pDNA Complexes Research Articles

Preparation and characterization of calcium-doped graphene oxide-chitosan Nanocarrier to enhance the gene delivery in MCF-7 cell line

Gene delivery has emerged as a novel and effective method in the treatment of malignancies within medical interventions by applying nanotechnology. Consequently, the development of appropriate nanocarriers is a key focus of this research. Dynamic light scattering (DLS), fourier transform infrared (FT-IR) spectroscopy, x-ray diffraction (XRD), and thermal gravimetric analysis (TGA) were employed for the characterization of the synthesized nanocarrier. Furthermore, to assess the gene transfer capability of the nanocarrier, various techniques such as gel retardation assay, nuclease resistance assay, cytotoxicity assay, flow cytometry, and transfection were employed. The average particle size and zeta potential of the GO-CS@Ca nanocarrier were obtained as 319.8 nm and + 92.8 mv, respectively. In the gel retardation test, it was observed that pDNA was effectively condensed by the GO-CS@Ca nanocarrier. The results of the MTT assay indicated that both GO-CS@Ca nanocarrier and the GO-CS@Ca/pDNA nanoplex with low toxicity. In flow cytometry analysis, it was observed that the complexation of pDNA with the GO-CS@Ca nanocarrier resulted in effective gene delivery to the MCF-7 cell line and consequently increased apoptosis induction.

A photothermal surface modified with polyelectrolyte multilayers for gene transfection and cell harvest

Gene transfection, which involves introducing nucleic acids into cells, is a pivotal technology in the life sciences and medical fields, particularly in gene therapy. Surface-mediated transfection, primarily targeting cells adhering to surfaces, shows promise for enhancing cell transfection by localizing and presenting surface-bound nucleic acids directly to the cells. However, optimizing endocytosis for efficient delivery remains a persistent challenge. Additionally, ensuring efficient and non-traumatic cell harvest capability is crucial for applications such as ex vivo cell-based therapy. To address these challenges, we developed a photothermal platform with enzymatic degradation capability for efficient gene transfection and cell harvest. This platform is based on carbon nanotubes (CNTs) doped with poly(dimethylsiloxane) and modified with polyelectrolyte multilayers (PEMs) containing hyaluronic acid and quaternized chitosan, allowing for substantial loading of poly(ethyleneimine)/plasmid DNA (pDNA) complexes through electrostatic interactions. Upon irradiation of near-infrared laser, the photothermal properties of CNTs enable high transfection efficiency by delivering pDNA into attached cells via a membrane disruption mechanism. The engineered cells can be harvested by treating with a non-toxic hyaluronidase solution to degrade PEMs, thus maintaining good viability for further applications. This platform has demonstrated remarkable efficacy across various cell lines (including Hep-G2 cells, Ramos cells and primary T cells), achieving a transfection efficiency exceeding 95 %, cell viability exceeding 90 %, and release efficiency surpassing 95 %, highlighting its potential for engineering living cells.

Dynamic Covalent Boronate Chemistry Accelerates the Screening of Polymeric Gene Delivery Vectors via In Situ Complexation of Nucleic Acids.

Gene therapy provides exciting new therapeutic opportunities beyond the reach of traditional treatments. Despite the tremendous progress of viral vectors, their high cost, complex manufacturing, and side effects have encouraged the development of nonviral alternatives, including cationic polymers. However, these are less efficient in overcoming cellular barriers, resulting in lower transfection efficiencies. Although the exquisite structural tunability of polymers might be envisaged as a versatile tool for improving transfection, the need to fine-tune several structural parameters represents a bottleneck in current screening technologies. By taking advantage of the fast-forming and strong boronate ester bond, an archetypal example of dynamic covalent chemistry, a highly adaptable gene delivery platform is presented, in which the polycation synthesis and pDNA complexation occur in situ. The robustness of the strategy entitles the simultaneous evaluation of several structural parameters at will, enabling the accelerated screening and adaptive optimization of lead polymeric vectors using dynamic covalent libraries.

