Long wavelength light excited triplet-triplet energy transfer and method thereof

Abstract

The present disclosure relates generally to various light-triggered drug release and light reactions, including reactions generally based on long wavelength light excited triplet-triplet energy transfer. In one aspect, the present disclosure relates to methods for absorbing energy in a photosensitizer, which energy is transferred through triplet-triplet energy transfer to cleave cleavable or other active moieties, thereby causing release of the releasable moieties. One aspect relates to a composition, a photosensitizer composition comprising a photosensitizer capable of activation from singlet-triplet (S-T) at a near infrared (NIR) wavelength, a cleavable moiety that accepts triplet-triplet energy transfer from a higher energy state photosensitizer to cause cleavage of the cleavable moiety, and a releasable moiety that can be released from the composition upon cleavage of the cleavable moiety. Such systems and methods can be used in a variety of biological or physical applications. Also disclosed are methods for making or using such systems, kits comprising such systems.

Description

Technical Field

[0001] The present disclosure generally relates to various light-triggered drug release and photoreactions, including reactions generally based on photoexcited triplet-triplet energy transfer. Background Art

[0002] Photolysis, also known as photouncaging or photocleavage reaction, has been used to control molecular functions or to release desired components using light irradiation. 1 It has been identified as a powerful method that is spatiotemporally controllable and has shown excellent strength in imaging, photocatalysis, photopharmacology, neuroscience, and drug delivery. Photocleavable prodrugs (PDs) with tailored structures consisting of photoremovable protecting groups (PPGs) and drug molecules have been developed for light-triggered precision drug excitation. 2,3 Following systemic administration of the photocleavable prodrug, localized photoirradiation can be applied to the disease lesion to specifically activate the prodrug in situ, reducing systemic toxicity and thereby increasing biocompatibility and efficacy. 4,5 In biomedical applications, near-infrared (NIR) light (650-900 nm) is ideal for photoactivated therapy due to its deep tissue penetration and low phototoxicity. 6,7 However, the low photon energy of NIR light cannot meet the requirements of commonly used PPGs such as nitrobenzene-based 8 , Coumarin 9-11 , and boron-dipyrromethene (BODIPY), etc. 12 The direct excitation threshold.

[0003] One of the strategies to achieve long-wavelength light-triggered prodrug photolysis is to develop photon upconversion systems. Lanthanide-doped upconversion nanoparticles (UCNPs) have emerged as a reliable platform to convert NIR light into UV / visible light, thus enabling long-wavelength light to excite short-wavelength photoresponsive PPGs. 13 ,14 However, since UCNPs show low absorption coefficient and cross-sectional area, and the efficiency of luminescence resonance energy transfer (LRET) between UCNPs and PPG is still unsatisfactory, the required excitation power density is usually high (10 1 ~10 4 W / cm 2 Triplet-triplet annihilation-based upconversion (TTA-UC) is another strategy for long-wavelength light-triggered photolysis that relies on multi-step energy transfer between a photosensitizer and an annihilator to generate upconverted photons ( Figure 1 A). 15,16 TTA-UC can utilize low irradiation long wavelength light (10 -3 ~10 -1 W / cm 2), however, in many cases, the internal energy consumption during the multi-step energy transfer process still leads to low quantum yield and photolysis efficiency.

[0004] To overcome these problems, the “single-photon upconversion photolysis” strategy can sensitize the prodrug to its triplet excited state with a single low-energy photon ( Figure 1 B). 17,18 As a mechanism, long-wavelength light activates the photosensitizer (PS) from the ground state to a singlet excited state (S1), followed by intersystem crossing (ISC) to its triplet excited state (T1), and activates the prodrug via triplet-triplet energy transfer. This process is similar to photon upconversion, but only involves a single photon. So far, the anti-Stokes shift of this process remains limited (i.e., we can only use red light to activate green light-responsive photolysis) because the photon energy needs to be higher than the S1 state of the PS, and only PS with a low singlet energy level can be used (e.g., platinum or palladium coordinated porphyrins). Therefore, it is important to develop a new mechanism for upconversion photolysis that overcomes the limitation that the photon energy must be higher than the S1 state of the PS by utilizing a PS with singlet to triplet (ST) absorption (STPS). Summary of the invention

[0005] The present disclosure generally relates to various light-triggered drug release and photoreactions based on photoexcited triplet-triplet energy transfer, preferably direct singlet-to-triplet activation of photosensitizers at near infrared (NIR) wavelengths.

[0006] In one aspect, the present invention generally relates to compositions. In one set of embodiments, the composition comprises: a photosensitizer capable of being activated from a singlet-triplet state (ST) at near infrared (NIR) wavelengths, a cleavable moiety capable of accepting triplet-triplet energy transfer from a higher energy state of the photosensitizer to cause cleavage of the cleavable moiety, and a releasable moiety that can be released from the composition after cleavage of the cleavable moiety.

[0007] In some embodiments, the present disclosure provides a composition comprising: a photosensitizer capable of being activated from a singlet-triplet state (ST) at a near infrared (NIR) wavelength, and a prodrug comprising a cleavable portion and a releasable portion, wherein the cleavable portion accepts triplet-triplet energy transfer from a higher energy state of the photosensitizer to cause cleavage of the cleavable portion and release of the releasable portion.

[0008] In some embodiments, the composition comprises a carrier material containing a photosensitizer and a prodrug comprising an active part (e.g., a drug) and a releasable part. In some embodiments, the absorption of incident photons by the photosensitizer causes energy transfer to the photosensitizer, which is then transferred to the active part, thereby causing a chemical reaction in the active part. In some embodiments, the active part is a prodrug comprising a cleavable part and a releasable part. In one group of embodiments, the composition comprises a photosensitizer, which can absorb photons under near-infrared (NIR) wavelengths, resulting in activation from singlet-triplet (ST) to produce a higher energy state, followed by triplet-triplet energy transfer from the photosensitizer to the cleavable part in the active part, thereby causing the cleavage of the cleavable part and releasing the releasable part from the composition. According to another group of embodiments, the composition comprises a photosensitizer, which can directly sensitize the cleavable part via a triplet-triplet transfer process (TTET).

[0009] In one embodiment, the composition comprises an annihilating agent. In one embodiment, the composition does not comprise an annihilating agent.

[0010] On the other hand, the present invention generally relates to a method. According to one group of embodiments, the method includes absorbing photons by a photosensitizer at a near infrared-NIR wavelength to cause activation of the photosensitizer from a singlet state to a triplet state (ST), transferring energy directly from the photosensitizer to an active portion via triplet-triplet energy transfer, generating an excited state of a cleavable portion in the active portion via triplet-triplet energy transfer, and using the transferred energy to cause a chemical reaction in the active portion. In some embodiments, the photosensitizer is activated from a singlet (S0) state to a triplet (T1) state. In some embodiments, the photosensitizer is directly activated from a singlet (S0) state to a triplet (T1) state.

[0011] In another set of embodiments, the method comprises applying to the subject a composition comprising a photosensitizer, a cleavable moiety capable of receiving triplet-triplet energy transfer from the photosensitizer to cause cleavage of the cleavable moiety, and applying light to at least a portion of the subject to cause cleavage of the cleavable moiety.

[0012] In another set of embodiments, the method comprises applying to the subject a composition comprising a photosensitizer capable of being activated from a singlet-triplet state (ST) when exposed to near infrared (NIR) wavelengths and a prodrug comprising a cleavable portion and a releasable portion, wherein the cleavable portion accepts triplet-triplet energy transfer from a higher energy state of the photosensitizer to cause cleavage of the cleavable portion and release of the releasable portion; and applying near infrared (NIR) light to the subject to cause cleavage of the cleavable portion and release of the releasable portion. In some embodiments, triplet-triplet energy is not transferred via an annihilator.

[0013] In still another set of embodiments, the method comprises applying to the subject a composition comprising a photosensitizer, a cleavable moiety, and a carrier material, and applying near infrared (NIR) wavelength light to the subject. In some embodiments, light absorption by the photosensitizer from a singlet state to a triplet state (ST) results in energy transfer from the triplet state of the photosensitizer to the cleavable moiety to cause cleavage of the cleavable moiety and release of the releasable moiety.

[0014] In still another set of embodiments, the method comprises applying a composition comprising a photosensitizer, a prodrug comprising a cleavable portion and a releasable portion, and a carrier material to a tumor of a subject, and applying light to at least a portion of the tumor, wherein absorption of light by the photosensitizer causes energy transfer to the cleavable portion to cause cleavage of the cleavable portion and release of the releasable portion. In one embodiment, the releasable portion is a drug. In one embodiment, the releasable portion comprises a drug. In one embodiment, the releasable portion is an anticancer drug.

[0015] In some embodiments applicable to all compositions and methods of the present disclosure, the photosensitizer is activated from a singlet (S0) state to a triplet (T1) state. In some embodiments, the photosensitizer is activated directly from a singlet (S0) state to a triplet (T1) state. In some embodiments, triplet-triplet energy transfer is from a triplet (T1) state of the photosensitizer to a cleavable moiety. In some embodiments, the triplet (T1) state of the photosensitizer is higher than the triplet state of the cleavable moiety.

[0016] In some embodiments applicable to all compositions and methods of the present disclosure, the photosensitizer is activated from a singlet-triplet state (ST) at a near infrared (NIR) wavelength or when exposed to near infrared (NIR) light, preferably from 650 to 900 nm. In some embodiments, the wavelength of the near infrared (NIR) light is between about 650 nm and about 750 nm. In some embodiments, the wavelength of the near infrared (NIR) light is about 690 nm. In some embodiments, the excitation wavelength of the photosensitizer is between about 650 nm and about 750 nm. In some embodiments, the excitation wavelength of the photosensitizer is about 690 nm.

[0017] In some embodiments applicable to all compositions and methods of the present disclosure, the photosensitizer comprises a transition metal-porphyrin. In some embodiments, the transition metal is osmium (Os). In one embodiment, the photosensitizer is Os(II) bromophenyl terpyridine complex (Os(bptpy)2 2+ ).

[0018] In some embodiments, the cleavable moiety in the prodrug is photocleavable. In some embodiments, the cleavable moiety is BODIPY. In some embodiments, the prodrug comprises a drug attached to BODIPY.

[0019] In some embodiments, the releasable portion in the prodrug is a drug. In some embodiments, the releasable portion in the prodrug comprises a drug or a drug derivative. In some embodiments, the drug is an anti-inflammatory drug, an anticancer drug or an anti-angiogenic drug. In some embodiments, the drug is selected from the group consisting of the following: chlorambucil (CAB), vadimezan, indomethacin, naproxen, ibuprofen, benzyloxycinnamic acid, tetracaine, dopamine, tyramine and homoveratrylamine.

[0020] In another aspect, the present invention encompasses methods of preparing one or more embodiments described herein, e.g., compositions comprising a photosensitizer and a cleavable moiety. In still another aspect, the present invention encompasses methods of using one or more embodiments described herein, e.g., compositions comprising a photosensitizer and a cleavable moiety. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 (A-D). Schematic diagram of the photolysis strategy reported in this work and the mechanism of NIR light-triggered upconversion-like photolysis with one-step energy transfer. (A) Mechanism of TTA-UC-mediated photolysis. (B) Mechanism of red light-triggered upconversion-like photolysis. (C) Mechanism of NIR light-triggered upconversion-like photolysis with one-step energy transfer. (D) Os(bptpy)2 2+ The S1 and T1 energy levels and chemical structures of BODIPY-Cb (PS: photosensitizer; An: annihilator; PD: prodrug).

[0022] Figure 2 (A~F). (A) Normalized BODIPY-Cb and Os(bptpy)2 2+ Absorption spectrum in dichloromethane. (B) Os(bptpy)2 2+ (10 μM) Phosphorescence quenching in the presence of different concentrations of BODIPY-Cb, ex = 492 nm, in N2-saturated toluene. (C) Stern-Volmer plot of phosphorescence quenching based on different concentrations of BODIPY-Cb. (D) With or without Os(bptpy)2 2+(expressed as Os) BODIPY-Cb photolysis reaction under 690nm light irradiation for different lengths of time. (E) BODIPY-Cb photolysis rate after 690nm light irradiation for different lengths of time. (F) Cb generation rate after 690nm light irradiation for different lengths of time. (Solution: N2-saturated methanol; BODIPY-Cb concentration: 10 -3 M; Light irradiation: 690nm, 100mW / cm 2 ; n=3).

