Polymeric composite nanoparticles, semiconducting polymers and uses thereof

Abstract

This invention relates to polymer composite nanoparticles, semiconductor polymers, and their uses. Specifically, this invention relates to a polymer composite nanoparticle emitting near-infrared afterglow luminescence, said nanoparticle comprising: (a) a semiconductor polymer of formula I; (b) optionally, an amphiphilic copolymer; and (c) optionally, a small molecule dye having near-infrared emission, wherein, when present, the amphiphilic copolymer encapsulates the semiconductor polymer of formula I and, when present, the small molecule dye. This invention relates to a semiconductor polymer of formula I. The invention also relates to the use of the polymer composite nanoparticles described herein in the preparation of imaging agents and the use of polymer composite nanoparticles as defined herein in the preparation of imaging agents for in vivo imaging of oxidative stress in the liver of subjects.

Description

[0001] This application is a divisional application of the invention patent application filed on August 1, 2018, entitled "Polymer Nanoparticles for Afterglow Molecular Imaging" with application number 201880045551.6. Technical Field

[0002] This invention relates to polymers and polymer nanoparticles that are particularly suitable for afterglow molecular imaging techniques. Background Technology

[0003] The listing or discussion of previously disclosed documents in this specification should not necessarily be construed as an admission that such documents are part of the prior art or common general knowledge.

[0004] Optical imaging plays a vital role in biology and medicine [see, for example, Ntziachristos, V., et al., Nat Biotechnol 23, 313-320 (2005)]. However, the need for real-time optical excitation during imaging results in tissue autofluorescence, which can impair imaging sensitivity and specificity in living subjects [e.g., see Smith, AM, et al., Nat Nanotechnol 4, 710-711 (2009)]. Optical imaging strategies such as bioluminescence and Cerenkov luminescence have eliminated the need for simultaneous optical excitation and have attracted great interest in molecular imaging [e.g., see Chu, J., et al., Nat Biotechnol 34, 760-767 (2016); Thorek, DL, et al., Nat Med 19, 1345-1350 (2013)]. However, bioluminescent probes require enzymes / substrates to produce light emission, and therefore their signals are often influenced by the enzyme microenvironment and substrate biodistribution in living animals [e.g., see So, MK, et al., J.]. Nat Biotechnol 24, 339-343 (2006)]. In contrast, Cherenkov probes rely on the release of charged particles from radioisotopes, which involves complex synthetic procedures and exhibits short luminescence lifetimes [e.g., see Liu, H., et al., J. Nucl. Med. 53, 1579-1584 (2012)].

[0005] Afterglow luminescence is an intrinsic luminescence process that occurs after photoexcitation has ended [e.g., see de Chermont, QL, et al., Proc. Natl. Acad. Sci. USA [104, 9266-9271 (2007)]. Afterglow luminescence is typically caused by the slow release of photons from energy traps after thermal simulation of the studied material. Although afterglow imaging holds great promise for in vivo imaging due to the lack of real-time excitation, only a few inorganic nanoparticles have been shown to produce afterglow luminescence under biologically relevant conditions [e.g., see Maldiney, T., et al., J Am Chem Soc 133, 11810-11815 (2011); Maldiney, T., et al., Nat Mater 13, 418-426 (2014); Li, ZJ, et al., J Am Chem Soc 137, 5304-5307 (2015)]. These articles relate to materials containing rare earth heavy metal ions (such as europium, praseodymium, and chromium) [see, for example, Maldiney, T., et al., Opt Mater Express 2, 261-268 (2012); Abdukayum, A., et al., J Am Chem Soc 135,14125-14133 (2013); Liu, F., et al. Sci Rep-Uk 3(2013); Maldiney, T., et al., Opt Mater 35, 1852-1858 (2013); Sharma, SK, et al., Opt Mater 36, 1901-1906(2014); Shi, JP, et al., Biomaterials 37, 260-270 (2015)]. Therefore, current-generation afterglow probes are severely hampered by toxicity issues [e.g., see Toppari, J. et al., Environ. Health Perspect. 104 Suppl 4, 741-803 (1996)].

[0006] Due to the difficulty in modifying the surface of such proteins, inorganic afterglow nanoparticles are currently used as accumulation probes and have limited targeting applications [e.g., see de Chermont, QL, et al., Proc. Natl. Acad. Sci. USA104, 9266-9271 (2007); Maldiney, T., et al., J Am Chem Soc 133, 11810-11815(2011); Maldiney, T., et al., Nat Mater 13, 418-426 (2014)]. Therefore, contrast is determined by the difference in probe concentration between the target tissue and adjacent normal tissue. Compared to concentration differences, intelligently activatable probes that undergo signal intensity changes when detecting molecular targets can provide high contrast and real-time information on pathological conditions at the molecular level [e.g., see Kobayashi, H. & Choyke, PL, ]. Chem. Res. 44, 83-90 (2011); Lovell, JF,et al., Chem Rev 110, 2839-2857 (2010)]. Summary of the Invention Surprisingly, a group of polymer materials has been found to exhibit afterglow luminescence in a manner suitable for biological applications. Therefore, in a first aspect of the invention, polymeric composite nanoparticles emitting near-infrared afterglow luminescence are provided, the nanoparticles comprising: (a) Semiconductor polymers of formula I:

[0007] (b) Optionally, an amphiphilic copolymer; and (c) Optionally, a small molecule dye having near-infrared emission, wherein: Amphiphilic copolymers (when present) encapsulating semiconductor polymers of formula I and small molecule dyes (when present); and In the polymer of formula I: R1 to R3 and R5 independently represent equation C q H 2q+1 Alkyl chains, wherein 1 ≤ q ≤ 50, R4 represents part of formula Ia or formula Ib:

[0008] Where 1≤s≤50 and 10≤t≤500;

[0009] Where 1≤u≤50 and 10≤v≤500; R6 represents C q H 2q+1 The alkyl chain, a portion of formula Ia or a portion of formula Ib, wherein q, s, t, u and v are as defined above; R7 represents the singlet oxygen sensitization region; Each of n, m, and o is greater than or equal to 0, and p is 0 or 1, wherein at least one of n, m, o, and p is greater than 0; A represents the part of Ic or Id:

[0010]

[0011] Among them, R8 and R 11 Independently represented by C q H 2q+1 alkyl chains, wherein: 1 ≤ q ≤ 50; R9 and R 12 Independently represented by C q H 2q+1 The alkyl chain, a portion of formula Ia or a portion of formula Ib, wherein q, s, t, u and v are as defined above; R 10 and R 13 It independently represents the singlet oxygen sensitization portion; When p is 1, then w, x, y, and z (if they exist) are independently greater than or equal to 0; and Small molecule dyes are present when o and p are 0, and are optionally present when: o is greater than or equal to 20; p is 1 and x or z is greater than or equal to 20; The sum of o and x is greater than or equal to 20; The sum of o and z is greater than or equal to 20; (o+x) / (n+m+o+w+x)>0.05; or (o+z) / (n+m+o+y+z)>0.05; Amphiphilic copolymers exist when m, o, and p are 0, and optionally exist when: (m+o+w) / (n+m+o+w+x)>0.1; or (m+o+y) / (n+m+o+y+z)>0.1; and The premise is that when n is greater than 0, one or more of m, o and p are also greater than 0.

[0012] In some embodiments of the first aspect of the invention, p can be 0. In embodiments where p is 0, the amphiphilic copolymer may optionally be present when: m is greater than or equal to 20, and m / (n+m+o) is greater than 0.1, and R 6 Cq H 2q+1 Where 1≤q≤50; m is greater than or equal to 20, and (m+o) / (x+y+z) is greater than 0.1, and R 6 It is a part of equation Ia or a part of equation Ib; or o is greater than or equal to 20, and (m+o) / (x+y+z) is greater than 0.1, and R 6 It is a part of formula Ia or a part of formula Ib.

[0013] In some embodiments of the first aspect of the invention: (a) n is 0; (b) An amphiphilic copolymer exists; (c) When present, each of n, m, o, w, x, y, and z can independently have a value from 5 to 1000; (d) The number average molecular weight of the polymer of formula I can be from 1,000 to 300,000 Daltons, such as from 1,000 to 100,000 Daltons. (e) When present, the singlet oxygen-sensitized components R7 and R 10 and R 13 It can be independently selected from one or more of the following groups: metalloporphyrins, metal phthalocyanines, naphthyl phthalocyanines, metal naphthyl phthalocyanines, dihydroporphyrin, rhodamine, anthocyanins, carotenoids, anthocyanins, rose bengal, methylene blue, and more particularly, 2,3-naphthyl phthalocyanine bis(trihexylsiloxy)silane, porphyrins (octaethylporphyrin, tetraphenylporphyrin), phthalocyanines, tetrapyrroles, transition metal complexes (Ir(III) complexes, Ru(II) complexes, Pt(II) complexes and Os(II) complexes) and boron-dipyrrole methylene (BODIPY) based photosensitizers; (f) The weight ratio of the amphiphilic copolymer to the polymer of formula I can be from 1:1 to 200:1, such as 1.5:1 to 100:1, such as 2:1 to 80:1; (g) When present, the small molecule dye with near-infrared radiation can be a singlet oxygen sensitizing compound selected from one or more of the following groups: metal porphyrins, metal phthalocyanines, naphthyl phthalocyanines, metal-naphthyl phthalocyanines, dihydroporphyrins, rhodamine, anthocyanins, carotenoids, anthocyanins, Bengal rose red, methylene blue, and more particularly, 2,3-naphthyl phthalocyanine bis(trihexylsiloxy)silanes, porphyrins (octaethylporphyrin, tetraphenylporphyrins), phthalocyanines, tetrapyrroles, transition metal complexes (Ir(III) complexes, Ru(II) complexes, Pt(II) complexes and Os(II) complexes) and boron-dipyrrole methylene (BODIPY) based photosensitizers; (h) When present, the amphiphilic copolymer may be selected from one or more of the following groups: alkyl-substituted chitosans, and more particularly, poly(alkyl)-b-poly(ethylene glycol), poly(ethylene glycol)- b -Poly(propylene glycol)- b - Poly(ethylene glycol), poly(ethylene glycol) methyl ether-block-poly(lactide-co-glycolic acid) (PEG-PLGA), poly(styrene)-block-poly(acrylic acid) (PS-PAA), poly(styrene-co-maleic anhydride) (PSMA), 1,2-dispalmitoyl-sn-glycerol-3-phosphocholine (DPPC), and 1,2-distearate-sn-glycerol-3-phosphoethanolamine-poly(ethylene glycol) (DSPE-PEG), optionally wherein the amphiphilic copolymer may have a number average molecular weight of 1,000 to 50,000 Daltons; and / or, when present, the amphiphilic copolymer may be selected from poly(ethylene glycol)-b-poly(propylene glycol)-b-poly(ethylene glycol) (e.g., (PEG)). 100 -b-(PPG) 65 -b-(PEG) 100 ), 1,2-distearyl-sn-glycerol-3-phosphate ethanolamine-poly(ethylene glycol) (DSPE-PEG), , , , And more specifically One or more of them.

[0014] In some embodiments of the invention, the amphiphilic copolymer (when present) may further include a quenching portion that can be cleaved by the reactive portion at the in vitro or in vivo test site. Suitable quenching portions may be selected from one or more of the following groups: Thiol-sensitive moieties, such as the 2,4-dinitrobenzenesulfonyl (DNBS) moiety and ; Sensitive parts of reactive oxygen species and ; pH-sensitive components, such as , , , , and ; In the peptide quencher portion, there are furin-sensitive Arg-Arg-Val-Arg-quenchers, Caspase-3-sensitive Asp-Val-Glu-Asp-quenchers, fibroblast activator protein-α (FAPα)-sensitive Gly-Pro-quenchers, matrix metalloproteinase-2 (MMP-2)-sensitive Gly-Arg-Val-Gly-Leu-Pro-quenchers, and MMP-7-sensitive Gly-Met-Trp-Ser-Leu-... -Pro-Val- quencher, MMP-13-sensitive Leu-Gly-Arg-Met-Gly-Leu-Pro- quencher, cathepsin B-sensitive Lys-lys- quencher, cathepsin D-sensitive Leu-Arg-Phe-Phe-Cys-Ile-Pro- quencher, cathepsin S-sensitive Arg-Leu- quencher, urokinase-sensitive Arg-Gly- quencher, and Legumain-sensitive Asn-Ala-Ala- quencher. The quencher is a dark quencher. Suitable dark quenchers can be selected from black hole quenchers (BHQ)-1, BHQ-2, BHQ-3 and QSY-7.

[0015] In some embodiments of the present invention, the amphiphilic copolymer containing the quenching portion may be: , , , And more specifically

[0016] (C) 18 PEG 12 -DNBS).

[0017] In some embodiments of the present invention, the polymer of Formula I may be selected from the following polymers: (i) Wherein, n and m are as defined above, optionally, wherein the number of n and m repeating units provides a polymer having a number average molecular weight of 25,000 to 200,000 Daltons, such as 45,000 to 150,000 Daltons, such as 50,000 to 100,000 Daltons, such as about 59,781 Daltons, and / or the molar ratio of n repeating units in the polymer is about 88%, and the molar ratio of m repeating units in the polymer is about 11% (e.g., the molar ratio of n repeating units in the polymer is 88.0% to 89.0%, and the molar ratio of m repeating units in the polymer is 11.0% to 12.0%). (ii) Wherein, m is as defined above, optionally, the number of m repeating units provides a polymer having a number average molecular weight of 15,000 to 100,000 Daltons, such as 20,000 to 75,000 Daltons, such as 20,000 to 50,000 Daltons, such as about 26,565 Daltons. (iii) Where p is 1, and the number of repeating units y and z provides that the polymer has a number average molecular weight of 5,000 to 20,000 Daltons, such as 7,000 to 18,000 Daltons, such as 8,900 to 15,000 Daltons, such as about 13,000 Daltons, and / or the molar ratio of y repeating units in the polymer is 85 to 99%, and the molar ratio of z repeating units in the polymer is 1 to 15% (e.g., the molar ratio of y repeating units in the polymer is 90.0 to 95.0%, and the molar ratio of z repeating units in the polymer is 5.0 to 10.0%).

[0018] In a second aspect of the invention, a semiconductor polymer of formula I is provided:

[0019] In the polymer of formula I: R1 to R3 and R5 independently represent equation C q H 2q+1 Alkyl chains, wherein 1 ≤ q ≤ 50, R4 represents part of formula Ia or formula Ib:

[0020] Where 1≤s≤50 and 10≤t≤500;

[0021] Where 1≤u≤50 and 10≤v≤500; R6 represents C q H 2q+1 The alkyl chain, a portion of formula Ia or a portion of formula Ib, wherein q, s, t, u and v are as defined above; R7 represents the singlet oxygen sensitization region; Each of n, m, and o is greater than or equal to 0; p is 0 or 1; A represents the part of Ic or Id:

[0022]

[0023] Among them, R8 and R11 Independently represented by C q H 2q+1 alkyl chains, wherein: 1 ≤ q ≤ 50; R9 and R 12 Independently represented by C q H 2q+1 The alkyl chain, a portion of formula Ia or a portion of formula Ib, wherein q, s, t, u and v are as defined above; R 10 and R 11 It independently represents the singlet oxygen sensitization portion; When p is 1, w, x, y, and z (if they exist) are independently greater than or equal to 0; The premise is: When n is greater than 0, one or more of m, o, and p are also greater than 0; and At least one of n, m, o, and p is greater than 0.

[0024] Embodiments of the second aspect of the invention include those in which the following situations occur: (i)n can be 0; (ii) p can be 0; (iii) The number average molecular weight of the polymer of formula I can be from 1,000 to 100,000 Daltons, such as 1,000 to 100,000 Daltons. (iv) When present, each of n, m, o, w, x, y, and z can independently have a value from 5 to 1000; (v) When present, the singlet oxygen-sensitized components R7 and R 10 and R 13 It can be independently selected from one or more of the following groups: metalloporphyrins, metal phthalocyanines, naphthyl phthalocyanines, metal-naphthyl phthalocyanines, dihydroporphyrin, rhodamine, anthocyanins, carotenoids, anthocyanins, Bengal rose, methylene blue, and more particularly, 2,3-naphthyl phthalocyanine bis(trihexylsiloxy)silane, porphyrins (octaethylporphyrin, tetraphenylporphyrin), phthalocyanines, tetrapyrroles, transition metal complexes (Ir(III) complexes, Ru(II) complexes, Pt(II) complexes and Os(II) complexes) and boron-dipyrrole methylene (BODIPY)-based photosensitizers.

[0025] In an embodiment of the second aspect of the invention, the polymer of formula I may be selected from the following polymers: (i) Wherein, n and m are as defined above, optionally, the number of n and m repeating units provided by the polymer having a number average molecular weight of 25,000 Daltons to 200,000 Daltons, such as 45,000 Daltons to 150,000 Daltons, such as 50,000 Daltons to 100,000 Daltons, such as about 59,781 Daltons, and / or the molar ratio of n repeating units in the polymer is about 88%, and the molar ratio of m repeating units in the polymer is about 12% (e.g., the molar ratio of n repeating units in the polymer is 88.0% to 89.0%, and the molar ratio of m repeating units in the polymer is 11.0% to 12.0%). (ii) Wherein, m is as defined above, optionally, the number of repeating units provided by the polymer having a number average molecular weight of 15,000 Daltons to 100,000 Daltons, such as 20,000 Daltons to 75,000 Daltons, such as 20,000 Daltons to 50,000 Daltons, such as about 26,565 Daltons; and (iii) Where p is 1, and the number of repeating units y and z provides that the polymer has a number average molecular weight of 5,000 to 20,000 Daltons, such as 7,000 to 18,000 Daltons, such as 8,900 to 15,000 Daltons, such as about 13,000 Daltons, and / or the molar ratio of y repeating units in the polymer is 85 to 99%, and the molar ratio of z repeating units in the polymer is 1 to 15% (e.g., the molar ratio of y repeating units in the polymer is 90.0 to 95.0%, and the molar ratio of z repeating units in the polymer is 5.0 to 10.0%).

[0026] In a third aspect of the invention, the use of any technically plausible combination of polymeric composite nanoparticles and embodiments thereof as defined in the first aspect of the invention is provided in the preparation of an imaging agent for the diagnosis of lesions or diseases in deep tissues and / or organs using afterglow luminescence. Imaging may be in vitro, or more particularly in vivo.

[0027] In a fourth aspect of the invention, a method is provided for in vivo imaging in deep tissues and / or organs using any technically plausible combination of polymer composite nanoparticles and embodiments thereof as defined in the first aspect of the invention. This method includes irradiating the polymer composite nanoparticles with an NIR laser before or after providing them to a living organism (e.g., injecting them subcutaneously, intradermally, or intravenously into a living organism) and detecting afterglow luminescence using an imaging system / device. In various embodiments of the invention, the afterglow luminescence of the injected polymer nanoparticles can be reactivated in vivo by subjecting a living organism or a portion of a living organism to NIR laser irradiation.

[0028] In a fifth aspect of the invention, any technically plausible combination of polymeric composite nanoparticles and embodiments thereof, as defined in the first aspect of the invention, is provided as an imaging agent for diagnosing lesions or diseases in deep tissues and / or organs using afterglow luminescence. Imaging may be in vitro, or more particularly in vivo.

[0029] In the implementation methods of the third to fifth aspects: (ai) In vivo imaging can be performed on living organisms (preferably animals or humans); (aii) In vivo imaging can be used for the purpose of locating lymph nodes and / or visualizing tumors (such as breast, lung, or liver tumors); (aiii) Imaging agents can be used for in vivo imaging for image-guided surgery. For example, polymeric composite nanoparticles may be one in which the amphiphilic copolymer also includes a quenching portion that can be reactively cleaved at the in vitro or in vivo test site as defined above.

[0030] In a sixth aspect of the invention, the use of polymeric composite nanoparticles as defined in the first aspect of the invention above in the preparation of an imaging agent for in vivo imaging of oxidative stress in the liver of a subject is provided, wherein the amphiphilic copolymer in the polymeric composite nanoparticles further includes a quenching portion as defined above that can be partially cleaved by the reactive portion at the test site in vitro or in vivo (e.g., the amphiphilic copolymer may be C...). 18 -PEG 12 -DNBS), the method includes the following steps: supplying polymer composite nanoparticles into a living organism, then irradiating the polymer composite nanoparticles with an NIR laser, and then using an imaging system / device to detect afterglow luminescence in the liver.

[0031] In a seventh aspect of the invention, a method is provided for in vivo imaging of oxidative stress in the liver of a subject using polymer composite nanoparticles as defined in the first aspect of the invention above, wherein the amphiphilic copolymer in the polymer composite nanoparticles further includes a quenching portion as defined above that can be partially cleaved by the reactive portion at the test site in vitro or in vivo (e.g., the amphiphilic copolymer may be C...). 18 -PEG 12 The method (DNBS) includes the following steps: intravenously injecting polymer composite nanoparticles into a living organism, irradiating the polymer composite nanoparticles with an NIR laser, and then detecting afterglow luminescence in the liver using an imaging system / device. In various embodiments of the invention, the afterglow luminescence of the injected polymer nanoparticles can be reactivated in vivo by subjecting a living organism or a portion of a living organism to NIR laser irradiation after being injected subcutaneously, intradermally, or intravenously with the polymer composite nanoparticles.

[0032] In an eighth aspect of the invention, polymeric composite nanoparticles as defined above in the first aspect of the invention are provided as imaging agents for determining oxidative stress in the liver of a subject using afterglow luminescence, wherein the amphiphilic copolymer in the polymeric composite nanoparticles further includes a quenching portion as defined above that can be partially cleaved by the reactive portion at the test site in vitro or in vivo (e.g., the amphiphilic copolymer may be C...). 18 -PEG 12 -DNBS). Examples of such tests performed in mice are provided in the Examples section.

[0033] In embodiments of the sixth to eighth aspects, oxidative stress in the liver can be used to determine drug-induced hepatotoxicity.

[0034] Aspects and embodiments of the invention are described in the following numbered clauses.

[0035] 1. A polymer composite nanoparticle emitting near-infrared afterglow luminescence, said nanoparticle comprising: (a) Semiconductor polymers of formula I:

[0036] (b) Optionally, an amphiphilic copolymer; and (c) Optionally, a small molecule dye having near-infrared emission, wherein: When present, the amphiphilic copolymer encapsulates the semiconductor polymer of Formula I and, when present, the small molecule dye; and In the polymer of Formula I: R1 to R3 and R5 independently represent equation C q H 2q+1 Alkyl chains, wherein 1 ≤ q ≤ 50, R4 represents part of formula Ia or formula Ib:

[0037] Where 1≤s≤50 and 10≤t≤500;

[0038] Where 1≤u≤50 and 10≤v≤500; R6 represents C q H 2q+1 The alkyl chain, a portion of formula Ia or a portion of formula Ib, wherein q, s, t, u and v are as defined above; R7 represents the singlet oxygen sensitization region; Each of n, m, and o is greater than or equal to 0, and p is 0 or 1, wherein at least one of n, m, o, and p is greater than 0; A represents the part of Ic or Id:

[0039]

[0040] Among them, R8 and R 11 Independently represented by C q H 2q+1 alkyl chains, wherein: 1 ≤ q ≤ 50; R9 and R 12 Independently represented by C q H 2q+1 The alkyl chain, a portion of formula Ia or a portion of formula Ib, wherein q, s, t, u and v are as defined above; R 10 and R 13 It independently represents the singlet oxygen sensitization portion; When p is 1, then w, x, y, and z, if they exist, are independently greater than or equal to 0; and The small molecule dye is present when o and p are 0, and optionally when: o is greater than or equal to 20; p is 1 and x or z is greater than or equal to 20; The sum of o and x is greater than or equal to 20; The sum of o and z is greater than or equal to 20; (o+x) / (n+m+o+w+x)>0.05; or (o+z) / (n+m+o+y+z)>0.05; The amphiphilic copolymer is present when m, o, and p are 0, and optionally present when: (m+o+w) / (n+m+o+w+x)>0.1; or (m+o+y) / (n+m+o+y+z)>0.1; and The premise is that when n is greater than 0, one or more of m, o and p are also greater than 0.

[0041] 2. The complex according to Clause 1, wherein n is 0.

[0042] 3. The complex according to Clause 1 or Clause 2, wherein p is 0.

[0043] 4. The composite pigment according to Clause 3, wherein the amphiphilic copolymer is optionally present when: m is greater than or equal to 20, and m / (n+m+o) is greater than 0.1 and R 6 C q H 2q+1 Where 1≤q≤50; m is greater than or equal to 20, and (m+o) / (x+y+z) is greater than 0.1, and R 6 It is a part of equation Ia or a part of equation Ib; or o is greater than or equal to 20, and (m+o) / (x+y+z) is greater than 0.1, and R 6 It is a part of formula Ia or a part of formula Ib.

[0044] 5. The complex according to any one of clauses 1 to 3, wherein the amphiphilic copolymer is present.

[0045] 6. The complex according to any one of the preceding clauses, wherein, when present, each of n, m, o, w, x, y and z independently has a value from 5 to 1000.

[0046] 7. The complex according to any one of the preceding clauses, wherein the polymer of Formula I has a number-average molecular weight of 1,000 to 300,000 Daltons, such as 1,000 to 100,000 Daltons.

