Effective cerebral tuberculosis treatment via nose-to-brain transport of anti-TB drugs using mucoadhesive nano-aggregates.

Central nervous system tuberculosis (CNS-TB) is a severe form of extra-pulmonary tuberculosis with high mortality and morbidity rates. The standard treatment regimen for CNS-TB parallels that of pulmonary TB, despite the challenge posed by the blood-brain barrier (BBB), which limits the efficacy of first-line anti-TB drugs (ATDs). Nose-to-brain (N2B) drug delivery offers a promising solution for achieving high ATD concentrations directly at infection sites in the brain while bypassing the BBB. This study aimed to develop chitosan nanoparticles encapsulating ATDs, specifically isoniazid (INH) and rifampicin (RIF). These nanoparticles were further processed into micro-sized chitosan nano-aggregates (NA) via spray drying. Both INH-NA and RIF-NA showed strong mucoadhesion and significantly higher permeation rates across RPMI 2650 cells compared to free ATDs. Intranasal administration of these NAs to TB-infected mice for four weeks resulted in a significant reduction of mycobacterial load by approximately ∼2.86 Log 10 CFU compared to the untreated group. This preclinical data highlights the efficacy of intranasal chitosan nano-aggregates in treating CNS-TB, demonstrating high therapeutic potential, and addressing brain inflammation challenges. To our knowledge, this study is the first to show nasal delivery of ATD nano-formulations for CNS-TB management.

Evaluation of Drug-Loaded and Surface-Adsorbed DNase/Tween-80 Solid Lipid Nanoparticles against Staphylococcus aureus Biofilms.

The aim of this study was to explore the suitability of Tween-80 or DNase I adsorbed onto the surface of gentamicin-loaded solid lipid nanoparticles (SLNs) to disrupt Staphylococcus aureus biofilms in vitro. We hypothesized that surface-adsorbed DNase I or Tween-80 of SLNs will degrade the biofilm component, extracellular DNA (e-DNA), and extracellular matrix (ECM) of S. aureus biofilms. The SLNs loaded with drug (core) and surface-adsorbed disruptors (Tween-80 or DNase I) to deliver biofilm disruptors first at the site of action, which will help to break down the biofilm, and further drug release from the core will easily penetrate the biofilm and facilitate the killing of bacteria residing in S. aureus biofilms. The SLNs were synthesized by the double emulsion method; the size was 287.3 ± 7.4 nm for blank SLNs and 292.4 ± 2.36 nm for drug-loaded SLNs. The ζ-potential of blank SLNs was -25.6 ± 0.26 mV and that of drug-loaded SLNs was -13.16 ± 0.51 mV, respectively. The successful adsorption of DNase I or Tween-80 was confirmed by the activity of DNase I in blank surface-adsorbed SLNs and the change in the ζ-potential of SLNs after adsorbing DNase I or Tween-80. The surface morphology and size of the SLNs were further characterized using scanning electron microscopy. The encapsulation efficiency of the drug was 16.85 ± 0.84%. The compatibility of the drug with the excipient was confirmed by Fourier transform infrared spectroscopy and the degree of crystallinity was confirmed by X-ray diffraction (XRD) analysis. SLNs showed a sustained release of the drug up to 360 h. SLNs were easily taken up by A549 cells with minimal or no toxicity. The present study showed that Tween-80- or DNase I-adsorbed SLNs efficiently disrupt S. aureus biofilms and possess no or minimal toxicity against cells and red blood cells (RBCs).

Staphylococcus aureus Biofilm Destabilization by Tween-80 and Lung Surfactants to Overcome Biofilm-Imposed Drug Resistance.

This study is aimed to evaluating the potential of tween-80 and artificial lung surfactant (ALS) to destabilize S. aureus biofilm. The biofilm destabilization was studied by crystal violet staining, bright field microscopy, and scanning electron microscopy (SEM). During the study, S. aureus biofilm was exposed with tween-80 along various concentrations (1%, 0.1%, and 0.05%) or LS (lung surfactant) at (2.5%, 5%, and 15%) for 2 hrs. It was observed that 0.1% of tween-80 destabilized 63.83 ± 4.35% and 15% ALS 77 ± 1.7% biofilm in comparison to without treatment. The combination of tween-80 and ALS was used and showed a synergistic effect to destabilize 83.4 ± 1.46% biofilm. These results showed the potential of tween-80 and ALS as biofilm disruptors, which further needs to explore in an in-vivo animal model to access the actual potential of biofilm disruption in natural conditions. This study could play a pivotal role to overcome the problem of antibiotic resistance imposed due to biofilm formation to combat antibiotic resistance imposed by bacteria.

