Gene Delivery Applications Research Articles

Liposome-based nanocapsules for the controlled release of dietary curcumin: PDDA and silica nanoparticle-coated DMPC liposomes enhance the fluorescence efficiency and anticancer activity of curcumin.

Nanosystems with various compositions and biological properties are being extensively investigated for drug and gene delivery applications. Many nanotechnology methods use novel nanocarriers, such as liposomes, in therapeutically targeted drug delivery systems. However, liposome matrices suffer from several limitations, including drug leakage and instability. Therefore, the surface modification of liposomes by coating them or adding polymers has advanced their application in drug delivery. Hence, the prevention of drug release from the liposome bilayers was the main focus of this work. For this purpose, liposomes were synthesized according to a thin film hydration method by applying various surface modifications. Three different nanocapsules, N1, N2, and N3, were prepared using 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), poly(diallyldimethylammonium)chloride (PDAA) polymer, and silica nanoparticles. PDDA and silica nanoparticles were coated on the surface of liposomes using a layer-by-layer assembly method, completely encapsulating curcumin into the core of the liposome. Fluorescence spectroscopy, TGA, DLS, XRD, SEM, and zeta potential methods were used to characterize the prepared nanocapsules. Interestingly, the fluorescence of curcumin showed a blue shift and the fluorescence efficiency was extraordinarily enhanced ∼25-, ∼54-, and ∼62-fold in the N1, N2, and N3 nanocapsules, respectively. Similarly, encapsulation efficiency, drug loading, and the anticancer activity of dietary curcumin were investigated for the different types of DMPC nanocapsules. The drug efficiencies of the liposomes were established according to the release of curcumin from the liposomes. The results showed that the release of curcumin from the nanocapsules decreased as the number of layers at the surface of the liposomes increased. The release of curcumin follows the Higuchi model; thus, a slow rate of diffusion is observed when a number of layers is added. The better encapsulation and higher anti-cancer activity of curcumin were also observed when more layers were added, which is due to electrostatic interactions inhibiting curcumin from being released.

Enhancing the in vivo stability of polycation gene carriers by using PEGylated hyaluronic acid as a shielding system

Abstract To increase the in vivo stability of cationic gene carriers and avoid the adverse effects of their positive charge, we synthesized a new shielding material by conjugating low molecular weight polyethylene glycol (PEG) to a hyaluronic acid (HA) core. The HA-PEG conjugate assembled with the positively charged complex, forming a protective layer through electrostatic interactions. DNA/polyetherimide/HA-PEG (DNA/PEI/HA-PEG) nanoparticles had higher stability than both DNA/polyethyleneimine (DNA/PEI) and DNA/PEI/HA complexes. Furthermore, DNA/PEI/HA-PEG nanoparticles also showed a diminished nonspecific response toward serum proteins in vivo. The in vivo transfection efficiency was also enhanced by the low cytotoxicity and the improved stability; therefore, this material might be promising for use in gene delivery applications.

Dicationic amphiphiles bearing an amino acid head group with a long-chain hydrophobic tail for in vitro gene delivery applications.

Amino acid-based cationic lipids, which have proven their efficacy as plasmid DNA nanocarriers, were employed as dicationic forms to transfect genes into cancer and non-cancer cells in this study. Proline, methionine, and serine amino acids are involved as hydrophilic moieties and the hydrocarbon long-chain serves as a hydrophobic tail. In a multicultural investigation, cationic lipids were employed as nano-vectors in conjunction with the helper lipid DOPE. To quantify the lipid efficient size, charge, and pDNA binding, biophysical analyses such as hydrodynamic diameter, zeta potential, agarose gel electrophoresis, and serum stability were done primarily. The liposomal particle composition was examined by scanning electron microscopy (SEM). Synthesized dicationic vector lipoplex formulations with reporter genes were found to be non-toxic to the cells investigated by MTT assay, and in addition, therapeutic gene p53 transfected into oral and brain cancer cells causing cell death was examined. In vitro investigations further validated that the proline-based lipid (C14-P) has high gene knockdown efficacy than methionine-based lipid (C14-M) and serine-based lipid (C14-S) at optimal N/P ratios as measured by β-galactosidase protein and eGFP expression. C14-P lipid shows superior cellular internalization compared to C14-M and C14-S in HEK-293 and CAL-27 cells attested by confocal study. These findings could include the proline-based lipid vector's exceptional gene delivery activity.

