RESUMEN
Inhalable nanomedicines are increasingly being developed to optimise the pharmaceutical treatment of respiratory diseases. Large lipid-based nanosystems at the forefront of the inhalable nanomedicines development pipeline, though, have a number of limitations. The objective of this study was, therefore, to investigate the utility of novel small lipidated sulfoxide polymers based on poly(2-(methylsulfinyl)ethyl acrylate) (PMSEA) as inhalable drug delivery platforms with tuneable membrane permeability imparted by differential albumin binding kinetics. Linear PMSEA (5 kDa) was used as a hydrophilic polymer backbone with excellent anti-fouling and stealth properties compared to poly(ethylene glycol). Terminal lipids comprising single (1C2, 1C12) or double (2C12) chain diglycerides were installed to provide differing affinities for albumin and, by extension, albumin trafficking pathways in the lungs. Albumin binding kinetics, cytotoxicity, lung mucus penetration and cellular uptake and permeability through key cellular barriers in the lungs were examined in vitro. The polymers showed good mucus penetration and no cytotoxicity over 24 h at up to 1 mg ml-1. While 1C2-showed no interaction with albumin, 1C12-PMSEA and 2C12-PMSEA bound albumin with KD values of approximately 76 and 10 µM, respectively. Despite binding to albumin, 2C12-PMSEA showed reduced cell uptake and membrane permeability compared to the smaller polymers and the presence of albumin had little effect on cell uptake and membrane permeability. While PMSEA strongly shielded these lipids from albumin, the data suggest that there is scope to tune the lipid component of these systems to control membrane permeability and cellular interactions in the lungs to tailor drug disposition in the lungs.
Asunto(s)
Lípidos , Humanos , Animales , Lípidos/química , Polímeros/química , Administración por Inhalación , Sistemas de Liberación de Medicamentos , Albúminas/química , Albúminas/metabolismo , Pulmón/metabolismo , Unión Proteica , Portadores de Fármacos/químicaRESUMEN
The increasing resistance of clinically relevant microbes against current commercially available antimicrobials underpins the urgent need for alternative and novel treatment strategies. Cationic lipidated oligomers (CLOs) are innovative alternatives to antimicrobial peptides and have reported antimicrobial potential. An understanding of their antimicrobial mechanism of action is required to rationally design future treatment strategies for CLOs, either in monotherapy or synergistic combinations. In the present study, metabolomics was used to investigate the potential metabolic pathways involved in the mechanisms of antibacterial activity of one CLO, C12-o-(BG-D)-10, which we have previously shown to be effective against methicillin-resistant Staphylococcus aureus (MRSA) ATCC 43300. The metabolomes of MRSA ATCC 43300 at 1, 3, and 6 h following treatment with C12-o-(BG-D)-10 (48 µg/mL, i.e., 3× MIC) were compared to those of the untreated controls. Our findings reveal that the studied CLO, C12-o-(BG-D)-10, disorganized the bacterial membrane as the first step toward its antimicrobial effect, as evidenced by marked perturbations in the bacterial membrane lipids and peptidoglycan biosynthesis observed at early time points, i.e., 1 and 3 h. Central carbon metabolism and the biosynthesis of DNA, RNA, and arginine were also vigorously perturbed, mainly at early time points. Moreover, bacterial cells were under osmotic and oxidative stress across all time points, as evident by perturbations of trehalose biosynthesis and pentose phosphate shunt. Overall, this metabolomics study has, for the first time, revealed that the antimicrobial action of C12-o-(BG-D)-10 may potentially stem from the dysregulation of multiple metabolic pathways.IMPORTANCEAntimicrobial resistance poses a significant challenge to healthcare systems worldwide. Novel anti-infective therapeutics are urgently needed to combat drug-resistant microorganisms. Cationic lipidated oligomers (CLOs) show promise as new antibacterial agents against Gram-positive pathogens like methicillin-resistant Staphylococcus aureus (MRSA). Understanding their molecular mechanism(s) of antimicrobial action may help design synergistic CLO treatments along with monotherapy. Here, we describe the first metabolomics study to investigate the killing mechanism(s) of CLOs against MRSA. The results of our study indicate that the CLO, C12-o-(BG-D)-10, had a notable impact on the biosynthesis and organization of the bacterial cell envelope. C12-o-(BG-D)-10 also inhibits arginine, histidine, central carbon metabolism, and trehalose production, adding to its antibacterial characteristics. This work illuminates the unique mechanism of action of C12-o-(BG-D)-10 and opens an avenue to design innovative antibacterial oligomers/polymers for future clinical applications.
