Photoresponsive peptide delivery systems and methods of use thereof

Abstract

Provided herein are photoresponsive prodrugs comprising an active agent conjugated to a photoresponsive group, and nanoparticles formed in which a photoresponsive prodrug is co-assembled with a polymer. Also provided are methods of treating a subject comprising administering a photoresponsive prodrug or nanoparticle to the subject and irradiating a target site with a light source.

Description

Technical Field

[0001] The present invention generally relates to the field of synthetic prodrugs, nanoparticles and their use in treating or preventing disease. Background Art

[0002] Posterior segment diseases such as age-related macular degeneration, diabetic retinopathy, and posterior uveitis have attracted widespread attention in recent decades, as damage to the retina and optic nerve is the main cause of severe vision loss. Due to the unique anatomy and physiology of the eye, delivering adequate doses of drugs to the posterior segment remains challenging, and systemic administration of high-dose drugs often results in severe systemic adverse events. Although intravitreal injections have been shown to be a good solution, local adverse events caused by repeated surgeries are sometimes observed, including cataracts, endophthalmitis, vitreous hemorrhage, not to mention the mental stress caused to some patients.

[0003] In recent years, light-responsive nanocarriers for targeted and sustainable drug delivery to the posterior segment of the eye have been widely studied because light can easily reach the posterior segment through anterior tissues (including the cornea, lens, and vitreous body, which are usually transparent in nature). Although it is widely believed that short-wavelength light can cause damage to multiple ocular tissues, especially the retina, due to phototoxicity, most reported nanocarriers still require short-wavelength light to trigger drug release because otherwise complex manufacturing processes or toxic ingredients are required, and two-photon excitation mechanisms or photon upconversion processes usually require pulsed lasers with high-power sources.

[0004] Currently, there is still a need for photoresponsive prodrug delivery systems with improved therapeutic efficacy, better cyclic stability, and lower phototoxicity. SUMMARY OF THE INVENTION

[0006] This overview describes several embodiments of the presently disclosed subject matter, and in many cases lists variations and permutations of these embodiments. This overview is merely an example of numerous different embodiments. Mentions of one or more representative features of a given embodiment are also exemplary. Such embodiments may generally exist with or without the features mentioned; similarly, these features may be applied to other embodiments of the presently disclosed subject matter, whether or not listed in this overview. In order to avoid excessive repetition, this overview does not list or suggest all possible functional combinations.

[0007] In one aspect, the present invention relates to a photoresponsive prodrug comprising an active agent conjugated to a photoresponsive group. Such a prodrug can be used to achieve light-controlled delivery of peptides and treatment and prevention of various diseases.

[0008] In some embodiments, the active agent is an anti-angiogenic agent. In some embodiments, the active agent is an anti-angiogenic peptide. In some embodiments, the active agent is an AS16 peptide having the sequence ATWLPPRAANLLMAAS (SEQ ID NO: 1) or an FM12 peptide comprising the amino acid sequence FPNWSLRPMNQM (SEQ ID NO: 2).

[0009] In some embodiments, the photoresponsive group is boron-dipyrromethene (BODIPY). In some embodiments, BODIPY is a moiety selected from the group consisting of Formula (VII), Formula (VIII), Formula (IX), Formula (X), Formula (XI), or Formula (XII). In some embodiments, BODIPY is a moiety of Formula (XII):

[0010]

[0011]

[0012]

[0013] In some embodiments, the photoresponsive prodrug is:

[0014]

[0015] In another aspect, provided herein are nanoparticles comprising a photoresponsive prodrug disclosed herein and one or more polymers.

[0016] In some embodiments, the polymer is selected from polylactic acid (PLA), polyethylene glycol (PEG), polydimethylsiloxane (PDMS), polyethyleneimine (PEI), polyamidoamine (PAMAM) and combinations thereof. In some embodiments, the polymer is a PLA-PEG copolymer. In some embodiments, the polymer is PLA5k-PEG2k or PEG3.4k-PLA5k.

[0017] In some embodiments, the nanoparticles have a diameter of about 20-200 nm. In some embodiments, the nanoparticles have a diameter of about 40-100 nm. In some embodiments, the nanoparticles have a diameter of about 50-70 nm.

[0018] In some embodiments, the nanoparticles have a polydispersity index (PDI) of 0.120-0.220. In some embodiments, the nanoparticles have a polydispersity index (PDI) of 0.150-0.200.

[0019] In some embodiments, the nanoparticles have a diameter of about 60 nm and a PDI of 0.170; or have a diameter of about 122 nm and a PDI of 0.2.

[0020] In some embodiments, the nanoparticles comprise a photoresponsive prodrug of the formula co-assembled with PLA5k-PEG2k

[0021]

[0022] or a photoresponsive prodrug of cFM12 coassembled with PEG3.4k-PLA5k.

[0023] In some embodiments, the nanoparticles further encapsulate an additional agent. In some embodiments, the additional agent is a hydrophobic drug. In some embodiments, the additional agent is an anticancer drug. In some embodiments, the additional agent is selected from the group consisting of tamoxifen, amsacrine, bexarotene, estramustine, irofoven, trabectedin, cetuximab, panitumumab, tositumomab, alemtuzumab, bevacizumab, edrecolomab, gemtuzumab, alvocidib, seliciclib, aminolevulinic acid, methyl aminolevulinate, efaproxiral, porfimer sodium, talaporfin, temoporfm, vituximab, tadalafil ... Tiporfin, alitretinoin, tretinoin, anagrelide, arsenic trioxide, atrasentan, bortezomib, carmofur, celecoxib, demecillin, elisimol, elsamitrucin, etoglu, lonidamine, lucanthone, masoprofol, dibromomannitol, mitoguanazone, mitotane, oblimersen sodium, omacetin, sitimagene, ceradenovec, tegafur, testolactone, thiazofurine, tipifarnib, vorinostat or iniparib. In some embodiments, the additional agent is elisimol.

[0024] In another aspect, provided herein is a pharmaceutical composition comprising: (i) a photoresponsive prodrug disclosed herein or a nanoparticle disclosed herein; and (ii) a pharmaceutically acceptable excipient.

[0025] In yet another aspect, provided herein is a method for delivering a drug to a target site in a subject, comprising: (i) administering to the subject a photoresponsive prodrug disclosed herein, a nanoparticle disclosed herein, or a pharmaceutical composition disclosed herein; and (ii) irradiating the self-assembled system at the target site with a light source.

[0026] In some embodiments, the light source has a wavelength of about 500-1300nm. In some embodiments, the light source has a wavelength of about 600-900nm. In some embodiments, the light source has a wavelength of about 600-700nm. In some embodiments, the light source has a wavelength of about 656nm.

[0027] In some embodiments, the photoresponsive prodrugs or nanoparticles disclosed herein are administered by at least one of oral administration, transdermal administration, inhalation, intranasal administration, topical administration, intravaginal administration, ocular administration, intramural administration, intracerebral administration, rectal administration, parenteral administration, intravenous administration, intraarterial administration, intramuscular administration, subcutaneous administration, intratumoral administration, and any combination thereof. In some embodiments, the photoresponsive prodrugs or nanoparticles disclosed herein are administered by intravenous administration.

[0028] In some embodiments, the target site is the eye, skin, or a tumor. In some embodiments, the target site is the posterior segment of the eye.

[0029] In another aspect, provided herein is a method for treating or preventing a disease characterized by abnormal angiogenesis, comprising: (i) administering to a subject a photoresponsive prodrug disclosed herein, a nanoparticle disclosed herein, or a pharmaceutical composition disclosed herein; and (ii) irradiating the self-assembled system at a target site with a light source.

[0030] In some embodiments, the light source has a wavelength of about 500-1300nm. In some embodiments, the light source has a wavelength of about 600-900nm. In some embodiments, the light source has a wavelength of about 600-700nm. In some embodiments, the light source has a wavelength of about 656nm.

[0031] In some embodiments, the photoresponsive drugs or nanoparticles disclosed herein are administered by at least one of: oral administration, transdermal administration, inhalation, intranasal administration, topical administration, intravaginal administration, ocular administration, intramural administration, intracerebral administration, rectal administration, parenteral administration, intravenous administration, intraarterial administration, intramuscular administration, subcutaneous administration, intratumoral administration, and any combination thereof. In some embodiments, the photoresponsive drugs or nanoparticles disclosed herein are administered by intravenous administration.

[0032] In some embodiments, the target site is the eye, skin, or a tumor. In some embodiments, the target site is the posterior segment of the eye.

[0033] In some embodiments, the method is used to treat or prevent cancer. In some embodiments, the method is used to treat or prevent kidney cancer, lung cancer, breast cancer, colon cancer, prostate cancer, brain cancer, chondrosarcoma or angiosarcoma.

[0034] In some embodiments, the method is used to treat or prevent ocular neovascular disease. In some embodiments, the method is used to treat or prevent age-related macular degeneration (AMD), choroidal neovascularization secondary to myopia, proliferative diabetic retinopathy, diabetic macular edema, retinal vascular occlusion such as retinal vein occlusion, eye tumors, Hipper-Lindau syndrome, retinopathy of prematurity and polypoidal choroidal vasculopathy. In some embodiments, the method is used to treat or prevent AMD, especially exudative AMD (wet AMD).

[0035] In another aspect, provided herein is a kit comprising a photoresponsive prodrug disclosed herein, a nanoparticle disclosed herein, or a pharmaceutical composition disclosed herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Schematic diagram of the NIR light-responsive AS16 peptide release nanosystem for the treatment of choroidal neovascularization. (a) 3D structural and functional illustration of the nanoLAMP composed of BODIPY-AS16 conjugate and PLA5k-PEG2k polymer. NIR light triggers the release of AS16 at the ideal location for targeted anti-angiogenesis and anti-M2 macrophage polarization therapy. (b) Synthesis route of BODIPY-AS16 conjugate.

[0037] Figure 2 . Uncaged (a) and BODIPY caged (b) AS16 peptide 1 H-NMR spectrum.

[0038] Figure 3 .Characterization of nanoLAMP. (3a) UV-Vis spectra of nanoLAMP, LAMP, BODIPY-OH and AS16 peptide. (3b) Dynamic size change of nanoLAMP after NIR light irradiation shown by DLS. (3c) Results of stability test of nanoLAMP in DMEM complete medium at 37°C by DLS. (3d) Quantitative photorelease curve of AS16 peptide obtained by HPLC analysis. (3e) TEM images of nanoLAMP before and after NIR light irradiation. (3f) Color change of nanoLAMP after NIR light irradiation for different time periods. (3g) Loading capacity and encapsulation efficiency of BODIPY-AS16 conjugate in nanoLAMP. (3h) HPLC spectrum showing the release of AS16 peptide. The data are expressed as mean ± SEM.

