A Protein Nanocarrier from Charge-Conversion Polymer in Response to Endosomal pH

A new type of polyion complex (PIC) micelle was prepared from lysozyme and the block copolymer, PEG-pAsp(EDA-Cit), that can switch the charge from anionic to cationic at the endosomal pH. The charge-conversion was due to the degradation of the citraconic amide side chain at pH 5.5. This abrupt charge-conversion can make the PIC micelles promptly release the internal protein in response to the endosomal pH. This pH-sensitive charge-conversion polymer is promising for the future design of nanocarriers for early endosomal release

PEGylated Polyplex Micelles from Triblock Catiomers with Spatially Ordered Layering of Condensed pDNA and Buffering Units for Enhanced Intracellular Gene Delivery

An A−B−C type triblock copolymer, tandemly aligning two types of polycations with different pKa values in a single polymer strand, was developed for the construction of novel polyplex micelles, satisfying a high DNA condensing ability as well as a proton buffering activity directed to elevating gene transfection. The micelle might feature the distinctive three-layered structure, where an inner polyplex layer of condensed pDNA with poly(l-lysine) (pKa ∼9.4) as the C segment is successively wrapped with an intermediate layer of poly[(3-morpholinopropyl)aspartamide] (B segment) with a comparatively low pKa of ∼6.2, to provide a buffering effect, and an outer PEG layer (A segment) as a biocompatible palisade

Preparation and Biological Characterization of Polymeric Micelle Drug Carriers with Intracellular pH-Triggered Drug Release Property: Tumor Permeability, Controlled Subcellular Drug Distribution, and Enhanced in Vivo Antitumor Efficacy

A novel intracellular pH-sensitive polymeric micelle drug carrier that controls the systemic, local, and subcellular distributions of pharmacologically active drugs has been developed in this study. The micelles were prepared from self-assembling amphiphilic block copolymers, poly(ethylene glycol)-poly(aspartate hydrazone adriamycin), in which the anticancer drug, adriamycin, was conjugated to the hydrophobic segments through acid-sensitive hydrazone linkers. By this polymer design, the micelles can stably preserve drugs under physiological conditions (pH 7.4) and selectively release them by sensing the intracellular pH decrease in endosomes and lysosomes (pH 5−6). In vitro and in vivo studies show that the micelles have the characteristic properties, such as an intracellular pH-triggered drug release capability, tumor-infiltrating permeability, and effective antitumor activity with extremely low toxicity. The acquired experimental data clearly elucidate that the optimization of both the functional and structural features of polymeric micelles provides a promising formulation not only for the development of intracellular environment-sensitive supramolecular devices for cancer therapeutic applications but also for the future treatment of intractable cancers with limited vasculature

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 <i>in vivo</i> 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

Polyplexes from Poly(aspartamide) Bearing 1,2-Diaminoethane Side Chains Induce pH-Selective, Endosomal Membrane Destabilization with Amplified Transfection and Negligible Cytotoxicity

Polyplexes assembled from poly(aspartamide) derivatives bearing 1,2-diaminoethane side chains, [PAsp(DET)] display amplified in vitro and in vivo transfection activity with minimal cytotoxicity. To elucidate the molecular mechanisms involved in this unique function of PAsp(DET) polyplexes, the physicochemical and biological properties of PAsp(DET) were thoroughly evaluated with a control bearing 1,3-diaminopropane side chains, PAsp(DPT). Between PAsp(DET) and PAsp(DPT) polyplexes, we observed negligible physicochemical differences in particle size and ζ-potential. However, the one methylene variation between 1,2-diaminoethane and 1,3-diaminopropane drastically altered the transfection profiles. In sharp contrast to the constantly high transfection efficacy of PAsp(DET) polyplexes, even in regions of excess polycation to plasmid DNA (pDNA) (high N/P ratio), PAsp(DPT) polyplexes showed a significant drop in the transfection efficacy at high N/P ratios due to the progressively increased cytotoxicity with N/P ratio. The high cytotoxicity of PAsp(DPT) was closely correlated to its strong destabilization effect on cellular membrane estimated by hemolysis, leakage assay of cytoplasmic enzyme (LDH assay), and confocal laser scanning microscopic observation. Interestingly, PAsp(DET) revealed minimal membrane destabilization at physiological pH, yet there was significant enhancement in the membrane destabilization at the acidic pH mimicking the late endosomal compartment (pH ∼5). Apparently, the pH-selective membrane destabilization profile of PAsp(DET) corresponded to a protonation change in the flanking diamine unit, i.e., the monoprotonated gauche form at physiological pH and diprotonated anti form at acidic pH. These significant results suggest that the protonated charge state of 1,2-diaminoethane may play a substantial role in the endosomal disruption. Moreover, this novel approach for endosomal disruption neither perturbs the membranes of cytoplasmic vesicles nor organelles at physiological pH. Thus, PAsp(DET) polyplexes, residing in late endosomal or lysosomal states, smoothly exit into the cytoplasm for successful transfection without compromising cell viability

Dynamic Stabilization of Unit Polyion Complexes Incorporating Small Interfering RNA by Fine-Tuning of Cationic Block Length in Two-Branched Poly(ethylene glycol)‑<i>b</i>‑poly(l‑lysine)

