International Journal of Pharmaceutics (v.525, #2)

Delivery of siRNAs by Gaetano Lamberti (291-292).

Targeted si-RNA with liposomes and exosomes (extracellular vesicles): How to unlock the potential by Sophia Antimisiaris; Spyridon Mourtas; Konstantina Papadia (293-312).
Display OmittedThe concept of RNA interference therapeutics has been initiated 18 years ago, and the main bottleneck for translation of the technology into therapeutic products remains the delivery of functional RNA molecules into the cell cytoplasm. In the present review article after an introduction about the theoretical basis of RNAi therapy and the main challenges encountered for its realization, an overview of the different types of delivery systems or carriers, used as potential systems to overcome RNAi delivery issues, will be provided. Characteristic examples or results obtained with the most promising systems will be discussed. Focus will be given mostly on the applications of liposomes or other types of lipid carriers, such as exosomes, towards improved delivery of RNAi to therapeutic targets. Finally the approach of integrating the advantages of these two vesicular systems, liposomes and exosomes, as a potential solution to realize RNAi therapy, will be proposed.
Keywords: Lipid nanoparticles; Exosomes; Extracellular vesicles; targeted delivery; RNAi; siRNA; Cationic lipid;

Polymeric nanoparticles for siRNA delivery: Production and applications by Gennara Cavallaro; Carla Sardo; Emanuela Fabiola Craparo; Barbara Porsio; Gaetano Giammona (313-333).
Ideally designed polymer-based siRNA carriers based on (a) amphiphilic or (b) water-soluble polymers for efficient targeted gene therapyDisplay OmittedGene therapy through the use of siRNA and a polymeric carrier are becoming an efficient therapeutic option to conventional pharmaceutical formulations for the treatment of deadly diseases, such as cancer, pulmonary, ocular and neurodegenerative diseases. However, several considerations regarding the stability, formulation, and efficacy have to be faced up until these systems could be considered to be a marketable pharmaceutical products for to extend siRNA application to clinical practice. This review is focused on the key challenges of siRNA therapeutics, with special attention on the faced obstacles and on the formulation-related difficulties, providing a list of requirements needed for obtaining an ideal carrier for siRNA. Moreover, the current non-viral polymers investigated for the realization of efficient carriers for siRNA are described, with a special attention on synthetic polyamines such as polyethylenimine (PEI), polysaccharides such as chitosan and inulin (INU), and polyaminoacids such as α,β-poly(N-2-hydroxyethyl)-d,l-aspartamide (PHEA) and poly-l-lysine (PLL).
Keywords: siRNA delivery; Polymeric non viral vectors; Polyaspartamide; Polyethylenimine; Chitosan; Inulin;

Aptamer delivery of siRNA, radiopharmaceutics and chemotherapy agents in cancer by Carlos E.B. de Almeida; Lais Nascimento Alves; Henrique F. Rocha; Januário Bispo Cabral-Neto; Sotiris Missailidis (334-342).
Aptamers are capable of specifically recognising tumour markers and deliver therapeutic agents like siRNA, Radiopharmaceuticals, Chemotherapy agents or nanoparticles with high efficiency, thus improving their therapeutic potential and minimising unwanted side effects.Display OmittedAptamers are oligonucleotide reagents with high affinity and specificity, which among other therapeutic and diagnostic applications have the capability of acting as delivery agents. Thus, aptamers are capable of carrying small molecules, nanoparticles, radiopharmaceuticals or fluorescent agents as well as nucleic acid therapeutics specifically to their target cells. In most cases, the molecules may possess interesting therapeutic properties, but their lack of specificity for a particular cell type, or ability to internalise in such a cell, hinders their clinical development, or cause unwanted side effects. Thus, chemotherapy or radiotherapy agents, famous for their side effects, can be coupled to aptamers for specific delivery. Equally, siRNA have great therapeutic potential and specificity, but one of their shortcomings remain the delivery and internalisation into cells. Various methodologies have been proposed to date, including aptamers, to resolve this problem. Therapeutic or imaging reagents benefit from the adaptability and ease of chemical manipulation of aptamers, their high affinity for the specific marker of a cell type, and their internalisation ability via cell mediated endocytosis. In this review paper, we explore the potential of the aptamers as delivery agents and offer an update on current status and latest advancements.
Keywords: Aptamers; siRNA; Radiopharmaceutics; Chemotherapy; Cancer;