[0047] 8. The complex according to any one of the preceding clauses, wherein, when present, the singlet oxygen-sensitizing moieties R7, R... 10 and R 13The photosensitizer is independently selected from one or more of the following groups: metalloporphyrins, metal phthalocyanines, naphthyl phthalocyanines, metal-naphthyl phthalocyanines, dihydroporphyrins, rhodamine, anthocyanins, carotenoids, anthocyanins, Bengal rose red, methylene blue, 2,3-naphthyl phthalocyanine bis(trihexylsiloxy)silane, porphyrins (octaethylporphyrin, tetraphenylporphyrin), phthalocyanines, tetrapyrroles, transition metal complexes (Ir(III) complexes, Ru(II) complexes, Pt(II) complexes and Os(II) complexes) and boron-dipyrrole methylene (BODIPY)-based photosensitizers.

[0048] 9. The complex according to any one of the preceding clauses, wherein the polymer of Formula I is selected from the polymers listed below: (i) Wherein, n and m are as defined in any of the preceding clauses, optionally, wherein the number of n and m repeating units provides a polymer having a number average molecular weight of 25,000 to 200,000 Daltons, such as 45,000 to 150,000 Daltons, such as 50,000 to 100,000 Daltons, such as about 59,781 Daltons, and / or the molar ratio of n repeating units in the polymer is about 88% and the molar ratio of m repeating units in the polymer is about 11% (e.g., the molar ratio of n repeating units in the polymer is 88.0 to 89.0% and the molar ratio of m repeating units in the polymer is 11.0 to 12.0%). (ii) Wherein, m is as defined in any of the preceding clauses, optionally, wherein the number of m repeating units provides a polymer having a number average molecular weight of 15,000 to 100,000 Daltons, such as 20,000 to 75,000 Daltons, such as 20,000 to 50,000 Daltons, such as about 26,565 Daltons; and (iii) , Wherein, p is 1, and the number of repeating units y and z provides that the polymer has a number average molecular weight of 5,000 to 20,000 Daltons, such as 7,000 to 18,000 Daltons, such as 8,900 to 15,000 Daltons, such as about 13,000 Daltons, and / or the molar ratio of y repeating units in the polymer is 85 to 99% and the molar ratio of z repeating units in the polymer is 1 to 15% (e.g., the molar ratio of y repeating units in the polymer is 90.0 to 95.0% and the molar ratio of z repeating units in the polymer is 5.0 to 10.0%).

[0049] 10. The complex according to any one of clauses 1 to 3 and 5 to 9, wherein the weight-to-weight ratio of the amphiphilic copolymer to the polymer of formula I is 1:1 to 200:1, such as 1.5:1 to 100:1, such as 2:1 to 80:1. 11. The complex according to any one of the preceding clauses, wherein, when present, the small molecule dye having near-infrared emission is a singlet oxygen sensitizing compound selected from one or more of the following groups: metalloporphyrins, metal phthalocyanines, naphthyl phthalocyanines, metal-naphthyl phthalocyanines, dihydroporphyrin, rhodamine, anthocyanins, carotenoids, anthocyanins, Bengal rose red, methylene blue, 2,3-naphthyl phthalocyanine bis(trihexylsiloxy)silane, porphyrins (octaethylporphyrin, tetraphenylporphyrin), phthalocyanines, tetrapyrroles, transition metal complexes (Ir(III) complexes, Ru(II) complexes, Pt(II) complexes and Os(II) complexes) and boron-dipyrrole methylene (BODIPY)-based photosensitizers.

[0050] 12. The complex according to any one of the preceding clauses, wherein, when present, the amphiphilic copolymer is selected from one or more of the group consisting of: alkyl-substituted chitosan, and more particularly, poly(alkyl)-b-poly(ethylene glycol), poly(ethylene glycol)-b-poly(propylene glycol)-b-poly(ethylene glycol), poly(ethylene glycol) methyl ether-block-poly(lactide-co-glycolic acid) (PEG-PLGA), poly(styrene)-block-poly(acrylic acid) (PS-PAA), poly(styrene-co-maleic anhydride) (PSMA), 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC), and 1,2-distearyl-sn-glycerol-3-phosphoethanolamine-poly(ethylene glycol) (DSPE-PEG), optionally, wherein the amphiphilic copolymer has a number average molecular weight of 1,000 to 50,000 Daltons.

[0051] 13. The complex according to any one of the preceding clauses, wherein the amphiphilic copolymer further comprises a quenching portion that can be cleaved by the reactive portion at the test site in vitro or in vivo.

[0052] 14. The complex according to clause 13, wherein the quenching portion is selectable from one or more of the group consisting of: Thiol-sensitive moieties, such as the 2,4-dinitrophenylsulfonyl (DNBS) moiety and ; Sensitive parts of reactive oxygen species and ; pH-sensitive components, such as , , , , and ; In the peptide quencher portion, there are quenchers such as furin-sensitive Arg-Arg-Val-Arg-quenchers, Caspase-3-sensitive Asp-Val-Glu-Asp-quenchers, fibroblast activator protein-α (FAPα)-sensitive Gly-Pro-quenchers, matrix metalloproteinase-2 (MMP-2)-sensitive Gly-Arg-Val-Gly-Leu-Pro-quenchers, and MMP-7-sensitive Gly-Met-Trp-Ser-Leu-Pr-quenchers. o-Val-quencher, MMP-13-sensitive Leu-Gly-Arg-Met-Gly-Leu-Pro-quencher, cathepsin B-sensitive Lys-lys-quencher, cathepsin D-sensitive Leu-Arg-Phe-Phe-Cys-Ile-Pro-quencher, cathepsin S-sensitive Arg-Leu-quencher, urokinase-sensitive Arg-Gly-quencher, and Legumain-sensitive Asn-Ala-Ala-quencher. The quenching agent is a dark quenching agent.

[0053] 15. The complex according to Clause 14, wherein the dark quencher is selected from black hole quencher (BHQ)-1, BHQ-2, BHQ-3 and QSY-7.

[0054] 16. The composite according to clause 13 or 14, wherein the amphiphilic copolymer comprising the quenched portion is selected from one or more of the following: (C 18 PEG 12 -DNBS), , , and For example, the amphiphilic copolymer containing the quenching portion can be C 18 PEG 12 -DNBS.

[0055] 17. The complex according to any one of clauses 1 to 3 and 5 to 16, wherein the amphiphilic copolymer is selected from one or more of poly(ethylene glycol)-b-poly(propylene glycol)-b-poly(ethylene glycol) (e.g., (PEG)100-b-(PPG)65-b-(PEG)100), 1,2-distearate-sn-glycerol-3-phosphate ethanolamine-poly(ethylene glycol) (DSPE-PEG), and .

[0056] 18. Semiconductor polymers of Formula I:

[0057] In the polymer of Formula I: R1 to R3 and R5 independently represent equation C q H 2q+1 Alkyl chains, wherein 1 ≤ q ≤ 50, R4 represents part of formula Ia or formula Ib:

[0058] Where 1≤s≤50 and 10≤t≤500;

[0059] Where 1≤u≤50 and 10≤v≤500; R6 represents C q H 2q+1 The alkyl chain, a portion of formula Ia or a portion of formula Ib, wherein q, s, t, u and v are as defined above; R7 represents the singlet oxygen sensitization region; Each of n, m, and o is greater than or equal to 0; p is 0 or 1; A represents the part of Ic or Id:

[0060]

[0061] Among them, R8 and R 11 Independently represented by C q H 2q+1 alkyl chains, wherein: 1 ≤ q ≤ 50; R9 and R 12 Independently represented by C q H 2q+1 The alkyl chain, a portion of formula Ia or a portion of formula Ib, wherein q, s, t, u and v are as defined above; R 10 and R 11 It independently represents the singlet oxygen sensitization portion; When p is 1, w, x, y, and z, if they exist, are independently greater than or equal to 0; The premise is: When n is greater than 0, one or more of m, o, and p are also greater than 0; and At least one of n, m, o, and p is greater than 0.

[0062] 19. The polymer according to Clause 18, wherein n is 0.

[0063] 20. The polymer according to Clause 18 or Clause 19, wherein p is 0.

[0064] 21. The polymer according to any one of clauses 18 to 20, wherein the polymer of formula I has a number-average molecular weight of 1,000 to 100,000 Daltons, such as 1,000 to 100,000 Daltons.

[0065] 22. The polymer according to any one of clauses 18 to 21, wherein, when present, each of n, m, o, w, x, y and z independently has a value of 5 to 1000.

[0066] 23. The polymer according to any one of clauses 18 to 22, wherein, when present, the singlet oxygen-sensitized moieties R7, R... 10 and R 13 The photosensitizer is independently selected from one or more of the following groups: metalloporphyrins, metal phthalocyanines, naphthyl phthalocyanines, metal-naphthyl phthalocyanines, dihydroporphyrins, rhodamine, anthocyanins, carotenoids, anthocyanins, Bengal rose red, methylene blue, 2,3-naphthyl phthalocyanine bis(trihexylsiloxy)silane, porphyrins (octaethylporphyrin, tetraphenylporphyrin), phthalocyanines, tetrapyrroles, transition metal complexes (Ir(III) complexes, Ru(II) complexes, Pt(II) complexes and Os(II) complexes) and boron-dipyrrole methylene (BODIPY)-based photosensitizers.

[0067] 24. The polymer according to any one of clauses 18 to 23, wherein the polymer of formula I is selected from the polymers listed below: (i) Wherein, n and m are as defined in any of the preceding clauses, optionally, wherein the number of n and m repeating units provides a polymer having a number average molecular weight of 25,000 Daltons to 200,000 Daltons, such as 45,000 Daltons to 150,000 Daltons, such as 50,000 Daltons to 100,000 Daltons, such as about 59,781 Daltons, and / or the molar ratio of n repeating units in the polymer is about 88% and the molar ratio of m repeating units in the polymer is about 12% (e.g., the molar ratio of n repeating units in the polymer is 88.0% to 89.0% and the molar ratio of m repeating units in the polymer is 11.0% to 12.0%). (ii) Wherein, m is as defined in any of the preceding clauses, optionally, wherein the number of m repeating units provides a polymer having a number average molecular weight of 15,000 Daltons to 100,000 Daltons, such as 20,000 Daltons to 75,000 Daltons, such as 20,000 Daltons to 50,000 Daltons, such as about 26,565 Daltons; (iii) , Wherein, p is 1, and the number of repeating units y and z provides that the polymer has a number average molecular weight of 5,000 to 20,000 Daltons, such as 7,000 to 18,000 Daltons, such as 8,900 to 15,000 Daltons, such as about 13,000 Daltons, and / or the molar ratio of y repeating units in the polymer is 85 to 99% and the molar ratio of z repeating units in the polymer is 1 to 15% (e.g., the molar ratio of y repeating units in the polymer is 90.0 to 95.0% and the molar ratio of z repeating units in the polymer is 5.0 to 10.0%).

[0068] 25. Use of polymer composite nanoparticles according to any one of Clauses 1 to 17 in the preparation of imaging agents for the purpose of diagnosing lesions or diseases in deep tissues and / or organs using afterglow luminescence.

[0069] 26. The use as described in Clause 25, wherein the use is in vivo or in vitro.

[0070] 27. The use as described in Clause 26, wherein the in vivo imaging is used for the purpose of locating lymph nodes and / or visualizing tumors.

[0071] 28. The use according to any one of clauses 25 to 27, wherein the formulation of the imaging agent is used for in vivo imaging for image-guided surgery.

[0072] 29. The use as described in Clause 28, wherein the polymer composite nanoparticles are as defined in any one of Clauses 13 to 17.

[0073] 30. Use of polymer composite nanoparticles as defined in any one of Clauses 13 to 17 in the preparation of an imaging agent for in vivo imaging of oxidative stress in the liver of a subject, the method comprising the steps of: supplying pre-irradiated polymer nanoparticles to a living organism, irradiating activatable polymer nanoparticles with an NIR laser, and detecting afterglow luminescence in the liver using an imaging system / device.

[0074] 31. The use as described in Clause 30, wherein oxidative stress in the liver is used to determine drug-induced hepatotoxicity. Attached Figure Description

[0075] Figure 1 a to 1h show the synthesis and characterization of SPN. Figure 1 a) SPs (MEHPP, POPPV, PFBT, MEHCPV, BOPPV, MDMOPPV, and MEHPPV) and amphiphilic triblock copolymers (PEG-) used for the synthesis of SPN b -PPG- b The chemical structure of PEG. Figure 1 b) Schematic diagram of SPN preparation via nanoprecipitation. Figure 1 c) The average hydrodynamic diameter of the SPN in 1×PBS buffer (pH = 7.4). Figure 1 d) Representative TEM images of SPN-MEHPPV. Figure 1 e) Images of white light (upper group), afterglow emission (middle group), and fluorescence (lower group) of SPN-MEHPP, SPN-POPPV, SPN-PFBT, SPN-MEHCPV, SPN-BOPPV, SPN-MDMOPPV, and SPN-MEHPPV in 1×PBS buffer (pH = 7.4). Fluorescence and afterglow images of the SPNs were captured at their respective maximum absorption intensities (absorbance 0.5 after 10-fold dilution). Except for SPN-MEHPP and SPN-POPPV (430 nm), fluorescence images of all SPNs were acquired after excitation at 465 nm. SPNs were processed at a power density of 0.1 W / cm². 2 After pre-illumination under white light for 1 min, afterglow images were acquired for 30 s. Figure 1 f) Normalized fluorescence spectrum of SPN in 1×PBS buffer (pH = 7.4). Figure 1 g) Normalized afterglow emission spectra of SPN-BOPPV, SPN-MDMOPPV, and SPN-MEHPPV in 1×PBS buffer (pH = 7.4). Figure 1 h) Figure 1 Quantification of fluorescence intensity and afterglow intensity of SPN in e.

[0076] Figure 2 a to 2f illustrate the mechanism of SPN afterglow. Figure 2 a) MEHPPV (10 µg / mL) irradiated in CHCl3 for 4 h (power: 0.1 W / cm²). 2 UV-Vis absorption spectra before and after ( ). Figure 2 b) MEHPPV irradiated with light in CHCl3 for 24 h (power: 0.1 W / cm²) 2 FTIR spectra before and after ( ). Figure 2 c) The change in fluorescence enhancement (F / F0) of SOSG (1 µM) with illumination time at 528 nm, in the absence or presence of SPN-MEHPPV (1.25 µg / mL). Figure 2 d) Decrease in afterglow luminescence of SPN-MEHPPV (62.5 µg / mL) at room temperature. Before collecting the afterglow signal, the nanoparticle solution was subjected to a power density of 0.1 W / cm². 2 Pre-irradiate with white light for 1 min. Figure 2 e) After purging with N2, O2 or in the presence of 50 w / w Afterglow images and intensities of SPN-MEHPPV (62.5 µg / mL) acquired at different temperatures (room temperature and 60℃) after treatment with % NaN3. Figure 2 f) The proposed mechanism of afterglow luminescence of SP based on PPV.

[0077] Figure 3 a to 3f show 1 NIR afterglow amplified by O2 sensitizer. Figure 3 a) for 1 A schematic diagram illustrating the proposed mechanism of NIR afterglow amplified by O2 sensitizer. Figure 3 b) Schematic diagram of SPN-NCBS pre-irradiated with an 808 nm laser for afterglow enhancement, compared to a 514 nm laser. Figure 3 c) Afterglow luminescence images of 12.5 µg / mL SPN-NCBS (based on MEHPPV mass) pre-irradiated at 514 nm (left) or 808 nm (right). Afterglow images were acquired under a bioluminescence model for 30 s. The nanoparticle solution was passed through an 808 or 514 nm laser (1 W / cm²). 2 Pre-irradiation for 1 min, followed by image acquisition for 5 s after removal of the laser source. Fluorescence of SPN-MEHPPV and SPN-NCBS5 in 1×PBS buffer (pH = 7.4) was measured. Figure 3 d) and NIR-induced afterglow emission spectrum ( Figure 3 e). ( Figure 3 f) Quantitative analysis of the absolute fluorescence and afterglow luminescence intensity of SPN-MEHPPV under different NCBS doping concentrations. Error bars represent standard deviation (n = 3).

[0078] Figure 4 a to 4e show tissue penetration studies of NIR afterglow luminescence. Figure 4 a) Afterglow luminescence imaging (top group) and fluorescence imaging (bottom group) of SPN-NCBS5 solution penetrating chicken tissue of varying thicknesses. Figure 4b) Changes in SBR (Sequencing Blowout Ratio) of SPN-NCBS5 afterglow luminescence and fluorescence with tissue depth. * The SBR at a depth of 4 cm through chicken tissue showed a statistically significant difference (n = 3, P < 0.01). Figure 4 c) Schematic diagram of afterglow luminescence imaging through a live mouse, with the SPN-NCBS5 solution located 1.7 cm below the mouse. Figure 4 d) Afterglow and fluorescence images of SPN-NCBS5 solution passing through live mice. Imaged using an 808 or 514 nm laser (1 W / cm²). 2 Pre-irradiation with SPN-NCBS5 solution (62.5 μg / mL, 50 μL) for 1 min, followed by image acquisition within 5 s after laser removal. Fluorescence images were acquired at 780 nm after excitation at 710 nm. Figure 4 e) Figure 4 SBR for afterglow luminescence and fluorescence imaging in d.

[0079] Figure 5 a through 5f show in vivo afterglow imaging of lymph nodes and tumors. Figure 5 a) Schematic diagram of afterglow luminescence imaging of lymph nodes. SPN-NCBS5 was subjected to an 808 nm laser (1 W / cm²). 2 Pre-irradiate for 1 min, then store at -20°C for one day, and then use directly for lymph node imaging. Figure 5 b) Fluorescence imaging (left) and afterglow luminescence imaging (right) of lymph nodes in live mice at t = 65 min after intradermal injection of SPN-NCBS5 (0.25 mg / mL, 0.05 mL) into the forepaw of mice. Figure 5 c) Changes in SBR (Sequencing Backscattering and Fluorescence) of lymph nodes in live mice over time after injection. This was achieved by imaging at 808 nm (1 W / cm²). 2 Irradiation was performed for 1 min, and in situ regeneration of afterglow of SPN-NCBS5 was performed at t = 65 min after injection. *In live mice, the SBR between fluorescence and afterglow luminescence was statistically significant at t = 130 min after SPN-NCBS5 injection (n = 3, P < 0.01). Figure 5 d) Schematic diagram of afterglow luminescence imaging of xenografted HeLa tumors in a mouse model. Figure 5 e) Afterglow luminescence (top group) and fluorescence images (bottom group) of tumors in live mice at representative time points following systemic administration of SPN-NCBS5 (0.25 mg / mL, 0.2 mL) via tail vein injection. The tumor is located on the right shoulder, indicated by the white dashed circle and arrow. Figure 5f) Changes in SBR over time for afterglow luminescence and NIR fluorescence imaging of tumors in live mice. Intensity values ​​are the mean ± sd for n = 3 mice. Error bars are based on standard deviation (n = 3 mice). At 808 nm (0.5 W / cm²) 2 After 1 min of irradiation, afterglow emission images were acquired for 180 s. After excitation at 710 nm, fluorescence images were acquired at 780 nm for 0.1 s.

[0080] Figure 6 a to 6h showed in vivo afterglow luminescence imaging of drug-induced hepatotoxicity. Figure 6 a) Schematic diagram of the design and activation mechanism of a bio-thiol-activated afterglow probe (SPN-thiol). Figure 6 b) Afterglow emission spectra of SPN-thiol (12.5 µg / mL) in 1×PBS buffer (pH = 7.4) with or without Cys (1 mM). Figure 6 c) Afterglow emission image of SPN-thiol (12.5 µg / mL) in 1×PBS (pH = 7.4) at 37°C in the presence of Cys, Hcy, GSH, and other amino acids (1 mM). Figure 6 d) Afterglow intensity of SPN-thiol in 1×PBS (pH = 7.4) at 37°C in the presence of Cys, Hcy, GSH, and other amino acids (1 mM). 1: Blank, 2: Arg, 3: Asn, 4: Gln, 5: Gly, 6: His, 7: Leu, 8: Lys, 9: Met, 10: Pro, 11: Ser, 12: Val, 13: GSH, 14: Hcy, 15: Cys. Figure 6 e) Fitted calibration curve of afterglow luminescence intensity of SPN-thiol as a function of Cys concentration. Figure 6 f) A schematic diagram of the APAP-induced toxicity mechanism, in which GSH depletion leads to afterglow inactivation, while NAC repairs this depletion to reactivate afterglow. Figure 6 g) Representative afterglow emission images of mice treated intraperitoneally with APAP (300 mg / kg), saline, or NAC (200 mg / kg) with APAP (300 mg / kg), followed by intravenous injection of SPN-thiol (0.25 mg / mL, 0.2 mL) at t = 20 min. Imaged at 808 nm (1 W / cm²). 2 After irradiation for 1 minute for in-situ regeneration, afterglow emission images were acquired for 180 seconds. Figure 6h) Changes in afterglow luminescence and SBR of NIR fluorescence imaging of the liver in live mice over time. Intensity values ​​are the mean ± sd of n = 3 mice. *At t = 2 h after SPN-thiol injection, there was a statistically significant difference in afterglow luminescence intensity between the saline and APAP-treated groups (n = 3, P < 0.01); **At t = 2 h after SPN-thiol injection, there was no statistically significant difference between the saline and APAP / NAC-treated groups (n = 3, P > 0.05).

[0081] Figure 7 The stability of SPN-MEHPPV (10 µg / mL) after 60 days of storage in the dark in 1×PBS (pH = 7.4) is shown. Error bars represent the standard deviation of three independent measurements.

[0082] Figure 8 The UV-Vis absorption spectra of SPN-MEHPP, SPN-POPPV, SPN-PFBT, SPN-MEHCPV, SPN-BOPPV, SPN-MDMOPPV and SPN-MEHPPV in 1×PBS buffer (pH = 7.4) are shown.

[0083] Figure 9 a to 9c show ( Figure 9 a) Fluorescence images (top) and afterglow emission images (bottom) of various SPs in THF solution at their respective maximum absorption wavelengths under the same optical density (absorbance was 0.5 after 10-fold dilution). Fluorescence images of all SPs were acquired for 0.1 s. Except for MEHCPV and POPPV, which were excited at 430 nm and acquired at 520 ± 10 nm, the others were excited at 465 ± 10 nm and emitted at 580 ± 10 nm. For the afterglow emission images, all SPs were captured at a power of 0.1 W / cm². 2 The light was applied for 1 minute, and the signal was collected for 30 seconds using an open filter. Figure 9 b) Normalized fluorescence spectra of SPs in THF solution, including BOPPV, MDMOPPV, and MEHPPV SPs excited at 455 nm; PFBT, MEHCPV, POPPV, and MEHPP excited at 440, 420, 360, and 320 nm, respectively. Figure 9 c) Quantitative analysis of fluorescence intensity and afterglow luminescence intensity of SP in THF. Error bars represent the standard deviation of three independent measurements. Compared with MEHPPV, MEHCPV with an electron-withdrawing cyano group on the vinylene backbone exhibits 5.1 times lower afterglow luminescence. This is because the electron-withdrawing substituent can slow down the afterglow luminescence. 1O2 oxidation reduces afterglow luminescence. Similarly, the substituents of POPPV have weaker electron-donating groups than those of BOPPV, MDMOPPV, and MEHPPV [e.g., see Abdukayum, A., et al., J Am Chem Soc [135, 14125-14133 (2013)]; they exhibit a weaker afterglow intensity.

[0084] Figure 10 a to 10f show MDMOPPV ( Figure 10 a) and BOPPV (10 μg / mL) ( Figure 10 d) Irradiation with light in CHCl3 (power: 0.1 W / cm²) 2 UV-Vis absorption spectra before and after 4 hours; MDMOPPV ( Figure 10 b) and BOPPV ( Figure 10 e) Irradiation in CDCl3 (power: 0.1 W / cm²) 2 Before and after overnight stay 1 H NMR spectrum, and MDMOPPV ( Figure 10 c) and BOPPV ( Figure 10 f) Irradiation with light in CHCl3 (power: 0.1 W / cm²) 2 Fourier transform infrared (FT-IR) spectra before and after overnight storage.

[0085] Figure 11 a to 11d show MEHPP ( Figure 11 a) POPPV ( Figure 11 b) PFBT Figure 11 c) and MEHCPV ( Figure 11 d) (10 µg / mL) irradiated with light in CHCl3 (power: 0.1 W / cm²) 2 UV-Vis absorption spectra before and after 4 hours.

[0086] Figure 12 The following images show the temperature-responsive afterglow emission of SPN-BOPPV (top, 180 µg / mL), SPN-MDMOPPV (middle, 83.3 µg / mL), and SPN-MEHPPV (bottom, 62.5 µg / mL) acquired for 30 s using an open filter via an IVIS spectral imaging system.

[0087] Figure 13 a and 13b show ( Figure 13 a) In PBS buffer (pH = 7.4), the UV-Vis absorption of SPN-MEHPPV and NCBS-only at various NCBS doping levels, and ( Figure 13b) Fluorescence spectrum of SPN-NCBS5 in 1×PBS buffer (pH = 7.4). Excitation: 710 nm.

[0088] Figure 14 a and 14b show ( Figure 14 a) Representative TEM images of SPN-NCBS5, and ( Figure 14 b) DLS of SPN-NCBS in 1×PBS buffer (pH = 7.4).

