Guidelines for the role of autophagy in drug delivery vectors uptake pathways.

The process of autophagy refers to the intracellular absorption of cytoplasm (such as proteins, nucleic acids, tiny molecules, complete organelles, and so on) into the lysosome, followed by the breakdown of that cytoplasm. The majority of cellular proteins are degraded by a process called autophagy, which is both a naturally occurring activity and one that may be induced by cellular stress. Autophagy is a system that can save cells' integrity in stressful situations by restoring metabolic basics and getting rid of subcellular junk. This happens as a component of an endurance response. This mechanism may have an effect on disease, in addition to its contribution to the homeostasis of individual cells and tissues as well as the control of development in higher species. The main aim of this study is to discuss the guidelines for the role of autophagy in drug delivery vector uptake pathways. In this paper, we discuss the meaning and concept of autophagy, the mechanism of autophagy, the role of autophagy in drug delivery vectors, autophagy-modulating drugs, nanostructures for delivery systems of autophagy modulators, etc. Later in this paper, we talk about how to deliver chemotherapeutics, siRNA, and autophagy inducers and inhibitors. We also talk about how hard it is to make a drug delivery system that takes nanocarriers' roles as autophagy modulators into account.

Cell-penetrating peptides with nanoparticles hybrid delivery vectors and their uptake pathways.

Cell-penetrating peptides (CPPs) are molecules that improve the cellular uptake of various molecular payloads that do not easily traverse the cellular membrane. CPPs can be found in pharmaceutical and medical products. The vast majority of cell-penetrating chemicals that are discussed in published research are peptide based. The paper also delves into the various applications of hybrid vectors. Because CPPs are able to carry cargo across the cellular membrane, they are a viable candidate for use as a suitable carrier for a wide variety of cargoes, such as siRNA, nanoparticles, and others. In which we discuss the CPPs, their classification, uptake mechanisms, hybrid vector systems, nanoparticles and their uptake mechanisms, etc. Further in this paper, we discuss CPPs conjugated to Nanoparticles, Combining CPPs with lipids and polymeric Nanoparticles in A Conjugated System, CPPs conjugated to nanoparticles for therapeutic purposes, and potential therapeutic uses of CPPs as delivery molecules. Also discussed the preclinical and clinical use of CPPS, intracellular trafficking of nanoparticles, and activatable and bioconjugated CPPs.

Uptake pathways of cell-penetrating peptides in the context of drug delivery, gene therapy, and vaccine development.

Cell-penetrating peptides have been extensively utilized for the purpose of facilitating the intracellular delivery of cargo that is impermeable to the cell membrane. The researchers have exhibited proficient delivery capabilities for oligonucleotides, thereby establishing cell-penetrating peptides as a potent instrument in the field of gene therapy. Furthermore, they have demonstrated a high level of efficiency in delivering several additional payloads. Cell penetrating peptides (CPPs) possess the capability to efficiently transport therapeutic molecules to specific cells, hence offering potential remedies for many illnesses. Hence, their utilization is imperative for the improvement of therapeutic vaccines. In contemporary studies, a plethora of cell-penetrating peptides have been unveiled, each characterized by its own distinct structural attributes and associated mechanisms. Although it is widely acknowledged that there are multiple pathways through which particles might be internalized, a comprehensive understanding of the specific mechanisms by which these particles enter cells has to be fully elucidated. The absorption of cell-penetrating peptides can occur through either direct translocation or endocytosis. However, it is worth noting that categories of cell-penetrating peptides are not commonly linked to specific entrance mechanisms. Furthermore, research has demonstrated that cell-penetrating peptides (CPPs) possess the capacity to enhance antigen uptake by cells and facilitate the traversal of various biological barriers. The primary objective of this work is to examine the mechanisms by which cell-penetrating peptides are internalized by cells and their significance in facilitating the administration of drugs, particularly in the context of gene therapy and vaccine development. The current study investigates the immunostimulatory properties of numerous vaccine components administered using different cell-penetrating peptides (CPPs). This study encompassed a comprehensive discussion on various topics, including the uptake pathways and mechanisms of cell-penetrating peptides (CPPs), the utilization of CPPs as innovative vectors for gene therapy, the role of CPPs in vaccine development, and the potential of CPPs for antigen delivery in the context of vaccine development.

