Understanding the adsorption of salmon calcitonin, antimicrobial peptide AP114 and polymyxin B onto lipid nanocapsules

The adsorption of therapeutic molecules, e.g., peptides, onto nanocarriers is influenced by the properties of the carrier, adsorbed molecule and continuous phase. Hence, through changes in the composition of the nanocarrier and the medium, it should be possible to tune the system to make it capable of efficiently adsorbing peptides. The adsorption of calcitonin, antimicrobial peptide AP114 and polymyxin B onto lipid nanocapsules was investigated. The adsorption data were fitted to a Langmuir isotherm. Dynamic light scattering and laser Doppler velocimetry were used to investigate the changes in the hydrodynamic diameter and zeta potential, respectively, of the nanocarrier. The peptide adsorption was primarily governed by electrostatic forces; however, even without the presence of an ionisable surfactant, a significant amount of each tested molecule was adsorbed due to the enormous surface area of the nanocarriers and to peptide-nanocarrier interactions. The addition of an ionisable lipophilic surfactant, lecithin, improved the adsorption yield, which reached values of up to 100%. The adsorption yield and the properties of the nanocarrier, particularly the zeta potential, depended on the carrier and peptide concentrations and their mixing ratio. The adsorption of all tested molecules obeyed the Langmuir model over a limited concentration range

Lipid-based nanoformulations for peptide delivery

Nanoformulations have attracted a lot of attention because of their size-dependent properties. Among the array of nanoformulations, lipid nanoformulations (LNFs) have evoked increasing interest because of the advantages of their high degree of biocompatibility and versatility. The performance of lipid nanoformulations is greatly influenced by their composition and structure. Therapeutic peptides represent a growing share of the pharmaceutical market. However, the main challenge for their development into commercial products is their inherent physicochemical and biological instability. Important peptides such as insulin, calcitonin and cyclosporin A have been incorporated into LNFs. The association or encapsulation of peptides within lipid-based carriers has shown to protect the labile molecules against enzymatic degradation. This review describes strategies used for the formulation of peptides and some methods used for the assessment of association efficiency. The advantages and drawbacks of such carriers are also described

Synergistic interactions between doxycycline and terpenic components of essential oils encapsulated within lipid nanocapsules against gram negative bacteria

The combination of essential oils (EOs) with antibiotics provides a promising strategy towards combating resistant bacteria. We have selected a mixture of 3 major components extracted from EOs: carvacrol (oregano oil), eugenol (clove oil) and cinnamaldehyde (cinnamon oil). These compounds were successfully encapsulated within lipid nanocapsules (LNCs). The EOs-loaded LNCs were characterised by a noticeably high drug loading of 20% and a very small particle diameter of 114nm. The in vitro interactions between EOs-loaded LNCs and doxycycline were examined via checkerboard titration and time-kill assay against 5 Gram-negative strains: Acinetobacter baumannii SAN, A. baumannii RCH, Klebsiella pneumoniae, Escherichia coli and Pseudomonas aeruginosa. No growth inhibition interactions were found between EOs-loaded LNCs and doxycycline (FIC index between 0.7 and 1.30). However, when bactericidal effects were considered, a synergistic interaction was observed (FBC index equal to 0.5) against all tested strains. A synergistic effect was also observed in time-kill assay (a difference of at least 3 log between the combination and the most active agent alone). Scanning electron microscopy (SEM) was used to visualise the changes in the bacterial membrane. The holes in bacterial envelope and leakage of cellular contents were observed in SE micrographs after exposure to the EOs-LNCs and the doxycycline combination

Internalisation par les oligodendrocytes de nanocapsules lipidiques vectorisées

Introduction Les nanoparticules lipidiques (LNC) constituent un vecteur potentiel de thérapeutiques ciblées à l’échelon cellulaire. Nous avons étudié leur intérêt pour atteindre spécifiquement les oligodendocytes (OL). Objectifs Étudier la pénétration éventuelle de LNC vectorisées avec un peptide (NFL-TBS) que nous avions identifié comme pénétrant dans les OL par endocytose et aux effets proremyélinisants (Fressinaud et Eyer, 2015). Patients et méthodes Les cultures d’OL de rats, en milieu chimiquement défini, étaient traitées par différentes concentrations de LNC identifiées par un fluorochrome (DiD) et adsorbées ou non avec le peptide NFL-TBS. Leur effet sur le développement des OL était observé. La colocalisation intracellulaire du DiD et des marqueurs immunocytochimiques des OL (A2B5, CNP, MBP) était détectée par microscopie confocale (triple/quadruple marquage) et les OL ayant incorporé les LNC étaient quantifiés. Trois expériences étaient tripliquées. Résultats L’adsorption de NFL-TBS permettait la pénétration des LNC dans la majorité des OL, la colocalisation du DiD et des différents marqueurs d’OL confirmant la situation intracellulaire des LNC. La présence des LNC DiD + était abondante et aisément identifiable après quelques heures dans le corps cellulaire. Aucun effet négatif de la présence des LNC n’était observé après plusieurs jours à concentration optimale. Discussion L’internalisation de LNC de taille et concentration optimales n’altère pas le développement des OL. Ces particules représentent de potentiels vecteurs de molécules à visée thérapeutique dans la SEP. Conclusion L’internalisation par les OL in vitro des nanocapsules lipidiques fonctionnalisées par le peptide NFL-TBS, suggère leur éventuel potentiel pour servir de vecteur délivrant des molécules thérapeutiques au cours de la SEP

Formulation and nebulization of fluticasone propionate-loaded lipid nanocarriers.