[0089] Figure 15 a to 15c ( Figure 15 a) Fluorescence images of SPN-MEHPPV at a concentration of 12.5 µg / mL (based on the mass of MEHPPV) at various NCBS doping levels (i.e., 0%, 1%, 2.5%, 5%, and 10%), excited at 465 ± 10 nm and emitted at 580 ± 10 nm or 780 ± 10 nm, acquired for 0.1 s; Figure 15 b) Normalized fluorescence spectra of SPN-MEHPPV in 1×PBS buffer (pH = 7.4) at various NCBS doping levels (i.e., 0%, 1%, 2.5%, 5%, and 10%). Excitation: 465 nm, and ( Figure 15 c) NCBS nanoparticles at a concentration of 0.625 µg / mL (based on the mass of NCBS) (precipitated with PEG- b -PPG- b Afterglow luminescence (left) and fluorescence (right) images of NCBS-PEG. Fluorescence images were acquired at 580±10 nm or 780±10 nm after excitation at 465±10 nm. Afterglow images were acquired under a bioluminescence model for 30 s. The nanoparticle solution was subjected to an 808 nm laser (1 W / cm²). 2 Pre-irradiate for 1 min, and then collect images for 5 s after removing the laser.

[0090] Figure 16 a and 16b show ( Figure 16 a) Afterglow luminescence of SPN-NCBS5 (12.5 µg / mL) after irradiation with an 808 nm laser at different power densities, and ( Figure 16 b) Through a power density of 1 W / cm 2 Afterglow emission of SPN-NCBS5 (12.5 µg / mL) under different illumination times by an 808 nm laser. Error bars represent the standard deviation of three independent measurements.

[0091] Figure 17 a and 17b show ( Figure 17a) Through 808 nm or 514 nm (1 W / cm²) 2 The fitting correction curve of the afterglow luminescence intensity of SPN-NCBS5 after laser irradiation as a function of concentration, and ( Figure 17 b) Fluorescence enhancement (F / F0) of SOSG (1 µM) at 528 nm with or without SPN-NCBS5 (1.25 µg / mL) as a function of exposure time to an 808 nm or 514 nm laser. Error bars represent the standard deviation of three independent measurements.

[0092] Figure 18 a to 18f show ( Figure 18 a) The chemical structure of meso-tetraphenylporphyrin (TPP); Figure 18 b) UV-Vis absorption of SPN-MEHPPV at various TPP doping levels in PBS buffer (pH = 7.4); Figure 18 c) Fluorescence (left) and afterglow luminescence (right) images of SPN-MEHPPV with various TPP doping levels in PBS buffer (pH = 7.4). Fluorescence images were acquired for 0.1 s, excited at 465 ± 10 nm and emitted at 580 ± 10 nm and 660 ± 10 nm. Bioluminescence images were acquired for 30 s at 580 ± 10 nm and 660 ± 10 nm. Fluorescence spectra of SPN-MEHPPV with various TPP doping levels in PBS buffer (pH = 7.4) are also shown. Figure 18 d) and afterglow emission spectrum ( Figure 18 e). Afterglow emission spectra were acquired for 30 s at each filter from 540 ± 10 nm to 840 ± 10 nm and analyzed by region of interest (ROI) analysis; Figure 18 f) Quantification of fluorescence and afterglow luminescence in SPN-MEHPPV at various TPP doping levels. Error bars represent the standard deviation of three independent measurements. Total fluorescence was obtained by integrating the area of ​​the fluorescence spectrum using Origin 9.0. Total afterglow luminescence was obtained using open filter and region of interest (ROI) analysis. For fluorescence spectra, based on MEHPPV composition, [SPN] = 2.5 µg / mL and excitation wavelength of 465 nm. For afterglow luminescence spectra, fluorescence and afterglow luminescence images were obtained based on MEHPPV composition, [SPN] = 62.5 µg / mL. All SPNs were irradiated with white light for 1 min (power: 0.1 W / cm²) before afterglow luminescence acquisition. 2 ).

[0093] Figure 19a to 19h show the SPN-MDMOPPV at various NCBS doping levels in PBS buffer (pH = 7.4). Figure 19 a) UV-Vis absorption spectrum, ( Figure 19 b) Fluorescence spectrum and ( Figure 19 c) Afterglow emission spectrum; Figure 19 d) Quantification of fluorescence intensity and afterglow luminescence intensity of SPN-MDMOPPV at various NCBS doping levels; in PBS buffer (pH = 7.4), the fluorescence intensity and afterglow luminescence intensity of SPN-MDMOPPV at various TPP doping levels. Figure 19 e) UV-Vis absorption spectrum, ( Figure 19 f) Fluorescence spectrum and ( Figure 19 g) Afterglow emission spectrum, and ( Figure 19 h) Quantification of fluorescence intensity and afterglow emission intensity of SPN-MDMOPPV with various TPP doping levels. Error bars represent the standard deviation of three independent measurements. Total fluorescence was obtained by integrating the area of ​​the fluorescence spectrum using Origin 9.0. Afterglow emission spectra were acquired for 30 s at each filter from 540 ± 10 nm to 840 ± 10 nm and then quantified by region of interest (ROI) analysis. Total afterglow emission was obtained with an open filter for 30 s and ROI analysis. For fluorescence spectra, based on the MDMOPPV composition, [SPN] = 3.3 µg / mL, with an excitation wavelength of 465 nm. For afterglow emission spectra, based on the MDMOPPV composition, [SPN] = 83.3 µg / mL. All SPNs were irradiated with white light for 1 min (power: 0.1 W / cm²) before afterglow emission acquisition. 2 ).

[0094] Figure 20 The cytotoxicity studies of SPN-MEHPPV and SPN-NCBS5 are shown. The in vitro viability of HeLa cells treated with SPN-MEHPPV and SPN-NCBS5 solutions at concentrations of 5, 10, 20, and 30 µg / mL for 24 h is presented. The percentage of viable cells after SPN treatment is calculated relative to cells treated with the same volume of saline (viability arbitrarily defined as 100%). Error bars represent the standard deviation of three independent measurements.

[0095] Figure 21 a and 21b show ( Figure 21 a) Afterglow luminescence imaging of chicken tissue of different thicknesses through SPN-NCBS5 solution pre-irradiated in situ at 808 nm (top) or 514 nm (bottom), and ( Figure 21 b) Variation of afterglow luminescence SBR with depth of chicken tissue. Error bars represent the standard deviation of three independent measurements.

[0096] Figure 22 a to 22e show ( Figure 22 a) at 808 nm (1 W / cm) 2 (Left) or (middle) after 514 nm in situ pre-irradiation, after which SPN-NCBS5 passes through a live mouse, and ( Figure 22 b) Fluorescence image excited at 465 nm (right); Figure 22 c) Figure 22 SBRs exhibiting afterglow luminescence and fluorescence in a&b. Figure 22 d) at 808 nm (1 W / cm) 2 The fitted correction curve of the afterglow luminescence intensity of SPN-NCBS5 in vivo through live mice after pre-irradiation as a function of nanoparticle concentration (LOD: 40 ng / mL), and ( Figure 22 e) Fitted calibration curve of in vivo fluorescence intensity of SPN-NCBS5 across live mice as a function of nanoparticle concentration (excitation: 710 nm, LOD: 8550 ng / mL). Error bars represent the standard deviation of three independent measurements.

[0097] Figure 23 a to 23d show ( Figure 23 a) SBR was calculated from in vivo afterglow luminescence images of mice with subcutaneous inclusions of SPN-NCBS5 (12.5 μg / mL, 50 μL); Figure 23 b) SBR of afterglow luminescence and fluorescence of SPN-NCBS5 (12.5 μg / mL, 50 μL) subcutaneous inclusions. SBR = [(afterglow signal, ROI 1) - (background 2, ROI 3)] / [(background 1, ROI 2) - (background 2, ROI 3)]; in vivo afterglow luminescence intensity of subcutaneous inclusions of nanoparticles ( Figure 23 c) and fluorescence intensity ( Figure 23 d) Variation with SPN-NCBS5 concentration. Error bars represent the standard deviation of three independent measurements. Figure 24 This shows the effect at 808 nm (power: 1 W / cm). 2 The afterglow intensity of SPN-NCBS (62.5 µg / mL) was measured over time after 1 min of irradiation and storage at 0°C or -20°C. Error bars represent the standard deviations of three independent measurements.

[0098] Figure 25The images show fluorescence (left) and afterglow emission (right) images of lymph nodes in live mice at t = 30 min after intradermal injection of SPN-NCBS5 (0.25 mg / mL, 0.05 mL) into the forepaw of mice. Fluorescence images were acquired at 780 ± 10 nm for 0.1 s after excitation at 465 ± 10 nm. Afterglow emission images were acquired for 180 s using an open filter without re-illumination.

[0099] Figure 26 a and 26b show ( Figure 26 a) At 808 nm (power: 1 W / cm) 2 The in vivo afterglow decay of SPN-NCBS5 was recorded 1 min after light irradiation, and ( Figure 26 b) The afterglow intensity of the subcutaneous inclusion SPN-NCBS5 as a function of the number of photoactivated cycles. Error bars represent the standard deviation of three independent measurements. Figure 27 The in vitro afterglow luminescence of major organs in mice is shown 48 h after systemic administration of SPN-NCBS5 (0.25 mg / mL, 0.2 mL) via tail vein injection. Values ​​are mean ± sd for n = 3 mice.

[0100] Figure 28 a and 28b show ( Figure 28 a) TEM image of SPN-thiol, and ( Figure 28 b) DLS of SPN-thiol in 1×PBS buffer (pH = 7.4). Error bars represent the standard deviation of three independent measurements.

[0101] Figure 29 a to 29d show ( Figure 29 a) Fluorescence spectra of SPN-thiol in 1×PBS buffer in the presence of different concentrations of Cys (pH = 7.4); Figure 29 b) The fluorescence intensity of SPN-thiol at 780 nm as a function of Cys concentration; Figure 29 c) The fluorescence intensity of SPN-thiol at 780 nm in 1×PBS at 37°C in the presence of Cys, Hcy, GSH and other amino acids (1 mM), and ( Figure 29 d) Fluorescence image of SPN-thiol (12.5 µg / mL) at 37 °C in the presence of Cys, Hcy, GSH, and other amino acids (1 mM). Error bars represent the standard deviation of three independent measurements. Figure 30 a and 30b show ( Figure 30a) Representative fluorescence images of mice treated with intraperitoneal injection of APAP (300 mg / kg), saline, or NAC (200 mg / kg) with APAP (300 mg / kg), followed by intravenous injection of SPN-thiol (0.25 mg / mL, 0.2 mL) at t = 20 min, and ( Figure 30 b) The ratios of fluorescence intensity and afterglow intensity in all groups (APAP-treated, untreated, and NAC / APAP-treated) to those in the untreated group. Values ​​are the mean ± sd for n = 3 mice. * There were statistically significant differences in fluorescence and afterglow intensity between the untreated and APAP-treated groups (n = 3, P < 0.05); ** There were no statistically significant differences between the untreated and APAP-treated groups and the APAP group with NAC repair (n = 3, P > 0.05). Figure 31 The in vitro afterglow luminescence of major organs in mice 3.5 h after systemic administration of SPN-thiol is shown. * There was a statistically significant difference in afterglow luminescence intensity between the untreated and APAP-treated groups (n = 3, P < 0.05); ** There was no statistically significant difference between the untreated and APAP-treated groups and the APAP group with NAC repair (n = 3, P > 0.05).

[0102] Figure 32 Representative histological and epithelial (H&E) findings of mouse livers at t = 3.5 h following treatment with APAP (300 mg / kg). Scale bar represents 50 μm.

[0103] Figure 33 a to 33e show ( Figure 33 a) Schematic diagram of SPN-MEHPPV degradation by myeloperoxidase (MPO); Figure 33 b) UV-Vis absorption spectra of SPN-NCBS5 (25 µg / mL) solutions in phosphate buffer (50 mM, pH = 7.0) containing NaCl (150 mM) before treatment (blank, left) and after treatment with 300 µM H2O2 alone (H2O2, middle) or with 300 µM H2O2 and 50 µg / mL MPO (H2O2 + MPO, right) at 37 °C for 8 h for four consecutive times; Figure 33c) White light images (top group), fluorescence images (middle group), and afterglow emission images (bottom group) of SPN-NCBS5 (25 µg / mL) treated for 8 h at 37°C in phosphate buffer (50 mM, pH = 7.0) containing NaCl (150 mM) and untreated (blank, left) or treated only with 300 µM H2O2 (H2O2, middle) or with 300 µM H2O2 and 50 µg / mL MPO (H2O2 + MPO, right). The nanoparticle solution was passed through an 808 nm laser (1 W / cm²) before the afterglow signal was collected. 2 Pre-irradiation for 1 min. Afterglow images were acquired under a bioluminescence model for 30 s. After excitation at 465 nm, fluorescence images were acquired at 780 nm for 0.1 s. Figure 33 d) Quantitative analysis of the afterglow intensity in c. Error bars are based on standard deviation (n = 3) and; Figure 33 e) Gel permeation chromatography (GPC) traces of nanoparticle inclusions. The freeze-dried sample was dissolved in THF solution for GPC testing. Wavelength: 500 nm.

[0104] Figure 34 a and 34b show ( Figure 34 a) Fluorescence images of live mouse livers at representative time points following systemic administration of SPN-NCBS5 (0.25 mg / mL, 0.2 mL) via tail vein injection. Fluorescence images were acquired at 780 nm for 0.1 s after excitation at 710 nm, and ( Figure 34 b) Quantitative analysis of NIR fluorescence changes over time in live mouse livers. Intensity values ​​are the mean ± SD of n = 3 mice. Error bars are based on standard deviation (n = 3 mice).

[0105] Figure 35 Representative histological (H&E) findings of mouse organs are shown 3 days after systemic administration of SPN-NCBS5 (0.25 mg / mL, 0.2 mL) or saline via tail vein injection. Scale bar represents 50 μm.

[0106] Figure 36 The synthetic routes for PPV-PEG1 and PPV-PEGL are shown. Reagents and conditions: (i) 1,10-dibromodecane, sodium methoxide, ethanol, reflux for 2 h. (ii) 2-ethylhexyl bromide, sodium methoxide, ethanol, reflux for 2 h. (iii) paraformaldehyde, HBr (33 wt% in acetic acid), acetic acid, 70°C, 4 h. (iv) potassium tert-butoxide, tetrahydrofuran (THF), 25°C, overnight. (v) sodium azide, THF / N,N-Dimethylformamide (DMF), 40°C, overnight. (vi)CuBr, N , N , N ', N '', N '''-Pentamethyldiethylenetriamine (PMDETA), PEG-acetylene, THF, 25°C, 48 h.

[0107] Figure 37 a to Figure 37 f illustrates a comparison of several properties between SPPVN and PPVP. Preparation ( Figure 37 a) SPPVN and ( Figure 37 b) Schematic diagram of PPVP; Figure 37 c) DLS of SPPVN and PPVP in 1×PBS buffer (pH = 7.4). Inset: Representative TEM images of SPPVN and PPVP. Scale bar represents 100 nm; DLS of SPPVN and PPVP in 1×PBS buffer (pH = 7.4). Figure 37 d) Absorption spectrum and ( Figure 37 e) Fluorescence spectrum. ( Figure 37 f) Afterglow emission spectra of SPPVN and PPVP at the same mass concentration (130 µg / mL). SPPVN and PPVP solutions were pre-irradiated with 514 nm light for 1 min before collecting the afterglow emission signal. The laser power used in the experiment was 1 W / cm². 2 The error bars represent the standard deviations of three independent measurements.

[0108] Figure 38 a through 38f illustrate in vivo tissue penetration studies and lymph node imaging. Figure 38 a) Fluorescence and afterglow images of SPPVN solution passing through live mice. Fluorescence images were acquired at 780 nm after excitation at 710 nm. Before collecting afterglow images, SPPVN solution (130 µg / mL, 50 µL) was excited at 514 or 808 nm (1 W / cm²). 2 ) for 1 min. ( Figure 38 b) Figure 38 SBR imaging of fluorescence and afterglow luminescence in a. Figure 38 c) Fluorescence and afterglow emission images of lymph nodes in live mice 60 min after intradermal injection of SPPVN (450 µg / mL, 50 µL) into the forepaw of mice. At 808 nm (0.3 W / cm²), fluorescence was observed. 2 After 1 minute of laser irradiation, an afterglow image was acquired for 30 seconds. Figure 38 d) Figure 38SBR imaging of fluorescence and afterglow luminescence in lymph nodes of type c. Figure 38 e) Fluorescence and afterglow images of the tumor and skin following local injection of SPPVN (130 µg / mL, 50 µL). Figure 38 f) Fluorescence intensity and afterglow intensity of tumor and skin following local injection of SPPVN (130 µg / mL, 50 µL). Error bars represent the standard deviation of three independent measurements (n = 3). ns.: not significant, ** statistically significant (p < 0.01, n = 3).

[0109] Figure 39 a to 39e illustrate in vivo tumor imaging. During intravenous injection ( Figure 39 a) SPPVN or ( Figure 39 b) Fluorescence and afterglow images of live mice at different time points following PPVP (450 µg / mL, 200 µL). Figure 39 c) Changes in SBR (Self-Brightness Ratio) of tumors in live mice after treatment with SPPVN or PPVP over time following injection. Figure 39 d) In vitro fluorescence quantification of major organs from mice 48 h after SPPVN or PPVP injection. Figure 39 e) In vitro fluorescence images of major mouse organs at 48 h post-SPPVN or PPVP injection. (Image taken at 808 nm, 0.3 W / cm²) 2 Afterglow emission images were acquired 30 s after 1 min of laser irradiation. Fluorescence images were acquired at 780 nm after excitation at 710 nm. Error bars represent the standard deviation of three independent measurements (n = 3). *Statistically significant differences were observed (p < 0.05, n = 3).

[0110] Figure 40 Images a through 40e show in vivo imaging of peritoneal metastatic tumors. Figure 40 a) A schematic diagram of the establishment of a metastatic 4T1 tumor model and imaging procedure. Figure 40 b) Changes in afterglow intensity in the lower quadrant of mice injected with SPPVN or PPVP over time. The injection doses of SPPVN and PPVP were 450 µg / mL and 200 µL, respectively. *Statistically significant differences were observed at t = 1.5 h (p < 0.01, n = 3). Figure 40 c) Fluorescence and afterglow images of mice after skin excision to expose the peritoneum 1.5 h following SPPVN or PPVP injection. Tumor areas are marked with white circles. Figure 40d) Afterglow intensity of tumor regions and background in mice marked in Figure (c) after being injected with SPPVN and PPVP. ns.: Not significant, ** statistically significant difference (p<0.01, n=3). Figure 40 e) Obtain H&E-stained sections and confocal images of peritoneal metastatic tumors from mice injected with SPPVN or PPVP. Tumor regions are marked with white boxes. Error bars represent the standard deviation of three independent measurements (n = 3).

[0111] Figure 41 Biodegradability and clearance rate studies were shown from day a to day 41. Figure 41 a) Schematic diagram of the degradation of PPV-PEGL in the presence of myeloperoxidase (MPO) and H2O2. Figure 41 b) Absorption spectra of PPV-PEGL solution (10 μg / mL) after treatment with MPO (40 μg / mL) and H2O2 (100 μM) for different times. Figure 41 c) Confocal fluorescence images of macrophages incubated with PPV-PEGL (30 μg / mL) and stimulated with LPS for different time periods. Macrophages were co-stained with Hoechst 33342. Figure 41 d) Quantification of fluorescence intensity over time in the livers of live mice injected with SPPVN (450 µg / mL, 200 µL). Fluorescence signals were acquired at 780 nm after excitation at 710 nm. Error bars represent the standard deviation of three independent measurements (n = 3).

[0112] Figure 42 a to 42f illustrate the mechanism of afterglow of PPV-PEG1. Figure 42 a) Representative DLS of PPV-PEG1. Inset: Representative TEM image of PPV-PEG1, scale bar represents 50 nm. Figure 42 b) Schematic diagram of fluorescence and afterglow luminescence of PPV-PEG1. Figure 42 c) Normalized absorption, fluorescence, and afterglow emission spectra of PPV-PEG1 in 1×PBS buffer (pH = 7.4). Figure 42 d) The fluorescence enhancement (F / F0) of SOSG (1 μM) at 528 nm with irradiation time at 514 nm, in the absence or presence of PPV-PEG1 (0.6 μg / mL). Figure 42e) Afterglow luminescence intensity and images of PPV-PEG1 (40 μg / mL) acquired at room temperature after O2 purging or in the presence of NaN3 (50 w / w%). **Statistically significant differences were observed (P < 0.01, n = 3, statistical significance calculated relative to the control). Figure 42 f) The proposed mechanism of afterglow luminescence of PPV-PEG1. Error bars represent the standard deviations of three independent measurements.

[0113] Figure 43 a to 43f show ( Figure 43 a) PPV-Br1 before and after 12 h of 514 nm light irradiation in CDCl3. 1 1H NMR spectrum. Figure 43 b) FTIR spectra of PPV-Br1 before and after 12 h of 514 nm light irradiation in THF. Absorption spectra of PPV-Br1 (12 μg / mL) before and after 12 h of 514 nm light irradiation in THF ( Figure 43 c) and fluorescence spectrum ( Figure 43 d). Absorption spectrum of PPV-PEG1 (12 μg / mL) in water before and after 12 h of 514 nm light irradiation ( Figure 43 e) and fluorescence spectrum ( Figure 43 f).

[0114] Figure 44 a to 44c show ( Figure 44 a) Absorption spectra of PPV-PEG1 in 1×PBS (pH = 7.4) at various NCBS doping levels (w / w %); Figure 44 b) Fluorescence spectra of PPV-PEG1 in 1×PBS (pH = 7.4) at various NCBS doping levels, and ( Figure 44 c) DLS of PPV-PEG1 in 1×PBS (pH = 7.4) at various NCBS doping levels. Error bars represent the standard deviation of three individual measurements.

[0115] Figure 45 a to 45f show the effects of doped photosensitizers on the physical and optical properties of nanoparticles. Figure 45 a) DLS of NCBS-doped PPV-PEGL with different NCBS doping levels in 1×PBS buffer (pH = 7.4). Absorption spectra of NCBS-doped PPV-PEGL with different NCBS doping levels in 1×PBS buffer (pH = 7.4). Figure 45 b) Fluorescence spectroscopy ( Figure 45 c) and afterglow emission spectrum ( Figure 45 d). ( Figure 45e) Quantitative analysis of the absolute fluorescence and afterglow luminescence intensity of NCBS-doped PPV-PEGL (70 μg / mL) with different NCBS doping concentrations. Figure 45 f) Decrease in afterglow luminescence of NCBS-doped PPV-PEGL (130 μg / mL) at room temperature. The SPPVN solution was pre-irradiated with 514 nm light for 1 min before collecting the afterglow luminescence signal. The optical power used in the experiment was 1 W / cm². 2 The error bars represent the standard deviations of three independent measurements.

[0116] Figure 46 a to 46d show the NIR afterglow amplified by the photosensitizer. Figure 46 a) Afterglow emission images of NCBS-doped PPV-PEGL (130 μg / mL) pre-irradiated with 514 or 808 nm. Figure 46 b) The ratio of the afterglow luminescence intensity of NCBS-doped PPV-PEGL pre-irradiated with NCBS at 808 nm to that of NCBS-doped PPV-PEGL pre-irradiated with 514 nm under different NCBS doping concentrations (I 808 / I 514 ). Figure 46 c) The fluorescence enhancement (F / F0) of SOSG with irradiation time via 808 or 514 nm laser in the presence of SPPVN at 528 nm. Figure 46 d) A schematic diagram illustrating the proposed mechanism of afterglow luminescence in NCBS-doped PPV-PEGL pre-irradiated with 514 or 808 nm lasers. The laser power used in the experiment was 1 W / cm². 2 . Figure 47 a to 47c show ( Figure 47 a) Average diameters of SPPVT and SPPVN in 1×PBS (pH = 7.4); absorption spectra of SPPVT (200 μg / mL) with TPP doping levels of 0% and 2% (w / w %). Figure 47 b) Fluorescence spectroscopy ( Figure 47 c) and afterglow emission spectrum ( Figure 47 d). The error bars represent the standard deviations of three individual measurements.

[0117] Figure 48 a and 48b show ( Figure 48 a) Changes in DLS levels of PPV-PEG1 and SPPVN with incubation time in 1×PBS (pH = 7.4). Figure 48 b) Cell viability of HeLa cells after incubation with various concentrations of SPPVN. Error bars represent the standard deviation of three individual measurements.

[0118] Figure 49 a to 49c show ( Figure 49 a) In vivo afterglow decay of SPPVN (130 μg / mL, 50 μL) recorded 1 min after irradiation with 808 nm laser light; Figure 49 b) The change in afterglow luminescence intensity of subcutaneously injected SPPVN (130 μg / mL, 50 μL) with the number of light irradiation cycles. The laser power used in the experiment was 0.3 W / cm². 2 ,as well as( Figure 49 c) SBR was calculated from in vivo afterglow luminescence images of mice with subcutaneous inclusions of SPPVN (130 μg / mL, 50 μL). Error bars represent the standard deviation of three independent measurements (n = 3).

[0119] Figure 50 a to 50c show sizes of 1 mm. 3 In vivo imaging of the tumor. Figure 50 a) Fluorescence and afterglow luminescence images of tumors in live mice at different time points following intravenous injection of SPPVN (450 μg / mL, 200 μL). Figure 50 b) Changes in the SBR of tumor fluorescence and afterglow luminescence imaging in live mice over time after injection. Figure 50 c) Quantitative fluorescence analysis of major organs in mice 48 h after intravenous injection of SPPVN. Error bars represent the standard deviation of three independent measurements (n = 3). The fluorescence was measured using an 808 nm laser (0.3 W / cm²). 2 After 1 min of irradiation, afterglow emission images were acquired for 30 s. Fluorescence images were acquired at 780 nm after excitation at 710 nm.

[0120] Figure 51 a and 51b show the treatment with SPPVN (450 μg / mL, 200 μL) to carry 5 and 1 mm cells. 3 The liver of mice with tumors ( Figure 51 a) Fluorescence intensity and ( Figure 51 b) Changes in afterglow intensity over time after injection.

