The Degree of Inulin Polymerization Is Important for Short-Term Amelioration of High-Fat Diet (HFD)-Induced Metabolic Dysfunction and Gut Microbiota Dysbiosis in Rats.

Inulin, a non-digestible polysaccharide, has gained attention for its prebiotic properties, particularly in the context of obesity, a condition increasingly understood as a systemic inflammatory state linked to gut microbiota composition. This study investigates the short-term protective effects of inulin with different degrees of polymerization (DPn) against metabolic health deterioration and gut microbiota alterations induced by a high-fat diet (HFD) in Sprague Dawley rats. Inulin treatments with an average DPn of 7, 14, and 27 were administered at 1 g/kg of bodyweight to HFD-fed rats over 21 days. Body weight, systemic glucose levels, and proinflammatory markers were measured to assess metabolic health. Gut microbiota composition was analyzed through 16S rRNA gene sequencing. The results showed that inulin27 significantly reduced total weight gain and systemic glucose levels, suggesting a DPn-specific effect on metabolic health. The study also observed shifts in gut microbial populations, with inulin7 promoting several beneficial taxa from the Bifidobacterium genera, whilst inducing a unique microbial composition compared to medium-chain (DPn 14) and long-chain inulin (DPn: 27). However, the impact of inulin on proinflammatory markers and lipid metabolism parameters was not statistically significant, possibly due to the short study duration. Inulin with a higher DPn has a more pronounced effect on mitigating HFD-induced metabolic health deterioration, whilst inulin7 is particularly effective at inducing healthy microbial shifts. These findings highlight the benefits of inulin as a dietary adjuvant in obesity management and the importance of DPn in optimizing performance.

Lipid Nanocarriers-Enabled Delivery of Antibiotics and Antimicrobial Adjuvants to Overcome Bacterial Biofilms.

The opportunistic bacteria growing in biofilms play a decisive role in the pathogenesis of chronic infectious diseases. Biofilm-dwelling bacteria behave differently than planktonic bacteria and are likely to increase resistance and tolerance to antimicrobial therapeutics. Antimicrobial adjuvants have emerged as a promising strategy to combat antimicrobial resistance (AMR) and restore the efficacy of existing antibiotics. A combination of antibiotics and potential antimicrobial adjuvants, (e.g., extracellular polymeric substance (EPS)-degrading enzymes and quorum sensing inhibitors (QSI) can improve the effects of antibiotics and potentially reduce bacterial resistance). In addition, encapsulation of antimicrobials within nanoparticulate systems can improve their stability and their delivery into biofilms. Lipid nanocarriers (LNCs) have been established as having the potential to improve the efficacy of existing antibiotics in combination with antimicrobial adjuvants. Among them, liquid crystal nanoparticles (LCNPs), liposomes, solid lipid nanoparticles (SLNs), and nanostructured lipid carriers (NLCs) are promising due to their superior properties compared to traditional formulations, including their greater biocompatibility, higher drug loading capacity, drug protection from chemical or enzymatic degradation, controlled drug release, targeted delivery, ease of preparation, and scale-up feasibility. This article reviews the recent advances in developing various LNCs to co-deliver some well-studied antimicrobial adjuvants combined with antibiotics from different classes. The efficacy of various combination treatments is compared against bacterial biofilms, and synergistic therapeutics that deserve further investigation are also highlighted. This review identifies promising LNCs for the delivery of combination therapies that are in recent development. It discusses how LNC-enabled co-delivery of antibiotics and adjuvants can advance current clinical antimicrobial treatments, leading to innovative products, enabling the reuse of antibiotics, and providing opportunities for saving millions of lives from bacterial infections.

Effective γδ T-cell clinical therapies: current limitations and future perspectives for cancer immunotherapy.