Determination of Optimum Ratio of Cationic Polymers and Small Interfering RNA with Agarose Gel Retardation Assay.

Nanomaterials have aroused attention in the recent years for their high potential for gene delivery applications. Most of the nanoformulations used in gene delivery are positively charged to carry negatively charged oligonucleotides. However, excessive positively charged carriers are cytotoxic. Therefore, the complexed oligonucleotide/nanoparticles should be well-examined before the application. In that manner, agarose gel electrophoresis, which is a basic method utilized for separation, identification, and purification of nucleic acid molecules because of its poriferous nature, is one of the strategies to determine the most efficient complexation rate. When the electric field is applied, RNA fragments can migrate through anode due to the negatively charged phosphate backbone. Because RNA has a uniform mass/charge ratio, RNA molecules run in agarose gel proportional according to their size and molecular weight. In this chapter, the determination of complexation efficiency between cationic polymer carriers and small interfering RNA (siRNA) cargos by using agarose gel electrophoresis is described. siRNA/cationic polymer carrier complexes are placed in an electric field and the charged molecules move through the counter-charged electrodes due to the phenomenon of electrostatic attraction. Nucleic acid cargos are loaded to cationic carriers via the electrostatic interaction between positively charged amine groups (N) of the carrier and negatively charged phosphate groups (P) of RNA. The N/P ratio determines the loading efficiency of the cationic polymer carrier. In here, the determination of N/P ratio, where the most efficient complexation occurs, by exposure to the electric field with a gel retardation assay is explained.

Advanced Materials in Cancer Therapy

The overview of this review article depends on the various techniques of formation of silver nanoparticles and different application take place in medicinal point of view.
 The branch of nanotechnology plays an important role in medical science research. In this different nanoparticle is synthesized which have various application in gene delivery, drug delivery and reduce the toxic effect of drugs in the human body and also act as an antibacterial in pharmaceutical industries.
 In recent days silver nanoparticles have had an important role due to their optical and catalytic properties. A large number of different particles or methods are used to prepare the different shapes of silver nanoparticles used in drug delivery. Different shapes of nanoparticles have increased their demand in various researches depend on medicinal uses. Silver nanoparticle preparation can be studied by 3 techniques related to irradiations, chemicals, bacteria, fungi, and plants. 
 Nanomedicine have a large number of advantages in treating various chronic diseases by using biological agents, chemotherapeutic agents, and used to deliver the drug to a specific site of the body. A silver nanoparticle is prepared for detection tool to detect the adverse effect of diseases on the target cell. Nanoparticles are used in cancer therapy to remove the damaged cell of the body.

ROS-responsive nanoparticle-mediated delivery of CYP2J2 gene for therapeutic angiogenesis in severe hindlimb ischemia.

With critical limb ischemia (CLI) being a multi-factorial disease, it is becoming evident that gene therapy with a multiple bio-functional growth factor could achieve better therapeutic outcomes. Cytochrome P450 epoxygenase-2J2 (CYP2J2) and its catalytic products epoxyeicosatrienoic acids (EETs) exhibit pleiotropic biological activities, including pro-angiogenic, anti-inflammatory and cardiovascular protective effects, which are considerably beneficial for reversing ischemia and restoring local blood flow in CLI. Here, we designed a nanoparticle-based pcDNA3.1-CYP2J2 plasmid DNA (pDNA) delivery system (nanoparticle/pDNA complex) composed of a novel three-arm star block copolymer (3S-PLGA-po-PEG), which was achieved by conjugating three-armed PLGA to PEG via the peroxalate ester bond. Considering the multiple bio-functions of CYP2J2-EETs and the sensitivity of the peroxalate ester bond to H2O2, this nanoparticle-based gene delivery system is expected to exhibit excellent pro-angiogenic effects while improving the high oxidative stress and inflammatory micro-environment in ischemic hindlimb. Our study reports the first application of CYP2J2 in the field of therapeutic angiogenesis for CLI treatment and our findings demonstrated good biocompatibility, stability and sustained release properties of the CYP2J2 nano-delivery system. In addition, this nanoparticle-based gene delivery system showed high transfection efficiency and efficient VEGF expression in vitro and in vivo. Intramuscular injection of nanoparticle/pDNA complexes into mice with hindlimb ischemia resulted in significant rapid blood flow recovery and improved muscle repair compared to mice treated with naked pDNA. In summary, 3S-PLGA-po-PEG/CYP2J2-pDNA complexes have tremendous potential and provide a practical strategy for the treatment of limb ischemia. Moreover, 3S-PLGA-po-PEG nanoparticles might be useful as a potential non-viral carrier for other gene delivery applications.

