Substrate-mediated nucleic acid delivery from self-assembled monolayers

Substrate-mediated nucleic acid (NA) delivery involves the immobilization of NAs or NA delivery vehicles to biomaterials for localized transfection of cells. Self-assembled monolayers (SAMs) offer an easy system to immobilize delivery vectors. SAMs form well-defined surfaces; therefore, the effect of surface composition on vector immobilization and transfection efficiency can also be studied. To date, the most effective SAM-mediated delivery systems have utilized nonspecific interactions for immobilization; however, systems that rely on specific interactions between vector and surface can impart higher control of spatial and/or temporal delivery. This review summarizes systems that use both specific and nonspecific interactions for gene delivery from SAMs; highlights progress and remaining challenges; and explores other specific recognition modalities that might be employed for future applications in surface-mediated NA delivery.     

Self-assembled surfactant nano-structures important in drug delivery: a review

Role of self assembled structures as a vehicle is significant over the years. Their applications have been found for all routes of drug delivery. These micro and nano structures are containers loaded with drugs, ideal for targeted and sustained release of the drug. Drug efficacy depends on the drug loaded into the vehicle, temperature, drug solubility, pH, release characteristics, additives and most significantly, the vehicle morphology. This in turn suggests that the same vehicle cannot be used with high efficiency for all types of drugs and locations where the drug delivery has to take place. The status of various self assembled structures and their applications in drug delivery is reviewed in this communication.     

Potential efficacy of cell-penetrating peptides for nucleic acid and drug delivery in cancer

Cell penetrating peptides (CPPs) are short amphipathic and cationic peptides that are rapidly internalized across cell membranes. They can be used to deliver molecular cargo, such as imaging agents (fluorescent dyes and quantum dots), drugs, liposomes, peptide/protein, oligonucleotide/DNA/RNA, nanoparticles and bacteriophage into cells. The utilized CPP, attached cargo, concentration and cell type, all significantly affect the mechanism of internalization. The mechanism of cellular uptake and subsequent processing still remains controversial. It is now clear that CPP can mediate intracellular delivery via both endocytic and non-endocytic pathways. In addition, the orientation of the peptide and cargo and the type of linkage are likely important. In gene therapy, the designed cationic peptides must be able to 1) tightly condense DNA into small, compact particles; 2) target the condensate to specific cell surface receptors; 3) induce endosomal escape; and 4) target the DNA cargo to the nucleus for gene expression. The other studies have demonstrated that these small peptides can be conjugated to tumor homing peptides in order to achieve tumor-targeted delivery in vivo. On the other hand, one of the major aims in molecular cancer research is the development of new therapeutic strategies and compounds that target directly the genetic and biochemical agents of malignant transformation. For example, cell penetrating peptide aptamers might disrupt protein-protein interactions crucial for cancer cell growth or survival. In this review, we discuss potential functions of CPPs especially for drug and gene delivery in cancer and indicate their powerful promise for clinical efficacy.     

Inferring consensus structure from nucleic acid sequences

This paper presents an unsupervised inference method for determiningthe higher-order structure from sequence data. The method isgeneral, but in this paper it is applied to nucleic acid sequencesin determining the secondary (2-D) and tertiary (3-D) structureof the macromolecule. The method evaluates position -positioninterdependence of the sequence using an information measureknown as expected mutual information. The expected mutual informationis calculated for each pair of positions and the chi-squaretest is used to screen statistically significant position pairs.In the calculation of expected mutual information, an unbiasedprobability estimator is used to overcome the problem associatedwith zero observation in conserved sites. A selection criterionbased on known structural constraints of the strongest interdependentposition pairs is applied yielding position pairs most indicativeof secondary and tertiary interactions. The method has beentested using tRNA and 5S rRNA sequences with very good results. Received on July 20, 1990; accepted on January 15, 1991     

Timely drug delivery from controlled-release devices: Dynamic analysis and novel design concepts

Design tools are provided to assist in the development of drug-release devices that can control the time to establish a steady-state flux in addition to a desired delivery rate. In this contribution, the primary focus is placed on passive, heat-aided, and electrically assisted transport through planar membranes. Approximate analytical methods, that describe process dynamics, were applied to appropriate mathematical models to derive relationships between the system properties (e.g., diffusion coefficient, temperature and voltage potential) and the flux response time. Three case studies were investigated to illustrate the theoretical results. A steady-state benzocaine flux through ethylene-vinyl acetate membranes was achieved in 40 min as predicted by a first-moment time constant approach. It took 5 h and 45 min to reach a constant delivery rate of amitriptyline HCl across human skin for a donor cell concentration of 0.032 M and an electric current of 0.4 mA/cm². Based on the upper limit and range estimates of the first eigenvalue, the onset of steady-state flux occurred between 3.4 and 3.5 min when the donor and receiver cells were maintained at 47 and 37 °C, respectively. These predictions were confirmed by simulation, experimental evidence and graphical examination of drug-release data.     

