pH-responsive high-density lipoprotein-like nanoparticles to release paclitaxel at acidic pH in cancer chemotherapy

Jae-Yoon Shin,1,* Yoosoo Yang,1,* Paul Heo,1 Ji-Chun Lee,1 ByoungJae Kong,1 Jae Youl Cho,1 Keejung Yoon,1 Cheol-Su Shin,2 Jin-Ho Seo,3 Sung-Gun Kim,4 Dae-Hyuk Kweon11Department of Genetic Engineering, College of Biotechnology and Bioengineering, and Center for Human Interface Nano Technology, Sungkyunkwan University, 2APTech Research Center, Suwon, 3Department of Agricultural Biotechnology, Seoul National University, Seoul, 4Department of Biomedical Science, Youngdong University, Chungbuk, South Korea*These authors contributed equally to this workBackground: Nanoparticles undergoing physicochemical changes to release enclosed drugs at acidic pH conditions are promising vehicles for antitumor drug delivery. Among the various drug carriers, high-density lipoprotein (HDL)-like nanoparticles have been shown to be beneficial for cancer chemotherapy, but have not yet been designed to be pH-responsive.Methods and results: In this study, we developed a pH-responsive HDL-like nanoparticle that selectively releases paclitaxel, a model antitumor drug, at acidic pH. While the well known HDL-like nanoparticle containing phospholipids, phosphatidylcholine, and apolipoprotein A-I, as well as paclitaxel (PTX-PL-NP) was structurally robust at a wide range of pH values (3.8–10.0), the paclitaxel nanoparticle that only contained paclitaxel and apoA-I selectively released paclitaxel into the medium at low pH. The paclitaxel nanoparticle was stable at physiological and basic pH values, and over a wide range of temperatures, which is a required feature for efficient cancer chemotherapy. The homogeneous assembly enabled high paclitaxel loading per nanoparticle, which was 62.2% (w/w). The molar ratio of apolipoprotein A-I and paclitaxel was 1:55, suggesting that a single nanoparticle contained approximately 110 paclitaxel particles in a spherical structure with a 9.2 nm diameter. Among the several reconstitution methods applied, simple dilution following sonication enhanced the reconstitution yield of soluble paclitaxel nanoparticles, which was 0.66. As a result of the pH responsiveness, the anticancer effect of paclitaxel nanoparticles was much more potent than free paclitaxel or PTX-PL-NP.Conclusion: The anticancer efficacy of both paclitaxel nanoparticles and PTX-PL-NP was dependent on the expression of scavenger receptor class B type I, while the killing efficacy of free paclitaxel was independent of this receptor. We speculate that the pH responsiveness of paclitaxel nanoparticles enabled efficient endosomal escape of paclitaxel before lysosomal break down. This is the first report on pH-responsive nanoparticles that do not contain any synthetic polymer.Keywords: pH responsiveness, nanoparticle, apolipoprotein A-I, paclitaxe

A Chemical Controller Of Snare-Driven Membrane Fusion That Primes Vesicles For Ca\u3csup\u3e2+\u3c/sup\u3e-Triggered Millisecond Exocytosis

Membrane fusion is mediated by the SNARE complex which is formed through a zippering process. Here, we developed a chemical controller for the progress of membrane fusion. A hemifusion state was arrested by a polyphenol myricetin which binds to the SNARE complex. The arrest of membrane fusion was rescued by an enzyme laccase that removes myricetin from the SNARE complex. The rescued hemifusion state was metastable and long-lived with a decay constant of 39 min. This membrane fusion controller was applied to delineate how Ca2+ stimulates fusion-pore formation in a millisecond time scale. We found, using a single-vesicle fusion assay, that such myricetin-primed vesicles with synaptotagmin 1 respond synchronously to physiological concentrations of Ca2+. When 10 μM Ca2+ was added to the hemifused vesicles, the majority of vesicles rapidly advanced to fusion pores with a time constant of 16.2 ms. Thus, the results demonstrate that a minimal exocytotic membrane fusion machinery composed of SNAREs and synaptotagmin 1 is capable of driving membrane fusion in a millisecond time scale when a proper vesicle priming is established. The chemical controller of SNARE-driven membrane fusion should serve as a versatile tool for investigating the differential roles of various synaptic proteins in discrete fusion steps

Soluble N-Ethylmaleimide-Sensitive Factor Attachment Protein Receptor-Derived Peptides for Regulation of Mast Cell Degranulation

