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1.
Int J Nanomedicine ; 19: 5419-5437, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38868592

RESUMO

Introduction: Acute myeloid leukemia (AML) remains difficult to treat due to its heterogeneity in molecular landscape, epigenetics and cell signaling alterations. Precision medicine is a major goal in AML therapy towards developing agents that can be used to treat patients with different 'subtypes' in combination with current chemotherapies. We have previously developed dextrin-colistin conjugates to combat the rise in multi-drug resistant bacterial infections and overcome dose-limiting nephrotoxicity. Recent evidence of colistin's anticancer activity, mediated through inhibition of intracellular lysine-specific histone demethylase 1 (LSD1/KDM1A), suggests that dextrin-colistin conjugates could be used to treat cancer cells, including AML. This study aimed to evaluate whether dextrin conjugation (which reduces in vivo toxicity and prolongs plasma half-life) could enhance colistin's cytotoxic effects in myeloid leukemia cell lines and compare the intracellular uptake and localization of the free and conjugated antibiotic. Results: Our results identified a conjugate (containing 8000 g/mol dextrin with 1 mol% succinoylation) that caused significantly increased toxicity in myeloid leukemia cells, compared to free colistin. Dextrin conjugation altered the mechanism of cell death by colistin, from necrosis to caspase 3/7-dependent apoptosis. In contrast, conjugation via a reversible ester linker, instead of an amide, had no effect on the mechanism of the colistin-induced cell death. Live cell confocal microscopy of fluorescently labelled compounds showed both free and dextrin-conjugated colistins were endocytosed and co-localized in lysosomes, and increasing the degree of modification by succinoylation of dextrin significantly reduced colistin internalization. Discussion: Whilst clinical translation of dextrin-colistin conjugates for the treatment of AML is unlikely due to the potential to promote antimicrobial resistance (AMR) and the relatively high colistin concentrations required for anticancer activity, the ability to potentiate the effectiveness of an anticancer drug by polymer conjugation, while reducing side effects and improving biodistribution of the drug, is very attractive, and this approach warrants further investigation.


Assuntos
Apoptose , Colistina , Dextrinas , Colistina/farmacologia , Colistina/química , Colistina/farmacocinética , Dextrinas/química , Dextrinas/farmacologia , Humanos , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Antibacterianos/farmacologia , Antibacterianos/química , Antibacterianos/farmacocinética , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/patologia , Sobrevivência Celular/efeitos dos fármacos
2.
RSC Pharm ; 1(1): 68-79, 2024 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-38646595

RESUMO

The acute kidney injury (AKI) and dose-limiting nephrotoxicity, which occurs in 20-60% of patients following systemic administration of colistin, represents a challenge in the effective treatment of multi-drug resistant Gram-negative infections. To reduce clinical toxicity of colistin and improve targeting to infected/inflamed tissues, we previously developed dextrin-colistin conjugates, whereby colistin is designed to be released by amylase-triggered degradation of dextrin in infected and inflamed tissues, after passive targeting by the enhanced permeability and retention effect. Whilst it was evident in vitro that polymer conjugation can reduce toxicity and prolong plasma half-life, without significant reduction in antimicrobial activity of colistin, it was unclear how dextrin conjugation would alter cellular uptake and localisation of colistin in renal tubular cells in vivo. We discovered that dextrin conjugation effectively reduced colistin's toxicity towards human kidney proximal tubular epithelial cells (HK-2) in vitro, which was mirrored by significantly less cellular uptake of Oregon Green (OG)-labelled dextrin-colistin conjugate, when compared to colistin. Using live-cell confocal imaging, we revealed localisation of both, free and dextrin-bound colistin in endolysosome compartments of HK-2 and NRK-52E cells. Using a murine AKI model, we demonstrated dextrin-colistin conjugation dramatically diminishes both proximal tubular injury and renal accumulation of colistin. These findings reveal new insight into the mechanism by which dextrin conjugation can overcome colistin's renal toxicity and show the potential of polymer conjugation to improve the side effect profile of nephrotoxic drugs.