Laser-assisted nucleic acid delivery: A systematic review

Gene therapy has become an effective treatment modality for some conditions. Laser light may augment or enhance gene therapy through photomechanical, photothermal, and photochemical. This review examined the evidence base for laser therapy to enhance nucleic acid transfection in mammalian cells. An electronic search of MEDLINE, Scopus, EMBASE, Web of Science, and Google Scholar was performed, covering all available years. The preferred reporting items for systematic reviews and meta-analyses guideline for systematic reviews was used for designing the study and analyzing the results. In total, 49 studies of laser irradiation for nucleic acid delivery were included. Key approaches were optoporation, photomechanical gene transfection, and photochemical internalization. Optoporation is better suited to cells in culture, photomechanical and photochemical approaches appear well suited to in vivo use. Additional studies explored the impact of photothermal for enhancing gene transfection. Each approach has merits and limitations. Augmenting nucleic acid delivery using laser irradiation is a promising method for improving gene therapy. Laser protocols can be non-invasive because of the penetration of desirable wavelengths of light, but it depends on various parameters such as power density, treatment duration, irradiation mode, etc. The current protocols show low efficiency, and there is a need for further work to optimize irradiation parameters.     

Self-assembled virus-like particles from rotavirus structural protein VP6 for targeted drug delivery

Proteins of viral capsid may self-assemble into virus-like particles (VLPs) that can find many biomedical applications such as platform for drug delivery. In this paper, we describe preparation of VLPs by self-assembly of VP6, a rotavirus capsid protein that was chemically conjugated with doxorubicin (DOX), an anticancer drug. VP6 was first highly expressed in E. Coli, followed by purification and renaturation. DOX was then covalently attached to VP6 to form DOX-VP6 (DVP6) conjugates, which were subsequently self-assembled into VLPs under appropriate condition. Next, lactobionic acid (LA) was chemically linked to the surface of the VLPs. We demonstrated that the aforementioned nanosystem shows specific targeting to hepatoma cell line HepG2. The chemically functionalized VLPs, a kind of biological nanoparticles with excellent biocompatibility and biodegradability, can be prepared in large scale from E. Coli through our method, which may find practical applications in biomedicine.     

Lipase-catalyzed copolymerization of lactic and glycolic acid with potential as drug delivery devices

The copolymerization of lactic and glycolic acid (PLGA) using Candida antarctica lipase B as biocatalyst has been achieved with the aim to generate useful biomedical materials. The influence of the reaction conditions, such as solvent and temperature, on the enzyme's catalytic activity was studied to optimize the synthetic procedure. The evaluated parameters were the conversion, the isolated PLGA and the number average molecular weight (M(n)). The identification and purity of the products were assessed by FTIR and NMR. The conversion was determined using analytical titration and the M(n) through end-group analysis. It was found that PLGA oligomers were obtained with satisfactory conversion levels when isopropyl ether was employed as solvent. The use of toluene increased the M(n) but decreased the isolated polyester. Higher percentages of recovered PLGA were reached increasing the temperature from 60 to 80 degrees C using toluene, while a reduction in the M(n) was evidenced under these conditions.     

Ab initio conformational analysis of nucleic acid components: intrinsic energetic contributions to nucleic acid structure and dynamics

In recent years, the use of high-level ab initio calculations has allowed for the intrinsic conformational properties of nucleic acid building blocks to be revisited. This has provided new insights into the intrinsic conformational energetics of these compounds and its relationship to nucleic acids structure and dynamics. In this article we review recent developments and present new results. New data include comparison of various levels of theory on conformational properties of nucleic acid building blocks, calculations on the abasic sugar, known to occur in vivo in DNA, on the TA conformation of DNA observed in the complex with the TATA box binding protein, and on inosine. Tests of the Hartree-Fock (HF), second-order M?ller-Plesset (MP2), and Density Functional Theory/Becke3, Lee, Yang and Par (DFT/B3LYP) levels of theory show the overall shape of backbone torsional energy profiles (for gamma, epsilon, and chi) to be similar for the different levels, though some systematic differences are identified between the MP2 and DFT/B3LYP profiles. The east pseudorotation energy barrier in deoxyribonucleosides is also sensitive to the level of theory, with the HF and DFT/B3LYP east barriers being significantly lower (approximately 2.5 kcal/mol) than the MP2 counterpart (approximately 4.0 kcal/mol). Additional calculations at various levels of theory suggest that the east barrier in deoxyribonucleosides is between 3.0 and 4.0 kcal/mol. In the abasic sugar, the west pseudorotation energy barrier is found to be slightly lower than the east barrier and the south pucker is favored more than in standard nucleosides. Results on the TA conformation suggest that, at the nucleoside level, this conformation is significantly destabilized relative to the global energy minimum, or relative to the A- and B-DNA conformations. Deoxyribocytosine would destabilize the TA conformation more than other bases relative to the A-DNA conformation, but not relative to the B-DNA conformation.     

