Yu Matsumoto

Improving Drug Potency and Efficacy by Nanocarrier-Mediated Subcellular Targeting

Polymeric micelles containing a chemotherapeutic drug carry it adjacent to the DNA target in tumor cells, enhancing the drug potency. Special Delivery to the Nucleus Micelles are useful in the washing machine, where they self-assemble from soaps, trap grease inside, and carry it away. These spheres, formed by linear molecules with hydrophobic tails that cluster in the core and hydrophilic heads sticking out, can carry cargo other than dirt. Micelles self-assembled in the presence of a chemotherapeutic drug can ensnare and carry it to tumors, where they are ingested by cells. By creating micelles that disperse in specific environment within the late endosome and lysosome, a region of the cell near the nucleus, Murakami et al. force these soapy spheres to release their deadly cargo—in this case a platinum-based drug called DACHPt [(1,2-diaminocyclohexane) platinum(II)]—right in the neighborhood of its target: DNA. This direct assault on the genome proves to be an effective antitumor strategy: Tumor cells growing in mice succumb more readily to a micelle-delivered derivative of platinum than they do to free drug. The authors’ micelle carriers are carefully assembled from block copolymers with properties suited to their task. A poly(ethylene glycol) polymer is linked to a string of glutamic acids, with a boron dipyrromethene at each end. By attaching fluorescent tags of different colors to the ends, the authors endowed their micelles with the ability to signal to an observer whether they are intact. When all the poly(glutamic acid) segments were clustered in the core, their red fluorescence was quenched and only the green surface dye on the poly(ethylene glycol) was visible. Once the micelle encountered specific conditions in the late endosome and lysosome, the core dispersed, releasing the drug and dequenching the red dye. By taking advantage of these visible markers of the micelle state, the authors showed by time-lapse confocal laser scanning microscopy that the micelles were taken up into tumor cells by endocytosis and that they traveled to the late endosomal/lysosomal compartment, where the micelles dispersed and the drug was released. This color-coded behavior was apparent both in cultured tumor cells and in tumor cells growing subcutaneously in mice, which the authors monitored in the animals, also by confocal laser scanning microscopy. But does the direct delivery of DACHPt to the nuclear area improve its effectiveness? A comparison of free DACHPt to the micelle-carried drug shows that it can help with one serious problem of cancer therapeutics—tumors that become drug-resistant. After repeated exposure to DACHPt, tumor cells develop defensive proteins, such as metallothionein and methionine synthase, in their cytoplasm that inactivate the drug, protecting the tumor cell DNA from damage. Tumors that have become resistant to DACHPt grow well in the presence of the drug, but the micelle-delivered version effectively inhibited the tumors’ growth, most likely by bypassing the cells’ cytoplasmic defenses. Therefore, with appropriate chemical modifications, micelles can be used to carry medicinal cargo right where it is needed. Nanocarrier-mediated drug targeting is an emerging strategy for cancer therapy and is being used, for example, with chemotherapeutic agents for ovarian cancer. Nanocarriers are selectively accumulated in tumors as a result of their enhanced permeability and retention of macromolecules, thereby enhancing the antitumor activity of the nanocarrier-associated drugs. We investigated the real-time subcellular fate of polymeric micelles incorporating (1,2-diaminocyclohexane) platinum(II) (DACHPt/m), the parent complex of oxaliplatin, in tumor tissues by fluorescence-based assessment of their kinetic stability. These observations revealed that DACHPt/m was extravasated from blood vessels to the tumor tissue and dissociated inside each cell. Furthermore, DACHPt/m selectively dissociated within late endosomes, enhancing drug delivery to the nearby nucleus relative to free oxaliplatin, likely by circumvention of the cytoplasmic detoxification systems such as metallothionein and methionine synthase. Thus, these drug-loaded micelles exhibited higher antitumor activity than did oxaliplatin alone, even against oxaliplatin-resistant tumors. These findings suggest that nanocarriers targeting subcellular compartments may have considerable benefits in clinical applications.

