Article pubs.acs.org/Biomac
Cite This: Biomacromolecules XXXX, XXX, XXX−XXX
Cellulose Abetted Assembly and Temporally Decoupled Loading of Cargo into Vesicles Synthesized from Functionally Diverse Lamellar Phase Forming Amphiphiles Alexander Li,†,‡ Joseph Pazzi,†,‡ Melissa Xu,† and Anand Bala Subramaniam*,† †
Department of Bioengineering, University of California, Merced, Merced, California 95343, United States S Supporting Information *
ABSTRACT: Self-assembled micrometer-scale vesicles composed of lamellar phase forming amphiphiles are useful as chemical microreactors, as minimal artificial cells, as protocell mimics for studies of the origins of life, and as vehicles for the targeted delivery of drugs. Given their varied uses, discovery of a universal mechanism that is simple, rapid, and that produces vesicles from a large variety of amphiphiles with different chemical and physical properties at high yield is extremely desirable. Here we show that cellulose, in the form of cellulose paper, facilitates the assembly of membranous vesicles 5−20 μm in diameter from scientifically and technologically important amphiphiles of diverse chemical structures and functionality such as fatty acids (fatty acid vesicles), amphiphilic diblock copolymers, and amphiphilic triblock copolymers (polymersomes). Assembly of vesicles occurred within 90 min of placing the amphiphile-coated cellulose paper into aqueous solutions. Varying thermal and chemical conditions, however, are required for the high-yield assembly of vesicles from the different amphiphiles. The vesicles, when attached to cellulose fibers, have membranes that remain unsealed. This topological characteristic of the vesicles grown on paper allowed the scalable separation of the process of growth from the process of loading cargo (temporally decoupled growth and loading). We demonstrate a temporally decoupled process to rapidly produce large quantities of protein-loaded polymersomes on the benchtop by using high temperatures to accelerate the growth of the polymersomes and subsequently milder temperatures during diffusive loading of the protein cargo. such as on the clay montomorillonite17 and on hydrocarbon covered glass.45 Polymersomes from diblock and triblock copolymers have been produced through gentle hydration,5,11,46,47 through electroformation5,46 through microfluidic methods,37,48 and through gel-assisted hydration.6,49−52 We have recently shown that dry phospholipid and sphingolipid films deposited on the cellulose fibers of chromatography and filter paper, when placed into aqueous solutions, spontaneously assemble into giant liposomes.53 Here we report that the phenomena of cellulose paper-abetted assembly is general to lamellar phase forming amphiphiles of diverse chemical structures and chemical complexities such as fatty acids, amphiphilic diblock copolymers, and amphiphilic triblock copolymers. We also show that the vesicles when attached to the cellulose fibers remain open to the environment, allowing for the diffusive loading of cargo from the external phase after the process of vesicle growth was complete (temporally decoupled growth and loading). Extraction of vesicles from the paper seals the membrane thus encapsulating the cargo. This easily scalable temporally decoupled process of
1. INTRODUCTION Vesicles with semipermeable membranes composed of amphiphilic molecules, that is, molecules with both a hydrophilic group and a hydrophobic group, can encapsulate and compartmentalize finite aqueous regions within a larger aqueous continuous phase. Amphiphiles with a hydrophilic/ lipophilic balance ratio, HLB, of about 10, or with packing v parameters, a l ∼ 1, where v is the volume of the hydrophobic 0c
group, lc is the critical length of the hydrophobic group, and a0 is the preferred area of the hydrophilic group, assemble into vesicles when present above their critical aggregation concentration1 in aqueous solutions. A variety of chemically distinct molecules such as phospholipids,2 sphingolipids,2 fatty acids,3,4 fatty alcohols,3,4 and amphiphilic block copolymers5−8 assemble into vesicles. Vesicles assembled from these amphiphiles have fundamental importance in basic research and applied technologies ranging from their use as chemical reactors,9−16 models for primitive cells,17−22 artificial cells,23−29 contrast agents,30,31 drug carriers,32−39 and artificial blood.40−42 Various methods have been reported for assembling vesicles. Fatty acid vesicles can grow in bulk through agitation,43,44 a change in pH4, or upon the addition of cosurfactants4 or fatty acid micelles.22 Fatty acid vesicles can also form on surfaces © XXXX American Chemical Society
