozyme that cleaves mRNA with a site-specific recognition sequence can be used for the destruction of mRNA encoding VEGF, but the use of hammerhead ribozyme is preferred. The hammerhead ribozyme cleaves mRNA at a position defined by an adjacent region that forms complementary base pairs with the target mRNA. The only requirement is that the target mRNA has the following two-base sequence: 5'-UG-3'. The construction and production of hammerhead ribozymes are well known in the art. The ribozymes of the present disclosure also include RNA endoribonucleases (hereinafter "Cech-type ribozymes") such as those that naturally occur in Tetrahymena thermophila (known as IVS or L-19 IVS RNA). The Cech-type ribozyme has an eight-base pair active site that hybridizes to the target RNA sequence after cleavage of the target RNA has occurred. The present disclosure encompasses Cech-type ribozymes that target an eight-base pair active site sequence present in the gene encoding VEGF.

[0099] In a further aspect, the therapeutic agent may include an antibody that inhibits VEGF (such as bevacizumab or ranibizumab). In yet a further aspect, the therapeutic agent includes an agent that inhibits VEGF activity (such as tyrosine kinase stimulated by VEGF), examples of which include, but are not limited to, lapatinib, sunitinib, sorafenib, axitinib, and pazopanib.

[0100] The term "anti-RAS agent" or "anti-renin angiotensin system agent" refers to an agent that reduces or inhibits, either partially or completely, the activity or production of molecules of the renin angiotensin system (RAS). Non-limiting examples of "anti-RAS" or "anti-renin angiotensin system" molecules are one or more of angiotensin converting enzyme (ACE) inhibitors, angiotensin receptor blockers, and renin inhibitors.

[0101] In some embodiments, the therapeutic agent may include a renin angiotensin system (RAS) inhibitor. In some embodiments, the renin angiotensin system (RAS) inhibitor is one or more of an angiotensin converting enzyme (ACE) inhibitor, an angiotensin receptor blocker, and a renin inhibitor.

[0102] Non-limiting examples of angiotensin-converting enzyme (ACE) inhibitors useful in the present invention include alacepril, alatriopril, altiorpril calcium, ancovenin, benazepril, benazepril hydrochloride, benazeprilate, benzazepril, benzoyl captopril, captopril, captopril cysteine, captopril glutathione, ceranapril, ceranopril, seranapril, cilazapril, cilazaprilate, converstatin, delapril, delapril diacid, enalapril, enalaprilat, enaliren, enapril, epicaptopril, horoxymicin, phenopril, fosinopril, fosinopril sodium, fosinopril, fosinopril sodium, fosinoprilate, fosinopril acid, glycopril, hemorphin-4, idapril, imidapril, indolapril, indolaprilat, lisinopril, lisinoprilat, mixanpril, moexipril, moexiprilate, moexiprilat, mobenzapril, murasein A, murasein B, murasein C, pentopril, perindopril, perindoprilate, pivalopril, pivopril, quinapril, quinapril hydrochloride, quinaprilat, ramipril, ramiprilate, spirapril, spirapril hydrochloride, spiraprilate, spiropril, spirapril hydrochloride, temocapril, temocapril hydrochloride, teprotide,trandolapril, trandolaprilate, utibapril, zafirlukast, zafirlukast sodium, zofenopril, zofenoprilate, pharmaceutically acceptable salts thereof, and mixtures thereof, but are not limited thereto.

[0103] Non-limiting examples of angiotensin receptor blockers useful in the present invention include irbesartan (U.S. Patent No. 5,270,317, which is incorporated herein by reference in its entirety), candesartan (U.S. Patents Nos. 5,196,444 and 5,705,517, which are incorporated herein by reference in their entireties), valsartan (U.S. Patent No. 5,399,578, which is incorporated herein by reference in its entirety), and losartan (U.S. Patent No. 5,138,069, which is incorporated herein by reference in its entirety), but are not limited thereto.

[0104] Non-limiting examples of renin inhibitors that can be used as therapeutic agents include aliskiren, ditikiren, enalkiren, remikiren, terlakiren, ciprokiren, and zankiren, their pharmaceutically acceptable salts, and mixtures thereof, but are not limited thereto.

[0105] The term "steroid" refers to compounds belonging to or related to the following exemplary families of compounds: corticosteroids, mineralicosteroids, and sex steroids (including, for example, potentially androgenic or estrogenic, or anti-androgenic and anti-estrogenic molecules). These include, for example, prednisone, prednisolone, methyl-prednisolone, triamcinolone, fluocinolone, aldosterone, spironolactone, danazol (otherwise known as OPTINA in other cases), and others. In some embodiments, the therapeutic agent may include a steroid.

[0106] The term "peroxisome proliferator-activated receptor γ agent" or "PPAR-γ agent" or "PPARG agent" or "PPAR-γ agent" refers to an agent that acts directly or indirectly on the peroxisome proliferator-activated receptor. This agent may also affect PPAR-α ("PPARA") activity.

[0107] In some embodiments, the therapeutic agent may include an agent that modulates macrophage polarization. Exemplary agents that modulate macrophage polarization include peroxisome proliferator-activated receptor gamma (PPAR-γ) modulators, including, for example, agonists, partial agonists, antagonists, or combinations of PPAR-γ / α agonists. In some embodiments, the therapeutic agent may include a PPARγ modulator (including a PPARγ modulator that is a full agonist or a partial agonist). In some embodiments, the PPARγ modulator is a member of the drug class of thiazolidinediones (TZD, or glitazone). By way of non-limiting example, the PPARγ modulator can be one or more of rosiglitazone (AVANDIA), pioglitazone (ACTOS), troglitazone (REZULIN), netoglitazone, riboglitazone, ciglitazone, rhodanine. In some embodiments, the PPARγ modulator is one or more of irbesartan and telmesartan. In some embodiments, the PPARγ modulator is a non-steroidal anti-inflammatory drug (e.g., an NSAID such as ibuprofen) or an indole. Known inhibitors include the experimental agent GW-9662. Further examples of PPARγ modulators are those described in WIPO Publication Nos. WO / 1999 / 063983, WO / 2001 / 000579, Nat Rev Immunol. 2011 Oct. 25;11(11):750-61, or agents identified using the method of WO / 2002 / 068386 (the contents of which are incorporated herein by reference in their entirety).

[0108] In some embodiments, the PPARγ modulator is a "dual" PPAR modulator or a "balanced" PPAR modulator or a "pan" PPAR modulator. In some embodiments, the PPARγ modulator is a glitazar that binds to two or more PPAR isoforms (e.g., muraglitazar (Pargluva) and tesaglitazar (Galida) and aleglitazar).

[0109] In some embodiments, the therapeutic agent may include semapimod (CNI-1493) as described in Bianchi, et al. (March 1995). Molecular Medicine (Cambridge, Mass.) 1(3):254-266, which is incorporated herein by reference in its entirety.

[0110] In some embodiments, the therapeutic agent may include a migration inhibitory factor (MIF) inhibitor. Exemplary MIF inhibitors are described in WIPO Publication Nos. WO2003 / 104203, WO2007 / 070961, WO2009 / 117706, and U.S. Patent Nos. 7,732,146, 7,632,505, 7,294,753, 7,294,753, which are incorporated herein by reference in their entirety. In some embodiments, the MIF inhibitor is (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazoleacetic acid methyl ester (ISO-1), isoxazoline, p425 (J. Biol. Chem., 287, 30653-30663), epoxyazadiron, or vitamin E.

[0111] In some embodiments, the therapeutic agent can include, for example, chemokine receptor 2 (CCR2) inhibitors as described in U.S. Patents and Patent Publications: U.S. Patent No. 7,799,824, U.S. Patent No. 8,067,415, US2007 / 0197590, US2006 / 0069123, US2006 / 0058289, and US2007 / 0037794, the contents of which are hereby incorporated by reference in their entirety. In some embodiments, the CCR2 inhibitor is maraviroc, cenicriviroc, CD192, CCX872, CCX140, 2-((isopropylaminocarbonyl)amino)-N-(2-((cis-2-((4-(methylthio)benzoyl)amino)cyclohexyl)amino)-2-oxoethyl)-5-(trifluoromethyl)-benzamide, vicriviroc, SCH351125, TAK779, Teijin, RS-504393, Compound 2, Compound 14, or Compound 19 (Plos ONE 7(3):e32864).

[0112] In some embodiments, the therapeutic agent can include an agent that modulates autophagy, microautophagy, mitophagy, or other forms of autophagy. In some embodiments, the therapeutic agent can include sirolimus, tacrolimis, rapamycin, everolimus, bafilomycin, chloroquine, hydroxychloroquine, spautin-1, metformin, perifosine, resveratrol, trichostatin, valproic acid, Z-VAD-FMK, or others known in the art. Without wishing to be bound by theory, an agent that modulates autophagy, microautophagy, mitophagy, or other forms of autophagy can alter the recycling of intracellular components (such as, but not limited to, organelles, mitochondria, endoplasmic reticulum, lipids, or others). Without further wishing to be bound by theory, this agent may or may not act via microtubule-associated protein 1A / 1B light chain 3 (LC3).

[0113] In some embodiments, the therapeutic agent can include an agent used for the treatment of cancer (i.e., an anticancer drug or an antineoplastic agent). Exemplary anticancer drugs can be selected for a subject from among antimetabolite anticancer agents and antimitotic anticancer agents, as well as combinations thereof. A variety of antimetabolite anticancer agents and antimitotic anticancer agents (including a single such agent or a combination of such agents) can be used in the methods and compositions described herein.

[0114] Antimetabolite anticancer agents typically structurally resemble natural metabolites that are involved in normal metabolic processes of cancer cells (such as nucleic acid and protein synthesis). However, the antimetabolites are sufficiently different from the natural metabolites such that they interfere with the metabolic processes of cancer cells. In cells, the antimetabolites are mistaken for similar metabolites and are processed by the cells in a manner similar to normal compounds. The presence of the "decoy" metabolites prevents the cells from performing biological functions, and the cells do not proliferate and survive. For example, antimetabolites can exert cytotoxic activity by substituting these incorrect nucleotides into cellular DNA, thereby interfering with cell division, or by inhibiting important cellular enzymes and preventing DNA replication.

[0115] Thus, in one aspect, the antimetabolite anticancer agent is a nucleotide or a nucleotide analog. In certain embodiments, for example, the antimetabolite can include a purine (such as guanine or adenosine) or an analog thereof, or a pyrimidine (cytidine or thymidine) or an analog thereof, with or without an attached sugar moiety.

[0116] Suitable antimetabolite anticancer agents used in the present disclosure are generally classified according to the metabolic processes they affect, and such anticancer agents can include, but are not limited to, analogs and derivatives of folic acid, pyrimidine, purine, and cytidine. Thus, in one aspect, the antimetabolite(s) is selected from the group consisting of cytidine analogs, folic acid analogs, purine analogs, pyrimidine analogs, and combinations thereof.

[0117] In one particular embodiment, for example, the antimetabolite is a cytidine analog. According to this embodiment, for example, the cytidine analog can be selected from the group consisting of cytarabine (cytosine arabinodside), azacitidine (5-azacitidine), and salts, analogs, and derivatives thereof.

[0118] In another particular embodiment, for example, the antimetabolite is a folic acid analog. Folic acid analogs or antifolate substances generally function by inhibiting dihydrofolate reductase (DHFR), an enzyme involved in nucleotide formation. When this enzyme is blocked, nucleotides are not formed, interfering with DNA replication and cell division. According to a particular embodiment, for example, the folic acid analog can be selected from the group consisting of denopterin, methotrexate (amethopterin), pemetrexed, pteropterin, raltitrexed, trimetrexate, and salts, analogs, and derivatives thereof.

[0119] In another specific embodiment, for example, the antimetabolite is a purine analog. Purine-based antimetabolites function by inhibiting DNA synthesis (e.g., by interfering with the production of purine-containing nucleotides (adenine and guanine), and this interference interrupts DNA synthesis and cell division). Purine analogs are also incorporated into the DNA molecule itself during DNA synthesis, which can interfere with cell division. According to certain embodiments, for example, the purine analog can be selected from the group consisting of acyclovir, allopurinol, 2-aminoadenosine, arabinosyladenine (araA), azacitidine, azathiprine, 8-aza-adenosine, 8-fluoro-adenosine, 8-methoxy-adenosine, 8-oxo-adenosine, cladribine, deoxycoformycin, fludarabine, gancylovir, 8-aza-guanosine, 8-fluoro-guanosine, 8-methoxy-guanosine, 8-oxo-guanosine, guanosine diphosphate, guanosine diphosphate-β-L-2-amino fucose, guanosine diphosphate-D-arabinose, guanosine diphosphate-2-fluorofucose, guanosine diphosphate fucose, mercaptopurine (6-MP), pentostatin, thiampurine, thioguanine (6-TG), as well as salts, analogs, and derivatives thereof.

[0120] In yet another specific embodiment, for example, the antimetabolite is a pyrimidine analog. Similar to the purine analogs discussed above, pyrimidine-based antimetabolites block the synthesis of pyrimidine-containing nucleotides (cytosine and thymine in DNA; cytosine and uracil in RNA). By acting as a "decoy", pyrimidine-based compounds can prevent the production of nucleotides and / or be incorporated into growing DNA strands, leading to termination. According to certain embodiments, for example, the pyrimidine analog can be selected from the group consisting of ancitabine, azacitidine, 6-azauridine, bromouracil (e.g., 5-bromouracil), capecitabine, carmofur, chlorouracil (e.g., 5-chlorouracil), cytarabine (cytosine arabinoside), cytosine, didoxuridine, 3'-azido-3'-deoxythymidine, 3'-dideoxycytidine-2'-ene, 3'-deoxy-3'-deoxythymidine-2'-ene, dihydrouracil, doxifluridine, enocitabine, floxuridine, 5-fluorocytosine, 2-fluorodeoxycytidine, 3-fluoro-3'-deoxythymidine, fluorouracil (e.g., 5-fluorouracil (also known as 5-FU)), gemcitabine, 5-methylcytosine, 5-propynylcytosine, 5-propynylthymine, 5-propynyluracil, thymine, uracil, uridine, and salts, analogs, and derivatives thereof. In one embodiment, the pyrimidine analog is other than 5-fluorouracil. In another embodiment, the pyrimidine analog is gemcitabine or a salt thereof.

[0121] In certain embodiments, the antimetabolite is selected from the group consisting of 5-fluorouracil, capecitabine, 6-mercaptopurine, methotrexate, gemcitabine, cytarabine, fludarabine, pemetrexed, and salts, analogs, derivatives, and combinations thereof. In other embodiments, the antimetabolite is selected from the group consisting of capecitabine, 6-mercaptopurine, methotrexate, gemcitabine, cytarabine, fludarabine, pemetrexed, and salts, analogs, derivatives, and combinations thereof. In one particular embodiment, the antimetabolite is other than 5-fluorouracil. In a particularly preferred embodiment, the antimetabolite is gemcitabine or a salt thereof (e.g., gemcitabine HCl (Gemzar®)).

[0122] Other antimetabolite anticancer agents can be selected from the group consisting of, but not limited to, acanthifolic acid, aminothiadiazole, brequinar sodium, Ciba-Geigy CGP-30694, cyclopentylcytosine, cytarabine phosphate stearate, cytarabine conjugate, Lilly DATHF, Merrel Dow DDFC, desaguanine, didox, Yoshitomi DMDC, Wellcome EHNA, Merck & Co. EX-015, fazarabine, fludarabine phosphate, N-(2'-furanyl)-5-fluorouracil, Daiichi Seiyaku FO-152, 5-FU-fibrinogen, isopropylpyrrolidine, Lilly LY-188011; in particular, Lilly LY-264618, metobenzaprim, Wellcome MZPES, norspermidine, NCI NSC-127716, NCI NSC-264880, NCI NSC-39661, NCI NSC-612567, Warner-Lambert PALA, pentostatin, pirimtrexate, plicamycin, Asahi Chemical PL-AC, Takeda TAC-788, thiazofurin, Erbamont TIF, tyrosine kinase inhibitors, Taiho UFT, and uricytin.

