Manipulation of Macrophage Uptake by Controlling the Aspect Ratio of Graft Copolymer Micelles.

Nanostructures of drug carriers play a crucial role in nanomedicine due to their ability to influence drug delivery. There is yet no clear consensus regarding the optimal size and shape (e.g., aspect ratio) of nanoparticles for minimizing macrophage uptake, given the difficulties in controlling the shape and size of nanoparticles while maintaining identical surface properties. Here, we employed graft copolymer self-assembly to prepare polymer micelles with aspect ratios ranging from 1.0 (spherical) to 10.8 (cylindrical) and closely matched interfacial properties. Notably, our findings emphasize that cylindrical micelles with an aspect ratio of 2.4 are the least susceptible to macrophage uptake compared with both their longer counterparts and spherical micelles. This reduced uptake of the short cylindrical micelles results in a 3.3-fold increase in blood circulation time compared with their spherical counterparts. Controlling the aspect ratio of nanoparticles is crucial for improving drug delivery efficacy through better nanoparticle design.

Influence of Polymer Side Chain Size and Backbone Length on the Self-Assembly of Supramolecular Polymer Bottlebrushes.

Hydrogen bonds are a versatile tool for creating fibrous, bottlebrush-like assemblies of polymeric building blocks. However, a delicate balance of forces exists between the steric repulsion of the polymer chains and these directed supramolecular forces. In this work we have systematically investigated the influence of structural parameters of the attached polymers on the assembly behaviour of benzene trisurea (BTU) and benzene tris(phenylalanine) (BTP) conjugates in water. Polymers with increasing main chain lengths and different side chain sizes were prepared by reversible addition-fragmentation chain-transfer (RAFT) polymerization of hydroxyethyl acrylate (HEA), tri(ethylene glycol) methyl ether acrylate (TEGA) and oligo(ethylene glycol) methyl ether acrylate (OEGA). The resulting structures were analyzed using small angle X-ray scattering (SAXS) and transmission electron microscopy (TEM). Both BTU and BTP formed fibres with PHEA attached, but a transition to spherical morphologies was observed at degrees of polymerisation (DP) of 70 and above. Overall, the main chain length appeared to be a dominating factor in inducing morphology transitions. Increasing the side chain size generally had a similar effect but mainly impeded any aggregation as is the case of POEGA. Interestingly, BTP conjugates still formed fibres, suggesting that the stronger intermolecular interactions can compensate partially for the steric repulsion.

Molecular Bottlebrushes for Immunostimulatory CpG ODN Delivery: Relationship among Cation Density, Complex Formation Ability, and Cytotoxicity.

Artificially designed short single-stranded DNA sequences containing unmethylated CG (CpG ODNs) are agonists for toll-like receptor 9 (TLR9); thus, they have great potential as vaccine adjuvants for cancer immunotherapy and preventing infectious diseases. To deliver effectively CpG ODNs into cells bearing TLR9, nanoparticle polyion complexes of cationic polymers that are able to ingest multiple CpG ODN molecules have been developed; however, their structures and synthesized polycations are hard to control and bioincompatible, respectively. To solve these issues, we designed cationic molecular bottlebrushes (CMBs) with branches that are made from copolymers of 2-methacryloyloxyethyl phosphorylcholine and 2-methacryloyloxyethyl trimethylammonium chloride. Several instrumental methods were carried out to determine the structure of a CMB and its complex with CpG ODNs. The complexation did not change the overall shape of the original CMB, and the bound CpG ODNs were captured by the outer layer of the CMB. The moderation of cations was important to reduce toxicity and improve secretion of inflammatory cytokines.

Polymeric core-crosslinked particles prepared via a nanoemulsion-mediated process: from particle design and structural characterization to in vivo behavior in chemotherapy.

