Noncovalent Stabilization of Vesicular Polyion Complexes with Chemically Modified/Single-Stranded Oligonucleotides and PEG-b-guanidinylated Polypeptides for Intracavity Encapsulation of Effector Enzymes Aimed at Cooperative Gene Knockdown.

For the simultaneous delivery of antisense oligonucleotides and their effector enzymes into cells, nanosized vesicular polyion complexes (PICs) were fabricated from oppositely charged polyion pairs of oligonucleotides and poly(ethylene glycol) (PEG)-b-polypeptides. First, the polyion component structures were carefully designed to facilitate a multimolecular (or secondary) association of unit PICs for noncovalent (or chemical cross-linking-free) stabilization of vesicular PICs. Chemically modified, single-stranded oligonucleotides (SSOs) dramatically stabilized the multimolecular associates under physiological conditions, compared to control SSOs without chemical modifications and duplex oligonucleotides. In addition, a high degree of guanidino groups in the polypeptide segment was also crucial for the high stability of multimolecular associates. Dynamic light scattering and transmission electron microscopy revealed the stabilized multimolecular associates to have a 100 nm sized vesicular architecture with a narrow size distribution. The loading number of SSOs per nanovesicle was determined to be ∼2500 using fluorescence correlation spectroscopic analyses with fluorescently labeled SSOs. Furthermore, the nanovesicle stably encapsulated ribonuclease H (RNase H) as an effector enzyme at ∼10 per nanovesicle through simple vortex-mixing with preformed nanovesicles. Ultimately, the RNase H-encapsulated nanovesicle efficiently delivered SSOs with RNase H into cultured cancer cells, thereby eliciting the significantly higher gene knockdown compared with empty nanovesicles (without RNase H) or a mixture of nanovesicles with RNase H without encapsulation. These results demonstrate the great potential of noncovalently stabilized nanovesicles for the codelivery of two varying bio-macromolecule payloads for ensuring their cooperative biological activity.

Systemic Brain Delivery of Antisense Oligonucleotides across the Blood-Brain Barrier with a Glucose-Coated Polymeric Nanocarrier.

Current antisense oligonucleotide (ASO) therapies for the treatment of central nervous system (CNS) disorders are performed through invasive administration, thereby placing a major burden on patients. To alleviate this burden, we herein report systemic ASO delivery to the brain by crossing the blood-brain barrier using glycemic control as an external trigger. Glucose-coated polymeric nanocarriers, which can be bound by glucose transporter-1 expressed on the brain capillary endothelial cells, are designed for stable encapsulation of ASOs, with a particle size of about 45 nm and an adequate glucose-ligand density. The optimized nanocarrier efficiently accumulates in the brain tissue 1 h after intravenous administration and exhibits significant knockdown of a target long non-coding RNA in various brain regions, including the cerebral cortex and hippocampus. These results demonstrate that the glucose-modified polymeric nanocarriers enable noninvasive ASO administration to the brain for the treatment of CNS disorders.

Glucose-linked sub-50-nm unimer polyion complex-assembled gold nanoparticles for targeted siRNA delivery to glucose transporter 1-overexpressing breast cancer stem-like cells.

Cancer stem-like cells (CSCs) treatment is a plausible strategy for enhanced cancer therapy. Here we report a glucose-installed sub-50-nm nanocarrier for the targeted delivery of small interfering RNA (siRNA) to CSCs through selective recognition of the glucose ligand to the glucose transporter 1 (GLUT1) overexpressed on the CSC surface. The siRNA nanocarrier was constructed via a two-step assembling process. First, a glucose-installed poly(ethylene glycol)-block-poly(l-lysine) modified with lipoic acid (LA) at the ω-end (Glu-PEG-PLL-LA) was associated with a single siRNA to form a unimer polyion complex (uPIC). Second, a 20 nm gold nanoparticle (AuNP) was decorated with ~65 uPICs through AuS bonding. The glucose-installed targeted nanoparticles (Glu-NPs) exhibited higher cellular uptake of siRNA payloads in a spheroid breast cancer (MBA-MB-231) cell culture compared with glucose-unconjugated control nanoparticles (MeO-NPs). Notably, the Glu-NPs became more efficiently internalized into the CSC fraction, which was defined by aldehyde dehydrogenase (ALDH) activity assay, than the other fractions, probably due to the higher GLUT1 expression level on the CSCs. The Glu-NPs elicited significantly enhanced gene silencing in a CSC-rich orthotopic MDA-MB-231 tumor tissue following systemic administration to tumor-bearing mice. Ultimately, the repeated administrations of polo-like kinase 1 (PLK1) siRNA-loaded Glu-NPs significantly suppressed the growth of orthotopic MDA-MB-231 tumors. These results demonstrate that Glu-NP is a promising nanocarrier design for CSC-targeted cancer treatment.