Production and Purification of Adeno-Associated Viral Vectors (AAVs) Using Orbitally Shaken HEK293 Cells.

Here, we describe methods for the production of adeno-associated viral (AAV) vectors by transient transfection of HEK293 cells grown in serum-free medium using orbital shaken bioreactors and the subsequent purification of vector particles. The protocol for expression of AAV components is based on polyethyleneimine (PEI)-mediated transfection of a three-plasmid system and is specified for production in milliliter-to-liter scales. After PEI and plasmid DNA (pDNA) complex formation, the diluted cell culture is transfected without a prior concentration step or medium exchange. Following a 7-day batch process, cell cultures are further processed using a set of methods for cell lysis and vector recovery. Methods for the purification of viral particles are described, including immunoaffinity and anion-exchange chromatography, ultrafiltration, as well as digital PCR to quantify the concentration of vector particles.

Controllable Star Cationic Poly(Disulfide)s Achieve Genetically Cascade Catalytic Therapy by Delivering Bifunctional Fusion Plasmids.

The absence of effective delivery vectors and suitable multifunctional plasmids limits cancer gene therapy development. The star cationic poly(disulfide)s with β-cyclodextrin cores (termed β-CD-g-PSSn ) for caveolae-mediated endocytosis are designed and prepared via mild and controllable disulfide exchange polymerization for high-efficacy cancer therapy. Then, β-CD-g-PSSn /pDNA complexes are transported to the Golgi apparatus and endoplasmic reticulum. Disulfides in β-CD-g-PSSn vectors are degraded by glutathione in tumor cells, which not only promotes intracellular pDNA release but also reduces in vitro and in vivo toxicity. One bifunctional fusion plasmid pCATKR, which expresses catalase (CAT) fused to KillerRed (KR) (CATKR) in the same target cell, is also proposed for genetically cascade catalytic therapy. When compared with pCAT-KR (plasmid expressing CAT and KR separately in the same cell), delivered pCATKR decomposes hydrogen peroxide, alleviates tumor hypoxia more effectively, generates stronger reactive oxygen species (ROS) capabilities under moderate irradiation, and leads to robust antitumor cascade photodynamic effects. These impressive results are attributed to fusion protein design, which shortens the distance between CAT and KR catalytic centers and leads to improved ROS production efficiency. This work provides a promising strategy by delivering a catalytic cascade functional plasmid via a high-performance vector with biodegradable and caveolae-mediated endocytosis characteristics.

Effects of Microfluidic Shear on the Plasmid DNA Structure: Implications for Polymeric Gene Delivery Vectors.

Microfluidic manufacturing of advanced gene delivery vectors necessitates consideration of the effects of microfluidic shear forces on the structural integrity of plasmid DNA (pDNA). In this paper, we expose pDNA to variable shear forces in a two-phase, gas-liquid microfluidic reactor and apply gel electrophoresis to analyze the products of on-chip shear-induced degradation. The effects of shear rate, solvent environment, pDNA size, and copolymer complexation on shear-induced degradation are investigated. We find that small naked pDNA (pUC18, 2.7 kb) exhibits shear rate-dependent shear degradation in the microfluidic channels in a mixed organic solvent (dioxane/water/acetic acid; 90/10/<0.1 w/w/w), with the extents of both supercoil isoform relaxation and complete fragmentation increasing as the maximum shear rates increase from 4 × 105 to 2 × 106 s-1. However, over the same range of shear rates, the same pDNA sample shows no evidence of microfluidic shear-induced degradation in a pure aqueous environment. Quiescent control experiments in the same mixed organic solvent prove that a combination of solvent and shear forces is involved in the observed shear-induced degradation. Furthermore, we show that shear degradation effects in mixed organic solvents can be significantly attenuated by complexation of pDNA with the block copolymer polycaprolactone-block-poly(2-vinylpyridine) prior to exposure to microfluidic shear. Finally, we demonstrate that medium (pDSK519, 8.1 kb) and large (pRK290, 20 kb) naked pDNA are more sensitive to shear-induced microfluidic degradation in the mixed organic solvent environment than small pDNA, with both plasmids showing complete fragmentation even at the lowest shear rate, although we found no evidence of shear-induced damage in water for the largest investigated naked pDNA even at the highest flow rate. The resulting understanding of the interplay of the solvent and shear effects during microfluidic processing should inform microfluidic manufacturing routes to new gene therapy formulations.