3.
Nanoscale ; 15(44): 18015-18032, 2023 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-37916389

RESUMO

LipoParticles, core-shell assemblies consisting of a polymer core coated by a lipid membrane, are promising carriers for drug delivery applications with intracellular targets. This is of great interest since it is actually challenging to treat infections involving intracellular bacteria such as bone and joint infections where the bacteria are hidden in osteoblast cells. The present work reports for the first time to the best of our knowledge the proof of enhanced internalization of particles in osteoblast cells thanks to a lipid coating of particles (= LipoParticles). The ca. 300 nm-sized assemblies were elaborated by reorganization of liposomes (composed of DPPC/DPTAP 10/90 mol/mol) onto the surface of poly(lactic-co-glycolic acid) (PLGA) particles, and were characterized by dynamic light scattering (DLS), transmission electron microscopy (TEM), and zetametry. Optimization of these assemblies was also performed by adding poly(ethylene glycol) (PEG) chains on their surface (corresponding to a final formulation of DPPC/DPTAP/DPPE-PEG5000 8/90/2 mol/mol/mol). Interestingly, this provided them colloidal stability after their 20-fold dilution in PBS or cell culture medium, and made possible their freeze-drying without forming aggregates after their re-hydration. Their non-cytotoxicity towards a human osteoblast cell line (MG63) was also demonstrated. The enhanced internalization of LipoParticles in this MG63 cell line, in comparison with PLGA particles, was proven by observations with a confocal laser scanning microscope, as well as by flow cytometry assays. Finally, this efficient internalization of LipoParticles in MG63 cells was confirmed by TEM on ultrathin sections, which also revealed localization close to intracellular Staphylococcus aureus.


Assuntos
Nanopartículas , Polímeros , Humanos , Polímeros/farmacologia , Polietilenoglicóis , Lipossomos , Osteoblastos , Lipídeos , Portadores de Fármacos
4.
Sci Rep ; 12(1): 4986, 2022 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-35322119

RESUMO

In a number of chronic respiratory diseases e.g. cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD), the production of viscous mucin reduces pulmonary function and represents an effective barrier to diffusion of inhaled therapies e.g. antibiotics. Here, a 2-compartment Transwell model was developed to study impaired diffusion of the antibiotic colistin across an artificial sputum (AS) matrix/medium and to quantify its antimicrobial activity against Pseudomonas aeruginosa NH57388A biofilms (alone and in combination with mucolytic therapy). High-performance liquid chromatography coupled with fluorescence detection (HPLC-FLD) revealed that the presence of AS medium significantly reduced the rate of colistin diffusion (> 85% at 48 h; p < 0.05). Addition of alginate oligosaccharide (OligoG CF-5/20) significantly improved colistin diffusion by 3.7 times through mucin-rich AS medium (at 48 h; p < 0.05). Increased diffusion of colistin with OligoG CF-5/20 was shown (using confocal laser scanning microscopy and COMSTAT image analysis) to be associated with significantly increased bacterial killing (p < 0.05). These data support the use of this model to study drug and small molecule delivery across clinically-relevant diffusion barriers. The findings indicate the significant loss of colistin and reduced effectiveness that occurs with mucin binding, and support the use of mucolytics to improve antimicrobial efficacy and lower antibiotic exposure.


