Acid-Sensitive Surfactants Enhance the Delivery of Nucleic Acids.

The development of endosomal disruptive agents is a major challenge in the field of drug delivery and pharmaceutical chemistry. Current endosomal disruptive agents are composed of polymers, peptides, and nanoparticles and have had limited clinical impact. Alternatives to traditional endosomal disruptive agents are therefore greatly needed. In this report, we introduce a new class of low molecular weight endosomal disruptive agents, termed caged surfactants, that selectively disrupt endosomes via reversible PEGylation under acidic endosomal conditions. The caged surfactants have the potential to address several of the limitations hindering the development of current endosomal disruptive agents, such as high toxicity and low excretion, and are amenable to traditional medicinal chemistry approaches for optimization. In this report, we synthesized three generations of caged surfactants and demonstrated that they can enhance the ability of cationic lipids to deliver mRNA into primary cells. We also show that caged surfactants can deliver siRNA into cells when modified with the RNA-binding dye thiazole orange. We anticipate that the caged surfactants will have numerous applications in pharmaceutical chemistry and drug delivery given their versatility.

A pH-sensitive eosin-block copolymer delivers proteins intracellularly.

There is great interest in developing strategies to deliver proteins into the cytoplasm of cells. We report here a PEG-poly-eosin block copolymer (PEG-pEosin) that can encapsulate proteins and release them in active form under mildly acidic conditions. A PEG-pEosin formulation composed of Cre and the endosomolytic protein LLO efficiently performed gene editing in cells and in the brains of mice after an intracranial injection.

Particle-Size-Dependent Delivery of Antitumoral miRNA Using Targeted Mesoporous Silica Nanoparticles.

Multifunctional core-shell mesoporous silica nanoparticles (MSN) were tailored in size ranging from 60 to 160 nm as delivery agents for antitumoral microRNA (miRNA). The positively charged particle core with a pore diameter of about 5 nm and a stellate pore morphology allowed for an internal, protective adsorption of the fragile miRNA cargo. A negatively charged particle surface enabled the association of a deliberately designed block copolymer with the MSN shell by charge-matching, simultaneously acting as a capping as well as endosomal release agent. Furthermore, the copolymer was functionalized with the peptide ligand GE11 targeting the epidermal growth factor receptor, EGFR. These multifunctional nanoparticles showed an enhanced uptake into EGFR-overexpressing T24 bladder cancer cells through receptor-mediated cellular internalization. A luciferase gene knock-down of up to 65% and additional antitumoral effects such as a decreased cell migration as well as changes in cell cycle were observed. We demonstrate that nanoparticles with a diameter of 160 nm show the fastest cellular internalization after a very short incubation time of 45 min and produce the highest level of gene knock-down.

The concept of foam as a drug carrier for intraperitoneal chemotherapy, feasibility, cytotoxicity and characteristics.

For decades, intraperitoneal chemotherapy (IPC) was delivered into the abdominal cavity as a liquid solution. This preliminary study aims to evaluate foam as a potential new drug carrier for IPC delivery. Foam-based intraperitoneal chemotherapy (FBIC) was produced with taurolidine, hydrogen peroxide, human serum, potassium iodide and doxorubicin/ oxaliplatin for both ex vivo and in vitro experiments. Analysis of FBIC efficacy included evaluation of cytotoxicity, tissue penetration, foam stability, temperature changes and total foam volume per time evaluation. FBIC showed penetration rates of about 275 ± 87 µm and higher cytotoxicity compared to controls and to conventional liquid IPC (p < 0.005). The volume of the generated foam was approximately 50-times higher than the initial liquid solution and temporarily stable. Foam core temperature was measured and increased to 47 °C after 9 min. Foam ingredients (total protein content) were evenly distributed within different locations. Our preliminary results are quite encouraging and indicate that FBIC is a feasible approach. However, in order to discuss a possible superior effect over conventional liquid or aerosolized chemo applications, further studies are required to investigate pharmacologic, pharmacodynamic and physical properties of FBIC.

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.

Sequence-Defined Cationic Lipo-Oligomers Containing Unsaturated Fatty Acids for Transfection.

