Results were processed using FlowJo software (v 7.6.5, Tree Star, Inc.). == In vitro cytotoxicity assay == MCF-7 and HCC1954 cells (1104) were plated into 96-well trays by sextuplicate under the culture conditions detailed earlier. and evaluate a drug delivery system based on protein A-functionalized ISCOMs to target HER2-overexpressing cells. We describe the preparation of ISCOMs, the loading with the drugs doxorubicin and paclitaxel, the binding of ISCOMs to alkyl vinyl sulfone-protein A, the coupling of Tmab, and the evaluation in both HER2-overexpressing breast cancer cells (HCC1954) and non-overexpressing cells (MCF-7) by flow cytometry and fluorescence microscopy. Results show that the uptake is dependent on the level of overexpression of HER2, and the analysis of the cell viability reveals that targeted drugs are selective toward HCC1954, whereas MCF-7 cells remain unaffected. == Conclusion == Protein A-functionalized ISCOMs are versatile carriers that can be coupled to antibodies that act as targeting agents to deliver drugs. When coupling to Tmab and loading Ibrutinib-biotin with paclitaxel or doxorubicin, they become efficient vehicles for the selective delivery of the drug to Tmab-resistant HER2-overexpressing breast cancer cells. These nanoparticles may pave the way for the development of novel therapies for poor prognosis resistant patients. Keywords:targeted drug delivery, doxorubicin, HER2, nanoparticle, paclitaxel, protein A, trastuzumab == Introduction == Nanomedicine is an expanding field of research KIAA1516 that allows the development of nanoparticles (NPs) capable of improving the stability, transport, bioavailability, and specificity during drug delivery to tumor cells.1Additionally, NPs may also be useful to increase the sensitivity of different diagnostic techniques, improving the early detection of the disease and the success of the treatment.2The first NPs used as vehicles of antitumor drugs were liposomes conjugated with doxorubicin (Dox) or with albumin and paclitaxel (Pac). Currently, there exist a wide diversity of NPs, including some of inorganic nature such as iron oxide and gold NPs.2 Breast cancer remains the leading cause of cancer death in women, with a frequency that increases with age until menopause.3Among the types of breast cancer, ~20%30% overexpress the proto-oncogene human epidermal growth factor receptor 2 (HER2), and they are characterized as being very invasive. Similarly, tumors in other locations such as colon, stomach, pancreas, bladder, lung, and testicle may also overexpress HER2.4,5At present, much research is focused on the study of the prognostic value of HER2 and the development of new therapies specifically directed against tumors that overexpress this receptor.6 Depending on the type of breast cancer and its stage, treatment can include surgical resection, radiotherapy, chemotherapy, and hormone therapy. However, there is still a high rate of recurrence and metastasis, due in part to the resistance that tumor cells develop to some drugs. In addition, it is necessary to find more specific drugs that Ibrutinib-biotin reduce or avoid side effects from conventional chemotherapy, such as, for example, the cardiac dysfunction associated with anthracycline-based therapy.5,7 The humanized antibody trastuzumab (Tmab) represented a great advance in the treatment of breast tumors that overexpress HER2. This antibody specifically binds to HER2 and is widely used to treat early, advanced, and metastatic stages of cancer.2,8Tmab interferes with mitogenic signaling induced by the receptor, and its action is highly specific against HER2-overexpressing cells.9However, a large proportion of patients develop primary or secondary resistance in the course of the treatment.10,11 It has been found that the combined treatment of Tmab and doxorubicin produces a synergistic antitumor effect against HER2-overexpressing breast tumor cells.12Unfortunately, there exists evidence that Dox may cause Ibrutinib-biotin cardiac toxicity, which limits its use. However, Tmab-conjugated NPs loaded with doxorubicin fortify the antitumor effect of the drug, reducing its cardiotoxicity.13 In this regard, the conjugation of drugs with antibodies that recognize specific antigens of target cells allows the improvement of the specificity of the treatment and, in many cases, reduces the undesired side effects. This strategy has tremendous promise as a new targeted cancer therapy, and a relatively large number of antibodydrug conjugates are already in clinical development.14In particular, Tmab conjugated to the Ibrutinib-biotin cytotoxic agent emtansine was approved by the US Food and Drug Administration in 2013 for the treatment of patients with HER2-positive breast cancer, and it is currently in the market.15 However, the linkage of the drug to the antibody is far from trivial, and in this context our group has already reported the construction of immunostimulating complexes (ISCOM) functionalized with vinyl sulfone lipid nanocapsules. The reactivity of the vinyl sulfone group Ibrutinib-biotin toward amine and thiol groups was exploited to obtain ISCOMs functionalized with protein A, the affinity of which for IgG allows the anchoring of an antibody that targets the ISCOMs to specific cells.16In this work, we go one step further: ISCOMs functionalized with protein A and loaded with Pac or Dox are conjugated to Tmab and assayed against Tmab-resistant HER2-overexpressing breast cancer cells (HCC1954) and non-overexpressing cells (MCF-7). == Materials and.