Development of Focused Ultrasound-Assisted Nanoplexes for RNA Delivery.

RNA-based therapeutics, including siRNA, have obtained recognition in recent years due to their potential to treat various chronic and rare diseases. However, there are still limitations to lipid-based drug delivery systems in the clinical use of RNA therapeutics due to the need for optimization in the design and the preparation process. In this study, we propose adaptive focused ultrasound (AFU) as a drug loading technique to protect RNA from degradation by encapsulating small RNA in nanoliposomes, which we term nanoplexes. The AFU method is non-invasive and isothermal, as nanoplexes are produced without direct contact with any external materials while maintaining precise temperature control according to the desired settings. The controllability of sample treatments can be effectively modulated, allowing for a wide range of ultrasound intensities to be applied. Importantly, the absence of co-solvents in the process eliminates the need for additional substances, thereby minimizing the potential for cross-contaminations. Since AFU is a non-invasive method, the entire process can be conducted under sterile conditions. A minimal volume (300 µL) is required for this process, and the treatment is speedy (10 min in this study). Our in vitro experiments with silencer CD44 siRNA, which performs as a model therapeutic drug in different mammalian cell lines, showed encouraging results (knockdown > 80%). To quantify gene silencing efficacy, we employed quantitative polymerase chain reaction (qPCR). Additionally, cryo-electron microscopy (cryo-EM) and atomic force microscopy (AFM) techniques were employed to capture images of nanoplexes. These images revealed the presence of individual nanoparticles measuring approximately 100-200 nm in contrast with the random distribution of clustered complexes observed in ultrasound-untreated samples of liposome nanoparticles and siRNA. AFU holds great potential as a standardized liposome processing and loading method because its process is fast, sterile, and does not require additional solvents.

Modifying the proton transfer of 3,5-bis(2-hydroxyphenyl)-1H-1,2,4-triazole by water, confinement and confined water.

The effect of water, confinement and confined water on the proton transfer of 3,5-bis(2-hydroxyphenyl)-1H-1,2,4-triazole (bis-HPTA) was investigated. Water alters the proton transfer process. At higher pH, an anion is formed in water and it undergoes intermolecular proton transfer and forms a keto tautomer. Confinement of molecule in ß-cyclodextrin affects the intramolecular proton transfer. It also prevents the intermolecular proton transfer of the anionic form. In reverse micelle, the molecule resides in the interfacial region and interacts with bound water. The intermolecular hydrogen bond of the surfactants opens the intramolecular hydrogen bond in the weaker ß-ring of bis-HPTA. It led to single tautomer emission from bis-HPTA. An increase in water amount enhances the relative amount of trans-enol, but predominantly tautomer emission is observed.

Advanced nanoengineered-customized point-of-care tools for prostate-specific antigen.

Change in the level of human prostate-specific antigen (PSA) is a major element in the development and progression of prostate cancer (PCa). Most of the methodologies are currently restricted to their application in routine clinical screening due to the scarcity of adequate screening tools, false reading, long assay time, and cost. Innovative techniques and the integration of knowledge from a variety of domains, such as materials science and engineering, are needed to provide sustainable solutions. The convergence of precision point-of-care (POC) diagnostic techniques, which allow patients to respond in real time to changes in PSA levels, provides promising possibilities for quantitative and quantitative detection of PSA. This solution could be interesting and relevant for use in PCa diagnosis at the POC. The approaches enable low-cost real-time detection and are simple to integrate into user-friendly sensor devices. This review focuses on the investigations, prospects, and challenges associated with integrating engineering sciences with cancer biology to develop nanotechnology-based tools for PCa diagnosis. This article intends to encourage the development of new nanomaterials to construct high-performance POC devices for PCa detection. Finally, the review concludes with closing remarks and a perspective forecast.

Nano-enabled strategies to combat methicillin-resistant Staphylococcus aureus.

