iRGD-TRP-PK1-modified red blood cell membrane vesicles as a new chemotherapeutic drug delivery and targeting system in head and neck cancer

Abstract

Background: Chemotherapy is essential for treating tumors, including head and neck cancer (HNC). However, the toxic side effects of chemotherapeutic drugs limit their widespread use. Therefore, a targeted delivery system that can transport the drug to the pathological site while minimizing damage to healthy tissues is urgently needed. Methods: Application of animal imaging, flow cytometry, fluorescence staining, cell activity assay, transmission electron microscopy, western blotting and immunohistochemistry to evaluate the targeting and killing effects of internalizing RGD peptide (iRGD)-transient receptor potential (TRP)-PK1-modified red blood cell vesicles (RBCVs) on HNC cells in vitro and in vivo. Results: TRP-PK1 was ligated to iRGD, enabling autonomous insertion into the lipid bilayer. Additionally, RBCVs were labeled with iRGD-TRP-PK1 to achieve tumor targeting. Based on the self-assembly capability of TRP-PK1 to form a "leakage potassium" channel on the biofilm, RBCVs were fragmented within the high-potassium (K⁺) environment inside tumor cells. This fragmentation facilitated the release of the drug loaded onto the RBCVs. Conclusion: The advantageous properties of TRP-PK1 are utilized in our design, resulting in a cost-effective and straightforward approach to drug delivery and release. Ultimately, the objective of suppressing tumor growth while minimizing side effects was accomplished by iRGD-TRP-PK1-modified RBCVs in our study. These findings provide novel insights into the enhancement of targeted delivery systems and present promising avenues for the treatment of HNC.

Keywords: Drug Delivery; Head and Neck Cancer; Red Blood Cell Membrane Vesicles; Targeting System; iRGD-TRP-PK1.

Figure 1

Preparation and characterization of iRGD-TRP-PK1-modified RBCVs. (A) Schematic illustration of the formation of iRGD-TRP-PK1-modified RBCVs. (B) Detection of Dox and CDDP loading efficiency by RBCVs in a hypotonic to isotonic transition environment. (C) Electron microscopy of iRGD-TRP-PK1-modified RBCVs and their loading with Dox and CDDP. (D) Particle size and concentration distribution of iRGD-TRP-PK1-modified RBCVs and their loading with Dox and CDDP. CDDP: cisplatin, Dox: doxorubicin, iRGD: internalizing RGD peptide, RBCV: red blood cell versicle.

Figure 2

iRGD-TRP-PK1 enhances the cellular uptake of RBCVs. (A) Representative images of integrin α_vβ₃ expression in NP69, CNE1, HN4, CNE2, HONE1, and 5-8F cells. (B) Representative images of iRGD-TRP-PK1-modified RBCVs uptake by NP69, HN4, and CNE2 cells. (C) Targeting of iRGD-TRP-PK1-modified RBCVs after integrin α_vβ₃ inhibitor--Cyclo(-RGDfK) treatment. (D) Representative flow images of the fraction of RBCVs taken up by HN4 cells after treatment with iRGD-, TRP-PK1-, iRGD-TRP-PK1-modified RBCVs and Cyclo(-RGDfK) treatment. (E) Percentage of HN4 cells after uptake of RCBVs. (F) Targeting of iRGD-TRP-PK1-modified RBCVs and their loading with Dox and CDDP in tumors established by HN4 cells in a CDX model. CDX: cell-derived xenograft (CDX), iRGD: internalizing RGD peptide.

Figure 2

iRGD-TRP-PK1 enhances the cellular uptake of RBCVs. (A) Representative images of integrin α_vβ₃ expression in NP69, CNE1, HN4, CNE2, HONE1, and 5-8F cells. (B) Representative images of iRGD-TRP-PK1-modified RBCVs uptake by NP69, HN4, and CNE2 cells. (C) Targeting of iRGD-TRP-PK1-modified RBCVs after integrin α_vβ₃ inhibitor--Cyclo(-RGDfK) treatment. (D) Representative flow images of the fraction of RBCVs taken up by HN4 cells after treatment with iRGD-, TRP-PK1-, iRGD-TRP-PK1-modified RBCVs and Cyclo(-RGDfK) treatment. (E) Percentage of HN4 cells after uptake of RCBVs. (F) Targeting of iRGD-TRP-PK1-modified RBCVs and their loading with Dox and CDDP in tumors established by HN4 cells in a CDX model. CDX: cell-derived xenograft (CDX), iRGD: internalizing RGD peptide.

Figure 3

iRGD-TRP-PK1 promoted anti-cancer drug release which loaded within RBCVs in HNC cells. (A) Electron microscopy morphology images of iRGD-TRP-PK1-modified RBCVs in a high-potassium environment. (B) Representative images of iRGD-TRP-PK1-modified RBCVs distribution within HN4 and CNE2 cells. (C) Rate of Dox release in high K⁺ and PBS as time progresses, *P<0.05 by two-way ANOVA. (D) Release of Dox from iRGD-TRP-PK1-modified RBCVs loaded with Dox after co-incubation with CNE2 cells. (E) Release of Dox from iRGD-TRP-PK1-modified RBCVs loaded with Dox after co-incubation with HN4 cells. Dox: doxorubicin, iRGD: internalizing RGD peptide, RBCVs: red blood cell vesicles.