[0123] In one aspect, the anti-mitotic agent is a microtubule inhibitor or a microtubule stabilizer. Generally, microtubule stabilizers (such as taxanes and epothilones) bind to the inner surface of the β - microtubule chain, promoting the nucleation and elongation phases of the polymerization reaction and reducing the critical concentration of tubulin subunits required for microtubule assembly, thereby promoting microtubule assembly. Different from microtubule inhibitors (such as vinca alkaloids) that prevent microtubule assembly, microtubule stabilizers (such as taxanes) reduce the lag time and dramatically shift the dynamic equilibrium between tubulin dimers and microtubule polymers towards polymerization. Thus, in one aspect, the microtubule stabilizer is a taxane or an epothilone. In another aspect, the microtubule inhibitor is a vinca alkaloid.

[0124] In some embodiments, the therapeutic agent may include a taxane or a derivative or analog thereof. The taxane can be a naturally occurring compound or related form, or a chemically synthesized compound or its derivative having anti-neoplastic properties. Taxanes are a family of terpenes (including but not limited to paclitaxel (Taxol®) and docetaxel (Taxotere®)), which are mainly derived from the Pacific yew tree (Taxus brevifolia) and have activity against certain tumors (especially breast tumors, ovarian tumors). In one aspect, the taxane is docetaxel or paclitaxel. Paclitaxel is a preferred taxane and is considered an anti-mitotic agent that enhances the assembly of microtubules from tubulin dimers and stabilizes microtubules by preventing depolymerization. This stability results in the inhibition of the normal dynamic reorganization of the microtubule network essential for interphase and mitotic cell functions related to life.

[0125] Also included are various known taxane derivatives that contain both hydrophilic and hydrophobic derivatives. Taxane derivatives include the galactose and mannose derivatives described in International Patent Application No. WO99 / 18113; the piperazino derivatives and other derivatives described in WO99 / 14209; the taxane derivatives described in WO99 / 09021, WO98 / 22451, and U.S. Patent No. 5,869,680; the 6-thio derivatives described in WO98 / 28288; the sulfenamide derivatives described in U.S. Patent No. 5,821,263; deoxygenated paclitaxel compounds (such as those described in U.S. Patent No. 5,440,056); and the taxol derivatives described in U.S. Patent No. 5,415,869, but are not limited thereto. As pointed out above, it further includes prodrugs of paclitaxel, including but not limited to those described in WO98 / 58927; WO98 / 13059; and U.S. Patent No. 5,824,701. A taxane can also be a taxane conjugate (e.g., paclitaxel-PEG, paclitaxel-dextran, paclitaxel-xylose, docetaxel-PEG, docetaxel-dextran, docetaxel-xylose, and the like). Other derivatives are also mentioned in other references, such as in “Synthesis and Anticancer Activity of Taxol Derivatives,” D.G.I. Kingston et al., Studies in Organic Chemistry, vol. 26, in the title “New Trends in Natural Products Chemistry” (1986), Atta-ur-Rabman, P.W. le Quesne, Eds. (Elsevier, Amsterdam 1986). Each of these references is hereby incorporated by reference in its entirety into this specification.

[0126] Various taxanes can be readily prepared using techniques known to those of ordinary skill in the art (see WO94 / 07882, WO94 / 07881, WO94 / 07880, WO94 / 07876, WO93 / 23555, WO93 / 10076; U.S. Patent Nos. 5,294,637; 5,283,253; 5,279,949; 5,274,137; 5,202,448; 5,200,534; 5,229,529; and EP590,267, each of which is incorporated herein by reference in its entirety) or can be obtained from various commercial sources (including, for example, Sigma-Aldrich Co., St. Louis, Mo).

[0127] Alternatively, the antimitotic agent can be a microtubule inhibitor. In one preferred embodiment, the microtubule inhibitor is a vinca alkaloid. Generally, vinca alkaloids are mitotic spindle poisons. Vinca alkaloid agents act during mitosis when the chromosomes are being distributed and begin to move along the tubules of the mitotic spindle toward one of the poles, prior to cell separation. Under the action of these spindle poisons, the spindle becomes disorganized by the dispersion of the chromosomes during mitosis, affecting cell proliferation. According to certain embodiments, for example, the vinca alkaloid is selected from the group consisting of vinblastine, vincristine, vindesine, and vinorelbine, and salts, analogs, and derivatives thereof.

[0128] The antimitotic agent can also be an epothilone. Generally, members of the epothilone class of compounds stabilize microtubule function according to mechanisms similar to those of taxanes. Epothilones also cause cell cycle arrest at the G2-M transition phase and can lead to cytotoxicity and ultimately apoptosis. Suitable epithiolones include epothilone A, epothilone B, epothilone C, epothilone D, epothilone E, and epothilone F, and salts, analogs, and derivatives thereof. One particular epothilone analog is ixabepilone (Ixempra™), an epothilone B analog.

[0129] In certain embodiments, the anti-mitotic anti-cancer agent is selected from the group consisting of taxanes, epothilones, vinca alkaloids, and salts and combinations thereof. Thus, for example, in one embodiment, the anti-mitotic agent is a taxane. More preferably, in this embodiment, the anti-mitotic agent is paclitaxel or docetaxel, even more preferably paclitaxel. In another embodiment, the anti-mitotic agent is an epothilone (e.g., an epothilone B analog). In another embodiment, the anti-mitotic agent is a vinca alkaloid.

[0130] Examples of anti-cancer drugs that can be used in the present disclosure include, but are not limited to, thalidomide; platinum coordination complexes (such as cisplatin (cis-DDP), oxaliplatin, and carboplatin); anthraquinones (such as mitoxantrone); substituted ureas (such as hydroxyurea); methylhydrazine derivatives (such as procarbazine (N-methylhydrazine, MIH)); adrenocortical suppressants (such as mitotane (o,p’-DDD) and aminoglutethimide); RXR agonists (such as bexarotene); and tyrosine kinase inhibitors (such as sunitinib and imatinib). Examples of additional anti-cancer drugs include alkylating agents, antimetabolites, natural products, hormones and antagonists, and various agents. Alternative names are shown in parentheses. Examples of alkylating agents include nitrogen mustards [mechlorethamine, cyclophosphainide, ifosfamide, melphalan (sarcolysin), and chlorambucil, etc.]; ethyleneimines and methylmelamines [hexamethylmelamine and thiotepa, etc.]; alkyl sulfonates [busulfan, etc.]; nitrosoureas [carmustine (BCNU), semustine (methyl-CCNU), lomustine (CCNU), and streptozocin (streptozotocin), etc.]; DNA synthesis antagonists [estramustine phosphate, etc.]; and triazines [dacarbazine (DTIC, dimethyl-triazenoimidazole carboxamide), and temozolomide, etc.]. Examples of antimetabolites include folic acid analogs [methotrexate (amethopterin), etc.]; pyrimidine analogs [fluorouracin (5-fluorouracil, 5-FU, SFU), floxuridine (fluorodeoxyuridine, FUdR), cytarabine (cytosine arabinoside), and gemcitabine, etc.]; purine analogs [mercaptopurine (6-mercaptopurine, 6-MP), thioguanine (6-thioguanine, TG) and pentostatin (2’-deoxycoformycin, deoxycoformycin), cladribine and fludarabine, etc.]; and topoisomerase inhibitors [amsacrine, etc.].Examples of natural products include vinca alkaloids [such as vinblastine (VLB) and vincristine]; taxanes [such as paclitaxel, protein-bound paclitaxel (Abraxane), and docetaxel (Taxotere)]; epipodophyllotoxins [such as etoposide and teniposide]; camptothecins [such as topotecan and irinotecan]; antibiotics [such as dactinomycin (actinomycin D), daunorubicin (daunomycin, rubidomycin), doxorubicin, bleomycin, mitomycin (mitomycin C), idarubicin, epirubicin]; enzymes [such as L-asparaginase]; and biological response modifiers [such as interferon α and interleukin 2]. Examples of hormones and antagonists include luteinizing releasing hormone agonists [such as buserelin]; corticosteroids [such as prednisone and related preparations]; progestins [such as hydroxyprogesterone caproate, medroxyprogesterone acetate, and megestrol acetate]; estrogens [such as diethylstilbestrol and ethinyl estradiol and related preparations]; estrogen antagonists [such as tamoxifen and anastrozole]; androgens [such as testosterone propionate and fluoxymesterone and related preparations]; androgen antagonists [such as flutamide and bicalutamide]; and gonadotropin releasing hormone analogs [such as leuprolide]. Alternative names and trade names for these and additional examples of anticancer drugs, as well as their methods of use including dosing and administration regimens, will be known to those of ordinary skill in the art.

[0131] In some embodiments, the anti-cancer agent may include a chemotherapeutic agent. Suitable chemotherapeutic agents include alkylating agents, antibiotics, antimetabolites, hormonal agents, plant-derived agents and their synthetic derivatives, anti-angiogenic agents, differentiation-inducing agents, cell growth arrest-inducing agents, apoptosis-inducing agents, cytotoxic agents, agents that affect cellular bioenergetics (i.e., affect cellular ATP levels and the molecules / activities that regulate these levels), biological agents (e.g., monoclonal antibodies), kinase inhibitors, as well as inhibitors of growth factors and their receptors, gene therapy agents, cell therapy (e.g., stem cells), or any combination thereof, but are not limited thereto.

[0132] According to these embodiments, the chemotherapeutic agent is selected from the group consisting of cyclophosphamide, chlorambucil, melphalan, mechlorethamine, ifosfamide, busulfan, lomustine, streptozocin, temozolomide, dacarbazine, cisplatin, carboplatin, oxaliplatin, procarbazine, uracil mustard, methotrexate, pemetrexed, fludarabine, cytarabine, fluorouracil, floxuridine, gemcitabine, capecitabine, vinblastine, vincristine, vinorelbine, etoposide, paclitaxel, docetaxel, doxorubicin, daunorubicin, epirubicin, idarubicin, mitoxantrone, bleomycin, mitomycin, hydroxyurea, topotecan, irinotecan, amsacrine, teniposide, erlotinib hydrochloride, and combinations thereof. Each possibility represents a separate embodiment of the invention.

[0133] According to certain embodiments, the therapeutic agent may include a biological drug (particularly an antibody). According to some embodiments, the antibody is selected from the group consisting of cetuximab, anti-CD24 antibody, panitumumab, and bevacizumab.

[0134] When used in the present disclosure, the therapeutic agent can include peptides, proteins (such as hormones, enzymes, antibodies, monoclonal antibodies, antibody fragments, monoclonal antibody fragments, and the like), nucleic acids (aptamers, siRNA, DNA, RNA, antisense nucleic acids or the like, antisense nucleic acid analogs or the like), low molecular weight compounds or high molecular weight compounds, receptor agonists, receptor antagonists, partial receptor agonists, and partial receptor antagonists.

[0135] Additional representative therapeutic agents can include, but are not limited to, peptide drugs, protein drugs, desensitizing agents, antigens, factors, growth factors, anti-infective agents (such as antibiotics, antimicrobials, antiviral substances, antibacterial substances, antiparasitic substances, antifungal substances, and combinations thereof), anti-allergic substances, steroids, androgenic steroids, decongestants, hypnotics, steroidal anti-inflammatory agents, anticholinergic substances, sympathomimetic substances, sedatives, miotics, psychostimulants, tranquilizers, vaccines, estrogens, progesterone agents, humoral agents, prostaglandins, analgesics, anti-seizure substances, anti-malarial substances, anti-histamine substances, cardiac agents, non-steroidal anti-inflammatory agents, anti-Parkinson's disease agents, anti-Alzheimer's disease agents, antihypertensive agents, β-adrenergic blockers, α-adrenergic blockers, nutrients, and benzophenanthridine alkaloids. The therapeutic agent can further be a substance that can act as a stimulant, sedative, hypnotic, analgesic, anti-convulsant, and the like.

[0136] The additional therapeutic agents may include CNS-active drugs, neuroactive drugs, anti-inflammatory and anti-inflammatory drugs, renal and cardiovascular drugs, gastrointestinal drugs, anti-neoplastic substances, immunomodulatory substances, immunosuppressive substances, hematopoietic agents, growth factors, anticoagulants, thrombolytic substances, antiplatelet agents, hormones, hormone activators, hormone antagonists, vitamins, ophthalmic agents, anabolic agents, antacids, anti-asthmatic agents, anti-cholesterol and anti-lipid agents, anticonvulsants, anti-diarrheal substances, anti-emetic substances, anti-manic agents, anti-metabolites, anti-emetic substances, anti-obesity agents, anti-pyretic and analgesic agents, anti-spasmodic agents, anti-thrombotic agents, anti-tussive agents, anti-hyperuricemic agents, anti-anginal agents, anti-histamine substances, appetite suppressants, biological agents, cerebral dilators, coronary dilators, bronchodilators, cytotoxic agents, congestion removers, diuretics, diagnostic agents, erythropoietic agents, expectorants, gastrointestinal sedatives, hyperglycemic agents, hypnotics, hypoglycemic agents, laxatives, mineral supplements, mucolytics, neuromuscular drugs, peripheral vasodilators, psychotropic substances, stimulants, thyroid and anti-thyroid agents, tissue growth agents, uterine relaxants, vitamins, antigenic materials, etc. Other classes of therapeutic agents include those cited in Goodman & Gilman’s The Pharmacological Basis of Therapeutics (McGraw Hill), and therapeutic agents included in Merck Index and The Physicians’ Desk Reference (Thompson Healthcare).

[0137] Other therapeutic agents include androgen inhibitors, polysaccharides, growth factors (e.g., vascular endothelial growth factor - VEGF), hormones, anti - angiogenesis inducers, dextromethorphan, dextromethorphan hydrobromide, noscapine, carbetapentane citrate, clofedanol hydrochloride, chlorpheniramine maleate, phenindamine tartrate, pyrilamine maleate, doxylamine succinate, phenyltoloxamine citrate, phenylephrine hydrochloride, phenylpropanolamine hydrochloride, pseudoephedrine hydrochloride, ephedrine, codeine phosphate, codeine sulfate morphine, mineral supplements, cholestryramine, N - acetylprocainamide, acetaminophen, aspirin, ibuprofen, phenylpropanolamine hydrochloride, caffeine, guaifenesin, aluminum hydroxide, magnesium hydroxide, peptides, polypeptides, proteins, amino acids, hormones, interferons, cytokines, and vaccines.

[0138] Further examples of therapeutic agents include peptide drugs, protein drugs, desensitizing agents, antigens, anti - infectious agents (antibiotics, antimicrobials, antiviral substances, antibacterial substances, antiparasitic substances, antifungal substances, and combinations thereof, etc.), anti - allergic substances, androgenic steroids, decongestants, hypnotics, steroidal anti - inflammatory agents, anticholinergic substances, sympathomimetic substances, sedatives, miotics, psychostimulants, tranquilizers, vaccines, estrogens, progesterone agents, humoral agents, prostaglandins, analgesics, anti - convulsants, antimalarial substances, anti - histamine substances, antiproliferative agents, anti - VEGF agents, cardiac agents, non - steroidal anti - inflammatory agents, anti - Parkinson's disease agents, anti - Alzheimer's disease agents, antihypertensive agents, β - adrenergic blocking agents, nutrients, and benzophenanthridine alkaloids, but are not limited thereto. The agents can further be stimulants, sedatives, hypnotics, analgesics, anticonvulsants, and substances that can act as such.