Various polymeric nanoparticles have been used as drug carriers in drug delivery systems (DDSs). Most of them were constructed from dynamic self-assembly systems formed via hydrophobic interactions and from structures that are unstable in an in vivo environment owing to their relatively weak formation forces. As a solution to this issue, physically stabilized core-crosslinked particles (CP) with chemically crosslinked cores have received attention as alternatives to the dynamic nanoparticles. This focused review summarizes recent advances in the construction, structural characterization, and in vivo behavior of polymeric CPs. First, we introduce a nanoemulsion-mediated method to create polyethylene glycol (PEG)-bearing CPs and their structural characterization. The relationship between the PEG chain conformations in the particle shell and the in vivo fate of the CPs is also discussed. After that, the development and advantages of zwitterionic amino acid-based polymer (ZAP)-bearing CPs are presented to address the poor penetration and the internalization of PEG-based CPs into tumor tissues and cells, respectively. Finally, we conclude and discuss prospects for application of polymeric CPs in the DDS field.

Zwitterionic Amino Acid Polymer-Grafted Core-Crosslinked Particle toward Tumor Delivery.

Zwitterionic amino acid polymers (ZAPs) exhibit biocompatibility and recognition capability for amino acid transporters (AATs) overexpressed on cancer cells. They are potential cancer-targeting ligands in nanoparticle-based nanomedicines utilized in cancer chemotherapy. Here, a poly(glutamine methacrylate) (pGlnMA)-grafted core-crosslinked particle (pGlnMA-CCP) is prepared through the formation of nanoemulsions stabilized using amphiphilic block copolymers comprising pGlnMA as the hydrophilic block. The chain conformation of the grafted polymer and the particle structure of pGlnMA-CCP are precisely elucidated by dynamic light scattering, X-ray scattering, and transmission electron microscopy. pGlnMA-CCP demonstrates active cellular uptake and deep penetration behaviors for cancer cells and spheroids, respectively, via an AAT-mediated mechanism. The in vivo pharmacokinetics of pGlnMA-CCP is practically comparable to those of a CCP covered with poly(polyethylene glycol methacrylate) (pPEGMA), which inhibits protein adsorption and prolongs blood retention, implying that the biocompatible properties of pGlnMA are similar to those of pPEGMA. Furthermore, pGlnMA-CCP accumulates in cancer tissues at a higher level than that of pPEGMA systems. The results demonstrate that the properties of cancer targetability, tumor permeability, efficient tumor accumulation, and biocompatibility can be obtained by grafting pGlnMA onto nanoparticles, suggesting a high potential of pGlnMA as a ligand for cancer-targeting nanomedicines.

Impact of Zwitterionic Polymers on the Tumor Permeability of Molecular Bottlebrush-Based Nanoparticles.

Biocompatible polymers possessing antifouling properties for biomolecules are necessary to be combined with nanoparticles for cancer chemotherapy to improve their retention in blood and subsequent tumor accumulation. However, these properties simultaneously lead to poor affinity to cells, and low tumor tissue permeability subsequently, which is one of the major barriers in achieving efficient anticancer efficacy. To address this, we try to elucidate the tumor permeability of nanoparticles using molecular bottlebrushes (MBs) as model polymeric nanoparticles composed of various biocompatible polymers. An MB comprising nonionic poly[(ethylene glycol) methyl ether methacrylate] (PEGMA) shows no tumor permeability at all, whereas zwitterionic MBs composed of poly(phosphobetaine methacrylate), poly(sulfobetaine methacrylate), or poly(carboxybetaine methacrylate) penetrate deeply into tumor tissues. The carboxybetaine-based MBs showed an efficient cellular uptake into cancer cells while the other MBs did not, which enable them to penetrate into tumor tissues via the transcytosis pathway. Additionally, their permeability is based on intercellular or intracellular pathways, which might be related to the zwitterionic betaine properties that recognize protein transporters on cancer cells. Surprisingly, incorporating only 10 mol % of the zwitterionic betaine polymers into PEGMA-based MBs significantly enhances their tissue permeability. This platform technology enables us to redesign the PEG-based nanoparticles developed for cancer chemotherapy in clinical applications.

Impact of Polyethylene Glycol (PEG) Conformations on the In Vivo Fate and Drug Release Behavior of PEGylated Core-Cross-Linked Polymeric Nanoparticles.

