Carbon-Based Nanostructures as Emerging Materials for Gene Delivery Applications.

Gene therapeutics are promising for treating diseases at the genetic level, with some already validated for clinical use. Recently, nanostructures have emerged for the targeted delivery of genetic material. Nanomaterials, exhibiting advantageous properties such as a high surface-to-volume ratio, biocompatibility, facile functionalization, substantial loading capacity, and tunable physicochemical characteristics, are recognized as non-viral vectors in gene therapy applications. Despite progress, current non-viral vectors exhibit notably low gene delivery efficiency. Progress in nanotechnology is essential to overcome extracellular and intracellular barriers in gene delivery. Specific nanostructures such as carbon nanotubes (CNTs), carbon quantum dots (CQDs), nanodiamonds (NDs), and similar carbon-based structures can accommodate diverse genetic materials such as plasmid DNA (pDNA), messenger RNA (mRNA), small interference RNA (siRNA), micro RNA (miRNA), and antisense oligonucleotides (AONs). To address challenges such as high toxicity and low transfection efficiency, advancements in the features of carbon-based nanostructures (CBNs) are imperative. This overview delves into three types of CBNs employed as vectors in drug/gene delivery systems, encompassing their synthesis methods, properties, and biomedical applications. Ultimately, we present insights into the opportunities and challenges within the captivating realm of gene delivery using CBNs.

Anti-Infective and Toxicity Properties of Carbon Based Materials: Graphene and Functionalized Carbon Nanotubes.

Recently, antimicrobial activities of various carbon-based nanomaterials against specific pathogens have become one of the most significant research interests in this field. Carbon nanotubes (CNTs) are promising multidisciplinary nanostructures in biomedicine, drug delivery, genetic engineering, biosensors, and artificial implants. However, the biomedical administration of CNTs is dependent on their solubility, toxicity, and biocompatibility, as well as novel drug-delivery applications through optimization of the drug's loading capacity, cellular absorption, and continuous release within the target cell. The usage of CNTs and Graphene materials as antimicrobial agents and nanocarriers for antibiotics delivery would possibly improve their bioavailability and facilitate better anti-infective therapy. However, it is worth mentioning that CNTs' antimicrobial activity and toxicity are highly dependent on their preparation and synthesis method. Various types of research have confirmed that diameter, length, residual catalyst, metal content, surface coating, electronic structure, and dispersibility would affect CNTs' toxicity toward bacteria and human cells. In this review article, a general study was performed on the antimicrobial properties of carbon-based nanomaterials, as well as their toxicity and applications in confronting different microorganisms. This study could be useful for researchers who are looking for new and effective drug delivery methods in the field of microbial resistance.

Design of double functionalized carbon nanotube for amphotericin B and genetic material delivery.

In the present work, single wall carbon nanotubes (SWCNT) were successively functionalized with phospholipid DSPE-PEG carboxylic acid, and then, with ethylenediamine (EDA), to obtain double functionalized single wall carbon nanotube (DFSWCNT). Then, DFSWCNT was applied as a carrier for delivering amphotericin B (Amb) and EGFP plasmid. FSWCNT's concentration obtained via UV-visible analysis was 0.99 mg/mL. The TGA analysis results provided the lost weights of DSPE-PEG-COOH, EDA, Amb and SWCNT impurities. XPS results showed that carbon atoms' percentage decreased during the functionalization processes from 97.2% (SWCNT) to 76.4% (FSWCNT) and 69.9% (DFSWNCT). Additionally, the oxygen atoms' percentage increased from 2.3% (SWCNT) to 21% and 22.5% for FSWCNT and DFSWCNT, respectively. New bonds such as C-N and N-C=O appeared in the synthesized nanocarrier. The IG/ID ratio in Raman analysis decreased from 7.15 (SWCNT) to 4.08 (FSWCNT). The amount of Amb released to phosphate buffer saline medium was about 33% at pH = 5.5 and 75% at pH = 7.4 after 48 h. CCK8 results confirmed that the toxicity of functionalized SWCNT had decreased. In a 2:1 ratio of DFSWCNT/EGFP plasmid, the cell viability (87%) and live transfected cells (56%) were at their maximum values. The results indicate that carbon nanotubes have the potential to be applied as drug/gene delivery systems with outstanding properties such as high loading capacity and easy penetration to cell membrane.

Preparation, characterization and in-vivo efficacy study of glatiramer acetate (GA)-hydrogel-microparticles as novel drug delivery system for GA in RRMS.

