Attenuated polyethylene glycol immunogenicity and overcoming accelerated blood clearance of a fully PEGylated dendrimer.

Polyethylene glycol (PEG) is a popular biocompatible polymer and PEGylated nanoparticles passively accumulate in tumor tissues because of their enhanced permeability and retention effects. Recently, the anti-PEG immunity of PEGylated nanoparticles has become an issue that needs to be solved for their clinical applications. Dendrimers are highly branched and well-defined polymers with many terminal groups, which act as potent drug carriers. In this study, we examined the pharmacokinetics, biodistribution, anti-PEG immunity, and tumor accumulation of a fully PEGylated polyamidoamine (PAMAM) dendrimer after the first and second injections and compared them to those of a PEGylated liposome with the same lipid component as Doxil®. The PEGylated dendrimer showed greater blood circulation than that of the PEGylated liposome after the first and second injections in rats. In mice injected with the PEGylated dendrimer, much less anti-PEG immunoglobulin M (IgM) was generated than that in mice injected with the PEGylated liposome. The PEGylated dendrimer accumulated in the tumor after both the first and second injections. Our results indicated that the PEGylated dendrimer with a small size and high PEG density showed attenuated anti-PEG immunity and overcame the accelerated blood clearance phenomenon, which is useful for drug delivery systems for cancer treatment.

Preparation and Characterization of Acrylic and Methacrylic Phospholipid-Mimetic Polymer Hydrogels and Their Applications in Optical Tissue Clearing.

The 2-methacryloyloxyethyl phosphorylcholine (MPC) polymers are mimetic to phospholipids, being widely used as biocompatible polymers. In our previous study, MPC polymer hydrogels proved more effective for optical tissue clearing compared to acrylamide (AAm) polymer hydrogels. In the present study, 2-acryloyloxyethyl phosphorylcholine (APC) was synthesized and employed to create hydrogels for a comparative analysis with methacrylic MPC-based hydrogels. APC, an acrylic monomer, was copolymerized with AAm in a similar reactivity. In contrast, MPC, as a methacrylic monomer, demonstrated higher copolymerization reactivity than AAm, leading to a spontaneously delayed two-step polymerization behavior. This suggests that the polymer sequences and network structures became heterogeneous when both methacrylic and acrylic monomers, as well as crosslinkers, were present in the copolymerization system. The molecular weight of the APC polymers was considerably smaller than that of the MPC polymers due to the formation of mid-chain radicals and subsequent ß-scission during polymerization. The swelling ratios in water and strain sweep profiles of hydrogels prepared using acrylic and methacrylic compounds differed from those of hydrogels prepared using only acrylic compounds. This implies that copolymerization reactivity influences the polymer network structures and crosslinking density in addition to the copolymer composition. APC-based hydrogels are effective for the optical clearing of tumor tissues and are applicable to both passive and electrophoretic methods.

In vivo transfection of cytokine genes into tumor cells using a synthetic vehicle promotes antitumor immune responses in a visceral tumor model.

The tumor microenvironment (TME) strongly affects the clinical outcomes of immunotherapy. This study aimed to activate the antitumor immune response by manipulating the TME by transfecting genes encoding relevant cytokines into tumor cells using a synthetic vehicle, which is designed to target tumor cells and promote the expression of transfected genes. Lung tumors were formed by injecting CT26.WT intravenously into BALB/c mice. Upon intravenous injection of the green fluorescent protein-coding plasmid encapsulated in the vehicle, 14.2% tumor-specific expression was observed. Transfection of the granulocyte-macrophage colony-stimulating factor (GM-CSF) and CD40 ligand (L)-plasmid combination and interferon gamma (IFNγ) and CD40L-plasmid combination showed 45.5% and 54.5% complete remission (CR), respectively, on day 60; alternate treatments with both the plasmid combinations elicited 66.7% CR, while the control animals died within 48 days. Immune status analysis revealed that the density of dendritic cells significantly increased in tumors, particularly after GM-CSF- and CD40L-gene transfection, while that of regulatory T cells significantly decreased. The proportion of activated killer cells and antitumoral macrophages significantly increased, specifically after IFNγ and CD40L transfection. Furthermore, the level of the immune escape molecule programmed death ligand-1 decreased in tumors after transfecting these cytokine genes. As a result, tumor cell-specific transfection of these cytokine genes by the synthetic vehicle significantly promotes antitumor immune responses in the TME, a key aim for visceral tumor therapy.

