SREKA-targeted liposomes for highly metastatic breast cancer therapy

Abstract

Chemotherapy is still a leading therapeutic approach in various tumor types that is often accompanied by a poor prognosis because of metastases. PEGylated liposomes with CREKA targeting moiety are well-known therapeutic agents, especially in highly metastatic experimental models. CREKA specifically targets tumor-associated ECM, which is present at the primary, as well as metastatic tumor sites. To better understand the function of the targeting moieties, we decided to design various liposome formulations with different amounts of targeting moiety attached to their DSPE-PEG molecules. Moreover, a new tumor-homing pentapeptide (SREKA) was designed, and a novel conjugation strategy between SREKA and DSPE-PEGs. First, the in vitro proliferation inhibition of drug-loaded liposomes and the cellular uptake of their cargo were investigated. Afterward, liposome stability in murine blood and drug accumulation in different tissues were measured. Furthermore, in vivo tumor growth, and metastasis inhibition potencies of the different liposome formulations were examined. According to our comparative studies, SREKA-liposomes have a uniform phenotype after formulation and have similar characteristics and tumor-homing capabilities to CREKA-liposomes. However, the exchange of the N-terminal cysteine to serine during conjugation results in a higher production yield and better stability upon conjugation to DSPE-PEGs. We also showed that SREKA-liposomes have significant inhibition on primary tumor growth and metastasis incidence; furthermore, increase the survival rate of tumor-bearing mice. Besides, we provide evidence that the amount of targeting moiety attached to DSPE-PEGs is largely responsible for the stability of liposomes, therefore it plays an important role in toxicity and targeting.

Keywords: SREKA peptide; Targeted cancer therapy; nanocarriers; tumor metastasis.

Figure 1.

The process of metastasis and the working principle of newly developed targeted liposomes. A: Metastatic breast cancer can develop when the basement membrane is broken down, and it allows malignant cells to migrate from their primary site into the bloodstream or lymphatic vessels (intravasation). When circulating tumor cells adhere to distant locations inside the vessel, extravasation occurs, and cells invade the distant tissue. If these tumor cells can survive in their new microenvironment and start to proliferate, metastatic sites are established. Cancer cells and tumor-associated fibroblasts produce an increased amount of fibrin, resulting in the accumulation of fibrin clots in proximity to the primary tumor and especially at metastases. PEGylated liposomes are stable drug carriers, which, once injected into patients, stay inside the bloodstream for an extended amount of time compared to the free drug. Modification of PEG molecules with SREKA enables liposomes to specifically bind to fibrin clots associated with primary and metastatic tumor sites resulting in the accumulation of liposomes at these sites and the release of their cargo. B: DSPE-PEG2000 conjugate of Aoa-SREKA-NH2 peptide derivative with oxime-bond (upper) and DSPE-PEG2000 conjugate of H-CREKA-NH2 peptide with thioether bond (lower).

Figure 2.

Characterization of liposome samples. Intensity weighted size distributions were measured by DLS (A) and Zeta potential values of the liposomes with different amounts of DSPE-PEG-CREKA and DSPE-PEG-SREKA (B). Representative TEM images of the Lipo-NP (C) and Lipo-100C (D) samples. Scale bars represent 200 nm.

Figure 3.

In vitro evaluation of liposome formulations and free drug using 4T1-Luc and NIH-3T3 cell lines. A and B: Cellular uptake of drug in either free from or encapsulated in liposomes. Cells were treated for 1, 4, and 24 h respectively, with an equal amount of drug either using the free drug or different liposome formulations. Drug uptake over time of 4T1-Luc (A) and NIH-3T3 (B) cell lines was measured by HPLC-MS/MS. Technical replicates (TR) = 1, biological replicate = 1.

Figure 4.

Pharmacokinetics and biodistribution of the free and liposome-encapsulated drug in murine allograft breast cancer model. Mice were treated with free drug and liposome formulations once. A: Whole blood was collected 1, 4, 24, 48, and 96 h after treatment. Total daunomycin in blood samples was extracted and measured by HPLC-MS/MS. B: Organs and primary tumors were collected from each mouse 96 h after treatment. Tissues were homogenized, and total daunomycin was extracted and detected using HPLC-MS/MS. Error bars represent the mean ± SEM. The level of daunomycin in the kidney was chosen to be 100% as it was the median value (TR = 3, BR = 3–5).

Figure 5.

Inhibition of primary tumor expansion. Change of primary tumor size of mice treated with liposome formulations and free drug. Tumor volume is represented in mm³. *SL = Significance level. Error bars represent mean ± SEM (BR > 5, ***p < .001).

Figure 6.

Inhibition of metastatic incidences in the lung. A: H&E staining of the lung of mice treated with liposome formulations and free drug. B: The number of metastatic nodules on the surface of the entire lung of mice treated with mock treatment, free daunomycin and liposomal formulations. *SL = Significance level. Error bars represent the mean ± SEM (n > 5, ***p < .001).

Figure 7.

Inhibition of metastatic incidences in the liver. A: Ki-67 staining of the liver obtained from mice treated with free daunomycin and liposomal formulations. B: The ratio of metastatic and healthy cells in the liver of mice from different treatment groups. *SL = Significance level. Error bars represent the mean ± SEM (n = 15, ***p < .001).

Figure 8.

Survival of murine allograft breast cancer model upon treatment with the free and liposome-encapsulated drug. The survival of mice (from the time tumor cells were injected until reaching cutoff values) is compared by the Mantel-Cox test between controls and groups treated with either free drug or drug encapsulated in liposomes. The median survival of different groups is shown in the table below. *MS = Median survival in days. **SL = Significance level. (n > 4, *p < .05).