[0121] Figure 52 It was shown that 48 h after systemic administration of SPPVN or PPVP, there were 5 or 1 mm 3 Ex vivo fluorescence images of major organs in mice with tumors.

[0122] Figure 53 a and 53b show ( Figure 53a) Confocal fluorescence images of multicellular tumor spheroids (MCTS) incubated with SPPVN or PPVP for 12 h. The fluorescence of SPPVN and PPVP was adjusted to be identical before incubation. Figure 53 b) Quantitative analysis of fluorescence intensity in multicellular tumor spheroids incubated with SPPVN and PPVP. **Statistically significant differences were observed (p<0.01, n=3).

[0123] Figure 54 The intestines excised from peritoneal metastatic tumor-bearing mice injected with SPPVN or PPVP are shown for H&E staining and confocal imaging.

[0124] Figure 55 Fluorescence images of live mice at different time points following systemic administration of SPPVN (450 μg / mL, 200 μL) are shown.

[0125] Figure 56 a through 56e illustrate the synthesis and characterization of SPN-PPV-TPP. Figure 56 a) PPV, PPV-TPP 2.5% and PPV-TPP 5% Synthetic route. Reagents and conditions: i) tris(dibenzylacetone)dipalladium(O)[Pd2(dba)3], tris(p-tolyl)phosphine (TP), chlorobenzene, 100℃, 24 h. Figure 56 (b) Schematic diagram of the preparation of SPN-PPV-TPP. Figure 56 c) DLS of SPN2.5 in 1×PBS buffer (pH = 7.4). Figure 56 d) TEM image of SPN2.5. Scale bar represents 100 nm. Figure 56 e) Cell viability of 4T1 cells after incubation with SPN2.5 solutions of various concentrations.

[0126] Figure 57 a through 57f illustrate the optical properties of SPN-PPV-TPP. Figure 57 a) Normalized UV-Vis absorption spectrum of SPN-PPV-TPP. Figure 57 b) Fluorescence spectra of SPN-PPV-TPP. In 1× PBS (pH = 7.4), the concentration of the PPV component of both SPs was 30 μg / mL. Figure 57 c) Afterglow emission spectrum of SPN-PPV-TPP (100 µg / mL). The SPN-PPV-TPP solution was pre-irradiated with white light for 1 min before collecting the afterglow emission signal. Error bars represent the standard deviation of three independent measurements. Figure 57d) Fluorescence image (top) and afterglow emission image (bottom) of SPN-PPV-TPP in 1×PBS (pH = 7.4). Fluorescence images were acquired after excitation at 430 nm and emission at 720 nm. The fluorescence was measured under white light at 1 W / cm². 2 After pre-irradiating SPN-PPV-TPP with a power density of 1 min, afterglow images were acquired for 30 s. Figure 57 e) Quantification of fluorescence and afterglow intensity of SPN-PPV-TPP. Error bars represent the standard deviation of three independent measurements. Figure 57 f) Normalized afterglow decay of SPN-PPV-TPP (100 µg / mL) at room temperature. The SPN-PPV-TPP solution was pre-irradiated with white light for 1 min before collecting the afterglow emission signal. The optical power used in the experiment was 1 W / cm². 2 .

[0127] Figure 58 a and 58b show in vivo imaging of tumor hypoxia. Figure 58 a) Fluorescence and afterglow luminescence images of the tumor and skin following local injection of SPN2.5 (100 µg / mL, 50 µL). Figure 58 b) Fluorescence and afterglow intensity of tumor and skin following local injection of SPPVN (130 µg / mL, 50 µL). Error bars represent the standard deviation of three independent measurements (n = 3). ns: not significant, ** statistically significant (p < 0.01, n = 3).

[0128] Figure 59 Images a through 59e show in vivo imaging of peritoneal metastatic tumors. Figure 59 a) Fluorescence and afterglow luminescence images of peritoneal metastatic tumors in live mice at different time points following intravenous injection of SPN2.5 (400 µg / mL, 200 µL). Liver sites are marked with black circles. Tumor sites are marked with white boxes. Figure 59 b) Fluorescence and afterglow luminescence images of mice whose skin was removed to expose the peritoneum 4 h after SPN2.5 injection. Tumor areas are marked by white circles. Figure 59 c) Figure 4 The afterglow intensity of the white-framed region in mice injected with SPN2.5 (400 µg / mL, 200 µL) varies with time post-injection. Error bars represent the standard deviation of three independent measurements (n = 3). Figure 59 d) H&E-stained sections of peritoneal metastatic tumors obtained from mice injected with SPN2.5. Tumor areas are marked by black dashed lines.

[0129] Figure 60DLS data for SPN0 and SPN5 incubated with PBS (pH = 7.4) are shown. The PDI for SPN0 is 0.31. The PDI for SPN5 is 0.346.

[0130] Figure 61 The DLS data for SPN2.5 are shown as a function of incubation time with PBS (pH = 7.4) and FBS.

[0131] Figure 62 The fluorescence enhancement (F / F0) of SOSG (1 µM) at 528 nm with white light irradiation time is shown in the absence or presence of SPN0, SPN2.5 and SPN5 (0.8 µg / mL).

[0132] Figure 63 The afterglow luminescence intensity and images of SPN2.5 (50 µg / mL) acquired at room temperature after purging with O2, N2 or in the presence of NaN3 (50 w / w%) are shown. Detailed Implementation

[0133] This article discloses the production and application of semiconductor polymer nanoparticles (SPN) as afterglow luminescence probes for molecular imaging in live mice.

[0134] The SPNs disclosed in this paper are constructed from optically active semiconductor polymers (SPs) and represent an alternative class of photonic nanomaterials. They are entirely organic and contain bio-benign components to overcome the toxicity caused by metal ions. Irradiation of PPV-based SPNs leads to the formation of unstable chemical defects (dioxetane units), which can spontaneously and slowly decompose to release photons that induce afterglow luminescence. Although the mechanism controlling the afterglow luminescence of PPV-based SPNs is similar to chemiluminescence, it does not require an exogenous ROS to trigger the reaction. Instead, the SPNs themselves can generate singlet oxygen upon light irradiation (…). 1 O2), which then causes afterglow luminescence. This afterglow mechanism also differs from that of rare-earth-doped inorganic nanoparticles, where the energy of the photons absorbed is stored in the intrinsic defect lattice rather than in photoinduced chemical defects.

[0135] Therefore, this paper discloses polymer composite nanoparticles that emit near-infrared afterglow luminescence, the nanoparticles comprising: (a) Semiconductor polymers of formula I:

[0136] (b) Optionally, an amphiphilic copolymer; and (c) Optionally, a small molecule dye having near-infrared emission, wherein: Amphiphilic copolymers (when present) encapsulating semiconductor polymers of formula I and small molecule dyes (when present); and In the polymer of formula I: R1 to R3 and R5 independently represent formula C q H 2q+1 Alkyl chains, wherein 1 ≤ q ≤ 50, R4 represents part of formula Ia or formula Ib:

[0137] Where 1≤s≤50 and 10≤t≤500;

[0138] Where 1≤u≤50 and 10≤v≤500; R6 represents C q H 2q+1 The alkyl chain, a portion of formula Ia or a portion of formula Ib, wherein q, s, t, u and v are as defined above; R7 represents the singlet oxygen sensitization region; Each of n, m, and o is greater than or equal to 0, and p is 0 or 1, wherein at least one of n, m, o, and p is greater than 0; A represents the part of Ic or Id:

[0139]

[0140] Among them, R8 and R 11 Independently represented by C q H 2q+1 alkyl chains, wherein: 1 ≤ q ≤ 50; R9 and R 12 Independently represented by C q H 2q+1 The alkyl chain, a portion of formula Ia or a portion of formula Ib, wherein q, s, t, u and v are as defined above; R 10 and R 13 It independently represents the singlet oxygen sensitization portion; When p is 1, then w, x, y, and z (if they exist) are independently greater than or equal to 0; and Small molecule dyes are present when o and p are 0, and are optionally present when: o is greater than or equal to 20; p is 1 and x or z is greater than or equal to 20; The sum of o and x is greater than or equal to 20; The sum of o and z is greater than or equal to 20; (o+x) / (n+m+o+w+x)>0.05; or (o+z) / (n+m+o+y+z)>0.05; Amphiphilic copolymers exist when m, o, and p are 0, and optionally exist when: (m+o+w) / (n+m+o+w+x)>0.1; or (m+o+y) / (n+m+o+y+z)>0.1; and The premise is that when n is greater than 0, one or more of m, o and p are also greater than 0.

[0141] It should be understood that the aforementioned composite nanoparticles cover the following situations: (aa) All three components (a), (b) and (c) exist; (ab) Only components (a) and (b) exist; (ac) contains only components (a) and (c); and (ad) Only component (a) exists.

[0142] In the presence of all three components (a), (b), and (c), an amphiphilic copolymer (component (b)) encapsulates a semiconductor polymer of formulation I (component (a)) and a small molecule dye (component (c)). When used herein, the term "encapsulation" means that other materials are completely trapped within the polymer matrix of the encapsulating material.

[0143] In the presence of only components (a) and (b), the amphiphilic copolymer (component (b)) encapsulates the semiconductor polymer of formula I (component (a)). As described above, a small molecule dye may not be required when: (o+x) / (n+m+o+w+x)>0.05; or (o+z) / (n+m+o+y+z)>0.05, or more specifically, p is 1 and x or z is greater than or equal to 20, or more specifically, o is greater than or equal to 20; the sum of o and x is greater than or equal to 20; or the sum of o and z is greater than or equal to 20. It should be understood that the reason why a small molecule dye can be excluded under the above conditions is because the semiconductor polymer of formula I is doped with an equivalent group that provides the same functionality (i.e., the singlet oxygen-sensitized portion). Therefore, in some embodiments, a small molecule dye is not present when any of the foregoing conditions are met.

[0144] In the presence of only components (a) and (c), the semiconductor polymer of Formula I (component (a)) encapsulates the small molecule dye (component (c)). As described above, when (m+o+w) / (n+m+o+w+x)>0.1 or (m+o+y) / (n+m+o+y+z)>0.1, the amphiphilic copolymer may not be required. In other embodiments, the amphiphilic copolymer may not be required when: m is greater than or equal to 20 and m / (n+m+o) is greater than 0.1 and R 6 C q H 2q+1 Where 1≤q≤50; m is greater than or equal to 20, and (m+o) / (x+y+z) is greater than 0.1, and R 6 It is a part of formula Ia or a part of formula Ib; or o is greater than or equal to 20, and (m+o) / (x+y+z) is greater than 0.1, and R 6 It is a part of formula Ia or a part of formula Ib. In some embodiments, when any of the foregoing conditions are met, there is no amphiphilic copolymer.

[0145] It should be understood that the above conditions regarding the absence of components (b) and (c) also apply to the case where only (a) is present. That is, when any combination of the above conditions regarding the absence of both the amphiphilic copolymer and the small molecule dye is satisfied, it may result in the absence of both components.

[0146] Preferred embodiments of the present invention include those listed above with (ac), more particularly (ab), and even more particularly (aa). In other words, preferred embodiments are those in which an amphiphilic copolymer is present.

[0147] In view of the foregoing, it should also be understood that the present invention also relates to semiconductor polymers of Formula I:

[0148] In the polymer of formula I: R1 to R3 and R5 independently represent formula C q H 2q+1 alkyl chains; wherein, 1≤q≤50, R4 represents part of formula Ia or formula Ib: Where 1≤s≤50 and 10≤t≤500;

[0149] Where 1≤u≤50 and 10≤v≤500;

[0150] R6 represents C q H 2q+1 The alkyl chain, a portion of formula Ia or a portion of formula Ib, wherein q, s, t, u and v are as defined above; R7 represents the singlet oxygen sensitization region; Each of n, m, and o is greater than or equal to 0; p is 0 or 1; A represents the part of Ic or Id:

[0151]

[0152] Among them, R8 and R 11 Independently represented by C q H 2q+1 alkyl chains, wherein: 1 ≤ q ≤ 50; R9 and R 12 Independently represented by C q H 2q+1 The alkyl chain, a portion of formula Ia or a portion of formula Ib, wherein q, s, t, u and v are as defined above; R 10 and R 11 It independently represents the singlet oxygen sensitization portion; When p is 1, w, x, y, and z (if they exist) are independently greater than or equal to 0; The premise is: When n is greater than 0, one or more of m, o, and p are also greater than 0; and At least one of n, m, o, and p is greater than 0.

[0153] In the embodiments described herein, the term "comprising / including" can be interpreted as requiring the mentioned features but not limiting the presence of other features. Alternatively, the term "comprising" can also refer to situations where only the listed components / features are present (e.g., the term "comprising / including" can be replaced by the phrases "consisting of" or "substantially consisting of"). It will be clearly understood that both broader and narrower interpretations can be applied to all aspects and embodiments of the invention. In other words, the term "comprising / including" and its synonyms can be replaced by the phrases "consisting of" or "substantially consisting of" or their synonyms, and vice versa.

[0154] Unless otherwise expressly stated, references to embodiments of the semiconductor polymer of Formula I may refer equally to the polymer itself or to the polymer as part of a composite material comprising one or more amphiphilic copolymers and small molecule dyes having near-infrared emission.

[0155] Unless otherwise stated, the term "alkyl" refers to a saturated, unbranched or branched acyclic hydrocarbon group. The alkyl group can be any C... 1-50 Alkyl groups, and more preferably C 1-10 Alkyl (such as ethyl, propyl (e.g., n-propyl or isopropyl), pentyl, or more particularly butyl (e.g., branched or unbranched butyl), octyl (e.g., unbranched or more particularly branched octyl (e.g., 2-ethylhexyl)) or methyl).

[0156] When used herein, the term “r” is used to indicate that the semiconductor polymer of Formula I is a random copolymer when two or more of n, m, o and p are greater than 0.

[0157] In some embodiments of the present invention, n and / or p can be 0.

[0158] In some embodiments of the invention, when one or more of n, m, o, and p are not zero, each of n, m, o, w, x, y, and z can independently have a value from 5 to 1000, such as 10 to 750, such as 15 to 500, such as 20 to 250, such as 50 to 100. When a list of ranges is provided herein, any technically reasonable combination of the provided endpoint values ​​can be used to provide other ranges. For example, other ranges explicitly included in the above list include: 5 to 10, 5 to 15, 5 to 20, 5 to 50, 5 to 100, 5 to 250, 5 to 500 and 5 to 750; 10 to 15, 10 to 20, 10 to 50, 10 to 100, 10 to 250, 10 to 500 and 10 to 1000; 15 to 20, 15 to 50, 15 to 100, 15 to 250, 15 to 750 and 5 to 1000; 20 to 50, 20 to 100, 20 to 500, 20 to 750, and 20 to 1000; 50 to 250, 50 to 500, 50 to 750, and 50 to 1000; 100 to 250, 100 to 500, 100 to 750, and 100 to 1000; 250 to 500, 250 to 750 and 250 to 1000; 500 to 750 and 500 to 1000; and 750 to 1000.

[0159] It should be understood that this will be clearly thought of for other combinations of the same type listed in the range.

[0160] In embodiments of the invention, the number-average molecular weight of the polymer of Formula I can be from 1,000 to 300,000 Daltons, such as from 1,000 to 100,000 Daltons, such as from 1,500 to 50,000 Daltons, such as from 25,000 to 75,000 Daltons.

[0161] When used herein, the term "singlet oxygen sensitization moiety" refers to any suitable organic or organometallic moiety capable of converting oxygen in the triplet state to one of its singlet states, and can be incorporated into the types of organic polymers disclosed herein. More particularly, the singlet oxygen sensitization moiety can be a photosensitive moiety. Suitable photosensitive compounds that can be incorporated as singlet oxygen sensitization moieties into polymers of Formula I are disclosed at DeRosa, MC and Crutchley, RJ. Photosensitized singlet oxygen and its applications , Coordination Chemistry Reviews 233 / 234 (2002) 351 / 371, specifically on pages 354–359 of the aforementioned review article, which is incorporated herein by reference. More specifically, R7, R 10 and R 13 The singlet oxygen-sensitized moiety can be independently selected from one or more of the following groups: metalloporphyrins, metal phthalocyanines, naphthyl phthalocyanines, metal-naphthyl phthalocyanines, dihydroporphyrin, rhodamine, anthocyanins, carotenoids, anthocyanins, Bengal rose red, methylene blue, and more particularly, 2,3-naphthyl phthalocyanine bis(trihexylsiloxy)silane, porphyrins (octaethylporphyrin, tetraphenylporphyrin), phthalocyanines, tetrapyrroles, transition metal complexes (Ir(III) complexes, Ru(II) complexes, Pt(II) complexes). And Os(II) complexes) and boron-dipyrrole methylene (BODIPY)-based photosensitizers (e.g., the group may consist of one or more of the following: 2,3-naphthylphthalocyanine bis(trihexylsiloxy)silane, porphyrin (octaethylporphyrin, tetraphenylporphyrin), phthalocyanine, tetrapyrrole, transition metal complexes (Ir(III) complexes, Ru(II) complexes, Pt(II) complexes and Os(II) complexes) and boron-dipyrrole methylene (BODIPY)-based photosensitizers).

[0162] Similarly, when used herein, the term "small molecule dye with near-infrared emission" refers to any suitable organic or organometallic molecule capable of converting oxygen in its triplet state to one of its singlet states, as an entity independent of the semiconductor polymer of Formula I. More specifically, small molecule dyes with near-infrared emission can be photosensitive moieties. Suitable photosensitive compounds are disclosed in DeRosa, MC and Crutchley, RJ. Photosensitized singlet oxygen and its applications , Coordination Chemistry Reviews233 / 234 (2002)351 / 371, specifically on pages 354–359 of the aforementioned review article, which is incorporated herein by reference. More particularly, small molecule dyes exhibiting near-infrared emission may be selected from one or more of the group consisting of: metalloporphyrins, metal phthalocyanines, naphthyl phthalocyanines, metal-naphthyl phthalocyanines, dihydroporphyrins, rhodamine, anthocyanins, carotenoids, anthocyanins, Bengal rose, methylene blue, and more particularly, 2,3-naphthyl phthalocyanine bis(trihexylsiloxy)silanes, porphyrins (octaethylporphyrin, tetraphenylporphyrin), phthalocyanines, tetrapyrroles, transition metal complexes (Ir(III) complexes, Ru(II) complexes, Pt(II) complexes). The group can consist of one or more of the following: 2,3-naphthylphthalocyanine bis(trihexylsiloxy)silane, porphyrin (octaethylporphyrin, tetraphenylporphyrin), phthalocyanine, tetrapyrrole, transition metal complexes (Ir(III) complexes, Ru(II) complexes, Pt(II) complexes and Os(II) complexes) and boron-dipyrrole methylene (BODIPY) photosensitizers.

[0163] In embodiments of the present invention, the polymer of Formula I may be selected from the following polymers: (i) Wherein, n and m are as defined above, optionally, wherein the number of n and m repeating units provides a polymer having a number average molecular weight of 25,000 to 200,000 Daltons, such as 45,000 to 150,000 Daltons, such as 50,000 to 100,000 Daltons, such as about 59,781 Daltons, and / or the molar ratio of n repeating units in the polymer is about 88%, and the molar ratio of m repeating units in the polymer is about 11% (e.g., the molar ratio of n repeating units in the polymer is 88.0% to 89.0%, and the molar ratio of m repeating units in the polymer is 11.0% to 12.0%). (ii) Wherein, m is as defined above, optionally, the number of m repeating units provides a polymer having a number average molecular weight of 15,000 to 100,000 Daltons, such as 20,000 to 75,000 Daltons, such as 20,000 to 50,000 Daltons, such as about 26,565 Daltons. (iii) , Wherein, p is 1, and the number of y and z repeating units provides that the polymer has a number average molecular weight of 5,000 to 20,000 Daltons, such as 7,000 to 18,000 Daltons, such as 8,900 to 15,000 Daltons, such as about 13,000 Daltons, and / or the molar ratio of y repeating units in the polymer is 85 to 99%, and the molar ratio of z repeating units in the polymer is 1 to 15% (e.g., the molar ratio of y repeating units in the polymer is 90.0 to 95.0%, and the molar ratio of z repeating units in the polymer is 5.0 to 10.0%).

[0164] When used herein, the term "amphiphilic copolymer" refers to a polymeric material (e.g., a random copolymer or more particularly a block copolymer) containing at least two monomeric units, wherein the resulting polymer has one or more inherently hydrophobic portions and one or more inherently hydrophilic portions. Examples of suitable amphiphilic copolymers include, but are not limited to, one or more of the following: alkyl-substituted chitosans, and more particularly poly(alkyl)- b -Poly(ethylene glycol), Poly(ethylene glycol)- b -Poly(propylene glycol)- b - Poly(ethylene glycol), poly(ethylene glycol) methyl ether-block-poly(lactide-co-glycolic acid) (PEG-PLGA), poly(styrene)-block-poly(acrylic acid) (PS-PAA), poly(styrene-co-maleic anhydride) (PSMA), 1,2-dispalmitoyl-sn-glycerol-3-phosphocholine (DPPC), and 1,2-distearate-sn-glycerol-3-phosphoethanolamine-poly(ethylene glycol) (DSPE-PEG). Any amphiphilic copolymer of suitable molecular weight can be used. For example, amphiphilic copolymers can have a number average molecular weight of 1,000 to 50,000 Daltons, such as 1,500 to 40,000 Daltons, such as 5,000 to 25,000 Daltons, such as 4,000 to 20,000 Daltons.

[0165] In some embodiments of the invention, the amphiphilic copolymer may also include a quenching portion that can be cleaved by the reactive portion at the in vitro or in vivo test site. When used herein, the term "quenching portion" is intended to mean any portion capable of absorbing excitation energy and re-emitting it as another form of energy, not light. Examples of suitable quenching portions include dark quenchers, including but not limited to dabsyl (dimethylaminoazobenzenesulfonic acid), black hole quenchers, Qxl quenchers, Iowa Black RQ, Iowa Black RQ, IRDye QC-1, and 2,4-dinitrophenylsulfonyl portions. Suitable dark quenchers include black hole quenchers (BHQ)-1, BHQ-2, BHQ-3, and QSY-7, and 2,4-dinitrophenylsulfonyl (DNBS) portions.

[0166] As described above, the quenching portion is attached to the amphiphilic copolymer in such a way that, under certain conditions present at the test / imaging site, it can be removed upon exposure to the reactive portion. In other words, the quenching portion comprises both a quenching group and a linking group, which incorporates the reactive group and can be cleaved under certain conditions. Examples of suitable quenching portions (with the linker incorporated) include, but are not limited to, thiol-sensitive portions, such as the 2,4-dinitrobenzenesulfonyl (DNBS) portion, and... ; Reactive oxygen species sensitive components and ; pH-sensitive components, such as , , , , and ; In the peptide quencher portion, there are quenchers such as furin-sensitive Arg-Arg-Val-Arg- quenchers, Caspase-3-sensitive Asp-Val-Glu-Asp- quenchers, fibroblast activator protein-α (FAPα)-sensitive Gly-Pro- quenchers, matrix metalloproteinase 2 (MMP-2)-sensitive Gly-Arg-Val-Gly-Leu-Pro- quenchers, MMP-7-sensitive Gly-Met-Trp-Ser-Leu-Pro-Val- quenchers, and MMP-13-sensitive Leu-Gly-Arg-Met- Gly-Leu-Pro quencher, cathepsin B-sensitive Lys-lys quencher, cathepsin D-sensitive Leu-Arg-Phe-Phe-Cys-Ile-Pro quencher, cathepsin S-sensitive Arg-Leu quencher, urokinase-sensitive Arg-Gly quencher, and Legumain-sensitive Asn-Ala-Ala quencher, wherein the quencher is a dark quencher (e.g., as defined above).

[0167] In a particular embodiment of the invention in which the amphiphilic copolymer further includes a quenching portion, the amphiphilic copolymer may be selected from one or more of the following: (C 18 PEG 12 -DNBS), , , ,as well as For example, an amphiphilic copolymer including a quenching portion can be C 18 PEG 12 -DNBS.

[0168] Therefore, in a specific embodiment of the present invention, the amphiphilic copolymer is selected from one or more of the following: poly(ethylene glycol)- b -Poly(propylene glycol)- b - Poly(ethylene glycol) (e.g., PEG) 100 -b-(PPG) 65 -b-(PEG) 100 ), 1,2-distearyl-sn-glycerol-3-phosphate ethanolamine-poly(ethylene glycol) (DSPE-PEG) and .

[0169] It should be understood that polymers of Formula I and polymer composite nanoparticles as defined herein are capable of emitting NIR afterglow luminescence. Therefore, composite materials and / or polymers of Formula I can be used in afterglow imaging techniques. With this in mind, the following is provided: (ia) The use of polymer composite nanoparticles as defined above in the preparation of imaging agents for the diagnosis of lesions or diseases in deep tissues and / or organs using afterglow luminescence; (ib) A method for in vivo imaging in deep tissues and / or organs using polymer composite nanoparticles as defined above, the method comprising irradiating the polymer composite nanoparticles with an NIR laser before or after delivery of the polymer composite nanoparticles to a living organism (e.g., subcutaneous, intradermal, or intravenous injection of the polymer composite nanoparticles into a living organism), and detecting afterglow luminescence using an imaging system / device; and (ic) Polymer composite nanoparticles as defined above are used as imaging agents for diagnosing conditions or diseases in deep tissues and / or organs using afterglow luminescence.