Gene Delivery into T-Cells Using Ternary Complexes of DNA, Lipofectamine, and Carboxy-Terminal Phenylalanine-Modified Dendrimers.

T-cells play critical roles in various immune reactions, and genetically engineered T-cells have attracted attention for the treatment of cancer and autoimmune diseases. Previously, it is shown that a polyamidoamine dendrimer of generation 4 (G4), modified with 1,2-cyclohexanedicarboxylic anhydride (CHex) and phenylalanine (Phe) (G4-CHex-Phe), is useful for delivery into T-cells and their subsets. In this study, an efficient non-viral gene delivery system is constructed using this dendrimer. Ternary complexes are prepared using different ratios of plasmid DNA, Lipofectamine, and G4-CHex-Phe. A carboxy-terminal dendrimer lacking Phe (G3.5) is used for comparison. These complexes are characterized using agarose gel electrophoresis, dynamic light scattering, and ζpotential measurements. In Jurkat cells, the ternary complex with G4-CHex-Phe at a P/COOH ratio of 1/5 shows higher transfection activity than other complexes, such as binary and ternary complexes with G3.5, without any significant cytotoxicity. The transfection efficiency of the G4-CHex-Phe ternary complexes decreases considerably in the presence of free G4-CHex-Phe and upon altering the complex preparation method. These results suggest that G4-CHex-Phe promotes the cellular internalization of the complexes, which is useful for gene delivery into T-cells.

T Cell-Association of Carboxy-Terminal Dendrimers with Different Bound Numbers of Phenylalanine and Their Application to Drug Delivery.

T cells play important roles in various immune reactions, and their activation is necessary for cancer immunotherapy. Previously, we showed that polyamidoamine (PAMAM) dendrimers modified with 1,2-cyclohexanedicarboxylic acid (CHex) and phenylalanine (Phe) underwent effective uptake by various immune cells, including T cells and their subsets. In this study, we synthesized various carboxy-terminal dendrimers modified with different bound numbers of Phe and investigated the association of these dendrimers with T cells to evaluate the influence of terminal Phe density. Carboxy-terminal dendrimers conjugating Phe at more than half of the termini exhibited a higher association with T cells and other immune cells. The carboxy-terminal Phe-modified dendrimers at 75% Phe density tended to exhibit the highest association with T cells and other immune cells, which was related to their association with liposomes. A model drug, protoporphyrin IX (PpIX), was encapsulated into carboxy-terminal Phe-modified dendrimers, which were then used for drug delivery into T cells. Our results suggest the carboxy-terminal Phe-modified dendrimers are useful for delivery into T cells.

Microporous Structure Formation of Poly(methyl methacrylate) via Polymerization-Induced Phase Separation in the Presence of Poly(ethylene glycol).

It has been demonstrated that nano- or micro-structured polymeric materials have huge potential as advanced materials. However, most of the current fabricating methods have limitations either in cost or in size. Here, we investigate the bulk polymerization of methyl methacrylate in the presence of poly(ethylene glycol) (PEG). We found that phase separation occurs during bulk polymerization. After removal of PEG via sonication, microscopic structures of poly(methyl methacrylate), including porous structures, co-continuous monolith structures, or particle aggregation structures, are formed. These structures can be controlled by the amount of PEG added and the reaction temperature. The results are summarized in phase diagrams. The addition of PEG significantly affects the reaction kinetics. Phase separation is coupled with the reaction acceleration known as the Trommsdorff effect. As a result, the reaction completes in a shorter time when the PEG amount is higher. We demonstrate surface coating to fabricate an amphiphobic surface, repelling both water and oil. The methods presented here have the potential to fabricate microscopic structures in large areas cost-effectively.