Asunto(s)
Antibacterianos , Metabolómica , Staphylococcus aureus Resistente a Meticilina , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Metabolómica/métodos , Antibacterianos/farmacología , Antibacterianos/química , Pruebas de Sensibilidad Microbiana , Cationes/química , Cationes/metabolismo , Cationes/farmacologíaRESUMEN
Delivery to peripheral lymphatics can be achieved following interstitial administration of nano-sized delivery systems (nanoparticles, liposomes, dendrimers etc) or molecules that hitchhike on endogenous nano-sized carriers (such as albumin). The published work concerning the hitchhiking approach has mostly focussed on the lymphatic uptake of vaccines conjugated directly to albumin binding moieties (ABMs such as lipids, Evans blue dye derivatives or peptides) and their subsequent trafficking into draining lymph nodes. The mechanisms underpinning access and transport of these constructs into lymph fluid, including potential interaction with other endogenous nanocarriers such as lipoproteins, have largely been ignored. Recently, we described a series of brush polyethylene glycol (PEG) polymers containing end terminal short-chain or medium-chain hydrocarbon tails (1C2 or 1C12, respectively), cholesterol moiety (Cho), or medium-chain or long-chain diacylglycerols (2C12 or 2C18, respectively). We evaluated the association of these materials with albumin and lipoprotein in rat plasma, and their intravenous (IV) and subcutaneous (SC) pharmacokinetic profiles. Here we fully detail the association of this suite of polymers with albumin and lipoproteins in rat lymph, which is expected to facilitate lymph transport of the materials from the SC injection site. Additionally, we characterise the thoracic lymph uptake, tissue and lymph node biodistribution of the lipidated brush PEG polymers following SC administration to thoracic lymph cannulated rats. All polymers had moderate lymphatic uptake in rats following SC dosing with the lymph uptake higher for 1C2-PEG, 2C12-PEG and 2C18-PEG (5.8%, 5.9% and 6.7% dose in lymph, respectively) compared with 1C12-PEG and Cho-PEG (both 1.5% dose in lymph). The enhanced lymph uptake of 1C2-PEG, 2C12-PEG and 2C18-PEG appeared related to their association profile with different lipoproteins. The five polymers displayed different biodistribution patterns in major organs and tissues in mice. All polymers reached immune cells deep within the inguinal lymph nodes of mice following SC dosing. The ability to access these immune cells suggests the potential of the polymers as platforms for the delivery of vaccines and immunotherapies. Future studies will focus on evaluating the lymphatic targeting and therapeutic potential of drug or vaccine-loaded polymers in pre-clinical disease models.
Asunto(s)
Polietilenglicoles , Animales , Polietilenglicoles/química , Polietilenglicoles/farmacocinética , Distribución Tisular , Masculino , Ratas Sprague-Dawley , Lípidos/química , Ganglios Linfáticos/metabolismo , Linfa/metabolismo , Ratones , Ratas , Albúminas/administración & dosificación , Albúminas/farmacocinética , Lipoproteínas/farmacocinética , Lipoproteínas/administración & dosificación , FemeninoRESUMEN
OBJECTIVES: Nanomedicines are being actively developed as inhalable drug delivery systems. However, there is a distinct utility in developing smaller polymeric systems that can bind albumin in the lungs. We therefore examined the pulmonary pharmacokinetic behavior of a series of lipidated brush-PEG (5 kDa) polymers conjugated to 1C2, 1C12 lipid or 2C12 lipids. METHODS: The pulmonary pharmacokinetics, patterns of lung clearance and safety of polymers were examined in rats. Permeability through monolayers of primary human alveolar epithelia, small airway epithelia and lung microvascular endothelium were also investigated, along with lung mucus penetration and cell uptake. RESULTS: Polymers showed similar pulmonary pharmacokinetic behavior and patterns of lung clearance, irrespective of lipid molecular weight and albumin binding capacity, with up to 30% of the dose absorbed from the lungs over 24 h. 1C12-PEG showed the greatest safety in the lungs. Based on its larger size, 2C12-PEG also showed the lowest mucus and cell membrane permeability of the three polymers. While albumin had no significant effect on membrane transport, the cell uptake of C12-conjugated PEGs were increased in alveolar epithelial cells. CONCLUSION: Lipidated brush-PEG polymers composed of 1C12 lipid may provide a useful and novel alternative to large nanomaterials as inhalable drug delivery systems.
