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.

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.

Cryopreservation of rat embryos at all developmental stages by small-volume vitrification procedure and rapid warming in cryotubes.

Intracellular ice formation during cryopreservation is lethal to the cell, including during warming. Here, we examined the effect of sample volume and warming rate on the cryopreservation success of 1-cell rat embryos based on successful development into blastocysts in vitro and to term in vivo following embryo transfer. Embryos were equilibrated in 5% propylene glycol solution for 10 min, held for 40 s at 0 °C in cryopreservation solution (5%PG + PEPeS), and cooled by immersion in liquid nitrogen. When 1-cell embryos were cryopreserved in a volume of 30-100 µL at a cooling rate of 5830-7160 °C/min and warmed at 35,480-49,400 °C/min by adding 1 mL of 0.3 M sucrose solution at 50 °C, 17.3-28.8% developed into blastocysts, compared with 57.0% of untreated embryos. However, when 1-cell embryos were cryopreserved in a smaller volume of 15 µl at 7950 °C/min and warmed at 68,850 °C/min, 58.8 ± 10.6% developed into blastocysts and 50.0 ± 7.4% developed to term, comparable to that of non-treated embryos (57.0 ± 5.4% and 51.4 ± 3.1%, respectively). Cryopreserved embryos at other developmental stages also showed high in vitro culture potential similar to that of the control. Using a conventional cryotube and a small-volume vitrification procedure with rapid warming, we achieved high levels of subsequent rat embryonic development at all developmental stages.

Enhancing the Mechanical Properties of Poly(vinyl alcohol) Fibers by Lithium Iodide Addition.

The effect of lithium iodide (LiI) on the mechanical strength, properties, and molecular orientation of poly(vinyl alcohol) (PVA) fibers spun by wet spinning and then heat-stretched was studied. The stretchability of LiI-PVA fibers was improved, and the rupture during stretching was suppressed compared to PVA fibers. In addition, the tensile strength and elastic modulus of the thermally stretched fibers have been significantly improved. It was also found that the addition of LiI improves the molecular orientation of PVA. This was achieved because LiI reduced the hydrogen bonds between the molecular chains of PVA, resulting in reduced crystallinity. Most of the LiI in the fiber could be removed by a coagulation bath and washing during the spinning process. This means that LiI is eventually removed, and the heat-treatment strengthens the hydrogen bonds, resulting in excellent mechanical strength.

Optogenetic Calcium Ion Influx in Myoblasts and Myotubes by Near-Infrared Light Using Upconversion Nanoparticles.

Bioactuators made of cultured skeletal muscle cells are generally driven by electrical or visible light stimuli. Among these, the technology to control skeletal muscle consisting of myoblasts genetically engineered to express photoreceptor proteins with visible light is very promising, as there is no risk of cell contamination by electrodes, and the skeletal muscle bioactuator can be operated remotely. However, due to the low biopermeability of visible light, it can only be applied to thin skeletal muscle films, making it difficult to realize high-power bioactuators consisting of thick skeletal muscle. To solve this problem, it is desirable to realize thick skeletal muscle bioactuators that can be driven by near-infrared (NIR) light, to which living tissue is highly permeable. In this study, as a promising first step, upconversion nanoparticles (UCNPs) capable of converting NIR light into blue light were bound to C2C12 myoblasts expressing the photoreceptor protein channelrhodopsin-2 (ChR2), and the myoblasts calcium ion (Ca2+) influx was remotely manipulated by NIR light exposure. UCNP-bound myoblasts and UCNP-bound differentiated myotubes were exposed to NIR light, and the intracellular Ca2+ concentrations were measured and compared to myoblasts exposed to blue light. Exposure of the UCNP-bound cells to NIR light was found to be more efficient than exposure to blue light in terms of stimulating Ca2+ influx.

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.

Design of self-assembled glycopolymeric zwitterionic micelles as removable protein stabilizing agents.

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.