Dually Stabilized Triblock Copolymer Micelles with Hydrophilic Shell and Hydrophobic Interlayer for Systemic Antisense Oligonucleotide Delivery to Solid Tumor.

For intravenous delivery of antisense oligonucleotides (ASOs) to solid tumors, a triblock copolymer was synthesized from poly(2-ethyl-2-oxazoline) (PEtOx), poly(2-n-propyl-2-oxazoline) (PnPrOx), and poly(l-lysine) (PLL) segments. The triblock copolymer, PEtOx-PnPrOx-PLL, was utilized to fabricate a compartmentalized polymeric micelle featuring a hydrophilic PEtOx shell, thermoresponsive PnPrOx interlayer, and ASO/PLL polyion complex (PIC) core. In this formulation, the PnPrOx-derived interlayer underwent the phase transition from hydrophilic elongated state to hydrophobic collapsed state at a lower critical solution temperature (LCST) to enhance the micelle stability. Three triblock copolymers comprising varying lengths of PEtOx segment (2k, 7k, and 12 kDa) were compared to investigate the effect of hydrophilic chain length on the micelle properties. The triblock copolymer micelles (TCMs) were prepared in a two-step manner: mixing between triblock copolymer and ASO in a buffer solution at 4 °C and then increasing the temperature of the solution up to 37 °C. This protocol was crucial for the fabrication of TCMs with both smaller size and narrower size distribution, probably due to the formation of the well-compartmentalized hydrophobic interlayer in the micelle structure. The presence of the PnPrOx segment dramatically enhanced the stability of TCMs in serum-containing media and elicited more efficient cellular uptake of ASO payloads, resulting in higher gene silencing efficiency in cultured prostate cancer (PC-3) cells, compared with a control diblock copolymer micelle (DCM). The blood circulation property of TCMs was prolonged with an increase in the length of PEtOx segment, permitting the efficient accumulation of ASO payloads in a subcutaneous PC-3 tumor model. Ultimately, the systemic delivery of ASO targeting a long noncoding RNA (lncRNA) by the TCMs significantly reduced the expression level of lncRNA in the subcutaneous PC-3 tumor in a sequence-specific manner. These results demonstrate the superiority of TCMs equipped with the hydrophilic shell and hydrophobic interlayer to the cancer-targeted systemic ASO delivery.

Tuned Density of Anti-Tissue Factor Antibody Fragment onto siRNA-Loaded Polyion Complex Micelles for Optimizing Targetability into Pancreatic Cancer Cells.

