pH-responsive high stability polymeric nanoparticles for targeted delivery of anticancer therapeutics.

The practical application of nanoparticles (NPs) as chemotherapeutic drug delivery systems is often hampered by issues such as poor circulation stability and targeting inefficiency. Here, we have utilized a simple approach to prepare biocompatible and biodegradable pH-responsive hybrid NPs that overcome these issues. The NPs consist of a drug-loaded polylactic-co-glycolic acid (PLGA) core covalently 'wrapped' with a crosslinked bovine serum albumin (BSA) shell designed to minimize interactions with serum proteins and macrophages that inhibit target recognition. The shell is functionalized with the acidity-triggered rational membrane (ATRAM) peptide to facilitate internalization specifically into cancer cells within the acidic tumor microenvironment. Following uptake, the unique intracellular conditions of cancer cells degrade the NPs, thereby releasing the chemotherapeutic cargo. The drug-loaded NPs showed potent anticancer activity in vitro and in vivo while exhibiting no toxicity to healthy tissue. Our results demonstrate that the ATRAM-BSA-PLGA NPs are a promising targeted cancer drug delivery platform.

The Effect of Phosphatidylserine on a pH-Responsive Peptide Is Defined by Its Noninserting End.

The acidity-triggered rational membrane (ATRAM) peptide was designed to target acidic diseases such as cancer. An acidic extracellular medium, such as that found in aggressive tumors, drives the protonation of the glutamic acids in ATRAM, leading to the membrane translocation of its C-terminus and the formation of a transmembrane helix. Compared to healthy cells, cancerous cells often increase exposure of the negatively charged phosphatidylserine (PS) on the outer leaflet of the plasma membrane. Here we use a reconstituted vesicle system to explore how PS influences the interaction of ATRAM with membranes. To explore this, we used two new variants of ATRAM, termed K2-ATRAM and Y-ATRAM, with small modifications at the noninserting N-terminus. We observed that the effect of PS on the membrane insertion pK and lipid partitioning hinged on the sequence of the noninserting end. Our data additionally indicate that the effect of PS on the insertion pK does not merely depend on electrostatics, but it is multifactorial. Here we show how small sequence changes can impact the interaction of a peptide with membranes of mixed lipid composition. These data illustrate how model studies using neutral bilayers, which do not mimic the negative charge found in the plasma membrane of cancer cells, may fail to capture important aspects of the interaction of anticancer peptides with tumor cells. This information can guide the design of therapeutic peptides that target the acidic environments of different diseased tissues.

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.

Anecdote or Reality: Are People From the South and/or Rural Areas of the USA More Empathetic?

Although numerous studies have analyzed empathy scores of allopathic and osteopathic medical students and how these scores fluctuate throughout undergraduate medical education, little is known about whether demographics have an impact on medical students' empathy scores. Using the Balanced Emotional Empathy Scale (BEES) and Jefferson Scale of Empathy (JSE) surveys to measure affective and cognitive empathy, respectively, this present study examined the relationship between empathy scores with the following demographic data from first and second year students at a southeastern US osteopathic medical school: the classification of whether students came from a rural or urban background, the region of the US students lived in prior to matriculation to medical school, and the town/city size of where they were currently living at the time of applying to medical school. Data analyses showed only one statistically significant data point (p < 0.04) that revealed first year osteopathic medical students (OMS-I) from towns with < 10,000 occupants had higher JSE scores in comparison to students coming from towns with 50,000-99,999 occupants. However, trends could be established. For example, anecdotally, it is often presumed people from the southern United States tend to be friendlier than those from the northern states; however, contrary to this, the data revealed OMS-I students from the southern United States had a slightly lower BEES scores than OMS-I students from the northeastern states. JSE scores were nearly identical across all four US census bureau regions. Additionally, OMS-I students coming from an urban background had a higher BEES scores than those coming from a rural background. Compared to population norms, combined male and female BEES scores for OMS-I and -II students were within ± 0.5 s.d. of the norm and are considered to be "average" scores. Combined OMS-I JSE scores were below the population norm at the 37th percentile, and combined OMS-II JSE scores were also at the 37th percentile, except for students from the western region at the 44th percentile.

A novel pH-dependent membrane peptide that binds to EphA2 and inhibits cell migration.

Misregulation of the signaling axis formed by the receptor tyrosine kinase (RTK) EphA2 and its ligand, ephrinA1, causes aberrant cell-cell contacts that contribute to metastasis. Solid tumors are characterized by an acidic extracellular medium. We intend to take advantage of this tumor feature to design new molecules that specifically target tumors. We created a novel pH-dependent transmembrane peptide, TYPE7, by altering the sequence of the transmembrane domain of EphA2. TYPE7 is highly soluble and interacts with the surface of lipid membranes at neutral pH, while acidity triggers transmembrane insertion. TYPE7 binds to endogenous EphA2 and reduces Akt phosphorylation and cell migration as effectively as ephrinA1. Interestingly, we found large differences in juxtamembrane tyrosine phosphorylation and the extent of EphA2 clustering when comparing TYPE7 with activation by ephrinA1. This work shows that it is possible to design new pH-triggered membrane peptides to activate RTK and gain insights on its activation mechanism.

