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Lipid-polymer chimeric (hybrid) nanosystems are promising platforms for the design of effective gene delivery vectors. In this regard, we developed DNA nanocarriers comprised of a novel poly[(stearyl methacrylate-co-oligo(ethylene glycol) methyl ether methacrylate] [P(SMA-co-OEGMA)] amphiphilic random copolymer, the cationic 1,2-dioleoyl-3-(trimethylammonium) propane (DOTAP), and the zwitterionic L-α-phosphatidylcholine, hydrogenated soybean (soy) (HSPC) lipids. Chimeric HSPC:DOTAP:P[(SMA-co-OEGMA)] nanosystems, and pure lipid nanosystems as reference, were prepared in several molar ratios of the components. The colloidal dispersions obtained presented well-defined physicochemical characteristics and were further utilized for the formation of lipoplexes with a model DNA of linear topology containing 113 base pairs. Nanosized complexes were formed through the electrostatic interaction of the cationic lipid and phosphate groups of DNA, as observed by dynamic, static, and electrophoretic light scattering techniques. Ultraviolet–visible (UV–Vis) and fluorescence spectroscopy disclosed the strong binding affinity of the chimeric and also the pure lipid nanosystems to DNA. Colloidally stable chimeric/lipid complexes were formed, whose physicochemical characteristics depend on the N/P ratio and on the molar ratio of the building components. Cryogenic transmission electron microscopy (Cryo-TEM) revealed the formation of nanosystems with vesicular morphology. The results suggest the successful fabrication of these novel chimeric nanosystems with well-defined physicochemical characteristics, which can form stable lipoplexes.

The gangliosidoses GM2 are a group of pathologies mainly affecting the central nervous system due to the impaired GM2 ganglioside degradation inside the lysosome. Under physiological conditions, GM2 ganglioside is catabolized by the β-hexosaminidase A in a GM2 activator protein-dependent mechanism. In contrast, uncharged substrates such as globosides and some glycosaminoglycans can be hydrolyzed by the β-hexosaminidase B. Monogenic mutations on HEXA, HEXB, or GM2A genes arise in the Tay–Sachs (TSD), Sandhoff (SD), and AB variant diseases, respectively. In this work, we validated a CRISPR/Cas9-based gene editing strategy that relies on a Cas9 nickase (nCas9) as a potential approach for treating GM2 gangliosidoses using in vitro models for TSD and SD. The nCas9 contains a mutation in the catalytic RuvC domain but maintains the active HNH domain, which reduces potential off-target effects. Liposomes (LPs)- and novel magnetoliposomes (MLPs)-based vectors were used to deliver the CRISPR/nCas9 system. When LPs were used as a vector, positive outcomes were observed for the β-hexosaminidase activity, glycosaminoglycans levels, lysosome mass, and oxidative stress. In the case of MLPs, a high cytocompatibility and transfection ratio was observed, with a slight increase in the β-hexosaminidase activity and significant oxidative stress recovery in both TSD and SD cells. These results show the remarkable potential of CRISPR/nCas9 as a new alternative for treating GM2 gangliosidoses, as well as the superior performance of non-viral vectors in enhancing the potency of this therapeutic approach.

The effective protection and delivery of antisense oligomers to its site of action is a challenge without an optimal strategy. Some of the most promising approaches encompass the complexation of nucleic acids, which are anionic, with liposomes of fixed or ionizable cationic charge. Thus, the main purpose of this work was to study the complexation of cationic liposomes with anti-EFG1 2′OMe oligomers and evaluate the complex efficacy to control Candida albicans filamentation in vitro and in vivo using a Galleria mellonella model. To accomplish this, cationic dioleoyl-trimethylammoniumpropane (DOTAP) was mixed with three different neutral lipids dioleoyl-phosphocholine (DOPC), dioleoyl-phosphatidylethanolamine (DOPE) and monoolein (MO) and used as delivery vectors. Fluorescence Cross Correlation Spectroscopy measurements revealed a high association between antisense oligomers (ASO) and cationic liposomes confirming the formation of lipoplexes. In vitro, all cationic liposome-ASO complexes were able to release the anti-EFG1 2′OMe oligomers and consequently inhibit C. albicans filamentation up to 60% after 72 h. In vivo, from all formulations the DOTAP/DOPC 80/20 ρchg = 3 formulation proved to be the most effective, enhancing the G. mellonella survival by 40% within 48 h and by 25% after 72 h of infection. In this sense, our findings show that DOTAP-based lipoplexes are very good candidates for nano-carriers of anti-EFG1 2′OMe oligomers., This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the strategic funding of UIDB/04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte and Daniela Eira Araújo [SFRH/BD/121417/2016] PhD grant. The authors also acknowledge the project funding by the “02/SAICT/2017 – Projetos de Investigação Científica e Desenvolvimento Tecnológico (IC&DT) – POCI-01-0145-FEDER-028893”. This research is also supported by Microfluidic Layer-by-layer Assembly of Cationic Liposome-Nucleic Acid Nanoparticles for Gene Delivery project (032520) co-funded by FCT and the ERDF through COMPETE2020., info:eu-repo/semantics/publishedVersion

Acute respiratory distress syndrome (ARDS) is a devastating clinical syndrome that leads to acute respiratory failure and accounts for over 70,000 deaths per year in the United States alone, even prior to the COVID-19 pandemic. While its molecular details have been teased apart and its pathophysiology largely established over the past 30 years, relatively few pharmacological advances in treatment have been made based on this knowledge. Indeed, mortality remains very close to what it was 30 years ago. As an alternative to traditional pharmacological approaches, gene therapy offers a highly controlled and targeted strategy to treat the disease at the molecular level. Although there is no single gene or combination of genes responsible for ARDS, there are a number of genes that can be targeted for upregulation or downregulation that could alleviate many of the symptoms and address the underlying mechanisms of this syndrome. This review will focus on the pathophysiology of ARDS and how gene therapy has been used for prevention and treatment. Strategies for gene delivery to the lung, such as barriers encountered during gene transfer, specific classes of genes that have been targeted, and the outcomes of these approaches on ARDS pathogenesis and resolution will be discussed.

Non-viral vectors offer the potential to deliver nucleic acids including mRNA and DNA into cells in vivo. However, designing materials that effectively deliver to target organs and then to desired compartments within the cell remains a challenge. Here we develop polymeric materials that can be optimized for either DNA transcription in the nucleus or mRNA translation in the cytosol. We synthesized poly(beta amino ester) terpolymers (PBAEs) with modular changes to monomer chemistry to investigate influence on nucleic acid delivery. We identified two PBAEs with a single monomer change as being effective for either DNA (D-90-C12-103) or mRNA (DD-90-C12-103) delivery to lung endothelium following intravenous injection in mice. Physical properties such as particle size or charge did not account for the difference in transfection efficacy. However, endosome co-localization studies revealed that D-90-C12-103 nanoparticles resided in late endosomes to a greater extent than DD-90-C12-103. We compared luciferase expression in vivo and observed that, even with nucleic acid optimized vectors, peak luminescence using mRNA was two orders of magnitude greater than pDNA in the lungs of mice following systemic delivery. This study indicates that different nucleic acids require tailored delivery vectors, and further support the potential of PBAEs as intracellular delivery materials.

TRAIL (tumor necrosis factor-related apoptosis-inducing ligand), also known as APO2L, belongs to the tumor necrosis factor family. By binding to the death receptor 4 (DR4) or DR5, TRAIL induces apoptosis of tumor cells without causing side toxicity in normal tissues. In recent years TRAIL-based therapy has attracted great attention for its promise of serving as a cancer drug candidate. However, the treatment efficacy of TRAIL protein was under expectation in the clinical trials because of the short half-life and the resistance of cancer cells. TRAIL gene transfection can produce a “bystander effect” of tumor cell killing and provide a potential solution to TRAIL-based cancer therapy. In this review we focus on TRAIL gene therapy and various design strategies of TRAIL DNA delivery including non-viral vectors and cell-based TRAIL therapy. In order to sensitize the tumor cells to TRAIL-induced apoptosis, combination therapy of TRAIL DNA with other drugs by the codelivery methods for yielding a synergistic antitumor efficacy is summarized. The opportunities and challenges of TRAIL-based gene delivery and therapy are discussed.

Gene supplementation therapy with plasmid DNA (pDNA) represents one of the most promising strategies for the treatment of monogenic diseases such as Fabry disease (FD). In the present work, we developed a solid lipid nanoparticles (SLN)-based non-viral vector with a size below 100 nm, and decorated with galactomannan (GM) to target the liver as an α-Galactosidase A (α-Gal A) production factory. After the physicochemical characterization of the GM-SLN vector, cellular uptake, transfection efficacy and capacity to increase α-Gal A activity were evaluated in vitro in a liver cell line (Hep G2) and in vivo in an animal model of FD. The vector showed efficient internalization and it was highly efficient in promoting protein synthesis in Hep G2 cells. Additionally, the vector did not show relevant agglutination of erythrocytes and lacked hemolytic activity. After the systemic administration to Fabry mice, it achieved clinically relevant α-Gal A activity levels in plasma, liver, and other organs, importantly in heart and kidneys, two of the most damaged organs in FD. This work shows the potential application of GM-decorated lipidic nanocarries for the treatment of FD by pDNA-based gene augmentation. This research was funded by MCIU/AEI/FEDER, UE (RTI2018-098672-B-I00) and by the University of the Basque Country UPV/EHU (GIU20/048).

