Formulation optimization of lyophilized aptamer-gold nanoparticles: Maintained colloidal stability and cellular uptake.

Anti-nucleolin (NCL) aptamer AS1411 is the first anticancer aptamer tested in clinical trials. Gold nanoparticles (AuNP) have been widely exploited for various biomedical applications due to their unique functional properties. In this study, we evaluated the colloidal stability and targeting capacity of AS1411-funtionalized AuNP (AuNP/NCL-Apt) against MCF-7 breast cancer cell line before and after lyophilization. Trehalose, mannitol, and sucrose at various concentrations were evaluated to determine their cryoprotection effects. Our results indicate that sucrose at 10 % (w/v) exhibits the best cryoprotection effect and minimal AuNP/NCL-Apt aggregation as confirmed by UV-Vis spectroscopy and dynamic light scattering (DLS) measurements. Moreover, the lyophilized AuNP/NCL-Apt at optimized formulation maintained its targeting and cytotoxic functionality against MCF-7 cells as proven by the cellular uptake assays utilizing flow cytometry and confocal laser scanning microscopy (CLSM). Quantitative PCR (qPCR) analysis of nucleolin-target gene expression also confirmed the effectiveness of AuNP/NCL-Apt. This study highlights the importance of selecting the proper type and concentration of cryoprotectant in the typical nanoparticle lyophilization process and contributes to our understanding of the physical and biological properties of functionalized nanoparticles upon lyophilization.

Doxorubicin conjugates: a practical approach for its cardiotoxicity alleviation.

INTRODUCTION: Doxorubicin (DOX) emerges as a cornerstone in the arsenal of potent chemotherapeutic agents. Yet, the clinical deployment of DOX is tarnished by its proclivity to induce severe cardiotoxic effects, culminating in heart failure and other consequential morbidities. In response, a panoply of strategies has undergone rigorous exploration over recent decades, all aimed at attenuating DOX's cardiotoxic impact. The advent of encapsulating DOX within lipidic or polymeric nanocarriers has yielded a dual triumph, augmenting DOX's therapeutic efficacy while mitigating its deleterious side effects. AREAS COVERED: Recent strides have spotlighted the emergence of DOX conjugates as particularly auspicious avenues for ameliorating DOX-induced cardiotoxicity. These conjugates entail the fusion of DOX through physical or chemical bonds with diminutive natural or synthetic moieties, polymers, biomolecules, and nanoparticles. This spectrum encompasses interventions that impinge upon DOX's cardiotoxic mechanism, modulate cellular uptake and localization, confer antioxidative properties, or refine cellular targeting. EXPERT OPINION: The endorsement of DOX conjugates as a compelling stratagem to mitigate DOX-induced cardiotoxicity resounds from this exegesis, amplifying safety margins and the therapeutic profile of this venerated chemotherapeutic agent. Within this ambit, DOX conjugates stand as a beacon of promise in the perpetual pursuit of refining chemotherapy-induced cardiac compromise.

Unlocking the Therapeutic Potential of BCL-2 Associated Protein Family: Exploring BCL-2 Inhibitors in Cancer Therapy.

Apoptosis, programmed cell death pathway, is a vital physiological mechanism that ensures cellular homeostasis and overall cellular well-being. In the context of cancer, where evasion of apoptosis is a hallmark, the overexpression of anti-apoptotic proteins like Bcl2, Bcl-xL and Mcl-1 has been documented. Consequently, these proteins have emerged as promising targets for therapeutic interventions. The BCL-2 protein family is central to apoptosis and plays a significant importance in determining cellular fate serving as a critical determinant in this biological process. This review offers a comprehensive exploration of the BCL-2 protein family, emphasizing its dual nature. Specifically, certain members of this family promote cell survival (known as anti-apoptotic proteins), while others are involved in facilitating cell death (referred to as pro-apoptotic and BH3-only proteins). The potential of directly targeting these proteins is examined, particularly due to their involvement in conferring resistance to traditional cancer therapies. The effectiveness of such targeting strategies is also discussed, considering the tumor's propensity for anti-apoptotic pathways. Furthermore, the review highlights emerging research on combination therapies, where BCL-2 inhibitors are used synergistically with other treatments to enhance therapeutic outcomes. By understanding and manipulating the BCL-2 family and its associated pathways, we open doors to innovative and more effective cancer treatments, offering hope for resistant and aggressive cases.