[0023] Figure 3 (A~B). Different prodrugs in Os(bptpy)2 2+ Upconversion photolysis in the presence of 690 nm light irradiation. (A) Reaction and conditions of the photolysis reaction. (B) Chemical structure of BODIPY prodrug. The percentage represents the photolysis yield of the corresponding prodrug.

[0024] Figure 4 (A-F). (A) Schematic diagram of NIR light-triggered drug release from nanoparticles (Os / BC nanoparticles). (B) Size distribution of various nanoparticles. (C) TEM image of Os / BC nanoparticles. (D) Normalized absorption of BC nanoparticles and Os / BC nanoparticles in aqueous solution. The red area indicates the NIR window. (E) The percentage of uncleaved BODIPY-Cb and Cb released from Os / BC nanoparticles after 690 nm light irradiation for 0-30 min (n=3). (F) The percentage of uncleaved BODIPY-Cb and Cb released from BC nanoparticles after 690 nm light irradiation for 0-30 min (n=3).

[0025] Figure 5 (A-E). (A) Schematic diagram of Os nanoparticles, BC nanoparticles and Os / BC nanoparticles, and the Jablonski diagram of their energy transfer process after NIR-light irradiation. (B) Cytotoxicity of Os nanoparticles, BC nanoparticles and Os / BC nanoparticles to HeLa cells when protected from light. (C) Cytotoxicity of Os nanoparticles, BC nanoparticles and Os / BC nanoparticles to HeLa cells under light irradiation (n=5). (D) Calcein-AM / PI staining of HeLa cells after treatment with Os nanoparticles, BC nanoparticles and Os / BC nanoparticles under light irradiation. Scale bar: 20 μm. (E) Apoptosis study of HeLa cells after treatment with Os nanoparticles, BC nanoparticles and Os / BC nanoparticles under light irradiation. Light irradiation: 690 nm, 100 mW / cm 2 , 30min.

[0026] Figure 6(AH). (A) Representative IVIS fluorescence images of mice within 24 h after injection of free DiR and DiR nanoparticles (n=3). The red dashed circle indicates the tumor area. (B) Quantitative analysis of biodistribution in major organs and tumors determined by IVIS. Tu, He, Lu, Sp, Li and Ki represent tumor, heart, lung, spleen, liver and kidney, respectively. ***p<0.005. (C) Schematic diagram of treatment plan. (D) Photographs of mice irradiated with NIR light at the tumor site. (E) Tumor volume of each group (n=5) (lamp parameters: 690 nm, 300 mW / cm 2 , 10min). (F) Photos of tumors removed on day 13 after different treatments. (G) Body weight of each group. (H) H&E staining of representative tumor sections of different treatment groups. Scale bar: 200μm.

[0027] Figure 7 (A~B). (A) Photolysis rate of BODIPY-Cb in air or N2-saturated solution after 530nm light irradiation for different time periods. (B) Cb generation rate in air or N2-saturated solution after 530nm light irradiation for different time periods. (BODIPY-Cb concentration: 10 -3 M; Light irradiation: 50mW / cm 2 ; n=3).

[0028] Figure 8 .Os(bptpy)2 at 0.1 2+ HPLC elution curves of BODIPY-Cb in the presence and absence of NIR light irradiation. (Prodrug concentration: 10 -3 M; lamp parameters: 690nm, 100mW / cm 2 , 30min; time length: 0-8min; solution: 88% methanol, 10% dichloromethane, 2% acetone, N2 saturated; λ abs =260nm).

[0029] Fig. 9 . At 0.1 equivalent Os(bptpy)2 2+ HPLC elution curves of free DMXAA and BODIPY-DMXAA in the presence and absence of NIR light irradiation. (Prodrug concentration: 10 -3 M; lamp parameters: 690nm, 100mW / cm 2 , 30min; time length: 5min; solution: 88% methanol, 10% dichloromethane, 2% acetone, N2 saturated; λ abs =254nm).

[0030] Fig.10 . At 0.1 equivalent Os(bptpy)22+ HPLC elution curves of free IDM and BODIPY-IDM in the presence and absence of NIR light irradiation. (Prodrug concentration: 10 -3 M; lamp parameters: 690nm, 100mW / cm 2 , 30min; time length: 5min; solution: 88% methanol, 10% dichloromethane, 2% acetone, N2 saturated, λ abs =254nm).

[0031] Fig.11 . At 0.1 equivalent Os(bptpy)2 2+ HPLC elution curves of free NPX and BODIPY-NPX in the presence and absence of NIR light irradiation. (Prodrug concentration: 10 -3 M; lamp parameters: 690nm, 100mW / cm 2 , 30min; time length: 5min; solution: 88% methanol, 10% dichloromethane, 2% acetone, N2 saturated; λ abs =260nm).

[0032] Fig.12 . At 0.1 equivalent Os(bptpy)2 2+ HPLC elution curves of free IBF and BODIPY-IBF in the presence and absence of NIR light irradiation. (Prodrug concentration: 10 -3 M; lamp parameters: 690nm, 100mW / cm 2 , 30min; time length: 5min; solution: 88% methanol, 10% dichloromethane, 2% acetone, N2 saturated; λ abs =254nm).

[0033] Fig.13 . At 0.1 equivalent Os(bptpy)2 2+ HPLC elution curves of free BCA and BODIPY-BCA in the presence and absence of NIR light irradiation. (Prodrug concentration: 10 -3 M; lamp parameters: 690nm, 100mW / cm 2 , 30min; time length: 5min; solution: 88% methanol, 10% dichloromethane, 2% acetone, N2 saturated; λ abs =254nm).

[0034] Fig.14 . At 0.1 equivalent Os(bptpy)2 2+ HPLC elution curves of free TCI and BODIPY-TCI in the presence and absence of NIR light irradiation. (Prodrug concentration: 10-3 M; lamp parameters: 690nm, 100mW / cm 2 , 30min; time length: 5min; solution: 88% methanol, 10% dichloromethane, 2% acetone, N2 saturated; λ abs =280nm).

[0035] Fig.15 . At 0.1 equivalent Os(bptpy)2 2+ HPLC elution curves of free DPA and BODIPY-DPA in the presence and absence of NIR light irradiation. (Prodrug concentration: 10 -3 M; lamp parameters: 690nm, 100mW / cm 2 , 30min; time length: 5min; solution: 88% methanol, 10% dichloromethane, 2% acetone, N2 saturated; λ abs =280nm).

[0036] Fig.16 . At 0.1 equivalent Os(bptpy)2 2+ HPLC elution curves of free TyrA and BODIPY-TyrA in the presence and absence of NIR light irradiation. (Prodrug concentration: 10 -3 M; lamp parameters: 690nm, 100mW / cm 2 , 30min; time length: 5min; solution: 88% methanol, 10% dichloromethane, 2% acetone, N2 saturated; λ abs =280nm).

[0037] Fig.17 . At 0.1 equivalent Os(bptpy)2 2+ HPLC elution curves of free HVA and BODIPY-HVA in the presence and absence of NIR light irradiation. (Prodrug concentration: 10 -3 M; lamp parameters: 690nm, 100mW / cm 2 , 30min; time length: 5min; solution: 88% methanol, 10% dichloromethane, 2% acetone, N2 saturated; λ abs =280nm).

[0038] Fig.18 . At 0.1 equivalent Os(bptpy)2 2+ HPLC elution curves of free Cb and BODIPY-Cb in the presence and absence of NIR light irradiation. (Prodrug concentration: 10 -3 M; lamp parameters: 690nm, 100mW / cm 2, 30min; time length: 5min; solution: 88% methanol, 10% dichloromethane, 2% acetone, N2 saturated; λ abs =260nm).

[0039] Fig.19 .Size and PDI of Os / BC nanoparticles in PBS at 37°C for 72h.

[0040] Fig. 20 . Representative fluorescence images of tumors and major organs 24 h after injection of free DiR and DiR-labeled nanoparticles.

[0041] Fig.21 .Tumor weight at the end of the in vivo experiment (day 13).

[0042] Fig. 22 .H&E staining of the main organ sections of mice after different treatments. Scale bar: 200μm.

[0043] Fig.23 .Os(bptpy)2·2PF6 1 H-NMR spectrum.

[0044] Fig.24 .MALDI-TOF MS spectrum of Os(bptpy)2·2PF6.

[0045] Fig.25 .BODIPY-Cb 1 H-NMR spectrum.

[0046] Fig.26 .BODIPY-DMXAA 1 H-NMR spectrum.

[0047] Fig. 27 .BODIPY-IDM 1 H-NMR spectrum.

[0048] Fig.28 .BODIPY-NPX 1 H-NMR spectrum.

[0049] Fig.29 .BODIPY-IBF 1 H-NMR spectrum.

[0050] Fig.30 .BODIPY-BCA 1 H-NMR spectrum.

[0051] Fig.31 .BODIPY-TCI 1 H-NMR spectrum.

[0052] Fig.32 .BODIPY-DPA 1 H-NMR spectrum.

[0053] Fig.33 .BODIPY-TyrA 1 H-NMR spectrum.

[0054] Fig.34 .BODIPY-HVA 1 H-NMR spectrum.

[0055] Fig.35 .BODIPY2-Cb 1 H-NMR spectrum.

[0056] Fig.36 (A-F). (A-E) BODIPY-OH photocage under 530 nm green light, 625 nm red light (with / without PtTPBP) and 690 nm NIR light (with / without Os(bptpy)2 2+ HPLC traces under 100mW / cm 2 , 0-7 min) (DAD detector, 540 nm). (F) Normalized residual amount of BODIPY-OH in different groups. DETAILED DESCRIPTION

[0057] Prodrug photolysis can control drug release in time and space at the desired lesion. For photoactivated therapy, near-infrared (NIR) light is preferred because of its deep tissue penetration and low phototoxicity. However, most photocleavable groups cannot be directly activated by NIR light. This article provides a new class of up-conversion processes that trigger prodrug photolysis by NIR light only via one-step energy transfer. Photosensitizers can be activated via singlet-triplet (ST) absorption, and photolysis of prodrugs based on boron-dipyrromethene (BODIPY) is achieved via triplet-triplet energy transfer. Using this strategy, NIR light can achieve green light response photolysis with a single-photon process. Design and show that a variety of drugs and bioactive molecules are released with high yields (up to 87%) under low-intensity NIR light (100mW / cm2, 5min). In addition, a micellar nanosystem encapsulating both photosensitizers and prodrugs has been developed to show the practicality of the present disclosure in a normoxic aqueous environment for cancer therapy. The present disclosure can be used to advance the development of photocleavable prodrugs and photoresponsive drug delivery systems for photoactivated therapy.

[0058] This paper discloses a novel mechanism of upconversion-like photolysis, which overcomes the limitation that the photon energy must be higher than the S1 state of PS by utilizing PS with singlet to triplet (ST) absorption (STPS). Since STPS can be directly activated from S0 to T1 state, photons with energy higher than T1 of STPS are sufficient to initiate upconversion-like photolysis, which greatly reduces the excitation energy level required to achieve NIR light activation ( Figure 1 C). Multiple steps of energy transfer can be bypassed, resulting in less internal energy loss and higher photolysis effect. In the presence of STPS, a series of short-wavelength light-responsive photocaged conjugated prodrugs, including chlorambucil, valdemethoxazole, indomethacin, naproxen, ibuprofen, benzyloxycinnamic acid, tetracaine, dopamine, tyramine and homoveratrol prodrugs were uncaged with high yield (up to 87%) by low-intensity and short-duration NIR light (100 mW / cm2, 5 min). Surprisingly, the drug release yield was even higher than that of drug release directly triggered by short-wavelength light. This strategy was validated in vivo using micellar nanosystems for light-triggered drug release and light-activatable cancer therapy. This study provides a new strategy to trigger prodrug photolysis with high yield using low-energy photons of long-wavelength light, indicating its potential for advanced light-activated therapies.

[0059] The present disclosure generally relates to various photoreactions, including reactions generally based on triplet-triplet energy transfer processes (TTET) from photosensitive and photocleavable groups. The present disclosure relates in one aspect to the following systems and methods, which are used to absorb energy (e.g., from photons) from a singlet state to a triplet state (preferably from an S0 state to a T1 state) in a photosensitizer, and transfer the energy to a prodrug comprising a cleavable portion and a releasable portion by triplet-triplet energy transfer, to result in cleavage of the cleavable portion and release of the releasable portion. In some embodiments, the releasable portion is or comprises a drug.

