Polyplex Micelles with Phenylboronate/Gluconamide Cross-Linking in the Core Exerting Promoted Gene Transfection through Spatiotemporal Responsivity to Intracellular pH and ATP Concentration.

Polyplexes as gene delivery carriers require integrated functionalities to modulate intracellular trafficking for efficient gene transfection. Herein, we developed plasmid DNA (pDNA)-loaded polyplex micelles (PMs) from poly(ethylene glycol)-based block catiomers derivatized with 4-carboxy-3-fluorophenylboronic acid (FPBA) and d-gluconamide to form pH- and ATP-responsive cross-linking in the core. These PMs exhibited robustness in the extracellular milieu and smooth endosomal escape after cellular uptake, and they facilitated pDNA decondensation triggered by increased ATP concentration inside of the cell. Laser confocal microscopic observation revealed that FPBA installation enhanced the endosomal escapability of the PMs; presumably, this effect resulted from the facilitated endo-/lysosomal membrane disruption triggered by the released block catiomers with hydrophobic FPBA moieties in the side chain from the PM at lower pH condition of endo-/lysosomes. Furthermore, the profile of intracellular pDNA decondensation from the PMs was monitored using Förster resonance energy transfer measurement by flow cytometry; these observations confirmed that PMs optimized for ATP-responsivity exerted effective intracellular decondensation of loaded pDNA to attain promoted gene transfection.

Synthetic Polyamines to Regulate mRNA Translation through the Preservative Binding of Eukaryotic Initiation Factor 4E to the Cap Structure.

Polyion complexes (PICs) of mRNA with synthetic polyamines are receiving increasing attention as mRNA delivery vehicles, and the search for polyamine structure maximizing the translational efficiency of complexed mRNA becomes a critical research topic. Herein, we discovered that fine-tuning of the protonation status of synthetic polyamines can regulate mRNA translation through the preservative binding of eukaryotic initiation factor 4E to m(7)GpppN (cap structure) on the 5' end of mRNA. A series of polyamines with varied numbers of aminoethylene repeats in their side chains were prepared by an aminolysis reaction of poly(ß-benzyl-l-aspartate) and paired with mRNA to form PICs. PICs formed from polyamines with higher numbers of aminoethylene repeats preserved the original translational efficiency to naked mRNA, whereas the efficiency significantly dropped by decreasing the number of aminoethylene repeats in the polyamines. Immunoprecipitation assays using anti-eIF4E antibodies revealed that the binding affinity of eIF4E to the cap structure of mRNA in the PIC was sensitive to the number of charged aminoethylene repeats in the polyamine side chain and was strongly correlated with their translational efficiency. These results indicate that the fine-tuning of the polyamine structure plays a critical role in maximizing the translational efficiency of mRNA in the PICs having potential utility as mRNA delivery vehicles.

Polyplex Micelles with Double-Protective Compartments of Hydrophilic Shell and Thermoswitchable Palisade of Poly(oxazoline)-Based Block Copolymers for Promoted Gene Transfection.

Improving the stability of polyplex micelles under physiological conditions is a critical issue for promoting gene transfection efficiencies. To this end, hydrophobic palisade was installed between the inner core of packaged plasmid DNA (pDNA) and the hydrophilic shell of polyplex micelles using a triblock copolymer consisting of hydrophilic poly(2-ethyl-2-oxazoline), thermoswitchable amphiphilic poly(2-n-propyl-2-oxazoline) (PnPrOx) and cationic poly(L-lysine). The two-step preparation procedure, mixing the triblock copolymer with pDNA below the lower critical solution temperature (LCST) of PnPrOx, followed by incubation above the LCST to form a hydrophobic palisade of the collapsed PnPrOx segment, induced the formation of spatially aligned hydrophilic-hydrophobic double-protected polyplex micelles. The prepared polyplex micelles exhibited significant tolerance against attacks from nuclease and polyanions compared to those without hydrophobic palisades, thereby promoting gene transfection. These results corroborated the utility of amphiphilic poly(oxazoline) as a molecular thermal switch to improve the stability of polyplex gene carriers relevant for physiological applications.