Pharmaco-toxicological effects of the novel tryptamine hallucinogen 5-MeO-MiPT on motor, sensorimotor, physiological, and cardiorespiratory parameters in mice-from a human poisoning case to the preclinical evidence.

RATIONALE: The 5-methoxy-N-methyl-N-isopropyltryptamine (5-MeO-MiPT, known online as "Moxy") is a new psychedelic tryptamine first identified on Italian national territory in 2014. Its hallucinogen effects are broadly well-known; however, only few information is available regarding its pharmaco-toxicological effects. OBJECTIVES: Following the seizure of this new psychoactive substances by the Arm of Carabinieri and the occurrence of a human intoxication case, in the current study we had the aim to characterize the in vivo acute effects of systemic administration of 5-MeO-MiPT (0.01-30 mg/kg i.p.) on sensorimotor (visual, acoustic, and overall tactile) responses, thermoregulation, and stimulated motor activity (drag and accelerod test) in CD-1 male mice. We also evaluated variation on sensory gating (PPI, prepulse inhibition; 0.01-10 mg/kg i.p.) and on cardiorespiratory parameters (MouseOx and BP-2000; 30 mg/kg i.p.). Lastly, we investigated the in silico ADMET (absorption, distribution, metabolism, excretion, toxicity) profile of 5-MeO-MiPT compared to 5-methoxy-N,N-diisopropyltryptamine (5-MeO-DIPT) and N,N-dimethyltryptamine (DMT). RESULTS: This study demonstrates that 5-MeO-MiPT dose-dependently inhibits sensorimotor and PPI responses and, at high doses, induces impairment of the stimulated motor activity and cardiorespiratory changes in mice. In silico prediction shows that the 5-MeO-MiPT toxicokinetic profile shares similarities with 5-MeO-DIPT and DMT and highlights a cytochrome risk associated with this compound. CONCLUSIONS: Consumption of 5-MeO-MiPT can affect the ability to perform activities and pose a risk to human health status, as the correspondence between the effects induced in mice and the symptoms occurred in the intoxication case suggests. However, our findings suggest that 5-MeO-MiPT should not be excluded from research in the psychiatric therapy field.

Dual-loaded liposomal carriers to combat chemotherapeutic resistance in breast cancer.

INTRODUCTION: The resistance to chemotherapy is a significant hurdle in breast cancer treatment, prompting the exploration of innovative strategies. This review discusses the potential of dual-loaded liposomal carriers to combat chemoresistance and improve outcomes for breast cancer patients. AREAS COVERED: This review discusses breast cancer chemotherapy resistance and dual-loaded liposomal carriers. Drug efflux pumps, DNA repair pathways, and signaling alterations are discussed as chemoresistance mechanisms. Liposomes can encapsulate several medicines and cargo kinds, according to the review. It examines how these carriers improve medication delivery, cancer cell targeting, and tumor microenvironment regulation. Also examined are dual-loaded liposomal carrier improvement challenges and techniques. EXPERT OPINION: The use of dual-loaded liposomal carriers represents a promising and innovative strategy in the battle against chemotherapy resistance in breast cancer. This article has explored the various mechanisms of chemoresistance in breast cancer, emphasizing the potential of dual-loaded liposomal carriers to overcome these challenges. These carriers offer versatility, enabling the encapsulation and precise targeting of multiple drugs with different modes of action, a crucial advantage when dealing with the complexity of breast cancer treatment.

Can we use normal saline stored under stress conditions? A simulated prehospital emergency medical setting.

