The effect of bovine milk lactoferrin-loaded exosomes (exoLF) on human MDA-MB-231 breast cancer cell line.

BACKGROUND: Cancer is still the most challenging disease and is responsible for many deaths worldwide. Considerable research now focuses on targeted therapy in cancer using natural components to improve anti-tumor efficacy and reduce unfavorable effects. Lactoferrin is an iron-binding glycoprotein found in body fluids. Increasing evidence suggests that lactoferrin is a safe agent capable of inducing anti-cancer effects. Therefore, we conducted a study to evaluate the effects of the exosomal form of bovine milk lactoferrin on a human MDA-MB-231 breast cancer cell line. METHODS: The exosomes were isolated from cancer cells by ultracentrifugation and incorporated with bovine milk lactoferrin through the incubation method. The average size of the purified exosome was determined using SEM imaging and DLS analysis. The maximum percentage of lactoferrin-loaded exosomes (exoLF) was achieved by incubating 1 mg/ml of lactoferrin with 30 µg/ml of MDA-MB-231 cells-derived exosomes. Following treatment of MDA-MB-231 cancer cells and normal cells with 1 mg/ml exoLF MTT assay applied to evaluate the cytotoxicity, PI/ annexin V analysis was carried out to illustrate the apoptotic phenotype, and the real-time PCR was performed to assess the pro-apoptotic protein, Bid, and anti-apoptotic protein, Bcl-2. RESULTS: The average size of the purified exosome was about 100 nm. The maximum lactoferrin loading efficiency of exoLF was 29.72%. MTT assay showed that although the 1 mg/ml exoLF treatment of MDA-MB-231 cancer cells induced 50% cell growth inhibition, normal mesenchymal stem cells remained viable. PI/ annexin V analysis revealed that 34% of cancer cells had late apoptotic phenotype after treatment. The real-time PCR showed an elevated expression of pro-apoptotic protein Bid and diminished anti-apoptotic protein Bcl-2 following exoLF treatment. CONCLUSION: These results suggested that exoLF could induce selective cytotoxicity against cancer cells compared to normal cells. Incorporating lactoferrin into the exosome seems an effective agent for cancer therapy. However, further studies are required to evaluate anti-tumor efficacy and the underlying mechanism of exoLF in various cancer cell lines and animal models.

Butyrophilins: Dynamic Regulators of Protective T Cell Immunity in Cancer.

The efficacy of current immunotherapies remains limited in many solid epithelial malignancies. Recent investigations into the biology of butyrophilin (BTN) and butyrophilin-like (BTNL) molecules, however, suggest these molecules are potent immunosuppressors of antigen-specific protective T cell activity in tumor beds. BTN and BTNL molecules also associate with each other dynamically on cellular surfaces in specific contexts, which modulates their biology. At least in the case of BTN3A1, this dynamism drives the immunosuppression of αß T cells or the activation of Vγ9Vδ2 T cells. Clearly, there is much to learn regarding the biology of BTN and BTNL molecules in the context of cancer, where they may represent intriguing immunotherapeutic targets that could potentially synergize with the current class of immune modulators in cancer. Here, we discuss our current understanding of BTN and BTNL biology, with a particular focus on BTN3A1, and potential therapeutic implications for cancer.

Characterization of fennel germplasm for physiological persistence and drought recovery: Association with biochemical properties.

In the perennial medicinal plant fennel (Foeniculum vulgare), persistence over years, production stability, and successful post-drought recovery are as important as plant productivity. Characterization of productivity, post-drought recovery, persistency and their association with phytochemical properties has not yet been performed in fennel. In this study, 64 fennel genotypes (from 23 different countries) from four subspecies/varieties including F. vulgare ssp. piperitum, F. vulgare var. vulgare and F. vulgare var. dulce and F. vulgare var. azoricum were evaluated in the field over four years (2015-2018), then was assessed for post-drought recovery over the next two years (2019-2020). High genotypic variation was observed among genotypes and subspecies. Based on the GC-MS analysis, trans-anethole (22.4-90.6%), estragole (2.1-25.8%), fenchone (4.9-19.8%), and limonene (0.5-11.9%) are major components in the essential oils of the studied germplasm. The highest persistence and the lowest average post-drought recovery belonged to ssp. piperitum. This subspecies also had the highest amount of limonene, fenchone, and estragole and the lowest amount of trans-anethole compared to other subspecies. The highest essential oil content and stability was observed in var. dulce. Seed yield and persistence were found to be negatively associated, suggesting that selection for more productivity may indirectly promote less persistent genotypes. Post-drought recovery was negatively associated with two main phytochemical compounds of essential oil, fenchone and estragole, but positively associated with trans-anethole. Persistence had negative correlation with estragole content. Results indicated that indirect selection for post-drought recovery and persistence may be possible through selection for phytochemical properties. The negative correlation between some essential oil components may indicate that some are isomers, which can limit the ability to select for certain combinations. Superior genotypes identified in this study can be used to construct populations for future studies and as parents of crosses to develop new varieties.