Asunto(s)
Polietilenglicoles , Polímeros , Ratas , Humanos , Animales , Polímeros/química , Polietilenglicoles/química , Peso Molecular , Sistemas de Liberación de Medicamentos , Pulmón/metabolismo , Lípidos/química , Albúminas/metabolismoRESUMEN
Polymeric prodrugs have been applied to control the delivery of various types of therapeutics. Similarly, conjugation of peptide therapeutics to lipids has been used to prolong systemic exposure. Here, we extend on these two approaches by conjugating brush polyethylene glycol (PEG) polymers with different lipid components including short-chain (1C2) or medium-chain (1C12) monoalkyl hydrocarbon tails, cholesterol (Cho), and diacylglycerols composed of two medium-chain (2C12) or long-chain (2C18) fatty acids. We uniquely evaluate the integration of these lipid-polymers into endogenous lipid trafficking pathways (albumin and lipoproteins) and the impact of lipid conjugation on plasma pharmacokinetics after intravenous (IV) and subcutaneous (SC) dosing to cannulated rats. The IV and SC elimination half-lives of Cho-PEG (13 and 22 h, respectively), 2C12-PEG (11 and 17 h, respectively) and 2C18-PEG (12 h for both) were prolonged compared to 1C2-PEG (3 h for both) and 1C12-PEG (4 h for both). Interestingly, 1C2-PEG and 1C12-PEG had higher SC bioavailability (40 % and 52 %, respectively) compared to Cho-PEG, 2C12-PEG and 2C18-PEG (25 %, 24 % and 23 %, respectively). These differences in pharmacokinetics may be explained by the different association patterns of the polymers with rat serum albumin (RSA), bovine serum albumin (BSA) and lipoproteins. For example, in pooled plasma (from IV pharmacokinetic studies), 2C18-PEG had the highest recovery in the high-density lipoprotein (HDL) fraction. In conclusion, the pharmacokinetics of brush PEG polymers can be tuned via conjugation with different lipids, which can be utilised to tune the elimination half-life, biodistribution and effect of therapeutics for a range of medical applications. STATEMENT OF SIGNIFICANCE: Lipidation of therapeutics such as peptides has been employed to extend their plasma half-life by promoting binding to serum albumin, providing protection against rapid clearance. Here we design and evaluate innovative biomaterials consisting of brush polyethylene glycol polymers conjugated with different lipids. Importantly, we show for the first time that lipidated polymeric materials associate with endogenous lipoprotein trafficking pathways and this, in addition to albumin binding, controls their plasma pharmacokinetics. We find that conjugation to dialkyl lipids and cholesterol leads to higher association with lipid trafficking pathways, and more sustained plasma exposure, compared to conjugation to short and monoalkyl lipids. Our lipidated polymers can thus be utilised as delivery platforms to tune the plasma half-life of various pharmaceuticals.
Asunto(s)
Polietilenglicoles , Polímeros , Ratas , Animales , Polietilenglicoles/farmacología , Distribución Tisular , Semivida , Péptidos/farmacología , Lipoproteínas HDL , Colesterol , Albúmina Sérica Bovina/farmacologíaRESUMEN
Soft polymer nanoparticles designed to disassemble and release an antagonist of the neurokinin 1 receptor (NK1R) in endosomes provide efficacious yet transient relief from chronic pain. These micellar nanoparticles are unstable and rapidly release cargo, which may limit the duration of analgesia. We examined the efficacy of stable star polymer nanostars containing the NK1R antagonist aprepitant-amine for the treatment of chronic pain in mice. Nanostars continually released cargo for 24 h, trafficked through the endosomal system, and disrupted NK1R endosomal signaling. After intrathecal injection, nanostars accumulated in endosomes of spinal neurons. Nanostar-aprepitant reversed mechanical, thermal and cold allodynia and normalized nociceptive behavior more efficaciously than free aprepitant in preclinical models of neuropathic and inflammatory pain. Analgesia was maintained for >10 h. The sustained endosomal delivery of antagonists from slow-release nanostars provides effective and long-lasting reversal of chronic pain.