Background: Data on stability and suitability to use normal saline stored under stress conditions in ambulances is lacking. Objective: We aimed to study the impact of exposure to extreme temperature variations on normal saline stability and compatibility with its packaging. Methods: Normal saline in 96 polyolefin bags were exposed to continuous temperature of 22, 50, and 70 °C or to a cyclic temperature of 70 °C per 8 h and 22 °C per 16 h. The bags were sampled at 12, 24, 48 and 72 h and at 1, 2, 3, and 4 weeks in the short- and long-term experiments, respectively. Solution inside the bags was evaluated for any evidence of crystallization, discoloration, turbidity, or pH changes. A sample of normal saline was withdrawn from each bag to analyze sodium and chloride levels. Results: Precipitation, discoloration, or turbidity were not observed in the solution inside normal saline bags. The average pH was 5.59 at 22 °C, 5.73 at 50 °C, 5.86 at 70 °C and 5.79 at cyclic exposure. In the short- and long-term experiments, sodium and chloride concentrations were within 100.2-111.27% and 99.04-110.95%, respectively. Leaching of the plastic components in the polyolefin bag into the normal saline solution was not detected. Conclusions: Sodium and chloride levels of normal saline were stable and compatible with polyolefin bags stored in simulated continuous and cyclic extreme temperatures for around one month. The effect of storage in the cabinet of operational ambulance vehicles during different seasons in arid countries is yet to be evaluated in real-world conditions, to further confirm our results.

Comprehensive evaluation of the pharmacological and toxicological effects of γ-valerolactone as compared to γ-hydroxybutyric acid: Insights from in vivo and in silico models.

Γ-valerolactone (GVL), marketed online as "Tranquilli-G" and "excellent Valium", is used as a legal substitute for γ-hydroxybutyric acid (GHB); however, until now, GVL has only been connected to one Drug-Facilitated Sexual Assault (DFSA) case. Moreover, the pharmaco-toxicological effects of GVL are poorly studied. The aim of this study was to investigate the 1) in vivo effects of gavage administration of GVL (100-3000 mg/kg) on neurological (myoclonia, convulsions), sensorimotor (visual, acoustic, and overall tactile) responses, righting reflex, thermoregulation, motor activity (bar, drag, and accelerod test) and cardiorespiratory changes (heart rate, breath rate, oxygen saturation, and pulse distension) in CD-1 male mice and the 2) in silico ADMET profile of GVL in comparison to GHB and the open active form γ-hydroxyvaleric acid (GHV). The present study demonstrates that GVL inhibits, in a dose-dependent manner, sensorimotor and motor responses and induces cardiorespiratory depression (at a dose of 3000 mg/kg) in mice. The determination of the ED50 in sensorimotor and motor responses revealed that GVL is about 4-5 times less potent than GHB. In silico prediction of ADMET profiles revealed toxicokinetic similarities between GHB and GHV, and differences with GVL. These results suggest that GVL could be used as a substitute for GHB and should be added to forensic toxicology screenings.

PLGA-Gold Nanocomposite: Preparation and Biomedical Applications.

A composite system consisting of both organic and inorganic nanoparticles is an approach to prepare a new material exhibiting "the best of both worlds". In this review, we highlight the recent advances in the preparation and applications of poly(lactic-co-glycolic acid)-gold nanoparticles (PLGA-GNP). With its current clinically use, PLGA-based nanocarriers have promising pharmaceutical applications and can "extract and utilize" the fascinating optical and photothermal properties of encapsulated GNP. The resulting "golden polymeric nanocarrier" can be tracked, analyzed, and visualized using the encapsulated gold nanoprobes which facilitate a better understanding of the hosting nanocarrier's pharmacokinetics and biological fate. In addition, the "golden polymeric nanocarrier" can reveal superior nanotherapeutics that combine both the photothermal effect of the encapsulated gold nanoparticles and co-loaded chemotherapeutics. To help stimulate more research on the development of nanomaterials with hybrid and exceptional properties, functionalities, and applications, this review provides recent examples with a focus on the available chemistries and the rationale behind encapsulating GNP into PLGA nanocarriers that has the potential to be translated into innovative, clinically applicable nanomedicine.

Quality by Design Approach in Liposomal Formulations: Robust Product Development.

