Добірка наукової літератури з теми "Lipid nanoparticles of nonlamellar lipids"

Nanostructure bicontinuous cubic phase self-assembled materials are receiving expanding applications as biocompatible delivery systems in various therapeutic fields. The functionalization of cubosome, spongosome, hexosome and liposome nanocarriers by pH-sensitive lipids and/or pH-sensitive polymer shells offers new opportunities for oral and topical drug delivery towards a new generation of cancer therapies. The electrochemical behavior of drug compounds may favor pH-triggered drug release as well. Here, we highlight recent investigations, which explore the phase behavior of mixed nonlamellar lipid/fatty acid or phospholipid systems for the design of pH-responsive and mucoadhesive drug delivery systems with sustained-release properties. X-ray diffraction and small-angle X-ray scattering (SAXS) techniques are widely used in the development of innovative delivery assemblies through detailed structural analyses of multiple amphiphilic compositions from the lipid/co-lipid/water phase diagrams. pH-responsive nanoscale materials and nanoparticles are required for challenging therapeutic applications such as oral delivery of therapeutic proteins and peptides as well as of poorly water-soluble substances. Perspective nanomedicine developments with smart cubosome nanocarriers may exploit compositions elaborated to overcome the intestinal obstacles, dual-drug loaded pH-sensitive liquid crystalline architectures aiming at enhanced therapeutic efficacy, as well as composite (lipid/polyelectrolyte) types of mucoadhesive controlled release colloidal cubosomal formulations for the improvement of the drugs’ bioavailability.

Due to their distinctive structural features, lyotropic nonlamellar liquid crystalline nanoparticles (LCNPs), such as cubosomes and hexosomes, are considered effective drug delivery systems. Cubosomes have a lipid bilayer that makes a membrane lattice with two water channels that are intertwined. Hexosomes are inverse hexagonal phases made of an infinite number of hexagonal lattices that are tightly connected with water channels. These nanostructures are often stabilized by surfactants. The structure’s membrane has a much larger surface area than that of other lipid nanoparticles, which makes it possible to load therapeutic molecules. In addition, the composition of mesophases can be modified by pore diameters, thus influencing drug release. Much research has been conducted in recent years to improve their preparation and characterization, as well as to control drug release and improve the efficacy of loaded bioactive chemicals. This article reviews current advances in LCNP technology that permit their application, as well as design ideas for revolutionary biomedical applications. Furthermore, we have provided a summary of the application of LCNPs based on the administration routes, including the pharmacokinetic modulation property.

Based on the recent reports of World Health Organization, increased antibiotic resistance prevalence among bacteria represents the greatest challenge to human health. In addition, the poor solubility, stability, and side effects that lead to inefficiency of the current antibacterial therapy prompted the researchers to explore new innovative strategies to overcome such resilient microbes. Hence, novel antibiotic delivery systems are in high demand. Nanotechnology has attracted considerable interest due to their favored physicochemical properties, drug targeting efficiency, enhanced uptake, and biodistribution. The present review focuses on the recent applications of organic (liposomes, lipid-based nanoparticles, polymeric micelles, and polymeric nanoparticles), and inorganic (silver, silica, magnetic, zinc oxide (ZnO), cobalt, selenium, and cadmium) nanosystems in the domain of antibacterial delivery. We provide a concise description of the characteristics of each system that render it suitable as an antibacterial delivery agent. We also highlight the recent promising innovations used to overcome antibacterial resistance, including the use of lipid polymer nanoparticles, nonlamellar liquid crystalline nanoparticles, anti-microbial oligonucleotides, smart responsive materials, cationic peptides, and natural compounds. We further discuss the applications of antimicrobial photodynamic therapy, combination drug therapy, nano antibiotic strategy, and phage therapy, and their impact on evading antibacterial resistance. Finally, we report on the formulations that made their way towards clinical application.