Intracellular Delivery of Charge-Converted Monoclonal Antibodies by Combinatorial Design of Block/Homo Polyion Complex Micelles.

Direct intracellular delivery of antibodies has gained much attention, although only a few agents have been developed, and none of them has reached clinical stages. The main obstacles here are the insufficient characteristics of delivery systems including stability and appropriate ability for intracellular antibody release. We tailored the structure of polyion complex (PIC) micelles by loading transiently charge-converted antibody derivatives for achieving enhanced stability, delivery to cytosol, and precise antigen recognition inside cells. Citraconic anhydride was used for the charge conversion of the antibody; the optimized degree of modification was identified to balance the stability of PIC micelles in the extracellular compartment and prompt pH-triggered disintegration after their translocation into the acidic endosomal compartment of target cells. The use of a mixture of homo- and block-catiomers in an appropriate ratio to construct PIC micelles substantially enhanced the endosomal escaping efficacy of the loaded antibody, leading to improved recognition of intracellular antigens.

Modulated protonation of side chain aminoethylene repeats in N-substituted polyaspartamides promotes mRNA transfection.

Fine-tuning of chemical structures of polycation-based carriers (polyplexes) is an attractive strategy for safe and efficient mRNA transfaction. Here, mRNA polyplexes comprising N-substituted polyaspartamides with varied numbers of side chain aminoethylene repeats were constructed, and their transfection ability against human hepatoma cells was examined. Transfection efficacy clearly correlated with the number of aminoethylene repeats: polyplexes with odd number repeats (PA-Os) produced sustained increases in mRNA expression compared with those with even number repeats (PA-Es). This predominant efficacy of PA-Os over PA-Es was contradictory to our previous findings for pDNA polyplexes prepared from the same N-substituted polyaspartamides, that is, PA-Es revealed superior transfection efficacy of pDNA than PA-Os. Intracellular FRET analysis using flow cytometry and polyplex tracking under confocal laser scanning microscopy revealed that overall transfection efficacy was determined through the balance between endosomal escaping capability and stability of translocated mRNA in cytoplasm. PA-Es efficiently transported mRNA into the cytoplasm. However, their poor cytoplasmic stability led to facile degradation of mRNA, resulting in a less durable pattern of transfection. Alternatively, PA-Os with limited capability of endosomal escape eventually protect mRNA in the cytoplasm to induce sustainable mRNA expression. Higher cytoplasmic stability of pDNA compared to mRNA may shift the limiting step in transfection from cytoplasmic stability to endosomal escape capacity, thereby giving an opposite odd-even effect in transfection efficacy. Endosomal escaping capability and nuclease stability of polyplexes are correlated with the modulated protonation behavior in aminoethylene repeats responding to pH, appealing the substantial importance of chemistry to design polycation structures for promoted mRNA transfection.

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.

PEGylated polyplex with optimized PEG shielding enhances gene introduction in lungs by minimizing inflammatory responses.

Safety is a critical issue in clinical applications of nonviral gene delivery systems. Safe and effective gene introduction into the lungs was previously achieved using polyplexes from poly(ethyleneglycol) (PEG)-block-polycation [PEG-block-PAsp(DET)] and plasmid DNA (pDNA). Although PEGylated polyplexes appeared to be safe, an excess ratio of polycation to pDNA was needed to obtain sufficient transgene expression, which may cause toxicities shortly after gene introduction. In the present study, we investigated the combined use of two polymers, PEG-block-PAsp(DET) (B) and homo PAsp(DET) (H) across a range of mixing ratios to construct polyplexes. Although transgene expressions following in vitro transfections increased in parallel with increased proportions of H, polyplexes with B/H = 50/50 formulation produced the highest expression level following in vivo intratracheal administration. Higher proportions of H elicited high levels of cytokine induction with significant inflammation as assessed by histopathological examinations. Based on the aggregation behavior of polyplexes in bronchoalveolar lavage fluids (BALFs), we suggested that rapid aggregation of polyplexes in the lung induced acute inflammatory responses, resulting in reduced transgene expression. B/H formulation of polyplex can help to improve gene therapy for the respiratory system because it achieves both effective PEG shielding of polyplexes and functioning of PAsp(DET) polycations to enhance endosomal escape.