A Method for Using Cell-Penetrating Peptides for Loading Plasmid DNA into Secreted Extracellular Vesicles.

The low bioavailability and high toxicity of plasmid DNA (pDNA)-based therapeutics pose challenges for their in vivo application. Extracellular vesicles (EVs) have great potential to overcome these limitations, as they are biocompatible native cargo carriers. Various methods for loading pDNA into EVs, including electroporation, sonication, and co-incubation, have been previously investigated, but their success has been questionable. In this study, we report a unique method for loading EVs with pDNA through transient transfection using cell-penetrating peptides (CPPs). With this method, we found a 104-fold increase in the expression levels of the luciferase reporter protein in recipient cells compared to the untreated cells. These data point to the high transfection efficacy and bioavailability of the delivered encapsulated nucleic acid. Furthermore, the in vivo experimental data indicate that the use of pDNA-loaded EVs as native delivery vehicles reduces the toxic effects associated with traditional nucleic acid (NA) delivery and treatment.

Gene-environment interactions that influence CVD, lipid traits, obesity, diabetes, and hypertension appear to be able to influence gene therapy.

Most mind boggling diseases are accepted to be impacted by both genetic and environmental elements. As of late, there has been a flood in the improvement of different methodologies, concentrate on plans, and measurable and logical techniques to examine gene-environment cooperations (G × Es) in enormous scope studies including human populaces. The many-sided exchange between genetic elements and environmental openings has long charmed the consideration of clinicians and researchers looking to grasp the complicated starting points of diseases. While single variables can add to disease, the blend of genetic variations and environmental openings frequently decides disease risk. The fundamental point of this paper is to talk about the Gene-Environment Associations That Impact CVD, Lipid Characteristics, Obesity, Diabetes, and Hypertension Have all the earmarks of being Ready to Impact Gene Therapy. This survey paper investigates the meaning of gene-environment collaborations (G × E) in disease advancement. The intricacy of genetic and environmental communications in disease causation is explained, underlining the multifactorial idea of many circumstances. The job of gene-environment cooperations in cardiovascular disease, lipid digestion, diabetes, obesity, and hypertension is investigated. This audit fixates on Gene by Environment (G × E) collaborations, investigating their importance in disease etiology.

Design and Synthesis of N-Doped Porous Carbons for the Selective Carbon Dioxide Capture under Humid Flue Gas Conditions.

The design of novel porous solid sorbents for carbon dioxide capture is critical in developing carbon capture and storage technology (CCS). We have synthesized a series of nitrogen-rich porous organic polymers (POPs) from crosslinking melamine and pyrrole monomers. The final polymer's nitrogen content was tuned by varying the melamine ratio compared to pyrrole. The resulting polymers were then pyrolyzed at 700 °C and 900 °C to produce high surface area nitrogen-doped porous carbons (NPCs) with different N/C ratios. The resulting NPCs showed good BET surface areas reaching 900 m2 g-1. Owing to the nitrogen-enriched skeleton and the micropore nature of the prepared NPCs, they exhibited CO2 uptake capacities as high as 60 cm3 g-1 at 273 K and 1 bar with significant CO2/N2 selectivity. The materials showed excellent and stable performance over five adsorption/desorption cycles in the dynamic separation of the ternary mixture of N2/CO2/H2O. The method developed in this work and the synthesized NPCs' performance towards CO2 capture highlight the unique properties of POPs as precursors for synthesizing nitrogen-doped porous carbons with a high nitrogen content and high yield.

Improvement of Transfection with PepFects Using Organic and Inorganic Materials.

