Sugar alcohol-modified polyester nanoparticles for gene delivery via selective caveolae-mediated endocytosis.

Nucleic acid-based drugs are changing the scope of emerging medicine in preventing and treating diseases. Nanoparticle systems based on lipids and polymers developed to navigate tissue-level and cellular-level barriers are now emerging as vector systems that can be translated to clinical settings. A class of polymers, poly(ß-amino esters) (PBAEs) known for their chemical flexibility and biodegradability, has been explored for gene delivery. These polymers are sensitive to changes in the monomer composition affecting transfection efficiency. Hence to add functionality to these polymers, we partially substituted ligands to an identified effective polymer chemistry. We report here a new series of statistical copolymers based on PBAEs where the backbone is modified with sugar alcohols to selectively facilitate the caveolae-mediated endocytosis pathway of cellular transport. These ligands are grafted at the polymer's backbone, thereby establishing a new strategy of modification in PBAEs. We demonstrate that these polymers form nanoparticles with DNA, show effective complexation and cargo release, enter the cell via selective caveolae-mediated endocytosis, exhibit low cytotoxicity, and increase transfection in neuronal cells.

Non-viral delivery of nucleic acid for treatment of rare diseases of the muscle.

Rare muscular disorders (RMDs) are disorders that affect a small percentage of the population. The disorders which are attributed to genetic mutations often manifest in the form of progressive weakness and atrophy of skeletal and heart muscles. RMDs includes disorders such as Duchenne muscular dystrophy (DMD), GNE myopathy, spinal muscular atrophy (SMA), limb girdle muscular dystrophy, and so on. Due to the infrequent occurrence of these disorders, development of therapeutic approaches elicits less attention compared with other more prevalent diseases. However, in recent times, improved understanding of pathogenesis has led to greater advances in developing therapeutic options to treat such diseases. Exon skipping, gene augmentation, and gene editing have taken the spotlight in drug development for rare neuromuscular disorders. The recent innovation in targeting and repairing mutations with the advent of CRISPR technology has in fact opened new possibilities in the development of gene therapy approaches for these disorders. Although these treatments show satisfactory therapeutic effects, the susceptibility to degradation, instability, and toxicity limits their application. So, an appropriate delivery vector is required for the delivery of these cargoes. Viral vectors are considered potential delivery systems for gene therapy; however, the associated concurrent immunogenic response and other limitations have paved the way for the applications of other non-viral systems like lipids, polymers, cellpenetrating peptides (CPPs), and other organic and inorganic materials. This review will focus on non-viral vectors for the delivery of therapeutic cargoes in order to treat muscular dystrophies.

The heparin-binding hemagglutinin protein of Mycobacterium tuberculosis is a nucleoid-associated protein.

Nucleoid-associated proteins (NAPs) regulate multiple cellular processes such as gene expression, virulence, and dormancy throughout bacterial species. NAPs help in the survival and adaptation of Mycobacterium tuberculosis (Mtb) within the host. Fourteen NAPs have been identified in Escherichia coli; however, only seven NAPs are documented in Mtb. Given its complex lifestyle, it is reasonable to assume that Mtb would encode for more NAPs. Using bioinformatics tools and biochemical experiments, we have identified the heparin-binding hemagglutinin (HbhA) protein of Mtb as a novel sequence-independent DNA-binding protein which has previously been characterized as an adhesion molecule required for extrapulmonary dissemination. Deleting the carboxy-terminal domain of HbhA resulted in a complete loss of its DNA-binding activity. Atomic force microscopy showed HbhA-mediated architectural modulations in the DNA, which may play a regulatory role in transcription and genome organization. Our results showed that HbhA colocalizes with the nucleoid region of Mtb. Transcriptomics analyses of a hbhA KO strain revealed that it regulates the expression of ∼36% of total and ∼29% of essential genes. Deletion of hbhA resulted in the upregulation of ∼73% of all differentially expressed genes, belonging to multiple pathways suggesting it to be a global repressor. The results show that HbhA is a nonessential NAP regulating gene expression globally and acting as a plausible transcriptional repressor.