In cancer chemotherapy, core-cross-linked particles (CCPs) are a promising drug carrier due to their high structural stability in an in vivo environment, resulting in improved tumor delivery. A biocompatible polymer of polyethylene glycol (PEG) is often utilized to coat the surface of CCPs to avoid nonspecific adsorption of proteins in vivo. The PEG density and conformation on the particle surface are important structural factors that determine the in vivo fate of such PEGylated nanoparticles, including their pharmacokinetics and pharmacodynamics. However, contrary to expectations, we found no significant differences in the in vivo pharmacokinetics and pharmacodynamics of the PEGylated CCPs with the different PEG densities including mushroom, brush, and dense brush conformations. On the contrary, the in vivo release kinetics of hydrophilic and hydrophobic model drugs from the PEGylated CCPs was strongly dependent on the PEG conformation and the drug polarity. This may be related to the water-swelling degree in the particle PEG layer, which promotes and inhibits the diffusion of hydrophilic and hydrophobic drugs, respectively, from the particle core to the water phase. Our results provide guidelines for the design of cancer-targeting nanomedicine based on PEGylated CCPs.

Inflammogenic effect of polyacrylic acid in rat lung following intratracheal instillation.

BACKGROUND: Some organic chemicals are known to cause allergic disorders such as bronchial asthma and hypersensitivity pneumonitis, and it has been considered that they do not cause irreversible pulmonary fibrosis. It has recently been reported, however, that cross-linked acrylic acid-based polymer, an organic chemical, might cause serious interstitial lung diseases, including pulmonary fibrosis. We investigated whether or not intratracheal instillation exposure to cross-linked polyacrylic acid (CL-PAA) can cause lung disorder in rats. METHODS: Male F344 rats were intratracheally instilled with dispersed CL-PAA at low (0.2 mg/rat) and high (1.0 mg/rat) doses, and were sacrificed at 3 days, 1 week, 1 month, 3 months and 6 months after exposure to examine inflammatory and fibrotic responses and related gene expressions in the lungs. Rat lungs exposed to crystalline silica, asbestos (chrysotile), and NiO and CeO2 nanoparticles were used as comparators. RESULTS: Persistent increases in total cell count, neutrophil count and neutrophil percentage, and in the concentration of the cytokine-induced neutrophil chemoattractant (CINC)-1, CINC-2 and C-X-C motif chemokine 5 (CXCL5), which correlated with lung tissue gene expression, were observed in bronchoalveolar lavage fluid (BALF) from 3 days until at least 1 month following CL-PAA intratracheal instillation. Persistent increases in heme oxygenase-1 (HO-1) in the lung tissue were also observed from 3 days to 6 months after exposure. Histopathological findings of the lungs demonstrated that extensive inflammation at 3 days was greater than that in exposure to silica, NiO nanoparticles and CeO2 nanoparticles, and equal to or greater than that in asbestos (chrysotile) exposure, and the inflammation continued until 1 month. Fibrotic changes also progressed after 1 month postexposure. CONCLUSION: Our results suggested that CL-PAA potentially causes strong neutrophil inflammation in the rat and human lung.

Effect of Different Molecular Weights of Polyacrylic Acid on Rat Lung Following Intratracheal Instillation.

BACKGROUND: We conducted intratracheal instillations of different molecular weights of polyacrylic acid (PAA) into rats in order to examine what kinds of physicochemical characteristics of acrylic acid-based polymer affect responses in the lung. METHODS: F344 rats were intratracheally exposed to a high molecular weight (HMW) of 598 thousand g/mol or a low molecular weight (LMW) of 30.9 thousand g/mol PAA at low and high doses. Rats were sacrificed at 3 days, 1 week, 1 month, 3 months and 6 months post exposure. RESULTS: HMW PAA caused persistent increases in neutrophil influx, cytokine-induced neutrophil chemoattractants (CINC) in the bronchoalveolar lavage fluid (BALF), and heme oxygenase-1 (HO-1) in the lung tissue from 3 days to 3 months and 6 months following instillation. On the other hand, LMW PAA caused only transient increases in neutrophil influx, CINC in BALF, and HO-1 in the lung tissue from 3 days to up to 1 week or 1 month following instillation. Histopathological findings of the lungs demonstrated that the extensive inflammation and fibrotic changes caused by the HMW PAA was greater than that in exposure to the LMW PAA during the observation period. CONCLUSION: HMW PAA induced persistence of lung disorder, suggesting that molecular weight is a physicochemical characteristic of PAA-induced lung disorder.

Crosslinked Structure of Polyacrylic Acid Affects Pulmonary Fibrogenicity in Rats.