Nanomedicine is an emerging field with continuous growth and differentiation. Liposomal formulations are a major platform in nanomedicine, with more than fifteen FDA-approved liposomal products in the market. However, as is the case for other types of nanoparticle-based delivery systems, liposomal formulations and manufacturing is intrinsically complex and associated with a set of dependent and independent variables, rendering experiential optimization a tedious process in general. Quality by design (QbD) is a powerful approach that can be applied in such complex systems to facilitate product development and ensure reproducible manufacturing processes, which are an essential pre-requisite for efficient and safe therapeutics. Input variables (related to materials, processes and experiment design) and the quality attributes for the final liposomal product should follow a systematic and planned experimental design to identify critical variables and optimal formulations/processes, where these elements are subjected to risk assessment. This review discusses the current practices that employ QbD in developing liposomal-based nano-pharmaceuticals.

Evaluation of the Benefits of Microfluidic-Assisted Preparation of Polymeric Nanoparticles for DNA Delivery.

An effective delivery vehicle of genetic materials to their target site is the key to a successful gene therapy. In many cases, nanoparticles are used as the vehicle of choice and the efficiency of the delivery relies heavily on the physicochemical properties of the nanoparticles. Microfluidics, although being a low throughput method, has been increasingly researched for the preparation of nanoparticles. A range of superior properties were claimed in the literature for microfluidic-prepared platforms, but no evidence on direct comparison of the properties of the nanoparticles prepared by microfluidics and conventional high throughput method exists, leaving the industry with little guidance on how to select effective large-scale nanoparticle manufacturing method. This study used plasmid DNA-loaded PLGA-Eudragit nanoparticles as the model system to critically compare the nanoparticles prepared by conventional and microfluidics-assisted nanoprecipitation. The PLGA-Eudragit nanoparticles prepared by microfluidics were found to be statistically significantly larger than the ones prepared by conventional nanoprecipitation. PLGA-Eudragit nanoparticle prepared conventionally showed higher DNA loading efficiency. Although the DNA-loaded nanoparticles prepared by both methods did not induce significant cytotoxicity, the transfection efficiency was found to be higher for the ones prepared conventionally which has good potential for plasmid delivery. This study for the first time provides a direct comparison of the DNA-loaded nanoparticles prepared by microfluidic and conventional methods. The findings bring new insights into critical evaluation of the selection of manufacturing methods of nanoparticles for future gene therapy.

Quercetin-gold nanorods incorporated into nanofibers: development, optimization and cytotoxicity.

Herein, a polymeric nanofiber scaffold loaded with Quercetin (Quer)-gold nanorods (GNR) was developed and characterized. Several parameters related to loading Quer into GNR, incorporating the GNR-Quer into polymeric solutions, and fabricating the nanofibers by electrospinning were optimized. GNR-Quer loaded into a polymeric mixture of poly(lactic-co-glycolic acid) (PLGA) (21%) and poloxamer 407 (23%) has produced intact GNR-Quer-nanofibers with enhanced physical and mechanical properties. GNR-Quer-nanofibers demonstrated a slow pattern of Quer release over time compared to nanofibers free of GNR-Quer. Dynamic mechanical thermal analysis (DMTA) revealed enhanced uniformity and homogeneity of the GNR-Quer-nanofibers. GNR-Quer-nanofibers demonstrated a high ability to retain water upon incubation in phosphate buffer saline (PBS) for 24 h compared to nanofibers free of GNR-Quer. A cellular toxicity study indicated that the average cellular viability of human dermal fibroblasts was 76% after 24 h of exposure to the nanofibers containing a low concentration of GNR-Quer.

Microfluidics for pharmaceutical nanoparticle fabrication: The truth and the myth.