In Vitro Cellular Uptake and Transfection of Oligoarginine-Conjugated Glycol Chitosan/siRNA Nanoparticles.

Chitosan and its derivatives have been extensively utilized in gene delivery applications because of their low toxicity and positively charged characteristics. However, their low solubility under physiological conditions often limits their application. Glycol chitosan (GC) is a derivative of chitosan that exhibits excellent solubility in physiological buffer solutions. However, it lacks the positive characteristics of a gene carrier. Thus, we hypothesized that the introduction of oligoarginine peptide to GC could improve the formation of complexes with siRNA, resulting in enhanced uptake by cells and increased transfection efficiency in vitro. A peptide with nine arginine residues and 10 glycine units (R9G10) was successfully conjugated to GC, which was confirmed by infrared spectroscopy, 1H NMR spectroscopy, and elemental analysis. The physicochemical characteristics of R9G10-GC/siRNA complexes were also investigated. The size and surface charge of the R9G10-GC/siRNA nanoparticles depended on the amount of R9G10 coupled to the GC. In addition, the R9G10-GC/siRNA nanoparticles showed improved uptake in HeLa cells and enhanced in vitro transfection efficiency while maintaining low cytotoxicity determined by the MTT assay. Oligoarginine-modified glycol chitosan may be useful as a potential gene carrier in many therapeutic applications.

Hybrid polymer/lipid vesicle synthesis: Association between cationic liposomes and lipoplexes with chondroitin sulfate

The association of cationic carriers with different anionic mucoadhesive biopolymers has been widely explored as an alternative to improve their delivery routes and specific targeting. This work presents a complete analysis of the association between chondroitin sulfate (CS) and cationic liposomes (CLs)/lipoplex (CL-pDNA). In this study, plasmid DNA (pDNA) was used as a genetic cargo for association with carriers. Firstly, we measured the stoichiometry of pseudo complexes and evaluated their colloidal properties, structural and morphological characteristics. Optimized CL-pDNA lipoplexes (positive z-potential) and CL-CS / CL-pDNA-CS (negative z-potential with CS mass ratio of 9% (w/w)) were further studied in detail. Small-angle X-ray scattering analysis and cryo-transmission electron microscopy micrographs revealed that the electrostatic interaction between CS and CL / CL-pDNA easily reorganized the lipid bilayers resulting in nanoscale uni/multilamellar vesicles. A high CS mass ratio (9% (w/w)) led to the reassembly of liposomal structure, wherein the pDNA was easily exchanged for CS chains, forming more than 50% of dense multilamellar vesicles. This data evidenced that the association between CS and CLs is not a conventional coating process since it generates complex and hybrid structures. We believe that these obtained colloidal data may be used in the future to investigate polymer-tailored nanocarriers and their production process. In brief, the colloidal study of hybrid structures may open interesting perspectives for developing novel carriers for drug and gene delivery applications.

Renaissance for Phage-Based Bacterial Control

Bacteriophages (phages) are an underutilized biological resource with vast potential for pathogen control and microbiome editing. Phage research and commercialization have increased rapidly in biomedical and agricultural industries, but adoption has been limited elsewhere. Nevertheless, converging advances in DNA sequencing, bioinformatics, microbial ecology, and synthetic biology are now poised to broaden phage applications beyond pathogen control toward the manipulation of microbial communities for defined functional improvements. Enhancements in sequencing combined with network analysis make it now feasible to identify and disrupt microbial associations to elicit desirable shifts in community structure or function, indirectly modulate species abundance, and target hub or keystone species to achieve broad functional shifts. Sequencing and bioinformatic advancements are also facilitating the use of temperate phages for safe gene delivery applications. Finally, integration of synthetic biology stands to create novel phage chassis and modular genetic components. While some fundamental, regulatory, and commercialization barriers to widespread phage use remain, many major challenges that have impeded the field now have workable solutions. Thus, a new dawn for phage-based (chemical-free) precise biocontrol and microbiome editing is on the horizon to enhance, suppress, or modulate microbial activities important for public health, food security, and more sustainable energy production and water reuse.