Fabry disease (FD) is a monogenic X-linked lysosomal storage disorder caused by a deficiency in the lysosomal enzyme α-Galactosidase A (α-Gal A). It is a good candidate to be treated with gene therapy, in which moderately low levels of enzyme activity should be sufficient for clinical efficacy. In the present work we have evaluated the efficacy of a non-viral vector based on solid lipid nanoparticles (SLN) to increase α-Gal A activity in an FD mouse model after intravenous administration. The SLN-based vector incremented α-Gal A activity to about 10%, 15%, 20% and 14% of the levels of the wild-type in liver, spleen, heart and kidney, respectively. In addition, the SLN-based vector significantly increased α-Gal A activity with respect to the naked pDNA used as a control in plasma, heart and kidney. The administration of a dose per week for three weeks was more effective than a single-dose administration. Administration of the SLN-based vector did not increase liver transaminases, indicative of a lack of toxicity. Additional studies are necessary to optimize the efficacy of the system, however, these results reinforce the potential of lipid-based nanocarriers to treat FD by gene therapy.

Gene therapy is an attractive therapeutic method for the treatment of genetic disorders for which the efficient delivery of nucleic acids into a target cell is critical. The present study is aimed at evaluating the potential of copolymers based on linear polyglycidol to act as carriers of nucleic acids. Functional copolymers with linear polyglycidol as a non-ionic hydrophilic block and a second block bearing amine hydrochloride pendant groups were prepared using previously synthesized poly(allyl glycidyl ether)-b-polyglycidol block copolymers as precursors. The amine functionalities were introduced via highly efficient radical addition of 2-aminoethanethiol hydrochloride to the alkene side groups. The modified copolymers formed loose aggregates with strongly positive surface charge in aqueous media, stabilized by the presence of dodecyl residues at the end of the copolymer structures and the hydrogen-bonding interactions in polyglycidol segments. The copolymer aggregates were able to condense DNA into stable and compact nanosized polyplex particles through electrostatic interactions. The copolymers and the corresponding polyplexes showed low to moderate cytotoxicity on a panel of human cancer cell lines. The cell internalization evaluation demonstrated the capability of the polyplexes to successfully deliver DNA into the cancer cells.

Research on the improvement and fabrication of polymeric systems as non-viral gene delivery carriers is required for their implementation in gene therapy. Random copolymers have not been extensively utilized for these purposes. In this regard, double hydrophilic poly[(2-(dimethylamino) ethyl methacrylate)-co-(oligo(ethylene glycol) methyl ether methacrylate] [P(DMAEMA-co-OEGMA)] random copolymers were synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization. The copolymers were further modified by quaternization of DMAEMA tertiary amine, producing the cationic P(QDMAEMA-co-OEGMA) derivatives. Fluorescence and ultraviolet–visible (UV–vis) spectroscopy revealed the efficient interaction of copolymers aggregates with linear DNAs of different lengths, forming polyplexes, with the quaternized copolymer aggregates exhibiting stronger binding affinity. Light scattering techniques evidenced the formation of polyplexes whose size, molar mass, and surface charge strongly depend on the N/P ratio (nitrogen (N) of the amine group of DMAEMA/QDMAEMA over phosphate (P) groups of DNA), DNA length, and length of the OEGMA chain. Polyplexes presented colloidal stability under physiological ionic strength as shown by dynamic light scattering. In vitro cytotoxicity of the empty nanocarriers was evaluated on HEK293 as a control cell line. P(DMAEMA-co-OEGMA) copolymer aggregates were further assessed for their biocompatibility on 4T1, MDA-MB-231, MCF-7, and T47D breast cancer cell lines presenting high cell viability rates.

Glioblastoma (GBM) is the most common and aggressive primary brain tumor in the adult population and it carries a dismal prognosis. Inefficient drug delivery across the blood brain barrier (BBB), an immunosuppressive tumor microenvironment (TME) and development of drug resistance are key barriers to successful glioma treatment. Since gliomas occur through sequential acquisition of genetic alterations, gene therapy, which enables to modification of the genetic make-up of target cells, appears to be a promising approach to overcome the obstacles encountered by current therapeutic strategies. Gene therapy is a rapidly evolving field with the ultimate goal of achieving specific delivery of therapeutic molecules using either viral or non-viral delivery vehicles. Gene therapy can also be used to enhance immune responses to tumor antigens, reprogram the TME aiming at blocking glioma-mediated immunosuppression and normalize angiogenesis. Nano-particles-mediated gene therapy is currently being developed to overcome the BBB for glioma treatment. Another approach to enhance the anti-glioma efficacy is the implementation of viro-immunotherapy using oncolytic viruses, which are immunogenic. Oncolytic viruses kill tumor cells due to cancer cell-specific viral replication, and can also initiate an anti-tumor immunity. However, concerns still remain related to off target effects, and therapeutic and transduction efficiency. In this review, we describe the rationale and strategies as well as advantages and disadvantages of current gene therapy approaches against gliomas in clinical and preclinical studies. This includes different delivery systems comprising of viral, and non-viral delivery platforms along with suicide/prodrug, oncolytic, cytokine, and tumor suppressor-mediated gene therapy approaches. In addition, advances in glioma treatment through BBB-disruptive gene therapy and anti-EGFRvIII/VEGFR gene therapy are also discussed. Finally, we discuss the results of gene therapy-mediated human clinical trials for gliomas. In summary, we highlight the progress, prospects and remaining challenges of gene therapies aiming at broadening our understanding and highlighting the therapeutic arsenal for GBM.

Cancer is the second cause of death worldwide, surpassed only by cardiovascular diseases, due to the lack of early diagnosis, and high relapse rate after conventional therapies. Chemotherapy inhibits the rapid growth of cancer cells, but it also affects normal cells with fast proliferation rate. Therefore, it is imperative to develop other safe and more effective treatment strategies, such as gene therapy, in order to significantly improve the survival rate and life expectancy of patients with cancer. The aim of gene therapy is to transfect a therapeutic gene into the host cells to express itself and cause a beneficial biological effect. However, the efficacy of the proposed strategies has been insufficient for delivering the full potential of gene therapy in the clinic. The type of delivery vehicle (viral or non viral) chosen depends on the desired specificity of the gene therapy. The first gene therapy trials were performed with therapeutic genes driven by viral promoters such as the CMV promoter, which induces non-specific toxicity in normal cells and tissues, in addition to cancer cells. The use of tumor-specific promoters over-expressed in the tumor, induces specific expression of therapeutic genes in a given tumor, increasing their localized activity. Several cancer- and/or tumor-specific promoters systems have been developed to target cancer cells. This review aims to provide up-to-date information concerning targeting gene therapy with cancer- and/or tumor-specific promoters including cancer suppressor genes, suicide genes, anti-tumor angiogenesis, gene silencing, and gene-editing technology, as well as the type of delivery vehicle employed. Gene therapy can be used to complement traditional therapies to provide more effective treatments.

Genetic diseases seriously threaten human health and have always been one of the refractory conditions facing humanity. Currently, gene therapy drugs such as siRNA, shRNA, antisense oligonucleotide, CRISPR/Cas9 system, plasmid DNA and miRNA have shown great potential in biomedical applications. To avoid the degradation of gene therapy drugs in the body and effectively deliver them to target tissues, cells and organelles, the development of excellent drug delivery vehicles is of utmost importance. Viral vectors are the most widely used delivery vehicles for gene therapy in vivo and in vitro due to their high transfection efficiency and stable transgene expression. With the development of nanotechnology, novel nanocarriers are gradually replacing viral vectors, emerging superior performance. This review mainly illuminates the current widely used gene therapy drugs, summarizes the viral vectors and non-viral vectors that deliver gene therapy drugs, and sums up the application of gene therapy to treat genetic diseases. Additionally, the challenges and opportunities of the field are discussed from the perspective of developing an effective nano-delivery system., Graphical abstract Types of gene therapy drugs, viral and non-viral delivery strategies, and disease treatment status of gene therapy drugs.Image, graphical abstract

Gene therapy by expression constructs or down-regulation of certain genes has shown great potential for the treatment of various diseases. The wide clinical application of nucleic acid materials dependents on the development of biocompatible gene carriers. There are enormous various compounds widely investigated to be used as non-viral gene carriers including lipids, polymers, carbon materials, and inorganic structures. In this review, we will discuss the recent discoveries on non-viral gene delivery systems. We will also highlight the in vivo gene delivery mediated by non-viral vectors to treat cancer in different tissue and organs including brain, breast, lung, liver, stomach, and prostate. Finally, we will delineate the state-of-the-art and promising perspective of in vivo gene editing using non-viral nano-vectors., Graphical abstractUnlabelled Image

Background Polymeric drug delivery systems have been achieved great development in the last two decades. Polymeric drug delivery has defined as a formulation or a device that enables the introduction of a therapeutic substance into the body. Biodegradable and bio-reducible polymers make the magic possible choice for lot of new drug delivery systems. The future prospects of the research for practical applications has required for the development in the field. Main body Natural polymers such as arginine, chitosan, dextrin, polysaccharides, poly (glycolic acid), poly (lactic acid), and hyaluronic acid have been treated for polymeric drug delivery systems. Synthetic polymers such as poly (2-hydroxyethyl methacrylate), poly(N-isopropyl acrylamide)s, poly(ethylenimine)s, dendritic polymers, biodegradable and bio-absorbable polymers have been also discussed for polymeric drug delivery. Targeting polymeric drug delivery, biomimetic and bio-related polymeric systems, and drug-free macromolecular therapeutics have also treated for polymeric drug delivery. In polymeric gene delivery systems, virial vectors and non-virial vectors for gene delivery have briefly analyzed. The systems of non-virial vectors for gene delivery are polyethylenimine derivatives, polyethylenimine copolymers, and polyethylenimine conjugated bio-reducible polymers, and the systems of virial vectors are DNA conjugates and RNA conjugates for gene delivery. Conclusion The development of polymeric drug delivery systems that have based on natural and synthetic polymers are rapidly emerging to pharmaceutical fields. The fruitful progresses have made in the application of biocompatible and bio-related copolymers and dendrimers to cancer treatment, including their use as delivery systems for potent anticancer drugs. Combining perspectives from the synthetic and biological fields will provide a new paradigm for the design of polymeric drug and gene delivery systems.