[0039] Figure 4HPLC spectra of LAMP at different time periods under NIR light irradiation, indicating the release of BODIPY in LAMP after NIR light irradiation.

[0040] Figure 5 . In vitro NIR-induced release of AS16 peptide from nanoLAMP for anti-angiogenic therapy. (5a) VEGF stimulates HUVEC tube formation. HUVEC suspension was treated with 20 ng / mL VEGF 165 treated and co-incubated with different preparations at a dose equivalent to 5 μM of AS16 peptide. The light-irradiated nanoLAMP group showed significantly less tube structure formation compared to the control group and the non-light-irradiated nanoLAMP group (n=3). (5b) Migration assay of each group at 24 h. The trend was similar to the tube formation assay (n=3). (5c) Representative images of wound healing assays of each group, showing that the light-irradiated nanoLAMP group had a superior therapeutic effect (n=3). (5d) Representative images of Western blot analysis of pro-angiogenic signaling proteins in HUVECs after different treatments (n=3). (5e) Statistical analysis of Western blot analysis calculated by ImageJ. (5f) Statistical analysis of the number of junctions in the tube formation assay. (5g) Statistical analysis of the total fragment length of each group. (5h) Statistical analysis of the cell counts in the bottom chamber of each group. (5i) Statistical analysis of the wound healing process of each group measured by ImageJ software. (*: p<0.05; **: p<0.01; ***: p<0.001). Data are presented as mean ± SEM.

[0041] Figure 6 .The therapeutic efficacy of nanoLAMP was investigated by imaging diagnosis. (6a) Representative images of OCTA (optical coherence tomography angiography) of each group. The bright signals located around the optic nerve represent CNV lesions in the posterior segment of the mouse eye (n=6). (6b) FFA (fluorescent fundus angiography) images of each group taken by Phoenix Micron IV system 5 minutes after intravenous injection of sodium fluorescein (n=6). (6c) Representative images of OCT (optical coherence tomography) of each group acquired by TowardPi tip swept source OCT (n=6). (6d) Statistical analysis of CNV area by OCTA. (6e) Statistical analysis of FFA of each group. Grade I: no hyperfluorescence; Grade II: hyperfluorescence, no leakage; Grade III: hyperfluorescence in the images of early or mid-transition images, late leakage; Grade IV: bright hyperfluorescence, increased intensity and size. (6f) Statistical analysis of CNV thickness of each group. (*: p<0.05; **: p<0.01; ***: p<0.001). Data are presented as mean ± SEM.

[0042] Figure 7.The anti-angiogenic efficacy of nanoLAMP was revealed by the study of isolated tissues. (7a) Representative images of choroidal plain mounts incubated with CD31 antibody and Ki-67 antibody (n=6). (7b) H&E images of each group, CNV lesions are marked with asterisks (n=6). (7c) Western blot strips used to study anti-angiogenic signals after each treatment (n=3). (7d) Statistical analysis of CNV size marked by CD31 antibody in choroidal plain mounts. (7e) Statistical analysis of Ki-67 positive cells in each group. (7f) Statistical analysis of CNV thickness by H&E sections. (7g) Statistical analysis of Western blot analysis after each treatment. (*: p<0.05; **: p<0.01; ***: p<0.001). Data are expressed as mean ± SEM.

[0043] Figure 8 .Anti-M2 macrophage polarization properties of nanoLAMP. (8a) Representative images of choroidal plain mounts stained with CD206 antibody and F4 / 80 antibody (n=6). (8b) Statistical analysis of CD206 positive area in each group. (8c) Statistical analysis of F4 / 80 positive area in each group. (8d) Western blot analysis of Arg-1 and GAPDH (n=3). (8e) Statistical analysis of Western blot analysis of Arg-1 after each treatment. (*: p<0.05; **: p<0.01). Data are expressed as mean ± SEM.

[0044] Fig. 9 .Ocular safety profile of nanoLAMP treatment. (9a) Representative images of the cornea and retina (near the optic nerve) of each group (n=3). (9b) TUNEL images of the control group and nanoLAMP-treated group (n=3). (9c) Statistical analysis of INL and ONL thickness of each group. (9d) Representative images of fERG (flash electroretinogram) at selected stimulation intensities of each group (n=3). (9e) Statistical analysis of scotopic a-wave of each group. (9f) Statistical analysis of scotopic b-wave of each group. (ns: not significant). Data are presented as mean ± SEM.

[0045] Fig.10 .Systemic safety profile of nanoLAMP treatment. (10a) Representative images of major organs in each group (n=3). (10b) Hematological parameters of each group after treatment (n=3). (10c) Biochemical blood characteristics of mice after treatment with different formulations (n=3). (10d) Statistical analysis of MTT assay of ARPE-19 and HUCEC in each group under dark conditions. (ns: not significant). Data are presented as mean ± SEM.

[0046] Fig.11.Preparation of BODIPY caged AS16 (cAS16) / PLA5k-PEG2k / elisemol-Cu NPs. (a) Size distribution of nanoparticles. (b) Encapsulation efficiency and loading of cAS16 and elisemol-Cu. (c) Cytotoxicity of free elisemol-Cu, cAS16 / PLA5k-PEG2k NPs (with light irradiation), cAS16 / PLA5k-PEG2k / elisemol-Cu NPs (with / without light irradiation), and PLA5k-PEG2k / elisemol-Cu NPs (with light irradiation). Light source: LED, 656 nm, 100 mW / cm 2 ,n=3,mean±SD.

[0047] Fig.12 .Tunnel image of a CNV lesion. Red fluorescence indicates apoptotic cells.

[0048] Fig.13 .BODIPY caged FM12 peptide (cFM12) nanoparticles for light-triggered PD-L1 blockade. (a) Standard curve of FM12 peptide shown by HPLC. (b) HPLC shows the rate of FM12 peptide release from nanoparticles triggered by 656 nm light irradiation at consecutive time points. (c) RT-qPCR shows the changes in IL-2 gene expression of Jurkat T cells treated with different concentrations of cFM12 nanoparticles (n=3). (d) RT-qPCR shows the changes in TNF-α gene expression of Jurkat T cells treated with different concentrations of cFM12 nanoparticles (n=3). (f) Schematic diagram of cFM12 nanoparticles. (*: p<0.05; **: p<0.01; ***: p<0.001). Data are presented as mean ± SEM. DETAILED DESCRIPTION OF THE INVENTION

[0050] definition

[0051] As used herein, the singular forms "a", "an", and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. In addition, to the extent the terms "including", "includes", "having", "has", "with" or variations thereof are used in the detailed description and / or the claims, these terms are intended to be inclusive in a manner similar to the term "comprising". The transitional terms / phrases (and any grammatical variations thereof) "comprising", "comprises", "comprise", "consisting essentially of", "consists essentially of", "consisting", and "consists of" may be used interchangeably.

[0052] The phrase "consisting essentially of" or "consists essentially of" means that the claim covers embodiments including the specified materials or steps and those that do not materially affect the basic and novel characteristics of the claim.

[0053] The term "about" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured, i.e., the limitations of the measurement system. In the context of compositions containing a certain amount of an ingredient using the term "about," these compositions contain the specified amount of the ingredient with a variation (error range) of 0-10% (X ± 10%) relative to the value. In other contexts, the term "about" provides a variation (error range) of 0-10% (X ± 10%) about a given value. Obviously, such a variation represents a range of up to 10% above or below a given value, such as X ± 1%, X ± 2%, X ± 3%, X ± 4%, X ± 5%, X ± 6%, X ± 7%, X ± 8%, X ± 9%, or X ± 10%.

[0054] In the present disclosure, ranges are expressed in abbreviated form to avoid having to elaborate and describe each value in the range. Where appropriate, any appropriate value in the range can be selected as the upper limit, lower limit or end point of the range. For example, the range 0.1-1.0 represents final values ​​0.1 and 1.0, and intermediate values ​​0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and all intermediate ranges included in 0.1-1.0, such as 0.2-0.5, 0.2-0.8, 0.7-1.0, etc. It is envisioned that there are values ​​of at least two significant figures in a range, for example, the range 5-10 represents all values ​​between 5.0 and 10.0 and between 5.00 and 10.00, including the final value. When ranges are used herein, combinations and sub-combinations of ranges (e.g., sub-ranges within the disclosed range) and specific embodiments thereof are clearly included.

[0055] As used herein, the term "subject" refers to an animal in need or desire of delivering the benefit provided by a therapeutic compound. The animal can be, for example, a human, pig, horse, goat, cat, mouse, rat, dog, ape, fish, chimpanzee, orangutan, guinea pig, hamster, cattle, sheep, bird, chicken, and any other vertebrate or invertebrate. These benefits can include, but are not limited to, treatment of health conditions, diseases, or conditions; prevention of health conditions, diseases, or conditions; immune health; enhancement of organ, tissue, or system function in the body. The preferred subject in the context of the present invention is a human. The subject can be of any age or developmental stage, including infants, young children, adolescents, teenagers, adults, or the elderly.

[0056] As used herein, the terms "therapeutically effective amount," "therapeutically effective dose," "effective amount," and "effective dose" are used to refer to an amount or dose of a compound or composition that, when administered to a subject, is capable of treating or ameliorating a condition, disease, or disorder in a subject, or is capable of enhancing the health or function of an organ, tissue, or body system. In other words, the amount is "therapeutically effective" when administered to a subject. The actual amount will vary depending on a variety of factors, including, but not limited to, the specific condition, disease, or disorder being treated or improved; the severity of the condition; the specific organ, tissue, or body system in which the health or function is to be enhanced; the patient's weight, height, age, and health; and the route of administration.

[0057] As used herein, the term "treat" refers to eradicating, reducing, ameliorating or reversing the signs or symptoms of a health condition, disease or disorder to any extent, and includes but does not require complete cure of the condition, disease or disorder. Treatment can be a cure, amelioration or partial amelioration of the condition. "Treatment" can also include improving or enhancing a condition or feature, for example, bringing the function of a particular system in the body to a state of high health or homeostasis.

[0058] As used herein, "preventing" a health condition, disease, or disorder means avoiding, delaying, preventing, or minimizing the onset of specific signs or symptoms of the condition, disease, or disorder. Prevention can be, but is not required to be, absolute or complete; that is, signs or symptoms may occur later. Prevention can include reducing the severity of the onset of such condition, disease, or disorder, and / or inhibiting the progression of the condition, disease, or disorder to a more serious condition, disease, or disorder.