Engineering approaches in siRNA delivery by Anna Angela Barba; Sara Cascone; Diego Caccavo; Gaetano Lamberti; Gianluca Chiarappa; Michela Abrami; Gabriele Grassi; Mario Grassi; Giovanna Tomaiuolo; Stefano Guido; Valerio Brucato; Francesco Carfì Pavia; Giulio Ghersi; Vincenzo La Carrubba; Roberto Andrea Abbiati; Davide Manca (343-358).
Display OmittedsiRNAs are very potent drug molecules, able to silence genes involved in pathologies development. siRNAs have virtually an unlimited therapeutic potential, particularly for the treatment of inflammatory diseases. However, their use in clinical practice is limited because of their unfavorable properties to interact and not to degrade in physiological environments. In particular they are large macromolecules, negatively charged, which undergo rapid degradation by plasmatic enzymes, are subject to fast renal clearance/hepatic sequestration, and can hardly cross cellular membranes. These aspects seriously impair siRNAs as therapeutics. As in all the other fields of science, siRNAs management can be advantaged by physical-mathematical descriptions (modeling) in order to clarify the involved phenomena from the preparative step of dosage systems to the description of drug-body interactions, which allows improving the design of delivery systems/processes/therapies. This review analyzes a few mathematical modeling approaches currently adopted to describe the siRNAs delivery, the main procedures in siRNAs vectors’ production processes and siRNAs vectors’ release from hydrogels, and the modeling of pharmacokinetics of siRNAs vectors. Furthermore, the use of physical models to study the siRNAs vectors’ fate in blood stream and in the tissues is presented. The general view depicts a framework maybe not yet usable in therapeutics, but with promising possibilities for forthcoming applications.
Keywords: siRNAs; Delivery vectors; in vitro models; Mathematical modeling; Physical modeling;

Fluorescence- and computed tomography for assessing the biodistribution of siRNA after intratracheal application in mice by Antonia Geyer; Cornelia Lorenzer; Sebastian Gehrig; Manuela Simlinger; Johannes Winkler; Haider Sami; Manfred Ogris (359-366).
Display OmittedPulmonary delivery of nucleic acids opens the possibility for direct treatment of lung diseases, like fibrosis, cancer, and infections. Lung retention and biodistribution of nucleic acids remain important issues for the development of suitable therapeutic approaches. Moreover, monitoring the dynamic biodistribution processes of siRNA after aerosol delivery can help in identifying bottlenecks and optimizing therapeutic concepts. We investigated dynamic biodistribution events after intratracheal application of chemically stabilized siRNA labelled with near infrared emitting dye AlexaFluor750 (AF750). Epifluorescence imaging was combined with spectral unmixing to improve the signal to noise ratio. Transillumination imaging has been utilized for quantitative fluorescence imaging tomography (FLIT) together with contrast agent enhanced X-ray absorption computed tomography (CT). Spectral unmixing allowed unambiguous detection of AF750 signals, which could be clearly distinguished from food derived autofluorescence. After successful delivery to the lung, fluorescent signals were also observed in kidneys and bladder, indicating renal excretion of AF750-siRNA. Gel electrophoresis of urine samples showed presence of intact siRNA, at least to a considerable extent. FLIT/CT allowed signal quantification and precise allocation to anatomical structures.
Keywords: siRNA delivery; Pulmonary delivery; Biodistribution; Fluorescence tomography; Near infrared imaging; Aerosolization;