Accelerated polymer-polymer click conjugation by freeze-thaw treatment.

Herein, we report a unique technique to accelerate polymer-SNA conjugation based on copper-free click chemistry: gradual freeze-thawing of the reaction solution substantially increases the conjugation rate possibly because of the reactant concentration at the microenvironment scale. This technique was applied to the conjugation between a small interfering RNA (siRNA) and PEG in an aqueous buffer at/below room temperature.

Dual environment-responsive polyplex carriers for enhanced intracellular delivery of plasmid DNA.

In this study, we describe a multifunctional, nontoxic delivery vehicle with dual-environment sensitivity to deliver plasmid DNA (pDNA) into the cytoplasm of cells. This delivery vehicle was designed to be destabilized by reduction of disulfide cross-links in the intracellular environment and also to contain pH-sensitive membrane-destabilizing activity in acidic late endosomal/lysosomal compartments to allow escape of pDNA into the cell cytoplasm. Polyion complex formation was used to form ternary polyplexes using ionic polymers containing specific chemistries to achieve functional demands. First, template binary polyplexes were formed by association of cationic poly(l-lysine) containing thiol groups (PLys(PDP)) with pDNA and were subsequently cross-linked by disulfide formation for increased stability. Then, binary cross-linked polyplexes were coated with a pH-sensitive membrane-active polyanion, poly(ethylene glycol)-b-poly(aspartamide(DET-Aco)) (PEG-PAsp(DET-Aco)), to produce ternary cross-linked polyplexes. PEG-PAsp(DET-Aco) comprises two repeating units of aminoethylene in PAsp side chains and primary amines modified with anionic cis-aconitic groups. PEG-PAsp(DET-Aco) degrades at acidic pH to generate the parent PEG-PAsp(DET) polymer, which is active toward late endosomal/lysosomal membranes and thus can assist in the endosomal escape of pDNA following endocytosis. Binary/ternary cross-linked polyplexes remained stable toward counter polyanion exchange with dextran sulfate, but released pDNA following disulfide reduction. Ternary cross-linked polyplexes formed by addition of PEG-PAsp(DET-Aco) resulted in enhanced gene transfection efficiency in cultured cells (Huh-7 and HUVEC) without associated cytotoxicity. The enhanced gene transfection was found to be correlated with improved endosomal escape by observation of intracellular trafficking using confocal laser scanning microscopy. This multifunctional ternary cross-linked polyplex demonstrates the successful design of a gene delivery vehicle utilizing intracellular stimuli, and is a promising platform for further development toward practical use.

Odd-even effect of repeating aminoethylene units in the side chain of N-substituted polyaspartamides on gene transfection profiles.

A series of the N-substituted polyaspartamides possessing repeating aminoethylene units in the side chain was prepared in this study to identify polyplexes with effective endosomal escape and low cytotoxicity. All cationic N-substituted polyaspartamides showed appreciably lower cytotoxicity than that of commercial transfection reagents. Interestingly, a distinctive odd-even effect of the repeating aminoethylene units in the polymer side chain on the efficiencies of endosomal escape and transfection to several cell lines was observed. The polyplexes from the polymers with an even number of repeating aminoethylene units (PA-Es) achieved an order of magnitude higher transfection efficiency, without marked cytotoxicity, than those of the polymers with an odd number of repeating aminoethylene units (PA-Os). This odd-even effect agreed well with the buffering capacity of these polymers as well as their capability to disrupt membrane integrity selectively at endosomal pH, leading to highly effective endosomal escape of the PA-E polyplexes. Furthermore, the formation of a polyvalent charged array with precise spacing between protonated amino groups in the polymer side chain was shown to be essential for effective disruption of the endosomal membrane, thus facilitating transport of the polyplex into the cytoplasm. These data provide useful knowledge for designing polycations to construct safe and efficient nonviral gene carriers.

pDNA/poly(L-lysine) Polyplexes Functionalized with a pH-Sensitive Charge-Conversional Poly(aspartamide) Derivative for Controlled Gene Delivery to Human Umbilical Vein Endothelial Cells.

