Nonviral Pulmonary Delivery of siRNA

RNA interference (RNAi) is an important part of the cell’s defenses against viruses and other foreign genes. Moreover, the biotechnological exploitation of RNAi offers therapeutic potential for a range of diseases for which drugs are currently unavailable. Unfortunately, the small interfering RNAs (siRNAs) that are central to RNAi in the cytoplasm are readily degradable by ubiquitous nucleases, are inefficiently targeted to desired organs and cell types, and are excreted quickly upon systemic injection. As a result, local administration techniques have been favored over the past few years, resulting in great success in the treatment of viral infections and other respiratory disorders.Because there are several advantages of pulmonary delivery over systemic administration, two of the four siRNA drugs currently in phase II clinical trials are delivered intranasally or by inhalation. The air–blood barrier, however, has only limited permeability toward large, hydrophilic biopharmaceuticals such as nucleic acids; in addition, the lung imposes intrinsic hurdles to efficient siRNA delivery. Thus, appropriate formulations and delivery devices are very much needed. Although many different formulations have been optimized for in vitro siRNA delivery to lung cells, only a few have been reported successful in vivo. In this Account, we discuss both obstacles to pulmonary siRNA delivery and the success stories that have been achieved thus far.The optimal pulmonary delivery vehicle should be neither cytotoxic nor immunogenic, should protect the payload from degradation by nucleases during the delivery process, and should mediate the intracellular uptake of siRNA. Further requirements include the improvement of the pharmacokinetics and lung distribution profiles of siRNA, the extension of lung retention times (through reduced recognition by macrophages), and the incorporation of reversible or stimuli-responsive binding of siRNA to allow for efficient release of the siRNAs at the target site. In addition, the ideal carrier would be biodegradable (to address difficulties with repeated administration for the treatment of chronic diseases) and would contain targeting moieties to enhance uptake by specific cell types. None of the currently available polymer- and lipid-based formulations meet every one of these requirements, but we introduce here several promising new approaches, including a biodegradable, nonimmunogenic polyester.We also discuss imaging techniques for following the biodistribution according to the administration route. This tracking is crucial for better understanding the translocation and clearance of nanoformulated siRNA subsequent to pulmonary delivery.In the literature, the success of pulmonary siRNA delivery is evaluated solely by relief from or prophylaxis against a disease; side effects are not studied in detail. It also remains unclear which cell types in the lung eventually take up siRNA. These are critical issues for the translational use of pulmonary siRNA formulations; accordingly, we present a flow cytometry technique that can be utilized to differentiate transfected cell populations in a mouse model that expresses transgenic enhanced green fluorescence protein (EGFP). This technique, in which different cell types are identified on the basis of their surface antigen expression, may eventually help in the development of safer carriers with minimized side effects in nontargeted tissues

Modular Synthesis of Folate Conjugated Ternary Copolymers: Polyethylenimine-<i>graft</i>-Polycaprolactone-<i>block</i>-Poly(ethylene glycol)-Folate for Targeted Gene Delivery

Folate receptor (FR) is overexpressed in a variety of human cancers. Gene delivery vectors conjugated with folate as a ligand could possibly deliver gene materials into target tumor cells via FR-mediated endocytosis. This study addresses novel folate-conjugated ternary copolymers based on polyethylenimine-graft-polycaprolactone-block-poly­(ethylene glycol) (PEI-<i>g</i>-PCL-<i>b</i>-PEG-Fol) as targeted gene delivery system using a modular synthesis approach including “click” conjugation of folate moieties with heterobifunctional PEG-<i>b</i>-PCL at PEG terminus and subsequently the introduction of PEI by a Michael addition between folate-PEG-<i>b</i>-PCL and PEI via active PCL terminus. This well-controlled synthetic procedure avoids tedious separation of byproduct. The structure of PEI-<i>g</i>-PCL-<i>b</i>-PEG-Fol was confirmed by ¹H NMR and UV spectra. DNA condensation of PEI-<i>g</i>-PCL-<i>b</i>-PEG-Fol was tested using a SYBR Gold quenching assay and agarose gel electrophoresis upon heparin competition assay. Although PEI-<i>g</i>-PCL-<i>b</i>-PEG-Fol could condense DNA completely at N/P ratio >2, polyplexes of N/P ratio 10 with sizes of about 120 nm and positive zeta potentials were selected for further biological evaluations due to polyplex stability. An enhancement of cellular uptake of PEI-<i>g</i>-PCL-<i>b</i>-PEG-Fol/pDNA polyplexes was observed in FR overexpressing KB cells in comparison to unmodified PEI-<i>g</i>-PCL-<i>b</i>-PEG, through flow cytometry analysis and confocal laser scanning imaging. Importantly, this enhanced cellular uptake could be inhibited by free folic acid and did not occur in FR-negative A549 cells, demonstrating specific cell uptake by FR-mediated endocytosis. Furthermore, the transfection efficiency of PEI-<i>g</i>-PCL-<i>b</i>-PEG-Fol/pDNA polyplexes was increased approximately 14-fold in comparison to folate-negative polyplexes. Therefore, the PEI-<i>g</i>-PCL-<i>b</i>-PEG-Fol merits further investigation under in vivo conditions for targeting FR overexpressing tumors