[0060] In some embodiments, the photosensitizer and the cleavable moiety can be contained in a suitable carrier material, for example, in a particle or micelle. Such systems and methods can be used for various applications, including various biological or physical applications. For example, such systems and methods can be used to deliver drugs or other releasable moieties to regions within a subject's body. Other aspects of the invention generally relate to methods for preparing or using such systems, kits comprising such systems, etc.

[0061] It should be understood that, as known to those of ordinary skill in the art, the term "triplet" generally refers to the electronic state of a molecule, rather than the number of electrons present in the molecule. For example, in a triplet state, a molecule may have unpaired electrons, so that the net spin of the molecule is 1. Molecules absorb energy, for example, by absorbing photons, which may cause electrons in the molecule to be "promoted" from a lower energy state (or shell) to a higher energy state (or shell), which may change the net spin of the molecule, and the emission or transfer of this energy may allow the higher energy electrons to return to a lower state. In some embodiments, absorbing photons causes electrons in the molecule to be promoted from a singlet (S) stage to a triplet (T) stage. In some embodiments, the singlet stage is a ground state stage, such as an S0 stage. In some embodiments, the triplet state is a T1 state. In some embodiments, the photosensitizer is activated directly from the S0 state to the T1 stage.

[0062] In some embodiments, the energy from the triplet state of the photosensitizer can be transferred to the cleavable part in the prodrug comprising a cleavable part and a releasable part. This type of energy transfer may involve a variety of mechanisms, such as triplet-triplet energy transfer (TTET) etc. For example, triplet-triplet energy transfer can be carried out by the exchange of electrons with different rotations and energies, for example, between two molecules (such as between a cleavable part and a photosensitizer, etc.). Then, due to the energy from the photosensitizer, the cleavable part can be cleaved, and preferably releases the releasable part, which is preferably a medicine. Energy transfer to the cleavable part can be carried out by a variety of processes.

[0063] In some embodiments, prodrug contains cleavable part and releasable part.In some embodiments, the cleavage of cleavable part can cause the rupture of one or more keys (for example, covalent bond) in or connected to the cleavable part.In some embodiments, the cleavage of cleavable part causes the rupture of the key between cleavable part and releasable part.In some embodiments, the cleavage of cleavable part can cause a part of the part to be separated from, for example, the releasable part.Therefore, in this way, absorbing photons (for example, via photosensitizer) can produce a series of events leading to the release of the releasable part of, for example, medicine.Therefore, by controlling the incident light, the release of the releasable part can be controlled as needed.However, it should be understood that the releasable part is not necessary, for example, the cleavage of the cleavable part may cause other chemical or structural changes in the cleavable part.In addition, it should be understood that energy can be transferred to other active parts instead of cleavable parts, for example, energy may cause photoisomerization, rearrangement, photocycloaddition or other chemical reactions.

[0064] Therefore, in one set of embodiments, a composition comprising a photosensitizer and a cleavable moiety (or other active moiety) can be applied to an area (e.g., within a sample, within a subject, etc.), and near infrared (NIR) light is applied to the area (or at least a portion of the area) to cause cleavage of the cleavable moiety, for example, to cause a chemical change to release the releasable moiety, etc. As described above, other active moieties can also be used. For example, if the active moiety is a cleavable moiety, the releasable moiety can be a drug, and light can be applied to cause the release of the drug. In one embodiment, the transferred energy causes a photoreaction of the active moiety (e.g., photoisomerization, rearrangement, photocleavage, or other chemical reaction). In one embodiment, the photoreaction further triggers a structural or compositional change in the composition containing the carrier material.

[0065] In certain embodiments, the encapsulated drug or other molecule / object can be released. As another non-limiting example, the releasable portion can be a tracer (e.g., a radioactive tracer, an inert molecule, a detectable entity, etc.) that can be introduced into a system (e.g., a biological system such as a cell or organism, etc., or a non-biological system such as a polymer, etc.), which is released at an appropriate time (e.g., by applying light), for example, rather than immediately releasing after applying or adding the composition. The tracer can then be detected using any suitable technology, for example, fluorescence, radioactivity, bioanalysis, chemistry or enzyme activity, etc.

[0066] In some embodiments, components such as photosensitizers and / or cleavable moieties can be contained in suitable carrier materials using physical encapsulation or chemical conjugation. In some embodiments, the component is on the surface of the carrier material. In some embodiments, the component is part of the carrier material. In some embodiments, the carrier material can keep the photosensitizer and / or cleavable moieties in close proximity to each other, for example, to allow electron and / or photon transfer as discussed herein. For example, in one embodiment, the photosensitizer and / or cleavable moieties can be contained in particles, such as microparticles or nanoparticles. In some embodiments, the particles can contain a certain environment (e.g., a hydrophobic or non-polar environment), for example, to keep the photosensitizer or cleavable moieties in close proximity, thereby promoting the transfer of electrons and / or photons, etc.

[0067] 5.1.1 Photosensitizers

[0068] In some embodiments of the present disclosure, the composition includes a photosensitizer. The photosensitizer can be any composition capable of absorbing photons to produce a higher energy state. Energy can be transferred to a cleavable portion. In some embodiments, the photosensitizer can absorb light of near infrared (NIR) wavelengths, for example, from about 650nm to about 900nm. In some embodiments, the excitation wavelength of the photosensitizer is at near infrared (NIR) energy, for example, from about 650nm to about 900nm. As a non-limiting example, the excitation wavelength of the photosensitizer (i.e., the photosensitizer is activated or undergoes activation from a singlet state-triplet state (ST)) is at a near infrared (NIR) wavelength of about 650nm to 700nm, about 700 to 750nm, about 750nm to 800nm, about 800nm ​​to 850nm, or about 850nm to 900nm.

[0069] In some embodiments, the photosensitizer is capable of absorbing near infrared (NIR) light (i.e., the excitation wavelength is at near infrared (NIR) energy), for example, about 600-650 nm, about 610-620 nm, about 620-630 nm, about 630-640 nm, about 640-650 nm, about 650-660 nm, about 660-670 nm, about 670-680 nm, about 680-690 nm, about 690-700 nm, about 700-710 nm, about 710-720 nm, about 720-730 nm, about 7 800nm, about 880nm-890nm, about 890nm-900nm, about 800nm-910nm, about 810nm-820nm, about 820nm-830nm, about 830nm-840nm, about 840-850nm, about 850-860nm, about 860-870nm, about 870-880nm, about 880-890nm, about 890-900nm, or about 900nm.

[0070] It will be appreciated that the photosensitizer may be excited by light of a single wavelength (eg, monochromatic light, such as would be supplied by a laser, etc.), or by light of different wavelengths (eg, from a light source producing a range of wavelengths).

[0071] In some embodiments, the photosensitizer is a fluorophore. In some embodiments, the photosensitizer comprises a transition metal. In some embodiments, the photosensitizer is a transition metal that can be activated from a singlet-triplet state (ST) at a near infrared (NIR) wavelength. In some embodiments, the photosensitizer is a transition metal that can be activated from a singlet state to a triplet state (S0-T1) at a near infrared (NIR) wavelength. In some embodiments, the photosensitizer is a transition metal that can be activated from a singlet state to a triplet state (S0-T1) at a near infrared (NIR) wavelength, that is, an electron is directly transferred from a ground state (S0) to a triplet state (e.g., T1) state. In some embodiments, the near infrared (NIR) wavelength is 690nm.

[0072] In some embodiments, the transition metal capable of near infrared (NIR) absorption or activation is osmium (Os) or its complex. In some embodiments, the transition metal capable of near infrared (NIR) absorption or activation is titanium (Ti), zirconium (Zr), vanadium (V) or nickel (Ni) or their complexes.

[0073] In one embodiment, the photosensitizer comprises a porphyrin or a porphyrin derivative, for example, a transition metal-porphyrin, such as Os porphyrin. Specific non-limiting examples of photosensitizers include osmium porphyrins, such as Os(II) bromophenyl terpyridine complex (Os(bptpy)2 2+ ), bis(2,2';6',2"-terpyridine)osmium(II) hexafluorophosphate, bis(4'-(pyridin-4-yl)-2,2':6',2"-terpyridine)osmium(II) hexafluorophosphate, bis(4,4',4"-tri-tert-butyl-2,2':6',2"-terpyridine)osmium(II) hexafluorophosphate, bis(4'-phenyl-2,2':6',2"-terpyridine)osmium(II) hexafluorophosphate, or bis(4'-(4-methoxyphenyl)-2,2':6',2"-terpyridine)osmium(II) hexafluorophosphate.

[0074] A variety of triplet photosensitizers are known to those of ordinary skill in the art; many of which are commercially available.

[0075] In some embodiments, the yield of the releasable moiety obtained by photolysis at near infrared (NIR) wavelengths is higher than the yield obtained when using a photosensitizer that absorbs green or red light irradiation or using red light. For example, in some embodiments, activation of the photosensitizer at near infrared wavelengths (i.e., by exposure to near infrared (NIR) wavelengths, e.g., 690 nm light) results in the release of about 50% to 55% of the cleavable moiety (e.g., drug). In some embodiments, about 55% to 60%, 60% to 65%, 65% to 70%, 70% to 75%, 75% to 80%, 80% to 81%, 81% to 82%, 82% to 83%, 83% to 84%, 84% to 85%, 85% to 86%, 86% to 87%, 87% to 88%, 88% to 89%, 89% to 90%, 90% to 91%, 91% to 92%, 92% to 93%, 93% to 94%, 94% to 95%, 95% to 96%, 96% to 97%, 97% to 98%, 98% to 99%, 99%, or about 100% of the cleavable moiety is released from the composition upon irradiation of the photosensitizer at a near infrared (NIR) wavelength.

[0076] For example, as contemplated in this paper, using Os(bptpy)2 2+ Near-infrared (NIR) light irradiation of BODIPY-Cb as a photosensitizer resulted in 84.17±4.21% release of free chlorambucil (Cb). In contrast, photolysis by green light irradiation (530 nm) or red light activation (625 nm) resulted in much lower yields of approximately 31.71% and 41.74%, respectively.

[0077] 5.1.2 Cleavable and releasable fractions

[0078] In some embodiments, the cleavable moiety receives triplet-triplet energy transfer from the photosensitizer. In some embodiments, the photosensitizer in the triplet state is in a higher energy state than the cleavable moiety in the triplet state. In some embodiments, the cleavable moiety receives triplet-triplet energy transfer to cause cleavage of the cleavable moiety and release of the releasable moiety.

[0079] In some embodiments, the cleavable moiety is photocleavable. In some embodiments, the cleavable moiety comprises boron-dipyrromethene (BODIPY) or a BODIPY derivative, or a fullerene or a fullerene derivative. In some embodiments, the cleavable moiety is a BODIPY derivative represented by the following structure:

[0080]

[0081] In some embodiments, energy transfer to the reactive moiety results in cleavage of a bond within or attached to the cleavable moiety, and / or a bond within or attached to a different moiety of the cleavable moiety, e.g., a releasable moiety.

[0082] In some embodiments, the composition comprises a prodrug comprising a cleavable portion and a releasable portion. In some embodiments, the composition comprises a therapeutically effective amount of the prodrug.

[0083] In some embodiments, provided herein are prodrugs comprising a cleavable moiety and a releasable moiety.

[0084] In some embodiments, the prodrug provided according to some embodiments of the present disclosure comprises a BODIPY derivative. In some embodiments, the prodrug provided according to some embodiments of the present disclosure is represented by the following structure:

[0085]

[0086] Wherein "cargo" means a releasable portion. In some embodiments, the "cargo" comprises a drug. In some embodiments, the cargo is derived from a drug.

[0087] In some embodiments, the cleavable moiety (e.g., BODIPY or a BODIPY derivative) is bonded to the drug via a cleavable chemical group such as an ester, carbamate, carbonate, amide, etc. In some embodiments, the prodrug is a BODIPY-ester prodrug represented by the following structure:

[0088]

[0089] wherein R is a drug or wherein R is derived from a drug.

[0090] In some embodiments, the prodrug is a BODIPY-carbamate prodrug represented by the following structure:

[0091]

[0092] wherein R is a drug or wherein R is derived from a drug.

[0093] In some embodiments, releasable part and cleavable part are connected by chemical groups such as ester, carbamate, carbonate, amide, etc. The cracking of the key (e.g., ester, carbamate) in the chemical group can cause the release of the releasable part, which can be a therapeutic agent or a medicine. However, it should be understood that in other embodiments, the cracking of a single bond does not necessarily require the release of the releasable part, for example, if more than one bond connects the multiple parts of the molecule together. In addition, in some embodiments, energy transfer to the active part can cause other chemical reactions in the active part, but does not necessarily cause the cracking of the cleavable bond. If present, the releasable part can be any suitable part that can be released, for example, during cracking (including photo-cracked).

