Design of Highly Selective Zn-Coordinated Polyampholyte for Cancer Treatment and Inhibition of Tumor Metastasis.

Developing anticancer agents with negligible cytotoxicity against normal cells while mitigating multidrug resistance and metastasis is challenging. Previously reported cationic polymers have effectively eradicated cancers but are clinically unsuitable due to their limited selectivity. Herein, a series of poly(l-lysine)- and nicotinic acid-based polymers were synthesized using varying amounts of dodecylsuccinic anhydride. Zn-coordinating polymers concealed their cationic charge and enhanced selectivity. These Zn-bound polymers were highly effective against liver and colon cancer cells (HepG2 and Colon 26, respectively) and prevented cancer cell migration. They also displayed potent anticancer activity against drug-resistant cell lines (COR-L23/R): their cationic structure facilitated cancer cell membrane disruption. Compared to these polymers, doxorubicin was less selective and less efficacious against drug-resistant cell lines and was unable to prevent cell migration. These polymers are potential cancer treatment agents, offering a promising solution for mitigating drug resistance and tumor metastasis and representing a novel approach to designing cancer therapeutics.

Design of an Ice Recrystallization-Inhibiting Polyampholyte-Containing Graft Polymer for Inhibition of Protein Aggregation.

Freezing-induced damage to proteins, through osmotic stress and ice recrystallization, during protein processing and long-term storage is a serious concern and may lead to loss of protein activity owing to denaturation. In this study, graft copolymers composed of a cryoprotective polymer (capable of preventing osmotic stress) and poly(vinyl alcohol) (PVA; known for its high ice recrystallization inhibition (IRI) property) were developed. The polymers had high IRI activity, albeit slightly lower than that of PVA alone, but substantially higher than that of succinylated ε-poly-l-lysine (PLLSA) alone. The graft polymers showed an efficiency higher than that of PVA or PLLSA alone in protecting proteins from multiple freeze-thaw cycles, as well as during prolonged freezing, indicating a synergy between PVA and PLLSA. The PLLSA-based graft polymer is a promising material for use in protein biopharmaceutics for the long-term storage of proteins under freezing conditions.

Cellular Flocculation Using Concentrated Polymer Brush-Modified Cellulose Nanofibers with Different Fiber Lengths.

In this study, concentrated polymer brush-modified cellulose nanofibers (CNFs) with different fiber lengths were used for the flocculation of cells for systematically studying the mechanism of this unique cellular flocculation based on colloidal flocculation theory. Concentrated poly(p-styrenesulfonic acid sodium salt) brush-grafted CNF (CNF-PSSNa) with different fiber lengths were cultured with three different cell types to examine their influence on floc (cell clusters formed by cellular flocculation) characteristics. The floc size and survival rate could be controlled by modifying the CNF-PSSNa fiber lengths. The three cell types showed the same flocculation tendency after culture, indicating the applicability of the method in different cell lines. After 2 weeks of culture, CNF-PSSNa increased the specific expression of hepatocytes compared to the two-dimensional cell culture. Thus, owing to its wide applicability, high cell viability, and ability to control cell size and improve cell function, this technology could be used as a new three-dimensional cell culture method.

Facile Photolithographic Fabrication of Zwitterionic Polymer Microneedles with Protein Aggregation Inhibition for Transdermal Drug Delivery.

Microneedle technology has received considerable attention in transdermal drug delivery system research owing to its minimally invasive and convenient self-administration with enhanced transdermal transport. The pre-drug loading microneedle method has been developed for several protein and chemical medicines. However, the protein activity and efficacy are severely affected owing to protein aggregation. Herein, we aim to develop non-degradable hydrogel photocross-linkable microneedles for suppressing protein aggregation. Four-point star-shaped microneedles are fabricated via a photolithography process, and sulfobetaine (SPB) monomer is combined with dextran-glycidyl methacrylate/acrylic acid to form the hydrogel network. Incorporating zwitterionic poly-sulfobetaine (poly-SPB) in the microneedles enables the protection of proteins from denaturation even under external stress, releases the proteins in their native state (without activity loss), and exhibits sufficient mechanical strength to penetrate porcine skin. The microneedles exhibit a high drug loading capacity along with an efficient drug release rate. The rhodamine B drug loading and release model shows that the microneedles can load 8 µg of drugs on one microneedle patch of 41 needles and release nearly 80% of its load within 1 h. We anticipate that this pre-drug loading platform and the advanced features of the microneedles can provide an effective option for administering therapeutic drugs.