We conducted intratracheal instillations of polyacrylic acid (PAA) with crosslinking and non-crosslinking into rats in order to examine what kinds of physicochemical characteristics of acrylic-acid-based polymers affect responses in the lung. F344 rats were intratracheally exposed to similar molecular weights of crosslinked PAA (CL-PAA) (degree of crosslinking: ~0.1%) and non-crosslinked PAA (Non-CL-PAA) at low and high doses. Rats were sacrificed at 3 days, 1 week, 1 month, 3 months, and 6 months post-exposure. Both PAAs caused increases in neutrophil influx, cytokine-induced neutrophil chemoattractants (CINC) in the bronchoalveolar lavage fluid (BALF), and heme oxygenase-1 (HO-1) in the lung tissue from 3 days to 6 months following instillation. The release of lactate dehydrogenase (LDH) activity in the BALF was higher in the CL-PAA-exposed groups. Histopathological findings of the lungs demonstrated that the extensive fibrotic changes caused by CL-PAA were also greater than those in exposure to the Non-CL- PAA during the observation period. CL-PAA has more fibrogenicity of the lung, suggesting that crosslinking may be one of the physicochemical characteristic factors of PAA-induced lung disorder.

Research and development of anti-maceration laparoscopic surgical cotton swabs.

INTRODUCTION: Although laparoscopic cotton swabs have been used in procedures such as blunt tissue dissection and elevation of organs, fluid maceration is widely known to reduce their original performance. Thus, we developed an anti-maceration laparoscopic surgical cotton swab that is expected to solve this problem by coating the cotton swab with water-resistant resin. This study aimed to determine whether anti-maceration cotton swabs perform better than conventional products. MATERIAL AND METHODS: Fine surface shape analysis of cotton swabs was performed using microfocus X-ray computed tomography, and changes due to fluid absorption of the anti-maceration cotton swabs and pre-existing products were quantitatively compared. As indices, the degree of expansion by maceration and SMD (surface roughness index of the fiber industry showing the size of irregularities on the surface) were evaluated. RESULTS: The degree of expansion was lower in anti-maceration swabs than in conventional products. Maceration reduced SMD in existing products, whereas the SMD in anti-maceration cotton swabs did not change. CONCLUSIONS: Anti-maceration cotton swabs have a superior performance over conventional products.

Ultra-thin surgical swab: its development and clinical application.

INTRODUCTION: Ultra-thin (diameter: 3 mm) surgical swabs have not been in practical use as it is difficult to manufacture these using the pre-existing methods; therefore, a new technology has been adopted. This study aimed to evaluate the safety and efficiency of ultra-thin surgical swabs in bench-top and preclinical settings. MATERIAL AND METHODS: We performed liquid particle counter (LPC) test, cotton strength test, shaft bending comparison and surgical vision test for checking the durability of swabs as bench-top investigation, and laparoscopic surgery in a porcine model as preclinical investigation. All tests were compared with conventional 5-mm swabs. RESULTS: The ultra-thin swabs had fewer eluted particles in the LPC test, endured a 5 kg pulling force in the cotton strength test, their shaft did not break at 100 mm bending in the shaft bend comparison test, and interfered less in the surgical vision test. They were used for manipulating organs with no damage in preclinical investigation. CONCLUSIONS: Three millimeters ultra-thin surgical swabs manufactured with the new manufacturing technology are effective and safe.

Phosphorylcholine-Grafted Molecular Bottlebrush-Doxorubicin Conjugates: High Structural Stability, Long Circulation in Blood, and Efficient Anticancer Activity.