Bioinspired synthesis of bioactive glass nanocomposites for hyaluronic acid delivery to bone and skin.

In this study, we present nanocomposites of bioactive glass (BG) and hyaluronic acid (HA) (nano-BGHA) for effective delivery of HA to skin and bone. The synthesis of the nanocomposites has been carried out through the bio-inspired method, which is a modification of the traditional Stober's synthesis as it avoids using ethanol, ammonia, synthetic surfactants, or high-temperature calcination. This environmentally friendly, bio-inspired route allowed the synthesis of mesoporous nanocomposites with an average hydrodynamic radius of ∼190 nm and an average net surface charge of ∼-21 mV. Most nanocomposites are amorphous and bioactive in nature with over 70 % cellular viability for skin and bone cell lines even at high concentrations, along with high cellular uptake (90-100 %). Furthermore, the nanocomposites could penetrate skin cells in a transwell set-up and artificial human skin membrane (StratM®), thus depicting an attractive strategy for the delivery of HA to the skin. The purpose of the study is to develop nanocomposites of HA and BG that can have potential applications in non-invasive treatments that require the delivery of high molecular weight HA such as in the case of osteoarthritis, sports injury treatments, eye drops, wound healing, and some anticancer treatments, if further investigated. The presence of BG further enhances the range to bone-related applications. Additionally, the nanocomposites can have potential cosmeceutical applications where HA is abundantly used, for instance in moisturizers, dermal fillers, shampoos, anti-wrinkle creams, etc.

Albumin Nanoparticles Surface Decorated with a Tumor-Homing Peptide Help in Selective Killing of Triple-Negative Breast Cancer Cells.

In this article, we describe a method of delivery of doxorubicin using a novel tumor-homing peptide-based albumin nanoparticle system to triple-negative breast cancer cells (TNBC). The absence and reduced expression of the hormone (estrogen, progesterone) and HER2 (human epidermal growth factor 2) receptors, respectively, render TNBC patients nonsusceptible to different available targeted therapies. These peptide-modified nanoparticles could be taken up by TNBC cells more effectively than their bare counterparts. The drug-loaded peptide-modified nanoparticles achieved an optimal but crucial balance between cell killing in cancerous cells and cell survival in the noncancerous ones. This appears to be because of different routes of entry and subsequent fate of the bare and peptide-modified nanoparticles in cancerous and noncancerous cells. In a TNBC mouse model, the peptide-modified system fared better than the free drug in mounting an antitumor response while not being toxic systemically.

Surface-Engineered Mucus Penetrating Nucleic Acid Delivery Systems with Cell Penetrating Peptides for the Lungs.

Nucleic acids, both DNA and small RNAs, have emerged as potential therapeutics for the treatment of various lung disorders. However, delivery of nucleic acids to the lungs is challenging due to the barrier property imposed by mucus, which is further reinforced in disease conditions such as chronic obstructive pulmonary disease and asthma. The presence of negatively charged mucins imparts the electrostatic barrier property, and the mesh network structure of mucus provides steric hindrance to the delivery system. To overcome this, the delivery system either needs to be muco-inert with a low positive charge such that the interactions with mucus are minimized or should have the ability to transiently dismantle the mucus structure for effective penetration. We have developed a mucus penetrating system for the delivery of both small RNA and plasmid DNA independently. The nucleic acid core consists of a nucleic acid (pDNA/siRNA) and a cationic/amphipathic cell penetrating peptide. The mucus penetrating coating consists of the hydrophilic biopolymer chondroitin sulfate A (CS-A) conjugated with a mucolytic agent, mannitol. We hypothesize that the hydrophilic coating of CS-A would reduce the surface charge and decrease the interaction with negatively charged mucins, while the conjugated mannitol residues would disrupt the mucin-mucin interaction or decrease the viscosity of mucus by increasing the influx of water into the mucus. Our results indicate that CS-A-mannitol-coated nanocomplexes possess reduced surface charge, reduced viscosity of artificial mucus, and increased diffusion in mucin suspension as well as increased penetration through the artificial mucus layer as compared to the non-coated ones. Further, the coated nanocomplexes showed low cytotoxicity as well as higher transfection in A-549 and BEAS-2B cells as compared to the non-coated ones.

