Development Of H₂S-activatable Near-infrared Probes For In Vivo Imaging

The design of optical imaging probes in response to biological functional molecules for in vivo analysis with high-sensitivity and high precision holds an important role in promoting the early diagnosis of diseases.For in vivo fluorescence imaging,reducing the background fluorescence signal of biological tissues and improving the signal-to-background ratio（SBR）is the key to achieve accurate detection and analysis of disease markers.H₂S is identified as the third gasotransmitter in biology,which can elicit diverse physiological and pathological functions.Detection of H₂S levels is invaluable for the study of H₂S-related biological processes and disease diagnosis.Over the past decades,although a large number of fluorescent probes have been reported to detect cellular H₂S,most of them reported so far for in vivo imaging exhibited some limitations:（1）most probes have emissions in the visible light region,thus suffering from strong tissue autofluorescence and limited tissue penetration depth,which largely restrict their applications in vivo.（2）most probes may be hampered by poor in vivo targeting ability,which makes it impossible to selectively deliver to target sites for H₂S detection in vivo.（3）the response time of most reported H₂S-activated probes exceed 10 minutes,which can not achieve fast,accurate,and reliable detection of H₂S due to the rapid diffusion and very short half-life of H₂S in vivo（1～2 min）.Electrochromic materials（EMs）that display distinct color changes during an electrochemistry-induced electron-transfer（redox）process have been widely used as optical memories and electrochromic devices,including auto-dimming mirrors,smart windows,and electronic displays.In this thesis,starting from EMs with organicπelectronic structure,we first construct an activatable near-infrared（NIR）fluorescent probe that is specifically activated by endogenous H₂S in vivo.The probe with significantly enhanced NIR fluorescence at830 nm realizes non-invasive and real-time detection of hepatic and tumor H₂S levels in mice.We next build a smart activatable photosensitizer（PS）specifically activated by endogenously generated H₂S in tumors,which is used for imaging-guided highly effective photodynamic therapy（PDT）of tumors.Additionally,we further construct a H₂S-activatable NIR afterglow probe with high SBR and high sensitivity for imaging of tiny tumors and orthotopic liver tumors,and delineation of tumor margins in clinical hepatic cancer specimens.The research contents are as follows:In the first part,we report the engineering of an organicπ-electron structure-based EM(dicationic 1,1,4,4-tetraarylbutadiene,1²⁺)as a unique H₂S-responsive chromophore amenable to build H₂S-activatable fluorescent probes(1²⁺-SPNs)for in vivo H₂S detection.We demonstrate that EM 1²⁺with a strong absorption（500–850 nm）efficiently quenches the fluorescence（580,700,or 830 nm）of different fluorophores within 1²⁺-SPNs.We demonstrate that H₂S-triggered fast reduction of 1²⁺into nearly colorless diene 2 via a two-electron transfer process within 1²⁺-SPNs can efficiently eliminate the fluorescence resonance energy transfer（FRET）process from semiconducting polymers to 1²⁺,thus recovering the fluorescence at either 580,700 or830 nm.Molecular imaging of endogenous H₂S in vivo is realized using NIR fluorescent probe(1²⁺-SPN830),revealing the increment of cystathionineγ-lyase（CSE）expression and H₂S production in livers during lipopolysaccharide（LPS）-induced inflammatory response.Moreover,integrating a targeting ligand FA on the1²⁺-SPN830 surface to prepare 1²⁺-SPN830-FA can realize real-time monitoring and noninvasive imaging of H₂S-related tumors.In the second part,encouraged by the results in the first part,we further develop a tumor-targeting and H₂S-activatable NIR photosensitizer(1²⁺-PSs-FA)for effective PDT of H₂S-related tumors in mice.Due to the strong and broad absorption of 1²⁺（500–850 nm）,it can quench the fluorescence and ¹O₂ generation of two different organic PSs silicon 2,3-naphthalocyanine bis(trihexylsilyloxide（NIR775）and rhodamine 6G（R6G）.The activation of 1²⁺-PSs-FA with H₂S not only simultaneously increases dual fluorescences（～557-fold at 555 nm and～335-fold at 780 nm）,bur also remarkably amplify the ¹O₂ generation.Activatable PDT guided by fluorescence imaging of H₂S-related tumors is realized in mice following i.v.injection of1²⁺-PSs-FA and near-infared light irradiation.This study demonstrates the promise of using EMs to build activatable probes for imaging guided of selective PDT of tumors.In the third part,to improve the reaction rate,SBR and tissue penetration,we optimize an organic EM F1²⁺and integrate it into NIR photosensitizer NIR775 and poly[2-methoxy-5-（2-ethylhexyloxy）-1,4-penylenevinylene]（MEH-PPV）containing nanoparticles,developing an H₂S-activatable NIR afterglow probe(F1²⁺-ANP).turn-on ratio（～122-fold）toward H₂S,enabling high-sensitivity and-specificity measurement of H₂S concentration in bloods from healthy persons,hepatic or colorectal cancer patients.Furthermore,constructing a hepatic-tumor-targeting and H₂S-activatable afterglow probe(F1²⁺-ANP-Gal)by introducingβ-galactose（β-Gal）on the surface of F1²⁺-ANP allows for noninvasive imaging of tiny subcutaneous Hep G2 tumors（<3 mm in diameter）and orthotopic liver tumors in mice.Strikingly,F1²⁺-ANP-Gal accurately delineates tumor margins in excised hepatic cancer specimens,which may facilitate intraoperative guidance of hepatic cancer surgery.This study presents the potential of using H₂S-activatable afterglow luminescence probes for in vivo imaging,which could detect H₂S levels in other diseases（e.g.,liver inflammation and hypertension）and promote early disease diagnosis in the future.