Nanovehicles as a novel target strategy for hyperthermic intraperitoneal chemotherapy: a multidisciplinary study of peritoneal carcinomatosis

Abstract

In general, detection of peritoneal carcinomatosis (PC) occurs at the late stage when there is no treatment option. In the present study, we designed novel drug delivery systems that are functionalized with anti-CD133 antibodies. The C1, C2 and C3 complexes with cisplatin were introduced into nanotubes, either physically or chemically. The complexes were reacted with anti-CD133 antibody to form the labeled product of A0-o-CX-chem-CD133. Cytotoxicity screening of all the complexes was performed on CHO cells. Data showed that both C2 and C3 Pt-complexes are more cytotoxic than C1. Flow-cytometry analysis showed that nanotubes conjugated to CD133 antibody have the ability to target cells expressing the CD133 antigen which is responsible for the emergence of resistance to chemotherapy and disease recurrence. The shortest survival rate was observed in the control mice group (K3) where no hyperthermic intraperitoneal chemotherapy procedures were used. On the other hand, the longest median survival rate was observed in the group treated with A0-o-C1-chem-CD133. In summary, we designed a novel drug delivery system based on carbon nanotubes loaded with Pt-prodrugs and functionalized with anti-CD133 antibodies. Our data demonstrates the effectiveness of the new drug delivery system and provides a novel therapeutic modality in the treatment of melanoma.

Keywords: carcinomatosis; hyperthermic intraperitoneal chemotherapy; intraperitoneal perfusion and nanovehicles; palliative.

Figure 1. Animal model of peritoneal carcinomatosis and chemotherapy procedures

A. To induce peritoneal carcinomatosis, the 1 × 10⁶ B16 cells were injected into the right lower abdomen quadrant of all animals from each group. B. Intraoperative picture of a mouse from the K3 group, one week after the B16 cells had been injected, the PC process at an advanced stage and exposed following preparation of the peritoneal and visceral tissue. C. Nanovehicles (DDS) were sonicated in 0.7 ml of sterile PBS for three minutes to obtain a homogenous solution. D. Continuous checking ensured that a consistent temperature was maintained across all study groups. E. A mouse from the A0-o-C1-chem-CD133 group just before the modified HIPEC procedure was performed. F. The same mouse as in E immediately after the application of liquid chemotherapy. G. The same mouse as in B just after the surgical cytoreduction of tumors. H. The same mouse as in E/F having sutures applied immediately after the experimental HIPEC procedure, a process consistently applied across all study and control groups.

Figure 2. Thermal analysis results for pure C2 (/C3), A0-o, A0-o-C2 (/C3)-phys, A0-o-C2 (/C3)-chem-n, and A0-o-C2 (/C3)-chem

Figure 3A. High-resolution transmission electron microscopy and z-TEM images of the A0-o-C2-phys sample

Figure 3B. High-resolution transmission electron microscopy and z-TEM images of the A0-o-C3-phys sample

Figure 4A. High-resolution transmission electron microscopy and z-TEM images of the A0-o-C2-chem sample

Figure 4B. High-resolution transmission electron microscopy and z-TEM images of the A0-o-C3-chem sample

Figure 5. The influence of C2 deposition on nitrogen adsorption-desorption isotherms recorded at a temperature of 77 K for the A0-o, A0-o-C2-phys, and A0-o-C2-chem samples

Figure 6. Cytotoxicity of drug delivery systems measured by MTT (A) and LDH (B) assays in CHO cells

Figure 7. Real time cell analysis of B16-F10 after treatment with drugs administered alone (C1, C2 and C3) and with drugs delivered chemically or physically via nanotubes (A0-o-C1, A0-o-C2 and A0-o-C3)

Figure 8. LT50 of tested compounds calculated on the basis of real time cell analysis

Figure 9. Flow cytometry analysis of the anti-CD133 antibody delivered with nanotubes

A.– 5.4% of positively labeled cells (gate P3). B. – Positively labeled cells (red) are small in size and have low granularity.

Figure 10. Survival rate analysis

A. K2;K3;K4 group vs. K1 group. B. C1;C2;C3 group vs. K1. C. AO-o-C1-chem; AO-o-C2-chem; AO-o-C3-chem group vs. K1 group.D. AO-o-C1-chem-CD133 and AO-o-C3-chem-CD133 group vs. K1 group.

Figure 11. H&E staining showing the changes in different organs from all mice groups by light microscopy

A. Focal capsule infiltration (single cells), dispersed infiltration in perirenal adipose tissue presented in a mouse kidney from an A0-o-C2-chem group. Magnification 10x. B. Kidney Capsule infiltration presented in a mouse from A0-o-C3-chem-CD133 group. Magnification 10x. C. Infiltration within the diaphragm in a mouse from A0-o-C3-chem group. Magnification 10x. D. Under capsule infiltration presented in mouse liver from A0-o-C2-chem group. Magnification 10x. E. Liver capsule infiltration presented in mouse from A0-o-C2-chem group. Magnification 10x. F. Liver capsule infiltration presented in mouse from control K2 group. Magnification 10x. G. Small foci of pleural infiltration, metastases to mediastinal lymph nodes presented in mouse from A0-o-C3-chem-CD133 group. Magnification 20x. H. Pleural infiltration presented in a mouse from A0-o-C3-chem-CD133 group. Magnification 20x.

Figure 12. Sample macroscopic pictures from autopsy

A. A mouse from the A0-o-C1-chem-CD133 group with metastatic PC in the peritoneal cavity. B. A mouse from the K1 group with metastatic PC in the peritoneal cavity after the partial removal (during dissection/post-mortem) of large tumor masses which covered the visceral organs.

Scheme 1. Outline of C2 synthesis

Scheme 2. Outline of C3 synthesis

Scheme 3. Outline of A0-o-C1-chem synthesis

Scheme 4. Outline of A0-o-C2-chem synthesis

Scheme 5. Outline of A0-o-C3-chem synthesis