Relapsing Remitting Multiple Sclerosis is a chronic Central nervous system autoimmune disease. There is no absolute treatment for MS and the available remedies are called disease modifying therapies (DMTs). Glatiramer acetate (GA) is one of the FDA approved DMTs. Currently, injection-site problems and unfavorable daily injection are the most common milestones in administration of GA. So that, the design of improved drug delivery systems with sustained release profile seem necessary and helpful in order to minimize GA adverse effects and improve patients' compliance. In this study, we have manufactured a novel chitosan-PLGA (poly (lactic-co-glycolic acid)) hydrogel-microparticles containing GA by double emulsion method. Hydrogel-microparticles' properties including size, morphology and GA loading were investigated. In-vitro drug release was studied during 30 days. In vivo efficacy of GA-hydrogel-microparticles was evaluated in experimental autoimmune encephalomyelitis (EAE) as an established animal model for MS. Pathological studies were performed through H&E (Hematoxylin and Eosin) staining of brain, spine, liver, skin and kidney tissues. Luxol fast blue staining of brain tissue was also done. The obtained results were applied for safety and efficacy evaluations. GA loading and Entrapment efficiency (EE %) of 60% and 95% were achieved, respectively. In- vitro release studies confirms a sustained release profile for GA-hydrogel-microparticles. Mean clinical scores and mean body weights obtained from EAE animal model for GA-hydrogel-microparticles were compared to the outcomes achieved from conventional Iranian brand-generic injection solution of GA (Copamer®, 20 mg/ml). EAE outcomes and pathological studies confirm similar therapeutic efficacy with longer dosing intervals possibility, improved safety through decreased adverse effects and elimination of site injection reactions for GA-hydrogel-microparticles. Further studies on pharmacokinetic and pharmacodynamics in human volunteers are still required to thoroughly examine different aspects of this newly developed GA- hydrogel-microparticles.

Genome-Wide Expression Analysis of Root Tips in Contrasting Rice Genotypes Revealed Novel Candidate Genes for Water Stress Adaptation.

Root system architecture (RSA) is an important agronomic trait with vital roles in plant productivity under water stress conditions. A deep and branched root system may help plants to avoid water stress by enabling them to acquire more water and nutrient resources. Nevertheless, our knowledge of the genetics and molecular control mechanisms of RSA is still relatively limited. In this study, we analyzed the transcriptome response of root tips to water stress in two well-known genotypes of rice: IR64, a high-yielding lowland genotype, which represents a drought-susceptible and shallow-rooting genotype; and Azucena, a traditional, upland, drought-tolerant and deep-rooting genotype. We collected samples from three zones (Z) of root tip: two consecutive 5 mm sections (Z1 and Z2) and the following next 10 mm section (Z3), which mainly includes meristematic and maturation regions. Our results showed that Z1 of Azucena was enriched for genes involved in cell cycle and division and root growth and development whereas in IR64 root, responses to oxidative stress were strongly enriched. While the expansion of the lateral root system was used as a strategy by both genotypes when facing water shortage, it was more pronounced in Azucena. Our results also suggested that by enhancing meristematic cell wall thickening for insulation purposes as a means of confronting stress, the sensitive IR64 genotype may have reduced its capacity for root elongation to extract water from deeper layers of the soil. Furthermore, several members of gene families such as NAC, AP2/ERF, AUX/IAA, EXPANSIN, WRKY, and MYB emerged as main players in RSA and drought adaptation. We also found that HSP and HSF gene families participated in oxidative stress inhibition in IR64 root tip. Meta-quantitative trait loci (QTL) analysis revealed that 288 differentially expressed genes were colocalized with RSA QTLs previously reported under drought and normal conditions. This finding warrants further research into their possible roles in drought adaptation. Overall, our analyses presented several major molecular differences between Azucena and IR64, which may partly explain their differential root growth responses to water stress. It appears that Azucena avoided water stress through enhancing growth and root exploration to access water, whereas IR64 might mainly rely on cell insulation to maintain water and antioxidant system to withstand stress. We identified a large number of novel RSA and drought associated candidate genes, which should encourage further exploration of their potential to enhance drought adaptation in rice.

In Vitro Evaluation of DSPE-PEG (5000) Amine SWCNT Toxicity and Efficacy as a Novel Nanovector Candidate in Photothermal Therapy by Response Surface Methodology (RSM).