Saponin and fluorine-modified polycation as a versatile gene delivery system.

Despite the development of many novel carriers for the delivery of various types of genetic material, the lack of a delivery system with high efficiency and low cytotoxicity is a major bottleneck. Herein, low molecular weight polyethylenimine (PEI1.8k) was functionalized with saponin residues using phenylboronic acid (PBA) as an ATP-responsive cross-linker, and a fluorinated side chain to construct PEI-PBA-SAP-F polycation as a highly efficient delivery vector. This vehicle could transfect small plasmid DNA (∼3 kb) with outstanding efficiency into various cells, including HEK 293T, NIH3T3, A549, PC12, MCF7 and HT-29, as well as robust transfection of a large plasmid (∼9 kb) into HEK 293T cells. The carrier indicated good transfection efficacy even at high concentration of serum and low doses of plasmid. The use of green fluorescent protein (GFP) knock-out analysis demonstrated transfection of different types of CRISPR/Cas9 complexes (Cas9/sgRNA ribonucleoproteins RNP, plasmid encoding Cas9 plus sgRNA targeting GFP, Cas9 expression plasmid plusin vitro-prepared sgRNA). In summary, we report an effective PEI-PBA-SAP-F gene carrier with the appropriate lipophilic/cationic balance for biomedical applications.

TRAIL/S-layer/graphene quantum dot nanohybrid enhanced stability and anticancer activity of TRAIL on colon cancer cells.

Tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL), known as a cytokine of the TNF superfamily, is considered a promising antitumor agent due to its ability to selectively induce apoptosis in a wide variety of cancer cells. However, failure of its successful translation into clinic has led to development of nano-based platforms aiming to improve TRAIL therapeutic efficacy. In this regard, we fabricated a novel TRAIL-S-layer fusion protein (S-TRAIL) conjugated with graphene quantum dots (GQDs) to benefit both the self-assembly of S-layer proteins, which leads to elevated TRAIL functional stability, and unique optical properties of GQDs. Noncovalent conjugation of biocompatible GQDs and soluble fusion protein was verified via UV-visible and fluorescence spectroscopy, size and ζ-potential measurements and transmission electron microscopy. The potential anticancer efficacy of the nanohybrid system on intrinsically resistant cells to TRAIL (HT-29 human colon carcinoma cells) was investigated by MTT assay and flow cytometry, which indicated about 80% apoptosis in cancer cells. These results highlight the potential of TRAIL as a therapeutic protein that can be extensively improved by taking advantage of nanotechnology and introduce S-TRAIL/GQD complex as a promising nanohybrid system in cancer treatment.

Antibacterial, antibiofilm, anti-inflammatory, and wound healing effects of nanoscale multifunctional cationic alternating copolymers.

Infectious diseases caused by new or unknown bacteria and viruses, such as anthrax, cholera, tuberculosis and even COVID-19, are a major threat to humanity. Thus, the development of new synthetic compounds with efficient antimicrobial activity is a necessity. Herein, rationally designed novel multifunctional cationic alternating copolymers were directly synthesized through a step-growth polymerization reaction using a bivalent electrophilic cross-linker containing disulfide bonds and a diamine heterocyclic ring. To optimize the activity of these alternating copolymers, several different diamines and cross-linkers were explored to find the highest antibacterial effects. The synthesized nanopolymers not only displayed good to excellent antibacterial activity as judged by minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa, and Escherichia coli, but also reduced the number of biofilm cells even at low concentrations, without killing mammalian cells. Furthermore, in vivo experiments using infected burn wounds in mice demonstrated good antibacterial activity and stimulated wound healing, without causing systemic inflammation. These findings suggest that the multifunctional cationic nanopolymers have potential as a novel antibacterial agent for eradication of multidrug resistant bacterial infections.

