Purpose Breast Cancer Stem Cells (BCSCs) resist conventional treatments and cause tumor recurrence. Almost 25% of breast cancers overexpress human epidermal growth factor receptor-2 (HER2). Here we developed a novel multi-targeted nanosystem to specifically eradicate HER2-positive BCSCs. Methods Plasmids containing CXCR1 promoter, PE38 toxin and 5′UTR of the basic fibroblast growth factor-2 (bFGF 5'UTR) were constructed. Polyamidoamine (PAMAM) dendrimers functionalized with an anti-HER2 VHH

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... used for plasmid delivery. Stem proportion of MCF-10A and MDA-MB-231/HER2+ (a cellular model of HER2 overexpression, developed in our lab) were evaluated by mammosphere formation assay. Hanging drop technique was used to produce spheroids. The uptake, gene expression and killing efficacy of the multi-targeted nanosystem were evaluated in both monolayer and spheroid culture. Results MDA-MB-231/HER2+ had more than 3 times higher ability to form mammosphere compared to MCF-10A, representing higher stem proportion in MDA-MB-231/HER2+. In hanging drop culture, MDA-MB-231/HER+ generated compact well-rounded spheroids, while MCF-10A failed to form compact cellular masses. The multi-targeted nanosystem showed much better uptake, higher PE38 expression and subsequent cell death in MDA-MB-231/HER2+ compared to MCF-10A. However efficacy of our targeted toxin gene therapy were lower in MDA-MB-231/HER2+ spheroid than those in monolayer. Conclusions Our novel multi-targeted nanosystem resulted in synergistic cytotoxic effects in HER2-positive BCSCs. This results demonstrate the necessity to use a combinatorial tumor targeting approach, to restrict the expression of a killer gene to target cells. Lower efficacy of our targeted gene therapy in spheroid models vs. monolayer culture suggest that, anticancer therapy assessments using spheroid might be more predictive of clinical efficacy than conventional monolayer culture.