Transfection Efficacy and Cellular Uptake of Lipid-Modified Polyethyleneimine Derivatives for Anionic Nanoparticles as Gene Delivery Vectors.

Cationic polyethylenimine (PEI)-based nonviral gene carriers have been desirable to overcome the limitations of viral vectors in gene therapy. A range of PEI derivatives were designed, synthesized, and evaluated for nonviral delivery applications of plasmid DNA (pDNA). Linolenic acid, lauric acid, and oleic acid were covalently conjugated with low-molecular-weight PEI (Mw ∼ 1200 Da) via two different linkers, gallic acid (GA) and p-hydroxybenzoic acid (PHPA), that allows a differential loading of lipids per modified amine (3 vs 1, respectively). 1H NMR spectrum confirmed the expected structure of the conjugates as well as the level of lipid substitution. SYBR Green binding assay performed to investigate the 50% binding concentration (BC50) of lipophilic polymers to pDNA revealed increased BC50 with an increased level of lipid substitution. The particle analysis determined that GA- and PHPA-modified lipopolymers gave pDNA complexes with ∼300 and ∼100 nm in size, respectively. At the polymer/pDNA ratio of 5.0, the ζ-potentials of the complexes were negative (-6.55 to -10.6 mV) unlike the complexes with the native PEI (+11.2 mV). The transfection experiments indicated that the prepared lipopolymers showed higher transfection in attachment-dependent cells than in suspension cells based on the expression of the reporter green fluorescent protein (GFP) gene. When loaded with Cy3-labeled pDNA, the lipopolymers exhibited effective cellular uptake in attachment-dependent cells while the cellular uptake was limited in suspension cells. These results demonstrate the potential of lipid-conjugated PEI via GA and PHPA linkers, which are promising for the modification of anchorage-dependent cells.

Plasmid containing VEGF-165 and ANG-1 dual genes packaged with fibroin-modified PEI to promote the regeneration of vascular network and dermal tissue

Reducing the cytotoxicity of cationic polymers is the major issue to their use as a gene delivery carrier. In this study, plasmids containing encoding vascular endothelial cell growth factor 165 and angiopoietin-1 were packaged with the conjugates of cationic fibroin (CSF) and polyethylenimine (PEI), instead of packaging pDNA with PEI alone, to prepare nanocomplexes (CSF+PEI)/pDNA. The complexes were loaded into a silk fibroin scaffold to enhance its function to induce microvascular network generation and dermal tissue regeneration. The results of transfecting EA.hy926 cells with the complexes in vitro showed that (CSF+PEI)/pDNA had a stronger transfection ability than PEI/pDNA. Importantly, compared with PEI as the gene carrier alone, the cell viability was significantly increased and the cytotoxicity was effectively reduced after the conjugate of CSF and PEI was used as the gene carrier. The results of angiogenesis in chick embryo chorioallantoic membranes showed that compared with scaffolds loaded with PEI/pDNA, the neovascularization ratio in scaffolds loaded with (CSF+PEI)/pDNA was significantly increased. In vivo experimental results of scaffolds implantation for full-thickness skin defects in SD rats showed that, compared with loading PEI/pDNA complex, loading (CSF+PEI)/pDNA complex in the scaffold more effectively promoted the formation of vascular network in the scaffold and accelerated the regeneration of dermal tissue. The gene delivery system established in this study has application potential not only in the regeneration of vascular-containing tissues, but also in tumor gene therapy.