Polyplex Micelles with Cyclic RGD Peptide Ligands and Disulfide Cross-Links Directing to the Enhanced Transfection via Controlled Intracellular Trafficking

Thiolated c(RGDfK)-poly(ethylene glycol)-block-poly(lysine) (PEG-PLys), a novel block polymer that has a cyclic RGD peptide in the PEG terminus and thiol groups in the PLys side chain, was prepared and applied to the preparation of targetable disulfide cross-linked polyplex micelles through ion complexation with plasmid DNA (pDNA). The obtained polyplex micelles achieved remarkably enhanced transfection efficiency against cultured HeLa cells possessing alpha(v)beta(3) integrin receptors, which are selectively recognized by cyclic RGD peptides, demonstrating the synergistic effect of cyclic RGD peptide ligands on the micelle surface and disulfide cross-links in the core to exert the smooth release of pDNA in the intracellular environment via reductive cleavage. This enhancement was not due to an increase in the uptake amount of polyplex micelles but to a change in their intracellular trafficking route. Detailed confocal laser scanning microscopic observation revealed that polyplex micelles with cyclic RGD peptide ligands were distributed in the perinuclear region in the early stages preferentially through caveolae-mediated endocytosis, which may be a desirable pathway for avoiding the lysosomal degradation of delivered genes. Hence, this approach to introducing ligands and cross-links into the polyplex micelles is promising for the construction of nonviral gene vectors that enhance transfection by controlling intracellular distribution.

Targeted polymeric micelles for siRNA treatment of experimental cancer by intravenous injection

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.

Three-layered polyplex micelle as a multifunctional nanocarrier platform for light-induced systemic gene transfer

Nanocarriers responding to light have great potential for pinpoint therapy, and recent studies have revealed promising in vivo activity. However, light-selective gene transfer still remains challenging in the systemic application. Here we report systemic light-responsive nanocarriers for gene delivery developed through the sequential self-assembly of ABC-type triblock copolymer/DNA/dendrimeric photosensitizer, forming polyplex micelles with three-layered functional nanocompartments. The DNA-packaged core is covered by the photosensitizer-incorporated intermediate layer, which is encompassed by an outer shielding shell. This three-layered structure permits multistep photosensitizer and DNA delivery into a solid tumour by a systemic route: the shielding layer minimizes unfavourable interactions with blood components, and the photosensitizer is delivered to endo-/lysosomal membranes to facilitate light-selective cytoplasmic translocation of the micelles, accomplishing DNA delivery into the nucleus to exert gene expression. The polyplex micelles display >100-fold photoenhanced gene expression in cultured cells and exhibit light-induced in vivo gene transfer in solid tumours following systemic administration.

In situ quantitative monitoring of polyplexes and polyplex micelles in the blood circulation using intravital real-time confocal laser scanning microscopy.

Surface modification using poly(ethylene glycol) (PEG) is a widely used strategy to improve the biocompatibility of cationic polymer-based nonviral gene vectors (polyplexes). A novel method based on intravital real-time confocal laser scanning microscopy (IVRTCLSM) was applied to quantify the dynamic states of polyplexes in the bloodstream, thereby demonstrating the efficacy of PEGylation to prevent their agglomeration. Blood flow in the earlobe blood vessels of experimental animals was monitored in a noninvasive manner to directly observe polyplexes in the circulation. Polyplexes formed distinct aggregates immediately after intravenous injection, followed by interaction with platelets. To quantify aggregate formation and platelet interaction, the coefficient of variation and Pearsons correlation coefficient were adopted. In contrast, polyplex micelles prepared through self-assembly of plasmid DNA with PEG-based block catiomers had dense PEG palisades, revealing no formation of aggregates without visible interaction with platelets during circulation. This is the first report of in situ monitoring and quantification of the availability of PEGylation to prevent polyplexes from agglomeration over time in the blood circulation. This shows the high utility of IVRTCLSM in drug and gene delivery research.

Precise Engineering of siRNA Delivery Vehicles to Tumors Using Polyion Complexes and Gold Nanoparticles

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.

Targeted gene delivery by polyplex micelles with crowded PEG palisade and cRGD moiety for systemic treatment of pancreatic tumors

Adequate retention in systemic circulation is the preliminary requirement for systemic gene delivery to afford high bioavailability into the targeted site. Polyplex micelle formulated through self-assembly of oppositely-charged poly(ethylene glycol) (PEG)-polycation block copolymer and plasmid DNA has gained tempting perspective upon its advantageous core-shell architecture, where outer hydrophilic PEG shell offers superior stealth behaviors. Aiming to promote these potential characters toward systemic applications, we strategically introduced hydrophobic cholesteryl moiety at the ω-terminus of block copolymer, anticipating to promote not only the stability of polyplex structure but also the tethered PEG crowdedness. Moreover, Mw of PEG in the PEGylated polyplex micelle was elongated up to 20 kDa for expecting further enhancement in PEG crowdedness. Furthermore, cyclic RGD peptide as ligand molecule to integrin receptors was installed at the distal end of PEG in order for facilitating targeted delivery to the tumor site as well as promoting cellular uptake and intracellular trafficking behaviors. Thus constructed cRGD conjugated polyplex micelle with the elevated PEG shielding was challenged to a modeled intractable pancreatic cancer in mice, achieving potent tumor growth suppression by efficient gene expression of antiangiogenic protein (sFlt-1) at the tumor site.