During apoptosis, Bax-type proteins permeabilize the outer mitochondrial membrane to release intermembrane apoptogenic factors into the cytosol via a poorly understood mechanism. We have proposed that Bax and ΔN76Bcl-xL(the Bax-like cleavage fragment of Bcl-xL) function by forming pores that are at least partially composed of lipids (lipidic pore formation). Since the membrane monolayer must bend during lipidic pore formation, we here explore the effect of intrinsic membrane monolayer curvature on pore formation. Nonlamellar lipids with positive intrinsic curvature such as lysophospholipids promoted membrane permeabilization, whereas nonlamellar lipids with negative intrinsic curvature such as diacylglycerol and phosphatidylethanolamine inhibited membrane permeabilization. The differential effects of nonlamellar lipids on membrane permeabilization were not correlated with lipid-induced changes in membrane binding or insertion of Bax or ΔN76Bcl-xL. Altogether, these results are consistent with a model whereby Bax-type proteins change the bending propensity of the membrane to form pores comprised at least in part of lipids in a structure of net positive monolayer curvature.

This study is the first report that demonstrates nonlamellar arrangements, or lipidic particles, of phosphatidate inserted in the lipid bilayer of liposomes using polyclonal antibodies from mice and flow cytometry. Sera immunoreactivity was analyzed using liposomes that displayed smooth bilayers or phosphatidate particles, as shown by electron microscopy. This cytofluorimetric analysis showed that immune mice sera have a specific immunoreactivity with the phosphatidate particles formed by Mn2+, which also cross-reacted with those formed by Ca2+ and with the cardiolipin particles formed by Mn2+. In addition, these immune sera hardly reacted with smooth bilayered liposomes, independently of the lipid composition studied. Thus, this new methodology can be applied to demonstrate nonlamellar molecular arrangements of lipids in biological membranes.Key words: nonlamellar lipid immunogenicity, lipidic particles immunogenicity, liposomes, flow citometry, anti-phospholipid antibodies.

L'objectif principal de cette thèse est d'étudier l'effet neuroprotecteur des plasmalogènes (Pls) et d'explorer le potentiel des nanoparticules lipidiques contre les maladies neurodégénératives. Notre stratégie vise à créer un système auto-assemblé, augmentant l'efficacité des plasmalogènes et d'un neuropeptide, le polypeptide activateur de l'adénylate cyclase hypophysaire (PACAP), pour la neuroprotection. Pls, un groupe distinctif de glycérophospholipides membranaires, contiennent généralement une chaîne d'acyle gras polyinsaturé en position sn-2 et une chaîne alkyle liée par une liaison éther-vinyle en position sn-1 du squelette glycérol. La correction du déclin des niveaux de plasmalogènes chez les personnes âgées offre des perspectives pour les thérapies liées à la maladie de Parkinson, à la maladie d'Alzheimer et à la démence. Nous résumons les progrès des nanoparticules lipidiques (LNPs) dans le ciblage de multiples mécanismes de neurodégénérescence. Notre recherche sur les LNPs chargées en plasmalogène explore leur impact in vitro/in vivo sur des modèles de neurodégénérescence. Notre étude montre la faisabilité d'améliorer l'efficacité du Pls avec les LNPs. Nous utilisons des plasmalogènes naturels pour créer des nanoformulations impliquant un excipient lipidique nonlamellaire (monooléine, divers agents tensioactifs et de petites quantités de vitamine E, curcumine ou coenzyme Q10. En utilisant la méthode SAXS, nous avons identifié des caractéristiques structurelles des LNPs (vésicules, cubosomes et hexosomes). Les évaluations in vitro utilisent des cellules SH-SY5Y, différenciées avec 10 µM d'acide rétinoïque pendant 5 jours. Les tests de viabilité cellulaire indiquent une absente de toxicité à une concentration totale en lipides de 10 µM pour une incubation de 24 heures. Nous avons étudié l'impact des nanoparticules chargées en Pls sur les cellules neuronales en utilisant la neurotoxine 6-OHDA comme modèle in vitro de la maladie de Parkinson. Nous explorons les mécanismes de dommages cellulaires (stress oxydatif et enzymes apoptotiques), identifiant la voie de signalisation ERK-Akt-CREB-BDNF. Cela suggère la nécessité d'adopter plusieurs stratégies dans le traitement des maladies neurodégénératives. Plusieurs composés neuroprotecteurs documentés ont été utilisés pour démontrer la capacité à restaurer les lésions neuronales causées par le 6-OHDA, offrant un modèle de conditions neurodégénératives pour élucider davantage les effets bénéfiques des Pls. Nous nous concentrons ensuite sur la protéine de liaison à