Light-triggered polymer-naloxone conjugate for opioid reversal

Abstract

A conjugate includes a polymer and an active agent. The active agent is linked to the polymer via a cleavable linker. The active agent is an agent capable of reversing or reducing an effect of an opioid in a subject.

Description

LIGHT-TRIGGERED POLYMER-NALOXONE CONJUGATEFOR OPIOID REVERSALCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This Application claims benefit under 35 U.S.C. § 119(e) of the U.S. Provisional Application No. 63 / 516, 190 filed July 28, 2023, the content of which is incorporated herein by reference in its entirety.TECHNICAL FIELD

[0002] This disclosure relates generally to compositions, methods, and kits for stimulus- triggered release of naloxone.GOVERNMENT SUPPORT

[0003] This invention was made with government support under grant numbers R35GM131728 and K99GM141269 awarded by the National Institutes of Health (NIH). The government has certain rights in the invention.BACKGROUND

[0004] Opioids are substances used for pain relief, but are often abused, leading to an epidemic of opioid-related deaths. Currently, there are more than 40,000 deaths each year in the United States related to opioid abuse. A common treatment for an opioid overdose is naloxone, a medicine that rapidly reverses and blocks the effects of opioids. However, there is limited access to naloxone formulations, and timely access to naloxone formulations is even more limited. Moreover, naloxone is only effective for a short period of time, so re-narcotization is possible as the administered naloxone wears off. One solution to these shortcomings is to have naloxone delivered through a sustained release system that would be placed into a patient at the time of contact with the healthcare system. However, prolonged constant release of naloxone could interfere with normal usage of opioids, such as in acute perioperative use, or in treatment of opioid use disorder. Additionally, constant drug release for a problem that is likely to be episodic (overdose) is inefficient and wastes the finite drug payload.

[0005] Therefore, a solution is needed that provides timely and controlled episodic release of naloxone within a patient’s body.SUMMARY

[0006] Described herein are compositions, methods, and kits for reducing or reversing an effect of an opioid.

[0007] According to some embodiments, a conjugate includes a polymer and an active agent. The active agent is linked to the polymer via a cleavable linker. In some embodiments, the active agent is an agent capable of reversing or reducing an effect of an opioid in a subject.

[0008] In some embodiments, a particle includes a conjugate. The conjugate includes a polymer and an active agent. The active agent is an agent capable of reversing or reducing an effect of an opioid in a subject. The active agent is linked to the polymer via a cleavable linker.

[0009] In some embodiments, a hydrogel includes the conjugate. In some embodiments, a depot suitable for implantation into a subject includes the conjugate, the particle, or the hydrogel. According to some embodiments, a pharmaceutical composition includes a therapeutically effective amount of the conjugate, the particle, the hydrogel, or the depot, and a pharmaceutically acceptable excipient.

[0010] According to some embodiments, a method for reducing or reversing an effect of an opioid in a subject includes applying a stimulus at a site of where the subject was administered an effective amount of a conjugate, a particle, a hydrogel, or a depot including an active agent. The active agent is an agent capable of reversing or reducing an effect of an opioid, and the stimulus is capable of cleaving the cleavable linker linking the polymer and the active agent.

[0011] In some embodiments, a method includes administering a conjugate, a particle, a hydrogel, a depot, or a pharmaceutical composition to a subject

[0012] In some embodiments, a kit includes a conjugate, a particle, a hydrogel, a depot, or a pharmaceutical composition.

[0013] According to some embodiments, a method includes administering a conjugate, a particle, a hydrogel, or a depot to a subject.

[0014] In some embodiments, a kit includes a conjugate, a particle, a hydrogel, a depot, or a pharmaceutical composition.BRIEF DESCRIPTION OF THE DRAWINGS

[0015] This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0016] Disclosed herein are embodiments of compositions, methods, and kits pertaining to stimulus-triggered release of naloxone. This description includes drawings, wherein:

[0017] FIG. 1 is a skeletal structure diagram of PLGA-CM-NLX (poly(lactic-co-glycolic acid)-Coumarin-naloxone).

[0018] FIG. 2 is a graph of a dynamic light scattering of PLGA-CM-NLX nanoparticles.

[0019] FIG. 3 is a TEM (transmission electron microscopy) image of PLGA-CM-NLX nanoparticles.

[0020] FIG. 4 is a UV-vis (ultraviolet-visible spectroscopy) graph of a PLGA-CM-NLX solution in acetone and PLGA-CM-NLX nanoparticles in water.

[0021] FIG. 5 is skeletal structure diagram of a scheme of photocleavage releasing naloxone from PLGA-CM-NLX.

[0022] FIG. 6 is a mass spectrum of a cleaved naloxone.

[0023] FIG. 7 is a graph showing the release of naloxone by photocleavage of PLGA-CM-NLX nanoparticles (10 mg / mL) over time under 400 nm LED (light emitting diode) at different irradiances (n=4, data are means ± SD (standard deviation)).

[0024] FIG. 8 is a graph of repeated photo-triggered naloxone release over time (n=4, arrows represent two minutes of irradiation by 400 nm LED at 30 mW / cm2, n=4, data are means ± SD).

[0025] FIG. 9 is a graph of hotplate latency of mice under morphine without (black line) or with (red line) naloxone injection as a function of time (n=4; data are means ± SD).

[0026] FIG. 10 is a graph of hotplate latency of mice under morphine with inj ected PLGA-CM-NLX nanoparticles, without (green line) or with (blue line) subsequent two minutes of irradiation by 400 nm LED at 300 mW / cm2as a function of time (n=4, data are means ± SD; * indicates p < 0.05 and NS indicates p > 0.05 compared to morphine alone;+indicates p < 0.05 and ns indicates p > 0.05 compared to morphine treated with free naloxone). The black dash line is the curve for morphine and the grey dash line is the baseline for untreated animals.

[0027] FIG. 11 is an illustration of an experimental timeline.

[0028] FIG. 12 is a graph of hotplate latency of mice treated with morphine (10 mg / kg) at the times indicated in FIG. 11 after injection of PLGA-CM-NLX nanoparticles, followed by hv. a single two-minute irradiation with a 400 nm LED at 300 mW / cm2, as a function of time. The black dashed line is for morphine not followed by irradiation. The grey dashed line indicates baseline latency for untreated animals. Data are means ± SD, n=4.

[0029] FIG. 13 is a graph of latency at thirty minutes after the irradiation (irradiation occurred 30 minutes after introduction of morphine, for a total latency of 60 minutes) in FIG.12 as a function of time after injection of PLGA-CM-NLX nanoparticles. The yellow dash line is the latency at this time point for morphine, and the blue dashed line is for the morphine treated with free naloxone group. * indicates p < 0.05 and p values in yellow are compared to latency in the morphine only group.+indicates p < 0.05 and p values in blue are compared to morphine treated with free naloxone.

[0030] FIG. 14 is a collection of In Vivo Imaging System (IVIS) images of mice injected with PLGA-CM-NLX nanoparticles covalently labeled with cyanine5.5 (cy5.5).

[0031] FIG. 15 is a graph of relative fluorescent intensity over time from IVIS (n=4, data are means ± SD).

[0032] FIG. 16 is an illustration of an experimental timeline for hotplate latency of mice injected with PLGA-CM-NLX nanoparticles. Morphine was administered at predetermined intervals followed by irradiation thirty minutes later.

[0033] FIG. 17 is a graph of hotplate latency as a function of time on day 3, according to the experimental timeline of FIG. 16. hv indicates two minutes of irradiation by 400 nm LED at 300 mW / cm2. The black dashed line is the curve for morphine and the grey dashed line is the baseline for untreated animals. Data are means ± SD, n=4.

[0034] FIG. 18 is a graph of hotplate latency as a function of time on day 5, according to the experimental timeline of FIG. 16. hv indicates two minutes of irradiation by 400 nm LED at 300 mW / cm2. The black dashed line is the curve for morphine and the grey dashed line is the baseline for untreated animals. Data are means ± SD, n=4.

[0035] FIG. 19 is a graph of hotplate latency as a function of time on day 10, according to the experimental timeline of FIG. 16. hv indicates two minutes of irradiation by 400 nm LED at 300 mW / cm2. The black dashed line is the curve for morphine and the grey dashed line is the baseline for untreated animals. Data are means ± SD, n=4.

[0036] FIG. 20 is a graph of hotplate latency as a function of time on day 20, according to the experimental timeline of FIG. 16. hv indicates two minutes of irradiation by 400 nm LED at 300 mW / cm2. The black dashed line is the curve for morphine and the grey dashed line is the baseline for untreated animals. Data are means ± SD, n=4.

[0037] FIG. 21 is a graph of latency at sixty minutes after morphine injection (from FIGS. 17-20) as a function of time after injection. * indicates p < 0.05 and p values in blue are compared to morphine treated with free naloxone.

[0038] FIG. 22 is a graph of latency of mice receiving two sequential doses of morphine without other injections as a function of time (the dashed curved line). The grey dashed line is the baseline for untreated animals. Data are means ± SD and n=4.

[0039] FIG. 23 is a graph of latency of mice receiving two sequential doses of morphine and a single injection of naloxone at thirty minutes as a function of time (the dashed curved line). The grey dashed line is the baseline for untreated animals. Data are means ± SD and n=4.

[0040] FIG. 24 is a graph of latency of mice receiving two sequential doses of morphine, an injection of PLGA-CM-NLX nanoparticles at time zero, and one irradiation at thirty minutes (hv: two minutes of irradiation by 400 nm LED at 300 mW / cm2) as a function of time (the dashed curved line). The grey dashed line is the baseline for untreated animals. Data are means ± SD and n=4.

[0041] FIG. 25 is a graph of latency of mice receiving two sequential doses of morphine, an injection of PLGA-CM-NLX nanoparticles at time zero, one irradiation at thirty minutes (hv two minutes of irradiation by 400 nm LED at 300 mW / cm2), and one irradiation at ninety minutes as a function of time (the dashed curved line). The grey dashed line is the baseline for untreated animals. Data are means ± SD and n=4.

[0042] FIG. 26 is a collection of images of hematoxylin and eosin (H&E) stained slides of the tissues around the site of injection, including epidermis, dermis, dermal white adipose tissue (dWAT), panniculus carnosus (PC) (a subcutaneous muscle layer occurring in mice), and interstitial connective tissue (ICT). Images a and b are normal tissues and images c-h show tissue reaction to the materials 4, 14, and 30 days after the injection. Mtox is myotoxicity and Infl is inflammation.

[0043] FIGS. 27-30 are skeletal structure diagrams of synthetic routes to make PLGA- coumarin-naloxone (PLGA-CM-NLX). PLGA is poly(lactic-co-glycolic acid), CM is coumarin, and NLX is naloxone.

[0044] FIG. 27 is a skeletal structure diagram of synthetic routes to make Compounds 1- 5. Compound 1 (DIBO-COOH) is created via step (i), which indicates addition of trimethyl silyl iodide and CHCh at 4°C. DIBO is dibenzocyclooctyne. Compound 2 is created via step (ii), which indicates addition of / / -butyl lithium and tetrahydrofuran (THF). Compound 3 is created via step (iii), which indicates addition of bromine and CHCI3. Compound 4 (DIBO-OH) is created via step (iv), which indicates addition of lithium diisopropylamide and THF. Compound 5 is created via step (v), which indicates addition of succinic anhydride, 4- dimethylaminopyridine, and THF.

[0045] FIG. 28 is a skeletal structure diagram of synthetic routes to make Compounds 6- 8. Compound 6 is created via step (vi), which indicates addition of SeO2 and 1,4-dioxane and undergoing reflux. Compound 7 is created via step (vii), which indicates addition of CH3NO2,A,A,A'A'-tetramethylethylenediamine, and THF. Compound 8 (NH2-CM-0H) is created via step (viii), which indicates addition of Zn and acetic acid.

[0046] FIG. 29 is a skeletal structure diagram of synthetic routes to make Compounds 9- 11. Compound 9 (DIBO-CM-OH) is created via step (ix), which indicates addition of N,N- diisopropylethylamine (DIPEA) and hexafluorophosphate azabenzotriazole tetramethyl uranium. Compound 10 (DIBO-CM-NH) is created via step (x), which indicates addition of 4- nitrophenyl chloroformate, DIPEA, A,A'-dimethylethylenediamine, and dichloromethane. Compound 11 (DIBO-CM-NLX) is created via step (xi), which indicates addition of triphosgene, DIPEA, and naloxone hydrochloride.

[0047] FIG. 30 is a skeletal structure diagram of synthetic routes to make Compound 12 (PLGA-CM-NLX) via SPAAC (strain-promoted azide-alkyne click chemistry reaction). Compound 12 is created via step (xiii), which indicates addition of dichloromethane at room temperature.

[0048] FIG. 31 is a Fourier-transform infrared (FT-IR) spectra of DIBO-CM-NLX, PLGA- N3, and PLGA-CM-NLX.

[0049] FIG. 32 is aJH NMR (nuclear magnetic resonance) of PLGA-CM-NLX.

[0050] FIG. 33 is a graph of gel permeation chromatography (GPC) curves of PLGA-N3 and PLGA-CM-NLX as a function of retention time.

[0051] FIG. 34 is a collection of IVIS images of mice injected with free dye cyanine 5.5 (cy5.5) at days 0, 1, 7, 14, and 28. Most fluorescent signals disappeared within one day.

[0052] FIG. 35 is a bar chart of cytotoxicity of PLGA-CM-NLX nanoparticles at various concentrations. Cell survival (%) of 3T3 cells with various concentrations of PLGA-CM-NLX nanoparticles, naloxone, and saline was determined by MTS (3-(4,5-dimethylthiazol-2-yl)-5- (3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay. Data are means ± SD and n=4. P values are compared to the saline group.

[0053] FIG. 36 is a collection of images of hematoxylin and eosin (H&E) stained slides of tissue reaction to subcutaneously injected PLGA-CM-NLX nanoparticles without irradiation. Images a and b are tissue at four days and images c and d are tissue at fourteen days. Mtox is myotoxicity and Infl is inflammation.

[0054] FIG. 37 is a skeletal structure diagram of synthesis of P407-CM-NLX (P407 is Poloxamer 407). The synthesis includes reacting a coumarin-naloxone conjugate with an azide moiety with an alkyne-terminated P407 through copper-catalyzed azide-alkyne cycloaddition (CuAAC).

[0055] FIG. 38 is a graph of storage moduli of P407-CM-NLX, in units of Pascals, as a function of temperature, in units of degrees Celsius.

[0056] FIG. 39 is an image depicting P407-CM-NLX as a liquid at 20°C.

[0057] FIG. 40 is an image depicting P407-CM-NLX as a solid (gel) at 37°C.

[0058] FIG. 41 is a graph of hotplate latency of mice in units of seconds as a function of time in units of minutes. The mice were injected subcutaneously with morphine and P407-CM- NLX at time = 0 minutes. The blue line (with square markers) represents mice that had no subsequent irradiation. The red line (with circle markers) represents mice that had subsequent irradiation at 30 minutes, hv indicates two minutes of irradiation by 400 nm LED at 300 mW / cm2.

[0059] FIG. 42 is a graph of hotplate latency of mice in units of seconds as a function of time in units of minutes. The mice were injected subcutaneously with morphine at time = 0 minutes. The blue line (with square markers) represents mice that were injected subcutaneously with NLX at time = 30 minutes and the red line (with circle markers) represents mice that were injected subcutaneously with P407-CM-NLX at time = 0 minutes and irradiated with light at time = 30 minutes, hv indicates two minutes of irradiation by 400 nm LED at 300 mW / cm2.

[0060] FIG. 43 is a graph of hotplate A latency of mice injected subcutaneously with morphine without (M, black line) or with (M+NLX, red line) naloxone injection at 30 min.

[0061] FIG. 44 is a graph of hotplate A latency of mice injected subcutaneously with morphine with injected NLX-NP at t = 0 min, without ((M+NLX-NP hv (-)), green line) or with ((M+NLX-NP hv (+)), blue line) subsequent irradiation at 30 min.

[0062] FIG. 45 is a bar chart of A Latency at 60 min, from data in FIGS. 43-44.

[0063] FIG. 46 is an illustration of experimental timeline for delayed triggering.

[0064] FIG. 47 is a graph of hotplate A latency of mice treated with morphine at the indicated time after injection of NLX-NP, followed by irradiation.

[0065] FIG. 48 is a graph of hotplate A Latency at 60 min after morphine and 30 min after irradiation (from data in FIGS. 44 and 47) as a function of days after injection of NLX-NP. p values in yellow compare the A latencies of irradiated animals to animals 60 min after morphine; p values in blue compare the A latencies of irradiated animals to those 30 min after free naloxone.

[0066] FIG. 49 is a collection of In Vivo Imaging System (IVIS) images of mice injected with NLX-NP containing PLGA covalently labeled with the fluorescent dye cyanine5.5 (PLGA-cy5.5).

[0067] FIG. 50 is a graph of quantitation of relative fluorescent intensity over time from IVIS data in FIG. 50. All irradiations (hv) in this figure were 2 min with 400 nm LED at 300 mW cm'2. All data are means ± SD, n = 4.

[0068] FIG. 51 is a graph of A Latency as a function of time. Time to onset of morphine (M) reversal by free naloxone (NLX) and NLX-NP with irradiation. NLX injection or light irradiation on NLX-NP was performed 30 min after the initial morphine injection (time zero).

[0069] FIG. 52 is a graph of hotplate A latency of mice treated with morphine (M), and mice treated with morphine at two months (in key: 2 months) after injection of NLX-NP, followed by irradiation, (hv: 2 min irradiation by 400 nm LED at 300 mW cm'2; data are means ± SD, n = 4). No significant difference between animals administered with morphine with or without triggering.

[0070] FIG. 53 is a collection of In Vivo Imaging System (IVIS) images of mice injected with free cy5.5.

[0071] FIG. 54 is a graph of latency of mice receiving two sequential doses of morphine without other injections as a function of time (in minutes), shown as the grey line labeled “M+M.” The dashed line labeled “M” represents latency of mice receiving a single dose of morphine without other injections as a function of time (in minutes). Data are means ± SD and n=4.

[0072] FIG. 55 is a bar chart of hotplate latency (in seconds) at 60 minutes from data in FIGS. 54 and 23-25.

[0073] FIG. 56 is a bar chart of hotplate latency (in seconds) at 120 minutes from data in FIGS. 54 and 23-25.

[0074] FIG. 57 is a skeletal structure diagram of a synthetic route to make CM-NLX (Compound 14).

[0075] FIG. 58 is a skeletal structure diagram of photocleavage of CM-NLX.

[0076] FIG. 59 is a graph of photocleavage of CM-NLX (1 mg / mL) over time with a 400 nm LED at different irradiances. Data are means ± SD and n=4.

[0077] FIG. 60 is a graph of hotplate latency (in seconds) as a function of time in minutes to show time to onset of morphine (M) reversal by free naloxone (NLX) and NLX-NP with irradiation. NLX injection or light irradiation on NLX-NP was performed 30 min after the initial morphine injection (time zero).

[0078] FIG. 61 is a graph of cell survival (determined by MTS assay) of a. 3T3 cells, and b. C2C12 cells exposed to various concentrations of NLX-NP, naloxone. Data are means ± SD (n = 4). P values are in comparison to the saline group.

[0079] FIG. 62 is a1H NMR spectrum of Compound 1.

[0080] FIG. 63 is a1H NMR spectrum of Compound 2.

[0081] FIG. 64 is aJH NMR spectrum of Compound 3.

[0082] FIG. 65 is aJH NMR spectrum of Compound 4 (DIBO-OH).

[0083] FIG. 66 is aJH NMR spectrum of Compound 5 (DIBO-COOH).

[0084] FIG. 67 is aJH NMR spectrum of Compound 9 (DIBO-CM-OH).

[0085] FIG. 68 is aJH NMR spectrum of Compound 10 (DIBO-CM-NH).

[0086] FIG. 69 isJH NMR spectrum of Compound 11 (DIBO-CM-NLX).

[0087] FIG. 70 is 'H NMR spectrum of Compound 13 (CM-NH).

[0088] FIG. 71 is 'H NMR spectrum of Compound 14 (CM-NLX).