Using micro-sized channels to manipulate fluids is the essence of microfluidics which has wide applications from analytical chemistry to material science and cell biology research. Recently, using microfluidic-based devices for pharmaceutical research, in particular for the fabrication of micro- and nano-particles, has emerged as a new area of interest. The particles that can be prepared by microfluidic devices can range from micron size droplet-based emulsions to nano-sized drug loaded polymeric particles. Microfluidic technology poses unique advantages in terms of the high precision of the mixing regimes and control of fluids involved in formulation preparation. As a result of this, monodispersity of the particles prepared by microfluidics is often recognised as being a particularly advantageous feature in comparison to those prepared by conventional large-scale mixing methods. However, there is a range of practical drawbacks and challenges of using microfluidics as a direct micron- and nano-particle manufacturing method. Technological advances are still required before this type of processing can be translated for application by the pharmaceutical industry. This review focuses specifically on the application of microfluidics for pharmaceutical solid nanoparticle preparation and discusses the theoretical foundation of using the nanoprecipitation principle to generate particles and how this is translated into microfluidic design and operation.

Identification of substandard drug products using electronic tongue: cefdinir suspension as a pilot example.

BACKGROUND: Electronic tongue (ET) is a well-established technology that is used to detect the taste of a food or a medicinal product and to differentiate between different products based on their tastes. In addition, it can be used to monitor environmental parameters and biochemical and biological processes. PURPOSE: This study aims to assess any correlation between the results of pharmacopeial quality control (ie, assay, impurities, and dissolution, etc) and ET analysis for reconstituted cefdinir (CR) suspension over 10 days (ie, shelf-life). METHODS: The reconstituted CR suspension was tested for several quality attributes such as dissolution behavior, pH, assay, related substances, and microbial contamination. An HPLC analytical method was verified and then used for chemical analysis. The taste of CR reconstituted suspension was followed over 10 days and was then compared with the quality control results. Moreover, Pearson's correlation test was used to find a correlation between chemical analysis results and ET results. RESULTS: Pearson's test of correlation showed a significant correlation (p-value <0.05) between the conventional chemical analysis results (% of CR, % of preservative, % of released CR, % of total impurities and % of total undefined impurities in the reconstituted suspension) with the change of their taste (ie, % pattern discrimination index). ET was able to correlate the results of stability of CR suspension with the change in the taste of the suspension during the shelf life of the reconstituted suspension. CONCLUSION: The obtained results may suggest the use of ET as a new tool for a rapid assessment of the general quality of a suspension. Moreover, such results would suggest the use of ET to identify fake or substandard products, especially those have been stored under inappropriate storage conditions.

Assembly and Degradation of Inorganic Nanoparticles in Biological Environments.

In solution, nanoparticles may be conceptually compartmentalized into cores and engineered surface coatings. Recent advances allow for simple and accurate characterization of nanoparticle cores and surface shells. After introduction into a complex biological environment, adsorption of biological molecules to the nanoparticle surface as well as a loss of original surface components occur. Thus, colloidal nanoparticles in the context of the biological environment are hybrid materials with complex structure, which may result in different chemical, physical, and biological outcomes as compared to the original engineered nanoparticles. In this review, we will discuss building up an engineered inorganic nanoparticle from its inside core to its outside surface and following its degradation in a biological environment from its outside to its inside. This will involve the way to synthesize selected inorganic nanoparticles. Then, we will discuss the environmental changes upon exposure of these nanoparticles to biological media and their uptake by cells. Next, the intracellular fate of nanoparticles and their degradation will be discussed. Based on these examples, the need to see nanoparticles in the context of the biological environment as dynamic hybrid materials will be highlighted.

Synergistic antibacterial activity of silver nanoparticles and hydrogen peroxide.

The increasing challenge of antibiotic resistance requires not only the discovery of new antibiotics, but also the development of new alternative approaches. Herein, the synergistic antibacterial activity of silver nanoparticles and hydrogen peroxide combination is reported. Unlike the bacteriostatic or slightly bactericidal activity achieved by using each agent alone, using these two agents in combination, even at relatively low concentrations, resulted in complete eradication of both the Gram negative Escherichia coli and the Gram positive Staphylococcus aureus in short treatment times indicating a clear synergistic effect between them. Modifying the surface chemistry of silver nanoparticles and the accompanied change in their surface charge enabled a further enhancement of such synergistic effect implying the importance of this aspect. Mechanistically, a Fenton-like reaction between silver nanoparticles and hydrogen peroxide is discussed and hypothesized to be the basis of the observed synergy. Achieving such a significant antibacterial activity at low concentrations reduces the potential toxicity of these agents and hence enables their utilization as an alternative antibacterial approach in wider range of applications.