γδ T cells are a unique subset of T lymphocytes, exhibiting features of both innate and adaptive immune cells and are involved with cancer immunosurveillance. They present an attractive alternative to conventional T cell-based immunotherapy due, in large part, to their lack of major histocompatibility (MHC) restriction and ability to secrete high levels of cytokines with well-known anti-tumour functions. To date, clinical trials using γδ T cell-based immunotherapy for a range of haematological and solid cancers have yielded limited success compared with in vitro studies. This inability to translate the efficacy of γδ T-cell therapies from preclinical to clinical trials is attributed to a combination of several factors, e.g. γδ T-cell agonists that are commonly used to stimulate populations of these cells have limited cellular uptake yet rely on intracellular mechanisms; administered γδ T cells display low levels of tumour-infiltration; and there is a gap in the understanding of γδ T-cell inhibitory receptors. This review explores the discrepancy between γδ T-cell clinical and preclinical performance and offers viable avenues to overcome these obstacles. Using more direct γδ T-cell agonists, encapsulating these agonists into lipid nanocarriers to improve their pharmacokinetic and pharmacodynamic profiles and the use of combination therapies to overcome checkpoint inhibition and T-cell exhaustion are ways to bridge the gap between preclinical and clinical success. Given the ability to overcome these limitations, the development of a more targeted γδ T-cell agonist-checkpoint blockade combination therapy has the potential for success in clinical trials which has to date remained elusive.

Antibiotic resistance and tolerance: What can drug delivery do against this global threat?

Antimicrobial resistance and tolerance (AMR&T) are urgent global health concerns, with alarmingly increasing numbers of antimicrobial drugs failing and a corresponding rise in related deaths. Several reasons for this situation can be cited, such as the misuse of traditional antibiotics, the massive use of sanitizing measures, and the overuse of antibiotics in agriculture, fisheries, and cattle. AMR&T management requires a multifaceted approach involving various strategies at different levels, such as increasing the patient's awareness of the situation and measures to reduce new resistances, reduction of current misuse or abuse, and improvement of selectivity of treatments. Also, the identification of new antibiotics, including small molecules and more complex approaches, is a key factor. Among these, novel DNA- or RNA-based approaches, the use of phages, or CRISPR technologies are some potent strategies under development. In this perspective article, emerging and experienced leaders in drug delivery discuss the most important biological barriers for drugs to reach infectious bacteria (bacterial bioavailability). They explore how overcoming these barriers is crucial for producing the desired effects and discuss the ways in which drug delivery systems can facilitate this process.

Combating Acute Myeloid Leukemia via Sphingosine Kinase 1 Inhibitor-Nanomedicine Combination Therapy with Cytarabine or Venetoclax.

MP-A08 is a novel sphingosine kinase 1 (SPHK1) inhibitor with activity against acute myeloid leukemia (AML). A rationally designed liposome-based encapsulation and delivery system has been shown to overcome the physicochemical challenges of MP-A08 and enable its effective delivery for improved efficacy and survival of mice engrafted with human AML in preclinical models. To establish therapies that overcome AML's heterogeneous nature, here we explored the combination of MP-A08-loaded liposomes with both the standard chemotherapy, cytarabine, and the targeted therapy, venetoclax, against human AML cell lines. Cytarabine (over the dose range of 0.1-0.5 µM) in combination with MP-A08 liposomes showed significant synergistic effects (as confirmed by the Chou-Talalay Combination Index) against the chemosensitised human AML cell lines MV4-11 and OCI-AML3. Venetoclax (over the dose range of 0.5-250 nM) in combination with MP-A08 liposomes showed significant synergistic effects against the chemosensitised human AML cell lines, particularly in venetoclax-resistant human AML cells. This strong synergistic effect is due to multiple mechanisms of action, i.e., inhibiting MCL-1 through SPHK1 inhibition, leading to ceramide accumulation, activation of protein kinase R, ATF4 upregulation, and NOXA activation, ultimately resulting in MCL-1 degradation. These combination therapies warrant further consideration and investigation in the search for a more comprehensive treatment strategy for AML.

Minimum Information for Conducting and Reporting In Vitro Intracellular Infection Assays.