Fibrillin: from domain structure to supramolecular assembly.

In the last 5 years, significant progress has been made in understanding the structure and function of all the major domains composing the fibrillins. A previous review [Meth. Enzymol. 245 (1994), 29] focused on the isolation of fibrillin monomers and fibrillin-containing polymers (microfibrils). In this article, information gained from recent studies which have further elucidated molecular structure and investigated effects of mutations on structural and functional properties will be summarized. In addition, studies of functional domains in fibrillins which may be important in assembling microfibrils will be discussed. Throughout this review, the authors have attempted to identify areas of research which have been controversial. In the conclusion, we raise important questions which remain unresolved.     

Ionic liquids: prospects for nucleic acid handling and delivery

Operations with nucleic acids are among the main means of studying the mechanisms of gene function and developing novel methods of molecular medicine and gene therapy. These endeavours usually imply the necessity of nucleic acid storage and delivery into eukaryotic cells. In spite of diversity of the existing dedicated techniques, all of them have their limitations. Thus, a recent notion of using ionic liquids in manipulations of nucleic acids has been attracting significant attention lately. Due to their unique physicochemical properties, in particular, their micro-structuring impact and tunability, ionic liquids are currently applied as solvents and stabilizing media in chemical synthesis, electrochemistry, biotechnology, and other areas. Here, we review the current knowledge on interactions between nucleic acids and ionic liquids and discuss potential advantages of applying the latter in delivery of the former into eukaryotic cells.     

Vaccines for COVID-19: perspectives from nucleic acid vaccines to BCG as delivery vector system

This article discusses standard and new disruptive strategies in the race to develop an anti-COVID-19 vaccine. We also included new bioinformatic data from our group mapping immunodominant epitopes and structural analysis of the spike protein. Another innovative approach reviewed here is the use of BCG vaccine as priming strategy and/or delivery system expressing SARS-CoV-2 antigens.     

Cationic lipid-mediated nucleic acid delivery: beyond being cationic

Realization of the potential of nucleic acids as drugs is intricately linked to their in vivo delivery. Cationic lipids demonstrated tremendous potential as safe, efficient and scalable in vitro carriers of nucleic acids. For in vivo delivery of nucleic acids, the extant two component liposomal preparations consisting of cationic lipids and nucleic acids have been largely found to be insufficient. Being a soft matter, liposomes readily respond to many physiological variables leading to complex component and morphological changes, thus confounding the efforts in a priori identification of a “competent” formulation. In the recent past many chemical moieties that provide advantage in facing the challenges of barriers in vivo, were incorporated into cationic lipids to improve the transfection efficiency. The cationic lipids, essential for DNA condensation and protection, definitely require additional components to be efficient in vivo. In addition, formulations of cationic lipid carriers with non-lipidic components, mainly peptides, have demonstrated success in in vivo transfection. The present review describes some recent successes of in vivo nucleic acid delivery by cationic lipids.     

SAND, a new protein family: from nucleic acid to protein structure and function prediction

As a result of genome, EST and cDNA sequencing projects, there are huge numbers of predicted and/or partially characterised protein sequences compared with a relatively small number of proteins with experimentally determined function and structure. Thus, there is a considerable attention focused on the accurate prediction of gene function and structure from sequence by using bioinformatics. In the course of our analysis of genomic sequence from Fugu rubripes, we identified a novel gene, SAND, with significant sequence identity to hypothetical proteins predicted in Saccharomyces cerevisiae, Schizosaccharomyces pombe, Caenorhabditis elegans, a Drosophila melanogaster gene, and mouse and human cDNAs. Here we identify a further SAND homologue in human and Arabidopsis thaliana by use of standard computational tools. We describe the genomic organisation of SAND in these evolutionarily divergent species and identify sequence homologues from EST database searches confirming the expression of SAND in over 20 different eukaryotes. We confirm the expression of two different SAND paralogues in mammals and determine expression of one SAND in other vertebrates and eukaryotes. Furthermore, we predict structural properties of SAND, and characterise conserved sequence motifs in this protein family.     

Targeted gene delivery: the role of peptide nucleic acid

Receptor-mediated endocytosis can be exploited to achieve efficient cell-specific gene delivery. Our laboratory has used two approaches for targeted gene delivery. One uses polycation as a carrier for plasmid DNA and the other uses peptide nucleic acid (PNA) as a carrier. Targeted gene delivery using polycation carriers has been widely utilized with some success. This approach mainly suffers from large particle size and non-specific interaction with blood components. These drawbacks have limited use of this type of vector forin vivo applications. Using PNA as a carrier, on the other hand, allows for smaller particle size and less non-specific interactions. The stability of this vector in the circulation may be a limiting factor. In addition, both types of vector lack mechanisms for endosome escape and nuclear transport. In this chapter, current developments and uses for targeted gene delivery of each approach are reviewed.