Double Click-Functionalized siRNA Polyplexes for Gene Silencing in Epidermal Growth Factor Receptor-Positive Tumor Cells.

Sequence-defined lipo-oligomers generated via solid-phase assisted synthesis have been developed as siRNA delivery systems for RNA-interference (RNAi) based gene silencing. Here, novel siRNA lipo-polyplexes were established, which were postmodified with monovalent or bivalent DBCO-PEG24 agents terminated with peptide GE11 (YHWYGYTPQNVI) for epidermal growth factor receptor (EGFR)-targeted siRNA delivery into EGFR-positive tumor cells. Lipo-oligomers containing eight cationizable succinoyltetraethylene-pentamine (Stp) units mediated higher siRNA nanoparticle core stability than those containing four Stp units, and the incorporation of histidines for enhanced endosomal buffer capacity resulted in an improved gene silencing efficiency. Lipo-polyplexes modified with monovalent or bivalent PEG-GE11 via the copper-free click reaction possessed significantly enhanced cellular internalization and transfection efficiency in EGF receptor-positive human cervical KB and hepatoma Huh7 cells in comparison with the corresponding lipo-polyplexes shielded with PEG24 without targeting. Furthermore, modification with the bivalent DBCO-PEG24-GE11 ligand resulted in higher gene silencing efficiency than modification with the same equivalents of the monovalent DBCO-PEG24-GE11 ligand.

Co-delivery of pretubulysin and siEG5 to EGFR overexpressing carcinoma cells.

Small interfering RNA (siRNA) represents a new class of therapeutic agents. Its successful intracellular delivery is a major challenge. Lipo-oligomeric carriers can complex siRNA into lipopolyplexes and thus mediate its cellular uptake. In this study, siRNA against the kinesin related mRNA EG5 gene (siEG5) and the microtubule inhibitor pretubulysin (PT) were co-formulated into polyplexes using azide-containing lipo-oligomer 1198. Nanoparticles were further modified by click reaction using shielding agent DBCO-PEG or EGFR targeting peptide GE11 (DBCO-PEG-GE11). Polyplexes displayed efficient payload incorporation and homogenous particle sizes of 200 nm. The biological effects of the unmodified and surface-functionalized polyplexes were investigated. The successful GE11-mediated intracellular delivery of siRNA into the EGFR overexpressing KB and Huh7 cell lines facilitated potent silencing of an EGFP-luciferase reporter gene by GFP siRNA. Specific downregulation of EG5 mRNA by siEG5 resulted in the expected antitumoral activity. The combination formulation 1198 siEG5 + PT provided superior antitumoral activity over free PT and 1198 siEG5.

Combination Chemotherapy of L1210 Tumors in Mice with Pretubulysin and Methotrexate Lipo-Oligomer Nanoparticles.

In the current study, nanoparticles containing the antimetabolite drug methotrexate (MTX) and the novel tubulin-binding drug pretubulysin (PT) were developed for combination chemotherapy. Polyelectrolyte complexes were formed based on ∼20 nm cationic nanomicelles of lipo-oligomer 454 with the anionic MTX at the molar ratio of 3:1, resulting in spherical nanoparticles with sizes of 150 nm (454 MTX). Particle formation in the presence of PT, which also interacts with 454, resulted in coloaded micelle complexes (454 PT+MTX) of 170 nm as demonstrated by transmission electron microscopy and dynamic light scattering measurements. Both drugs were incorporated to a high extent (∼85% for MTX, ∼70% for PT). Nanoparticles were stable in up to 20% serum and physiological NaCl solution. Cellular internalization of 454 PT+MTX into L1210 leukemia and KB cervix carcinoma cells was determined by confocal light scattering microscopy. The antitumor activity of the drug combination PT+MTX in both cell lines was strongly increased by drug formulation with 454 with IC50 values of PT+MTX decreasing 11-fold from 0.22 nM to 19 pM on L1210 cells and 6-fold from 2.8 to 0.48 nM on KB cervix carcinoma cells. Systemic treatment of NMRI nu/nu mice bearing subcutaneous L1210 tumors with 454 PT+MTX nanoparticles resulted in a more effective delay of tumor growth in comparison to the free drug combination of PT+MTX without 454. Importantly, nanoparticle formulation of PT+MTX with 454 increased the survival of mice by more than 100% compared to that of the buffer treated group and more than 40% compared to that of the free drug group.

Combined antitumoral effects of pretubulysin and methotrexate.