Nowadays, finding a novel, effective, biocompatible, and minimally invasive cancer treatment is of great importance. One of the most promising research fields is the development of biocompatible photothermal nanocarriers. PTT (photothermal therapy) with an NIR (near-infrared) wavelength range (700-2000 nm) would cause cell death by increasing intercellular and intracellular temperature. PTT could also be helpful to overcome drug resistance during cancer treatments. In this study, an amine derivative of phospholipid poly ethylene glycol (DSPE-PEG (5000) amine) was conjugated with SWCNTs (single-walled carbon nanotubes) to reduce their intrinsic toxicity. Toxicity studies were performed on lung, liver, and ovarian cancer cell lines that were reported to show some degree of drug resistance to cisplatin. Toxicity results suggested that DSPE-PEG (5000) amine SWCNTs might be biocompatible photothermal nanocarriers in PTT. Therefore, our next step was to investigate the effect of DSPE-PEG (5000) amine SWCNT concentration, cell treatment time, and laser fluence on the apoptosis/necrosis of SKOV3 cells post-NIR exposure by RSM and experimental design software. It was concluded that photothermal efficacy and total apoptosis would be dose-dependent in terms of DSPE-PEG (5000) amine SWCNT concentration and fluence. Optimal solutions which showed the highest apoptosis and lowest necrosis were then achieved.

PEGylated single-walled carbon nanotubes as co-adjuvants enhance expression of maturation markers in monocyte-derived dendritic cells.

Aim: This study investigated the application of phospholipid-PEGylated single-walled carbon nanotubes (PL-PEG-SWCNTs) as a safe co-adjuvant for the commercial recombinant hepatitis B virus vaccine to enhance induction of monocyte-derived dendritic cells (MDDCs) differentiation and activation in vitro as an immune response initiator cell to prompt a long-term immune response after a single dose injection. Methods: Immature MDDCs were exposed to PL-PEG-SWCNTs alone and in combination with hepatitis B vaccine. Results & conclusion: Study results confirm the enhanced expression of maturation markers in human immature MDDCs after PL-PEG-SWCNT exposure. The results suggest that PL-PEG-SWCNT is an efficient co-adjuvant for the commercial recombinant hepatitis B virus vaccine to enhance dendritic cell response stimulation in vitro.

Differential adaptation strategies to different levels of soil water deficit in two upland and lowland genotypes of rice: a physiological and metabolic approach.

BACKGROUND: Upland genotypes of rice are less sensitive to soil water deficit (SWD), making them suitable candidates for revealing the strategies underlying plant tolerance. The physiological factors, the biochemical traits needed to withstand oxidative stress, and the metabolite fluctuations of an upland genotype (Azucena) and an intolerant lowland genotype (IR64) genotype were measured under two levels of SWD (withholding water for 7- or 14 days) to identify SWD-responsive strategies associated with tolerance. RESULTS: After withholding water for 7 days, no significant changes in physiological and biochemical traits of Azucena were observed, whereas in IR64, significant decreases in physiological factors were recorded along with increases in oxidative-stress indicators. However, the root length of Azucena increased significantly, showing a clear stress avoidance strategy. Under a prolonged treatment (14 days), IR64 entered an oxidative-damage stage, whereas Azucena exhibited a highly efficient antioxidant system. Our metabolite analysis also revealed two different enriched pathways. After a 7-day SWD, the sugar levels were decreased in the leaves of Azucena but increased in IR64. The reduction in the sugar levels (up to 1.79-log2FC) in the Azucena leaves may be indicative of their transport to the roots, supplying the carbon source needed for root elongation. Under a 14-day treatment, proline and aspartate family members accumulated to the highest levels in Azucena, whereas an increase in the levels of aromatic amino acids with key roles in the biosynthesis of secondary metabolites was detected in IR64. CONCLUSION: The adaptation strategies identified in two types of rice genotypes in confronting SWD may assist researchers in finding the proper indicators for screening more tolerant genotypes. © 2019 Society of Chemical Industry.

Trend Analysis of Medicine Consumption Based on Therapeutic Categories in Iran: 2000-2016.