Cell-penetrating peptides (CPPs) are a promising non-viral vector for gene and drug delivery. CPPs exhibit high cell transfection, and are biocompatible. They can be also conjugated with organic and inorganic nanomaterials, such as magnetic nanoparticles (MNPs), graphene oxide (GO), metal-organic frameworks (MOFs), and chitosan. Nanomaterials offered a high specific surface area and provided relatively straightforward methods to be modified with biomolecules including CPPs and oligonucleotides (ONs). Novel nanomaterials conjugates with CPP/ONs complexes are therefore of interest for cell transfection with high efficiency. In this chapter, we described a summary of the non-viral vectors consisting of CPPs and nanomaterials. The book chapter also included a protocol to generate hybrid biomaterials consisting of CPPs and nanoparticles (NPs) for the delivery of oligonucleotides. The conjugation of NPs with CPPs serves as an effective platform for gene therapy with high cell transfection efficiency. The protocol is simple, offers high cell transfection compared to the CPPs-ONs complexes, and can be used for further improvements using external stimuli.

Transcriptional Profiling Reveals Ribosome Biogenesis, Microtubule Dynamics and Expression of Specific lncRNAs to be Part of a Common Response to Cell-Penetrating Peptides.

Cell-penetrating peptides (CPPs) are short peptides that are able to efficiently penetrate cellular lipid bilayers. Although CPPs have been used as carriers in conjugation with certain cargos to target specific genes and pathways, how rationally designed CPPs per se affect global gene expression has not been investigated. Therefore, following time course treatments with 4 CPPs-penetratin, PepFect14, mtCPP1 and TP10, HeLa cells were transcriptionally profiled by RNA sequencing. Results from these analyses showed a time-dependent response to different CPPs, with specific sets of genes related to ribosome biogenesis, microtubule dynamics and long-noncoding RNAs being differentially expressed compared to untreated controls. By using an image-based high content phenotypic profiling platform we confirmed that differential gene expression in CPP-treated HeLa cells strongly correlates with changes in cellular phenotypes such as increased nucleolar size and dispersed microtubules, compatible with altered ribosome biogenesis and cell growth. Altogether these results suggest that cells respond to different cell penetrating peptides by alteration of specific sets of genes, which are possibly part of the common response to such stimulus.

Chitosan enhances gene delivery of oligonucleotide complexes with magnetic nanoparticles-cell-penetrating peptide.

Gene-based therapies, including the delivery of oligonucleotides, offer promising methods for the treatment of cancer cells. However, they have various limitations including low efficiency. Herein, cell-penetrating peptides (CPPs)-conjugated chitosan-modified iron oxide magnetic nanoparticles (CPPs-CTS@MNPs) with high biocompatibility as well as high efficiency were tested for the delivery of oligonucleotides such as plasmid pGL3, splice correction oligonucleotides, and small-interfering RNA. A biocompatible nanocomposite, in which CTS@MNPs was incorporated in non-covalent complex with CPPs-oligonucleotide, is developed. Modifying the surface of magnetic nanoparticles with cationic chitosan-modified iron oxide improved the performance of magnetic nanoparticles-CPPs for oligonucleotide delivery. CPPs-CTS@MNPs complexes enhance oligonucleotide transfection compared to CPPs@MNPs or CPPs. The hydrophilic character of CTS@MNPs improves complexation with plasmid pGL3, splice correction oligonucleotides, and small-interfering RNA payload, which consequently resulted in not only strengthening the colloidal stability of the constructed complex but also improving their biocompatibility. Transfection using PF14-splice correction oligonucleotides-CTS@MNPs showed sixfold increase of the transfection compared to splice correction oligonucleotides-PF14 that showed higher transfection than the commercially available lipid-based vector Lipofectamine™ 2000. Nanoscaled CPPs-CTS@MNPs comprise a new family of biomaterials that can circumvent some of the limitations of CPPs or magnetic nanoparticles.

Uptake Mechanism of Cell-Penetrating Peptides.