[0089] FIG. 72 is an expanded version of the symmetrical skeletal structure diagram ofPLGA-CM-NLX (poly(lactic-co-glycolic acid)-Coumarin-naloxone) in FIG. 1.DETAILED DESCRIPTION

[0090] The present invention is described with reference to the attached figures. The figures are not drawn to scale, and they are provided merely to illustrate the instant invention. Several aspects of the invention are described below with reference to example applications for illustration. It should be understood that numerous specific details, relationships, and methods are set forth to provide a full understanding of the invention. One having ordinary skill in the relevant art, however, will readily recognize that the invention can be practiced without one or more of the specific details or with other methods. In other instances, well- known structures or operations are not shown in detail to avoid obscuring the invention. The present invention is not limited by the illustrated ordering of acts or events, as some acts may occur in different orders and / or concurrently with other acts or events. Furthermore, not all illustrated acts or events are required to implement a methodology in accordance with the present invention.Definitions

[0091] As used herein, the term “comprising” means that other elements can also be present in addition to the defined elements presented. The use of “comprising” indicates inclusion rather than limitation.

[0092] As used herein the term “consisting essentially of’ refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.

[0093] The term “consisting of’ refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

[0094] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, references to “the method” includes one or more methods, and / or steps of the type described herein and / or which will become apparent to those persons skilled in the art upon reading this disclosure and so forth. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise.

[0095] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term “about.”

[0096] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within one or more than one standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 15%, up to 10%, up to 5%, or up to 1% of a given value.

[0097] Where a range of values is provided, each numerical value between the upper and lower limits of the range is contemplated and disclosed herein.

[0098] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art to which this disclosure belongs. It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims. Definitions of common terms in molecular biology can be found in THE MERCK MANUAL OF DIAGNOSIS AND THERAPY, 19th Edition, published by Merck Sharp & Dohme Corp., 2011 (ISBN 978-0-911910-19-3); ROBERT S. PORTER ET AL. (EDS.), THE ENCYCLOPEDIA OF MOLECULAR CELL BIOLOGY AND MOLECULAR MEDICINE,published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and ROBERT A. MEYERS (ED.), MOLECULAR BIOLOGY AND BIOTECHNOLOGY: A COMPREHENSIVE DESK REFERENCE, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); IMMUNOLOGY BY WERNER LUTTMANN, published by Elsevier, 2006; LEWIN’S GENES XI, published by Jones & Bartlett Publishers, 2014 (ISBN-1449659055); MICHAEL RICHARD GREEN AND JOSEPH SAMBROOK, MOLECULAR CLONING: A LABORATORY MANUAL, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012) (ISBN 1936113414); DAVIS ET AL., BASIC METHODS IN MOLECULAR BIOLOGY, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X); LABORATORY METHODS IN ENZYMOLOGY: DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, 2014 (ISBN 047150338X, 9780471503385), and CURRENT PROTOCOLS IN PROTEIN SCIENCE (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005 (ISBN 0471142735), the contents of which are all incorporated by reference herein in their entireties.

[0099] Compounds described herein can include one or more asymmetric centers, and thus can exist in various isomeric forms, e.g., enantiomers and / or diastereomers. For example, the compounds described herein can be in the form of an individual enantiomer, diastereomer or geometric isomer, or can be in the form of a mixture of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomer. Isomers can be isolated from mixtures by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers can be prepared by asymmetric syntheses. See, e.g., JACQUES ET AL., ENANTIOMERS, RACEMATES AND RESOLUTIONS (Wiley Interscience, New York, 1981); WILEN ET AL., TETRAHEDRON 33:2725 (1977); ELIEL, STEREOCHEMISTRY OF CARBON COMPOUNDS (McGraw — Hill, NY, 1962); and WILEN, TABLES OF RESOLVING AGENTS AND OPTICAL RESOLUTIONS 268 (E.L. Eliel, Ed., Univ, of Notre Dame Press, Notre Dame, IN 1972). The invention additionally encompasses compounds described herein as individual isomers substantially free of other isomers, and alternatively, as mixtures of various isomers.

[0100] The term “pharmaceutically acceptable salt” refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, BERGE ET AL., describe pharmaceutically acceptable salts in detail in J. PHARMACEUTICAL SCIENCES, 1977, 66, 1-19, incorporated herein byreference. Pharmaceutically acceptable salts of the compounds of this invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid or by using other methods known in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3 -phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium andN+(Ci-4 alkyljy salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate, and aryl sulfonate.

[0101] The term “solvate” refers to forms of the compound that are associated with a solvent, usually by a solvolysis reaction. This physical association may include hydrogen bonding. Conventional solvents include water, methanol, ethanol, acetic acid, DMSO, THF, diethyl ether, and the like. The compounds of Formula (I) may be prepared, e.g., in crystalline form, and may be solvated. Suitable solvates include pharmaceutically acceptable solvates and further include both stoichiometric solvates and non-stoichiometric solvates. In certain instances, the solvate will be capable of isolation, for example, when one or more solvent molecules are incorporated in the crystal lattice of a crystalline solid. “Solvate” encompasses both solution-phase and isolable solvates. Representative solvates include hydrates, ethanolates, and methanolates.

[0102] The term “hydrate” refers to a compound that is associated with water. Typically, the number of the water molecules contained in a hydrate of a compound is in a definite ratio to the number of the compound molecules in the hydrate. Therefore, a hydrate of acompound may be represented, for example, by the general formula R»x H2O, wherein R is the compound and wherein x is a number greater than 0. A given compound may form more than one type of hydrates, including, e.g., monohydrates (x is 1), lower hydrates (x is a number greater than 0 and smaller than 1, e.g., hemihydrates (R»0.5 H20)), and polyhydrates (x is a number greater than 1, e.g., dihydrates (R»2 H2O) and hexahydrates (R»6 H20)).

[0103] The term “tautomers” refer to compounds that are interchangeable forms of a particular compound structure, and that vary in the displacement of hydrogen atoms and electrons. Thus, two structures may be in equilibrium through the movement of TI electrons and an atom (usually H). For example, enols and ketones are tautomers because they are rapidly interconverted by treatment with either acid or base. Another example of tautomerism is the aci- and nitro- forms of phenylnitromethane, that are likewise formed by treatment with acid or base.

[0104] Tautomeric forms may be relevant to the attainment of the optimal chemical reactivity and biological activity of a compound of interest.

[0105] It is also to be understood that compounds that have the same molecular formula but differ in the nature or sequence of bonding of their atoms or the arrangement of their atoms in space are termed “isomers.” Isomers that differ in the arrangement of their atoms in space are termed “stereoisomers.”

[0106] Stereoisomers that are not mirror images of one another are termed “diastereomers” and those that are non-superimposable mirror images of each other are termed “enantiomers.” When a compound has an asymmetric center, for example, it is bonded to four different groups, a pair of enantiomers is possible. An enantiomer can be characterized by the absolute configuration of its asymmetric center and is described by the R- and S-sequencing rules of Cahn and Prelog, or by the manner in which the molecule rotates the plane of polarized light and designated as dextrorotatory or levorotatory (i.e., as (+) or (-)-isomers respectively). A chiral compound can exist as either individual enantiomer or as a mixture thereof. A mixture containing equal proportions of the enantiomers is called a “racemic mixture.”

[0107] The terms “composition” and “formulation” are used interchangeably.

[0108] A “subject” to which administration is contemplated includes, but is not limited to, humans (i.e., a male or female of any age group, e.g., a pediatric subject (e.g., infant, child, adolescent) or adult subject (e.g., young adult, middle-aged adult, or senior adult)) and / or other non-human animals, for example, mammals (e.g., primates (e.g., cynomolgus monkeys, rhesus monkeys); commercially relevant mammals such as cattle, pigs, horses, sheep, goats, cats, and / or dogs) and birds (e.g., commercially relevant birds such as chickens,ducks, geese, and / or turkeys). In certain embodiments, the animal is a mammal. The animal may be a male or female and at any stage of development. A non-human animal may be a transgenic animal.

[0109] A “patient” refers to a human subject in need of treatment of a disease.

[0110] In jurisdictions that forbid the patenting of methods that are practiced on the human body, the meaning of “administering” to a human patient shall be restricted to prescribing a controlled substance that will be administer to the patent by any technique (e.g., orally, inhalation, topical application, injection, insertion, etc.). The broadest reasonable interpretation that is consistent with laws or regulations defining patentable subject matter is intended. In jurisdictions that do not forbid the patenting of methods that are practiced on the human body, the “administering” includes both methods practiced on the human body and also the foregoing activities.

[0111] The terms “administer,” “administering,” or “administration,” refers to implanting, absorbing, ingesting, injecting, inhaling, or otherwise introducing an inventive compound, or a pharmaceutical composition thereof. Conjugates, particles and compositions described herein can be administered by any appropriate route known in the art including, but not limited to, oral or parenteral routes, including intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, nasal, rectal, and topical (including buccal and sublingual) administration. Exemplary modes of administration include, but are not limited to, injection, infusion, instillation, inhalation, or ingestion. “Injection” includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal, intracerebro spinal, and intrastemal injection and infusion.

[0112] In some embodiments, the conjugate, particle, or composition is administered parenterally, i.e., parenteral administration. The phrases “parenteral administration” and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal, intracerebro spinal, and intrastemal injection, infusion and other injection or infusion techniques, without limitation.

[0113] The terms “treatment,” “treat,” and “treating” refer to reversing, alleviating, delaying the onset of, or inhibiting the progress of a “pathological condition” (e.g., a disease,disorder, or condition, or one or more signs or symptoms thereof) described herein. In some embodiments, treatment may be administered after one or more signs or symptoms have developed or have been observed. In other embodiments, treatment may be administered in the absence of signs or symptoms of the disease or condition. For example, treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and / or in light of genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, for example, to delay or prevent recurrence.

[0114] The terms “condition,” “disease,” and “disorder” are used interchangeably.

[0115] An “effective amount” of an agent described herein refers to an amount sufficient to elicit the desired biological response. In certain embodiments, an effective amount is an amount of the prodrug agent sufficient to elicit the desired biological response, upon cleavage of the cleavable linker between the agent X and the polymer P. An effective amount of an agent described herein may vary depending on such factors as the desired biological endpoint, the pharmacokinetics of the agent, the condition being treated, the mode of administration, and the age and health of the subject. In certain embodiments, an effective amount is a therapeutically effective amount. In certain embodiments, an effective amount is a prophylactic treatment. In certain embodiments, an effective amount is the amount of an agent described herein in a single dose. In certain embodiments, an effective amount is the combined amounts of an agent described herein in multiple doses. When an effective amount of a composition is referred herein, it means the amount is prophylactically and / or therapeutically effective, depending on the subject and / or the disease to be treated. Determining the effective amount or dosage is within the abilities of one skilled in the art.

[0116] A “therapeutically effective amount” of an agent described herein is an amount sufficient to provide a therapeutic benefit in the treatment of a condition or to delay or minimize one or more symptoms associated with the condition. A therapeutically effective amount of an agent means an amount of therapeutic agent, alone or in combination with other therapies, which provides a therapeutic benefit in the treatment of the condition. In certain embodiments, a therapeutically effective amount is an amount of the prodrug agent, alone or in combination with other therapies, which provides a therapeutic benefit in the treatment of the condition, upon cleavage of the cleavable linker between the agent X and the polymer P. The term “therapeutically effective amount” can encompass an amount that improves overall therapy, reduces or avoids symptoms, signs, or causes of the condition, and / or enhances the therapeutic efficacy of another therapeutic agent. In certain embodiments, a therapeutically effectiveamount is an amount of the prodrug agent sufficient to elicit the desired biological response, upon cleavage of the cleavable linker between the agent X and the polymer P.

[0117] Embodiments of the various aspects described herein include a photocleavable linker. The term “photocleavable” is used herein in a manner well known to those skilled in the art and refers to a group, e.g., a linker having a specific bond that can be cleaved by irradiation at a specific wavelength. As used herein, the term “photocleavable linker” refers to any chemical group that attaches or operably links two molecules, e.g., a polymer and naloxone. A photocleavable linker is a linker that can be cleaved by exposure to electromagnetic radiation (e.g., visible light, UV light, infrared, etc.). The wavelength of light necessary to photocleave the linker is dependent upon the structure of the photocleavable linker used. Any art-recognized photocleavable linker can be used for the conjugates described herein. Exemplary photocleavable linker include, but are not limited to, chemical molecules containing a coumarin moiety, an o-nitrobenzyl moiety, a p-nitrobenzyl moiety, a m- nitrobenzyl moiety, a nitoindoline moiety, a bromo hydroxycoumarin moiety, a bromo hydroxyquinoline moiety, a hydroxyphenacyl moiety, a dimethozybenzoin moiety, or any combinations thereof.

[0118] Exemplary photocleavable linkers are described, for example, in PCT / US2021 / 013628; US Pat. No. 430,175; US Pat. No. 5,635,608; US Pat. No. 5,872,243; US Pat. No. 5,888,829; US Pat. No. 6,043,065; US PGPUB No. 2010 / 0105120; Olejnik et al; Photocleavable Peptide-DNA Conjugates: Synthesis and Applications to DNA Analysis Using MALDI-MS, Nucleic Acids Research 27(2): 4626-4631, 1999; J. Olejnik and K. Rothschild, Methods Enzymol 291 : 135-154, 1998; Pelliccioli et al., Photoremovable protecting groups: reaction mechanisms and applications, Photochem. Photobiol. Sci. 1 441-458 (2002); Goeldner and Givens, Dynamic Studies in Biology, Wiley-VCH, Weinheim (2005); Marriott, Methods in Enzymology, Vol. 291, Academic Press, San Diego (1998); Morrison, Bioorganic Photochemistry, Vol. 2, Wiley, New York (1993); Adams and Tsien, Annu. Rev. Physiol. 55 755-784 (1993); Mayer et al., Biologically Active Molecules with a Light Switch, Angew. Chem. Int. Ed. 45 4900-4921 (2006); Pettit et al., Neuron 19 465-471 (1997); and Furuta et al., Brominated 7- hydroxycoumarin-4-ylmethyls: Photolabile protecting groups with biologically useful cross- sections for two photon photolysis, Proc. Natl. Acad. Sci. USA 96 1193-1200 (1999), contents of all of which are incorporated by reference herein.

[0119] In some embodiments of any one of the aspects described herein, the photocleavable linkers is based on the coumarin system, such as BHC (Furuta and Iwamura, Methods Enzymol. 291 50-63 (1998); Furuta et al., Proc. Natl. Acad. Sci. USA 96 1 193-1200(1999); Suzuki et al., Org. Lett. 5:4867 (2003); U.S. Patent No. 6,472,541, the disclosure of which is incorporated by reference herein). The DMACM linkage photocleaves in nanoseconds (Hagen et al., [7-(Dialkylamino)coumarin-4-yl]methyl-caged Compounds as Ultrafast and Effective Long-Wavelength Phototriggers of 8-Bromo-Substituted Cyclic Nucleotides, Chem Bio Chem 4 434-442 (2003)) and is cleaved by visible light (U.S. Patent Application Serial No. 11 / 402,715 the disclosure of which is incorporated by reference herein). Coumarin-based photolabile linkages are also available for linking to aldehydes and ketones (Lu et al., BHC- diol as a photolabile protecting group for aldehydes and ketones, Org. Lett. 5 2119-2122 (2003)). Closely related analogues, such as BHQ, are also suitable (Fedoryak et al., Brominated hydroxyquinoline as a photolabile protecting group with sensitivity to multiphoton excitation, Org. Lett. 4 3419-3422 (2002)). Another suitable photocleavable linker comprises the pHP group (Park and Givens, J. Am. Chem. Soc. 119:2453 (1997), Givens et al., New Phototriggers 9: p -Hydroxyphenacyl as a CTerminal Photoremovable Protecting Group for Oligopeptides, J. Am. Chem. Soc. 122 2687-2697 (2000); Zhang et aL, J. Am. Chem. Soc. 121 5625-5632, (1999); Conrad et al., J. Am. Chem. Soc. 122 9346-9347 (2000); Conrad et al., Org. Lett. 2 1545-1547 (2000)). A ketoprofen derived photolabile linkage is also suitable (Lukeman et al., Carbanion-Mediated Photocages: Rapid and Efficient Photorelease with Aqueous Compatibility, J. Am. Chem. Soc. 127 7698-7699 (2005)).

[0120] In some embodiments, the photocleavable linker is a chromophore. Examples of chromophores which are photoresponsive include, but are not limited to, acridines, nitroaromatics, and arylsulfonamides. The efficiency and wavelength at which the chromophore becomes photoactivated and thus releases naloxone will vary depending on the particular functional group(s) attached to the chromophore. For example, when using nitroaromatics, such as derivatives of onitrobenzylic compounds, the absorption wavelength can be significantly lengthened by addition of methoxy groups.

[0121] In some embodiments, the photocleavable linker can comprise a nitro-aromatic compound. Exemplary photocleavable linkers having an ortho-nitro aromatic core scaffold include, but are not limited to, ortho-nitro benzyl (ONB), l-(2-nitrophenyl)ethyl (NPE), alpha- carboxy2-nitrobenzyl (CNB), 4,5-dimethoxy-2-nitrobenzyl (DMNB), 1 -(4, 5 -dimethoxy - 2nitrophenyl)ethyl (DMNPE), 5-carboxymethoxy-2-nitrobenzyl (CMNB) and ((5carboxymethoxy-2-nitrobenzyl)oxy)carbonyl (CMNCBZ) photolabile cores. It will be appreciated that the substituents on the aromatic core are selected to tailor the wavelength of absorption, with electron donating groups (e.g., methoxy) generally leading to longer wavelength absorption. For example, nitrobenzyl (NB) and nitrophenyl ethyl (NPE) aremodified by addition of two methoxy residues into 4,5-dimethoxy-2-nitrobenzyl and l-(4,5- dimethoxy-2-nitrophenyl)ethyl, respectively, thereby increasing the absorption wavelength range to 340-360 nm.

[0122] Further, other ortho-nitro aromatic core scaffolds include those that trap nitroso byproducts in a hetero Diels Alder reaction as generally discussed in U.S. Patent Publication No. 2010 / 0105120 and Pirrung et al., J. Org. Chem. 68: 1 138 (2003). The nitrodib enzofurane (NDBF) chromophore offers an extinction coefficient significantly higher in the near UV region but it also has a very high quantum yield for the deprotection reaction and it is suitable for twophoton activation (Momotake et al, The nitrodibenzofuran chromophore: a new caging group for ultra-efficient photolysis in living cells, Nat. Methods 3 35-40 (2006)). The NPP group is an alternative introduced by Pfleiderer et al. that yields a less harmful nitrostyryl species (Walbert et al., Photolabile Protecting Groups for Nucleosides: Mechanistic Studies of the 2-(2-Nitrophenyl)ethyl Group, Helv. Chim. Acta 84 1601 -161 1 (2001)).

[0123] In exemplary embodiments involving UV light, the photocleavable linkers can be selected from the group consisting of alpha-carboxy-2-nitrobenzyl (CNB, 260 nm), 1- (2nitrophenyl)ethyl (NPE, 260 nm), 4,5-dimethoxy-2-nitrobenzyl (DMNB, 355 nm), 1- (4,5dimethoxy-2-nitrophenyl)ethyl (DMNPE, 355 nm), (4,5-dimethoxy-2- nitrobenzoxy)carbonyl (NVOC, 355 nm), 5-carboxymethoxy-2-nitrobenzyl (CMNB, 320 nm), ((5-carboxymethoxy- 235 nitrobenzyl)oxy)carbonyl (CMNCBZ, 320 nm), desoxybenzoinyl (desyl, 360 nm), and anthraquino2-ylmethoxy carbonyl (AQMOC, 350 nm).

[0124] A photocleavable linker is cleaved by exposure to light of an appropriate wavelength, e.g., blue light. For example, the photocleavable linker is cleaved by exposure to a wavelength ranging from about 380 to 780 nm (the exact wavelength or wavelength range will depend on the specific photocleavable linker used, and could be, for example, 380, 400, 450, 500, 550, 600, 650, 700, 750, or 780, or some range therebetween)..

[0125] An “acid-cleavable linker” refers to a linker which is capable of being split in response to a change (decrease) in the pH (e.g., increased acidity). Examples of acid-cleavable linkers include, but are not limited to, hydrazones, cis-Aconityl, esters, esters of amino acids, — C=NN — and — OC(O) — , appropriately substituted benzyloxycarbonyl groups, and appropriately substituted diphenyl- methylamino groups.

[0126] A “base-cleavable linker” refers to a linker which is capable of being split in response to a change (increase) in the pH (e.g., decreased acidity). Examples of base-cleavable linkers include, but are not limited to, groups containing ester linkages.