The emergence of methicillin-resistant Staphylococcus aureus (MRSA) has become a threat to global health because of limited treatments. MRSA infections are difficult to treat due to increasingly developing resistance in combination with protective biofilms of Staphylococcus aureus (S. aureus). Nanotechnology-based research revealed that effective MRSA treatments could be achieved through targeted nanoparticles (NPs) that withstand biological films and drug resistance. Thus, the principal aim towards improving MRSA treatment is to advance drug delivery tools, which successfully address the delivery-related problems. These potential delivery tools would also carry drugs to the desired sites of therapeutic action to overcome the adverse effects. This review focused on different types of nano-engineered carriers system for antimicrobial agents with improved therapeutic efficacy of entrapped drugs. The structural characteristics that play an essential role in the effectiveness of delivery systems have also been addressed with a description of recent scientific advances in antimicrobial treatment, emphasizing challenges in MRSA treatments. Consequently, existing gaps in the literature are highlighted, and reported contradictions are identified, allowing for the development of roadmaps for future research.

Enhanced gene expression by a novel designed leucine zipper endosomolytic peptide.

An endosomal trap is a major barrier in gene therapy. We have designed an endosomolytic peptide based on the leucine zipper sequence and characterized it both structurally and functionally. The results illustrated that leucine zipper endosomolytic peptide (LZEP) exhibited appreciable hemolysis of human red blood cells (hRBCs) at pH 5.0, but negligible hemolysis at pH 7.4. Calcein release experiment indicated that only at pH 5.0 but not at pH 7.4, LZEP was able to permeabilize hRBCs. LZEP revealed significant self-assembly as well as peptide induced α-helical structure at pH 5.0. Unlike at pH 5.0, LZEP failed to self-assemble and showed a random coil structure at pH 7.4. Transfection data depicted that lipoplexes modified with LZEP resulted in significantly higher gene expression as compared to lipoplexes without LZEP. Interestingly, the transfection efficacy of LZEP modified lipid nanoparticles reached the levels of Lipofectamine 2000 (LF 2000), without any cellular toxicity as observed by MTT assay. The results suggest a novel approach for designing endosomolytic peptides by employing the leucine zipper sequence and simultaneously the designed peptides could be utilized for enhancing gene delivery into mammalian cells.

Autophagy Paradox of Cancer: Role, Regulation, and Duality.

Autophagy, a catabolic process, degrades damaged and defective cellular materials through lysosomes, thus working as a recycling mechanism of the cell. It is an evolutionarily conserved and highly regulated process that plays an important role in maintaining cellular homeostasis. Autophagy is constitutively active at the basal level; however, it gets enhanced to meet cellular needs in various stress conditions. The process involves various autophagy-related genes that ultimately lead to the degradation of targeted cytosolic substrates. Many factors modulate both upstream and downstream autophagy pathways like nutritional status, energy level, growth factors, hypoxic conditions, and localization of p53. Any problem in executing autophagy can lead to various pathological conditions including neurodegeneration, aging, and cancer. In cancer, autophagy plays a contradictory role; it inhibits the formation of tumors, whereas, during advanced stages, autophagy promotes tumor progression. Besides, autophagy protects the tumor from various therapies by providing recycled nutrition and energy to the tumor cells. Autophagy is stimulated by tumor suppressor proteins, whereas it gets inhibited by oncogenes. Due to its dynamic and dual role in the pathogenesis of cancer, autophagy provides promising opportunities in developing novel and effective cancer therapies along with managing chemoresistant cancers. In this article, we summarize different strategies that can modulate autophagy in cancer to overcome the major obstacle, i.e., resistance developed in cancer to anticancer therapies.

Preparation and in vivo evaluation of red blood cell membrane coated porous silicon nanoparticles implanted with 155Tb.