Nature of bilayer lipids affects membranes deformation and pore resealing during nanoparticle penetration

Interactions of nanoparticles (NPs) with lipid membranes have enormous biological implications especially for gene delivery applications. In this work, using all-atom steered- and molecular dynamics simulations, we investigated deformation of lipid membranes and pore closure during a NP penetration process. Three membrane bilayer models built from 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC), dipalmitoylphosphatidylcholine (DPPC) and dilauroylphosphatidylcholine (DLPC), and a NP formed by 2 short interfering RNA (siRNA) and 6 polyethylenimine (PEI) molecules were used. Our results showed that different membrane lipids could lead to differences in pore formation (symmetric vs. asymmetric), and could undergo different levels of pore-mediated flip-flops during the closure. DLPC showed the largest number of flip-flops among the three lipid membranes. In addition, introduction of hydrophobic linoleic acid (LA) substitution onto the PEIs was found to facilitate pore formation, since the long LA tails could insert themselves into the hydrophobic region of the membrane where the lipid tails were less aligned. Compared with DPPC, POPC and DLPC membranes had less alignment of lipid tails in the bilayer, which promoted the insertion of LA tails and hence NP entry into the cell. Our observations provide valuable insight into the membrane deformations and pore dynamics during NP penetration and will be important for the design of NP carriers for effective gene delivery.

Interaction of calf thymus DNA and glucose-based gemini cationic surfactants with different spacer length: A spectroscopy and DLS study

The interactions between calf thymus DNA and a series of glucose-based cationic gemini surfactants 1a-1c with different spacer length, n = 4, 6 and 8, were studied by UV absorption, fluorescence spectroscopy, circular dichroism, FT-IR, dynamic light scattering and zeta potential measurements. The results showed that all the surfactants could interact with DNA efficiently. On addition of increasing concentration of the surfactants, UV absorption hypochromicity with insignificant blue shift were observed, until the DNA signal disappeared. The surfactant 1c was more efficient in the reduction of absorption intensity of DNA. According to the fluorescence quenching experiments by ethidium bromide exclusion, 1c exhibited the highest binding properties, with the binding constant at 3.25 × 108 L·mol−1. The spectroscopy study indicated that the surfactants bound with the DNA by a non-intercalative mode, mainly electrostatic interaction between the positively charged headgroups of the surfactants and negatively charged phosphate groups of DNA at low concentration, and the hydrophobic interaction among the alkyl chains at high concentration. The conformation of DNA during the interaction process could be kept B-form of DNA. For 1c, the DNA molecules can be compacted to about 103 nm in hydrodynamic diameter at 0.2 mM, while the minimum sizes of DNA were 140 nm and 133 nm, respectively, in the presence of 1a and 1b. The impact of the cationic gemini surfactants on the DNA compaction and condensation would shed light on their potential applications in gene delivery.

Preparation of poly(DMA-co-DBA) and its application in gene delivery

(2-dimethyl amino)ethyl methacrylate (DMA) was co-polymerized with (2-dibutyl amino)ethyl methacrylate (DBA) to form random copolymer P(DMA-co-DBA) with DMA/DBA molar ratios in the range of 9:1 to 1:9 by Atom Transfer Radical Polymerization (ATRP). The (co)polymers were characterized with techniques such as GPC, 1HNMR and acid-base titration. The cytotoxicity of the polymers was assessed by MTT. Size and zeta potential of P(DMA-co-DBA)/DNA polyplex were analyzed by dynamic light scattering. In vitro gene transfection was performed for P(DMA-co-DBA)/DNA polyplex in the medium with and without serum. In comparison with PDMA/DNA and PEI/DNA, P(DMA-co-DBA) 8:2/DNA showed much higher transfection efficiency than those of PDMA/DNA and PEI/DNA within the range of 0.125 − 1.25 μg·mL−1 of DNA concentrations in the medium with 10% FBS. Confocal images showed that P(DMA-co-DBA) 8:2/DNA was superior to PDMA/DNA and PEI/DNA in the speed and capacity for endosomal release, which might be the main cause of its higher transfection efficiency.

