Cryopreservation enables long-term preservation of cells at ultralow temperatures. Current cryoprotective agents (CPAs) have several limitations, making it imperative to develop CPAs with advanced ...properties. Previously, we developed a novel synthetic polyampholyte-based CPA, copolymer of 2-(dimethylamino)ethyl methacrylate (DMAEMA) and methacrylic acid(MAA) (poly(MAA-DMAEMA)), which showed excellent efficiency and biocompatibility. Introduction of hydrophobicity increased its efficiency significantly. Herein, we investigated the activity of other polyampholytes. We prepared two zwitterionic polymers, poly(sulfobetaine) (SPB) and poly(carboxymethyl betaine) (CMB), and compared their efficiency with poly(MAA-DMAEMA). Poly-SPB showed only intermediate property and poly-CMB showed no cryoprotective property. These data suggested that the polymer structure strongly influences cryoprotection, providing an impetus to elucidate the molecular mechanism of cryopreservation. We investigated the mechanism by studying the interaction of polymers with cell membrane, which allowed us to identify the interactions responsible for imparting different properties. Results unambiguously demonstrated that polyampholytes cryopreserve cells by strongly interacting with cell membrane, with hydrophobicity increasing the affinity for membrane interaction, which enables it to protect the membrane from various freezing-induced damages. Additionally, cryoprotective polymers, especially their hydrophobic derivatives, inhibit the recrystallization of ice, thus averting cell death. Hence, our results provide an important insight into the complex mechanism of cryopreservation, which might facilitate the rational design of polymeric CPAs with improved efficiency.
Protein aggregation is a process by which misfolded proteins polymerizes into aggregates and forms fibrous structures with a β-sheet conformation, known as amyloids. It is an undesired outcome, as it ...not only causes numerous neurodegenerative diseases, but is also a major deterrent in the development of protein biopharmaceuticals. Here, we report a rational design for the synthesis of novel zwitterionic polymer-based core-shell nanogels via controlled radical polymerization. Nanogels with different sizes and functionalities in the core and shell were prepared. The nanogels exhibit remarkable efficiency in the protection of lysozyme against aggregation. Addition of nanogels suppresses the formation of toxic fibrils and also enables lysozyme to retain its enzymatic activity. Increasing the molecular weight and degree of hydrophobicity markedly increases its overall efficiency. Investigation of higher order structures revealed that lysozyme when heated without any additive loses its secondary structure and transforms into a random coil conformation. In contrast, presence of nanogels facilitates the retention of higher order structures by acting as molecular chaperones, thereby reducing molecular collisions. The present study is the first to show that it is possible to design zwitterionic nanogels using appropriate polymerization techniques that will protect proteins under conditions of extreme stress and inhibit aggregation.
Graft copolymers consisting of two different zwitterionic blocks were synthesized via reversible addition fragmentation chain transfer polymerization. These polymers showed dual properties of thermo- ...and pH-responsiveness in an aqueous solution. Ultraviolet–visible spectroscopy and dynamic light scattering were employed to study the phase behavior under varying temperatures and pH values. Unlike the phase transition temperatures of other graft copolymers containing nonionic blocks, the phase transition temperature of these polymers was easily tuned by changing the polymer concentration. Owing to the biocompatible and stimuli-responsive nature of the polymers, this system was shown to effectively release proteins (lysozyme) while simultaneously protecting them against denaturation. The positively charged lysozyme was shown to bind with the negatively charged polymer at the physiological pH (pH 7.4). However, it was subsequently released at pH 3, at which the polymer exhibits a positive charge. Protein aggregation studies using a residual enzymatic activity assay, circular dichroism, and a Thioflavin T assay revealed that the secondary structure of the lysozyme was retained even after harsh thermal treatment. The addition of these polymers helped the lysozyme retain its enzymatic activity and suppressed its fibrillation. Both polymers showed excellent protein protection properties, with the negatively charged polymer exhibiting slightly superior protein protection properties to those of the neutral polymer. To the best of the authors’ knowledge, this is the first study to develop a graft copolymer system consisting of two different zwitterionic blocks that shows dual thermo- and pH-responsive properties. The presence of the polyampholyte structure enables these polymers to act as protein release agents, while simultaneously protecting the proteins from severe stress.
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Cancer is one of the leading causes of death worldwide. As the cancer burden continues to increase globally, it exerts tremendous physical, emotional, and financial strain on ...individuals, families, communities, and health care systems. Cancer can affect any part of the body and is characterized by its uncontrollable growth. Numerous treatments, such as radiation therapy and chemotherapy which utilize various drugs, are currently in use; however, their harmful side effects and the development of drug resistance have resulted in major roadblocks when treating cancer. With advancements in synthetic and polymer chemistry, the use of nanoparticle-based drug delivery systems and chemotherapeutic macromolecules have garnered increasing attention in the previous decade. This review discusses the recent advancements in the use of nanoparticle-based drug delivery systems as well as the development of synthetic biodegradable polypeptides and polymers for cancer treatment (both in vivo and in vitro). Additionally, we outline the potent selectivity and efficacy for immunotherapies and bacteria-based therapies that are used to treat various cancers.
