Carbon Dots with Tunable Charge as Mucus-Penetrating Gene Carriers

Adhesion Receptors Mediate Efficient Non-viral Gene Delivery

Engineered carbon dots for mucosal gene delivery.

AimThis study aims to compare the mucus permeating and mucoadhesive properties of three generations of thiolated cyclodextrins (CDs). MethodsFree thiol groups of thiolated γ-CDs (CD-SH) were S-protected with 2-mercaptonicotinic acid (MNA), leading to a second generation of thiolated CDs (CD-SS-MNA) and with 2 kDa polyethylene glycol (PEG) bearing a terminal thiol group leading to a third generation of thiolated CDs (CD-SS-PEG). The structure of these thiolated CDs was confirmed and characterized by FT-IR, 1H NMR and colorimetric assays. Thiolated CDs were evaluated regarding viscosity, mucus diffusion, and mucoadhesion. ResultsThe viscosity of the mixture of CD-SH, CD-SS-MNA, or CD-SS-PEG with mucus increased up to 11-, 16-, and 14.1-fold compared to unmodified CD within 3 hours, respectively. Mucus diffusion increased in the following rank order: unprotected CD-SH < CD-SS-MNA < CD-SS-PEG. The residence time of CD-SH, CD-SS-MNA, and CD-SS-PEG on porcine intestine was up to 9.6-, 12.55-, and 11.2-fold prolonged compared to native CD, respectively. ConclusionAccording to these results, S-protection of thiolated CDs can be a promising approach to improve their mucus permeating and mucoadhesive properties. Statement of significanceThree generations of thiolated cyclodextrins (CDs) with different types of thiol ligands have been synthesized to improve mucus interaction. 1st generation of thiolated CDs was synthesized by converting hydroxyl groups into thiols by reaction with Thiourea. For 2nd generation, free thiol groups were S-protected by reaction with 2-mercaptonicotinic acid (MNA), resulting in high reactive disulfide bonds. For 3rd generation, terminally thiolated short PEG chains (2 kDa) were used for S-protection of thiolated CDs. Mucus penetrating properties were found to be increased as follows: 1st generation < 2nd generation < 3rd generation. Furthermore, mucoadhesive properties were improved in the following rank order: 1st generation < 3rd generation < 2nd generation. This study suggests that the S-protection of thiolated CDs can enhance mucus penetrating and mucoadhesive properties.

Mineralized polyplexes for gene delivery: Improvement of transfection efficiency as a consequence of calcium incubation and not mineralization

Gene electrotransfer upregulate DNA pattern recognition receptors or DNA sensors, which are part of the innate immune system. In this study, we tested if addition of the cocktail of innate immune system inhibitors to the cells during gene electrotransfer (GET) can increase transfection efficiency and cell survival. The results indicate that this cocktail can decrease cytosolic DNA sensors expression after GET, and consequently increase cell survival and transfection efficiency in B16 cells, but only in highly metastatic B16F10 subtype. We demonstrated that DNA sensors expression during the transfection methods needs to be downregulated if higher transfection efficiency and better cells’ survival is needed. The inhibition of the receptors of the innate immune system can improve the transfection efficiency also for GET of malignant melanoma B16 cells, but only of highly metastatic subtype.

Gene electrotransfer upregulate DNA pattern recognition receptors or DNA sensors, which are part of the innate immune system. In this study, we tested if addition of the cocktail of innate immune system inhibitors to the cells during gene electrotransfer (GET) can increase transfection efficiency and cell survival. The results indicate that this cocktail can decrease cytosolic DNA sensors expression after GET, and consequently increase cell survival and transfection efficiency in B16 cells, but only in highly metastatic B16F10 subtype. We demonstrated that DNA sensors expression during the transfection methods needs to be downregulated if higher transfection efficiency and better cells' survival is needed. The inhibition of the receptors of the innate immune system can improve the transfection efficiency also for GET of malignant melanoma B16 cells, but only of highly metastatic subtype.

Microtubule Acetylation Through HDAC6 Inhibition Results in Increased Transfection Efficiency

Conventional therapies of several diseases, in particular cancer therapy, have been insufficient clinically for effective and safe treatment of these important diseases. The major cause of side effects is insufficient uptake and non-selective delivery of therapeutic molecules. In order to overcome this problem, colloidal, nano-sized carrier systems have been developed for gene and drug delivery. These novel delivery systems have a wide range of modification capabilities, such as controllable particle size and surface charge or grafting of different molecules for active or passive targeting to cells. A variety of modification or formulation approaches ensure the efficacy, equality and safety of the system. In this context, cationic nano-sized drug delivery systems have a net positive surface charge, suggesting strong cellular interactions with negatively charged biological membranes. This electrostatic interaction between cationic nanoparticles and cell membranes brings with it enhanced uptake of nanoparticles by cells. Another important advantage of cationic nanocarriers is that they are able to condense DNA, siRNA, nucleotides, peptides and proteins to form polyplexes that are able to deliver their load intracellularly, resulting in increased transfection efficiency. In this chapter the surface properties, cellular interaction and uptake mechanism of nano-sized drug carrier systems and the innovations in treatment are described using examples from the literature. In addition, various cationic polymers commonly used in drug and gene delivery and their characteristics are summarized. Positively charged nanocarrier systems emerge as a promising option for effective drug or gene therapy and extensive research is being carried out in this field worldwide.

827. A Novel Bone Gene Therapy Platform Based on Nonvirally Transfected Human Mesenchymal Stem Cells

Development of lipid membrane based assays to accurately predict the transfection efficiency of cell-penetrating peptide-based gene nanoparticles.