Antibody fragment (Fab')-installed polyion complex (PIC) micelles were constructed to improve targetability of small interfering RNA (siRNA) delivery to pancreatic cancer cells. To this end, we synthesized a block copolymer of azide-functionalized poly(ethylene glycol) and poly(l-lysine) and prepared PIC micelles with siRNA. Then, a dibenzylcyclooctyne (DBCO)-modified antihuman tissue factor (TF) Fab' was conjugated to azido groups on the micellar surface. A fluorescence correlation spectroscopic analysis revealed that 1, 2, or 3 molecule(s) of Fab'(s) were installed onto one micellar nanoparticle according to the feeding ratio of Fab' (or DBCO) to micelle (or azide). The resulting micelles exhibited ∼40 nm in hydrodynamic diameter, similar to that of the parent micelles before Fab' conjugation. Flow cytometric analysis showed that three molecules of Fab'-installed PIC micelles (3(Fab')-micelles) had the highest binding affinity to cultured pancreatic cancer BxPC3 cells, which are known to overexpress TF on their surface. The 3(Fab')-micelles also exhibited the most efficient gene silencing activity against polo-like kinase 1 mRNA in the cultured cancer cells. Furthermore, the 3(Fab')-micelles exhibited high penetrability and the highest cellular internalization amounts in BxPC3 spheroids compared with one or two molecule(s) of Fab'-installed PIC micelles. These results demonstrate the potential of anti-TF Fab'-installed PIC micelles for active targeting of stroma-rich pancreatic tumors.

Chemical gas-generating nanoparticles for tumor-targeted ultrasound imaging and ultrasound-triggered drug delivery.

Although there is great versatility of ultrasound (US) technologies in the real clinical field, one main technical challenge is the compromising of high quality of echo properties and size engineering of ultrasound contrast agents (UCAs); a high echo property is offset by reducing particle size. Herein, a new strategy for overcoming the dilemma by devising chemical gas (CO2) generating carbonate copolymer nanoparticles (Gas-NPs), which are clearly distinguished from the conventional gas-encapsulated micro-sized UCAs. More importantly, Gas-NPs could be readily engineered to strengthen the desirable in vivo physicochemical properties for nano-sized drug carriers with higher tumor targeting ability, as well as the high quality of echo properties for tumor-targeted US imaging. In tumor-bearing mice, anticancer drug-loaded Gas-NPs showed the desirable theranostic functions for US-triggered drug delivery, even after i.v. injection. In this regard, and as demonstrated in the aforementioned study, our technology could serve a highly effective platform in building theranostic UCAs with great sophistication and therapeutic applicability in tumor-targeted US imaging and US-triggered drug delivery.

Systemic delivery of siRNA by actively targeted polyion complex micelles for silencing the E6 and E7 human papillomavirus oncogenes.

Human papillomavirus (HPV) E6 and E7 oncogenes are essential for the immortalization and maintenance of HPV-associated cancer and are ubiquitously expressed in cervical cancer lesions. Small interfering RNA (siRNA) coding for E6 and E7 oncogenes is a promising approach for precise treatment of cervical cancer, yet a delivery system is required for systemic delivery to solid tumors. Here, an actively targeted polyion complex (PIC) micelle was applied to deliver siRNAs coding for HPV E6/E7 to HPV cervical cancer cell tumors in immune-incompetent tumor-bearing mice. A cell viability assay revealed that both HPV type 16 and 18 E6/E7 siRNAs (si16E6/E7 and si18E6/E7, respectively) interfered with proliferation of cervical cancer cell lines in an HPV type-specific manner. A fluorescence imaging biodistribution analysis further revealed that fluorescence dye-labeled siRNA-loaded PIC micelles efficiently accumulated within the tumor mass after systemic administration. Ultimately, intravenous injection of si16E6/E7 and si18E6/E7-loaded PIC micelles was found to significantly suppress the growth of subcutaneous SiHa and HeLa tumors, respectively. The specific activity of siRNA treatment was confirmed by the observation that p53 protein expression was restored in the tumors excised from the mice treated with si16E6/E7- and si18E6/E7-loaded PIC micelles for SiHa and HeLa tumors, respectively. Therefore, the actively targeted PIC micelle incorporating HPV E6/E7-coding siRNAs demonstrated its therapeutic potential against HPV-associated cancer.

Theranostic gas-generating nanoparticles for targeted ultrasound imaging and treatment of neuroblastoma.

