Akihiro Kishimura

Spontaneous formation of nanosized unilamellar polyion complex vesicles with tunable size and properties.

Fabrication of monodispersed, submicrometer-sized vesicles (nanosomes) that form through self-assembly possessing a thin and permeable membrane remains a significant challenge. Conventional fabrication of nanosomes through self-assembly of amphiphilic molecules often requires cumbersome processes using organic solvents combined with physical procedures (e.g., sonication, thermal treatment, and membrane filtration) to obtain unilamellar structures with a controlled size distribution. Herein, we report the first example of spontaneously formed submicrometer-sized unilamellar polyion complex vesicles (Nano-PICsomes) via self-assembly of a pair of oppositely charged PEG block aniomer and homocatiomer in an aqueous medium. Detailed dynamic light scattering and transmission electron microscopic analysis revealed that vesicle sizes can be controlled in the range of 100-400 nm with a narrow size distribution, simply by changing the total polymer concentration. Also, each Nano-PICsome was composed of a uniform single PIC membrane, the thickness of which is around 10-15 nm, regardless of its size. Fluorescence correlation spectroscopy measurement verified that Nano-PICsomes were able to encapsulate water-soluble fluorescent macromolecules in the inner water phase and release them slowly into the exterior. Moreover, cross-linking of the vesicle membrane allows tuning of permeability, enhancement in stability under physiological conditions, and preservation of size and structure even after freeze-drying and centrifugation treatment. Finally, Nano-PICsomes showed a long circulation time in the bloodstream of mice. Precise control of the particle size and structure of hollow capsules through simple aqueous self-assembly and easy modification of their properties by cross-linking is quite novel and fascinating in terms of ecological, low-cost, and low-energy fabrication processes as well as the potential utility in the biomedical arena.

Visible Drug Delivery by Supramolecular Nanocarriers Directing to Single-Platformed Diagnosis and Therapy of Pancreatic Tumor Model

Nanoparticle therapeutics are promising platforms for cancer therapy. However, it remains a formidable challenge to assess their distribution and clinical efficacy for therapeutic applications. Here, by using multifunctional polymeric micellar nanocarriers incorporating clinically approved gadolinium (Gd)-based magnetic resonance imaging contrast agents and platinum (Pt) anticancer drugs through reversible metal chelation of Pt, simultaneous imaging and therapy of an orthotopic animal model of intractable human pancreatic tumor was successfully performed without any serious toxicity. The strong tumor contrast enhancement achieved by the micelles correlated with the 24 times increase of r(1) of the Gd chelates, the highest for the formulations using clinically approved Gd chelates reported to date. From the micro-synchrotron radiation X-ray fluorescence spectrometry scanning of the lesions, we confirmed that both the Gd chelates and Pt drugs delivered by the micelles selectively colocalized in the tumor interior. Our study provides new insights for the design of theranostic micelles with high contrast enhancement and site-specific clinical potential.

Size-controlled long-circulating PICsome as a ruler to measure critical cut-off disposition size into normal and tumor tissues

Selective disposition of nanocarriers into target tissue is an essential issue in drug delivery. Critical size of nanocarriers (∼150 nm) discriminating the permeability into normal and tumor tissues was determined by the use of size-tunable, polyion complex hollow vesicles (PICsome) as a ruler.

Hydrothermally synthesized PEGylated calcium phosphate nanoparticles incorporating Gd-DTPA for contrast enhanced MRI diagnosis of solid tumors.

Organic-inorganic hybrid nanoparticles with calcium phosphate (CaP) core and PEGylated shell were developed to incorporate magnetic resonance imaging (MRI) contrast agent diethylenetriaminepentaacetic acid gadolinium (III) (Gd-DTPA) for noninvasive diagnosis of solid tumors. A two-step preparation method was applied to elaborate hybrid nanoparticles with a z-average hydrodynamic diameter about 80nm, neutral surface ξ-potential and high colloidal stability in physiological environments by self-assembly of poly(ethylene glycol)-b-poly(aspartic acid) block copolymer, Gd-DTPA, and CaP in aqueous solution, followed with hydrothermal treatment. Incorporation into the hybrid nanoparticles allowed Gd-DTPA to show significant enhanced retention ratio in blood circulation, leading to high accumulation in tumor positions due to enhanced permeability and retention (EPR) effect. Moreover, Gd-DTPA revealed above 6 times increase of relaxivity in the nanoparticle system compared to free form, and eventually, selective and elevated contrast enhancements in the tumor positions were observed. These results indicate the high potential of Gd-DTPA-loaded PEGylated CaP nanoparticles as a novel contrast agent for noninvasive cancer diagnosis.

Bioactive polymeric metallosomes self-assembled through block copolymer-metal complexation

Spontaneous formation of polymeric metallosomes with uniform size (~100 nm) was found to occur in aqueous medium through the reaction of an anticancer agent, (1,2-diaminocyclohexane)platinum(II) (DACHPt), with a Y-shaped block copolymer of ω-cholesteroyl-poly(L-glutamic acid) and two-armed poly(ethylene glycol) (PEGasus-PLGA-Chole). Circular dichroism spectrum measurements revealed that the PLGA segment forms an α-helix structure within the metallosomes, suggesting that secondary-structure formation of metallocomplexed PLGA segment may drive the self-assembly of the system into vesicular structure. These metallosomes can encapsulate water-soluble fluorescent macromolecules into their inner aqueous phase and eventually deliver them selectively into tumor tissues in mice, owing to the prolonged blood circulation. Accordingly, fluorescent imaging of the tumor was successfully demonstrated along with an appreciable antitumor activity by DACHPt moieties retained in the vesicular wall of the metallosomes, indicating the potential of metallosomes as multifunctional drug carriers.

pH-dependent permeability change and reversible structural transition of PEGylated polyion complex vesicles (PICsomes) in aqueous media

The acidic pH-sensitivity of polyion complex vesicles (PICsomes) was investigated, using dynamic light scattering (DLS) and confocal laser scanning microscopy (CLSM). PICsomes showed pH-dependent and reversible structural transition, and also underwent a change in permeability by sensing acidic pH. Increased membrane permeability at the pH corresponding to cellular endosomes may be useful for future applications of PICsomes as a delivery vehicle of biologically active compounds to intracellular compartment.