Asunto(s)
Dolor Crónico , Antagonistas del Receptor de Neuroquinina-1 , Animales , Aprepitant/farmacología , Aprepitant/uso terapéutico , Dolor Crónico/tratamiento farmacológico , Endosomas , Ratones , Antagonistas del Receptor de Neuroquinina-1/farmacología , Antagonistas del Receptor de Neuroquinina-1/uso terapéutico , Polímeros/farmacologíaRESUMEN
PURPOSE: Directing nanoparticles to cancer cells without using antibodies is of great interest. Subtle changes to the surface chemistry of nanoparticles can significantly affect their biological fate, including their propensity to associate with different cell populations. For instance, nanoparticles functionalized with thiol-reactive groups can potentially enhance association with cells that over-express cell-surface thiol groups. The potential of such an approach for enhancing drug delivery for childhood acute lymphoblastic leukemia (ALL) cells has not been investigated. Herein, we investigate the impact of thiol-reactive star polymers on the cellular association and the mechanisms of uptake of the nanoparticles. METHODS: We prepared fluorescently labeled star polymers functionalized with an mPEG brush corona and pyridyl disulfide to examine how reactivity to exofacial thiols impacts cellular association with ALL cells. We also studied how variations to the mPEG brush composition could potentially be used as a secondary method for controlling the extent of cell association. Specifically, we examined how the inclusion of shorter diethylene glycol brush moieties into the nanoparticle corona could be used to further influence cell association. RESULTS: Star polymers incorporating both thiol-reactive and diethylene glycol brush moieties exhibited the highest cellular association, followed by those functionalized solely with thiol reactive groups compared to control nanoparticles in T and B pediatric ALL patient-derived xenografts harvested from the spleens and bone marrow of immunodeficient mice. Transfection of cells with an early endosomal marker and imaging with correlative light and electron microscopy confirmed cellular uptake. Endocytosis inhibitors revealed dynamin-dependent clathrin-mediated endocytosis as the main uptake pathway for all the star polymers. CONCLUSION: Thiol-reactive star polymers having an mPEG brush corona that includes a proportion of diethylene glycol brush moieties represent a potential strategy for improved leukemia cell delivery.
Asunto(s)
Sistemas de Liberación de Medicamentos/métodos , Nanopartículas/administración & dosificación , Nanopartículas/química , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Compuestos de Sulfhidrilo/química , Animales , Línea Celular Tumoral , Endocitosis/efectos de los fármacos , Glicoles de Etileno/química , Humanos , Ratones , Polietilenglicoles/química , Polímeros/síntesis química , Polímeros/química , Polímeros/farmacocinética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
This report details the synthesis of lipidated 2-vinyl-4,4-dimethyl-5-oxazolone (VDM) oligomers via an optimised Cu(0)-mediated reversible-deactivation radical polymerisation approach, and the use of these oligomers as a versatile functional platform for the rapid generation of antimicrobial materials. The relative amounts of CuBr2 and Me6TREN were optimised to allow the fast and controlled polymerisation of VDM. These conditions were then used with the initiators ethyl 2-bromoisobutyrate, dodecyl 2-bromoisobutyrate, and (R)-3-((2-bromo-2-methylpropanoyl)oxy)propane-1,2-diyl didodecanoate to synthesise a library of oligo(VDM) (degree of polymerisation = 10) with ethyl, dodecyl or diglyceride end-groups. Subsequently, ring-opening of the pendant oxazolone group with various amines (i.e., 2-(2-aminoethyl)-1,3-di-Boc-guanidine, 1-(3-aminopropyl)imidazole, N-Boc-ethylenediamine, or N,N-dimethylethylenediamine) expanded the library to give 12 functional oligomers incorporating different cationic and lipid elements. The antimicrobial activities of these oligomers were assessed against a palette of bacteria and fungi: i.e. Staphylococcus aureus, Escherichia coli, Candida albicans, and Cryptococcus neoformans. The oligomers generally exhibited the greatest activity against the fungus, C. neoformans, with a minimum inhibitory concentration of 1 µg mL-1 (comparable to the clinically approved antifungal fluconazole). To assess haemocompatibility, the oligomers were assayed against erythrocytes, with the primary amine or guanidine containing C12 and 2C12 oligomers exhibiting greater lysis against the red blood cells (HC10 values between 7.1 and 43 µg mL-1) than their imidazole and tertiary amine counterparts (HC10 of >217 µg mL-1). Oligomers showed the greatest selectivity for C. neoformans, with the C12- and 2C12-tertiary amine and C12-imidazole oligomers possessing the greatest selectivity of >54-109. These results demonstrate the utility of reactive oligomers for rapidly assessing structure-property relationships for antibacterial and antifungal materials.