An efficient endosome-escaping function was integrated into the polyplex of plasmid DNA (pDNA) with poly(L-lysine) (PLys) to improve its gene transfection efficiency through electrostatic coating with charge-conversional polymer (CCP). CCP showed charge-conversional function responding to endosomal pH, leading to the release of pDNA/PLys polyplex into the cytoplasm. The cells took up the intact CCP-integrated ternary polyplex, which exerted appreciably higher transfection efficiency with lower cytotoxicity than pDNA/PLys polyplex against human umbilical vein endothelial cells (HUVECs). This is consistent with the facilitated endosomal escape of the CCP-integrated ternary polyplex compared to the pDNA/PLys polyplex as directly observed with confocal laser-scanning microscopy.

Intratracheal gene transfer of adrenomedullin using polyplex nanomicelles attenuates monocrotaline-induced pulmonary hypertension in rats.

Pulmonary arterial hypertension (PAH) is a life-threatening disease characterized by progressive PAH and right ventricular failure. Despite recent advances in therapeutic approaches using prostanoids, endothelin antagonists, and so on, PAH remains a challenging condition. To develop a novel therapeutic approach, we have established a nonviral gene delivery system of poly(ethylene glycol) (PEG)-based block catiomers, which form a polyplex nanomicelle with a nanoscaled core-shell structure in the presence of DNA. The polyplex nanomicelle from PEG-b-poly{N-[N-(2-aminoethyl)-2-aminoethyl]aspartamide} (PEG-b-P[Asp(DET)]), having ethylenediamine units at the side chain, showed ~100-fold increase in luciferase transgene expression activity in mouse lung via intratracheal administration with a minimal toxicity compared with the polyplex from linear poly(ethylenimine) (LPEI). The transfection activity was highest on day 3 after administration and remained detectable until day 14. PEG-b-P[Asp(DET)] polyplex nanomicelles were formulated with a therapeutic plasmid bearing the human adrenomedullin (AM) gene and intratracheally administered to rats with monocrotaline-induced pulmonary hypertension. The right ventricular pressure significantly decreased 3 days after administration as confirmed by a notable increase of pulmonary human AM mRNA levels. Intratracheal administration of PEG-b-P[Asp-(DET)] polyplex nanomicelles showed remarkable therapeutic efficacy with PAH animal models without compromising biocompatibility.

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).

Glucose transporter 1-mediated vascular translocation of nanomedicines enhances accumulation and efficacy in solid tumors.

Nanomedicine modification with ligands directed to receptors on tumor blood vessels has the potential for selectively enhancing nanomedicine accumulation in malignant tissues by overcoming the vascular barrier of tumors. Nevertheless, the development of broadly applicable ligand approaches capable of promoting the transvascular transport of nanomedicines in a wide spectrum of tumors has been elusive so far. By considering the indispensable and persistent glycolytic fueling of tumors, we developed glucose-installed polymeric micelles loading cisplatin (Gluc-CDDP/m) targeting the glucose transporter 1 (GLUT1), which is overexpressed in most tumors and present on vascular endothelial cells, toward improving the delivery efficiency and therapeutic efficacy. The design of the glucose ligands on Gluc-CDDP/m was engineered to control the conjugation via the carbon 6 of the glucose moieties, as well as the ligand density on the poly (ethylene glycol) (PEG) shell of the micelles. The series of micelles was then studied in vitro and in vivo against GLUT1-high human squamous cell carcinoma of the head and neck OSC-19 cells and GLUT1-low human glioblastoma-astrocytoma U87MG cells. Our results showed that precisely tuning the micelles to have glucose ligands on 25% of their PEG chains increased the efficacy against the tumors by significantly enhancing the tumor accumulation, even in GLUT1-low U87MG tumors. The enhancement of the intratumoral levels of these micelles was hindered by concomitant administration of glucose, or the GLUT1 inhibitor STF-31, confirming a GLUT1/glucose-mediated increment of the accumulation. Intravital confocal laser scanning microscopy imaging of tumor tissues further demonstrated the rapid extravasation and penetration of Gluc-CDDP/m in OSC-19 tumors compared to non-targeted CDDP/m. These findings indicate GLUT1-targeting as a promising approach for overcoming the vascular barrier and boosting the delivery of nanomedicine in tumors.