Synthesis and Evaluation of BSA-Loaded PLGA-Chitosan Composite Nanoparticles for the Protein-Based Drug Delivery System.

The purpose of this study was to synthesize composite nanoparticles (NPs) based on poly(d,l-lactic-co-glycolic acid) (PLGA) and chitosan (CS) and evaluate their suitability for the delivery of protein-based therapeutic molecules. Composite NPs possess a unique property which is not exhibited by any other polymer. Unlike other polymers, only the composite NPs lead to improved transfection efficiency and sustained release of protein. The composite NP were prepared by grafting CS on the surface of PLGA NPs through EDC-NHS coupling reaction. The size of bovine serum albumin (BSA)-loaded PLGA NPs and BSA-loaded PLGA-CS composite NPs was 288 ± 3 and 363 ± 4 nm, respectively. The zeta potential of PLGA NPs is -18 ± 0.23, and that of composite particles is 19 ± 0.40, thus confirming the successful addition of CS on the surface of PLGA NPs. Composite NPs were characterized using dynamic light scattering, scanning/transmission electron microscopy, Fourier transform infrared spectroscopy, X-ray diffraction, release profile, and gel electrophoresis. The encapsulation efficiency of PLGA NPs was 88%. These composite NPs were easily uptaken by the A549 cell line with no or minimal cytotoxicity. The present study emphasizes that the composite NPs are suitable for delivery of BSA into the cells with no cytotoxicity or very little cytotoxicity, while maintaining the integrity of the encapsulated BSA.

Bioengineered Ciprofloxacin-Loaded Chitosan Nanoparticles for the Treatment of Bovine Mastitis.

Mastitis is the most devastating economic disease in dairy cattle. Mastitis in dairy cattle frequently occurs during the dry period or during early lactation. Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus)are the main causative agents of mastitis in India. S. aureus can form microabscesses in the udder and develop a subclinical form of mastitis. This bacterial property hinders an effective cure during the lactation period. Antimicrobials used for treatments have a short half-life at the site of action because of frequent milking; thereforethey are unable to maintain the desired drug concentration for effective clearance of bacteria. We demonstrated the potential of ciprofloxacin-encapsulated nanocarriersthat can improve the availability of drugs and provide an effective means for mastitis treatment. These drug-loaded nanoparticles show low toxicity and slow clearance from the site of action. Antimicrobial activity against clinical strains of E. coli and S. aureus showed that the zone of inhibition depended on the dose (0.5 mg to 2 mg/mL nanoparticle solution from 11.6 to 14.5 mm and 15 to 18 mm). These nanoparticles showed good antimicrobial activity in broth culture and agar diffusion assay against bacteria.

Nanoparticle and Nanostructure Synthesis and Controlled Growth Methods.

Nanomaterials are materials with one or more nanoscale dimensions (internal or external) (i.e., 1 to 100 nm). The nanomaterial shape, size, porosity, surface chemistry, and composition are controlled at the nanoscale, and this offers interesting properties compared with bulk materials. This review describes how nanomaterials are classified, their fabrication, functionalization techniques, and growth-controlled mechanisms. First, the history of nanomaterials is summarized and then the different classification methods, based on their dimensionality (0-3D), composition (carbon, inorganic, organic, and hybrids), origin (natural, incidental, engineered, bioinspired), crystal phase (single phase, multiphase), and dispersion state (dispersed or aggregated), are presented. Then, the synthesis methods are discussed and classified in function of the starting material (bottom-up and top-down), reaction phase (gas, plasma, liquid, and solid), and nature of the dispersing forces (mechanical, physical, chemical, physicochemical, and biological). Finally, the challenges in synthesizing nanomaterials for research and commercial use are highlighted.

Review on Nanoparticles and Nanostructured Materials: Bioimaging, Biosensing, Drug Delivery, Tissue Engineering, Antimicrobial, and Agro-Food Applications.

In the last few decades, the vast potential of nanomaterials for biomedical and healthcare applications has been extensively investigated. Several case studies demonstrated that nanomaterials can offer solutions to the current challenges of raw materials in the biomedical and healthcare fields. This review describes the different nanoparticles and nanostructured material synthesis approaches and presents some emerging biomedical, healthcare, and agro-food applications. This review focuses on various nanomaterial types (e.g., spherical, nanorods, nanotubes, nanosheets, nanofibers, core-shell, and mesoporous) that can be synthesized from different raw materials and their emerging applications in bioimaging, biosensing, drug delivery, tissue engineering, antimicrobial, and agro-foods. Depending on their morphology (e.g., size, aspect ratio, geometry, porosity), nanomaterials can be used as formulation modifiers, moisturizers, nanofillers, additives, membranes, and films. As toxicological assessment depends on sizes and morphologies, stringent regulation is needed from the testing of efficient nanomaterials dosages. The challenges and perspectives for an industrial breakthrough of nanomaterials are related to the optimization of production and processing conditions.