Polyurethane/Liquid Crystal Microfibers with pDNA Polyplex Loadings for the Optimal Release and Promotion of HUVEC Proliferation.

Fiber structures with connected pores resemble the natural extracellular matrix (ECM) in tissues, and show high potential for promoting the formation of natural functional tissue. The geometry of composite fibers produced by electrospinning is similar to that of the living-tissue ECM, in terms of structural complexity. The introduction of liquid crystals does not affect the morphology of fibers. The composite mat shows better hydrophilicity, with higher content of liquid crystal. At the same time, the higher the content of liquid crystal, the lower the modulus and tensile strength, and the higher the breaking energy and the elongation at break. Additionally, the factors affecting fibers are also investigated in this study. The addition of liquid crystals to the fibers' matrix can slow down the release of pDNA, which is the most common vehicle for genetic engineering, and the encapsulation of pDNA polymer into the fiber matrix can maintain biological activity. The continued release of the pDNA complex was achieved in this study through liquid crystals, and the effective release is controllable. In addition, the integration of liquid crystals into fibers with pDNA polymers can cause a faster transfection rate and promote HUVEC (Human Umbilical Vein Endothelial Cells) growth. It is possible to combine electrospun fibers containing LC (liquid crystal) with pDNA condensation technology to achieve the goal of a sustained release. The production of inductable tissue-building equipment can manipulate the required signals at an effective level in the local tissue microenvironment.

Gelatin-Based Electrospun Nanofibers Cross-Linked Using Horseradish Peroxidase for Plasmid DNA Delivery.

The delivery of nucleic acids is indispensable for tissue engineering and gene therapy. However, the current approaches involving DNA/RNA delivery by systemic and local injections face issues such as clearance, off-target distribution, and tissue damage. In this study, we report plasmid DNA (pDNA) delivery using gelatin electrospun nanofibers obtained through horseradish peroxidase (HRP)-mediated insolubilization. The nanofibers were obtained through the electrospinning of an aqueous solution containing gelatin possessing phenolic hydroxyl (Ph) moieties (Gelatin-Ph) and HRP with subsequent HRP-mediated cross-linking of the Ph moieties by exposure to air containing 16 ppm H2O2 for 30 min. Then, Lipofectamine/pDNA complexes were immobilized on the nanofibers through immersion in the solution containing the pDNA complexes, resulting in transfection and sustained delivery of pDNA. Cells cultured on the resultant nanofibers expressed genome-editing molecules including Cas9 protein and guide RNA (gRNA), resulting in targeted gene knock-in and knock-out. These results demonstrated the potential of Gelatin-Ph nanofibers obtained through electrospinning and subsequent HRP-mediated cross-linking for gene therapy and tissue regeneration by genome editing.

Cationic RGD peptidomimetic nanoconjugates as effective tumor targeting gene delivery vectors with antimicrobial potential

Gene delivery combined with systemic targeting approach has shown promising potential in cancer gene therapy. Peptides are ideal functional motif for constructing biocompatible non-viral gene delivery vehicles. RGD peptides, in particular, are known to recognize the integrin αVβ3, which is expressed specifically on angiogenic blood vessels and, therefore, is considered vital for anti-angiogenesis strategies and cancer treatment. In recent times, several RGD peptide-based non-viral gene delivery vectors have been utilized for targeted gene delivery, however, lack in proteolytic stability. In the current study, we have investigated a series of non-naturally modified RGD peptide mimic (MOH) nanoconjugates with low molecular weight branched polyethylenimine (bPEI 1.8 kDa). The projected peptide mimic, Fmoc-FFARKA (MOH), has already been demonstrated to have high binding efficiency for αVβ3 integrins and enhanced cell adhesive ability with high stability compared to the natural RGD counterpart. The polymer-peptide, PEI-MOH (PMOH), nanoconjugate vectors have been designed to enhance the tumor targeting ability, therapeutic proficiency, transfection efficiency and proteolytic stability. The synthesized nanoconjugates showed the ability to protect the bound DNA with low cytotoxicity and their pDNA complexes displayed enhanced transfection efficiency. Furthermore, a competitive study confirmed their selective behavior towards liver cancer cells, HepG2. Lastly, PMOH nanoconjugates also exerted significant antimicrobial effects against drug-resistant pathogens. Altogether, the data suggest that nanosized non-naturally modified RGD peptide mimic-based gene vectors hold great potential as efficient biomaterials for targeted gene delivery and antimicrobial applications.