[0139] Further representative therapeutic agents include analgesics (such as acetaminophen, acetylsalicylic acid, and the like); anesthetics (such as lidocaine, xylocaine, and the like); anorectics (such as dextroamphetamine, phentermine tartrate, and the like); anti-arthritis agents (such as methylprednisolone, ibuprofen, and the like); anti-asthma agents (such as terbutaline sulfate, theophylline, ephedrine, and the like); antibiotics (such as sulfisoxazole, penicillin G, ampicillin, cephalosporin, amikacin, gentamicin, tetracycline, chloramphenicol, erythromycin, clindamycin, isoniazid, rifampin, and the like); antifungal agents (such as amphotericin B, nystatin, ketoconazole, and the like); antiviral agents (such as acyclovir, amantadine, and the like); anti-cancer agents (such as cyclophosphamide, methotrexate, etretinate, paclitaxel, taxol, and the like); anticoagulants (such as heparin, warfarin, and the like); anti-convulsants (such as phenyloin sodium, diazepam, and the like); antidepressants (such as isocarboxazid, amoxapine, and the like); antihistamines (such as diphenhydramine HCl, chlorpheniramine maleate, and the like); hormones (such as insulin, progestin, estrogen, corticosteroid, glucocorticoid, androgen, and the like); tranquilizers (such as thorazine, diazepam, chlorpromazine HCl, reserpine, chlordiazepoxide HCl, and the like); anti-emetics (such as belladonna alkaloids, dicyclomine hydrochloride, and the like); vitamins and minerals (such as essential amino acids, calcium, iron, potassium, zinc, vitamin B12, and the like); cardiovascular agents (such as prazosin HCl, nitroglycerin, propranolol HCl, hydralazine HCl, pancreatin, succinate dehydrogenase, and the like);Peptides and proteins (LHRH, somatostatin, calcitonin, growth hormone, glucagon-like peptide, growth hormone releasing factor, angiotensin, FSH, EGF, bone morphogenetic protein (BMP), erythropoeitin (EPO), interferon, interleukin, collagen, fibrinogen, insulin, factor VIII, factor IX, Enbrel®, Rituxam®, Herceptin®, α-glucosidase, Cerazyme / Ceredose®, vasopressin, ACTH, human serum albumin, γ-globulin, structural proteins, blood product proteins, complex proteins, enzymes, antibodies, monoclonal antibodies, and the like); prostaglandins; nucleic acids; carbohydrates; fats; narcotics (morphine, codeine, and the like, psychotherapy, etc.); antimalarial substances, L-dopa, diuretics (furosemide, spironolactone, and the like, etc.); antiulcer drugs (rantidine HCl, cimetidine HCl, and the like, etc.) are mentioned but not limited thereto.;

[0140] Therapeutic agents include immunomodulatory substances (such as cytokines, interleukins, interferons, colony-stimulating factors, tumor necrosis factors, and the like); immunosuppressive substances (such as rapamycin, tacrolimus, and the like); allergens (such as cat dander, oak tree pollen, house dust mites, grass pollen, and the like); antigens of bacterial organisms (Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Streptococcus pyrogenes, Corynebacterium diphteriae, Listeria monocytogenes, Bacillus anthracis, Clostridium tetani, Clostridium botulinum, Clostridium perfringens, Neisseria meningitides, Neisseria gonorrhoeae, Streptococcus mutans, Pseudomonas aeruginosa, Salmonella typhi, Haemophilus parainfluenzae, Bordetella pertussis, Francisella tularensis, Yersinia pestis, Vibrio cholerae, Legionella pneumophila, Mycobacterium tuberculosis, Mycobacterium leprae, Treponema pallidum, Leptspirosis interrogans, Borrelia burgddorferi, Campylobacter jejuni, and the like).Antigens of viruses (such as variola virus, influenza A and B viruses, respiratory syncytial virus, parainfluenza virus, measles virus, human immunodeficiency virus, severe acute respiratory syndrome virus, varicella-zoster virus, herpes simplex virus types 1 and 2, cytomegalovirus, Epstein-Barr virus, rotavirus, rhinovirus, adenovirus, papillomavirus, poliovirus, mumps virus, rabies virus, rubella virus, coxsackievirus, equine encephalitis virus, Japanese encephalitis virus, yellow fever virus, Rift Valley fever virus, lymphocytic choriomeningitis virus, hepatitis B virus, and the like); antigens that may also be of such fungal organisms, protozoan organisms, and parasitic organisms (such as Cryptococcus neoformans, Histoplasma capsulatum, Candida albicans, Candida tropicalis, Nocardia asteroids, Rickettsia ricketsii, Rickettsia typhi, Mycoplasma pneumoniae, Chlamydia psittaci, Chlamydia trachomatis, Plasmodium falciparum, Trypanasoma brucei, Entamoeba histolytica, Toxoplasma gondii, Trichomonas vaginalis, Schistosoma mansoni, and the like). These antigens can be in the form of whole dead organisms, peptides, proteins, glycoproteins, carbohydrates, or combinations thereof.;

[0141] In a further specific embodiment, the therapeutic agent may include an antibiotic. Antibiotics include, for example, amikacin, gentamicin, kanamycin, neomycin, netilmicin, streptomycin, tobramycin, paromomycin, ansamycin, geldanamycin, herbimycin, carbacephems, loracarbef, carbapenems, ertapenem, doripenem, imipenem / cilastatin, meropenem, cephalosporins (first generation), cefadroxil, cefazolin, cefalotin, cefalothin, cephalexin, cephalosporins (second generation), cefaclor, cefamandole, cefoxitin, cefprozil, cefuroxime, cephalosporins (third generation), cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, cefibuten, ceftezoxime, ceftriaxone, cephalosporins (fourth generation), cefepime, cephalosporins (fifth generation), ceftobiprole, glycopeptides, teicoplanin, vancomycin, macrolides, azithromycin, clarithromycin, dirithromycin, erythromycin, Roxithromycin, troleandomycin, telithromycin, spectinomycin, monobactams, aztreonam, penicillins, amoxicillin, ampicillin, azlocillin, carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, mezlocillin, meticillin, nafcillin, oxacillin, penicillin, piperacillin, ticarcillin, polypeptides, bacitracin, colistin, polymyxin B, quinolones, ciprofloxacin, enoxacin, gatifloxacin, levofloxacin, lomefloxacin, moxifloxacin, norfloxacin, ofloxacin, trovafloxacin, sulfonamides, mafenide, prontosil (classical), sulfacetamide, sulfamethizole, sulfanilimide (classical), sulfasalazine, sulfisoxazole, trimethoprim, trimethoprim-sulfamethoxazole (co-trimoxazole) (TMP-SMX), tetracyclines (demeclocycline, doxycycline, minocycline,Examples include oxytetracycline, tetracycline, and others); arsphenamine, chloramphenicol, clindamycin, lincomycin, ethambutol, fosfomycin, fusidic acid, furazolidone, isoniazid, linezolid, metronidazole, mupirocin, nitrofurantoin, platensimycin, pyrazinamide, quinupristin / dalfopristin, rifapentine (rifampin in the United States), timidazole, or one or more combinations thereof. In one aspect, the therapeutic agent can be a combination of rifapentine (rifampin in the United States) and minocycline.,

[0142] Growth factors useful as therapeutic agents include transforming growth factor-α (“TGF-α”), transforming growth factor (“TGF-β”), platelet-derived growth factor (“PDGF”), fibroblast growth factor (“FGF”) (including FGF acidic isoforms 1 and 2, FGF basic type 2, and FGF 4, 8, 9, and 10), nerve growth factor (“NGF”) (including NGF 2.5, NGF 7.0 and βNGF and neurotrophins, brain-derived neurotrophic factor), cartilage-derived factor, bone growth factor (BGF), basic fibroblast growth factor, insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), granulocyte colony-stimulating factor (G-CSF), insulin-like growth factor (IGF) I and II, hepatocyte growth factor, glial neurotrophic growth factor (GDNF), stem cell factor (SCF), keratinocyte growth factor (KGF), transforming growth factor (TGF) (including TGFα, β, β1, β2, β3), skeletal growth factor, bone matrix-derived growth factor, and bone-derived growth factor, and mixtures thereof, but are not limited thereto.

[0143] Cytokines useful as therapeutic agents include, but are not limited to, cardiotrophin, stromal cell-derived factor, macrophage-derived chemokine (MDC), melanoma growth-stimulating activity (MGSA), macrophage inflammatory protein 1α (MIP-1α), 2, 3α, 3β, 4, and 5, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, TNF-α, and TNF-β. Immunoglobulins useful in the present disclosure include, but are not limited to, IgG, IgA, IgM, IgD, IgE, and mixtures thereof. Some preferred growth factors include VEGF (vascular endothelial growth factor), NGF (nerve growth factor), PDGF-AA, PDGF-BB, PDGF-AB, FGFb, FGFa, and BGF.

[0144] Other molecules useful as therapeutic agents include, but are not limited to, growth hormone, leptin, leukemia inhibitory factor (LIF), tumor necrosis factor α and β, endostatin, thrombospondin, osteogenic protein-1, osteogenic proteins 2 and 7, osteonectin, somatomedin-like peptide, osteocalcin, interferon α, interferon αA, interferon β, interferon γ, interferon 1α, and interleukins 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, and 18.

[0145] In some embodiments, the therapeutic agent is present in the disclosed drug delivery composition in an amount of about 10, about 20, about 30, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, about 300, about 310, about 320, about 330, about 340, about 350, about 360, about 370, about 380, about 390, about 400, about 410, about 420, about 430, about 440, about 450, about 460, about 470, about 480, about 490, or about 500 (μg of therapeutic agent per mg of body weight of the disclosed drug delivery composition); or in a range of amounts of the therapeutic agent encompassed by any of the foregoing values; or in any combination of the foregoing values.

[0146] In some embodiments, the therapeutic agent exhibits a substantially zero-order release kinetics over a period of at least 30 days (e.g., 45 days, 60 days, 3 months, 6 months, 9 months, 1 year, or more). The therapeutic agent may exhibit substantially zero-order release kinetics at the time of implantation of the drug delivery composition or at a later period (e.g., the therapeutic agent may begin to exhibit substantially zero-order release kinetics about 30 days after implantation of the drug delivery composition). In other embodiments, the drug delivery composition may exhibit a kinetics that deviates from zero-order kinetics.

[0147] Method for preparing the disclosed drug delivery composition In various aspects, the disclosed drug delivery devices are prepared by the methods disclosed hereinbelow and as described in certain aspects of the following representative examples.

[0148] Thus, in one aspect, a method for preparing a drug delivery device described herein comprises forming a first layer comprising a first polymer on a conductive rod; and forming a second layer comprising a second polymer on the first layer.

[0149] In some embodiments, the formation of the first layer includes electrospinning using a solution of a first polymer and a voltage difference of about 10 kV to about 30 kV.

[0150] In some embodiments, the first polymer solution is about 1 w / v% to about 10 w / v% in at least one organic solvent. In some embodiments, at least one organic solvent in the first polymer solution includes trifluoroacetic acid, dichloromethane, hexafluoroisopropanol, or a combination thereof. In some embodiments, trifluoroacetic acid and dichloromethane are present in a ratio of about 1:10 to about 10:1 (e.g., a ratio of about 5:3 to about 10:3). In some embodiments, trifluoroacetic acid and dichloromethane are present in a ratio of about 7:3.

[0151] In some embodiments, the formation of the second layer includes electrospinning onto the formed first layer using a solution comprising a second polymer and optionally a porogen, and the voltage difference used for electrospinning is about 20 kV to about 30 kV.

[0152] In some embodiments, the solution comprising the second polymer and optionally a porogen is about 1 w / v% to about 10 w / v% (e.g., about 2.5 w / v% to about 10 w / v% or about 5 w / v% to about 10 w / v%) of the second polymer and porogen, based on the total weight. In some embodiments, the solution comprising the second polymer and porogen is a 1,1,1,3,3,3-hexafluoropropan-2-ol solution.

[0153] In some embodiments, the weight ratio of the second polymer to the porogen is from about 90:100 to about 100:1 (e.g., from about 90:100 to 99.9:0.1, from about 90:100 to 95:5, or from about 95:5 to 99.9:0.1). In some embodiments, the weight ratio of the second polymer to the porogen is about 99:1, about 95:5, about 92.5:7.5, or about 90:10. In some embodiments, the weight ratio of the second polymer to the porogen ranges from about 50:50 to about 100:0.

[0154] "Porogen", as used herein, refers to any material that can be used to produce a porous material (e.g., porous polycaprolactone as described herein). In some embodiments, the porogen comprises a water-soluble compound, i.e., such that when the drug delivery device is washed with water, the porogen is substantially removed from the outer layer. In some embodiments, the porogen comprises a compound selected from ([Tris(hydroxymethyl)methylaminopropanesulfonic acid])(TAPS), (2-(Bis(2-hydroxyethyl)amino)acetic acid)(Bicine), (Tris(hydroxymethyl)aminomethane) or (2-Amino-2-(hydroxymethyl)propane-1,3-diol)(Tris), (N-[Tris(hydroxymethyl)methyl]glycine)(Tricine), (3-[N-Tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid)(TAPSO), (4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid)(HEPES), (2-[[1,3-Dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid)(TES), (3-(N-Morpholino)propanesulfonic acid)(MOPS), (Piperazine-N,N'-bis(2-ethanesulfonic acid))(PIPES), dimethylarsinic acid, (2-(N-Morpholino)ethanesulfonic acid)(MES), or a salt thereof (such as its sodium salt). In some embodiments, the porogen comprises the sodium salt of HEPES. In some embodiments, the porogen comprises a water-soluble polymer such as polyethylene glycol, polyoxyethylene copolymer, acrylate copolymer (including quaternary ammonium groups, polyacrylamide, polyvinyl alcohol, hyaluronic acid, and polyvinylpyrrolidone). In other embodiments, the porogen comprises gelatin, polyethylene glycol (PEG), chitosan, polyvinylpyrrolidone (PVP), polyvinyl alcohol, or agarose.

[0155] In some embodiments, the method further includes sintering the drug delivery device following the formation of the outer layer. In some embodiments, the sintering is conducted at a temperature of about 50°C to about 150°C (e.g., about 90°C to about 110°C). In some embodiments, the sintering includes heating for a period of about 1 minute to about 6 hours (e.g., about 30 minutes to about 6 hours).

[0156] In some embodiments, the method further includes washing the drug delivery device following the sintering. In some embodiments, the drug delivery device is washed with a saturated sodium bicarbonate solution followed by deionized water. In some embodiments, the porogen is substantially removed from the drug delivery device when washing with deionized water.

[0157] In some embodiments, the method further includes drying the drug delivery device following the washing. In some embodiments, the drying is under vacuum. In some embodiments, the drying is at a temperature of about 50°C to about 150°C (e.g., about 90°C to 110°C). In some embodiments, the drying occurs for a period of about 1 minute to about 6 hours (e.g., about 30 minutes to about 6 hours).

[0158] In other embodiments, the disclosed capsules can be manufactured by any suitable method as would be readily understood by one of ordinary skill in the art. In some embodiments, the disclosed capsules can be manufactured by asymmetric membrane formation. Representative examples of such methods are provided in Yen, C. et al. “Synthesis and characterization of nanoporous polycaprolactone membranes via thermally- and nonsolvent-induced phase separations for biomedical device application” Journal of Membrane Science 2009, 343:180-88 (which is hereby incorporated by reference in its entirety for all purposes). In some embodiments, the disclosed capsules can be manufactured using three-dimensional printing. In some embodiments, the disclosed capsules can be manufactured around methylcellulose, which can subsequently be removed to form the lumen compartment. In some embodiments, the disclosed capsules can be manufactured by the methods described by Envisia Therapeutics in WO2015 / 085251, WO2016 / 144832, WO2016 / 196365, WO2017 / 015604, WO2017 / 015616, or WO2017 / 015675 (each of which is hereby incorporated by reference in its entirety for all purposes). In yet other embodiments, the disclosed capsules can be manufactured by methods similar to those used in the manufacture of hollow fiber membranes (phase inversion such as nonsolvent-induced phase inversion (NIPS), (solvent) evaporation-induced phase inversion (EIPS), vapor adsorption-induced phase inversion (VIPS), and thermally-induced phase inversion method (TIPS)). In some embodiments, the disclosed capsules can be manufactured using methods similar to those described in US2015 / 232506 (which is hereby incorporated by reference in its entirety for all purposes). In some embodiments, the pores can, instead, be formed by laser diffraction of the capsules.

[0159] In some embodiments, the two tube-shaped ends of the capsule are closed. The ends can be closed by any sealing technique as appropriately selected by one of ordinary skill in the art. In some embodiments, the two ends are sealed using a high-frequency tube sealing technique. In such a technique, the high frequency generates eddy currents in the wall, which heats at least the polymer layer. When the temperature reaches the melting point of the polymer, the clamp is closed and the melted polymer is cooled and formed. In some embodiments, the two ends are sealed using hot-jaw tube sealing, where a heated jaw applies heat to the outside of the tube shape to heat the inside for sealing. In some embodiments, the two ends can be sealed using ultrasonic tube sealing. In such a technique, the polymer composition of the inner layer is heated and melted by the high-frequency frictional force introduced from an ultrasonic horn. The clamp is then closed around the section intended to be sealed, cooled, and formed to seal the ends. In some embodiments, the two ends are sealed using hot air sealing, where the system heats the seal area inside the capsule with hot air and then subsequently presses and cools the ends in a subsequent station.