A Combination of Synthetic Molecules Acts as Antifreeze for the Protection of Skin against Cold-Induced Injuries.

Seasonal and occupational exposure of the human body to extreme cold temperatures can result in cell death in the exposed area due to the formation of ice crystals. This leads to superficial or deep burn injury and compromised functionality. Currently available therapeutics can be ineffective in extreme cases, and thus, it is necessary to develop prophylactic strategies. In this study, we have devised a combination of known synthetic cryopreservative agents (termed SynAFP) and evaluated their potential antifreeze applications on skin. The prophylactic activity of SynAFP in vitro is indicated by improved cellular revival and cell viability, retention of the cytoskeleton, and normal cell cycle progression even after cold stress. A comprehensive whole-cell proteomic approach revealed that in the presence of SynAFP, cold-induced downregulation of proteins involved in cell-cell adhesion and upregulation of those related to mitochondrial stress were ameliorated. Pre-application of SynAFP in mice facing a frostbite challenge prevents their skin from incurring significant injury as confirmed through macroscopic and histological examination. Moreover, multiple applications of SynAFP on mouse skin at room temperature did not compromise skin integrity. SynAFP was also formulated in anAloe vera-based cream (referred to as fSynAFP), which offered similar protection under cold stress conditions. Thus, SynAFP can be considered as a potential candidate for formulating a topical intervention for protection from cold-induced injuries to skin.

Nonimmunogenic Hydrogel-Mediated Delivery of Antibiotics Outperforms Clinically Used Formulations in Mitigating Wound Infections.

Treatment of chronic wound infections caused by Gram-positive bacteria such as Staphylococcus aureus is highly challenging due to the low efficacy of existing formulations, thereby leading to drug resistance. Herein, we present the synthesis of a nonimmunogenic cholic acid-glycine-glycine conjugate (A6) that self-assembles into a supramolecular viscoelastic hydrogel (A6 gel) suitable for topical applications. The A6 hydrogel can entrap different antibiotics with high efficacy without compromising its viscoelastic behavior. Activities against different bacterial species using a disc diffusion assay demonstrated the antimicrobial effect of the ciprofloxacin-loaded A6 hydrogel (CPF-Gel). Immune profiling and gene expression studies after the application of the A6 gel to mice confirmed its nonimmunogenic nature to host tissues. We further demonstrated that topical application of CPF-Gel clears S. aureus-mediated wound infections more effectively than clinically used formulations. Therefore, cholic acid-derived hydrogels are an efficacious matrix for topical delivery of antibiotics and should be explored further.

Functions of, and replenishment strategies for, chondroitin sulfate in the human body.

Chondroitin sulfate (CS) belongs to a class of molecules called glycosaminoglycans (GAGs). These are long, linear chains of polysaccharides comprising alternating amino sugars and hexuronic acid. Similar to other GAGs, CS is important in a multitude of biological activities. Alteration of CS levels has been implicated in several pathological conditions, including osteoarthritis (OA) and other inflammatory diseases, as well as physiological conditions, such as aging. Therefore, devising replenishment strategies for this molecule is an important area of research. In this review, we discuss the nature of CS, its function in different organs, and its implications in health and disease. We also describe different methods for the exogenous administration of CS.

Topical Application of Peptide-Chondroitin Sulfate Nanoparticles Allows Efficient Photoprotection in Skin.