Dissecting the role of the elongation factor 1A isoforms in hepatocellular carcinoma cells by liposome-mediated delivery of siRNAs by Rossella Farra; Bruna Scaggiante; Chiara Guerra; Gabriele Pozzato; Mario Grassi; Fabrizio Zanconati; Francesca Perrone; Cinzia Ferrari; Francesco Trotta; Gabriele Grassi; Barbara Dapas (367-376).
Display OmittedEukaryotic elongation factor 1A (eEF1A), a protein involved in protein synthesis, has two major isoforms, eEF1A1 and eEF1A2. Despite the evidences of their involvement in hepatocellular carcinoma (HCC), the quantitative contribution of each of the two isoforms to the disease is unknown.We depleted the two isoforms by means of siRNAs and studied the effects in three different HCC cell lines. Particular care was dedicated to select siRNAs able to target each of the two isoform without affecting the other one. This is not a trivial aspect due to the high sequence homology between eEF1A1 and eEF1A2.The selected siRNAs can specifically deplete either eEF1A1 or eEF1A2. This, in turn, results in an impairment of cell vitality, growth and arrest in the G1/G0 phase of the cell cycle. Notably, these effects are quantitatively superior following eEF1A1 than eEF1A2 depletion. Moreover, functional tests revealed that the G1/G0 block induced by eEF1A1 depletion depends on the down-regulation of the transcription factor E2F1, a known player in HCC.In conclusion, our data indicate that the independent targeting of the two eEF1A isoforms is effective in reducing HCC cell growth and that eEF1A1 depletion may result in a more evident effect.
Keywords: siRNA; eEF1A1; eEF1A2; HCC; E2F1; Liposome delivery;

In vitro and ex vivo delivery of tailored siRNA-nanoliposomes for E2F1 silencing as a potential therapy for colorectal cancer by Sabrina Bochicchio; Barbara Dapas; Ilaria Russo; Carolina Ciacci; Ornella Piazza; Stefaan De Smedt; Eline Pottie; Anna Angela Barba; Gabriele Grassi (377-387).
Display OmittedTailored developed nanoliposomes loaded with a siRNA against the transcription factor E2F1 (siE2F1), were produced and delivered to human colorectal adenocarcinoma cell lines and to intestinal human biopsies. siE2F1 loaded nanoliposomes were produced through a dedicated ultrasound assisted technique producing particles with about 40 nm size (Small Unilamellar Vesicles, SUVs) and 100% siRNA encapsulation efficiency. Compared to other production methods, the one proposed here can easily produce particles in the nanometric scale by suitable ultrasonic duty cycle treatments. Furthermore, SUVs have a high degree of size homogeneity, a relevant feature for uniform delivery behaviour.siE2F1-loaded SUVs demonstrated a very low cytotoxicity in cells when compared to a commercial transfection agent. Moreover, SUVs loaded with siE2F1 were effective in the down regulation of the target in cultured colon carcinoma cells and in the consequent reduction of cell growth. Finally, a remarkable uptake and target silencing efficiencies were observed in cultured human biopsy of colonic mucosa. In conclusion, whereas further studies in more complex models are required, the siE2F1-SUVs generated have the potential to contribute to the development of novel effective inflammatory bowel diseases-associated colorectal cancer therapies for a future personalized medicine.
Keywords: Nanoliposome; siRNA delivery; E2F1; Colonic mucosa; Colorectal cancer;

Small nanosized poly(vinyl benzyl trimethylammonium chloride) based polyplexes for siRNA delivery by Bo Lou; Nataliia Beztsinna; Grigoris Mountrichas; Joep B. van den Dikkenberg; Stergios Pispas; Wim E. Hennink (388-396).
Display OmittedThe success of siRNA gene therapy requires the availability of safe and efficient delivery systems. In the present study, we investigated poly(vinyl benzyl trimethylammonium chloride) (PVTC) and its block copolymer with poly(oligo(ethyleneglycol) methacrylate) (POEGMA) as delivery vector for siRNA. Small polyplexes ranging from 8 to 25 nm in diameter were formed in aqueous solution by spontaneous self-assembly of both the homopolymer and block copolymer with siRNA and the formed particles were stable at physiological ionic strength. It was shown that when human ovarian adenocarcinoma cells were transfected, siRNA polyplexes based on PVTC (40 kDa) and PVTC-POEGMA-4 (PP4, 34 kDa) efficiently induced luciferase gene silencing to the same extent as the formulation based on a commercial lipid (Lipofectamine®) (∼80%), and showed higher gene silencing than the linear polyethylenimine formulation linear polyethylenimine (∼35%). Importantly, the POEGMA block polymers displayed a significantly lower cytotoxicity as compared to L-pEI. siRNA polyplexes based on the block polymers displayed high cellular uptake resulting in ∼50% silencing of luciferase expression also in the presence of serum. These results demonstrate that PVTC-based polymers are promising siRNA delivery vectors.
Keywords: Quaternized polymer; siRNA; Polyplexes; Polymeric gene delivery; Gene therapy;