Pretubulysin (PT), a potent tubulin-binding antitumoral drug, and the well-established antimetabolite methotrexate (MTX) were tested separately or in combination (PT+MTX) for antitumoral activity in L1210 leukemia cells or KB cervix carcinoma cells in vitro and in vivo in NMRI-nu/nu tumor mouse models. In cultured L1210 cells, treatment with PT or MTX displays strong antitumoral effects in vitro, and the combination PT+MTX exceeds the effect of single drugs. PT also potently kills the MTX resistant KB cell line, without significant MTX combination effect. Cell cycle analysis reveals the expected arrest in G1/S by MTX and in G2/M by PT. In both cell lines, the PT+MTX combination induces a G2/M arrest which is stronger than the PT-triggered G2/M arrest. PT+MTX does not change rates of apoptotic L1210 or KB cells as compared to single drug applications. Confocal laser scanning microscopy images show the microtubule disruption and nuclear fragmentation induced by PT treatment of L1210 and KB cells. MTX changes the architecture of the F-actin skeleton. PT+MTX combines the toxic effects of both drugs. In the in vivo setting, the antitumoral activity of drugs differs from their in vitro cytotoxicity, but their combination effects are more pronounced. MTX on its own does not display significant antitumoral activity, whereas PT reduces tumor growth in both L1210 and KB in vivo models. Consistent with the cell cycle effects, MTX combined at moderate dose boosts the antitumoral effect of PT in both in vivo tumor models. Therefore, the PT+MTX combination may present a promising therapeutic approach for different types of cancer.

Folate receptor-directed orthogonal click-functionalization of siRNA lipopolyplexes for tumor cell killing in vivo.

The delivery of small interfering RNA (siRNA) and its therapeutic usage as an anti-cancer agent requires a carrier system for selective internalization into the cytosol of tumor cells. We prepared folate-bearing formulations by first complexing siRNA with the novel azido-functionalized sequence-defined cationizable lipo-oligomer 1106 (containing two cholanic acids attached to an oligoaminoamide backbone in T-shape configuration) into spherical, ∼100-200 nm sized lipopolyplexes, followed by surface-functionalization with various folate-conjugated DBCO-PEG agents. Both the lipo-oligomer and the different defined shielding and targeting agents with mono- and bis-DBCO and varying PEG length were generated by solid phase supported synthesis. A bivalent DBCO surface agent with a PEG24 spacer was identified as the optimal formulation in terms of nanoparticle size, folate receptor (FR) targeting, cellular uptake and gene silencing in vitro. Notably, near-infrared fluorescence bioimaging studies showed that double-click incorporation of bivalent DBCO-PEG24 resulted in siRNA/1106/DBCO2-ss2-PEG24-FolA lipopolyplexes with extended biodistribution and intratumoral delivery in a subcutaneous FR-positive leukemia mouse model. Intravenous administration of analogous therapeutic siRNA lipopolyplexes (directed against the kinesin spindle motor protein EG5) mediated tumoral EG5 mRNA knockdown by ∼60% and, in combination with the novel antitubulin drug pretubulysin, significantly prolonged survival of aggressive leukemia bearing mice without noticeable side effects.

Efficient Shielding of Polyplexes Using Heterotelechelic Polysarcosines.

Shielding agents are commonly used to shield polyelectrolyte complexes, e.g., polyplexes, from agglomeration and precipitation in complex media like blood, and thus enhance their in vivo circulation times. Since up to now primarily poly(ethylene glycol) (PEG) has been investigated to shield non-viral carriers for systemic delivery, we report on the use of polysarcosine (pSar) as a potential alternative for steric stabilization. A redox-sensitive, cationizable lipo-oligomer structure (containing two cholanic acids attached via a bioreducible disulfide linker to an oligoaminoamide backbone in T-shape configuration) was equipped with azide-functionality by solid phase supported synthesis. After mixing with small interfering RNA (siRNA), lipopolyplexes formed spontaneously and were further surface-functionalized with polysarcosines. Polysarcosine was synthesized by living controlled ring-opening polymerization using an azide-reactive dibenzo-aza-cyclooctyne-amine as an initiator. The shielding ability of the resulting formulations was investigated with biophysical assays and by near-infrared fluorescence bioimaging in mice. The modification of ~100 nm lipopolyplexes was only slightly increased upon functionalization. Cellular uptake into cells was strongly reduced by the pSar shielding. Moreover, polysarcosine-shielded polyplexes showed enhanced blood circulation times in bioimaging studies compared to unshielded polyplexes and similar to PEG-shielded polyplexes. Therefore, polysarcosine is a promising alternative for the shielding of non-viral, lipo-cationic polyplexes.

Sequence-Defined Oligoamide Drug Conjugates of Pretubulysin and Methotrexate for Folate Receptor Targeted Cancer Therapy.

