Horacio Cabral

Progress of drug-loaded polymeric micelles into clinical studies

Targeting tumors with long-circulating nano-scaled carriers is a promising strategy for systemic cancer treatment. Compared with free small therapeutic agents, nanocarriers can selectively accumulate in solid tumors through the enhanced permeability and retention (EPR) effect, which is characterized by leaky blood vessels and impaired lymphatic drainage in tumor tissues, and achieve superior therapeutic efficacy, while reducing side effects. In this way, drug-loaded polymeric micelles, i.e. self-assemblies of amphiphilic block copolymers consisting of a hydrophobic core as a drug reservoir and a poly(ethylene glycol) (PEG) hydrophilic shell, have demonstrated outstanding features as tumor-targeted nanocarriers with high translational potential, and several micelle formulations are currently under clinical evaluation. This review summarizes recent efforts in the development of these polymeric micelles and their performance in human studies, as well as our recent progress in polymeric micelles for the delivery of nucleic acids and imaging.

Charge‐Conversional Polyionic Complex Micelles—Efficient Nanocarriers for Protein Delivery into Cytoplasm

Special delivery! Polyionic complex (PIC) micelles that contain the charge-conversional moieties citaconic amide or cis-aconitic amide were developed for cytoplasmic protein delivery. The increase of the charge density on the protein cargo helped the stability of the PIC micelles without cross-linking, and the charge-conversion in endosomes induced the dissociation of the PIC micelles to result in efficient endosomal release (see picture).

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.

Phenylboronic acid-installed polymeric micelles for targeting sialylated epitopes in solid tumors

Ligand-mediated targeting of nanocarriers to tumors is an attractive strategy for increasing the efficiency of chemotherapies. Sialylated glycans represent a propitious target as they are broadly overexpressed in tumor cells. Because phenylboronic acid (PBA) can selectively recognize sialic acid (SA), herein, we developed PBA-installed micellar nanocarriers incorporating the parent complex of the anticancer drug oxaliplatin, for targeting sialylated epitopes overexpressed on cancer cells. Following PBA-installation, the micelles showed high affinity for SA, as confirmed by fluorescence spectroscopy even at intratumoral pH conditions, i.e., pH 6.5, improving their cellular recognition and uptake and enhancing their in vitro cytotoxicity against B16F10 murine melanoma cells. In vivo, PBA-installed micelles effectively reduced the growth rate of both orthotopic and lung metastasis models of melanoma, suggesting the potential of PBA-installed nanocarriers for enhanced tumor targeting.

Assessment of Tumor Metastasis by the Direct Determination of Cell-Membrane Sialic Acid Expression

Sialic acid (SA) is an anionic monosaccharide that frequently occurs at the termini of glycan chains and provides many opportunities for the assessment of both normal and pathological cell processes. It is generally present in tumorassociated carbohydrate antigens, including those clinically approved as tumor markers. Accordingly, the overexpression of SA on cell membranes has been implicated in the malignant and metastatic phenotypes of various types of cancer. Therefore, SA is an important molecular target for diagnostic and therapeutic approaches. The installation of SA-specific ligands enables reagents to target highly sialylated or tumor cells. Alternatively, monitoring of the cellsurface expression of SA should provide rational indexes of dynamic changes in pathological conditions and other SAassociated biological events. We previously developed a method for the potentiometric detection of SA by exploiting the reversible and specific nature of the binding between phenylboronic acid (PBA) and SA. A gold electrode modified with PBA and with a carefully optimized dissociation constant (or pKa value) was able to quantify SA present in the free state as well as cell-surface SA under physiological aqueous conditions. The observed ability of the electrode to differentiate altered levels of SA expression on the surface of rabbit erythrocytes is relevant to the diagnosis of insulin-dependent diabetes mellitus (IDDM). The approach provided a new rationale for the label-free, noninvasive (enzyme-free and operative on living cells), and real-time determination of SA. Herein we show that the technique can also be applied to the analysis of tumor malignancy and the degree of metastasis. PBA derivatives are able to form reversible cyclic boronates with 1,2-diols, 1,3-diols, and polyols: hallmark structures of the majority of glycans. Because of this property, PBA has quite a history as a synthetic ligand for these molecules. It is usually observed that these complexes have a stabilizing effect only if PBA is disassociated (at pH values above the pKa value), [6] whereas those formed between nondissociated PBA and sugars are unstable with high susceptibility to hydrolysis. However, as an exception, a complex formed between nondissociated PBA and SA is remarkably stable owing to its special binding modalities, some aspects of which have been clarified previously. As a result, a PBA with an appropriate pKa value can provide a molecular basis for selective recognition of SA among other saccharides under physiological conditions (see the Supporting Information). A procedure for the surface modification of a gold electrode with PBA was described previously. Briefly, a self-assembled monolayer (SAM) of 10-carboxy-1-decanethiol was first formed on a gold electrode. Next, a reaction between the terminal carboxyl groups and 3-aminophenylboronic acid resulted in the introduction of meta-amidesubstituted PBA on the SAM surface. Both quartz crystal microbalance (QCM) and ellipsometric measurements confirmed stoichiometric monolayer formation at each step of the reaction (see the Supporting Information). The surface PBA moiety had an apparent pKa value of about 9.5, as judged from pH-dependent changes in its threshold voltage (VT; see the Supporting Information). We could therefore safely conclude that it was not dissociated at the physiological pH value (7.4) and would be SA-specific under such conditions. The electrode was then linked to a field-effect-transistor (FET) gate for the real-time monitoring of charge-density changes on the electrode. In this configuration, a carboxyl anion of SA can be detected as a positive-direction shift of the VT value of the FET. Owing to the nature of the field effect, FET-based charge detection is possible only within a distance corresponding to the electrical double layer or the Debye length, which is no greater than a few nanometers even under conditions of minimized ionic strength. This requirement should be compatible with the purpose of detecting cellsurface SA moieties, which generally dominate the termini of the glycan chains, as described earlier. Besides, the tumoror metastasis-associated overexpression of SA is usually found in the form of polysialylation. Such a sequential arrangement of target SA units (as an SA homopolymer) on the glycanchain termini may help to enable the precise reflection of altered levels of SA expression. Moreover, the fact that the technique is limited to short detection distances could beneficially restrict charge detection to molecules that are truly (covalently) bound to the electrode surface within the vicinity of the Debye length (i.e., PBA-bound SA) and exclude other charges bound through nonspecific or noncovalent interactions. To demonstrate the ability of the electrode to assess malignancy or metastasis of a tissue specimen, metastatic murine melanoma cells expressing luciferase (B16-F10-Luc[*] Dr. A. Matsumoto, Dr. H. Cabral, N. Sato, Dr. K. Kataoka, Dr. Y. Miyahara Centre for NanoBio Integration, The University of Tokyo Hongo 7-3-1, Bunkyo-ku, Tokyo (Japan) Fax: (+ 81)29-860-4506 E-mail: miyahara.yuji@nims.go.jp