[0059] In some embodiments of the present invention, the method includes administering multiple doses of the compounds of the present invention. The method may include administering 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40 or more therapeutically effective doses of a composition comprising a compound of the present invention as described herein. In some embodiments, the dose is administered over a period of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 14 days, 21 days, 30 days or more than 30 days. In addition, treating a subject with a therapeutically effective amount of the compounds of the present invention may include a single treatment or may include a series of treatments. It should also be understood that the effective dose of the compound for treatment may increase or decrease during a specific treatment. The change in dose may be caused and become apparent by the results of diagnostic analysis or imaging techniques known in the art for detecting tumor size. In some embodiments of the present invention, the method includes administering the compound multiple times a day, including but not limited to 2 times a day, 3 times a day, and 4 times a day.

[0060] As used herein, "drug" refers to a chemical compound manufactured for use as a medicine and / or therapeutic agent.

[0061] As used herein, the term "pharmaceutically acceptable" means compatible with the other ingredients of the pharmaceutical composition and not deleterious to the recipient thereof.

[0062] As used herein, a "photoresponsive prodrug" is a compound that can be converted into a specific drug compound or a pharmaceutically acceptable salt of such a compound by light irradiation. Preferably, the photoresponsive prodrug is a compound covalently bonded to a photocleavable group.

[0063] As used herein, a "photoresponsive group" or "photocleavable group" is a chemical group that can be removed or detached by irradiation with light through a photocleavage reaction.

[0064] The recitation of a list of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of the listed groups. The recitation of an embodiment of a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof.

[0065] Any composition or method provided herein can be combined with one or more of any other compositions and methods provided herein.

[0066] Other features and advantages of the invention will become apparent from the following description of preferred embodiments thereof, and from the claims.All references cited herein are incorporated herein by reference.

[0067] Photoresponsive prodrugs

[0068] The subject matter of the present disclosure includes photoresponsive prodrugs. Specifically, in certain embodiments, the photoresponsive prodrug comprises an active agent conjugated to a photoresponsive group. Under light irradiation, the photoresponsive prodrug is cleaved, thereby promoting the release of the active agent. In some embodiments, the photoresponsive prodrug can respond to NIR light with deeper penetration and less phototoxicity to the retina compared to short-wavelength light.

[0069] Active agents of the present disclosure, such as proteins, include but are not limited to enzymes, organic catalysts, ribozymes, organometallic compounds, proteins, glycoproteins, peptides, polyamino acids, antibodies, nucleic acids, steroid molecules, antibiotics, antiviral agents, antifungal agents, anticancer agents, analgesics, antirejection agents, immunosuppressants, cytokines, carbohydrates, oleophobes, lipids, extracellular matrix and / or individual components thereof, decalcified bone matrix, drugs, chemotherapeutic agents, viruses, viral vectors, and prions.

[0070] In some embodiments, the active agent of the present disclosure is an anti-angiogenic agent. As used herein,

[0071] "Anti-angiogenic agent" refers to a compound that blocks or interferes with the development of blood vessels to a certain extent. For example, an anti-angiogenic agent can be a small molecule, peptide or antibody that binds to a growth factor or growth factor receptor involved in promoting angiogenesis. In some embodiments, the anti-angiogenic agent is a peptide that inhibits VEGF signaling. In some embodiments, the anti-angiogenic agent is a peptide that inhibits M2 macrophage polarization. In some embodiments, the anti-angiogenic agent is a peptide that inhibits VEGF signaling and M2 macrophage polarization. In some embodiments, the anti-angiogenic agent is an AS16 peptide, which consists of the amino acid sequence ATWLPPRAANLLMAAS (SEQ ID NO: 1).

[0072] In some embodiments, the active agent of the present disclosure targets programmed death ligand 1 (PD-L1). In some embodiments, the active agent is a FM12 peptide consisting of the amino acid sequence FPNWSLRPMNQM (SEQ ID NO: 2).

[0073] In some embodiments, the photoresponsive group comprises a boron-dipyrromethene (BODIPY) unit. As used herein, the term "BODIPY" refers to a structural subunit having the following boron-dipyrromethene (BODIPY) core structure:

[0074]

[0075] wherein each R is independently selected from the group consisting of F, OH, H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, alkynyl, and substituted alkynyl.

[0076] In some embodiments, the photoresponsive group comprises a BODIPY unit of the formula:

[0077]

[0078] in:

[0079] The wavy lines represent the attachment points of the photoresponsive groups; R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are each independently hydrogen, halogen, optionally substituted C 1 -C 6 -alkyl, optionally substituted C 2 -C 6 -alkenyl or (C 6 -C 14 -aryl)-(C 2 -C 6 -alkenyl-), wherein (C 6 -C 14 -aryl)-(C 2 -C 6 -alkenyl-) is optionally in C 2 -C 6 -alkenyl or C 6 -C 14 -aryl is substituted. In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are each independently hydrogen, halogen, optionally substituted C 1 -C 6 -alkyl, optionally substituted C 2 -C 6 -alkenyl, (C 6 -C 14 -aryl)-(C 2 -C 6 -alkenyl-), wherein (C 6 -C 14 -aryl)-(C 2 -C 6-alkenyl-) is optionally in C 2 -C 6 -alkenyl or C 6 -C 14 - substituted or optionally substituted (C 2 -C 8 -heterocyclyl-), and

[0080] Each R is independently selected from the group consisting of F, OH, H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, alkynyl, and substituted alkynyl.

[0081] As used herein, the term “C 1-6 "-alkyl" refers to a monovalent saturated aliphatic hydrocarbon group having 1 to 6 carbon atoms. The term is not limited to straight chain and branched hydrocarbon groups, such as methyl (CH 3 —), ethyl (CH 3 CH 2 —), n-propyl (CH 3 CH 2 CH 2 —), isopropyl ((CH 3 ) 2 CH—), n-butyl (CH 3 CH 2 CH 2 CH 2 —), isobutyl ((CH 3 ) 2 CHCH 2 —), sec-butyl ((CH 3 )(CH 3 CH 2 )CH—), tert-butyl ((CH 3 ) 3 C—), n-pentyl (CH 3 CH 2 CH 2 CH 2 CH 2 —) and neopentyl ((CH 3 ) 3 CCH 2 —). Terminology C 1-6 -Alkyl also includes cycloalkyl groups, including but not limited to cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0082] The term "C 1 -C 20"-alkylene" refers to a straight-chain and branched-chain saturated divalent group having 1 to 20 carbon atoms (such as 10 to 20 carbon atoms, 12 to 18 carbon atoms, 1 to about 20 carbon atoms, 1 to 10 carbon atoms, 1 to 8 carbon atoms, 1 to 6 carbon atoms, or 2 to 4 carbon atoms). Straight-chain C 1 -C 6 -alkylene examples include those having 1 to 6 carbon atoms, such as —CH 2 —, —CH 2 CH 2 —, —CH 2 CH 2 CH 2 —, —CH 2 CH 2 CH 2 CH 2 — and —CH 2 CH 2 CH 2 CH 2 CH 2 —. Branched-chain (C 1 -C 20 )-alkylene examples include —CH(CH 3 )CH 2 — and —CH 2 CH(CH 3 )CH 2 —.

[0083] As used herein, the term "C 2-6 -alkenyl" refers to a monovalent and divalent unsaturated hydrocarbon group having 2 to 6 carbon atoms. This term includes, but is not limited to, straight-chain and branched-chain hydrocarbon groups, such as vinyl (CH 2 ═CH—), propenyl (CH 2 ═CH 2 CH 2 —), isopropenyl ((CH 3 )(CH 2 )C—), —CH═CH—, —CH═CH—CH 2 —, —CH═CH—CH═CH—, etc. The term C 2-6 -alkenyl also includes cycloalkenyl, including but not limited to cyclopentenyl and cyclohexenyl.

[0084] As used herein, the term "C 6 -C 14 -aryl" refers to a cyclic aromatic hydrocarbon having 6 to 14 carbon atoms (for example, 6 to 12 carbon atoms or 6 to 10 carbon atoms). Such aryl may be substituted or unsubstituted. Aryl includes, but is not limited to, phenyl, biphenyl, fluorenyl, phenanthryl, and naphthyl.

[0085] As used throughout the specification, "substituted" refers broadly to substitution of a group (e.g., C 1-6 -alkyl, C 2-6 -Alkenyl, C 6 -C 14 -Aryl and C 2 -C 8 -heterocyclyl) and produce stable compounds as described herein. Examples of suitable substituents include, but are not limited to, alkyl (e.g., C 1-6 -alkyl), alkenyl (e.g., C 2-6 -alkenyl), aryl (e.g., C 6 -C 14 -aryl), alkaryl (e.g., C 1-6 -alkyl-C 6 -C 14 -aryl), hydroxy, alkoxy (e.g., C 1-6 -alkyl-O—), aryloxy (e.g., C 6 -C 14 -aryl-O—), carboxyl (i.e. CO 2 H), alkylcarboxyl (e.g., C 1-6 -alkyl-C(O)O—), arylcarboxyl (e.g., C 6 -C 14 -aryl-C(O)O—), cyano, cyanate (i.e., —OCN group), silyl, siloxy, phosphine, halogen (e.g., F, Cl, Br, and I), nitro, and C 2 -C 8 -heterocyclyl-. Other suitable substituents include -N(R 8 ) 2 , where each R 8 is hydrogen, alkyl (e.g., C 1-6 -alkyl), aryl (e.g., C 6 -C 14 -aryl) or alkaryl (e.g., C 1-6 -alkyl-C 6 -C 14 -aryl), wherein each alkyl, aryl or alkaryl group may be substituted; and -[O-R 9 —] p O—R 10 , where R 9 is an alkylene group (e.g., C 1 -C 20 -alkylene) or cycloalkylene (e.g., (C 3 -C 20 )-cycloalkylene), R 10 is an alkyl group (e.g., C 1-6-alkyl), and p is an integer from 1 to about 10 (e.g., an integer from 1 to 5, 2 to 8, 2 to 5, or 2 to 4).

[0086] In some embodiments, R 1 and R 4 Each independently is (C 6 -C 14 -aryl)-(C 2 -C 6 -alkenyl-), wherein (C 6 -C 14 -aryl)-(C 2 -C 6 -alkenyl-) is optionally in C 2 -C 6 -alkenyl or C 6 -C 14 - is substituted on the aryl group; that is, (C 6 -C 14 -aryl)-(C 2 -C 6 -alkenyl-) is optionally in C 2 -C 6 -Alkenyl, C 6 -C 14 -aryl or both are substituted. In some embodiments, R 1 and R 4 Each independently is (C 6 -C 10 -aryl)-(C 2 -C 4 -alkenyl-), wherein (C 6 -C 10 -aryl)-(C 2 -C 4 -alkenyl-) is optionally in C 2 -C 4 -alkenyl or C 6 -C 10 - is substituted on the aryl group; that is, (C 6 -C 10 -aryl)C 2 -C 4 -alkenyl-) is optionally in C 2 -C 4 -Alkenyl, C 6 -C 10 -aryl or both are substituted. In other embodiments, R 1 and R 4 Each is -CH═CH—C 6 -C 10 -aryl, wherein C 6 -C 10-aryl is optionally substituted. In other embodiments, R 1 and R 4 Each is -CH=CH-phenyl, wherein the phenyl group is optionally substituted. In other embodiments, R 1 and R 4 Each is -CH=CH-phenyl, wherein the phenyl group is optionally substituted with a methoxy group.