Design and Biophysical Characterization of Bioresponsive Degradable Poly(dimethylaminoethyl methacrylate) Based Polymers for In Vitro DNA Transfection

Water-soluble, degradable polymers based on poly­(<i>N</i>,<i>N</i>-dimethylaminoethyl methacrylate) (PDMAEMA) with low cytotoxicity and good p-DNA transfection efficiency are highlighted in this article. To solve the nondegradability issue of PDMAEMA, new polymers based on DMAEMA and 5,6-benzo-2-methylene-1,3-dioxepane (BMDO) for gene transfection were synthesized. A poly­(ethylene oxide) (PEO) azo-initiator was used as free-radical initiator. PEGylation was performed to improve water solubility and to reduce cytotoxicity of the polymers. The resulting polymers contain hydrolyzable ester linkages in the backbone and were soluble in water even with very high amounts of ester linkages. These degradable copolymers showed significantly less toxicity with a MTT assay using L929 cell lines and demonstrated promising DNA transfection efficiency when compared with the gold standard poly­(ethyleneimine). Bioresponsive properties of the corresponding quaternized DMAEMA based degradable polymers were also studied. Although the quaternized DMAEMA copolymers showed enhanced water solubility, they were inferior in gene transfection and toxicity as compared to the unquaternized copolymers

Efficient and Tumor Targeted siRNA Delivery by Polyethylenimine-<i>graft</i>-polycaprolactone-<i>block</i>-poly(ethylene glycol)-folate (PEI–PCL–PEG–Fol)

Efficient delivery of functional nucleic acids into specific cells or tissues is still a challenge for gene therapy and largely depends on targeted delivery strategies. The folate receptor (FR) is known to be overexpressed extracellularly on a variety of human cancers and is therefore an outstanding gate for tumor-targeted Trojan horse-like delivery of therapeutics. In this study, an amphiphilic and biodegradable ternary copolymer conjugated with folate as ligand, polyethylenimine-<i>graft</i>-polycaprolactone-<i>block</i>-poly­(ethylene glycol)-folate (PEI–PCL–PEG–Fol) was synthesized and evaluated for targeted siRNA delivery via folate–FR recognition. The amphiphilic character of similar polymers was shown previously to support endosomal release of endocytosed nanocarriers and to promote formation of long circulating micelles. The obtained PEI–PCL–PEG–Fol exhibited less cytotoxicity in comparison with the corresponding ternary copolymer without folate (PEI–PCL–PEG) and with unmodified PEI25kDa. Stable micelle-like polyplexes with hydrodynamic diameters about 100 nm were found to have a zeta potential of +8.6 mV, which was lower than that of micelleplexes without folate-conjugation (+13–16 mV). Nonetheless, increased cellular uptake and in vitro gene knockdown of PEI–PCL–PEG–Fol/siRNA micelleplexes were observed in SKOV-3 cells, an FR overexpressing cell line, in comparison with the nonfolate-conjugated ones. Moreover, PEI–PCL–PEG–Fol/siRNA micelleplexes exhibited excellent stability <i>in vivo</i> during the analysis of 120 min and a longer circulation half life than hyPEI25kDa/siRNA polyplexes. Most interestingly, the targeted delivery system yielded 17% deposition of the i.v. injected siRNA per gram in the tumor after 24 h due to the effective folate targeting and the prolonged circulation