New Insights into the Biosensing of Parkinson's Disease Biomarkers: A Concise Review.

BACKGROUND: Parkinson's disease (PD) is a long-term, degenerative, and neurological disease in which a person loses control of certain body functions. The formulation of novel effective therapeutics for PD as a neurodegenerative disease requires accurate and efficient diagnosis at the early stages. OBJECTIVE: Analyzing data gathered by measurable signals converted from biological reactions allows for qualitative and quantitative evaluations. Among various approaches reported so far, biosensors are powerful analytical tools that have been used in detecting the biomarkers of PD. METHODS: Biosensor's biological recognition components include antibodies, receptors, microorganisms, nucleic acids, enzymes, cells and tissues, and biomimetic structures. This review introduces electrochemical, optical, and optochemical detection of PD biomarkers based on recent advances in nanotechnology and material science, which resulted in the development of high-performance biosensors in this field. RESULTS: PD biomarkers such as α-synuclein protein, dopamine (DA), urate, ascorbic acid, miRNAs, and their biological roles are summarized. Additionally, the advantages and disadvantages of the usual standard methods are reviewed. We compared electrochemical, optical, and optochemical biosensors' properties and novel strategies for higher sensitivity and selectivity. CONCLUSION: The development of novel biosensors is required for the early diagnosis of PD as sensitive, rapid, reliable, and cost-effective systems.

Smart arginine-equipped polycationic nanoparticles for p/CRISPR delivery into cells.

An efficient and safe delivery system for the transfection of CRISPR plasmid (p/CRISPR) into target cells can open new avenues for the treatment of various diseases. Herein, we design a novel nonvehicle by integrating an arginine-disulfide linker with low-molecular-weight PEI (PEI1.8k) for the delivery of p/CRISPR. These PEI1.8k-Arg nanoparticles facilitate the plasmid release and improve both membrane permeability and nuclear localization, thereby exhibiting higher transfection efficiency compared to native PEI1.8kin the delivery of nanocomplexes composed of PEI1.8k-Arg and p/CRISPR into conventional cells (HEK 293T). This nanovehicle is also able to transfect p/CRISPR in a wide variety of cells, including hard-to-transfect primary cells (HUVECs), cancer cells (HeLa), and neuronal cells (PC-12) with nearly 5-10 times higher efficiency compared to the polymeric gold standard transfection agent. Furthermore, the PEI1.8k-Arg nanoparticles can edit the GFP gene in the HEK 293T-GFP reporter cell line by delivering all possible forms of CRISPR/Cas9 system (e.g. plasmid encoding Cas9 and sgRNA targeting GFP, and Cas9/sgRNA ribonucleoproteins (RNPs) as well as Cas9 expression plasmid andin vitro-prepared sgRNA) into HEK 293T-GFP cells. The successful delivery of p/CRISPR into local brain tissue is also another remarkable capability of these nanoparticles. In view of all the exceptional benefits of this safe nanocarrier, it is expected to break new ground in the field of gene editing, particularly for therapeutic purposes.

Synthesis and characterization of vitamin D3-functionalized carbon dots for CRISPR/Cas9 delivery.

Aim: To develop a novel nanovector for the delivery of genetic fragments and CRISPR/Cas9 systems in particular. Materials & methods: Vitamin D3-functionalized carbon dots (D/CDs) fabricated using one-step microwave-aided methods were characterized by different microscopic and spectroscopic techniques. The 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide assay and flow cytometry were employed to determine the cell viability and transfection efficiency. Results: D/CDs transfected CRISPR plasmid in various cell lines with high efficiency while maintaining their remarkable efficacy at high serum concentration and low plasmid doses. They also showed great potential for the green fluorescent protein disruption by delivering two different types of CRISPR/Cas9 systems. Conclusion: Given their high efficiency and safety, D/CDs provide a versatile gene-delivery vector for clinical applications.

Highly Photoluminescent Nitrogen- and Zinc-Doped Carbon Dots for Efficient Delivery of CRISPR/Cas9 and mRNA.