[0087] In some embodiments, R 2 and R 5 are each independently hydrogen, halogen or optionally substituted C 1 -C 6 -alkyl. In some embodiments, R 2 and R 5 Each is independently hydrogen, methyl or ethyl. In other embodiments, R 2 and R 5 All are hydrogen.

[0088] In some embodiments, R 3 and R 6 are each independently optionally substituted C 1 -C 6 -alkyl. In some embodiments, R 3 and R 6 Each is a methyl group.

[0089] In some embodiments, each R is independently F or optionally substituted C 1 -C 6 -alkyl. In some embodiments, each R is F. In some embodiments, each R is methyl.

[0090] In some embodiments, the photoresponsive group is a moiety selected from the group consisting of Formula (VII), Formula (VIII), Formula (IX), Formula (X), Formula (XI), or Formula (XII):

[0091]

[0092]

[0093]

[0094] In some embodiments, the photoresponsive group has formula (XII):

[0095]

[0096] In some embodiments, the active agent is present via a covalent bond, -O-, -NR'-, -S-, -C(=O)-, -C(=O)NR'-, C(=O)O-, -NR'C(=O)NR'-, -O(C=O)NR'-, optionally substituted C 1 -C 6 Alkynyl, optionally substituted C 2-6 -alkenyl or a combination thereof is conjugated to a photoresponsive group, wherein R' is each independently selected from the group consisting of hydrogen, halogen, OH, H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, alkynyl, and substituted alkynyl. In some embodiments, the active agent is conjugated to the photoresponsive group by a covalent bond, -O-, -NR'-, -C(=O)NR'-, -C(=O)O-, -O(C)=O)NR'-, or a combination thereof. In a further embodiment, the active agent is conjugated to the photoresponsive group by -O(C=O)NR'-.

[0097] In some embodiments, the light-responsive prodrugs of the invention have the following formula (LAMP):

[0098]

[0099] In some embodiments, the photoresponsive prodrug of the invention is cFM12:

[0100]

[0101] In some embodiments, the photoresponsive prodrug can be tuned to be photoactivated at a specific wavelength and / or within a given wavelength range. In some embodiments, the photoresponsive prodrug can be tuned to be photoactivated at certain wavelengths by appropriately selecting the phosphorus responsive group included in the compound.

[0102] In some embodiments, the photoresponsive prodrug comprises an active agent, and the compound can remain inert until activated by light having a specific wavelength, thereby cleaving the active agent from the photoresponsive prodrug.

[0103] As used herein, the term "light" is used herein to refer to any electromagnetic radiation that can activate a compound. In some embodiments, light includes ultraviolet light, visible light, near infrared light (NIR) or infrared light (IR). Compared with other types of stimulation, light can be used to control drug release in time and space, thereby improving therapeutic efficacy and reducing adverse events. Due to the transparent nature of the cornea, lens and vitreous body, the fundus is more susceptible to external light irradiation. Some embodiments of photoresponsive prodrugs have the surprising and unexpected advantage of being activated by light with a wavelength greater than 500nm. Other embodiments of the compounds of the present invention can be photoactivated by light with a wavelength greater than 650nm.

[0104] More specifically, as used herein, light may refer to energy having a wavelength of about 350nm to about 1300nm. In specific embodiments, light may refer to energy having a wavelength of about 500nm to about 1300nm. In some embodiments, light includes a wavelength of about 500nm to about 1000nm. In some embodiments, light includes a wavelength of about 600 to about 900nm. In some embodiments, light includes a wavelength of about 600 to about 700nm. In some embodiments, light includes a wavelength of about 656nm.

[0105] Photoresponsive nanoparticles

[0106] In some embodiments, the photoresponsive prodrugs of the present invention can self-assemble into drug delivery polymers to form nanoparticles. In some embodiments, the nanoparticles can be prepared by nanoprecipitation. Nanoparticles can protect and guide drugs (such as AS16 peptides) to the target area to enhance their therapeutic efficacy and inhibit therapeutic efficacy in other tissues and organs without light irradiation.

[0107] In some embodiments, the polymer forming the nanoparticles of the present invention can be selected from: protein-based polymers, such as collagen, albumin, gelatin; polysaccharides, such as agarose, alginate, carrageenan, hyaluronic acid, dextran, chitosan, cyclodextrin; polyesters, such as poly (lactic acid), poly (glycolic acid), poly (hydroxybutyrate), poly (β-caprolactone), poly (β-malic acid), poly (dioxanone); polyanhydrides, such as poly (sebacic acid), poly (adipic acid), poly (terephthalic acid); polyamides, such as poly (imino carbonate), polyamino acids; phosphorus-based Polymers, such as polyphosphates, polyphosphonates, polyphosphazenes; other synthetic biodegradable polymers, such as poly(cyanoacrylates), polyurethanes, polyorthoesters, polydihydropyrans, polyacetals; cellulose derivatives, such as carboxymethyl cellulose, ethyl cellulose, cellulose acetate, cellulose acetate propionate, hydroxypropyl methylcellulose; silicones, such as polydimethylsiloxane, colloidal silica; acrylic polymers, such as polymethacrylates, poly(methyl methacrylate), polyhydro(ethyl methacrylate); polyvinylpyrrolidone; poloxamers; poloxamines; and combinations thereof.

[0108] In some embodiments, the polymer forming the nanoparticles of the present invention is selected from polylactic acid (PLA), polyethylene glycol (PEG), polydimethylsiloxane (PDMS), polyethyleneimine (PEI), polyamidoamine (PAMAM) and combinations thereof; preferably, the polymer is selected from PLA-PEG copolymers; more preferably, the polymer is PLA5k-PEG2k.

[0109] In some embodiments, the nanoparticles of the present invention are formed by self-assembly of LAMP and PLA5k-PEG2k (nanoLAMP). The 3D structure and functional mechanism of nanoLAMP are as follows Figure 1 a. After administration, the AS16 peptide is released from the nanoLAMP at the site of NIR light irradiation and binds to the VEGF receptor and tyrosine kinase receptor 2 (TIE-2) on the cell membrane, thereby inhibiting angiogenesis and M2 macrophage polarization.

[0110] In some embodiments, the nanoparticles of the present invention are formed by co-assembly of cFM12 and PEG3.4k-PLA5k. A schematic diagram of such nanoparticles is shown in FIG. Fig.13 f.

[0111] Pharmaceutical compositions and kits

[0112] The subject matter of the present disclosure also includes pharmaceutical compositions comprising the photoresponsive prodrugs or nanoparticles disclosed herein. Such pharmaceutical compositions may include at least one pharmaceutically acceptable carrier. In this regard, the term "pharmaceutically acceptable carrier" refers to sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, as well as sterile powders that are reconstituted into sterile injectable solutions or dispersions before use. Appropriate fluidity can be maintained, for example, by using coating materials such as lecithin, by maintaining the desired particle size in the case of dispersions, and by using surfactants. These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifiers and dispersants. Prevention of microbial action can be ensured by including various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, etc. It may also be necessary to include isotonic agents such as sugars, sodium chloride, etc. The injection preparation can be sterilized, for example, by filtering through a bacteria-retaining filter or by incorporating a sterilizing agent in the form of a sterile solid composition, which can be dissolved or dispersed in sterile water or other sterile injection media before use. Suitable inert carriers may include sugars such as lactose.

[0113] Suitable formulations include aqueous and non-aqueous sterile injection solutions which may contain antioxidants, buffers, bacteriostats, bactericidal antibiotics and solutes which render the formulation isotonic with the body fluids of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.

[0114] The composition can take the form of a suspension, solution or emulsion in an oily or aqueous vehicle, and can contain a preparatant, such as a suspending agent, a stabilizing agent and / or a dispersing agent. Alternatively, the active ingredient can be constituted with a suitable carrier (e.g., sterile pyrogen-free water) in powder form before use.

[0115] Formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a frozen or freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier immediately prior to use.

[0116] Prodrugs or nanoparticles can also be formulated into preparations for implantation or injection. Thus, for example, prodrugs or nanoparticles can be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins or as sparingly soluble derivatives (e.g., as sparingly soluble salts).

[0117] The subject matter of the present disclosure also includes a kit, which may include a photoresponsive prodrug, nanoparticle or pharmaceutical composition as described herein packaged with a device for administration. As will be appreciated by those skilled in the art or ordinary skill, the appropriate administration aid will depend on the formulation of the selected compound or composition and / or the ideal site of administration. For example, if the formulation of the compound or composition is suitable for injection into a subject, the device may be a syringe. For another example, if the ideal site of administration is a cell culture medium, the device may be a sterile pipette.

[0118] How to use

[0119] In certain embodiments, prodrugs and nanoparticles, such as, for example, LAMPS or nanoLAMPS can be administered to a subject.Any mode of administration that allows the nanoparticles to contact cells is contemplated in the subject methods, including, for example, oral, intravenous, intraperitoneal, intramuscular, intrathecal, or subcutaneous.

[0120] As used herein, the term "administering" refers to any method of providing a compound and / or its pharmaceutical composition to a subject. Such methods are well known to those skilled in the art and include, but are not limited to, oral administration, transdermal administration, inhalation administration, intranasal administration, topical administration, intravaginal administration, ocular administration, intraauricular administration, intracerebral administration, rectal administration, and parenteral administration, including injection such as intravenous administration, intraarterial administration, intramuscular administration, subcutaneous administration. Administration may be continuous or intermittent. In various aspects, the formulation may be administered therapeutically; that is, administered to treat an existing disease or condition. In further various aspects, the formulation may be administered prophylactically; that is, administered to prevent a disease or condition.