Development of delivery carriers for plasmid DNA by conjugation of a helical template to oligoarginine

Arginine (Arg)-rich peptides can penetrate the cell membrane and deliver nucleic acid-based therapeutics into cells. In this study, a helical template designed with a repeating sequence composed of two l-leucines (l-Leu) and a 2-aminoisobutyric acid (Aib) (l-Leu-l-Leu-Aib) was conjugated to nona-arginine on either the C- or N- terminus, designated as Block 1 and Block 2. Each terminal modification induced helical structure formation and improved the physicochemical properties of peptide/plasmid DNA (pDNA) complexes, resulting in efficient intracellular pDNA delivery. The introduction of a helical template may be effective for the endosomal escape of pDNA and pDNA release from complexes in cells. These results emphasized the potency of a helical template for the development of novel cell-penetrating peptides for pDNA delivery.

PH-sensitive packaging of cationic particles by an anionic block copolymer shell

Cationic non-viral vectors show great potential to introduce genetic material into cells, due to their ability to transport large amounts of genetic material and their high synthetic versatility. However, designing materials that are effective without showing toxic effects or undergoing non-specific interactions when applied systemically remains a challenge. The introduction of shielding polymers such as polyethylene glycol (PEG) can enhance biocompatibility and circulation time, however, often impairs transfection efficiency. Herein, a multicomponent polymer system is introduced, based on cationic and hydrophobic particles (P(nBMA46-co-MMA47-co-DMAEMA90), (PBMD)) with high delivery performance and a pH-responsive block copolymer (poly((N-acryloylmorpholine)-b-(2-(carboxy)ethyl acrylamide)) (P(NAM72-b-CEAm74), PNC)) as shielding system, with PNAM as alternative to PEG. The pH-sensitive polymer design promotes biocompatibility and excellent stability at extracellular conditions (pH 7.4) and also allows endosomal escape and thus high transfection efficiency under acidic conditions. PNC shielded particles are below 200 nm in diameter and showed stable pDNA complexation. Further, interaction with human erythrocytes at extracellular conditions (pH 7.4) was prevented, while acidic conditions (pH 6) enabled membrane leakage. The particles demonstrate transfection in adherent (HEK293T) as well as difficult-to-transfect suspension cells (K-562), with comparable or superior efficiency compared to commercial linear poly(ethylenimine) (LPEI). Besides, the toxicity of PNC-shielded particles was significantly minimized, in particular in K-562 cells and erythrocytes. In addition, a pilot in vivo experiment on bone marrow blood cells of mice that were injected with PNC-shielded particles, revealed slightly enhanced cell transfection in comparison to naked pDNA. This study demonstrates the applicability of cationic hydrophobic polymers for transfection of adherent and suspension cells in culture as well as in vivo by co-formulation with pH-responsive shielding polymers, without substantially compromising transfection performance.Graphical

Bioreducible polyethylenimine core–shell nanostructures as efficient and non-toxic gene and drug delivery vectors