Carboxy-terminal dendrimers with phenylalanine for a pH-sensitive delivery system into immune cells including T cells.

Although T cells play important roles in various immune reactions, there are only a few reports on delivery systems into T cells. Our previous study showed that carboxy-terminal phenylalanine (Phe)-modified polyamidoamine (PAMAM) dendrimers have both temperature- and pH-sensitive properties, which are affected by the chemical structure. The self-assembled structures of Phe, observed in phenylketonuria, enhance the protein aggregation, the association with the cell membrane and the membrane permeability. In this study, we applied the Phe-modified dendrimers to a pH-sensitive drug delivery system into T cells. Dendrimers with different amino acids and acid anhydrides were synthesized, and their pH-responsive association with T cells and their subsets was investigated. The dendrimers modified with Phe and cyclohexanedicarboxylic acid (CHex) showed higher uptake into various cells, including Jurkat cells, CD3+ T cells, CD3 + CD4+ helper T cells and CD3 + CD8+ killer T cells. These dendrimers were internalized into T cells via endocytosis, and their cellular uptake was enhanced under weak acidic conditions (pH 6.5). Our results showed that Phe- and CHex-modified dendrimers have a delivery potential to T cells and their subsets, which may be useful for cancer immunotherapy.

Solubilization of Paclitaxel by Self-Assembled Amphiphilic Phospholipid-Mimetic Polymers with Varied Hydrophobicity.

2-Methacryloyloxyethyl phosphorylcholine (MPC) polymers have been used as a coating agent on medical devices and as a carrier in drug delivery systems (DDSs). Paclitaxel (PTX) is a water-insoluble anticancer drug whose solubilizer is necessary for administration. Block and random copolymers composed of hydrophilic MPC and butyl methacrylate, named PMB, show different properties, depending on the polymer sequence and MPC content. In the present study, we used amphiphilic MPC polymers comprising hydrophobic dodecyl methacrylate (DMA). The self-assembling properties and PTX solubilization of random and block poly(MPC-co-DMA)s (rPMDs and bPMDs) with different compositions were examined and compared. rPMDs with high DMA content formed large and relatively loose self-assembled structures, which solubilized PTX. However, bPMDs formed small and compact self-assembled structures with poor PTX solubilization. PTX solubilized by PMB with small and loose self-assembled structures showed efficient drug action, similar to free PTX; however, rPMDs fell short of demonstrating PTX efficiency. Our results suggest that the self-assembling properties and the hydrophobicity of amphiphilic MPC polymers largely affect PTX solubilization as well as drug action, which is required to be controlled by the polymer sequence, as well as the structure and composition of the hydrophobic monomer for efficient DDS.

Application of Zwitterionic Polymer Hydrogels to Optical Tissue Clearing for 3D Fluorescence Imaging.

Zwitterionic polymers have both anion and cation groups in the side chain and have been used in various biomedical applications because of the unique properties. In this study, zwitterionic polymer hydrogels are applied to optical tissue clearing for 3D fluorescence imaging. Polyacrylamide hydrogels have been employed in Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/Immunostaining/In situ-hybridization-compatible Tissue-hYdrogel method. Zwitterionic polymer hydrogels are produced using zwitterionic monomers, such as 3-[(3-acrylamidopropyl)dimethylammonio]propane-1-sulfonate (DAPS) and 2-methacryloyloxyethyl phosphorylcholine (MPC), and crosslinkers. The hydrogels made from poly(DAPS-co-acrylamide) and MPC homopolymers afford the most transparent tumor tissues. However, the tissues cleared using DAPS copolymers-containing hydrogels became turbid in a refractive index-matching solution, which are unable to obtain clear 3D fluorescence images. In contrast, the 3D fluorescence imaging is achieved in the MPC polymer-treated 2-mm-thick brain slices after immunostaining. The 3D fluorescence imaging of lung metastasis that is cleared by the MPC hydrogel to demonstrate the possible application to cancer diagnosis is performed. The results indicate the increased potentials of zwitterionic polymer hydrogels, especially MPC polymer hydrogels, in biomedical applications.