[0121] In some embodiments, after administration, the photoresponsive prodrug can release an active agent, such as an AS16 peptide, when the subject is irradiated with light. In some embodiments, the wavelength of the light can be about 100nm to about 1000nm, about 500nm to about 900nm, about 620nm to about 750nm, or preferably about 656nm. In some embodiments, the subject, nanoparticles and / or prodrugs can be irradiated for about 1s to about 24 hours, about 10 seconds to about 12 hours, about 15 seconds to about 1 hour, about 30 seconds to about 30 minutes, about 45 seconds to about 10 minutes, or about 7 minutes. In some embodiments, the entire subject can be irradiated. Alternatively, a specific part of the subject can be irradiated, such as, for example, the eyes, head, face, legs, arms, wrists, chest, abdomen, neck or calves. In some embodiments, the subject can be irradiated before, during or after the prodrug, nanoparticle or composition of the subject invention is administered. In some embodiments, after administering the compounds or compositions of the subject invention, the irradiation of the prodrug, nanoparticles and / or subject can occur for at least 1s, 2s, 5s, 10s, 15s, 30s, 45s, 1 minute, 2 minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 5 hours or 10 hours. In some embodiments, the subject, nanoparticles and / or prodrug can be irradiated after the prodrug is located in a specific location (such as a tumor, an organ, including the eye or tissue). In certain embodiments, the irradiance can be about 1mW / cm 2 About 1000mW / cm 2 , 5mW / cm 2 To about 500mW / cm 2 、10mW / cm 2 to about 250mW / cm 2 、15mW / cm 2 to about 150mW / cm 2 , 25mW / cm 2 to about 125mW / cm 2 , or about 100mW / cm 2 The light may be delivered by any natural or artificial source capable of providing light of the wavelength and irradiance within the stated amount of time, including, for example, a laser, an incandescent lamp, a halogen lamp, a fluorescent lamp, or a light emitting diode (LED).

[0122] In some embodiments, the subject prodrugs, nanoparticles or compositions can be irradiated to photoactivate a photoresponsive group, such as, for example, BODIPY. In some embodiments, irradiation of BODIPY can produce cleavage of the photoresponsive prodrug. Cleavage can trigger a cascade of anti-angiogenic agent release, resulting in a combined anti-angiogenic therapy for CNV.

[0123] In certain embodiments, the prodrug and / or nanoparticle is non-toxic before being activated by light. Upon exposure of the prodrug and / or nanoparticle to light, preferably at 656 nm, the anti-angiogenic agent can be released from the prodrug or a composition containing the nanoparticle or prodrug.

[0124] Further, the subject matter disclosed at present includes methods for treating diseases such as cancer and age-related macular degeneration (AMD). In some embodiments, the method includes administering a prodrug, nanoparticle or composition disclosed herein, and then exposing the application site of the subject to light after administering the compound. As mentioned above, the light in some embodiments can be light with a wavelength of about 500nm to about 1300nm.

[0125] All patents, patent applications, provisional applications, and publications mentioned or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.

[0126] The following are examples illustrating procedures for practicing the present invention. These examples should not be construed as limiting. Unless otherwise indicated, all percentages are by weight and all solvent mixture ratios are by volume. Example

[0127] Materials and methods

[0128] Reagents. AS16 peptide was synthesized and purchased from Genescript (Nanjing, China). PLA5k-PEG2k was purchased from PonsureBiotech (Shanghai, China). Human umbilical vein endothelial cells (HUVEC) and human retinal pigment epithelial cells (ARPE-19) were obtained from the American Type Culture Collection (Manassas, VA, USA). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin solution and trypsin-EDTA 0.25% solution, collagen I (rat tail) were purchased from Thermo Fisher Scientific, Inc. (Eugene, OR, USA). BCA protein detection kit was purchased from Thermo Fisher Scientific, Inc. (Eugene, OR, USA). Table 1 summarizes the main antibodies involved in this study.

[0129] Table.1 Summary of the main antibodies used in this study

[0130]

[0131] Instruments. Dynamic light scattering (DLS) analysis was performed by Nano-ZS (Malvern Instruments, UK). TEM images were captured by a CM100 transmission electron microscope (Philips, USA). UV-vis absorption spectra and cell viability were measured by a SpectraMax M4 multi-mode microplate reader (Molecular Devices, USA). Drug loading, encapsulation efficiency, and drug concentration were detected by 1260 Infinity II HPLC (Agilent Technologies, USA). Photolysis experiments and irradiation were performed using a commercial diode laser system (Laserwave, Canada). Light irradiance was measured by a PM100USB power energy meter (Thorlabs, USA) equipped with an S142C integral sphere photodiode power sensor (silicon, 350-1100nm, Thorlabs, USA). Fluorescence images were captured by an LSM 980 confocal microscope (ZEISS, Germany). An experimental CNV mouse model was constructed using an OcuLight infrared laser system (IRIDEX, USA). OCTA and OCT images were captured by TowardPi cutting-edge swept-source OCT (TowardPi Medical Technology Ltd, China). FFA images were captured by Phoenix Micron IV retinal imaging system (Phoenix Research Laboratories, USA). ERG results were obtained by Celeris-Diagnosys system (Diagnosys, USA).

[0132] Animals. Animal studies were performed under the approval and supervision of the Committee on the Use of Live Animals in Teaching and Research of the Zhongshan Eye Center of Sun Yat-sen University. C57BL / 6J mice (18–22 g, 6–8 weeks, male) were obtained from Shanghai Model Organisms Technology Co., Ltd. (Shanghai, China). All mice were housed in a conventional experimental housing area with alternating 12-h light / dark cycle control, regulated temperature at 21 ± 4 °C, relative humidity at 50 ± 10%, and fed with UV-treated food and 1-μm filtered water ad libitum.

[0133] Statistical analysis. Unless otherwise specified, all data are presented as mean ± SD. Statistical analysis was performed using GraphPad Prism 9.0.1 software. Unpaired two-tailed student's t test was used for comparison between groups, while one-way ANOVA test combined with Dunnett's post hoc test was used for multiple group analysis. p < 0.05 was considered statistically significant.

[0134] Example 1. Preparation and characterization of nanoparticles.

[0135] 1.1 Synthesis of BODIPY-NPC

[0136] BODIFY-NPC is based on Figure 1 The obtained BODIPY-NPC was synthesized by the synthetic route shown in b. 1 H spectrum Figure 2 As shown in a.

[0137] 1.1.1 (5,5-difluoro-1,3,7,9-tetramethyl-5H-4λ 4 , 5λ 4 -Bipyrrolo[1,2-c:2′,1′-f][1,3,2]diazaboridine-10-yl)methyl acetate (BODIPY-F 2 Synthesis of -OAc)(1) (A)

[0138] 2-Chloro-2-oxoethyl acetate (0.6 mL, 5.6 mmol, 1.2 eq) was added to 2,4-dimethylpyrrole (1.0 mL, 9.3 mmol, 2.0 eq) in anhydrous dichloromethane (DCM) (40 mL) under nitrogen atmosphere. The reaction was stirred at reflux for 3 hours. Thereafter, DIPEA (3.1 mL, 18.6 mmol, 4 eq) was added. The resulting mixture was stirred at room temperature for another 30 minutes. Boron trifluoride etherate (2.3 mL, 18.6 mmol, 4 eq) was then added and the reaction solution was stirred for 30 minutes. Silica was then added to the flask and the solvent was evaporated. Systematic purification of BODIPY-F by flash chromatography 2 -OAc. The product was obtained as red-gold crystals (814 mg, 45.4% yield).

[0139] 1.1.2 (5,5-difluoro-3,7-bis((E)-4-methoxyphenylvinyl)-1,9-dimethyl-5H-4λ 4 , 5λ 4 Synthesis of dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaboridine-10-yl)methyl acetate (B)

[0140] Compound A (200 mg, 0.652 mmol, 1 eq.) was reacted with 4-anisaldehyde (4 ml, 32.8 mmol, 53 eq.) under nitrogen atmosphere at 60 °C in the dark for 2 hours. The reaction was monitored by TLC. During this period, the color of the mixture changed from red to purple and then to dark green. The mixture was cooled to room temperature and heated to 40 °C. The system was purified by flash chromatography to obtain the product as a dark red powder (258 mg, 74.2% yield).

[0141] 1.1.3 (5,5-difluoro-3,7-bis((E)-4-methoxyphenylvinyl)-1,9-dimethyl-5H-4λ 4 , 5λ 4 Synthesis of dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaboridine-10-yl)methanol (C)

[0142] A mixture of NaOH aqueous solution (43mg, 0.5ml, 1.076mmol, 4 equivalents) and methanol (9.5ml) was added dropwise to a solution of compound B (150mg, 0.269mmol, 1 equivalent) in DCM. The reaction was monitored by TLC and stirred at room temperature in dark for 2 hours. Thereafter, the solvent was evaporated and redissolved with DCM. Then, the solution was extracted with 0.01M HCl. The organic layer was collected and hydrated with anhydrous sodium sulfate. The product in a dark red powder form was obtained by evaporation without further purification (126mg, 90.87% yield).

[0143] 1.1.4 (3,7-bis ((E)-4-methoxyphenylvinyl) -1,5,5,9-tetramethyl-5H-4λ 4 , 5λ 4 Synthesis of dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaboridine-10-yl)methanol (D)

[0144] Compound C (100 mg, 0.194 mmol, 1 equivalent) was dispersed in 5 ml of Et 2 O. Methylmagnesium bromide (3M, tetrahydrofuran (THF) solution, 0.3 ml, 0.97 mmol) was added dropwise to the compound C solution in a dark place under a nitrogen atmosphere. The reaction was monitored by TLC. The color of the solution changed from dark green to dark blue. After stirring for 2 hours, the reaction was terminated by adding 0.5 ml of distilled water dropwise to the mixture. THF and Et were removed by evaporation. 2 O. The residue was redissolved in DCM. The DCM solution was extracted 3 times with saturated sodium chloride aqueous solution. The organic layer was collected and dried over anhydrous sodium sulfate. The resulting product (48 mg, 48.9% yield) was then obtained as a dark blue solid by flash chromatography.

[0145] 1.1.5(3,7-bis((E)-4-methoxyphenylvinyl)-1,5,5,9-tetramethyl-5H-4λ 4 , 5λ 4 Synthesis of 1-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaboridine-10-yl)methyl(4-nitrophenyl)carbonate (E)

[0146] Under nitrogen atmosphere, compound D (40 mg, 0.079 mmol, 1 equivalent) was dissolved in anhydrous DCM (2 ml) containing DIPEA (80 μl, 0.39 mmol, 5 equivalents) and pyridine (30 μl, 0.312 mmol, 4 equivalents). A 1 ml DCM solution of 4-nitrophenyl chloroformate (158 mg, 0.78 mmol, 10 equivalents) was added dropwise to the compound D solution in an ice bath. The reaction was stirred at room temperature in the dark for 4 hours. Thereafter, the mixture was purified by flash chromatography. The product (41 mg, 77.3% yield) was obtained as a dark powder.

[0147] 1.2 Synthesis of BODIPY-AS16 conjugate (LAMP)

[0148] 20 mg of AS16 peptide (1 eq., 0.012 mmol) was dissolved in 500 μL of anhydrous DMSO. 2 μL of DIPEA was then added to the AS16 solution. Subsequently, BODIPY-NPC (3 eq., 0.036 mmol) was dissolved in 500 μL of anhydrous DCM and added dropwise to the AS16 solution. The mixture was heated at N 2 The mixture was stirred at room temperature overnight under protection. DCM was removed by evaporation. The mixture was diluted with ACN and the NIR light-responsive BODIPY-AS16 conjugate was purified by HPLC. 1 H spectrum Figure 2 b shows the UV-vis spectrum of the obtained LAMP. Figure 3 As shown in a. The UV-vis spectrum of LAMP shows that AS16 and BODIPY have characteristic absorption peaks at 220nm and 656nm, respectively, which indicates that LAMP is successfully synthesized.

