Extracellular vesicles containing fullerene derivatives prepared by an exchange reaction for photodynamic therapy.

Extracellular vesicles (EVs) have excellent biocompatibility and long retention times in the circulation and have consequently been expected to be useful as drug-delivery systems. However, their applications have been limited because of the inability to introduce hydrophobic compounds to EVs without the use of harmful organic solvents. Herein, we developed an organic-solvent-free drug-loading technique based on the host exchange reaction. We demonstrated that the exchange reaction enabled quantitative loading of EVs with highly concentrated (0.1 mM) hydrophobic fullerene derivatives. Fullerene derivative-loaded EVs (EVs/C60) could eliminate cancer cell lines more efficiently than fullerene derivative-loaded liposomes (Lip/C60). Moreover, the photodynamic activity of EVs/C60 was fivefold higher than that of the clinically available photosensitizer photofrin. EVs/C60 could efficiently suppress tumor growth in tumor-xenograft model mice.

Extracellular Vesicles Comprising Carborane Prepared by a Host Exchanging Reaction as a Boron Carrier for Boron Neutron Capture Therapy.

With their low immunogenicity and excellent deliverability, extracellular vesicles (EVs) are promising platforms for drug delivery systems. In this study, hydrophobic molecule loading techniques were developed via an exchange reaction based on supramolecular chemistry without using organic solvents that can induce EV disruption and harmful side effects. To demonstrate the availability of an exchanging reaction to prepare drug-loading EVs, hydrophobic boron cluster carborane (CB) was introduced to EVs (CB@EVs), which is expected as a boron agent for boron neutron capture therapy (BNCT). The exchange reaction enabled the encapsulation of CB to EVs without disrupting their structure and forming aggregates. Single-particle analysis revealed that an exchanging reaction can uniformly introduce cargo molecules to EVs, which is advantageous in formulating pharmaceuticals. The performance of CB@EVs as boron agents for BNCT was demonstrated in vitro and in vivo. Compared to L-BPA, a clinically available boron agent, and CB delivered with liposomes, CB@EV systems exhibited the highest BNCT activity in vitro due to their excellent deliverability of cargo molecules via an endocytosis-independent pathway. The system can deeply penetrate 3D cultured spheroids even in the presence of extracellular matrices. The EV-based system could efficiently accumulate in tumor tissues in tumor xenograft model mice with high selectivity, mainly via the enhanced permeation and retention effect, and the deliverability of cargo molecules to tumor tissues in vivo enhanced the therapeutic benefits of BNCT compared to the L-BPA/fructose complex. All of the features of EVs are also advantageous in establishing anticancer agent delivery platforms.

Improving the Photodynamic Activity of Water-Soluble Porphyrin-Polysaccharide Complexes by Folic Acid Modification.

Studies have shown that folate receptors are highly expressed in various cancer cells. Here, we synthesized folic acid-conjugated pullulan (FAPL) as a solubilizing agent to improve the photodynamic activity of porphyrin derivative-polysaccharide complexes. The porphyrin derivative-FAPL complex exhibited long-term stability in an aqueous solution, attributed to the folic acid modification. Furthermore, in vitro and in vivo experiments highlighted the enhanced photodynamic activity of the porphyrin derivative-FAPL complex toward 4T1 breast-cancer cells, compared with the activities of the porphyrin derivative-pullulan complex and Photofrin. This enhanced activity is attributed to the improvement of intracellular uptake by the folate receptor.

Boron agent delivery platforms based on natural products for boron neutron capture therapy.

Boron neutron capture therapy (BNCT) is one of the most promising modalities for cancer treatment due to its minimal invasiveness. Although two types of boron agents are clinically used, several issues persist in their delivery, including poor water solubility, instability in aqueous media, selectivity toward cancer cells, accumulation in cancer cells, retention time in tumor tissue, and efficiency in achieving the boron neutron capture reaction. Addressing these challenges, numerous groups have explored various boron agents to enhance the therapeutic benefits of BNCT. This review summarizes delivery platforms based on natural products for BNCT.