Targeting the Blind Spot of Polycationic Nanocarrier-Based siRNA Delivery

Polycationic nanocarriers attract increasing attention to the field of siRNA delivery. We investigated the self-assembly of siRNA <i>vs</i> pDNA with polycations, which are broadly used for nonviral gene and siRNA delivery. Although polyethyleneimine (PEI) was routinely adopted as siRNA carrier based on its efficacy in delivering pDNA, it has not been investigated yet why PEI efficiently delivers pDNA to cells but is controversially discussed in terms of efficacy for siRNA delivery. We are the first to investigate the self-assembly of PEI/siRNA <i>vs</i> PEI/pDNA and the steps of complexation and aggregation through different levels of hierarchy on the atomic and molecular scale with the novel synergistic use of molecular modeling, molecular dynamics simulation, isothermal titration calorimetry, and other characterization techniques. We are also the fist to elucidate atomic interactions, size, shape, stoichiometry, and association dynamics for polyplexes containing siRNA <i>vs</i> pDNA. Our investigation highlights differences in the hierarchical mechanism of formation of related polycation–siRNA and polycation–pDNA complexes. The results of fluorescence quenching assays indicated a biphasic behavior of siRNA binding with polycations where molecular reorganization of the siRNA within the polycations occurred at lower N/P ratios (nitrogen/phosphorus). Our results, for the first time, emphasize a biphasic behavior in siRNA complexation and the importance of low N/P ratios, which allow for excellent siRNA delivery efficiency. Our investigation highlights the formulation of siRNA complexes from a thermodynamic point of view and opens new perspectives to advance the rational design of new siRNA delivery systems

Targeting the Blind Spot of Polycationic Nanocarrier-Based siRNA Delivery

Polycationic nanocarriers attract increasing attention to the field of siRNA delivery. We investigated the self-assembly of siRNA <i>vs</i> pDNA with polycations, which are broadly used for nonviral gene and siRNA delivery. Although polyethyleneimine (PEI) was routinely adopted as siRNA carrier based on its efficacy in delivering pDNA, it has not been investigated yet why PEI efficiently delivers pDNA to cells but is controversially discussed in terms of efficacy for siRNA delivery. We are the first to investigate the self-assembly of PEI/siRNA <i>vs</i> PEI/pDNA and the steps of complexation and aggregation through different levels of hierarchy on the atomic and molecular scale with the novel synergistic use of molecular modeling, molecular dynamics simulation, isothermal titration calorimetry, and other characterization techniques. We are also the fist to elucidate atomic interactions, size, shape, stoichiometry, and association dynamics for polyplexes containing siRNA <i>vs</i> pDNA. Our investigation highlights differences in the hierarchical mechanism of formation of related polycation–siRNA and polycation–pDNA complexes. The results of fluorescence quenching assays indicated a biphasic behavior of siRNA binding with polycations where molecular reorganization of the siRNA within the polycations occurred at lower N/P ratios (nitrogen/phosphorus). Our results, for the first time, emphasize a biphasic behavior in siRNA complexation and the importance of low N/P ratios, which allow for excellent siRNA delivery efficiency. Our investigation highlights the formulation of siRNA complexes from a thermodynamic point of view and opens new perspectives to advance the rational design of new siRNA delivery systems

Angiographic comparison of the sham-operated and the subarachnoid hemorrhage (SAH)-only group, the angiographic comparison of the sham-operated group and the subarachnoid hemorrhage (SAH)-only group revealed a significant difference of arterial filling (p = 0.004, Kruskal-Walis-test).

<p>Angiographic comparison of the sham-operated and the subarachnoid hemorrhage (SAH)-only group, the angiographic comparison of the sham-operated group and the subarachnoid hemorrhage (SAH)-only group revealed a significant difference of arterial filling (p = 0.004, Kruskal-Walis-test).</p

Representative digital subtraction angiographies of A: Scham-operated group, B: SAH group and C: Treatment-group (40%) demonstrating the internal carotid artery (ICA) to external carotid artery (ECA).

<p>Representative digital subtraction angiographies of A: Scham-operated group, B: SAH group and C: Treatment-group (40%) demonstrating the internal carotid artery (ICA) to external carotid artery (ECA).</p

Preparation of <i>in situ</i> forming nimodipine depot system.

<p>Preparation of <i>in situ</i> forming nimodipine depot system.</p

Cortical microinfarct in a control animal.

<p>In the H&E stained section, a small infarct in the deeper cortical layers is visible (a). At higher magnification, a mononuclear infiltrate can be distinguished (b, arrows; inset from a), indicating ongoing resorption. Iba1 immunohistochemistry (c; d inset from c) reveals activated microglia in and around the lesion (d, arrows). Scale bar corresponds to 1000 µm (a,c) and 200 µm (b,d), respectively.</p