Low molecular weight branched polyethylenimine (LMW bPEIs 1.8 kDa) have received considerable attention for the fabrication of nucleic acid carriers due to their biocompatible and non-toxic nature. However, due to the inadequate nucleic acid complexation ability and transportation across the cell membrane, these show poor transfection efficacy, limiting their clinical applications. Therefore, to overcome these challenges, in this study, we have grafted bPEI 1.8 kDa with a disulfide bond containing hydrophobic moiety, 3-(2-pyridyldithio) propionic acid (PDPA), via amide linkages through EDC/NHS-mediated coupling to obtain N-[3-(2-pyridyldithio)] propionoyl polyethylenimine (PDPP) conjugates. The best formulation for nucleic acid transfection was evaluated after preparing a series of PDPP conjugates by varying the amount of PDPA. In an aqueous environment, these PDPP conjugates self-assembled to form spherical shaped core–shell PDPP nanostructures with size ranging from ∼188–307 nm and zeta-potential from ∼ +3 to +19 mV. The positively charged surface of the core–shell nanocomposites helps in the binding of plasmid DNA (pDNA), its transportation inside the cell, and protection against enzymes. Evaluation of PDPP/pDNA complexes on mammalian cells revealed that all these complexes showed significantly improved transfection efficacy without hampering cytocompatibility. Amongst all, the pDNA complex of PDPP-2 exhibited the best transfection efficiency (i.e. >6-fold) in comparison to pDNA complex of the native bPEI. The nanocomposites exhibited the redox responsive behavior advantageous for therapeutic delivery to the tumor cells. The core of the nanostructures facilitate the encapsulation of a hydrophobic model drug, ornidazole. In vitro drug release analysis showed a faster release rate in response to a reductant mimicking the cellular environment. Altogether, these nanostructures have great potential to co-deliver both drug and gene simultaneously in response to tumor cell reductive microenvironment in vitro and could be used as the next-generation delivery system.

Gelatin nanofiber mats with Lipofectamine/plasmid DNA complexes for in vitro genome editing

Gelatin electrospun nanofiber mats are gaining interest for applications in biomaterials science, such as tissue engineering and drug/gene delivery systems. In this study, we report the use of electrospun gelatin nanofiber mats for plasmid DNA (pDNA) delivery. Gelatin nanofiber mats were insolubilized via cross-linking with glutaraldehyde. On the cross-linked mats, human embryonic kidney-derived HEK293 cells demonstrated high viability for 7 days of culture (>95%) and were able to proliferate during that time. The Lipofectamine/pDNA complexes were immobilized on the mats through immersion in a solution, and HEK293 cells cultured on these mats expressed GFP for 7 days. Furthermore, HEK293 cells did not express GFP via the pDNA complexes released from the mats because the ability to deliver pDNA into the cells was lost. Since the mats could be used to transfect multiple types of pDNA into the cells simultaneously, we have achieved targeted genome editing using the mats. These data highlight the potential of gelatin nanofiber mats with Lipofectamine/pDNA complexes for local gene therapy via pDNA delivery as well as genome editing.

Co‐delivery of paclitaxel and CXCL1 shRNA via cationic polymeric micelles for synergistic therapy against ovarian cancer

AbstractThe preparation of polymer therapeutics capable of synergistic co‐delivery systems for chemotherapeutic drugs and genes remains a tough problem in cancer therapy. Herein, we develop a novel co‐delivery system for chemotherapeutic drugs and genes. Paclitaxel (PTX) was covalently grafted onto the cationic copolymer poly(ethylene glycol)‐b‐poly(l‐lysine) (PEG‐b‐PLL) via a disulfide bond, and then CXCL1 shRNA was loaded on the cationic micelles PEG‐b‐PLL‐SS‐PTX by electrostatic interaction to construct PEG‐b‐PLL‐SS‐PTX/pDNA complex. The chemical structure of the complex was confirmed by gel permeation chromatography, 1H NMR and Fourier transform infrared spectroscopy. The binding ability of the cationic micelles and pDNA was detected by gel retardation assay. In vitro gene transfection studies revealed an enhanced gene transfection efficiency due to the co‐delivery of PTX and pDNA to ovarian cancer cells. In vitro and in vivo data demonstrated that the co‐delivery complex exhibited a synergistic role of chemotherapy and gene therapy and possessed enhanced antitumor effects. The strategy of using drug‐loaded cationic micelles as gene delivery carriers is a versatile approach to construct a co‐delivery system, which might result in more efficient cancer therapy. © 2022 Society of Industrial Chemistry.