[0127] A “chemically cleavable linker” refers to a linker which is capable of being split in response to the presence of a chemical (e.g., acid, base, oxidizing agent, reducing agent, Pd(O)). Examples of chemically cleavable linkers include, but are not limited to, 3,3'- dithiobis(sulfosuccinimidyl propionate), and dialkoxydiphenylsilane (DADPS) and azobenzene (AZO) moieties.

[0128] An “enzymatically cleavable linker” refers to a linker which is subject to degradation by one or more enzymes. Examples of enzymatically cleavable linkers include, but are not limited to, peptide-based linkers such as a valine-citrulline(Val-Cit) dipeptide linker and a phenylalanine-lysine (Phe-Lys) dipeptide linker, — NHCHRAC(O)NHCHRBC(O) — , where RA and RB are the R groups of the two adjacent amino acids, and P-glucuronide linkers.

[0129] A “ribozyme-cleavable linker” refers to a linker which is capable of being split in response to a ribozyme (an RNA molecule capable of acting as an enzyme).

[0130] A “redox-cleavable linker” refers to a linker which is capable of being split in response to a redox reaction (oxidation-reduction reaction). Examples of redox-cleavable linkers include, but are not limited to, — S — S — and — C(R)2 — S — S — , wherein R is H or C1-C6 alkyl and at least one R is C1-C6 alkyl such as CH3 or CH2CH3).

[0131] A “nucleophile-cleavable linker” refers to a linker which is capable of being split in response to a nucleophile (e.g., halogen ions, hydroxide ions, azides, amines). Examples of nucleophile-cleavable linkers include, but are not limited to, phenyl sulfonate, dimedoneenamines (Dde)-type, and keto-acyl linkers.

[0132] An “electrophile-cleavable linker” refers to a linker which is capable of being split in response to an electrophile (e.g., hydronium ions, boron trifluoride, aluminum chloride, halogen molecules).

[0133] A “temperature-cleavable linker” refers to a linker which is capable of being split in response to an increase and / or decrease in temperature.

[0134] A “spontaneous-hydrolysis-cleavable linker” refers to a linker which is capable of being split in response to a spontaneous-hydrolysis-reaction.

[0135] A “reactive-oxygen-species-cleavable linker” refers to a linker which is capable of being split in response to a reactive oxygen species (an oxygen-containing unstable molecule; e.g., superoxide anion (O2'), hydrogen peroxide, hydroxyl radical HO»).

[0136] An “ultrasound-cleavable linker” refers to a linker which is capable of being split in response to sound or other vibrations having an ultrasonic frequency.

[0137] An “electromagnetic-radiation-cleavable linker” refers to a linker which is capable of being split in response to electromagnetic radiation (e.g., radio waves, gamma-rays, visible light).

[0138] Embodiments of the various aspects described herein include a polymer. As used herein, the term “polymer” refers to oligomers, co-oligomers, polymers and co-polymers, e.g., random block, multiblock, star, grafted, gradient copolymers and combination thereof.

[0139] In some embodiments, the polymer is a biocompatible polymer. The term “biocompatible polymer” refers to polymers which are non-toxic, chemically inert, and substantially non-immunogenic when used internally in a subject and which are substantially insoluble in blood. The biocompatible polymer can be either non-biodegradable or biodegradable. Preferably, the biocompatible polymer is also noninflammatory when employed in situ.

[0140] Biodegradable polymers are disclosed in the art. Examples of suitable biodegradable polymers include, but are not limited to, linear-chain polymers such as polylactides, polyglycolides, polycaprolactones, copolymers of polylactic acid and polyglycolic acid, polyanhydrides, polyepsilon caprolactone, polyamides, polyurethanes, polyesteramides, polyorthoesters, polydioxanones, polyacetals, polyketals, polycarbonates, polyorthocarbonates, polydihydropyrans, polyphosphazenes, polyhydroxybutyrates, polyhydroxy valerates, polyalkylene oxalates, polyalkylene succinates, poly(malic acid), poly(amino acids), polyvinylpyrrolidone, polyethylene glycol, polyhydroxycellulose, polymethyl methacrylate, chitin, chitosan, copolymers of polylactic acid and polyglycolic acid, poly(glycerol sebacate) (PGS), and copolymers, terpolymers, and copolymers including one or more of the foregoing. Other biodegradable polymers include, for example, gelatin, collagen, silk, chitosan, alginate, cellulose, poly-nucleic acids, etc.

[0141] Suitable non-biodegradable biocompatible polymers include, by way of example, cellulose acetates (including cellulose diacetate), polyethylene, polypropylene, polybutylene, polyethylene terphthalate (PET), polyvinyl chloride, polystyrene, polyamides, nylon, polycarbonates, polysulfides, polysulfones, hydrogels (e.g., acrylics), polyacrylonitrile, polyvinylacetate, cellulose acetate butyrate, nitrocellulose, copolymers of urethane / carb onate, copolymers of styrene / maleic acid, poly(ethylenimine), Poloxamers (e.g. Pluronic such as Pol oxamers 407 and 188), Hyaluron, heparin, agarose, Pullulan, and copolymers including one or more of the foregoing, such as ethylene / vinyl alcohol copolymers (EVOH).

[0142] In some embodiments, the biocompatible polymer is a copolymer of polylactic acid and polyglycolic acid, poly(glycerol sebacate) (PGS), poly(ethylenimine), Pluronic (Pol oxamers 407, 188), Hyaluron, heparin, agarose, or Pullulan.

[0143] The term “block polymer” described herein means a polymer including at least two different blocks and preferably at least three different blocks.

[0144] “Naloxone” (“NLX”) described herein means 17-allyl-4,5a-epoxy-3,14- dihydroxymorphinan-6-one (CAS Number 465-65-6, also referred to as “naloxone free base”) and / or pharmaceutically acceptable salts thereof.

[0145] “Hydrogel” described herein means a hydrophilic or amphiphilic polymeric network composed of homopolymers or copolymers, which is insoluble due to the presence of covalent chemical crosslinks. The crosslinks provide the network structure and physical integrity. Hydrogels exhibit a thermodynamic compatibility with water which allows them to swell in aqueous media. Generally, a hydrogel is a three-dimensional polymeric structure that is insoluble in water or some other liquid but which is capable of absorbing and retaining large quantities of water or some other liquid to form a stable, often soft and pliable, structure. In some embodiments, water or some other liquid can penetrate in between the polymer chains of the polymer network, subsequently causing swelling and the formation of a hydrogel.

[0146] As used herein, the term “opioid receptor antagonist” refers compounds which are capable to block or even reverse the biological responses induced by the opioid receptor activity. The opioid receptor is activated by opioid like substances of the body. These opioid like substances are peptides which are capable of binding to the opioid receptors, e.g., the endorphins, endomorphins, enkephalines, dynorphins, and deltorphins, as well as nociceptine, P-casomorphin-5, morphiceptin, and dermorphin. Exemplary opioid receptor antagonists include, but are not limited to, naloxone, naltrexone, nalmefene, naloxonazine, nor-binaltorphimine, naltrindole or combinations thereof.

[0147] As used herein, the term “competitive receptor opioid antagonist” refers to a compound that binds to an opioid receptor site; its effects may be overcome by increased concentration of the agonist (e.g., an opioid).

[0148] As used herein, the term “opioid” refers to a compound that exhibits opium or morphine-like properties, and includes active and antagonist activity, which compounds include the brain and interact with stereospecific and saturated binding sites in other tissues. Generally, opioids bind to the opioid receptors. Exemplary opioids include, but are not limited to, morphine, hydromorphone, oxymorphone, dihydromorphine, diacetyl morphine (diamorphine or heroin), codeine, hydrocodone, oxycodone, methadone, theabine, pethidine(meperidine), alfentanil, fentanyl, remifentanil, sufentanil, etorphine, carefentanil, buorenophine, tramadol, venlafaxine, or a pharmaceutically acceptable salt thereof, or an analogue or derivative thereof. In some embodiments, the opioid is a synthetic or semisynthetic molecule.

[0149] As used herein, an “opiate” refers to any compound having a morphine-based ring structure such that the structure-activity relationships of the compound results in physiological binding affinity to an opiate receptor (i.e., for example, Kd =10'3to 10'12M).Conjugates

[0150] According to some embodiments, a conjugate includes a polymer; and an active agent. The active agent is an agent capable of reversing or reducing an effect of an opioid in a subject. The active agent is linked to the polymer via a cleavable linker. In some embodiments, the agent capable of reversing or reducing an effect of an opioid is a competitive opioid receptor antagonist.

[0151] In some embodiments, the opioid receptor has a structure similar to an opioid without an agonistic effect on the opioid receptor itself but binds to a receptor or competitively binds to an analgesic agent. In some embodiments, the agent capable of reversing or reducing an effect of an opioid is a competitive opioid receptor antagonist.

[0152] In some embodiments, the agent capable of reversing or reducing an effect of an opioid selectively blocks an opioid receptor of any type (e.g., p, 5, K, etc.) or subtype (e.g., p 1 or p 2).

[0153] In some embodiments, the agent capable of reversing or reducing an effect of an opioid is naloxone (NLX) or a pharmaceutically acceptable salt thereof, or an analogue or derivative thereof. In some embodiments, the opioid is an opiate.

[0154] According to some embodiments, the cleavable linker is a photocleavable linker. In some embodiments, the photocleavable linker is cleaved by exposure to light having an average wavelength of between about 300 nm and about 900 nm, of between about 350 nm and about 530 nm, or of between about 375 nm and about 475 nm. In some embodiments, the photocleavable linker is cleaved by exposure to light having an average wavelength of about 400 nm.

[0155] In some embodiments, the photocleavable linker includes a coumarin (“CM”) moiety, a o-nitrobenzyl moiety, a p-nitrobenzyl moiety, a m-nitrobenzyl moiety, a benzoin moiety, a nitroindoline moiety, a quinoline moiety, a hydroxyphenacyl moiety, or any combination thereof. In some embodiments, the photocleavable linker includes a coumarinmoiety, a o-nitrobenzyl moiety, a benzoin moiety, a 7-nitroindoline moiety, a p- hydroxyphenacyl moiety, a hydroxyquinoline moiety, or any combination thereof. According to some embodiments, the photocleavable linker includes a coumarin moiety. In some embodiments, the photocleavable linker includes 7- (diethylamino) coumarin-4-yl] methyl carboxyl.

[0156] In some embodiments, the photocleavable linker has the following structure:, where each R is independently a polymer and R’ is the agent capable of reversing or reducing an effect of an opioid.

[0157] The cleavable linker may be cleaved in any suitable fashion. In some embodiments, the cleavable linker is a chemically cleavable linker, an enzymatically cleavable linker, a ribozyme-cleavable linker, a redox-cleavable linker, an acid-cleavable linker, a base-cleavable linker, a nucleophile-cleavable linker, an electrophile-cleavable linker, a temperature-cleavable linker, a spontaneous-hydrolysis-cleavable linker, a reactive-oxygen-species-cleavable linker, an ultrasound-cleavable linker, and / or an electromagnetic-radiation-cleavable linker.

[0158] In some embodiments, the polymer is a hydrophilic polymer of an amphiphilic polymer. In some embodiments, the polymer is selected from one or more of the following groups: polylactides, polyglycolides, polycaprolactones, polyanhydrides, polyepsilon caprolactone, polyamides, polyurethanes, polyesteramides, polyorthoesters, polydioxanones, polyacetals, polyketals, polycarbonates, polyorthocarbonates, polydihydropyrans, polyphosphazenes, polyhydroxybutyrates, polyhydroxyvalerates, polyalkylene oxalates, polyalkylene succinates, poly(malic acid), poly(amino acids), polyvinylpyrrolidone, polyetheres (e.g., polyethylene glycol), polyhydroxy cellulose, polymethyl methacrylate, chitin, chitosan, poly(glycerol sebacate) (PGS), cellulose acetates (including cellulose diacetate), polyethylene, polypropylene, polybutylene, polyethylene terphthalate (PET), polyvinyl chloride, polystyrene, polyamides, nylon, polycarbonates, polysulfides, polysulfones, hydrogels (e.g., acrylics), polyacrylonitrile, polyvinylacetate, cellulose acetate butyrate,23SUBSTITUTE SHEET (RULE 26)nitrocellulose, copolymers of ur ethane / carb onate, copolymers of styrene / maleic acid, poly(ethylenimine), poloxamers (e.g., poloxamer P407, poloxamer P331, poloxamer P188, pol oxamer 182, poloxamer Pl 24, poloxamer P338, or poloxamer P237), and copolymers or terpolymers, thereof.

[0159] In some embodiments, the polymer is poly(lactic-co-glycolic acid) (PLGA), a copolymer including PLGA, or a poloxamer. In some embodiments, a ratio of lactide to glycolide in the PLGA is between 50:50 and 100:0, between 50:50 and 90: 10, between 50:50 and 75:25, and / or any other suitable ratio. In some embodiments, the polymer is a biocompatible polymer and / or is biodegradable.

[0160] In some embodiments, the conjugate is of Formula (I):P-(L-X)n(Formula I). n is an integer of 1 or more. Each instance of X is independently the agent capable of reversing or reducing an effect of an opioid. Each instance of L is independently the cleavable linker; and P is the polymer.

[0161] In some embodiments, n is an integer between 1 and 100,000, inclusive. For example, in some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, n is 2. In some embodiments, a first agent capable of reversing or reducing an effect of an opioid is linked to a first end of the polymer and a second agent capable of reversing or reducing an effect of an opioid is linked to a second end of the polymer.

[0162] In some embodiments, the conjugate is:, the full symmetrical structure of which is provided below:

[0163] In some embodiments, the conjugate is formulated in a particle. In some embodiments, the conjugate is formulated in a hydrogel.24SUBSTITUTE SHEET (RULE 26)Particles

[0164] The conjugates described herein can be included in a particle. Accordingly, in another aspect provided herein is a particle comprising a conjugate described herein. In some embodiments, a particle includes any of the conjugates described herein.

[0165] A particle is not limited to a particular shape and size and can include spherical, rod like, faceted, plates, shells, oviods, or other shapes. Particles can be monodisperse or polydisperse. The size distribution of particles can be characterized by Poly dispersity index (PDI). PDI of particle size distribution is determined by methods commonly known by one of ordinary skill in the art, for example, by dynamic light scattering (DLS) measurement. With regard to DLS used for particle size determinations, the common use of second or third order cumulant analyses to fit the autocorrelation function leads to the values of PDI. The absolute value of PDI determined from this method ranges from zero and higher, with small values indicating narrower distributions. For example, PDI ranging from 0 to about 0.3 or from 0 to about 0.4 presents relatively monodisperse particle size distributions. This criterion has been generally accepted in the art of dynamic light scattering for particle size determinations. In some embodiments the particles are monodisperse. For example, the particles have a narrow particle size distribution such as having a poly dispersity index below about 0.5, such as below about 0.4, below about 0.3 or below about 0.2.

[0166] In some embodiments, the size and morphology (e.g., diameter, sphericity, and porosity) of particles may be characterized by techniques including, but not limited to, dynamic light scattering, coulter counter, microscopy, sieve analysis, dynamic image analysis, static image analysis, and laser diffraction.

[0167] The particle may be of any suitable size. For example, the particle may be a nanoparticle or a microparticle. In some embodiments, the particle is a microparticle. As used herein, the term “microparticle” refers to particles that are from about 1pm to about 500 pm in size. For example, a microparticle is from about 1pm to about 500 pm in size. In some embodiments, the microparticle is from about 1 pm to about 250 pm, from about 1 pm to about 200 pm, from about 1 pm to about 150 pm or from about 1 pm to about 100 pm in size. For example, the microparticle is from about 1 pm to about 75 pm or from about 1 pm to about 100 pm in size.

[0168] In some embodiments, the particle is a nanoparticle. As used herein, the term “nanoparticle” refers to particles that are from about 1 nm to about 950 nm in size. For example, a nanoparticle is from about 10 nm to about 500 nm in size. In some embodiments,the nanoparticle is from about 10 nm to about 250 nm, from about 25 nm to about 200 nm, from about 50 nm to about 175 nm or from about 75 nm to about 150 nm in size. For example, the nanoparticle is from about 80 nm to about 125 nm or from about 90 nm to about 120 nm in size.

[0169] In some embodiments, the particle has a hydrodynamic diameter of less than about 1000 nm, of less than about 750 nm, of less than about 700 nm, of less than about 600 nm, of less than about 550 nm, and / or of less than about 550 nm. In some embodiments, the particle has a hydrodynamic diameter of from about 25 nm to about 400 nm, of from about 50 nm to about 300 nm, of from about 75 nm to about 200 nm, of from about 80 nm to about 150 nm, of about 85 nm to about 125 nm, and / or of from about 90 nm to about 110 nm. The particle may be of any suitable shape. For example, the particle may be substantially spherical, substantially ovoid-shaped, oblong, irregularly shaped, conical, and / or any other suitable shape. In some embodiments, the particle further includes a dye. A benefit of including a dye is, inter alia, providing a visible indicator of where the particle is located in a subject.Hydrogel

[0170] In some embodiments, a conjugate or particle described herein is comprised in a hydrogel, the particle is a hydrogel particle. In some embodiments, the hydrogel is a crosslinked polysaccharide hydrogel. In some embodiments, the hydrogel comprises a conjugate, such as any one of the conjugates described herein. In some embodiments, hydrogel comprises a particle comprising a conjugate described herein, such as any one of the particles described herein. In some embodiments, the hydrogel includes a dye. The dye may be useful for providing a visual indication of a location of the hydrogel.Depot

[0171] In some embodiments, a depot suitable for implantation into a subject includes any one of the conjugates described herein, any one of the particles described herein, or any one of the hydrogels described herein.Pharmaceutical Compositions

[0172] In some embodiments, a pharmaceutical composition includes a therapeutically effective amount of any of the conjugates described herein, any one of the particles described herein, any one of the hydrogels described herein, any of the depots described herein, and / or a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical compositionis formulated for systemic administration. In some embodiments, the pharmaceutical composition is formulated for parenteral administration. In some embodiments, the pharmaceutical composition is formulated for intravenous, intramuscular, subcutaneous, and / or transdermal administration. However, the pharmaceutical composition is not limited to these forms of administration, and the pharmaceutical administration may be administered by any suitable means, such as via microneedles and / or via a microneedle patch. In some embodiments, the pharmaceutical composition further includes a dye. The dye is useful for providing a visual indicator of a location of the pharmaceutical composition.Methods of Treatment

[0173] In some embodiments, a method for reducing or reversing an effect of an opioid in a subject includes applying a stimulus at a site of where the subject was administered an effective amount of any one of the conjugates described herein, any one of the particles described herein, any one of the hydrogels described herein, any one of the depots described herein, and / or any one of the pharmaceutical compositions described herein. The active agent is an agent capable of reversing or reducing an effect of an opioid, and the stimulus is capable of cleaving the cleavable linker linking the polymer and the active agent.

[0174] The stimulus may be applied for any suitable amount of time. For example, the stimulus may be applied for 30 minutes or less, for 15 minutes or less, for 10 minutes or less, for 5 minutes or less, for three minutes or less, and / or until at least an effective amount of the conjugate, particle, hydrogel and / or the depot is administered.

[0175] In some embodiments, the method includes applying the stimulus for a first period of time, waiting for a first period of time, and applying the stimulus for a second period of time. The first and second periods of time may be any suitable amount of time. For example, the first and second periods of time may be at least cumulatively long enough to administer an effective amount of the conjugate, particle, hydrogel, depot, and / or pharmaceutical composition. As another example, the first and second periods of time may each be short enough to avoid, or mitigate, any negative effects (e.g., burns for a temperature stimulus) caused by the stimulus. The first and second periods of time may be the same or similar but may also be different. In some embodiments, the first period of time and / or the second period of time are less than six hours, less than two hours, less than one hour, less than 45 minutes, less than 30 minutes, less than 20 minutes, less than 15 minutes, less than 10 minutes, less than 5 minutes, less than 3 minutes, less than 2 minutes, less than 1 minute, and / or less than 30 seconds.

[0176] In some embodiments, the cleavable linker is a photocleavable linker, and the stimulus is light. In some such embodiments, the light includes near-infrared (NIR) light, and / or visible light. In some embodiments, the light may be of a wavelength capable of cleaving the cleavable linker. In some embodiments, the light has an average wavelength of between about 300 nm and about 900 nm, between about 350 nm and about 530 nm, between about 375 nm and about 475 nm, and / or about 400 nm. In some embodiments, the light is blue light. In some embodiments, the light is red light, green light, yellow light, and / or any other suitable color light.