In August 2017, for the first time, a gene therapy was approved for market release in the United States. That approval was followed by two others before the end of the year. This article cites primary literature, review articles concerning particular biotechnologies, and press releases by the FDA and others in order to provide an overview of the current status of the field of gene therapy with respect to its translation into practice. Technical hurdles that have been overcome in the past decades are summarized, as are hurdles that need to be the subject of continued research. Then, some social and practical challenges are identified that must be overcome if the field of gene therapy, having survived past failures, is to achieve not only technical and clinical but also market success. One of these, the need for an expanded capacity for the manufacturing of viral vectors to be able to meet the needs of additional gene therapies that will be coming soon, is a challenge that the talents of current and future bioengineers may help address.

Gene therapy is a promising strategy for treatments of various diseases. Efficient and safe introduction of therapeutic genes into targeted cells is essential to realize functions of the genes. High-molecular-weight polyethylenimines (HMW PEIs) including 25 kDa branched PEI and 22 kDa linear PEI are widely used for in vitro gene transfection. However, high-gene transfection efficiency is usually accompanied with high cytotoxicity, which hampers their further clinical study. On the contrary, low-molecular-weight polyethylenimines (LMW PEIs) such as 1.8 kDa PEI and 800 Da PEI show good biocompatibility but their applications are limited by the poor DNA condensation capability. In this study, we find that 1.8 kDa PEI, but not 800 Da PEI combined with low-dose 25 kDa PEI could significantly promote gene transfection with low cytotoxicity. Plasmids encoding enhanced green fluorescence protein (EGFP) were delivered by the combined PEI and gene transfection efficiency was evaluated by microscopic observation and flow cytometry. Parameters including concentrations of 25 kDa PEI and 1.8 kDa PEI and preparation ways were further optimized. This study presents an efficient and safe combined PEI-based non-viral gene delivery strategy with potential for in vivo applications.

Gene therapy and DNA vaccination are among the most expected biotechnological and medical advances for the coming years. However, the lack of cost-effective large-scale production and purification of pharmaceutical-grade plasmid DNA (pDNA) still hampers their wide application. Downstream processing, which is mainly chromatography-based, of pDNA remains the key manufacturing step. Despite its high resolution, the scaling-up of chromatography is usually difficult and presents low capacity, resulting in low yields. Alternative methods that are based on aqueous two-phase systems (ATPSs) have been studied. Although higher yields may be obtained, its selectivity is often low. In this work, modified polymers based on poly(ethylene glycol) (PEG) derivatisation with amino groups (PEGamine) or conjugation with positively charged amino acids (PEGlysine, PEGarginine, and PEGhistidine) were studied to increase the selectivity of PEGdextran systems towards the partition of a model plasmid. A two-step strategy was employed to obtain suitable pure formulations of pDNA. In the first step, a PEGdextran system with the addition of the affinity ligand was used with the recovery of the pDNA in the PEG-rich phase. Then, the pDNA was re-extracted to an ammonium-sulphate-rich phase in the second step. After removing the salt, this method yielded a purified preparation of pDNA without RNA and protein contamination., Thanks are due to the Fundação para a Ciência e Tecnologia (FCT, Portugal) for financial support to the NMR Portuguese network (PTNMR, Bruker Avance III 400-Univ. Minho) and strategic funding for CQUM (UID/QUI/00686/2020)., info:eu-repo/semantics/publishedVersion

One of the greatest challenges for the development of genetic therapies is the efficient targeted delivery of therapeutic nucleic acids. Towards this goal, we have introduced a new engineering initiative in self-assembly of biologically safe and stable nanovesicle complexes (∼90 to 140 nm) derived from giant unilamellar vesicle (GUV) precursors and comprising plasmid DNA or siRNA and targeting peptide ligands. The biological performance of the engineered nanovesicle complexes were studied both in vitro and in vivo and compared with cationic liposome-based lipopolyplexes. Compared with cationic lipopolyplexes, nanovesicle complexes did not show advantages in transfection and cell uptake. However, nanovesicle complexes neither displayed significant cytotoxicity nor activated the complement system, which are advantageous for intravenous injection and tumour therapy. On intravenous administration into a neuroblastoma xenograft mouse model, nanovesicle complexes were found to distribute throughout the tumour interstitium, thus providing an alternative safer approach for future development of tumour-specific therapeutic nucleic acid interventions. On oropharyngeal instillation, nanovesicle complexes displayed better transfection efficiency than cationic lipopolyplexes. The technological advantages of nanovesicle complexes, originating from GUVs, over traditional cationic liposome-based lipopolyplexes are discussed. Statement of Significance The efficient targeted delivery of nucleic acids in vivo provides some of the greatest challenges to the development of genetic therapies. Giant unilamellar lipid vesicles (GUVs) have been used mainly as cell and tissue mimics and are instrumental in studying lipid bilayers and interactions. Here, the GUVs have been modified into smaller nanovesicles. We have then developed novel nanovesicle complexes comprising self-assembling mixtures of the nanovesicles, plasmid DNA or siRNA, and targeting peptide ligands. Their biophysical properties were studied and their transfection efficiency was investigated. They transfected cells efficiently without any associated cytotoxicity and with targeting specificity, and in vivo they resulted in very high and tumour-specific uptake and in addition, efficiently transfected the lung. The peptide-targeted nanovesicle complexes allow for the specific targeted enhancement of nucleic acid delivery with improved biosafety over liposomal formulations and represent a promising tool to improve our arsenal of safe, non-viral vectors to deliver therapeutic cargos in a variety of disorders.

Nucleic acid-based therapeutics are evaluated for their potential of treating a plethora of diseases, including cancer and inflammation. Short nucleic acids, such as miRNAs, have emerged as versatile regulators for gene expression and are studied for therapeutic purposes. However, their inherent instability in vivo following enteral and parenteral administration has prompted the development of novel methodologies for their delivery. Although research on the oral delivery of siRNAs is progressing, with the development and utilization of promising carrier-based methodologies for the treatment of a plethora of gastrointestinal diseases, research on miRNA-based oral therapeutics is lagging behind. In this review, we present the potential role of miRNAs in diseases of the GI tract, and analyze current research and the cardinal features of the novel carrier systems used for nucleic acid oral delivery that can be expanded for oral miRNA administration.

A series of multifunctional compounds (MFCs) 1a–1d based on 1,8-naphthalimide moiety were designed and synthesized. Due to the good fluorescence property and nucleic acid binding ability of 1,8-naphthalimide, these MFCs were applied in Cu2+ ion recognition, lysosome staining as well as RNA delivery. It was found that these MFCs exhibited highly selective fluorescence turn-off for Cu2+ in aqueous solution. The fluorescence emission of 1a−1d was quenched by a factor of 116-, 20-, 12-, and 14-fold in the presence of Cu2+ ions, respectively. Most importantly, 1a-Cu and 1b-Cu could be used as imaging reagents for detection of lysosome in live human cervical cancer cells (HeLa) using fluorescence microscopy. Furthermore, in order to evaluate the RNA delivery ability of 1a−1d, cellular uptake experiments were performed in HeLa, HepG2, U2Os, and MC3T3-E1 cell lines. The results showed that all the materials could deliver Cy5-labled RNA into the targeted cells. Among them, compound 1d modified with long hydrophobic chain exhibited the best RNA delivery efficiency in the four tested cell lines, and the performance was far better than lipofectamine 2000 and 25 kDa PEI, indicating the potential application in non-viral vectors.

Background Gene delivery systems are essentially necessary for the gene therapy of human genetic diseases. Gene therapy is the unique way that is able to use the adjustable gene to cure any disease. The gene therapy is one of promising therapies for a number of diseases such as inherited disorders, viral infection and cancers. The useful results of gene delivery systems depend open the adjustable targeting gene delivery systems. Some of successful gene delivery systems have recently reported for the practical application of gene therapy. Main body The recent developments of viral gene delivery systems and non-viral gene delivery systems for gene therapy have briefly reviewed. The viral gene delivery systems have discussed for the viral vectors based on DNA, RNA and oncolytic viral vectors. The non-viral gene delivery systems have also treated for the physicochemical approaches such as physical methods and chemical methods. Several kinds of successful gene delivery systems have briefly discussed on the bases of the gene delivery systems such as cationic polymers, poly(L-lysine), polysaccharides, and poly(ethylenimine)s. Conclusion The goal of the research for gene delivery system is to develop the clinically relevant vectors such as viral and non-viral vectors that use to combat elusive diseases such as AIDS, cancer, Alzheimer, etc. Next step research will focus on advancing DNA and RNA molecular technologies to become the standard treatment options in the clinical area of biomedical application.

The development of efficient and safe nucleic acid delivery vectors remains an unmet need holding back translation of gene therapy approaches into bedside. Graphene oxide (GO) could help bypass such bottleneck thanks to its large surface area, versatile chemistry and biocompatibility, which could overall enhance transfection efficiency while abolishing some of the limitations linked to the use of viral vectors. Here, we aimed to assess the capacity of bare GO, without any further surface modification, to complex a short double-stranded nucleic acid of biological relevance (siRNA) and mediate its intracellular delivery. GO formed stable complexes with siRNA at 10:1, 20:1 and 50:1 GO:siRNA mass ratios. Complexation was further corroborated by atomistic molecular dynamics simulations. GO:siRNA complexes were promptly internalized in a primary mouse cell culture, as early as 4 h after exposure. At this time point, intracellular siRNA levels were comparable to those provided by a lipid-based transfection reagent that achieved significant gene silencing. Time-lapse tracking of internalized GO and siRNA evidenced a sharp decrease of intracellular siRNA from 4 to 12 h, while GO was sequestered in large vesicles, which may explain the lack of biological effect (i.e. gene silencing) achieved by GO:siRNA complexes. This study underlines the potential of non-surface modified GO flakes to act as 2D siRNA delivery platforms, without the need for cationic functionalization, but warrants further vector optimization to allow effective release of the nucleic acid and achieve efficient gene silencing.