[0170] It should be understood that the imaging mentioned in (ia) and (ic) can be external or, more specifically, internal.

[0171] When used herein, with reference to the mode of use, the term "polymer composite nanoparticle" may refer to the nanoparticles described in items (aa) through (ad) above. That is, polymer composite nanoparticles may comprise only the semiconductor polymer of Formula I, or additionally one or both of an amphiphilic copolymer and a small molecule dye. In specific embodiments mentioned herein, in addition to the polymer of Formula I, polymer composite nanoparticles may also comprise at least one of an amphiphilic copolymer and a small molecule dye, and preferably contain both components. In a preferred embodiment of the invention, polymer composite nanoparticles may be those in which an amphiphilic copolymer is present and which also include a quenched portion that can be cleaved by the reactive portion at the in vitro or in vivo test site, as described in detail above.

[0172] When used herein, the term "deep" in relation to tissues and / or organs refers to tissues and / or organs with an imaging depth greater than 2 cm. Organs and / or tissues that may be mentioned herein include, but are not limited to, the brain, lungs, liver, stomach, intestines, kidneys, and bladder.

[0173] The term "organism" organism )","organism( organisms )","patient( patient ")" and "patient ( patients The term "subject" includes mammalian (e.g., human) patients. As used herein, the terms "subject" or "patient" are well known in the art and are used interchangeably herein to refer to mammals, including dogs, cats, rats, mice, monkeys, cattle, horses, goats, sheep, pigs, camels, and most preferably humans. In some embodiments, a subject is a subject in need of treatment or a subject suffering from a disease or ailment. However, in other embodiments, a subject may be a healthy subject. The term does not indicate a specific age or sex. Therefore, it is intended to cover adult and juvenile subjects, whether male or female.

[0174] When used herein, the term “near-infrared” or “NIR” refers to a wavelength of 700 to 1,400 nm, such as between 700 and 1,400 nm. When used herein, “NIR laser” may refer to a laser having a wavelength of 700 to 1,400 nm, such as 750 to 1,000 nm, such as 800 to 900 nm, such as 808 nm.

[0175] The term "effective amount" refers to the amount of a compound that imparts a therapeutic effect to the treated patient (e.g., sufficient to treat or prevent disease). The effect can be objective (i.e., measurable by some test or marker) or subjective (i.e., the subject gives instructions or feels the effect).

[0176] Polymer composite nanoparticles can be administered in pharmaceutically acceptable dosage forms, comprising compounds, via any suitable route, but specifically via: oral, intravenous, intramuscular, skin, subcutaneous, mucosal (e.g., sublingual or buccal), rectal, transdermal, nasal, pulmonary (e.g., trachea or bronchus), topical, or any other parenteral route. Specific routes of administration that may be mentioned include subcutaneous, intradermal, or intravenous administration. In alternative embodiments (e.g., during surgery), administration may be by spraying the composition containing the polymer composite nanoparticles onto the site of interest of the subject.

[0177] Polymer composite nanoparticles are typically administered as drug formulations mixed with pharmaceutically acceptable adjuvants, diluents, or carriers, which can be appropriately selected considering the intended route of administration and standard pharmaceutical practice. These pharmaceutically acceptable carriers can be chemically inert to the active compound and may not cause harmful side effects or toxicity under the conditions of use. Suitable drug formulations can be developed, for example, by Remington. The Science and Practice of Pharmacy The following can be found: , 19th ed., Mack Printing Company, Easton, Pennsylvania (1995). For parenteral administration, parenteral-acceptable aqueous solutions can be used, which are pyrogen-free and have the necessary pH, isotonicity, and stability. Suitable solutions are well known to those skilled in the art, and many methods are described in the literature. A brief overview of drug delivery methods can also be found, for example, in Langer, Science (1990) 249 The same principle found in 1527 can also be widely applied to this invention.

[0178] In addition, the preparation of suitable formulations can be routinely carried out by technicians using conventional techniques and / or according to standards and / or recognized pharmaceutical practices.

[0179] The amount of polymer composite nanoparticle formulation used according to the present invention will depend on various factors, such as the size of the tissue and / or organ to be imaged, the specific patient, and the nanoparticles used. In any case, the amount of polymer composite nanoparticles in the formulation can be routinely determined by a technician.

[0180] For example, solid oral compositions (such as tablets or capsules) may contain 1 to 99% (w / w) of polymeric composite nanoparticles; 0 to 99% (w / w) of diluents or fillers; 0 to 20% (w / w) of disintegrants; 0 to 5% (w / w) of lubricants; 0 to 5% (w / w) of glidants; 0 to 50% (w / w) of granulators or binders; 0 to 5% (w / w) of antioxidants; and 0 to 5% (w / w) of colorants.

[0181] Parenteral preparations (such as solutions or suspensions for injection or solutions for infusion) may contain 1 to 50% (w / w) of polymeric composite nanoparticles; and 50% (w / w) to 99% (w / w) of a liquid or semi-solid carrier or solvent (e.g., a solvent such as water); and 0 to 20% (w / w) of one or more other excipients, such as buffers, antioxidants, suspension stabilizers, tension modifiers, and preservatives.

[0182] Depending on the organ / tissue to be imaged, the patient to be treated, and the route of administration, polymeric composite nanoparticles can be administered to subjects in need at varying doses. When used herein, the term "dose" is intended to refer to the amount of polymeric composite nanoparticles delivered to the organism / subject to provide the desired image. It is not intended to imply any therapeutic efficacy.

[0183] However, in the context of this invention, the dosage administered to mammals, particularly humans, should be sufficient to achieve imaging of organs and / or tissues in mammals within a reasonable timeframe. Those skilled in the art will recognize that, among other things, the selection of the exact dosage and composition, as well as the most suitable delivery method, will also be influenced by: the pharmacological properties of the formulation, the nature of the organ and / or tissue to be imaged, and the recipient's physical condition and mental acuity, the subject's age, illness, weight, sex, and the stage / severity of the disease that may affect said organ / tissue.

[0184] Administration can be continuous or intermittent (e.g., by bolus injection). The dosage can also be determined by the timing and frequency of administration. In the case of oral or parenteral administration, the dose of the polymer composite nanoparticles according to the invention per imaging cycle can vary between about 0.01 mg and about 1000 mg.

[0185] In any case, a physician or other technician can routinely determine the actual dose that will be most suitable for an individual subject. The doses described above are examples of average cases; of course, higher or lower dose ranges may be available in individual instances, and this is also within the scope of the invention.

[0186] The nanoparticles according to the invention can be administered in any suitable formulation via any suitable route of administration (e.g., subcutaneous, intradermal, or intravenous injection into a living subject (e.g., mice)). As described above, activation of the polymer composite nanoparticles can be completed after administration and delivery to the site of action or, in some cases, before. Pre-administration activation can be accomplished by pre-irradiating the composition with an NIR laser (e.g., an 808 nm laser) for an appropriate period of time. If the pre-irradiated nanoparticles lose afterglow illumination before the imaging test is completed, they can be reactivated by irradiating the site to be imaged for an appropriate period of time (e.g., for mice, with an 808 nm laser for 1 minute) before continuing to acquire afterglow emission images. In the case of nanoparticles supplied in an inactive state, they can be activated by pre-irradiating the site to be imaged for an appropriate period of time (e.g., for mice, with an 808 nm laser for 1 minute) before starting to acquire afterglow emission images.

[0187] It should be understood that when polymeric composite nanoparticles include a quenching portion as part of an amphiphilic copolymer, a pre-activation step is not required because the activation energy provided is absorbed by the quencher. In this case, there is no pre-activation before administration; instead, initial activation occurs in vivo once the polymeric composite nanoparticles reach the site of interest. It should be noted that a relevant advantage of polymeric composite nanoparticles including a quenching portion as part of an amphiphilic copolymer is that the tissue and / or organ to be imaged can be the only tissue and / or organ exhibiting afterglow luminescence. More specifically, tissues and / or organs exhibiting afterglow luminescence may only exhibit afterglow luminescence if they suffer from a disease or condition that causes rapid breakage of the linking groups (as defined above) in the quenching portion. Such diseases and conditions can include cancer / tumor or oxidative stress in the liver.

[0188] In a typical instance, when the subject is a mouse, afterglow images can be acquired using the IVIS Spectrum imaging system at different time points (e.g., t = 0.5, 1, 2, 4, 8, 12, 24, 36, and 48 h post-injection), where the polymer composite nanoparticles are (re)activated as needed. It should be understood that the timing and frequency of imaging can vary depending on the subject's organ and / or tissue being imaged. As discussed in more detail below in the Examples section, once afterglow imaging has occurred, the intensity of the afterglow is analyzed using a t-test, and guiding diagnostic results are summarized.

[0189] One specific application of the aforementioned imaging techniques is the localization of lymph nodes and / or visualization of tumors. Tumors that may be mentioned herein include, but are not limited to, breast tumors, lung tumors, and liver tumors. It should be understood that these tissues / organs can exhibit enhanced metabolic activity, thereby enabling the rapid removal of the quenching portion described in certain embodiments of the invention (i.e., polymer composite nanoparticles in which the amphiphilic copolymer includes a quencher portion). Therefore, in a particular embodiment of the invention, when the polymer composite nanoparticles comprise an amphiphilic copolymer containing a quencher, imaging techniques can be performed concurrently with surgery on a subject (e.g., to remove a tumor). In this case, the polymer composite nanoparticles can be sprayed directly onto the site of interest, or delivered to the site of action as described above, to determine whether all diseased organs / tissues have been removed during the surgical procedure.

[0190] Finally, when the composite nanoparticle polymer composite nanoparticles include an amphiphilic copolymer, the amphiphilic copolymer also includes a quenching portion as defined above that can be cleaved by the reactive portion at the in vitro or in vivo test site (e.g., the amphiphilic copolymer can be C...). 18 -PEG 12When DNBS is used, the resulting material is particularly suitable for determining oxidative stress in the liver of subjects. Therefore, the following is provided: (iia) Polymer composite nanoparticles as defined above in the first aspect of the invention (wherein, the amphiphilic copolymer further includes a quenching portion as defined above that can be partially cleaved by the reactive portion at the in vitro or in vivo test site (e.g., the amphiphilic copolymer may be C 18 -PEG 12 -DNBS) is used in a method for preparing an imaging agent for in vivo imaging of oxidative stress in the liver of a subject, the method comprising the steps of: supplying polymer composite nanoparticles into a living organism, irradiating the polymer composite nanoparticles with an NIR laser, and detecting afterglow luminescence in the liver using an imaging system / device. (ib) Using polymer composite nanoparticles as defined above in the first aspect of the invention (wherein, the amphiphilic copolymer further includes a quenching portion as defined above that can be partially cleaved by the reactive portion at the in vitro or in vivo test site (e.g., the amphiphilic copolymer may be C 18 -PEG 12 -DNBS) is a method for in vivo imaging of oxidative stress in the liver of subjects, comprising the steps of: intravenously injecting polymer composite nanoparticles into a living organism; irradiating the polymer composite nanoparticles with an NIR laser; and then detecting afterglow luminescence in the liver using an imaging system / device. In various embodiments of the invention, the afterglow luminescence of the injected polymer nanoparticles can be reactivated in vivo by subjecting a living organism or a portion of a living organism to NIR laser irradiation after being injected subcutaneously, intradermally, or intravenously with the polymer composite nanoparticles; and (ic) Polymer composite nanoparticles as defined above in the first aspect of the invention, wherein the amphiphilic copolymer further includes a quenching portion as defined above that can be partially cleaved by the reactive portion at the in vitro or in vivo test site (e.g., the amphiphilic copolymer may be C 18 -PEG 12 -DNBS), used as an imaging agent, is employed to determine oxidative stress in the liver of a subject using afterglow luminescence. Examples of such tests performed in mice are provided in the Examples section.

[0191] Although oxidative stress can be any oxidative stress experienced by the liver of a subject, it can be particularly useful in determining drug-induced hepatotoxicity.

[0192] Other aspects and embodiments of the present invention and disclosure are provided through the following non-limiting examples.

[0193] Example The invention will be further described in conjunction with the following embodiments, which are presented for illustrative purposes only.

[0194] experiment Materials and methods Chemicals and other materials Unless otherwise stated, all chemicals used in the experiments were purchased from Sigma-Aldrich. Poly(2,5-dioctyl-1,4-phenylenevinylene) (POPPV), poly[(9,9'-dioctylfluorenyl-2,7-diyl)-alt(benzo[2,1,3]thiadiazole-4,7-(diyl)] (PFBT), poly(5-(2-ethylhexyloxy)-2-methoxycyano-terephthalimide) (MEHCPV), xylyl-terminated poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylene] (MEHPP), and poly[2,5-bisoctyloxy]-1,4-phenylenevinylene] (BOPPV) were purchased from Luminescence Technology Corp. Paraformaldehyde was purchased from VWR Singapore Pte Ltd. 1,10-dibromodecane and 2-ethylhexyl bromide were purchased from TCI Ltd. Dialysis membranes with a 3 kDa MWCO were purchased from Spectrum Labs.

[0195] Instruments and characterization Proton nuclear magnetic resonance was recorded using CDCl3 or D2O as solvents via Bruker Avance II 300 MHz NMR. 1 ¹H NMR spectra. Spectra were obtained with the tetramethylsilane signal at 0 ppm as an internal reference. Afterglow signals and images were acquired using an IVIS Spectrum imaging system in bioluminescent (unexcited) mode. Fourier transform infrared (FT-IR) spectra were obtained using a Nicolet 8700 FT-IR spectrometer. GPC results were measured using a Shimadzu LC-VP system with polystyrene as the standard and THF as the eluent. Dynamic light scattering (DLS) measurements were performed on a Malvern Nano-ZS particle size analyzer. Transmission electron microscopy (TEM) images were captured from a JEM 1400 TEM with accelerating voltages ranging from 40 to 120 kV.

[0196] Absorption spectra were recorded on a Shimadzu UV-2450 spectrophotometer. Fluorescence measurements were performed on a Fluorolog 3-TCSPC spectrofluorometer (Horiba Jobin Yvon). Confocal fluorescence images of the cells were obtained using an LSM510 confocal laser scanning microscope (Carl Zeiss, Germany) with an excitation wavelength of 488 nm.

[0197] Fluorescence and afterglow emission images were acquired using the IVIS Spectrum imaging system. 514 nm laser excitation was obtained using a green laser with a 514 nm filter (Stellar-Pro ML / 150, Modu-Laser, Centerville, UT, USA). Unless otherwise specified, an 808 nm high-power NIR laser (operating mode: CW, fiber-backed output power: 2.5 W, LED display: diode current, multimode fiber, fiber core diameter: 400 μm, fiber connector: SMA905, with adjustable laser drive module: 0) was used. 100%, laser spot size: 1 cm 2 Irradiate the sample or other irradiated areas for 1 minute to produce afterglow luminescence.

[0198] For in vitro imaging of nanoparticles, unless otherwise specified, fluorescence images are acquired for 0.1 s by excitation at 465±10 nm or 430±10 nm and emission at 580±10 nm or 780±10 nm. The intensity of the fluorescence image is calculated by integrating over the area. In some experiments, emission is at 520±20 nm or 720±20 nm. For in vivo experiments, fluorescence imaging is performed for 0.1 s by excitation at 465±10 nm or 710±10 nm and emission at 780±10 nm. For afterglow imaging, unless otherwise specified, a power density of 1 W / cm² is used for in vitro experiments. 2 The power density for in vivo experiments was 0.3 W / cm². 2 The sample was pre-irradiated with 514 nm or 808 nm for 1 min. For in vivo experiments, the laser output was equipped with a concave lens and positioned approximately 10 cm above the mouse, allowing the output laser to cover the entire body of the mouse. In vitro acquisition of afterglow emission images was performed using an open filter or a specific emission filter for 0.1 s. In vivo acquisition of afterglow emission images was performed using an open filter for 30 s.

[0199] Biological experiments Lymph node imaging For SPN-NCBS5: To balance sufficient replication of results with reduced mouse numbers, the experimental size was 3 mice per treatment. All mouse images were included in the analysis. Cages of mice were randomly selected for the following treatment. A solution of SPN-NCBS5 (0.25 mg / mL, 0.05 mL) was irradiated and stored at -20°C for one day. Immediately afterwards, warm SPN-NCBS5 was administered intradermally to the forepaw of live mice anesthetized with oxygen containing 2% isoflurane. Afterglow and fluorescence images were collected at t = 30 min post-injection. At t = 65 min post-injection, images were obtained by lasering at 1 W / cm² using an 808 nm laser. 2 Mice were irradiated with a power density of 1 min. Afterglow luminescence and fluorescence images were then collected again at 70, 100, and 130 min post-injection using darkness. During the imaging process, the mice were warmed with a heating pad under continuous isoflurane anesthesia.

[0200] For SPPVN: SPPVN (450 μg / mL, 0.05 mL) was administered intradermally to the forepaw of live mice anesthetized with oxygen containing 2% isoflurane. At t = 60 min post-injection, mice were irradiated with an 808 nm laser for 1 min, and afterglow and fluorescence images were acquired. Afterglow images were collected using an open filter for 30 s. Fluorescence images were acquired for 0.1 s by excitation at 710 ± 10 nm and emission at 780 ± 10 nm.

[0201] Tumor mouse model All animal experiments were conducted in accordance with guidelines established by the Sing Health Animal Care and Use Committee (IACUC).

[0202] To establish tumors in eight-week-old BALB / c mice, HeLa cells (3-5 × 10⁶ cells per mouse) were used. 6 Each mouse was given 0.1 mL of DMEM supplemented medium (1 mL, 10% FBS, 1% penicillin / streptomycin antibiotic) and injected subcutaneously into the right shoulder. Prior to imaging experiments, tumors were allowed to grow to a single aspect ratio of 6. 8 mm (approximately 10-15 days).

[0203] To establish a tumor-bearing mouse model, 4T1 cell suspension (200 μL, 1×10⁻⁶ cells) was prepared. 6Subcutaneous injection was administered to the left shoulder of nude mice. The tumor was allowed to grow for approximately 7 days prior to imaging experiments. To establish a small-sized tumor-bearing mouse model for sensitive tumor imaging, two million 4T1 cells suspended in 50 ml of matrix gel in a 50% v / v mixture supplemented with DMEM (10% FBS, 1% penicillin / strep (100 U / mL penicillin and 100 μg / mL streptomycin)) were subcutaneously injected into the shoulder of mice to establish a tumor model in six-week-old female nu / nu mice. The tumor-bearing mice were then divided into two groups of three mice each. For one group, the tumor was allowed to grow to approximately 2 mm in a single aspect prior to in vivo imaging experiments. For the other group, the tumor was allowed to grow to approximately 1 mm in a single aspect prior to use.

[0204] Tumor volume is calculated as follows: volume = (½) 2 In the equation, D represents the maximum diameter of the tumor, while d represents the minimum diameter of the tumor.

[0205] To establish a mouse model of peritoneal metastasis, 4T1 cell suspension (100-200 μL, 2-4 × 10⁻⁴ cells) was prepared. 5 The tumor was injected intraperitoneally into nude mice. The tumor was allowed to grow for approximately 3-4 days prior to imaging experiments.

[0206] Animal and tumor imaging For SPN-NCBS5: To balance sufficient replication of results with reduced mouse numbers, the experimental size was 3 mice per treatment. All mouse images are included in the analysis. Cages of tumor-bearing mice were randomly selected for the following treatment. SPN-NCBS5 (0.25 mg / mL, 0.2 mL) was administered systematically via tail vein (n = 3). Afterglow and fluorescence images were acquired at t = 0.5, 1, 2, 4, 8, 12, 24, 36, and 48 h post-injection. Fluorescence images were captured with an acquisition time of 0.1 s, excited at 710 ± 10 nm and emitted at 780 ± 10 nm. Before acquiring afterglow images, a power density of 0.5 W / cm² was used. 2 Mice were irradiated with an 808 nm laser for 1 min (the output power of the 808 nm high-power NIR laser was adjusted to 5.5 W and used to irradiate the whole body of the mice at a distance of 15 cm). Afterglow images were captured using an open filter with an acquisition time of 180 s.

[0207] For SPPVN or PPVP: SPPVN or PPVP (450 μg / mL, 200 μL) was systematically injected via tail vein into 4T1 tumor-bearing mice with varying tumor volumes. Afterglow and fluorescence images were then acquired at different time points post-injection. Fluorescence images were acquired for 0.1 s by excitation at 710 ± 10 nm and emission at 780 ± 10 nm. Mice were irradiated with an 808 nm laser for 1 min before capturing afterglow images. Afterglow images were then acquired using an open filter for 30 s. For in vitro biodistribution studies, mice were euthanized by CO2 asphyxiation, and tumors, liver, spleen, intestine, kidney, lung, and heart were collected for fluorescence imaging to assess the tissue distribution of SPPVN or PPVP.

[0208] In vivo imaging of drug-induced hepatotoxicity To balance adequate replication of results with reduced mouse numbers, the experimental group size was 3 mice per treatment. At this sample size, the large difference in projected signal of drug-induced hepatotoxicity ensured sufficient efficacy (derived from G*Power analysis: d = 4.2, α = 0.003, efficacy = 0.90) [e.g., see Liu, F. et al., Sci Rep-Uk 3 (2013)]. All mouse images were included in the analysis. Mice were fasted for 8 hours prior to imaging for all drug-induced hepatotoxicity. Cages of mice were randomly selected for the following treatments. Mice were treated with APAP (300 mg / kg), saline, or N-acetyl-L-cysteine ​​(NAC, 200 mg / kg) via intraperitoneal injection, followed by APAP (300 mg / kg) treatment. After 20 min, nude mice treated with APAP, saline, and NAC / APAP were anesthetized with oxygen containing 2% isoflurane and then systematically injected with SPN-thiol (0.25 mg / mL, 0.2 mL) via the tail vein. Fluorescence and afterglow images were collected 2 h after SPN-thiol injection. Fluorescence images were captured with an excitation at 710 ± 10 nm and an emission at 780 ± 10 nm, with an acquisition time of 0.1 s. Before acquiring afterglow images, the mice were treated with an 808 nm laser at 1 W / cm². 2 Mice were irradiated with an 808 nm laser at a power density of 1 W / cm² for 1 min. Afterglow images were captured with an acquisition time of 180 s and an open filter. To determine the biodistribution of SPN-thiol, mice were euthanized 4 h later. The heart, lungs, liver, kidneys, and spleen were removed and placed on black paper. 2All organs were pre-irradiated for 1 min, and afterglow emission images were acquired for 180 s. The afterglow emission intensity of each individual organ was analyzed using ROI analysis with Living Image 4.0 software.

[0209] Cell culture and cytotoxicity assay HeLa cervical adenocarcinoma epithelial cells were purchased from the American Type Culture Collection (ATCC). HeLa cells were cultured in DMEM (GIBCO) with 10% FBS (GIBCO) at a humid environment of 37°C containing 5% CO2 and 95% air. HeLa cells were then seeded into 96-well plates (200 μL per well, 5000 cells) and cultured for 24 h. SPPVN solutions (final concentrations: 0, 5, 10, 20, and 40 μg / mL) were then added to the wells. Cells were then incubated for another 24 h, followed by the addition of MTS (100 μL, 0.1 mg / mL) for another 4 h. The absorbance of MTS was measured at 490 nm using a microplate reader. Cell viability was calculated as the ratio of the absorbance of cells incubated with SPPVN to that of cells incubated with cell culture medium only.

[0210] In vitro cytotoxicity was measured in the 4T1 cell line using the [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazonium (MTS) viability assay. 4T1 cells were cultured in DMEM containing 10% FBS at 37°C under a humidified environment of 5% CO2 and 95% air. 4T1 cells were then cultured at a rate of 3 × 10⁻⁶ cells / year. 4 Cells were seeded at a concentration of 10 cells / mL into 96-well plates (Costar, IL, USA). After 24 h of incubation, the medium was replaced with fresh medium containing different concentrations (0, 2.5, 5, 10, 20, 30 μg / mL) of SPN-PPV-TPP suspension, and the cells were then incubated for another 24 h. At specified time intervals, MTS reagent was added to the cell medium at a volume ratio of 1 to 10 for cell incubation. After 3 h, UV measurements (490 nm) were performed in an incubator, and the results were normalized for untreated samples to obtain cell viability.

[0211] Confocal fluorescence imaging of multicellular tumor spheroids (MCTS) To generate MCTS, a poly(hydroxyethyl methacrylate) (PHEMA) membrane was coated onto the bottom of a tissue culture flask (T25). PHEMA (450 mg) was dissolved in 30 mL of 95% ethanol, and the mixture was slowly shaken at 37°C for 24 h. After the PHEMA was completely dissolved, 4 mL of the solution was added to the tissue culture flask. The flask was then dried at 37°C for 48 h. For sterilization, the PHEMA-coated flask must be exposed to UV light for 1 h before use. 4T1 monolayer cells were digested with trypsin to ensure a single-cell suspension, and the cell count was performed using a hemocytometer. 5 × 10⁵ cells were added to 5 mL of fresh DMEM medium. 5 4T1 cells were placed in PHEMA-coated flasks. The cells were incubated at 37°C in a humidified atmosphere containing 5% CO2, with the culture medium replaced every other day. 4T1 MCTS (approximately 400 μm in diameter) spontaneously formed within about 7 days. The uptake of nanoparticles by the MCTS was observed using confocal laser scanning microscopy (CLSM). For each experiment, approximately 20 4T1 MCTS were manually examined using a Pasteur pipette and then transferred to 5 mL eppendorf tubes. SPPVN or PPVP (25 μg / mL) was added to the MCTS suspension, and the cells were co-cultured at 37°C for 12 h. The culture medium was then removed, and the MCTS were washed with PBS (pH = 7.4) and observed using CLSM.