Cell-penetrating peptides have been extensively used since their discovery for delivering cargoes unable to cross the cell membrane. Among other transported cargoes, they have shown very efficient delivery for oligonucleotides making cell-penetrating peptides a powerful tool for gene therapy. Numerous cell-penetrating peptides have now been discovered offering a wide library of structures and mechanisms of actions. Nevertheless, if it is known that different pathways are available for particles to be taken up, most mechanisms by which these particles enter cells are still to be characterized more precisely. Indeed it is admitted that cell-penetrating peptides are taken up either by direct translocation or by endocytosis but classes of cell-penetrating peptides are usually not related to specific entrance mechanisms. Actually, for most particles, different pathways can be detected during their uptake which makes the literature sometimes contradictory. Recent studies have nevertheless shown convergent uptake patterns for individual structures. Acetylated cell-penetrating peptides complexed with oligonucleotides have been shown to interact to scavenger receptor class A to induce caveolae-mediated endocytosis whereas antimicrobial peptides create pores in the cell membrane for direct translocation. Arginine-rich peptides have presented concentration-dependent mechanisms, being taken up either by membrane destabilization or clathrin-mediated endocytosis. Relating the structure of cell-penetrating peptides or their particles to distinct mechanisms would allow this delivery platform to become even more specific by using rational design to fit to the desired uptake pathway.

Role of autophagy in cell-penetrating peptide transfection model.

Cell-penetrating peptides (CPPs) uptake mechanism is still in need of more clarification to have a better understanding of their action in the mediation of oligonucleotide transfection. In this study, the effect on early events (1 h treatment) in transfection by PepFect14 (PF14), with or without oligonucleotide cargo on gene expression, in HeLa cells, have been investigated. The RNA expression profile was characterized by RNA sequencing and confirmed by qPCR analysis. The gene regulations were then related to the biological processes by the study of signaling pathways that showed the induction of autophagy-related genes in early transfection. A ligand library interfering with the detected intracellular pathways showed concentration-dependent effects on the transfection efficiency of splice correction oligonucleotide complexed with PepFect14, proving that the autophagy process is induced upon the uptake of complexes. Finally, the autophagy induction and colocalization with autophagosomes have been confirmed by confocal microscopy and transmission electron microscopy. We conclude that autophagy, an inherent cellular response process, is triggered by the cellular uptake of CPP-based transfection system. This finding opens novel possibilities to use autophagy modifiers in future gene therapy.

Magnetic Nanoparticle Assisted Self-assembly of Cell Penetrating Peptides-Oligonucleotides Complexes for Gene Delivery.

Magnetic nanoparticles (MNPs, Fe3O4) incorporated into the complexes of cell penetrating peptides (CPPs)-oligonucleotides (ONs) promoted the cell transfection for plasmid transfection, splice correction, and gene silencing efficiencies. Six types of cell penetrating peptides (CPPs; PeptFect220 (denoted PF220), PF221, PF222, PF223, PF224 and PF14) and three types of gene therapeutic agents (plasmid (pGL3), splicing correcting oligonucleotides (SCO), and small interfering RNA (siRNA) were investigated. Magnetic nanoparticles incorporated into the complexes of CPPs-pGL3, CPPs-SCO, and CPPs-siRNA showed high cell biocompatibility and efficiently transfected the investigated cells with pGL3, SCO, and siRNA, respectively. Gene transfer vectors formed among PF14, SCO, and MNPs (PF14-SCO-MNPs) showed a superior transfection efficiency (up to 4-fold) compared to the noncovalent PF14-SCO complex, which was previously reported with a higher efficiency compared to commercial vector called Lipofectamine™2000. The high transfection efficiency of the new complexes (CPPs-SCO-MNPs) may be attributed to the morphology, low cytotoxicity, and the synergistic effect of MNPs and CPPs. PF14-pDNA-MNPs is an efficient complex for in vivo gene delivery upon systemic administration. The conjugation of CPPs-ONs with inorganic magnetic nanoparticles (Fe3O4) may open new venues for selective and efficient gene therapy.

Graphene oxide nanosheets in complex with cell penetrating peptides for oligonucleotides delivery.