Galactosylated polyaspartamide copolymers for siRNA targeted delivery to hepatocellular carcinoma cells by Gennara Cavallaro; Rossella Farra; Emanuela Fabiola Craparo; Carla Sardo; Barbara Porsio; Gaetano Giammona; Francesca Perrone; Mario Grassi; Gabriele Pozzato; Gabriele Grassi; Barbara Dapas (397-406).
Display OmittedThe limited efficacy of available treatments for hepatocellular carcinoma (HCC) requires the development of novel therapeutic approaches. We synthesized a novel cationic polymer based on α,β-poly-(N-2-hydroxyethyl)-d,L-aspartamide (PHEA) for drug delivery to HCC cells. The copolymer was synthesized by subsequent derivatization of PHEA with diethylene triamine (DETA) and with a polyethylene glycol (PEG) derivative bearing galactose (GAL) molecules, obtaining the cationic derivative PHEA-DETA-PEG-GAL. PHEA-DETA-PEG-GAL has suitable chemical-physical characteristics for a potential systemic use and can effectively deliver a siRNA (siE2F1) targeted against the transcription factor E2F1, a gene product involved in HCC. The presence of GAL residues in the polyplexes allows the targeting of HCC cells that express the asialo-glycoprotein receptor (ASGP-R). In these cells, but not in ASGP-R non-expressing cells, PHEA-DETA-PEG-GAL/siE2F1 polyplexes induce the reduction of the mRNA and protein levels of E2F1 and of E2F1-regulated genes, all involved in the promotion of the G1/S phase transition. This results in a decrease of cell proliferation with a G1/G0 phase cells accumulation. Notably, removal of GAL residue almost completely abrogates the targeting capacity of the developed polyplexes.In conclusion, the generated polyplexes demonstrate the potential to effectively contributing to the development of novel anti-HCC therapeutic approaches via a siRNA-targeted delivery.
Keywords: Polyplexes; HCC; siRNA; E2F1; PHEA-DETA-PEG-GAL;

Effective melanoma cancer suppression by iontophoretic co-delivery of STAT3 siRNA and imatinib using gold nanoparticles by Suman Labala; Anup Jose; Sumeet Rajesh Chawla; Mohammed Shareef Khan; Shubhmita Bhatnagar; Onkar Prakash Kulkarni; Venkata Vamsi Krishna Venuganti (407-417).
Display OmittedCo-delivery of chemotherapeutic agents improve anti-tumor efficacy and reduce cancer resistance. Here, we report development of layer-by-layer assembled gold nanoparticles (LbL-AuNP) containing anti-STAT3 siRNA and imatinib mesylate (IM) to treat melanoma. The combination treatment with STAT3 siRNA and IM in B16F10 melanoma cells showed greater suppression of STAT3 protein, decreased cell viability and increased apoptotic events compared with LbL-AuNP containing either STAT3 siRNA or IM. In vivo efficacy studies in melanoma tumor bearing mice showed that non-invasive topical iontophoretic administration (0.5 mA/cm2) of LbL-AuNP was comparable with intratumoral administration. Co-delivery of STAT3 siRNA and IM using LbL-AuNP showed significant (p < 0.05) reduction in percentage tumor volume, tumor weight and suppressed STAT3 protein expression compared with either STAT3 siRNA or IM loaded LbL-AuNP. Taken together, LbL-AuNP can be developed as nanocarrier system for co-delivery of siRNA and small molecule drugs for topical iontophoretic delivery.
Keywords: Gold nanoparticle; Melanoma; STAT3 siRNA; Imatinib mesylate; Iontophoresis; Co-delivery;