[0160] Method of treatment using the disclosed drug delivery device Also provided herein is a method of treating a clinical condition by administration of the disclosed drug delivery composition. The clinical condition can be a clinical disorder, disease, dysfunction, or other pathological condition that can be alleviated by the therapeutic composition.

[0161] The term "administering" or "administration" to a subject of the disclosed drug delivery device encompasses any route by which the device is introduced or delivered to the subject to perform its intended function. Administration can be effected by any suitable route, including oral, intranasal, parenteral (intravenous, intramuscular, intraperitoneal, or subcutaneous), or topical. Administration includes self-administration and administration by another person. In some instances, administration is via injection into the eye (including intraocular injection). In other instances, e.g., in the treatment of cancer, administration can be via injection of the disclosed drug delivery composition into, in contact with, adjacent to, or proximal to a tumor or other mass of cancer cells.

[0162] It is intended that the various modes of treatment or prevention of the medical diseases and conditions described as "substantially" mean, which includes total and less than total treatment or prevention, and it will also be recognized that a certain degree of biologically or medically significant result is achieved. Treatment can be continuous extended treatment for chronic diseases, or single or multiple administrations for the treatment of acute conditions.

[0163] The term "separate" administration refers to the administration of at least two active ingredients by different routes at the same time or substantially the same time.

[0164] The term "sequential" administration refers to the administration of at least two active ingredients at different times, where the route of administration is the same or different. More particularly, sequential use refers to the whole of administering one of the active ingredients before the administration of the other(s) begins. Thus, it is possible to administer one of the active ingredients for minutes, hours, or days before the administration of the other active ingredient(s). Thus, the term "sequential" is different from "simultaneous" administration.

[0165] The term "simultaneous" administration refers to the administration of at least two active ingredients by the same route at the same time or substantially the same time.

[0166] As used herein, the term "therapeutic" means treatment and / or prophylaxis. A therapeutic effect is obtained by suppression, remission, or eradication of a disease state.

[0167] The present disclosure further provides a method of treating an ophthalmic disease or disorder by administering a therapeutically effective amount of a composition described herein. In some embodiments, the disclosed method involves treating an ophthalmic disorder, including injecting a therapeutically effective amount of the disclosed composition into the eye of a subject. The subject can be a patient who has been diagnosed with an ophthalmic disorder. In some aspects, the method can further include diagnosing the subject with an ophthalmic disorder.

[0168] Ophthalmic disorders include acute macular neuroretinopathy; Behcet's disease; angiogenesis (including choroidal angiogenesis); diabetic uveitis; histoplasmosis; infections (such as those caused by fungi or viruses); macular degeneration (including acute macular degeneration (AMD), exudative AMD, non-exudative AMD, and exudative AMD); edema (such as macular edema, cystoid macular edema, and diabetic macular edema); multifocal choroiditis; ocular trauma affecting posterior eye sites or locations; eye tumors; retinal disorders (such as central retinal vein occlusion, diabetic retinopathy (including proliferative diabetic retinopathy), proliferative vitreoretinopathy (PVR), retinal arterial occlusive diseases, retinal detachment, uveitis retinal diseases, etc.); sympathetic ophthalmia; Vogt Koyanagi-Harada (VKH) syndrome; choroidal exudation; posterior eye pathologies caused by or affected by eye laser treatment; posterior eye pathologies that can be caused by or affected by photodynamic therapy, photocoagulation, radiation retinopathy, epiretinal membrane disorders, branch retinal vein occlusion, anterior ischemic optic neuropathy, non-retinopathy diabetic retinal dysfunction, retinitis pigmentosa, cancer, and glaucoma. In certain examples, the ophthalmic disorder is exudative age-related macular degeneration (exudative AMD), cancer, angiogenesis, macular edema, or edema. In further specific aspects, the ophthalmic disorder is exudative age-related macular degeneration (exudative AMD).

[0169] In various aspects, an injection for the treatment of an ophthalmic disorder can be an injection into the vitreous chamber of the eye. In some cases, the injection is a vitreous injection, a subconjunctival injection, a sub-Tenon's injection, a retrobulbar injection, or a suprachoroidal injection.

[0170] The "ocular region" or "ocular site" means any region of the ocular globe (eyeball) that includes the anterior and posterior segments of the eye, and generally includes any functional (e.g., for vision) or structural tissue found within the eye, or a tissue or cell layer that partially or completely lines the interior or exterior of the eye, but is not limited thereto. Specific examples of regions of the ocular globe within the ocular region include, but are not limited to, the anterior chamber, the posterior chamber, the vitreous cavity, the choroid, the suprachoroidal space, the conjunctiva, the subconjunctival space, the episcleral space, the intracorneal space, the subretinal space, the sub-Tenon's space, the epicorneal space, the sclera, the pars plana, surgically induced avascular regions, the macula, and the retina.

[0171] "Ophthalmic disorder" can mean a disease, illness, or condition that affects or involves the eye, or a part or region of the eye. Broadly speaking, the eye includes the ocular globe (including the cornea), as well as other tissues and fluids that make up the ocular globe, the muscles around the eye (such as the oblique and rectus muscles), and portions of the optic nerve within or adjacent to the eye.

[0172] "Glaucoma" means primary glaucoma, secondary glaucoma, and / or congenital glaucoma. Primary glaucoma can include open-angle glaucoma and closed-angle glaucoma. Secondary glaucoma can occur as a complication of various other conditions (injuries, inflammation, pigment dispersion, vascular diseases, and diabetes, etc.). The increased intraocular pressure in glaucoma damages the optic nerve that enters the eye, causing blindness. Thus, in one non-limiting embodiment, by reducing reactive oxygen species, STC-1, or MSCs that express an increased amount of STC-1, are used in the treatment of glaucoma and can prevent or delay the onset of blindness.

[0173] As related to eye conditions, "inflammation-mediated" means any eye condition from which benefit can be obtained by treatment with anti-inflammatory agents, and is intended to include, but not be limited to, uveitis, macular edema, acute macular degeneration, retinal detachment, eye tumors, fungal or viral infections, multifocal choroiditis, diabetic retinopathy, uveitis, proliferative vitreoretinopathy (PVR), sympathetic ophthalmia, Vogt-Koyanagi-Harada (VKH) syndrome, histoplasmosis, and uveal effusion.

[0174] Regarding eye conditions, "injury" or "damage" are interchangeable and refer to the cellular and morphological signs and symptoms resulting from inflammation-mediated conditions (such as inflammation, etc.), tissue damage caused by means other than inflammation (such as chemical damage including chemical burns), and damage caused by infection (including but not limited to bacterial, viral, or fungal infections).

[0175] "Intraocular" means within or beneath the eye tissues. Examples of intraocular administration of a drug delivery system include administration of the drug delivery system to the sub-Tenon's space, subconjunctival, suprachoroidal, subretinal, vitreal, anterior chamber of the eye, and similar locations. Intraocular administration of a drug delivery system excludes administration of the drug delivery system to local, systemic, intramuscular, subcutaneous, intraperitoneal, and similar locations.

[0176] "Macular degeneration" refers to any of a number of disorders and pathologies in which the macula degenerates or loses its functional activity. The degeneration or loss of functional activity can result, for example, from cell death, decreased cell proliferation, loss of normal biological function, or a combination of the foregoing. Macular degeneration can lead to, and / or manifest as, a change in the structural integrity of the cells and / or extracellular matrix of the macula, a change in normal cell matrix construction and / or extracellular matrix construction, and / or a loss of function of macular cells. The cells can be any cell type normally present in or near the macula, including RPE cells, photoreceptors, and capillary endothelial cells. Age-related macular degeneration (or ARMD) is the primary macular degeneration-related pathology, although a number of others are known, including but not limited to Best macular degeneration, Stargardt macular degeneration, Sorsby fundus dystrophy, Mallatia Leventinese / Doyne honeycomb retinal dystrophy, and RPE pattern dystrophy. Age-related macular degeneration (AMD) is described as either "atrophic" or "exudative". The wet exudative neovascular form of AMD affects approximately 10-20% of those affected by AMD, is characterized by abnormal blood vessels that grow under or through the retinal pigment epithelium (RPE), and results in hemorrhage, exudation, scarring, or serous retinal detachment. Eighty to ninety percent of AMD patients are of the atrophic type, characterized by atrophy of the retinal pigment epithelium and loss of macular photoreceptors. Drusen may or may not be present in the macula. There can also be atrophy of areas of the retinal pigment epithelium in the macula that cause visual loss. At present, there is no cure for any form of AMD, although some success in attenuating exudative AMD has been achieved with photodynamic therapy and particularly anti-VEGF therapy.

[0177] Drusen are debris-like materials that accumulate with age under the RPE. Drusen are observed using fundus examination. A normal eye has a macula without drusen, although drusen may be abundant in the peripheral retina. The presence of soft drusen in the macula is judged as an early stage of AMD in the absence of any loss of macular vision. Drusen contain various lipids, polysaccharides, and glycosaminoglycans together with multiple proteins, modified proteins, or protein adducts. There is no generally accepted treatment method to address drusen formation and thereby manage the progressive nature of AMD.

[0178] "Ocular neovascularization" (ONV) is used herein to refer to choroidal neovascularization or retinal neovascularization, or both.

[0179] "Retinal neovascularization" (RNV) refers to, for example, abnormal occurrence, sprouting, and / or proliferation of retinal blood vessels on the retinal surface.

[0180] "Subretinal neovascularization" (SRNVM) refers to abnormal occurrence, sprouting, and / or proliferation of blood vessels under the surface of the retina.

[0181] "Cornea" refers to the transparent structure that forms the anterior part of the fibrous tunic of the eye. The cornea consists of five layers, specifically: 1) the anterior corneal epithelium (continuous with the conjunctiva); 2) the anterior limiting lamina (Bowman layer); 3) the substantia propria or stromal cell layer; 4) the posterior limiting lamina (Descemet membrane); and 5) the endothelium of the anterior chamber or corneal endothelium (keratoderma).

[0182] "Retina" refers to the innermost layer of the eye that surrounds the vitreous body and is continuous posteriorly with the optic nerve. The retina is composed of layers including: 1) the inner limiting membrane; 2) the nerve fiber layer; 3) the layer of ganglion cells; 4) the inner plexiform layer; 5) the inner nuclear layer; 6) the outer plexiform layer; 7) the outer nuclear layer; 8) the outer limiting membrane; 9) the layer containing the rod and cone layers.

[0183] "Retinal degeneration" refers to any hereditary or acquired degeneration of the retina and / or retinal pigment epithelium. Non-limiting examples include retinitis pigmentosa, Best disease, RPE pattern dystrophy, and age-related macular degeneration.

[0184] In various aspects, methods of treating ophthalmic disorders include treating various diseases or conditions of the retina of the eye, such diseases or conditions including the following: Macular disease / retinal degeneration: Macular degeneration (including age-related macular degeneration (ARMD) such as non-exudative age-related macular degeneration and exudative age-related macular degeneration); Choroidal neovascularization; Retinopathy (including diabetic retinopathy, acute macular neuroretinopathy and chronic macular neuroretinopathy, central serous chorioretinopathy); and Macular edema (including cystoid macular edema and diabetic macular edema); Uveitis / retinitis / choroiditis: Acute multifocal placoid pigment epitheliopathy, Behcet's disease, shotgun choroiditis, infectious (syphilis, Lyme disease, tuberculosis, toxoplasmosis), uveitis (including intermediate uveitis (pars planitis) and anterior uveitis), multifocal choroiditis, multiple evanescent white dot syndrome (MEWDS), ocular sarcoidosis, posterior scleritis, serpiginous choroiditis, subretinal fibrosis, uveitis syndrome, and Vogt-Koyanagi-Harada syndrome; Vascular diseases / exudative diseases: Retinal arterial occlusive diseases, central retinal vein occlusion, disseminated intravascular coagulation syndrome, branch retinal vein occlusion, hypertensive changes, ocular ischemia syndrome, retinal arteriolar microaneurysms, Coats' disease, parafoveal telangiectasia, hemi-central retinal vein occlusion, papillophlebitis, central retinal artery occlusion, branch retinal artery occlusion, carotid artery disease (CAD), vasospastic angitis, sickle cell retinopathy and other abnormal hemoglobinopathies, pigmentary streaks, familial exudative vitreoretinopathy, Eales' disease; Traumatic / surgical diseases: Sympathetic ophthalmia, uveitic retinal diseases, retinal detachment, trauma, laser, PDT, photocoagulation, decreased blood flow during surgery, radiation retinopathy, bone marrow transplant retinopathy; Proliferative disorders: Proliferative vitreoretinopathy and epiretinal membrane, proliferative diabetic retinopathy; Infectious disorders: Ocular histoplasmosis, ocular toxocariasis, ocular histoplasmosis syndrome (OHS), endophthalmitis, toxoplasmosis, retinal diseases associated with HIV infection, choroidal diseases associated with HIV infection, uveitis diseases associated with HIV infection, viral retinitis, acute retinal necrosis, progressive outer retinal necrosis, fungal retinal diseases, ocular syphilis, ocular tuberculosis, diffuse unilateral subacute neuroretinitis, and myiasis;Genetic disorders: retinitis pigmentosa, systemic disorders associated with retinal dystrophy, congenital stationary night blindness, cone dystrophy, Stargardt disease, macular fundus, Best disease, pattern dystrophy of the retinal pigment epithelium, X-linked retinal detachment, Sorsby fundus dystrophy, benign central macular dystrophy, Bietti crystalline dystrophy, elastic fibrovascular pseudoxanthoma; Retinal tears / retinal holes: retinal detachment, macular hole, giant retinal tear; Tumors: retinal diseases associated with tumors, congenital hypertrophy of the RPE, posterior uveal melanoma, choroidal hemangioma, choroidal osteoma, choroidal metastasis, retinal / retinal pigment epithelial hamartoma, retinoblastoma, vascular proliferative tumors of the fundus, retinal astrocytoma, intraocular lymphocytic tumors; Others: punctate inner choroidopathy, acute posterior multifocal placoid pigment epitheliopathy, myopic retinal degeneration, acute retinal pigment epitheliitis, and the like can be mentioned.;

[0185] Anterior eye diseases are diseases, illnesses, or conditions that affect or involve the area or site in the front part of the eye (i.e., the front of the eye), including the muscles around the eye, eyelids, or tissues or fluids in the eye located in front of the posterior wall of the lens capsule or ciliary muscle. Therefore, anterior eye diseases mainly affect or involve the conjunctiva, cornea, anterior chamber, iris, posterior chamber (behind the iris but in front of the posterior wall of the lens capsule), lens or lens capsule, and the blood vessels and nerves that vascularize or innervate the anterior eye area or site.;

[0186] Therefore, examples of anterior eye diseases can include aphakia; pseudophakia; astigmatism; blepharospasm; cataract; conjunctival diseases; conjunctivitis (including but not limited to atopic keratoconjunctivitis); corneal injury (including but not limited to injury to the corneal stromal region); corneal diseases; corneal ulcer; dry eye syndrome; eyelid diseases; lacrimal gland diseases; lacrimal duct obstruction; myopia; presbyopia; pupillary disorders; refractive disorders and strabismus and other diseases, illnesses, or conditions. Since the clinical goal of glaucoma treatment can be to reduce the high pressure of the aqueous humor in the anterior chamber (i.e., reduce intraocular pressure), glaucoma can be determined to be an anterior eye disease.;

[0187] Other eye diseases or disorders that can be treated according to the present invention include, but are not limited to, ocular cicatricial pemphigoid (OCP), Stevens Johnson syndrome, and cataracts.