Water Solubilization and Guest Molecule-Induced Fluorescence Changes of Porous Host Molecules via Complexing with Polysaccharide or Polypeptide.

To detect small aromatic molecules in water, we prepared functional host molecules based on water-soluble N,N'-bis(2-aminobenzophenone)-1,4,5,8-naphthalenetetracarboxylic diimide (1) and a solubilizing agent using a high-speed vibration milling apparatus. The fluorescence response of host 1-solubilizing agent complexes before and after extraction of small aromatic guest molecules was large and the fluorescence maxima were dependent on the small aromatic guest molecules.

HER-2-Targeted Boron Neutron Capture Therapy with Carborane-integrated Immunoliposomes Prepared via an Exchanging Reaction.

Boron neutron capture therapy (BNCT) is a promising modality for cancer treatment because of its minimal invasiveness. To maximize the therapeutic benefits of BNCT, the development of efficient platforms for the delivery of boron agents is indispensable. Here, carborane-integrated immunoliposomes were prepared via an exchanging reaction to achieve HER-2-targeted BNCT. The conjugation of an anti-HER-2 antibody to carborane-integrated liposomes successfully endowed these liposomes with targeting properties toward HER-2-overexpressing human ovarian cancer cells (SK-OV3); the resulting BNCT activity toward SK-OV3 cells obtained using the current immunoliposomal system was 14-fold that of the l-BPA/fructose complex, which is a clinically available boron agent. Moreover, the growth of spheroids treated with this system followed by thermal neutron irradiation was significantly suppressed compared with treatment with the l-BPA/fructose complex.

HER-2-targeted boron neutron capture therapy using an antibody-conjugated boron nitride nanotube/ß-1,3-glucan complex.

The development of boron agents with integrated functionality, including biocompatibility, high boron content, and cancer cell targeting, is desired to exploit the therapeutic efficacy of boron neutron capture therapy (BNCT). Here, we report the therapeutic efficacy of BNCT using a HER-2-targeted antibody-conjugated boron nitride nanotube/ß-1,3-glucan complex. The anticancer effect of BNCT using our system was 30-fold that of the clinically available boron agent l-BPA/fructose complex.

Controlled Release of Drug-Encapsulated Protein Films with Various Sodium Dodecyl Sulfate Concentrations.

Protein-based drug carriers are ideal drug-delivery platforms because of their biocompatibility, biodegradability, and low toxicity. Many types and shapes of protein-based platforms, including nanoparticles, hydrogels, films, and minipellets, have been prepared to deliver drug molecules. In this study, protein films containing the desired amounts of doxorubicin (DOX) as cancer drugs were developed using a simple mixing method. The release ratio and rate of DOXs were dependent on the surfactant concentration. The drug release ratio was controlled within the range of 20-90% depending on the amount of the surfactant used. The protein film surface was analyzed using a microscope before and after drug release, and the relationship between the degree of film swelling and the drug release ratio was discussed. Moreover, the effects of cationic surfactants on the protein film were investigated. Non-toxic conditions of the protein films were confirmed in normal cells, while the toxicity of the drug-encapsulated protein film was confirmed in cancer cells. Remarkably, it was observed that the drug-encapsulated protein film could eliminate 10-70% of cancer cells, with the extent of efficacy varying based on the surfactant amount.

Selective Photodynamic Activity of Tetrakis(4-aminophenyl)porphyrins with and without Acetyl Protecting Groups on Cancer and Normal Cells.