PL, TL and Persistent Luminescence Characterization of Undoped and Eu Doped Nanostructured Hydroxyapatite

Hydroxyapatite (HAp, Ca10(PO4)6(OH)2) possesses the closest mineralized structure to natural bone tissue and has been widely using in as bone substitutes and for various biomedical applications. Undoped and doped with rare earth elements HAps are the basis of promising biomedical imaging, bone tissue engineering, gene and drug delivery applications. In the present work, we present the results of investigation of the photoluminescence (PL), thermoluminescence (TL) and persistent luminescence (PersL) of synthetic nanostructured HAp (n-HAp). Undoped and Eudoped (0.0, 0.1, 0.5, 1.0, 2.0 mol %) n-HAp powders were synthesized by solution-precipitation method and characterized by SEM-EDS, FTIR and Raman techniques. The 3D excitation-emission spectrum of undoped n-HAp exhibits a broad band with excitation/emission maximum at 376/454 nm, respectively. The maximum position slightly shifts to shorter excitation and emission wavelengths with increases of the Eu concentration that could be due to a possible overlapping between the Eu emission band and intrinsic emission of undoped n-HAp. It was found that (similarly to behavior of PL in Si nanocrystals, Xin et al., RSC Adv., 2019, 9, 8310) the emission peak is red-shifted as the excitation wavelength red-shifts. The typical Eu3+ emission bands were observed for high Eu doping concentrations. TL glow curves of n-HAp samples exposed to beta radiation from Sr-Y source display a broad TL peak with maxima in-between 145 and 165 oC depending on the Eu concentration. The highest TL intensity is observed for undoped n-HAp, showing quenching effect of Eu on TL emission. This TL quenching effect contrasted with the PersL decay emission measured immediately after irradiation exposure, which was highest for 0.1 mol % Eu. The intrinsic defects in n-HAp host is related to the PL, TL and PersL emissions; however, the nature of the radiative and non-radiative recombination mechanisms requires further investigation. The PL, TL and PersL of undoped HAp may have prospective application in the fields of radiation detection, ionizing radiation dosimetry and biomedical imaging. Keywords: Hydroxylapatite, thermoluminescence, persistent luminescence.

Exploring phosphonium and ammonium chitosan polymers and their PEGylated analogs for high performance gene delivery

• New chitosan derivatives containing triethylammonium chloride or triethylphosphonium chloride and their PEGylated analogs have been synthesized and characterized. • The derivatives efficiently bind pDNA at low weight ratio of derivative:pDNA of 2.8:1. • Apoptosis measurements by flow cytometry indicated that the derivatives containing triethylammonium chloride or triethylphosphonium chloride and PEG induce no perceptible and mild apoptosis with their concentration at 100 µg/mL. • The present study provides evidence that these PEGylated derivatives are safe and effective vectors for efficient nucleic acid delivery. Among cationic polymers, chitosan (CS) is a promising non-viral vector due to its biocompatibility, biodegradability and safety profile. However, its application for gene delivery is hindered by its poor solubility at physiological pH. In this study, novel water-soluble chitosan derivatives containing triethylphosphonium chloride ((N-(1-carboxybutyl-4-triethylphosphonium chloride) chitosan = TEPB-CS)) or triethylammonium chloride ((N-(1-carboxybutyl-4-triethylammonium chloride) chitosan = TEAB-CS)) with 4-carbon spacers separating the cationic units from the chitosan backbone were synthesized by carbodiimide mediated coupling reactions. Two different degrees of substitution of quaternary phosphonium (43% and 21%) and ammonium (40.6% and 26%) were synthesized. The polymers with higher degrees of substitutions displayed higher solubility in aqueous media and were further grafted with mPEG (TEAB-CS-g-mPEG and TEPB-CS-g-mPEG) and used for DNA complexation, cytotoxicity, apoptosis, and gene transfection studies. Chemical characterization of the above polymers was performed by FTIR, 1 H NMR, and 31 P NMR, and the degree of substitution of the grafted phosphonium and ammonium and mPEG was calculated by 1 H NMR. The polymers efficiently bound pDNA at weight ratio of polymer:pDNA of 2.8:1. From dynamic light scattering experiments, the mean diameters of the calf thymus DNA complexes with the polymers, in water and phosphate buffer (pH 7.4), were in the range of 140 – 330 nm. Zeta potential of these complexes in water became positive as the weight ratios of the polymers/DNA exceeded 2:1. Most of these polymers mildly affect the viability of HeLa cells with a final concentration of 10 µg/mL or higher. Cell viability was only modestly compromised in the presence of 100 µg/mL of TEPB-CS-g-mPEG, and this derivative was also found to display the highest transfection efficiencies among all four polymers. Apoptosis measurements by flow cytometry indicated that both TEAB-CS-g-mPEG and TEPB-CS-g-mPEG induce no perceptible and mild apoptosis with their concentration at 100 µg/mL, respectively. Together, the present study provides evidence that these PEGylated TEPB-CS and TEAB-CS are safe and effective vectors for efficient nucleic acid delivery.

