Extracellular vesicles (EVs) are highly specialized nanoscale assemblies that deliver complex biological cargos to mediate intercellular communication. EVs are heterogeneous, and characterization of ...this heterogeneity is paramount to understanding EV biogenesis and activity, as well as to associating them with biological responses and pathologies. Traditional approaches to studying EV composition generally lack the resolution and/or sensitivity to characterize individual EVs, and therefore the assessment of EV heterogeneity has remained challenging. We have recently developed an atomic force microscope IR spectroscopy (AFM-IR) approach to probe the structural composition of single EVs with nanoscale resolution. Here, we provide a step-by-step procedure for our approach and show its power to reveal heterogeneity across individual EVs, within the same population of EVs and between different EV populations. Our approach is label free and able to detect lipids, proteins and nucleic acids within individual EVs. After isolation of EVs from cell culture medium, the protocol involves incubation of the EV sample on a suitable substrate, setup of the AFM-IR instrument and collection of nano-IR spectra and nano-IR images. Data acquisition and analyses can be completed within 24 h, and require only a basic knowledge of spectroscopy and chemistry. We anticipate that new understanding of EV composition and structure through AFM-IR will contribute to our biological understanding of EV biology and could find application in disease diagnosis and the development of EV therapies.
Nanomedicine seeks to apply nanoscale materials for the therapy and diagnosis of diseased and damaged tissues. Recent advances in nanotechnology have made a major contribution to the development of ...multifunctional nanomaterials, which represents a paradigm shift from single purpose to multipurpose materials. Multifunctional nanomaterials have been proposed to enable simultaneous target imaging and on-demand delivery of therapeutic agents only to the specific site. Most advanced systems are also responsive to internal or external stimuli. This approach is particularly important for highly potent drugs (
e.g.
chemotherapeutics), which should be delivered in a discreet manner and interact with cells/tissues only locally. Both advances in imaging and precisely controlled and localized delivery are critically important in cancer treatment, and the use of such systems - theranostics - holds great promise to minimise side effects and boost therapeutic effectiveness of the treatment. Among others, mesoporous silica nanoparticles (MSNPs) are considered one of the most promising nanomaterials for drug delivery. Due to their unique intrinsic features, including tunable porosity and size, large surface area, structural diversity, easily modifiable chemistry and suitability for functionalization, and biocompatibility, MSNPs have been extensively utilized as multifunctional nanocarrier systems. The combination or hybridization with biomolecules, drugs, and other nanoparticles potentiated the ability of MSNPs towards multifunctionality, and even smart actions stimulated by specified signals, including pH, optical signal, redox reaction, electricity and magnetism. This paper provides a comprehensive review of the state-of-the-art of multifunctional, smart drug delivery systems centered on advanced MSNPs, with special emphasis on cancer related applications.
Effectiveness of the delivery of anticancer drugs and the efficacy of cancer therapy can be enhanced using smart multifunctional mesoporous nanoparticles.
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Raman spectroscopy was used to evaluate the effects of temperature and humidity on the physicochemical stability of inhalable spray-dried (SD) powders containing budesonide (BUD) and ...crystalline or amorphous lactose. Powders prepared by spray drying of BUD-lactose solution or suspension containing lactose crystals in BUD solution were stored for 0, 1 and 7 days at 25 °C/60 RH or 40 °C/75 RH. Bulk powders along with the large and small particle size fractions collected on stages 2 and 5, respectively, of the Next Generation Impactor (NGI) were chemically characterised. SD powder from solution contained BUD and lactose in amorphous form and both components were homogeneously distributed in bulk and in the particles collected from the two NGI stages. In contrast, SD powder from suspension showed heterogeneous distribution of lactose and drug in the particles containing crystalline lactose. After 1 day of storage at either condition, recrystallisation and changes in the chemical composition of the particles for the SD powder from solution occurred. The number of drug-only particles increased by 70 on stage 5, whereas most particles on stage 2 still contained both drug and lactose. These changes were not observed in the SD powder from suspension after storage, confirming superior stability of the SD powder obtained from suspension.
The toxicity of nanomaterials raises major concerns because of the impact that nanomaterials may have on health, which remains poorly understood. We need to explore the fate of individual ...nanoparticles in cells at nano and molecular levels to establish their safety. Conformational changes in secondary protein structures are one of the main indicators of impaired biological function, and hence, the ability to identify these changes at a nanoscale level offers unique insights into the nanotoxicity of materials. Here, we used nanoscale infrared spectroscopy and demonstrated for the first time that nanodiamond-induced alterations in both extra- and intracellular secondary protein structures lead to the formation of antiparallel β-sheet, β-turns, intermolecular β-sheet, and aggregation of proteins. These conformational changes of the protein structure may result in the loss of functionality of proteins and in turn lead to adverse effects.
The interaction between gut microbiota and host plays a central role in health. Dysbiosis, detrimental changes in gut microbiota and inflammation have been reported in non-communicable diseases. ...While diet has a profound impact on gut microbiota composition and function, the role of food additives such as titanium dioxide (TiO
), prevalent in processed food, is less established. In this project, we investigated the impact of food grade TiO
on gut microbiota of mice when orally administered via drinking water. While TiO
had minimal impact on the composition of the microbiota in the small intestine and colon, we found that TiO
treatment could alter the release of bacterial metabolites
and affect the spatial distribution of commensal bacteria
by promoting biofilm formation. We also found reduced expression of the colonic mucin 2 gene, a key component of the intestinal mucus layer, and increased expression of the beta defensin gene, indicating that TiO
significantly impacts gut homeostasis. These changes were associated with colonic inflammation, as shown by decreased crypt length, infiltration of CD8
T cells, increased macrophages as well as increased expression of inflammatory cytokines. These findings collectively show that TiO
is not inert, but rather impairs gut homeostasis which may in turn prime the host for disease development.