Sequence-defined cationic lipo-oligomers containing unsaturated fatty acids are potent nucleic acid carriers that are produced by solid-phase supported synthesis. However, the trifluoroacetic acid (TFA)-mediated removal of acid-labile protecting groups and cleavage from the resin can be accompanied by side products caused by an addition of TFA to the double bonds of unsaturated fatty acids. These TFA adducts are converted into hydroxylated derivatives under aqueous conditions. Here we describe an optimized cleavage protocol (precooling cleavage solution to 4 °C, 20 min cleavage at 22 °C), which minimizes TFA adduct formation, retains the unsaturated hydrocarbon chain character, and ensures high yields of the synthesis.

Targeting APLN/APLNR Improves Antiangiogenic Efficiency and Blunts Proinvasive Side Effects of VEGFA/VEGFR2 Blockade in Glioblastoma.

Antiangiogenic therapy of glioblastoma (GBM) with bevacizumab, a VEGFA-blocking antibody, may accelerate tumor cell invasion and induce alternative angiogenic pathways. Here we investigate the roles of the proangiogenic apelin receptor APLNR and its cognate ligand apelin in VEGFA/VEGFR2 antiangiogenic therapy against distinct subtypes of GBM. In proneural GBM, apelin levels were downregulated by VEGFA or VEGFR2 blockade. A central role for apelin/APLNR in controlling GBM vascularization was corroborated in a serial implantation model of the angiogenic switch that occurs in human GBM. Apelin and APLNR are broadly expressed in human GBM, and knockdown or knockout of APLN in orthotopic models of proneural or classical GBM subtypes significantly reduced GBM vascularization compared with controls. However, reduction in apelin expression led to accelerated GBM cell invasion. Analysis of stereotactic GBM biopsies from patients as well as from in vitro and in vivo experiments revealed increased dissemination of APLNR-positive tumor cells when apelin levels were reduced. Application of apelin-F13A, a mutant APLNR ligand, blocked tumor angiogenesis and GBM cell invasion. Furthermore, cotargeting VEGFR2 and APLNR synergistically improved survival of mice bearing proneural GBM. In summary, we show that apelin/APLNR signaling controls GBM angiogenesis and invasion and that both pathologic features are blunted by apelin-F13A. We suggest that apelin-F13A can improve the efficiency and reduce the side effects of established antiangiogenic treatments for distinct GBM subtypes. SIGNIFICANCE: Pharmacologic targeting of the APLNR acts synergistically with established antiangiogenic treatments in glioblastoma and blunts therapy resistance to current strategies for antiangiogenesis.See related commentary by Amoozgar et al., p. 2104.

Increased Tissue Penetration of Doxorubicin in Pressurized Intraperitoneal Aerosol Chemotherapy (PIPAC) after High-Intensity Ultrasound (HIUS).

BACKGROUND: High-intensity ultrasound (HIUS) has been studied for the past two decades as a new therapeutic option for solid tumor direct treatment and a method for better chemotherapy delivery and perfusion. This treatment approach has not been tested to our knowledge in peritoneal metastatic therapy, where limited tissue penetration of intraperitoneal chemotherapy has been a main problem. Both liquid instillations and pressurized aerosols are affected by this limitation. This study was performed to evaluate whether HIUS improves chemotherapy penetration rates. METHODS: High-intensity ultrasound (HIUS) was applied for 0, 5, 30, 60, 120, and 300 seconds on the peritoneal tissue samples from fresh postmortem swine. Samples were then treated with doxorubicin via pressurized intraperitoneal aerosol chemotherapy (PIPAC) under 12 mmHg and 37°C temperature. Tissue penetration of doxorubicin was measured using fluorescence microscopy on frozen thin sections. RESULTS: Macroscopic structural changes, identified by swelling of the superficial layer of the peritoneal surface, were observed after 120 seconds of HIUS. Maximum doxorubicin penetration was significantly higher in peritoneum treated with HIUS for 300 seconds, with a depth of 962.88 ± 161.4 µm (p < 0.05). Samples without HIUS had a penetration depth of 252.25 ± 60.41. Tissue penetration was significantly increased with longer HIUS duration, with up to 3.8-fold increased penetration after 300 sec of HIUS treatment. CONCLUSION: Our data indicate that HIUS may be used as a method to prepare the peritoneal tissue for intraperitoneal chemotherapy. Higher tissue penetration rates can be achieved without increasing chemotherapy concentrations and preventing structural damage to tissue using short time intervals. More studies need to be performed to analyze the effect of HIUS in combination with intraperitoneal chemotherapy.