Biomedical Applications of Carbon Nanomaterials: Fullerenes, Quantum Dots, Nanotubes, Nanofibers, and Graphene.

Carbon nanomaterials (CNMs) have received tremendous interest in the area of nanotechnology due to their unique properties and flexible dimensional structure. CNMs have excellent electrical, thermal, and optical properties that make them promising materials for drug delivery, bioimaging, biosensing, and tissue engineering applications. Currently, there are many types of CNMs, such as quantum dots, nanotubes, nanosheets, and nanoribbons; and there are many others in development that promise exciting applications in the future. The surface functionalization of CNMs modifies their chemical and physical properties, which enhances their drug loading/release capacity, their ability to target drug delivery to specific sites, and their dispersibility and suitability in biological systems. Thus, CNMs have been effectively used in different biomedical systems. This review explores the unique physical, chemical, and biological properties that allow CNMs to improve on the state of the art materials currently used in different biomedical applications. The discussion also embraces the emerging biomedical applications of CNMs, including targeted drug delivery, medical implants, tissue engineering, wound healing, biosensing, bioimaging, vaccination, and photodynamic therapy.

Single nucleotide polymorphic macrophage cytokine regulation by Mycobacterium tuberculosis and drug treatment.

AIM: To investigate the survival of Mycobacterium tuberculosis in primary macrophages with SNPs affecting cytokine secretion under treatment with drugs in solution or microparticles. MATERIALS & METHODS: Volunteers were typed for TNF (-308G/A), IL-10 (-1082A/G) and IL-4 (-590C/T). Monocyte-derived macrophages (MDMs) were infected in vitro. Cytokine secretion and survival of intracellular bacilli were estimated. RESULTS: IL-10 AG associated with high secretion in uninfected and infected MDMs (p < 0.05) and was reduced more effectively by microparticles than drugs, irrespective of genotype (p < 0.05). Differences were observed between IL-4 secretion by MDMs of CC and TT genotypes (p = 0.1). Bacteria proliferated more in MDMs from volunteers with higher IL-4 levels (p = 0.05). Microparticles showed higher efficacy (p = 0.05) than drugs. CONCLUSION: IL-4 and IL-10 SNPs affect the ability of macrophages to counter infection with M. tuberculosis. Microparticles elicit favorable macrophage cytokines regardless of SNPs.

The devil's advocacy: when and why inhaled therapies for tuberculosis may not work.

Factors that are inimical to the success of inhaled therapies for tuberculosis (TB) include: (i) lack of access of inhaled therapies to poorly-aerated areas of the tubercular lung; (ii) limited ability to penetrate biofilms formed by extracellular bacilli; (iii) selection for resistant bacilli on account of administration of low doses of anti-TB agents; (iv) induction of inflammation and/or immunopathology in the airways and lungs; and (v) anomalies in antigen processing and presentation of vaccines delivered to the lungs. Further, the claim that inhaled therapies rescue alternatively-activated macrophages may not be applicable to all individuals. Fortunately, there are ways and means to address each of the above factors.

Inhalable microparticles modify cytokine secretion by lung macrophages of infected mice.

Inhalable microparticles containing a large payload of isoniazid (INH) and rifabutin (RFB) in equal proportions show extremely high efficacy against experimental animal tuberculosis (TB). It was investigated whether inhaled microparticles affect the cytokine environment in the lung lumen, and cytokine secretion by airway and lung macrophages recovered from mice infected with Mycobacterium tuberculosis (Mtb). We attempted to determine whether the cytokine environment of the mouse lung receives significant contribution by lung macrophages, and whether these macrophages maintain a profile of cytokine secretion that is consistent with the cytokine environment of the lung. Groups of mice were infected intravenously with Mtb H37Ra and treated with (a) 5 mg/Kg each of INH and RFB administered by oral gavage; or, (b) 2.5 mg/Kg of the same and an additional ∼2.5 mg/Kg in the form of inhaled microparticles; or, (c) ∼2.5 mg/Kg by inhalation alone. Bronchioalveolar lavage (BAL) was carried out and recovered macrophages cultured. BAL Fluid and culture supernatants were assayed for tumor necrosis factor (TNF-α), interferon (IFN-γ), interleukin (IL)-12 and IL-10 by ELISA and amounts compared with both infected and uninfected, untreated controls. Inhaled microparticles enhanced secretion of TNF-α and supported IFN-γ secretion despite upregulated IL-10. Oral chemotherapy with the same drugs enhanced IL-12 and downregulated TNF-α. Differences in cytokine profiles suggest distinct effects of drug delivery modalities on innate immune strategies mobilized during host response. These differences might account, in part, for the extraordinary efficacy of the microparticles.

