Mechanisms of Cup-Shaped Vesicle Formation Using Amphiphilic Diblock Copolymer

A cup shape is a dynamic morphology of cells and organelles. With the aim of elucidating the formation of the biotic cup-shaped morphology, this study investigated cup-shaped vesicles consisting of an amphiphilic diblock copolymer from the aspect of synthetic polymer chemistry. Cup-shaped vesicles were obtained by the polymerization-induced self-assembly of poly(methacrylic acid)-block-poly(n-butyl methacrylate-random-methacrylic acid), PMAA-b-P(BMA-r-MAA), in an aqueous methanol solution using the photo nitrox-ide-mediated controlled/living radical polymerization technique. Field emission scanning electron microscopic observations demonstrated that the cup-shaped vesicles were suddenly formed during the late stage of the polymerization due to the extension of the hydrophobic P(BMA-r-MAA) block chain. During the early stage, the polymerization produced spherical vesicles rather than a cup shape. As the hydrophobic block chain was extended by the polymerization progress, the spherical vesicles reduced the size and were accompanied by the generation of small particles that were attached to the vesicles. The vesicles continued to reduce the size due to further extension of the hydrophobic chain; however, they suddenly grew into cup-shaped vesicles. This growth was accounted for by a change in the critical packing shape of the copolymer due to the hydrophobic chain extension. These findings are helpful for a better understanding of the biotic cup-shaped vesicle


Introduction
Cup-shaped vesicles, often seen during cytosis, are a dynamic morphology of the How to cite this paper: Yoshida, E. (2022) Mechanisms of Cup-Shaped Vesicle Formation Using Amphiphilic Diblock Copolymer. Open Journal of Polymer Chemistry, metabolisms in the cytoplasm. Examples of cup-shaped vesicles include phagophores, which are formed during the early stage of autophagy to initiate the autophagous metabolism [1] [2] [3]. The phagophores take damaged organelles and harmful components into their cavity of the cup by extending their relative protein-poor membrane and form the closed phagosomes with a double membrane structure for catabolism and recycling of these useless organelles and components [4] [5]. Exosomes that are involved in exocytosis are also cup-shaped structures [6]. The exosomes transport materials, such as proteins, lipids, mRNA, and DNA, out of the cell for life activity maintenance and transmit information for cell-to-cell communication. These cup-shaped vesicles temporarily appear to play their roles, then disappear after finishing their tasks. However, the mechanisms of the cup-shaped vesicle generation have not been completely clarified, although the proteins and lipids participating in the cup-shaped vesicle formation have been discovered [3].
Some of these vesicles serve in drug delivery as effective carriers that load drugs into the cavity of the cup [13]. The synthetic cup-shaped vesicles are also dynamic because they are the intermediates of their final structures, such as worm-like vesicles [14].
Micron-sized giant vesicles composed of amphiphilic poly(methacrylic acid)-block-poly(alkyl methacrylate)-random-methacrylic acid) produced unique artificial biomembrane models for cells and organelles based on many similarities to the biomembranes regarding their morphologies [15] [16], stimuli-responsiveness [17], and membrane permeability [18]. Examples of the models are the spherical vesicles with the erythrocyte-like morphology transformation [19], the perforated vesicles for the nuclear envelope [20], the villus-like structure [21], the anastomosed tubular networks following a fenestrated sheet for the endoplasmic reticulum and Golgi apparatus [22], the tubule extension from the vesicle surface for the neurons [23], and a polyelectrolyte that induces the budding separation for the membrane protein for endocytosis [24]. These models facilitated a better understanding of the biotic morphology formations. This study investigated the cup-shaped vesicle formation using amphiphilic poly(methacrylic acid)-blockpoly(n-butyl methacrylate-random-methacrylic acid), PMAA-b-P(BMA-r-MAA), with the aim of elucidating the biotic cup-shaped vesicle formation from the aspect of synthetic polymer chemistry and molecular self-assembly. The PMAA-b-P(BMA-r-MAA) has been reported to produce flexible vesicles due to the butyl chain stretchiness [25]. This paper describes the formation and mechanism of 2. Experimental

