Therapeutic modulation of gene expression in the disease state: Treatment strategies and approaches for the development of next-generation of the epigenetic drugs.

Epigenetic dysregulation is an important determinant of many pathological conditions and diseases. Designer molecules that can specifically target endogenous DNA sequences provide a means to therapeutically modulate gene function. The prokaryote-derived CRISPR/Cas editing systems have transformed our ability to manipulate the expression program of genes through specific DNA and RNA targeting in living cells and tissues. The simplicity, utility, and robustness of this technology have revolutionized epigenome editing for research and translational medicine. Initial success has inspired efforts to discover new systems for targeting and manipulating nucleic acids on the epigenetic level. The evolution of nuclease-inactive and RNA-targeting Cas proteins fused to a plethora of effector proteins to regulate gene expression, epigenetic modifications and chromatin interactions opened up an unprecedented level of possibilities for the development of "next-generation" gene therapy therapeutics. The rational design and construction of different types of designer molecules paired with viral-mediated gene-to-cell transfers, specifically using lentiviral vectors (LVs) and adeno-associated vectors (AAVs) are reviewed in this paper. Furthermore, we explore and discuss the potential of these molecules as therapeutic modulators of endogenous gene function, focusing on modulation by stable gene modification and by regulation of gene transcription. Notwithstanding the speedy progress of CRISPR/Cas-based gene therapy products, multiple challenges outlined by undesirable off-target effects, oncogenicity and other virus-induced toxicities could derail the successful translation of these new modalities. Here, we review how CRISPR/Cas-based gene therapy is translated from research-grade technological system to therapeutic modality, paying particular attention to the therapeutic flow from engineering sophisticated genome and epigenome-editing transgenes to delivery vehicles throughout efficient and safe manufacturing and administration of the gene therapy regimens. In addition, the potential solutions to some of the obstacles facing successful CRISPR/Cas utility in the clinical research are discussed in this review. We believe, that circumventing these challenges will be essential for advancing CRISPR/Cas-based tools towards clinical use in gene and cell therapies.

Comparative analysis of PEG-liposomes and RBCs-derived nanovesicles for anti-tumor therapy.

Lipid-based vesicular nanoparticles, for instance liposomes, conjugated with polyethylene glycol (PEG) have proven to be the closest to an ideal drug delivery vehicle, making way for several PEG-liposomes based nanomedicines in market. However, the synthetic nature of the nanomaterial poses a threat to stimulate immune system. Alternatively, nanovesicles derived from mammalian cells, such as RBCs, have gained interests as they may not elicit much immune response due to the presence of host specific self-recognition markers on their surface. While several reports demonstrating the superior efficacy of these naturally derived vesicles have come out in the last few years, a comparison with clinically established liposomes is still missing. Thus, we conducted an in-vitro and in-vivo comparative studies between PEG-Liposomes and nanovesicles (NVEs) derived from red blood cell (RBC) membrane with an aim to establish a biocompatible nanocarrier for efficient delivery of chemotherapeutic drugs and photothermal agents.

Red blood cells membrane-derived nanoparticles: Applications and key challenges in their clinical translation.

Cellular membrane-derived nanoparticles, particularly of red blood cells (RBCs), represent an emerging class of drug delivery systems. The lack of nucleus and organelles in these cells makes them easy to process and empty out intracellular contents. The empty vesicle membranes can then be either used as a coating on nanoparticles or can be reassembled into a nanovesicle. Engineered RBCs membrane has unique ability to retain its lipid bilayer architecture with host's proteins during top-down approach, thus allowing it to form stable nanoformulations mimicking RBCs stealth properties. In addition, its core-shell structure allows loading of different drug molecules, and its surface chemistry can be manipulated by facile conjugation with ligands on the shell. The remarkable ability of RBCs membrane to fuse with membranes of other cells enables the formation of hybrid nanovesicles. In this review, we highlight the biomedical applications of such vesicles and discuss the potential challenges related to its clinical translation. Although nano-RBCs retain much of the host's proteins, which may give an edge over synthetic nanoparticles in terms of lower immunogenicity, its production at industrial level is more challenging. This review gives the critical analysis of barriers involved in the translation of RBCs-derived nanoparticles from preclinical to clinical level. This article is categorized under: Therapeutic Approaches and Drug Discovery > Emerging Technologies Biology-Inspired Nanomaterials > Lipid-Based Structures Toxicology and Regulatory Issues in Nanomedicine > Regulatory and Policy Issues in Nanomedicine.