Systemic Targeting of Lymph Node Metastasis through the Blood Vascular System by Using Size-Controlled Nanocarriers.

Occult nodal metastases increase the risk of cancer recurrence, demoting prognosis and quality of life of patients. While targeted drug delivery by using systemically administered nanocarriers can potentially control metastatic disease, lymph node metastases have been mainly dealt by locally injecting nanocarriers, which may not always be applicable. Herein, we demonstrated that sub-50 nm polymeric micelles incorporating platinum anticancer drugs could target lymph node metastases in a syngeneic melanoma model after systemic injection, even after removing the primary tumors, limiting the growth of the metastases. By comparing these micelles with clinically used doxorubicin-loaded liposomes (Doxil) having 80 nm, as well as a 70 nm version of the micelles, we found that the targeting efficiency of the nanocarriers against lymph node metastases was associated with their size-regulated abilities to extravasate from the blood vasculature in metastases and to penetrate within the metastatic mass. These findings indicate the potential of sub-50 nm polymeric micelles for developing effective conservative treatments against lymph node metastasis capable of reducing relapse and improving survival.

Identification of DBC1 as a transcriptional repressor for BRCA1

Background:DBC1/KIAA1967 (deleted in breast cancer 1) is a putative tumour-suppressor gene cloned from a heterozygously deleted region in breast cancer specimens. Caspase-dependent processing of DBC1 promotes apoptosis, and depletion of endogenous DBC1 negatively regulates p53-dependent apoptosis through its specific inhibition of SIRT1. Hereditary breast and ovarian cancer susceptibility gene product BRCA1, by binding to the promoter region of SIRT1, is a positive regulator of SIRT1 expression.Methods:A physical interaction between DBC1 and BRCA1 was investigated both in vivo and in vitro. To determine the pathophysiological significance of DBC1, its role as a transcriptional factor was studied.Results:We found a physical interaction between the amino terminus of DBC1 and the carboxyl terminus of BRCA1, also known as the BRCT domain. Endogenous DBC1 and BRCA1 form a complex in the nucleus of intact cells, which is exported to the cytoplasm during ultraviolet-induced apoptosis. We also showed that the expression of DBC1 represses the transcriptional activation function of BRCT by a transient expression assay. The expression of DBC1 also inhibits the transactivation of the SIRT1 promoter mediated by full-length BRCA1.Conclusion:These results revealed that DBC1 may modulate the cellular functions of BRCA1 and have important implications in the understanding of carcinogenesis in breast tissue.

Light-induced cytosolic activation of reduction-sensitive camptothecin-loaded polymeric micelles for spatiotemporally controlled in vivo chemotherapy.

Nanomedicines capable of smart operation at the targeted site have the potential to achieve the utmost therapeutic benefits. Providing nanomedicines that respond to endogenous stimuli with an additional external trigger may improve the spatiotemporal control of their functions, while avoiding drawbacks from their inherent tissue distribution. Herein, by exploiting the permeabilization of endosomes induced by photosensitizer agents upon light irradiation, we complemented the intracellular action of polymeric micelles incorporating camptothecin (CPT), which can sharply release the loaded drug in response to the reductive conditions of the cytosol, as an effective strategy for precisely controlling the function of these nanomedicines in vivo, while advancing toward a light-activated chemotherapy. These camptothecin-loaded micelles (CPT/m) were stable in the bloodstream, with minimal drug release in extracellular conditions, leading to prolonged blood circulation and high accumulation in xenografts of rat urothelial carcinoma. With the induction of endosomal permeabilization with the clinically approved photosensitizer, Photofrin, the CPT/m escaped from the endocytic vesicles of cancer cells into the cytosol, as confirmed both in vitro and in vivo by real-time confocal laser microscopies, accelerating the drug release from the micelles only in the irradiated tissues. This spatiotemporal switch significantly enhanced the in vivo antitumor efficacy of CPT/m without eliciting any toxicity, even at a dose 10-fold higher than the maximum tolerated dose of free CPT. Our results indicate the potential of reduction-sensitive drug-loaded polymeric micelles for developing safe chemotherapies after activation by remote triggers, such as light, which are capable of permeabilizing endosomal compartments.