OBJECTIVE: Iranian healthcare system cost has been remarkably growing during the recent decades. Drug shortages and the economic effects of the sanctions have also exposed the pitfalls and shortcomings of the pharmaceutical system. Moreover, the pharmaceutical system is a major expenditure source for the health system. Pharmaceutical market trend analysis serves as a reliable tool to gather and analyze market and consumption data to take account of the past policies and forecast the future of the market and disease trends. METHODS: The present work tries to quantitatively describe and analyze past 17 years of Iranian pharmaceutical market sales data from 2000 to 2016. The anatomical therapeutic chemical-classified drug utilization data obtained from the Iranian Food and Drug Administration were used to analyze the market trends for each therapeutic class and categorize them. FINDINGS: The size of Iranian pharmaceutical market has grown dramatically during the last 5 years reaching to $5.183 billion in 2016 from $3.847 billion in 2011 with a compound annual growth rate of 6.14%. Antidiabetics and antineoplastic agents were one of the most prominent market growth drivers. Introduction of new drug molecules to the market has also contributed to the trend. However, the noncommunicable diseases are gaining momentum in the national disease burden. CONCLUSION: The market growth necessitates policies to prevent the overwhelming cost burden on the healthcare system which in turn requires well-informed decision-making. Pharmaceutical market trend analysis tries to supply the evidence feed for informed policy-making to forecast, prioritize, and contain the cost burden imposed on the healthcare system by the pharmaceutical sector.

Optimized Transferosomal Bovine Lactoferrin (BLF) as a Promising Novel Non-Invasive Topical Treatment for Genital Warts Caused by Human Papiluma Virus (HPV).

Human papillomavirus (HPV) cause common warts, laryngeal papilloma, and genital condylomata and might lead to development of cervical cancer. Lactoferrin (LF) is a member of the transferrin family, which has antiviral activity against HPV-16.  LF is an important player in the defense against pathogenic microorganisms and has also been shown to have activity against several viruses including herpesvirus, adenovirus, rotavirus, and poliovirus. Bovine LF (BLF) has been reported to be a more potent inhibitor of HPV entry in comparison to human LF. The goal of the present study is to formulate, evaluate and optimize transfersomal vesicles as a non-invasive transdermal delivery system which assumed to be a suitable for treatment of genital warts. Transfersomes have been prepared by two methods including reverse phase evaporation and thin film hydration with different ratios of cholesterol: lecithin: DOTAP in the presence of SDS or Tween 80. The transferosomes were then evaluated regarding size, polydispersity, and LF loading. In-vitro release studies in pH 5.3 and 7.4, stability evaluation in 4 °C and 25 °C, and TEM imaging hve been performed on optimized transferosomal lactoferrin. The optimized transferosomes were found to have 100 nm sizes with good polydispersity index and encapsulation efficiency of 91% for lactoferrin as well as sustained release of lactoferrin during 24 h. Transferosomal lactoferrin efficacy was evaluated by MTT assay. It was seen that the viral inhibitory concentration (IC50) of transfersomal lactoferrin has been significantly improved to nearly one tenth in comparison to free lactoferrin.

PEGylated single-walled carbon nanotubes as nanocarriers for cyclosporin A delivery.

Single-walled carbon nanotubes (SWCNTs) have attracted the attention of many researchers due to their remarkable physicochemical features and have been found to be a new family of nanovectors for the delivery of therapeutic molecules. The ability of these nanostructures to load large amounts of drug molecules on their outer surface has been considered as the main advantage by many investigators. Here, we report the development of a PEGylated SWCNT-mediated delivery system for cyclosporin A (CsA) as a potent immunosuppressive agent. The available OH group in the CsA structure was first linked to a bi-functional linker (i.e., succinic anhydride) in order to provide a COOH terminal group. CsA succinylation process was optimized by using the modified simplex method. The resulting compound, CsA-CO-(CH(2))(2)-COOH, was then grafted onto the exterior surface of SWCNTs, previously PEGylated with phospholipid-PEG(5000)-NH(2) conjugates, through the formation of an amide bond with the free amine group of PEGylated SWCNTs. Drug loading, stability of the PEGylated SWCNT-CsA complex, and in vitro release of the drug were evaluated. Loading efficiencies of almost 72% and 68% were achieved by UV spectrophotometry and elemental analysis methods, respectively. It was observed that 57.3% of cyclosporine was released from CsA-Pl-PEG(5000)-SWCNTs after 3 days. In this investigation, we conjugated CsA to an amine-terminated phospholipid-polyethylene glycol chain attached on SWCNTs via a cleavable ester bond and demonstrated the possible potential of PEGylated SWCNT-based systems for CsA delivery.

Evaluation of the Effect of PEGylated Single-Walled Carbon Nanotubes on Viability and Proliferation of Jurkat Cells.

