Oral delivery of aptamer-decorated SICTERS Raman probes for colonoscopy-guided resection and photothermal immunization of microtumors

Abstract

Endoscopic resection of colon cancer represents an advanced technique in early intervention, but faces the challenges in identifying tumor margins and removing residual lesions. Here, we developed orally administered Raman probes (i.e., BBT-Apt@CS NPs) that enable precise tumor imaging and photothermal ablation. Protected by an enteric coating for colon-targeted release, the inner nanoparticles use a stacking-induced charge transfer-enhanced Raman scattering (SICTERS) mechanism and are functionalized with tumor-targeting aptamers. In a proof-of-concept study using the endoscopic optical fiber, we achieved in situ precise detection of microtumors and effective photothermal ablation of residual lesions at the resection margins, with simultaneous induction of immune memory. In a rechallenge model, mice treated with photothermal therapy on residual lesions exhibited no tumor recurrence. We have optimized endoscopic resection strategies through theranostic Raman probes, thus providing an innovative solution for the minimally invasive precision intervention in colorectal cancer.

Fig. 1.. Schematic illustration of an orally delivered, aptamer-decorated SICTERS-based Raman probe (BBT-Apt@CS NPs).

(A) BBT-Apt@CS NPs were constructed on aptamer-decorated BBT NPs by coating chitosan and sodium alginate layer by layer. (B) The process of the coating slowly degrades and realizes Raman imaging–assisted detection and photothermal immunity. After oral delivery of BBT-Apt@CS NPs, the coating degrades slowly in colon and the exposed aptamer identifies overexpressed nucleolins on tumor surfaces. Because of the photothermal properties of BBT NPs, the dying tumor cells release tumor-associated antigens to promote maturation of DC and proliferation of T cell.

Fig. 2.. Construction and characterization of BBT-Apt@CS NPs.

(A) Schematic diagram of the preparation of BBT-Apt@CS NPs. BBT NPs were modified with aptamers to form BBT-Apt NPs, which were coated with chitosan to yield BBT-Apt@C NPs, and then further coated with sodium alginate to obtain BBT-Apt@CS NPs. (B) The particle size of BBT NPs, BBT-Apt NPs, BBT-Apt@C NPs, and BBT-Apt@CS NPs. (C) The change of zeta potential as the coating layer increases. Data are presented as mean ± SD. (D) Agarose gel electrophoresis was used to monitor the modification of aptamers. (E) The absorption spectra of BBT NPs, BBT-Apt NPs, BBT-Apt@C NPs, and BBT-Apt@CS NPs remained consistent. (F) The TEM image of BBT-Apt@CS NPs. Scale bar, 500 nm. (G and H) The Raman signal stability of BBT-Apt NPs (G) and BBT-Apt@CS NPs (H) in simulated gastric fluid (SGF), simulated intestinal fluid (SIF), and simulated colonic fluid (SCF). (I to K) Particle size of BBT-Apt NPs and BBT-Apt@CS NPs in SGF (I), SIF (J), and SCF (K). Data are presented as mean ± SD. (L) Agarose gel electrophoresis was used to monitor the degradation of aptamers from free aptamer, BBT-Apt NPs, and BBT-Apt@CS NPs in SGF, SIF, and SCF.

Fig. 3.. Selective recognition of tumor cells and tissues by BBT-Apt NPs.

(A) Schematic illustration of BBT NPs and BBT-Apt NPs. (B) Raman images (894 cm⁻¹) of BBT NPs and BBT-Apt NPs in NCM460 and CT26. Raman image (894 cm⁻¹) of CT26 tumor spheroids (C) and their quantitative Raman intensity (D) after 6-hour incubation with BBT-Apt NPs. Data are presented as mean ± SD (n = 3), and statistical significance was calculated via unpaired two-tailed t test, **P < 0.01. (E and F) The recognition of NCM460 and CT26 cells by BBT-Apt NPs (Cy5 labeled) over time and their quantitative relative fluorescence intensity (G). Data are presented as Mean ± SD (n = 3), and statistical significance was calculated via two-way analysis of variance (ANOVA) with Sidak’s post hoc test, ****P < 0.0001. (H) Representative Raman images of health colon and tumor after treat with BBT-Apt NPs (0.1 mg/ml) for 0.5 hours. (I) Raman spectra of site 1 and site 2 in (H).

Fig. 4.. Orally delivery of BBT-Apt@CS NPs for sensitive tumor detection in vivo.

(A) Raman imaging was performed on tumor-bearing mice (inoculated CT26 cells for 5 days) after oral administration of BBT-Apt@CS NPs or BBT-Scrambled Apt@CS NPs (BBT-Scr@CS NPs). (B) The BBT-Apt@CS NPs and BBT-Scr@CS NPs tumor areas were randomly selected and quantified. (C) Tumor section from (A) BBT-Apt@CS NPs group to perform HE staining and Raman imaging. (D) Raman imaging was performed on tumor-bearing mice (inoculated CT26 cells for 7 days) after oral administration of BBT-Apt@CS NPs or BBT-Scr@CS NPs. (E) Pharmacokinetics of oral BBT-Apt@CS NPs (20 mg kg⁻¹). (F) Raman imaging of colon on APC^Min/+ model and HE staining of tumor area. (G) Schematic diagram of Raman endoscopy detection. (H and I) Raman endoscopy detection after oral administration of BBT-Apt@CS NPs (20 mg kg⁻¹), showing Raman spectra of tumor area and health area. (J) HE staining of tumor area and health area.

Fig. 5.. Raman imaging–guided photothermal therapy after surgical resection.

(A) Thermal imaging of photothermally treated mice after surgical resection and (B) the heating curves. (C) Schematic diagram of Raman imaging–guided photothermal therapy. (D) Kaplan-Meier survival rate curve of tumor bearing mice treated with indicated therapy for 57 days (n = 5). Statistical significance was calculated via the log-rank Mantel-Cox test, *P < 0.05, **P < 0.01, ns means no significant difference. (E) Representative IVIS bioluminescence imaging of mice bearing orthotopic colon tumor after different treatment in 21 days. (F) The fold changes in Radiance relative to day 0 were visualized using a heatmap.

Fig. 6.. Immune effects induced by photothermal therapy.

(A) Representative IVIS bioluminescence imaging of mice bearing orthotopic colon tumor after different treatment and re-inoculation in the armpits on day 22. (B) Tumor growth curve of the CT26 tumors in the armpits. Data are presented as mean ± SD (n = 5), and statistical significance was calculated via unpaired two-tailed t test, *P < 0.05. (C) Representative flow cytometric plot of CD4⁺ and CD8⁺ T cells (gated on L/D⁻ CD45⁺ CD3⁺ cells) in tumor and lymph gland, and cytometric plot of CD80⁺ and CD86⁺ DC cells (gated on L/D⁻ CD45⁺ CD80⁺ CD86⁺ cells) in lymph nodes. (D) Quantitative analysis of CD4⁺ and CD8⁺ T cells in tumor, and CD80⁺ and CD86⁺ DC cells in lymph nodes. Data are presented as mean ± SD (n = 5), and statistical significance was calculated via unpaired two-tailed t test. **P < 0.01, ****P < 0.0001.