Bottom-Up Preparation of Phase-Separated Polymersomes

A bottom-up approach to fabricating monodisperse, two-component polymersomes that possess phase-separated (“patchy”) chemical topology is presented. This approach is compared with already-existing top-down preparation methods for patchy polymer vesicles, such as film rehydration. These findings demonstrate a bottom-up, solvent-switch self-assembly approach that produces a high yield of nanoparticles of the target size, morphology, and surface topology for drug delivery applications, in this case patchy polymersomes of a diameter of ≈50 nm. In addition, an image processing algorithm to automatically calculate polymersome size distributions from transmission electron microscope images based on a series of pre-processing steps, image segmentation, and round object identification is presented.     

Reactive Oxygen,Nitrogen, and Sulfur Species (RONSS)‐Responsive Polymersomes for Triggered Drug Release

Reactive oxygen, nitrogen, and sulfur species (RONSS) are cross‐reacting and involved in a myriad of physiological and pathological processes. Similar to acidic pH, overexpressed enzymes, and other specific stimuli found in pathological microenvironments, RONSS are recognized as a category of emerging triggering events and have been employed to design activatable theranostic nanomaterials. In this regard, a plethora of RONSS‐responsive nanovectors including polymeric micelles and vesicles (also referred to as polymersomes) are constructed. In comparison with micelles, polymersomes comprising aqueous interiors enclosed by hydrophobic membranes show intriguing applications in synergistic delivery of both hydrophobic and hydrophilic drugs, nanoreactors, and artificial organelles. This feature article focuses on the recent developments in the fabrication of RONSS‐responsive polymersomes and their potential biomedical applications in terms of triggered drug delivery.

     

Pentafluorophenyl Ester‐based Polymersomes as Nanosized Drug‐Delivery Vehicles

In this work, activated ester chemistry is employed to synthesize biocompatible and readily functionalizable polymersomes. Via aminolysis of pentafluorophenyl methacrylate‐based precursor polymers, an N‐(2‐hydroxypropyl) methacrylamide (HPMA)‐analog hydrophilic block is obtained. The precursor polymers can be versatile functionalized by simple addition of suitable primary amines during aminolysis as demonstrated using a fluorescent dye. Vesicle formation is proven by cryoTEM and light scattering. High encapsulation efficiencies for hydrophilic cargo like siRNA are achieved using dual centrifugation and safe encapsulation is demonstrated by gel electrophoresis. In vitro studies reveal low cytotoxicity and no protein adsorption‐induced aggregation in human blood serum occurs, making the vesicles interesting candidates as nanosized drug carriers.

     

In Vitro Drug and Gene Delivery Using Random Cationic Copolymers Forming Stable and pH‐Sensitive Polymersomes

Stimuli‐sensitive polymeric vesicles or polymersomes as self‐assembled colloidal nanocarriers have received paramount importance for their integral role as delivery system for therapeutics and biotherapeutics. This work describes spontaneous polymersome formation at pH 7, as evidenced by surface tension, steady state fluorescence, dynamic light scattering, and microscopic studies, by three hydrophilic random cationic copolymers synthesized using N ,N‐(dimethylamino)ethyl methacrylate (DMAEM) and methoxy poly(ethylene glycol) monomethacrylate in different mole ratios. The results suggest that methoxy poly(ethylene glycol) chains constitute the bilayer membrane of the polymersomes and DMAEM projects toward water constituting the positively charged surface. The polymersomes have been observed to release their encapsulated guest at acidic pH as a result of transformation into polymeric micelles. All these highly biocompatible cationic polymers show successful gene transfection ability as nonviral vector on human cell line with different potential. Thus these polymers prove their utility as a potential delivery system for hydrophilic model drug as well as genetic material.

     

Polymersomes Prepared from Thermoresponsive Fluorescent Protein–Polymer Bioconjugates: Capture of and Report on Drug and Protein Payloads

Polymersomes provide a good platform for targeted drug delivery and the creation of complex (bio)catalytically active systems for research in synthetic biology. To realize these applications requires both spatial control over the encapsulation components in these polymersomes and a means to report where the components are in the polymersomes. To address these twin challenges, we synthesized the protein–polymer bioconjugate PNIPAM‐b‐amilFP497 composed of thermoresponsive poly(N‐isopropylacrylamide) (PNIPAM) and a green‐fluorescent protein variant (amilFP497). Above 37 °C, this bioconjugate forms polymersomes that can (co‐)encapsulate the fluorescent drug doxorubicin and the fluorescent light‐harvesting protein phycoerythrin 545 (PE545). Using fluorescence lifetime imaging microscopy and Förster resonance energy transfer (FLIM‐FRET), we can distinguish the co‐encapsulated PE545 protein inside the polymersome membrane while doxorubicin is found both in the polymersome core and membrane.     