[0177] In some embodiments, the light has an intensity of at least 50 mW / cm2, at least 100 mW / cm2, at least 150 mW / cm2, at least 200 mW / cm2, at least 250 mW / cm2, at least 300 mW / cm2, at least 350 mW / cm2, at least 400 mW / cm2, at least 500 mW / cm2, at least 750 mW / cm2, and / or at least 1000 mW / cm2. In some embodiments, the light is applied by a LED (light emitting diode).

[0178] In some embodiments, the cleavable linker is a chemically cleavable linker and the stimulus is a reagent for cleaving the chemically cleavable linker. In some embodiments, the cleavable linker is an enzymatically cleavable linker and the stimulus is an enzyme. In some embodiments, the cleavable linker is a ribozyme-cleavable linker and the stimulus is a ribozyme. In some embodiments, the cleavable linker is a redox-cleavable linker and the stimulus is a redox agent. In some embodiments, the cleavable linker is an acid-cleavable linker and the stimulus is an acid. In some embodiments, the cleavable linker is a base-cleavable linker and the stimulus is a base.

[0179] In some embodiments, the cleavable linker is a nucleophile-cleavable linker and the stimulus is a nucleophile. In some embodiments, the cleavable linker is an electrophile- cleavable linker and the stimulus is an electrophile. In some embodiments, the cleavable linker is a temperature-cleavable linker and the stimulus is a change in temperature. In some embodiments, the cleavable linker is a spontaneous-hydrolysis-cleavable linker and the stimulus is a hydrolysis reagent. In some embodiments, the cleavable linker is a reactive- oxygen-species-cleavable linker and the stimulus is a reactive-oxygen-species or a reagent capable of generating a reactive-oxygen-species. In some embodiments, the cleavable linker is an ultrasound-cleavable linker and the stimulus is ultrasound. In some embodiments, the cleavable linker is an electromagnetic-radiation-cleavable linker and the stimulus is electromagnetic radiation.

[0180] In some embodiments, the method further includes administering any one of the conjugates described herein, any one of the particles described herein, any one of the hydrogelsdescribed herein, any one of the depots described herein, and / or any one of the pharmaceutical compositions described herein, to the subject prior to applying the stimulus. In some embodiments, said administering includes parenteral administration. In some embodiments, said administering includes intravenous, intramuscular, subcutaneous or transdermal administration. In some embodiments, said administering includes subcutaneous injection. In some embodiments, said administering is via microneedles. In some embodiments, said administering is via a microneedle patch. In some embodiments, said administering is in a wrist of the subject.

[0181] In some embodiments, the subject is overdosed on an opioid. In some embodiments, the subject has an opioid addiction and / or abuses opioids. In some embodiments, the subject is human.Kits

[0182] In some embodiments, a kit includes any one of the conjugates described herein, any one of the particles described herein, any one of the hydrogels described herein, any one of the depots described herein, and / or any one of the pharmaceutical compositions described herein. In some embodiments, the kit includes a means for administering the conjugate, the particle, the hydrogel, the depot, and / or the pharmaceutical composition to a subject. In some embodiments, the kit includes a means for cleaving the cleavable linker.

[0183] In some embodiments, the cleavable linker is a photocleavable linker and the kit includes a light emitting device. The light emitting device may be used for cleaving the photocleavable linker. In some embodiments, the light emitting device emits near-infrared light. In some embodiments, the light emitting device emits visible light. In some embodiments, the light emitting device emits a light with an average wavelength of between about 300 nm and about 900 nm, between about 350 nm and about 530 nm, between about 375 nm and about 475 nm, and / or about 400 nm. In some embodiments, the light is blue light. In some embodiments, the light has an intensity of at least 50 mW / cm2, at least 100 mW / cm2, at least 150 mW / cm2, at least 200 mW / cm2, at least 250 mW / cm2, at least 300 mW / cm2, at least 350 mW / cm2. In some embodiments, the light emitting device includes a LED. In some embodiments, the light emitting device is in form of a wearable. Examples of wearables include wristbands, ankle bands, arm bands, leg bands, and belts. In some embodiments, the light emitting device is in form of a bracelet.

[0184] In some embodiments, the kit includes instructions for use. In some embodiments, the cleavable linker is a chemically cleavable linker and the kit includes a reagent for cleavingthe chemically cleavable linker. In some embodiments, the cleavable linker is an enzymatically cleavable linker and the kit includes an enzyme. In some embodiments, the cleavable linker is a ribozyme cleavable linker and the kit includes a ribozyme. In some embodiments, the cleavable linker is a redox-cleavable linker and the kit includes a redox agent. In some embodiments, the cleavable linker is an acid-cleavable linker and the kit includes an acid. In some embodiments, the cleavable linker is a base-cleavable linker and the kit includes a base.

[0185] In some embodiments, the cleavable linker is a nucleophile-cleavable linker and the kit includes a nucleophile. In some embodiments, the cleavable linker is an electrophile- cleavable linker and the kit includes an electrophile. In some embodiments, the cleavable linker is a temperature-cleavable linker and the kit includes a means for changing the temperature at the site of the administration. The means for changing the temperature may be capable of increasing temperature, decreasing temperature, or both. In some embodiments, the cleavable linker is a spontaneous-hydrolysis-cleavable linker and the kit includes a hydrolysis reagent. In some embodiments, the cleavable linker is a reactive-oxygen-species-cleavable linker and the kit includes a reactive-oxygen-species or a reagent capable of generating a reactive-oxygenspecies. In some embodiments, the cleavable linker is an ultrasound-cleavable linker and the kit includes a device for generating ultrasound. In some embodiments, the cleavable linker is an electromagnetic-radiation-cleavable linker and the kit includes a device for generating electromagnetic radiation.

[0186] Some exemplary aspects of the disclosure are described by one or more of following numbered Embodiments:

[0187] Embodiment 1 : A conjugate comprising: (i) a polymer; and (ii) an active agent, wherein the active agent is an agent capable of reversing or reducing an effect of an opioid in a subject, and wherein the active agent is linked to the polymer via a cleavable linker.

[0188] Embodiment 2: The conjugate of Embodiment 1, wherein the agent capable of reversing or reducing an effect of an opioid is a competitive opioid receptor antagonist.

[0189] Embodiment s: The conjugate of any one of Embodiments 1-2, wherein the agent capable of reversing or reducing an effect of an opioid is naloxone or a pharmaceutically acceptable salt thereof, or an analogue or derivative thereof.

[0190] Embodiment 4: The conjugate of any one of Embodiments 1-3, wherein the opioid is morphine, hydromorphone, oxymorphone, dihydromorphine, diacetyl morphine (diamorphine or heroin), codeine, hydrocodone, oxycodone, methadone, theabine, pethidine (meperidine), alfentanil, fentanyl, remifentanil, sufentanil, etorphine, carefentanil,buorenophine, tramadol, venlafaxine, or a pharmaceutically acceptable salt thereof, or an analogue or derivative thereof.

[0191] Embodiment s: The conjugate of any one of Embodiments 1-4, wherein the opioid is a synthetic or semi-synthetic molecule.

[0192] Embodiment 6: The conjugate of any one of Embodiments 1-5, wherein the opioid is an opiate.

[0193] Embodiment ?: The conjugate of any one of Embodiments 1-6, wherein the cleavable linker is a photocleavable linker.

[0194] Embodiment 8: The conjugate of Embodiment 7, wherein the photocleavable linker is cleaved by exposure to light having an average wavelength of between about 300 nm and about 900 nm.

[0195] Embodiment 9: The conjugate of any one of Embodiments 7-8, wherein the photocleavable linker is cleaved by exposure to light having an average wavelength of between about 350 nm and about 530 nm.

[0196] Embodiment 10: The conjugate of any one of Embodiments 7-9, wherein the photocleavable linker is cleaved by exposure to light having an average wavelength of between about 375 nm and about 475 nm.

[0197] Embodiment 11 : The conjugate of any one of Embodiments 7-10, wherein the photocleavable linker is cleaved by exposure to light having an average wavelength of about 400 nm.

[0198] Embodiment 12: The conjugate of any one of Embodiments 7-11, wherein the photocleavable linker comprises a coumarin moiety, a o-nitrobenzyl moiety, a p-nitrobenzyl moiety, a m-nitrobenzyl moiety, a benzoin moiety, a nitroindoline moiety, a quinoline moiety, a hydroxyphenacyl moiety, or any combination thereof.

[0199] Embodiment 13: The conjugate of any one of Embodiments 7-12, wherein the photocleavable linker comprises a coumarin moiety, a o-nitrobenzyl moiety, a benzoin moiety, a 7-nitroindoline moiety, a p-hydroxyphenacyl moiety, a hydroxyquinoline moiety, or any combination thereof.

[0200] Embodiment 14: The conjugate of any one of Embodiments 7-13, wherein the photocleavable linker comprises a coumarin moiety.

[0201] Embodiment 15: The conjugate of any one of Embodiments 7-14, wherein the photocleavable linker comprises 7- (diethylamino) coumarin-4-yl] methyl carboxyl.

[0202] Embodiment 16: The conjugate of any one of Embodiments 7-15, wherein the photocleavable linker comprises: , wherein R is the polymer; and R’ is the agent capable of reversing or reducing an effect of an opioid.

[0203] Embodiment 17: The conjugate of any one of Embodiments 1-6, wherein the cleavable linker is a chemically cleavable linker.

[0204] Embodiment 18: The conjugate of any one of Embodiments 1-6, wherein the cleavable linker is an enzymatically cleavable linker.

[0205] Embodiment 19: The conjugate of any one of Embodiments 1-6, wherein the cleavable linker is a ribozyme-cleavable linker.

[0206] Embodiment 20: The conjugate of any one of Embodiments 1-6, wherein the cleavable linker is a redox-cleavable linker.

[0207] Embodiment 21 : The conjugateany one of Embodiments 1-6, wherein the cleavable linker is an acid-cleavable linker.

[0208] Embodiment 22: The conjugate of any one of Embodiments 1-6, wherein the cleavable linker is a base-cleavable linker.

[0209] Embodiment 23 : The conjugate of any one of Embodiments 1-6, wherein the cleavable linker is a nucleophile-cleavable linker.

[0210] Embodiment 24: The conjugate of any one of Embodiments 1-6, wherein the cleavable linker is an electrophile-cleavable linker.

[0211] Embodiment 25: The conjugate of any one of Embodiments 1-6, wherein the cleavable linker is a temperature-cleavable linker.

[0212] Embodiment 26: The conjugate of any one of Embodiments 1-6, wherein the cleavable linker is a spontaneous-hydrolysis-cleavable linker.

[0213] Embodiment 27: The conjugate of any one of Embodiments 1-6, wherein the cleavable linker is a reactive-oxygen-species-cleavable linker.

[0214] Embodiment 28: The conjugate of any one of Embodiments 1-6, wherein the cleavable linker is an ultrasound-cleavable linker.

[0215] Embodiment 29: The conjugate of any one of Embodiments 1-6, wherein the cleavable linker is an electromagnetic-radiation-cleavable linker.

[0216] Embodiment 30: The conjugate of any one of Embodiments 1-29, wherein the polymer is a hydrophilic polymer of an amphiphilic polymer.

[0217] Embodiment 31 : The conjugate of any one of Embodiments 1-30, wherein the polymer is selected from the group consisting of polylactides, polyglycolides, polycaprolactones, polyanhydrides, polyepsilon caprolactone, polyamides, polyurethanes,polyesteramides, polyorthoesters, polydioxanones, polyacetals, polyketals, polycarbonates, polyorthocarbonates, polydihydropyrans, polyphosphazenes, polyhydroxybutyrates, polyhydroxyvalerates, polyalkylene oxalates, polyalkylene succinates, poly(malic acid), poly(amino acids), polyvinylpyrrolidone, polyetheres (e.g., polyethylene glycol), polyhydroxycellulose, polymethyl methacrylate, chitin, chitosan, poly(glycerol sebacate) (PGS), cellulose acetates (including cellulose diacetate), polyethylene, polypropylene, polybutylene, polyethylene terphthalate (PET), polyvinyl chloride, polystyrene, polyamides, nylon, polycarbonates, polysulfides, polysulfones, hydrogels (e.g., acrylics), polyacrylonitrile, polyvinylacetate, cellulose acetate butyrate, nitrocellulose, copolymers of urethane / carb onate, copolymers of styrene / maleic acid, poly(ethylenimine), poloxamers (e.g., poloxamer P407, poloxamer P331, poloxamer P188, poloxamer 182, poloxamer P124, poloxamer P338, or poloxamer P237), and copolymers or terpolymers, thereof.

[0218] Embodiment 32: The conjugate of any one of Embodiments 1-31, wherein polymer is poly(lactic-co-glycolic acid) (PLGA), a copolymer comprising PLGA or a poloxamer.

[0219] Embodiment 33: The conjugate of Embodiment 32, wherein a ratio of lactide to glycolide in the PLGA is between 50:50 and 100:0.

[0220] Embodiment 34: The conjugate of Embodiment 32 or 33, wherein a ratio of lactide to glycolide in the PLGA is between 50:50 and 90: 10.

[0221] Embodiment 35: The conjugate of any of Embodiments 32-34, wherein a ratio of lactide to glycolide in the PLGA is between 50:50 and 75:25.

[0222] Embodiment 36: The conjugate of any one of Embodiments 1-35, wherein the polymer is a biocompatible polymer.

[0223] Embodiment 37: The conjugate of any one of Embodiments 1-366, wherein the polymer is biodegradable.

[0224] Embodiment 38: The conjugate of any one of Embodiments 1-37, wherein the conjugate is of Formula (I), P-(L-X)n, wherein: n is an integer of 1 or more; each instance of X is independently the agent capable of reversing or reducing an effect of an opioid; each instance of L is independently the cleavable linker; and P is the polymer.

[0225] Embodiment 39: The conjugate Embodiment 38, wherein n is an integer between 1 and 100,000, inclusive.

[0226] Embodiment 40: The conjugate of any one of 38-39, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0227] Embodiment 41 : The conjugate of any one of Embodiments 38-40, wherein n is 2.

[0228] Embodiment 42: The conjugate of any one of Embodiments 1-41, wherein a first agent capable of reversing or reducing an effect of an opioid is linked to a first end of the polymer and a second agent capable of reversing or reducing an effect of an opioid is linked to a second end of the polymer.

[0229] Embodiment 43 : The conjugate of any one of Embodiments 1-42, wherein the conjugate is: .

[0230] Embodiment 44: The conjugate of any one of Embodiments 1-43, wherein the conjugate is formulated in a particle.

[0231] Embodiment 45: The conjugate of any one of Embodiments 1-44, wherein the conjugate is formulated in a hydrogel, optionally, the hydrogel is a cross-linked polysaccharide hydrogel.

[0232] Embodiment 46: A particle comprising a conjugate of any one of Embodiments 1-45.

[0233] Embodiment 47: The particle of Embodiment 46, wherein the particle is comprised in a hydrogel, e.g., the particle is a hydrogel particle.

[0234] Embodiment 48: The particle of Embodiment 47, wherein the hydrogel is a crosslinked polysaccharide hydrogel, e.g., the particle is a cross-linked polysaccharide hydrogel particle.

[0235] Embodiment 49: The particle of any one of Embodiments 46-48, wherein the particle is a nanoparticle or microparticle.

[0236] Embodiment 50: The particle of any one of Embodiments 46-49, wherein the particle is a nanoparticle.

[0237] Embodiment 51 : The particle of any one of Embodiment 46-50, wherein the particle has a hydrodynamic diameter of less than about 1000 nm.

[0238] Embodiment 52: The particle of any one of Embodiment 46-51, wherein the particle has a hydrodynamic diameter of less than about 750 nm.

[0239] Embodiment 53: The particle of any one of Embodiment 46-52, wherein the particle has a hydrodynamic diameter of less than about 700 nm.

[0240] Embodiment 54: The particle of any one of Embodiment 46-53, wherein the particle has a hydrodynamic diameter of less than about 600 nm.

[0241] Embodiment 55: The particle of any one of Embodiment 46-54, wherein the particle has a hydrodynamic diameter of less than about 500 nm or less than about 550 nm.

[0242] Embodiment 56: The particle of any one of Embodiments 46-55, wherein the particle has a hydrodynamic diameter of from about 25 nm to about 400 nm.

[0243] Embodiment 57: The particle of any one of Embodiments 46-56, wherein the particle has a hydrodynamic diameter of from about 50 nm to about 300 nm.

[0244] Embodiment 58: The particle of any one of Embodiments 46-57, wherein the particle has a hydrodynamic diameter of from about 75 nm to about 200 nm.

[0245] Embodiment 59: The particle of any one of Embodiments 46-58, wherein the particle has a hydrodynamic diameter of from about 80 nm to about 150 nm.

[0246] Embodiment 60: The particle of any one of Embodiments 46-59, wherein the particle has a hydrodynamic diameter of about 85 nm to about 125 nm.

[0247] Embodiment 61 : The particle of any one of Embodiments 46-60, wherein the particle has a hydrodynamic diameter of from about 90 nm to about 110 nm.

[0248] Embodiment 62: The particle of any one of Embodiments 46-61, wherein the particle is substantially spherical.

[0249] Embodiment 63 : The particle of any one of Embodiments 46-62, wherein the particle further comprises a dye.

[0250] Embodiment 64: A hydrogel comprising a conjugate of any one of Embodiments 1-45.

[0251] Embodiment 65: The hydrogel of Embodiment 64, wherein the conjugate is present in a particle.

[0252] Embodiment 66: The hydrogel of Embodiment 65, wherein the particle is a particle of any one of Embodiments 46-63.

[0253] Embodiment 67: The hydrogel of any one of Embodiments 64-66, wherein the hydrogel comprises a dye.

[0254] Embodiment 68: A depot suitable for implantation into a subject comprising a conjugate of any one of Embodiments 1-45, or a particle of any one of Embodiments 46-63, or a hydrogel of any one of Embodiments 64-67.

[0255] Embodiment 69: A pharmaceutical composition comprising a therapeutically effective amount of a conjugate of any one of Embodiments 1-45, or a particle of any one of Embodiments 46-63, or a hydrogel of any one of Embodiments 64-67, or a depot of Embodiment 68, and a pharmaceutically acceptable excipient.

[0256] Embodiment 70: The pharmaceutical composition of Embodiment 69, wherein the pharmaceutical composition is formulated for systemic administration.

[0257] Embodiment 71 : The pharmaceutical composition of any one of Embodiments 69-70, wherein the pharmaceutical composition is formulated for parenteral administration.

[0258] Embodiment 72: The pharmaceutical composition of any one of Embodiments 69-71, wherein the pharmaceutical composition is formulated for intravenous, intramuscular, subcutaneous or transdermal administration.

[0259] Embodiment 73 : The pharmaceutical composition of any one of Embodiments 69-72, wherein the pharmaceutical composition further comprises a dye.

[0260] Embodiment 74: A method for reducing or reversing an effect of an opioid in a subject, the method comprises: applying a stimulus at a site of where the subject was administered an effective amount of a conjugate of any one of Embodiments 1-45, or a particle of any one of Embodiments 46-63, or a hydrogel of any one of Embodiments 64-67, or a depot of Embodiment 68, or a pharmaceutical composition of any one of Embodiments 69-73, wherein the active agent is an agent capable of reversing or reducing an effect of an opioid, and wherein the stimulus is capable of cleaving the cleavable linker linking the polymer and the active agent.

[0261] Embodiment 75: The method of Embodiment 74, wherein the stimulus is applied for 30 minutes or less.

[0262] Embodiment 76: The method of any one of Embodiments 74-75, wherein the stimulus is applied for 15 minutes or less.

[0263] Embodiment 77: The method of any one of Embodiments 74-76, wherein the stimulus is applied for 10 minutes or less.

[0264] Embodiment 78: The method of any one of Embodiments 74-77, wherein the stimulus is applied for 5 minutes or less.

[0265] Embodiment 79: The method of any one of Embodiments 74-78, wherein the stimulus is applied for 3 minutes or less.

[0266] Embodiment 80: The method of any one of Embodiments 74-79, wherein the method comprises applying the stimulus for a first period of time, waiting for a first period of time, and applying the stimulus for a second period of time.