Vesicle-associated V-soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins and target membrane-associated T-SNAREs (syntaxin 4 and SNAP-23) assemble into a core trans-SNARE complex that mediates membrane fusion during mast cell degranulation. This complex plays pivotal roles at various stages of exocytosis from the initial priming step to fusion pore opening and expansion, finally resulting in the release of the vesicle contents. In this study, peptides with the sequences of various SNARE motifs were investigated for their potential inhibitory effects against SNARE complex formation and mast cell degranulation. The peptides with the sequences of the N-terminal regions of vesicle-associated membrane protein 2 (VAMP2) and VAMP8 were found to reduce mast cell degranulation by inhibiting SNARE complex formation. The fusion of protein transduction domains to the N-terminal of each peptide enabled the internalization of the fusion peptides into the cells equally as efficiently as cell permeabilization by streptolysin-O without any loss of their inhibitory activities. Distinct subsets of mast cell granules could be selectively regulated by the N-terminal-mimicking peptides derived from VAMP2 and VAMP8, and they effectively decreased the symptoms of atopic dermatitis in mouse models. These results suggest that the cell membrane fusion machinery may represent a therapeutic target for atopic dermatitis

Synthesis and physicochemical characterization of acyl myricetins as potential anti-neuroexocytotic agents

Abstract Acyl myricetins (monopropionyl-, dipropionyl-, and monooctanoyl-myricetin, termed as MP1, MP2, and MO1, respectively) were synthesized through enzymatic or non-enzymatic esterification reaction of myricetin aglycone. Structure study indicated the hydroxyl group at C4′ in B-ring was highly susceptible to acylation. Over its parental myricetin, acylated compounds showed enhanced lipophilicity (from 7.4- to 26.3-fold) and oxidative stability (from 1.9- to 3.1-fold) on the basis of logP and decay rate, respectively. MO1, presenting the physicochemical superiority compared to the others, provided lowest EC50 value of 2.51 μM on inhibition of neutrotransmitter release and CC50 value of 59.0 μM, leading to widest therapeutic window. All myricetin esters did not show any irritation toxicity when assessed with a chicken embryo assay. This study describes information on acylation of myricetin that has not yet been explored, and suggests that MO1 has membrane fusion-arresting and anti-neuroexocytotic potential for industrial application due to its enhanced biological properties

A Chemical Controller of SNARE-Driven Membrane Fusion That Primes Vesicles for Ca²⁺-Triggered Millisecond Exocytosis

Membrane fusion is mediated by the SNARE complex which is formed through a zippering process. Here, we developed a chemical controller for the progress of membrane fusion. A hemifusion state was arrested by a polyphenol myricetin which binds to the SNARE complex. The arrest of membrane fusion was rescued by an enzyme laccase that removes myricetin from the SNARE complex. The rescued hemifusion state was metastable and long-lived with a decay constant of 39 min. This membrane fusion controller was applied to delineate how Ca²⁺ stimulates fusion-pore formation in a millisecond time scale. We found, using a single-vesicle fusion assay, that such myricetin-primed vesicles with synaptotagmin 1 respond synchronously to physiological concentrations of Ca²⁺. When 10 μM Ca²⁺ was added to the hemifused vesicles, the majority of vesicles rapidly advanced to fusion pores with a time constant of 16.2 ms. Thus, the results demonstrate that a minimal exocytotic membrane fusion machinery composed of SNAREs and synaptotagmin 1 is capable of driving membrane fusion in a millisecond time scale when a proper vesicle priming is established. The chemical controller of SNARE-driven membrane fusion should serve as a versatile tool for investigating the differential roles of various synaptic proteins in discrete fusion steps

A Chemical Controller of SNARE-Driven Membrane Fusion That Primes Vesicles for Ca²⁺-Triggered Millisecond Exocytosis

Membrane fusion is mediated by the SNARE complex which is formed through a zippering process. Here, we developed a chemical controller for the progress of membrane fusion. A hemifusion state was arrested by a polyphenol myricetin which binds to the SNARE complex. The arrest of membrane fusion was rescued by an enzyme laccase that removes myricetin from the SNARE complex. The rescued hemifusion state was metastable and long-lived with a decay constant of 39 min. This membrane fusion controller was applied to delineate how Ca²⁺ stimulates fusion-pore formation in a millisecond time scale. We found, using a single-vesicle fusion assay, that such myricetin-primed vesicles with synaptotagmin 1 respond synchronously to physiological concentrations of Ca²⁺. When 10 μM Ca²⁺ was added to the hemifused vesicles, the majority of vesicles rapidly advanced to fusion pores with a time constant of 16.2 ms. Thus, the results demonstrate that a minimal exocytotic membrane fusion machinery composed of SNAREs and synaptotagmin 1 is capable of driving membrane fusion in a millisecond time scale when a proper vesicle priming is established. The chemical controller of SNARE-driven membrane fusion should serve as a versatile tool for investigating the differential roles of various synaptic proteins in discrete fusion steps