Inhalable microparticles containing isoniazid and rifabutin target macrophages and 'stimulate the phagocyte' to achieve high efficacy.

Macrophage responses to infection with Mycobacterium tuberculosis (MTB) and treatment with soluble isoniazid (INH) plus rifabutin (RFB) versus microparticles containing equivalent amounts of drugs were compared. It was investigated whether macrophages driven to alternative activation upon infection with MTB could be rescued to display the classical activation phenotype. It was established that microparticles sustain high levels of drugs in cytosol of macrophages for longer period as compared to soluble drugs. Microparticles co-localized with intracellular bacteria, and induced a variety of innate bactericidal responses, including induction of free radicals, alteration of mitochondrial membrane potential and apoptosis. The data strongly suggest that additional benefit may be derived from the nature of the drug delivery system, which fulfils Koch's dictum 'stimulate the phagocyte' for curing tuberculosis.

Intracellular time course, pharmacokinetics, and biodistribution of isoniazid and rifabutin following pulmonary delivery of inhalable microparticles to mice.

Intracellular concentrations of isoniazid and rifabutin resulting from administration of inhalable microparticles of these drugs to phorbol-differentiated THP-1 cells and the pharmacokinetics and biodistribution of these drugs upon inhalation of microparticles or intravenous administration of free drugs to mice were investigated. In cultured cells, both microparticles and dissolved drugs established peak concentrations of isoniazid ( approximately 1.4 and 1.1 microg/10(6) cells) and rifabutin ( approximately 2 microg/ml and approximately 1.4 microg/10(6) cells) within 10 min. Microparticles maintained the intracellular concentration of isoniazid for 24 h and rifabutin for 96 h, whereas dissolved drugs did not. The following pharmacokinetic parameters were calculated using WinNonlin from samples obtained after inhalation using an in-house apparatus (figures in parentheses refer to parameters obtained after intravenous administration of an equivalent amount, i.e., 100 microg of either drug, to parallel groups): isoniazid, serum half-life (t(1/2)) = 18.63 +/- 5.89 h (3.91 +/- 1.06 h), maximum concentration in serum (C(max)) = 2.37 +/- 0.23 microg x ml(-1) (3.24 +/- 0.57 microg x ml(-1)), area under the concentration-time curve from 0 to 24 h (AUC(0-24)) = 55.34 +/- 13.72 microg/ml(-1) h(-1) (16.64 +/- 1.80 microg/ml(-1) h(-1)), and clearance (CL) = 63.90 +/- 13.32 ml x h(-1) (4.43 +/- 1.85 ml x h(-1)); rifabutin, t(1/2) = 119.49 +/- 29.62 h (20.18 +/- 4.02 h), C(max) = 1.59 +/- 0.01 microg x ml(-1) (3.47 +/- 0.33 microg x ml(-1)), AUC(0-96) = 109.35 +/- 14.78 microg/ml(-1) h(-1) (90.82 +/- 7.46 microg/ml(-1) h(-1)), and CL = 11.68 +/- 7.00 ml x h(-1) (1.03 +/- 0.11 ml.h(-1)). Drug targeting to the lungs in general and alveolar macrophages in particular was observed. It was concluded that inhaled microparticles can reduce dose frequency and improve the pharmacologic index of the drug combination.

A hand-held apparatus for "nose-only" exposure of mice to inhalable microparticles as a dry powder inhalation targeting lung and airway macrophages.