Controlling the particle structure of tumor-targeting nanomedicines in vivo remains challenging but must be achieved to control their in vivo fate and functions. Molecular bottlebrushes (MBs), where brush side chains are densely grafted from a main chain, have recently received attention as building blocks of polymer-based prodrugs because their rigid structure would be expected to demonstrate high structural stability in vivo. Here, we synthesized a poly(methacryloyloxyethyl phosphorylcholine) (pMPC)-grafted molecular bottlebrush (PCMB) conjugated with a cancer drug, doxorubicin (DOX), via an acid-cleavable hydrazone bond. A pMPC-based linear polymer (LP) conjugated with DOX was also prepared for comparison. We confirmed the lack of structural transition in the PCMB between before and after conjugation with DOX using small-angle light and X-ray scattering techniques, whereas the structure of LP was significantly influenced by DOX conjugation and transformed from a random-coil structure to a large agglomerate via hydrophobic interactions among DOXs. Although PCMB-DOX and LP-DOX showed comparable tissue permeability, pharmacokinetics, and ability to accumulate in tumor tissues, the antitumor efficacy of PCMB-DOX was better than that of LP-DOX. This was presumably due to the formation of LP-DOX agglomerates. The diffusion of cleaved DOX would be restricted in the hydrophobic core of the agglomerate, resulting in the DOX release at the tumor site being compromised. In contrast to LP-DOX, DOX release from PCMB-DOX was not compromised after accumulation in tumor tissues because it did not form such an agglomerate, resulting in the strong antitumor effect. We have demonstrated the potential of MBs as building blocks of drug carriers and believe that these findings can contribute to the design of polymer-based nanomedicines.

Polymer Micelles Composed of Molecular-Bottlebrush-Based Surfactants: Precisely Controlling Aggregation Number Corresponding to Polyhedral Structures.

Over the past few decades, there has been remarkable progress in the construction of self-assemblies in the field of supramolecular chemistry, such as micelles with precisely controlled and refined structures. One promising approach represents the previously proposed concept of Platonic micelles, in which the aggregation number (Nagg ) is discretized in accordance with vertexes of regular polyhedra (i.e., Platonic solids), i.e., 4, 6, 8, 12, and 20 units. Herein, attempt is made to construct Platonic polymer micelles using rigid and persistent architecture of molecular-bottlebrush-based surfactant (MBS). The structure of MBS micelles is carefully elucidated using small-angle X-ray and light scattering and analytical centrifugation measurements. This study shows that the Nagg of MBS micelles is consistent with one of the Platonic numbers when Nagg is intentionally set in the range of 4-20. In addition, some of the MBS micelles demonstrate a discontinuous change in Nagg , when the salt concentration is changed, which is an important factor in controlling micellar Nagg . This is one of the characteristic aggregation behaviors of Platonic micelles in surfactant systems, which strongly indicates the formation of Platonic micelles from macromolecular surfactants.

A ROCK Inhibitor Promotes Graft Survival during Transplantation of iPS-Cell-Derived Retinal Cells.

Currently, retinal pigment epithelium (RPE) transplantation includes sheet and single-cell transplantation, the latter of which includes cell death and may be highly immunogenic, and there are some issues to be improved in single-cell transplantation. Y-27632 is an inhibitor of Rho-associated protein kinase (ROCK), the downstream kinase of Rho. We herein investigated the effect of Y-27632 in vitro on retinal pigment epithelium derived from induced pluripotent stem cells (iPS-RPE cells), and also its effects in vivo on the transplantation of iPS-RPE cell suspensions. As a result, the addition of Y-27632 in vitro showed suppression of apoptosis, promotion of cell adhesion, and higher proliferation and pigmentation of iPS-RPE cells. Y-27632 also increased the viability of the transplant without showing obvious retinal toxicity in human iPS-RPE transplantation into monkey subretinal space in vivo. Therefore, it is possible that ROCK inhibitors can improve the engraftment of iPS-RPE cell suspensions after transplantation.

Characterizing an siRNA-Containing Lipid-Nanoparticle Prepared by a Microfluidic Reactor: Small-Angle X-ray Scattering and Cryotransmission Electron Microscopic Studies.

A new cationic-lipid/siRNA particle that was designed to deliver siRNA was investigated by the combination of small-angle X-ray scattering (SAXS), asymmetric field flow fractionation coupled with multiangle light scattering, and cryotransmission electron microscopy (cryo-TEM). The particle was prepared through two-step mixing using a microfluidic technique. In the first step, siRNA was premixed with a cationic lipid in an EtOH-rich solution. In the second step, the premixed solution was mixed with other lipids, followed by solvent exchange with water. SAXS showed formation of a siRNA/cationic lipid pair in the first step, and this pair consisted of the major part of the core in the final particle. The relationship between the hydrodynamic radius and the radius of gyration indicated the formation of a densely packed core and PEG-rich shell, confirming a well-known core-shell model. SAXS and cryo-TEM showed that the ordering of the core structure enhanced as the siRNA content increased.

