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.

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.

Mimicking the Gastrointestinal Mucus Barrier: Laboratory-Based Approaches to Facilitate an Enhanced Understanding of Mucus Permeation.

The gastrointestinal mucus layer plays a significant role in maintaining gut homeostasis and health, offering protective capacities against the absorption of harmful pathogens as well as commensal gut bacteria and buffering stomach acid to protect the underlying epithelium. Despite this, the mucus barrier is often overlooked during preclinical pharmaceutical development and may pose a significant absorption barrier to high molecular weight or lipophilic drug species. The complex chemical and physical nature of the dynamic mucus layer has proven problematic to reliably replicate in a laboratory setting, leading to the development of multiple mucus models with varying complexity and predictive capacity. This, coupled with the wide range of analysis methods available, has led to a plethora of possible approaches to quantifying mucus permeation; however, the field remains significantly under-represented in biomedical research. For this reason, the development of a concise collation of the available approaches to mucus permeation is essential. In this review, we explore widely utilized mucus mimics ranging in complexity from simple mucin solutions to native mucus preparations for their predictive capacity in mucus permeation analysis. Furthermore, we highlight the diverse range of laboratory-based models available for the analysis of mucus interaction and permeability with a specific focus on in vitro, ex vivo, and in situ models. Finally, we highlight the predictive capacity of these models in correlation with in vivo pharmacokinetic data. This review provides a comprehensive and critical overview of the available technologies to analyze mucus permeation, facilitating the efficient selection of appropriate tools for further advancement in oral drug delivery.

Design and implementation of a program wide pharmaceutical compounding curriculum using the scaffold learning approach.

BACKGROUND AND PURPOSE: We evaluated the design and implementation of a program wide pharmaceutical compounding curriculum covering five modules over four years using the scaffold learning approach in a pharmacy degree program. EDUCATIONAL ACTIVITY AND SETTING: A programmatic approach was taken in the development of compounding expertise, which required moving away from a compartmentalized course design to a multi-course approach spanning all four years of the pharmacy program. FINDINGS: Since the intervention began in 2014, course failure rates, which were around 34% (2012-2014), have significantly decreased to 1.5% (2015-2019), and the percentage of students achieving distinction and above has increased four-fold from 20% (2012-2014) to 80% (2015-2019). SUMMARY: A program wide scaffold learning approach was more effective in the development of compounding skills throughout the pharmacy program than teaching compounding techniques in different modules without clear vertical integration.

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.

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.

Understanding the interfacial properties of nanostructured liquid crystalline materials for surface-specific delivery applications.

Nonlamellar liquid crystalline dispersions such as cubosomes and hexosomes have great potential as novel surface-targeted active delivery systems. In this study, the influence of internal nanostructure, chemical composition, and the presence of Pluronic F127 as a stabilizer, on the surface and interfacial properties of different liquid crystalline particles and surfaces, was investigated. The interfacial properties of the bulk liquid crystalline systems with coexisting excess water were dependent on the internal liquid crystalline nanostructure. In particular, the surfaces of the inverse cubic systems were more hydrophilic than that of the inverse hexagonal phase. The interaction between F127 and the bulk liquid crystalline systems depended on the internal liquid crystalline structure and chemical composition. For example, F127 adsorbed to the surface of the bulk phytantriol cubic phase, while for monoolein cubic phase, F127 was integrated into the liquid crystalline structure. Last, the interfacial adsorption behavior of the dispersed liquid crystalline particles also depended on both the internal nanostructure and the chemical composition, despite the dispersions all being stabilized using F127. The findings highlight the need to understand the specific surface characteristics and the nature of the interaction with colloidal stabilizer for understanding and optimizing the behavior of nonlamellar liquid crystalline systems in surface delivery applications.

Optimisation of a High-Throughput Model for Mucus Permeation and Nanoparticle Discrimination Using Biosimilar Mucus.

High-throughput permeation models are essential in drug development for timely screening of new drug and formulation candidates. Nevertheless, many current permeability assays fail to account for the presence of the gastrointestinal mucus layer. In this study, an optimised high-throughput mucus permeation model was developed employing a highly biorelevant mucus mimic. While mucus permeation is primarily conducted in a simple mucin solution, the complex chemistry, nanostructure and rheology of mucus is more accurately modelled by a synthetic biosimilar mucus (BSM) employing additional protein, lipid and rheology-modifying polymer components. Utilising BSM, equivalent permeation of various molecular weight fluorescein isothiocyanate-dextrans were observed, compared with native porcine jejunal mucus, confirming replication of the natural mucus permeation barrier. Furthermore, utilising synthetic BSM facilitated the analysis of free protein permeation which could not be quantified in native mucus due to concurrent proteolytic degradation. Additionally, BSM could differentiate between the permeation of poly (lactic-co-glycolic) acid nanoparticles (PLGA-NP) with varying surface chemistries (cationic, anionic and PEGylated), PEG coating density and size, which could not be achieved by a 5% mucin solution. This work confirms the importance of utilising highly biorelevant mucus mimics in permeation studies, and further development will provide an optimal method for high-throughput mucus permeation analysis.