Electrostatic Coating of Viral Particles for Gene Delivery Applications in Muscular Dystrophies: Influence of Size on Stability and Antibody Protection

Duchenne Muscular Dystrophy (DMD) is one of the most common muscular dystrophies, caused by mutated forms of the dystrophin gene. Currently, the only treatment available is symptoms management. Novel approximations are trying to treat these patients with gene therapy, namely, using viral vectors. However, these vectors can be recognized by the immune system decreasing their therapeutic activity and making impossible a multidose treatment due to the induction of the humoral immunity following the first dose. Our objective is to demonstrate the feasibility of using a hybrid vector to avoid immune clearance, based on the electrostatic coating of adeno-associated virus (AAVs) vectors with our proprietary polymers. We coated model adeno-associated virus vectors by electrostatic interaction of our cationic poly (beta aminoester) polymers with the viral anionic capsid and characterized biophysical properties. Once the nanoformulations were designed, we studied their in vivo biodistribution by bioluminescence analysis and we finally studied the capacity of the polymers as potential coatings to avoid antibody neutralization. We tested two polymer combinations and we demonstrated the need for poly(ethylene glycol) addition to avoid vector aggregation after coating. In vivo biodistribution studies demonstrated that viral particles are located in the liver (short times) and also in muscles (long times), the target organ. However, we did not achieve complete antibody neutralization shielding using this electrostatic coating. The null hypothesis stands: although it is feasible to coat viral particles by electrostatic interaction with a proprietary polymer, this strategy is not appropriate for AAVs due to their small size, so other alternatives are required as a novel treatment for DMD patients.

Adeno-Associated Virus Vector Gene Delivery Elevates Factor I Levels and Downregulates the Complement Alternative Pathway In Vivo.

The complement system is a key component of innate immunity, but impaired regulation influences disease susceptibility, including age-related macular degeneration and some kidney diseases. While complete complement inhibition has been used successfully to treat acute kidney disease, key unresolved challenges include strategies to modulate rather than completely inhibit the system and to deliver therapy potentially over decades. Elevating concentrations of complement factor I (CFI) restricts complement activation in vitro and this approach was extended in the current study to modulate complement activation in vivo. Sustained increases in CFI levels were achieved using an adeno-associated virus (AAV) vector to target the liver, inducing a 4- to 5-fold increase in circulating CFI levels. This led to decreased activity of the alternative pathway as demonstrated by a reduction in the rate of inactive C3b (iC3b) deposition and more rapid formation of C3 degradation products. In addition, vector application in a mouse model of systemic lupus erythematosus (NZBWF1), where tissue injury is, in part, complement dependent, resulted in reduced complement C3 and IgG renal deposition. Collectively, these data demonstrate that sustained elevation of CFI reduces complement activation in vivo providing proof-of-principle support for the therapeutic application of AAV gene delivery to modulate complement activation.

Enhancement of Antioxidant and Hydrophobic Properties of Alginate via Aromatic Derivatization: Preparation, Characterization, and Evaluation.