Safe and efficient delivery of CRISPR/Cas9 systems is still a challenge. Here we report the development of fluorescent nitrogen- and zinc-doped carbon dots (N-Zn-doped CDs) using one-step microwave-aided pyrolysis based on citric acid, branched PEI25k, and different zinc salts. These versatile nanovectors with a quantum yield of around 60% could not only transfect large CRISPR plasmids (∼9 kb) with higher efficiency (80%) compared to PEI25k and lipofectamine 2000 (Lipo 2K), but they also delivered mRNA into HEK 293T cells with the efficiency 20 times greater than and equal to that of PEI25k and Lipo 2K, respectively. Unlike PEI25k, N-Zn-doped CDs exhibited good transfection efficiency even at low plasmid doses and in the presence of 10% fetal bovine serum (FBS). Moreover, these nanovectors demonstrated excellent efficiency in GFP gene disruption by transferring plasmid encoding Cas9 and sgRNA targeting GFP as well as Cas9/sgRNA ribonucleoproteins into HEK 293T-GFP cells. Hence, N-Zn-doped CDs with remarkable photoluminescence properties and high transfection efficiency in the delivery of both CRISPR complexes and mRNA provide a promising platform for developing safe, efficient, and traceable delivery systems for biological research.

Polyethylenimine-Functionalized Carbon Dots for Delivery of CRISPR/Cas9 Complexes.

Carbon dots (CDs) have become the focus of many studies due to their outstanding optical properties and good biocompatibility. We investigated their potential application to produce a smart and highly efficient yet nontoxic nanovector for gene delivery. This was achieved by conjugating PEI1.8k-functionalized CDs (synthesized by one-step microwave-assisted pyrolysis) with arginine-disulfide linkers to produce CD-PEI1.8k-Arg nanoparticles. This nanovector could deliver p-CRISPR (9.3 kb) into different types of cell lines with higher efficiency compared to native PEI1.8k or PEI25k. CD-PEI1.8k-Arg also maintained its outstanding transfection efficiency at a high serum concentration and low p-CRISPR dose, compared to PEI25k, which was ineffective under those conditions. Additionally, CD-PEI1.8k-Arg could knock out the GFP gene with great efficiency by delivering the required components of CRISPR/Cas9, including a plasmid encoding Cas9, sgRNA targeting GFP, and Cas9/sgRNA ribonucleoproteins (RNPs) into the HEK 293T-GFP cells. Moreover, the nanoparticles showed potential for the local delivery of p-CRISPR into brain tissue. The remarkable properties of CD-PEI1.8k-Arg could enable the development of a safe, highly efficient gene-delivery nanovector for the treatment of various diseases in the near future.

The Lumiptosome, an engineered luminescent form of the apoptosome can report cell death by using the same Apaf-1 dependent pathway.

Detection of the apoptosis signature becomes central in understanding cell death modes. We present here a whole-cell biosensor that detects Apaf-1 association and apoptosome formation using a split-luciferase complementary assay. Fusion of N-terminal (Nluc) and C-terminal (Cluc)-fragments of firefly luciferase to the N-terminus of human Apaf-1 was performed in HEK293 cells by using CRISPR-Cas9 technology. This resulted in a luminescent form of the apoptosome that we named 'Lumiptosome'. During Apaf-1 gene editing, a high number of knock-in events were observed without selection, suggesting that the Apaf-1 locus is important for the integration of exogenous transgenes. Since activation of caspase-9 is directly dependent on the apoptosome formation, measured reconstitution of luciferase activity should result from the cooperative association of Nluc-Apaf-1 and Cluc-Apaf-1. Time-response measurements also confirmed that formation of the apoptosome occurs prior to activation of caspase-3. Additionally, overexpression of the Bcl2 apoptosis regulator in transgenic and normal HEK293 cells confirmed that formation of the Lumiptosome depends on release of cytochrome c Thus, HEK293 cells that stably express the Lumiptosome can be utilized to screen pro- and anti-apoptotic drugs, and to examine Apaf-1-dependent cellular pathways.

Development and Clinical Translation of Approved Gene Therapy Products for Genetic Disorders.

The field of gene therapy is striving more than ever to define a path to the clinic and the market. Twenty gene therapy products have already been approved and over two thousand human gene therapy clinical trials have been reported worldwide. These advances raise great hope to treat devastating rare and inherited diseases as well as incurable illnesses. Understanding of the precise pathomechanisms of diseases as well as the development of efficient and specific gene targeting and delivery tools are revolutionizing the global market. Currently, human cancers and monogenic disorders are indications number one. The elevated prevalence of genetic disorders and cancers, clear gene manipulation guidelines and increasing financial support for gene therapy in clinical trials are major trends. Gene therapy is presently starting to become commercially profitable as a number of gene and cell-based gene therapy products have entered the market and the clinic. This article reviews the history and development of twenty approved human gene and cell-based gene therapy products that have been approved up-to-now in clinic and markets of mainly North America, Europe and Asia.