Porphyrin Containing Polymersomes with Enhanced ROS Generation Efficiency: In Vitro Evaluation

Porphyrins are molecules possessing unique photophysical properties making them suitable for application in photodynamic therapy. The incorporation of porphyrins into natural or synthetic nano‐assemblies such as polymersomes is a strategy to improve and prolong their therapeutic capacities and to overcome their limitations as therapeutic and diagnostic agents. Here, 5,10,15,20‐tetrakis(1‐(6‐ethoxy‐6‐oxohexyl)‐4‐pyridin‐1‐io)‐21H,23H‐porphyrin tetrabromide porphyrin is inserted into polymersomes in order to demonstrate that the encapsulation enhances its ability to generate highly reactive singlet oxygen (¹O₂) upon irradiation in vitro. The photoactivation of the free and polymersome‐encapsulated porphyrin is evaluated by electron spin resonance and cell viability assays on three different mammalian cell lines. The results indicate that by encapsulating the porphyrin, a controlled ROS delivery within the cells is achieved, at the same time avoiding side effects such as dark toxicity, non‐specific porphyrin release and over time decreased activity in vitro. This work focuses on showing a not‐toxic model system for modern therapeutic nanomedicine, which works under mild irradiation and dosage conditions.     

Electrospun Polymeric Fibers Decorated with Silk Microcapsules via Encapsulation and Surface Immobilization for Drug Delivery

Hollow polymer microcapsules as drug carriers have the advantages of drug protection, storage, and controlled release. Microcapsules combined with tissue engineering scaffolds such as electrospun microfibers can enhance long-term local drug retention. However, the combination methods of microcapsules and fibers still need to be further explored. Here, different technical approaches to functionalize electrospun polycaprolactone (PCL) microfibers with silk fibroin (SF) microcapsules through encapsulation and surface immobilization are developed, including direct blending and emulsion electrospinning for encapsulation, as well as covalent and cleavable disulfide-linkage for surface immobilization. The results of “blending” approach show that silk microcapsules with different sizes could be uniformly encapsulated inside electrospun fibers without aggregation. To further reduce the use of organic solvents, the microcapsules in the aqueous phase can be uniformly distributed in the PCL organic phase and successfully electrospun into fibers using surfactant span-80. For surface immobilization, silk microcapsules are efficiently covalent binding to the surface of electrospun PCL fibers via click chemistry and exhibited noncytotoxic. Based on this method, with the incorporation of a disulfide bond, the linkages between microcapsule and fiber could be cleaved under reducing conditions. These microcapsule-electrospun fiber combination methods provide sufficient options for different drug delivery requirements.     

Novel PEGylated Amphiphilic Copolymers as Nanocarriers for Drug Delivery: Synthesis,Characterization and Curcumin Encapsulation

Amphiphilic polymers can self assemble into micellar nano-particles and can be effectively used as nano carriers for drug delivery. A number of macromolecular delivery systems are under investigation to improve the efficacy of prospective drugs. In this study, seven new co-polymers were synthesized under mild reaction conditions in bulk (without solvent) by chemoenzymatic approach using Candida antarctica lipase (Novozyme 435) and molecular sieves, subsequently these polymers were treated with different long chain bromoalkanes and acid chlorides for attachment of the lipophilic moieties to the backbone polymer via an ether or an ester linkage, respectively in order to make them amphiphilic. These synthesized nano-particles demonstrated high drug loading capacity and have the potential to encapsulate hydrophobic drugs.     

Sequestration of Proteins by Fatty Acid Coacervates for Their Encapsulation within Vesicles

Encapsulating biological materials in lipid vesicles is of interest for mimicking cells; however, except in some particular cases, such processes do not occur spontaneously. Herein, we developed a simple and robust method for encapsulating proteins in fatty acid vesicles in high yields. Fatty acid based, membrane‐free coacervates spontaneously sequester proteins and can reversibly form membranous vesicles upon varying the pH value, the precrowding feature in coacervates allowing for protein encapsulation within vesicles. We then produced enzyme‐enriched vesicles and show that enzymatic reactions can occur in these micrometric capsules. This work could be of interest in the field of synthetic biology for building microreactors.     

In Vitro Study of Calcium Microsecond Electroporation of Prostate Adenocarcinoma Cells

Electroporation, applied as a non-thermal ablation method has proven to be effective for focal prostate treatment. In this study, we performed pre-clinical research, which aims at exploring the specific impact of this so-called calcium electroporation on prostate cancer. First, in an in-vitro study of DU 145 cell lines, microsecond electroporation (μsEP) parameters were optimized. We determined hence the voltage that provides both high permeability and viability of these prostate cancer cells. Subsequently, we compared the effect of μsEP on cells’ viability with and without calcium administration. For high-voltage pulses, the cell death’s mechanism was evaluated using flow-cytometry and confocal laser microscopy. For lower-voltage pulses, the influence of electroporation on prostate cancer cell mobility was studied using scratch assays. Additionally, we applied calcium-binding fluorescence dye (Fluo-8) to observe the calcium uptake dynamic with the fluorescence microscopy. Moreover, the molecular dynamics simulation visualized the process of calcium ions inflow during μsEP. According to our results calcium electroporation significantly decreases the cells viability by promoting apoptosis. Furthermore, our data shows that the application of pulsed electric fields disassembles the actin cytoskeleton and influences the prostate cancer cells’ mobility.     