MicroRNAs (miRNAs) represent a family of short non-coding regulatory RNA molecules that are produced in a tissue and time-specific manner to orchestrate gene expression post-transcription. MiRNAs hybridize to target mRNA(s) to induce translation repression or mRNA degradation. Functional studies have demonstrated that miRNAs are engaged in virtually every physiological process and, consequently, miRNA dysregulations have been linked to multiple human pathologies. Thus, miRNA mimics and anti-miRNAs that restore miRNA expression or downregulate aberrantly expressed miRNAs, respectively, are highly sought-after therapeutic strategies for effective manipulation of miRNA levels. In this regard, carrier vehicles that facilitate proficient and safe delivery of miRNA-based therapeutics are fundamental to the clinical success of these pharmaceuticals. Here, we highlight the strengths and weaknesses of current state-of-the-art viral and non-viral miRNA delivery systems and provide perspective on how these tools can be exploited to improve the outcomes of miRNA-based therapeutics.

The era of the aging society has arrived, and this is accompanied by an increase in the absolute numbers of patients with neurological disorders, such as Alzheimer’s disease (AD) and Parkinson’s disease (PD). Such neurological disorders are serious costly diseases that have a significant impact on society, both globally and socially. Gene therapy has great promise for the treatment of neurological disorders, but only a few gene therapy drugs are currently available. Delivery to the brain is the biggest hurdle in developing new drugs for the central nervous system (CNS) diseases and this is especially true in the case of gene delivery. Nanotechnologies such as viral and non-viral vectors allow efficient brain-targeted gene delivery systems to be created. The purpose of this review is to provide a comprehensive review of the current status of the development of successful drug delivery to the CNS for the treatment of CNS-related disorders especially by gene therapy. We mainly address three aspects of this situation: (1) blood-brain barrier (BBB) functions; (2) adeno-associated viral (AAV) vectors, currently the most advanced gene delivery vector; (3) non-viral brain targeting by non-invasive methods.

Stimuli-responsive, “smart” polymeric materials used in the biomedical field function in a bio-mimicking manner by providing a non-linear response to triggers coming from a physiological microenvironment or other external source. They are built based on various chemical, physical, and biological tools that enable pH and/or temperature-stimulated changes in structural or physicochemical attributes, like shape, volume, solubility, supramolecular arrangement, and others. This review touches on some particular developments on the topic of stimuli-sensitive molecular tools for biomedical applications. Design and mechanistic details are provided concerning the smart synthetic instruments that are employed to prepare supra- and macro-molecular architectures with specific responses to external stimuli. Five major themes are approached: (i) temperature- and pH-responsive systems for controlled drug delivery; (ii) glycodynameric hydrogels for drug delivery; (iii) polymeric non-viral vectors for gene delivery; (iv) metallic nanoconjugates for biomedical applications; and, (v) smart organic tools for biomedical imaging.

Genome editing holds the promise of one-off and potentially curative therapies for many patients with genetic diseases. This is especially true for patients affected by mucopolysaccharidoses as the disease pathophysiology is amenable to correction using multiple approaches. Ex vivo and in vivo genome editing platforms have been tested primarily on MSPI and MPSII, with in vivo approaches having reached clinical testing in both diseases. Though we still await proof of efficacy in humans, the therapeutic tools established for these two diseases should pave the way for other mucopolysaccharidoses. Herein, we review the current preclinical and clinical development studies, using genome editing as a therapeutic approach for these diseases. The development of new genome editing platforms and the variety of genetic modifications possible with each tool provide potential applications of genome editing for mucopolysaccharidoses, which vastly exceed the potential of current approaches. We expect that in a not-so-distant future, more genome editing-based strategies will be established, and individual diseases will be treated through multiple approaches.

The incorporation of chloroquine within nano formulations, rather than as a co-treatment of the cells, could open a new avenue for in vivo retinal gene delivery. In this manuscript, we evaluated the incorporation of chloroquine diphosphate into the cationic niosome formulation composed of poloxamer 188, polysorbate 80 non-ionic surfactants, and 2,3-di (tetradecyloxy) propan-1-amine (hydrochloride salt) cationic lipid, to transfect rat retina. Niosome formulations without and with chloroquine diphosphate (DPP80, and DPP80-CQ, respectively) were prepared by the reverse phase evaporation technique and characterized in terms of size, PDI, zeta potential, and morphology. After the incorporation of the pCMS-EGFP plasmid, the resultant nioplexes -at different cationic lipid/DNA mass ratios- were further evaluated to compact, liberate, and secure the DNA against enzymatic digestion. In vitro procedures were achieved in ARPE-19 cells to assess transfection efficacy and intracellular transportation. Both nioplexes formulations transfected efficiently ARPE-19 cells, although the cell viability was clearly better in the case of DPP80-CQ nioplexes. After subretinal and intravitreal injections, DPP80 nioplexes were not able to transfect the rat retina. However, chloroquine containing vector showed protein expression in many retinal cells, depending on the administration route. These data provide new insights for retinal gene delivery based on chloroquine-containing niosome non-viral vectors., This project was supported by the Basque Country Government (CGIC10/172), Spanish Ministry of Education (Grant CTQ2017-84415-R, MAT2015-69967-C3-1R), the Generalitat de Catalunya (2014/SGR/624) and the Instituto de Salud Carlos III (CB06_01_0019, CB06_01_1028). The authors also wish to thank the intellectual and technical assistance from the ICTS “NANBIOSIS”, more specifically by the Drug Formulation Unit (U10) of the CIBER in Bioengineering, Biomaterials, and Nanomedicine (CIBER-BBN) at the University of Basque Country (UPV/EHU). Technical and human support provided by SGIker (UPV/EHU) is acknowledged.

The emergence of genetic engineering at the beginning of the 1970′s opened the era of biomedical technologies, which aims to improve human health using genetic manipulation techniques in a clinical context. Gene therapy represents an innovating and appealing strategy for treatment of human diseases, which utilizes vehicles or vectors for delivering therapeutic genes into the patients' body. However, a few past unsuccessful events that negatively marked the beginning of gene therapy resulted in the need for further studies regarding the design and biology of gene therapy vectors, so that this innovating treatment approach can successfully move from bench to bedside. In this paper, we review the major gene delivery vectors and recent improvements made in their design meant to overcome the issues that commonly arise with the use of gene therapy vectors. At the end of the manuscript, we summarized the main advantages and disadvantages of common gene therapy vectors and we discuss possible future directions for potential therapeutic vectors.

Lipids contain hydrocarbons and are the building blocks of cells. Lipids can naturally form themselves into nano-films and nano-structures, micelles, reverse micelles, and liposomes. Micelles or reverse micelles are monolayer structures, whereas liposomes are bilayer structures. Liposomes have been recognized as carriers for drug delivery. Solid lipid nanoparticles and lipoplex (liposome-polycation-DNA complex), also called lipid nanoparticles, are currently used to deliver drugs and genes to ocular tissues. A solid lipid nanoparticle (SLN) is typically spherical, and possesses a solid lipid core matrix that can solubilize lipophilic molecules. The lipid nanoparticle, called the liposome protamine/DNA lipoplex (LPD), is electrostatically assembled from cationic liposomes and an anionic protamine-DNA complex. The LPD nanoparticles contain a highly condensed DNA core surrounded by lipid bilayers. SLNs are extensively used to deliver drugs to the cornea. LPD nanoparticles are used to target the retina. Age-related macular degeneration, retinitis pigmentosa, and diabetic retinopathy are the most common retinal diseases in humans. There have also been promising results achieved recently with LPD nanoparticles to deliver functional genes and micro RNA to treat retinal diseases. Here, we review recent advances in ocular drug and gene delivery employing lipid nanoparticles.

Despite effective gene silencing in vitro, in vivo delivery and transfection of siRNA remain challenging due to the lack of carriers that protect siRNA stably in the body. This study is focused to elucidate the correlation between complex stability and transfection efficiency of siRNA carriers. The carriers were prepared by using polymer nanoassemblies made of a cationic branched polymer [poly(ethylene imine): bPEI] to which hydrophilic poly(ethylene glycol) polymers were tethered covalently. These polymer tethered nanoassemblies (TNAs) were further modified with lipophilic chains (palmitate: PAL) in the core to stabilize siRNA TNAs complexes through ionic and hydrophobic interactions in combination. The effects of PAL in the core of TNAs were investigated with respect to in vitro transfection, intracellular gene delivery, and toxicity of the complexes, using a human colon cancer HT29 cell line stably expressing a luciferase reporter gene. A commercial transfection agent (RNAiMax) was used as a control. TNAs entrapping siRNA showed the greatest complex stability in the absence of PAL although TNAs with a greater PAL content induced effective intracellular siRNA delivery, while luciferase expression decreased as the amount of PAL increased in the core of TNAs. These results demonstrate that lipophilic components in carriers affect not only complex stability but also intracellular distribution and transfection of siRNA in cancer cells.