This study describes a method to synthesize compounds with an arylidene group at the alpha position of N‐methyl‐2‐pyrrolidone (NMP) and an alcohol group at the gamma position in a single‐step ...reaction using NMP and aldehyde. The highest yields were obtained with Ni(glyme)Cl2 as a nickel catalyst and tert‐butyl hydroperoxide as the oxidant, and various aryl aldehydes reacted under optimal conditions to yield the target compounds.
The reaction of N‐methyl‐2‐pyrrolidone (NMP) and aldehydes in the presence of tert‐butyl hydroperoxide and nickel catalyst gave the alpha‐arylidene and gamma hydroxy‐substituted‐Substituted NMPs in good yields.
Developing stabilizers that protect proteins from denaturation under stress, and are easy to remove from solutions, is a challenge in protein therapeutics. In this study, micelles made of trehalose, ...a zwitterionic polymer (poly-sulfobetaine; poly-SPB), and polycaprolactone (PCL) were synthesized by a one-pot reversible addition-fragmentation chain-transfer (RAFT) polymerization reaction. The micelles protect lactate dehydrogenase (LDH) and human insulin from denaturation due to stresses like thermal incubation and freezing, and help them retain higher-order structures. Importantly, the protected proteins are readily isolated from the micelles by ultracentrifugation, with over 90% recovery, and almost all enzymatic activity is retained. This suggests the great potential of poly-SPB-based micelles for use in applications requiring protection and removal as required. The micelles may also be used to effectively stabilize protein-based vaccines and drugs.
Polysulfobetaine, trehalose, and polycaprolactone were combined to create micelles that stabilize proteins and can be easily removed by ultracentrifugation. The three components work synergistically to enhance protein protection within the micelles.
Protein storage and delivery are crucial for biomedical applications such as protein therapeutics and recombinant proteins. Lack of proper protocols results in the denaturation of proteins, rendering ...them inactive and manifesting undesired side effects. In this study, polyampholyte‐based (succinylated ε‐poly‐l‐lysine) hydrogels containing polyvinyl alcohol and polyethylene glycol polymer matrices to stabilize proteins are developed. These hydrogels facilitated the loading and release of therapeutic amounts of proteins and withstood thermal and freezing stress (15 freeze–thaw cycles and temperatures of −80 °C and 37 °C), without resulting in protein denaturation and aggregation. To the best of our knowledge, this strategy has not been applied to the design of hydrogels constituting polymers, (in particular, polyampholyte‐based polymers) which have inherent efficiency to stabilize proteins and protect them from denaturation. Our findings can open up new avenues in protein biopharmaceutics for the design of materials that can store therapeutic proteins long‐term under severe stress and safely deliver them.
Polyampholyte‐based hydrogels are prepared using protein‐stabilizing polymers to encapsulate and release therapeutic proteins. The hydrogels ameliorate thermal and freezing stress‐induced denaturation and facilitate the retention of their native higher‐order structure and functions. This promotes a new dimension in therapeutic protein storage and delivery.
A completely synthetic polyampholyte cryoprotectant was developed with cationic and anionic monomers by reversible addition-fragmentation chain transfer polymerization. The neutralized random ...polyampholyte, which had an equal composition ratio of monomers, showed high cryoprotective properties in mammalian cells. Introduction of a small amount of hydrophobic monomer enhanced cell viability after cryopreservation, indicating the importance of hydrophobicity. Leakage experiments confirmed that these polyampholytes protected the cell membrane during cryopreservation. Due to low cytotoxicity, this polyampholyte has the potential to replace the convention cryoprotective agent dimethyl sulfoxide. The present study is the first to show that we can design a polymeric cryoprotectant that will protect the cell membrane during freezing using appropriate polymerization techniques.
Adaptive plasticity of Breast Cancer stem cells (BCSCs) is strongly correlated with cancer progression and resistance, leading to a poor prognosis. In this study, we report the expression profile of ...several pioneer transcription factors of the
network associated with tumor initiation and metastasis. In the triple negative breast cancer cell line (MDA-MB-231) stably transfected with human Oct3/4-GFP, differentially expressed genes (DEGs) were identified using qPCR and microarray, and the resistance to paclitaxel was assessed using an MTS assay. The tumor-seeding potential in immunocompromised (NOD-SCID) mice and DEGs in the tumors were also assessed along with the intra-tumor (CD44+/CD24-) expression using flow cytometry. Unlike 2-D cultures, the Oct3/4-GFP expression was homogenous and stable in 3-D mammospheres developed from BCSCs. A total of 25 DEGs including
and
were identified in Oct3/4 activated cells coupled with a significantly increased resistance to paclitaxel. In mice, the higher Oct3/4 expression in tumors correlated with enhanced tumorigenic potential and aggressive growth, with metastatic lesions showing a >5-fold upregulation of DEGs compared to orthotopic tumors and variability in different tissues with the highest modulation in the brain. Serially re-implanting tumors in mice as a model of recurrence and metastasis highlighted the sustained upregulation of
and
genes in metastatic lesions with a 2-fold higher expression of stem cell markers (CD44+/CD24-). Thus,
transcriptome may drive the differentiation and maintenance of BCSCs, promoting their tumorigenic potential, metastasis and resistance to drugs such as paclitaxel with tissue-specific heterogeneity.
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