Instrumentation
The photo-NMP was performed using an Ushio optical modulex BA-H502, an illuminator OPM2-502H with a high-illumination lens UI-OP2SL, and a 500W super high-pressure UV lamp USH-500SC2. 1 H NMR measurements were conducted using Jeol ECS400 and ECS500 FT NMR spectrometers. Gel permeation chromatography (GPC) was performed at 40˚C using a Tosoh GPC-8020 instrument equipped with a DP-8020 dual pump, a CO-8020 column oven, and a RI-8020 refractometer. Two gel columns of Tosoh TSK-GEL α-M were used with N,Ndimethylformamide (DMF) containing 30 mM LiBr and 60 mM H 3 PO 4 as the eluent. Field emission scanning electron microscopy (FE-SEM) measurements were performed using a Hitachi SU8000 scanning electron microscope.
Methanol (MeOH) was refluxed over magnesium with iodine for several hours, and then distilled. Distilled water purchased from Wako Pure Chemical Industries was further purified by distillation. Extrapure Ar gas with over 99.999 vol% purity was purchased from Taiyo Nippon Sanso Corporation.

Preparation of PMAA End-Capped with MTEMPO
The PMAA end-capped with MTEMPO was prepared as reported previously  (4 mL) were placed in a 30-mL test tube joined to a high vacuum valve. The contents were degassed several times using a freeze-pump-thaw cycle and then charged with Ar. The polymerization was carried out at room temperature for 5.5 h with irradiation at 9.3 amperes with a 500W super high-pressure UV lamp using a reflective light from a mirror in order to avoid any thermal polymerization caused by the direct irradiation [28]. MeOH (11 mL) and distilled water (5 mL) degassed by bubbling Ar for 15 min were added to the product under a flow of Ar.
After the product was completely dissolved in the aqueous MeOH solution, part the PMAA molecular weight. The conversion was calculated to be 76% by 1

Photopolymerization-Induced Self-Assembly
The

FE-SEM Observations
The vesicles were dried in air and subjected to the FE-SEM measurements at 1.0 kV without coating. Morphologies and size of vesicles were determined by the FE-SEM observations, while the size distribution was calculated as reported previously [29].

Results and Discussion
The polymerization-induced self-assembly of PMAA-b-P(BMA-r-MAA) was performed in an aqueous methanol solution (MeOH/water = 3/1 v/v) at room temperature through the photo-NMP of BMA and MAA using a PMAA endcapped with MTEMPO ( Figure 1). The monomer conversions were estimated by 1 H NMR. The 1 H NMR spectrum of the copolymer is shown in Figure 2    The polymerization produced a negligible change in the molar ratios of the monomer units for the random copolymer block throughout the polymerization.
The molecular weight of the copolymer was determined by GPC based on PMAA standards. As shown in Figure 4, the GPC curve of the copolymer shifted to the higher molecular weight side with the reaction time. The molecular weight of the copolymer and DP of the P(BMA-r-MAA) block chain linearly increased with the BMA conversion, although the molecular weight distribution showed a slight increase with the conversion (Figure 5). The linear increases in the molecular weight and DP with the monomer conversion verified the living nature of the polymerization.
FE-SEM observations clarified the mechanism of the cup-shaped vesicle formation by the polymerization progress. Figure 6 shows the FE-SEM images of the vesicles for each BMA conversion. The vesicles were spherical rather than cup shaped during the early stage of the polymerization. The size of the vesicles decreased by the P(BMA-r-MAA) block chain extension (Figure 7). Some vesicles were joined to each other directly or via small particles, suggesting that they are structurally unstable. By further extending the hydrophobic chain, the E. Yoshida     for the phagophore formation that the phosphatidylethanolamine-containing proteins and phosphatidylinositol 3 phosphate-binding proteins alter the lipid composition of the source-derived bilayer to form phagophores [3]. These lipids of phosphatidylethanolamine and phosphatidylinositol 3 phosphate have inverted cone-like and cylindrical critical packing shapes, which extend the surface area and produce curvature of the bilayer membrane [30]. The sources of the bilayer are considered to be the endoplasmic reticulum and the outer leaflet of mitochondria rich in these lipids [31] [32]. The findings in this study indicate that the phagophore formation is caused by introducing these lipids into the source-derived lipid bilayer to produce the membrane curvature and extension.