A convenient cytosolic drug delivery constitutes a very powerful tool for the treatment and/or prevention of several relevant human diseases. Along with recent advances in therapeutic technologies based on biomacromolecules (e.g. oligonucleotides or proteins), we also require the development of technologies which improve the transport of therapeutic molecules to the cell of choice. This has led to the emergence of a variety of promising methods over the last 20 years. Despite significant progress, these methods still suffer from several shortcomings including low/variable delivery efficiency, high cytotoxicity, and perhaps most importantly, ineffective endosomal/lysosomal escape. In this context, Polymer Therapeutics (PT) have emerged as an exciting alternative to overcome such limitations. Specifically, well defined polypeptide-based therapeutics could be considered excellent candidates for drug delivery due to their suitable biodegradability, versatility, multivalence and high drug loading capacity. On the other hand, a comprehensive understanding of therapeutic molecules is also required for the rational selection and design of an appropriate intracellular delivery carrier. The application of new, robust, and sophisticated characterisation techniques has complemented existing techniques to meet the challenge of working under physiological or near-physiological conditions. The remarkable development in the design of drug delivery systems has forced the establishment of design guidelines to achieve the specific therapeutic effect. The importance of an exhaustive physicochemical characterisation has given rise to more efficient therapeutic strategies via better control of the pharmacokinetics and biodistribution of such nanomedicines. On this basis, the main aim of this thesis is focused on two main topics: (i) the design, development, and validation of nanosized polypeptide-based carriers capable of facilitating the cytosolic transport of bioactive agents which are not able to cross biological membranes by themselves or exhibit low lysosomal stability, such as plasmid DNA (pDNA), small interfering RNA (siRNA), or proteins, and (ii) the exhaustive physicochemical characterisation of polymeric drug delivery systems to determine their solution conformation and its correlation with their therapeutic output. In order to accomplish the above-mentioned goals and based on well-established properties of poly-L-glutamic acid (PGA) as a polymer carrier, firstly, we synthesised and evaluated different conjugates of PGA with succinyl tetraethylene pentamine (Stp) oligoaminoamides as non-viral carriers for pDNA or siRNA delivery. We hypothesised that these zwitterionic bioresponsive and biodegradable carriers may achieve similar transfection efficiencies as those achieved for analogous polycations, but with greater safety in biological scenarios by avoiding polycation-triggered side effects. After physicochemical characterisation of the obtained conjugates, we evaluated cytotoxicity in both N2a and 4T1 cell lines to assess cell viability. We also performed transfection and cell internalisation assays to assess conjugate functionality. We also continued exploring different alternatives within the field of polypeptides. We synthesised and characterised multifunctional polymeric platforms based on natural or synthetic polyaminoacids, such as PGA, poly-L-arginine (PArg), poly-L-ornithine (P(Orn)), and their derivatives to find an encouraging vehicle for effective siRNA delivery as anticancer treatment. We obtained several oligonucleotide conjugates and complexes and performed preliminary in vitro studies in B16F10-luc-G5 cell line. Upon comparing the obtained results of silencing, we established that P(Orn)-based systems offered the most promising results. Additionally, the feasibility of the delivery of a protein, alanine:glyoxylate aminotransferase (AGT), in order to promote enzyme-replacement therapy in a rare disease Hyperoxaluria Type I by conjugating the enzyme with a polyethyleneglycol (PEG)-PGA block-co-polymer based nanocarrier was also evaluated. This conjugation strategy does not significantly alter the functional properties of AGT and endows the protein with the ability to cross the plasma membrane and localise in the cytosol of a cellular model of PH1. Engineering AGT by the insertion of a stronger peroxisomal targeting sequence (PTS) and the mutation of one of the polymer anchoring points located on the “extended PTS1” partly decreases the conjugation efficiency. However, this allows peroxisomal targeting of the conjugates, resulting in the enhanced ability to detoxify intraperoxisomal glyoxylate with respect to the wild-type protein. We obtained all mentioned systems from polymers with very low polydispersity (Ɖ~1.2) and, therefore, precise and well-defined structures, so allowing reproducibility and the determination of a clear structure-activity relationship (SAR). We also exhaustively investigated physicochemical properties of all obtained polypeptides in terms of size and solution conformation. Finally, in order to deeply understand the importance of solution conformation in conjugate therapeutic output, highly advanced physicochemical techniques such as small angle neutron scattering (SANS) or small angle X-ray scattering (SAXS) were used always trying to mimic physiological conditions. A rigorous and detailed structural investigation of polymeric nanosystems already classified as successful drug delivery nanocarriers were performed.

Gene therapy has long been heralded as the new hope to evolve from symptomatic care of genetic pathologies to a full cure. Recent successes in using gene therapy for treating several ocular and haematopoietic pathologies have shown the great potential of this approach that, in the early days, relied on the use of viral vectors, which were considered by many to be undesirable for human treatment. Therefore, there is considerable interest and effort in developing non-viral vectors, with efficiency close to that of viral vectors. The aim of this study was to develop suitable non-viral carriers for gene therapy to treat pathologies affecting the retina. In this study poly(2-(N,N-dimethylamino)ethyl methacrylate), PDMAEMA was synthesized by reversible addition-fragmentation chain transfer (RAFT) and the in vitro cytocompatibility and transfection efficiency of a range of polymer:DNA ratios evaluated using a retinal cell line; in vivo biocompatibility was evaluated by ocular injection in C57BL/6 mice. The results showed that through RAFT, it is possible to produce a defined-size polymer that is compatible with cell viability in vitro and capable of efficiently directing gene expression in a polymer–DNA ratio-dependent manner. When injected into the eyes of mice, these vectors induced a transient, mild inflammation, characteristic of the implantation of medical devices. These results form the basis of future studies where RAFT-synthesized PDMAEMA will be used to deliver gene expression systems to the retina of mouse models of retinal pathologies., Funding was provided by the Portuguese Foundation for Science and Technology via SFRH/BD/70318/2010 and SFRH/BPD/78404/2011 scholarships to A.V.O. and S.S., respectively, PTDC/SAU-BEB/098475/2008 to G.A.S., Pest-OE/EQB/LA0023/2011 to I.B.B. L.A. and PEst-OE_QUI_UI4023_2011 to C.I.Q.A., PIRG05-GA-2009–249314 – EyeSee to G.A.S.

Cancer has been, from the beginning, a target of intense research for gene therapy approaches. Currently, more than 60% of all on-going clinical gene therapy trials worldwide are targeting cancer. Indeed, there is a clear unmet medical need for novel therapies. This is further urged by the fact that current conventional cancer therapies are frequently troubled by their toxicities. Different gene therapy strategies have been employed for cancer, such as pro-drug activating suicide gene therapy, anti-angiogenic gene therapy, oncolytic virotherapy, gene therapy-based immune modulation, correction/compensation of gene defects, genetic manipulation of apoptotic and tumor invasion pathways, antisense, and RNAi strategies. Cancer types, which have been targeted with gene therapy, include brain, lung, breast, pancreatic, liver, colorectal, prostate, bladder, head and neck, skin, ovarian, and renal cancer. Currently, two cancer gene therapy products have received market approval, both of which are in China. In addition, the stimulation of the host’s immune system, using gene therapeutic approaches, has gained vast interest. The intention of this review is to point out the most commonly viral and non-viral vectors and methods used in cancer gene therapy, as well as highlight some key results achieved in clinical trials.

Small interfering RNA (siRNA) has proved to be a powerful tool for target-specific gene silencing via RNA interference (RNAi). Its ability to control targeted gene expression gives new hope to gene therapy as a treatment for cancers and genetic diseases. However, siRNA shows poor pharmacological properties, such as low serum stability, off-targeting, and innate immune responses, which present a significant challenge for clinical applications. In addition, siRNA cannot cross the cell membrane for RNAi activity because of its anionic property and stiff structure. Therefore, the development of a safe, stable, and efficient system for the delivery of siRNA therapeutics into the cytoplasm of targeted cells is crucial. Several nanoparticle platforms for siRNA delivery have been developed to overcome the major hurdles facing the therapeutic uses of siRNA. This review covers a broad spectrum of non-viral siRNA delivery systems developed for enhanced cellular uptake and targeted gene silencing in vitro and in vivo and discusses their characteristics and opportunities for clinical applications of therapeutic siRNA.

Cholesteric Liquid Crystal Polymers (ChLCP), synthetized in our laboratory through a stereoselective polycondensation reaction1 as multifunctional optically active materials, have been extensively characterized 2 by NMR, Raman spectroscopy, steady-state fluorescence, molecular modeling, and SAXS/WAXS. These ChLCP behave both as thermotropic and lyotropic, confering interesting macromolecular properties indicative of potential application on the biomedical and engineering field. They have shown to be biocompatible against macrophages and fibroblasts cellular lines, and able to interact with biomacromolecules such as lipids (both neutral and cationi) and nucleic acids, the structures of the complexes being identified by synchrotron radiation source 3, 4, 5. Cationic liposomial/surfactant systems based on our CLCP were developed which entrapped DNA plasmids, acting as non viral cationic vectors for gene therapy, which successfully transfected in several tumor cell lines 6, 7. Cationic functionalized ChLCP have been synthesized, dispersed in TAE (0,04M TRIS; 0,001M EDTA) and complexed directly with commercial DNA of increasing complexity: [Poly-A]; [Poly-C]; [Poly-G]; [PolydT]; [PolyC-PolyG]; [PolyAC-PolydT]; commercial calf thymus DNA and plasmid. Three different proportions ChLCP:DNA were prepared: (1:2), (1:1), and (2:1) respectively by mixing and digesting for 12h in a swinging shaker. Neutron scattering experiments, had shown sufficient contrast (scattering length density difference) between new cholesteric PTOBEE-Ammonium (1.5 to 1.9 x 1010 /cm2) and polynucleotide [PolyC-PolyG] (3.32 x 1010 /cm2) for contrast variation SANS experiments. This experiment was successfully performed at NIST 8. The structure of the cationic complexes has been studied by SAXS at the BM16 beamline at ESRF, at room temperature. A monochromatized beam at = 0,9795 Å was used. Two-dimensional data recorded by an image-plate detector was placed at 5975 cm from the sample. The program Fit2D was employed to evaluate the beam centre position and to generate a mask file. Binary data are normalized by the detector response and pixels are radially averaged into 1D. Silver behenate (d= 58.3 Å) was used to calibrate the angular axis. Structural analysis of the cationic complexes is proposed.