Assuntos
Fibrose Cística , Infecções por Pseudomonas , Alginatos/química , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Biofilmes , Colistina/farmacologia , Colistina/uso terapêutico , Fibrose Cística/microbiologia , Humanos , Mucinas/metabolismo , Oligossacarídeos/química , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa
5.
Sci Rep ; 11(1): 10600, 2021 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-34011987

RESUMO

Attachment of polysaccharide carriers is increasingly being used to achieve precision delivery and improved effectiveness of protein and peptide drugs. Although it is clear that their clinical effectiveness relies on the purity and integrity of the conjugate in storage, as well as following administration, instability of polysaccharide-based conjugates can reduce the protective efficacy of the polymer, which may adversely affect the bioactive's potency. As a model, these studies used dextrin-colistin conjugates, with varying degrees of polymer modification (1, 2.5 and 7.5 mol% succinoylation) to assess the effect of storage temperature (- 20, 4, 21 and 37 °C) and duration (up to 12 months) on saccharide and colistin release and antimicrobial activity. Estimation of the proportion of saccharide release (by comparison of area under the curve from size exclusion chromatograms) was more pronounced at higher temperatures (up to 3 and 35% at - 20 °C and 37 °C, respectively after 12 months), however, repeated freeze-thaw did not produce any measurable release of saccharides, while addition of amylase (20, 100, 500 IU/L) caused rapid release of saccharides (> 70% total within 24 h). At all temperatures, conjugates containing the lowest degree of succinoylation released the highest proportion of free colistin, which increased with storage temperature, however no trend in saccharide release was observed. Despite the clear physical effects of prolonged storage, antimicrobial activity of all samples was only altered after storage at 37 °C for 12 months (> threefold decreased activity). These results demonstrate significant release of saccharides from dextrin-colistin conjugates during prolonged storage in buffered solution, especially at elevated temperature, which, in most cases, did not affect antimicrobial activity. These findings provide vital information about the structure-activity relationship of dextrin-colistin conjugates, prior to full-scale commercial development, which can subsequently be applied to other polysaccharide-protein and -peptide conjugates.


Assuntos
Fenômenos Químicos , Colistina/química , Dextrinas/química , Temperatura , Amilases/metabolismo , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Refratometria , Açúcares/análise
6.
J Pharm Sci ; 109(8): 2527-2535, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32428534

RESUMO

To understand and predict the efficacy and toxicity of nanoparticle-based drugs in vivo, the free and entrapped forms of the drug have to be determined using suitable characterization methods. Herein, a solid-phase extraction (SPE) method combined with high-performance liquid chromatography (HPLC) measurements were used to separately quantify free and entrapped cyclosporine A (CsA) in 50 and 120 nm-sized lipid nanoparticles (NPs). Combined with colloidal stability measurements, HPLC quantification of the free and entrapped drug, separated using SPE, was used to monitor the stability of the nanotherapeutics under storage or physiological conditions. The SPE method was proven not to alter the core-shell template of the lipid nanocarriers. Method validation demonstrated suitable linearity, repeatability, accuracy, and specificity to quantify the free, entrapped, and total drug. Under storage conditions, the %free and %entrapped CsA remained constant over 9 weeks for both NPs. Under physiological conditions, the release profile was similar for both buffers/mediums used, indicating a biphasic mode of release. The validated SPE method was proven to be suitable for the determination of a wide range of free versus entrapped compounds.


Assuntos
Nanopartículas , Preparações Farmacêuticas , Cromatografia Líquida de Alta Pressão , Lipídeos , Reprodutibilidade dos Testes , Extração em Fase Sólida
7.
Int J Mol Sci ; 20(22)2019 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-31726778

RESUMO

Nanoparticles have been extensively studied for drug delivery and targeting to specific organs. The functionalization of the nanoparticle surface by site-specific ligands (antibodies, peptides, saccharides) can ensure efficient recognition and binding with relevant biological targets. One of the main challenges in the development of these decorated nanocarriers is the accurate quantification of the amount of ligands on the nanoparticle surface. In this study, nanostructured lipid carriers (NLC) were functionalized with N-acetyl-D-galactosamine (GalNAc) units, known to target the asialoglycoprotein receptor (ASGPR). Different molar percentages of GalNAc-functionalized surfactant (0%, 2%, 5%, and 14%) were used in the formulation. Based on ultra-high-performance liquid chromatography separation and evaporative light-scattering detection (UPLC-ELSD), an analytical method was developed to specifically quantify the amount of GalNAc units present at the NLC surface. This method allowed the accurate quantification of GalNAc surfactant and therefore gave some insights into the structural parameters of these multivalent ligand systems. Our data show that the GalNAc decorated NLC possess large numbers of ligands at their surface and suitable distances between them for efficient multivalent interaction with the ASGPR, and therefore promising liver-targeting efficiency.