[0149] 1.3 Preparation of NIR light-responsive BODIPY-AS16 conjugated nanoparticles (nanoLAMP)

[0150] NanoLAMP was prepared by nanoprecipitation. Specifically, 4 μL of 100 mg / ml PLA5k-PEG2k was mixed with 4 μL of 25 mg / ml BODIPY-AS16 conjugate and added to ddHO under vigorous stirring. 2 O. Size distribution and zeta potential were determined by Malvern Zetasizer (Nano ZS90, Malvern, UK). Morphological studies were performed using a transmission electron microscope (TEM, Hitachi S-4800, Tokyo, Japan).

[0151] Dynamic light scattering (DLS) showed that the size of nanoLAMP was about 60.74 nm and the polydispersity index (PdI) was about 0.17. Figure 3 As shown in b, the zeta potential of nanoLAMP is about -1.43 mV.

[0152] Example 2. Evaluation of the colloidal stability of nanoLAMP.

[0153] The stability of nanoLAMP was evaluated by DLS analysis and TEM imaging after incubation with PBS buffer (pH 7.4) and serum containing DMEM medium (10%) at 37°C for at least 48 h. The size and morphology changes of the nanoparticles can indicate their colloidal stability. By DLS detection, nanoLAMP showed satisfactory stability characteristics in DMEM complete medium containing 10% FBS, as no obvious size and PdI changes were observed at 37°C for 48 h ( Figure 3 c). The encapsulation efficiency and drug loading of LAMP in the nanoparticles were 72.69%±2.49% and 21.78%±1.78%, respectively. Figure 3 g).

[0154] Example 3. NIR light triggered drug release.

[0155] At 100mW / cm 2 After 656 nm laser irradiation for different time intervals (0, 1, 2, 5 and 7 min), the photocleavage products released from the nanoLAMP solution were separated by centrifugation, completely dissolved in acetonitrile / methanol mixture (1:1) and further analyzed by HPLC. The cumulative drug release in the dark was evaluated as a control for drug leakage calibration before illumination. The nanoLAMP degradation rate (%) was calculated as the percentage of decomposed LAMP to the initial LAMP content (t = 0). The AS16 recovery rate (%) was determined as the molar ratio of the generated AS16 peptide to the initial AS16 content.

[0156] The dimensional changes after 656 nm light irradiation were investigated by DLS and transmission electron microscopy (TEM). Figure 3 As shown in b, under 656nm light irradiation (100mw / cm 2 A significant change in the size of the nanoparticles from 60.74 nm to 44.94 nm was observed at 50 °C (5 min), and the TEM results showed consistent results ( Figure 3 b and 3e). The recovery of free AS16 peptide was also detected by HPLC analysis at 220 nm, and the results showed that about 30% of the free AS16 peptide was released after light irradiation ( Figure 3 d and 3h). After light irradiation, obvious color changes can be observed with the naked eye ( Figure 3 f). In addition, the release of free BODIPY molecules was detected at 656 nm ( Figure 4). These results confirmed that nanoLAMP could respond rapidly to NIR light irradiation within 5 min and trigger the release of the encapsulated AS16 peptide at the desired location.

[0157] Example 4. In vitro study of nanoLAMP for anti-angiogenic therapy.

[0158] 4.1 Tube formation experiments

[0159] Apply 60 μL of melted matrix gel to the bottom of a 96-well plate and wait for it to solidify. Then add 100 μL of 2*10 4 The complete medium of HUVEC cells was added. The wells were divided into 5 groups (PBS, BODIPY plus light irradiation, AS16 peptide, nanoLAMP, nanoLAMP plus light irradiation) and received corresponding treatments. The concentration of AS16 peptide was set to 5 μM, and the light irradiation parameters were 656 nm, 100 mw / cm 2 The number of connections and total fragment length of each group were measured by Image J software (1.53e). Data analysis was performed by GraphPad Prism software (9.0.0).

[0160] 4.2 Wound healing experiment

[0161] 5*10 5 HUVEC cells were seeded in 24-well plates and incubated to 90% confluence. A 200 μL pipette tip was then passed through each well. The cells were then washed twice with PBS to remove detached cells and incubated with 1 ml of DMEM medium containing 5% FBS. The grouping and treatment methods were the same as above. The data of each group were measured and analyzed by Image J and GraphPad Prism, respectively.

[0162] 4.3 Migration experiment

[0163] 200 μL containing 2*10 4 The serum-free medium of HUVEC cells was inoculated into the upper chamber of the transwell, and 600 μL of full medium was added to the lower chamber. Different drugs were added to the upper chamber. The grouping, treatment method and calculation were the same as above.

[0164] 4.4 Results

[0165] To verify the in vitro anti-angiogenic properties of nanoLAMP, tube formation assays, migration assays, and wound healing assays were also performed on HUVECs, and the concentration of each group was set equal to that of nanoLAMP containing 5 μM AS16 peptide. After NIR light irradiation, nanoLAMP exerted a similar or even better therapeutic effect than the AS16-only group, almost completely inhibiting the formation of tubular networks between HUVECs. In contrast, the therapeutic effect of nanoLAMP on the tube formation process under dark conditions was negligible because when there was no NIR light irradiation, the AS16 peptide was imprisoned inside the nanoparticles by BODIPY due to the long PEG chain and away from the receptors on the cell surface ( Figure 5 a). This result indicates that nanoLAMP is photoresponsive and can release functional AS16 peptide upon NIR light irradiation.

[0166] Likewise, migration experiments showed similar trends as tube formation experiments, e.g. Figure 5 As shown in (b), the number of migrated HUVECs was minimal in the nanoLAMP plus light irradiation group and the AS16 peptide group. However, when there was no light, the number of cells in the lower chamber remained the same as that in the control group. This result suggests that nanoLAMP successfully inhibited HUVEC migration under light irradiation, while maintaining a negligible therapeutic effect in the absence of light.

[0167] Finally, the wound healing assay showed consistent results, indicating significantly impaired recovery of scratches generated in the nanoLAMP plus light irradiation group and the AS16 peptide group ( Figure 5 c) Statistical analysis of tube formation, migration and wound healing was performed using GraphPad Prism software. Figure 5 f-5i, showing the significantly superior anti-angiogenic properties of nanoLAMP under light irradiation.

[0168] Since the AS16 peptide was previously designed to inhibit VEGF signaling, its therapeutic effect was further evaluated by western blot analysis. According to previous studies, phosphorylation of AKT and MAPK plays a key role in cell proliferation and vascular sprouting. Figure 5 As shown in Figure d, nanoLAMP under light irradiation successfully inhibited the upregulation of VEGF phosphorylation of AKT and MAPK, in which the phosphorylation of AKT and MAPK proteins was significantly reduced, and its trend was similar to that of the AS16 peptide group. This result shows that nanoLAMP successfully released functional AS16 peptide under NIR light irradiation and inhibited VEGF signaling and angiogenesis.

[0169] Example 5. Therapeutic efficacy of nanoLAMP for anti-angiogenic therapy in a laser-induced CNV mouse model assessed by imaging.

[0170] 5.1 Construction of laser-induced CNV mouse model.

[0171] CNV was induced by producing 4 laser spots in the mouse fundus by the laser of 810nm. Specifically, C57BL / 6 mice (6-8 weeks) were anesthetized with 1% sodium pentobarbital (40mg / kg). Then the pupil was dilated with 0.5% tropicamide eye drops, local anesthesia was performed with tetracaine eye drops, and then sodium carboxymethylcellulose eye drops were used to prevent ocular surface dehydration. Four lasers (six times if western blot analysis) were burned (810nm wavelength, 140mW power, 75μm spot size, 75ms duration) to the eyes of each mouse at a distance almost equal to the optic disc using the OcuLight infrared laser system. Subsequently, mice received chloramphenicol eye drops to prevent infection, and rested on a heating pad until awake. All animal procedures were carried out in accordance with the guidelines of the Living Animal Teaching and Research Committee of the Zhongshan Eye Center of Sun Yat-sen University.

[0172] 5.2 Treatment of mice.

[0173] Mice with laser lesions of equal size were selected and randomly divided into five groups: saline group, BODIPY plus light irradiation group, AS16 peptide group, nanoLAMP group, and nanoLAMP plus light irradiation group. On the third and fifth days after laser photocoagulation, 2 mg / kg AS16 peptide and an equal amount of nanoLAMP were injected via tail vein. Mice in the nanoLAMP plus light irradiation group were irradiated with light (656 nm, 50 mw / cm 2 , 5min), while the group without light irradiation was carefully placed in a dark room to avoid accidental light sources. On day 7 after CNV induction, FFA (fluorescent fundus angiography) and OCTA (optical coherence tomography angiography) images of each group were collected, and then the mice were euthanized. The eyes were enucleated for further investigation.

[0174] 5.3 OCTA

[0175] Optical coherence tomography angiography (OCTA) images were captured using a Beiming ultra-wide swept source OCT (TowardPiMedical Technology Ltd). CNV lesions were defined as spindle-shaped hyperreflective areas with their long axis aligned with the level of the RPE. Only scans through the center of the lesion were used to measure the thickness and length of CNV lesions, and ImageJ software and GraphPad Prism were used for analysis and calculation.

[0176] 5.4 FFA

[0177] For FFA, sodium fluorescein (0.3 mL 2% sodium fluorescein) was injected intraperitoneally, and continuous real-time FFA images were captured by Pheonix Micron IV 4–5 min after fluorescein injection. The leakage grade of CNV was evaluated independently by two experts, and the grading criteria of FFA images were as follows: grade I indicated no strong fluorescence; grade II indicated hyperfluorescence with no leakage; grade III indicated hyperfluorescence in early or mid-transition images with late leakage; and grade IV indicated bright hyperfluorescence with an increase in intensity and size during the transition phase of angiography.

[0178] 5.5 Results

[0179] like Figure 6 As shown in a and 6d, OCTA showed that nanoLAMP with light exposure significantly reduced the CNV area compared with the other groups. AS16 peptide, BODIPY plus light exposure, and nanoLAMP without light exposure showed negligible effects. In addition, FFA testing showed that nanoLAMP plus light exposure successfully suppressed the leakage of CNV lesions, which is the main cause of severe visual loss in patients with exudative AMD ( Figure 6 b and 6e). In addition, OCT images showed a similar trend in the thickness of CNV, with the thickness in the nanoLAMP plus light irradiation group significantly reduced by more than 70% compared with the control group ( Figure 6 c and 6f). These imaging assessments showed that nanoLAMP effectively reduced the severity of laser-induced CNV in mice when light irradiation was applied, whereas the AS16 peptide was blocked in the nanoparticles when light irradiation was absent.