In this article, we describe a method of delivery of chondroitin sulfate to skin as nanoparticles and demonstrate its anti-inflammatory and antioxidant role using UV irradiation as a model condition. These nanoparticles, formed through electrostatic interactions of chondroitin sulfate with a skin-penetrating peptide, were found to be homogenous with positive surface charges and stable at physiological and acidic pH under certain conditions. They were able to enter into the human keratinocyte cell line (HaCaT), artificial skin membrane (mimicking the human skin), and mouse skin tissue unlike free chondroitin sulfate. The preapplication of nanoparticles also exhibited reduced levels of oxidative stress, cyclobutane pyrimidine dimer formation, TNF-α, and so on in UV-B-irradiated HaCaT cells. In an acute UV-B irradiation mouse model, their topical application resulted in reduced epidermal thickness and sunburn cells, unlike in the case of free chondroitin sulfate. Thus, a completely noninvasive method was used to deliver a bio-macromolecule into the skin without using injections or abrasive procedures.

Higher order assembling of the mycobacterial polar growth factor DivIVA/Wag31.

DivIVA or Wag31, which is an essential pole organizing protein in mycobacteria, can self-assemble at the negatively curved side of the membrane at the growing pole to form a higher order structural scaffold for maintaining cellular morphology and localizing various target proteins for cell-wall biogenesis. The structural organization of polar scaffold formed by polymerization of coiled-coil rich Wag31, which is implicated in the anti-tubercular activities of amino-pyrimidine sulfonamides, remains to be determined. A single-site phosphorylation in Wag31 regulates peptidoglycan biosynthesis in mycobacteria. We report biophysical characterizations of filaments formed by mycobacterial Wag31 using circular dichroism, atomic force microscopy and small angle solution X-ray scattering. Atomic force microscopic images of the wild-type, a phospho-mimetic (T73E) and a phospho-ablative (T73A) form of Wag31 show mostly linear filament formation with occasional curving, kinking and apparent branching. Solution X-ray scattering data indicates that the phospho-mimetic forms of the Wag31 polymers are on average more compact than their phospho-ablative counterparts, which is likely due to the extent of bending/branching. Observed structural features in this first view of Wag31 filaments suggest a basis for higher order Wag31 scaffold formation at the pole.

Role of Cellular Retention and Intracellular State in Controlling Gene Delivery Efficiency of Multiple Nonviral Carriers.

Nonviral gene delivery has seen major progress in the last two decades owing to facile synthesis, low toxicity, and ease of modification of nanocarriers that take nucleic acids to cells and tissues. Gene delivery nanocomplexes need to reach the target locations in significant amounts by overcoming multiple barriers. While the importance of nanocomplex stability, cellular uptake, intracellular trafficking, and nuclear localization has been studied extensively, the role of cellular retention and recycling of these nanocomplexes is less understood in the context of gene delivery. In this study, we used different DNA carriers and made efforts to understand the role played by cellular retention in determining their gene delivery efficiency across multiple cell lines. In addition, we also analyzed whether state of complexation and localization of the nanocomplexes play a role in conjunction with cellular retention. We observed higher transfection efficiencies for nanocomplexes showing better retention, lower unpackaging, and low recycling. Our data also suggests that nanocomplexes made of peptides with terminal cysteine modification show enhanced retention and transfection efficiency compared to their counterparts with no terminal cysteine. Overall, the work highlights myriad of factors to be considered for improving gene delivery efficiency of nanocomplexes.

Silymarin-loaded nanostructured lipid carrier gel for the treatment of skin cancer.

Aim: The present study was aimed at determining the antiproliferative, antioxidant, anti-inflammatory and antitumor activity of developed silymarin-nanostructured lipid carrier (NLC) gel. Materials & methods: B16 melanoma cell line and albino mice were used as ex vivo and in vivo models, respectively, to evaluate the aforementioned pharmacological activities. Results: The volume of large tumors significantly (p < 0.05) reduced from 5.02 to 3.05 mm3, levels of IL-1α and TNF-α were significantly (p < 0.001) lower and levels of superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH) significantly (p < 0.0001) increased in the group treated with silymarin-NLC gel. Furthermore, in skin treated with placebo and conventional gels, a basosquamous carcinoma and squamous cell carcinoma were noticed, respectively. Conclusion: Silymarin-NLC gel presented better treatment outcomes compared with silymarin-conventional gel.