Precise Enzymatic Cleavage Sites for Improved Bioactivity of siRNA Lipo-Polyplexes.

Sequence-defined cationic lipo-oligomers are potent siRNA carriers, forming stable lipo-polyplexes based on both electrostatic and hydrophobic interactions and, after endocytosis and endosomal protonation, facilitating the delivery of siRNA into the cytosol. After completion of the nucleic acid delivery process, carriers should be readily biodegradable to ensure minimum accumulation of amphiphilic molecules that are harmful to lysosomes and other intracellular organelles. Endolysosomal enzymes may degrade a surplus of carrier molecules left over in lysosomes and thereby facilitate the generation and rapid excretion of cleavage products. By solid-phase supported synthesis, a library of sequence-defined lipo-oligomers was generated containing artificial and natural amino acids comprising precise enzymatic cleavage sites. Incorporating either short cleavable l-arginine sequences (RR), noncleavable d-arginine linkers (rr), or varieties of both tailored the degradability of lipo-oligomers, as demonstrated upon incubation with the endolysosomal protease cathepsin B. Cleavage products were identified by MALDI-TOF mass spectrometry. The effect of improved intracellular degradation on cell tolerability was studied by transfecting Huh7-eGFPLuc and DU145-eGFPLuc cells. Positioning of enzymatic cleavage sites between a lipophilic diacyl domain and an ionizable oligocationic siRNA binding unit enabled efficient enzymatic degradation of the carrier and reduced the lytic potential under lysosomal conditions. Highly degradable carriers containing at least one l-arginine dipeptide linker significantly improved the viability of transfected cells without hampering gene silencing activity. Therefore, the precise integration of enzymatic cleavage sites in lipo-oligomers is a promising strategy toward biocompatible nucleic acid carriers.

Optimized Solid-Phase-Assisted Synthesis of Oleic Acid Containing siRNA Nanocarriers.

Cationic lipo-oligomers containing unsaturated oleic acid are potent siRNA carriers based on electrostatic and hydrophobic lipo-polyplex formation and endosomal membrane destabilization. Lipo-oligomers can be produced by solid-phase-supported synthesis in sequence-defined form. However, the trifluoroacetic acid (TFA)-mediated removal of acid-labile protecting groups and cleavage from the resin can be accompanied by side products caused by the addition of TFA to the double bonds of oleic acid. Under aqueous conditions, these TFA adducts of oleic acid are converted into hydroxystearic acid derivatives. The cleavage protocol was optimized to decrease TFA adducts. The pure oleic acid (C18:1) containing lipo-oligomer was compared with analogous structures containing saturated or modified hydrophobic moieties (stearic acid (C18:0), hydroxystearic acid, and 8-nonanamidooctanoic acid). The structure containing intact oleic acid shows favorable pH dependency of lytic activity, efficient gene silencing, and excellent cell tolerability relative to its counterparts.

Antitumoral Cascade-Targeting Ligand for IL-6 Receptor-Mediated Gene Delivery to Glioma.

The effective treatment of glioma is largely hindered by the poor transfer of drug delivery systems across the blood-brain barrier (BBB) and the difficulty in distinguishing healthy and tumorous cells. In this work, for the first time, an interleukin-6 receptor binding I6P7 peptide was exploited as a cascade-targeting ligand in combination with a succinoyl tetraethylene pentamine (Stp)-histidine oligomer-based nonviral gene delivery system (I6P7-Stp-His/DNA). The I6P7 peptide provides multiple functions, including the cascade-targeting potential represented by a combined BBB-crossing and subsequent glioma-targeting ability, as well as a direct tumor-inhibiting effect. I6P7-Stp-His/DNA nanoparticles (NPs) mediated higher gene expression in human glioma U87 cells than in healthy human astrocytes and a deeper penetration into glioma spheroids than scrambled peptide-modified NPs. Transport of I6P7-modified, but not the control, NPs across the BBB was demonstrated in vitro in a transwell bEnd.3 cell model resulting in transfection of underlying U87 cells and also in vivo in glioma-bearing mice. Intravenous administration of I6P7-Stp-His/plasmid DNA (pDNA)-encoding inhibitor of growth 4 (pING4) significantly prolonged the survival time of orthotopic U87 glioma-bearing mice. The results denote that I6P7 peptide is a roborant cascade-targeting ligand, and I6P7-modified NPs might be exploited for efficient glioma therapy.