[0212] In vitro biodegradability study of PPV-PEGL PPV-PEGL solution (10 μg / mL) was treated with H2O2 (100 μM) and MPO (40 μg / mL) in phosphate buffer (50 mM, pH = 7.0) containing NaCl (150 mM). Due to enzyme activity loss, H2O2 and MPO were replenished three times after every 36 h of incubation. RAW264.7 macrophage cells were used for in vitro biodegradation studies. RAW264.7 cells were purchased from ATCC. Cells were cultured in DMEM supplemented with 10% FBS and penicillin / streptomycin antibiotics (1%) at a humid environment of 37°C containing 5% CO2 and 95% air. After seeding cells into imaging dishes, cells were cultured for 12 h in medium containing PPV-PEGL (30 μg / mL). Cells were stimulated with medium containing lipopolysaccharide (LPS) (1 μg / mL) for 0, 4, and 12 h. Prior to imaging, cells were stained with Hoechst 33342 (NucBlue Live ReadyProbes reagent) and fixed with 4% paraformaldehyde. Confocal microscopy images of the cells were obtained using CLSM. In vivo tissue penetration of fluorescence and NIR afterglow luminescence For fluorescence imaging, a solution of SPPVN (50 μL, 130 μg / mL) was placed under the abdomen of a live mouse. Fluorescence images were acquired for 0.1 s by excitation at 710 nm and emission at 780 nm. For afterglow imaging, the SPPVN (50 μL, 130 μg / mL) solution was pre-irradiated with a 514 or 808 nm laser for 1 min, and then the solution was placed under the abdomen of a live mouse. Afterglow images were acquired for 30 s using an open filter.

[0213] In vivo imaging for differentiating between hypoxic and normoxic environments Prior to injection, the SPPVN solution was purged with N2 for 5 min to remove oxygen. 4T1 tumor-bearing mice were treated with the purged SPPVN (130 μg / mL, 5 μL) via intratumoral and subcutaneous injections, respectively. Fluorescence images were acquired for 0.1 s by excitation at 710 ± 10 nm and emission at 780 ± 10 nm. Mice were irradiated with an 808 nm laser for 1 min before capturing afterglow images. Afterglow images were acquired using an open filter with an acquisition time of 30 s.

[0214] Following anesthesia, 50 μL of SPN2.5 (100 μg / mL, oxygen removed by purging the solution with nitrogen) was injected orally into the pre-implanted tumor in the left shoulder of nude mice (n = 2). The same SPN2.5 solution was subcutaneously injected into the right shoulder of these mice. Fluorescence images of the mice were acquired at 720 nm after excitation at 500 nm. After pre-irradiation with white light for 1 min, afterglow images were acquired using an open filter with an acquisition time of 30 s.

[0215] In vivo peritoneal metastatic tumor imaging For SPPVN or PPVP: SPPVN or PPVP (450 μg / mL, 200 μL) was systematically injected into peritoneally metastatic 4T1 tumor-bearing mice via tail vein. Afterglow and fluorescence images were then acquired at different time points post-injection. 1.5 h post-injection, the skin and peritoneum of the injected mice were excised, and afterglow and fluorescence images in the lower quadrant region of the mice were acquired. Fluorescence images were acquired for 0.1 s by excitation at 710 ± 10 nm and emission at 780 ± 10 nm. Mice were irradiated with an 808 nm laser for 1 min before capturing afterglow images. Afterglow images were then acquired using an open filter with an acquisition time of 30 s.

[0216] SPN2.5: SPN2.5 (400 μg / mL, 200 μL) was intravenously injected into peritoneal metastatic 4T1 tumor-bearing mice. Fluorescence images of the mice were acquired at 720 nm after excitation at 500 nm at different time points post-injection (0 h, 20 min, 40 min, 1 h, 2 h, 4 h), followed by afterglow images acquired using an open filter for 30 s after 1 min of white light irradiation. The mice were euthanized 4 h post-injection, and fluorescence and afterglow images of organs and tumors after removal of skin and peritoneum were acquired.

[0217] SPPVN clearance rate SPPVN (450 μg / mL, 20 μL) was systematically injected into mice via the tail vein. Fluorescence images of the mice were captured at t = 0 h, 1 h, 2 h, 4 h, 6 h, 1 day, 2 day, 4 day, 6 day, 10 day, 13 day, 16 day, and 20 days post-injection. Fluorescence images were acquired at a collection time of 0.1 s by excitation at 710 ± 10 nm and emission at 780 ± 10 nm.

[0218] Histological analysis To confirm peritoneal metastatic tumor cells, mice were euthanized and tumor (tissue) was harvested and fixed in 4% paraformaldehyde. The tumor (tissue) was then embedded in paraffin S15 and cut into 10 µm thick sections for hematoxylin and eosin (H&E) staining according to standard protocols. Images of the stained sections were captured using a Nikon ECLIPSE 80i microscope (Nikon Corporation, Towa Optics, New Delhi, India).

[0219] Data Analysis Fluorescence and afterglow images were quantified using ROI analysis with Living Image 4.0 software. Unless otherwise stated, results are expressed as mean ± SD bias. Statistical comparisons between two groups were determined using Student's t-test. For all tests, p < 0.05 was considered statistically significant. All statistical calculations were performed using GraphPad Prismv.6 (GraphPad Software Inc., CA, USA).

[0220] Preparation of components and / or intermediates Preparation 1: Synthesis of propargyl-terminated poly(ethylene glycol) methyl ether (M n = 2000)(PEG-acetylene) Gathering under the ice bath ( Ethylene glycol ) Methyl ether ((M) n = 2000 g / mol) (1.0 g, 0.5 mmol) was dissolved in anhydrous THF (20 mL), and then sodium hydride (24 mg, 1 mmol) was added. The resulting mixture was stirred at 0 °C for 1 h. Subsequently, propargyl bromide (80 wt%, 110 μL) from toluene was added to the mixture, and the reaction was carried out at room temperature for 24 h. The mixture was then filtered to remove any precipitate. The resulting solution was concentrated and precipitated in excess diethyl ether to obtain a white solid. The solid was dissolved in water and dialyzed with water to remove impurities. The product was obtained after lyophilization. 1 H NMR (300 MHz, CDCl3,δ): 4.20 (d, 2H), 3.65 (s, 165H), 3.38 (s, 3H), 2.44 (t, 1H).

[0221] Preparation 2: Synthesis of 1-(10-bromodecoxy)-4-methoxybenzene (Compound 2) 4-Methoxyphenol (0.5 g, 4 mmol) and sodium methoxide (0.24 g, 4.4 mmol) were dissolved in ethanol (10 mL) and stirred at ambient temperature for 10 min. 1,10-Dibromodecane (6 g, 20 mmol) was added to the solution, and the reaction was carried out under reflux for 2 h. After cooling the solution to room temperature, water (50 mL) was added to dilute the solution. The resulting solution was then extracted with diethyl ether (50 mL × 3), and the combined organic phases were dried over anhydrous sodium sulfate overnight. After removing the diethyl ether, the crude product was purified by column chromatography using petroleum ether / dichloromethane (DCM) (100:0 to 100:25) as the eluent to obtain the product (separation yield: 92.4%). 1 H NMR (300 MHz, CDCl3, δ): 6.84 (s, 4H), 3.91 (t, 2H), 3.78 (s, 3H), 3.42 (t, 2H), 1.86 (m, 2H), 1.76 (m, 2H), 1.51-1.39 (m, 4H), 1.37-1.27(m, 8H).

[0222] Preparation 3: Synthesis of 1-(2-ethylhexyloxy)-4-methoxybenzene (Compound 3) 4-Methoxyphenol (0.5 g, 4 mmol) and sodium methoxide (0.24 g, 4.4 mmol) were dissolved in ethanol (10 mL) and stirred at ambient temperature for 10 min. 2-Ethylhexyl bromide (0.96 g, 5 mmol) was added to the solution, and the reaction was carried out under reflux for 2 h. After cooling the solution to room temperature, water (50 mL) was added to dilute the solution. The resulting solution was extracted with diethyl ether (50 mL × 3), and the combined organic phases were dried over anhydrous sodium sulfate overnight. After removing the diethyl ether, the crude product was purified by column chromatography using petroleum ether / DCM (100:0 to 100:25) as the eluent to obtain the product (separation yield: 87.5%). 1 H NMR (300 MHz, CDCl3, δ): 6.84 (s, 4H), 3.78 (s, 5H), 1.71 (m, 1H), 1.56-1.36 (m, 4H), 1.36-1.25 (m, 4H), 0.97-0.84 (m, 6H).

[0223] Preparation 4: Synthesis of 1-(10-bromodecoxy)-2,5-bis(bromomethyl)-4-methoxybenzene (Compound 4) Compound 2 (0.5 g, 1.5 mmol), paraformaldehyde (0.21 g), and hydrobromic acid (33 wt%, 0.7 mL) from acetic acid were added to acetic acid (2.8 mL). The reaction was carried out at 70 °C under a nitrogen atmosphere for 4 h. Then, DCM (40 mL) was added to the solution, and the organic phase was washed successively with water (30 mL × 2), saturated sodium bicarbonate (30 mL), and brine (30 mL). The organic phase was dried over anhydrous sodium sulfate overnight. After removing DCM, the crude product was purified by column chromatography using petroleum ether / DCM (100:0 to 100:20) as the eluent to obtain the product (separation yield: 91.1%). 1 H NMR (300 MHz, CDCl3, δ): 6.86 (s, 2H), 4.54 (s, 4H), 4.00 (t, 2H), 3.87 (s, 3H), 3.42 (t, 2H), 1.86 (m, 4H), 1.54-1.29 (m, 12H).

[0224] Preparation 5: Synthesis of 1-(2-ethylhexyloxy)-2,5-bis(bromomethyl)-4-methoxybenzene (Compound 5) Compound 3 (0.34 g, 1.4 mmol), paraformaldehyde (0.21 g), and hydrobromic acid (33 wt%, 0.7 mL) from acetic acid were added to acetic acid (2.8 mL). The reaction was carried out at 70 °C under a nitrogen atmosphere for 4 h. DCM (40 mL) was then added to the solution, and the organic phase was washed successively with water (30 mL × 2), saturated sodium bicarbonate (30 mL), and brine (30 mL). The organic phase was dried over anhydrous sodium sulfate overnight. After removing DCM, the crude product was purified by column chromatography using petroleum ether / DCM (100:0 to 100:20) as the eluent to obtain the product (separation yield: 93.6%). 1 H NMR (300 MHz, CDCl3, δ): 6.86 (s, 2H), 4.53 (s, 4H), 3.87 (s, 5H), 3.87 (s, 3H), 1.71 (m, 1H), 1.56-1.36 (m, 4H), 1.41-1.29 (m, 4H), 0.96-0.79 (m, 6H).

[0225] Preparation 6: Synthesis of C 18 PEG 12 -DNBS

[0226] Dodecyl polyethylene glycol octadecyl etheramine (C 18 -PEG 12-NH2 (80 mg, 0.1 mmol) and Et3N (27.5 µL, 0.2 mmol) were dissolved in anhydrous CH2Cl2. 2,4-Dinitrobenzenesulfonyl chloride (53 mg, 0.2 mmol) was added dropwise to the above solution at 0 °C, and the solution was then stirred under nitrogen at room temperature for 8 h. After the reaction, the mixture was diluted with CH2Cl2 and washed with water. The organic solvent was removed under reduced pressure, and the reaction mixture was purified by column chromatography (CH3OH / CH2Cl2, 1:8) to give pure product C. 18 PEG 12 -DNBS (103 mg, 80%). C 18 PEG 12 - DNBS quality: C 48 H 90 N3O 18 The calculated value of S, [(M+H)] + [C]: 1028.59, Measured value (obsvd.) ESI / MS: 1028.18. 18 PEG 12 -DNBS 1 H NMR (300 MHz, CDCl3) δ (ppm): 8.52 (dd, J1= 8.4 Hz, J2= 2.1 Hz, 1 H), 8.35 (d, J = 8.4 Hz, 1H), 6.66 (t, 1 H), 3.61 (m, 51 H), 3.45 – 3.55 (m, 8 H), 3.42 (t, 2 H), 3.33 (m, 2H), 1.54 (t, 2 H), 1.22 (m, 47 H), 0.84 (m, 3 H). C 18 PEG 12 -DNBS 13 C NMR (75 MHz, CDCl3) δ (ppm): 149.61, 147.97, 139.80, 132.54, 126.92, 120.54, 71.56, 70.55, 70.42, 70. 34, 70.04, 69.12, 43.79, 31.92, 29.69, 29.65, 29.50, 29.35, 26.09, 22.68 and 14.11.

[0227] Preparation 7: Synthesis of PPV-Br1 and PPV-BrL Compound 4 (50 mg for PPV-Br1, 5 mg for PPV-BrL) and compound 5 (0 mg for PPV-Br1, 50 mg for PPV-BrL) were dissolved in anhydrous THF (5 mL) under a nitrogen atmosphere. A solution of potassium tert-butoxide in THF (1 M, 0.4 mL) was added dropwise to this solution over 40 min. The reaction was carried out at room temperature for 8 h. The solution was filtered to remove any precipitate. The resulting solution was precipitated in excess methanol to give a red solid, which was washed twice with methanol. The collected solid was dried under vacuum to obtain PPV-Br1 or PPV-BrL. PPV-Br1: 1 H NMR (300 MHz, CDCl3, δ): 7.50, 7.18, 6.66, 4.65, 4.09, 3.96, 3.75, 3.40, 1.86, 1.48-0.93. PPV-BrL: 1HNMR (300 MHz, CDCl3, δ): 7.53, 7.19, 6.62, 5.13, 4.65, 4.16-3.66, 3.39, 2.04, 1.83, 1.68, 1.37, 1.25, 1.08-0.75.

[0228] Preparation 8: Synthesis of PPV-N 3 1 and PPV-N 3 L Dissolve PPV-Br1 or PPV-BrL (5 mg) in THF (2.5 mL) and N,N A mixture of dimethylformamide (1 mL) was added. Sodium azide (2 equivalents for the bromide group of PPV-Br1 or PPV-BrL) was added to the solution. The reaction was carried out overnight at 40 °C. The solvent was then removed under reduced pressure, and DCM (40 mL) was added to dissolve the residue. The resulting solution was washed with water (40 mL × 3), and the collected organic phase was dried overnight with anhydrous sodium sulfate. The resulting solution was concentrated and precipitated in excess methanol to give a red solid, which was washed twice with methanol. The collected solid was dried under vacuum to obtain PPV-N31 or PPV-N3L. PPV-N31: 1 H NMR (300 MHz, CDCl3, δ): 7.52, 7.19, 5.36, 5.12, 4.55, 4.09, 3.96, 3.76, 3.24, 1.86, 1.47-0.99. PPV-N3L: 1H NMR (300 MHz, CDCl3, δ): 7.53, 7.19, 6.63, 5.12, 5.01, 4.64, 4.14-3.58, 3.23, 2.04, 1.83, 1.68, 1.37, 1.26, 1.09-0.71.

[0229] Preparation 9: Synthesis of 1,4-dibromo-2,5-bis((2-ethylhexyl)oxy)benzene Add 2,5-dibromohydroquinone (500 mg, 1.87 mmol), potassium carbonate (780 mg, 5.61 mmol), and dimethylformamide (DMF) to a 50 mL round-bottom flask, then add 3- ( bromomethyl ) Heptane (0.8 mL, 4.58 mmol). The reaction was carried out at 80 °C for 12 h. The product was cooled to room temperature and extracted with dichloromethane (DCM). The organic layer was washed with water / salt water and dried over anhydrous sodium sulfate. The solvent was removed under vacuum, and the crude product was purified by column chromatography on silica gel / hexane to give 1,4-dibromo-2,5-bis((2-ethylhexyl)oxy)benzene (697.5 mg, 76.3% yield) as a light viscous oil. 1 HNMR (300 MHz, CDCl3) δ 7.08 (s, 2H), 3.82 (d, J = 5.6 Hz, 4H), 1.73 (dd, J =12.1, 6.0 Hz, 2H), 1.61 – 1.20 (m, 16H), 0.93 (t, J = 7.5 Hz, 12H).

[0230] Preparation 10: Synthesis of PPV-TPP 1,4-Dibromo-2,5-bis((2-ethylhexyl)oxy)benzene, 7,18-dibromo-5,10,15,20-tetraphenylporphyrin, trans-1,2-bis(tributyltin)ethylene, tris(dibenzylacetone)dipalladium(O), and tris(p-tolyl)phosphine were added to a 50 mL Schlenk tube, followed by the addition of chlorobenzene via a syringe (after degassing). The tube was purged with argon three times using a freeze-pump-thaw cycle. The reaction was carried out at 100 °C with vigorous stirring for 24 h. The mixture was cooled to room temperature, and the solvent was removed under vacuum. The crude product was poured into methanol, and the resulting brown solid was washed three times with methanol. PPV: 1H NMR (300 MHz, CDCl3) δ 7.46 (s, 1H), 7.12 (d, J = 21.9 Hz, 2H), 3.95 (t, J = 29.7 Hz, 4H), 3.40 (s, 1H), 1.89 (s, 2H), 1.30 (d, J = 26.2 Hz, 3H), 0.90 (s, 4H). PPV-TPP 2.5% : 1 HNMR (300 MHz, CDCl3) δ 8.22 (s, 1H), 7.77 (s, 2H), 7.42 (d, J = 25.4 Hz, 4H), 7.12 (d, J = 18.6 Hz, 4H), 4.03 (d, J = 30.0 Hz, 7H), 3.43 (d, J = 6.7 Hz, 8H), 1.90 (s, 14H), 1.26 (s, 6H), 0.96 – 0.69 (m, 5H). PPV-TPP 5% : 1 H NMR (300 MHz, CDCl3) δ 8.21 (s, 1H), 7.77 (s, 1H), 7.49 (s, 2H), 7.23 – 6.97 (m, 2H), 3.95 (t,J = 31.0 Hz, 4H), 3.28 (s, 1H), 1.96 (d, J = 47.6 Hz, 2H), 1.62 (d, J = 34.3 Hz, 6H), 1.27 (t, J = 18.3 Hz, 5H), 1.08 – 0.67 (m, 5H).

[0231] Example 1 Synthetic semiconductor polymer nanoparticles (SPNs): SPN-MEHPPV, SPN-PFBT, SPN-MEHPP, SPN-MEHCPV, SPN-POPPV, SPN-BOPPV, SPN-MDMOPPV, SPN-NCBS, and SPN-thiols Using a probe-type sonicator equipped with a microtip (Branson, W-150) at an output power of 2 watts RMS, the mixture containing MEHPPV (0.25 mg / mL) (Sigma-Aldrich) and PEG- was subjected to continuous sonication by rapidly injecting the mixture into distilled deionized water (9 mL, Milli-Q water) for 2 min. b -PPG- bA mixed tetrahydrofuran (THF) solution (1 mL) of PEG (20 mg / mL) (Sigma-Aldrich) was used to prepare SPN-MEHPPV. For SPN-NCBS, the mixed THF solution (1 mL) contained MEHPPV (0.25 mg / mL) (Sigma-Aldrich), PEG- b -PPG- b -PEG (20 mg / mL) (Sigma-Aldrich) and NCBS (from 0 to 0.025 mg / mL depending on the doping amount). Other SPNs, such as SPN-PFBT, SPN-MEHPP, SPN-MEHCPV, SPN-POPPV, SPN-BOPPV, and SPN-MDMOPPV, were prepared in a similar manner.

[0232] For tetraphenylporphyrin (TPP)-doped SPN-MEHPPV, a mixed THF solution (1 mL) contains MEHPPV (0.25 mg / mL) (Sigma-Aldrich), PEG- b -PPG- b -PEG (20 mg / mL) (Sigma-Aldrich) and TPP (from 0 to 0.025 mg / mL depending on the doping amount). For NCBS- or TPP-doped SPN-MDMOPPV, a mixed THF solution (1 mL) contains MDMOPPV (0.25 mg / mL) (Sigma-Aldrich), PEG- b -PPG- b -PEG (20 mg / mL) (Sigma-Aldrich) and NCBS or TPP (depending on the doping amount, from 0 to 0.025 mg / mL).

[0233] For SPN-thiols, the mixed THF solution (1 mL) consisted of MEHPPV (0.24 mg / mL), NCBS (12.5 μg / mL) (Sigma-Aldrich), and C 18 PEG 12 -DNBS (0.5 mg / mL) (Preparation 6) and DSPE-PEG (0.125 mg / mL) (Sigma-Aldrich) were used. After sonication, THF was evaporated at 65°C under a nitrogen atmosphere.

[0234] The aqueous solution was filtered using a polyethersulfone (PES) syringe-driven filter (0.22 µm) (Millipore) and washed three times by centrifugation at 3500 rpm for 15 min at 4 °C using a 50 K centrifuge filter unit (Millipore). The concentration of the SPN-MEHPPV or SPN-NCBS solution was determined by UV-Vis absorption based on its absorption coefficient. Finally, the SPN solution was concentrated to 0.1 mg / mL (based on the mass of MEHPPV) by ultrafiltration and stored in the dark at ~4 °C. All SPN concentrations are based on the mass of SP.

[0235] Example 2 Screening for semiconductor polymers (SPs) for afterglow. Based on the procedure described in Example 1, semiconductor polymers (SPs) with different molecular structures, such as MEHPP (Luminescence Technology Corp.), POPPV (Luminescence Technology Corp.), PFBT (Luminescence Technology Corp.), MEHCPV (Luminescence Technology Corp.), BOPPV (Luminescence Technology Corp.), MDMOPPV (Sigma-Aldrich), and MEHPPV (Sigma-Aldrich), were tested to identify structures favorable for afterglow luminescence. Figure 1 a). In the presence of amphiphilic triblock copolymers (PEG- b -PPG- b In the case of PEG (Sigma-Aldrich), seven SPs were converted into water-soluble nanoparticles using a nanoprecipitation method. Figure 1 b). The hydrodynamic diameter of the SPN, as measured by DLS, is similar, ranging from 30 to 40 nm. Figure 1 c). Transmission electron microscopy (TEM) imaging further confirmed the spherical morphology, with an average diameter of 33.9 ± 4.3 nm. Figure 1 d), which is almost identical to the DLS data. The nanoparticle solution is translucent ( Figure 1 e), even after two months of storage, there was no sedimentation or size change ( Figure 7 This indicates excellent stability in aqueous solution. Fluorescence and afterglow signals of SPNs were collected in both fluorescence (excited) and bioluminescence (unexcited) modes. SPNs based solely on PPV (such as SPN-BOPPV, SPN-MDMOPPV, and SPN-MEHPPV) (based on synthesis in Example 1) exhibited significant afterglow luminescence. Figure 1 e, h), although their emission spectral characteristics are similar to those of fluorescence spectra ( Figure 1 f, g). However, all SPNs exhibit high fluorescence (f, g). Figure 1 e, f). The fluorescence of SPN-MEHPP (based on the synthesis in Example 1) was undetectable because its absorption wavelength was too short to be excited by the IVIS Spectrum imaging system. Figure 8 The afterglow signal was only observed in PPV-based SPs, indicating that phenylene vinylenes play a crucial role in the generation of this real-time, unexcited luminescence. However, some PPV-based SPs (based on the synthesis in Example 1), including SPN-MEHCPV and SPN-POPPV, did not emit detectable afterglow luminescence, suggesting that substituents on the PPV backbone are also important. Similar afterglow behavior was observed for SPs dispersed in THF. Figure 9 This confirms that the chemical structure of SP, rather than the nanoparticle structure, controls the afterglow of SPN.

[0236] Example 3 Mechanism study of afterglow To determine the potential mechanism controlling the afterglow luminescence of SPN, the effect of light irradiation on the chemical structure of SP was investigated. The absorption peak of MEHPPV at 493 nm showed a significant blue shift, and the intensity decreased after irradiation, indicating a break in the conjugation length and thus the decomposition of MEHPPV (Sigma-Aldrich). Figure 2 a). Proton nuclear magnetic resonance (NMR) 1 ¹H NMR analysis revealed two new peaks at 9.88 and 10.47 ppm after light irradiation. These peaks were assigned to aldehyde and carboxyl groups, respectively. The peak broadening and splitting were caused by different chemical environments and indicated the formation of inhomogeneous fragments. Fourier transform infrared spectroscopy (FTIR) showed peaks at 1728 cm⁻¹. -1 Characteristic peaks of oxidized MEHPPV fragments were also detected at [location missing]. Additionally, a peak corresponding to the ethylene-1,2-dimethyl group was observed at 3053 cm⁻¹. -1 The peak at that location weakened after light irradiation, further confirming the oxidation of the vinylidene bond. For BOPPV (Luminescence Technology Corp.) and MDMOPPV (Sigma-Aldrich) (… Figure 10 Similar spectral changes were also observed, but other SPs did not show significant changes. Figure 11 These data clearly demonstrate that light irradiation oxidizes some of the vinyl bonds in PPV, thereby breaking them down into fragments with non-uniform oxidation.