Encapsulation and/or release behavior of bovine serum albumin within and from polylactide-grafted dextran microspheres

Polylactide (PLA)-grafted dextran (Dex-graft-PLA) of various contents of sugar units was synthesized by anionic polymerization of L-lactide (L-LA) using the alkoxide of partially trimethylsilylated dextran (TMSDex) and subsequently removing the trimethylsilyl (TMS) groups. The copolymer showed different solubility from L-LA homopolymer with increasing the content of sugar units. We prepared bovine serum albumin (BSA)-loaded microspheres (MS)s according to a water-in-oil-in-water emulsion-solvent evaporation/extraction method using methylene chloride/DMSO as an organic cosolvent. MSs prepared from Dex-graft-PLA [MS(Dex-graft-PLA)s] exhibited higher loading efficiency of BSA than MSs prepared from PLLA [MS(PLLA)s]. The in vitro release rate of BSA from MS(Dex-graft-PLA) was faster than that from MS(PLLA). BSA released from MS(Dex-graft-PLA) maintained the secondary structure of native BSA to a great extent, compared with BSA released from MS(PLLA).     

Encapsulation of proteins by Electro Hydro Dynamic Atomization

Summary: Aerosol reactor for Electro Hydro Dynamic Atomization has been used to encapsulate water soluble proteins in poly(ε-caprolactone). As a raw material w/o emulsion of BSA water solution emulsified in the organic solution of polymer was applied. Stability of the spraying process itself for particles production was investigated. Also raw material emulsion stability has been examined and emulsion stabiliser has been found. Protein release rate from produced particles was determined.     

Encapsulation of Arenes within a Porous Molybdenum Oxide {Mo132} Nanocapsule

The use of confined space to modulate chemical reactivity and to sequester organic compounds spans significant disciplines in chemistry and biology. Here, the inclusion and assembly of arenes into a water‐soluble porous metal oxide nanocapsule [{(Mo^VI)Mo^V₅O₂₁(H₂O)₆}₁₂{Mo^V₂O₄(CH₃COO)}₃₀]^42? (Mo₁₃₂) is reported. The uptake of benzene, halobenzenes, alkylbenzenes, phenols, and other derivatives was studied by NMR, where it was possible to follow the encapsulation process from the outside of the capsule through its pores and then into the interior. The importance of size or shape of the arenes, and various intermolecular bond interactions contributed by the benzene substituent on the encapsulation process was studied, showing the importance of π–π stacking and CH–π interactions. Furthermore, by using NOESY, ROESY, and HOESY NMR techniques it was possible to understand the interaction of the encapsulated arenes and the acetate linkers or ligands that line the interior of the Mo₁₃₂ capsule.     

Enzyme Degradable Polymersomes from Chitosan‐g‐[poly‐l‐lysine‐block‐ε‐caprolactone] Copolymer

The scope of this study includes the synthesis of chitosan‐g‐[peptide‐poly‐ε‐caprolactone] and its self‐assembly into polymeric vesicles employing the solvent shift method. In this way, well‐defined core–shell structures suitable for encapsulation of drugs are generated. The hydrophobic polycaprolactone side‐chain and the hydrophilic chitosan backbone are linked via an enzyme‐cleavable peptide. The synthetic route involves the functionalization of chitosan with maleimide groups and the preparation of polycaprolactone with alkyne end‐groups. A peptide functionalized with a thiol group on one side and an azide group on the other side is prepared. Thiol‐ene click‐chemistry and azide–alkyne Huisgen cycloaddition are then used to link the chitosan and poly‐ε‐caprolactone chains, respectively, with this peptide. For a preliminary study, poly‐l ‐lysin is a readily available and cleavable peptide that is introduced to investigate the feasibility of the system. The size and shape of the polymersomes are studied by dynamic light scattering and cryo‐scanning electron microscopy. Furthermore, degradability is studied by incubating the polymersomes with two enzymes, trypsin and chitosanase. A dispersion of polymersomes is used to coat titanium plates and to further test the stability against enzymatic degradation.     

Modular Redesign of a Cationic Lytic Peptide To Promote the Endosomal Escape of Biomacromolecules

Endocytosis is an important route for the intracellular delivery of biomacromolecules, wherein their inefficient endosomal escape into the cytosol remains a major barrier. Based on the understanding that endosomal membranes are negatively charged, we focused on the potential of cationic lytic peptides for developing endosomolysis agents to release such entrapped molecules. As such, a venom peptide, Mastoparan X, was employed and redesigned to serve as a delivery tool. Appending a tri‐glutamate unit to the N‐terminus attenuates the cytotoxicity of Mastoparan X by about 40 fold, while introduction of a Ni^II‐dipicolylamine complex enhances cellular uptake of the peptide by about 17 fold. Using the optimized peptide, various fluorescently labeled macromolecules were successfully delivered to the cytosol, enabling live‐cell imaging of acetylated histones.