The development of safe and efficient diagnostic/therapeutic agents for treating cancer in clinics remains challenging due to the potential toxicity of conventional agents. Although the annual incidence of neuroblastoma is not that high, the disease mainly occurs in children, a population vulnerable to toxic contrast agents and therapeutics. We demonstrate here that cancer-targeting, gas-generating polymeric nanoparticles are useful as a theranostic tool for ultrasound (US) imaging and treating neuroblastoma. We encapsulated calcium carbonate using poly(d,l-lactide-co-glycolide) and created gas-generating polymer nanoparticles (GNPs). These nanoparticles release carbon dioxide bubbles under acidic conditions and enhance US signals. When GNPs are modified using rabies virus glycoprotein (RVG) peptide, a targeting moiety to neuroblastoma, RVG-GNPs effectively accumulate at the tumor site and substantially enhance US signals in a tumor-bearing mouse model. Intravenous administration of RVG-GNPs also reduces tumor growth in the mouse model without the use of conventional therapeutic agents. This approach to developing theranostic agents with disease-targeting ability may provide useful strategy for the detection and treatment of cancers, allowing safe and efficient clinical applications with fewer side effects than may occur with conventional agents.

Echogenic Glycol Chitosan Nanoparticles for Ultrasound-Triggered Cancer Theranostics.

Theranostic nanoparticles hold great promise for simultaneous diagnosis of diseases, targeted drug delivery with minimal toxicity, and monitoring of therapeutic efficacy. However, one of the current challenges in developing theranostic nanoparticles is enhancing the tumor-specific targeting of both imaging probes and anticancer agents. Herein, we report the development of tumor-homing echogenic glycol chitosan-based nanoparticles (Echo-CNPs) that concurrently execute cancer-targeted ultrasound (US) imaging and US-triggered drug delivery. To construct this novel Echo-CNPs, an anticancer drug and bioinert perfluoropentane (PFP), a US gas precursor, were simultaneously encapsulated into glycol chitosan nanoparticles using the oil in water (O/W) emulsion method. The resulting Echo-CNPs had a nano-sized particle structure, composing of hydrophobic anticancer drug/PFP inner cores and a hydrophilic glycol chitosan polymer outer shell. The Echo-CNPs had a favorable hydrodynamic size of 432 nm, which is entirely different from the micro-sized core-empty conventional microbubbles (1-10 µm). Furthermore, Echo-CNPs showed the prolonged echogenicity via the sustained microbubble formation process of liquid-phase PFP at the body temperature and they also presented a US-triggered drug release profile through the external US irradiation. Interestingly, Echo-CNPs exhibited significantly increased tumor-homing ability with lower non-specific uptake by other tissues in tumor-bearing mice through the nanoparticle's enhanced permeation and retention (EPR) effect. Conclusively, theranostic Echo-CNPs are highly useful for simultaneous cancer-targeting US imaging and US-triggered delivery in cancer theranostics.

pH-controlled gas-generating mineralized nanoparticles: a theranostic agent for ultrasound imaging and therapy of cancers.

We report a theranostic nanoparticle that can express ultrasound (US) imaging and simultaneous therapeutic functions for cancer treatment. We developed doxorubicin-loaded calcium carbonate (CaCO3) hybrid nanoparticles (DOX-CaCO3-MNPs) through a block copolymer templated in situ mineralization approach. The nanoparticles exhibited strong echogenic signals at tumoral acid pH by producing carbon dioxide (CO2) bubbles and showed excellent echo persistence. In vivo results demonstrated that the DOX-CaCO3-MNPs generated CO2 bubbles at tumor tissues sufficient for echogenic reflectivity under a US field. In contrast, the DOX-CaCO3-MNPs located in the liver or tumor-free subcutaneous area did not generate the CO2 bubbles necessary for US contrast. The DOX-CaCO3-MNPs could also trigger the DOX release simultaneously with CO2 bubble generation at the acidic tumoral environment. The DOX-CaCO3-MNPs displayed effective antitumor therapeutic activity in tumor-bearing mice. The concept described in this work may serve as a useful guide for development of various theranostic nanoparticles for US imaging and therapy of various cancers.

Tumor-targeting glycol chitosan nanoparticles as a platform delivery carrier in cancer diagnosis and therapy.