INTRODUCTION: Porous silicon (PSi) nanoparticles are capable of delivering therapeutic payloads providing targeted delivery and sustained release of the payloads. In this work we describe the development and proof-of-concept in vivo evaluation of thermally hydrocarbonized porous silicon (PSi) nanoparticles that are implanted with radioactive 155Tb atoms and coated with red blood cell (RBC) membrane (155Tb-THCPSi). The developed nanocomposites can be utilized as an intravenous delivery platform for theranostic radionuclides. METHODS: THCPSi thin films were implanted with 155Dy ions that decay to 155Tb at the ISOLDE radioactive ion-beam (RIB) facility at CERN. The films were processed to nanoparticles by ball-milling and sonication, and subsequently coated with either a solid lipid and RBC membrane or solely with RBC membrane. The nanocomposites were evaluated in vitro for stability and in vivo for circulation half-life and ex vivo for biodistribution in Balb/c mice. RESULTS: Nanoporous THCPSi films were successfully implanted with 155Tb and processed to coated nanoparticles. The in vitro stability of the particles in plasma and buffer solutions was not significantly different between the particle types, and therefore the RBC membrane coated particles with less laborious processing method were chosen for the biological evaluation. The RBC membrane coating enhanced significantly the blood half-life compared to bare THCPSi particles. In the ex vivo biodistribution study a pronounced accumulation to the spleen was found, with lower uptake in the liver and a minor uptake in the lung, gall bladder and bone marrow. CONCLUSIONS: We have demonstrated, using 155Tb RIB-implanted PSi nanoparticles coated with mouse RBC membranes, the feasibility of using such a theranostic nanosystem for the delivery of RIB based radionuclides with prolonged circulation time. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE: For the first time, the RIB implantation technique has been utilized to produce PSi nanoparticle with a surface modified for better persistence in circulation. When optimized, these particles could be used in targeted radionuclide therapy with a combination of chemotherapeutic payload within the PSi structure.

Porous Silicon as a Platform for Radiation Theranostics Together with a Novel RIB-Based Radiolanthanoid.

Mesoporous silicon (PSi) is biocompatible and tailorable material with high potential in drug delivery applications. Here, we report of an evaluation of PSi as a carrier platform for theranostics by delivering a radioactive ion beam- (RIB-) based radioactive lanthanoid into tumors in a mouse model of prostate carcinoma. Thermally hydrocarbonized porous silicon (THCPSi) wafers were implanted with 159Dy at the facility for radioactive ion beams ISOLDE located at CERN, and the resulting [159Dy]THCPSi was postprocessed into particles. The particles were intratumorally injected into mice bearing prostate cancer xenografts. The stability of the particles was studied in vivo, followed by ex vivo biodistribution and autoradiographic studies. We showed that the process of producing radionuclide-implanted PSi particles is feasible and that the [159Dy]THCPSi particles stay stable and local inside the tumor over seven days. Upon release of 159Dy from the particles, the main site of accumulation is in the skeleton, which is in agreement with previous studies on the biodistribution of dysprosium. We conclude that THCPSi particles are a suitable platform together with RIB-based radiolanthanoids for theranostic purposes as they are retained after administration inside the tumor and the radiolanthanoid remains embedded in the THCPSi.

Drug-Loaded Multifunctional Nanoparticles Targeted to the Endocardial Layer of the Injured Heart Modulate Hypertrophic Signaling.

Ischemic heart disease is the leading cause of death globally. Severe myocardial ischemia results in a massive loss of myocytes and acute myocardial infarction, the endocardium being the most vulnerable region. At present, current therapeutic lines only ameliorate modestly the quality of life of these patients. Here, an engineered nanocarrier is reported for targeted drug delivery into the endocardial layer of the left ventricle for cardiac repair. Biodegradable porous silicon (PSi) nanoparticles are functionalized with atrial natriuretic peptide (ANP), which is known to be expressed predominantly in the endocardium of the failing heart. The ANP-PSi nanoparticles exhibit improved colloidal stability and enhanced cellular interactions with cardiomyocytes and non-myocytes with minimal toxicity. After confirmation of good retention of the radioisotope 111-Indium in relevant physiological buffers over 4 h, in vivo single-photon emission computed tomography (SPECT/CT) imaging and autoradiography demonstrate increased accumulation of ANP-PSi nanoparticles in the ischemic heart, particularly in the endocardial layer of the left ventricle. Moreover, ANP-PSi nanoparticles loaded with a novel cardioprotective small molecule attenuate hypertrophic signaling in the endocardium, demonstrating cardioprotective potential. These results provide unique insights into the development of nanotherapies targeted to the injured region of the myocardium.

Laser-ultrasonic delivery of agents into articular cartilage.