Design of Poly-l-Glutamate-Based Complexes for pDNA Delivery.

Due to the polyanionic nature of DNA, typically cationic or neutral delivery vehicles have been used for gene delivery. As a new approach, this study focuses on the design, development, and validation of nonviral polypeptide-based carriers for oligonucleotide delivery based on a negatively charged poly-l-glutamic acid (PGA) backbone partly derivatized with oligoaminoamide residues. To this end, PGA-derivatives modified with different pentameric succinyl tetraethylene pentamines (Stp5 ) are designed. Optionally, histidines for modulation of endosomal buffer capacity and cysteines for pDNA complex stabilization are included, followed by characterization of biophysical properties and gene transfer efficiency in N2a neuroblastoma or 4T1 breast cancer cells.

How to Tackle the Challenge of siRNA Delivery with Sequence-Defined Oligoamino Amides.

RNA interference (RNAi) as a mechanism of gene regulation provides exciting opportunities for medical applications. Synthetic small interfering RNA (siRNA) triggers the knockdown of complementary mRNA sequences in a catalytic fashion and has to be delivered into the cytosol of the targeted cells. The design of adequate carrier systems to overcome multiple extracellular and intracellular roadblocks within the delivery process has utmost importance. Cationic polymers form polyplexes through electrostatic interaction with negatively charged nucleic acids and present a promising class of carriers. Issues of polycations regarding toxicity, heterogeneity, and polydispersity can be overcome by solid-phase-assisted synthesis of sequence-defined cationic oligomers. These medium-sized highly versatile nucleic acid carriers display low cytotoxicity and can be modified and tailored in multiple ways to meet specific requirements of nucleic acid binding, polyplex size, shielding, targeting, and intracellular release of the cargo. In this way, sequence-defined cationic oligomers can mimic the dynamic and bioresponsive behavior of viruses.

Precise redox-sensitive cleavage sites for improved bioactivity of siRNA lipopolyplexes.

Lipo-oligomers have been proven as potent siRNA carriers based on stable electrostatic and hydrophobic complex formation and endosomal membrane destabilization. Although high stability of siRNA polyplexes is desirable in the extracellular space and cellular uptake, intracellular disassembly is important for the cytosolic release of siRNA and RNA-induced silencing complex formation. To improve the release, bioreducible sequence-defined lipo-oligomers were synthesized by solid-phase assisted synthesis using the disulfide building block Fmoc-succinoyl-cystamine for precise positioning of a disulfide unit between a lipophilic diacyl (bis-myristyl, bis-stearyl or bis-cholestanyl) domain and an ionizable oligocationic siRNA binding unit. Reducible siRNA polyplexes show higher gene silencing efficacy and lower cytotoxicity than their stable analogs, consistent with glutathione-triggered siRNA release and reduced lytic activity.

Targeted siRNA Delivery Using a Lipo-Oligoaminoamide Nanocore with an Influenza Peptide and Transferrin Shell.

Developing RNA-interference-based therapeutic approaches with efficient and targeted cytosolic delivery of small interfering RNA (siRNA) is remaining a critical challenge since two decades. Herein, a multifunctional transferrin receptor (TfR)-targeted siRNA delivery system (Tf&INF7) is designed based on siRNA complexes formed with the cationic lipo-oligoamino amide 454, sequentially surface-modified with polyethylene glycol-linked transferrin (Tf) for receptor targeting and the endosomolytic peptide INF7 for efficient cytosolic release of the siRNA. Effective Tf&INF7 polyplex internalization and target gene silencing are demonstrated for the TfR overexpressing tumor cell lines (K562, D145, and N2a). Treatment with antitumoral EG5 siRNA results in a block of tumor cell growth and triggered apoptosis. Tf-modified polyplexes are far more effective than the corresponding albumin- (Alb) or nonmodified 454 polyplexes. Competition experiments with excess of Tf demonstrate TfR target specificity. As alternative to the ligand Tf, an anti-murine TfR antibody is incorporated into the polyplexes for specific targeting and gene silencing in the murine N2a cell line. In vivo distribution studies not only demonstrate an enhanced tumor residence of siRNA in N2a tumor-bearing mice with the Tf&INF7 as compared to the 454 polyplex group but also a reduced siRNA nanoparticle stability limiting the in vivo performance.