Nanomaterials enabling clinical translation of antimicrobial photodynamic therapy.

Antimicrobial photodynamic therapy (aPDT) has emerged as a promising approach to aid the fight against looming antibiotic resistance. aPDT harnesses the energy of light through photosenstizers to generate highly reactive oxygen species that can inactivate bacteria and fungi with no resistance. To date aPDT has shown great efficacy against microbes causing localized infections in the skin and the oral cavity. However, its wide application in clinical settings has been limited due to both physicochemical and biological challenges. Over the past decade nanomaterials have contributed to promoting photosensitizer performance and aPDT efficiency, yet further developments are required to establish accredited treatment options. In this review we discuss the challenges facing the clinical application of aPDT and the opportunities that nanotechnology may offer to promote the safety and efficiency of aPDT.

Gallium Protoporphyrin Liquid Crystalline Lipid Nanoparticles: A Third-Generation Photosensitizer against Pseudomonas aeruginosa Biofilms.

The looming antimicrobial resistance pandemic has encouraged the investigation of antimicrobial photodynamic therapy (aPDT) as a promising technology to combat recalcitrant bacterial infections caused by antibiotic resistant strains. Here, we report on the optimization and effective application of gallium protoporphyrin liquid crystalline lipid nanoparticles (GaPP-LCNP) as a photosensitizer for aPDT against the Gram-negative bacteria P. aeruginosa in both planktonic and biofilm modes of growth. LCNP significantly enhanced the performance of GaPP as photosensitizer by two-fold, which was correlated with higher antibacterial activity, reducing the viability of planktonic P. aeruginosa by 7 log10 using 0.8 µM GaPP-LCNP and a light dose of 17 J.cm-2. Importantly, GaPP-LCNP also reduced the viability of biofilms by 6 log10 at relatively low light dose of 34.2 J.cm-2 using only 3 µM GaPP-LCNP. The high antibiofilm activity of GaPP-LCNP at low GaPP-LCNP dose indicated the high efficiency and safety profile of GaPP-LCNP as a promising platform for photodynamic inactivation of recalcitrant infections.

Liquid crystalline lipid nanoparticle promotes the photodynamic activity of gallium protoporphyrin against S. aureus biofilms.

Antimicrobial photodynamic therapy (aPDT) has emerged as an innovative strategy to combat antibiotic resistant microbes; yet aPDT efficacies against biofilms are sub-optimal due to inability of photosenstizers to reach microbes embedded in biofilm matrix. To overcome this challenge, liquid crystal lipid nanoparticles (LCNP) were employed in this study as a smart, biocompatible and triggerable delivery system for the new photosensitizer gallium protoporphyrin (GaPP), due to their capabilities in promoting efficient antimicrobial delivery to biofilms. The relationship between GaPP loading of LCNP, reactive oxygen species (ROS) production and the in vitro antibacterial activity against two antibiotic resistant Staphylococcus aureus strains was established. LCNP substantially improved the antibacterial activity of GaPP, completely eradicating S. aureus and MRSA planktonic cultures, using a GaPP concentration of 0.8 µM and light dose 1.9 J/cm2. At the same concentration and light dose, unformulated GaPP triggered only a 4 log10 and 2 log10 reduction in respective planktonic cultures. Most importantly, the activity of GaPP against biofilms was enhanced by 2-fold compared to unformulated GaPP, reducing the viability of S. aureus and MRSA biofilms by 8 log10 and 5 log10, respectively. The biosafety of photoactivated GaPP-LCNP was evaluated against human fibroblasts, which indicated a high safety profile of the treatment. Therefore, these findings encourage further investigations of GaPP-LCNP as a potential treatment for localized chronic infections.

A Comparison of Chitosan, Mesoporous Silica and Poly(lactic-co-glycolic) Acid Nanocarriers for Optimising Intestinal Uptake of Oral Protein Therapeutics.