[0267] Embodiment 81 : The method of Embodiment any one of Embodiments 74-80, wherein the cleavable linker is a photocleavable linker, and wherein the stimulus is light.

[0268] Embodiment 82: The method of Embodiment 81, wherein the light comprises near-infrared light.

[0269] Embodiment 83 : The method of any one of Embodiments 81-82, wherein the light comprises visible light.

[0270] Embodiment 84: The method of any one of Embodiments 81-83, wherein the light has an average wavelength of between about 300 nm and about 900 nm.

[0271] Embodiment 85 : The method of any one of Embodiments 81-84, wherein the light has an average wavelength of between about 350 nm and about 530 nm.

[0272] Embodiment 86: The method of any one of Embodiments 81-85, wherein the light has an average wavelength of between about 375 nm and about 475 nm.

[0273] Embodiment 87 : The method of any one of Embodiments 81-86, wherein the light has an average wavelength of about 400 nm.

[0274] Embodiment 88: The method of any one of Embodiments 81-87, wherein the light is blue light.

[0275] Embodiment 89: The method of any one of Embodiments 81-88, wherein the light has an intensity of at least 50 mW / cm2.

[0276] Embodiment 90: The method of any one of Embodiments 81-89, wherein the light has an intensity of at least 100 mW / cm2.

[0277] Embodiment 91 : The method of any one of Embodiments 81-90, wherein the light has an intensity of at least 150 mW / cm2.

[0278] Embodiment 92: The method of any one of Embodiments 81-91, wherein the light has an intensity of at least 200 mW / cm2.

[0279] Embodiment 93 : The method of any one of Embodiments 81-92, wherein the light has an intensity of at least 250 mW / cm2.

[0280] Embodiment 94: The method of any one of Embodiments 81-93, wherein the light has an intensity of at least 300 mW / cm2.

[0281] Embodiment 95 : The method of any one of Embodiments 81-94, wherein the light has an intensity of at least 350 mW / cm2.

[0282] Embodiment 96: The method of any one of Embodiment 81-95, wherein the light is applied by a LED.

[0283] Embodiment 97: The method of any one of Embodiments 74-80, wherein the cleavable linker is a chemically cleavable linker and the stimulus is a reagent for cleaving the chemically cleavable linker.

[0284] Embodiment 98: The method of any one of Embodiments 74-80, wherein the cleavable linker is an enzymatically cleavable linker and the stimulus is an enzyme.

[0285] Embodiment 99: The method of any one of Embodiments 74-80, wherein the cleavable linker is a ribozyme-cleavable linker and the stimulus is a ribozyme.

[0286] Embodiment 100: The method of any one of Embodiments 74-80, wherein the cleavable linker is a redox-cleavable linker and the stimulus is a redox agent.

[0287] Embodiment 101 : The method of any one of Embodiments 74-80, wherein the cleavable linker is an acid-cleavable linker and the stimulus is an acid.

[0288] Embodiment 102: The method of any one of Embodiments 74-80, wherein the cleavable linker is a base-cleavable linker and the stimulus is a base.

[0289] Embodiment 103: The method of any one of Embodiments 74-80, wherein the cleavable linker is a nucleophile-cleavable linker and the stimulus is a nucleophile.

[0290] Embodiment 104: The method of any one of Embodiments 74-80, wherein the cleavable linker is an electrophile-cleavable linker and the stimulus is an electrophile.

[0291] Embodiment 105: The method of any one of Embodiments 74-80, wherein the cleavable linker is a temperature-cleavable linker and the stimulus is a change in temperature.

[0292] Embodiment 106: The method of any one of Embodiments 74-80, wherein the cleavable linker is a spontaneous-hydrolysis-cleavable linker and the stimulus is a hydrolysis reagent.

[0293] Embodiment 107: The method of any one of Embodiments 74-80, wherein the cleavable linker is a reactive-oxygen-species-cleavable linker and the stimulus is a reactive- oxygen-species or a reagent capable of generating a reactive-oxygen-species.

[0294] Embodiment 108: The method of any one of Embodiments 74-80, wherein the cleavable linker is an ultrasound-cleavable linker and the stimulus is ultrasound.

[0295] Embodiment 109: The method of any one of Embodiments 74-80, wherein the cleavable linker is an electromagnetic-radiation-cleavable linker and the stimulus is electromagnetic radiation.

[0296] Embodiment 110: The method of any one of Embodiments 74-109, wherein the method further comprises: administering a conjugate of any one of Embodiments 1-45, or a particle of any one of Embodiments 46-63, or a hydrogel of any one of Embodiments 64-67, or a depot of Embodiment 68, or a pharmaceutical composition of any one of Embodiments 69-73, to the subject prior to applying the stimulus.

[0297] Embodiment 111 : A method comprising administering a conjugate of any one of Embodiments 1-45, or a particle of any one of Embodiments 46-63, or a hydrogel of any one of Embodiments 64-67, or a depot of Embodiment 68, or a pharmaceutical composition of any one of Embodiments 69-73, to a subject.

[0298] Embodiment 112: The method of Embodiment 110 or 111, wherein said administering comprises parenteral administration.

[0299] Embodiment 113: The method of any one of Embodiments 110-112, wherein said administering comprises intravenous, intramuscular, subcutaneous or transdermal administration.

[0300] Embodiment 114: The method of any one of Embodiments 110-113, wherein said administering comprises subcutaneous injection.

[0301] Embodiment 115: The method of any one of Embodiments 110-114, wherein said administering is via microneedles.

[0302] Embodiment 116: The method of any one of Embodiments 110-115, wherein said administering is via a microneedle patch.

[0303] Embodiment 117: The method of any one of Embodiments 110-116, wherein said administering is in a wrist of the subject.

[0304] Embodiment 118: The method of any one of Embodiments 74-117, wherein the subject is overdosed on an opioid.

[0305] Embodiment 119: The method of any one of Embodiments 74-118, wherein the subject is human.

[0306] Embodiment 120: A kit comprising a conjugate of any one of Embodiments 1-45, or a particle of any one of Embodiments 46-63, or a hydrogel of any one of Embodiments 64- 67, or a depot of Embodiment 68, or a pharmaceutical composition of any one of Embodiments 69-73.

[0307] Embodiment 121 : The kit of Embodiment 120, further comprising a means for administering the conjugate, the particle, the hydrogel, the depot, or the pharmaceutical composition to a subject.

[0308] Embodiment 122: The kit of any one of Embodiments 120-121, further comprising a means for cleaving the cleavable linker.

[0309] Embodiment 123: The kit of any one of Embodiments 120-122, wherein the cleavable linker is a photocleavable linker and the kit further comprises a light emitting device.

[0310] Embodiment 124: The kit of Embodiment 123, wherein the light emitting device emits near-infrared light.

[0311] Embodiment 125: The kit of any one of Embodiments 123-124, wherein the light emitting device emits visible light.

[0312] Embodiment 126: The kit of any one of Embodiments 123-125, wherein the light has an average wavelength of between about 300 nm and about 900 nm.

[0313] Embodiment 127: The kit of any one of Embodiments 123-126, wherein the light has an average wavelength of between about 350 nm and about 530 nm.

[0314] Embodiment 128: The kit of any one of Embodiments 123-127, wherein the light has an average wavelength of between about 375 nm and about 475 nm.

[0315] Embodiment 129: The kit of any one of Embodiments 123-128, wherein the light has an average wavelength of about 400 nm.

[0316] Embodiment 130: The kit of any one of Embodiments 123-129, wherein, wherein the light is blue light.

[0317] Embodiment 131 : The kit of any one of Embodiments 123-130, wherein the light has an intensity of at least 50 mW / cm2.

[0318] Embodiment 132: The kit of any one of Embodiments 123-131, wherein the light has an intensity of at least 100 mW / cm2.

[0319] Embodiment 133: The kit of any one of Embodiments 123-132 wherein the light has an intensity of at least 150 mW / cm2.

[0320] Embodiment 134: The kit of any one of Embodiments 123-133, wherein the light has an intensity of at least 200 mW / cm2.

[0321] Embodiment 135: The kit of any one of Embodiments 123-134, wherein the light has an intensity of at least 250 mW / cm2.

[0322] Embodiment 136: The kit of any one of Embodiments 123-135, wherein the light has an intensity of at least 300 mW / cm2.

[0323] Embodiment 137: The kit of any one of Embodiments 123-136, wherein the light has an intensity of at least 350 mW / cm2.

[0324] Embodiment 138: The kit of any one of Embodiments 123-137, wherein the light emitting device comprises a LED.

[0325] Embodiment 139: The kit of any one of Embodiments 123-138, wherein the light emitting device is in form of a wearable.

[0326] Embodiment 140: The kit of any one of Embodiments 123-139, wherein the light emitting device is in form of a bracelet.

[0327] Embodiment 141 : The kit of any one of Embodiments 123-140, wherein the kit further comprises instructions for use.

[0328] Embodiment 142: The kit of any one of Embodiments 120-122, wherein the cleavable linker is a chemically cleavable linker and the kit further comprises a reagent for cleaving the chemically cleavable linker.

[0329] Embodiment 143: The kit of any one of Embodiments 120-122, wherein the cleavable linker is an enzymatically cleavable linker and the kit further comprises an enzyme.

[0330] Embodiment 144: The kit of any one of Embodiments 120-122, wherein the cleavable linker is a ribozyme-cleavable linker and the kit further comprises a ribozyme.

[0331] Embodiment 145: The kit of any one of Embodiments 120-122, wherein the cleavable linker is a redox-cleavable linker and the kit further comprises a redox agent.

[0332] Embodiment 146: The kit of any one of Embodiments 120-122, wherein the cleavable linker is an acid-cleavable linker and the kit further comprises an acid.

[0333] Embodiment 147: The kit of any one of Embodiments 120-122, wherein the cleavable linker is a base-cleavable linker and the kit further comprises a base.

[0334] Embodiment 148: The kit of any one of Embodiments 120-122, wherein the cleavable linker is a nucleophile-cleavable linker and the kit further comprises a nucleophile.

[0335] Embodiment 149: The kit of any one of Embodiments 120-122, wherein the cleavable linker is an electrophile-cleavable linker and the kit further comprises an electrophile.

[0336] Embodiment 150: The kit of any one of Embodiments 120-122, wherein the cleavable linker is a temperature-cleavable linker and the kit further comprises means for changing the temperature at the site of the administration.

[0337] Embodiment 151 : The kit of any one of Embodiments 120-122, wherein the cleavable linker is a spontaneous-hydrolysis-cleavable linker and the kit further comprises a hydrolysis reagent.

[0338] Embodiment 152: The kit of any one of Embodiments 120-122, wherein the cleavable linker is a reactive-oxygen-species-cleavable linker and the kit further comprises a reactive-oxygen-species or a reagent capable of generating a reactive-oxygen-species.

[0339] Embodiment 153: The kit of any one of Embodiments 120-122, wherein the cleavable linker is an ultrasound-cleavable linker and the kit further comprises a device for generating ultrasound.

[0340] Embodiment 154: The kit of any one of Embodiments 120-122, wherein the cleavable linker is an electromagnetic-radiation-cleavable linker and the kit further comprises a device for generating electromagnetic radiation.Examples

[0341] In order for the present disclosure to be more fully understood, the following examples are set forth. The synthetic and biological examples described in this application are offered to illustrate the compositions, pharmaceutical compositions, methods, and kits provided herein and are not to be construed in any way as limiting their scope.Methods Used

[0342] Instruments and Characterizations of Materials.1H and13C NMR experiments were measured on a Varian 400 M or 500 NMR spectrometer. The spectra were referenced to the residual solvent peak in CDCh at h 7.27 ppm for 1H proton and h 77.0 ppm for13C, respectively. Gel permeation chromatography (GPC) was performed in N,N- dimethylformamide with 0.04M LiBr on a Tosoh EcoSEC HLC-8320 system (Tokyo, Japan) equipped with a TSKGel GMHHR-H 7.8X300 mm column and Rl / UV-vis detectors, with polystyrene standards. UV-vis absorption was measured on a NanoDrop™-Onecspectrometer (Thermo Fisher Scientific Inc., Waltham, MA). Dynamic light scattering (DLS) was measured on a Zetasizer Nano-Pro instrument (Malvern Panalytical Inc., Westborough, MA) equipped with He-Ne laser ( = 633 nm) Liquid chromatograph-mass spectrometry (LC-MS) experiments was conducted on an Agilent (Santa Clara, CA) 1260 Infinity II Series LC-MS. Transmission electron microscopy (TEM) images were taken with a Tecnai G2 Spirit BioTWIN transmission electron microscope operating at 80 kV (FEI company, Hillsboro, OR).

[0343] Preparation of PLGA-CM-NLX Nanoparticles . Nanoprecipitation method was used to prepare the PLGA-CM-NLX nanoparticles. One milliliter of polymer solution in acetone at 10 mg / mL was added dropwise into 10 mL water under vigorous stirring. The formed nanoparticles centrifuged and washed with water.

[0344] Photocleavage of PLGA-CM-NLX Nanoparticles In Vitro. To demonstrate the capability of PLGA-CM-NLX to release naloxone in the native form, 100 pL of PLGA-CM- NLX nanoparticles (10 mg / mL) was placed in a 2 mL centrifuge tube and irradiated with 400 nm LED light at irradiances and for durations described in the body of the paper. The formulation was then diluted to 1 mL and centrifuged. The supernate was used to verify the release of naloxone and determine its concentrations. For experiments of multiple times of photo triggering, the procedure was repeated at predetermined time points.

[0345] In Vitro Cytotoxicity. 3T3 cells were used to assess the cytotoxicity of the materials. Different compounds were directly added into the cell culture media (final concentrations: 0.01 mg / mL to 1 mg / mL) and incubated in the media bathing the cells in 96-well plates. After 24 hours, cell viability was evaluated with the MTS assay, and their survival expressed as percentages of results in untreated cells.

[0346] Animal Studies. Animal studies were performed according to protocols approved by the Boston Children’s Hospital Animal Care and Use Committee. CD1 mice (20-35 grams, from Charles River Laboratories) were housed in groups under a 12-hour / 12-hour light / dark cycle. The doses used were 10 mg / kg for morphine, 0.1 mg / kg for naloxone and 100 mg / kg for PLGA-CM-NLX nanoparticles throughout the work. All injections were subcutaneous. In lighttriggering experiments, hair was removed. The animals were restrained under brief isoflurane- oxygen and received 400 nm light irradiation at 300 mW / cm2for two minutes.

[0347] In neurobehavioral testing, mice were assessed for response to heat stimuli by measuring the time (latency) in seconds on a 50°C hotplate until they flinch, lick one of the hind paws, or jump. Before injection, animals underwent baseline testing. After injection, testing was performed every 30 minutes. Mice were removed from the hotplate at 60 seconds even if there was no response to avoid thermal injury.

[0348] In Vivo Imaging System (IVIS) experiments were conducted on a PerkinElmer (Waltham, MA) Lumina Series III instrument with excitation at 675 nm and emission at 720 nm for cy5.5-containing formulations. The fluorescence signal was analyzed with Livingimage solftware.

[0349] Histology. The mice were euthanized with carbon dioxide at determined time points. Skin surrounding tissues from the injected sites were dissected, fixed in formalin, embedded in paraffin and underwent standard processing to produce hematoxylin and eosin (H&E) stained slides.

[0350] Statistics. Data were described with means and standard deviations (SD) calculated from Microsoft Excel (365ProPlus). F-test was used to determine whether two samples have different variance, then unpaired t-test was used to calculate the p-values from the software of Microsoft Excel. Measurements were taken from distinct samples. OriginPro was used for plotting.Example 1 - Synthesis and Characterization of An Exemplary Conjugate (PLGA-CM-NLX)

[0351] Conjugates of (Formula I) may be prepared in view of the following synthetic schemes, and by using synthetic schemes and procedures recognized by one of ordinary skill in the art. A skeletal structure diagram of PLGA-CM-NLX, the exemplary conjugate prepared by the following synthetic scheme, is depicted in FIG. 1.

[0352] Unless otherwise noted, all reagents and solvents were used as received from Sigma-Aldrich (Burlington, MA), Thermo-Fisher (Waltham, MA) or VWR (Radnor, PA) Morphine was from Hikma (Eatontown, NJ). Dibenzocyclooctyne (DIBO) with a hydroxyl (DIBO-OH) was synthesized according to previously reported methods with some modifications. Bi-functionalized coumarin derivative NH2-CM-0H was prepared as reported.

[0353] Compounds of (Formula I) may be prepared using the synthetic scheme shown in FIGS. 27-30 and described below and procedures known by one of ordinary skill in the art.

[0354] Synthesis of Compound 1 (DIBO-COOH). Trimethylsilyl iodide (TMS-I, 25 g, 0.125 mol) was added to a solution of phenylacetaldehyde (12.5 g, 0.104 mol) in anhydrous chloroform (100 mL) in an ice-water bath. The mixture was stirred at 4°C in a cold room for a week. Then sodium sulfite aqueous solution (1.0 M, 200 mL) was added to the reaction mixture, which was vigorously stirred until the iodine was completely reduced. Chloroform (200 mL) was used to extract the reaction mixture. The organic phase was washed by saturated NaCl aqueous solution (200 mL) and then dried by anhydrous ISfeSCU and concentrated by vacuum. The residue was purified by silica gel chromatography using dichloromethane as the eluent to give the product as a white powder (4.6 g, 40%).JH NMR (CDCh, 400MHz, ppm, b) 7.09-6.95 (m, 8H), 5.29-5.27 (d, 2H), 3.58-3.50 (dd, 2H), 2.78-2.73 (d, 2H).13C NMR (CDCh, 100MHz, ppm, ci) 137.7, 131.5, 129.0, 126.8, 125.9, 125.1, 69.5, 36.1. ESLMS: m / z calculated for CI6HI5O [M+H]+: 223.1; observed: 223.1.

[0355] Synthesis of Compound 2 (5, 6-Dihydro-dibenzo[a,e]cycloocten-5-ol). Compound 1(4.6 g, 20.7 mmol) was dissolved in anhydrous tetrahydrofuran (100 mL) and placed in an icewater bath. Under a nitrogen atmosphere, n-butyl lithium (2.5 M, 16.5 mL) was added. After 4 hours, 100 mL water was added slowly to quench the reaction, followed by extraction with dichloromethane (100 mL x 3). The organic phase was dried by anhydrous Na2SO4 and concentrated by vacuum. The residue was purified by silica gel chromatography using di chloromethane as the eluent to give the product as a white powder (3.7 g, 80%).JH NMR (CDCh, 400MHz, ppm, 3) 7.48-7.46 (m, 1H), 7.26-7.10 (m, 7H), 6.90-6.82 (q, 2H), 5.32-5.28 (m, 1H), 3.49-3.45 (dd, 1H), 3.36-3.32 (dd, 1H), 1.84-1.83 (d, 1H).13C NMR (CDCh, 100MHz, ppm, 3) 140.8, 136.8, 136.2, 134.5, 131.6, 131.5, 130.1, 129.9, 129.3, 128.6, 127.4, 127.1, 127.0, 125.9, 74.5, 42.6. CI6HI5O [M+H]+: 223.1; observed: 223.1.

[0356] Synthesis of Compound 3 (11,12 -Dibromo-5, 6,11,12 -tetrahydro- dibenzo[a,e]cycloocten-5-ol). To a solution of Compound 2 (3.7 g, 16.7 mmol) in chloroform (50 mL), bromine (2.67 g, 16.7 mmol) was added slowly at 0°C. The reaction was stirred for 1 hour, and then quenched by the addition of sodium sulfite aqueous solution (1.0 M, 50 mL). The organic phase was separated and dried by anhydrous Na2SO4. The crude product was purified by silica gel chromatography using dichloromethane and hexane as the eluent. The light-yellow oil product (3.1 g, 49%) was used immediately for next step. 'H NMR (CDCh, 400MHz, ppm, 3) 7.70-7.59 (m, 2H), 7.41-6.87 (m, 6H), 5.89-5.77 (m, 1H), 5.47-5.29 (m, 1H), 3.78-3.58 (m, 1H), 3.12-3.08 (m, 1H). ESLMS: m / z calculated for Ci6Hi5Br2O [M+H]+: 380.9; observed: 380.9.