Different hydration states and passive tumor targeting ability of polyethylene glycol-modified dendrimers with high and low PEG density.

It has been reported that the amount of intermediate water, defined as water molecules loosely bound to a material, is a useful index of the material's bio-inert properties. Polyethylene glycol (PEG) is a well-known biocompatible polymer with a large amount of intermediate water. Many researchers have showed that PEGylated nanoparticles are passively accumulated in tumor tissues owing to their enhanced permeability and retention (EPR) effects. Dendrimers are regularly branched polymers with highly controllable size and structure, which can be exploited as potent drug carriers. In this study, we investigated the tripartite relationship among the PEG density, the hydration state, and the passive tumor targeting property, using PEGylated dendrimers. The fully PEGylated dendrimer, PEG64-den, showed similar hydration behavior to PEG and a passive tumor targeting property. In contrast, the hydration state of the partly PEGylated dendrimer, PEG5-den, was different from that of PEG64-den, and the passive tumor targeting property was not observed. This is the first report to show the hydration state of a drug carrier as well as discuss a relationship between the hydration state and biodistribution.

Association of Hydrophobic Carboxyl-Terminal Dendrimers with Lymph Node-Resident Lymphocytes.

Delivery systems to lymph node-resident T cells around tumor tissues are essential for cancer immunotherapy, in order to boost the immune responses. We previously reported that anionic dendrimers, such as carboxyl-, sulfonyl-, and phosphate-terminal dendrimers, were efficiently accumulated in lymph nodes via the intradermal injection. Depending on the terminal structure, their cell association properties were different, and the carboxyl-terminal dendrimers did not associate with any immune cells majorly. In this study, we investigated the delivery of carboxyl-terminal dendrimers with different hydrophobicity to lymph node-resident lymphocytes. Four types of carboxyl-terminal dendrimers-succinylated (C) and 2-carboxy-cyclohexanoylated (Chex) dendrimers with and without phenylalanine (Phe)-were synthesized and named C-den, C-Phe-den, Chex-den, and Chex-Phe-den, respectively. Chex-Phe-den was well associated with lymphocytes, but others were not. Chex-Phe-den, intradermally injected at the footpads of mice, was accumulated in the lymph node, and was highly associated with the lymphocytes, including T cells. Our results suggest that Chex-Phe-den has the potential for delivery to the lymph node-resident T cells, without any specific T cell-targeted ligands.

Formation of Hydrophobic Domains on the poly(MPC-co-Dodecyl Methacrylate)-Coated Surface Recognized by Macrophage-like Cells.

Copolymers comprising 2-methacryloyloxyethyl phosphorylcholine (MPC) and hydrophobic methacrylic esters were used as biomembrane-mimetic polymers to provide blood compatibility. In the present study, we compared the surfaces coated with two MPC polymers with different alkyl groups, namely, poly(MPC-co-butyl methacrylate) (PMB) and poly(MPC-co-dodecyl methacrylate) (PMD), to clarify the effect of their hydrophobic units. Various substrates, such as poly(ethylene terephthalate), polycarbonate, polypropylene, acrylonitrile-butadiene-styrene copolymer, and stainless steel, were coated with ethanol solutions containing various concentrations of PMD or PMB. The solubility of PMD in ethanol changed depending on the water content. Scanning probe microscopy and rhodamine 6G staining revealed heterogeneous microstructures on the PMD-coated surface but not on the PMB-coated surface. Adhesion of various cells was efficiently suppressed by the PMD coating at lower concentration than the PMB coating, except regarding the adhesion of macrophage-like RAW264.7 cells. Our results suggest that the dodecyl groups in PMD increased its affinity for the substrates and simultaneously induced the formation of hydrophobic domains recognized by RAW264.7 cells.