Recent heightened interest in inhaled bacteriophage (phage) therapy for combating antibacterial resistance in pulmonary infections has led to the development of phage powder formulations. Although ...phages have been successfully bioengineered into inhalable powders with preserved bioactivity, the stabilization mechanism is yet unknown. This paper reports the first study investigating the stabilization mechanism for phages in these powders. Proteins and other biologics are known to be preserved in dry state within a glassy sugar matrix at storage temperatures (Ts) at least ~50°C below the glass transition temperature (Tg). This is because at (Tg − Ts) >50°C, molecules are sufficiently immobilized with reduced reactivity. We hypothesized that this glass stabilization mechanism may also be applicable to phages comprising mostly of proteins. In this study, spray dried powders of Pseudomonas phage PEV20 containing lactose and leucine as excipients were stored at 5, 25 or 50°C and 15 or 33% relative humidity (RH), followed by assessment of bioactivity (PEV20 stability) and physical properties. PEV20 was stable with negligible titer loss after storage at 5°C/15% RH for 250 days, while storage at 33% RH caused increased titer losses of 1 log10 and 3 log10 at 5 and 25°C, respectively. The plasticizing effect of water at 33% RH lowered the Tg by 30°C, thus narrowing the gap between Ts and Tg to 19–28°C, which was insufficient for glass stabilization. In contrast, the (Tg − Ts) values were higher (range, 46–65°C) under the drier condition of 15% RH, resulting in the improved stability which corroborated with the vitrification hypothesis. Furthermore, phage remained stable (≤1 log10) when the (Tg − Ts) value lay between 26–48°C, but became inactivated as the value fell below 20°C. In conclusion, this study demonstrated that phage can be sufficiently stabilized in spray dried powders by keeping the (Tg − Ts) value above 46°C, thus supporting the vitrification hypothesis that phages are stabilized by immobilization inside a rigid glassy sugar matrix. These findings provide a guide to better manufacture and storage practices of inhaled phage powder products using for translational medicines.
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Therapeutic biologics such as genes, peptides, proteins, virus and cells provide clinical benefits and are becoming increasingly important tools in respiratory medicine. Pulmonary ...delivery of therapeutic biologics enables the potential for safe and effective treatment option for respiratory diseases due to high bioavailability while minimizing absorption into the systemic circulation, reducing off-target toxicity to other organs. Development of inhalable powder formulation requires stabilization of complex biological materials, and each type of biologics may present unique challenges and require different formulation strategy combined with manufacture process to ensure biological and physical stabilities during production and over shelf-life. This review examines key formulation strategies for stabilizing proteins, nucleic acids, virus (bacteriophages) and bacterial cells in inhalable powders. It also covers characterization methods used to assess physicochemical properties and aerosol performance of the powders, biological activity and structural integrity of the biologics, and chemical analysis at the nanoscale. Furthermore, the review includes manufacture technologies which are based on lyophilization and spray-drying as they have been applied to manufacture Food and Drug Administration (FDA)-approved protein powders. In perspective, formulation and manufacture of inhalable powders for biologic are highly challenging but attainable. The key requirements are the stability of both the biologics and the powder, along with the powder dispersibility. The formulation to be developed depends on the manufacture process as it will subject the biologics to different stresses (temperature, mechanical and chemical) which could lead to degradation by different pathways. Stabilizing excipients coupled with the suitable choice of process can alleviate the stability issues of inhaled powders of biologics.
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The development of effective drug delivery systems requires in-depth characterization of the micro- or nanostructure of the material vectors with high spatial resolution, resulting in ...a deep understanding of the design-function relationship and maximum therapeutic efficacy. Atomic force microscopy-infrared spectroscopy (AFM-IR) combines the high spatial resolution of AFM and the capabilities of IR spectroscopy to identify chemical composition and it has emerged as a powerful tool for the detailed characterization of a drug delivery system at the nanoscale. In addition, the instruments also allow thermal and mechanical evaluation at the nanoscale. In this review, we highlight the applications of AFM-IR in various drug delivery systems, including polymer-based carriers, lipid-contained nanocarriers, and metal-based nanocarriers. The existing challenges as well as the future perspectives for the application of AFM-IR for drug delivery vector characterization are also discussed.
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Intestinal Pseudomonas aeruginosa is highly problematic in immunocompromised patients such as those in intensive care units in hospitals. Phage therapy is an attractive alternative or ...supplementary therapy to antibiotics as it not only kills multidrug-resistant bacteria, but also minimises the disruption of gut microflora. Solid oral dosage forms (i.e., tablets) have the potential to effectively deliver viable phages to the gastrointestinal tract, but formulation studies have been scarce. In this study, Pseudomonas-targeting phage PEV20 was used as a model to produce tablets suitable for oral delivery by utilising industry-scale tablet compression and tablet coating machines. Phage tablets were produced by (i) spray drying of phages, (ii) direct compression of the phage powders into tablets, and then (iii) tablet coating. The resulting phage tablets had negligible phage titre reduction throughout the process and passed the British Pharmacopeia tests, including friability, weight variation, disintegration and dissolution of the tablets as well as weight gain and disintegration (in 0.1 M HCl and pH 7.4 phosphate buffer) of coated tablets. The developed formulation method can be utilised to produce tablets containing other phages and phage cocktails that are effective against enteric bacterial infections.
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