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Enhanced permeability in tumours is thought to result from malformed vascular walls with leaky cell-to-cell junctions. This assertion is backed by studies using electron microscopy and polymer casts that show incomplete pericyte coverage of tumour vessels and the presence of intercellular gaps. However, this gives the impression that tumour permeability is static amid a chaotic tumour environment. Using intravital confocal laser scanning microscopy we show that the permeability of tumour blood vessels includes a dynamic phenomenon characterized by vascular bursts followed by brief vigorous outward flow of fluid (named 'eruptions') into the tumour interstitial space. We propose that 'dynamic vents' form transient openings and closings at these leaky blood vessels. These stochastic eruptions may explain the enhanced extravasation of nanoparticles from the tumour blood vessels, and offer insights into the underlying distribution patterns of an administered drug.

A core-shell-type polyion complex (PIC) micelle with a disulfide cross-linked core was prepared through the assembly of iminothiolane-modified poly(ethylene glycol)-block-poly(L-lysine) [PEG-b-(PLL-IM)] and siRNA at a characteristic optimum mixing ratio. The PIC micelles showed a spherical shape of approximately 60 nm in diameter with a narrow distribution. The micellar structure was maintained at physiological ionic strength but was disrupted under reductive conditions because of the cleavage of disulfide cross-links, which is desirable for siRNA release in the intracellular reductive environment. Importantly, environment-responsive PIC micelles achieved 100-fold higher siRNA transfection efficacy compared with non-cross-linked PICs prepared from PEG-b-poly(L-lysine), which were not stable at physiological ionic strength. PICs formed with PEG-b-(PLL-IM) at nonoptimum ratios did not assemble into micellar structure and did not achieve gene silencing following siRNA transfection. These findings show the feasibility of core cross-linked PIC micelles as carriers for therapeutic siRNA and show that stable micellar structure is critical for effective siRNA delivery into target cells.

Small interfering ribonucleic acid (siRNA) cancer therapies administered by intravenous injection require a delivery system for transport from the bloodstream into the cytoplasm of diseased cells to perform the function of gene silencing. Here we describe nanosized polymeric micelles that deliver siRNA to solid tumors and elicit a therapeutic effect. Stable multifunctional micelle structures on the order of 45 nm in size formed by spontaneous self-assembly of block copolymers with siRNA. Block copolymers used for micelle formation were designed and synthesized to contain three main features: a siRNA binding segment containing thiols, a hydrophilic nonbinding segment, and a cell-surface binding peptide. Specifically, poly(ethylene glycol)-block-poly(L-lysine) (PEG-b-PLL) comprising lysine amines modified with 2-iminothiolane (2IT) and the cyclo-Arg-Gly-Asp (cRGD) peptide on the PEG terminus was used. Modification of PEG-b-PLL with 2IT led to improved control of micelle formation and also increased stability in the blood compartment, while installation of the cRGD peptide improved biological activity. Incorporation of siRNA into stable micelle structures containing the cRGD peptide resulted in increased gene silencing ability, improved cell uptake, and broader subcellular distribution in vitro and also improved accumulation in both the tumor mass and tumor-associated blood vessels following intravenous injection into mice. Furthermore, stable and targeted micelles inhibited the growth of subcutaneous HeLa tumor models and demonstrated gene silencing in the tumor mass following treatment with antiangiogenic siRNAs. This new micellar nanomedicine could potentially expand the utility of siRNA-based therapies for cancer treatments that require intravenous injection.

Dramatic advances in biological research have revealed the mechanisms underlying many diseases at the molecular level. However, conventional techniques may be inadequate for direct application of this new knowledge to medical treatments. Nanobiotechnology, which integrates biology with the rapidly growing field of nanotechnology, has great potential to overcome many technical problems and lead to the development of effective therapies. The use of nanobiotechnology in drug delivery systems (DDS) is attractive for advanced treatment of conditions such as cancer and genetic diseases. In this review paper for a special issue on biomaterial research in Japan, we discuss the development of DDS based on polymeric micelles mainly in our group for anti-cancer drug and gene delivery, and also address our challenges associated with developing polymeric micelles as super-functionalized nanodevices with intelligent performance.

For small interfering RNA (siRNA)-based cancer therapies, we report an actively-targeted and stabilized polyion complex micelle designed to improve tumor accumulation and cancer cell uptake of siRNA following systemic administration. Improvement in micelle stability was achieved using two stabilization mechanisms; covalent disulfide cross-linking and non-covalent hydrophobic interactions. The polymer component was designed to provide disulfide cross-linking and cancer cell-targeting cyclic RGD peptide ligands, while cholesterol-modified siRNA (Chol-siRNA) provided additional hydrophobic stabilization to the micelle structure. Dynamic light scattering confirmed formation of nano-sized disulfide cross-linked micelles (<50 nm in diameter) with a narrow size distribution. Improved stability of Chol-siRNA-loaded micelles (Chol-siRNA micelles) was demonstrated by resistance to both the dilution in serum-containing medium and counter polyion exchange with dextran sulfate, compared to control micelles prepared with Chol-free siRNA (Chol-free micelles). Improved stability resulted in prolonged blood circulation time of Chol-siRNA micelles compared to Chol-free micelles. Furthermore, introduction of cRGD ligands onto Chol-siRNA micelles significantly facilitated accumulation of siRNA in a subcutaneous cervical cancer model following systemic administration. Ultimately, systemically administered cRGD/Chol-siRNA micelles exhibited significant gene silencing activity in the tumor, presumably due to their active targeting ability combined with the enhanced stability through both hydrophobic interactions of cholesterol and disulfide cross-linking.

For systemic delivery of siRNA to solid tumors, a size-regulated and reversibly stabilized nanoarchitecture was constructed by using a 20 kDa siRNA-loaded unimer polyion complex (uPIC) and 20 nm gold nanoparticle (AuNP). The uPIC was selectively prepared by charge-matched polyionic complexation of a poly(ethylene glycol)-b-poly(L-lysine) (PEG-PLL) copolymer bearing ∼40 positive charges (and thiol group at the ω-end) with a single siRNA bearing 40 negative charges. The thiol group at the ω-end of PEG-PLL further enabled successful conjugation of the uPICs onto the single AuNP through coordinate bonding, generating a nanoarchitecture (uPIC-AuNP) with a size of 38 nm and a narrow size distribution. In contrast, mixing thiolated PEG-PLLs and AuNPs produced a large aggregate in the absence of siRNA, suggesting the essential role of the preformed uPIC in the formation of nanoarchitecture. The smart uPIC-AuNPs were stable in serum-containing media and more resistant against heparin-induced counter polyanion exchange, compared to uPICs alone. On the other hand, the treatment of uPIC-AuNPs with an intracellular concentration of glutathione substantially compromised their stability and triggered the release of siRNA, demonstrating the reversible stability of these nanoarchitectures relative to thiol exchange and negatively charged AuNP surface. The uPIC-AuNPs efficiently delivered siRNA into cultured cancer cells, facilitating significant sequence-specific gene silencing without cytotoxicity. Systemically administered uPIC-AuNPs showed appreciably longer blood circulation time compared to controls, i.e., bare AuNPs and uPICs, indicating that the conjugation of uPICs onto AuNP was crucial for enhancing blood circulation time. Finally, the uPIC-AuNPs efficiently accumulated in a subcutaneously inoculated luciferase-expressing cervical cancer (HeLa-Luc) model and achieved significant luciferase gene silencing in the tumor tissue. These results demonstrate the strong potential of uPIC-AuNP nanoarchitectures for systemic siRNA delivery to solid tumors.

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