A natural based polymer, chitosan has received widespread attention in drug delivery systems due to its valuable physicochemical and biological characteristics. In particular, hydrophobic moiety-conjugated glycol chitosan can form amphiphilic self-assembled glycol chitosan nanoparticles (GCNPs) and simultaneously encapsulate hydrophobic drug molecules inside their hydrophobic core. This GCNP-based drug delivery systems exhibit excellent tumor-homing efficacy, attributed to the long blood circulation and the enhanced permeability and retention effect; this tumor-targeting drug delivery results in improved therapeutic efficiency. In this review, we describe the requisite properties of GCNPs for cancer therapy as well as imaging for diagnosis, such as their basic characteristics, in vitro delivery efficiency and in vivo tumor-targeting ability.

Chemical tumor-targeting of nanoparticles based on metabolic glycoengineering and click chemistry.

Tumor-targeting strategies for nanoparticles have been predominantly based on optimization of physical properties or conjugation with biological ligands. However, their tumor-targeting abilities remain limited and insufficient. Furthermore, traditional biological binding molecules have intrinsic limitations originating from the limited amount of cellular receptors and the heterogeneity of tumor cells. Our two-step in vivo tumor-targeting strategy for nanoparticles is based on metabolic glycoengineering and click chemistry. First, an intravenous injection of precursor-loaded glycol chitosan nanoparticles generates azide groups on tumor tissue specifically by the enhanced permeation and retention (EPR) effect followed by metabolic glycoengineering. These 'receptor-like' chemical groups then enhance the tumor-targeting ability of drug-containing nanoparticles by copper-free click chemistry in vivo during a second intravenous injection. The advantage of this protocol over traditional binding molecules is that there are significantly more binding molecules on the surface of most tumor cells regardless of cell type. The subsequent enhanced tumor-targeting ability can significantly enhance the cancer therapeutic efficacy in animal studies.

Tumor-targeting transferrin nanoparticles for systemic polymerized siRNA delivery in tumor-bearing mice.

Transferrin (TF) is widely used as a tumor-targeting ligand for the delivery of anticancer drugs because the TF receptor is overexpressed on the surface of various fast-growing cancer cells. In this article, we report on TF nanoparticles as an siRNA delivery carrier for in vivo tumor-specific gene silencing. To produce siRNA carrying TF nanoparticles (NPs), both TF and siRNA were chemically modified with sulfhydryl groups that can build up self-cross-linked siRNA-TF NPs. Self-polymerized 5'-end thiol-modified siRNA (poly siRNA, psi) and thiolated transferrin (tTF) were spontaneously cross-linked to form stable NPs (psi-tTF NPs) under optimized conditions, and they could be reversibly degraded to release functional monomeric siRNA molecules under reductive conditions. Receptor-mediated endocytosis of TF induced rapid tumor-cell-specific uptake of the psi-tTF NPs, and the internalized NPs resulted in a downregulation of the target protein in red-fluorescent-protein-expressing melanoma cancer cells (RFP/B16F10) with negligible cytotoxicity. After systemic administration, the psi-tTF NPs showed marked accumulation at the tumor, leading to successful target-gene silencing in vivo. This psi-tTF NP system provided a safe and effective strategy for in vivo systemic siRNA delivery for cancer therapy.

Cancer cell-specific photoactivity of pheophorbide a-glycol chitosan nanoparticles for photodynamic therapy in tumor-bearing mice.

