Mechanistic insights into the pH-dependent membrane peptide ATRAM.

pH-responsive peptides are promising therapeutic molecules that can specifically target the plasma membrane in the acidified extracellular medium that bathes cells in tumors. We designed the acidity-triggered rational membrane (ATRAM) peptide to have a pH-responsive membrane interaction. At physiological pH, ATRAM binds to the membrane surface in a largely unstructured conformation, while in acidic conditions it inserts into lipid bilayers forming a transmembrane helix. However, the molecular mechanism ATRAM uses to target and insert into tumor cells remains poorly understood. Here, we determined that ATRAM inserts into cancer cells with a preferential membrane orientation, where the C-terminus of the peptide traverses the plasma membrane and explores the cytoplasm. Using biophysical techniques, we determined that the membrane interaction of ATRAM is contingent on the concentration of the peptide. Kinetic studies showed that membrane insertion occurs in at least three steps, where only the first step was affected by the membrane density of ATRAM. These observations, combined with membrane binding and leakage data, indicate that the interaction of ATRAM with lipid membranes is dependent on its oligomerization state. SPECT/CT imaging in mice revealed that ATRAM accumulates in the blood pool, where it has a prolonged circulation time (> 4 h). Since fast peptide clearance and degradation in circulation are major problems for clinical development, we studied the mechanism ATRAM uses to remain in the blood stream. Using binding and transfer assays, we determined that ATRAM binds reversibly to human serum albumin. We propose that ATRAM uses albumin as a carrier in the blood stream to evade clearance and proteolysis before interacting with the plasma membrane of cancer cells. We also show that ATRAM is able to be deliver liposomes to cells in a pH dependent way. Our data highlight the potential of ATRAM as a specific therapeutic agent for diseases that lead to acidic tissues, including cancer.

Development and evaluation of the Sinomenine transdermal patch.

Sinomenine transdermal patch was prepared and its properties were studied. The patches were produced by salivation method. The releasing rate in vitro of the patch was determined by HPLC. Peel test was used to evaluate the adhesion. Acute skin irritation test was performed in comparison with formalin (0.8%) by using mouse model. The Sinomenine TDDS Patch was prepared. The releasing rate in vitro followed the Higuchi equation (r>0.99), the releasing amount was beyond 90% in 24h. The peel adhesion to steel (N/25 mm) is 10 or above. The skin irritation tests showed negligible erythema and edema. The Sinomenine transdermal patch was prepared successfully and it may be beneficial for topical use.

Liposomal Nanoparticles Carrying anti-IL6R Antibody to the Tumour Microenvironment Inhibit Metastasis in Two Molecular Subtypes of Breast Cancer Mouse Models.

Tumour microenvironment (TME) contributes significantly towards potentiating the stemness and metastasis properties of cancer cells. IL6-Stat3 is one of the important cell signaling pathways in mediating the communication between tumour and immune cells. Here, we have systematically developed a novel anti-CD44 antibody-mediated liposomal nanoparticle delivery system loaded with anti-IL6R antibody, which could specifically target the TME of CD44+ breast cancer cells in different mouse models for triple negative and luminal breast cancer. This nanoparticle had an enhanced and specific tumour targeting efficacy with dramatic anti-tumour metastasis effects in syngeneic BALB/c mice bearing 4T1 cells as was in the syngeneic MMTV-PyMT mice. It inhibited IL6R-Stat3 signaling and moderated the TME, characterized by the reduced expression of genes encoding Stat3, Sox2, VEGFA, MMP-9 and CD206 in the breast tissues. Furthermore, this nanoparticle reduced the subgroups of Sox2+ and CD206+ cells in the lung metastatic foci, demonstrating its inhibitory effect on the lung metastatic niche for breast cancer stem cells. Taken together, the CD44 targeted liposomal nanoparticles encapsulating anti-IL6R antibody achieved a significant effect to inhibit the metastasis of breast cancer in different molecular subtypes of breast cancer mouse models. Our results shed light on the application of nanoparticle mediated cancer immune-therapy through targeting TME.