A pH-activatable nanoparticle with signal-amplification capabilities for non-invasive imaging of tumour malignancy

Engineered nanoparticles that respond to pathophysiological parameters, such as pH or redox potential, have been developed as contrast agents for the magnetic resonance imaging (MRI) of tumours. However, beyond anatomic assessment, contrast agents that can sense these pathological parameters and rapidly amplify their magnetic resonance signals are desirable because they could potentially be used to monitor the biological processes of tumours and improve cancer diagnosis. Here, we report an MRI contrast agent that rapidly amplifies magnetic resonance signals in response to pH. We confined Mn(2+) within pH-sensitive calcium phosphate (CaP) nanoparticles comprising a poly(ethylene glycol) shell. At a low pH, such as in solid tumours, the CaP disintegrates and releases Mn(2+) ions. Binding to proteins increases the relaxivity of Mn(2+) and enhances the contrast. We show that these nanoparticles could rapidly and selectively brighten solid tumours, identify hypoxic regions within the tumour mass and detect invisible millimetre-sized metastatic tumours in the liver.

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.

Bundled Assembly of Helical Nanostructures in Polymeric Micelles Loaded with Platinum Drugs Enhancing Therapeutic Efficiency against Pancreatic Tumor

Supramolecular assemblies of amphiphilic block copolymers having polypeptide segments offer significant advantages for tailoring spatial arrangement based on secondary structures in their optically active backbones. Here, we demonstrated the critical effect of α-helix bundles in cisplatin-conjugated poly(L- (or D-)glutamate) [P(L(or D)Glu)-CDDP] segment on the packaging of poly(ethylene glycol) (PEG)-P(L(or D)Glu)-CDDP block copolymers in the core of polymeric micelles (CDDP/m) and enhanced micelle tolerability to harsh in vivo conditions for accomplishing appreciable antitumor efficacy against intractable pancreatic tumor by systemic injection. CDDP/m prepared from optically inactive PEG-poly(D,L-glutamate) (P(D,LGlu)), gradually disintegrated in the bloodstream, resulting in increased accumulation in liver and spleen and reduced antitumor efficacy. Alternatively, CDDP/m from optically active PEG-P(L(or D)Glu) maintained micelle structure during circulation, and eventually attained selective tumor accumulation while reducing nonspecific distribution to liver and spleen. Circular dichroism and small-angle X-ray scattering measurements indicated regular bundled assembly of α-helices in the core of CDDP/m from PEG-P(L(or D)Glu), which is suggested to stabilize the micelle structure against dilution in physiological condition. CDDP/m suffered corrosion by chlorides in medium, yet the optically active micelles with α-helix bundles kept the micelle structure for prolonged time, with slowly releasing unimers and dimers from the surface of the bundled core in an erosion-like process, as verified by ultracentrifugation analysis. This is in sharp contrast with the abrupt disintegration of CDDP/m from PEG-P(D,LGlu) without secondary structures. The tailored assembly in the core of the polymeric micelles through regular arrangement of constituting segments is key to overcome their undesirable disintegration in bloodstream, thereby achieving efficient delivery of loaded drugs into target tissues.

Enhanced in vivo Magnetic Resonance Imaging of Tumors by PEGylated Iron-Oxide-Gold Core-Shell Nanoparticles with Prolonged Blood Circulation Properties

High-density poly(ethylene glycol) (PEG)-coated iron-oxide-gold core-shell nanoparticles (AuIONs) were developed as T(2) -weighted magnetic resonance imaging (MRI) contrast agents for cancer imaging. The PEG-coated iron-oxide-gold core-shell nanoparticles (PEG-AuIONs) were approximately 25 nm in diameter with a narrow distribution. Biodistribution experiments in mice bearing a subcutaneous colon cancer model prepared with C26 murine colon adenocarcinoma cells showed high accumulation of the PEG-AuIONs within the tumor mass and low nonspecific accumulation in the liver and spleen, resulting in high specificity to solid tumors. T(2) -weighted MR images following intravenous injection of PEG-AuIONs showed selective negative enhancement of tumor tissue in an orthotopic pancreatic cancer model prepared with MiaPaCa-2 human pancreatic adenocarcinoma cells. These results indicate that PEG-AuIONs are a promising MRI contrast agent for diagnosis of malignant tumors, including pancreatic cancer.

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.