Engineering Cellular Membrane for Dual Mode Therapy Using NIR Responsive Photosensitizer and Reversible Topoisomerase Inhibition Activity.

Extensive research over past few decades has highlighted the challenges of chemotherapy and prompted the need for multimodality therapy because chemotherapy alone cannot fully eradicate the tumor due to physiological barriers in its effective delivery and systemic side effects. It can be mitigated by adopting nanoparticles as more effective delivery method, but none of them completely prevents drug toxicities. Utilizing multiple therapeutic modes such as phototherapy that can act synergistically with chemotherapy in controlling tumor growth, while reducing the overall dosage, could become a preferred route for cancer management. Careful selection of nanoparticle system, which can simultaneously deliver both drug and photosensitizer, can significantly enhance the therapeutic outcome. Therefore, in this paper, we report development and potential of immune-compatible and long circulating nanoerythrosomes for enhancing the therapeutic potential of camptothecin and indocyanine green against murine cancer model. The RBCs membrane simultaneously loaded the nonpolar drug and amphiphilic photosensitizer in its lipid bilayer, which self-assembled to form stable nanoparticles. These nano constructs absorbed light in the near-infrared region and hence are suitable for targeting deep seated tissues. The dual chemo-phototherapy had great effect on cell viability and had therapeutic value.

Polymeric micelles coated with hybrid nanovesicles enhance the therapeutic potential of the reversible topoisomerase inhibitor camptothecin in a mouse model.

Nanoparticles with longer blood circulation, high loading capacity, controlled release at the targeted site, and preservation of camptothecin (CPT) in its lactone form are the key characteristics for the effective delivery of CPT. In this regard, natural membrane-derived nanovesicles, particularly those derived from RBC membrane, are important. RBC membrane can be engineered to form vesicles or can be coated over synthetic nanoparticles, without losing their basic structural features and can have prolonged circulation time. Here, we developed a hybrid system to encapsulate CPT inside the amphiphilic micelle and coat it with RBC membrane. Thus, it uses the dual ability of polymeric micelles to preserve CPT in its active form, while maintaining its "stealth" effect due to conserved RBC membrane coating. The hybrid system stabilized 60% of the drug in its active form even after 30 h of incubation in serum, in contrast to 15% active form present in free drug formulation after 1 h of incubation. It showed strong retention inside the Ehrlich Ascites Carcinoma (EAC) mice models for at least 72 h, suggesting camouflaging ability conferred by RBC membrane. Additionally, the nano formulation retarded the tumor growth rate more efficiently than free drug, with no evident signs of necrotic skin lesions. Histopathological analysis showed a significant reduction in cardiac atrophy, hepato-renal degeneration, and lung metastasis, which resulted in the increased overall survival of mice treated with the nano formulation. Hence, CPT-loaded polymeric micelles when coated with RBC membrane can prove to be a better system for the delivery of poorly soluble drug camptothecin.

Dual functionalized chitosan based composite hydrogel for haemostatic efficacy and adhesive property.

The objective of this study was to devise a dual functionalized chitosan based hydrogel dressing to control haemorrhage/ bleeding. The haemostatic hydrogel was formulated by amalgamation of a definite ratio of quaternized chitosan and phosphorylated chitosan along with tannic acid which acted as adjuvant hemostat and a crosslinker. Additionally, the hydrogel contained poly-Ɛ-lysine to impart the elastic and adhesive properties. The optimized hydrogel exhibited superior haemostatic activity (clotting time, 225 ± 5 s), platelet activation (soluble P-selectin concentration 2098 ± 150.19 ng mL-1), adhesion strength (almost 3 times higher in comparison to Axiostat), higher fluid absorption (approx. 14 times in 12 h) in addition to better mechanical properties, faster coagulation attributes (Prothrombin time, 12.6 s and activated partial thromboplastin time, 30.1 s) and lower proinflammatory potential (almost 3 times lower Tumor Necrosis Factor- α levels and 45 times lower InterLeukin-6 levels at 48 h against control) over marketed chitosan based dressing (clotting time, 300 ± 25 s). Cytotoxicity studies using L929 fibroblasts cells and in-vivo studies using Wistar rats confirmed that the optimized hydrogel was non-toxic, cytocompatible and biocompatible.

Red Blood Cells-Derived Vesicles for Delivery of Lipophilic Drug Camptothecin.