Dissimilar Materials Bonding Using Epoxy Monolith.

The epoxy monolith with a highly porous structure is fabricated by the thermal curing of 2,2-bis(4-glycidyloxyphenyl)propane and 4,4'-methylenebis(cyclohexylamine) in the presence of poly(ethylene glycol) as the porogen via polymerization-induced phase separation. In this study, we demonstrated a new type of dissimilar material bonding method for various polymers and metals coated with the epoxy monolith. On the basis of scanning electron microscopy (SEM) observations, the pore size and number of epoxy monoliths were evaluated to be 1.1-114 µm and 8.7-48 200 mm-2, respectively, depending on the ratio of the epoxy resin and cross-linking agent used for the monolith fabrication. Various kinds of thermoplastics, such as polyethylene, polypropylene, polyoxymethylene, acrylonitrile-butadiene-styrene copolymer, polycarbonate bisphenol-A, and poly(ethylene terephthalate), were bonded to the monolith-modified metal plates by thermal welding. The bond strength for the single lap-shear tensile test of stainless steel and copper plates with the thermoplastics was in the range of 1.2-7.5 MPa, which was greater than the bond strength value for each bonding system without monolith modification. The SEM observation of fractured test pieces directly confirmed an anchor effect on this bonding system. The elongated deformation of the plastics that filled in the pores of the epoxy monolith, was observed. It was concluded that the bond strength significantly depended on the intrinsic strength of the used thermoplastics. The epoxy monolith bonding of hard plastics, such as polystyrene and poly(methyl methacrylate), was performed by the additional use of adhesives, solvents, and a reactive monomer. The epoxy monolith sheets were also successfully fabricated and applied to dissimilar material bonding.

Facile synthesis of main-chain degradable block copolymers for performance enhanced dismantlable adhesion.

Block copolymers consisting of readily degradable polyperoxides and non-degradable vinyl polymers as the block segments were successfully synthesized by reversible chain transfer catalyzed polymerization, which is one of living radical polymerization techniques. The block copolymers showed characteristic morphology and wettability being different from the polymer blends. When block copolymers containing polyperoxide and polymethacrylate blocks were heated below 150 °C, the polyperoxide blocks were completely degraded and the polymethacrylate blocks were recovered without degradation. Block copolymers containing a poly(2-ethylhexyl methacrylate) block were then investigated as a dismantlable adhesion material, which requires adequate bonding strength during use and easy debonding on demand. Among the several block copolymers, the one consisting of poly(2-ethylhexyl methacrylate) and polyperoxide from methyl sorbate (PPMS) (M(n) = 4900) exhibited good performance as a pressure-sensitive adhesive (PSA). After heating the test specimens in a temperature range from 60 to 100 °C, PSA performance, which was evaluated by 180° peel strength and shear holding power measurements, was significantly diminished. Especially, after heating at 100 °C for 1 h, spontaneous debonding of some test specimens was observed because of the evolution of volatile acetaldehyde from PPMS.

Cohesive force change induced by polyperoxide degradation for application to dismantlable adhesion.

Polyperoxides containing peroxy bonds as the main-chain repeating units are a new class of degradable polymers because of significant changes in their molecular weight and physical properties during a degradation process. In this study, the application of linear and network polyperoxides to dismantlable adhesion was investigated. When the linear polyperoxide obtained from methyl sorbate and oxygen (PP-MS) was used as a pressure-sensitive adhesive (PSA), its shear holding power and 180° peel strength immediately decreased upon heating at 70 °C or under UV irradiation. Low-molecular-weight products, which were generated by the degradation of PP-MS, behaved as a plasticizer to effectively reduce the cohesive force. The adhesive properties of two types of polyperoxides-based network polymers, the cross-linking point and main-chain degradable network polymers, were evaluated. A cross-linking point degradable network polymer was produced by the oxygen cross-linking of dienyl-functionalized poly(ethylene glycol). A main-chain degradable network polymer was formed by the diisocyanate cross-linking of a hydroxy-functionalized polyperoxide. Both network polymers showed a higher adhesive strength than PP-MS due to their three-dimensional network structure. Noteworthy, the adhesive strength of the main-chain degradable network polymer was varied from the level of PSA to structural adhesives by increasing the added amount of the diisocyanate cross-linker. After heating at 110 °C, the cohesive and adhesive strengths significantly decreased. The linear and network polyperoxides are shown to be promising materials for dismantlable adhesion.