A new strategy for gene transfection using the nanocarrier of cell penetrating peptides (CPPs; PepFect14 (PF14) or PepFect14 (PF14) (PF221)) in complex with graphene oxide (GO) is reported. GO complexed with CPPs and plasmid (pGL3), splice correction oligonucleotides (SCO) or small interfering RNA (siRNA) are performed. Data show adsorption of CPPs and oligonucleotides on the top of the graphenic lamellar without any observed change of the particle size of GO. GO mitigates the cytotoxicity of CPPs and improves the material biocompatibility. Complexes of GO-pGL3-CPPs (CPPs; PF14 or PF221) offer 2.1-2.5 fold increase of the cell transfection compared to pGL3-CPPs (CPPs; PF14 or PF221). GO-SCO-PF14 assemblies effectively transfect the cells with an increase of >10-25 fold compared to the transfection using PF14. The concentration of GO plays a significant role in the material nanotoxicity and the transfection efficiency. The results open a new horizon in the gene treatment using CPPs and offer a simple strategy for further investigations.

ACE I/D and eNOS E298D gene polymorphisms in Saudi subjects with hypertension.

BACKGROUND: Hypertension has a multifactorial background based on genetic and environmental interactive factors. OBJECTIVES: We aimed to test for the association of the angiotensin-converting enzyme (ACE) and endothelial nitric oxide synthase (eNOS) gene polymorphisms with hypertension. SUBJECTS AND METHODS: Participants included 120 Saudi patients with hypertension and 250 normal healthy controls. For all participants, DNA was processed for characterization of ACE I/D and eNOS E298D gene polymorphisms. RESULTS: Hypertensive cases showed a significantly higher frequency of the ACE mutant D allele carriage (98.3% vs. 92.4%, p = 0.028, OR = 4.8). Cases with hypertension associated with diabetes and obesity showed 100% mutant D allele carriage. Regarding the eNOS E298D polymorphism, the frequency of the mutant D allele carriage was only observed to be higher among cases with hypertension associated with diabetes and obesity, in comparison with controls, yet not reaching statistical significance (41.2% vs. 34%, p > 0.05). CONCLUSIONS: There is increased frequency of ACE and eNOS mutant allele carriage among Saudi patients affected with hypertension, particularly if accompanied by obesity and diabetes.

Association of MTHFR C677T and A1298C gene polymorphisms with hypertension.

OBJECTIVES: To check for the association of genetic polymorphisms related to the methylenetetrahydrofolate reductase (MTHFR) gene namely C677T and A1298C with hypertension in Saudi affected subjects from Qassim region. SUBJECTS AND METHODS: Participants included 123 Saudi hypertensive cases (83 males and 40 females) in addition to 250 (142 males and 108 females) unrelated healthy controls from the same locality. Their age mean ±SD was 50.93 ± 15.43 years. For all subjects, DNA was extracted followed by real-time PCR amplifications for characterization of genotypes and alleles related to MTHFR C677T and A1298C gene polymorphisms RESULTS: Total cases showed significantly higher carriage rate for the mutant allele 677T compared to controls (40.7% vs. 26%, OR=1.9, 95% CI= 1.2-3.1) with a lower frequency of the wild type 677CC genotype (59.3% vs. 74%, p=0.004). The same was observed among cases-subgroups of hypertension associated with obesity with a notably higher odds ratio (OR=2.6, 95% CI=1.3-5.01, p=0.004). Total cases showed also significantly higher frequency of mutant 1298 C allele carriage rate compared to controls (59.3% vs. 42.4%, OR=1.98, 95% CI= 1.3-3.1) with a lower frequency of the normal AA genotype (40.7% vs. 57.6%, p=0.003). The same was observed among cases-subgroups of hypertension associated with both diabetes and obesity and among cases of hypertension with obesity, also with higher odds ratio (OR=2.6 and 2.2 respectively). CONCLUSION: This work showed that genetic polymorphisms related to the MTHFR gene are associated with the risk of hypertension particularly when accompanied with obesity and diabetes among Saudi subjects.

Frequency of thrombophilic genetic polymorphisms among Saudi subjects compared with other populations.