BackgroundPolyethyleneimine (PEI), which can interact with negatively charged DNA through electrostatic interaction to form nanocomplexes, has been widely attempted to use as a gene delivery system. However, PEI has some defects that are not fit for keeping on gene expression. Therefore, some modifications against PEI properties have been done to improve their application value in gene delivery. In this study, three modified PEI derivatives, including poly(ε-caprolactone)-pluronic-poly(ε-caprolactone) grafted PEI (PCFC-g-PEI), folic acid-PCFC-isophorone diidocyanate-PEI (FA-PEAs) and heparin-PEI (HPEI), were evaluated in terms of their cytotoxicity and transfection efficiency in vitro and in vivo in order to ascertain their potential application in gene therapy.MethodsMTT assay and a marker GFP gene, encoding green fluorescent protein, were used to evaluate cell toxicity and transfection activity of the three modified PEI in vitro. Renal cell carcinoma (RCC) models were established in BALB/c nude mice inoculated with OS-RC-2 cells to detect the gene therapy effects using the three PEI-derived nanoparticles as gene delivery vehicles. The expression status of a target gene Von Hippel-Lindau (VHL) in treated tumor tissues was analyzed by semiquantitative RT-PCR and immunohistochemistry.ResultsEach of three modified PEI-derived biomaterials had an increased transfection efficiency and a lower cytotoxicity compared with its precursor PEI with 25-kD or 2-kD molecule weight in vitro. And the mean tumor volume was obviously decreased 30% by using FA-PEAs to transfer VHL plasmids to treat mice RCC models. The VHL gene expression was greatly improved in the VHL-treated group. While there was no obvious tumor inhibition treated by PCFC-g-PEI:VHL and HPEI:VHL complexes.ConclusionsThe three modified PEI-derived biomaterials, including PCFC-g-PEI, FA-PEAs and HPEI, had an increased transfection efficiency in vitro and obviously lower toxicities compared with their precursor PEI molecules. The FA-PEAs probably provide a potential gene delivery system to treat RCC even other cancers in future.

The delivery of drugs directly from the nose to the brain has been explored for the treatment of neurological diseases, such as glioblastoma, by overcoming the blood-brain barrier. Nanocarriers have demonstrated outstanding ability to enhance drug bioavailability in the brain, following intranasal administration. However, the performance of these nanosystems may be hindered by inadequate interactions with the nasal mucosa, limiting their effectiveness in reaching the olfactory region, and consequently, the translocation of particles to the brain. Here, we designed hybrid lipid-polymer nanoparticles (LPNP), containing the cationic lipid DOTAP and the triblock copolymer Pluronic® F127 to combine the mucoadhesiveness and mucus-penetrating properties. Perillyl alcohol (POH), a molecule currently under clinical trials against glioblastoma, via intranasal route, was entrapped in the nanoparticles. LPNP-POH exhibited a balanced profile of mucus adhesion and penetration, suggesting that the formulation may enhance mucosal retention while maintaining effective mucus diffusivity. In vivo evaluations displayed higher translocation of LPNP-POH from the nasal cavity to the brain. LPNP-POH resulted in a 2.5-fold increase in the concentration of perillyl acid (a primary metabolite of POH) in the cerebral tissue compared to the free drug. In vitro assays demonstrated that LPNP-POH increased the cytotoxicity and reduced the tumor growth of U87MG glioma cells. These results highlighted that the engineered formulation, with optimized mucoadhesiveness and mucus penetration properties, improved nose-to-brain delivery of POH, offering a promising potential for glioblastoma therapy.

Genes are attractive candidates as therapeutic agents, and the development of safe and effective gene carriers is essential for the success of human gene therapy. To develop a gene delivery vector that shows low cytotoxicity and high efficiency, we synthesized poly-L-lysine-g-pluronic by conjugating poly-L-lysine (PLL) to pluronic, which is partially functionalized with para-nitrophenyl carbonate groups, and evaluated for its efficiency as a possible nonviral gene carrier candidate. Structural analysis of synthesized polymer was performed by using 1H-NMR. Gel retardation assay, zeta potential and size measurement confirmed that the new gene carrier made a compact complex with plasmid DNA. pCMV-beta-gal was used as a reporter gene, and the in vitro transfection efficiency was measured in HeLa cells by using the o-nitrophenyl-beta-D-galactopyranoside assay. The highest transfection efficiency among those tested was achieved at the 1:1 weight ratio of polymer:DNA, and a 3-fold increase in transfection efficiency was achieved by treatment of a lysosomotropic agent, chloroquine. Compared with unmodified PLL, PLL-g-pluronic showed about 2-fold increase in transfection efficiency with similar cytotoxicity specifically at the 1:1 weight ratio of polymer:DNA.

A non-viral gene therapy for treatment of recessive dystrophic epidermolysis bullosa.

Transfection efficiency is a critical parameter in gene therapy and molecular biology, representing the success rate at which nucleic acids are introduced and expressed in target cells. The combination of aptamers with nanotechnology-based delivery systems has demonstrated remarkable improvements in the transfection efficiency of therapeutic agents and holds significant potential for advancing gene therapy and the development of targeted treatments for various diseases, including cancer. In this work, cationic carbosilane dendritic systems are presented as an alternative to commercial transfection agents, demonstrating an increase in transfection efficiency when used for the internalization of apMNKQ2, an aptamer selected against a target in cancer. Their potential therapeutic use has been evaluated in breast cancer cell lines, MDA-MB-468 and MDA-MB-231, studying the cytotoxicity of the nanoconjugate, the internalization process, and its effect on cellular migration processes.