Living unimodal growth of polyion complex vesicles via two-dimensional supramolecular polymerization

Understanding the dynamic behavior of molecular self-assemblies with higher-dimensional structures remains a key challenge to obtaining well-controlled and monodispersed structures. Nonetheless, there exist few systems capable of realizing the mechanism of supramolecular polymerization at higher dimensions. Herein, we report the unique self-assembling behavior of polyion complexes (PICs) consisting of poly(ethylene glycol)-polyelectrolyte block copolymer as an example of two-dimensional supramolecular living polymerization. Monodispersed and submicrometer unilamellar PIC vesicles (nano-PICsomes) displayed time-dependent growth while maintaining a narrow size distribution and a unilamellar structure. Detailed analysis of the system revealed that vesicle growth proceeded through the consumption of unit PICs (uPICs) composed of a single polycation/polyanion pair and was able to restart upon the further addition of isolated uPICs. Interestingly, the resulting vesicles underwent dissociation into uPICs in response to mechanical stress. These results clearly frame the growth as a two-dimensional supramolecular living polymerization of uPICs.

SPIO-PICsome: Development of a highly sensitive and stealth-capable MRI nano-agent for tumor detection using SPIO-loaded unilamellar polyion complex vesicles (PICsomes)

Size controllable polyion complex vesicles (PICsomes), composed of biocompatible poly(ethylene glycol) (PEG) and poly(amino acid)s, have an extremely prolonged lifetime in the bloodstream that enables them to accumulate effectively in tumors via the enhanced permeability and retention (EPR) effect. The purpose of this study was to use PICsomes to synthesize a highly sensitive MRI contrast agent for more precise tumor detection. We synthesized SPIO-Cy5-PICsomes (superparamagnetic iron oxide nanoparticle-loaded Cy5-cross-linked Nano-PICsomes) and characterized them using dynamic light scattering and transmission electron microscopy in vitro and evaluated their ability to detect subcutaneously grafted tumors in vivo with MRI. The transverse relaxivity (r2) of the SPIO-Cy5-PICsomes (r2=663±28mM(-1)s(-1)) was 2.54 times higher than that of bare clinically-used SPIO. In in vivo MRI experiments on mice subcutaneously grafted with colon-26 tumor cells, the tumor signal was significantly altered at 3h after SPIO-Cy5-PICsome administration and persisted for at least 24h. Small and early-stage in vivo tumors (3days after grafting, approximately 4mm(3)) were also clearly detected with MRI. SPIO-loaded PICsomes are sensitive MRI contrast agents that can act as a powerful nanocarrier to detect small tumors for early diagnosis.

Multicompartment micelles with adjustable poly(ethylene glycol) shell for efficient in vivo photodynamic therapy

We describe the preparation of well-defined multicompartment micelles from polybutadiene-block-poly(1-methyl-2-vinyl pyridinium methyl sulfate)-block-poly(methacrylic acid) (BVqMAA) triblock terpolymers and their use as advanced drug delivery systems for photodynamic therapy (PDT). A porphyrazine derivative was incorporated into the hydrophobic core during self-assembly and served as a model drug and fluorescent probe at the same time. The initial micellar corona is formed by negatively charged PMAA and could be gradually changed to poly(ethylene glycol) (PEG) in a controlled fashion through interpolyelectrolyte complex formation of PMAA with positively charged poly(ethylene glycol)-block-poly(L-lysine) (PLL-b-PEG) diblock copolymers. At high degrees of PEGylation, a compartmentalized micellar corona was observed, with a stable bottlebrush-on-sphere morphology as demonstrated by cryo-TEM measurements. By in vitro cellular experiments, we confirmed that the porphyrazine-loaded micelles were PDT-active against A549 cells. The corona composition strongly influenced their in vitro PDT activity, which decreased with increasing PEGylation, correlating with the cellular uptake of the micelles. Also, a PEGylation-dependent influence on the in vivo blood circulation and tumor accumulation was found. Fully PEGylated micelles were detected for up to 24 h in the bloodstream and accumulated in solid subcutaneous A549 tumors, while non- or only partially PEGylated micelles were rapidly cleared and did not accumulate in tumor tissue. Efficient tumor growth suppression was shown for fully PEGylated micelles up to 20 days, demonstrating PDT efficacy in vivo.

Spontaneous Formation of Giant Unilamellar Vesicles from Microdroplets of a Polyion Complex by Thermally Induced Phase Separation

Water pump: Polyion complex (PIC) vesicles are spontaneously formed from PIC microdroplets, which are formed by mixing cationic and anionic polymers (see picture). The formation process can be reversibly controlled by local heating with a focused infrared laser that triggers microphase separation and subsequent water influx. The size of the resulting giant unilamellar vesicles is determined by the initial size of the PIC droplets.