Nucleic acid delivery to differentiated retinal pigment epithelial cells using cell-penetrating peptide as a carrier.

Nucleic acid delivery to the eye is a promising treatment strategy for many retinal disorders. In this manuscript, retinal gene delivery with non-coated and chondroitin sulphate (CS) coated amphipathic and cationic peptides was tested. The transfection and gene knockdown efficiencies were evaluated in different retinal pigment epithelial (RPE) cell models including both dividing and differentiated cells. In addition, the mobility of peptide-based gene delivery systems was examined in porcine vitreous by particle tracking analysis. The results indicate that amphipathic and cationic peptides are safe in vitro and are capable of high transgene expression and gene knockdown in dividing cells. We further demonstrate that incorporation of CS improves the efficiency of gene delivery of peptide-based systems. Most importantly, the transgene expression mediated by both non-coated and CS coated peptides was high in differentiated as well as in human primary RPE cells which are typically difficult to transfect. Coating of peptide-based gene delivery systems with CS improved diffusion in the vitreous and enhanced the stability of the polyplexes. The results indicate that a peptide-based system can be fine-tuned as a promising approach for retinal gene delivery.

Exocytosis - a putative road-block in nanoparticle and nanocomplex mediated gene delivery.

Gene and drug delivery mediated by nanostructures has seen tremendous growth over the last decade. However, the efficiency of these delivery approaches needs to be improved for better effects. Amongst various factors, cellular retention is expected to play a critical role. Nanoparticles and nanocomplexes internalized by the cells can be recycled back to the outside through the process of exocytosis. Although it sounds reasonable that the efficiency of these delivery systems should not only depend on their cellular uptake but also on the ability of cells to retain them, the process of cellular retention and exocytosis is relatively less studied in the literature. In the context of gene delivery, both inorganic nanoparticles and organic nanocomplexes are used, but there is limited information on how these nanoparticles and nanocomplexes are recycled and what could be the possible effect of such recycling on the efficiency of these delivery vectors. In this review we try to summarize the existing literature in this area, putative mechanisms involved in recycling of the nanoparticles, methods used to quantify exocytosis and factors affecting exocytosis. The possibility of enhancing cellular retention by blocking recycling pathways as well as the in vivo implications is also discussed here.

ZnO Nanoparticles Modified with an Amphipathic Peptide Show Improved Photoprotection in Skin.

ZnO nanoparticles of different sizes were functionalized with an amphipathic peptide, and its effect on nanoparticle stabilization and UV photoprotective activity was studied in this article. The peptide-modified nanoparticles exhibited lower aggregation, significant reduction in Zn2+ leaching in vitro and even inside the cells for smaller particle sizes, reduced photocatalytic activity, and reduced cellular toxicity under UV-B treated conditions. In addition, the peptide-modified 60 nm ZnO nanoparticles showed lower genotoxicity, lower oxidative stress induction levels, less DNA damage responses, and less immunogenic potential than the bare counterparts in the presence of UV-B rays. They localized more in the stratum corneum and epidermis ex vivo, indicating better retention in epidermis, and demonstrated improved UV-B protection and/or skin integrity in SKH-1 mice in vivo compared to unmodified nanoparticles and commercial UV-protective agents tested. To our knowledge, this is the first report on the application of peptide-modified ZnO nanoparticles for improved photoprotection.

Deciphering the Role of Chondroitin Sulfate in Increasing the Transfection Efficiency of Amphipathic Peptide-Based Nanocomplexes.

Glycosaminoglycans, both cell-surface and exogenous, can interfere with DNA delivery efficiency of nonviral carrier systems. In this work, we report an extensive comparative study to explore the effect of exogenously added chondroitin sulfate on biophysical characteristics, cellular uptake, transfection efficiency, and intracellular trafficking of nanocomplexes formed using primary and secondary amphipathic peptides developed in our laboratory. Our results indicate that the presence of exogenous chondroitin sulfate exhibits differential enhancement in transfection efficiency of the amphipathic peptides depending upon their chemical nature. The enhancement was more pronounced in primary amphipathic peptide-based nanocomplexes as compared to the secondary counterpart. This difference can be attributed to possible alteration of the intracellular entry pathway in addition to increased extracellular stability, less cellular toxicity, and assistance in nuclear accumulation. These results imply potential use of glycosaminoglycans such as chondroitin sulfate to improve the transfection efficiency of primary amphipathic peptides for possible in vivo applications.