[0180] Example 6. Therapeutic efficacy of nanoLAMP for anti-angiogenic therapy via isolated tissue in a laser-induced CNV mouse model.

[0181] Further studies were conducted to conclusively determine the therapeutic efficacy of nanoLAMP.

[0182] 6.1 Immunofluorescence staining of choroidal flat mounts.

[0183] To confirm the efficacy of nanoLAMP in inhibiting neovascularization, CNV mice receiving relevant treatments were anesthetized and their eyes were subsequently removed on the 7th day after laser induction. The cornea, lens, and vitreous were carefully removed after fixation in 4% paraformaldehyde at room temperature for 30 minutes, and the RPE-choroid-sclera complex was carefully separated. Then, the ocular tissue was stained with the primary antibody overnight at 4°C. After washing six times with PBS buffer (5 minutes per round), the RPE-choroid-sclera complex was incubated with the secondary antibody at room temperature for 2 hours, followed by 8 rounds of washing with PBS (5 minutes per round). Flat-mounted RPE-choroid slides were prepared by making 4 cuts from the edge to the optic nerve, and fluorescent images were acquired by confocal microscopy (LMS980 Carl Zeiss). The CNV lesion area was quantitatively analyzed by ImageJ software.

[0184] 6.2 Western blot analysis

[0185] After treatment, choroidal tissues of each group were isolated and dissolved with RIPA buffer containing 1% protease and phosphatase inhibitors. Protein samples were normalized by BCA protein assay, loaded with loading buffer and boiled for 10 minutes (95°C), and then separated by 5% / 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Then transferred to polyvinylidene fluoride filter (PVDF) membrane at 250mA constant current for 1.5h and incubated with 5% bovine serum albumin (BSA) blocking buffer for 1.5h, the protein membrane was incubated with primary antibody in WB primary antibody diluent (Servicbio, Wuhan, China) at 4°C overnight, and then washed with Tris buffered saline Tween (TBST) for 6 rounds, each for 5 minutes. Horseradish peroxidase (HRP)-secondary antibody was diluted in TBST and incubated with test strips for 2 hours at room temperature, and then washed with PBS. After reaction with ECL Western blotting substrate, the protein membrane was visualized by chemiluminescence imaging system. GAPDH staining was used for sample normalization.

[0186] 6.3 Histological evaluation

[0187] The enucleated eyeballs and major organs were collected, fixed with FAS eyeball fixative (for eyes) / 4% paraformaldehyde (for major organs), and then embedded in paraffin. Visual axis-oriented eyeball sections and histological structure-oriented organ sections were prepared and then stained with hematoxylin and eosin (H&E) for histological examination.

[0188] 6.4 Results

[0189] like Figure 7As shown in Figures 7a and 7d, the size of CNV lesions labeled with CD31 antibody in the nanoLAMP plus light irradiation group was significantly reduced compared with the other groups, which is consistent with other results. In addition, a significant decrease in Ki-67-positive cells in CNV lesions in the nanoLAMP administration plus light irradiation group was observed compared with the other groups, indicating that nanoLAMP has anti-angiogenic activity in CNV lesions ( Figure 7 Consistently, a significant reduction in the vertical size of CNV was observed in the hematoxylin and eosin (H&E)-stained cross-sections of the eyeballs in the nanoLAMP plus irradiation group ( Figure 7 b and 7f). It is worth mentioning that in all in vivo tests, the AS16 peptide group showed negligible therapeutic effects compared with the control group, which is estimated to be due to the rapid clearance and degradation of the AS16 peptide in the bloodstream after systemic administration. More importantly, the nanoparticles in the nanoLAMP group without light irradiation also did not show obvious therapeutic efficacy, indicating that the AS16 peptide was successfully blocked and prevented from exerting its effect in the absence of light irradiation. This phenomenon further confirms that our nanosystem reduces the risk of potential systemic adverse events and undesirable therapeutic effects of the AS16 peptide. In addition, western blot analysis showed that nanoLAMP in vivo successfully inhibited the activity of VEGF signaling when light irradiation was applied ( Figure 7 c and 7g), which showed that the phosphorylation of AKT and MAPK proteins was significantly inhibited. Taken together, these results indicate that nanoLAMP can effectively inhibit angiogenesis in vivo when NIR light is applied, and the in vivo photoresponsiveness of nanoLAMP is also confirmed.

[0190] Example 7. In vivo anti-M2 macrophage polarization properties of nanoLAMP.

[0191] The inhibitory activity of NanoLAMP on M2 macrophage polarization in vivo was also studied, because AS16 was reported to successfully inhibit M2 macrophage polarization by binding to the TIE2 receptor, which was previously reported to be involved in macrophage-induced angiogenesis. M2 macrophages were labeled with CD206 antibody, and the fluorescence signal of their choroidal spread was collected using a confocal microscope, and the operation process was the same as in Example 6.1. Figure 8As shown in Figures 8a and 8b, the CD206-positive area of ​​nanoparticles in the light-irradiated nanoLAMP group was significantly reduced compared with the other groups, indicating that our treatment successfully inhibited the M2 polarization of macrophages in CNV lesions. The CD206 signal colocalized well with F4 / 80, a clear marker for mature and activated macrophages, indicating that M2 macrophage polarization dominated the angiogenesis process in the late stage of laser-induced CNV, which is consistent with previous literature. Moreover, western blot analysis (using the same protocol as Example 6.2) showed that compared with the other groups, the nanoLAMP plus light-irradiation group significantly inhibited the expression of Arg-1 protein, which is defined as a specific marker for M2 macrophages ( Figure 8 d and 8e). These results indicate that our nanoparticles can effectively and accurately inhibit M2 macrophage polarization in vivo upon light irradiation.

[0192] Example 8. In vitro and in vivo biocompatibility of nanoLAMP.

[0193] 8.1 In vitro dark cell viability analysis.

[0194] The cytotoxicity of nanoLAMP to normal cells was tested using the MTT assay, and the in vitro biocompatibility of nanoLAMP under light irradiation was evaluated. HUVEC and ARPE-19 cells were incubated in 96-well plates and subjected to the relevant treatments. MTT assay was performed after 24 h of incubation.

[0195] 8.2 In vivo safety profile of nanoLAMP.

[0196] Each group of mice in the laser-induced CNV mouse model received the treatment described in Example 5.2. At the end of the treatment, blood from each mouse was collected by intracardiac puncture under deep anesthesia into a Microvette capillary blood collection tube (~0.1 mL) for CBC / w testing (RBC, WBC, Hb, platelets) and a micro-blood collection tube serum separator for biochemical testing (~0.8 mL). The serum of each group was separated by centrifugation, and the whole blood was stored at 4°C overnight. AST, ALT, UREA, and CREA levels were tested to determine kidney and liver function. At the end of the study, the mice were killed by cardiac perfusion with 0.9% saline followed by 4% paraformaldehyde. Major organs and tumors were removed, imaged and weighed, and the tissues were cut into 10 μm thick cryosections for H&E staining to evaluate in vivo biocompatibility.

[0197] 8.3 Electroretinogram

[0198] The mice were randomly divided into two groups, a control group and a treatment group. The treatment group was intravenously injected with nanoLAMP (containing 2 mg AS16 peptide) twice, with an interval of 1 day, and the control group was intravenously injected with PBS at the same time point. After another day, the mice were carefully placed in a dark room overnight to adapt to the darkness. Subsequently, the mice were anesthetized, mydriasis was dilated with 0.5% tropicamide, and then topical anesthesia was performed with tetracaine eye drops. A gold-plated wire ring electrode in contact with the corneal surface was used as the active electrode to record fERG. The mice were exposed to intensities of 0.003, 0.01, 0.03, 0.1, 0.3, 1.0, 3.0 and 10.0 log cd.s / m 2 Full-field scotopic flash.

[0199] 8.4 TUNEL assay

[0200] Mice received nanoLAMP intravenous injection and were euthanized one day after treatment. The eyes were harvested and paraffin sections were made. The sections of the nanoLAMP plus light irradiation group and the saline group were then incubated with the TUNEL detection kit (Servicebio Wuhan, China) according to the instructions, and images were captured by confocal microscopy.

[0201] 8.5 Results

[0202] like Fig. 9 As shown in a, NIR light and nanoLAMP treatment did not cause significant damage to the cornea, as no obvious neovascularization or edema was observed. In addition, one day after the second treatment, TUNEL assay of the retina showed no significant difference between the control group and the nanoLAMP treatment group ( Fig. 9 b), indicating that our treatment did not induce apoptosis in the retina. TUNEL assay of CNV lesions was used as a positive control ( Fig.12 ). In addition, nanoLAMP treatment did not reduce the thickness of the inner nuclear layer (INL) and outer nuclear layer (ONL) of the retina, further confirming that our treatment modality should be safe for ocular use ( Fig. 9 More importantly, the flash electroretinogram (fERG) results, which have high sensitivity for detecting retinal damage, showed no significant differences in scotopic a-wave and b-wave between the nanoLAMP-treated and control groups ( Fig. 9 d-9f). These results suggest that our nanoLAMP treatment does not cause significant damage to the cornea and retina and should be safe for ocular application.

[0203] In addition, H&E sections of major organs in each group (including heart, liver, spleen, lung, and kidney) were performed to conclusively determine the safety of our nanoLAMP treatment. Fig.10As shown in a, no obvious morphological changes were observed in all treatment groups. Whole blood samples and serum were also collected and separated for biocompatibility studies. Fig.10 As shown in b, there was no significant change in RBC, WBC, and platelet counts in each group, and the difference in Hb concentration among the groups was negligible ( Fig.10 c). In addition, the concentrations of ALT, AST, UREA, and CREA remained the same in each group after each treatment, indicating that liver and kidney functions were well preserved after treatment with our nanoLAMP. In addition, the MTT assays of HUVEC and ARPE-19 cell lines showed that our nanoparticles did not exert significant cytotoxic effects under dark conditions ( Fig.10 d), which indicates that our nanoparticles should be safe for systemic administration. Finally, the body weights of mice in each group did not show significant differences during the 15-day observation period ( Fig.10 e). These results all indicate that our nanoLAMP treatment is safe to use, with no obvious systemic or local adverse events.

[0204] Example 9. Nanoparticle encapsulation additives.