Tetrakis(4-aminophenyl)porphyrin (1) and tetrakis(4-acetamidophenyl)porphyrin (2) were dissolved in water with the incorporation of a polysaccharide (λ-carrageenan (CGN)) as a water-solubilizing agent. Although the photodynamic activity of the CGN-2 complex was considerably lower than that of the CGN-1 complex, the selectivity index (SI; IC50 in a normal cell/IC50 in a cancer cell) of the CGN-2 complex was considerably higher than that of the CGN-1 complex. This is because the photodynamic activity of the CGN-2 complex was significantly affected by the intracellular uptakes by the normal and cancer cells. During in vivo experiments, the CGN-2 complex inhibited tumor growth under light irradiation with high blood retention compared with the CGN-1 complex and Photofrin, which exhibited lower blood retention. This study showed that the photodynamic activity and SI are influenced by substituent groups of arene in the meso-positions of porphyrin analogs.

Phospholipid-Coated Boronic Oxide Nanoparticles as Boron Agents for Boron Neutron Capture Therapy.

Minimally invasive boron neutron capture therapy (BNCT) is an elegant approach for cancer treatment. The highly selective and efficient deliverability of boron agents to cancer cells is the key to maximizing the therapeutic benefits of BNCT. In addition, enhancement of the frequencies to achieve boron neutron capture reaction is also significant in improving therapeutic efficacy by providing a highly concentrated boron agent in each boron nanoparticle. As the density of the thermal neutron beam remains low, it is unable to induce high-efficiency cell destruction. Herein, we report phospholipid-coated boronic oxide nanoparticles as agents for BNCT that can provide a highly concentrated boron atom in each nanoparticle. The current system exhibited in vitro BNCT activity seven times higher than that of commercial boron agents. Furthermore, the system could penetrate cancer spheroids deeply, efficiently suppressing thermal neutron irradiation-induced growth.

Carborane bearing pullulan nanogel-boron oxide nanoparticle hybrid for boron neutron capture therapy.

Boron neutron capture therapy shows is a promising approach to cancer therapy, but the delivery of effective boron agents is challenging. To address the requirements for efficient boron delivery, we used a hybrid nanoparticle comprising a carborane = bearing pullulan nanogel and hydrophobized boron oxide nanoparticle (HBNGs) enabling the preparation of highly concentrated boron agents for efficient delivery. The HBNGs showed better anti-cancer effects on Colon26 cells than a clinically boron agent, L-BPA/fructose complex, by enhancing the accumulation and retention amount of the boron agent within cells in vitro. The accumulation of HBNGs in tumors, due to the enhanced permeation and retention effect, enabled the delivery of boron agents with high tumor selectivity, meeting clinical demands. Intravenous injection of boron neutron capture therapy (BNCT) using HBNGs decreased tumor volume without significant body weight loss, and no regrowth of tumor was observed three months after complete regression. The therapeutic efficacy of HBNGs was better than that of L-BPA/fructose complex. BNCT with HBNGs is a promising approach to cancer therapeutics.

Drug-Loaded Biocompatible Chitosan Polymeric Films with Both Stretchability and Controlled Release for Drug Delivery.

Chitosan is a natural polysaccharide with the advantageous qualities of biocompatibility and biodegradability, and it has recently been spotlighted as a soft material for a sustainable society. Advantages such as these are in demand for application in various biomaterials. Although extensive studies have been conducted on the preparation of chitosan films, overcoming the problems of weak mechanical properties remains a significant barrier. In the present study, we developed stretchable doxorubicin-loaded biocompatible chitosan films by adding acetic acid in controlled concentrations. The stretchable properties of doxorubicin-loaded chitosan film at various concentrations of acetic acid were measured. Elongation to the point of breakage reached 27% with a high concentration of acetic acid, which could be described as high stretchability. The release ratio of doxorubicin from chitosan film reached 70% with a high acetic acid concentration. The cytotoxicity of doxorubicin-loaded chitosan films was measured, and cancer spheroids had completely collapsed after 7 days. According to the results of skin permeability testing, use of the doxorubicin-loaded chitosan film is a plausible choice for a drug sealant.

Development of a Water-Dispersible Supramolecular Complex of Polyphenol with Polypeptides for Attenuation of the Allergic Response using a Mechanochemical Strategy.