Bacterial pathogens are constantly evolving to outsmart the host immune system and antibiotics developed to eradicate them. One key strategy involves the ability of bacteria to survive and replicate within host cells, thereby causing intracellular infections. To address this unmet clinical need, researchers are adopting new approaches, such as the development of novel molecules that can penetrate host cells, thus exerting their antimicrobial activity intracellularly, or repurposing existing antibiotics using nanocarriers (i.e., nanoantibiotics) for site-specific delivery. However, inconsistency in information reported across published studies makes it challenging for scientific comparison and judgment of experiments for future direction by researchers. Together with the lack of reproducibility of experiments, these inconsistencies limit the translation of experimental results beyond pre-clinical evaluation. Minimum information guidelines have been instrumental in addressing such challenges in other fields of biomedical research. Guidelines and recommendations provided herein have been designed for researchers as essential parameters to be disclosed when publishing their methodology and results, divided into four main categories: (i) experimental design, (ii) establishing an in vitro model, (iii) assessment of efficacy of novel therapeutics, and (iv) statistical assessment. These guidelines have been designed with the intention to improve the reproducibility and rigor of future studies while enabling quantitative comparisons of published studies, ultimately facilitating translation of emerging antimicrobial technologies into clinically viable therapies that safely and effectively treat intracellular infections.

Self-emulsifying drug delivery systems (SEDDS) disrupt the gut microbiota and trigger an intestinal inflammatory response in rats.

Self-emulsifying drug delivery systems (i.e. SEDDS, SMEDDS and SNEDDS) are widely employed as solubility and bioavailability enhancing formulation strategies for poorly water-soluble drugs. Despite the capacity for SEDDS to effectively facilitate oral drug absorption, tolerability concerns exist due to the capacity for high concentrations of surfactants (typically present within SEDDS) to induce gastrointestinal toxicity and mucosal irritation. With new knowledge surrounding the role of the gut microbiota in modulating intestinal inflammation and mucosal injury, there is a clear need to determine the impact of SEDDS on the gut microbiota. The current study is the first of its kind to demonstrate the detrimental impact of SEDDS on the gut microbiota of Sprague-Dawley rats, following daily oral administration (100 mg/kg) for 21 days. SEDDS comprising a lipid phase (i.e. Type I, II and III formulations according to the Lipid Formulation Classification Scheme) induced significant changes to the composition and diversity of the gut microbiota, evidenced through a reduction in operational taxonomic units (OTUs) and alpha diversity (Shannon's index), along with statistically significant shifts in beta diversity (according to PERMANOVA of multi-dimensional Bray-Curtis plots). Key signatures of gut microbiota dysbiosis correlated with the increased expression of pro-inflammatory cytokines within the jejunum, while mucosal injury was characterised by significant reductions in plasma citrulline levels, a validated biomarker of enterocyte mass and mucosal barrier integrity. These findings have potential clinical ramifications for chronically administered drugs that are formulated with SEDDS and stresses the need for further studies that investigate dose-dependent effects of SEDDS on the gastrointestinal microenvironment in a clinical setting.

Battle of the milky way: Lymphatic targeted drug delivery for pathogen eradication.

Many viruses, bacteria, and parasites rely on the lymphatic system for survival, replication, and dissemination. While conventional anti-infectives can combat infection-causing agents in the bloodstream, they do not reach the lymphatic system to eradicate the pathogens harboured there. This can result in ineffective drug exposure and reduce treatment effectiveness. By developing effective lymphatic delivery strategies for antiviral, antibacterial, and antiparasitic drugs, their systemic pharmacokinetics may be improved, as would their ability to reach their target pathogens within the lymphatics, thereby improving clinical outcomes in a variety of acute and chronic infections with lymphatic involvement (e.g., acquired immunodeficiency syndrome, tuberculosis, and filariasis). Here, we discuss approaches to targeting anti-infective drugs to the intestinal and dermal lymphatics, aiming to eliminate pathogen reservoirs and interfere with their survival and reproduction inside the lymphatic system. These include optimized lipophilic prodrugs and drug delivery systems that promote lymphatic transport after oral and dermal drug intake. For intestinal lymphatic delivery via the chylomicron pathway, molecules should have logP values >5 and long-chain triglyceride solubilities >50 mg/g, and for dermal lymphatic delivery via interstitial lymphatic drainage, nanoparticle formulations with particle size between 10 and 100 nm are generally preferred. Insight from this review may promote new and improved therapeutic solutions for pathogen eradication and combating infective diseases, as lymphatic system involvement in pathogen dissemination and drug resistance has been neglected compared to other pathways leading to treatment failure.