Research is ongoing to develop drug therapies to manage osteoarthritis (OA) and articular cartilage (AC) injuries. However, means to deliver drug to localized AC lesions are highly limited and not clinically available. This study investigates the capability of laser ultrasound (laser-induced plasma sound source) to deliver agents (methylene blue, MB, in PBS) into bovine AC. Treatment samples (n = 10) were immersed in MB solution simultaneously with LU exposure, while adjacent control 1 tissue (n = 10) was pre-treated with LU followed by immersion in MB and adjacent control 2 tissue (n = 10) was only immersed in MB. AC exposed (n = 22) or not exposed (n = 27) to LU were characterized for anomalies in structure, composition, viability or RNA expression. Optically detected MB content was significantly (p < 0.01) higher in treatment samples up to a depth of 500 µm from AC surface as compared to controls. No major unwanted short-term effects on AC structure, proteoglycan or collagen contents, chondrocyte viability or RNA expression levels were detected. In conclusion, LU can deliver agents into AC without major short-term concerns on safety. LU could reveal new strategies for the development of localized drug therapies in AC.

Microparticles to enhance delivery of drugs and growth factors into wound sites.

Microparticles with controlled size and morphology are of significant interest in the field of drug delivery. Although advanced nanoparticles have been the object of a substantial number of reviews, fewer have focused on microparticles, especially for the delivery of drugs and growth factors to the wound site. Microparticles show distinct advantages, including ease of production and characterization, extended release properties, high drug loading and little concern about the toxicity as compared with the nanosized systems. This review presents an introduction to the pathophysiology of wound healing and provides an overview of some of the recent advances in microparticle-based drugs and growth factors delivery to wound sites.

Lac dye as a potential anti-neoplastic agent.

OBJECTIVE: Certain anthraquinone compounds are effectively used for treatment of cancer. The present study describes the inhibitory activity of lac dye, a mixture of polyhydroxy anthraquinone compounds (Laccaic acid A, B, C, D and E), produced by the Indian lac insect, Kerria lacca (Kerr). MATERIALS AND METHODS: In vitro testing for anticancer activity of lac dye was done at four concentrations (10, 20, 40, 80 µg/ml) on ten human malignant cell lines including six human leukemia cell lines, U973, Raji, K562, Jurkat, MOLT 4 and HL60 using SRB assay. RESULTS AND CONCLUSION: Both crimson and yellow lac dye inhibits proliferation of Human leukemia cell lines, Raji, U937, K562, HL60 and Jurkat, therefore can be considered as a potential anticancer agent for leukemia.

In vitro and in vivo assessment of heart-homing porous silicon nanoparticles.

Chronic heart failure, predominantly developed after myocardial infarction, is a leading cause of high mortality worldwide. As existing therapies have still limited success, natural and/or synthetic nanomaterials are emerging alternatives for the therapy of heart diseases. Therefore, we aimed to functionalize undecylenic acid thermally hydrocarbonized porous silicon nanoparticles (NPs) with different targeting peptides to improve the NP's accumulation in different cardiac cells (primary cardiomyocytes, non-myocytes, and H9c2 cardiomyoblasts), additionally to investigate the behavior of the heart-targeted NPs in vivo. The toxicity profiles of the NPs evaluated in the three heart-type cells showed low toxicity at concentrations up to 50 µg/mL. Qualitative and quantitative cellular uptake revealed a significant increase in the accumulation of atrial natriuretic peptide (ANP)-modified NPs in primary cardiomyocytes, non-myocytes and H9c2 cells, and in hypoxic primary cardiomyocytes and non-myocytes. Competitive uptake studies in primary cardiomyocytes showed the internalization of ANP-modified NPs takes place via the guanylate cyclase-A receptor. When a myocardial infarction rat model was induced by isoprenaline and the peptide-modified [(111)In]NPs administered intravenously, the targeting peptides, particularly peptide 2, improved the NPs' accumulation in the heart up to 3.0-fold, at 10 min. This study highlights the potential of these peptide-modified nanosystems for future applications in heart diseases.

Variability of resistance in Black Bengal goats naturally infected with Haemonchus contortus.