Several advanced in vitro studies have proved the broad potential of cationic solid lipid nanoparticles (cSLNs) as synthetic nucleic acid vectors that have been proposed as an alternative to liposomes. Certainly, results regarding their transfection performances [1] are encouraging but are sometimes not as good as expected, even though the carrier cytotoxicity is always quite low. It is hoped that in the coming years these carriers may allow for higher efficiency over delivery of gene materials and that the development of upscalable and reproducible production of stable systems can be harnessed successfully. Nanovectors already play a very important role in pharmaceutical applications for the delivery of drugs or other biologically active materials. Over the past couple of decades, the incredible number of in vitro studies in chemical and biological engineering on nanocarrier systems provide several important aspects that relate to optimization of particle structures and characterize the mechanisms by which these particles interact with cells, and describing their behavior at the cellular, biochemical and molecular level.

Cancer is one of the most wide-spread diseases of modern times, with an estimated increase in the number of patients diagnosed worldwide, from 11.3 million in 2007 to 15.5 million in 2030 (www.who.int). In many cases, due to the delay in diagnosis and high increase of relapse, survival rates are low. Current therapies, including surgery, radiation and chemotherapy, have made significant progress, but they have many limitations and are far from ideal. Although immunotherapy has recently offered great promise as a new approach in cancer treatment, it is still very much in its infancy and more information on this approach is required before it can be widely applied. For these reasons effective, safe and patient-acceptable cancer therapy is still largely an unmet clinical need. Recent knowledge of the genetic basis of the disease opens up the potential for cancer gene therapeutics based on siRNA. However, the future of such gene-based therapeutics is dependent on achieving successful delivery. Extensive research is ongoing regarding the design and assessment of non-viral delivery technologies for siRNA to treat a wide range of cancers. Preliminary results on the first human Phase I trial for solid tumours, using a targeted non-viral vector, illustrate the enormous therapeutic benefits once the issue of delivery is resolved. In this review the genes regulating cancer will be discussed and potential therapeutic targets will be identified. The physiological and biochemical changes caused by tumours, and the potential to exploit this knowledge to produce bio-responsive 'smart' delivery systems, will be evaluated. This review will also provide a critical and comprehensive overview of the different non-viral formulation strategies under investigation for siRNA delivery, with particular emphasis on those designed to exploit the physiological environment of the disease site. In addition, a section of the review will be dedicated to pre-clinical animal models used to evaluate the stability, safety and efficacy of the delivery systems.

Covalent modification with polyethylene glycol (PEG), a non-toxic polymer used in food, cosmetic and pharmaceutical preparations for over 60 years, can profoundly influence the pharmacokinetic, pharmacologic and toxciologic profile of protein and peptide-based therapeutics. This review summarizes the history of PEGylation and PEG chemistry and highlights the value of this technology in the context of the design and development of recombinant viruses for gene transfer, vaccination and diagnostic purposes. Specific emphasis is placed on the application of this technology to the adenovirus, the most potent viral vector with the most highly characterized toxicity profile to date, in several animal models.

Background Gemini-lipid nanoparticles have been received major attention recently as non-viral delivery systems due to their successful non-invasive gene delivery through tough barriers such as eye and skin. The aim of this study was to evaluate non-viral gene delivery by a series of dicationic gemini surfactant-phospholipid nanoparticles (GL-NPs) and to explore their mechanism of interaction with cellular membranes of murine PAM212 epidermal keratinocytes. Methods NPs containing pCMV-tdTomato plasmid encoding red fluorescent protein (RFP) were prepared using 12 different gemini surfactants (m-s-m, with m = 12, 16 and 18C alkyl tail and s = 3 and 7C polymethylene spacer group and 7C substituted spacers with 7NH and 7NCH3) and dioleoylphosphatidylethanolamine helper lipid. RFP gene expression and cell viability status were evaluated using flow cytometry. MitoTracker Deep Red mitochondrial stain and the cell impermeable Sytox red nuclear stain were used as indicators of cell viability and cell membrane integrity, respectively. Results No significant viability loss was detected in cells transfected with 18-3-18, 18-7-18, 18-7NH-18, and 18-7NCH3-18 NPs, whereas a significant reduction of viability was detected in cells treated with 12-3-12, 12-7-12, 12-7NH-12, 16-7NH-16, or 16-7NCH3-16 GL-NPs. Compared to Lipofectamine Plus, 18-3-18 GL-NPs showed higher transfection efficiency and comparable viability profile by evaluation using MitoTracker Deep Red in PAM212 cells. Flow cytometric analysis of PAM212 cells stained with Sytox red revealed two cell populations with low and high fluorescent intensity, representing cells with partially-porated and highly-porated membranes, respectively. Additional combined staining with MitoTracker and ethidium homodimer showed that that 18-3-18 GL-NPs disturbed cell membrane integrity, while cells were still alive and had mitochondrial activity. Conclusion Taken together, this study demonstrated that 18-3-18 GL-NPs have higher transfection efficiency and comparable viability profile to the commercial Lipofectamine Plus, and the interaction of 18-3-18 GL-NPs with PAM212 cell membranes involves a permeability increase, possibly through the formation of nanoscale pores, which could explain efficient gene delivery. This novel nanoconstruct appears to be a promising delivery system for further skin gene therapy studies in vivo.

MicroRNAs are important class of nucleic acids as therapeutic agents. Intracellular delivery of microRNAs can be achieved through the employment of viral vectors. However, there are major safety concerns associated with this nucleic acid delivery approach, such as the increased risk of causing undesirable insertional mutation of the host genome. Synthetic particles provide an alternative approach for microRNA delivery. Inspired by nature, these artificial particles can be engineered to mimic the structure of viruses in packaging or attaching nucleic acids for therapeutic delivery. For effective non-viral delivery, the synthetic particles must possess the appropriate physical and chemical properties, as well as corresponding functionalities. Owing to their high versatilities, polymeric materials are good candidates for manufacturing artificial particles in the delivery of microRNAs. The properties and functionalities of particles can be customised with the appropriate choice of polymers. Incorporating polymeric materials also enables versatile modification of particles to improve the cellular uptake, targeting and release of microRNAs. This thesis presents the preparation of polyacrylamide-stabilised polystyrene latex particles as microRNA delivery vectors by surfactant-free Reversible Addition-Fragmentation Chain Transfer (RAFT) emulsion polymerisation. RAFT polymerisation techniques enable the synthesis of polymers with predetermined molecular weights, narrow molecular weight distributions and well-defined structures. The application of RAFT polymerisation to heterogeneous systems enables the fabrication of well-defined functional polymeric particles. The surface of these particles was post-modified with a disulfide-bearing linker for conjugation to a microRNA model, which can be released from the latex particles under reducing conditions. Additionally, the latex particles synthesised in this study were modified with a fluorescent dye to explore their in vitro uptake and biodistribution on a cell line and animal model.

Increasing attention has been paid to technology used for the delivery of genetic materials into cells for gene therapy and the generation of genetically engineered cells. So far, viral vectors have been mainly used because of their inherently high transfection efficiency of gene. However, there are some problems to be resolved for the clinical applications, such as the pathogenicity and immunogenicity of viral vectors themselves. Therefore, many research trials with non-viral vectors have been performed to enhance their efficiency to a level comparable to the viral vector. Two directions of these trials exist: Material improvement of non-viral vectors and their combination with various external physical stimuli. In this study gelatin was selected as a non-viral carrier for DNA. To give a positive charge to gelatin, different extents introduction of ethylenediamine (Ed), spermidine (Sd), and spermine (Sm) were reacted with gelatin in the presence of a water-soluble carbodiimide. When positively charged gelatin derivatives (Ed, Sd, and Sm) were mixed with negatively charged DNA, a self assembly of DNA nanoparticle (complex) was formed within few minutes through electrostatic interaction. Irrespective of the type of gelatin derivatives, the apparent molecular size of DNA was reduced by increasing the gelatin/DNA mixing ratio to attain a saturated value of about 150 nm. The condensed gelatin/DNA complexes showed the zeta potential of 10-15 mV. The amount of DNA internalized into the cells was significantly increased by the complexation with every gelatin derivative. The cells incubated with the gelatin/DNA complexes exhibited significantly stronger luciferase activities than naked plasmid DNA. This study clearly demonstrates and self-assembled DNA complexes has potential as a gene delivery vechile and are stable to transfer genetic materials to cells.

This paper investigates a series of dendrons based on the Newkome dendritic scaffold that displays a naturally occurring polyamine (spermine) on their surface. These dendrons have previously been shown to interact with DNA in a generation dependent manner with the more highly branched dendrons exhibiting a strong multivalency effect for the spermine surface groups. In this paper, we investigate the ability of these dendrons to transfect DNA into cells (human breast carcinoma cells, MDA-MB-231, and murine myoblast cells, C2C12) as determined by the luciferase assay. Although the dendrons are unable to transfect DNA in their own right, they are capable of delivering DNA in vitro when administered with chloroquine, which assists with escape from endocytic vesicles. The cytotoxicity of the dendrons was determined using the XTT assay, and it was shown that the dendrons were nontoxic either alone or in the presence of DNA. However, when administered with DNA and chloroquine, the most highly branched dendron did exhibit some cytotoxicity. This paper elucidates the relationship between in vitro transfection efficiency and toxicity. While transfection efficiencies are modest, the low toxicity of the dendrons, both in their own right, and in the presence of DNA, provides encouragement that this type of building block, which has a relatively high affinity for DNA, will provide a useful starting point for the further synthetic development of more effective gene transfection agents.