Membrane Bending Moduli of Coexisting Liquid Phases Containing Transmembrane Peptide.

A number of highly curved membranes in vivo, such as epithelial cell microvilli, have the relatively high sphingolipid content associated with "raft-like" composition. Given the much lower bending energy measured for bilayers with "nonraft" low sphingomyelin and low cholesterol content, observing high curvature for presumably more rigid compositions seems counterintuitive. To understand this behavior, we measured membrane rigidity by fluctuation analysis of giant unilamellar vesicles. We found that including a transmembrane helical GWALP peptide increases the membrane bending modulus of the liquid-disordered (Ld) phase. We observed this increase at both low-cholesterol fraction and higher, more physiological cholesterol fraction. We find that simplified, commonly used Ld and liquid-ordered (Lo) phases are not representative of those that coexist. When Ld and Lo phases coexist, GWALP peptide favors the Ld phase with a partition coefficient of 3-10 depending on mixture composition. In model membranes at high cholesterol fractions, Ld phases with GWALP have greater bending moduli than the Lo phase that would coexist.

Theoretical study of the interactions between the first transmembrane segment of NS2 protein and a POPC lipid bilayer.

Non-structural protein 2 (NS2) plays a crucial role in the hepatitis C virus (HCV) assembly. NS2 was predicted to be composed of three transmembrane (TM) segments. However, the mechanism of interactions between TM segments of NS2 and surrounding lipid environment remains unclear. Molecular dynamics simulations were applied to investigate the conformation and orientation of the first transmembrane segment (TM1) as well as the interactions of TM1 with a zwitterionic POPC lipid bilayer which identifies several key residues that stabilize the position of TM1 within the membrane. Along with the charged residues R3 and K27, the S23 and H25 were found to be the key elements in establishing the conformation of TM1 inside the membrane. The peptide forms a stable α-helix (the sequence 12-21) connected to N-terminal haft in POPC bilayer. The results also reveal that TM1 induces the ordering of lipid and does not destabilize the lipid bilayer system. The hydrophobic mismatch in which the segment tilts an angle along the membrane normal was observed in this system. The binding free energy profile of TM1 to the membrane was also estimated using umbrella sampling.

A Novel Soluble Peptide with pH-Responsive Membrane Insertion.

Several diseases, such as cancer, are characterized by acidification of the extracellular environment. Acidosis can be employed as a target to specifically direct therapies to the diseased tissue. We have used first principles to design an acidity-triggered rational membrane (ATRAM) peptide with high solubility in solution that is able to interact with lipid membranes in a pH-dependent fashion. Biophysical studies show that the ATRAM peptide binds to the surface of lipid membranes at pH 8.0. However, acidification leads to the peptide inserting into the lipid bilayer as a transmembrane α-helix. The insertion of ATRAM into membranes occurs at a moderately acidic pH (with a pK of 6.5), similar to the extracellular pH found in solid tumors. Studies with human cell lines showed a highly efficient pH-dependent membrane targeting, without causing toxicity. Here we show that it is possible to rationally design a soluble peptide that selectively targets cell membranes in acidic environments.

The negative charge of the membrane has opposite effects on the membrane entry and exit of pH-low insertion peptide.

The pH-low insertion peptide (pHLIP) targets acidic diseases such as cancer. The acidity of the environment causes key aspartic acids in pHLIP to become protonated, causing the peptide to insert into membranes. Here we investigate how the negative charge of the membrane influences how pHLIP enters and exits the lipid bilayer. We found that electrostatic repulsion affected differently the membrane entry and exit of pHLIP for negatively charged membranes. As a consequence, a large hysteresis was observed. We propose this is not a consequence of structural changes but results from local changes in the environment of aspartic acids, shifting their pK values.

Antimicrobial activity of water-soluble triazole phenazine clickamers against E. coli.

The antimicrobial potency of phenazine derivatives is attenuated by their inherently hydrophobic nature, complicating their use as antibiotic drugs. We have analyzed the cytotoxicity and mode of action of water-soluble bis-triazolyl phenazines against E. coli and a human epithelial (HaCat) cell line. We observed complete inhibition of bacterial growth over concentration ranges that do not affect the viability of human epithelial cells. Confocal fluorescence microscopy revealed a high degree of interaction between the phenazine compounds and E. coli, as well as evidence of membrane damage in phenazine-treated E. coli. Additional data suggests that the potency of these particular water-soluble phenazine compounds does not result from the production of reactive oxygen species, but rather from cytotoxic interference with metabolic electron-transfer cascades.