Recently, cell membrane-derived nanoparticles, particularly of RBCs, have been explored for delivery of hydrophilic solutes of varied size and complexities. So far, these naturally derived nanoparticles show a significant overlap with liposomes in terms of stability, solute encapsulation, and release. Unlike hydrophilic molecules, which are loaded inside the aqueous core, hydrophobic moieties largely partition inside the lipophilic shell, hence fate of these nanocarriers may be different. Since vesicles have more complex membrane architecture (due to natural lipids and additional proteins and glycoproteins), ease of loading hydrophobic drug, its release pattern, and overall particle stability cannot be compared to those of synthetic lipid-based carriers. Therefore, we derived nanovesicles (NVEs) from RBC membrane, loaded with hydrophobic drug camptothecin (CPT) and labeled noncovalently with amphiphilic fluorophore (CM-DiI). Although both CPT and CM-DiI are known to partition inside the membrane, the overall stability of NVEs and composition of membrane proteins, particularly CD47, "marker of self", did not change. Additionally, the developed NVEs were found to be nonphagocytic even in the presence of serum and showed minimal stimulation of macrophages to release cytokines. Further, this system showed slow release but strong retention of CPT and CM-DiI, respectively, over 24 h, hence appropriate for theranostic applications. Also, NVEs were internalized by lung carcinoma cells and possessed slightly higher toxicity than free CPT. When injected intravenously in balb/c mice, these nanovesicles showed higher retention in blood over 48 h and insignificant accumulation in vital organs like heart and kidneys, thus suggesting its potential for in vivo application. We believe that this system has superior stealth and comparable physicochemical properties to synthetic lipid-based nanocarriers; hence, it can be further developed as personalized medicine.

Antimicrobial and toxicological behavior of montmorillonite immobilized metal nanoparticles.

With increasing demand for novel and potent antimicrobial agents to combat cross-infections and infectious diseases, silver and copper based nanoparticles (NPs) deposited over supports such as montmorillonite (MMT) are playing a crucial role in shaping the current research scenario. Although materials based on Ag NP and Cu NP on MMT have been reported, its toxicological properties on human cell lines have not been accounted for. This paper reports a comparative study on synthesis, antibacterial, antifungal and toxicological behavior of Ag and Cu NPs deposited over MMT nanosheets synthesized by employment of different reduction media. The effect of synthesized NP-MMT hybrids on human erythrocytes and fibroblast cells has been evaluated. The NP formation was facilitated using borohydride and ethyl alcohol (wet chemical route) and photo-reduction and thermal treatment (physical reduction route). The NP-MMT hybrids showed NP formation over supporting silicate layers with particle size ~10-50 nm confirmed by TEM micrographs and loading of ~6-22 wt% of metallic element by EDX analysis. The MMT layers were peeled apart to accommodate NPs inside its galleries, confirmed by increased d-value in powder WAXD. The NP-hybrids showed excellent inhibition zone against bacteria E.coli and S. aureus and fungi A. niger. RBC hemolysis and cytocompatibility assay were performed in vitro to advocate its safety to live human cells. These hybrid materials are potential candidates for new generation advanced antimicrobial materials with less toxicity and highly potent behavior.

Experimental measurement of settling velocity of spherical particles in unconfined and confined surfactant-based shear thinning viscoelastic fluids.

An experimental study is performed to measure the terminal settling velocities of spherical particles in surfactant based shear thinning viscoelastic (VES) fluids. The measurements are made for particles settling in unbounded fluids and fluids between parallel walls. VES fluids over a wide range of rheological properties are prepared and rheologically characterized. The rheological characterization involves steady shear-viscosity and dynamic oscillatory-shear measurements to quantify the viscous and elastic properties respectively. The settling velocities under unbounded conditions are measured in beakers having diameters at least 25x the diameter of particles. For measuring settling velocities between parallel walls, two experimental cells with different wall spacing are constructed. Spherical particles of varying sizes are gently dropped in the fluids and allowed to settle. The process is recorded with a high resolution video camera and the trajectory of the particle is recorded using image analysis software. Terminal settling velocities are calculated from the data. The impact of elasticity on settling velocity in unbounded fluids is quantified by comparing the experimental settling velocity to the settling velocity calculated by the inelastic drag predictions of Renaud et al.(1) Results show that elasticity of fluids can increase or decrease the settling velocity. The magnitude of reduction/increase is a function of the rheological properties of the fluids and properties of particles. Confining walls are observed to cause a retardation effect on settling and the retardation is measured in terms of wall factors.