Efficacious oral delivery of therapeutic proteins remains challenging and nanoparticulate approaches are gaining interest for enhancing their permeability. In this study, we explore the ability for three comparably sized nanocarriers, with diverse physicochemical properties [i.e., chitosan (CSNP), mesoporous silica nanoparticles (MSNP) and poly(lactic-co-glycolic) acid (PLGA-NP)], to successfully facilitate epithelial uptake of a model protein, ovalbumin (OVA). We report the effect of nanoparticle surface chemistry and nanostructure on protein release, cell toxicity and the uptake mechanism in a Madin Darby Canine Kidney (MDCK) cell model of the intestinal epithelium. All nanocarriers exhibited bi-phasic OVA release kinetics with sustained and incomplete release after 4 days, and more pronounced release from MSNP than either polymeric nanocarriers. CSNP and MSNP displayed the highest cellular uptake, however CSNP was prone to significant dose-dependent toxicity attributed to the cationic surface charge. Approximately 25% of MSNP uptake was governed by a clathrin-independent endocytic mechanism, while CSNP and PLGA-NP uptake was not controlled via any endocytic mechanisms investigated herein. Furthermore, endosomal localisation was observed for CSNP and MSNP, but not for PLGA-NP. These findings may assist in the optimal choice and engineering of nanocarriers for specific intestinal permeation enhancement for oral protein delivery.

Oxidized mesoporous silicon microparticles for improved oral delivery of poorly soluble drugs.

Surface functionalized mesoporous silicon (pSi) microparticles are reported as a solid dispersion carrier for improving dissolution and enhancing the orally administered pharmacokinetics (fasted rat model) of indomethacin (IMC), employed as a model poorly soluble BCS type II drug. IMC was loaded via immersion/solvent evaporation onto the thermally oxidized pSi particles, which provide a stable hydrophilic matrix with a nanoporous structure. The solid state properties of IMC loaded pSi were characterized by Fourier transform infrared spectroscopy, X-ray powder diffraction, differential scanning calorimetry and thermogravimetric analysis. IMC molecules are encapsulated in a noncrystalline state due to geometric confinement in the nanopores; stability of the noncrystalline state has been demonstrated for several months under accelerated storage conditions. The pSi carrier facilitates accelerated immediate release of IMC and enhanced oral delivery performance in comparison with crystalline indomethacin and Indocid i.e. a 4-times reduction on T(max), a 200% increase on C(max) and a significant increase in bioavailability. The in vitro-in vivo correlation is discussed based on the noncompartment model and gives insight into the delivery mechanism for the pSi carrier.

Thermal oxidation for controlling protein interactions with porous silicon.

Thermal oxidation of porous silicon (pSi) has been used to control interactions with three proteins; lysozyme, papain, and human serum albumin (HSA) enabling the influences of protein structure, molecular weight, and charge to be elucidated. Adsorption behavior was assessed via adsorption isotherms while the structures of adsorbed proteins were investigated using a bioactivity assay, FTIR, and zeta potential. Time-of-flight secondary ion mass spectrometry was used to examine protein pore penetration. High protein adsorption onto unoxidized pSi (240-610 microg/m(2)) was attributed to predominately hydrophobic interactions which resulted in structural changes of the adsorbed proteins and significant loss of bioactivity. Thermal oxidation at 400 and 800 degrees C significantly reduced protein adsorption (80-485 microg/m(2)) by reducing hydrophobicity. Oxidation of pSi modified the protein adsorption mechanisms to solely electrostatic attraction for positively charged proteins and structural rearrangement for negatively charged proteins. Adsorption via electrostatic attraction preserved protein bioactivity and zeta potential, thus inferring a retention of their native structure. In contrast, the negative charge and globular structure of HSA resulted in a loss of structure. We have demonstrated that thermal oxidation of pSi can be used to control protein interactions, adsorbed structure, and bioactivity.

Detecting the presence of denatured human serum albumin in an adsorbed protein monolayer using TOF-SIMS.

We demonstrate the application of time-of-flight secondary ion mass spectrometry (TOF-SIMS) in conjunction with multivariate statistics to differentiate trace levels of denatured proteins in adsorbed monolayers; specifically, human serum albumin (HSA) on oxidized silicon substrates. Subtle differences in protein conformation due to thermal denaturation of HSA, unable to be determined by dynamic light scattering nor circular dichroism, were differentiated by TOF-SIMS. The fragmentation pattern is highly sensitive to protein conformation, allowing assessment of relative amounts of proteins in mixtures and quantifying amounts of denatured protein in a sample. Discussion is presented on ascribing orientation and conformational differences between samples based upon TOF-SIMS spectra. This has implications for detecting denatured protein in biotechnology and medical applications.

Mechanistic insight into cell growth, internalization, and cytotoxicity of PAMAM dendrimers.

We report on the role of PAMAM dendrimer concentration and generation (G2, G4, G6) on cell growth and cytotoxicity in HEK293T and HeLa cell lines and make comparisons with dendrimer-induced leakage from liposomes to probe the mechanisms in action. Specifically, we observed a striking transition from cell growth enhancement to a reduction in cell viability at a critical PAMAM dendrimer concentration, that is, approximately 500 nM. Confocal microscopy studies show evidence of a transition from cell membrane adhesion to cell internalization and cell nucleus interaction at equivalent dendrimer concentrations. A dendrimer concentration window of 500-700 nM was identified for effective cell internalization without significant cytotoxicity. Though liposome leakage correlated with cytotoxicity, no quantitative agreement was observed, that is, cells are 100 times (based on surface coverage) more resistant to dendrimers than liposomes. These findings have significant implications in the design of effective drug/gene delivery vehicles based on dendrimers.