[0237] Next, a singlet oxygen sensor (SOSG) was used to test singlet oxygen during photo-induced oxidation. 1 The generation of O2. After irradiating the SPN-MEHPPV (based on the synthesis in Example 1) solution with light for 5 min, the fluorescence intensity of SOSG at 528 nm increased by 1.69 times ( Figure 2 c). This proves that during irradiation, 1 O2, and caused the oxidation of MEHPPV. Based on these results, the proposed mechanism of afterglow luminescence of PPV-based SPN is as follows: Figure 2 As shown in f. Light irradiation of PPV produces... 1 O2, which passes through π 2 -π 2 Cycloaddition is used to oxidize the vinylidene bond (C=C) to form the PPV-dioxane intermediate. This intermediate is unstable [see, for example, Scurlock, RD, et al., J Am Chem Soc [117, 10194-10202 (1995)], and can spontaneously degrade into PPV-aldehyde and generate photons. Further oxidation of PPV-aldehyde produces PPV-carboxyl groups as the final product of the photoirradiation reaction. Therefore, the key step in afterglow luminescence is... 1 O2-induced formation of PPV-dioxane is determined by the oxidation sensitivity of the vinyl bonds in PPV. This explains why not all PPV-based SPNs exhibit afterglow and the effect of substituents. In fact, only PPVs with electron-donating substituents (alkoxy groups) (such as BOPPV, MDMOPPV, and MEHPPV) (synthesized according to Example 1) show detectable afterglow luminescence, while derivatives with weak electron-donating substituents (alkyl groups for POPPV) (synthesized according to Example 1) or strong electron-withdrawing substituents (cyano groups for MEHCPV) (synthesized according to Example 1) do not exhibit detectable afterglow luminescence.

[0238] Under biologically relevant conditions (pH = 7.4 at 37°C), the afterglow luminescence of PPV-based SPN is persistent, with a half-life of 6.6 min. Figure 2 c). Afterglow conditions can be improved by altering reaction conditions such as temperature and oxygen levels, as well as by adding... 1 O2 scavenger is used for control. This is achieved by regulating the first pre-light irradiation step (…). Figure 2e) When measured in solutions saturated with O2- and N2-, the afterglow of SPN-MEHPPV (based on the synthesis in Example 1) could increase by 1.25 times or decrease by 2.82 times, respectively. Furthermore, the addition of... 1 O2 scavenger (NaN3) can reduce afterglow intensity by 2.06 times. This can be achieved by regulating the second decomposition step (…). Figure 2 e), when the temperature is increased from 37°C to 60°C, the afterglow intensity can be increased by 5 times. However, at 0°C, the afterglow is almost completely suppressed ( Figure 12 Similar afterglow behavior was observed for SPN-MDMOPPV and SPN-BOPPV (based on the synthesis in Example 1). Figure 12 In summary, these data not only further validate the proposed afterglow mechanism, but also highlight... 1 The important role of O2 species in determining the afterglow brightness of SPN.

[0239] Although the mechanism controlling the afterglow luminescence of PPV-based SPNs is similar to that of chemiluminescence [e.g., see Dodeigne, C., et al., Talanta [51, 415-439 (2000)], but it does not require exogenous ROS to trigger the reaction. Instead, SPN itself can be generated under light irradiation. 1 O2 is absorbed, subsequently triggering afterglow luminescence. This afterglow mechanism also differs from that of rare-earth-doped inorganic nanoparticles, where the absorbed photon energy is stored in the intrinsic defect lattice rather than in photoinduced chemical defects [e.g., see Maldiney, T., et al., J Am Chem Soc 133, 11810-11815 (2011)].

[0240] Example 4 Optimization of afterglow NIR light in the 700 to 2,500 nm range penetrates biological tissue more efficiently than visible light because tissue scattering is reduced and biological autofluorescence is minimal in this region [e.g., see Smith, AM, et al., Nat Nanotechnol 4,710-711 (2009)]. To amplify the afterglow and redshift within the ideal NIR optical imaging window, nanoprecipitation was used to... 1 O2 sensitizer and 2,3-naphthylphthalocyanine bis(trihexylsiloxy)silane (NCBS) were doped into SPN-MEHPPV (synthesized according to Example 1). Figure 3 a and Figure 37 b). Because NCBS can absorb in the NIR region ( Figure 13a), so it induces afterglow by pre-irradiation at 808 nm. Therefore, according to the synthesis of SPN-NCBS in Example 1, NCBS with different weight percentages (1, 2.5, 5 and 10) were prepared. w / w SPNs containing % (%) were named SPN-NCBS1, SPN-NCBS2.5, SPN-NCBS5, and SPN-NCBS10, respectively. Doping had no significant effect on the size and morphology of the SPNs. Figure 14 The MEHPPV fluorescence at 580 nm (based on the synthesis in Example 1) gradually decreased with increasing doping concentration, while the NCBS emission at 775 nm gradually increased. Figure 3 d, f and Figure 15 This spectral change confirms efficient energy transfer from MEHPPV to NCBS. Saturation occurs at 5%, and further increases in doping concentration decrease the emission of NCBS. Figure 15 This is due to the self-quenching of NCBS when the local concentration within the nanoparticles increases.

[0241] Regardless of the pre-irradiation laser wavelength (808 or 514 nm), the afterglow intensity at 590 and 775 nm continuously increases with increasing doping concentration. Figure 3 c, e, f). Figure 16 The results of optimized laser irradiation conditions are shown. Signal quantification indicates that when comparing the optimal SPN (SPN-NCBS5) with the undoped control SPN (SPN-MEHPPV) (based on the synthesis in Example 1), the absolute afterglow intensity induced by pre-irradiation at 514 nm was increased by 6.8 times. Figure 3 f). Furthermore, at the same power density, the afterglow of SPN-NCBS5 at 808 nm can be further enhanced by 11 times compared to 514 nm. Figure 3 (b, c, e, f). This is attributed to the stronger production capacity of NCBS compared to MEHPPV. 1 O2's ability ( Figure 17 No afterglow was detected in nanoparticles composed solely of NCBS5. Figure 15 c). For tetraphenylporphyrin (TPP)-doped SPN-MEHPPV and NCBS- or TPP-doped SPN-MDMOPPV (synthesized as described in Example 1), further observations were also made. 1 The amplified afterglow of O2 sensitizer ( Figure 18 and Figure 19 These data indicate 1 O2 sensitizer is an in-particle promoter that can effectively amplify the afterglow of SPN and adjust its emission wavelength.

[0242] Example 5 In vivo and in vitro evaluation of SPN-NCBS5 Next, SPN-NCBS5 was evaluated in vivo and in vitro according to the protocol in the biological experiments section. Based on the good cell compatibility of SPN-NCBS5 (synthesized in Example 4), Figure 20 The amplified afterglow nanoparticles (SPN-NCBS5) are suitable for biological applications.

[0243] Yu Hui's tissue penetration research The penetration depth and imaging sensitivity of the afterglow of SPN-NCBS5 (synthesized in Example 4) were examined both in vitro and in vivo. This is because SPN-NCBS5 exhibits absorption and emission in the NIR region (…). Figure 13 and Figure 15 Therefore, after excitation at 710 nm, fluorescence signals are collected at 780 nm. Figure 13 (b) The afterglow was caused by pre-irradiation at 808 nm.

[0244] Next, a thicker layer of chicken tissue was placed on top of the sample. Both afterglow and fluorescence signals decreased with increasing penetration depth. Figure 4 a, b). However, due to the fluorescence (2.53 × 10⁻⁶), 7 ± 1.76×10 6 p / s / cm 2 ( / sr) compared to afterglow (824 ± 109 p / s / cm) 2 The background noise of NIR fluorescence (p / s / cm²) is very low; therefore, at a thickness of 1.5 cm, the SBR of the afterglow (291 ± 18) is 67 times higher than that of fluorescence (4.33 ± 0.96). Furthermore, at a thickness of 4 cm, the NIR fluorescence is close to the background noise, while the SBR of the afterglow remains at 17.7 ± 0.27. Similarly, the background noise of afterglow luminescence imaging in live mice is as low as 867 ± 80 p / s / cm². 2 / sr, because tissue autofluorescence is eliminated in the absence of real-time excitation. Therefore, when detecting NIR-induced afterglow signal from SPN-NCBS5 at a depth of 1.7 cm in live mice ( Figure 4 The c–e SBR reached 237 ± 22, which was 4.7 times and 120 times higher than that of visible light-induced afterglow (50.7 ± 4.5) and NIR fluorescence (1.98 ± 0.09), respectively. Importantly, the afterglow could be repeatedly loaded by in situ irradiation at 808 nm through chicken tissue or live mice. Figure 21 and 22 This confirms the feasibility of long-term in vivo imaging.

[0245] The afterglow signal of subcutaneously implanted SPN-NCBS5 (synthesized in Example 4) in live mice was linearly correlated with its concentration. Figure 23 c). Due to the high SBR of the afterglow, the limit of detection (LOD) for SPN-NCBS5 in live mice was 1.35 ng / mL. Figure 23 c), which is 80 times lower than the detection limit of NIR fluorescence ( Figure 23 d). Furthermore, after pre-irradiation, the afterglow of SPN-NCBS5 can be stored at -20°C, and the intensity decreased by only 3.8% after one day of storage. Figure 24 This demonstrates the feasibility of in vivo imaging using afterglow SPNs derived directly from storage without any optical preprocessing.

[0246] Afterglow imaging of lymph nodes and tumors The storable and NIR-reproducible afterglow of amplified SPN (SPN-NCBS5) (synthesized in Example 4) was used for real-time localization of lymph nodes in live mice. Figure 5 a) and follow the protocol described in the biological experiments section. Lymph node localization has important clinical significance in guiding the surgical resection of tumor tissue [e.g., see Kim, S., et al., Nat Biotechnol [22, 93-97 (2004)], but this had not been previously accomplished using afterglow imaging. Pre-irradiated SPN-NCBS5 was stored at -20°C for one day, warmed to room temperature, and then injected directly into the forepaw of live mice for continuous imaging without re-irradiation. Afterglow and fluorescence images were acquired at t = 30 min post-injection. Both afterglow and fluorescence imaging clearly depicted the axillary lymph nodes ( Figure 25 This indicates effective accumulation and retention of SPN-NCBS5 in sentinel lymph nodes. Although the afterglow decreased after 30 min at 37°C in live mice ( Figure 26 a), but the SBR of the afterglow image was still 7.8 ± 1.2, which was twice as high as that of the fluorescence image (3.9 ± 0.3). Figure 5 c). After in-situ afterglow regeneration by irradiation at 808 nm for 1 min at t = 65 min following injection, the SBR of the afterglow image was substantially increased to 419 ± 32: 127 times higher than that of the fluorescence. Figure 5 (b, c). Therefore, the NIR afterglow of the SPN-NCBS5 can locate lymph nodes with high contrast without real-time excitation during imaging. The afterglow luminescence of SPN-NCBS5 was also tested in passive targeted imaging of tumors in live mice and compared with NIR fluorescence. Afterglow and NIR fluorescence signals were acquired in real time after tail vein injection of SPN-NCBS5. Both signals gradually increased over time, but the SBR of the afterglow image was higher than that of the NIR fluorescence at all time points. Figure 5 d, e, f). Due to the low background of the afterglow, the tumor was visible at t = 1 h post-injection and clearly visible at t = 2 h post-injection for afterglow imaging. Figure 5 e). In contrast, for NIR fluorescence imaging, the tumor was only visible at t = 8 h post-injection. At t = 2 h, the SBR of the afterglow image was 149.7 ± 9.0, which was 23.3 times higher than that of the NIR fluorescence image (6.4 ± 0.9). Figure 5 f). At t = 36 h post-injection, both afterglow and fluorescence signals reached a plateau, indicating effective accumulation of SPN-NCBS5 in the tumor. In vitro data further showed that SPN-NCBS5 uptake was highest in the liver, followed by tumors, lungs, and other major organs. Figure 27 Therefore, compared to NIR fluorescence imaging, the afterglow of SPN-NCBS5 allows for faster, higher-contrast imaging of tumors in live mice. Furthermore, the ultrasensitive NIR afterglow of SPN-NCBS5 allows for faster tumor mapping within 2 hours, which is impossible with NIR fluorescence imaging. SPN-NCBS5 has an emission of 780 nm and a half-life of 396 s. This strong NIR afterglow allows for a SBR of 3387 ± 39 for subcutaneously implanted SPN-NCBS5 in live mice at a concentration of 12.5 µg / mL. Due to this high SBR, afterglow SPN allows for in vivo tumor imaging following systemic administration at doses (50 µg per mouse) that are much lower than other existing afterglow agents (200 to 1000 µg per mouse), while still providing a high SBR. Figure 5 ).

[0247] Activated afterglow probes for imaging drug-induced hepatotoxicity Drug-induced hepatotoxicity has long been a concern in modern medicine [e.g., see Nasr, A., et al., Adv. Ther. [28, 842-856 (2011)], and is one of the most common reasons why the Food and Drug Administration (FDA) refuses to approve drugs [see, for example, Kola, I. & Landis, J.]. Nat Rev Drug Discov3, 711-715 (2004)]. Evaluating potential hepatotoxicity prior to regulatory approval is challenging because current safety assays are only applicable to in vitro studies and have low predictive power [e.g., see Willmann, JK, et al., Nat Rev Drug Discov 7, 591-607 (2008)]. Oxidative stress and depletion of antioxidants in the liver are early complications of hepatotoxicity [e.g., see Pessayre, D., et al., Handb. Exp. Pharmacol. [311-365 (2010)]. Among antioxidants in living organisms, biothiols, including cysteine ​​(Cys), homocysteine ​​(Hcy), and glutathione (GSH), constitute the major component of systemic antioxidants that combat oxidative stress. Therefore, real-time in situ imaging of biothiols levels may be a feasible method for evaluating drug-induced hepatotoxicity.

[0248] To develop an activatable afterglow probe for biothiol imaging, according to Example 1, a probe was synthesized that interacts with an electron-withdrawing quencher (C). 18 PEG 12 -DNBS)(Preparation 6) conjugated amphiphilic oligomers, and co-precipitated with NCBS and MEHPPV ( Figure 6 a). The resulting activatable nanoprobe (SPN-thiol) has a similar size and morphology to other SPNs. Figure 28 Due to efficient electron transfer from the nucleus to the quencher, the afterglow of SPN-thiol is essentially quenched in its initial "afterglow-off" state. Figure 6 a). However, in the presence of bio-thiols (including GSH, Cys, and Hcy), the sulfonamide bonds on the SPN-thiol surface can be broken, thereby releasing DNBS from the nanoparticle surface. Therefore, electron transfer is eliminated, leading to afterglow activation (“afterglow-on” state). After activation by Cys, the afterglow of SPN-thiol at 780 nm increased by 8.3 times ( Figure 6 b). This is 1.75 times and 1.41 times higher than GSH and Hcy, respectively. Conversely, for other amino acids, the signal is still almost undetectable. Figure 6 c, d). This indicates that SPN-thiols exhibit high selectivity for biothiols, particularly Cys. Similar activation was observed in the fluorescence of SPN-thiols ( Figure 29 Furthermore, a linear relationship was observed between afterglow intensity and Cys concentration, with a limit of detection (LOD) of 0.60 μM. Figure 6 e), which is sufficient for the in vivo biological concentration of biothiols (~0.1 to 10 mM).

[0249] SPN-thiols (based on synthesis in Example 1) were used for in vivo imaging of drug-induced hepatotoxicity. Figure 6 f), and according to the protocol in the biological experiments section. Acetaminophen (APAP) is an antipyretic and analgesic drug, and is used as a model drug because the mechanism of APAP-induced hepatotoxicity is well established. Excessive APAP induces oxidative stress and nitrosation stress, which in turn depletes biothiols, triggering a signaling cascade that leads to necrotic cell death. Mice were first treated with toxic doses of APAP or saline, and then SPN-thiols were administered systemically via intravenous injection at t = 20 min after APAP treatment. Afterglow and fluorescence signals were acquired in real time, and they gradually increased over time ( Figure 6 h). At t = 2 h after SPN-thiol injection, the afterglow of APAP-treated mice was 1.99 times lower than that of saline-treated control mice due to the decreased level of biothiols in the liver. Figure 6 g). In contrast, when using APAP processing before... N -acetyl- L When β-cysteine ​​(NAC, an FDA-approved antioxidant) was used to protect mice, the afterglow signal was comparable to that of saline-treated control mice. This is because NAC has the ability to effectively scavenge ROS, maintaining the level of antioxidants in the liver. Histological studies further showed that extensive liver necrosis occurred 3 h after APAP treatment, while no liver damage was found in saline-treated control mice or NAC-protected mice. Figure 32 The decreased afterglow intensity following APAP treatment and the increased afterglow intensity following NAC retrieval confirm that SPN-thiols can be used for longitudinal imaging of hepatotoxicity in vivo. Furthermore, the SBR ratio of the afterglow was ~25 times higher than that of NIR fluorescence at all time intervals. Figure 6 (h), indicating that afterglow imaging has higher sensitivity for drug-induced hepatotoxicity.

[0250] The structural variability of SPN has also facilitated the development of smart, activatable afterglow probes (SPN-thiols) for drug-induced hepatotoxicity in live mice. Figure 6This is the first demonstration that a persistent luminescence system alters signal intensity in response to molecules of interest in vivo. Existing persistent luminescence nanoparticles have only been used for passive or active tumor targeting. The NIR persistent luminescence of the bio-thiol-activated probe (SPN-thiol) at 780 nm can be fully activated by bio-thiols such as Cys, Hcy, and GSH. Since these bio-thiols are important antioxidants against oxidative stress, SPN-thiol can detect antioxidant levels in the liver of live mice, and thus enables real-time persistent luminescence imaging of drug-induced hepatotoxicity and repair, with an SBR level 25 times higher than that of NIR fluorescence imaging. More importantly, SPN-thiol can detect hepatotoxicity within 20 min of drug attack, which is much shorter than the observation time for histological changes in the liver (~3 h) [e.g., see Shuhendler, AJ, et al., Nat Biotechnol 32, 373-380 (2014)].

[0251] Example 6 Biodegradability and biocompatibility of SPN Afterglow SPN can be degraded by enzymes. For example... Figure 33 As shown, the degradation of SPN-NCBS5 (synthesized in Example 4) can be catalyzed by H2O2 and MPO produced by immune cells (such as neutrophils) in living animals. Upon incubation with H2O2 and MPO, the vinylidene bonds of MEHPPV (based on the synthesis in Example 1) are cleaved, leading to the formation of PPV-aldehyde fragments. The degradation was confirmed by a decrease in the absorbance, fluorescence, and afterglow intensity of the nanoparticles, as well as a significant decrease in molecular weight.

[0252] Figure 31 In vitro biodistribution data showed that afterglow SPNs accumulated more extensively in the liver because their size (~33 nm) was greater than 5 nm. Therefore, afterglow SPNs are primarily cleared by the liver. Figure 34 As shown, SPN-NCBS5 (based on the synthesis in Example 4) underwent enzymatic degradation in the liver over a period of more than 18 days, eventually leading to almost complete clearance.

[0253] The toxicity of SPN afterglow was evaluated through histological analysis. Figure 35 As shown, no histopathological changes were observed in organs from mice treated with SPN-NCBS5 (synthesized in Example 4) compared to the control.

[0254] Example 7 Synthesis and characterization of amphiphilic PPV derivatives To ensure good water solubility, PPV is designed to have PEG as a graft chain. Figure 36First, 4-methoxyphenol was reacted with 1,10-dibromodecane (Sigma-Aldrich) and 2-ethylhexyl bromide (Sigma-Aldrich), respectively, to give compound 2 (Preparation 2) and compound 3 (Preparation 2). Compounds 2 and 3 were then treated with paraformaldehyde and HBr to give compound 4 (Preparation 4) and compound 5 (Preparation 5), respectively. Compound 4 was then polymerized in the presence of potassium tert-butoxide to give a PPV polymer (PPV-Br1) with bromide groups on its side chain (Preparation 7). To synthesize PPVs with a lower PEG grafting density, compounds 4 and 5 were copolymerized in a molar ratio of 8 to 1 to produce PPV-BrL (Preparation 7). These bromide PPVs were then reacted with sodium azide to replace the bromide with azide, giving PPV-N31 and PPV-N3L (Preparation 8). The results were obtained by proton nuclear magnetic resonance (NMR) assay. 1 ¹H NMR spectroscopy (data not shown) confirmed the conversion of bromide to azide, indicating that for both PPV-N31 and PPV-N3L, The resonance peak of CH2Br (3.40 ppm for PPV-Br1 and 3.39 ppm for PPV-BrL) transforms into... The resonance peak of CH2N3 (3.24 ppm for PPV-N31 and 3.23 ppm for PPV-N3L).

[0255] PPV-PEG1 and PPV-PEGL were prepared by a copper (I)-catalyzed alkyne-azide cycloaddition (CuAAC) reaction. PPV-N31 or PPV-N3L (2 mg) (Preparation 8) was dissolved in THF (3 mL), and then copper (I) bromide (2 equivalents for the azide group of PPV-N31 or PPV-N3L), PEG-alkyne (2 equivalents for the azide group of PPV-N31 or PPV-N3L) (Preparation 1) and... N,N,N',N'',N''' - Pentamethyldiethylenetriamine (PMDETA) (8 equivalents for the azide group of PPV-N31 or PPV-N3L) (Sigma-Aldrich). The reaction was carried out at room temperature under a nitrogen atmosphere for 48 h. Water was added to the mixture, and the resulting solution was dialyzed with pure water to remove any impurities and excess PEG-acetylene. After lyophilization, PPV-PEG1 or PPV-PEGL was obtained. PPV-PEG1: 1H NMR (300 MHz, CDCl3, δ): 7.50, 7.12, 4.56, 4.18, 3.86, 3.62, 3.54, 3.38, 3.36, 1.42-1.07, 0.92-0.69. PPV-PEGL: 1H NMR (300 MHz, CDCl3, δ): 7.54, 7.19, 5.34, 4.67, 4.14-3.79, 3.65, 3.57, 3.38, 1.68, 1.45-1.11, 1.09-0.66.

[0256] Gel permeation chromatography (GPC) showed that both PPV-PEG1 and PPV-PEGL had higher molecular weights than their corresponding precursors (PPV-Br1 and PPV-BrL), thus confirming that PEG was grafted onto the PPV backbone (Table 1).

[0257] Table 1. GPC of PPV polymers

[0258] Due to its amphiphilic properties, PPV-PEG1 can be directly dissolved in phosphate-buffered saline (PBS) and self-assemble into small nanoparticles. The optical properties and afterglow mechanism of PPV were investigated. Figure 42 and Figure 43 PPV-PEG1 exhibits strong afterglow luminescence, and its spectrum is almost identical to the fluorescence spectrum with maximum emission at 580 nm. Figure 42 c). The underlying mechanism of afterglow luminescence ( Figure 42 f) Same as previously reported nanoparticles: singlet oxygen generated from PPV upon light irradiation ( 1 O2 can react with vinyl bonds to form dioxane units that generate photons upon degradation (as discussed in Example 3). To amplify and redshift the afterglow luminescence into the NIR region, 1 O2 sensitizer and 2,3-naphthylphthalocyanine bis(trihexylsiloxy)silane (NCBS) doped into PPV-PEG1 Figure 44 However, fluorescence spectroscopy and DLS results showed poor fluorescence resonance energy transfer (FRET) from the PPV fragment to NCBS and low encapsulation efficiency, which should be attributed to the high grafting density of PEG to PPV-PEG1.

[0259] To better encapsulate NCBS, NCBS-doped nanoparticles were prepared using PPV-PEGL, which has a lower PEG grafting density compared to PPV-PEG1. Figure 37 a). According to the synthesis of SPN-NCBS in Example 1, in the amphiphilic triblock copolymer (PEG- b -PPG-b PPVP was prepared via nanoprecipitation of MEHPPV (Sigma-Aldrich) and NCBS (2 w / w% relative to MEHPPV) in the presence of PEG. Figure 3 a) Preparation of SPPVN and SPPVT via nanoprecipitation. Briefly, PPV-PEGL (20 mg), NCBS, or TPP (2 w / w% relative to PPV fragment) was dissolved in THF (1 mL). The solution was rapidly injected into a mixture of THF (1 mL) and water (9 mL) after vigorous sonication at 110 W for 1 min. The THF in the resulting solution was removed under a gentle nitrogen flow, and the solution was purified by filtration through a 0.22 μm PVDF syringe-driven filter. The prepared SPPVN (2 w / w% NCBS) or SPPVT (2 w / w% TPP) solutions were concentrated by ultrafiltration and stored at 4°C for subsequent use.

[0260] DLS results, UV and fluorescence spectroscopy showed successful encapsulation of NCBS, and efficient FRET from PPV fragment to NCBS. Figure 45 a, 45b, and 45c). The afterglow spectrum of the NCBS-doped nanoparticles is similar to that of the fluorescence spectrum. Figure 45 d). In particular, among all the nanoparticles tested, SPPVN exhibited the highest afterglow luminescence intensity ( Figure 45 e). At room temperature, the afterglow of SPPVN in 1×PBS buffer is persistent, with a half-life of 4.8 min (e). Figure 45 f). Furthermore, the afterglow intensity of the SPPVN induced by pre-irradiation at 808 nm was 8 times that induced by pre-irradiation at 514 nm, because more afterglow can be generated from NCBS after irradiation at 808 nm. 1 O2 (2.6 times) ( Figure 46 When another photosensitizer, TPP, was used, a similar amplified afterglow phenomenon was detected. Figure 47 Compared to SPPVT, SPPVN exhibits a longer emission wavelength (780 vs. 660 nm), higher energy transfer efficiency (51% vs. 37%), and a significantly enhanced afterglow intensity compared to undoped nanoparticles (20x vs. 1.8x). The selection of nanoparticles with 2... w / w SPPVN of %NCBS is used for further research.