Tunable Dual-Thermoresponsive Core-Shell Nanogels Exhibiting UCST and LCST Behavior.

Thermoresponsive polymers change their physical properties as the temperature is changed and have found extensive use in a number of fields, especially in tissue engineering and in the development of drug delivery systems. The synthesis of a novel core-shell nanogel composed of N-isopropylacrylamide and sulfobetaine by reversible addition fragmentation chain transfer polymerization is reported. The core-shell architecture of the nanogels is confirmed using energy dispersive X-ray spectroscopy in scanning transmission electron microscopy. These nanogels exhibit dual thermoresponsive behavior, i.e., the core of the nanogel exhibits lower critical solution temperature, while the shell displays upper critical solution temperature behavior. Transition temperatures can be easily tuned by changing the molecular weight of the constituent polymer. These nanogels can be efficiently used in temperature-triggered delivery of therapeutic proteins and drugs.

Toward a Molecular Understanding of the Mechanism of Cryopreservation by Polyampholytes: Cell Membrane Interactions and Hydrophobicity.

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.

Liposome promotion of tumor growth is associated with angiogenesis and inhibition of antitumor immune responses.

Liposomes have tremendous potential as drug carriers in the treatment of cancer. However, despite enhanced tumor drug delivery and decreased toxicity, patient survival rates have not improved significantly compared to corresponding free drug treatments. Importantly, we found that a liposomal nanoparticle currently used as a drug carrier in cancer patients enhanced tumor growth in an immune competent murine model of cancer. This was associated with increased tumor angiogenesis and suppression of antitumor immune responses as indicated by decreased cytokine production by tumor macrophages and cytotoxic T cells, diminished tumor infiltration of tumor-specific T cells, and decreased number of dendritic cells in tumor draining lymph nodes. These results suggest that carrier-induced immunosuppression and angiogenesis have the potential to reduce the antitumor effects of drugs loaded within. These findings may have significant implications for the current use and future development of anticancer nanoparticles and further investigations are urgently needed. FROM THE CLINICAL EDITOR: This study discusses important implications of nanoliposome-based drug delivery systems in cancer therapy, and demonstrates that nanoliposomes may have immunosuppressive and angiogenetic properties, directly counterbalancing their anti-cancer activity, which may also have important clinical implications related to more widespread applications of such systems.

Enhancement of cryopreservation with intracellularly permeable zwitterionic polymers.

A novel copolymer containing zwitterionic and methylsulfinyl structures was developed, which enhanced cryoprotective efficacy by enabling intracellular cytoplasmic permeation without relying on mediated endocytosis and diffused out of the cells within approximately 30 min, making it more advantageous than polymeric nanoparticles for the transport of membrane-impermeable cryoprotectants such as trehalose.

Polyampholytes and Their Hydrophobic Derivatives as Excipients for Suppressing Protein Aggregation.

Protein aggregation, which occurs under various physiological conditions, can affect cell function and is a major issue in the field of protein therapeutics. In this study, we developed a polyampholyte composed of ε-poly-l-lysine and succinic anhydride and evaluated its protein protection efficacy. This polymer was able to protect different proteins from thermal stress and its performance significantly exceeded that of previously reported zwitterionic polymers. In addition, we synthesized derivatives with varying degrees of hydrophobicity, which exhibited remarkably enhanced efficiency; thus, the polymer concentration required for protein protection was very low. By facilitating the retention of protein enzymatic activity and stabilizing the higher-order structure, these polymers enabled the protein to maintain its native state, even after being subjected to extreme thermal stress. Thus, such polyampholytes are extremely effective in protecting proteins from extreme stress and may find applications in protein biopharmaceuticals and drug delivery systems.

Polyampholyte-Based Polymer Hydrogels for the Long-Term Storage, Protection and Delivery of Therapeutic Proteins.

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.

Mechanistic insights and importance of hydrophobicity in cationic polymers for cancer therapy.