Oral delivery of protein-based therapeutics: Gastroprotective strategies, physiological barriers and in vitro permeability prediction.

The number of biological molecules emerging as therapeutics is growing exponentially due to their higher specificity and tolerability profiles compared to small molecules. Despite this, their traditionally parenteral delivery often results in poor patient compliance and incomplete treatment. Current research is focussed on developing effective oral delivery strategies to facilitate administration of these biomolecules, however no universal method exists to simultaneously provide gastric protection as well as enhance transport across the gastrointestinal epithelium. Furthermore, for efficient formulation development it is imperative that we can reliably analyse permeability of biomolecules through the gastrointestinal tract, highlighting the importance of the continual development and ongoing evaluation of in vitro predictive permeability tools. Here, we review the physiological obstacles associated with peptide and protein delivery throughout the gastrointestinal tract. Furthermore, we highlight methods utilised to circumvent these barriers and promote improved intestinal permeability. Lastly, we explore in vitro models employed to predict epithelial transport. Key findings highlight the need to carefully understand gastrointestinal physiology, allowing specific engineering of oral delivery systems for biomolecules. Significant importance is placed upon understanding enzymatic degradation susceptibility as well as uptake mechanisms for particulate and protein-based therapeutics for the development of successful oral protein delivery platforms.

A liposome-micelle-hybrid (LMH) oral delivery system for poorly water-soluble drugs: Enhancing solubilisation and intestinal transport.

A novel liposome-micelle-hybrid (LMH) carrier system was developed as a superior oral drug delivery platform compared to conventional liposome or micelle formulations. The optimal LMH system was engineered by encapsulating TPGS micelles in the aqueous core of liposomes and its efficacy for oral delivery was demonstrated using lovastatin (LOV) as a model poorly soluble drug with P-gp (permeability glycoprotein) limited intestinal absorption. LOV-LMH was characterised as unilamellar, spherical vesicles encapsulating micellar structures within the interior aqueous core and showing an average diameter below 200 nm. LMH demonstrated enhanced drug loading, water apparent solubility and extended/controlled release of LOV compared to conventional liposomes and micelles. LMH exhibited enhanced LOV absorption and transportation in a Caco-2 cell monolayer model of the intestine by inhibiting the P-gp transporter system compared to free LOV. The LMH system is a promising novel oral delivery approach for enhancing bioavailability of poorly water-soluble drugs, especially those presenting P-gp effluxes limited absorption.

The encapsulation and release of guanosine from PEGylated liposomes.

The encapsulation and release kinetics of guanosine from liposomes and polyethylene glycol (PEG)-modified liposomes are reported. Specifically, the influence of PEG chain length, PEGylation level, lipid type, drug-loading level, temperature, and solution conditions (i.e., salt and pH effects) on the rate and mechanism for release have been determined. Increasing PEGylation significantly reduced the guanosine release kinetics; this is more significant for greater molecular weight PEG and is correlated with the PEG layer thickness. Further, the mechanism for guanosine release changed from diffusion to interfacial control as the PEG level increased. The interfacial structure introduced by PEG also increased the activation energy required for guanosine transport across the lipid bilayer from 14 to 22 kJ mol(-1). Findings from this study provide further insight into optimizing the formulation of Stealth liposomes.

Controlling and Predicting the Dissolution Kinetics of Thermally Oxidised Mesoporous Silicon Particles: Towards Improved Drug Delivery.

Porous silicon (pSi) continues to receive considerable interest for use in applications ranging from sensors, biological scaffolds, therapeutic delivery systems to theranostics. Critical to all of these applications is pSi degradation and stabilization in biological media. Here we report on progress towards the development of a mechanistic understanding for the dissolution behavior of native (unoxidized) and thermally oxidized (200-600 °C) pSi microparticles. Fourier transform infrared (FTIR) spectroscopy was used to characterize the pSi surface chemistry after thermal oxidation. PSi dissolution was assessed using a USP method II apparatus by monitoring the production of orthosilicic acid, and the influence of gastro-intestinal (GI) fluids were examined. Fitting pSi dissolution kinetics with a sum of the exponential model demonstrated that the dissolution process strongly correlates with the three surface hydride species and their relative reactivity, and was supported by the observed FTIR spectral changes of pSi during dissolution. Finally, the presence of GI proteins was shown to hamper pSi dissolution by adsorption to the pSi surface acting as a barrier preventing water attack. These findings are significant in the optimal design of pSi particles for oral delivery and other controlled drug delivery applications.