PEG-detachable polyplex micelles based on disulfide-linked block catiomers as bioresponsive nonviral gene vectors.

PEG-based polyplex micelles, which can detach the surrounding PEG chains responsive to the intracellular reducing environment, were developed as nonviral gene vectors. A novel block catiomer, PEG-SS-P[Asp(DET)], was designed as follows: (i) insertion of biocleavable disulfide linkage between PEG and polycation segment to trigger PEG detachment and (ii) a cationic segment based on poly(aspartamide) with a flanking N-(2-aminoethyl)-2-aminoethyl group, P[Asp(DET)], in which the Asp(DET) unit acts as a buffering moiety inducing endosomal escape with minimal cytotoxicity. The polyplex micelles from PEG-SS-P[Asp(DET)] and plasmid DNA (pDNA) stably dispersed in an aqueous medium with a narrowly distributed size range of approximately 80 nm due to the formation of hydrophilic PEG palisades while undergoing aggregation by the addition of 10 mM dithiothreitol (DTT) at the stoichiometric charge ratio, indicating the PEG detachment from the micelles through the disulfide cleavage. The PEG-SS-P[Asp(DET)] micelles showed both a 1-3 orders of magnitude higher gene transfection efficiency and a more rapid onset of gene expression than PEG-P[Asp(DET)] micelles without disulfide linkages, due to much more effective endosomal escape based on the PEG detachment in endosome. These findings suggest that the PEG-SS-P[Asp(DET)] micelle may have promising potential as a nonviral gene vector exerting high transfection with regulated timing and minimal cytotoxicity.

Binding enhancement of antigen-functionalized PEGylated gold nanoparticles onto antibody-immobilized surface by increasing the functionalized antigen using alpha-sulfanyl-omega-amino-PEG.

We established a technique for constructing PEGylated gold nanoparticles (GNP) that have small compounds with almost complete functionalities on their surfaces using a newly synthesized hetero-telechelic poly(ethylene glycol) (PEG), and their association/dissociation behavior on an antibody-immobilized surface was evaluated using a surface plasmon resonance (SPR) sensor.

Enhanced Intracellular Delivery of siRNA by Controlling ATP-Responsivity of Phenylboronic Acid-Functionalized Polyion Complex Micelles.

Intracellular delivery of small interfering RNA (siRNA) is a long-standing challenge in oligonucleotide therapeutics. Herein, adenosine triphosphate (ATP)-responsive polyion complex micelles assembled from poly(ethylene glycol)-block-poly(l-lysine) (PEG-PLys) bearing 4-carboxy-3-fluorophenylboronic acid (FPBA) moiety in the PLys side chains (FPBA micelle) for the delivery of cholesterol-modified siRNA (Chol-siRNA) are described. The pKa of FPBA moiety is 7.2 and, therefore, it exists in equilibrium between negatively charged tetravalent and noncharged hydrophobic trivalent forms in physiological pH conditions. Each form cooperatively stabilizes the micelle in distinct modes, that is, a covalent ester-linkage between charged boronate and ribose functionality at 3' ends of Chol-siRNA and a hydrophobic interaction between noncharged boronic acid and Chol-siRNA. When exposed to ATP at a concentration associated with the intracellular environment, the Chol-siRNA/boronate linkage is readily cleaved to facilitate the release of Chol-siRNA into cytoplasm. In order to further optimize this switching capability, the effect of FPBA modification rate is studied for the resulting ATP-responsive behavior of the micelles. As a result, the range of 23-35% in the modification rate is found suitable to maximize the gene silencing efficiency, demonstrating the potential of the FPBA-modified micelles as ATP-responsive smart siRNA carrier systems.