Lyophilized Lipid Liquid Crystalline Nanoparticles as an Antimicrobial Delivery System.

Lipid liquid crystalline nanoparticles (LCNPs) are unique nanocarriers that efficiently deliver antimicrobials through biological barriers. Yet, their wide application as an antimicrobial delivery system is hindered by their poor stability in aqueous dispersions. The production of dried LCNP powder via lyophilization is a promising approach to promote the stability of LCNPs. However, the impact of the process on the functionality of the loaded hydrophobic cargoes has not been reported yet. Herein, we investigated the potential of lyophilization to produce dispersible dry LCNPs loaded with a hydrophobic antimicrobial compound, gallium protoporphyrin (GaPP). The effect of lyophilization on the physicochemical characteristics and the antimicrobial activity of rehydrated GaPP-LCNPs was studied. The rehydrated GaPP-LCNPs retained the liquid crystalline structure and were monodisperse (PDI: 0.27 ± 0.02), with no significant change in nanoparticle concentration despite the minor increase in hydrodynamic diameter (193 ± 6.5 compared to 173 ± 4.2 prior to freeze-drying). Most importantly, the efficacy of the loaded GaPP as an antimicrobial agent and a photosensitizer was not affected as similar MIC values were obtained against S. aureus (0.125 µg/mL), with a singlet oxygen quantum yield of 0.72. These findings indicate the suitability of lyophilization to produce a dry form of LCNPs and pave the way for future studies to promote the application of LCNPs as an antimicrobial delivery system.

Targeting the gut microbiome to control drug pharmacomicrobiomics: the next frontier in oral drug delivery.

INTRODUCTION: The trillions of microorganisms that comprise the gut microbiome form dynamic bidirectional interactions with orally administered drugs and host health. These relationships can alter all aspects of drug pharmacokinetics and pharmacodynamics (PK/PD); thus, there is a desire to control these interactions to maximize therapeutic efficacy. Attempts to modulate drug-gut microbiome interactions have spurred advancements within the field of 'pharmacomicrobiomics' and are poised to become the next frontier of oral drug delivery. AREAS COVERED: This review details the bidirectional interactions that exist between oral drugs and the gut microbiome, with clinically relevant case examples outlining a clear motive for controlling pharmacomicrobiomic interactions. Specific focus is attributed to novel and advanced strategies that have demonstrated success in mediating drug-gut microbiome interactions. EXPERT OPINION: Co-administration of gut-active supplements (e.g. pro- and pre-biotics), innovative drug delivery vehicles, and strategic polypharmacy serve as the most promising and clinically viable approaches for controlling pharmacomicrobiomic interactions. Targeting the gut microbiome through these strategies presents new opportunities for improving therapeutic efficacy by precisely mediating PK/PD, while mitigating metabolic disturbances caused by drug-induced gut dysbiosis. However, successfully translating preclinical potential into clinical outcomes relies on overcoming key challenges related to interindividual variability in microbiome composition and study design parameters.

Targeting Acute Myeloid Leukemia Using Sphingosine Kinase 1 Inhibitor-Loaded Liposomes.

Acute myeloid leukemia (AML) kills 75% of patients and represents a major clinical challenge with a need to improve on current treatment approaches. Targeting sphingosine kinase 1 with a novel ATP-competitive-inhibitor, MP-A08, induces cell death in AML. However, limitations in MP-A08's "drug-like properties" (solubility, biodistribution, and potency) hinder its pathway to the clinic. This study demonstrates a liposome-based delivery system of MP-A08 that exhibits enhanced MP-A08 potency against AML cells. MP-A08-liposomes increased MP-A08 efficacy against patient AML cells (>140-fold) and significantly prolonged overall survival of mice with human AML disease (P = 0.03). The significant antileukemic property of MP-A08-liposomes could be attributed to its enhanced specificity, bioaccessibility, and delivery to the bone marrow, as demonstrated in the pharmacokinetic and biodistribution studies. Our findings indicate that MP-A08-liposomes have potential as a novel treatment for AML.

Liposome-Micelle-Hybrid (LMH) Carriers for Controlled Co-Delivery of 5-FU and Paclitaxel as Chemotherapeutics.