Flower Necklaces of Controllable Length Formed From N-(2-Hydroxypropyl) Methacrylamide-Based Amphiphilic Statistical Copolymers.

N-(2-Hydroxypropyl)methacrylamide (HPMA)-based statistical copolymers bearing anticancer drugs have attracted attention for their efficacy in cancer treatments. However, controlling the size and morphology of aggregates of this type of polymer has been challenging and is far from being understood. In this study, small-angle X-ray scattering and asymmetric-flow field-flow fractionation with multiangle light scattering were used to investigate the structure of aggregates formed in aqueous solutions of HPMA-based statistical copolymers of different molecular weights with the model drug pyrene borne in different amounts. The analysis revealed that spherical objects (flower micelles) were formed by the assembly of pyrene moieties in low-molecular-weight copolymers, and the flower micelles connected linearly to form string-of-pearls assemblies (flower necklaces) in high-molecular-weight copolymers. The number of pyrene moieties per polymer chain likely dominates the size and morphology of the copolymer micelles. This study shows how to alter the aggregate structure by changing the molecular weight and composition of copolymers.

Structural Polymorphism of Resorcinarene Assemblies.

In 1997, a study based on X-ray crystallography revealed that resorcinarenes adopt a hexameric capsule-like structure. The function of resorcinarenes has been discussed on the basis of this structure; however, our recent study showed that the hexamer may be only one of resorcinarenes' polymorphic members. Here, we present the solvent dependence of the aggregation number of C-undecylresorcinarene in water-saturated toluene and chloroform using small-angle neutron and X-ray scattering and analytical ultracentrifugation measurements. We found that a new octamer was formed in toluene where the eight resorcinarene units were placed at the vertices of a regular cube; this contrasts to the previous structure in chloroform, namely, a hexamer with the six resorcinarenes located at the vertices of a regular octahedron that has a cavity inside where chloroform molecules are pooled.

Monodisperse Micelles with Aggregation Numbers Related to Platonic Solids.

Recent studies show that calix[4]arene-based micelles are monodisperse with defined Nagg values chosen from 4, 6, 8, 12, 20, and 32. Interestingly, all these numbers coincide with the face numbers of Platonic solids, so they are called "Platonic micelles." As long as a certain geometric condition is fulfilled, any amphiphilic molecule can form a Platonic micelle. The preferred Nagg values are explained in relation to the mathematical Tammes problem, namely, how to obtain the best coverage of a sphere's surface with multiple identical circles. The coverage ratio can be calculated and produces maxima at 4, 6, 12, 20, and 32, coinciding with the observed Nagg values. In this feature article, Platonic micelles as well as their morphological transition by controlling pH, salt, temperature, and electrostatic interactions are summarized.

A Strategy for Personalized Treatment of iPS-Retinal Immune Rejections Assessed in Cynomolgus Monkey Models.

Recently, we successfully transplanted an autograft, or major histocompatibility complex (MHC)-matched allografts, from induced-pluripotent-stem-cell-derived retinal pigment epithelial (iPSC-RPE) cells in patients with age-related macular degeneration. However, there was an issue regarding immune rejection after transplantation. In this study, we established a preoperational in vitro "drug-lymphocytes-grafts immune reaction (Drug-LGIR)" test to determine the medication for immune rejection using host immunocompetent cells (lymphocytes) and transplant cells (target iPSC-RPE cells) together with different medications. The adequacy of the test was assessed by in vivo transplantation in monkey models together with medication based on in vitro data. In the results of Drug-LGIR tests, some drugs exhibited significant suppression of RPE cell-related allogeneic reactions, while other drugs did not, and the efficacy of each drug differed among the recipient monkeys. Based on the results of Drug-LGIR, we applied cyclosporine A or local steroid (triamcinolone) therapy to two monkeys, and successfully suppressed RPE-related immune rejections with RPE grafts, which survived without any signs of rejection under drug administration. We propose that our new preoperational in vitro Drug-LGIR test, which specifies the most efficacious medication for each recipient, is useful for controlling immune attacks with personalized treatment for each patient after retinal transplantation.