[0094] In some embodiments, the releasable portion may include a portion of a prodrug or a portion of a cleavable portion. Different releasable portions may be used in various embodiments, depending on the application. For example, the releasable portion may include a drug, a tracer (e.g., a fluorescent or radioactive compound), a caged substance, a peptide or protein, a small molecule (e.g., a molecular weight less than about 1 kDa or about 2 kDa), etc. In some embodiments, the exact form of the releasable portion is not important, for example, if it is connected by a cleavable bond of a cleavable portion that is itself cleaved as described above; then the cleavage of the cleavable bond can thus cause the separation of the releasable portion, regardless of the exact composition of the releasable portion.

[0095] As a non-limiting example, the releasable moiety is or comprises a drug, such as an anticancer drug selected from the group consisting of chlorambucil (CAB), valdemethoxane, indomethacin, naproxen, ibuprofen, benzyloxycinnamic acid, tetracaine, dopamine, tyramine, and homoveratamine.

[0096] In some embodiments, the prodrug is represented by any of the following structures:

[0097]

[0098]

[0099]

[0100] In some embodiments, the present disclosure provides a compound represented by any of the following structures:

[0101]

[0102]

[0103]

[0104] In some embodiments, the compound may be a prodrug comprising a cleavable moiety and a releasable moiety.

[0105] In some embodiments, the present disclosure provides a compound represented by the structure of Formula 5. In some embodiments, the present disclosure provides a compound represented by the structure of Formula 6. In some embodiments, the present disclosure provides a compound represented by the structure of Formula 7. In some embodiments, the present disclosure provides a compound represented by the structure of Formula 8. In some embodiments, the present disclosure provides a compound represented by the structure of Formula 9. In some embodiments, the present disclosure provides a compound represented by the structure of Formula 10. In some embodiments, the present disclosure provides a compound represented by the structure of Formula 11. In some embodiments, the present disclosure provides a compound represented by the structure of Formula 12. In some embodiments, the present disclosure provides a compound represented by the structure of Formula 13.

[0106] In other embodiments, the releasable portion may include anti-angiogenic drugs, such as TNP-470 or Combretastatin A4, etc. In another embodiment, the releasable portion may include anti-inflammatory drugs, such as dexamethasone, etc. In still another group of embodiments, the releasable portion includes anticancer drugs and / or chemotherapeutics, such as chlorambucil, doxorubicin, topotecan, or verteporfin. In still another group of embodiments, the releasable portion may include fluorescent proteins, such as GFP or YFP, etc. In still another group of embodiments, the releasable portion may include fluorescent compounds, such as fluorescein, rhodamine, or calcein, etc. In still another group of embodiments, the releasable portion includes peptides or proteins, such as RGD peptides, etc. In another embodiment, the releasable portion may include radioactive atoms.

[0107] 5.1.3 Carrier

[0108] In some embodiments, the photosensitizer, active moiety (e.g., cleavable moiety), and / or releasable moiety (if present) are contained in a suitable carrier material. The carrier material can hold some or all of these substances in close proximity to each other (e.g., as discussed above). In some embodiments, the carrier material can create an environment that is conducive to the compound, such as those discussed herein as fluorescent, etc. For example, the carrier material can create an aqueous environment, a hydrophobic environment, a polar or non-polar environment, etc. In some embodiments, the carrier material creates a water-repellent environment.

[0109] In one group of embodiments, the carrier material is formed by a polymer. Any suitable polymer can be used. Examples of polymers include, but are not limited to, polylactic acid, polyglycolic acid, polyethylene oxide, polystyrene, polyethylene, polypropylene, etc. In some embodiments, the polymer can be biodegradable or biocompatible, for example, for various medical or biological applications. In some embodiments, more than one polymer can be used, and the polymer can be physically blended and / or chemically combined, for example, as a copolymer. As a non-limiting example, the carrier material can include a copolymer, such as poly (D, L-lactic acid)-poly (ethylene oxide) etc.

[0110] However, it should be understood that the carrier material need not be limited to polymeric materials. For example, in other embodiments, the carrier material may include silica, ceramics, or other materials.

[0111] The carrier material can be present in any suitable form. For example, the carrier material can be present as a film, as a block of material, as particles, as micelles, etc. In some embodiments, components such as photosensitizers, active moieties, and / or releasable moieties can be added or chemically conjugated to the carrier material during and / or after the carrier material is formed. The carrier material can be formed using any suitable technique; for example, techniques for producing polymers, silica gels, ceramics are known to those of ordinary skill in the art.

[0112] The average diameter of non-spherical particles can be taken as the volume of the perfect sphere with the same particle volume. If the carrier material exists as a film, the film can have any cross-sectional thickness. For example, the average thickness of the film can be less than about 1 mm, less than about 500 microns, less than about 300 microns, less than about 100 microns, less than about 50 microns, less than about 30 microns, less than about 10 microns, less than about 5 microns, less than about 3 microns, less than about 1 micron, less than about 500nm, less than about 300nm, less than about 100nm, less than about 50nm, less than about 30nm, less than about 10nm, etc.

[0113] The carrier material can also include one or more polymer micelles. The polymer micelle can have any suitable average diameter. For example, the average diameter of the micelle can be less than about 1 mm, less than about 500 microns, less than about 300 microns, less than about 100 microns, less than about 50 microns, less than about 30 microns, less than about 10 microns, less than about 5 microns, less than about 3 microns, less than about 1 micron, less than about 500nm, less than about 300nm, less than about 100nm, less than about 50nm, less than about 30nm, less than about 10nm, etc.

[0114] 5.1.4 Treatment methods

[0115] As mentioned, compositions such as those discussed herein can be used in a variety of applications, including biological and medical applications, as well as non-biological or non-medical applications. As a non-limiting example, in one set of embodiments, the compositions discussed herein can be applied to a subject. The subject can be a human or a non-human. For example, the subject can be a rat, a mouse, a rabbit, a goat, a cat, a dog, etc. The composition can also be applied to any suitable sample, for example, a biological sample, a physical sample, a chemical sample, etc.

[0116] Light can be applied to the composition to cause release of the releasable portion, if present. The light can be monochromatic light (e.g., laser or coherent light), or in some embodiments, the light can be non-monochromatic or incoherent. The light can have any suitable frequency, for example, including frequencies discussed herein, such as near infrared (NIR) wavelengths, etc.

[0117] In one set of embodiments, the composition is administered to a subject to treat a tumor.

[0118] The composition can be applied directly to the tumor, and / or systemically applied to the subject's body so that at least some of the composition can travel to the tumor (e.g., via the blood), so that light can be applied to the tumor (or a portion thereof) to, for example, cause the release of a releasable portion for diagnosis and / or treatment of the tumor. The composition may include, for example, anti-angiogenic drugs, anti-inflammatory drugs, radioactive substances, anticancer drugs, and / or chemotherapeutic drugs, and light can be applied to the tumor to cause release. Such applications can be directed, for example, by applying light directly to the tumor (or at least a portion thereof); therefore, the release elsewhere in the subject's body can be minimized by not applying light elsewhere. In this way, by applying light directly to the tumor (or a portion thereof), or at least near the tumor, the release of the drug (or other suitable release portion) can be controlled or confined to the tumor or near the tumor. In some embodiments, there may be more than one composition.

[0119] Other parts of the subject can also be treated in various embodiments. For example, the composition can be applied directly to a specific location in the subject's body, or systemically applied to the subject so that at least some of the composition can travel to the location where the light is applied. For example, the composition can be applied to the skin, eyes, body cavity (or blood), and light can be applied to a portion of the skin, eyes, body cavity or blood to cause local release of the releasable portion.

[0120] In various aspects, the compositions described herein can be applied by any suitable method, for example, contained in a solution or suspension, such as an inhalation solution, topical drops, eye drops, intranasal introduction agents, ointments for epidermal application, intravenous solutions, injection solutions (e.g., subcutaneous or intravenous), or suppositories, etc. In one group of embodiments, the composition is introduced parenterally or topically. For example, the composition can be contained in a cream, gel, or ointment applied to the skin. In some embodiments, the composition can be applied once or more per day, by less than once per day, or by continuous application, etc., once or more per day, until the desired therapeutic effect is achieved.

[0121] As mentioned, certain aspects of the present invention provide methods of administering any of the compositions disclosed herein to a subject. When administered, the compositions of the present invention are applied as a pharmaceutically acceptable formulation in a therapeutically effective, pharmaceutically acceptable amount. As used herein, the term "pharmaceutically acceptable" is given its ordinary meaning. Pharmaceutically acceptable compositions are generally compatible with the other materials of the formulation and are generally harmless to the subject. Any of the compositions of the present invention can be administered to a subject in a therapeutically effective dose. As used herein, a "therapeutically effective" amount means an amount necessary to delay the onset of a particular condition to be treated, inhibit the progression of a particular condition to be treated, completely stop the onset or progression of a particular condition to be treated, diagnose a particular condition to be treated, or otherwise achieve a medically desired result. When administered to a subject, the effective amount will depend on the particular condition to be treated and the desired outcome. The therapeutically effective dose can be determined by a person of ordinary skill in the art, for example, using factors such as those further described below and using only routine experimentation.

[0122] Any medically acceptable method can be used to administer the composition to the subject. Administration can be localized (i.e., to a specific area, physiological system, tissue, organ, or cell type) or systemic, depending on the condition to be treated. For example, the composition can be administered orally, vaginally, rectally, orally, or topically, or nasally, or transdermally, or by parenteral injection or implantation, or by surgical administration, or by any other method of administration. Examples of parenteral administration methods that can be used in the present invention include intravenous, intradermal, subcutaneous, intracavitary, intramuscular, intraperitoneal, epidural, or intrathecal. Examples of implantation methods include any implantable or injectable drug delivery system. The use of implants is particularly suitable in some embodiments of the present invention. Implants containing the composition can be constructed and configured to remain in the body for at least 2 to 4 hours, 4 to 12 hours, 12 to 24 hours, 24 to 48 hours, 1 to 7 days, 7 to 15 days, at least 30 or 45 days, preferably at least 60 or 90 days, or even longer in some cases. Long-term release implants are known to those of ordinary skill in the art.

[0123] In certain embodiments of the present invention, the composition can be combined with a suitable pharmaceutically acceptable carrier, for example, incorporated into a liposome, incorporated into a polymer release system, or suspended in a liquid, for example, in a dissolved form, or a colloidal form, or a micellar form. Generally, pharmaceutically acceptable carriers suitable for use in the present invention are known to those of ordinary skill in the art. Pharmaceutically acceptable carriers may include non-toxic materials that do not significantly interfere with the effectiveness of the biological activity of the active compound to be administered, but are used as, for example, to stabilize or protect the active compound in the composition before use, as a formulation component. The carrier can be organic or inorganic, and can be natural or synthetic, and one or more active compounds of the present invention are combined with the carrier to promote the application of the composition. The carrier can be soluble or insoluble, depending on the application.

[0124] In some embodiments, the kit of the present invention may include any form of instructions, which are provided together with the composition of the present invention in a manner that allows one of ordinary skill in the art to recognize that the instructions are associated with the composition of the present invention. For example, the instructions may include instructions for using, changing, mixing, diluting, preserving, applying, assembling, storing, packaging and / or preparing the composition and / or other compositions associated with the kit. In some embodiments, the instructions may also include instructions for the delivery and / or application of the composition.

[0125] The following examples are intended to illustrate certain embodiments of the invention, but are not intended to illustrate the full scope of the invention.

[0126] Example

[0127] 5.1 Preparation and characterization of osmium-based STPS and BODIPY-Cb prodrugs

[0128] BODIPY-chlorambucil prodrug (BODIPY-Cb) was prepared, which can release chlorambucil as an anticancer agent for photoactivated therapy under green light irradiation. 17 As STPS, the osmium(II) complex shows strong singlet to triplet absorption and can be excited directly from its ground state to the T1 state. 19 Here, the osmium-containing photosensitizer Os(II) bromophenyl terpyridine complex (Os(bptpy)2 2+ ) (see Supporting Information , Scheme S2).