Assuntos
Portadores de Fármacos/química , Galactosamina/química , Lipídeos/química , Nanopartículas/química , Tensoativos/química
8.
Langmuir ; 35(27): 8984-8995, 2019 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-31244247

RESUMO

Cisplatin ( cis-diaminedichloroplatinum(II), CDDP) plays a crucial role in the treatment of various malignant tumors. However, its clinical efficacy and applicability are restricted by issues of toxicity and resistance. Here, for drug delivery purposes, the outer surface of MCM-41 mesoporous silica nanoparticles (MSNs) was functionalized with poly(ethylene glycol) ( Mw = 10 000 g/mol) or low-molecular-weight ( Mw = 1800 g/mol) branched polyethyleneimine (PEI). Given the strong affinity of sulfur for platinum, thiol-functionalized MSNs were synthesized for comparison by co-condensation with (3-mercaptopropyl)triethoxysilane. CDDP loading was performed either by adsorption or impregnation in aqueous media without the use of dimethyl sulfoxide as a solubilizer. CDDP loading capacities obtained by impregnation were higher than those obtained by adsorption and varied from 3.9 to 16.1 wt %, depending on the functional group. Loaded nanomaterials were characterized by scanning electron microscopy, scanning transmission electron microscopy-high-angle annular dark-field, and Raman spectroscopy. Depending on the functional groups, platinum-based species were either dispersed in the nanomaterials as nanocrystals or uniformly distributed as molecular species. The spectral signature of CDDP was strongly modified when platinum species were homogeneously distributed within the nanomaterials. Preliminary drug release studies performed at 37 °C showed that the behavior of CDDP-loaded MSNs strongly depends on the nature of the present functional groups. Among the functionalization routes investigated in this paper, PEI-based functionalization showed the most promising results for further applications in controlled drug release with the absence of burst release and a sustained release over 72 h.


Assuntos
Antineoplásicos/química , Cisplatino/química , Nanopartículas/química , Dióxido de Silício/química , Estrutura Molecular , Tamanho da Partícula , Porosidade , Propriedades de Superfície
9.
Mol Pharm ; 16(7): 3199-3207, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31125239

RESUMO

Polymer masked-unmasked protein therapy (PUMPT) uses conjugation of a biodegradable polymer, such as dextrin, hyaluronic acid, or poly(l-glutamic acid), to mask a protein or peptide's activity; subsequent locally triggered degradation of the polymer at the target site regenerates bioactivity in a controllable fashion. Although the concept of PUMPT is well established, the relationship between protein unmasking and reinstatement of bioactivity is unclear. Here, we used dextrin-colistin conjugates to study the relationship between the molecular structure (degree of unmasking) and biological activity. Size exclusion chromatography was employed to collect fractions of differentially degraded conjugates and ultraperformance liquid chromatography-mass spectrometry (UPLC-MS) employed to characterize the corresponding structures. Antimicrobial activity was studied using a minimum inhibitory concentration (MIC) assay and confocal laser scanning microscopy of LIVE/DEAD-stained biofilms with COMSTAT analysis. In vitro toxicity of the degraded conjugate was assessed using an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. UPLC-MS revealed that the fully "unmasked" dextrin-colistin conjugate composed of colistin bound to at least one linker, whereas larger species were composed of colistin with varying lengths of glucose units attached. Increasing the degree of dextrin modification by succinoylation typically led to a greater number of linkers bound to colistin. Greater antimicrobial and antibiofilm activity were observed for the fully "unmasked" conjugate compared to the partially degraded species (MIC = 0.25 and 2-8 µg/mL, respectively), whereas dextrin conjugation reduced colistin's in vitro toxicity toward kidney cells, even after complete unmasking. This study highlights the importance of defining the structure-antimicrobial activity relationship for novel antibiotic derivatives and demonstrates the suitability of LC-MS to aid the design of biodegradable polymer-antibiotic conjugates.