To stabilize small interfering RNA (siRNA) in the bloodstream for systemic RNAi therapeutics, we previously fabricated ultrasmall siRNA nanocarriers that were sub-20 nm in hydrodynamic diameter, named as unit polyion complexes (uPICs), using two-branched poly­(ethylene glycol)-b-poly­(l-lysine) (bPEG-PLys). The blood retention time of uPICs is dramatically increased in the presence of free bPEG-PLys, suggesting dynamic stabilization of uPICs by free bPEG-PLys based on their equilibrium. Herein, we examined how the degree of polymerization of PLys (DPPLys) affected the dynamic stability of uPICs in the bloodstream during prolonged circulation. We prepared a series of bPEG-PLys with DPPLys values of 10, 13, 20, 40, and 80 for the uPIC formation and siRNA with 40 negative charges. These bPEG-PLys were then evaluated in physicochemical characterization and pharmacokinetic analyses. Structural analyses revealed that the uPIC size and association numbers were mainly determined by the molecular weights of PEG and DPPLys, respectively. Under bPEG-PLys-rich conditions, the hydrodynamic diameters of uPICs were 15–20 nm, which were comparable to that of the bPEG block (i.e., ∼18 nm). Importantly, DPPLys significantly affected the association constant of bPEG-PLys to siRNA (Ka) and blood retention of free bPEG-PLys. A smaller DPPLys resulted in a lower Ka and a longer blood retention time of free bPEG-PLys. Thus, DPPLys can control the dynamic stability of uPICs, i.e., the balance between Ka and blood concentration of free bPEG-PLys. Ultimately, the bPEG-PLys with DPPLys values of 14 and 19 prolonged the blood circulation of siRNA-loaded uPICs with relatively small amounts of free bPEG-PLys. This study revealed that the uPIC formation between siRNA and bPEG-PLys can be controlled by their charges, which may be helpful for designing PIC-based delivery systems

<em>In Vivo</em> Messenger RNA Introduction into the Central Nervous System Using Polyplex Nanomicelle

<div><p>Messenger RNA (mRNA) introduction is a promising approach to produce therapeutic proteins and peptides without any risk of insertion mutagenesis into the host genome. However, it is difficult to introduce mRNA <em>in vivo</em> mainly because of the instability of mRNA under physiological conditions and its strong immunogenicity through the recognition by Toll-like receptors (TLRs). We used a novel carrier based on self-assembly of a polyethylene glycol (PEG)-polyamino acid block copolymer, polyplex nanomicelle, to administer mRNA into the central nervous system (CNS). The nanomicelle with 50 nm in diameter has a core-shell structure with mRNA-containing inner core surrounded by PEG layer, providing the high stability and stealth property to the nanomicelle. The functional polyamino acids possessing the capacity of pH-responsive membrane destabilization allows smooth endosomal escape of the nanomicelle into the cytoplasm. After introduction into CNS, the nanomicelle successfully provided the sustained protein expression in the cerebrospinal fluid for almost a week. Immune responses after mRNA administration into CNS were effectively suppressed by the use of the nanomicelle compared with naked mRNA introduction. <em>In vitro</em> analyses using specific TLR-expressing HEK293 cells confirmed that the nanomicelle inclusion prevented mRNA from the recognition by TLRs. Thus, the polyplex nanomicelle is a promising system that simultaneously resolved the two major problems of <em>in vivo</em> mRNA introduction, the instability and immunogenicity, opening the door to various new therapeutic strategies using mRNA.</p> </div

Comparison of mRNA, plasmid DNA (pDNA) and protein.

<p>mRNA or pDNA that expressed GLuc was incorporated in the polyplex nanomicelle and injected into the subarachnoid space of rats by intrathecal injection. The proteinous form of GLuc was also used for intrathecal injection. GLuc expression was evaluated from cerebrospinal fluid (CSF) collected at the indicated time points. The data are presented as the mean ± standard error of the mean (s.e.m.) (N = 4). RLU, relative luminescence units.</p

In vitro analysis of Toll-like receptor (TLR) signalling after mRNA introduction.

<p>To evaluate mRNA-mediated TLR signaling, HEK293 cells expressing TLR7 (grey bars) were used. HEK293 cells expressing TLR9 (white bars), which does not recognise mRNA, were also used as a negative control. Cells were treated with naked mRNA, polyplex nanomicelle or Lipofectamine 2000 using unmodified mRNA (a, b) or modified mRNA (c, d). Expression of interleukin (IL)-8 (a, c) and interferon (IFN)-β1 (b, d) was measured at transcriptional levels using real-time quantitative PCR (RT-PCR) 4 h after mRNA introduction. The data are presented as the mean ± standard error of the mean (s.e.m.) (N = 6). Statistical significance was assessed by 2-tailed Student's t-test, *, P<0.05, ***, P<0.001.</p

Nanomedicines Eradicating Cancer Stem-like Cells <i>in Vivo</i> by pH-Triggered Intracellular Cooperative Action of Loaded Drugs

Nanomedicines capable of control over drug functions have potential for developing resilient therapies, even against tumors harboring recalcitrant cancer stem cells (CSCs). By coordinating drug interactions within the confined inner compartment of core–shell nanomedicines, we conceived multicomponent nanomedicines directed to achieve synchronized and synergistic drug cooperation within tumor cells as a strategy for enhancing efficacy, overcoming drug resistance, and eradicating CSCs. The approach was validated by using polymeric micellar nanomedicines co-incorporating the pan-kinase inhibitor staurosporine (STS), which was identified as the most potent CSC inhibitor from a panel of signaling-pathway inhibitors, and the cytotoxic agent epirubicin (Epi), through rationally contriving the affinity between the drugs. The micelles released both drugs simultaneously, triggered by acidic endosomal pH, attaining concurrent intracellular delivery, with STS working as a companion for Epi, down-regulating efflux transporters and resistance mechanisms induced by Epi. These features prompted the nanomedicines to eradicate orthotopic xenografts of Epi-resistant mesothelioma bearing a CSC subpopulation