The conjugation of small molecule drugs to ligand containing carrier systems facilitates receptor targeted delivery. The folate receptor (FR) constitutes an ideal target for tumor selective therapy, being overexpressed on several tumor types. It can be targeted using the vitamin folic acid (FolA) or the structurally related drug methotrexate (MTX). Several sequence-defined oligoamides with mono- and multivalent FolA or MTX ligands and an additional thiol conjugation site are synthesized via solid-phase assisted synthesis. Their structure activity relationships are assessed in respect to dihydrofolate reductase inhibition, receptor mediated endocytosis, and cytotoxicity. Then, the tubulin-binding agent pretubulysin (PT), a highly potent drug exhibiting antitumoral, antiangiogenic, and antimetastatic properties, is conjugated via an activated mercaptane derivative to the set of FR-targeting oligoamides. In a combined PT/MTX cytotoxicity study in FR-overexpressing KB and L1210 cells, a 2-arm MTX-PT construct or the 4-arm analog displays the highest potency in the respective cell lines.

Perceived social support in adults with autism spectrum disorder and attention-deficit/hyperactivity disorder.

Perceived social support (PSS) has been related to physical and mental well-being in typically developing individuals, but systematic characterizations of PSS in autism spectrum disorder (ASD) are limited. We compared self-report ratings of the multidimensional scale of PSS (MSPSS) among age- and IQ-matched groups of adults (18-58 years) with cognitively high-functioning ASD (N = 41), or attention-deficit/hyperactivity disorder (ADHD; N = 69), and neurotypical controls (NC; N = 69). Accompanying group comparisons, we used machine learning random forest (RF) analyses to explore predictors among a range of psychopathological and socio-emotional variables. Relative to both ADHD and NC, adults with ASD showed lower MSPSS ratings, specifically for the friends subscale (MSPSS-f). Across ASD and ADHD, interindividual differences in autism severity, affective empathy, symptoms of anxiety related to social interactions, hyperactivity/impulsivity, and somatization best predicted MSPSS-f. These relationships did not differ between clinical groups. While group comparisons demonstrated greater impairment in individuals with ASD, analyzing individuals' characteristics revealed cross-diagnoses similarities in regard to their MSPSS-f relationships. This is consistent with the Research Domain Criteria framework, supporting a trans-diagnostic approach as on the path toward "precision medicine." Autism Res 2017, 10: 866-877. © 2017 International Society for Autism Research, Wiley Periodicals, Inc.

Influence of Defined Hydrophilic Blocks within Oligoaminoamide Copolymers: Compaction versus Shielding of pDNA Nanoparticles.

Cationic polymers are promising components of the versatile platform of non-viral nucleic acid (NA) delivery agents. For a successful gene delivery system, these NA vehicles need to comprise several functionalities. This work focuses on the modification of oligoaminoamide carriers with hydrophilic oligomer blocks mediating nanoparticle shielding potential, which is necessary to prevent aggregation or dissociation of NA polyplexes in vitro, and hinder opsonization with blood components in vivo. Herein, the shielding agent polyethylene glycol (PEG) in three defined lengths (12, 24, or 48 oxyethylene repeats) is compared with two peptidic shielding blocks composed of four or eight repeats of sequential proline-alanine-serine (PAS). With both types of shielding agents, we found opposing effects of the length of hydrophilic segments on shielding and compaction of formed plasmid DNA (pDNA) nanoparticles. Two-arm oligoaminoamides with 37 cationizable nitrogens linked to 12 oxyethylene units or four PAS repeats resulted in very compact 40⁻50 nm pDNA nanoparticles, whereas longer shielding molecules destabilize the investigated polyplexes. Thus, the balance between sufficiently shielded but still compact and stable particles can be considered a critical optimization parameter for non-viral nucleic acid vehicles based on hydrophilic-cationic block oligomers.

CD25(+)CD4(+) regulatory T cells from the peripheral blood of asymptomatic HIV-infected individuals regulate CD4(+) and CD8(+) HIV-specific T cell immune responses in vitro and are associated with favorable clinical markers of disease status.

Human immunodeficiency virus (HIV) disease is associated with loss of CD4(+) T cells, chronic immune activation, and progressive immune dysfunction. HIV-specific responses, particularly those of CD4(+) T cells, become impaired early after infection, before the loss of responses directed against other antigens; the basis for this diminution has not been elucidated fully. The potential role of CD25(+)CD4(+) regulatory T cells (T reg cells), previously shown to inhibit immune responses directed against numerous pathogens, as suppressors of HIV-specific T cell responses was investigated. In the majority of healthy HIV-infected individuals, CD25(+)CD4(+) T cells significantly suppressed cellular proliferation and cytokine production by CD4(+) and CD8(+) T cells in response to HIV antigens/peptides in vitro; these effects were cell contact dependent and IL-10 and TGF-beta independent. Individuals with strong HIV-specific CD25(+) T reg cell function in vitro had significantly lower levels of plasma viremia and higher CD4(+): CD8(+) T cell ratios than did those individuals in whom this activity could not be detected. These in vitro data suggest that CD25(+)CD4(+) T reg cells may contribute to the diminution of HIV-specific T cell immune responses in vivo in the early stages of HIV disease.