Among the numerous nanosized drug delivery systems currently under investigation, carbon nanotubes (CNTs), regardless of being single or multiple-walled, offer several advantages and are considered as promising candidates for drug targeting. Despite the valuable potentials of CNTs in drug delivery, their toxicity still remains an important issue. After the PEGylation of single-walled CNTs (SWCNTs) with phospholipid-PEG (Pl-PEG) conjugates to prepare water-dispersible nanostructures, the present study was designed to evaluate whether the functionalization with Pl-PEG derivatives could alter the cytotoxic response of cells in culture, affect their viability and proliferation. In-vitro cytotoxicity screens were performed on cultured Jurkat cells. The SWCNTs samples used in this exposure were pristine SWCNTs, Pl-PEG 2000/5000-SWCNTs at various concentrations. Jurkat cells were first incubated for 3 h at 37°C with test materials and seeded in 6-well culture plates at a given concentration. The plates were then incubated for 24, 48 and 72 h at 37°C in a 5% CO2 humidified incubator. Cell Viability and proliferation assay were performed using trypan blue exclusion test and the cell cycle kinetic status of Jurkat cells was analyzed by flow cytometry. Cell morphology was finally studied using double staining technique and a fluorescence microscope. We found that, regardless of the duration of exposure, functionalized SWCNTs were substantially less toxic, compared to pure SWCNTs and that the molecular weight of Pl-PEGs played an important role at higher concentrations. In conclusion, our noncovalent protocol seemed to be effective for increasing SWCNTs biocompatibility.

Optimization of single-walled carbon nanotube solubility by noncovalent PEGylation using experimental design methods.

In this study, noncovalent functionalization of single-walled carbon nanotubes (SWCNTs) with phospholipid-polyethylene glycols (Pl-PEGs) was performed to improve the solubility of SWCNTs in aqueous solution. Two kinds of PEG derivatives, ie, Pl-PEG 2000 and Pl-PEG 5000, were used for the PEGylation process. An experimental design technique (D-optimal design and second-order polynomial equations) was applied to investigate the effect of variables on PEGylation and the solubility of SWCNTs. The type of PEG derivative was selected as a qualitative parameter, and the PEG/SWCNT weight ratio and sonication time were applied as quantitative variables for the experimental design. Optimization was performed for two responses, aqueous solubility and loading efficiency. The grafting of PEG to the carbon nanostructure was determined by thermogravimetric analysis, Raman spectroscopy, and scanning electron microscopy. Aqueous solubility and loading efficiency were determined by ultraviolet-visible spectrophotometry and measurement of free amine groups, respectively. Results showed that Pl-PEGs were grafted onto SWCNTs. Aqueous solubility of 0.84 mg/mL and loading efficiency of nearly 98% were achieved for the prepared Pl-PEG 5000-SWCNT conjugates. Evaluation of functionalized SWCNTs showed that our noncovalent functionalization protocol could considerably increase aqueous solubility, which is an essential criterion in the design of a carbon nanotube-based drug delivery system and its biodistribution.

Development and optimization of a sublingual tablet formulation for physostigmine salicylate.

This study is aimed to design and optimize a sublingual tablet formulation of physostigmine salicylate, an effective drug in Alzheimer's disease and nerve gas poisoning, by means of the D-optimal experimental design methodology. Polyvinyl pyrrolidone, lactose, starch 1500 and sodium starch glycolate were used in the formulations as independent variables. Tablets were prepared by the direct compression method and evaluated for their physical properties (tablet hardness, disintegration time and friability), which were regarded as responses in a D-optimal design. Due to the significance of the special cubic model for data fitted, compared to other models, it was used to examine the obtained results. Response surface plots were plotted to study the tablet properties and the optimized overlay plot was generated based on the results and targets considered for the responses. After verification of the optimum checkpoint formulations, an optimized formulation was chosen due to its desirable physical properties and closely observed and predicted values. Drug assay, content uniformity of the dosage unit, drug dissolution and accelerated stability studies were done on the optimum formulation as further experiments. All the obtained results complied with the requirements of a sublingual tablet formulation.

Analgesic and anti-inflammatory activity of Lactuca sativa seed extract in rats.

Lactuca sativa (Lettuce) is a member of Compositae family. In folk medicine of Iran, the seeds of this plant were used for relieving of inflammation and osteodynia. In this study, anti-nociceptive and anti-inflammatory activities of a crude methanol/petroleum ether (70/30, v/v) extract of the seeds have been evaluated. The extract exhibited a time- and dose-dependent analgesic effect in formalin test and also a dose-dependent anti-inflammatory activity in a carrageenan model of inflammation. The extract had no analgesic effect in tail-flick test up to the highest dose used (6 g/kg). No abnormal behavior and lethality was observed by the extract up to 6 g/kg. Preliminary phytochemical analysis showed the presence of triterpenoids, saponins and simple phenols in the extract.