Microparticles containing isoniazid and rifabutin were aerosolised using a simple apparatus fabricated from a 15-ml centrifuge tube. The dose available for inhalation by rodents was determined by collecting microparticles emitted at the delivery port. The dose available for inhalation was proportional to durations of exposure ranging from 10 to 90 s (10.5-13.5 CV%) and the weight of powder taken for fluidization (10-50 mg, r2=0.982). The apparatus was then used to administer inhalations of microparticles to mice. Other groups of mice received free rifabutin orally, or by i.v. injection. Rifabutin was estimated in serum and tissues of dosed mice by HPLC. When approximately 20 mg of microparticles were loaded in the apparatus, approximately 2.5 mg were collected at the delivery port in 30 s of operation. Mice inhaled approximately 300 microg of the 2.5 mg emitted at the delivery port. Airway and lung macrophages of mice receiving inhalations for 30 s accumulated 0.38 microg of rifabutin, while the amount in blood serum of these mice was 0.62 microg. In mice receiving 83 microg rifabutin i.v. or orally, the intracellular amounts were 0.06 and 0.07 microg respectively, while the amounts in serum were 1.02 and 0.80 microg. These observations confirmed that inhalation of microparticles targeted airway and lung macrophages.

Inhalable microparticles containing large payload of anti-tuberculosis drugs.

Microparticles containing large payloads of two anti-tuberculosis (TB) drugs were prepared and evaluated for suitability as a dry powder inhalation targeting alveolar macrophages. A solution containing one part each of isoniazid and rifabutin, plus two parts poly(lactic acid) (L-PLA) was spray-dried. Drug content and in vitro release were assayed by HPLC, and DSC was used to elucidate release behaviour. Particle size was measured by laser scattering and aerosol characteristics by cascade impaction using a Lovelace impactor. Microparticles were administered to mice using an in-house inhalation apparatus or by intra-tracheal instillation. Drugs in solution were administered orally and by intra-cardiac injection. Flow cytometry and HPLC were used to investigate the specificity and magnitude of targeting macrophages. Microparticles having drug content approximately 50% (w/w), particle size approximately 5 microm and satisfactory aerosol characteristics (median mass aerodynamic diameter, MMAD=3.57 microm; geometric standard deviation, GSD=1.41 microm; fine particle fraction, FPF(<4.6 microm)=78.91+/-8.4%) were obtained in yields of >60%. About 70% of the payload was released in vitro in 10 days. Microparticles targeted macrophages and not epithelial cells on inhalation. Drug concentrations in macrophages were approximately 20 times higher when microparticles were inhaled rather than drug solutions administered. Microparticles were thus deemed suitable for enhanced targeted drug delivery to lung macrophages.

Enhancement of apoptosis of THP-1 cells infected with Mycobacterium tuberculosis by inhalable microparticles and relevance to bactericidal activity.

The relevance of apoptosis to killing of intracellular Mycobacterium tuberculosis was studied with phorbol-differentiated THP-1 cells. Microparticles containing isoniazid and rifabutin induced intrinsic apoptosis and bacterial killing equivalent to that with dissolved drugs and maximally enhanced purinergic P2 receptor activity, while drug-free microparticles induced apoptosis via a different mechanism without killing bacteria.

Uptake of inhalable microparticles affects defence responses of macrophages infected with Mycobacterium tuberculosis H37Ra.

OBJECTIVES: To investigate whether inhalable microparticles containing two anti-tuberculosis agents, isoniazid and rifampicin, evoke host-defence strategies in macrophages in addition to targeting the incorporated drugs. METHODS: Microparticles were prepared by spray-drying a homogeneous solution of drugs and poly(lactic acid) (PLA; apparent viscosity 1.1 cP). Four parts PLA and three parts rifampicin were dissolved in dichloromethane. One part isoniazid was dissolved in methanol. The two solutions were mixed in the ratio 22 : 3 at which none of the solutes precipitated. These were administered as 'nose-only' inhalations to mice or exposed to cultured J774 mouse macrophages. Targeting to lung macrophages was investigated by transmission electron microscopy. Reactive oxygen species (ROS) were estimated by a cytochrome c assay and flow cytometry. Reactive nitrogen intermediates (RNI) were assayed using Griess reagent. Cytokines in culture supernatants were estimated by ELISA. RESULTS: Treatment with inhalable microparticles targeted lung macrophages in vivo and induced intense Golgi activity in the vicinity of microparticle-containing phagosomes. Microparticles induced a respiratory burst involving NADPH oxidase and enhanced NO production by infected macrophages. Microparticle-induced NADPH oxidase activation required optimal calcium ions. Microparticles efficiently induced tumour necrosis factor-alpha (TNF-alpha) secretion by macrophages recovered from infected mice. CONCLUSIONS: Microparticle phagocytosis induces responses in infected murine macrophages that are indicative of activation of innate bactericidal mechanisms, and are inimical to bacterial survival. It is likely that such responses augment straightforward drug action on the bacterium and contribute to the unexpectedly high efficacy of microparticles in experimental tuberculosis.