[0357] Synthesis of Compound 4 (DIBO-OH) (5,6-Dihydro-l 1,12-didehydro- dibenzo[a,e]cycloocten-5-ol). To a solution of Compound 3 (3.1 g, 8.18 mmol) in anhydrous tetrahydrofuran (60 mL), lithium diisopropylamide (LDA, 2M, 15 mL) in tetrahydrofuran was added dropwise at 0°C. The reaction was quenched after 2 hours by addition of water. The mixture was extracted by di chloromethane (100 mL x 2), and the organic phase was washed by saturated NaCl aqueous solution (100 mL x 3), dried by anhydrous ISfeSCU and concentrated by vacuum. The residue was purified by silica gel chromatography to give the product as a white solid (800 mg, 45%). 'HNMR (CDC13, 400MHz, ppm, 3) 7.77-7.74 (m, 1H), 7.44-7.29 (m, 7H), 4.64 (s, 1H), 3.14-3.09 (dd, 1H), 2.97-2.91 (dd, 1H), 2.14-2.12 (d, 1H).13C NMR (CDCI3, 100MHz, ppm, 3) 155.6, 151.6, 129.6, 128.0, 127.9, 126.9, 126.8, 126.1, 124.1, 123.7, 121.2, 112.9, 110.6, 75.2, 48.8. ESI-MS: m / z calculated for CI6HI3O [M+H]+: 221.1; observed: 221.1.

[0358] Synthesis of Compound 5 (DIBO-COOH). Compound 5 (DIBO-OH, 660 mg, 3.00 mmol), and succinic anhydride (1.50 g, 15.0 mmol) were dissolved in anhydrous tetrahydrofuran (20 mL), followed by the addition of 4-dimethylaminopyridine (1.83 g, 15.0 mmol). The mixture was stirred at ambient temperature for 4 hours. Excess HC1 (2 M) was added dropwise into the solution until the pH value became acidic. The solution was extracted by large amount of di chloromethane and washed by water (100 mL) for three times. The organic phase was dried by anhydrous ISfeSCU and concentrated by vacuum. The residue was purified by silica gel chromatography (di chloromethane: ethyl acetate 3: 1) to give the product as white a powder (780 mg, 81%). 'H NMR (CDCI3, 400MHz, ppm, 3) 7.54-7.53 (m, 1H), A - 9 (m, 7H), 5.61 (t, 1H), 3.18-3.15 (dd, 1H), 2.97-2.93 (dd, 1H), 2.88-2.78 (m, 4H).13C NMR (CDC13, 100MHz, ppm, 3) 177.3, 170.8, 150.9, 150.8, 129.9, 128.0, 127.3, 127.1, 126.3, 125.9, 123.9, 123.8, 121.4, 113.0, 109.8, 74.24, 46.26, 29.07, 28.93. ESI-MS: m / z calculated for C20H15O4 [M-H]’: 319.1; observed: 319.1.

[0359] Synthesis of Compound 6 (CM-CHO). 7-Diethylamino-4-methylcoumarin (2.31 g, 10 mmol) was dissolved in 1,4-dioxane (50 mL). Selenium dioxide (2.00 g, 18 mmol) was added. The mixture was refluxed overnight before filtered through celite. The filtrate was concentrated and purified by silica gel chromatography (dichloromethane: hexane 3: 1) to give the product as a red crystal (860 mg, 35%). 'HNMR (CDCI3, 400MHz, ppm, 5) 9.95 (s, 1H), 8.21-8.19 (d, 1H), 6.60-6.57 (d, 1H), 6.46 (s, 1H), 6.37 (s, 1H), 3.40-3.34 (m, 4H), 1.18-1.14 (m, 6H).13C NMR (CDCI3, 100MHz, ppm, 5) 192.39, 161.60, 157.15, 150.57, 143.68, 126.93, 117.27, 109.83, 104.06, 97.98, 45.04, 12.34. ESI-MS: m / z calculated for C14H16NO3 [M+H]+: 246.1; observed: 246.1.

[0360] Synthesis of Compound 7 (NO2-CM-OH). Compound 1 (245 mg, 1.0 mmol) was dissolved in tetrahydrofuran (THF, 2 mL). In an ice-water bath, a solution of nitromethane (610 mg, 10 mmol) in THF (2 mL) was added dropwise. Then, a solution of N, N, N ’N tetramethylethylenediamine (35 mg, 0.3 mmol) in THF (2 mL) was added dropwise. The reaction mixture was allowed to warm up to ambient temperature and stirred for another 4 hours. The solvent was removed by a rotatory evaporator. The residue was re-dissolved in a small amount of dichloromethane and purified by silica gel chromatography (dichloromethane: ethyl acetate 10: 1) to give the product as an orange solid (220 mg, 76%). 'H NMR (CDCh, 400MHz, ppm, 5) 7.42-7.40 (d, 1H), 6.64-6.62 (dd, 1H), 6.54 (s, 1H), 6.38 (s, 1H), 5.82-5.80 (m, 1H), 4.66-4.59 (m, 2H), 3.47-3.41 (m, 4H), 3.18-3.17 (d, 1H), 1.24-1.21 (t, 6H).13C NMR (CDCh, 100MHz, ppm, 5) 161.97, 156.50, 151.84, 150.84, 123.95, 109.12, 106.64, 104.96, 98.13, 79.70, 67.06, 44.82, 12.41. ESLMS: m / z calculated for C15H19N2O5 [M+H]+: 307.1; observed: 307.1.

[0361] Synthesis of Compound 8 (NH2-CM-0H). Compound 2 (920 mg, 3.2 mmol) was suspended in acetic acid (15 mL). The suspension was placed in an ice-water bath. Then zinc powder (1.35 g, 20.8 mmol) was added. After 30 minutes, the reaction mixture was allowed to warm up to ambient temperature and stirred for another 2 hours. The solid was removed by filtration. The filtrate was neutralized by saturated Na2CCh aqueous solution. The mixture was extracted with dichloromethane (200 mL) and then washed with saturated Na2CCh aqueous solution (100 mL) and saturated NaCl aqueous solution (100 mL x 2). The organic phase was dried by anhydrous Na2SC>4 and concentrated by vacuum. The residue was purified by silica gel chromatography (dichloromethane: methanol 5 : 1) to give the product as a yellow solid (550 mg, 65%). 'HNMR (CDCh, 400MHz, ppm, 5) 7.38-7.36 (d, 1H), 6.53-6.52 (dd, 1H), 6.43 (s, 1H), 6.30 (s, 1H), 5.03-5.01 (m, 1H), 3.44-3.34 (m, 6H), 3.16-3.13 (m, 1H), 2.87-2.85 (m, 1H), 1.17-1.15 (t, 6H).13C NMR (CDCh, 100MHz, ppm, 5) 162.65, 156.46, 156.30, 150.36, 124.75, 108.66, 106.12, 105.54, 97.75, 68.86, 46.77, 44.47, 44.65, 12.43. ESI-MS: m / z calculated for C15H21N2O3 [M+H]+: 277.2; observed: 277.1.

[0362] Synthesis of Compound 9 (DIBO-CM-OH). To a solution of DIBO-COOH (80 mg, 0.25 mmol) in dichloromethane (2 mL) in an ice-water bath, A,A-diisopropylethylamine (DIPEA, 65 mg, 0.50 mmol) is added, followed by hexafluorophosphate azabenzotri azole tetramethyl uronium (HATU, 95 mg, 0.25 mmol). After 5 min, NH2-CM-OH (70 mg, 0.25 mmol) in dichloromethane (5 mL) is added. The mixture was then stirred for 2 hours at ambient temperature. Dichloromethane (80 mL) was used to dilute the reaction mixture, which was then washed by saturated NaCl aqueous solution (100 mL x 3). The organic phase was dried byanhydrous Na2SO4 and concentrated using a rotatory evaporator. The residue was purified by silica gel chromatography (dichloromethane: methanol 20: 1) to give the product as a yellow solid (120 mg, 83%). *HNMR (CDC13, 400MHz, ppm, 3) 7.69-7.63 (t, 1H), 7.53-7.51 (d, 1H), 7.37-7.15 (m, 7H), 6.86-6.82 (m, 1H), 6.70-6.65 (m, 1H), 6.60-6.50 (m, H), 6.32-6.30 (d, 1H), 5.5 (m, 1H), 5.13-5.06 (dd, 1H), 3.90-3.70 (m, 2H), 3.40-4.35 (t, 4H), 3.15-3.00 (m, 2H), 2.95- 2.87 (m, 2H), 2.62-2.57 (m, 2H), 1.19-1.15 (t, 6H).13C NMR (CDCI3, 100MHz, ppm, 3) 173.0, 171.9, 162.8, 156.3, 156.1, 150.8, 132.1, 131.4, 129.9, 129.1, 128.1 128.0, 126.9. 127.2, 127.0, 126.1, 125.2, 125.1, 125.8, 125.2, 123.9, 123.6, 121.9, 109.8, 76.8, 68.3, 46.9, 46.5, 46.1, 30.5, 29.6, 12.3. ESI-MS: m / z calculated for C35H35N2O6 [M+H]+: 579.2; observed: 579.2.

[0363] Synthesis of Compound 10 (DIBO-CM-NH). DIBO-CM-OH (100 mg, 0.17 mmol) and DIPEA (65 mg, 0.50 mmol) were dissolved in anhydrous dichloromethane (3 mL) and placed in an ice-water bath. Then, 4-nitrophenyl chloroformate (100 mg, 0.50 mmol) in di chloromethane (5 mL) was added. After 3 hours, A,A'-dimethylethylenediamine (132 mg, 1.50 mmol) in di chloromethane (5mL) was added slowly. After 2 hours, the reaction mixture was diluted by di chloromethane (100 mL) and washed by saturated NaCl aqueous solution (100 mL x 3). The organic phase was dried by anhydrous ISfeSCU and concentrated by vacuum. The residue was purified by silica gel chromatography (dichloromethane: methanol 20: 1) to give the product as a yellow solid (85 mg, 75%).JH NMR (CDCI3, 400MHz, ppm, 3 8.07- 7.80 (m, 1H), 7.85-7.76 (dd, 1H), 7.58-7.50 (dd, 1H), 7.45-7.20 (m, 7H), 6.65-6.63 (t, 1H), 6.45-6.41 (d, 1H), 6.17-5.93 (m, 2H), 5.56-5.52 (m, 1H), 4.15-3.85 (m, 2H), 3.40-3.35 (q, 4H), 3.66-2.44 (m, 17H), 1.20-1.15 (t, 6H).13C NMR (CDCI3, 100MHz, ppm, 3) 172.9, 172.6, 162.3, 156.5, 156.3, 152.1, 150.9, 130.1, 129.5, 128.3, 128.2, 127.4, 127.2, 126.2, 125.9, 125.2, 124.0, 123.6, 121.1, 115.3, 112.9, 109.9, 109.3, 105.5, 105.1, 97.6, 71.8, 48.5, 48.4, 45.9, 44.7, 43.4, 34.6, 34.4, 33.9, 30.1, 29.4, 12.4. ESI-MS: m / z calculated for C40H45N4O7 [M+H]+: 693.3; observed: 693.4.

[0364] Synthesis of Compound 11 (DIBO-CM-NLX). To a solution of DIBO-CM-NH (70 mg, 0.10 mmol) in anhydrous dichloromethane (2 mL), a solution of triphosgene (12 mg, 0.04 mmol) in di chloromethane (2 mL) was added, followed by DIPEA (20 mg, 0.15 mmol). After 1 hour, naloxone hydrochloride (40 mg, 0.11 mmol), DIPEA (40 mg, 0.30 mmol) was added. The reaction mixture then was stirred for 48 hours. Dichloromethane (100 mL) was used to dilute the reaction mixture. The solution was washed by saturated NaCl aqueous solution (100 mL x 3). The organic phase was dried by anhydrous Na2SO4 and concentrated by vacuum. The residue was re-dissolved in a small amount of dichloromethane and purified by silica gel chromatography (dichloromethane: methanol 20: 1) to give the product as a yellow solid (65mg, 62%). 'H NMR (CDCh, 400MHz, ppm, 3) 8.13-7.70 (m, 1H), 7.57-7.50 (m, 1H), 7.45- 7.20 (m, 8H), 6.90-6.60 (m, 3H), 6.50-6.48 (m, 1H), 6.35-6.01 (m, 2H), 5.88-5.70 (m, 1H), 5.59-5.53 (m, 1H), 5.26-5.14 (m, 2H), 4.73-4.62 (m, 1H), 3.99-3.49 (m, 2H), 3.43-3.39 (m, 4H), 3.20-2.90 (m, 21H), 2.84-1.53 (m, 8H) 30H, 1.20-1.17 (t, 6H).13C NMR (CDCh, 100MHz, ppm, 3) 207.8, 207.3, 172.4, 172.3, 171.2, 162.2, 156.4, 155.0, 154.9, 153.2, 151.2, 151.0, 150.8, 150.7, 148.0, 147.9, 134.9, 133.1, 130.6, 130.3, 130.0, 128.1, 128.0, 127.2, 127.1, 127.0, 126.2, 126.1, 125.9, 124.2, 124.1, 123.7, 123.0, 122.9, 121.3, 119.7, 119.6, 119.4, 119.3, 118.2, 112.9, 109.9, 109.8, 109.3, 109.2, 106.0, 104.5, 97.6, 90.7, 90.6, 76.4, 76.3, 72.2, 70.0, 62.0, 57.6, 57.3, 50.7, 50.5, 47.4, 46.1, 44.7, 43.9, 43.1, 36.2, 26.0, 34.6, 34.5, 31.5, 30.6, 29.7, 29.6, 29.0, 25.3, 22.9, 22.6, 20.7, 18.7, 14.1, 12.4. ESI-MS: m / z calculated for C60H64N5O12 [M+H]+: 1046.4; observed: 1046.4.

[0365] Synthesis of Compound 12 (PLGA-CM-NLX). PLGA-N3 (molecular weight 2kDa., lactide:glycolide 50:50) and DIBO-CM-OH were dissolved together in dichloromethane with 1 : 1 ratio of azide and cyclooctyne moieties. After 24 hours, the mixture was precipitated three times to give the product as a yellow solid.JH NMR (CDCh, 400MHz, ppm, 3) 8.2-8.05 (m, 4H), 7.70-5.70 (m, 36H), 5.30-5.10 (m, 20H), 4.4-4.32 (m, 6H), 5.00-4.55 (m, 32H), 4.20-3.60 (br, 4H), 3.45-3.32 (m, 8H), 3.26-1.78 (m, 58H), 1.65-1.45 (m, 48H), 1.20-1.12 (m, 12H). GPC: Mn= 3.4 kg / mol, D = 1.2.

[0366] Synthesis of Compound 13 (CM-NH). CM-OH (100 mg, 0.40 mmol) and DIPEA (160 mg, 1.23 mmol) were dissolved in anhydrous dichloromethane (3 mL) and placed in an ice- water bath. Then, 4-nitrophenyl chloroformate (160 mg, 0.80 mmol) in dichloromethane (5 mL) was added. After 3 h, A, A' -dimethyl ethylenediamine (352 mg, 4.00 mmol) in dichloromethane (5mL) was added slowly. After 2 h, the reaction mixture was diluted by dichloromethane (100 mL) and washed by saturated NaCl aqueous solution (100 mL x 3). The organic phase was dried by anhydrous Na2SO4 and concentrated by vacuum. The residue was purified by silica gel chromatography (dichloromethane: methanol 10: 1) to give the product as a yellow solid (140 mg, 95%). 'H NMR (CDCh, 400MHz, ppm, <5) 7.28-7.26 (d, 1H), 6.54- 6.52 (d, 1H), 6.46-6.45 (d, 1H), 6.07 (s, 1H), 5.21 (s, 2H), 3.50-3.45 (m, 2H), 3.39-3.35 (q, 4H), 3.26 (m, 1H), 3.01-2.97 (d, 3H), 2.87-2.77 (m, 2H), 2.50-2.43 (d, 3H), 1.18-1.15 (t, 6H).13C NMR (CDCh, 100MHz, ppm, <5) 161.92, 156.00, 155.66, 124.31, 124.25, 108.51, 105.88, 105.51, 97.54, 62.30, 49.29, 48.78, 48.21, 44.56, 35.96, 35.66, 35.21, 34.58, 31.41, 29.52, 22.47, 12.28. ESLMS: m / z calculated for C19H28N3O4 [M+H]+: 362.2; observed: 362.2.

[0367] Synthesis of Compound 14 (CM-NLX). To a solution of CM-NH (140 mg, 0.39 mmol) in anhydrous dichloromethane (2 mL), a solution of triphosgene (49 mg, 0.16 mmol) in di chloromethane (2 mL) was added, followed by DIPEA (20 mg, 0.15 mmol). After 1 h, naloxone hydrochloride (40 mg, 0.11 mmol), DIPEA (65 mg, 0.66 mmol) and 4- dimethylaminopyridine (226 mg, 0.62 mmol) was added. The reaction mixture then was stirred for 48 h. Di chloromethane (100 mL) was used to dilute the reaction mixture. The solution was washed by saturated NaCl aqueous solution (100 mL x 3). The organic phase was dried by anhydrous ISfeSCU and concentrated by vacuum. The residue was re-dissolved in a small amount of dichloromethane and purified by silica gel chromatography (dichloromethane: methanol 20: 1) to give the product as a yellow solid (215 mg, 77%).JH NMR (CDCh, 400MHz, ppm, 3) 7.31-7.28 (m, 1H), 6.88-6.81 (m, 1H), 6.66-6.63 (m, 1H), 6.56-6.54 (d, 1H), 6.48-6.47 (d, 1H), 6.10-6.03 (m, 1H), 5.81-5.75 (m, 1H), 5.29-5.13 (m, 5H), 4.69-4.61 (m, 1H), 3.67-3.53 (m, 2H), 3.41-3.36 (q, 4H), 3.14-2.93 (m, 13H), 2.59-1.56 (m, 8H), 1.19-1.16 (t, 6H).13C NMR (CDCh, 100MHz, ppm, 3) 207.73, 161.94, 161.81, 156.22, 156.17, 155.59, 155.29, 154.12, 153.68, 150.58, 150.44, 150.39, 147.94, 135.03, 133.30, 129.91, 129.75, 124.47, 124.38, 123.22, 119.35, 119.29, 118.16, 108.63, 106.33, 106.00, 97.7, 90.60, 90.41, 70.13, 62.69, 6247,62.06, 57.58, 50.63, 47.71, 47.43, 47.03, 44.70, 43.16, 36.05, 35.62, 35.05, 34.84, 31.14, 30.58, 28.25, 22.98, 12.44. ESLMS: m / z calculated for C39H47N4O9 [M+H]+: 715.3; observed: 715.3.

[0368] Characterization of PLGA-CM-NLX. After the reaction between DIBO-CM-OH and the azide (PLGA-N3), disappearance of the characteristic azide group at 2100 cm'1in Fourier-transform infrared (FT-IR) spectra, as shown in FIG. 31, indicated the success of the coupling. Representative peaks from the polymer and the drug (NLX) were observed in the NMR spectrum shown in FIG. 32, e.g., 5.2 ppm and 4.8 ppm from the lactic and glycolic moieties of PLGA, respectively, and 5.8 ppm from the vinyl moiety of naloxone. The retention time on gel permeation chromatography (GPC) curve of PLGA-CM-NLX was also shortened compared to PLGA-azide (as shown in FIG. 33), indicating an increase in molecular size and molecular weight. SPAAC was used rather than copper-catalyzed azide-alkyne cycloaddition because it is highly efficient for macromolecules and biomaterials. It involves mild reaction conditions, and does not require catalysts, so there is no metal residue.