l'élément de réponse au cAMP (CREB) et sa phosphorylation conduisant à l'expression des neurotrophines, cruciale pour prévenir les troubles neurologiques. À travers des nano-assemblages lipidiques-peptiques, nous avons étudié l'impact des différentes organisations structurelles des LNPs sur la phosphorylation de CREB dans un modèle in vitro de la maladie de Parkinson. Dans un modèle murin de la maladie de Parkinson, les LNPs de structure vésiculaire et hexosomale ont démontré une efficacité distincte dans la restauration de la fonction motrice. L'intervention intranasale a influencé la régulation génétique liée à la maladie de Parkinson et rééquilibré les profils lipidiques. L'administration nasale de LNPs chargées en Pls a amélioré les symptômes comportementaux de la maladie et a régulé à la baisse des gènes tels que IL33 et Tnfa. Nos résultats indiquent l'impact significatif des nanoformulations hexosomales sur l'atténuation de la maladie, le métabolisme lipidique et les modifications génétiques réactives potentiellement impliquées dans la régénération
The primary aim of this thesis is to investigate the neuroprotective effect of plasmalogens (Pls) and explore the potential of lipid nanoparticles against neurodegenerative diseases. Our strategy aims to create a self-assembled system, enhancing the efficacy of plasmalogens and the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) for neuroprotection. The Pls, a distinctive group of membrane glycerophospholipids, typically contain a polyunsaturated fatty acyl chain at the sn-2 position and an alkyl chain linked by a vinyl-ether bond at the sn-1 position of the glycerol backbone. Pls, with their unique structure featuring a vinyl ether bond, possess free radical scavenging capabilities and antioxidant properties. Addressing the decline in plasmalogen levels in aging individuals holds promise for therapies related to Parkinson's disease, Alzheimer's disease, and dementia. Recent research has expanded our understanding of their antioxidant effects, anti-inflammation, and their involvement in ferroptosis. However, challenges persist in implementing plasmalogens in treatments of neurodegenerative diseases and in developing suitable drug delivery systems. We summarize the progress in lipid nanoparticles (LNPs) for targeting multiple neurodegeneration mechanisms. Our research on plasmalogen-loaded LNPs explores their fabrication mechanism and in vitro/in vivo impacts on neurodegenerative models. Our study shows the feasibility of enhancing Pls efficacy using LNPs as carriers. We employ natural plasmalogens from scallops to create nanoformulations involving a non-lamellar lipid excipient (MO) for structural stabilization, various surfactants, and small amounts of vitamin E, curcumin, or coenzyme Q10. Using small-angle X-ray scattering (SAXS), we identified the structural features of various LNPs (vesicles, cubosomes, and hexosomes). Our in vitro evaluations utilized human neuroblastoma SH-SY5Y cells, differentiated with 10 µM retinoic acid for 5 days. Cell viability tests indicated non-toxicity of the LNPs at a total lipid concentration of 10 µM for 24-hour incubation. We study the impact of Pls nanoparticles on an in vitro model of Parkinson's disease using neuronal cells induced by the neurotoxin 6-OHDA. Using the SH-SY5Y cell line, we explore cellular damage mechanisms (oxidative stress and apoptotic enzymes) via identifying the impact on the ERK-Akt-CREB-BDNF signaling pathway. Several documented neuroprotective compounds were used to demonstrate the ability to restore neuronal lesions caused by 6-OHDA, offering a model of neurodegenerative conditions to further elucidate the beneficial effects of the Pls-based LNPs. We then focus on the cAMP response element binding protein (CREB) and its phosphorylation leading to neurotrophin expression, crucial in preventing neurological disorders. Through lipid peptide nano-assemblies, we studied the impact of different structural organizations of the LNPs on CREB phosphorylation in an in vitro model of Parkinson's disease. Notably, liquid crystalline lipid nanoparticles loaded with plasmalogens prolonged CREB activation under neurodegenerative conditions, showing potential for enhanced neuroregeneration through sustained CREB activation in response to the neurotrophic nanoassemblies. In a mouse model of Parkinson's disease, vesicle and hexosome LNPs demonstrated distinct effectiveness in restoring motor function. The nanomedicine-mediated intervention influenced Parkinson's disease-related gene regulation and rebalanced lipid profiles. Nasal administration of Pls-loaded LNPs improved disease behavioral symptoms and downregulated genes like IL33 and Tnfa. The obtained results indicated the significant impact of hexosomal LNP nanomedicines on disease attenuation, lipid metabolism, and responsive gene modifications potentially involved in regeneration