Facile Hydrophobication of Glutathione-Protected Gold Nanoclusters and Encapsulation into Poly(lactide-co-glycolide) Nanocarriers.

We report a simple surface functionalization of glutathione-capped gold nanoclusters by hydrophobic ion pairing with alkylamine followed by a complete phase transfer to various organic solvents with maintained colloidal stability and photoluminescence properties. The described surface hydrophobication enables efficient encapsulation of gold nanoclusters into PLGA nanocarriers allowing their visualization inside cultured cells using confocal fluorescent microscopy. The simplicity and efficiency of the described protocols should extend the biomedical applications of these metallic nanoclusters as a fluorescent platform to label hydrophobic polymeric nanocarriers beyond conventional organic dyes.

Ligand density on nanoparticles: A parameter with critical impact on nanomedicine.

Nanoparticles modified with ligands for specific targeting towards receptors expressed on the surface of target cells are discussed in literature towards improved delivery strategies. In such concepts the ligand density on the surface of the nanoparticles plays an important role. How many ligands per nanoparticle are best for the most efficient delivery? Importantly, this number may be different for in vitro and in vivo scenarios. In this review first viruses as "biological" nanoparticles are analyzed towards their ligand density, which is then compared to the ligand density of engineered nanoparticles. Then, experiments are reviewed in which in vitro and in vivo nanoparticle delivery has been analyzed in terms of ligand density. These results help to understand which ligand densities should be attempted for better targeting. Finally synthetic methods for controlling the ligand density of nanoparticles are described.

Tunable sustained release drug delivery system based on mononuclear aqueous core-polymer shell microcapsules.

Poly(d,l-lactide-co-glycolide) (PLGA) and poly(d,l-lactide) (PLA) polymers were used successfully in the preparation of polymer shell microcapsules with mononuclear aqueous cores by the internal phase separation method. These microcapsules were prepared with varying amounts of polymer and water and loaded with fluorescein sodium as a model water soluble drug. Evaluation of drug loading and encapsulation efficiency reveals an optimum polymer to water ratio of around 1:3. Prepared PLGA and PLA microcapsules exhibit sustained drug release over 7 and 49 days, respectively. Drug release from both microcapsule types follow zero order kinetics over the first 90% release. Further tuning of release rate is found possible by preparing microcapsules with mixtures of PLGA and PLA polymers at varying ratios. These results suggest that aqueous core-PLGA and PLA microcapsules would be promising platforms for a wide range of sustained drug delivery systems for many hydrophilic drugs.

Controlling the internal morphology of aqueous core-PLGA shell microcapsules: promoting the internal phase separation via alcohol addition.

The ability to control the internal core architecture of polymeric microcapsules has a direct impact on their applications. However, this task, especially to produce microcapsules with a high percentage of mononuclear aqueous cores, proved to be challenging. In this work, and in continuation to our previous studies, we report a facile protocol to prepare poly(D,L-lactide-co-glycolide) (PLGA) microcapsules with unprecedented percentage (almost 100%) of mononuclear aqueous cores by the internal phase separation method via adding alcohols. Different types of alcohols (methanol, ethanol, propanol, isopropanol, butanol, and octanol) were incorporated into the internal phase solution and then emulsified into mineral oil. In situ monitoring of emulsion droplets was performed by phase contrast microscopy at different time points and the percentage of mononuclear droplets was measured. While alcohol-free formulation ended up with only around 51% of mononuclear microcapsules, incorporating alcohols resulted in the formation of more than 90% of mononuclear microcapsules. Octanol, in particular, exhibited an outstanding performance as its incorporation led to an immediate (at 0 h) formation of almost entirely mononuclear microcapsules. Final microcapsules exhibited spherical shape with mean particle size in the range of 1-2 µm as depicted by scanning electron microscopy and dynamic light scattering analysis.