With the rapid development of nanotechnology in the recent decade, novel DNA and RNA delivery systems for gene therapy have become available that can be used instead of viral vectors. These non-viral vectors can be made of a variety of materials, including inorganic nanoparticles, carbon nanotubes, liposomes, protein and peptide-based nanoparticles, as well as nanoscale polymeric materials. They have as advantages over viral vectors a decreased immune response, and additionally offer flexibility in design, allowing them to be functionalized and targeted to specific sites in a biological system with low cytotoxicity. The focus of this review is to provide an overview of novel nanotechnology-based methods to deliver DNA and small interfering RNAs into biological systems.

At the basis of gene therapy stays as fundamental process the delivery of proper nucleic acids into the cell nucleus. This process needs efficient and safe vectors able to condense and protect DNA filaments, masking their phosphate negative charges and thus promoting membrane crossing. Due to their intrinsic properties, Cell Penetrating Peptides, and in particular the arginine-rich ones, have attracted attention in this field as adjuvant for DNA cargo in cell membrane crossing. The work reported in this PhD thesis concerns the synthesis of new potential non-viral vectors designed and built on macrocyclic scaffolds with well-defined cationic and lipophilic domains. They are functionalized with arginines, lysines, guanidinylated amino acids or ammonium ions. Their ability in DNA binding and condensation and their transfection properties in vitro were investigated also looking for establishing relationships between activity and structural features.

227 págs., [EN] Over the past few years and due to biotechnology development, gene therapy has emerged as a promising therapeutic tool because of the use of exogenous nucleic acids as active ingredients. These nucleic acids, i.e. Deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), can be delivered into the organism in order to modulate the expression of proteins modified in particular diseases.These acquired or genetic diseases include pathologies such as cancer, AIDS, neurological disorders or autoimmune diseases. Ocular gene therapy is one of the most investigated fields and its progress lies, among other things, in the easy accessibility into the eye and the convenient methods for its evaluation. Thus, several research groups use gene therapy as alternative to treat diseases such as retinitis pigmentosa, macular degeneration and X-linked juvenile retinoschisis. The vast majority of these studies are carried out using viral vectors to deliver genetic material, as they have proved to be the most effective to date. Nevertheless, the safety-related risks that involve their use have triggered the development of non-viral vectors, and the improvement of their efficacy has become a key challenge in gene therapy. In order to design non-viral vectors, an understanding of the extra and intracellular barriers conditioning the transfection process is needed: interaction and subsequent passage through cellular membrane, intracellular trafficking, resistance against degradation, genetic material release, passage through nuclear membrane and finally, expression of the protein codified by the plasmid that vector transports.In the present work hybrid gene delivery systems have been developed and evaluated. These systems, based on solid lipid nanoparticles (SLN), also include compounds of different nature to favor the transfection process by mans of several mechanisms. Formulations were characterized in terms of size, surface charge, protection capacity against DNAses and plasmid release from the nanoparticle. Furthermore, “in vitro” transfection capacity, cellular uptake, endocytosis mechanisms and intracellular trafficking were studied by means of confocal laser microscopy and flow cytometry. These processes were carried out over two cell lines (ARPE-19 and HEK-293) and were related to transfection capacity of the formulations developed.Firstly, the influence of a peptide, protamine, in the transfection capacity of SLN was evaluated. The presence of protamine in SLN increased the “in vitro” transfection capacity in ARPE-19 cells, with respect to SLN without protamine, but it caused a decrease in HEK-293 cells. It was observed that this different behavior is conditioned by the cellular uptake mechanism of nanoparticles, which depends on cell line as well as vector composition. Next, a polysaccharide, dextran, was included in the formulation. When lipid nanoparticles were prepared with protamine and the dextran, a high transfection capacity was achieved in ARPE-19 cells with the plasmid that codifies green fluorescent protein (EGFP) as well as the one that codifies retinoschisin, a protein related to X-linked juvenile retinoschisis. Once the vector was optimized by means of “in vitro” studies, the “in vivo” evaluation was conducted. For that purpose, a vector prepared with SLN, protamine, dextran and the plasmid that codified EGFP was administrated by different ocular routes in rats. After intravitreal injection, retina ganglion cells were mainly transfected, and when administrated by subretinal injection, retinal pigment epithelium cells as well as photoreceptors were the most transfected cells. After topical application, the vector was also able to transfect corneal cells. Finally, transfection capacity after intravenous administration into mice was also evaluated, detecting green fluorescent protein in liver, spleen and lung; the expression was maintained for at least seven days.