Received: November 23, 2017 Revised: February 1, 2018
A
DOI: 10.1021/acs.biomac.7b01645 Biomacromolecules XXXX, XXX, XXX−XXX
Article
Biomacromolecules
2. EXPERIMENTAL SECTION
loading (i.e., as opposed to simultaneous growth and loading) of self-assembled vesicles, allowed for conditions that were dissimilar during vesicle growth and during vesicle loading. We find that a large number of vesicles can be produced within 90 min from paper coated with nominal surface concentrations of O (0.1−1) nmol/mm2 of these amphiphiles. The conditions for the formation of vesicles on cellulose paper, however, is different for each of the amphiphiles. To quantify systematically the properties of the vesicles produced from the different amphiphiles on cellulose paper, we developed a method of analysis that used confocal fluorescence microscopy and digital image analysis. We produced vesicles that had a higher buoyant density than the surrounding solution by (i) encapsulating sucrose or polymers of sucrose in the vesicles and (ii) suspending the vesicles in a continuous phase consisting of an isomolar solution of glucose. Over the course of 2 h, tens of thousands of vesicles sedimented to a single plane at the bottom of an imaging chamber and their motion in the solution dampened. We then obtained centimeter square fluorescence tile scan images of the solution at this plane, at a pixel resolution of 457 nm, and used digital image analysis to quantify the diameters and the degree of lamellarity of the vesicles. Compared to previously reported methods for fabricating vesicles, the cellulose paper-abetted method uses sustainable and easily manipulated paper, does not require a power source, leaves the membranes free of contamination by cellulose,53 and is rapid. These features of the method suggest significant advantages for scalability and general applicability. Fatty acid vesicles formed at room temperature in a buffer with a pH of 8.5 when the nominal surface concentration of oleic acid on the paper was between 1−3 nmol/mm2. About 70% of the fatty acid vesicles were between 5 and 10 μm in diameter, and 66% of the vesicles were unilamellar. For the amphiphilic diblock copolymer poly(butadiene-b-ethylene oxide) PBD46PEO30, vesicle growth, when limited to 90 min, required buffer temperatures of 80 °C. For growth at lower temperatures, these polymer vesicles, referred to as polymersomes, required incubation times of 12 h or more. The median sizes of polymersomes grown at 80 °C was 9 μm for nominal surface concentrations between 0.1 and 0.6 nmol/mm2. Ninety six percent of the polymersomes were unilamellar. To demonstrate that growth at high temperatures does not limit the practical application of PBD46PEO30 polymersomes produced on cellulose paper, we encapsulated a model protein, fluorescein isothiocyanate conjugated with bovine serum albumin (FITC-BSA), at room temperature after growing the PBD46PEO30 polymersomes at 80 °C. Polymersomes also formed on cellulose paper coated with 0.6−1 nmol/mm2 of the amphiphilic triblock copolymer polyoxyethylene−polyoxypropylene−polyoxyethylene (PEO5PPO67PEO5, Pluronic L121), at room temperature, within 90 min. Incubating the paper at higher temperatures resulted in the formation of Pluronic L121 droplets rather than polymersomes. We show that this result is due to the Pluronic L121 membranes condensing irreversibly to form droplets upon heating. Along with single Pluronic L121 polymersomes, we also observed doublets, triplets, and clusters of polymersomes with shapes reminiscent of soap bubbles. Some of the Pluronic L121 polymersomes also had polymer accretions on the membrane. These structural properties of the Pluronic L121 polymersomes differed from those of fatty acid vesicles and PBD46PEO30 polymersomes.