[0129] As shown in the UV-visible spectrum, BODIPY-Cb prodrug exhibits a single absorption peak in the visible light region (peak at 543 nm, ε = 8.94 × 10 4 M -1 cm -1 ), indicating green light excitation. Os(bptpy)2 2+ It shows a broad peak in the visible to NIR region ( Figure 2 A). The absorption peak at about 492 nm is identified as the singlet metal to ligand charge transfer ( 1 MLCT) absorption, also known as singlet-singlet (SS) absorption. It is worth noting that the peak in the far infrared region (peak at 688nm, ε = 2.66×10 4 M -1 cm -1 ) was identified as triplet metal to ligand charge transfer ( 3 The two rotatable bromophenyl groups of the bromophenyl terpyridine ligand extend the ST absorption from 650 to 750 nm, accompanied by an increase in the absorption coefficient. BODIPY-Cb and Os(bptpy)2 2+ And more detailed photophysical properties of BODIPY-Cb are recorded in Table 1 of the Supporting Information.

[0130] Table 1. Photophysical properties of BODIPY-Cb and Os(bptpy)2·2PF6

[0131]

[0132] a In a dichloromethane solution containing 2% acetone, 10 μM;

[0133] b λ ex =543nm; c λ ex =491nm; d λex =688nm;

[0134] e,f,g In toluene, TD-DFT calculations were performed according to previous studies. 37,38 ;

[0135] h By the maximum S1 emission wavelength (λ ex ) calculation, E = 1240 / λ em,S1 ;

[0136] i By the maximum T1 emission wavelength (λ em ) calculation, E = 1240 / λ em,T1 .

[0137] Based on phosphorescence emission, Os(bptpy)2 2+ The T1 energy level of α was determined to be 1.69 eV, which is consistent with the density functional theory (DFT) calculations (Table 1). 20 Therefore, low energy NIR photons can be used to activate it.BODIPY-Cb shows a high singlet excited state (S1 = 2.18 eV) and a low triplet state (T1 = 1.52 eV). 17 Based on the energy level, the molecule is 2+ and BODIPY-Cb meet the energy requirements of TTET (T1(PS)>T1(PD)) and the energy requirements of the upconversion process (T1(PS)<S1(PD)). Figure 1 D). Apply NIR light and convert Os(bptpy)2 2+ After activation to the triplet state (1.69 eV), the photon energy can be transferred to the triplet state (1.52 eV) of the BODIPY prodrug and trigger its photolysis. 2+ TTET between BODIPY-Cb prodrug

[0138] Stern-Volmer phosphorescence quenching experiments were used to verify the 2+ TTET to BODIPY-Cb. It was found that the Os(bptpy)2 was quenched by titration of the BODIPY-Cb prodrug into its N2-saturated toluene solution. 2+ It was observed that as the concentration of BODIPY-Cb increased, Os(bptpy)2 2+ The phosphorescence reduction confirmed 3 Os(bptpy)2 2+* arrive 3 BODIPY1-Cb * Energy transfer ( Figure 2, B and C). TTET rate constant (k TTET ) is calculated as (2.72±0.14)×10 11 M -1 s -1 , indicating that from Os(bptpy)2 2+* Effective energy transfer from the T1 state of to the T1 state of BODIPY-Cb.

[0139] 6.3 Photolysis of BODIPY-based prodrugs via an upconversion-like process

[0140] The photolysis of BODIPY-Cb was studied by irradiating the solution with a 530 nm or 690 nm lamp and analyzing the products by high performance liquid chromatography (HPLC). Figure 2 D). It was observed that BODIPY-Cb can be cleaved under 530 nm green light because high-energy photons can directly activate the prodrug to the S1 state of BODIPY1-Cb, followed by cleavage relaxation and free drug generation ( Figure 7 ). It is noteworthy that photolysis is delayed in air-saturated solutions, suggesting that relaxation of the cleavage may occur from the T1 state quenched by oxygen. 2+ In the presence of N2, the decomposition of the prodrug and the generation of free Cb were observed after irradiation with 690 nm light in a solution saturated with N2 ( Figure 2 EF, Figure 8 This can be explained by the TTET process, in which BODIPY-Cb receives the 2+ The energy of T1 is then raised to the T1 state. In addition, when the solution contains Os(bptpy)2 2+ At the same time, the molar ratio of Os(bptpy)2 2+ In the presence of BODIPY-Cb at 100 mW / cm 2 After 5 min of photoirradiation, the decomposition was nearly complete (96.74 ± 1.26%), and free Cb was generated in a relatively high yield of 84.17 ± 4.21%. It should be noted that the yield of free drug was significantly higher than that of BODIPY-Cb photolysis with shorter wavelength light, including the yield of direct photolysis by green light irradiation (maximum yield of 31.71%) ( Figure 7 ) and the yield of single-photon-like upconversion photolysis by red light irradiation using a platinum photosensitizer (the maximum yield was 41.74%). 17This result demonstrates the advantages of utilizing low-energy NIR photons and simplifying the energy transfer process, i.e., reducing the photodamage of the prodrug and the unexpected relaxation of the excited state. The high photolysis yield of the free drug may lead to better efficacy of the prodrug after photoirradiation. In addition, as expected, the decomposition of BODIPY-Cb and the generation of free Cb in air-saturated solutions are greatly delayed because the T1 state is quenched by oxygen molecules (green line, Figure 2 E~F). For details, the quantum yield of photolysis (Ф p ), quantum yield of drug release (Ф r ) and cross-sectional areas are reported in Table S3.

[0141] Table S3. Quantum yield and cross-sectional area of ​​BODIPY-Cb prodrugs under different conditions

[0142]

[0143] Prodrug photolysis has emerged as a spatiotemporally controllable process for photochemical control of biological processes, and related research largely depends on the precise deprotection of bioactive molecules under light irradiation. 21-23 Inspired by the NIR-light-triggered photolysis of BODIPY-Cb, we extended this concept to the NIR-light-triggered release of different bioactive molecules, including: a) anticancer drugs, e.g., chlorambucil (Cb) and dimethoate (DMXAA); b) anti-inflammatory drugs, e.g., indomethacin (IDM), naproxen (NPX), ibuprofen (IBF), and benzyloxycinnamic acid (BCA); c) anesthetics, e.g., tetracaine (TCI); d) biogenic amines, e.g., dopamine (DPA), tyramine (TyrA), and homoveratrol (HVA) ( Figure 3 ). BODIPY photocages are conjugated to free drug molecules to form photoactivatable prodrugs with photolabile linkages (including esters and carbamates). Illumination can be performed with NIR lamps at 100 mW / cm 2 , 690nm for 5min, in the presence of 0.1 equivalent of Os(bptpy)2 2+ and 1 equivalent of prodrug in a N2-saturated solution. For BODIPY-chlorambucil (BODIPY-Cb, compound 4), the photolysis yield was measured to be 84.17±4.21%, as described above. For BODIPY-valdemexin (BODIPY-DMXAA, compound 5), the prodrug was completely consumed and the yield of free DMXAA was 82.52±7.22% ( Fig. 9For BODIPY-indomethacin (BODIPY-IDM, compound 6), BODIPY-naproxen (BODIPY-NPX, compound 7), BODIPY-ibuprofen (BODIPY-IBF, compound 8) and BODIPY-benzyloxycinnamic acid (BODIPY-BCA, compound 9), the photolysis yields were 68.95±4.69%, 84.32±5.79%, 48.49±4.69% and 87.12±3.17%, respectively ( Figures 10 to 13 ). It should be noted that the above-mentioned prodrugs (compounds 4 to 9) were constructed by combining BODIPY photocages and drug molecules having carboxyl groups with photolabile ester bonds. In addition, the photocages were conjugated with drug molecules having amine groups to prepare prodrugs having photolabile carbamate bonds. Thus, BODIPY-tetracaine (BODIPY-TCI, compound 10) showed a photolysis yield of 41.34 ± 4.61% after 5-min NIR-light irradiation ( Fig.14 For BODIPY-dopamine (BODIPY-DPA, compound 11), BODIPY-tyramine (BODIPY-TyrA, compound 12), and BODIPY-homoveratamine (BODIPY-HVA, compound 13), the photolysis yields were 69.04±1.99%, 46.57±2.37%, and 74.17±3.97%, respectively ( Figures 15 to 18 In addition, the structure of BODIPY2-Cb prodrug (compound 14) is similar to that of BODIPY-Cb, but without iodine insertion at positions 2 and 6, which is located at Os(bptpy)2 2+ The absence of iodine atoms in the presence of iodine did not show any photolysis yield upon exposure to NIR light, which could be explained by the lack of iodine atoms leading to low ISC efficacy and shortened T1 lifetime. 24 In addition, the quantum yields and cross-sectional areas of different prodrugs are listed in Table 4.

[0144] Table S4. Quantum yield and cross-sectional area of ​​BODIPY prodrug photolysis under NIR light

[0145]

[0146] a ε(λ ex )Here is the absorption coefficient of Os(bptpy)2·2PF6 at 688nm (ε=26600M -1 cm -1 ).

[0147] Compared with the content disclosed in US2021 / 0228719, the improvement is that the photolysis yield of free drugs is increased. In the present disclosure, 0.1 equivalent of Os(bptpy)2 2+ In the case of BODIPY-Cb at 100mW / cm 2 After 5 min of photoirradiation, it was completely decomposed, and free Cb was generated with a relatively high yield of 84.17 ± 4.21%. The yield of free drug was significantly higher than that of BODIPY-Cb photolysis with shorter wavelength light, including the yield of direct photolysis by green light irradiation (maximum yield of 31.71%) and the yield of upconversion photolysis by red light irradiation using PtTPBP photosensitizer (maximum yield of 41.74%) (disclosed in US2021 / 0228719). This observation can be explained by the reduction in photobleaching caused by photolysis using 690 nm NIR light instead of 530 nm green light or 625 nm red light. Fig.36 The different photobleaching rates of BODIPY-Cb under different light are shown.

[0148] 6.4 Photoactivatable Nanosystems for NIR Light-Triggered Drug Release

[0149] NIR light-triggered prodrug activation was then investigated in biological systems for medical applications. Considering that both prodrugs and photosensitizers are hydrophobic and the energy transfer process is oxygen-sensitive, it would be challenging to activate photolabile prodrugs in normoxic aqueous solution via an upconversion-like mechanism. Therefore, we loaded the prodrug and photosensitizer into polymer nanoparticles that can protect the triplet state from oxygen quenching and thus enable TTET-based photolysis and drug release in biological environments. The FDA-approved block copolymer, poly(lactic acid)-poly(ethylene glycol) (PLA) 5000 -mPEG 5000 ), for the construction of biocompatible and biodegradable nanoparticles ( Figure 4 A). 25 Dynamic light scattering (DLS) recorded the blank nanoparticles (blank nanoparticles), loaded with Os(bptpy)2 2+ Nanoparticles (Os nanoparticles), BODIPY-Cb loaded nanoparticles (BC nanoparticles) and Os (bptpy) 2 2+ The size of the nanoparticles with BODIPY-Cb (Os / BC nanoparticles) is about 50 nm (Table 5), which is also verified by TEM imaging ( Figure 4 B and C). Excellent colloidal stability of the nanoparticles was observed, and the size of these nanoparticles remained stable for at least 72 h at 37 °C ( Fig.19In addition, the absorption spectrum of Os / BC nanoparticles showed peaks in both the visible region (540 nm) and the NIR region (690 nm), indicating that Os(bptpy)2 2+ and BODIPY-Cb were successfully encapsulated in nanoparticles ( Figure 4 D, Table 6).

[0150] Table 5. Size and PDI of Os nanoparticles, BC nanoparticles and / or Os / BC nanoparticles

[0151]

[0152] Table 6. Composition of Os / BC nanoparticles

[0153]

[0154] Light-triggered drug release has attracted much attention in drug delivery and precision disease treatment. 26～28 At 100mW / cm 2 The prodrug activation and drug release from Os / BC nanoparticles were studied under 690 nm light irradiation. The release of free drug Cb and the consumption of BODIPY-Cb were clearly observed after light irradiation, indicating that upconversion-like photolysis occurred in Os / BC nanoparticles dispersed in normoxic aqueous solution. Quantitatively, the decomposition of prodrug BODIPY-Cb and the release of Cb increased with irradiation time from 0 to 30 min ( Figure 4 E). After 30 min of light irradiation, the percentage of drug release was detected to be 62.24%, while 79.52% of BODIPY-Cb was consumed. In comparison, nanoparticles encapsulating only BODIPY-Cb (BC nanoparticles) showed negligible Cb release after NIR-light irradiation, indicating that the prodrug photolysis within the nanoparticles depends on the release of Cb from Os(bptpy)2 2+ Energy transfer to BODIPY-Cb ( Figure 4 F). Overall, these observations confirm that Os / BC nanoparticles are capable of NIR light-triggered drug release in normoxic aqueous solution.