[0205] To further investigate whether this system can be used to deliver hydrophobic drugs, the anticancer drug ilisimol-Cu was incorporated to prepare nanoparticles with BODIPY caged-AS16 (cAS16) and PLA5k-PEG2k. 0.5 μL of 10 mg / mL ilisimol was added to 200 μL of 0.1 mg / mL CuCl 2 ·2H 2 O solution to prepare the ilisimol-Cu complex. Subsequently, the cAS16 & PLA5k-PEG2k DMSO mixture (4 μL 20 mg / ml cAS16 + 4 μL 100 mg / ml PLA5k-PEG2k) was added to the above solution under vigorous stirring to allow the components to self-assemble into nanoparticles. The nanoparticles were then concentrated by centrifugation at 18000 rpm for 30 minutes. Aggregates were also removed by centrifugation at 4000 rpm for 5 minutes. The supernatant was used for characterization and cell experiments. Fig.11 As shown in a, ilisimol-Cu can be successfully encapsulated by cAS16 / PLA5k-PEG2k. Nanoparticles were prepared by nanoprecipitation. Free peptides and drugs were removed by ultrafiltration. The encapsulation efficiency and drug loading of cAS16 were 75.34% and 20.08%, respectively. The encapsulation efficiency and drug loading of ilisimol-Cu were 13.25% and 8.82%, respectively ( Fig.11 b). After the nanoparticles are successfully prepared, CT26 cells are incubated with the nanoparticles. Fig.11As shown in c, cAS16 showed no cytotoxicity after light irradiation. Nanoparticles loaded with elisimol-Cu showed similar cytotoxicity to free elisimol-Cu with or without light irradiation. These results suggest that this system can be used to encapsulate hydrophobic small molecules for combined or synergistic therapy.

[0206] Example 10. BODIPY caged FM12 peptide for light-triggered PD-L1 blockade

[0207] To further explore whether BODIPY photocage molecules can be used to deliver other hydrophilic peptides, a peptide inhibitor targeting programmed death ligand 1 (PD-L1) (FPNWSLRPMNQM, SEQ ID NO: 2, FM12) was conjugated to BODIPY at the N-terminus to synthesize BODIPY caged FM12 (cFM12). The conjugate can also form stable polymer nanoparticles with the help of PEG3.4K-PLA5K. Fig.13 a and 13b, under red light irradiation (656 nm, 30 mW / cm 2 ), FM12 peptides could be successfully released from cFM12 in a time-dependent manner. In addition, after co-culture with MDA-MB-231 cells, the released FM12 peptides could promote the gene expression levels of pro-inflammatory cytokines such as IL-2 and TNF-α in Jurkat T cells compared with the control group treated with an equal amount of PBS ( Fig.13 c and 13d). For the preparation of nanoparticles, briefly, 2 μL of 10 mg / mL cFM12 was mixed with 2 μL of 100 mg / mL PEG3.4K-PLA5K in DMSO solution. Subsequently, 4 μL of cFM12 and PEG3.4K-PLA5K DMSO mixture was added to 200 μL of aqueous solution under vigorous stirring to allow the components to self-assemble into nanoparticles. The nanoparticles were then concentrated by centrifugation at 18000 rpm for 30 min. Aggregates were removed by centrifugation at 4000 rpm for 5 min. The supernatant was used for characterization and cell experiments. Fig.13 As shown in Figures e and 13f, the prepared nanoparticles have a well-dispersed size distribution with a diameter of approximately 122 nm and a PDI of approximately 0.2. These results suggest that this strategy can also be applied to other peptides for successful light harvesting and nanoparticle formation to achieve red light-triggered PD-L1 blockade and cancer immunotherapy.

[0208] The foregoing description of the specific embodiments will so fully reveal the general nature of the present disclosure that others can easily modify and / or adapt the application of various such specific embodiments without departing from the general concepts of the present disclosure, without undue experimentation, by applying knowledge within the technical scope of the relevant art (including the contents of the cited documents and incorporated herein by reference). Therefore, based on the teachings and guidance presented herein, such adaptations and modifications are intended to be within the meaning and scope of equivalents of the disclosed embodiments. It should be understood that the wording or terminology herein is for the purpose of description rather than limitation, so that the terms or wording of this specification will be interpreted by those skilled in the art based on the teachings and guidance presented herein, combined with the knowledge of those skilled in the relevant art.

[0209] Although various embodiments of the present disclosure have been described above, it should be understood that they are presented by way of example rather than limitation. It is apparent to those skilled in the relevant art that various changes may be made in form and detail without departing from the spirit and scope of the present disclosure. Therefore, the present disclosure should not be limited to any of the exemplary embodiments described above, but should only be limited according to the attached claims and their equivalents.

[0210] All references cited herein are incorporated by reference in their entirety and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

[0211]

Claims

1. A photoresponsive prodrug compound comprising a peptide conjugated to a photoresponsive group.

2. The compound of claim 1, wherein the peptide is an anti-angiogenic peptide, more preferably an AS16 peptide comprising the amino acid sequence of SEQ ID NO: 1 (ATWLPPRAANLLMAAS) or an FM12 peptide comprising the amino acid sequence of SEQ ID NO: 2 (FPNWSLRPMNQM).

3. The compound of claim 1 or 2, wherein the photoresponsive group is a boron-dipyrromethene (BODIPY), preferably, the BODIPY is a moiety selected from the group consisting of: formula (VII), formula (VIII), formula (IX), formula (X), formula (XI) or formula (XII), more preferably, BODIPY is a moiety of formula (XII): formula (VII) Formula (VIII) Formula (IX) Formula (X) Formula (XI) Formula (XII) 4. The compound of any one of claims 1-3, wherein the photoresponsive prodrug is or cFM12:

5. Nanoparticles comprising the photoresponsive prodrug of any one of claims 1 to 4 and one or more polymers.

6. The nanoparticles of claim 5, wherein the polymer is selected from the group consisting of polylactic acid (PLA), polyethylene glycol (PEG), polydimethylsiloxane (PDMS), polyethyleneimine (PEI), polyamidoamine (PAMAM) and combinations thereof; preferably, the polymer is selected from the group consisting of PLA-PEG copolymers; more preferably, the polymer is PLA5k-PEG2k or PEG3.4k-PLA5k.

7. The nanoparticle of claim 5 or 6, having a diameter of about 20-200 nm, more preferably about 40-100 nm, even more preferably about 50-70 nm.

8. Nanoparticles according to claim 7, having a polydispersity index of 0.120-0.220, preferably 0.150-0.

200.

9. The nanoparticle of claim 7 or 8, having a diameter of about 60 nm and a PDI of 0.170; Or having a diameter of about 122 nm and a PDI of 0.

2.

10. The nanoparticles of any one of claims 5 to 9, comprising a photoresponsive prodrug of the formula co-assembled with PLA5k-PEG2k Or a photoresponsive prodrug of cFM12 of the formula co-assembled with PLA3.4k-PLA5k 11. The nanoparticle of any one of claims 5-10, further encapsulating an additive; preferably, the additive is a hydrophobic drug; preferably, the additive is an anticancer drug; more preferably, the additive is selected from tamoxifen, amsacrine, bexarotene, estramustine, irofoven, trabectedin, cetuximab, panitumumab, tositumomab, alemtuzumab, bevacizumab, edrecolomab, gemtuzumab, alvocidib, seliciclib, aminolevulinic acid, methyl aminolevulinate, efaproxiral, Porfimer sodium, talaporfin, temoporfm, verteporfin, alitretinoin, tretinoin, anagrelide, arsenic trioxide, atrasentan, bortezomib, carmofur, celecoxib, demeclofenac, elisimol, elsamitrucin, etoglu, lonidamine, lucanthone, masoprofol, dibromomannitol, mitoguanidine, mitotane, oblimersen sodium, omacetin, sitimagene, ceradenovec, tegafur, testolactone, thiazofurine, tipifarnib, vorinostat or iniparib; further more preferably, the additional agent is elisimol.

12. A pharmaceutical composition comprising: (i) the photoresponsive prodrug of any one of claims 1-4 or the nanoparticle of any one of claims 5-11; and (ii) a pharmaceutically acceptable excipient.

13. A method for delivering a drug to a target site in a subject, comprising: (i) administering to the subject a photoresponsive prodrug according to any one of claims 1 to 4, a nanoparticle according to any one of claims 5 to 11, or a pharmaceutical composition according to claim 12; and (ii) irradiating the self-assembled system at the target site with a light source.

14. The method of claim 13, wherein the light source has a wavelength of about 500-1300 nm, preferably about 600-900 nm, more preferably about 600-700 nm, further preferably about 656 nm.

15. The method of claim 13 or 14, wherein the photoresponsive prodrug or nanoparticle is administered by at least one of: oral administration, transdermal administration, inhalation, intranasal administration, topical administration, intravaginal administration, ocular administration, intramural administration, intracerebral administration, rectal administration, parenteral administration, intravenous administration, intraarterial administration, intramuscular administration, subcutaneous administration, intratumoral administration, and any combination thereof; preferably by intravenous administration.

16. The method of any one of claims 13-15, wherein the target site is the eye, skin or a tumor, preferably the posterior segment of the eye.

17. Use of the compound of any one of claims 1-4, the nanoparticle of any one of claims 5-11 or the pharmaceutical composition of claim 12 in the preparation of a medicament for treating or preventing a disease characterized by abnormal angiogenesis, preferably, wherein the self-assembled system at a target site is irradiated with a light source.

18. The use according to claim 17, wherein the light source has a wavelength of about 500-1300 nm, preferably about 600-900 nm, more preferably about 600-700 nm, further preferably about 656 nm.

19. The use of claim 17 or 18, wherein the compound or drug delivery is administered by at least one of: oral administration, transdermal administration, inhalation, intranasal administration, topical administration, intravaginal administration, ocular administration, intramural administration, intracerebral administration, rectal administration, parenteral administration, intravenous administration, intraarterial administration, intramuscular administration, subcutaneous administration, intratumoral administration, and any combination thereof; preferably by intravenous administration.

20. The use of any one of claims 17-19, wherein the target site is the eye, the skin or a tumor, preferably the posterior segment of the eye.

21. The use according to any one of claims 17 to 20, wherein the use is for treating or preventing cancer, preferably renal cancer, lung cancer, breast cancer, colon cancer, prostate cancer, brain cancer, chondrosarcoma or angiosarcoma.

22. The use of any one of claims 17-20, wherein the use is for treating or preventing ocular neovascular diseases; preferably age-related macular degeneration (AMD), choroidal neovascularization secondary to myopia, proliferative diabetic retinopathy, diabetic macular edema, retinal vascular occlusions such as retinal vein occlusions, ocular tumors, Hipper-Lindau syndrome, retinopathy of prematurity and polypoidal choroidal vasculopathy; more preferably AMD, especially exudative AMD (wet AMD).

23. A kit comprising the compound of any one of claims 1-14, the nanoparticle of any one of claims 5-11, or the pharmaceutical composition of claim 12.

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