[0261] Example 8 Compare the properties of SPPVN and PPVP SPPVN (synthesized in Example 7) and PPVP (based on Example 1 and Figure 3 a and Figure 37 The properties of SPPVN (synthesized as shown in diagram b) are compared. SPPVN has a much smaller hydrodynamic size than PPVP (24 vs. 34 nm). Figure 37 c). SPPVN and PPVP have similar UV absorption spectra, with two maximum peaks at ~500 and 775 nm, corresponding to the absorption of PPV and NCBS, respectively. Figure 37 d). Both SPPVN and PPVP exhibited stronger NCBS fluorescence at 775 nm than the PPV fragment at 590 nm. However, the ratio of SPPVN's emission intensity at 780 nm to that at 590 nm was 2.1 times higher than that of PPVP, indicating that SPPVN had a higher FRET efficiency than PPVP (51% vs. 24%). Figure 37 e). At the same mass concentration, the afterglow emission spectra of SPPVN and PPVP are similar to their fluorescence spectra, but the afterglow intensity of SPPVN is 1.3 times higher than that of PPVP. Figure 37 f). This improvement in FRET and enhanced afterglow can be attributed to the tighter contact between the PPV fragments and NCBS in the SPPVN compared to the PPVP. The results also indicate that the SPPVN possesses excellent physiological stability ( Figure 48 a) and cell compatibility ( Figure 48 b).

[0262] Example 9 In vivo evaluation of SPPVN, PPVP and PPV-PEGL Next, SPPVN (synthesized in Example 7), PPVP (synthesized in Example 1), and PPV-PEGL (synthesized in Example 7) were evaluated by in vivo imaging experiments according to the protocol in the biological experiment section.

[0263] Tissue Penetration Research The tissue penetration capabilities of afterglow imaging and NIR fluorescence imaging were compared by detecting SPPVN solution at a depth of 1.6 cm in live mice. Figure 38 a). Fluorescence signals were acquired at 780 nm after excitation at 710 nm, and after pre-irradiation at 514 or 808 nm for 1 min, afterglow luminescence signals were acquired in bioluminescence mode. This was due to strong tissue autofluorescence (2.06 × 10⁻⁶). 7 ± 1.90×10 6 p / s / cm 2 / sr), the fluorescence signal of the SPPVN solution is almost indistinguishable from the background signal ( Figure 38 a). Conversely, due to the elimination of real-time optical excitation, the background signal in afterglow imaging is very low (2900 ± 420 p / s / cm).2 Therefore, the afterglow signal can be clearly detected under pre-illumination at both 514 and 808 nm. The SBR of afterglow imaging induced by pre-illumination at 808 nm is 404 ± 63 ( sr). Figure 38 (b) These values ​​represent ~5.0 times and ~304 times that of afterglow imaging (80.4 ± 12.5) induced by pre-illumination at 514 nm and NIR fluorescence imaging (1.3 ± 0.2), respectively. These results indicate that afterglow imaging offers significantly higher penetration depth and imaging sensitivity compared to NIR fluorescence imaging.

[0264] Because the ambient temperature is higher in live mice, the afterglow emission of SPPVN decays faster in vivo than in vitro. Figure 49 a and 45f). Nevertheless, afterglow of SPPVN could be induced at least 6 times in live mice by pre-irradiation at 808 nm without a significant decrease in afterglow intensity (a and 45f). Figure 49 b). This demonstrates its suitability for long-term in vivo imaging. SPPVN exhibits a long NIR emission of 780 nm and a half-life of 288 s. Furthermore, the in vivo afterglow intensity of subcutaneously injected SPPVN (1.36 × 10⁻⁶) is [not specified in the original text]. 5 (p / s / cm 2 / sr) / (µg / mL)) is inorganic persistent nanoparticles (such as ZnGa2O4:Cr 3+ 27.2 times higher than that of nanoparticles Figure 49 c). The SBR of SPPVN administered subcutaneously at 130 µg / mL (4170 ± 179) can be tested even at higher concentrations (2 mg / mL) of ZnGa2O4:Cr. 3+ The nanoparticles (275) are 15.2 times higher. [See, for example, Li, ZJ, et al., J Am Chem Soc 137, 5304-5307 (2015)].

[0265] Lymph node imaging study The efficacy of SPPVN for lymph node imaging was tested after injection into the forepaw of live mice. Figure 38 c). NIR fluorescence and afterglow luminescence images were acquired at t = 60 min post-injection. Axillary lymph nodes were depicted using both afterglow and fluorescence imaging. Afterglow imaging alone showed a low-background image of the lymph nodes. Figure 38 Quantitative imaging showed that the SBR of afterglow imaging (622 ± 104) was 41 times higher than that of fluorescence imaging (15 ± 3). Figure 38i). These results demonstrate that, compared to NIR fluorescence imaging, afterglow imaging using SPPVN allows for the localization of lymph nodes with significantly higher contrast.

[0266] In vivo imaging for differentiating between hypoxic and normoxic environments Most tumor cells exist in a hypoxic environment due to the rapid oxygen consumption required for angiogenesis and cell proliferation [e.g., see Carmeliet P. and Jain RK]. Nature. [473, 298-307 (2011)]. Tumor hypoxia is also associated with an increased risk of invasion and metastasis. Therefore, imaging of tumor hypoxia can aid in the diagnosis and treatment of cancer. Since afterglow is sensitive to oxygen, the ability of SPPVN to distinguish between hypoxia and normoxic conditions was tested in vivo. Figure 42 e). First, purge the SPPVN solution with N2 to remove residual oxygen, and then inject it locally into the tumor or under the skin. Figure 38 e). The fluorescence intensity of locally injected tumors and skin is almost identical ( Figure 38 e). Conversely, the afterglow intensity of locally injected skin is 2.4 times higher than that of tumor afterglow intensity (e). Figure 38 (e and 38f). This difference in afterglow intensity is due to the hypoxic environment of the tumor, where the low oxygen levels reduce the afterglow intensity of the SPPVN. Therefore, these results suggest that the oxygen-sensitive afterglow of the SPPVN can be used to monitor hypoxia and normoxic conditions.

[0267] The afterglow luminescence of SPPVN was compared with that of PPVP in in vivo tumor imaging. Both SPPVN and PPVP were exposed to air prior to injection. To test their early detection capabilities, a tumor with a payload size of only ~5 mm was used. 3 Imaging experiments were conducted on live mice with xenografted tumors. Following systemic administration of the nanoparticles via tail vein injection, fluorescence and afterglow signals were acquired longitudinally. For both SPPVN and PPVP, afterglow and fluorescence signals gradually increased and reached saturation at 24 h post-injection; however, the afterglow imaging images at each time point exhibited a higher SBR (Sequencing Batch Ratio) than the fluorescence images. Figure 39 (a, 39b, and 39c). At 24 h, the SBR in afterglow imaging of mice injected with SPPVN was 306 ± 20, which was 11 times higher than the SBR (28 ± 1) in NIR fluorescence imaging. Figure 39 c). Due to such a high SBR, tumors can be detected by afterglow imaging as early as 40 minutes after SPPVN injection; in contrast, fluorescence can only be detected 4 hours after injection. Figure 39 a). For mice injected with PPVP, afterglow imaging and fluorescence imaging could only visualize the tumor at t = 4 and 8 h post-injection, respectively. Figure 39b). At t = 40 min after nanoparticle injection, the SBR of afterglow imaging in SPPVN-injected mice was 40 ± 3, which was ~22 times higher than the SBR of NIR fluorescence imaging (1.8 ± 0.2) and ~2.5 times higher than the afterglow imaging of PPVP (15.7 ± 1.5). Figure 39 c). Due to the high sensitivity of SPPVN, the afterglow allows for a size as small as approximately ~1 mm with an SBR of 63 ± 10 Hz 4 h post-injection. 3 The tumor can be detected. This is only possible for NIR fluorescence imaging, which is only possible 24 hours after injection. Figure 50 It should be noted that for different tumor sizes (5 and 1 mm)... 3 At each time point, the real-time fluorescence and afterglow emission signals of the livers from tumor-bearing mice injected with SPPVN were almost identical. Figure 51 This confirms that tumor size does not affect the biodistribution of nanoparticles. Furthermore, the tumor size detected by SPPVN (1 mm) 3 Compared to other reported NIR fluorescence imaging probes (50 to 500 mm) 3 Much smaller [for example, see Li Y., et al., Nat. Commun. 5 , 4712 (2014; Yang K., et al., Adv. Mater. 24, 1868-1872 (2012)].

[0268] In vivo peritoneal metastatic tumor imaging To determine why SPPVN delineates tumors more rapidly than PPVP, in vitro biodistribution and uptake in multicellular tumor spheroids (MCTS) were investigated. Unlike PPVP, which showed the highest uptake in the liver, SPPVN exhibited the highest uptake in tumors, being 1.4 times higher than in the liver. Figure 39 d and 3e, Figure 52 This can be attributed to the more stable, non-separable nanostructure of SPPVN compared to the binary micelle structure of PPVP. The calculated PEG density of SPPVN nanoparticles is ~1.7 times higher than that of PPVP (0.30 vs. 0.18 / nm). 2 According to literature reports, this should be another reason why SPPVN has better biodistribution [e.g., see Du X., et al., Biomaterials 69, 1-11 (2015)]. Furthermore, MCTS uptake studies showed that, after the same incubation time, SPPVN exhibited higher uptake (1.8 times) and deeper penetration than PPVP. Figure 53This is likely due to its smaller size compared to PPVP (24 vs. 34 nm). These data suggest that, in addition to the higher afterglow of SPPVN compared to PPVP, the better biodistribution and deeper penetration of SPPVN compared to PPVP should also be the reason for the faster detection of tumors in live mice after systemic administration.

[0269] To test the efficacy of SPPVN over PPVP in detecting metastatic tumor tissue, imaging experiments were performed on mice carrying peritoneal metastatic 4T1 tumors. 4T1 cancer cells (2 × 10⁻⁶) were... 5 4T1 cells were injected intraperitoneally into mice to establish a metastatic tumor model. Mice were randomly divided into two groups and treated with SPPVN or PPVP via the tail vein only 3 days after injection. Figure 40 a). Fluorescence and afterglow images were acquired after systemic administration of SPPVN or PPVP via tail vein injection. In mice injected with SPPVN, the afterglow signal gradually increased in the lower quadrant region (excluding the liver), but not in mice injected with PPVP, indicating significantly stronger accumulation of SPPVN in tumor tissue. Figure 40 b). At t = 1.5 h post-injection, the skin and peritoneum of the mice were excised, and the lower quadrant region of the mice was imaged. For both SPPVN and PPVP-injected mice, autofluorescence was detected only in the lower quadrant region. Conversely, for SPPVN-injected mice, strong afterglow spots were detected in the intestine, while for PPVP-injected mice, none were detected. Figure 40 c). Quantitative analysis of the afterglow signal showed that, in mice injected with SPPVN, the afterglow intensity in the tumor region was 6.1 times higher than the background signal; while in mice injected with PPVP, there was no statistically significant difference between the signal and the background signal. Figure 40 d). Histological examination confirmed the formation of micrometastases on the intestinal surface that were invisible to the naked eye. Figure 40 e and Figure 54 These results indicate that SPPVN is faster at detecting small peritoneal metastatic tumor tissues compared to PPVP.

[0270] Biodegradability Research The biodegradability of PPV-PEGL in solution and cells was studied under in vivo simulated conditions. Figure 41 a). Myeloperoxidase (MPO) and H2O2 can be produced in immune cells [e.g., see Klebanoff SJ, J. Leukoc. Biol.[77, 598-625 (2005)] was used to simulate physiological conditions, and the concentration of MPO used (40 µg / mL) was within the physiological concentration range (35.4–82.6 µg / mL) calculated according to previous literature [e.g., see Christensen RD and Rothstein G., Pediatr. Res. 19, 1278-1282 (1985)]. Incubation of PPV-PEGL with MPO and H2O2 resulted in a gradual decrease in absorption and a blue shift ( Figure 41 b). This confirmed the enzymatic oxidation of the double bond and the degradation of PPV-PEGL into small fragments. PPV-PEGL was then incubated with lipopolysaccharide (LPS)-activated RAW264.7 macrophages for different times to investigate intracellular biodegradability. With increasing incubation time, the fluorescence of PPV-PEGL in the cells gradually decreased ( Figure 41 c). At 12 h, the fluorescence was 2.3 times lower than that of unstimulated cells. These data indicate that PPV-PEGL is biodegradable under relevant conditions.

[0271] To investigate the in vivo clearance of SPPVN, SPPVN was systemically administered to live mice via tail vein, and fluorescence images were recorded at different time points. Following intravenous injection, SPPVN gradually accumulated in the liver, reaching its peak accumulation one day post-injection. Figure 41 d and Figure 55 Then, the fluorescence signal in the liver gradually decreased, and by 20 days post-injection, it was almost undetectable. These results indicate that in live mice, SPPVN can be degraded in the liver and cleared within 20 days via hepatobiliary excretion.

[0272] Example 10 Synthesis and characterization of SPN-PPV-TPP.

[0273] 1,4-Dibromo-2,5-bis((2-ethylhexyl)oxy)benzene (Preparation 8) was copolymerized with trans-1,2-bis(tributyltin)ethylene and 7,18-dibromo-5,10,15,20-tetraphenylporphyrin (TPP-Br) in different molar ratios via a Pd-catalyzed Stille coupling reaction to generate PPV and PPV-TPP. 2.5% and PPV-TPP 5% ( Figure 56 (a) Preparation 10. The molecular weight and polydispersity (PDI) of PPV-TPP were further characterized by GPC, and the molecular weight of the PPV polymers ranged from 8900 to 13000 (Table 2).

[0274] PPV-TPP (1 mg) was dissolved in 1 mL of THF. Then, in the presence of 0.75 mL of a THF solution containing the amphiphilic triblock copolymer ((PEG-b-PPG-b-PEG) (20 mg), PPV and PPV-TPP were separated using a nanoprecipitation method. 2.5% and PPV-TPP 5% (0.25 mL) was converted into water-soluble nanoparticles (SPN0, SPN2.5, and SPN5). Figure 56 (b) The obtained solution was rapidly injected into a mixture of DI water (9 mL) and THF (1 mL) under continuous sonication. THF was removed by a gentle stream of nitrogen. The resulting solution was purified by filtration through a 0.22 μm polyvinylidene fluoride syringe-driven filter (Millipore). The obtained nanoparticle solution was concentrated by ultrafiltration and then diluted with 1×PBS (pH = 7.4) to prepare solutions of different concentrations.

[0275] Table 2. GPC data for PPV polymers

[0276] DLS showed that the average hydrodynamic diameters of SPN0, SPN2.5, and SPN5 were similar, ranging from 25 to 30 nm. Figure 56 c and Figure 60 Taking SPN2.5 as an example, TEM imaging shows a uniform spherical morphology with an average diameter of ~25 nm, consistent with DLS data. Figure 56 d). For SPN2.5, no precipitation or significant size change was observed after 20 days of storage in PBS (pH = 7.4) or FBS. Figure 61 MTS analysis (as described in the biological experiments section) showed that SPN2.5 was not cytotoxic to 4T1 cells. Figure 56 e). These results demonstrate that these SPN-PPV-TPPs possess ideal water stability and cell compatibility for biological applications.

[0277] The optical properties of SPN-PPV-TPP were tested in PBS (pH = 7.4) solution. UV-Vis spectroscopy showed that all SPN-PPV-TPP exhibited similar maximum absorption in the range of 430 to 450 nm, corresponding to the PPV fragments ( Figure 57a). With increasing TPP content, a new absorption band appeared in the 600–700 nm range, which was assigned to the TPP-containing fragment. This absorption change confirmed the incorporation of TPP into the PPV-TPP framework. The fluorescence of TPP-free nanoparticles (SPNO) exhibited a significant maximum emission at 580 nm. With increasing TPP content, the fluorescence of the PPV fragment at 580 nm decreased, while NIR emission increased in the 650–750 nm range. Figure 57 b), and saturation occurred at a TPP content of 2.5% (SPN2.5). This confirms the occurrence of FRET from PPV fragments to TPP. Compared to SPN2.5, SPN5 showed a decrease in emission from 650 nm to 750 nm, which is likely due to local fluorescence quenching caused by the higher amount of TPP. This phenomenon was also confirmed by fluorescence images of SPN-PPV-TPP at 720 nm captured by IVIS, indicating that SPN2.5 had the highest fluorescence intensity among these nanoparticles (b). Figure 57 d). The afterglow spectrum and images of SPN-PPV-TPP were collected after illumination in bioluminescent mode (without real-time excitation). Figure 57 (c and 57e), showing that the afterglow spectrum is similar to their fluorescence spectrum.

[0278] Example 11 Study on the afterglow mechanism of SPN-PPV-TPP Similar to Example 3, using 1 O2 sensor green (SOSG) test light illumination during O2 sensor period 1 O2 generation. After 4 min of light irradiation, in the presence of SPN0, SPN2.5, and SPN5 (based on the synthesis in Example 10), the fluorescence intensity of SOSG at 528 nm increased by 6.44, 7.78, and 9.96 times, respectively, compared to before light irradiation. Figure 62 This data demonstrates that the higher the amount of TPP, the more it is produced during light irradiation. 1 The more O2, the better. Furthermore, when measured under O2 saturation conditions, the afterglow intensity of SPN2.5 can be increased by 1.90 times, while when measured under N2 saturation conditions, it can be decreased by 4.06 times. Figure 63 ).join in 1 O2 scavenger (NaN3) can reduce afterglow intensity by 1.58 times. Therefore, singlet oxygen (NaN3) generated under light irradiation 1O2 oxidizes the vinylidene bonds of PPV, forming an unstable PPV-dioxane intermediate that generates photons upon degradation. Quantitative data further indicate that the afterglow intensity of SPN2.5 is ~6.12 and ~2.14 times that of SPN0 and SPN5, respectively, consistent with their fluorescence intensity at 720 nm. Figure 57 e). Additionally, the afterglow luminescence half-life of SPN2.5 at room temperature is 5 min ( Figure 57 f), for which the imaging acquisition time is sufficiently long. These data indicate that incorporating TPP into the PPV framework amplifies and red-shifts the afterglow signal. SPN2.5 was chosen for the in vivo imaging experiments because it has the brightest afterglow intensity among SPN-PPV-TPP.

[0279] Example 12 In vivo evaluation of SPN-PPV-TPP Next, the in vivo evaluation of SPN-PPV-TPP (based on the synthesis in Example 10) was carried out according to the protocol in the biological experiment section.

[0280] In vivo imaging for differentiating between hypoxic and normoxic environments The deoxygenated SPN2.5 solution was injected locally into the tumor or under the skin. Figure 58 a). After excitation at 500 nm, fluorescence images of mice were acquired at 720 nm, while afterglow emission images were obtained using an open filter with a 30 s acquisition time after pre-irradiation with white light for 1 min. Figure 58 a). Signal quantification clearly showed that the afterglow intensity of the locally injected skin was 3.56 times higher than that of the tumor, while the fluorescence intensity between the skin and the tumor was almost the same ( Figure 58 b). This phenomenon is attributed to the hypoxic environment of the tumor, which has a low oxygen level that reduces... 1 The generation of O2 reduces the afterglow intensity of SPN2.5. These data suggest that the afterglow luminescence of SPN2.5 can be used to distinguish between hypoxic and normoxic environments in live mice.

[0281] In vivo peritoneal metastatic tumor imaging To test the ability of SPN2.5 in imaging metastatic tumor tissues, 4T1 cell suspensions (200 μL, 4 × 10⁻⁶ cells) were used. 5A mouse model of peritoneal metastatic tumors was established by intraperitoneal injection into nude mice. Four days after injection of cancer cells, mice were treated with SPN2.5 via the tail vein. Fluorescence and afterglow images were acquired immediately after the tail vein injection of SPN2.5. At t = 20 min post-injection, a significant afterglow signal (indicated by black circles) was observed from the mouse liver, while the fluorescence signal from the liver was only detectable after 1 h. Figure 59 a). This phenomenon is attributed to the higher tissue penetration of afterglow imaging compared to fluorescence imaging. Over time, the afterglow intensity (indicated by the white box) in the lower quadrant of the mouse gradually increases due to the accumulation of SPN2.5 at the tumor site. Figure 59 a and 59c). At t = 4 h post-injection, a significant afterglow signal was detected in the lower quadrant region of the mice, while no significant fluorescence signal was detected. Figure 59 a). At t = 4 h post-injection, the skin and peritoneum of the mice were removed, and the lower quadrant region of the mice was imaged using both afterglow and fluorescence. Due to the high sensitivity of afterglow, strong afterglow spots could be detected in the intestine, while only autofluorescence could be detected in the peritoneum. Figure 59 b). Histological examination further confirmed the presence of very small metastatic tumors on the surface of the intestine that were not visible to the naked eye. Figure 59 d). These results suggest that SPN2.5 could be used as a potential afterglow imaging agent for in vivo detection of metastatic tumors.

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Claims

1. A polymer composite nanoparticle emitting near-infrared afterglow luminescence, said nanoparticle comprising: (a) Semiconductor polymers of formula I: (b) Optionally, an amphiphilic copolymer; and (c) Optionally, a small molecule dye having near-infrared emission, wherein: When present, the amphiphilic copolymer encapsulates the semiconductor polymer of Formula I and, when present, the small molecule dye; and In the polymer of Formula I: R1 to R3 and R5 independently represent equation C q H 2q+1 alkyl chains, wherein 1 ≤ q ≤ 50, R4 represents part of formula Ia or formula Ib: Where 1≤s≤50 and 10≤t≤500; Where 1≤u≤50 and 10≤v≤500; R6 represents C q H 2q+1 The alkyl chain, a portion of formula Ia or a portion of formula Ib, wherein q, s, t, u and v are as defined above; R7 represents the singlet oxygen sensitization region; Each of n, m, and o is greater than or equal to 0, and p is 0 or 1, wherein at least one of n, m, o, and p is greater than 0; A represents the part of Ic or Id: Among them, R8 and R 11 Independently represented by C q H 2q+1 alkyl chains, wherein: 1 ≤ q ≤ 50; R9 and R 12 Independently represented by C q H 2q+1 The alkyl chain, a portion of formula Ia or a portion of formula Ib, wherein q, s, t, u and v are as defined above; R 10 and R 13 It independently represents the singlet oxygen sensitization portion; When p is 1, then w, x, y, and z, if they exist, are independently greater than or equal to 0; and The small molecule dye is present when o and p are 0, and optionally when: o is greater than or equal to 20; p is 1 and x or z is greater than or equal to 20; The sum of o and x is greater than or equal to 20; The sum of o and z is greater than or equal to 20; (o+x) / (n+m+o+w+x)>0.05; or (o+z) / (n+m+o+y+z)>0.05; The amphiphilic copolymer is present when m, o, and p are 0, and optionally present when: (m+o+w) / (n+m+o+w+x)>0.1; or (m+o+y) / (n+m+o+y+z)>0.1; and The premise is that when n is greater than 0, one or more of m, o and p are also greater than 0.

2. The complex according to claim 1, wherein, n is 0.

3. The complex according to claim 1 or claim 2, wherein, p is 0.

4. The composite pigment according to claim 3, wherein, The amphiphilic copolymer is optionally present when the following conditions are met: m is greater than or equal to 20, and m / (n+m+o) is greater than 0.1 and R 6 C q H 2q+1 Where 1≤q≤50; m is greater than or equal to 20, and (m+o) / (x+y+z) is greater than 0.1, and R 6 It is a part of formula Ia or a part of formula Ib; or o is greater than or equal to 20, and (m+o) / (x+y+z) is greater than 0.1, and R 6 It is a part of formula Ia or a part of formula Ib.

5. The complex according to any one of claims 1 to 3, wherein, The amphiphilic copolymer is present.

6. The complex according to any one of the preceding claims, wherein, When present, each of n, m, o, w, x, y, and z independently has a value from 5 to 1000.

7. The complex according to any one of the preceding claims, wherein, The amphiphilic copolymer also includes a quenching portion that can be cleaved by the reactive portion at the test site, either in vitro or in vivo.

8. Semiconductor polymers of Formula I: In the polymer of Formula I: R1 to R3 and R5 independently represent equation C q H 2q+1 alkyl chains, wherein 1 ≤ q ≤ 50, R4 represents part of formula Ia or formula Ib: in, 1 ≤ s ≤ 50 and 10 ≤ t ≤ 500; Where 1≤u≤50 and 10≤v≤500; R6 represents C q H 2q+1 The alkyl chain, a portion of formula Ia or a portion of formula Ib, wherein q, s, t, u and v are as defined above; R7 represents the singlet oxygen sensitization region; Each of n, m, and o is greater than or equal to 0; p is 0 or 1; A represents the part of Ic or Id: Among them, R8 and R 11 Independently represented by C q H 2q+1 alkyl chains, wherein: 1 ≤ q ≤ 50; R9 and R 12 Independently represented by C q H 2q+1 The alkyl chain, a portion of formula Ia or a portion of formula Ib, wherein q, s, t, u and v are as defined above; R 10 and R 11 It independently represents the singlet oxygen sensitization portion; When p is 1, w, x, y, and z, if they exist, are independently greater than or equal to 0; The premise is: When n is greater than 0, one or more of m, o, and p are also greater than 0; and At least one of n, m, o, and p is greater than 0.

9. Use of the polymer composite nanoparticles according to any one of claims 1 to 7 in the preparation of an imaging agent, said imaging agent being used to diagnose lesions or diseases in deep tissues and / or organs using afterglow luminescence.

10. Use of the polymer composite nanoparticles as defined in claim 7 in a method for preparing an imaging agent for in vivo imaging of oxidative stress in the liver of a subject, the method comprising the steps of: Pre-irradiated polymer nanoparticles were supplied to a living organism, activated polymer nanoparticles were irradiated with an NIR laser, and afterglow luminescence in the liver was detected using an imaging system / device.

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