Inhaled fluticasone propionate (FP) is often prescribed as a first-line therapy for the effective management of pulmonary diseases such as asthma. As nanocarriers offer many advantages over other drug delivery systems, this study investigated the suitability of lipid nanocapsules (LNCs) as a carrier for fluticasone propionate, examining the drug-related factors that should be considered in the formulation design and the behaviour of LNCs with different compositions and properties suspended within aerosol droplets under the relatively hostile conditions of nebulization. By adjusting the formulation conditions, particularly the nanocarrier composition, FP was efficiently encapsulated within the LNCs with a yield of up to 97%, and a concentration comparable to commercially available preparations was achieved. Moreover, testing the solubility of the drug in oil and water and determining the oil/water partition coefficient proved to be useful when assessing the encapsulation of the FP in the LNC formulation. Nebulization did not cause the FP to leak from the formulation, and no phase separation was observed after nebulization. LNCs with a diameter of 100nm containing a smaller amount of surfactant and a larger amount of oil provided a better FP-loading capacity and better stability during nebulization than 30 or 60nm LNCs

Chondroitin-based nanoplexes as peptide delivery systems-Investigations into the self-assembly process, solid-state and extended release characteristics

YesA new type of self-assembled polyelectrolyte complex nanocarrier composed of chondroitin (CHON) and protamine (PROT) was designed and the ability of the carriers to bind salmon calcitonin (sCT) was examined. The response of sCT-loaded CHON/PROT NPs to a change in the properties of the liquid medium, e.g. its pH, composition or ionic strength was studied and in vitro peptide release was assessed. The biocompatibility of the NPs was evaluated in Caco-2 cells. CHON/PROT NPs were successfully obtained with properties that were dependent on the concentration of the polyelectrolytes and their mixing ratio. X-ray diffraction determined the amorphous nature of the negatively charged NPs, while those with the positive surface potential were semi-crystalline. sCT was efficiently associated with the nanocarriers (98-100%) and a notably high drug loading (13-38%) was achieved. The particles had negative zeta potential values and were homogenously dispersed with sizes between 60 and 250 nm. CHON/PROT NPs released less than 10% of the total loaded peptide in the first hour of the in vitro release studies. The enthalpy of the decomposition exotherm correlated with the amount of sCT remaining in NPs after the release experiments. The composition of medium and its ionic strength was found to have a considerable influence on the release of sCT from CHON/PROT NPs. Complexation to CHON markedly reduced the toxic effects exerted by PROT and the NPs were compatible and well tolerated by Caco-2 cells

Reverse micelle-lipid nanocapsules: a novel strategy for drug delivery of the plectasin derivate AP138 antimicrobial peptide

Introduction: Resistance to traditional antibiotics is an increasingly serious problem. Antimicrobial peptides (AMPs) have emerged as a new therapeutic class with great potential against infectious diseases, as they are less prone to induce resistance. Nanotechnology-based delivery strategies can improve the efficiency and stability of AMPs, particularly against proteolytic degradation. Lipid nanocapsules (LNCs) are a new generation of biomimetic nanocarriers and were used in this study to deliver peptides. Methods: AMP-loaded reverse micelles (RM) were developed and incorpo rated into LNCs by the phase inversion process and the antimicrobial activity of the AMPs-loaded LNC was evaluated by the minimum inhibitory concentration method. We studied the activity of AMP solutions and AMP-loaded LNCs against Gram-positive and Gram-negative bacterial strains and then evaluated the encapsulation of a new cationic AMP called AP138. Finally, we analyzed the effect of enzymatic attack on AP138 and AP138-RM-LNCs after incubation with trypsin. Results: AP138 was efficiently encapsulated in the LNCs (encapsulation efficiency = 97.8% at a drug loading of 0.151%), resulting in protection against degradation by proteases and the preservation of antimicrobial activity against , including . Conclusion: This study shows that RM-LNCs are an excellent candidate system to deliver AMPs