Assuntos
Amilases/metabolismo , Colistina/química , Colistina/metabolismo , Dextrinas/química , Dextrinas/metabolismo , Composição de Medicamentos/métodos , Sistemas de Liberação de Medicamentos/métodos , Antibacterianos/química , Antibacterianos/metabolismo , Biofilmes/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cromatografia em Gel , Escherichia coli/efeitos dos fármacos , Humanos , Túbulos Renais Proximais/citologia , Espectrometria de Massas , Testes de Sensibilidade Microbiana , Microscopia Confocal , Estrutura Molecular
10.
Int J Pharm ; 566: 11-23, 2019 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-31112794

RESUMO

Lipid nanocarriers incorporating glycerides, polyethylene glycol (PEG)-stearates and phospholipids have attracted great attention for in vivo diagnostic, in vivo imaging, activated or non-activated targeted drug delivery. For quality control purposes, the development of appropriate methods for the quantification of their lipid components is needed. In the present study, we developed an analytical method for lipid quantification in formulated nanoparticles. PEG-stearates and glycerides were analyzed in a single run by RP-UPLC-ELSD using a two-step gradient elution program, while the analysis of phospholipids was accomplished by HILIC-UPLC-ELSD after isolation using an SPE silica column. Using both isolated compounds and commercial lipid standards, calibration curves were produced using second-order polynomials to attain the quantitative evaluation of each lipid excipient. Relative standard deviation of all analytes was between 0.9% and 5.3% for intra-day precision and recovery ranged from 83.5% to 112.2%. The presented method was successfully implemented to study the manufacturing process and stability of the formulated lipid excipients during long-term storage and accelerated conditions. The formulation lipid yield was determined and found equal to 82.5%.


Assuntos
Portadores de Fármacos/química , Excipientes/química , Lipídeos/química , Nanopartículas/química , Calibragem , Cromatografia Líquida de Alta Pressão , Luz , Reprodutibilidade dos Testes , Espalhamento de Radiação
11.
Int J Pharm ; 532(2): 779-789, 2017 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-28619458

RESUMO

A reversed-phase (RP) high-performance liquid chromatography (HPLC) method for the content determination of IR780-oleyl (IRO) dye in lipid nanoparticles was developed and validated. Chromatographic separation was performed on a RP C18 column with a gradient program of water and acetonitrile both with 0.1% (v/v) TFA, at a flow rate of 1.0mL/min and a total run of 21min. IRO dye detection was made by fluorescence at emission wavelength of 773nm (excitation wavelength: 744nm). According to ICH guidelines, the developed method was shown to be specific, linear in the range 3-8µg/mL (R2=0.9998), precise at the intra-day and inter-day levels as reflected by the coefficient of variation (CV≤1.98%) at three different concentrations (4, 6 and 8 µg/mL) and accurate, with recovery rates between 98.2-101.6% and 99.2-100.5%. The detection and quantitation limits were 0.41 and 1.24µg/mL, respectively. Stability studies of sample processing showed that IRO dye was stable after 24h in the autosampler or after three freeze/thaw cycles. Combined with fluorescence measurements, the developed method was successfully applied to optimize the loading capacity of IRO dye in the core of lipid nanoparticles.


Assuntos
Corantes Fluorescentes/análise , Indóis/análise , Nanopartículas/química , Cromatografia Líquida de Alta Pressão/métodos , Fluorescência , Corantes Fluorescentes/química , Indóis/química , Lipídeos/química , Reprodutibilidade dos Testes
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