Improved fertility of frozen-thawed porcine spermatozoa with 3,3-dimethylglutaric anhydride poly-L-lysine as a novel cryoprotectant.

In this study, we determined the efficacy of 3,3-dimethylglutaric anhydride poly-L-lysine (DMGA-PLL) as a cryoprotectant for porcine spermatozoa. Porcine spermatozoa were cryopreserved in a freezing extender containing 3% (v/v) glycerol and various concentrations of DMGA-PLL. At 12 h after thawing, the motility index of spermatozoa cryopreserved with 0.25% (v/v) DMGA-PLL (25.9) was significantly (P < 0.01) higher than that of spermatozoa cryopreserved with 0%, 0.125%, or 0.5% DMGA-PLL (10.0-16.3). In addition, the blastocyst formation rate of embryos derived from spermatozoa cryopreserved with 0.25% DMGA-PLL (22.8%) was significantly (P < 0.01) higher than that of embryos derived from spermatozoa cryopreserved with 0%, 0.125%, or 0.5% DMGA-PLL (7.9%-10.9%). The mean number of total piglets born to sows inseminated with spermatozoa cryopreserved without DMGA-PLL (9.0) was significantly (P < 0.05) lower than that of total piglets born to sows inseminated with spermatozoa stored at 17°C (13.8). However, when spermatozoa cryopreserved with 0.25% DMGA-PLL were used for artificial insemination, the mean number of total piglets (11.7) was not significantly different from that obtained following artificial insemination using spermatozoa stored at 17°C. The results showed the usefulness of DMGA-PLL as a cryoprotectant in the cryopreservation of porcine spermatozoa.

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.

Successful production of offspring derived from mouse zygotes vitrified with carboxylated ε-poly-L-lysine and polyvinyl alcohol without serum.

The vitrification of zygotes is important for their use as donors for generating genome-edited mice. We previously reported the successful vitrification of mouse zygotes using carboxylated ε-poly-L-lysine (COOH-PLL). However, this vitrification solution contains fetal calf serum (FCS), which contains unknown factors and presents risks of pathogenic viral and microbial contamination. In this study, we examined whether polyvinyl alcohol (PVA) can be used as an alternative to FCS in vitrification solutions for mouse zygotes. When COOH-PLL was added to the vitrification solutions, zygotes vitrified with solutions containing 0.01% PVA (PV0.01) and those vitrified in a control solution containing FCS (75.6%) developed into blastocysts (78.4%). In addition, there were no significant differences in the ability to develop to term between the control solution (46.6%) and PV0.01 (44.1%) groups. In conclusion, we clearly demonstrated that PVA can replace FCS in our vitrification solution supplemented with COOH-PLL for mouse zygotes.

Cryopreservation of pig spermatozoa using carboxylated poly-L-lysine as cryoprotectant.

In this study, we cryopreserved pig spermatozoa using carboxylated poly-L-lysine (CPLL) as the cryoprotectant to determine its efficacy. Pig spermatozoa were placed in a freezing extender containing 3% (v/v) glycerol and different CPLL concentrations. The motility indices of the spermatozoa cryopreserved with 0.25% (v/v) CPLL at 6 (59.3), 9 (53.7), and 12 (26.2) h after thawing were significantly higher (P < 0.01 or P < 0.05) than those of the spermatozoa cryopreserved without CPLL (53.7, 40.1, and 17.5 at 6, 9, and 12 h after thawing, respectively). The concentration of CPLL in the freezing extender did not affect the ability of frozen-thawed spermatozoa to fertilize oocytes in vitro. However, the blastocyst formation rate of embryos derived from spermatozoa cryopreserved with 0.25% CPLL (24.6%) was significantly higher (P < 0.01) than that of embryos derived from spermatozoa cryopreserved without CPLL (11.2%). The conception rate of the sows inseminated with spermatozoa cryopreserved with 0.25% CPLL (72.2%) was not significantly different from that of the sows inseminated with spermatozoa stored at 17°C (81.3%). However, the mean number of total piglets born to the former (10.0) was significantly lower (P < 0.05) than that of total piglets born to the latter (13.4). The results showed that CPLL in the freezing extender maintained the motility of frozen-thawed pig spermatozoa and improved the in vitro development of embryos produced by in vitro fertilization. In addition, we have demonstrated that piglets could be obtained with artificial insemination using spermatozoa cryopreserved with CPLL.