Development of molecules that can be effectively used for killing cancer cells remains a research topic of interest in drug discovery. However, various limitations of small molecules and nanotechnology-based drug-delivery systems hinder the development of chemotherapeutics. To resolve this issue, this study describes the potential application of polymeric molecules as anticancer drug candidates. We describe the design and synthesis of novel anticancer polymers containing hydrophobic groups. We established the fact that the cationic homopolymer (PAMPTMA) does not show any anticancer activity on its own; however, the insertion of hydrophobic moieties in copolymers (PAMPTMA-r-BuMA, PAMPTMA-r-HexMA, and PAMPTMA-r-OctMA) enhances their anticancer activity with a very low IC50 value (60 µg mL-1 for HepG2 cells). Mechanistic investigations were carried out using LDH leakage assay, cellular uptake, DOSY NMR and molecular dynamics to study the interaction between the polymer and the cell membrane as well as the role of hydrophobicity in enhancing this interaction. The results demonstrated that polymers are attracted by the anionic cancer cell membrane, which then leads to the insertion of hydrophobic groups inside the cell membrane, causing its disruption and ultimate lysis of the cell. This study demonstrates a novel and better approach for the rational design and discovery of new polymeric anticancer agents with improved efficacy.

Dual Thermo- and pH-Responsive Behavior of Double Zwitterionic Graft Copolymers for Suppression of Protein Aggregation and Protein Release.

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.

Cytosolic delivery of quantum dots mediated by freezing and hydrophobic polyampholytes in RAW 264.7 cells.

Quantum dots (QDs) can be delivered efficiently inside macrophages using a freeze-concentration approach. In this study, we introduced a new, facile, high concentration-based freezing technology of low toxicity. We also developed QD-conjugated new hydrophobic polyampholytes using poly-l-lysine (PLL), a naturally derived polymer, which showed sustained biocompatibility, stability over one week, and enhanced intracellular delivery. When freeze-concentration was applied, the QD-encapsulated hydrophobic polyampholytes showed a higher tendency to adsorb onto the cell membrane than the non-frozen molecules. Interestingly, we observed that the efficacy of adsorption of QDs on RAW 264.7 macrophages was higher than that on fibroblasts. Furthermore, the intracellular delivery of QDs using hydrophobic polyampholytes was higher than those of PLL and QDs. In vitro studies revealed the efficient endosomal escape of QDs in the presence of hydrophobic polyampholytes and freeze-concentration. Collectively, these observations indicated that the promising combination of freeze-concentration and hydrophobic polyampholytes may act as an effective and versatile strategy for the intracellular delivery of QDs, which can be used for biological diagnosis and therapeutic applications.

Liposome-induced immunosuppression and tumor growth is mediated by macrophages and mitigated by liposome-encapsulated alendronate.

Liposomal nanoparticles are the most commonly used drug nano-delivery platforms. However, recent reports show that certain pegylated liposomal nanoparticles (PLNs) and polymeric nanoparticles have the potential to enhance tumor growth and inhibit antitumor immunity in murine cancer models. We sought herein to identify the mechanisms and determine whether PLN-associated immunosuppression and tumor growth can be reversed using alendronate, an immune modulatory drug. By conducting in vivo and ex vivo experiments with the immunocompetent TC-1 murine tumor model, we found that macrophages were the primary cells that internalized PLN in the tumor microenvironment and that PLN-induced tumor growth was dependent on macrophages. Treatment with PLN increased immunosuppression as evidenced by increased expression of arginase-1 in CD11b+Gr1+ cells, diminished M1 functionality in macrophages, and globally suppressed T-cell cytokine production. Encapsulating alendronate in PLN reversed these effects on myeloid cells and shifted the profile of multi-cytokine producing T-cells towards an IFNγ+ perforin+ response, suggesting increased cytotoxic functionality. Importantly, we also found that PLN-encapsulated alendronate (PLN-alen), but not free alendronate, abrogated PLN-induced tumor growth and increased progression-free survival. In summary, we have identified a novel mechanism of PLN-induced tumor growth through macrophage polarization and immunosuppression that can be targeted and inactivated to improve the anticancer efficacy of PLN-delivered drugs. Importantly, we also determined that PLN-alen not only reversed protumoral effects of the PLN carrier, but also had moderate antitumor activity. Our findings strongly support the inclusion of immune-responsive tumor models and in-depth immune functional studies in the preclinical drug development paradigm for cancer nanomedicines, and the further development of chemo-immunotherapy strategies to co-deliver alendronate and chemotherapy for the treatment of cancer.

Inhibition of protein aggregation by zwitterionic polymer-based core-shell nanogels.

Protein aggregation is a process by which misfolded proteins polymerizes into aggregates and forms fibrous structures with a ß-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.

Cryoprotective properties of completely synthetic polyampholytes via reversible addition-fragmentation chain transfer (RAFT) polymerization and the effects of hydrophobicity.

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.