The prevalence of allergic disorders has increased worldwide in recent decades. Polyphenols, including resveratrol and curcumin, are posited to have potential as therapeutic agents for allergy; however, their use has been limited by poor water solubility. Accordingly, a highly concentrated, water dispersible, supramolecular complexes of polyphenols with polypeptides (poly-L-lysine, poly-γ-glutamic acid) and gelatin using high-speed vibration milling are developed. The complex exhibits resistance to photobleaching and thermal radiation. Treatment of a rat basophilic leukemia cell line (RBL-2H3) with polypeptide complexes containing resveratrol is suppressed allergic responses in vitro. Moreover, aerosolized administration of polypeptide complexes demonstrates excellent bioavailability and inhibition of immediate hypersensitivity reactions in ear tissue in vivo. Furthermore, the method avoids the use of organic solvent and therefore reduces undesirable biological responses.

Formulation of water-dispersible hydrophobic compound nanocomplexes with polypeptides via a supramolecular approach using a high-speed vibration milling technique.

Polypeptides were used to solubilize functional hydrophobic molecules via a high-speed vibrational milling method. Poly-l-lysine and poly-γ-glutamic acid, which are polypeptides, were able to prepare more highly concentrated water-dispersible complexes of hydrophobic compounds, including fullerenes, organic dyes, and porphyrin derivatives, than conventional water solubilizers, such as cyclodextrins and pullulan. In addition, the polypeptide systems endowed the complexes with long-term stability and resistance against thermal stress, which is advantageous for industrial applications. Furthermore, complexes of polypeptides and porphyrin derivatives showed a photodynamic activity against cancer cells, and the current system improved the dispersibility and storability of guest molecules without compromising their functionality.

Cyclodextrin-induced release of drug-entrapping liposomes associated with the solation of liposome gels.

In this work, we demonstrate that liposome gels in which liposomes are connected by polyethylene glycol terminated by cholesterol groups at both ends can store hydrophilic and hydrophobic drugs in the gel interiors, inner aqueous phases, and lipid membranes. The addition of cyclodextrins (CDxs) as extrinsic stimuli led to the release of drug-entrapping liposomes due to the interactions between CDxs and cholesteryl groups and/or the alkyl chains of lipids. The addition of aqueous solutions of ß-CDx, dimethyl-ß-CDx, trimethyl-ß-CDx, and γ-CDx (final concentration: 7.5 mM) induced the solation of liposome gels and the release of liposomes accompanying the solation. Furthermore, the addition of ß-CDx led to the partial release of hydrophilic drugs encapsulated in the liposomes, although the drug release was scarcely observed in other CDxs. In particular, the addition of trimethyl-ß-CDx, which has low cytotoxicity, accelerated solation, and cationic liposomes released from the gels were effectively taken up by murine colon cancer (Colon26) cells. Thus, we propose that liposomes released from liposome gels can function as drug carriers.

Theranostic Agent Combining Fullerene Nanocrystals and Gold Nanoparticles for Photoacoustic Imaging and Photothermal Therapy.

Developing photoactivatable theranostic platforms with integrated functionalities of biocompatibility, targeting, imaging contrast, and therapy is a promising approach for cancer diagnosis and therapy. Here, we report a theranostic agent based on a hybrid nanoparticle comprising fullerene nanocrystals and gold nanoparticles (FGNPs) for photoacoustic imaging and photothermal therapy. Compared to gold nanoparticles and fullerene crystals, FGNPs exhibited stronger photoacoustic signals and photothermal heating characteristics by irradiating light with an optimal wavelength. Our studies demonstrated that FGNPs could kill cancer cells due to their photothermal heating characteristics in vitro. Moreover, FGNPs that are accumulated in tumor tissue via the enhanced permeation and retention effect can visualize tumor tissue due to their photoacoustic signal in tumor xenograft model mice. The theranostic agent with FGNPs shows promise for cancer therapy.

Simple Formation of Cancer Drug-Containing Self-Assembled Hydrogels with Temperature and pH-Responsive Release.