Cholesterol-rich lipid-mediated nanoparticles boost of transfection efficiency, utilized for gene editing by CRISPR-Cas9.

Purpose: Gene therapy has become a promising remedy to treat disease by modifying the person's genes. The therapeutic potential of related tools such as CRISPR-Cas9 depends on the efficiency of delivery to the targeted cells. Numerous transfection reagents have been designed and lots of efforts have been devoted to develop carriers for this purpose. Therefore, the aim of the present study was to develop novel cholesterol-rich lipid-based nanoparticles to enhance transfection efficiency and serum stability. Materials and methods: We constructed two-, three- and four-component cationic liposomes (CLs) to evaluate the combined effect of cholesterol domain and DOPE (dioleoyl phosphatidylethanolamine), a fusogenic lipid, and the PEG (polyethylene glycol) moiety location inside or outside of the cholesterol domain on transfection efficiency and other properties of the particle. Lipoplex formation and pDNA (plasmid DNA) entrapment were assessed by gel retardation assay at different N/P ratios (3, 5, 7). Physicochemical characteristics, cytotoxicity, serum stability and endosomal escape capability of the lipoplexes were studied and transfection potential was measured by firefly luciferase assay. Next, HEK293 cell line stably expressing GFP was utilized to demonstrate the editing of a reporter through Cas9 and sgRNA plasmids delivery by the selected CL formula, which showed the highest transfection efficiency. Results: Among the designed CLs, the four-component formula [DOTAP (1,2-dioleoyl-3-trimethylammoniumpropane)/DOPE/cholesterol/Chol-PEG (cholesterol-polyethylene glycol)] showed the highest rate of transfection at N/P 3. Finally, transfection of Cas9/sgRNA by this formulation at N/P 3 resulted in 39% gene-editing efficiency to knockout GFP reporter. The results also show that this CL with no cytotoxicity effect can totally protect the plasmids from enzymatic degradation in serum. Conclusion: The novel PEGylated cholesterol domain lipoplex providing serum stability, higher transfection efficiency and endosomal release can be used for in vivo Cas9/sgRNA delivery and other future gene-therapy applications.

Apoptosome formation upon overexpression of native and truncated Apaf-1 in cell-free and cell-based systems.

Apaf-1 is a cytosolic multi-domain protein in the apoptosis regulatory network. When cytochrome c releases from mitochondria; it binds to WD-40 repeats of Apaf-1 molecule and induces oligomerization of Apaf-1. Here in, a split luciferase assay was used to compare apoptosome formation in cell-free and cell-based systems. This assay uses Apaf-1 tagged with either N-terminal fragment or C-terminal fragment of P. pyralis luciferase. In cell based-system, the apoptosome formation is induced inside the cells which express Apaf-1 tagged with complementary fragments of luciferase while in cell-free system, the apoptosome formation is induced in extracts of the cells. In cell-free system, cytochrome c dependent luciferase activity was observed with full length Apaf-1. However, luciferase activity due to apoptosome formation was much higher in cell based system compared to cell-free system. The truncated Apaf-1 which lacks WD-40 repeats (ΔApaf-1) interacted with endogenous Apaf-1 in a different fashion compared to native form as confirmed by different retention time of eluate in gel filtration and binding to affinity column. The interactions between endogenous Apaf-1 and ΔApaf-1 is stronger than its interaction with native exogenous Apaf-1 as indicated by dominant negative effect of ΔApaf-1 on caspase-3 processing.

Bio-nano hybrid materials based on bacteriorhodopsin: Potential applications and future strategies.

This review presents an overview of recent progress in the development of bio-nano hybrid materials based on the photoactive protein bacteriorhodopsin (bR). The interfacing of bR with various nanostructures including colloidal nanoparticles (such as quantum dots and Ag NPs) and nanoparticulate thin films (such as TiO2 NPs and ZnO NPs,) has developed novel functional materials. Applications of these materials are comprehensively reviewed in two parts: bioelectronics and solar energy conversion. Finally, some perspectives on possible future strategies in bR-based nanostructured devices are presented.