2.1. Materials. We purchased polystyrene well inserts (CellCrown 24 Scaffdex) from Sigma-Aldrich and Whatman Ashless Grade 42 Filtration Paper, 24-well polystyrene multiwell plates (Corning Costar), glass microscope slides (Thermo Scientific), and glass coverslips (No. 1 thickness, Thermo Scientific) from Thermo Fisher Scientific (Waltham, MA). 2.2. Chemicals. We purchased methanol (HPLC grade, purity ≥99.9%), oleic acid (purity ≥99%), sodium oleate (purity ≥95%), poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) (Pluronic L121, Pluronic F68, Pluronic L61), Triton X-100 (BioXtra grade), sodium dodecyl sulfate (BioXtra grade, ≥99.0%), myristoleic acid (purity ≥99%), myristic acid (purity ≥99%), α-hemolysin, fluorescein sodium salt (BioReagent grade), Direct Red 23 (Dye content 30%), Nile Red (microscopy grade), sucrose (BioXtra grade, purity ≥99.5%), glucose (BioXtra grade, purity ≥99.5%), albumin−fluorescein isothiocyanate conjugate (FITC-BSA; albumin from bovine, ≥7 mol FITC/mol albumin), and casein from bovine milk from Sigma-Aldrich. We purchased chloroform with 0.75% ethanol as preservative (ACS grade, purity ≥99.8%), Bicine buffer, pH 8.5, low endotoxin, and 20× phosphate buffered saline (PBS; 200 mM sodium phosphate, 3 M sodium chloride, Ultra Pure grade) from Thermo Fisher Scientific, and poly(butadiene-b-ethylene oxide) (P9095-BdEO, lot# P9757) from Polymer Source Inc. (Montreal, Canada). We refer to this block copolymer as PBD46PEO30 in this paper. We obtained 18.2 MOhm ultrapure water from an ELGA Pure-lab Ultra water purification system (Woodrige, IL). 2.3. PAPYRUS Method. Similar to our previously published protocol for producing liposomes,53 we first prepared a solution of the desired amphiphile in a good solvent. We used chloroform to dissolve oleic acid, PBD46PEO30, or Pluronic L121 along with 0.5 mol % Nile Red to serve as a fluorescent indicator for the membranes. Figure 1a−c shows the structural formulas, molecular weights, and schematic representations of the structures of the membranes that assemble from the members of the three classes of lamellar phase forming amphiphiles that we used in this work. We cut a piece of Whatman Grade 42 filter paper into a rough circle 15 mm in diameter (Figure 1d(i)). We molded the paper into the shape of the CellCrown insert and then deposited 10 μL of the solution of amphiphile onto the center of the molded paper (Figure 1d(ii)). The diameter of the paper that spanned the opening of the CellCrown insert was 12 mm. The nominal surface concentration of the amphiphile on the paper was the moles of the amphiphile that we deposited over the area of the paper that spanned the opening of the CellCrown (π(6)2 mm2). We placed the paper in a vacuum chamber for 60 min to evaporate all traces of solvent. We then affixed the paper to the CellCrown insert, the 3 mm overhang of the paper slipped under the elastic, securely affixing the amphiphile-coated region over the opening (Figure 1d(iii)). We added 1.0 mL of the growth buffer into the wells of a 24-well plate and then gently inserted the CellCrown into the well, careful to ensure that the amphiphile coated region was fully submerged in the growth buffer. The composition of the buffer used to grow the fatty acid vesicles was 0.2 M Bicine + 250 mM sucrose + 1 mM sodium oleate. The pH of the buffer was 8.5. We used sodium oleate in the buffer to ensure that the samples were above the critical aggregation concentration of oleic acid. For the PBD46PEO30 polymersomes, the composition of the buffer used for growth was 250 mM sucrose. For the Pluronic L121 polymersomes, the composition of the buffer used for the growth was 25 μM of Ficoll 400. We typically allowed the paper to incubate for 90 min. After 90 min, we carefully removed the CellCrown from the well and quickly (