Thrombophilic mutations increase the tendency toward thromboembolic disease. The aim of this study was to estimate the prevalence of the genetic variants related to thrombophilia among Saudis compared with other populations. Real-time polymerase chain reaction (PCR) genotyping was carried out to determine the polymorphic variants of factor V Leiden 1695G/A, prothrombin 20210G/A, plasmin activator inhibitor 1 4G/5G, methylene tetrahydrofolate reductase (MTHFR) 677C/T, MTHFR 1298A/C, and angiotensin-converting enzyme (ACE) insertion/deletion (I/D) among a representative sample of healthy Saudi subjects. Carraige rate for each of the mutant variants of factor V Leiden (FVL) and FII genes constituted 2% of the surveyed subjects giving an allele frequency of 0.01, homozygous forms of plasminogen activator inhibitor-1 (PAI-1) gene 4G/4G, MTHFR 677TT, 1298CC, and ACE DD were present among 7.7, 2.55, 7, and 51.8% of subjects with a mutant allele frequency of 0.4, 0.19, 0.29, and 0.73, respectively. This study showed that the Saudi population has a peculiar pattern regarding thrombophilic mutations that might warrant additional considerations for prophylaxis.

CYP2J2 -50 G/T and ADRB2 G46A gene polymorphisms in Saudi subjects with hypertension.

BACKGROUND: Hypertension is a result of complex factors including multiple genetic polymorphisms. OBJECTIVE: This study aims to check for the association of genetic polymorphisms of the cytochrome P450 2J2 (CYP 2J2) and beta-2-adrenergic receptor (ADRB2) genes with hypertension among Saudi subjects. SUBJECTS AND METHODS: This study included 116 cases with documented hypertension of at least 1 year duration. Their data were compared to that of 250 unrelated healthy nonhypertensive subjects from the same locality. For all participants, DNA was extracted and analyzed using real time polymerase chain reaction technique for the identification of genotypic and allelic variants of the CYP2J2 -50 G/T and ADRB2 G46A genes. RESULTS: Hypertensive cases showed a significantly higher frequency of mutant CYP2J2 -50 T allele carriage (TT and GT genotypes) compared with controls (odds ratio [OR]=3.7, p=0.0003). The same was observed among subgroups of hypertension associated with diabetes and obesity (OR=3.6, p=0.007) and cases with isolated hypertension (OR=8.4, p=0.0002). On the other hand, hypertensive cases, whether being isolated or associated with obesity and/or diabetes, showed a nonsignificant difference from controls in relation to all genotypic variants related to the ADRB2 G46A polymorphism (p>0.05). CONCLUSION: This study showed positive association of CYP2J2 gene polymorphism with hypertension among Saudi cases.

Prevalence of α-1-antitrypsin gene mutations in Saudi Arabia.

BACKGROUND/AIM: α-1 antitrypsin (AAT) deficiency results from mutations of the protease inhibitor (PI). The AAT gene is mapped on chromosome 14 and has been associated with chronic liver disease and chronic obstructive pulmonary disease (COPD). OBJECTIVE: To determine the frequency of AAT mutations on S and Z carrier alleles in healthy Saudi individuals from Qassim Province in Saudi Arabia. PATIENTS AND METHODS: A total of 158 healthy, unrelated participants from Qassim Province were recruited. They were genotyped for the two AAT-deficiency alleles, PIFNx01S and PIFNx01Z, using polymerase chain reaction, with primers designed throughout to mediate site-directed mutagenesis. RESULTS: Of the 158 subjects, 11.39% were carriers for the S mutation (i.e., had the MS genotype), whereas 2.53% were carriers for the Z mutation (i.e., had the MZ genotype). The SZ genotype was present in 3.8% of subjects, while the homozygous genotype SS was present in 1.9% of subjects. No subjects showed the ZZ mutant genotype. Accordingly, frequency of the mutant S and Z alleles of AAT gene was 9.49% and 3.19%, respectively. CONCLUSION: The results obtained showed a high prevalence of the AAT deficiency allele in the Saudi population. This probably warrants adoption of a screening program for at-risk individuals, so that they might initiate adequate prophylactic measures.