The research undertaken in these studies aimed to investigate the feasibility of developing and manufacturing innovative solid lipid carriers, such as solid lipid nanoparticles (SLN) and/or nanostructured lipid carriers (NLC) using a hot high pressure homogenization method, for didanosine(DDI). In addition, studies using in vitro differential protein adsorption were undertaken to establish whether the SLN and/or NLC have the potential to deliver DDI to the central nervous system (CNS). Prior to initiating pre-formulation, formulation development and optimization studies of DDI-Ioaded SLN and/or NLC, it was necessary to develop and validate an analytical method for the in vitro quantitation and analysis of DDI. An accurate, precise and sensitive RP-HPLC method with UV detection set at 248 nm was developed, optimized and validated for the quantitative in vitro analysis of DDI in formulations. Pre-formulation studies were designed to evaluate the thermal stability of DDI and to select and characterize lipid excipients that may be used for the manufacture of the nanocarriers. It was established that DDI is thermostable at temperatures not exceeding 163°C and therefore a hot high pressure homogenization technique could be used to manufacture DDI-loaded SLN and/or NLC. Lipid screening studies revealed that DDI is poorly soluble in both solid and liquid lipids. A combination of Precirol® ATO 5 and Transcutol® HP was found to have the best solubilizing-potential for DDI of all lipids investigated. The inclusion of Transcutol® HP into Precirol® ATO 5 changed the polymorphic form of the solid lipid from the stable 13-modification to a material that exhibited the co-existence between α- and β-polymorphic forms. The relatively high solubility of DDI in Transcutol® HP compared to Precirol® ATO 5 was an indication that a solid lipid matrix prepared from a binary mixture of Precirol® ATO 5 and Transcutol® HP was likely to have a higher loading capacity and encapsulation efficiency for DDI than a matrix consisting of Precirol® ATO 5 alone. Furthermore, the potential for the solid lipid matrix to exist in the α- and/or β-modifications when Transcutol® HP was added to Precirol® ATO 5 suggested that expulsion of DDI from a solid lipid matrix during prolonged storage periods was likely to be minimal. Therefore it was considered logical to investigate the feasibility of incorporating DDI into NLC and not in SLN. However, due to the limited solubility of DDI in lipids, formulation development of DDI-loaded NLC commenced using small quantities of DDI. Formulation development and optimization studies of DDI-loaded NLC were initially aimed at selecting a surfactant system that was capable of stabilizing NLC in an aqueous environment. Solutol® HS alone or a ternary mixture consisting of Solutol® HS, Tween® 80 and Lutrol® F68 was found to stabilize the nanoparticles in terms of particle size and the polydispersity index. The use of the ternary mixture as the surfactant system was preferred to using Solutol® HS alone as Lutrol® F68 and especially Tween® 80 have been successfully used to target the delivery of API to the brain. Aqueous DDI-free and DDI-Ioaded NLC containing increasing amounts of DDI were manufactured using hot high pressure homogenization at 800 bar for three cycles. The NLC formulations were characterized in terms of particle size, polydispersity index, zeta potential, and polymorphism, degree of crystallinity, encapsulation efficiency (EE), shape and surface morphology. The mean particle size for all formulations was below 250 nm with narrow polydispersity indices, indicating that narrow particle size distribution had been achieved. The d99% values for all formulations tested, were generated using laser diffractometry, and were below 400 nm, with span values ranging from 0.84 - 1.19 also suggesting that a narrow particle size distribution had been achieved. The zeta potential values measured in double distilled water with the conductivity adjusted to 50 μS/cm ranged from -18.4 to -11.4 mV. In addition, all the formulations showed a decrease in the degree of crystallinity as compared to the bulk lipid material and WAXS shows that the formulations existed in a single β-modification form. Furthermore DDI that had been incorporated into the NLC appeared to be molecularly dispersed in the lipid matrices. These parameters remained unaffected for most formulations following storage for two months at 25°C. In addition these formulations contained a mixture of spherical and non-spherical particles irrespective