[0369] PLGA-CM-NLX was dissolved in acetone and nanoparticles (PLGA-CM-NLX- NP) prepared by nanoprecipitation into water. The resulting particle size was 103 ± 29 nm by dynamic light scattering (DLS) and a graphical representation of intensity (%) and diameter (nm) of the PLGA-CM-NLX-NP is shown in FIG. 2. Spherical nanoparticles were observedby transmission electron microscopy (TEM), as shown in FIG. 3. PLGA-CM-NLX in acetone solution showed a representative absorption peak at 378 nm from the coumarin moiety in the UV-vis spectrum. The peak shifted to 392 nm when the polymer formed nanoparticles, as depicted in FIG. 4. The red-shift in absorption from solution to particle can be attributed to aggregation of molecules in the solid form.Example 2 In Vitro Photocleavage of An Exemplary Conjugate (PLGA-CM-NLX)

[0370] PLGA-CM-NLX irradiated with 400 nm light cleaves to an intermediate of a naloxone derivative, which subsequently undergoes self-immolative cyclization, releasing unmodified naloxone. A skeletal structure diagram of the cleavage and self-immolative cyclization is depicted in FIG. 5. Light-triggered release of naloxone in its native form (m / z of 328.2) (mass spectrum of the cleaved naloxone is shown in FIG. 6) was confirmed by liquid chromatography -mass spectroscopy (LC-MS). Naloxone release by photocleavage of PLGA- CM-NLX nanoparticles (10 mg / mL) increased with increasing duration or intensity of irradiation (400 nm LED), as shown graphically in FIG. 7. In addition, naloxone release could be repeatedly triggered by repeated irradiation (400 nm LED at 30 mW / cm2), as displayed in FIG. 8Example 3 In Vivo Efficacy of An Exemplary Conjugate (PLGA-CM-NLX)

[0371] The effectiveness of light-triggered naloxone was assessed in mice using a hotplate test, a widely used model for testing analgesia by opioids. At predetermined intervals (every 30 minutes) animals were placed on a 50°C hotplate, and the latency (time in seconds) to a response (flinching, licking their paws or jumping) was recorded every 30 minutes. Baseline latency was 27.8 ± 4.7 seconds. Animals treated with morphine (10 mg / kg, subcutaneously between the scapulae on the back of the mice) developed a latency of 60 seconds. Animals were removed from the hotplate at a maximum time of 60 seconds to avoid thermal injury. 60 s was the maximum latency. Therefore, the maximum A latency from the test was 32.2 s.) The A latency remained 32.2 s at 60 min, and gradually returned to baseline at a difference in 174.5 ± 50.0 min.

[0372] Animals were given naloxone treatments morphine, which had caused a maximum latency of 60 seconds. Free naloxone (0.1 mg / kg) injected subcutaneously between the scapulae of the mice, reduced the latency to 28.9 ± 3.3 seconds (p = 0.000001 compared to the morphine only group) at the next time point (60 minutes), indicating the injected free naloxone reversed the effect of morphine. Latency remained at baseline afterwards, indicating that naloxone had reversed the effect of morphine for the entirety of the opioid’s duration of effect. The latency as a function of time for mice injected with free naloxone is depicted in FIG. 9.

[0373] The ability of PLGA-CM-NLX-NP to provide triggered opioid reversal was also tested. PLGA-CM-NLX-NP (100 mg / kg, containing 300 pg NLX) were injected subcutaneously at the middle back part of mice. Morphine was given right after the nanoparticles. In the absence of irradiation of the site of PLGA-CM-NLX-NP injection, latency rose to 58.5 ± 3.0 seconds at 30 minutes after morphine injection (green line with upwards pointing triangles in FIG. 10), similar to latencies in mice injected with morphine only (p = 0.4; dashed line in FIG. 10), indicating that naloxone linked to the polymer was not released. When the site of PLGA-CM-NLX-NP injection was irradiated with a 400 nm LED for two minutes at 300 mW / cm2, latency decreased to 32.6 ± 5.7 seconds at 60 minutes (blue line with downwards pointing triangles in FIG. 10), significantly reduced compared to latency in the morphine only group at the same time (60 seconds, p = 0.00007), and similar to that in the free naloxone group (p=0.3). These data indicated that irradiation released naloxone and reversed the effect of the opioid.Example 4 In Vivo Efficacy of An Exemplary Conjugate (PLGA-CM-NLX) Depot System

[0374] The potential for clinical application (a depot system) was also tested. In clinical application, a depot system would be injected subcutaneously in the healthcare system, and only triggered in emergencies at a remote time. The potential for such an application was assessed by injecting PLGA-CM-NLX nanoparticles on Day 0 without any irradiation. At predetermined intervals (2 days, 1 week, or 1 month, as shown in FIG. 11), separate groups of animals were first injected with morphine (10 mg / kg), and then were irradiated with a 400 nm LED at 300 mW / cm2for 2 minutes at the site of the PLGA-CM-NLX-NP injection 30 minutes later, after the latency had risen to 60 seconds, as shown in the graph of FIG. 12. Latency was measured again 30 minutes later, to assess the effect of photo-triggered release of naloxone. Irradiation 2 days after injection of PLGA-CM-NLX-NP reduced latency from morphine from 60 seconds to 33.6±7.9 seconds, (p = 0.0005 compared to morphine alone) similar to the latency with morphine treated with free naloxone (p = 0.3) (as depicted in FIGS. 12 and 13). Irradiation at 1 week reduced latency from 60 seconds to 30.3±5.1 seconds (p = 0.00002 compared to morphine alone; p=0.6 compared to the free naloxone group), and at 1 month to 48.2±6.3 seconds, a lesser but still statistically significant reduction in latency (p = 0.0005 compared to morphine alone; p=0.00005 compared to the free naloxone group). The delayed triggerability was consistent with the demonstration with an in vivo imaging system (IVIS) that subcutaneously injected PLGA-CM-NLX / PLGA-cyanine5.5 nanoparticles covalently labeled with the fluorescent dye cyanine5.5 (cy5.5) remained in tissue for at least a month (as can beseen in FIG. 14 (In Vivo Imaging System (IVIS) images of mice injected with PLGA-CM- NLX nanoparticles covalently labeled with cyanine5.5 (cy5.5)), FIG. 15 (graph of relative fluorescent intensity over time from IVIS), and FIG. 34 (IVIS images of mice injected with free dye cyanine 5.5)).Example 5 In Vivo Efficacy of Repeated Dosing of An Exemplary Conjugate (PLGA-CM- NLX)

[0375] Repeated dosing with naloxone would also be important for eventual clinical application, to extend the useful lifetime of the device / depot. Repeated dosing was studied by injecting mice with PLGA-CM-NLX nanoparticles and testing the ability to trigger reversal of morphine on days 3, 5, 10, and 20 in the same animals. The experimental timeline for this is illustrated in FIG. 16. The latencies after triggering for days 3, 5, 10 were 33.6±7.9 seconds, 38.7±6.7 seconds, 43.3±12.9 seconds, respectively, which were all statistically significantly reduced latencies compared to the morphine only group (p < 0.05, p = 0.0005, 0.0007, 0.04, respectively, compared to morphine alone). FIGS. 17-20 are graphical representations of the latencies as a function of time for days 3, 5, 10, and 20, respectively. FIG. 21 is a graph of latency at sixty minutes after morphine injection as a function of time after injection, summarizing the data from FIGS. 17-20. Their differences from the free naloxone group gradually increased with time (p = 0.3, 0.04, 0.07 respectively, compared to morphine treated with free naloxone). The fourth triggering on day 20 (54.3±11.4 seconds, p > 0.05, p = 0.36 compared to morphine) did not reduce latency.

[0376] Repeated dosing with naloxone would also be important in case of opioid overdose with a dose too large to be reversed by a single triggered dose of naloxone. This situation was mimicked by repeated administration of morphine. After the initial dose of morphine, a second dose was given at 90 minutes, while the latency remained near maximal. The combined morphine doses produced analgesia for five hours until the latency returned to no significant difference to the baseline (p > 0.05), compared to two hours by a single dose of morphine (as shown in FIG. 22). A dose of free naloxone injected 30 minutes after the first dose of morphine decreased latency from 60 seconds to 29.5±5.0 seconds (p = 0.00002 compared to morphine). However, with a second dose of morphine at 90 minutes, latency rose to 60 seconds, i.e., it was not impeded by the previously administered naloxone (as shown in FIG. 23). Similarly, in animals injected with PLGA-CM-NLX-NP at the same time as the initial dose of morphine (t = 0 minutes), irradiation of the nanoparticles at 30 minutes reversed the effect of morphine but did not prevent the effect of the second dose (as shown in FIG. 24). A second irradiation at 90minutes did prevent the effect of the second dose of morphine (as shown in FIG. 25) with the latency decreased to 34.8±7.0 seconds (p = 0.0003 compared to morphine) at the next time point (i.e., 120 minutes), demonstrating that prolonged morphine effect can be reversed by repeated triggering.

[0377] When animals treated with morphine (A latency = 32.2 s) were administered free naloxone subcutaneously 30 min after the morphine (but at a different site in the lower back), the A latency decreased to 1.1 ± 3.3 s at the next time point (60 min; p = 0.000001, FIG. 43), and remained at baseline afterwards, indicating that free naloxone reversed the effect of morphine for the entirety of the opioid’s duration of effect.

[0378] The ability of NLX-NP to provide triggered opioid reversal was tested. NLX-NP (100 mg kg'1, containing 300 pg NLX) were injected subcutaneously in the lower back of mice, followed immediately by injection of morphine subcutaneously between the scapulae. In the absence of irradiation of the site of NLX-NP injection, A latency remained at 30.7 ± 3.0 s (FIG. 44) 30 minutes after morphine injection, similar to the latencies in mice injected with morphine only, indicating that naloxone linked to the polymer was not released. When the site of NLX- NP injection was irradiated with a 400 nm LED for two minutes at 300 mW cm'230 min after morphine, A latency decreased to 4.8 ± 5.7 s at 60 min (FIG. 44; p = 0.00007), and similar to that in morphine-treated animals treated with free naloxone at the same time point (p = 0.3). These data indicated that irradiation released naloxone and reversed the effect of the opioid. We measured that approximately 6.7 ± 0.7% of light from the LED could penetrate skin to the subcutaneous site of injection.

[0379] To determine effect onset time, animals were injected with morphine, then 30 min later injected with free naloxone or NLX-NP and then triggered with 400 nm light and tested every 5 min (FIG. 51). The onset time of reversal by naloxone triggered from NLX-NP was slightly slower than from free naloxone. With free naloxone, it took 5 min for A latency to become different from that of animals treated with morphine alone (p = 0.03); with triggered release of naloxone, this took 10 min (p = 0.04). With free naloxone, latency was similar to baseline within 10 min (p = 0.08); with triggered release of naloxone, this took 20 min (p = 0.06).

[0380] In clinical applications, the depot would be injected subcutaneously by healthcare practitioners, and only triggered in emergencies at a later time. The potential for late triggering was assessed by injecting NLX-NP on Day 0 without irradiation. At predetermined intervals after injection of NLX-NP (2 days, 1 week or 1 month, FIG. 46), separate groups of animals were first injected with morphine, then 30 min later, after the A latency had risen to themaximum (32.2 s) they were irradiated with a 400 nm LED at 300 mW cm'2for 2 min at the site of NLX-NP injection. Latency was measured again 30 minutes later, to assess the effect of photo-triggered release of naloxone (FIGS. 47-48). 2 days after injection of NLX-NP, irradiation reduced A latency from morphine from 32.2 s to 5.8 ± 7.9 s (p = 0.0006), similar to the A latency with morphine treated with free naloxone (p = 0.3, from FIG. 43) (FIGS. 47-48). Irradiation at 1 week reduced A latency from 32.2 s to 2.5 ± 5.1 s (p = 0.00002; p = 0.6 compared to the free naloxone group, from FIG. 43), and at 1 month from 32.2 s to 23.1 ± 2.7 s, a lesser but statistically significant reduction in A latency (p = 0.0005; p = 0.00005 compared to the free naloxone group). NLX-NP did not show triggered opioid reversal if irradiation was performed two months after injection (FIG. 52). The ability to trigger long after injection was consistent with the demonstration with an in vivo imaging system (IVIS) that subcutaneously injected NLX-NP containing PLGA covalently labeled with the fluorescent dye cyanine5.5 (PLGA-cy5.5) remained in tissue for at least a month (FIGS. 49-50). This prolonged retention can be attributed to the nanoparticles, since most of the fluorescent signal of injected free y5.5 disappeared within one day (FIG. 53).Example 6 - Biocompatibility of An Exemplary Conjugate (PLGA-CM-NLX)

[0381] PLGA-CM-NLX-NP showed minimal or no cytotoxicity in 3T3 fibroblasts (as shown in FIG. 35). Naloxone also showed minimal cytotoxicity below 0.5 mg / mL (p > 0.05 compared to the saline group), but started showing toxicity at high concentration (1 mg / mL, p = 0.007 compared to the saline group). To assess tissue reaction in vivo, animals were euthanized 4, 14, and 30 days following subcutaneous injection of PLGA-CM-NLX-NP. The tissues around the site of injection, including epidermis, dermis, dermal white adipose tissue (dWAT), panniculus carnosus (a subcutaneous muscle layer occurring in mice), and interstitial connective tissue, were processed into hematoxylin and eosin (H&E) stained slides, as shown in FIG. 26

[0382] Four days post-injection and irradiation, a mild to moderate inflammatory response with occasional neutrophils and a larger population of lymphocytes and macrophages occurred in tissues adjacent to the area of particle deposition, specifically the panniculus carnosus and interstitial connective tissue (c and d of FIG. 26). The depth of injection was measured as 664 ± 79 pm. The more superficial tissues, including the epidermis, dermis, and dWAT, did not demonstrate any inflammatory infiltration or cell injury. The inflammatory infiltration was diminished by days 14 and 30 (e, f, g, and h of FIG. 26), consisting principally of lymphocytes and foamy macrophages (reflecting the uptake of injected particles). Tissues from animalsinjected with PLGA-CM-NLX-NP without irradiation demonstrated a similar inflammatory response (as shown in FIG. 36), indicating that irradiation was not the source of inflammation.

[0383] On day 4, there was evidence of very mild myotoxicity of the panniculus carnosus, consisting of regenerating myocytes adjacent to the area of particle deposition (i.e., interstitial connective tissue) as well as myocytes with nuclear internalization (d of FIG. 26). The superficial layers of the panniculus carnosus (i.e., those further away from PLGA-CM-NLX- NP) did not have myotoxicity. In addition, the panniculus muscle is not relevant in human subcutaneous injections given the lack of panniculus muscle tissue in this layer in humans. By days 14 (f of FIG. 26) and 30 (h of FIG. 26), the degree of myotoxicity was overall diminished with myocytes demonstrating evidence of regeneration. Histology from animals injected with PLGA-CM-NLX-NP but not irradiated were similar to those that were (FIG. 36).

[0384] NLX-NP showed minimal or no cytotoxicity in C2C12 muscle cells (FIG. 61, p > 0.05 compared to the saline group). Naloxone also showed minimal cytotoxicity below 0.5 mg mL'1in both cell lines (p > 0.05 compared to the saline group), but cell survival was reduced to 76.0 ± 10.5 % at 1 mg mL'1for 3T3 cells (p = 0.007 compared to the saline group) and 80.0 ± 5.2 % cell survival for C2C12 cells (p = 0.0005 compared to the saline group).Example 6 - Hydrogel of An Exemplary Conjugate (P407-CM-NLX)

[0385] Poloxamer 407 (P407) is a polymer with thermal gelation property. A coumarinnaloxone conjugate with an azide (N3) moiety was synthesized according to the skeletal structure diagram of FIG. 37. The azide-containing coumarin-nal oxone conjugate was reacted with an alkyne-terminated P407 through copper-catalyzed azide-alkyne cycloaddition (CuAAC).

[0386] The thermal gelation property of P407-CM-NLX (20 wt% in aqueous) was verified by oscillatory shear rheology (10 rads'1, 1% strain, 1 °C / min). The material behaved as a liquid at 20°C (storage modulus G’ was lower than loss modulus), and formed a solid (gel) at 37°C (G’ was greater than G”) as shown in FIGS. 38-40.

[0387] In absence of irradiation, subcutaneously inj ected P407-CM-NLX did not affect the analgesia by morphine. A latency remained at the maximum at 32.2 ± 0 seconds, 60 minutes after morphine injection, similar to the latencies in mice injected with morphine only. When the site of P407-CM-NLX injection was irradiated with a 400 nm LED for two minutes at 300 mW / cm230 min after morphine, A latency decreased to -3.6 ± 11.4 seconds at 60 min, indicating its ability to reverse opioids.

[0388] The time to onset reversal by naloxone triggered from P407-CM-NLX was similar to that from free naloxone (no significant difference, p > 0.05 at all the time points), faster than PLGA-CM-NLX. With P407-CM-NLX, it took 5 minutes for A latency to become different from that of animals treated with morphine alone, and the latency was similar to baseline within 10 minutes. With PLGA-CM-NLX, it took 10 minutes for A latency to become different from that of animals treated with morphine alone, and the latency was similar to baseline within 20 min. Graphical representations of the A latency as a function of time can be seen in FIGS. 41- 42Examples Overview and Summary

[0389] A polymer-drug conjugate system was developed as a depot for providing externally triggered naloxone opioid reversal. For many externally triggered drug delivery systems, substantial untriggered drug release is not uncommon; it may result in untriggered therapeutic effects. Here, the conjugate PLGA-CM-NLX itself was confirmed to be inert and did not interrupt the effect of morphine in the hotplate test. It can be attributed to the fact that the drug was chemically conjugated on the polymer, which provided a stronger force to bound the drug in the depot compared with using physical encapsulation methods. The system only became activated when irradiated with the LED, and could produce opioid reversal as an analog to subcutaneously injected free naloxone. Minimal untriggered drug release from a depot is desired for the design of an externally triggered naloxone-releasing system, so that no continuously released naloxone would affect the normal usage of opioids.

[0390] Since the therapeutic index of naloxone is high, any possible over-triggering would not be a concern. Accidentally triggering of the system by the patient or ambient light would also not course harm to the patient, because naloxone does not have therapeutic effect in the absence of opioids. Besides, if the materials were to be used in a potential real-world scenario, ideally, it would be covered to protect it from ambient light by a wristband, equipped with an LED for triggering. The design would provide convenience for patients.

[0391] While inactive itself, the materials remained triggerable for up to a month of delay after its initial administration. This was attributed to the long retention time of the PLGA-based drug delivery system. In potential clinical applications, patients would be administered with materials when they leave the healthcare system, together with any opioid medications that would have normally been given to them for pain management. The naloxone depot system would only be activated when needed at a remote time as the exact time when an overdose would happen is unpredictable. Besides, overdose could happen more than once for the samepatient, a system that can be used for more than once would be beneficial. Such situations were mimicked by triggering the depot on multiple days in the same animals. The triggering can be performed three times on days 3, 5, 10, respectively. On-demand activation would provide flexibility and convenience for the patients.

[0392] Furthermore, the half-life of naloxone is known to be shorter than most opioids, and repeated naloxone administration may be sometimes needed to achieve adequate opioid reversal, particularly for long-acting opioid formations. Therefore, this situation was mimicked by using two sequential doses of morphine and assessing the possibility of the materials against prolonged morphine. One time triggering blocked the first dose of morphine, but renarcotization occurred when the second dose of morphine was given. A similar phenomenon was observed in the case of free naloxone control group. However, a second time triggering of the system successfully blocked the second dose of morphine. Therefore, with our system, the patients would not need to carry multiple naloxone devices (e.g., nasal sprays) all the time.

[0393] In summary, a novel light-responsive polymer-naloxone conjugate was developed. The conjugate was processed into injectable nanoparticles and was able to release naloxone in its native form upon blue light irradiation. It is worth noting that the materials did not affect normal opioid usage in the absence of photo-triggering. It only enabled opioid reversal by light irradiation. And such triggering can be performed weeks after the administration of the material, over multiple days and for multiple times. The exemplary conjugate in this study could provide a timely and on-demand opioid antidote administration method for combating the opioid crisis.REFERENCES Florence, C.; Luo, F.; Rice, K., The economic burden of opioid use disorder and fatal opioid overdose in the United States, 2017. Drug and Alcohol Dependence 2021, 218, 108350. Wermeling, D. P., Review of naloxone safety for opioid overdose: practical considerations for new technology and expanded public access. 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[0394] In the claims articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The disclosure includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The disclosure includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

[0395] Furthermore, the disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the disclosure, or aspects described herein, is / are referred to as comprising particular elements and / or features, certain embodiments described herein or aspects described herein consist, or consist essentially of, such elements and / or features. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein.

[0396] It is also noted that the terms “comprising” and “containing” are intended to be open and permit the inclusion of additional elements or steps. Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different embodiments described herein, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0397] This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. If there is a conflict between any of the incorporated references and the instant specification, the specification shall control. In addition, any particular embodiment of the present disclosure that falls within the prior art may be explicitly excluded from any one ormore of the claims. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein.

[0398] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description, but rather is as set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present disclosure, as defined in the following claims.

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A conjugate comprising: i. a polymer; and ii. an active agent, wherein the active agent is an agent capable of reversing or reducing an effect of an opioid in a subject, and wherein the active agent is linked to the polymer via a cleavable linker.