[0188] Posterior segment diseases are diseases, illnesses, or conditions that mainly affect or involve the posterior segment region or site of the eye (choroid or sclera (posterior to the surface through the posterior wall of the lens capsule), vitreous, vitreous chamber, retina, optic nerve (i.e., optic nerve head), and blood vessels and nerves that vascularize or innervate the posterior segment region or site, etc.). Thus, examples of posterior segment diseases include, for example, acute macular neuroretinopathy; Behcet's disease; choroidal neovascularization; diabetic retinopathy; uveitis; ocular histoplasmosis; infections (such as infections caused by fungi or viruses); macular degeneration (such as acute macular degeneration, non-exudative age-related macular degeneration, and exudative age-related macular degeneration, etc.); edema (such as macular edema, cystoid macular edema, and diabetic macular edema, etc.); multifocal choroiditis; ocular trauma affecting the posterior segment site or location; eye tumors; retinal disorders (such as central retinal vein occlusion, diabetic retinopathy (including proliferative diabetic retinopathy), proliferative vitreoretinopathy (PVR), retinal arterial or venous occlusive diseases, retinal detachment, uveitis retinal diseases, etc.); sympathetic ophthalmia; Vogt-Koyanagi-Harada (VKH) syndrome; uveal effusion; posterior segment diseases caused by or affected by laser treatment of the eye; diseases, illnesses, or conditions such as posterior segment diseases caused by or affected by photodynamic therapy, photocoagulation, radiation retinopathy, epiretinal membrane disorders, retinal vein branch occlusion, anterior ischemic optic neuropathy, non-retinopathy diabetic retinal dysfunction, retinitis pigmentosa, and glaucoma. Glaucoma can be considered a posterior segment disease since the treatment goal can be to prevent or reduce the loss of vision (i.e., neuroprotection) due to damage or loss of retinal ganglion cells or retinal nerve fibers.

[0189] In some embodiments, the ophthalmic disorder is an ocular inflammation resulting from, for example, iritis, conjunctivitis, seasonal allergic conjunctivitis, acute and chronic endophthalmitis, anterior uveitis, uveitis associated with systemic disease, posterior uveitis, retinitis, pars planitis, masquerade syndromes (including ocular lymphoma), pemphigoid, scleritis, keratitis, severe ocular allergy, corneal detachment, and disruption of the blood-aqueous barrier. In yet another embodiment, the ophthalmic disorder is a postoperative ocular inflammation resulting from, for example, laser refractive keratectomy, cataract removal surgery, intraocular lens implantation, vitrectomy, corneal transplantation, forms of superficial keratectomy (such as DSEK), and radial keratotomy.

[0190] In various aspects, an injection for the treatment of an ophthalmic disorder can be an injection into the vitreous humor of the eye. In some cases, the injection is an intravitreal injection, a subconjunctival injection, a sub-Tenon's injection, a retrobulbar injection, or a suprachoroidal injection.

[0191] In various aspects, a method for the treatment of an ophthalmic disorder includes administration of a disclosed drug delivery device containing, for example, a therapeutic agent in an amount from about 0.01 mg to about 25 mg; or from about 1 mg to about 15 mg, via injection. In some embodiments, the drug delivery composition can release an amount of drug that maintains a concentration of from about 10 picomoles to about 500 picomoles in the vitreous humor of the eye over a period of from about 10 days to about 12 months. The amount of therapeutic substance in the drug delivery composition will depend on the amount of therapeutic agent that can be present in one or more capsules and the amount necessary to achieve the desired therapeutic effect.

[0192] In some embodiments, the disclosed drug delivery may protect the bioactivity of the encapsulated therapeutic substance over a period of up to 12 months. The level of protection of bioactivity will depend on both the therapeutic agent used and the selected composition of the disclosed capsules, but can be quantified by HPLC (for determination of the amount and form of the drug present in the eye), a cellular assay of activity against a positive control (such as use of the therapeutic agent alone), and methods such as ELISA that characterize the form of other therapeutic substances or assay changes in biological activity (such as transcription factor expression).

[0193] Kit The present disclosure also relates to a kit comprising (a) a drug delivery composition described herein; (b) a drug delivery composition described herein in a sterile package; or (c) one of a pre-filled syringe or needle containing a drug delivery composition described herein; and instructions for administering a drug delivery composition described herein for treating a clinical condition or pathology.

[0194] In a further aspect, the disclosed kit may be packaged in a daily dosing regimen (e.g., packaged on a card, packaged with a dosing card, packaged on a blister or blow-molded plastic, etc.). Such packaging improves the product and increases ease of use for administration by healthcare professionals. Such packaging can also reduce potential medical errors. The present invention also features such kits that further contain instructions for use.

[0195] In a further aspect, the present disclosure also provides a pharmaceutical pack or kit comprising one or more packages containing a disclosed drug delivery composition. What is associated with such a package can be a notice in a form prescribed by a government agency that regulates the manufacture, use, or sale of pharmaceutical or biological products, and the notice reflects approval by an office for manufacture, use, or sale for human administration.

[0196] In various aspects, the disclosed kits may further include therapeutic agents, compounds, and / or products that are co-packaged, co-formulated, and / or co-delivered with other components. For example, a drug manufacturer, drug reseller, physician, compounding facility, or pharmacist can provide a kit that includes the disclosed drug delivery composition and another component for delivery to a patient.

[0197] The disclosed kits are intended to be used in connection with the disclosed methods of making the disclosed compositions, the disclosed methods of using or treating the disclosed compositions, and / or the disclosed compositions.

[0198] From the foregoing, it will be understood that the aspects herein are well adapted to attain all of the ends and objects above described, together with other advantages that are obvious and inherent to the structure, and are well suited to be combined with other advantages.

[0199] While certain elements and steps are considered in relation to each other, any element and / or step provided herein is contemplated to be combinable with other elements and / or steps, without regard to the explicit provision of that same combination (which is still within the scope provided herein).

[0200] It will be understood that certain characteristics and partial combinations are beneficial and can be used without regard to other characteristics and partial combinations. This is contemplated by the claims and is within their scope.

[0201] It will be understood that all matters set forth in the accompanying drawings and the description herein, which are shown or described in connection with the forms of carrying out the invention, are to be construed in an illustrative, and not a limiting, sense, as many possible aspects can be made without departing from the scope thereof.

[0202] It should also be understood that the terms used in this specification are for the purpose of describing particular embodiments only and are not intended to be limiting. Those skilled in the art will recognize many variations and adaptations of the embodiments described herein. These variations and adaptations are included within the teachings of this disclosure and are intended to be covered by the claims of this specification.

[0203] Aspects of the present disclosure have been described, and the following examples generally describe some additional aspects of the present disclosure. The aspects of the present disclosure are described in relation to the following examples as well as the corresponding text and figures, but it is not intended to limit the aspects of the present disclosure to this description. On the contrary, it is intended to cover all alternatives, modifications, and equivalents that are included within the spirit and scope of the present disclosure.

Examples

[0204] The following examples are set forth to provide those skilled in the art with a complete disclosure and description of how the compounds, compositions, products, devices, and / or methods claimed in this specification are made and evaluated, and are intended as a pure illustration of the present disclosure and are not intended to limit the scope that the inventors regard as their disclosure. Efforts have been made to ensure accuracy with respect to numerical values (e.g., amounts, temperatures, etc.), but some error and deviation will be present. Unless otherwise indicated, parts are by weight, temperature is in °C or at ambient temperature, and pressure is at or near atmospheric pressure.

[0205] Materials Chitosan (DD>75%, Mw 310,000 - 375,000 Da), polycaprolactone (Mn 80,000), trifluoroacetic acid (TFA), 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) sodium salt, and Tween 20 were purchased from Sigma-Aldrich Inc. (St. Louis, MO). 1,1,1,3,3,3-Hexafluoro-2-propanol (HFP) was purchased from Oakwood Products Inc. (Estill, SC). Dichloromethane (DCM), chromatographically purified bovine serum albumin (BSA), and VEGF recombinant human protein were purchased from Fisher Scientific International Inc. (Hampton (NH)). Bevacizumab (Avastin) was purchased from Genentech, Inc. (San Francisco, CA). Bicinchoninic acid (BCA) protein assay kit and colorimetric quantitative 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay, horseradish peroxidase (HRP) conjugated goat anti-human immunoglobulin G (IgG) crystalline fragment (Fc) secondary antibody, and 3,3’,5,5’-tetramethylbenzidine (TMB) were purchased from Thermo Fisher Scientific Inc. (Waltham, MA). Human retinal pigment epithelial cell line (ARPE-19 cells, CRL2302) and DMEM:F-12 medium were purchased from American Type Culture Collection (Rockville, MD). Human umbilical vein endothelial cells (HUVEC), medium 200PRF and low serum growth supplement and lactose dehydrogenase elevated virus (LDEV) free growth factor reduced basement membrane matrix were purchased from Thermo Fisher Scientific Inc. (Waltha, MA). All other reagents used were of analytical grade.

[0206] Capsule preparation Two sizes of capsules with different inner diameters (260 μm and 1645 μm) were fabricated in this study. The 1.645 mm-sized capsules were mainly used as a preliminary model for the smaller capsules to optimize the processing conditions. The 260-μm-sized capsules were used for subsequent studies.

[0207] The capsule fabrication process is shown in Fig. 1A. The chitosan fiber layer was prepared via electrospinning based on previous modified studies (see Gu, B.K., et al., Fabrication of sonicated chitosan nanofiber mat with enlarged porosity for use as hemostatic materials. 2013. 97(1): p. 65-73). Briefly, a 5.0% (w / v) chitosan solution prepared in a mixture of TFA and DCM at a volume ratio of 7:3 was extruded through a 20-gauge stainless-steel needle connected to the cathode of a high-voltage DC generator. The substrate was attached to a rotating drum collector at a speed of 500 rpm, and the electrospun fibers were deposited thereon. To obtain capsules with two different inner diameters, 315 stainless-steel rods with a diameter of either 1.645 mm or 260 μm were used for fiber collection. The solution was continuously supplied at a feed rate of 3.0 mL / h for the 1.645-mm drum collector and 1.0 mL / h for the 260-μm drum collector at a voltage of 25.0 kV. The humidity during electrospinning was controlled at 30% using a nitrogen-filled glove box.

[0208] To prepare the PCL nanoporous layer (see Cipitria, A., et al., Design, fabrication and characterization of PCL electrospun scaffolds - a review. 2011. 21(26): p. 9419 - 9453; Chaparro, F.J., et al., Sintered electrospun polycaprolactone for controlled model drug delivery. 2019; Nam, J., et al., Modulation of embryonic mesenchymal progenitor cell differentiation via control over pure mechanical modulus in electrospun nanofibers. 2011. 7(4): p. 1516 - 1524; and Chaparro, F.J., et al., Sintered electrospun poly(ε - caprolactone) - poly(ethylene terephthalate) for drug delivery. Journal of Applied Polymer Science, 2019. 0(0): p. 47731), a combination of 0.5 g of PCL and sodium HEPES salt was dissolved in 10.0 g of HFP, and the solution was continuously stirred at 40 °C overnight. Five mass ratios of PCL to sodium HEPES salt (100:0; 99:1; 95:5; 92.5:7.5; 90:10) were studied to evaluate the effect of the salt - induced porous structure of the PCL film on drug release. Using a syringe pump at a feed rate of 3.0 mL / h for a 1.645 - mm drum collector and 1.0 mL / h for a 260 - μm drum collector, the PCL solution was continuously supplied. A high - voltage DC generator was set to 24.0 kV to produce PCL nanofibers deposited on 315 stainless - steel rods with diameters of 1.645 mm and 260 μm with or without an undrawn chitosan layer, forming bilayer films and monolayer films, respectively.

[0209] Electrospun capsules were sintered under vacuum at 100 °C for 3 h using an AccuTemp digital vacuum oven to remove surface porosity, and then the capsules were gently removed from the rods (see Chaparro, F. J., et al., Sintered electrospun polycaprolactone for controlled model drug delivery. 2019). Samples were washed with saturated sodium bicarbonate solution to neutralize TFA and then with deionized water to dissolve and remove HEPES sodium salt. The capsules were dried under vacuum overnight. The outer diameters of the capsules before and after sintering prepared using a 1.645 mm rod were measured using a digital micrometer (Keyence). The film thickness was calculated as [outer diameter of sintered capsule - 1.645 mm] / 2. Images of the capsules prepared using a 260 μm diameter rod were acquired using an optical microscope (Cole-Parmer). The images were analyzed by Motic Image Plus to determine the outer diameter of the capsules. The film thickness was calculated as [outer diameter of sintered capsule - 260 μm] / 2.

[0210] Characterization of Capsules The morphological characteristics of the capsules were investigated by scanning electron microscopy (SEM) (FEI, Quanta 200). Cross-sections of the chitosan fiber layer, PCL fiber layer, and bilayer and monolayer films before and after salt elution were attached to carbon tape placed on an aluminum stub mount and sputter-coated with a layer of gold-palladium. The capsules were immersed in liquid nitrogen and fractured to obtain cross-sections for imaging. The average fiber size and pore size of the PCL layer and chitosan layer were characterized and quantified from SEM images of three samples using ImageJ (NIH).

[0211] Chemical analysis of the surface of electrospun samples was performed in attenuated total reflection (ATR) mode using a Fourier transform infrared (FTIR) spectrometer (Thermo Scientific, Nicolet Nexus 670). A germanium crystal was placed in contact with the sample and 100 scans were collected at 8 cm -1 resolution. Standard peak positions at 1727 cm -1 and 1590 cm -1 were used to identify PCL (carbonyl peak) and chitosan (amine band), respectively (see Elzein, T., et al., FTIR study of polycaprolactone chain organization at interfaces. Journal of Colloid and Interface Science, 2004. 273(2): p. 381-387; and Osman, Z. and A.K. Arof, FTIR studies of chitosan acetate based polymer electrolytes. Electrochimica Acta, 2003. 48(8): p. 993-999).

[0212] Drug release profiles and loading / encapsulation efficacy A hollow bilayer capsule with two open ends was obtained by removal of the drum collector. For capsules with an inner diameter of 1.645 mm, 2.0 mg of BSA powder (model protein) or 2.0 mg of lyophilized bevacizumab powder (Avastin, anti-VEGF) dissolved in phosphate buffered saline (PBS) at a concentration of 0.1 mg / μL was loaded into the capsules sealed at the ends using a tube sealer (Doug Care Equipment, TTS-8C) (see Chaparro, F.J., et al., Sintered electrospun polycaprolactone for controlled model drug delivery. 2019; and Bernards, D.A., et al., Nanostructured thin film polymer devices for constant-rate protein delivery. 2012.12(10):p.5355-5361). For capsules with an inner diameter of 260 μm, considering the limited volume inside the capsules, a slurry of 1.0 mg of concentrated BSA or 1.0 mg of bevacizumab at a concentration of 1.0 mg / μL was loaded into the capsules using a 31-gauge needle.

[0213] As described in the following steps, in vitro BSA release profiles from PCL single-layer capsules and PCL-chitosan bilayer capsules were obtained. Capsules were immersed in 1 mL of PBS in a 1.5 mL low-binding centrifuge tube to reduce binding of the centrifuge tube to the eluted protein. The centrifuge tube with the immersed capsules was incubated at 37 °C to mimic physiological conditions. Eluates were collected at 1 hour, 3 hours, 6 hours, 12 hours, 24 hours, 3 days, 1 week, 2 weeks, 1 month, and then monthly (see Sousa, F., et al., A new paradigm for antiangiogenic therapy through controlled release of bevacizumab from PLGA nanoparticles. 2017.7(1):p. 3736; Yandrapu, S.K., et al., Nanoparticles in Porous Microparticles Prepared by Supercritical Infusion and Pressure Quench Technology for Sustained Delivery of Bevacizumab. Molecular Pharmaceutics, 2013.10(12):p. 4676-4686; and Tyagi, P., et al., Light-activated, in situ forming gel for sustained suprachoroidal delivery of bevacizumab. Molecular pharmaceutics, 2013.10(8):p. 2858-2867). Fresh 1.0 mL of PBS was then added and maintained under incubation. BSA release profiles were obtained by determining the absorption of the eluted BSA by BCA assay and quantifying the concentration using a BSA protein-based standard curve. The same protocol was applied to obtain bevacizumab eluates for in vitro bevacizumab release from PCL single-layer capsules and PCL-chitosan bilayer capsules.The characteristic absorbance of bevacizumab was identified at 277 nm by UV-visible spectroscopy (Agilent, Cary 100 UV-Vis), and the release rate of bevacizumab from the capsules was determined at 277 nm by a microplate reader (BioTek, Synergy HT) and quantified based on the standard curve of the stock bevacizumab solution at different concentrations (see Li, F., et al., Controlled release of bevacizumab through nanospheres for extended treatment of age-related macular degeneration. 2012.6: p. 54). The experiments were performed in triplicate.