Asunto(s)
Antibacterianos/uso terapéutico , Cloruro de Polivinilo/química , Antibacterianos/farmacología , Estructura Molecular , Relación Estructura-ActividadRESUMEN
There is growing interest in synthetic polymers which co-opt the structural features of naturally occurring antimicrobial peptides. However, our understanding of how macromolecular architecture affects antibacterial activity remains limited. To address this, we investigated whether varying architectures of a series of block and statistical co-oligomers influenced antibacterial and hemolytic activity. Cu(0)-mediated polymerization was used to synthesize oligomers constituting 2-(Boc-amino)ethyl acrylate units and either diethylene glycol ethyl ether acrylate (DEGEEA) or poly(ethylene glycol) methyl ether acrylate units with varying macromolecular architecture; subsequent deprotection produced primary amine functional oligomers. Further guanylation provided an additional series of antimicrobial candidates. Both chemical composition and macromolecular architecture were shown to affect antimicrobial activity. A broad spectrum antibacterial oligomer (containing guanidine moieties and DEGEEA units) was identified that possessed promising activity (MIC = 2 µg mL-1) toward both Gram-negative and Gram-positive bacteria. Bacterial membrane permeabilization was identified as an important contributor to the mechanism of action.
Asunto(s)
Alquilantes/química , Antibacterianos/química , Péptidos Catiónicos Antimicrobianos/química , Estructura Molecular , Acrilatos/química , Acrilatos/farmacología , Alquilantes/farmacología , Alquilación , Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Cationes/química , Cationes/farmacología , Sustancias Macromoleculares/química , Sustancias Macromoleculares/farmacología , Pruebas de Sensibilidad Microbiana , Polimerizacion/efectos de los fármacos , Polímeros/química , Relación Estructura-ActividadRESUMEN
Cu(0)-mediated polymerization was employed to synthesize a library of structurally varied cationic polymers and their application as antibacterial peptide mimics was assessed. Eight platform polymers were first synthesized with low degrees of polymerization (DP) using (2-Boc-amino)ethyl acrylate as the monomer and either ethyl α-bromoisobutyrate or dodecyl 2-bromoisobutyrate as the initiator (thus providing hydrocarbon chain termini of C2 or C12, respectively). A two-step modification strategy was then employed to generate the final sixteen-member polymer library. Specifically, an initial deprotection was employed to reveal the primary amine cationic polymers, followed by guanylation. The biocidal activity of these cationic polymers was assessed against various strains of Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus pneumoniae. Polymers having a short segment of guanidine units and a C12 hydrophobic terminus were shown to provide the broadest antimicrobial activity against the panel of isolates studied, with MIC values approaching those for Gram-positive targeting antibacterial peptides: daptomycin and vancomycin. The C12-terminated guanidine functional polymers were assayed against human red blood cells, and a concomitant increase in haemolysis was observed with decreasing DP. Cytotoxicity was tested against HEK293 and HepG2 cells, with the lowest DP C12-terminated polymer exhibiting minimal toxicity over the concentrations examined, except at the highest concentration. Membrane disruption was identified as the most probable mechanism of bacteria cell killing, as elucidated by membrane permeability testing against E. coli.
RESUMEN
Lipidated polymers, like their protein counterparts, may be useful in fields as diverse as biochemistry and drug delivery. As such, strategies for preparing lipidated polymers with defined molecular architecture are clearly warranted. Herein, we describe a broadly-applicable methodology for synthesizing such lipidated materials, and demonstrate how they can be applied to the preparation of nanostructured drug delivery vehicles.
Asunto(s)
Sistemas de Liberación de Medicamentos , Lípidos/química , Metacrilatos/química , Nanoestructuras/química , Polietilenglicoles/química , Ácidos Polimetacrílicos/química , Portadores de Fármacos/química , Humanos , Lípidos/síntesis química , Metacrilatos/síntesis química , Polietilenglicoles/síntesis química , Ácidos Polimetacrílicos/síntesis químicaRESUMEN
The balance of cationicity and hydrophobicity can profoundly affect the performance of antimicrobial polymers. To this end a library of 24 cationic polymers with uniquely low degrees of polymerization was synthesized via Cu(0)-mediated polymerization, using three different cationic monomers and two initiators: providing two different hydrocarbon chain tail lengths (C2 and C12). The polymers exhibited structure-dependent antibacterial activity when tested against a selection of bacteria, viz, Staphylococcus aureus ATCC 29213, Klebsiella pneumoniae ATCC 13883, Acinetobacter baumannii ATCC 19606, and Pseudomonas aeruginosa ATCC 27853 as a representative palette of Gram-positive and Gram-negative ESKAPE pathogens. The five best-performing polymers were identified for additional testing against the polymyxin-resistant A. baumannii ATCC 19606R strain. Polymers having the lowest DP and a C12 hydrophobic tail were shown to provide the broadest antimicrobial activity against the bacteria panel studied as evidenced by lower minimum inhibitory concentrations (MICs). An optimal polymer composition was identified, and its mechanism of action investigated via membrane permeability testing against Escherichia coli. Membrane disruption was identified as the most probable mechanism for bacteria cell killing.