Synthesis and Characterization of Poly (β-amino Ester) and Applied PEGylated and Non-PEGylated Poly (β-amino ester)/Plasmid DNA Nanoparticles for Efficient Gene Delivery.

Polymer-based nanocarriers require extensive knowledge of their chemistries to learn functionalization strategies and understand the nature of interactions that they establish with biological entities. In this research, the poly (β-amino ester) (PβAE-447) was synthesized and characterized, aimed to identify the influence of some key parameters in the formulation process. Initially; PβAE-447 was characterized for aqueous solubility, swelling capacity, proton buffering ability, and cytotoxicity study before nanoparticles formulation. Interestingly, the polymer-supported higher cell viability than the Polyethylenimine (PEI) at 100 μg/ml. PβAE-447 complexed with GFP encoded plasmid DNA (pGFP) generated nanocarriers of 184 nm hydrodynamic radius (+7.42 mV Zeta potential) for cell transfection. Transfection assays performed with PEGylated and lyophilized PβAE-447/pDNA complexes on HEK-293, BEAS-2B, and A549 cell lines showed better transfection than PEI. The outcomes toward A549 cells (above 66%) showed the highest transfection efficiency compared to the other cell lines. Altogether, these results suggested that characterizing physicochemical properties pave the way to design a new generation of PβAE-447 for gene delivery.

Systematic development and characterization of inhalable dry powder containing Polymeric Lipid Hybrid Nanocarriers co-loaded with ABCB1 shRNA and docetaxel using QbD approach

The main objective of this research work was to extend the principles of the Quality by Design approach in optimization and development of dry powder inhalers containing hybrid nanocarriers for effective treatment of multidrug-resistant lung cancer. Two statistical designs were used to justify the feasibility and advantages of the Qbd approach for the development of PLHNCs (Polymeric Lipid Hybrid Nanocarriers). The particle size and encapsulation efficiency were selected as quality target profile. Plackett-Burman design (PBD) was used as the first design to evaluate 7 high-risk variables that have significant effect on the characteristics of PLHNCs. Among the 7 high-risk variables, 3 variables (i.e., concentration of polymer, lipid to polymer ratio and drug input) had a significant effect on encapsulation efficiency of PLHNCs. Box-Behnken design (BBD) was employed as the second statistical design to fully elucidate the formulation development parameters. It was found that the optimized range for the parameters showed promising results to achieve a quality target profile. Further, the optimized PLHNCs formulation was subjected to lyophilization using various cryoprotectants to increase drug retention and dry powder inhaler was developed using different grades of modified lactose to achieve better aerodymic properties. The PLHNCs developed using PBD and BBD showed significant increase in Docetaxel encapsulation along with high shRNA pDNA complexation. The developed product had an higher fine particle fraction (68.3 ± 2.5%) along with high drug retention (98.3 ± 3.1%) which showed how efficiently the developed dry powder of PLHNCs would be able to target the respiratory zone of the lungs.