The purpose of a drug delivery system is to efficiently deliver drugs to a desired target, while simultaneously reducing the side effects caused by these drugs and maximizing their efficacy. However, in the manufacture of a drug delivery system, it is difficult to control the amount of drug encapsulation. In this study, we developed a simple formation process of self-assembled hydrogels that made it easier to package the desired amount of anticancer drugs. A self-assembled hydrogel was prepared by simply mixing transferrin, dithiothreitol, and an anticancer drug in a salt solvent. The structural conditions of the hydrogel were determined in order to control the concentration of the transferrin protein, dithiothreitol, and salt in the solvent. The self-assembled hydrogels contained the desired amount of anticancer drugs. With this system, changes in pH and temperature control the release rate and the release ratio of anticancer drugs. The cytotoxicity of the drug-loaded hydrogel was evaluated, which showed that 80% of the treated cells had been killed following 48 h of incubation.

Photodynamic therapy using self-assembled nanogels comprising chlorin e6-bearing pullulan.

With minimal invasiveness and spatiotemporal therapeutic effects, photodynamic therapy is one of the most promising candidates for cancer treatment. Here, we developed a facile self-assembled nanogel using photosensitizer-grafted polysaccharides called chlorin e6-bearing pullulan. Chlorin e6 is used as a photosensitizer in cancer therapy. The anti-cancer effect of photodynamic therapy with our nanogel system was 780 times higher than that of the commercially available photosensitizer Photofrin. Finally, we demonstrated that actively growing cancer cell spheroids can be completely suppressed after treatment. Our system could efficiently induce tumor regression in tumor xenograft mice.

Tumor-targeting hyaluronic acid/fluorescent carborane complex for boron neutron capture therapy.

In cancer therapeutics, boron neutron capture therapy (BNCT) requires a platform for selective and efficient 10B delivery into tumor tissues for a successful treatment. However, the use of carborane, a promising candidate with high boron content and biostability, has significant limitations in the biomedical field due to its poor water-solubility and tumor-selectivity. To overcome these hurdles, we present in this study a fluorescent nano complex, combining fluorescent carborane and sodium hyaluronate for high boron concentration and tumor-selectivity. Tumor cells actively internalized the complex through binding hyaluronan to CD44, overexpressed on the tumor cell surface. Furthermore, the subcellular distribution of this complex could also be detected due to its fluorescent properties. Moreover, after thermal neutron irradiations, the complex produced excellent cytotoxicity, equal to or greater than that of the clinically-used BPA-fructose. Therefore, this novel complex could be potentially more suitable for BNCT than the boron agent.

Magnetically Navigated Protein Transduction In Vivo using Iron Oxide-Nanogel Chaperone Hybrid.

Systems for "protein transduction," intracellular delivery of functional proteins, are needed to address deliverability challenges of protein therapeutics. However, in vivo protein transduction remains challenging because of instability in serum, extracellular protease digestion and rapid excretion from the bloodstream. Here, a magnetically guided in vivo protein transduction using magnetic nanogel chaperone (MC) composed of iron oxide nanoparticles and a polysaccharide nanogel, a protein carrier inspired by "catch and release" mechanisms of molecular chaperones is demonstrated. The MC system enables efficient delivery of anti-cancer proteins, saporin and RNaseA, into cultured tumor lines and inhibits cell proliferation, mainly via apoptosis. Magnetic in vivo protein transduction via intravenous whole body administration is demonstrated in a fibrosarcoma model. By in vivo optical imaging, MC accumulated in tumor tissues under magnetic field three times more than without irradiation. With subcutaneous injection, saporin is delivered by MC to the cytoplasm in magnetically targeted tissues. In an oral cancer model, MC-delivered magnetically targeted saporin decreased tumor volume without significant body weight changes and no regrowth of tumor at 3 months after complete regression. Protein transduction with MC shows promise for cancer therapeutics and, potentially, for regenerative medicine and other biomedical applications.