Paclitaxel (PTX) and 5-fluorouracil (5-FU) are clinically relevant chemotherapeutics, but both suffer a range of biopharmaceutical challenges (e.g., either low solubility or permeability and limited controlled release from nanocarriers), which reduces their effectiveness in new medicines. Anticancer drugs have several major limitations, which include non-specificity, wide biological distribution, a short half-life, and systemic toxicity. Here, we investigate the potential of liposome-micelle-hybrid (LMH) carriers (i.e., drug-loaded micelles encapsulated within drug-loaded liposomes) to enhance the co-formulation and delivery of PTX and 5-FU, facilitating new delivery opportunities with enhanced chemotherapeutic performance. We focus on the combination of liposomes and micelles for co-delivery of PTX and 5_FU to investigate increased drug loading, improved solubility, and transport/permeability to enhance chemotherapeutic potential. Furthermore, combination chemotherapy (i.e., containing two or more drugs in a single formulation) may offer improved pharmacological performance. Compared with individual liposome and micelle formulations, the optimized PTX-5FU-LMH carriers demonstrated increased drug loading and solubility, temperature-sensitive release, enhanced permeability in a Caco-2 cell monolayer model, and cancer cell eradication. LMH has significant potential for cancer drug delivery and as a next-generation chemotherapeutic.

Liquid crystalline lipid nanoparticles improve the antibacterial activity of tobramycin and vancomycin against intracellular Pseudomonas aeruginosa and Staphylococcus aureus.

The intracellular survival of bacteria is a significant challenge in the fight against antimicrobial resistance. Currently available antibiotics suffer from limited penetration across host cell membranes, resulting in suboptimal treatment against the internalised bacteria. Liquid crystalline nanoparticles (LCNP) are gaining significant research interest in promoting the cellular uptake of therapeutics due to their fusogenic properties; however, they have not been reported for targeting intracellular bacteria. Herein, the cellular internalisation of LCNPs in RAW 264.7 macrophages and A549 epithelial cells was investigated and optimized through the incorporation of a cationic lipid, dimethyldioctadecylammonium bromide (DDAB). LCNPs displayed a honeycomb-like structure, while the inclusion of DDAB resulted into an onion-like organisation with larger internal pores. Cationic LCNPs enhanced the cellular uptake in both cells, reaching up to âˆ¼ 90% uptake in cells. Further, LCNPs were encapsulated with tobramycin or vancomycin to improve their activity against intracellular gram-negative, Pseudomonas aeruginosa (P. aeruginosa) and gram-positive, Staphylococcus aureus (S. aureus) bacteria. The enhanced cellular uptake of cationic LCNP resulted in significant reduction of intracellular bacterial load (up to 90% reduction), compared to antibiotic dosed in its free form; with reduced performance observed for epithelial cells infected with S. aureus. Specifically engineered LCNP can re-sensitise antibiotics against both intracellular Gram positive and negative bacteria in diverse cell lines.

Mesoporous Organosilica Nanoparticles to Fight Intracellular Staphylococcal Aureus Infections in Macrophages.

Intracellular bacteria are inaccessible and highly tolerant to antibiotics, hence are a major contributor to the global challenge of antibiotic resistance and recalcitrant clinical infections. This, in tandem with stagnant antibacterial discovery, highlights an unmet need for new delivery technologies to treat intracellular infections more effectively. Here, we compare the uptake, delivery, and efficacy of rifampicin (Rif)-loaded mesoporous silica nanoparticles (MSN) and organo-modified (ethylene-bridged) MSN (MON) as an antibiotic treatment against small colony variants (SCV) Staphylococcus aureus (SA) in murine macrophages (RAW 264.7). Macrophage uptake of MON was five-fold that of equivalent sized MSN and without significant cytotoxicity on human embryonic kidney cells (HEK 293T) or RAW 264.7 cells. MON also facilitated increased Rif loading with sustained release, and seven-fold increased Rif delivery to infected macrophages. The combined effects of increased uptake and intracellular delivery of Rif by MON reduced the colony forming units of intracellular SCV-SA 28 times and 65 times compared to MSN-Rif and non-encapsulated Rif, respectively (at a dose of 5 µg/mL). Conclusively, the organic framework of MON offers significant advantages and opportunities over MSN for the treatment of intracellular infections.