[0214] To determine the release rate of reactive bevacizumab from capsules with an inner diameter of 260 μm, enzyme-linked immunosorbent assay (ELISA) was performed as reported (see Tyagi, P., et al., Light-activated, in situ forming gel for sustained suprachoroidal delivery of bevacizumab. Molecular pharmaceutics, 2013.10(8): p. 2858-2867; and Varshochian, R., et al., Albuminated PLGA nanoparticles containing bevacizumab intended for ocular neovascularization treatment. Journal of Biomedical Materials Research Part A, 2015.103(10): p. 3148-3156). Briefly, 1 μg / mL of recombinant human VEGF protein in 100 μL of sodium carbonate buffer solution at pH 9.6 was immobilized overnight at 4 °C on a 96-well Nunc maxisorp plate (Thermo Fisher Scientific). The plate was blocked with 200 μL of 2% BSA solution in PBS / T (0.05% v / v Tween 20 in PBS at pH 7.4) for 2 hours at room temperature and washed three times with 300 μL of PBS / T. Then, bevacizumab eluted from the capsules was diluted in a 0.1% BSA-PBS / T solution between 0 ng / mL and 10 ng / mL (determined by the standard curve), 100 μL of the sample was added to each well, and further incubated for 2 hours at room temperature. Thereafter, the plate was washed three times with PBS / T, and 100 μL of HRP goat anti-human IgG Fc secondary antibody PBS / T solution (1:1000) was added to each well. The whole plate was incubated in the dark for 1 hour at room temperature and washed five times with PBS / T. Color development was induced by the addition of 100 μL of TMB and stopped by 100 μL of 1N sulfuric acid. The concentration of active bevacizumab in each test sample was determined by comparing the absorbance at 450 nm with the standard curve.

[0215] The drug payload was determined by the disruption of single- and double-layer capsules loaded with three BSA of different sizes and bevacizumab in PBS. Briefly, single- and double-layer capsules loaded with three BSA and bevacizumab were disrupted and immersed in a solution of 1 mL of PBS. The device was washed strongly 5 times with 1 mL of PBS using a vortex mixer. Each wash took at least 10 minutes. The collected eluates of BSA and reactive bevacizumab were determined by BCA assay, UV-visible spectroscopy, and ELISA. The drug encapsulation efficiency was calculated as free drug in eluate / total amount of drug * 100%. The drug loading efficiency was calculated as drug payload / capsule weight * 100%. The cumulative release % was calculated as the cumulative amount of drug eluted from the capsule / [drug payload * encapsulation efficiency] * 100%.

[0216] Biodegradation of capsules The long-term in vitro degradation and erosion of PCL single-layer capsules and PCL-chitosan double-layer capsules with an inner diameter of 260 μm were determined by morphological changes. Briefly, capsules with two sealed ends and double-layer capsules with two open ends incubated in PBS at physiological temperature for 9 months and 3 weeks were each recovered and then vacuum dried for characterization. The cross-sections of the PCL outer layer, chitosan inner layer, and both single- and double-layer capsules were investigated using SEM. Large disruptions and cracks were evaluated, and the average pore size of the PCL layer prepared with different ratios of HEPES sodium salt was quantified by analyzing three different images using Image J and compared with the initial pore size of the capsules before incubation by one-way ANOVA with post hoc Tukey test at a significance level of 0.05. The data are presented as mean ± standard deviation.

[0217] Cytotoxicity The in vitro cytotoxicity of PCL single-layer capsules and PCL-chitosan bilayer capsules was evaluated by an MTS assay performed with human retinal pigment epithelial (ARPE-19) cells (see Sur, A., et al., Pharmacological protection of retinal pigmented epithelial cells by sulindac involves PPAR-α. 2014. 111(47): p. 16754-16759; Andres-Guerrero, V., et al., Novel biodegradable polyesteramide microspheres for controlled drug delivery in ophthalmology. 2015. 211: p. 105-117; and Huhtala, A., et al., In vitro biocompatibility of degradable biopolymers in cell line cultures from various ocular tissues: extraction studies. Journal of Materials Science: Materials in Medicine, 2008. 19(2): p. 645-649). ARPE-19 cells were used at 4×10 for all experiments 4Seeded in a 48-well plate at a cell / well density. The cytotoxicity assay was performed by both the direct contact method and the extract exposure method. For the direct contact method, a 1-cm PCL monolayer capsule or a PCL-chitosan bilayer capsule was placed in the well plate seeded with cells for 24 hours. For the extract exposure method, the PCL monolayer capsule or the PCL-chitosan bilayer capsule was immersed in 1 mL of fresh medium for 1 day, 3 days, 1 week, 2 weeks, and 1 month. At each time point, the capsule-conditioned medium was transferred to the ARPE-19 cell culture, and each sample was incubated with the cells for 24 hours for measurement. To perform the cytotoxicity assay, the cell culture medium was mixed with 20 μL of MTS reagent and then incubated at 37 °C for 3 hours. The absorbance measurement of the supernatant was obtained using a microplate reader at 490 nm. The cell viability of the experimental group was normalized against the control group (untreated). All experiments were repeated in triplicate, and the data were analyzed by one-way ANOVA with post hoc Tukey's test at a significance level of 0.05. The data are presented as mean ± standard deviation.

[0218] Evaluation of Aggregation and Anti-angiogenic Activity The stability of bevacizumab was determined by an ultra-high performance liquid chromatography (UHPLC) 3000 system (Thermo Fisher Scientific Inc., Waltham, MA) using a SEC-1000 column. To determine the stability of bevacizumab during the lyophilization process, 500 μL of 25 mg / mL bevacizumab (Avastin) was lyophilized by a lyophilizer (Labconco), and the powder was rediluted in 500 μL of PBS. The instability of concentrated bevacizumab was also assessed by diluting the bevacizumab slurry from the device to 25 mg / mL in PBS. The original free bevacizumab before and after lyophilization, concentrated bevacizumab, and bevacizumab eluted from single- and double-layer capsules at specific time points were filtered through a 0.2 μm Whatman SPARTAN HPLC Syringe Filter (VWR International, Radnor, PA) prior to injection. The fractions of the original bevacizumab monomer, aggregates, and fragments were analyzed by HPLC spectral deconvolution and separated into individual elution peaks. The integrated areas of the monomer, aggregates, and fragments were normalized to the total area of the HPLC peaks to obtain the percentage of each component. The average molecular weight was then calculated from the fraction % and molecular weight of each component.

[0219] The anti-angiogenic activity of bevacizumab released from PCL single-layer capsules and PCL-chitosan bilayer capsules was further evaluated using a capillary-like tube formation assay (see Elsaid, N., et al., PLGA microparticles entrapping chitosan-based nanoparticles for the ocular delivery of ranibizumab. 2016. 13(9): p. 2923-2940; Arnaoutova, I. and H.K.J.N. p. Kleinman, In vitro angiogenesis: endothelial cell tube formation on gelled basement membrane extract. 2010. 5(4): p. 628; and DeCicco-Skinner, K.L., et al., Endothelial cell tube formation assay for the in vitro study of angiogenesis. 2014(91)). More specifically, HUVECs were exposed to VEGF (5 ng / mL), an angiogenesis promoter, mixed with i) 10 μg / mL of the original bevacizumab; ii) 10 μg / mL bevacizumab released from PCL single-layer capsules; and iii) 10 μg / mL bevacizumab released from PCL-chitosan bilayer capsules at 1 week, 2 weeks, 1 month, 3 months, 6 months, and 9 months. After 6 hours, Calcein AM was added to the cells and incubated for an additional 30 minutes. The cells were then visualized directly using a fluorescence microscope (Nikon, Eclipse TS100) equipped with a digital camera (Qimaging). Three images were analyzed using Image J to quantify the length of the capillary structures formed. The total tube length from the experimental groups was normalized to the VEGF-treated control group for each sample, and all experiments were repeated three times. The data were analyzed by one-way ANOVA with a post hoc Tukey test at a significance level of 0.05. The data are presented as mean ± standard deviation.

[0220] Injection feasibility Fresh porcine eyes obtained from a regional meat processing plant (Delaware Meats, Delaware, Ohio) were used for the assessment of the injectability of the device (see Hoshi, S., et al., In Vivo and In Vitro Feasibility Studies of Intraocular Use of Polyethylene Glycol-Based Synthetic Sealant to Close Retinal Breaks in Porcine and Rabbit Eyes. 2015. 56(8): p. 4705-4711). Capsules were pre-loaded into a 21-gauge hypodermic needle connected to a 1 mL syringe. The 21-gauge needle used in this study had an inner diameter similar to that of a commercial intraocular lens syringe (Ozurdex applicator) (see Lee, S.S., et al., Biodegradable implants for sustained drug release in the eye. 2010. 27(10): p. 2043-2053). The intraocular injection was placed 3 mm posterior to the limbus using the syringe needle. A small volume (100 μL) of PBS was used to extrude the capsule into the porcine vitreous humor to reduce the effect of increased intraocular pressure. After injection, the needle was removed and the sclera was cut around the middle of the eye to check the placement of the capsule in the vitreous humor.

[0221] Electrospinning of chitosan and PCL nanofibers as building blocks for IBB capsules The disclosed strategy for fabricating IBB capsules is based on a two-step coating of chitosan and PCL films on a rod-shaped mold, followed by removal from the mold. Electrospinning is used to generate a hollow bilayer structure with a porous core, which provides a high surface area-to-volume ratio for protein chemisorption and adjustable porosity for drug diffusion to obtain the desired functions. Electrospinning as a method for nanofiber fabrication is based on using an electric force to draw a charged polymer solution into nanofibers. Processing parameters (including humidity and voltage) were optimized to synthesize chitosan nanofibers. For example, either high humidity (above 30%) or low voltage (less than 24 kV) caused a significant loss of charge from the spinning head and prevented the threads of the chitosan solution from forming fibers. Therefore, low humidity and a relatively high voltage were used. On the other hand, the addition of TFA better dissolved the chitosan and DCM enabled timely evaporation of the solvent, both of which were required for electrospinning, so the chitosan precursor was dissolved in TFA and DCM. To place the chitosan nanofibers onto the steel rod mold, the chitosan nanofibers were collected directly onto the rotating steel rod. From the SEM images shown in Figure 2, the diameter of the chitosan fibers was 331.61 ± 186.19 nm, and these fibers were highly interconnected to form a highly porous structure, enabling efficient drug diffusion. However, the chitosan fiber mat was found to be fragile, which is consistent with reports of low mechanical flexibility (see Jayakumar, R., et al., Biomedical applications of chitin and chitosan based nanomaterials - A short review. 2010. 82(2): p. 227-232). For this purpose, a second layer of PCL was added, which not only provided physical trapping of the drug but also imparted improved flexibility.More specifically, PCL nanofibers with a diameter of 932.57 ± 399.42 nm were coated on chitosan nanofibers (see Baker, S.R., et al., Determining the mechanical properties of electrospun poly-ε-caprolactone (PCL) nanofibers using AFM and a novel fiber anchoring technique. 2016. 59: p. 203-212). For this purpose, nanofiber-based cylinders with a high surface area, high mechanical flexibility, and strong adhesion between different layers were constructed as building blocks for IBB capsules.

[0222] Synthesis and Characterization of Injectable Two-Layer Microcapsules Two nanofiber layers were utilized as building blocks to form a hollow capsule structure by directly removing the steel rod mold after electrospinning as shown in Figure 3. The bilayer PCL-chitosan nanofiber structure can provide significant physical and electrostatic interactions with protein therapeutics, but burst release can still occur considering that the size of the continuous porous structure of the nanofibers is significantly larger compared to the size of the protein (see Chaparro, F.J., et al., Sintered electrospun polycaprolactone for controlled model drug delivery. 2019). Sintering was used to melt the PCL nanofiber layer to reduce porosity and reduce burst release of the drug. On the other hand, directly coating the PCL layer without starting with the initial nanofiber structure made it difficult to achieve a thin layer structure on chitosan, which is important for the fabrication of injectable small capsules. The mechanism of the sintering-based formation of the bilayer structure was based on the relatively low melting point of PCL at 60 °C compared to chitosan nanofibers at 220 °C. Thus, PCL became almost non-porous during the process of physically holding the drug, while chitosan remained porous for electrostatically binding the drug. Additionally, this process also well integrated the two layers during the melting process. From the SEM images shown in Figure 3, the melting of the PCL nanofibers increased the adhesion between the two layers, so the chitosan fiber layer adhered to the PCL outer layer, which stabilized the bilayer structure. However, the framework composed of large fibers can still be observed on the surface of the PCL after sintering. During the sintering process, the film thickness decreased by 80% due to compression and increased density, so the capsule size can be controlled by adjusting the thickness of the chitosan and PCL fiber layers during the electrospinning process.

[0223] After sintering the fiber films of chitosan and PCL, bilayer microcapsules with a hollow structure were generated by a template strategy and by taking advantage of the mechanical robustness of the PCL outer layer. By controlling the shape and size of the template rod, the size and structure of the capsules could be effectively controlled. As a proof of concept, two sizes of single-layer PCL capsules and bilayer chitosan-PCL capsules were prepared: those with a larger inner diameter of 1.645 mm (previous model) that could be transplanted as scaffolds, and those with a smaller inner diameter of 260 μm (final model) that could be injected via a 21-gauge needle. The hollow structure with the steel rod as the template enabled a high volume mainly for drug loading, while the bilayer membrane provided physical trapping and chemical non-covalent bond formation to achieve sustained release over a long period.

[0224] In the device used for drug release studies, the outer diameter of the capsule with an inner diameter of 1.645 mm was approximately 1.815 mm, and the wall thickness was 89.36 ± 11.52 μm. Similarly, the outer diameter of the capsule with an inner diameter of 260 μm was approximately 430 μm, the membrane thickness was 89.85 ± 4.27 μm, and it was designed to be injectable via a 21-gauge needle. The increase in the capsule thickness promoted the mechanical properties of the capsule, which prevented rupture during injection. However, the increase in the capsule size might hinder intravitreal injection. Therefore, 80 - 90 μm was determined as the wall thickness that balanced mechanical robustness and injectability. Also, the membrane thickness was closely related to the drug diffusion rate, so the difference in thickness between the single-layer and bilayer capsules was controlled and minimized to reduce the effect of thickness on drug release.

[0225] Regulation of the nanoporous structure of the bilayer membrane After sintering, the PCL layer becomes non-porous and the drug release rate is significantly limited (see Chaparro, F.J., et al., Sintered electrospun polycaprolactone for controlled model drug delivery. 2019). However, without sintering, the bilayer capsules are highly porous and would result in an undesirably high drug release rate. Therefore, the salt leaching method was used to precisely regulate the three-dimensional porous structure of the bilayer membrane, enabling long-term sustainable drug release. More specifically, varying amounts of a water-soluble salt (HEPES) were mixed into the nanofibers during electrospinning. Incubation of the capsules in water before drug loading led to the elution of HEPES inside the film, thereby forming a porous structure again on the bilayer membrane. By adjusting the concentration of HEPES in the PCL nanofibers, the porosity could be effectively controlled. As shown in Figure 4, the pore size and distribution in the PCL sintered film strongly depended on the mass ratio of salt to PCL. For example, the pores were smaller at lower salt concentrations and tended to be more dispersed across the entire surface of the film. However, low amounts of salt also hindered the formation of interconnected pores for the diffusion and release of large molecules. Table 1 shows the results of the analytical pore sizes for different ratios of PCL to sodium salt of HEPES. For this purpose, PCL films prepared with salt concentrations exceeding 5.0% were used in capsule manufacturing and drug release studies as the interconnected porous structure was observed in cross-section via SEM. TIFF2025093918000002.tif69150

[0226] To assess the changes in the bilayer structure before and after salt elution, SEM imaging was used to observe the inner surface, outer surface, and cross-section of the bilayer capsules. Before salt elution, the PCL sintered film was roughened by some crystals of HEPES sodium salt embedded therein. After salt elution, a porous structure appeared in the PCL layer, and the chitosan layer lost its fibrous structure and formed a porous layer. The average pore size of the chitosan layer was 802.47 ± 501.02 nm, and this size enabled the three-dimensional diffusion of proteins and their interaction for sustainable release by maximizing the interaction with proteins via electrostatic interactions. The inner chitosan layer showed a more nanoporous structure with a thickness of 25 μm, which may be due to the relatively high melting point of chitosan. In contrast, the outer PCL layer had a denser structure with a total thickness of 65 μm with nanopores passing through, physically trapping the drug while supporting protein diffusion. These results are consistent with the morphology collected on the individual layers of PCL and chitosan.