[0155] 6.5 In vitro and in vivo photoactivated cancer therapy

[0156] In vitro and in vivo studies of light-controllable cancer therapy using Os / BC nanoparticles were further conducted. Chlorambucil is an FDA-approved antitumor drug that has been used in clinical cancer treatment since 1950. 29 Several chlorambucil prodrugs have been developed to reduce its systemic side effects by preventing off-target toxicity and enhance its efficacy through precise activation at the lesion site. 30,31Here, the cytotoxicity of light-triggered chlorambucil released from nanoparticles was investigated by 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT) assay against human cervical cancer (HeLa) cells. 2 , 30 min) showed a significant antiproliferative effect on HeLa cells, while BC nanoparticles showed negligible toxicity ( Figure 5 C). Os nanoparticles slightly inhibited cell growth after NIR-light exposure, indicating limited phototoxicity without prodrug activation. In addition, in a light-protected environment, Os nanoparticles, BC nanoparticles, and Os / BC nanoparticles all showed minimal cytotoxicity ( Figure 5 B). To further confirm this, live-dead cell staining analysis was performed by Calcein AM / PI co-staining. A large number of dead cells showing red fluorescence were observed in the Os / BC nanoparticles plus light treatment group, while no obvious cell death was induced in the other groups ( Figure 5 D) The results are in good agreement with the cytotoxicity studies, indicating that light-triggered prodrug activation and drug release from Os / BC nanoparticles effectively inhibited the growth of cancer cells.

[0157] To further investigate the mechanism of cell death triggered by prodrug photolysis, HeLa cells treated with Os nanoparticles, BC nanoparticles, and Os / BC nanoparticles and subsequently irradiated with NIR light were stained with Annexin-V FITC / PI to investigate the apoptotic process ( Figure 5 E). The results showed that about 63.33% of the cells underwent apoptosis after treatment with Os / BC nanoparticles (BC equivalent concentration of 10 μM) and NIR-light irradiation, which was mainly late apoptosis (59.48%). A much smaller proportion of apoptotic cells was observed in the groups treated with Os nanoparticles and BC nanoparticles. Overall, the results confirmed that the cytotoxicity of Os / BC nanoparticles is mainly based on the apoptosis-induced effect after light-triggered Cb release.

[0158] It was reported that PLA-mPEG micellar nanoparticles exhibited circulation stability and tumor accumulation ability based on the enhanced permeability and retention (EPR) effect in tumors after systemic administration. 32,33 To confirm this, we labeled the nanoparticles with 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindoltricarbocyanine iodide (DiR) dye and examined the biodistribution of DiR-loaded nanoparticles (DiR nanoparticles) after intravenous injection into HeLa tumor-bearing mice. Based on the fluorescence observation results of the in vivo imaging system (IVIS), it was found that the nanoparticles showed both longer circulation time and tumor accumulation ability compared with free DiR ( Figure 6A). As time increased from 0h to 24h, the fluorescence signal representing DiR nanoparticles in the tumor area increased significantly, while the free dye was completely metabolized within the first 8h. Tumors and major organs were removed 24h after injection for ex vivo fluorescence imaging. As a result, the nanoparticles showed the ability to preferentially accumulate and retain in tumors ( Figure 6 B and Fig. 20 ). It should be noted that the prodrugs can be selectively activated by NIR light in tumors, while those nanoparticles in normal tissues and organs will not be activated, which can alleviate the side effects of chemotherapy.

[0159] Encouraged by the antiproliferative effects and tumor retention ability, Os / BC nanoparticles are considered as agents that can be used for light-triggered drug release and light-activated cancer chemotherapy. 3 At about 14 days, mice bearing HeLa tumors were randomly divided into six groups (n=5). On day 1, PBS, free Cb (8 mg / kg), Os nanoparticles (at an equivalent concentration of Os in Os / BC nanoparticles), BC nanoparticles (at an equivalent concentration of Cb), and Os / BC nanoparticles (at an equivalent concentration of Cb) were intravenously injected. Figure 6 C). 24 h after injection, NIR-light irradiation (690 nm, 300 mW / cm 2 , 10min)( Figure 6 D). The formulation injection and light irradiation were repeated on the 6th and 7th days, respectively. To evaluate the efficacy, the tumor volume was recorded during the treatment. Obviously, group 6 (Os / BC nanoparticles + hv) showed the most significant tumor growth inhibition effect compared with other groups ( Figure 6 E). Group 3 (Os NPs + hv) showed a slight inhibitory effect on tumor volume. However, no statistical difference was found between Group 3 and Groups 1, 2, 4, and 5. This result is consistent with the above findings of low cytotoxicity of Os NPs after light irradiation, which can be explained by the Os(bptpy)2 2+ Systemic administration of free chlorambucil (Group 2) showed no detectable antitumor effect, which can be attributed to its short circulation time and rapid hydrolysis in the blood. 34 On day 13, we sacrificed the mice and excised tumors and organs for ex vivo characterization ( Figure 6 F). The tumor weight of group 6 was significantly lower than that of other groups, indicating the excellent anti-tumor efficacy of Os / BC nanoparticles and NIR light ( Fig.21 ). Hematoxylin and eosin (H&E) staining experiments were performed to study the pathology of tumors and organs. Significant necrosis was found in tumor tissues treated with Os / BC nanoparticles + hv, while negligible apoptosis / tissue necrosis was observed in other groups ( Figure 6H). No obvious tissue damage was observed in the major organs of all treated mice, including the heart, lung, liver, spleen, and kidney, indicating that the in vivo toxicity of the treatment was negligible ( Fig. 22 In addition, no significant changes in body weight were observed, further indicating that our system has low systemic side effects ( Figure 6 G).

[0160] 6.6 Discussion

[0161] In summary, we developed an upconversion-like prodrug photolysis method based on a one-step energy transfer from STPS to BODIPY-based prodrugs, overcoming the limitation that the photon energy must be higher than the S1 state of the photosensitizer. This method depends on the direct activation of STPS followed by energy transfer from the T1 state of STPS to the T1 state of the prodrug. Such a strategy allows the use of low-energy photons, such as NIR photons. In addition to extending the excitation wavelength, this strategy also exhibits many other advantages, such as low illumination and high photolysis yield, which may be responsible for the reduced energy loss and photodamage during the photolysis reaction. Notably, this upconversion-like photolysis strategy can be highly modular. A series of prodrugs were constructed for NIR light-triggered photochemical control of a range of bioactive molecules, including anticancer drugs, anti-inflammatory drugs, anesthetics, and biogenic amines. In addition, PLA-mPEG micellar nanoparticles can encapsulate photosensitizers and prodrugs, allowing NIR light-triggered drug release for effective cancer therapy. Overall, we demonstrate a new mechanism for NIR light-triggered upconversion-like photolysis via one-step energy transfer and a nanoparticle-based approach for biomedical applications. This study provides new insights into the development of light-activatable systems for photopharmacology and light-responsive drug delivery.

[0162] Materials and methods

[0163] 6.7.1. p-Nitrophenyl chloroformate was obtained from Sigma-Aldrich (Steinheim, Germany). Osmium(III) chloride hydrate, 4′-(4-bromophenyl)-2,2′:6′,2″-terpyridine, acetoxy-acetyl chloride, ammonium hexafluorophosphate, boron trifluoride etherate, 2,4-dimethylpyrrolidinium, triethylamine, hydrochloric acid, N,N-diisopropylethylamine, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), and all other chemicals were obtained from Dieckman (Shenzhen, China). Chlorambucil, dimethyl-xanthoneacetic acid, naproxen, ibuprofen, indomethacin, 4-benzyloxycinnamic acid, tetracaine, dopamine, tyramine, and homoveratramine were purchased from Bide Pharm (Shanghai, China). 2′,7′-Dichlorofluorescein diacetate (DCFH-DA) and 4′,6-diamidino-2-phenylindole (DAPI) were obtained from Thermo Fisher Scientific. Fisher (Haytham, Lancashire, UK). Dichloromethane, N,N-dimethylformamide, dimethyl sulfoxide, acrylonitrile, methanol, hexene, ethyl acetate, and tetrahydrofuran were obtained from Oriental (Hong Kong, China). Silica gel columns were purchased from Teledyne ISCO (Lincoln, USA). PLA 5k -mPEG 5k Supplied by Ponsure Biotechnology Co., Ltd. (Shanghai, China).

[0164] 6.7.2 Characterization

[0165] CombiFlash Rf chromatograph was purchased from Teledyne ISCO (Lincoln, USA) for chemical purification. The light sources used in this study were purchased from Yuanming Laser (Ningbo, China) and Mightex (California, USA). Purity verification and photolysis studies were performed using a high performance liquid chromatography (HPLC) system (Agilent technologies, 1260) connected to a C-18 column (obtained from Agilent (Santa Clara, California, USA). Nuclear magnetic resonance (NMR) spectra were collected by a Bruker AVANCE IIIHD 500 spectrometer (Massachusetts, USA). Fluorescence, phosphorescence and UV-visible spectra were collected by a SpectraMax M4 microplate reader (Molecular Devices, California, USA). Particle size and surface charge were recorded by a dynamic light scattering (DLS) device Zetasizer Nano-ZS90 (Malvern instruments, UK). Transmission electron microscopy (TEM) imaging was performed on a Hitachi HT7700 TEM (Tokyo, Japan). ACEA NovoCyte Quanteon flow cytometer (ACEA Biosciences, California, USA) was used for flow cytometry. In vivo and ex vivo imaging of mice was performed using the In Vivo Imaging System (PerkinElmer, USA). The Center for Comparative Medicine Research (CCMR) of the University of Hong Kong provided equipment such as balances and vernier calipers for animal studies.

[0166] 6.7.3 Construction and Characterization of Nanoparticles

[0167] The rapid nanoprecipitation method was used to construct nanoparticles loaded with photosensitizers / prodrugs. 5k -mPEG 5k (200 mg / mL in acetone), BODIPY-Cb (3×10 -2 M, 24.2 mg / mL in DMSO), and Os(bptpy)2·2PF6(10 -3 M, 1.26 mg / mL, in DMSO). Different formulations (Os nanoparticles, BC nanoparticles, and Os / BC nanoparticles) were then prepared using the optimized component ratios. Briefly, 224 μL of the mixed stock solution was added to 1.8 mL of N2-saturated water under vortexing. The resulting solution was added to a dialysis bag (M W:3400Da) and dialyzed against 4L water for 24h. The water outside the dialysis bag was replaced every 8h. The resulting nanoparticle solution was collected and filtered with a 220nm filter. Finally, the nanoparticle solution was concentrated by ultrafiltration (Mw=100kDa, 2000×g, 10min) and stored at 4°C until use. The concentration of each component was measured using HPLC. The loading capacity and encapsulation efficiency were calculated as follows:

[0168]

[0169]

[0170] 6.7.4 Measurement of Prodrug Photolysis in Nanoparticles

[0171] The aqueous solutions of Os nanoparticles, BC nanoparticles and Os / BC nanoparticles were diluted to 10 -3 M (based on BODIPY-Cb) and added to a 1.5 mL tube. 690 nm light (100 mW / cm 2 ) for 0 to 30 min. At each time point, 100 μL of sample was collected and dispersed with an equal volume (100 μL) of acetonitrile. The prodrug consumption and drug release yield were then analyzed using HPLC.

[0172] 6.7.5 Cell culture

[0173] Human cervical cancer cells (HeLa) were purchased from the Chinese Academy of Sciences Stem Cell Bank. The cells were cultured in DMEM (Gibco) supplemented with 10% FBS (Gibco) and 100 units / mL antibiotics (penicillin-streptomycin, Gibco) at 37°C in a 5% CO2 humidified atmosphere.

[0174] 6.7.6 Cytotoxicity analysis

[0175] Cell viability was determined by MTT assay. HeLa cells were cultured in 100 μL complete DMEM medium at a preliminary density of 5000 cells / well on a 96-well plate and incubated for 24 h. The medium was replaced with medium containing formulations (Os nanoparticles, BC nanoparticles, and Os / BC nanoparticles) at different concentrations (0-50 μM, based on BODIPY-Cb). After 4 h incubation, the cells were illuminated with NIR light (690 nm, 100 mW / cm 2 ) irradiated for 30 min. After 24 h incubation, MTT solution (10 μL / well) was added. After 3 h, the culture medium was discarded and DMSO (100 μL) was added to each well. The OD490 value was recorded by an ELISA instrument to calculate the cell survival rate.