Tez (Yüksek Lisans) -- İstanbul Teknik Üniversitesi, Fen Bilimleri Enstitüsü, 2012, Thesis (M.Sc.) -- İstanbul Technical University, Institute of Science and Technology, 2012, Gen tedavisi, genetik kaynaklı birçok hastalığa çözüm sunma potansiyeli açısından son yıllarda oldukça rağbet gören bir çalışma alanı olmuştur. Gen tedavisinin ana amacı hastalıkların ortaya çıkardığı semptomları iyileştirmeye çalışmak yerine, hastalığa sebep olan genetik bozuklukları ortadan kaldırmaktır. Bu tedavide, tedavi edici özelliği olan genler, en güvenli ve verimli bir taşıyıcı ile hedef dokuya ya da hücreye transfer edilmektedir. Bu amaçla kullanılan taşıyıcılar viral ve viral olmayan taşıyıcılar olmak üzere ikiye ayrılır. Viral taşıyıcılar, hedefe ulaşma verimliliği açısından bilinen en verimli taşıyıcılar olmakla birlikte güvenilirlik ve üretim aşamaları açısından uygulamaları çok kısıtlıdır. Bu sebeple, güvenli ve üretimi kolay alternatif bir metod olarak katyonik polimerler araştırılmaktadır. Literatürde, katyonik polimerlerin transfeksiyon verimliliklerini artırmak için yapılmış pek çok çalışmaya ulaşılabilir. Bu tez çalışmasında, gen tedavisi potensiyelleri açısından iki farklı polimer türü sentezlendi ve çeşitli özellikleri bakımından incelendi. Bu polimerlerin ilki, katyonik fakat biyobozunur olmayan poli(2-(dimetilamino)etil metakrilat) (pDMAEMA) polimeridir. İkinci tür taşıyıcı sistem ise katyonik ve biyobozunur disülfür bağı içeren, lineer poli(amidoamin) (SS-PAA) polimer türevleridir. SS-PAA ailesine ait farklı yan grup ve kaplama stratejileri ile sentezlenen 4 farklı polimer bu çalışmada incelendi. Bunlar poli-(N,N’-sistamin bisakrilamit-4-amino bütanol) p(CBA-ABOL), PEG’leme stratejisi ile geliştirilen p(CBA-ABOL/DMEDA/PEG), hiyaluronik asit (HA) kaplanmış p(CBA-ABOL) ve son olarak da poli-(N,N’-sistamin bisakrilamit-histamin) (p(CBA-HIS))’dir. Bu çalışmanın ilk aşaması olarak farklı molekül ağırlıklı ve farklı polimer oranlarına sahip polimer/pDNA (plazmit DNA) kompleksleri (polipleks) fizikokimyasal özellikleri açısından incelendi. Bu amaçla boyut, polidispersite ve yüzey yükü ölçümleri DLS cihazı ile yapıldı. İkinci olarak poliplekslerin pDNA’yı kaplayarak kararlı kompleksler oluşturma etkinlikleri agaroz jel elektroforez ile belirlendi. Bu karakterizasyonları takiben, polimer/pDNA komplekslerinin ARPE-19 hücreleri ile in vitro biyolojik değerlendirmeleri amaçlandı. Farklı molekül ağırlığına (MA) ve farklı polimer oranlarına sahip polipleksler hazırlanarak hücrelere transfer edildi ve hücre içine alınma ve genetik materyali hedefe ulaştırma verimlilikleri Hücre Sitometresi (Flow Cytometry) cihazı ile ölçüldü. Son olarak, poliplekslerin hücreler üzerindeki sitotoksik etkisi MTT Testi ile analiz edildi. pDMAEMA polimeri ile oluşturulan 2 farklı MA ve farklı polimer oranına sahip poliplekslerin HEPES tamponu ortamında yapılan boyut ve yük tayini sonuçlarına göre bu ortamda pDNA ile kararlı komplekler yapabildiği görüldü. Yapılan ölçümlerin fizyolojik ortama uygunluğunun denenmesi için aynı seri ölçümler bir de serum içermeyen transfeksiyon ortamı olan OptiMEM ortamında gerçekleştirildi. OptiMEM ortamında kompleks kararlılığının azaldığı ve agregasyon oluşumu gözlendi. Bu ölçümleri takiben pDMAEMA polimerinin pDNA’yı kondanse edebilme özelliğinin analizi için pDMAEMA polipleksleri agaroz jel elektroforeze uygulandı. Her iki MA ve polimer oranındaki kompleksler pDNA kaplamaya muktedirdi. Polimer MA ve oranının polipleks kararlılığına bariz bir etkisinin olmadığı gözlendi fakat yüksek polimer oranına sahip kompleksler daha küçük parçacık boyutu gösterdiler. pDMAEMA poliplekslerinin in vitro biyolojik değerlendirilmesi sonuçlarına göre pDMAEMA/pDNA komplekslerinin hücre içine alınma verimlilikleri oldukça tatmin ediciydi. Fakat hücre içine alınan poliplekslerin çekirdeğe ulaşma potansiyelleri oldukça düşük ve ancak hücreler için toksik olan polimer miktarlarında ilgi çekici sonuçlar verdi. SS-PAA polimerleri içerdikleri tekrarlanan sülfür köprüleri sayesinde biyobozunur polimerler olduklarından gen salımı konusunda iyi bir potansiyele sahiptirler. Bu ailenin ilk üyesi olarak p(CBA-ABOL) polimeri gen taşıyıcısı olarak değerlendirildi. pDMAEMA polipleksleri için yapılan tüm işlemler p(CBA-ABOL)/pDNA komplekleri için de denendi. Farklı polimer oranlarıyla oluşturulan p(CBA-ABOL) komplekslerinin yüzey yükü ve boyut tayini HEPES ve OptiMEM ortamlarında gerçekleştirildi. HEPES ortamında artan polimer miktarının daha kararlı kompleksler oluşturmasına karşılık, aynı komplekslerde OptiMEM ortamında agregasyon gözlendi. Agaroz jel elektroforez sonuçlarına göre yüksek polimer oranları 48/1 ve 96/1 ile oluşturulan kompleksler pDNA’yı kaplama açısından daha elverişli idiler. p(CBA-ABOL) poliplekslerinin in vitro biyolojik değerlendirilmelerinin sonucunda ise p(CBA-ABOL) poliplekslerinin genetik materyalin hücre içine alınması ve ardından çekirdeğe ulaştırılması açısından pDMAEMA komplekslerine göre daha verimli olduğu ayrıca ARPE-19 hücreleri için yalnızca ihmal edilebilir düzeyde bir toksisiteye sebep olduğu görüldü. p(CBA-ABOL) komplekslerinin fizyolojik ortamdaki agregasyonunu engellemek amacıyla evrensel bir strateji olan PEG’leme stratejisi kullanıldı. PEG’lenen p(CBA-ABOL) polimeri ile oluşturulan kompleksler de daha önceki ölçümlere tabi tutuldu. PEG’leme sonucunda komplekslerin OptiMEM ortamındaki kolloidal kararlılığında artış gözlenirken in vitro biyolojik değerlendirme açısından PEG’lenen p(CBA-ABOL) polimeri ile yapılan ölçümler istenilen verimi göstermedi. Hücre içine alınma verimliliği açısından PEG’lenmiş komplekslerin yok denecek kadar az bir potansiyellerinin olduğu gözlendi. Kompleksler hücre içine alınmadığından sitotoksisite analizine gerek duyulmadı. p(CBA-ABOL) komplekslerinin hücre içine alınma kapasitelerinin artırılması için kompleksler bir hücre zarı proteini olan HA ile elektrostatik olarak farklı HA/pDNA oranlarında kaplanarak önceki polimerler ile aynı şekilde fizikokimyasal ve biyolojik değerlendirmelere tabi tutuldu. Komplekslerin dış kabuğu anyonik HA ile kaplandığından yüzey yükünün pozitiften negatife döndüğü gözlendi. Parçacık boyutu ve kolloidal stabilite açısından ise kaplanmamış p(CBA-ABOL) komplekslerine göre OptiMEM ortamında daha kararlı kompleksler elde edildi. Biyolojik değerlendirme sonuçlarında, poliplekslerin hücre içine alımında HA kaplama ile elde edilmek istenen artış gözlenmedi. En son taşıyıcı sistem olarak SS-PAA polimerlerinin bir türevi olan p(CBA-HIS) polimeri ile oluşturulan kompleksler gen taşıma kapasiteleri açısından değerlendirildi. HIS yan grubu, ABOL yan grubuna göre daha asidik olduğundan komplekslerin hücre içine endozomlar vasıtasıyla alınmasını takiben endozomlardan çıkışı artıracağı ve buna bağlı olarak çekirdeğe ulaşan kompleks miktarının artacağı amaçlandı. p(CBA-HIS) kompleksleri kolloidal karalılık ve pDNA kondenzasyonu açısından p(CBA-ABOL) kompleksleri ile benzer özellikler gösterdiler. Aynı şekilde in vitro ölçümler sonucuda p(CBA-HIS) komplekslerinin hücre içine alınma kapasitelerinin daha yüksek olmasına rağmen genetik materyalin çekirdeğe ulaşma verimliliği açısından amaçlanan artış gözlenmedi. Sonuç olarak pDMAEMA polimer/pDNA komplekslerinin artan molekül ağırlıkları ve polimer miktarları hücreler üzerinde toksik etki yaparken, SS-PAA polimerlerinin transfeksiyon açısından iyi bir potansiyellerinin olduğu tespit edildi. Bu gruptan son olarak incelenen p(CBA-HIS) poliplekslerinin HEPES tamponu ortamında pDNA’yı kondanse etmesi ve transfekte ettiği hücreler üzerinde sitotoksik aktivite göstermemesine rağmen agregasyon oluşumu tamamen engellenemedi. Bu nedenle bu poliplekslerin fizyolojik şartlardaki davranışlarının daha ileri araştırmalarla incelenmesi gerekmektedir., Gene therapy has been a promising way to treat human genetic disorders. The major goal of gene therapy is to repair the genetic cause underlying the disorder by transferring the therapeutic nucleic acid with a safe and efficient carrier. For this purpose, two main classes of vectors are explained: viral and non-viral vectors. Even though, viral vectors are the most efficient carriers known, they have restrictions about their safety and production process. Cationic polymers have been the mostly investigated non-viral vector systems as a safe and easy-to-produce alternative to viral carriers. Moreover, numerous research have been done to increase their transfection efficiency. In this thesis study, two different types of cationic polymers were synthesized and investigated for their non-viral gene carrier potentials. One of them is a cationic, non-biodegradable poly(2-(dimethylamino)ethyl methacrylate) (pDMAEMA). The second type of carriers is cationic and biodegradable linear, disulfide linkage containing poly(amidoamine)s (SS-PAAs) that include four different polymers with different side groups and coating strategies. The SS-PAA family consists of p(CBA-ABOL), p(CBA-ABOL/DMEDA/PEG) with PEGylation strategy, p(CBA-ABOL) with HA-coating and lastly, p(CBA-HIS). The main objectives of this study were first of all, physicochemical characterization of the polymer/pDNA complexes with different polymer Mw and polymer/pDNA ratio in terms of size, polydispersity and surface charge which were measured by DLS. Secondly, the pDNA complexation ability of polyplexes was investigated by performing agarose gel electrophoresis. Thirdly, in vitro biological evaluation of polymers on ARPE-19 cells were performed. For this purpose, uptake and transfection efficiencies of the polymers and the effect of different polymer Mw’s and different polymer/pDNA ratios on uptake and transfection efficiency of polyplexes were evaluated by Flow Cytometry. Finally, the effect of polyplexes on the viability of ARPE-19 cells was analyzed with MTT Assay. While evaluating the pDMAEMA complexes, they showed the ability to form stable complexes in HEPES buffer and were able to efficiently complex pDNA. Nevertheless, the cationic nanoparticles formed with these polymers only exhibited signs of transfection in concentrations already toxic for the cells. This is why a new family of biodegradable polymers- the disulfide-containing PAAs, were investigated. p(CBA-ABOL) particles were also able to form stable polyplexes with efficiently complexed pDNA and also showed very high transfection efficiency, without significant effect on cell viability. Unfortunately, these nanoparticles were not stable in mimicked physiological conditions, which is why the same polymer was investigated with a PEG-chain as a universal coating strategy. Even though these particles were stable under physiological conditions, their uptake was drastically hindered. A different coating strategy with hyaluronic acid showed promising preliminary results, but the same results could not be observed with the research grade purity low Mw HA. Subsequently, another member of SS-PAA family with a different side chain and buffering capacity was evaluated. p(CBA-HIS) polymer was able to condense the pDNA in HEPES buffer and transfected the cells with almost no cytotoxic effect. However, the colloidal stability of p(CBA-HIS) polyplexes in physiological conditions should be further increased., Yüksek Lisans, M.Sc.

Dincer, Sevil/0000-0002-6887-6549 WOS: 000290672200006 PubMed: 20677903 In this work, positively charged, micelle-forming polymers were synthesized and used as a model vector to deliver antisense oligodeoxynucleotide (ASODN) into melanoma cells. Polymers and polymer/ASODN complexes were characterized by DLS according to size, charge, and critical micelle concentration. Nanosize and spherical complexes were observed by AFM. Complexes did not reveal significant toxicity to melanoma cells. Antiproliferative effect of the complexes was observed by immunocytochemical staining and estimated as 56.8% with N/P:9. High amount of apoptosis and very small amount of necrosis were estimated. According to the results, these positively charged polymers forming micelle-like structures seem promising as ASODN carriers. [TUBITAK-107T864] This study was supported financially by TUBITAK-107T864. We thank Res. Asist. Yeliz Basaran for AFM images, and Res. Asist. Eray Dalgakiran for DLS analysis.

The successful clinical application of nucleic acid-based therapeutic strategies has been limited by the poor delivery efficiency achieved by existing vectors. The development of alternative delivery systems for improved biological activity is, therefore, mandatory. Since the seminal observations two decades ago that the Tat protein, and derived peptides, can translocate across biological membranes, cell-penetrating peptides (CPPs) have been considered one of the most promising tools to improve non-invasive cellular delivery of therapeutic molecules. Despite extensive research on the use of CPPs for this purpose, the exact mechanisms underlying their cellular uptake and that of peptide conjugates remain controversial. Over the last years, our research group has been focused on the S413-PV cell-penetrating peptide, a prototype of this class of peptides that results from the combination of 13-amino-acid cell penetrating sequence derived from the Dermaseptin S4 peptide with the SV40 large T antigen nuclear localization signal. By performing an extensive biophysical and biochemical characterization of this peptide and its analogs, we have gained important insights into the mechanisms governing the interaction of CPPs with cells and their translocation across biological membranes. More recently, we have started to explore this peptide for the intracellular delivery of nucleic acids (plasmid DNA, siRNA and oligonucleotides). In this review we discuss the current knowledge of the mechanisms responsible for the cellular uptake of cell-penetrating peptides, including the S413-PV peptide, and the potential of peptide-based formulations to mediate nucleic acid delivery.