[0227] To further confirm the chemical properties of the bilayer capsules after sintering and washing, FTIR spectroscopy was performed on the final capsules shown in Figure 5. In the spectra shown, a significant peak at 1727 cm -1 was assigned to the carbonyl group in PCL. Peaks at 2963 cm -1 and 2995 cm -1 were C-H stretches in the backbone of PCL. A broad band at 3478 cm -1 due to the O-H stretching vibration from the abundant hydroxyl groups in the backbone of chitosan could be observed. Furthermore, a peak at 1571 cm -1Characteristic peaks for chitosan in [the relevant context] were assigned to N-H stretching. Such peaks provide strong evidence of the chemical nature of the chitosan layer and the PCL layer even after exposure to sintering. Thus, cationic chitosan remains active and is capable of chemically non-covalently binding anionic proteins (bevacizumab). SEM and FTIR demonstrated the feasibility of the disclosed bottom-up approach for the synthesis of hybrid nanofabricated capsules with widely tunable pore sizes, aspect ratios, dimensions, and providing optimal physical and chemical properties for drug loading and controlled release of protein therapeutics.

[0228] High payload and long-term sustainable drug release of bevacizumab To confirm the high payload of protein drugs enabled by the hollow structure of the capsules, drug encapsulation efficacy was determined by the destruction of drug-loaded capsules and the respective assessment of the amounts of BSA and bevacizumab leaking from the capsules. BSA was used as a model protein drug, and bevacizumab is a clinically used anti-VEGF therapeutic substance for the treatment of AMD. Considering that BSA and bevacizumab can adsorb to the chitosan layer of the bilayer capsules, both single-layer and bilayer capsules were used for the assessment of drug payload. No significant difference in drug payload was found between the two types of capsules. Based on this study, the BSA encapsulation efficacies of three large capsules and three small capsules were 100.39 ± 6.46% and 69.64 ± 7.15%, respectively. Lower encapsulation efficacies were observed for the loading of bevacizumab in both large and small capsules, which was assessed to be 52.66 ± 6.47% by UV-visible spectroscopy (a commonly used instrument that determines the concentration of proteins with characteristic absorption around 280 nm), as shown in Figure 12. However, a higher amount of reactive bevacizumab (729.02 ± 84.67 μg) was quantified by ELISA, which gave an encapsulation efficacy of approximately 70% for bevacizumab. The lower encapsulation efficiency may be due to the decreased sensitivity of UV-visible spectroscopy for lower concentrations of bevacizumab and the fact that cumulative release can be effectively detected by ELISA.The loading capacity of the capsule is approximately 26.60 ± 1.90% w / w, which is higher than most of the reported devices with a loading capacity of 10 - 15% (see Li, F., et al., Controlled release of bevacizumab through nanospheres for extended treatment of age-related macular degeneration. 2012.6: p. 54; and Badiee, P., et al., Ocular implant containing bevacizumab-loaded chitosan nanoparticles intended for choroidal neovascularization treatment. Journal of Biomedical Materials Research Part A, 2018.106(8): p. 2261-2271).

[0229] Using the model drug BSA, as shown in Figure 6, the ability of exemplary capsules to regulate the drug release profile by changing the surface morphology and porosity of the capsules was demonstrated. As a proof of concept, single- and double-layer capsules with inner diameters of 1.645 mm and 260 μm were prepared, and samples prepared under salt elution conditions of 5%, 7.5%, and 10% salts were also investigated. Figure 6 shows the BSA release profiles of single- and double-layer capsules for both sizes. As expected, burst release occurred in the 1.645 mm inner diameter PCL single-layer capsules during the first month, with more than 75% of the loaded BSA eluting from the capsules, which significantly limited the duration of drug release. Due to the increase in the surface area to volume ratio, the duration of burst release was shortened to two weeks in the 260 μm inner diameter capsules, and the cumulative release percentage was similar to that of the 1.645 mm inner diameter capsules during this period. Therefore, the drug diffusion rate of the small capsules was improved. However, there was burst release following stable release in both capsule sizes. The maximum drug release period for the single-layer capsules was approximately five months for the 1.645 mm inner diameter capsules and three months for the 260 μm inner diameter capsules, both prepared with 10% salt. The effect of HEPES sodium salt was also investigated. Higher salt concentrations resulted in faster release rates due to increased pore interconnectivity. In capsules with lower salt concentrations, burst release was delayed but still uncontrollable.

[0230] In contrast, the PCL-chitosan bilayer capsules did not show any obvious evidence of burst release. The bilayer capsules significantly retarded the release of BSA. The release profile of the bilayer capsules showed high linearity, which is summarized in Figure 7. After one month, the bilayer capsules with an inner diameter of 1.645 mm released approximately 15% of the loaded BSA, which was 60% lower than the release of the single-layer PCL capsules during the same period, indicating a higher ability to retain BSA inside the device. Similarly, the bilayer capsules with an inner diameter of 260 μm significantly reduced the burst release. Only 25% of the BSA eluted from the bilayer capsules with an inner diameter of 260 μm, which was higher than that of the 1.645 mm capsules due to the relatively larger surface area to volume for diffusion. The chitosan layer was effective in limiting drug diffusion, and the porosity of the PCL shell did not play a significant role in controlling the BSA release. There was no significant difference (p > 0.05) between the diameters of the single-layer and bilayer capsules, and thus the effect of thickness on drug release was negligible in these studies. Theoretically, the bilayer structure has the ability to control drug release for at least one year for both sizes of capsules based on the cumulative release data.

[0231] After the porous structure of the bilayer capsules was comprehensively investigated and optimized, capsules with a bilayer structure were used for the loading and release of bevacizumab (a targeted drug for clinical AMD treatment) and eluted with 10% HEPES. Consistent with the BSA drug release experiment, a sustainable release profile over 1 year and 9 months without an obvious initial burst release was successfully achieved in both 1.645 mm capsules and 260 μm capsules, respectively. Interestingly, during the drug release from the bilayer capsule-based, there was a further reduction in the burst release of bevacizumab compared to BSA. This may be due to the increased molecular weight and lower effective charge of bevacizumab compared to BSA. Under this condition, the pore size dominates the diffusion rate of bevacizumab. It would be difficult for the higher molecular weight bevacizumab to elute from the capsules with limited porous channels. This explains why the total release of both monolayer capsules and bilayer capsules prepared with 5% HEPES salt has a similar release kinetics. Also, compared to the other two capsules with larger pores inside the membrane, the capsules prepared with 5% HEPES salt have the lowest release rate. As shown in Figure 7, it is noteworthy that an almost zero-order release kinetics was achieved after the burst release with the 260 μm inner diameter bilayer capsules loaded with bevacizumab (P < 0.05).

[0232] The amount of bevacizumab was assayed and quantified using simple UV absorption, but it could not distinguish between reactive bevacizumab and the background of degraded polymers over time, nor could it distinguish the capsules. Therefore, the bevacizumab eluted from the 260 μm capsules was determined again by ELISA. Figure 13 shows that the general trend of bevacizumab release was consistent with previous results determined by UV-visible light. For example, the long-term cumulative release of bevacizumab from monolayer capsules made with 5% HEPES salt was estimated to be approximately 160 μg by UV-visible light, which was the same as that characterized by ELISA over 9 months. Therefore, by comparing the release results from UV-visible light and ELISA, the release profiles obtained by UV-visible light were reliable and could provide the general trend of bevacizumab release from monolayer and bilayer capsules. Similarly, the capsules prepared with 5% salt had a relatively slower release rate over 9 months compared to those with 7.5% and 10% salt. Based on these considerations, bilayer capsules with higher (7.5% and 10%) HEPES salt concentrations were then identified and used for the long-term release of anti-VEGF. At the same time, the high drug loading capacity and stable drug release profile over a 9-month period strongly indicate the potential of exemplary bilayer capsules as a versatile platform for the delivery of anti-VEGF therapeutic agents.

[0233] Biodegradation of capsules The in vitro degradation of PCL single-layer capsules and chitosan-PCL bilayer capsules was studied. Capsules with two closed ends were placed in PBS at 37 °C for 9 months, the capsules were recovered and characterized by SEM. The mechanical integrity of the whole device is mainly determined by the PCL layer that undergoes slow hydrolysis at the ester bond (see Darwis, D., et al., Enzymatic degradation of radiation crosslinked poly(ε-caprolactone). Polymer Degradation and Stability, 1998. 62(2): p. 259-265). Therefore, it is important to assess the erosion and degradation of the PCL layer over time. From the characteristic SEM images shown in Figure 11, both the single-layer capsules and the bilayer capsules remained intact after 9 months of incubation. However, after 9 months, the pores on the surface of the PCL membrane became larger and more dispersed compared to the initial capsules, indicating slow degradation of the PCL membrane. Table 2 summarizes the analytical pore sizes of the capsules under different conditions of HEPES salts. The pores on the PCL surface increased significantly by approximately 180 nm in diameter on average (p ≤ 0.05), but the whole device maintained its integrity without obvious cracks and fractures. The bilayer capsules were also characterized. After 9 months, the chitosan layer still adhered tightly on the PCL layer and the fibers were still well-defined and intact. The fiber framework of the chitosan surface layer was still evident without significant changes. Furthermore, the membrane thicknesses of both the single-layer capsules and the bilayer capsules were in the range of 80 μm to 90 μm, which is similar to their original thickness before incubation. However, as shown in Figure 15, when capsules with two open ends were immersed in PBS at physiological temperature for 3 weeks, a significant decrease in thickness and loss of chitosan fibers were observed.This is probably caused by the slow degradation of chitosan when exposed directly to water over a long period (see Kean, T. and M. Thanou, Biodegradation, biodistribution and toxicity of chitosan. Advanced Drug Delivery Reviews, 2010. 62(1): p. 3-11; and Onishi, H. and Y. Machida, Biodegradation and distribution of water-soluble chitosan in mice. Biomaterials, 1999. 20(2): p. 175-182). Furthermore, due to the weak mechanical properties of chitosan, the inner chitosan fiber membrane becomes vulnerable under shear forces during long-term incubation in water, which can lead to a significant loss of chitosan (see Sangsanoh, P. and P. J. B. Supaphol, Stability improvement of electrospun chitosan nanofibrous membranes in neutral or weak basic aqueous solutions. 2006. 7(10): p. 2710-2714; and Chen, Z., et al., Mechanical properties of electrospun collagen-chitosan complex single fibers and membrane. Materials Science and Engineering: C, 2009. 29(8): p. 2428-2435). Therefore, the hydrophobic PCL layer can protect the inner chitosan layer from degradation and further reduce the deterioration of the entire device. TIFF2025093918000003.tif61150

[0234] Cytotoxicity One of the most important characteristics of any drug delivery device is acceptable biocompatibility in the presence of the target cells or tissues, both short - term and long - term. For this purpose, the cytotoxicity of an exemplary bilayer capsule was investigated using retinal pigment epithelial (ARPE - 19) cells because they are the most predominant cells in the retina and are highly sensitive to toxic and foreign materials by both direct contact and extract exposure methods. The cell viability of RPE cells was measured using a standard mitochondrial activity assay, with and without treatment of monolayer and bilayer capsules. From the results shown in Figure 8, both PCL monolayer capsules and PCL - chitosan bilayer capsules showed only slight toxicity to RPE cells during 24 - hour direct incubation. Both PCL and chitosan have been reported to have good biocompatibility in intraocular applications (see Kim, J., et al., Long - term intraocular pressure reduction with intracameral polycaprolactone glaucoma devices that deliver a novel anti - glaucoma agent. 2018. 269: p. 45 - 51; and Wassmer, S., et al., Chitosan microparticles for delivery of proteins to the retina. Acta Biomaterialia, 2013. 9(8): p. 7855 - 7864). Similarly, the extracts of both capsules also did not affect cell viability over one month, as shown in Figure 8. Even at the one - month time point, a viability of over 95% was found for the bilayer capsules. Collectively, these results show negligible cytotoxicity to retinal pigment epithelial cells and suggest the potential of bilayer capsules for pre - clinical evaluation of ophthalmic models in future research.

[0235] Stability of the released VEGF inhibitor and therapeutic effect on anti - angiogenesis One of the main problems hindering the development of long-term sustained protein delivery systems is the aggregation and degradation of proteins in an aqueous environment. Bevacizumab is unstable under physiological conditions and tends to degrade and aggregate over time in the body (see Courtois, F., et al., Rational design of therapeutic mAbs against aggregation through protein engineering and incorporation of glycosylation motifs applied to bevacizumab. mAbs, 2016. 8(1): p. 99-112; Oliva, A., M. Llabres, and J. B. Farina, Capability measurement of size-exclusion chromatography with a light-scattering detection method in a stability study of bevacizumab using the process capability indices. Journal of Chromatography A, 2014. 1353: p. 89-98; Latypov, R. F., et al., Elucidation of acid-induced unfolding and aggregation of human immunoglobulin IgG1 and IgG2 Fc. Journal of Biological Chemistry, 2012. 287(2): p. 1381-1396; and Bakri, S. J., et al., Six-month stability of bevacizumab (Avastin) binding to vascular endothelial growth factor after withdrawal into a syringe and refrigeration or freezing. Retina, 2006. 26(5): p. 519-522). Further, the device is loaded with highly concentrated bevacizumab obtained from lyophilization.Aggregation and loss of activity can occur at high concentrations or during the lyophilization process (see Varshochian, R., et al., The protective effect of albumin on bevacizumab activity and stability in PLGA nanoparticles intended for retinal and choroidal neovascularization treatments. European Journal of Pharmaceutical Sciences, 2013. 50(3): p. 341-352). This is important because bevacizumab aggregates may not be released from implantation at the same rate as the monomer. Therefore, bevacizumab stability studies were required to assess the aggregation and fragmentation of bevacizumab during device fabrication and device incubation over time using HPLC. The analytical aggregation and fragmentation of bevacizumab are summarized in Table 3 and the HPLC spectra are shown in Figure 14. To confirm the stability of bevacizumab during lyophilization, the HPLC spectra of lyophilized bevacizumab were compared to those of commercially available bevacizumab (Avastin). 16% aggregates were formed in the original free bevacizumab and a slight increase in bevacizumab aggregates was observed during the lyophilization cycle. Aggregation in concentrated solutions was also assessed by diluting in PBS followed immediately by HPLC characterization. However, no change in bevacizumab aggregates was observed, indicating that the protein is fairly stable in concentrated solutions and further demonstrating that exemplary devices can delay the release of bevacizumab monomer. The efficacy of bevacizumab released over the long term from single-layer and bilayer capsules was also assessed. The percentage of aggregates ranged between 11% and 16% for both capsules over 3 months and it was also found that bevacizumab underwent fragmentation during long-term incubation. However, the efficacy of bevacizumab was still maintained at a high level over this period as shown in Table 3. The monomer of bevacizumab eluted from PCL single-layer capsules with an inner diameter of 260 μm accounted for 84% in 1 month and this value decreased slightly to 79% in 3 months.Similarly, the bevacizumab monomer released from chitosan-PCL bilayer capsules with an inner diameter of 260 μm was 82% over the first three months. This enhanced stability may be attributed to the adhesion of chitosan by ionic binding to glycoproteins and the increased bioavailability thereof. In addition, the hydrophobic PCL layer retarded the fragmentation process by reducing the liquid exchange across the capsules. Therefore, the efficacy of the long-term released bevacizumab was well maintained, suggesting a high potential of the exemplary capsules for the treatment of AMD without frequent injections. TIFF2025093918000004.tif135150

[0236] HPLC provides clear information about the bevacizumab monomer and aggregation delivered by the exemplary capsules, while ELISA characterizes the efficacy and amount of bevacizumab reactive to long-term released VEGF and ensures its effect on angiogenesis. Therefore, a bevacizumab ELISA was performed to determine the reactive bevacizumab released over time from capsules with an inner diameter of 260 μm. After one month, the release rate of the active bevacizumab was maintained at approximately 20 μg / mL per month, which was similar to the amount of bevacizumab determined by UV-visible light. Furthermore, the percentage of the biological activity of the eluted bevacizumab was also calculated by comparing the cumulative release percentage measured by ELISA to that determined by UV / visible light. From this result, the bevacizumab released from the single-layer capsules was able to maintain more than 90% biological activity during the nine-month period, which indicates the potential in protein protection. Fluctuations in biological activity were observed in the bilayer capsules, which were maintained around 80%. An increase in the background of the UV-visible light absorbance effect due to the slow biodegradation of the inner layer over the long incubation period as described above may cause a lower percentage of biological activity. However, both results strongly support the high biological activity of the proteins protected by the single-layer and bilayer capsules. In this regard, the exemplary hollow bilayer capsules that physically protect the drug have the potential to overcome this obstacle to sustained release.