Figure 3

iRGD-TRP-PK1 promoted anti-cancer drug release which loaded within RBCVs in HNC cells. (A) Electron microscopy morphology images of iRGD-TRP-PK1-modified RBCVs in a high-potassium environment. (B) Representative images of iRGD-TRP-PK1-modified RBCVs distribution within HN4 and CNE2 cells. (C) Rate of Dox release in high K⁺ and PBS as time progresses, *P<0.05 by two-way ANOVA. (D) Release of Dox from iRGD-TRP-PK1-modified RBCVs loaded with Dox after co-incubation with CNE2 cells. (E) Release of Dox from iRGD-TRP-PK1-modified RBCVs loaded with Dox after co-incubation with HN4 cells. Dox: doxorubicin, iRGD: internalizing RGD peptide, RBCVs: red blood cell vesicles.

Figure 4

iRGD-TRP-PK1-modified RBCVs loaded with chemotherapeutic agents significantly inhibit HNC cell proliferation and promote apoptosis. (A) Inhibition efficiency assay of cells after treatment of HN4 cells with different concentration of Dox, RBCV+Dox, iRGD-RBCV+Dox, iRGD-TRP-PK1-RBCV+Dox or CDDP, RBCV+CDDP, iRGD-RBCV+CDDP, iRGD-TRP-PK1-RBCV+CDDP. (B) Inhibition efficiency assay of cells after treatment of CNE2 cells with different concentration of Dox, RBCV+Dox, iRGD-RBCV+Dox, iRGD-TRP-PK1-RBCV+Dox or CDDP, RBCV+CDDP, iRGD-RBCV+CDDP, iRGD-TRP-PK1-RBCV+CDDP. (C) Cell apoptosis of HN4 cells after treated with CDDP, RBCV+CDDP, iRGD-RBCV+CDDP, iRGD-TRP-PK1-RBCV+CDDP and Cyclo(-RGDfK)+iRGD-TRP-PK1-RBCV+CDDP. (D) Cell apoptosis of CNE2 cells after treated with CDDP, RBCV+CDDP, iRGD-RBCV+CDDP, iRGD-TRP-PK1-RBCV+CDDP and Cyclo(-RGDfK)+iRGD-TRP-PK1-RBCV+CDDP. CDDP: cisplatin, CCK-8: cell counting kit-8, Dox: doxorubicin, OD: optical density, iRGD: internalizing RGD peptide, RBCVs: red blood cell vesicles. *P < 0.05 by Student's t-test.

Figure 4

iRGD-TRP-PK1-modified RBCVs loaded with chemotherapeutic agents significantly inhibit HNC cell proliferation and promote apoptosis. (A) Inhibition efficiency assay of cells after treatment of HN4 cells with different concentration of Dox, RBCV+Dox, iRGD-RBCV+Dox, iRGD-TRP-PK1-RBCV+Dox or CDDP, RBCV+CDDP, iRGD-RBCV+CDDP, iRGD-TRP-PK1-RBCV+CDDP. (B) Inhibition efficiency assay of cells after treatment of CNE2 cells with different concentration of Dox, RBCV+Dox, iRGD-RBCV+Dox, iRGD-TRP-PK1-RBCV+Dox or CDDP, RBCV+CDDP, iRGD-RBCV+CDDP, iRGD-TRP-PK1-RBCV+CDDP. (C) Cell apoptosis of HN4 cells after treated with CDDP, RBCV+CDDP, iRGD-RBCV+CDDP, iRGD-TRP-PK1-RBCV+CDDP and Cyclo(-RGDfK)+iRGD-TRP-PK1-RBCV+CDDP. (D) Cell apoptosis of CNE2 cells after treated with CDDP, RBCV+CDDP, iRGD-RBCV+CDDP, iRGD-TRP-PK1-RBCV+CDDP and Cyclo(-RGDfK)+iRGD-TRP-PK1-RBCV+CDDP. CDDP: cisplatin, CCK-8: cell counting kit-8, Dox: doxorubicin, OD: optical density, iRGD: internalizing RGD peptide, RBCVs: red blood cell vesicles. *P < 0.05 by Student's t-test.

Figure 5

iRGD-TRP-PK1-modified RBCVs loaded with chemotherapeutic agents significantly increase tumor suppression and reduce side effects. (A) Representative images depicting tumors after treatment with different formulations. (B) Statistical analysis of tumor weight/body weight after treatment with different formulations. (C) Statistical analysis of tumor volume after treatment with different formulations. (D) Representative images of PCNA expression by IHC. (E) Toxic analysis of organs from mice after treatment with different formulations by the pathological HE staining. HE: hematoxylin and eosin, iRGD: internalizing RGD peptide, RBCVs: red blood cell vesicles. *P < 0.05 using Student's t-test.

Figure 5

iRGD-TRP-PK1-modified RBCVs loaded with chemotherapeutic agents significantly increase tumor suppression and reduce side effects. (A) Representative images depicting tumors after treatment with different formulations. (B) Statistical analysis of tumor weight/body weight after treatment with different formulations. (C) Statistical analysis of tumor volume after treatment with different formulations. (D) Representative images of PCNA expression by IHC. (E) Toxic analysis of organs from mice after treatment with different formulations by the pathological HE staining. HE: hematoxylin and eosin, iRGD: internalizing RGD peptide, RBCVs: red blood cell vesicles. *P < 0.05 using Student's t-test.