We designed a cancer-cell specific photosensitizer nano-carrier by synthesizing pheophorbide a (PheoA) conjugated glycol chitosan (GC) with reducible disulfide bonds (PheoA-ss-GC). The amphiphilic PheoA-ss-GC conjugates self-assembled in aqueous condition to form core-shell structured nanoparticles (PheoA-ss-CNPs) with good colloidal stability and switchable photoactivity. The photoactivity of PheoA-ss-CNPs in an aqueous environment was greatly suppressed by the self-quenching effect, which enabled the PheoA-ss-CNPs to remain photo-inactive and in a quenched state. However, after the cancer cell-specific uptake, the nanoparticular structure instantaneously dissociated by reductive cleavage of the disulfide linkers, followed by an efficient dequenching process. Compared to non-reducible PheoA-conjugated GC-NPs with stable amide linkages (PheoA-CNPs), PheoA-ss-CNPs rapidly restored their photoactivity in response to intracellular reductive conditions, thus presenting higher cytotoxicity with light treatment. In addition, the PheoA-ss-CNPs presented prolonged blood circulation in vivo compared to free PheoA, demonstrating enhanced tumor specific targeting behavior through the enhanced permeation and retention (EPR) effect. The enhanced tumor accumulation of PheoA-ss-CNPs enabled tumor therapeutic efficacy that was more efficient than free PheoA in tumor-bearing mice. Based on the enhanced intracellular release for cytosolic high dose and switchable photoactivity mechanism for reduced side effects, these results suggest that PheoA-ss-CNPs have good potential for photodynamic therapy (PDT) in cancer treatment.

Gas-generating polymeric microspheres for long-term and continuous in vivo ultrasound imaging.

Ultrasound (US) imaging is one of the most common biomedical imaging methods, due to the easy assessment and noninvasive way. For more precise and accurate US imaging, many contrast agents have been developed in a form of microbubbles composed of inner gas and shell materials. However, microbubbles showed undesirable short half-life under acoustic field during US imaging and insufficient in vivo stability in blood flow due to diffusion or bubble destruction. Therefore, the improvement of the half-life and stability of microbubbles under in vivo condition is highly needed for long-term in vivo US imaging. Herein, we developed rationally designed gas-generating polymeric microsphere (GGPM) that can produce microbubbles without encapsulation of gas for long-term and continuous US imaging. The poly(cholesteryl γ-butyrolactone-b-propylene oxide), poly(CB-PO), with carbonate side chains was synthesized as gas-generating polymer by ring-opening polymerization of cholestryl γ-butyrolactone (CB) and propylene oxide (PO). As optimal structure for intense US signal generation, porous GGPMs (p-GGPMs) with the average size about 3-5 µm were prepared with poly(CB-PO) by double emulsion method. These p-GGPMs generated continuous US signals over 70 min, while the signals from Sonovue(®), a commercial US contrast agent were completely attenuated within 15 min. This long-term signal duration of p-GGPM was also reproduced when they were subcutaneously injected under the skin of mouse. Moreover, as advanced in vivo application, the fine US imaging of heart in rat was enabled by intravenous injection of p-GGPM. Therefore, these overall results showed the great potential of p-GGPM as gas-generating US contrast agent for in vivo biomedical imaging and diagnosis.

A new hydrotropic block copolymer micelle system for aqueous solubilization of paclitaxel.

Paclitaxel (PTX), a potent anti-cancer drug, is poorly soluble in water, and this has been a major limitation in developing patient friendly formulations for clinical applications. Recent studies on polymeric micelles, especially hydrotropic polymer micelles, have suggested an alternative formulation of PTX based on their high loading capacity and physical stability in aqueous media. The present study aims at aqueous solubilization of PTX in polymer micelles without using any organic solvents that is usually required for solubilization in polymer micelles. Poly(ethylene glycol) was used as a hydrophilic block and, as a hydrotropic block, poly(4-(2-vinylbenzyloxy-N-picolylnicotinamide)) (P(2-VBOPNA)) was synthesized by atom transfer radical polymerization. The hydrotropic block copolymers did not form a micellar structure at pH 2 or below due to protonation of PNA groups, but the aqueous solubility of PTX increased significantly by the hydrotropic activity of P(2-VBOPNA). At pH values higher than 2, the PTX solubility increased even further due to deprotonation of 2-VBOPNA, leading to effective polymer micellization. A longer hydrotropic block resulted in higher aqueous PTX solubility, and slightly slower release rate from the micelles. The hydrotropic block copolymers synthesized in this study are able to form PTX-loaded polymeric micelles in aqueous solution without using any organic solvents.