[0237] In addition, bevacizumab eluted from single-layer PCL and PCL-chitosan bilayer capsules was assayed for its inhibitory effect on VEGF-induced capillary growth in a tube formation assay using HUVEC, as shown in Fig. 9. At a concentration of 10 μg / mL, the original bevacizumab of the positive control resulted in a 93.15 ± 1.49% inhibition of capillary length. Bevacizumab eluted from single-layer capsules and bilayer capsules after 1 month led to approximately 13.33 ± 6.51% and 12.33 ± 4.63% tube formation in capsules with an inner diameter of 260 μm and capsules with an inner diameter of 1.645 mm, respectively, which was more effective compared to the conventional injection of the original bevacizumab. A slight increase in tube length formation appeared after 3 months due to the loss of bioactivity caused by long-term incubation at physiological temperature. However, there was no significant difference in the anti-angiogenic induction properties between bevacizumab eluted from bilayer capsules and bevacizumab eluted from single-layer capsules (p > 0.05). Slow drug diffusion was expected to delay the process of enzyme-catalyzed bevacizumab degradation, which significantly protected the proteins inside the capsules. Overall, the anti-angiogenic bioactivity was well maintained at a high level over 9 months, suggesting the potential of the protective effect of the capsules for long-term drug release.

[0238] Injection feasibility In addition to high drug loading capacity, sustained release of protein therapeutic substances, and maintenance of anti-VEGF bioactivity, these capsules can also be made injectable. To demonstrate this, as shown in Figure 10, an injection feasibility test was performed by delivering 10-mm-long capsules through the sclera into the vitreous humor of ex vivo pig eyes via a 21-gauge needle. Capsules with an outer diameter of 430 μm were used in this study because they are similar in size to the commercially available intravitreal lens (Ozurdex) with a diameter of 460 μm and a length of 6 mm. The Ozurdex applicator is equipped with a 22-gauge TSK needle (see Chan, A., L.-S. Leung, and M. S. Blumenkranz, Critical appraisal of the clinical utility of the dexamethasone intravitreal implant (Ozurdex®) for the treatment of macular edema related to branch retinal vein occlusion or central retinal vein occlusion. Clinical Ophthalmology (Auckland, NZ), 2011. 5: p. 1043; Arcinue, C. A., O. M. Ceron, and C. S. Foster, A comparison between the fluocinolone acetonide (Retisert) and dexamethasone (Ozurdex) intravitreal implants in uveitis. Journal of ocular pharmacology and therapeutics, 2013. 29(5): p. 501-507; and Querques, L., et al., Repeated intravitreal dexamethasone implant (Ozurdex®) for retinal vein occlusion. Ophthalmologica, 2013. 229(1): p. 21-25).The inner diameter of the needle is approximately 500 μm, which was able to fit an exemplary capsule (see Meyer, C.H., et al., Penetration force, geometry, and cutting profile of the novel and old Ozurdex needle: the MONO study. Journal of Ocular Pharmacology and Therapeutics, 2014. 30(5): p. 387-391). More specifically, in clinical applications, typically an anti-VEGF loaded capsule can be delivered intravitreally by a similar applicator, which can avoid invasive incision surgery. Thus, an advanced drug delivery system based on the exemplary bilayer capsule would be quite compatible with currently used clinical approaches.

[0239] In this study, to address an important challenge of long-term therapy for the treatment of exudative AMD, a polymer-based microstructured delivery platform was designed and developed to achieve sustained release of anti-VEGF in vitro. Sustained protein release was achieved by designing and optimizing the structure of chitosan-PCL bilayer microcapsules through the use of composite chemistry and engineering methods. Important properties of these chitosan-PCL microcapsules include size, porosity of the PCL shell, and a hollow structure for simple and convenient drug loading.

[0240] PCL-chitosan microcapsules were synthesized by a novel combination of electrospinning, sintering, and salt leaching. In preliminary studies, it was shown that chitosan fibers lost their structure after salt leaching. The formation of trifluoroacetate during fiber preparation was thought to accelerate the process of chitosan elution while using TFA and DCM as solvents, and thus the required step of neutralization with sodium bicarbonate solution was required during washing to reduce the effect of acidic salts on the bioactivity of bevacizumab (see Sangsanoh, P. and P.J.B. Supaphol, Stability improvement of electrospun chitosan nanofibrous membranes in neutral or weak basic aqueous solutions. 2006. 7(10): p. 2710-2714).

[0241] The thickness of the membrane was correlated with the drug release period. Theoretically, a thicker membrane would result in slower drug diffusion. Even though increasing the size of the capsule might help achieve slower drug release, increasing the size of the microcapsules would make injection through a small-gauge needle impossible. Therefore, a thinner membrane was required to make the capsules injectable for clinical applications. The chitosan layer was added to address this issue. In this study, all capsules had a thickness between 80 and 95 μm, which minimized the effect of thickness in the exploration of the relationship between drug release rate and the chitosan-PCL composite.

[0242] After optimizing these important factors to control drug release, the performance of the microcapsules was evaluated and optimized for sustained release of anti-VEGF. Bevacizumab has been clinically used in the treatment of exudative AMD since 2004 (see Michels, S., et al., Systemic bevacizumab (Avastin) therapy for neovascular age-related macular degeneration: twelve-week results of an uncontrolled open-label clinical study. 2005. 112(6): p. 1035-1047.e9). Theoretically, the isoelectric point (pI) of bevacizumab is 7.8 (see Nomoto, H., et al., Pharmacokinetics of bevacizumab after topical, subconjunctival, and intravitreal administration in rabbits. 2009. 50(10): p. 4807-4813). Its net charge calculated from the pI should be slightly positive at pH 7.4 as reported by a number of studies.However, protein aggregates in water and other organic solvents typically used during device manufacturing have the potential to reduce bioactivity and cause undesired side effects (see Varshochian, R., et al., Albuminated PLGA nanoparticles containing bevacizumab intended for ocular neovascularization treatment. Journal of Biomedical Materials Research Part A, 2015. 103(10): p. 3148-3156; Courtois, F., et al., Rational design of therapeutic mAbs against aggregation through protein engineering and incorporation of glycosylation motifs applied to bevacizumab. mAbs, 2016. 8(1): p. 99-112; and Varshochian, R., et al., The protective effect of albumin on bevacizumab activity and stability in PLGA nanoparticles intended for retinal and choroidal neovascularization treatments. European Journal of Pharmaceutical Sciences, 2013. 50(3): p. 341-352). Therefore, PBS is widely used for suspending bevacizumab to maintain stability and bioactivity.Bevacizumab has been reported to be net negatively charged in PBS at pH 7.4, which suggests binding to chitosan and may provide more sustainable release from exemplary capsules (see Li, S.K., et al., Effective electrophoretic mobilities and charges of anti-VEGF proteins determined by capillary zone electrophoresis. 2011. 55(3): p. 603-607; and Garcia-Quintanilla, L., et al., Pharmacokinetics of Intravitreal Anti-VEGF Drugs in Age-Related Macular Degeneration. Pharmaceutics, 2019. 11(8): p. 365). Binding of buffer ions in PBS to bevacizumab increases its hydrophilicity, which further increases its stability and causes a difference between the theoretical and experimental net charges of the protein (see Li, S.K., et al., Effective electrophoretic mobilities and charges of anti-VEGF proteins determined by capillary zone electrophoresis. 2011. 55(3): p. 603-607; and Chopra, P., J. Hao, and S.K.J.I.j.o.p.Li, Iontophoretic transport of charged macromolecules across human sclera. 2010. 388(1-2): p. 107-113). Therefore, the hypothesis is proposed that bevacizumab has a negative charge in the vitreous and capsules and can thus retain this protein via electrostatic attraction by positively charged chitosan. Similarly, BSA is a negatively charged protein in water with an isoelectric point near 4.7. BSA was able to bind to cationic ions and increase its surface charge under physiological conditions (in PBS).However, as previously reported, since these ions have little effect on the charge of BSA, BSA remains negatively charged in PBS (see Li, S.K., et al., Effective electrophoretic mobilities and charges of anti-VEGF proteins determined by capillary zone electrophoresis. 2011. 55(3): p. 603-607; and alis, A., et al., Measurements and Theoretical Interpretation of Points of Zero Charge / Potential of BSA Protein. Langmuir, 2011. 27(18): p. 11597-11604). By providing the combined electrostatic interaction between bevacizumab or BSA and chitosan and the protective effect from the PCL shell, the desired sustained drug release profile could be achieved.

[0243] In this study, the release rate for a payload similar to bevacizumab was also significantly improved. The average payload of the reported devices was in the range of 500 μg to 1000 μg (see Li, F., et al., Controlled release of bevacizumab through nanospheres for extended treatment of age-related macular degeneration. 2012.6: p.54; and Varshochian, R., et al., Albuminated PLGA nanoparticles containing bevacizumab intended for ocular neovascularization treatment. Journal of Biomedical Materials Research Part A, 2015.103(10): p.3148-3156). However, these devices had limitations including not being injectable and not sustaining release over three months. The drug loading capacity of these devices was not as expected. Also, the anti-VEGF bioactivity could be affected during the fabrication process in these devices due to the interaction of the therapeutic substance with the solvent or high temperature. However, drug loading was processed after device fabrication, which avoided the drug loss and inactivation that generally occur using conventional preparation methods (such as emulsions). Therefore, the capsule designed herein guaranteed a drug payload of 700 μg of bevacizumab due to the narrow space in the injectable capsule and the high molecular weight of bevacizumab. The inner space can be expanded by selectively increasing the template rod to facilitate drug loading. Also, in medicine, the dry powder of bevacizumab can be accurately replaced and loaded under a microscope, which could further promote drug loading efficiency and bevacizumab stability.In addition, other therapeutic substances with lower molecular weights comparable to the model drug BSA were evaluated using the exemplary device and may be able to significantly further increase the drug payload (see Rosenfeld, P.J., et al., Optical coherence tomography findings after an intravitreal injection of bevacizumab (Avastin®) for neovascular age-related macular degeneration. 2005. 36(4): p. 331-335).

[0244] The bilayer capsule can efficiently control the drug release rate by utilizing the electrostatic interaction between the protein therapeutic substance and the polymer, and can address many of the current problems associated with the clinical treatment of exudative AMD. It also provides an alternative approach for several diseases that require long-term treatment with protein therapeutic substances (such as colorectal and breast cancer, and some brain tumors, etc.). However, the device manufacturing method still needs to be optimized for the requirements of different protein therapeutic substances and may have great potential for eye, cancer, and other biomedical applications.

[0245] In conclusion, a polymer-based delivery platform was developed for the controlled release of anti-VEGF, which provides an effective means to regulate the polymer-protein interaction for the controlled release of therapeutic substances based on a bilayer microstructure that synergistically combines the electrostatic binding between chitosan and anti-VEGF with the protective hydrophobic layer of PCL. The bilayer structure was characterized in detail and the capsule performance for protein delivery was further determined. Most importantly, the exemplary designed delivery platform significantly improved the long-term in vitro release of anti-VEGF compared to the most recent devices, supporting the potential to treat AMD. In future studies, it is required to evaluate and re-optimize the therapeutic effect of the anti-VEGF loaded device in an in vivo AMD model.

[0246] It will be apparent to those skilled in the art that various modifications and variations can be made to the present disclosure without departing from the scope or spirit of the present disclosure. Other embodiments of the present disclosure will be apparent to those skilled in the art from consideration of the specification and practice of the disclosure disclosed herein. The specification and examples are considered to be merely illustrative, and it is intended that the true scope and spirit of the present disclosure be indicated by the following claims.

Claims

1. A method for preparing a drug delivery capsule for injection into the eye of a subject, comprising: forming an inner layer containing a first polymer having a net positive charge under physiological conditions on a conductive rod; and forming an outer layer on the inner layer, the outer layer containing a second polymer different from the first polymer, wherein: the forming of the inner layer includes electrospinning using a solution of the first polymer and a voltage difference of about 10 kV to about 30 kV; the solution of the first polymer is about 1 w / v% to about 10 w / v% in at least one organic solvent; the forming of the outer layer includes electrospinning a solution containing the second polymer and optionally a porogen onto the formed inner layer; the voltage difference used for electrospinning is about 10 kV to about 30 kV; the solution containing the second polymer and optionally the porogen is about 1 w / v% to about 10 w / v% of the second polymer and the porogen based on the total weight; the weight ratio of the second polymer to the optional porogen is about 50:50 to about 100:0, the method.

2. The method according to claim 1, wherein the first polymer includes chitosan or a derivative thereof.

3. The method according to any one of claims 1 or 2, wherein the second polymer includes PCL or a derivative thereof.

4. The method according to any one of claims 1 to 3, wherein at least one organic solvent in the solution of the first polymer is a mixture of trifluoroacetic acid and dichloromethane; the trifluoroacetic acid and the dichloromethane are present in a ratio of about 1:10 to about 10:

1.

5. The method according to claim 4, wherein the trifluoroacetic acid and the dichloromethane are present in a ratio of about 5:3 to about 10:

3.

6. The method according to claim 5, wherein the trifluoroacetic acid and the dichloromethane are present in a ratio of about 7:

3.

7. The method according to any one of claims 1 to 6, wherein the weight ratio of the second polymer to the porogen is about 90:100 to about 99.9:0.

1.

8. The method according to claim 7, wherein the weight ratio of the second polymer to the porogen is about 90:100 to about 95:

5.

9. The method according to claim 7, wherein the weight ratio of the second polymer to the porogen is from about 95:5 to about 99.9:0.

1.

10. The method according to any one of claims 1 to 9, wherein the solution comprising the second polymer and the porogen is about 2.5 w / v% to about 10 w / v% of the second polymer and the porogen, based on the total weight.

11. The method according to claim 10, wherein the solution comprising the second polymer and the porogen is about 5 w / v% to about 10 w / v% of the second polymer and the porogen, based on the total weight.

12. The method according to any one of claims 1 to 11, further comprising sintering the drug delivery capsule following the formation of the outer layer.

13. The method according to claim 12, wherein the sintering comprises heating for a period of about 1 minute to about 6 hours at a temperature of about 50 °C to about 150 °C.

14. The method according to claim 12, wherein the sintering comprises heating for a period of about 30 minutes to about 6 hours at a temperature of about 90 °C to about 110 °C.

15. The method according to any one of claims 12 to 14, further comprising washing the drug delivery capsule following the sintering.

16. The method according to claim 15, wherein the washing comprises washing the drug delivery capsule with a basic solution, an aqueous solution, or a combination thereof.

17. The method according to claim 15, wherein the washing comprises washing with a saturated sodium bicarbonate solution followed by washing with deionized water.

18. The method according to any one of claims 15 to 17, further comprising drying the drug delivery capsule following the washing.

19. The method according to claim 18, wherein the drying is for a period of about 1 minute to about 6 hours at a temperature of about 50 °C to about 150 °C under vacuum.

20. The method according to claim 18, wherein the drying is for a period of about 30 minutes to about 6 hours at a temperature of about 90 °C to about 110 °C under vacuum.

21. A drug delivery capsule produced by the method according to any one of claims 1 to 20.

22. The drug delivery capsule according to claim 21, further comprising a therapeutic agent.

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