Evaluation of mAb 2C5-modified dendrimer-based micelles for the co-delivery of siRNA and chemotherapeutic drug in xenograft mice model

Abstract

Combination therapy with small interfering RNA (siRNA) and chemotherapeutic drug is proven to be effective in downregulating cancer resistance proteins, such as P-glycoprotein (P-gp). These proteins are involved in multidrug resistance (MDR) of tumors. A targeted formulation capable of delivering siRNA and chemotherapeutic drug will not only downregulate P-gp but also increase the concentration of the chemotherapeutic drug at the site of tumor thereby increasing the therapeutic effect and lowering the systemic exposure. In this study, monoclonal antibody 2C5-modified dendrimer-based micelles were used to co-deliver siRNA and doxorubicin (DOX) to the tumor site in both male and female xenograft mouse model. The nucleosome-specific 2C5 antibody recognizes the cancer cells via the cell-surface bound nucleosomes. The ability of ability of the 2C5-modified formulation to affect the metastasis of highly aggressive triple negative breast cancer cell migration in (MDA-MB-231) was assessed by a wound healing. Further, the therapeutic efficacy of the formulation was assessed by measuring the tumor volume progression in which the 2C5-modified nanoparticle group had a similar tumor volume to the free drug group at the end of the study, although a 50% increase in DOX concentrations in blood was observed after the last dose of nanoparticle. The free drug group on the other hand showed body weight reduction as well as the visible irritation around the injection spot. The treatment group with 2C5-modified micelles has shown to be safe at the current dose of DOX and siRNA. Furthermore, the siRNA mediated P-gp downregualtion was studied using western blotting assay. We observed a 29% reduction of P-gp levels in both males and females with respect to the control (BHG). We also conclude that the dose of DOX and siRNA should be further optimized to have a better efficacy in a metastatic tumor model, which will be the subject of our future studies.

Keywords: Breast cancer; Co-delivery; Dendrimer; In vivo; Nanoparticles; Preclinical model; siRNA.

Fig. 1

In vivo study design. Created by BioRender.com

Fig. 2

Evaluation of tumor metastatic potential in vitro using wound healing assay, A: Figure shows the start and end points of cell migration in the treatment groups, B: The graph shows the coverage area of the cell with respect to time in hours

Fig. 3

Tumor progression (size) of the xenografted tumors in both male and female mice, A: Female mice, B: Male mice

Fig. 4

Body weight change of both male and female mice, A: The data for female mice, B: The data for male mice

Fig. 5

Dermatological toxicities observed in free DOX treated group, A: Open wounds on the mice in DOX group, B: Skin peeling observed on the tail region of the mice after repeated dosing. No similar toxicities were observed in other treatment group

Fig. 6

Weights of harvested tumors and livers in female and male mice. The tumors weights show the effectiveness of the treatment and the weights of the liver show the safety of the treatment, A: Tumor weights in female mice, B: Liver weights in female mice, C: Tumor weights in male mice, D: Liver weights in male mice

Fig. 7

Liver enzyme Aspartate Aminotransferase Activity (AST) activity indicates the safety of the treatment and liver toxicity caused by the treatment, A: AST activity in male mice, B: AST activity in female mice. Results indicate ± SD (n = 3), and significance was calculated with one-way ANOVA comparisons. **** p ≤ 0.0001, *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05

Fig. 8

Plasma concentrations of DOX prior to administration, A: Plasma concentrations of DOX with time in male mice, B: Plasma concentration of DOX with respect to time in female mice. Results indicate ± SD (n = 3), and significance was calculated with one-way ANOVA comparisons. ** p ≤ 0.05

Fig. 9

DOX accumulation from the ex vivo imaging data of the tumors and livers of female and male animals, A: Female tumors, B: Female livers, C: Male tumors, D: Male livers. Results indicate ± SD (n = 3), and significance was calculated with two-way ANOVA comparisons. **** p ≤ 0.0001, *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05

Fig. 10

Tumor metastasis to the liver and other regions in the body, A and B: multiple tumors in the neck region that were metastasized from the primary tumor and a blood vessel connecting them, C and D: Livers with metastatic tumor. White Arrows: Primary and Metastasized tumors, Red Arrows: Blood vessel connecting the primary tumor to the secondary tumor

Fig. 11

P-gp levels in harvested tumors measured using western blot. (A) P-gp levels female tumors, (B) P-gp levels in male tumors. The above data shows the densitometry analysis. Results indicate ± SD (n = 3)