A Smart Intracellular Self‐Assembling Bioorthogonal Raman Active Nanoprobe for Targeted Tumor Imaging

Abstract Inspired by the principle of in situ self‐assembly, the development of enzyme‐activated molecular nanoprobes can have a profound impact on targeted tumor detection. However, despite their intrinsic promise, obtaining an optical readout of enzyme activity with high specificity in native milieu has proven to be challenging. Here, a fundamentally new class of Raman‐active self‐assembling bioorthogonal enzyme recognition (nanoSABER) probes for targeted tumor imaging is reported. This class of Raman probes presents narrow spectral bands reflecting their vibrational fingerprints and offers an attractive solution for optical imaging at different bio‐organization levels. The optical beacon harnesses an enzyme‐responsive peptide sequence, unique tumor‐penetrating properties, and vibrational tags with stretching frequencies in the cell‐silent Raman window. The design of nanoSABER is tailored and engineered to transform into a supramolecular structure exhibiting distinct vibrational signatures in presence of target enzyme, creating a direct causality between enzyme activity and Raman signal. Through the integration of substrate‐specific for tumor‐associated enzyme legumain, unique capabilities of nanoSABER for imaging enzyme activity at molecular, cellular, and tissue levels in combination with machine learning models are shown. These results demonstrate that the nanoSABER probe may serve as a versatile platform for Raman‐based recognition of tumor aggressiveness, drug accumulation, and therapeutic response.

by recrystallization with diethyl ether.Isobutyl chloroformate (IBCF,20.48 mg,0.15 mmol) was added to a mixture of compound A (500 mg, 0.15 mmol) and 4-methylmorpholine (MMP, 30.3 mg, 0.3 mmol) in tetrahydrofuran (THF,10.0mL) at 0 °C under N2 gas atmosphere.The reaction mixture was stirred for 40 minutes.A solution of 2-cyano-6-aminobenzothiazole (CBT, 31.5 mg, 0.18 mmol) and additional IBCF (6.8 mg, 0.05 mmol) was added to the reaction mixture with continued stirring for 1 h at 0 °C, then stirred overnight at RT.The synthesized compound B (400 mg) was precipitated from the reaction mixture using diethyl ether, dried, and used for further steps.

Synthesis of C:
The peptide Ac-[Arg(Pbf)]6-Ala-Ala-Cys(StBu)-Pra-Lys(Boc)-COOH (C) was synthesized with the SPPS method on CTC resins.After sequential washing with DMF and DCM, resins were treated with 2% TFA/DCM to cleave the peptide from the resin without removing the protecting groups.Compound C was produced after concentrating the solvent using a rotary evaporator followed by recrystallization with diethyl ether.IBCF (20.48 mg, 0.15 mmol) was added to a mixture of compound C (500 mg, 0.15 mmol) and MMP (30.3 mg, 0.3 mmol in THF (10.0 mL) at 0 °C under N2 gas atmosphere.The reaction mixture was stirred for 40 minutes.A solution of CBT (31.5 mg, 0.18 mmol) and additional IBCF (6.8 mg, 0.05 mmol) was added to the reaction mixture with continued stirring for 1 h at 0°C, then stirred overnight at RT.The synthesized compound D (450 mg) was precipitated from the reaction mixture using diethyl ether, dried, and used for further steps.

Figure S1 .Figure
Figure S1.HR-MALDI-TOF/MS spectrum of nanoSABER.The spectrum reflects representative data from in vitro experiments repeated three times.

Figure S3 .
Figure S3.(a) Particle size distribution of alkyne-dimer nanoparticles as obtained from TEM.(b) TEM of nanoSABER solution showing the absence of self-assembled structures.Data are representative of three independent in vitro experiments.

Figure S10 .
Figure S10.In vitro cellular Raman mapping of DU145 cells without any treatment.(a) Brightfield image of a DU145 cell.(b) The 1440 cm −1 signal (green) corresponds to the CH2 bending mode from intrinsic cellular components.(c) and (d) Raman signal of alkyne and nitrile groups,

Figure S11 .
Figure S11.(a) HR-MALDI-TOF/MS spectrum of Alexa-nanoSABER.The spectrum reflects representative data from in vitro experiments repeated three times.

Figure S13 .Figure S14 .
Figure S13.(a) HPLC chromatogram of Alexa-nanoSABER showing a retention time of 25 minutes and (b) corresponding UV-Vis spectrum with an absorbance maximum at 498 nm.(c) Raman spectrum of Alexa-nanoSABER showing the presence of alkyne and nitrile Raman peaks, shown as mean±SD (n=22 independent in vitro measurements).The HPLC chromatogram and UV-Vis spectrum represent data from in vitro experiments repeated two times.

Figure S15 .
Figure S15.In vivo Raman imaging of (a) DU145 and (b) LNCaP tumor bearing mice 2 h post IV injection of PBS.Subpanels show representative data from three mice in each experimental group.

Figure S16 .
Figure S16.Multivariate Curve Resolution (MCR) was performed on the collected spectral matrix.MCR analysis breaks down the spectral matrix into a score and component matrix.Seven different MCR components were created.MCR1 has spectral features dominated by quartz, MCR2 is dominated by tissue-like spectra with alkyne/nitrile feature in the high wavenumber region, MCR3 is dominated by tissue-like spectra in the fingerprint region (with a few peaks in the high-wavenumber region) and MCR4-MCR7 have varied tissue-like