A total 290 Black Bengal goats (6 buck, 109 doe and 175 kids born from 11 sires) were studied to evaluate the variability of resistance in Black Bengal goats naturally infected with Haemonchus contortus. The variability of resistance in Black Bengal goat was studied for both genetic and non-genetic factors like village, sex, age dam, sire, dam resistance group and offspring resistance group. Male kids have slightly higher resistance than female kids although it was not significant. Resistance of kids was increased as age increases and kid population showed significantly different resistance status among the offspring resistant groups. The doe population showed significantly different LEPG as per the resistance group in all the collections. The present study found that the resistance of kids under sire were varied significantly and observed that the kids under sire 1, 6-8 were significantly more resistant than the kids of the sire 2, 5 and 11 in 3rd collection and it is also noticed that maternal genetic effect has a very little impact on resistance of kids. Males (buck) were most resistant and the kids were least resistant and the resistance of dam was in between the male and kids population.

Novel endosomolytic peptides for enhancing gene delivery in nanoparticles.

Trapping in the endosomes is currently believed to represent the main barrier for transfection. Peptides, which allow endosomal escape have been demonstrated to overcome this barrier, similarly to the entry of viruses. However, the design principles of such endosomolytic peptides remain unclear. We characterized three analogs derived from membrane disrupting antimicrobial peptides (AMP), viz. LL-37, melittin, and bombolitin V, with glutamic acid substituting for all basic residues. These analogs are pH-sensitive and cause negligible membrane permeabilization and insignificant cytotoxicity at pH7.4. However, at pH5.0, prevailing in endosomes, membrane binding and hemolysis of human erythrocytes become evident. We first condensed the emerald green fluorescent protein (emGFP) containing plasmid by protamine, yielding 115 nm diameter soluble nanoplexes. For coating of the nanoplex surface with a lipid bilayer we introduced a hydrophobic tether, stearyl-octa-arginine (SR8). The indicated peptides were dissolved in methanol and combined with lipid mixtures in chloroform, followed by drying at RT under a nitrogen flow. The dry residues were hydrated with nanoplexes in Hepes, pH7.4 yielding after a 30 min incubation at RT,rather monodisperse nanoparticles having an average diameter of 150-300 nm, measured by DLS and cryo-TEM. Studies with cell cultures showed the above peptides to yield expression levels comparable to those obtained using Lipofectamine 2000. However, unlike the polydisperse aggregates formed upon mixing Lipofectamine 2000 and plasmid, the procedure described yields soluble, and reasonably monodisperse nanoparticles, which can be expected to be suitable for gene delivery in vivo, using intravenous injection.

Association and expression analysis of single nucleotide polymorphisms of partial tumor necrosis factor alpha gene with mastitis in crossbred cattle.

A total of 129 crossbred cows were selected to explore the genotypic and expression profiling of partial TNF-α gene and its association with mastitis susceptibility. Two exon spanning region of TNF-α gene (221 bp and 239 bp) were amplified by Polymerase Chain Reaction (PCR). The different genotypic analysis by SSCP revealed that 221 bp fragment was monomorphic, whereas 239 bp was polymorphic. Association studies revealed that AA genotypes of 239 bp were more prevalent in mastitis group and the mRNA expression of TNF-α was significantly (P < 0.05) higher in AA genotypic animals compare to AB and BB. This suggested that genotypes AB and BB may be used as candidate markers for mastitis resistance selection in dairy cattle.

Polarized THG microscopy identifies compositionally different lipid droplets in mammalian cells.

Cells store excess lipids as two major compounds, triacylglycerols (TAGs) and cholesteryl esters (CEs), inside lipid droplets (LDs). The degree of lipid ordering is considered to play a major role in the mobility and enzymatic processing of lipids in LDs. Here, we provide evidence that polarized third-harmonic generation (THG) microscopy distinguishes between native TAG- and CE-enriched LDs in cells due to the different ordering of the two lipid species. We first demonstrate that the responses from synthetic TAG- and CE-enriched LDs using THG microscopy with linear and circular polarizations differ according to their different intrinsic ordering. We then employ simulations to dissect how polarization effects influence the THG from an isotropic LD. Finally, we induce TAG- and CE-enriched LDs in murine macrophages and demonstrate that polarized THG responses increase in a nonlinear fashion with increasing CE/TAG ratio. This suggests that with an increasing CE content, there is a rather sharp transition toward increased LD ordering. Our results demonstrate that polarized THG microscopy enables label-free quantitative analysis of LD ordering and discriminates between compositionally different LDs in intact mammalian cells.

