EDITORIAL

Ligand-mediated drug delivery systems have enormous potential for improving the efficacy of cancer treatment. In particular, Arg-Gly-Asp peptides are promising ligand molecules for targeting α_vβ₃/α_vβ₅ integrins, which are overexpressed in angiogenic sites and tumors, such as intractable human glioblastoma (U87MG). We here achieved highly efficient drug delivery to U87MG tumors by using a platinum anticancer drug-incorporating polymeric micelle (PM) with cyclic Arg-Gly-Asp (cRGD) ligand molecules. Intravital confocal laser scanning microscopy revealed that the cRGD-linked polymeric micelles (cRGD/m) accumulated rapidly and had high permeability from vessels into the tumor parenchyma compared with the PM having nontargeted ligand, “cyclic-Arg-Ala-Asp” (cRAD). As both cRGD/m- and cRAD-linked polymeric micelles have similar characteristics, including their size, surface charge, and the amount of incorporated drugs, it is likely that the selective and accelerated accumulation of cRGD/m into tumors occurred <i>via</i> an active internalization pathway, possibly transcytosis, thereby producing significant antitumor effects in an orthotopic mouse model of U87MG human glioblastoma

Aggregation Behavior of Cationic Nanohydrogel Particles in Human Blood Serum

For systemic siRNA delivery applications, well-defined drug carriers are required that guarantee stability for both carrier and cargo. Among various concepts progressing in market or final development, cationic nanohydrogel particles may serve as novel transport media especially designed for siRNA-in vivo experiments. In this work, the interaction of nanohydrogel particles with proteins and serum components was studied via dynamic light scattering in human blood serum as novel screening method prior to applications in vivo. The formation of larger aggregates mostly caused by charge interaction with albumin could be suppressed by nanogel loading with siRNA affording a neutral zeta potential for the complex. Preliminary in vivo studies confirmed the results inside the light-scattering cuvette. Although both carrier and cargo may have limited stability on their own under physiological relevant conditions, they can form safe and stable complexes at a charge neutralized ratio and thus making them applicable to systemic siRNA delivery

Aggregation Behavior of Cationic Nanohydrogel Particles in Human Blood Serum

For systemic siRNA delivery applications, well-defined drug carriers are required that guarantee stability for both carrier and cargo. Among various concepts progressing in market or final development, cationic nanohydrogel particles may serve as novel transport media especially designed for siRNA-in vivo experiments. In this work, the interaction of nanohydrogel particles with proteins and serum components was studied via dynamic light scattering in human blood serum as novel screening method prior to applications in vivo. The formation of larger aggregates mostly caused by charge interaction with albumin could be suppressed by nanogel loading with siRNA affording a neutral zeta potential for the complex. Preliminary in vivo studies confirmed the results inside the light-scattering cuvette. Although both carrier and cargo may have limited stability on their own under physiological relevant conditions, they can form safe and stable complexes at a charge neutralized ratio and thus making them applicable to systemic siRNA 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

Light-Induced Cytosolic Activation of Reduction-Sensitive Camptothecin-Loaded Polymeric Micelles for Spatiotemporally Controlled <i>in Vivo</i> 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 <i>in vivo</i>, 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 <i>in vitro</i> and <i>in vivo</i> by real-time confocal laser microscopies, accelerating the drug release from the micelles only in the irradiated tissues. This spatiotemporal switch significantly enhanced the <i>in vivo</i> 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

Tethered PEG Crowdedness Determining Shape and Blood Circulation Profile of Polyplex Micelle Gene Carriers

Surface modification by poly­(ethylene glycol) (PEG) onto gene carrier prepared through the electrostatic assembly of pDNA and polycation (polyplex) is a widely acknowledged strategy to advance their systemic application. In this regard, PEG crowdedness on the polyplex surface should give important contribution in determining blood circulation property; however its accurate quantification has never been demonstrated. We report here the first successful determination of PEG crowdedness for PEGylated polyplexes (polyplex micelle) formed from PEG–poly­(l-lysine) block copolymers (PEG–PLys) and plasmid DNA (pDNA). Tethered PEG chains were found to adopt mushroom and even squeezed conformation by modulating PEG crowdedness through PLys segment length. Energetic analysis was conducted on the polyplex micelle to elucidate effect of PEG crowdedness on shape and clarify its essential role in regulating packaging structure of pDNA within the polyplex micelle. Furthermore, the PEG crowdedness significantly correlated to blood retention profile, approving its critical role on both shape and systemic circulation property

Multicompartment Micelles with Adjustable Poly(ethylene glycol) Shell for Efficient <i>in Vivo</i> Photodynamic Therapy

We describe the preparation of well-defined multicompartment micelles from polybutadiene-<i>block</i>-poly(1-methyl-2-vinyl pyridinium methyl sulfate)-<i>block</i>-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)-<i>block</i>-poly(l-lysine) (PLL-<i>b</i>-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 <i>in vitro</i> cellular experiments, we confirmed that the porphyrazine-loaded micelles were PDT-active against A549 cells. The corona composition strongly influenced their <i>in vitro</i> PDT activity, which decreased with increasing PEGylation, correlating with the cellular uptake of the micelles. Also, a PEGylation-dependent influence on the <i>in vivo</i> 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 <i>in vivo</i>

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)-<i>b</i>-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>i.e.</i>, 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

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