The preparation of bioactive polymeric molecules requires the attention of scientists as it has a potential function in biomedical applications. In the current study, functional substitution of alginate with a benzoyl group was prepared via coupling its hydroxyl group with benzoyl chloride. Fourier transform infrared spectroscopy indicated the characteristic peaks of aromatic C=C in alginate derivative at 1431 cm−1. HNMR analysis demonstrated the aromatic protons at 7.5 ppm assigned to benzoyl groups attached to alginate hydroxyl groups. Wetting analysis showed a decrease in hydrophilicity in the new alginate derivative. Differential scanning calorimetry and thermal gravimetric analysis showed that the designed aromatic alginate derivative demonstrated higher thermo-stability than alginates. The aromatic alginate derivative displayed high anti-inflammatory properties compared to alginate. Finally, the in vitro antioxidant evaluation of the aromatic alginate derivative showed a significant increase in free radical scavenging activity compared to neat alginate against DPPH (2,2-diphenyll-picrylhydrazyl) and ABTS free radicals. The obtained results proposed that the new alginate derivative could be employed for gene and drug delivery applications.

DNA adsorption on like-charged surfaces mediated by polycations

In gene delivery applications, one of the first steps to ensure transfection of the genetic material is promoting the interaction with the negatively charged cell membranes. Due to the like-charged character of DNA and cell membranes, polycations are often used to mediate this interaction. The behaviour of these systems is sometimes counterintuitive and dependent on a number of interrelated factors, including membrane charge and composition, that are, in turn, dependent on cell type and pathology. For instance, upon tumorogenesis the membrane charge becomes more negative. Other features such as polycation structure, charge and concentration also impact on the polyplex-membrane interaction.Due to the inherent complexity of the system, much remains to clarify, especially because most previous work was focused on the cation-mediated adsorption with surfaces with charge densities much higher than those of cell membranes.In this work Monte Carlo simulations within the primitive model allows to establish the optimal conditions for the adsorption of DNA-polyplexes, ranging from undercharged to overcharged, upon responsive surfaces of different charge density mimicking healthy and tumoral cell membranes. Briefly, it was found that polyplex adsorption is favoured in the more negatively charged membranes, for polyplexes formed with longer polycations and at intermediate polycation concentrations.

Delivery of Bioactive Gene Particles via Gelatin-Collagen-PEG-Based Electrospun Matrices.

The fabrication of fiber mats via electrospinning has been adopted in the last decades to produce high quality scaffolds for tissue engineering. However, an effective combination of electrospinning methods with gene delivery therapies remains a challenge. In this study, we describe how the delivery of gene complexes via electrospun mats that contain different volumes of gelatin (Gel), collagen (Col), and polyethylene glycol (PEG) can affect gene expression by transfected cells. Non-viral complexes were formulated by using lipid modified polyethylenimine (PEI) polymer and plasmid DNAs (pDNA) like the reporter Green Fluorescent Protein (GFP) and the therapeutically relevant Bone Morphogenetic Protein-2 (BMP-2) and electrospuned after being mixed with different volumes of Gel-Col-PEG mats and delivered to human myoblast (C2C12) and mouse osteoblast cells (MC3T3). The entrapment of GFP complexes via different homogeneous electrospun fiber mats revealed that a high fraction of collagen in the mats affected the quality of the fibers and led to reduced transfection efficiency on target cells. On the other hand, the fabrication of double-layered mats that contained collagen without complexes as a first layer and gelatin-collagen-PEG with complexes as a second layer successfully induced GFP expression and ALP activity in C2C12 cells. We conclude that this study has established the advantage of formulating multilayered bioactive collagen-based mats for gene delivery applications.

Hydrophobic and hydrophilic modifications of polyethylenimine towards gene delivery applications

AbstractNon‐viral gene delivery has emerged as a promising approach for therapy in cancer treatment. Polyethylenimine (PEI) is a prominent transfecting agent, but due to toxicity and poor hemocompatibility, its usage for in vivo applications is limited. However, modification of PEI with different chemical groups can lead to conjugates with good transfection efficiency and better cytocompatibility. In this study, the efficiency of PEI derivatives, namely, PEI succinate (PEIS), PEI lauryl succinate (PEILS), PEI laurate (PEIL) was analyzed for gene delivery applications. Apart from biophysical characterization such as size, zeta potential, nanoplex stability and buffering capacity, cellular internalization, polymer trafficking and p53 transgene expression in C6 cell lines were also investigated. The results indicate that PEI conjugated with lauryl succinate act as better transfecting agent with high efficiency compared to other derivatives, and such balanced hydrophilic‐hydrophobic modification of PEI can render it to be a cytocompatible and effective nucleic acid delivery system.