High-Throughput Screening of pH-Dependent ß-sheet Self-Assembling Peptide.

pH-dependent peptide biomaterials hold tremendous potential for cell delivery and tissue engineering. However, identification of responsive self-assembling sequences with specified secondary structure remains a challenge. In this work, An experimental procedure based on the one-bead one-compound (OBOC) combinatorial library is developed to rapidly screen self-assembling ß-sheet peptides at neutral aqueous solution (pH 7.5) and disassemble at weak acidic condition (pH 6.5). Using the hydrophobic fluorescent molecule thioflavin T (ThT) as a probe, resin beads displaying self-assembling peptides show fluorescence under pH 7.5 due to the insertion of ThT into the hydrophobic domain, and are further cultured in pH 6.5 solution. The beads with extinguished fluorescence are selected. Three heptapeptides are identified that can self-assemble into nanofibers or nanoparticles at pH 7.5 and disassemble at pH 6.5. P1 (LVEFRHY) shows a rapid acid response and morphology transformation with pH modulation. Changes in the charges of histidine and hydrophobic phenyl motif of phenylalanine may play important roles in the formation of pH-responsive ß-sheet nanofiber. This high-throughput screening method provides an efficient way to identify pH-dependent ß-sheet self-assembling peptide and gain insights into structural design of such nanomaterials.

Localized Instillation Enables In Vivo Screening of Targeting Peptides Using One-Bead One-Compound Technology.

The One-Bead One-Compound (OBOC) library screening is an efficient technique for identifying targeting peptides. However, due to the relatively large bead size, it is challenging for the OBOC method to be applied for in vivo screening. Herein, we report an in vivo Localized Instillation Beads library (LIB) screening method to discover targeting peptides with the OBOC technique. Inspired by localized instillation, we constructed a cavity inside of a transplanted tumor of a mouse. Then, the OBOC heptapeptide library was injected and incubated inside the tumor cavity. After an efficient elution process, the retained beads were gathered, from which three MDA-MB-231 tumor-targeting heptapeptides were discovered. It was verified that the best peptide had 1.9-fold higher tumor accumulation than the commonly used targeting peptide RGD in vivo. Finally, two targeting proteins were discovered as potential targets of our targeting peptide to the MDA-MB-231 tumor. The in vivo LIB screening method expands the scope of OBOC peptide screening applications to discover targeting peptides in vivo feasibly and reliably.

Polymer-KLAK Peptide Conjugates Induce Cancer Cell Death through Synergistic Effects of Mitochondria Damage and Autophagy Blockage.

Nanoscaled polymer-peptide conjugates (PPCs) containing both functional peptides and synthetic polymer comprise a new family of biomaterials that can circumvent the limitation of peptides alone. Our previous work showed that PPCs with the therapeutic peptide KLAK, especially PPCs with shorter PEG spacers and a higher degree of polymerization, exhibit enhanced antitumor effects through disrupting mitochondrial membranes. However, as PPCs have a spherical nanostructure (45-60 nm), this may have other effects besides the conjugated therapeutic peptide KLAK itself when they enter cancer cells. In this research, we compared the proteome differences of U87 cells treated with KLAK, polymer, and their conjugates (P-KLAK) through quantitative proteomics technology. The result reveals that proteins involved in oxidative stress response and the Nrf2/ARE pathway were significantly up-regulated after P-KLAK treatment. Moreover, the overexpression of sequestosome 1, a protein substrate that is selectively incorporated into the formation of autophagosome and degraded by autophagy, is found in our study and has not been reported previously in the study of KLAK toxicity. Additional experiments suggest that upon endocytosis, P-KLAK causes lysosome impairment and results in autophagosomes accumulation. Hence, P-KLAK might induce U87 cell death by autophagy blockage due to lysosome impairment as well as mitochondria damage synergistically.

A General Strategy for Facile Synthesis and In Situ Screening of Self-Assembled Polymer-Peptide Nanomaterials.

A universal strategy for efficient, mild, and purification-free synthesis and in situ screening of functional polymer-peptide nanomaterials is described. More than 1000 polymer-peptide conjugates (PPCs) with various chemical structures, compositions, and therapeutic efficacy are created. According to this strategy, the structure-function relationship of the PPCs is revealed, and the antitumor efficacies of the top performing PPCs are evaluated in vivo.