Redox-dependent condensation of the mycobacterial nucleoid by WhiB4.

Oxidative stress response in bacteria is mediated through coordination between the regulators of oxidant-remediation systems (e.g. OxyR, SoxR) and nucleoid condensation (e.g. Dps, Fis). However, these genetic factors are either absent or rendered non-functional in the human pathogen Mycobacterium tuberculosis (Mtb). Therefore, how Mtb organizes genome architecture and regulates gene expression to counterbalance oxidative imbalance is unknown. Here, we report that an intracellular redox-sensor, WhiB4, dynamically links genome condensation and oxidative stress response in Mtb. Disruption of WhiB4 affects the expression of genes involved in maintaining redox homeostasis, central metabolism, and respiration under oxidative stress. Notably, disulfide-linked oligomerization of WhiB4 in response to oxidative stress activates the protein's ability to condense DNA. Further, overexpression of WhiB4 led to hypercondensation of nucleoids, redox imbalance and increased susceptibility to oxidative stress, whereas WhiB4 disruption reversed this effect. In accordance with the findings in vitro, ChIP-Seq data demonstrated non-specific binding of WhiB4 to GC-rich regions of the Mtb genome. Lastly, data indicate that WhiB4 deletion affected the expression of ~ 30% of genes preferentially bound by the protein, suggesting both direct and indirect effects on gene expression. We propose that WhiB4 structurally couples Mtb's response to oxidative stress with genome organization and transcription.

Zinc Oxide Nanoparticles Dispersed in Ionic Liquids Show High Antimicrobial Efficacy to Skin-Specific Bacteria.

Zinc oxide (ZnO) nanoparticles have been shown in the literature to have antibacterial properties and have been widely used in antibacterial formulations. However, one of the problems with ZnO nanoparticles is their tendency to aggregate, thereby causing damage to normal cells and lowering their antibacterial efficacy during application. In this work, we have attempted to avoid this by using a combination of ZnO nanoparticles and ionic liquids, a class of low melting salts containing organic cations and organic/inorganic anions that show antibacterial property as well, and tested the antibacterial activity of this dispersion. ZnO nanoparticles of 60 nm were dispersed in two different ionic liquids-choline acetate (IL1) and 1-butyl-3-methylimidazolium chloride (IL2)-to achieve high dispersibility, whereas ZnO dispersed in phosphate-buffered saline was taken as a control. These dispersions were tested on four strains- Escherichia coli, Bacillus subtilis, Klebsiella pneumoniae, and Staphylococcus epidermidis. Maximum efficiency was obtained for ZnO nanoparticles dispersed in imidazolium-based ionic liquids against skin-specific S. epidermidis. Skin infections induced by S. epidermidis are prevalent in hospital-acquired diseases. In most cases, traditional antibiotic-based therapies fail to combat such infections. Our strategy of developing a dispersion of ZnO nanoparticles in ionic liquids shows superior antibacterial efficacy in comparison to that shown individually by ZnO nanoparticles or ionic liquids. We have also established that the mechanism of killing this skin-specific bacterium is possibly through the production of reactive oxygen species leading to bacterial cell lysis. Further, we showed that this formulation is biocompatible and nontoxic to normal keratinocyte cells even under coculture conditions.

Dendrimeric amide- and carbamate-linked lysine-based efficient molecular transporters.

Amide- and carbamate-linked dendrimeric oligomers are reported as molecular transporters. They effectively complex with pDNA and transport it into cells at an efficiency superior to Lipofectamine, when complexation is carried out by incubation overnight. The carbamate-linked K2C is superior to amide-linked K2A; their pDNA complexes have very low associated cytotoxicity.