Cellular Flocculation Driven by Concentrated Polymer Brush-Modified Cellulose Nanofibers with Different Surface Charges.

This study examined the effect of the surface charge of concentrated polymer brush (CPB)-grafted cellulose nanofibers (CNFs) on HepG2 cell flocculation. Four polyelectrolytes, poly(p-styrenesulfonic acid sodium salt) (PSSNa), poly(acrylic acid) (PAA), poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA), and poly([(2-methacryloyloxy)ethyl]trimethylammonium chloride) (PMTAC), were grafted onto the CNF surface via surface-initiated atom transfer radical polymerization to form CNF-CPBs. The floc size of HepG2 cells depended on the surface charge of CNF-CPBs, where the anionic CNF-PSSNa formed larger flocs than CNF-PAA; due to the electrostatic repulsive forces, CNF-CPBs with a lower ζ-potential yielded smaller floc sizes. Contrastingly, the cytotoxic cationic CNF-PDMAEMA and CNF-PMTAC limited the floc size growth. Thus, appropriate electrostatic interactions are essential for floc formation and improved cell function in three-dimensional (3D) cell culture systems. Interestingly, while developing a novel 3D cell culture system, we reveal that colloidal flocculation theory is the driving mechanism behind this unique phenomenon.

Enhanced proliferation and differentiation of human mesenchymal stem cells in the gravity-controlled environment.

BACKGROUND: Human bone marrow mesenchymal stem cells (hMSCs) present a promising cell source with the potential to be used for curing various intractable diseases, and it is expected that the development of regenerative medicine employing cell-based therapy would be significantly accelerated when such methods are established. For that, powerful methods for selective growth and differentiation of hMSCs should be developed. METHODS: We developed an efficient method for hMSC proliferation and differentiation into osteoblasts and adipocytes using gravity-controlled environments. RESULTS: The results indicate that the average doubling time of hMSCs cultured in a regular maintenance medium under microgravity conditions (0.001 G) was 1.5 times shorter than that of cells cultured under natural gravity conditions (1.0 G). Furthermore, 99.2% of cells grown in the microgravity environment showed the expression of hMSC markers, as indicated by flow cytometry analysis. Osteogenic and adipogenic differentiation of hMSCs expanded in the microgravity environment was enhanced under microgravity and hypergravity conditions, respectively, as evidenced by the downregulation of hMSC markers and upregulation of osteoblast and adipocyte markers, respectively. Most cells differentiated into osteoblasts in the microgravity environment after 14 days (~80%) and adipocytes in the hypergravity environment after 12 days (~90%). CONCLUSIONS: Our results indicate that hMSC proliferation and selective differentiation into specific cell lineages could be promoted under microgravity or hypergravity conditions, suggesting that cell culture in the gravity-controlled environment is a useful method to obtain cell preparations for potential clinical applications.

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.

Quick and Mild Isolation of Intact Lysosomes Using Magnetic-Plasmonic Hybrid Nanoparticles.