[0176] 6.7.7 Live / dead cell staining

[0177] HeLa cells were seeded at a density of 10,000 cells / well in confocal microplates and treated with PBS, Os nanoparticles, BC nanoparticles, and Os / BC nanoparticles, where the equivalent concentration of BODIPY-Cb was 10 μM. For light irradiation, cells were illuminated with 690 nm light (100 mW / cm 2 ) for 30 min, followed by 24 h incubation. After washing with PBS and replacing the culture medium, calcein-AM and PI were added, and the cells were observed using a confocal laser scanning microscope (λex = 488 nm, 560 nm).

[0178] 6.7.8 Apoptosis analysis

[0179] HeLa cells were seeded at a density of 50,000 cells / well in 6-well plates and treated with free Cb or IR783 / BC nanoparticles in PBS, Os nanoparticles, BC nanoparticles, and Os / BC nanoparticles for 4 h, with BODIPY-Cb equivalent concentration of 10 μM. The cells in the irradiated group were then illuminated with light (690 nm, 100 mW / cm 2 ) for 30 min. After 24-h incubation, cells were washed three times with PBS, collected by trypsinization, and stained with Annexin-V / FITC apoptosis kit. Cell suspensions were analyzed by flow cytometry (λex

[0180] =488nm, 560nm).

[0181] 6.7.9 Animals

[0182] BALB / c nude mice (female, 4 weeks, 15-20 g) were obtained from the University's Laboratory Animal Center with free access to food and water. Animal experiments and procedures were approved by the Committee on the Use of Animals in Teaching and Research (CULATR) of the University of Hong Kong (protocol number 4381-17).

[0183] Mice bearing HeLa tumors were obtained by subcutaneous injection of HeLa cells for tumor implantation. HeLa cancer cells were collected by cell culture and trypsinization. The cells were washed with PBS and resuspended in serum-free DMEM medium supplemented with collagen and Matrigel. 2×10 6 The cells were dispersed in 100 μL of the mixture, which was injected subcutaneously into the axillary region of the mice. After 7 days of incubation, the tumor-bearing mice were randomly divided into multiple groups for in vivo experiments.

[0184] 6.7.10 Biodistribution in vivo

[0185] The biodistribution of free drugs or nanoparticles in HeLa tumor-bearing mice was measured by an in vivo fluorescence imaging system. DiR (1,1-dioctadecyl-3,3,3,3-tetramethylindoltricarbocyanine iodide) was used as a fluorescent dye to label PLA-mPEG nanoparticles during in vivo imaging. Six HeLa tumor-bearing mice (volume of about 200 mm 3 ) were randomly divided into two groups and treated with free DiR or DiR in PLA-mPEG nanoparticles via intravenous injection, where the DiR dose was 100 μg / kg. In vivo fluorescence imaging was performed 1, 2, 8 and 24 hours after injection, and anesthesia was used at each time point. At 24 h, all mice were sacrificed. Tumors and major organs (heart, liver, spleen, lung, kidney) were removed for ex vivo imaging (λex=710nm, λem=780nm).

[0186] 6.7.11 In vivo light-triggered prodrug activation and tumor inhibition

[0187] The antitumor efficacy of the formulations in the presence or absence of light was studied in mice bearing HeLa tumors. Seven days after tumor implantation, mice bearing a volume of 100 mm 3 Mice with tumors of about 20 μg / cm2 were randomly divided into 6 groups (5 mice in each group). Different treatments were applied to each group: (1) PBS; (2) free chlorambucil; (3) Os nanoparticles plus light irradiation; (4) BC nanoparticles plus light irradiation; (5) Os / BC nanoparticles; (6) Os / BC nanoparticles plus light irradiation. On the 1st and 6th day, the formulation was injected intravenously at a dose of 8 mg / kg. For the light irradiation group, light irradiation (690 nm, 300 mW / cm2) was performed 24 h after injection (day 2 and day 7). 2 During this period, tumor size and body weight were measured, and tumor volume was calculated as V = 1 / 2 × width 2 × length. On day 13, all mice were sacrificed, tumors and major organs were removed, and sections were processed for H&E staining and histochemical analysis. All histological studies were performed blindly by pathologists from the Department of Pathology, University of Hong Kong.

[0188] 6.7.12 Statistical analysis

[0189] All experiments were performed independently three times or more (n≥3). Data are expressed as mean ± standard deviation (SD). One-way ANOVA-LSD and independent sample t-test were used to determine the statistical significance of the differences using GraphpadPrism 8.0 software (***p<0.001, **p<0.01, and *p<0.05).

[0190] 6.7.13 Synthesis 6.7.13.1

[0192] The synthetic procedures for two BODIPY photocaged compounds 1 and 2 have been reported in our previous study (Scheme S1).

[0193]

[0194] Scheme S1. Chemical structures of BODIPY photocages 1 and 2.

[0195] 6.7.13.2 Synthesis of [Os(bptpy)2]·2PF6 (Compound 3)

[0196] The synthesis of [Os(bptpy)2]·2PF6 was performed based on a reported method with modifications. [ref]

[0197]

[0198] Scheme S2. Synthesis of [Os(bptpy)2]·2PF6 (Compound 3).

[0199] Osmium(III) chloride hydrate (314.6 mg, 1 mmol) and 4'-(4-bromophenyl)-2,2':6',2"-terpyridine (766.5 mg, 2 mmol) were dissolved in 50 mL of ethylene glycol and protected with nitrogen. The solution was heated to 200°C and refluxed overnight with continuous stirring. After the reaction, the solution turned dark purple. The solution was added dropwise to an excess of water (500 mL) with vigorous stirring. The obtained purple precipitate was collected by filtration, washed with water and THF, and dried. The obtained powder was then dissolved in ACN (150 mL) and heated at 70°C. Ammonium hexafluorophosphate (16.3 g, 10 mmol) was dissolved in water and added with stirring. After 2 h, the solution was cooled, and the product as a dark purple precipitate was collected by filtration, washed with water and ACN without further purification. 1 H NMR (500MHz, DMSO-d6): δ (ppm), 9.52 (s, 4H), 9.09-9.10 (d, 4H), 8.38-8.40 (d, 4H), 7.99-8. 01(d,4H),7.92-7.95(t,4H),7.43-7.44(d,4H),7.20-7.23(t,4H).m / z=968.03, actual measurement 968.00.

[0200] 6.7.13.3 General Procedure for Preparation of BODIPY Ester Prodrugs (4-9, 14)

[0201]

[0202] Scheme S3. Synthesis of BODIPY ester prodrugs.

[0203] 7.5 mg BODIPY light cage lock 2 (0.014 mmol) was dissolved in 300 μL dry dichloromethane and protected from light. Drugs with carboxyl groups (0.021 mmol), N, N'-dicyclohexylcarbodiimide (DCC) (6.9 mg, 0.0334 mmol), 4-dimethylaminopyridine (DMAP) (0.17 mg, 0.0014 mmol) were added to a sealable flask under nitrogen protection. 500 μL dry dichloromethane was added to the flask and stirred for 30 min, and then 2 drops of solution were injected. The flask was placed in a light-proof environment and covered on the surface with tin foil. After 12 h, thin layer chromatography confirmed that the reaction was complete, and the solvent was evaporated to remove. The residue was subjected to column chromatography on silica gel for purification.

[0204]

[0205] (CH3).[M+H] + :809.17, measured 809.20. 89% yield.

[0206]

[0207] 6H),2.35(s,6H),2.03(s,6H),1.38-1.40(d,3H)(CH3).[M+H] + :735.22, measured 735.20. 88% yield.

[0208]

[0209] 1.12(s,1H),0.73-0.75(d,6H)(CH3).[M+H] + :711.25, measured 711.20. 88% yield.

[0210]

[0211] (CH2),2.43(m,9H),2.34(m,6H),1.75-1.79(m,3H)(CH3).[M+H] + :557.38, measured 557.40. 78% yield.

[0212] 6.7.13.4 General Procedure for Preparation of BODIPY Carbamate Prodrugs (10-13)

[0213]

[0214] Scheme S3. Synthesis of BODIPY carbamate prodrugs.

[0215] BODIPY light cage lock 2 (50.0 mg, 0.096 mmol) was added to a 50 mL sealable flask. The flask was then evacuated with a pump and refilled with nitrogen. Dry dichloromethane (8 mL) was added under stirring to dissolve the solid. The flask was cooled to 0 ° C in an ice-water bath. N, N-diisopropylethylamine (0.168 mL, 0.96 mmol) was added with a syringe with a needle. Then p-nitrophenyl chloroformate (193.1 mg, 0.96 mmol) was dissolved in dry dichloromethane (2 mL) and added dropwise to the above solution. Pyridine (31 μL, 0.38 mmol) was added with a syringe with a needle. The flask was placed in a light-proof environment and covered on the surface with tin foil. After 12 h, thin layer chromatography confirmed that the reaction was complete, and the solvent was evaporated to remove. The product (15, BODIPY-p-nitrobenzene) was purified by chromatography on a silica column and flowed by vacuum drying for at least 24 h. Yield: 58%.

[0216] BODIPY-p-nitrobenzene (15, 20 mg, 0.029 mmol) was dissolved in tetrahydrofuran (2 mL) in a 25 mL sealable flask, evacuated with a pump and refilled with nitrogen. Under stirring at room temperature, the drug with an amino group (0.087 mmol) was dissolved in 0.5 mL DMF and added to the solution with a syringe with a needle. N, N-diisopropylethylamine (18 μL) was then added to the mixture. The flask was placed in a dark environment and covered on the surface with tin foil. After 4 h, the solvent was evaporated and the product was collected by high performance liquid chromatography.

[0217]

[0218] 0.07 (m, 6H) (CH3). 26% yield.

[0219]

[0220] Rate.

[0221]

[0222] 1.10 (s, 9H) (CH3). 37% yield.

[0223]

[0224] 6.7.13.5 Energy transfer quenching rate constant (k TTET )

[0225] The energy transfer quenching rate constant (k) was measured by Stern-Volmer experiment. TTETAs a representative BODIPY prodrug, BODIPY-Cb (compound 4) was used as an energy acceptor, while Os(bptpy)2 2+ 10 μM Os(bptpy)2 was prepared in the presence of different concentrations of BODIPY-Cb (0, 1, 2, 3, 5, 7.5 μM). 2+ The solution, wherein the solvent is a mixture of 88% methanol, 2% acetone and 10% dichloromethane, was deoxygenated by passing nitrogen (50 mL / min) for 10 min.

[0226] Os(bptpy)2 2+ The quenching constant (k q ) is calculated by the following equation (1):

[0227]

[0228] (I0:Os(bptpy)2 2+ Phosphorescence intensity of solution; I: Os(bptpy)2 2+ Phosphorescence intensity of the solution in the presence of BODIPY-Cb; [Q]: BODIPY-Cb concentration. )

[0229] The energy transfer quenching rate constant of this TTET process is quantitatively expressed as k TTET , can be calculated based on the following equation (2):

[0230]

[0231] (τ0:Os(bptpy)2 2+ Phosphorescence lifetime in N2-saturated solution without quencher. )

[0232] For Os(bptpy)2 2+ ,τ0=0.20μs. [ref]Os(bptpy)2 2+ k in the presence of BODIPY-Cb q Determined as (5.491±0.282)×10 4 M -1 Therefore, Os(bptpy)2 2+ The energy transfer quenching rate constant of TTET between BODIPY-Cb and Cb was calculated to be (2.718±0.140)×10 11 M - 1s -1 .

[0233] 6.7.13.6 Quantitative analysis of BODIPY prodrug photolysis

[0234] The photolysis efficiency of BODIPY prodrug was compared under different conditions, including different excitation wavelengths (690 nm or 530 nm), different Os(bptpy)2 2+ ratio, and different oxygen contents. BODIPY-Cb (compound 4) (100 μM) and Os(bptpy)2 2+ 100 μL of the mixture of 0 equivalent (0 μM), 0.05 equivalent (5 μM), 0.1 equivalent (10 μM)) was saturated with nitrogen or not saturated with nitrogen and illuminated with 690 nm light (100 mW / cm 2 , 5min) or 530nm light irradiation. The solvent is 88% methanol mixed with 10% dichloromethane and 2% acetone. For the N2 saturated group, after the molecule is dissolved, the solution is degassed with a vacuum pump for 10min, and then N2 is gently blown. After light irradiation, the resulting mixture is filled to 100μL with methanol to avoid errors caused by solvent evaporation. The resulting solution is loaded onto a sampler and analyzed by high performance liquid chromatography. Free drug, BODIPY light cage and prodrug are separated on a C18 column using the attached elution method (Table S2).