2. The conjugate of claim 1, wherein the agent capable of reversing or reducing an effect of an opioid is a competitive opioid receptor antagonist.

3. The conjugate of any one of claims 1-2, wherein the agent capable of reversing or reducing an effect of an opioid is naloxone or a pharmaceutically acceptable salt thereof, or an analogue or derivative thereof.

4. The conjugate of any one of claims 1-3, wherein the opioid is morphine, hydromorphone, oxymorphone, dihydromorphine, diacetyl morphine (diamorphine or heroin), codeine, hydrocodone, oxycodone, methadone, theabine, pethidine (meperidine), alfentanil, fentanyl, remifentanil, sufentanil, etorphine, carefentanil, buorenophine, tramadol, venlafaxine, or a pharmaceutically acceptable salt thereof, or an analogue or derivative thereof.

5. The conjugate of any one of claims 1-4, wherein the opioid is a synthetic or semisynthetic molecule.

6. The conjugate of any one of claims 1-5, wherein the opioid is an opiate.

7. The conjugate of any one of claims 1-6, wherein the cleavable linker is a photocleavable linker.

8. The conjugate of claim 7, wherein the photocleavable linker is cleaved by exposure to light having an average wavelength of between about 300 nm and about 900 nm.

9. The conjugate of any one of claims 7-8, wherein the photocleavable linker is cleaved by exposure to light having an average wavelength of between about 350 nm and about 530 nm.

10. The conjugate of any one of claims 7-9, wherein the photocleavable linker is cleaved by exposure to light having an average wavelength of between about 375 nm and about 475 nm.

11. The conjugate of any one of claims 7-10, wherein the photocleavable linker is cleaved by exposure to light having an average wavelength of about 400 nm.

12. The conjugate of any one of claims 7-11, wherein the photocleavable linker comprises a coumarin moiety, a o-nitrobenzyl moiety, a p-nitrobenzyl moiety, a m-nitrobenzyl moiety, a benzoin moiety, a nitroindoline moiety, a quinoline moiety, a hydroxyphenacyl moiety, or any combination thereof.

13. The conjugate of any one of claims 7-12, wherein the photocleavable linker comprises a coumarin moiety, a o-nitrobenzyl moiety, a benzoin moiety, a 7-nitroindoline moiety, a p-hydroxyphenacyl moiety, a hydroxyquinoline moiety, or any combination thereof.

14. The conjugate of any one of claims 7-13, wherein the photocleavable linker comprises a coumarin moiety.

15. The conjugate of any one of claims 7-14, wherein the photocleavable linker comprises 7- (diethylamino) coumarin-4-yl] methyl carboxyl.

16. The conjugate of any one of claims 7-15, wherein the photocleavable linker comprises:wherein R is the polymer; andR’ is the agent capable of reversing or reducing an effect of an opioid.

17. The conjugate of any one of claims 1-6, wherein the cleavable linker is a chemically cleavable linker.64SUBSTITUTE SHEET (RULE 26)18. The conjugate of any one of claims 1-6, wherein the cleavable linker is an enzymatically cleavable linker.

19. The conjugate of any one of claims 1-6, wherein the cleavable linker is a ribozyme- cleavable linker.

20. The conjugate of any one of claims 1-6, wherein the cleavable linker is a redox- cleavable linker.

21. The conjugate of any one of claims 1-6, wherein the cleavable linker is an acid- cleavable linker.

22. The conjugate of any one of claims 1-6, wherein the cleavable linker is a base-cleavable linker.

23. The conjugate of any one of claims 1-6, wherein the cleavable linker is a nucleophile- cleavable linker.

24. The conjugate of any one of claims 1-6, wherein the cleavable linker is an electrophile- cleavable linker.

25. The conjugate of any one of claims 1-6, wherein the cleavable linker is a temperature- cleavable linker.

26. The conjugate of any one of claims 1-6, wherein the cleavable linker is a spontaneous- hydrolysis-cleavable linker.

27. The conjugate of any one of claims 1-6, wherein the cleavable linker is a reactive- oxygen-species-cleavable linker.

28. The conjugate of any one of claims 1-6, wherein the cleavable linker is an ultrasound- cleavable linker.

29. The conjugate of any one of claims 1-6, wherein the cleavable linker is an electromagnetic-radiation-cleavable linker.

30. The conjugate of any one of claims 1-29, wherein the polymer is a hydrophilic polymer of an amphiphilic polymer.

31. The conjugate of any one of claims 1-30, wherein the polymer is selected from the group consisting of polylactides, polyglycolides, polycaprolactones, polyanhydrides, polyepsilon caprolactone, polyamides, polyurethanes, polyesteramides,polyorthoesters, polydioxanones, polyacetals, polyketals, polycarbonates, polyorthocarbonates, polydihydropyrans, polyphosphazenes, polyhydroxybutyrates, polyhydroxyvalerates, polyalkylene oxalates, polyalkylene succinates, poly(malic acid), poly(amino acids), polyvinylpyrrolidone, polyetheres (e.g., polyethylene glycol), polyhydroxycellulose, polymethyl methacrylate, chitin, chitosan, poly(glycerol sebacate) (PGS), cellulose acetates (including cellulose diacetate), polyethylene, polypropylene, polybutylene, polyethylene terphthalate (PET), polyvinyl chloride, polystyrene, polyamides, nylon, polycarbonates, polysulfides, polysulfones, hydrogels (e.g., acrylics), polyacrylonitrile, polyvinylacetate, cellulose acetate butyrate, nitrocellulose, copolymers of urethane / carbonate, copolymers of styrene / maleic acid, poly(ethylenimine), poloxamers (e.g., poloxamer P407, poloxamer P331, poloxamer P188, poloxamer 182, poloxamer P124, poloxamer P338, or poloxamer P237), and copolymers or terpolymers, thereof.

32. The conjugate of any one of claims 1-31, wherein polymer is poly(lactic-co-glycolic acid) (PLGA), a copolymer comprising PLGA or a poloxamer.

33. The conjugate of claim 32, wherein a ratio of lactide to glycolide in the PLGA is between 50:50 and 100:0.

34. The conjugate of claim 32 or 33, wherein a ratio of lactide to glycolide in the PLGA is between 50:50 and 90: 10.

35. The conjugate of any of claims 32-34, wherein a ratio of lactide to glycolide in the PLGA is between 50:50 and 75:25.

36. The conjugate of any one of claims 1-35, wherein the polymer is a biocompatible polymer.

37. The conjugate of any one of claims 1-36, wherein the polymer is biodegradable.

38. The conjugate of any one of claims 1-37, wherein the conjugate is of Formula (I):P-(L-X)n(Formula I), wherein: n is an integer of 1 or more; each instance of X is independently the agent capable of reversing or reducing an effect of an opioid;each instance of L is independently the cleavable linker; and P is the polymer.

39. The conjugate claim 38, wherein n is an integer between 1 and 100,000, inclusive.

40. The conjugate of any one of 38-39, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

41. The conjugate of any one of claims 38-40, wherein n is 2.

42. The conjugate of any one of claims 1-41, wherein a first agent capable of reversing or reducing an effect of an opioid is linked to a first end of the polymer and a second agent capable of reversing or reducing an effect of an opioid is linked to a second end of the polymer.

43. The conjugate of any one of claims 1-42, wherein the conjugate is:

44. The conjugate of any one of claims 1-43, wherein the conjugate is formulated in a particle.

45. The conjugate of any one of claims 1-44, wherein the conjugate is formulated in a hydrogel, optionally, the hydrogel is a cross-linked polysaccharide hydrogel.

46. A particle comprising a conjugate of any one of claims 1-45.

47. The particle of claim 46, wherein the particle is comprised in a hydrogel, e.g., the particle is a hydrogel particle.

48. The particle of claim 47, wherein the hydrogel is a cross-linked polysaccharide hydrogel, e.g., the particle is a cross-linked polysaccharide hydrogel particle.

49. The particle of any one of claims 46-48, wherein the particle is a nanoparticle or microparticle.

50. The particle of any one of claims 46-49, wherein the particle is a nanoparticle.67SUBSTITUTE SHEET (RULE 26)51. The particle of any one of claim 46-50, wherein the particle has a hydrodynamic diameter of less than about 1000 nm.

52. The particle of any one of claim 46-51, wherein the particle has a hydrodynamic diameter of less than about 750 nm.

53. The particle of any one of claim 46-52, wherein the particle has a hydrodynamic diameter of less than about 700 nm.

54. The particle of any one of claim 46-53, wherein the particle has a hydrodynamic diameter of less than about 600 nm.

55. The particle of any one of claim 46-54, wherein the particle has a hydrodynamic diameter of less than about 500 nm or less than about 550 nm.

56. The particle of any one of claims 46-55, wherein the particle has a hydrodynamic diameter of from about 25 nm to about 400 nm.

57. The particle of any one of claims 46-56, wherein the particle has a hydrodynamic diameter of from about 50 nm to about 300 nm.

58. The particle of any one of claims 46-57, wherein the particle has a hydrodynamic diameter of from about 75 nm to about 200 nm.

59. The particle of any one of claims 46-58, wherein the particle has a hydrodynamic diameter of from about 80 nm to about 150 nm.

60. The particle of any one of claims 46-59, wherein the particle has a hydrodynamic diameter of about 85 nm to about 125 nm.

61. The particle of any one of claims 46-60, wherein the particle has a hydrodynamic diameter of from about 90 nm to about 110 nm.

62. The particle of any one of claims 46-61, wherein the particle is substantially spherical.

63. The particle of any one of claims 46-62, wherein the particle further comprises a dye.

64. A hydrogel comprising a conjugate of any one of claims 1-45.

65. The hydrogel of claim 64, wherein the conjugate is present in a particle.

66. The hydrogel of claim 65, wherein the particle is a particle of any one of claims 46-63.

67. The hydrogel of any one of claims 64-66, wherein the hydrogel comprises a dye.

68. A depot suitable for implantation into a subject comprising a conjugate of any one of claims 1-45, or a particle of any one of claims 46-63, or a hydrogel of any one of claims 64-67.

69. A pharmaceutical composition comprising a therapeutically effective amount of a conjugate of any one of claims 1-45, or a particle of any one of claims 46-63, or a hydrogel of any one of claims 64-67, or a depot of claim 68, and a pharmaceutically acceptable excipient.

70. The pharmaceutical composition of claim 69, wherein the pharmaceutical composition is formulated for systemic administration.

71. The pharmaceutical composition of any one of claims 69-70, wherein the pharmaceutical composition is formulated for parenteral administration.

72. The pharmaceutical composition of any one of claims 69-71, wherein the pharmaceutical composition is formulated for intravenous, intramuscular, subcutaneous or transdermal administration.

73. The pharmaceutical composition of any one of claims 69-72, wherein the pharmaceutical composition further comprises a dye.

74. A method for reducing or reversing an effect of an opioid in a subject, the method comprises: applying a stimulus at a site of where the subject was administered an effective amount of a conjugate of any one of claims 1-45, or a particle of any one of claims 46-63, or a hydrogel of any one of claims 64-67, or a depot of claim 68, or a pharmaceutical composition of any one of claims 69-73, wherein the active agent is an agent capable of reversing or reducing an effect of an opioid, and wherein the stimulus is capable of cleaving the cleavable linker linking the polymer and the active agent.

75. The method of claim 74, wherein the stimulus is applied for 30 minutes or less.

76. The method of any one of claims 74-75, wherein the stimulus is applied for 15 minutes or less.

77. The method of any one of claims 74-76, wherein the stimulus is applied for 10 minutes or less.

78. The method of any one of claims 74-77, wherein the stimulus is applied for 5 minutes or less.

79. The method of any one of claims 74-78, wherein the stimulus is applied for 3 minutes or less.

80. The method of any one of claims 74-79, wherein the method comprises applying the stimulus for a first period of time, waiting for a first period of time, and applying the stimulus for a second period of time.

81. The method of claim any one of claims 74-80, wherein the cleavable linker is a photocleavable linker, and wherein the stimulus is light.

82. The method of claim 81, wherein the light comprises near-infrared light.

83. The method of any one of claims 81-82, wherein the light comprises visible light.

84. The method of any one of claims 81-83, wherein the light has an average wavelength of between about 300 nm and about 900 nm.

85. The method of any one of claims 81-84, wherein the light has an average wavelength of between about 350 nm and about 530 nm.

86. The method of any one of claims 81-85, wherein the light has an average wavelength of between about 375 nm and about 475 nm.

87. The method of any one of claims 81-86, wherein the light has an average wavelength of about 400 nm.

88. The method of any one of claims 81-87, wherein the light is blue light.

89. The method of any one of claims 81-88, wherein the light has an intensity of at least 50 mW / cm2.

90. The method of any one of claims 81-89, wherein the light has an intensity of at least 100 mW / cm2.

91. The method of any one of claims 81-90, wherein the light has an intensity of at least 150 mW / cm2.

92. The method of any one of claims 81-91, wherein the light has an intensity of at least 200 mW / cm2.

93. The method of any one of claims 81-92, wherein the light has an intensity of at least 250 mW / cm2.

94. The method of any one of claims 81-93, wherein the light has an intensity of at least 300 mW / cm2.

95. The method of any one of claims 81-94, wherein the light has an intensity of at least 350 mW / cm2.

96. The method of any one of claim 81-95, wherein the light is applied by a LED.

97. The method of any one of claims 74-80, wherein the cleavable linker is a chemically cleavable linker and the stimulus is a reagent for cleaving the chemically cleavable linker.

98. The method of any one of claims 74-80, wherein the cleavable linker is an enzymatically cleavable linker and the stimulus is an enzyme.

99. The method of any one of claims 74-80, wherein the cleavable linker is a ribozyme- cleavable linker and the stimulus is a ribozyme.

100. The method of any one of claims 74-80, wherein the cleavable linker is a redox- cleavable linker and the stimulus is a redox agent.

101. The method of any one of claims 74-80, wherein the cleavable linker is an acid- cleavable linker and the stimulus is an acid.

102. The method of any one of claims 74-80, wherein the cleavable linker is a base- cleavable linker and the stimulus is a base.

103. The method of any one of claims 74-80, wherein the cleavable linker is a nucleophile- cleavable linker and the stimulus is a nucleophile.

104. The method of any one of claims 74-80, wherein the cleavable linker is an electrophile- cleavable linker and the stimulus is an electrophile.

105. The method of any one of claims 74-80, wherein the cleavable linker is a temperature- cleavable linker and the stimulus is a change in temperature.

106. The method of any one of claims 74-80, wherein the cleavable linker is a spontaneous- hydrolysis-cleavable linker and the stimulus is a hydrolysis reagent.

107. The method of any one of claims 74-80, wherein the cleavable linker is a reactive- oxygen-species-cleavable linker and the stimulus is a reactive-oxygen-species or a reagent capable of generating a reactive-oxygen-species.

108. The method of any one of claims 74-80, wherein the cleavable linker is an ultrasound- cleavable linker and the stimulus is ultrasound.

109. The method of any one of claims 74-80, wherein the cleavable linker is an electromagnetic-radiation-cleavable linker and the stimulus is electromagnetic radiation.

110. The method of any one of claims 74-109, wherein the method further comprises: administering a conjugate of any one of claims 1-45, or a particle of any one of claims 46-63, or a hydrogel of any one of claims 64-67, or a depot of claim 68, or a pharmaceutical composition of any one of claims 69-73, to the subject prior to applying the stimulus.

111. A method comprising administering a conjugate of any one of claims 1-45, or a particle of any one of claims 46-63, or a hydrogel of any one of claims 64-67, or a depot of claim 68, or a pharmaceutical composition of any one of claims 69-73, to a subject.

112. The method of claim 110 or 111, wherein said administering comprises parenteral administration.

113. The method of any one of claims 110-112, wherein said administering comprises intravenous, intramuscular, subcutaneous or transdermal administration.

114. The method of any one of claims 110-113, wherein said administering comprises subcutaneous injection.

115. The method of any one of claims 110-114, wherein said administering is via microneedles.

116. The method of any one of claims 110-115, wherein said administering is via a microneedle patch.

117. The method of any one of claims 110-116, wherein said administering is in a wrist of the subject.

118. The method of any one of claims 74-117, wherein the subjectis overdosed on an opioid.

119. The method of any one of claims 74-118, wherein the subject is human.

120. A kit comprising a conjugate of any one of claims 1-45, or a particle of any one of claims 46-63, or a hydrogel of any one of claims 64-67, or a depot of claim 68, or a pharmaceutical composition of any one of claims 69-73.

121. The kit of claim 120, further comprising a means for administering the conjugate, the particle, the hydrogel, the depot, or the pharmaceutical composition to a subject.

122. The kit of any one of claims 120-121, further comprising a means for cleaving the cleavable linker.

123. The kit of any one of claims 120-122, wherein the cleavable linker is a photocleavable linker and the kit further comprises a light emitting device.

124. The kit of claim 123, wherein the light emitting device emits near-infrared light.

125. The kit of any one of claims 123-124, wherein the light emitting device emits visible light.

126. The kit of any one of claims 123-125, wherein the light has an average wavelength of between about 300 nm and about 900 nm.

127. The kit of any one of claims 123-126, wherein the light has an average wavelength of between about 350 nm and about 530 nm.

128. The kit of any one of claims 123-127, wherein the light has an average wavelength of between about 375 nm and about 475 nm.

129. The kit of any one of claims 123-128, wherein the light has an average wavelength of about 400 nm.

130. The kit of any one of claims 123-129, wherein, wherein the light is blue light.

131. The kit of any one of claims 123-130, wherein the light has an intensity of at least 50 mW / cm2.

132. The kit of any one of claims 123-131, wherein the light has an intensity of at least 100 mW / cm2.

133. The kit of any one of claims 123-132 wherein the light has an intensity of at least 150 mW / cm2.

134. The kit of any one of claims 123-133, wherein the light has an intensity of at least 200 mW / cm2.

135. The kit of any one of claims 123-134, wherein the light has an intensity of at least 250 mW / cm2.

136. The kit of any one of claims 123-135, wherein the light has an intensity of at least 300 mW / cm2.

137. The kit of any one of claims 123-136, wherein the light has an intensity of at least 350 mW / cm2.

138. The kit of any one of claims 123-137, wherein the light emitting device comprises a LED.

139. The kit of any one of claims 123-138, wherein the light emitting device is in form of a wearable.

140. The kit of any one of claims 123-139, wherein the light emitting device is in form of a bracelet.

141. The kit of any one of claims 123-140, wherein the kit further comprises instructions for use.

142. The kit of any one of claims 120-122, wherein the cleavable linker is a chemically cleavable linker and the kit further comprises a reagent for cleaving the chemically cleavable linker.

143. The kit of any one of claims 120-122, wherein the cleavable linker is an enzymatically cleavable linker and the kit further comprises an enzyme.

144. The kit of any one of claims 120-122, wherein the cleavable linker is a ribozyme- cleavable linker and the kit further comprises a ribozyme.

145. The kit of any one of claims 120-122, wherein the cleavable linker is a redox-cleavable linker and the kit further comprises a redox agent.

146. The kit of any one of claims 120-122, wherein the cleavable linker is an acid-cleavable linker and the kit further comprises an acid.

147. The kit of any one of claims 120-122, wherein the cleavable linker is a base-cleavable linker and the kit further comprises a base.

148. The kit of any one of claims 120-122, wherein the cleavable linker is a nucleophile- cleavable linker and the kit further comprises a nucleophile.

149. The kit of any one of claims 120-122, wherein the cleavable linker is an electrophile- cleavable linker and the kit further comprises an electrophile.

150. The kit of any one of claims 120-122, wherein the cleavable linker is a temperature- cleavable linker and the kit further comprises means for changing the temperature at the site of the administration.

151. The kit of any one of claims 120-122, wherein the cleavable linker is a spontaneous- hydrolysis-cleavable linker and the kit further comprises a hydrolysis reagent.

152. The kit of any one of claims 120-122, wherein the cleavable linker is a reactive-oxygenspecies-cleavable linker and the kit further comprises a reactive-oxygen-species or a reagent capable of generating a reactive-oxygen-species.

153. The kit of any one of claims 120-122, wherein the cleavable linker is an ultrasound- cleavable linker and the kit further comprises a device for generating ultrasound.

154. The kit of any one of claims 120-122, wherein the cleavable linker is an electromagnetic-radiation-cleavable linker and the kit further comprises a device for generating electromagnetic radiation.

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