Application of polymeric dimethylaminoethyl methacrylate based carriers of plasmid DNA for genetic transformation of Ceratodon purpureus moss

Introduction. Genetic engineering in plants is of great importance for agriculture, biotechnology and medicine, and nanomaterials are widely used for genetic engineering. The aim of present study was to evaluate the potential of poly(2-dimethylamino)ethyl methacrylate (DMAEMA)-based comb-like polymers as gene delivery systems in moss Ceratodon purpureus (Hedw.) Brid. protoplasts and determine the level of phytotoxicity of these polymers. Materials and Methods. In order to confirm the formation of complex of poly-DMAEMA carrier with plasmid DNA pSF3, gel retardation assay was used. The PEG-mediated transformation protocol was adapted to transform the protoplasts of C. purpureus moss with poly-DMAEMA carriers. Light microscopy was used to study a toxicity of polymers for moss protoplasts. The level of the polymers toxicity was estimated as IC50 value. Results and Discussion. The formation of pDNA complex with DMAEMA-based carriers took place at 0.03% concentration of the polymers BGA-21, BGA-22(2ph), BG-24, BG-25, BG-26 or 0.1% concentration of the BGA-22 polymer. Poly-DMAEMA carriers were able to deliver plasmid DNA pSF3 into protoplasts of C. purpureus moss. Three stable transformants of C. purpureus were obtained at using BGA-22 polymer, 2 clones – at using BGA-21 carrier, and 1 clone – at using BGA-22(2ph), BG-24, BG-25, BG-26 polymers. The poly-DMAEMA carriers at the working 0.0025% dose were relatively non-toxic for protoplasts of C. purpureus moss. 83.1-88.4% of viable protoplasts of C. purpureus moss were detected after treatment with studied carriers at 0.0025% dose. A survival ratio of protoplasts reached 66.7-72.9% under the effect of these polymers at 0.025% dose, which is 10 times higher than their working concentration. The IC50 value of poly-DMAEMA carriers was in the range of 0.113-0.164%, that was approximately 10 times higher than that of the PEG-6000 used for gene delivery in plants. Conclusion. Novel synthetic poly-DMAEMA carriers delivered the gene of interest into moss C. purpureus protoplasts and possessed a low phytotoxicity. Thus, these carriers can be useful for gene delivery into plant cells.

Biomimetic cell membrane-coated DNA nanoparticles for gene delivery to glioblastoma

Gene therapy has been introduced as an alternative to radiation and chemical therapy for glioblastoma. Biomimetic nanoparticles coated with cell membranes (CM) have advantages such as high biocompatibility and prolong half-life. To apply CM coated nanoparticles to gene delivery, the polyethylenimine (PEI25k)/plasmid DNA (pDNA) complexes were coated with CM from C6 rat glioblastoma cells. With the CM covering, the PEI25k/pDNA complexes formed stable nanoparticles with negative surface charge. The PEI25k/pDNA/CM nanoparticles had high colloidal stability and could be stored for approximately 20 days without aggregation. The transfection efficiency of the PEI25k/pDNA/CM nanoparticles was higher than that of the PEI25k/pDNA complex in serum-containing medium. This suggests that serum does not interfere with transfection efficiency of the nanoparticles. Moreover, the PEI25k/pDNA/CM nanoparticles had lower toxicity than the PEI25k/DNA complex in vitro and in vivo. The PEI25k/pDNA/CM nanoparticles prepared with CMs of different types of cells were transfected into cells. The results showed that the PEI25k/pDNA/CM nanoparticles with the C6 CM had the highest transfection efficiency to C6 cells, suggesting the homotypic targeting effect. The therapeutic effects of the nanoparticles were evaluated in intracranial C6 transplanted glioblastoma animal models. The PEI25k/pDNA/CM nanoparticles were prepared with herpes simplex virus thymidine kinase plasmid (pHSVtk) and injected into the tumor locally. The results showed that the PEI25k/pHSVtk/CM nanoparticles induced higher HSVtk expression compared with the PEI25k/pHSVtk complex. Furthermore, tumor size was reduced more efficiently by the PEI25k/pHSVtk/CM nanoparticles than by the PEI25k/pHSVtk complex. Overall results indicate that PEI25k/pDNA/CM nanoparticles are suitable for pDNA delivery to glioblastoma.