Facile synthesis of functional polyperoxides by radical alternating copolymerization of 1,3-dienes with oxygen.

We have developed a facile synthesis of degradable polyperoxides by the radical alternating copolymerization of 1,3-diene monomers with molecular oxygen at an atmospheric pressure. In this review, the synthesis, the degradation behavior, and the applications of functional polyperoxides are summarized. The alkyl sorbates as the conjugated 1,3-dienes gave a regiospecific alternating copolymer by exclusive 5,4-addition during polymerization and the resulting polyperoxides decomposed by the homolysis of a peroxy linkage followed by successive beta-scissions. The preference of 5,4-addition was well rationalized by theoretical calculations. The degradation of the polyperoxides occurred with various stimuli, such as heating, UV irradiation, a redox reaction with amines, and an enzyme reaction. The various functional polyperoxides were synthesized by following two methods, one is the direct copolymerization of functional 1,3-dienes, and the other is the functionalization of the precursor polyperoxides. Water soluble polyperoxides were also prepared, and the LCST behavior and the application to a drug carrier in the drug delivery system were investigated. In order to design various types of degradable polymers and gels we developed a method for the introduction of dienyl groups into the precursor polymers. The resulting dienyl-functionalized polymers were used for the degradable gels. The degradable branched copolymers showed a microphase-separated structure, which changed owing to the degradation of the polyperoxide segments.

Self-assembly and cellular uptake of degradable and water-soluble polyperoxides.

Water-soluble polyperoxides (PPs) as a new type of degradable and polymeric material were synthesized by the radical alternating copolymerization of sorbic derivatives containing a tetra(ethylene oxide) unit in the ester group using molecular oxygen. The obtained PPs showed a lower critical solution temperature (LCST)-type phase separation, and the transition temperature decreased according to the content of the hydrophobic ester group in the PPs. The PPs formed nanoaggregates with a diameter of 250-370 nm in water under the LCST. These PP aggregates were revealed to include 1-anilinonaphthalene-8-sulfonic acid as the fluorescence probe and epirubicin as the anticancer drug in their hydrophobic compartment. We evaluated the cytotoxicity and cellular uptake of the PPs in order to test their ability as a carrier used for the delivery of anticancer drugs. The cell viability in the presence of the PPs was comparable to those for the other biodegradable polymers, and epirubicin was taken up into the A549 efficiently with the PPs via an endocytosis mechanism.

Regiospecific radical polymerization of a tetrasubstituted ethylene monomer with molecular oxygen for the synthesis of a new degradable polymer.

A new class of degradable polymers is obtained from a diene monomer and molecular oxygen as the starting materials via a highly controlled radical copolymerization process. We now report the regiospecific copolymerization of a tetrasubstituted ethylene monomer with oxygen. Theoretical calculations support the highly selective propagations observed during the polymerization. The key steps are the regiospecific reactions of a peroxy radical to diene monomers and an allyl radical to molecular oxygen. The well-controlled molecular structure of the resulting polymer leads to the aldehyde-free degradation products during degradation by various stimuli, such as heating.

First disyndiotactic polymer from a 1,4-disubstituted butadiene by alternate molecular stacking in the crystalline state.

We report the first synthesis of a disyndiotactic polymer through topochemical polymerization using di(4-methoxybenzyl)muconate as the 1,3-diene dicarboxylate monomer. It provides a tritactic polymer under photoirradiation in the crystalline state, as a result of the alternate molecular stacking in a column formed in the crystals with aid of weak hydrogen bonds such as CH/pi and CH...O intermolecular interactions.