Rapid and efficient isolation of intact lysosomes is necessary to study their functions and metabolites by proteomic analysis. We developed a swift and robust nanoparticle-based magnetic separation method in which magnetic-plasmonic hybrid nanoparticles (MPNPs) conjugated with amino dextran (aDxt) were targeted to the lumen of lysosomes via the endocytosis pathway. For well-directed magnetic separation of the lysosomes, it is important to trace the intracellular trafficking of the aDxt-conjugated MPNPs (aDxt-MPNPs) in the endocytosis pathway. Therefore, we analyzed the intracellular transport process of the aDxt-MPNPs by investigating the time-dependent colocalization of plasmonic scattering of aDxt-MPNPs and immunostained marker proteins of organelles using the threshold Manders' colocalization coefficient (Rt). Detailed analysis of time variations of Rt for early and late endosomes and lysosomes allowed us to derive the transport kinetics of aDxt-MPNPs in a cell. After confirming the incubation time required for sufficient accumulation of aDxt-MPNPs in lysosomes, the lysosomes were magnetically isolated as intact as possible. By varying the elapsed time from homogenization to complete isolation of lysosomes (tdelay) and temperature (T), the influences of tdelay and T on the protein composition of the lysosomes were investigated by polyacrylamide gel electrophoresis and amino acid analysis. We found that the intactness of lysosomes could become impaired quite quickly, and to isolate lysosomes as intact as possible with high purity, tdelay = 30 min and T = 4 °C were optimal settings.

Elucidating the degradation mechanism of a self-degradable dextran-based medical adhesive.

We developed a self-degradable medical adhesive, LYDEX, consisting of periodate-oxidized aldehyde-functionalized dextran (AD) and succinic anhydride-treated ε-poly-l-lysine (SAPL). After gelation and adhesion of LYDEX by Schiff base bond formation between the AD aldehyde groups and SAPL amino groups, molecular degradation associated with the Maillard reaction is initiated, but the detailed degradation mechanism remains unknown. Herein, we elucidated the degradation mechanism of LYDEX by analyzing the main degradation products under typical solution conditions in vitro. The degradation of the LYDEX gel with a sodium periodate/dextran content of 2.5/20 was observed using gel permeation chromatography and infrared and 1H NMR spectroscopy. The AD ratio in the AD-SAPL mixture increased as the molecular weight decreased with the degradation time. This discovery of LYDEX self-degradability is useful for clarifying other polysaccharide hydrogel degradation mechanisms, and valuable for the use of LYDEX in medical applications, such as hemostatic or sealant materials.

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.

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.

Gene expression analysis of human induced pluripotent stem cells cryopreserved by vitrification using StemCell Keep.

In recent years, regenerative medicine research using human somatic and induced pluripotent stem cells has advanced considerably, promoting clinical applications. However, it is essential that these cells are cryopreserved safely and effectively. Most cryopreservation solution agents contain dimethyl sulfoxide (DMSO), which exhibits strong toxicity and can potentially promote cell differentiation. Hence, it is important to explore substitutes for DMSO in cryoprotectant solutions. One such alternative is StemCell Keep (SCK), a DMSO-free solution that has been reported to effectively cryopreserve human induced pluripotent stem cells (hiPS cells). To clarify the effect of cryopreservation agents on cells, DNA microarray analysis is useful, as it can identify a large number of gene expression differences in cryopreserved cells, as well as functional increases in gene groups. In this study, we performed gene expression analysis of SCK-cryopreserved hiPS cells using a DNA microarray gene chip. The hiPS cells vitrified with SCK or DMSO-based vitrification solutions were thawed and cultured on Matrigel under feeder-free conditions, and RNA was extracted for DNA microarray analysis. Genes obtained from DNA microarray data were classified by the keywords of Gene Ontology Biological Process Term, and their relationships were analyzed using DAVID or the GeneMANIA database. SCK-cryopreserved hiPS cells expressed several anti-apoptotic genes, as well as genes related to cell adhesion or proliferation at levels that were nearly equivalent to those of non-frozen hiPS cells. Gene enrichment analysis with selected genes of SCK-cryopreserved hiPS cells whose expression differences were superior to those of DAP-cryopreserved showed strong interactions of negative regulation of apoptotic process, cell adhesion and positive regulation of cell proliferation in DAVID analysis. We demonstrated that SCK successfully maintained the key functions of hiPS cells, including anti-apoptosis, cell adhesion, and cell proliferation, during cryopreservation.