Amorphous polymeric drug salts as ionic solid dispersion forms of ciprofloxacin

yesCiprofloxacin (CIP) is a poorly soluble drug that also displays poor permeability. Attempts to improve the solubility of this drug to date have largely focused on the formation of crystalline salts and metal complexes. The aim of this study was to prepare amorphous solid dispersions (ASDs) by ball milling CIP with various polymers. Following examination of their solid state characteristics and physical stability, the solubility advantage of these ASDs was studied, and their permeability was investigated via parallel artificial membrane permeability assay (PAMPA). Finally, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the ASDs were compared to those of CIP. It was discovered that acidic polymers, such as Eudragit L100, Eudragit L100C==, Carbopol and HPMCAS, were necessary for the amorphization of CIP. In each case, the positively charged secondary amine of CIP was found to interact with carboxylate groups in the polymers, forming amorphous polymeric drug salts. Although the ASDs began to crystallize within days under accelerated stability conditions, they remained fully XCray amorphous following exposure to 90% RH at 25 oC, and demonstrated higher than predicted glass transition temperatures. The solubility of CIP in water and simulated intestinal fluid was also increased by all of the ASDs studied. Unlike a number of other solubility enhancing formulations, the ASDs did not decrease the permeability of the drug. Similarly, no decrease in antibiotic efficacy was observed, and significant improvements in the MIC and MBC of CIP were obtained with ASDs containing HPMCASC") and HPMCASCMG. Therefore, ASDs may be a viable alternative for formulating CIP with improved solubility, bioavailability and antimicrobial activity

Levofloxacin in nanostructured lipid carriers: preformulation and critical process parameters for a highly incorporated formulation

Available online 13 September 2022The first step of a successful nanoformulation development is preformulation studies, in which the best excipients, drug-excipient compatibility and interactions can be identified. During the formulation, the critical process parameters and their impact must be studied to establish the stable system with a high drug entrapment efficiency (EE). This work followed these steps to develop nanostructured lipid carriers (NLCs) to deliver the antibiotic levofloxacin (LV). The preformulation studies covered drug solubility in excipients and thorough characterization using thermal analysis, X-ray diffraction and spectroscopy. A design of experiment based on the process parameters identified nanoparticles with 71%) and an acceptable level of LV degradation products (0.37-1.13%). To the best of our knowledge, this is the first time that a drug degradation is reported and studied in work on nanostructured lipids. LV impurities following the NLC production were detected, mainly levofloxacin N-oxide, a degradation product that has no antimicrobial activity and could interfere with LV quantification in spectrophotometric experiments. Also, the achievement of the highest EE in lipid nanoparticles than those described in the literature to date and the apparent protective action of NLC of entrapped-LV against degradation are important findings.This study was part-financed by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001, CAPES-PrInt, Santander – Program of International Mobility number 31/2018 and Sao Paulo Research Foundation (FAPESP) grant numbers 2018/03666-3, 2019/09719-4 and 2020/08059-8. LT and AU acknowledge funding from Science Foundation Ireland, grants 15/CDA/3602 and 12/RC/2275_P2.info:eu-repo/semantics/publishedVersio

Formulation and characterisation of polyelectrolyte complex nanoparticles for the delivery of salmon calcitonin

THESIS 10003The studies in this thesis explored the formulation of various polyelectrolyte complex nanoparticles as potential carriers for the delivery of macromolecules. The complexes were formed between a polyanion (hyaluroninc acid, HA or chondroitin, CHON) and a polycation (chitosan, CS, or protamine, PROT). Salmon calcitonin (sCT) was selected as a macromolecule to be encapsulated in the nanoparticles due to its clinical use in bone diseases and potential application in arthritis. Different factors influencing the formation and properties of the nanoparticles (NPs) were examined, e.g. concentration of the polymers, polyanion/polycation mixing ratio, molecular weight of polymers. The molecular weight of the polymers was determined by GPC (Gel Permeation Chromatography). The properties of the NPs were characterised with the use of different techniques. Particle size and size distribution were analysed with the employment of dynamic light scattering (DLS), zeta potential was measured with the use of laser Doppler velocimetry (LDV), while morphology was investigated by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Physical stability of the nanoparticle suspensions upon storage at room temperature were performed for a period up to 4 weeks. sCT was loaded into each type of NPs: HA/CS, CHON/CS, HA/PROT and CHON/PROT, and the possibility of formation of NPs between sCT and polyanionsiHA and CHON was also examined. Non-associated sCT was separated from NPs by a combined ultrafiltration-centrifugation technique, and concentration of sCT was analysed by HPLC (high performance liquid chromatography). The release of sCT from the polyelectrolyte complex nanoparticles was also examined. Selected formulations were converted into solid state forms by freeze-drying with the employment of trehalose and poly(ethylene glycol) polymers (PEGs) as cryoprotectants. The toxicity of the NPs, polymers and cryoprotectants was examined on Caco-2 cells with the use of MTS assay and flow cytometry. The in vitro (cell culture) and in vivo (in rats) performance of HA/CS/sCT NPs was also examined. Uncomplicated, organic solvent and surfactant-free production method by simply mixing aqueous solutions of polylelctrolytes at room temperature was successfully employed to fabricate novel, cross-linker-free and non-sedimenting nanoparticles