Protein adsorption determines pulmonary cell uptake of lipid-based nanoparticles.

The inhalable administration of lipid nanoparticles is an effective strategy for localised delivery of therapeutics against various lung diseases. Of this, improved intracellular delivery of pharmaceuticals for infectious disease and cancer management is of high significance. However, the influence of lipid nanoparticle composition and structure on uptake in pulmonary cell lines, especially in the presence of biologically relevant media is poorly understood. Here, the uptake of lamellar (liposomes) versus non-lamellar (cubosomes) lipid nanoparticles in macrophages and lung epithelial cells was quantified and the influence of bronchoalveolar lavage fluid (BALF), containing native pulmonary protein and surfactant molecules is determined. Cubosome uptake in both macrophages and epithelial cells was strongly mediated by a high percentage of molecular function regulatory and binding proteins present within the protein corona. In contrast, the protein corona did not influence the uptake of liposomes in epithelial cells. In macrophages, the proteins mediated a rapid internalisation, followed by exocytosis of liposomes after 6 h incubation. These findings on the influence of biological fluid in regulating lipid nanoparticle uptake mechanisms may guide future development of optimal intracellular delivery systems for therapeutics via the pulmonary route.

Lipid Liquid Crystal Nanoparticles: Promising Photosensitizer Carriers for the Treatment of Infected Cutaneous Wounds.

Cutaneous chronic wounds impose a silent pandemic that affects the lives of millions worldwide. The delayed healing process is usually complicated by opportunistic bacteria that infect wounds. Staphylococcus aureus is one of the most prevalent bacteria in infected cutaneous wounds, with the ability to form antibiotic-resistant biofilms. Recently, we have demonstrated the potential of gallium protoporphyrin lipid liquid crystalline nanoparticles (GaPP-LCNP) as a photosensitizer against S. aureus biofilms in vitro. Herein, we investigate the potential of GaPP-LCNP using a pre-clinical model of infected cutaneous wounds. GaPP-LCNP showed superior antibacterial activity compared to unformulated GaPP, reducing biofilm bacterial viability by 5.5 log10 compared to 2.5 log10 in an ex vivo model, and reducing bacterial viability by 1 log10 in vivo, while unformulated GaPP failed to reduce bacterial burden. Furthermore, GaPP-LCNP significantly promoted wound healing through reduction in the bacterial burden and improved early collagen deposition. These findings pave the way for future pre-clinical investigation and treatment optimizations to translate GaPP-LCNP towards clinical application.

The Influence of Blonanserin Supersaturation in Liquid and Silica Stabilised Self-Nanoemulsifying Drug Delivery Systems on In Vitro Solubilisation.

Reformulating poorly water-soluble drugs as supersaturated lipid-based formulations achieves higher drug loading and potentially improves solubilisation and bioavailability. However, for the weak base blonanserin, silica solidified supersaturated lipid-based formulations have demonstrated reduced in vitro solubilisation compared to their liquid-state counterparts. Therefore, this study aimed to understand the influence of supersaturated drug load on blonanserin solubilisation from liquid and silica solidified supersaturated self-nanoemulsifying drug delivery systems (super-SNEDDS) during in vitro lipolysis. Stable liquid super-SNEDDS with varying drug loads (90-300% of the equilibrium solubility) were solidified by imbibition into porous silica microparticles (1:1 lipid: silica ratio). In vitro lipolysis revealed greater blonanserin solubilisation from liquid super-SNEDDS compared to solid at equivalent drug saturation levels, owing to strong silica-BLON/lipid interactions, evidenced by a significant decrease in blonanserin solubilisation upon addition of silica to a digesting liquid super-SNEDDS. An increase in solid super-SNEDDS drug loading led to increased solubilisation, owing to the increased drug:silica and drug:lipid ratios. Solidifying SNEDDS with silica enables the fabrication of powdered formulations with higher blonanserin loading and greater stability than liquid super-SNEDDS, however at the expense of drug solubilisation. These competing parameters need careful consideration in designing optimal super-SNEDDS for pre-clinical and clinical application.

Inulin-lipid hybrid (ILH) microparticles promote pH-triggered release of rifampicin within infected macrophages.