Evaluation of synovial inflammation in juvenile idiopathic arthritis by power color Doppler and Spectral Doppler ultrasonography.

OBJECTIVES: To compare Power Color Doppler and Spectral Doppler ultrasonography indices (Resistive index and color fraction) in cases of Juvenile Idiopathic Arthritis (JIA) with healthy controls and evaluate their correlation with clinical and laboratory parameters. METHODS: A cross sectional study was done over a period of 16 mo. Thirty patients of JIA and 30 age and sex matched healthy children were enrolled. Swelling and tenderness scores were evaluated and hemoglobin, total leukocyte count, erythrocyte sedimentation rate and C-reactive protein were done. A total of 112 diseased joints and 135 healthy joints were evaluated by USG by the same radiologist, and color fraction and RI were recorded. RESULTS: Statistically significant higher color fraction and lower RI (Resistive Index) were found in diseased joints as compared to healthy joints. Also the value of color fraction increased significantly with increasing grade of tenderness and RI decreased significantly with increasing grade of swelling. CONCLUSIONS: Power Color Doppler and Spectral Doppler ultrasonography (USG) indices (RI and color fraction) are significantly different in JIA patients. These might find a place in early diagnosis, monitoring of disease activity and response to therapy in JIA patients.

Peptide-mediated targeting of liposomes to TrkB receptor-expressing cells.

BACKGROUND: The neurotrophic receptor tyrosine kinase B (TrkB) has diverse signaling roles in neurons and tumor cells. Accordingly, its suppressive targeting is of interest in neuroblastoma and other tumors, whereas its role in improving survival is focused in neurons. Here we describe targeting of TrkB-binding peptide-conjugated liposomes (PCL) to the TrkB-expressing mouse macrophage-like cell line RAW264, and to all-trans-retinoic acid-treated neuron-like TrkB⁺ SH-SY5Y human neuroblastoma cells. METHODS: Binding and internalization of PCL was monitored by flow cytometry and confocal fluorescence microscopy. RESULTS: Internalization of TrkB-targeted PCL by RAW264 cells was enhanced and faster when compared with PCL having the corresponding scrambled peptide. Likewise, binding and augmented uptake were confirmed for TrkB⁺ SH-SY5Y cells, with targeted PCL appearing in the cytoplasm after 20 minutes of incubation. CONCLUSION: We demonstrate here the feasibility of targeting liposomes to TrkB-expressing cells by 18-mer peptides, promoting cellular uptake (at least partly into endosomes) via receptor-mediated pathways.

Size-dependent passage of liposome nanocarriers with preserved posttransport integrity across the middle-inner ear barriers in rats.

OBJECTIVE: The goal of this study was to evaluate the impact of liposome nanocarrier size on the efficacy of its transport across the middle-inner ear barriers. MATERIALS AND METHODS: The dynamic distribution of liposome nanocarriers encapsulating gadolinium-tetra-azacyclo-dodecane-tetra-acetic acid (LPS+Gd-DOTA) of sizes 95, 130, and 240 nm were observed with a 4.7 T magnetic resonance machine after transtympanic injection in Wistar rats. Histology was performed with confocal microscopy using TRITC conjugated LPS+Gd-DOTA. The integrity of the LPS+Gd-DOTA after transportation was evaluated using cryo-transmission electron microscopy (Cryo-TEM). RESULTS: Size-dependent transport of the LPS+Gd-DOTA across the middle-inner ear barriers was shown using magnetic resonance imaging, which indicated that the 95-nm nanocarrier showed the significantly highest transport percentage, that the 130-nm nanocarrier showed moderate transport, and that the 240 nm nanocarrier showed the lowest transport. Histologic examinations showed that the LPS+Gd-DOTA were distributed in the epithelial cells of the utricle, capillaries of the spiral ligament, and the spiral ganglion cells. LPS+Gd-DOTA remained intact in the perilymph after transportation. CONCLUSION: The nanocarrier delivery strategy used in this work could be effective in the development of novel inner ear treatments.