[0235] Table 2. HPLC methods used for photolysis studies

[0236]

[0237] 6.7.13.7 Measurement of Photoreaction Quantum Yield

[0238] The quantum yield (Ф) of the photoreaction is defined as:

[0239]

[0240] The amount of reacted molecules (e.g., prodrug consumption or free drug generation) was determined by HPLC. The number of photons absorbed was determined by Reinecke salt photometry at excitation wavelengths of 530 nm and 690 nm. 35，36 . Power density is 50mW / cm 2 (530nm) and 100mW / cm 2 (690nm).

[0241] Exemplary products, systems and methods are listed in the following items:

[0242] 1. A composition comprising:

[0243] a photosensitizer capable of being activated from a singlet-triplet state (ST) at near infrared (NIR) wavelengths; a cleavable moiety that accepts triplet-triplet energy transfer from a higher energy state of the photosensitizer to cause cleavage of the cleavable moiety; and a releasable moiety that is releasable from the composition upon cleavage of the cleavable moiety,

[0244] wherein the composition does not contain an annihilating agent.

[0245] 2. The composition of item 1, wherein the singlet to triplet activation is from a singlet (S0) state to a triplet (T1) state.

[0246] 3. The composition of item 2, wherein the singlet to triplet activation is directly from the singlet (S0) state to the triplet (T1) state.

[0247] 4. The composition of item 2, wherein the triplet-triplet energy transfer is from the triplet (T1) state of the photosensitizer to the cleavable moiety.

[0248] 5. The composition of any one of items 2 to 4, wherein the triplet (T1) state of the photosensitizer is higher than the triplet state of the cleavable moiety.

[0249] 6. The composition of any one of items 1 to 5, wherein the photosensitizer comprises a transition metal-porphyrin.

[0250] 7. The composition of item 6, wherein the transition metal is osmium (Os).

[0251] 8. The composition of any one of items 1 to 6, wherein the photosensitizer is Os(II) bromophenyl terpyridine complex (Os(bptpy)2 2+ ).

[0252] 9. The composition of any one of items 1 to 8, wherein the near infrared (NIR) wavelength is between about 650 nm and about 750 nm.

[0253] 10. The composition of item 9, wherein the near infrared (NIR) wavelength is about 690 nm.

[0254] 11. The composition of item 1, wherein the excitation wavelength of the photosensitizer is between about 650 nm and about 750 nm.

[0255] 12. The composition of item 11, wherein the excitation wavelength of the photosensitizer is about 690 nm.

[0256] 13. The composition of any one of items 1 to 12, wherein the cleavable moiety is photocleavable.

[0257] 14. The composition of any one of items 1 to 13, wherein the cleavable moiety is BODIPY.

[0258] 15. The composition of any one of items 1 to 14, wherein the releasable portion comprises a drug.

[0259] 16. The composition of item 15, wherein the drug is an anti-inflammatory drug, an anti-cancer drug or an anti-angiogenesis drug.

[0260] 17. The composition of item 15 or 16, wherein the drug is selected from the group consisting of chlorambucil (CAB), valdemethoxane, indomethacin, naproxen, ibuprofen, benzyloxycinnamic acid, tetracaine, dopamine, tyramine and homoveratamine.

[0261] 18. The composition of any one of items 1 to 17, wherein the composition comprises a prodrug comprising the cleavable portion and the releasable portion.

[0262] 19. The composition of claim 18, wherein the prodrug comprises a drug linked to BODIPY.

[0263] 20. The composition of claim 18, wherein the cleavable portion is BODIPY, and wherein the releasable portion comprises a drug selected from the group consisting of chlorambucil, valdemethoxazole, indomethacin, naproxen, ibuprofen, benzyloxycinnamic acid, tetracaine, dopamine, tyramine, and homoveratamine.

[0264] 21. The composition of claim 18, wherein the prodrug is represented by the following structure:

[0265]

[0266] The “Cargo” includes the releasable part.

[0267] 22. The composition of claim 21, wherein the carrier comprises a drug.

[0268] 23. The composition of any one of items 18 to 22, wherein the prodrug is selected from the group consisting of:

[0269]

[0270]

[0271]

[0272] 24. The composition of any one of items 1 to 23, wherein at least about 50% of the cleavable moieties are released.

[0273] 25. The composition of any one of items 1 to 24, wherein at least about 80% of the cleavable moieties are released.

[0274] 26. The composition of any one of items 1 to 25, wherein at least about 90% of the cleavable moieties are released.

[0275] 27. The composition of any one of items 1 to 26, further comprising a carrier material.

[0276] 28. The composition of item 27, wherein the carrier material comprises the photosensitizer, the releasable portion and the cleavable portion.

[0277] 29. The composition of item 27 or 28, wherein the carrier material comprises a polymer.

[0278] 30. The composition of any one of items 27 to 29, wherein the carrier material comprises particles.

[0279] 31. The composition of item 30, wherein the average diameter of the particles is less than about 1 mm.

[0280] 32. The composition of any one of items 27 to 31, wherein the carrier material comprises a film.

[0281] 33. The composition of any one of items 27 to 32, wherein the carrier material comprises polymer micelles.

[0282] 34. A composition comprising:

[0283] a photosensitizer capable of activating from a singlet-triplet state (ST) upon exposure to near infrared (NIR) wavelengths; and

[0284] an active moiety that accepts triplet-triplet energy transfer from a higher energy state photosensitizer to induce a chemical reaction within the active moiety;

[0285] wherein the energy transferred from the photosensitizer is sufficient to cause a chemical reaction within the active moiety; and

[0286] wherein the composition does not contain an annihilating agent.

[0287] 35. A method comprising:

[0288] applying to a subject a composition comprising a photosensitizer, a cleavable moiety, and a releasable moiety, wherein the photosensitizer is capable of being activated from a singlet-triplet state (ST) upon exposure to near infrared (NIR) wavelengths; the cleavable moiety accepts triplet-triplet energy transfer from a higher energy state of the photosensitizer to cause cleavage of the cleavable moiety; and the releasable moiety is releasable from the composition upon cleavage of the cleavable moiety;

[0289] applying near infrared (NIR) light to the subject to cause cleavage of the cleavable moiety and release of the releasable moiety,

[0290] Wherein the triplet-triplet energy is not transferred via an annihilator.

[0291] 36. The method of item 35, wherein the light is coherent.

[0292] 37. The method of item 35, wherein the light is incoherent.

[0293] 38. The method of any one of items 35 to 37, wherein the composition comprises a prodrug comprising the cleavable portion and the releasable portion.

[0294] 39. A prodrug comprising a cleavable moiety and a releasable moiety, wherein the cleavable moiety is BODIPY, and wherein the releasable moiety comprises a drug selected from the group consisting of: valdecameron, indomethacin, naproxen, ibuprofen, benzyloxycinnamic acid, tetracaine, dopamine, tyramine, and homoveratamine.

[0295] 40. A compound selected from the group consisting of:

[0296]

[0297]

[0298] 41. The compound of item 40, which is selected from the group consisting of formula 5, 6, 8, and 13.

[0299] 42. The compound of formula 5.

[0300] 43. The compound of formula 6.

[0301] 44. The compound of formula 7.

[0302] 45. The compound of formula 8.

[0303] 46. ​​The compound of formula 9.

[0304] 47. The compound of formula 10.

[0305] 48. A compound of formula 11.

[0306] 49. The compound of formula 12.

[0307] 50. The compound of formula 13.

[0308] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the appended claims.

[0309] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference in their entirety into the specification, to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

[0310] Citation and discussion of references herein shall not be construed as an admission that these are prior art to the present invention.

[0311] References:

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Claims

1. A composition comprising: a photosensitizer capable of being activated from a singlet-triplet state (ST) at near infrared (NIR) wavelengths; a cleavable moiety that accepts triplet-triplet energy transfer from a higher energy state of the photosensitizer to cause cleavage of the cleavable moiety; and a releasable moiety that is releasable from the composition upon cleavage of the cleavable moiety, wherein the composition does not contain an annihilating agent.

2. The composition of claim 1, wherein the singlet-triplet activation is from a singlet (S0) state to a triplet (T1) state.

3. The composition of claim 2, wherein the triplet-triplet energy transfer is from the triplet (T1) state of the photosensitizer to the cleavable moiety.

4. The composition of claim 3, wherein the triplet (T1) state of the photosensitizer is higher than the triplet state of the cleavable moiety.

5. The composition of claim 1, wherein the photosensitizer comprises a transition metal-porphyrin.

6. The composition of claim 5, wherein the transition metal is osmium (Os).

7. The composition of claim 1, wherein the photosensitizer is Os(II) bromophenyl terpyridine complex (Os(bptpy)2 2+ ).

8. The composition of claim 1, wherein the near infrared (NIR) wavelength is between about 650 nm and about 750 nm.

9. The composition of claim 1, wherein the photosensitizer has an excitation wavelength between about 650 nm and about 750 nm.

10. The composition of claim 1, wherein the cleavable moiety is photocleavable.

11. The composition of claim 1, wherein the cleavable moiety is BODIPY.

12. The composition of claim 1, wherein the releasable portion comprises a drug.

13. The composition of claim 12, wherein the drug is an anti-inflammatory drug, an anti-cancer drug or an anti-angiogenesis drug.

14. The composition of claim 12, wherein the drug is selected from the group consisting of chlorambucil (CAB), valdemethoxane, indomethacin, naproxen, ibuprofen, benzyloxycinnamic acid, tetracaine, dopamine, tyramine, and homoveratamine.

15. The composition of claim 1, wherein said composition comprises a prodrug comprising said cleavable portion and said releasable portion.

16. The composition of claim 15, wherein the prodrug comprises a drug linked to BODIPY.

17. The composition of claim 15, wherein the cleavable moiety is BODIPY, and wherein the releasable moiety comprises a drug selected from the group consisting of chlorambucil CAB, valdemethoxane, indomethacin, naproxen, ibuprofen, benzyloxycinnamic acid, tetracaine, dopamine, tyramine, and homoveratamine.

18. The composition of claim 15, wherein the prodrug is represented by the following structure: The "Cargo" includes the releasable part.

19. The composition of claim 18, wherein the cargo comprises a drug.

20. The composition of claim 15, wherein the prodrug is selected from the group consisting of:

21. The composition of claim 1, wherein at least about 50% of said cleavable moiety is released.

22. The compound of claim 1, further comprising a carrier material.

23. The composition of claim 22, wherein said carrier material comprises said photosensitizer, said releasable moiety, and said cleavable moiety.

24. The composition of claim 22, wherein the carrier material comprises a polymer.

25. The composition of claim 22, wherein the carrier material comprises particles.

26. The composition of claim 25, wherein the particles have an average diameter of less than about 1 mm.

27. The composition of claim 22, wherein the carrier material comprises a film.

28. The composition of claim 22, wherein the carrier material comprises polymer micelles.

29. A composition comprising: a photosensitizer capable of activating from a singlet-triplet state (ST) upon exposure to near infrared (NIR) wavelengths; and an active moiety that accepts triplet-triplet energy transfer from a higher energy state photosensitizer to induce a chemical reaction within the active moiety; wherein the energy transferred from the photosensitizer is sufficient to cause a chemical reaction within the active moiety; and wherein the composition does not contain an annihilating agent.

30. A method comprising: applying to a subject a composition comprising a photosensitizer, a cleavable moiety, and a releasable moiety, wherein the photosensitizer is capable of being activated from a singlet-triplet state (ST) upon exposure to near infrared (NIR) wavelengths; the cleavable moiety accepts triplet-triplet energy transfer from a higher energy state of the photosensitizer to cause cleavage of the cleavable moiety; and the releasable moiety is releasable from the composition upon cleavage of the cleavable moiety; applying near infrared (NIR) light to the subject to cause cleavage of the cleavable moiety and release of the releasable moiety, Wherein the triplet-triplet energy is not transferred via an annihilator.

31. The method of claim 30, wherein the light is coherent.

32. The method of claim 30, wherein the light is incoherent.

33. The method of claim 30, wherein said composition comprises a prodrug comprising said cleavable portion and said releasable portion.

34. A prodrug comprising a cleavable moiety and a releasable moiety, wherein the cleavable moiety is BODIPY, and wherein the releasable moiety comprises a drug selected from the group consisting of: valdecameron, indomethacin, naproxen, ibuprofen, benzyloxycinnamic acid, tetracaine, dopamine, tyramine, and homoveratamine.

35. A compound selected from the group consisting of:

Images