Intracellular bacteria serve as a problematic source of infection due to their ability to evade biological immune responses and the inability for conventional antibiotics to efficiently penetrate cellular membranes. Subsequently, new treatment approaches are urgently required to effectively eradicate intracellular pathogens residing within immune cells (e.g. macrophages). In this study, the poorly soluble and poorly permeable antibiotic, rifampicin, was re-purposed via micro-encapsulation within inulin-lipid hybrid (ILH) particles for the treatment of macrophages infected with small colony variants of Staphylococcus aureus (SCV S. aureus). Rifampicin-encapsulated ILH (Rif-ILH) microparticles were synthesized by spray drying a lipid nano-emulsion, with inulin dissolved throughout the aqueous phase and rifampicin pre-loaded within the lipid phase. Rif-ILH were strategically designed and engineered with pH-responsive properties to promote lysosomal drug release upon cellular internalization, while preventing premature rifampicin release in plasma-simulating media. The pH-responsiveness of Rif-ILH was controlled by the acid-mediated hydrolysis of the inulin coating, where exposure to acidic media simulating the lysosomal environment of macrophages triggered hydrolysis of the oligofructose chain and the subsequent diffusion of rifampicin from Rif-ILH. This pH-provoked release mechanism, as well as the ability for ILH microparticles to be more readily internalized by macrophages, was found to be influential in triggering a 2.9-fold increase in intracellular rifampicin concentration within infected macrophages, compared to the pure drug. The subsequent increase in exposure of intracellular pathogens to rifampicin leads to a ~ 2-log improvement in antibacterial activity for Rif-ILH, at a rifampicin dose of 2.5 µg/mL. Thus, the reduction in viability of intracellular SCV S. aureus, in the absence of cellular toxicity, is indicative of ILH microparticles serving as a unique approach for the safe and efficacious delivery of antibiotics to phagocytic cells for the treatment of intracellular infections.

Investigation of TLR4 Antagonists for Prevention of Intestinal Inflammation.

Activation of toll-like receptor 4 (TLR4) has been shown to be a major influence on the inflammatory signalling pathways in intestinal mucositis (IM), as demonstrated by TLR4 knock-out mice. Pharmacological TLR4 inhibition has thus been postulated as a potential new therapeutic approach for the treatment of IM but specific TLR4 inhibitors have yet to be investigated. As such, we aimed to determine whether direct TLR4 antagonism prevents inflammation in pre-clinical experimental models of IM. The non-competitive and competitive TLR4 inhibitors, TAK-242 (10 µM) and IAXO-102 (10 µM), respectively, or vehicle were added to human T84, HT-29, and U937 cell lines and mouse colonic explants 1 h before the addition of lipopolysaccharide (LPS) (in vitro: 100 µg/mL; ex vivo: 10 µg/mL), SN-38 (in vitro: 1 µM or 1 nM; ex vivo: 2 µM), and/or tumour necrosis factor-alpha (TNF-α) (5 µg/mL). Supernatant was collected for human IL-8 and mouse IL-6 enzyme-linked immunosorbent assays (ELISAs), as a measure of inflammatory signalling. Cell viability was measured using XTT assays. Explant tissue was used in histopathological and RT-PCR analysis for genes of interest: TLR4, MD2, CD14, MyD88, IL-6, IL-6R, CXCL2, CXCR1, CXCR2. SN-38 increased cytostasis compared to vehicle (P < 0.0001). However, this was not prevented by either antagonist (P > 0.05) in any of the 3 cell lines. Quantitative histological assessment scores showed no differences between vehicle and treatment groups (P > 0.05). There were no differences in in vitro IL-8 (P > 0.05, in all 3 cells lines) and ex vivo IL-6 (P > 0.05) concentrations between vehicle and treatment groups. Transcript expression of all genes was similar across vehicle and treatment groups (P > 0.05). TLR4 antagonism using specific inhibitors TAK-242 and IAXO-102 was not effective at blocking IM in these pre-clinical models of mucositis. This work indicates that specific epithelial inhibition of TLR4 with these compounds is insufficient to manage mucositis-related inflammation. Rather, TLR4 signalling through immune cells may be a more important target to prevent IM.