of the amount of DDI that was added during the manufacture of the formulations. These studies showed that it was feasible to develop and incorporate small amounts of DDI into NLC. However in order to use these delivery systems for oral administration of DDI to paediatric patients, strategies to improve the amount of DDI that could be loaded into the particles and to achieve high encapsulation efficiencies had to be developed. The limited solubility of DDI in lipid media was identified as a major factor that affected the loading capacity and encapsulation efficiency of DDI in the NLC. Therefore, a novel strategy aimed at increasing the saturation solubility of DDI in the lipid by attempting to increase the dissolution velocity of the drug in the lipid using a particle size reduction approach, was designed and investigated. DDI was dispersed in Transcutol® HP and the particle size of DDI in the liquid lipid medium was reduced gradually using hot high pressure homogenization and the product obtained from these studies was used to manufacture DDI-loaded NLC using a cold high pressure homogenization procedure. Although the encapsulation efficiency and drug loading following use of this approach was relatively high, the particles were large and showed a tendency to grow in size leading to the formation of microparticles after storage for two months at 25°C. In addition, the degree of crystallinity of the nanoparticles increased rapidly over the same storage period which led to expulsion of DDI nanoparticles for the NLC, despite the DDI loading in NLC being unaffected. It was clearly evident that this new approach of manufacturing solid lipid nanocarriers could be used as a platform not only for enhancing the loading capacity of DDI in solid lipid nanocarriers but also for other hydrophilic drugs. Differential protein adsorption patterns of DDI-loaded NLC were generated in vitro using two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) in order to establish the potential for these systems to deliver DDI to the CNS. NLC formulations containing small amounts of DDI were used as these formulations showed a better stability profile than the formulation with a higher encapsulation efficiency and drug loading capacity. Furthermore, the encapsulation efficiency and drug loading of DDI were considered sufficient for use in 2-D PAGE studies. Data obtained from 2-D PAGE analysis reveal that DDI-loaded NLC preferentially adsorb proteins in vitro that are responsible for specific brain targeting in vivo. More importantly, these studies reveal that in addition to Tween® 80 that has already been shown to have the potential to target CDDS to the brain, Solutol® HS 15 has the potential to achieve a similar objective. Consequently, DDI-loaded NLC have the potential to deliver DDI to the brain and these results may be used as a platform for conducting in vivo studies to establish whether DDI can cross the blood brain barrier and enter the CNS when administered in NLC which may in turn lead to a major breakthrough in the management of HIV/AIDS and Aids Dementia Complex (ADC).

Nanopartículas lipídicas têm sido desenvolvidas para aplicação tópica de fármacos e ativos cosméticos. Neste trabalho, foi proposta a primeira aplicação de um lipídeo natural não-refinado biodegradável e biocompatível - manteiga de cupuaçu (Theobroma grandiflorum) - para a preparação de nanopartículas lipídicas, as quais foram denominadas teosferas. As teosferas foram preparadas por emulsificação-evaporação do solvente (EES) e por homogeneização à alta pressão (HAP), apresentando tamanho nanométrico e distribuição granulométrica estreita quando preparadas por ambos os métodos. O trabalho teve continuidade com a preparação de teosferas pelo método de EES utilizando manteiga de cupuaçu ou sua mistura com óleo de castanha do Brasil (Bertholletia excelsa) - também derivado da biodiversidade Amazônica - visando a incorporação de antioxidantes. Idebenona (IDB) foi selecionada por sua conhecida ação antioxidante e pela sua utilização em formulações cosméticas antienvelhecimento. IDB foi incorporada nas teosferas com eficiência de encapsulação superior a 99%, sendo que os estudos de liberação in vitro mostraram que a liberação de IDB a partir das teosferas foi mais lenta em comparação à IDB livre. Estes experimentos foram capazes ainda de demonstrar as características elásticas das teosferas. Além disso, foi evidenciada in vitro a atividade antioxidante superior das teosferas contendo IDB em relação ao ativo livre. Visando possibilitar a aplicação tópica de teosferas contendo IDB, em um trabalho subseqüente, suspensões de teosferas preparadas por HAP foram incorporadas em géis hidrofílicos. As formulações apresentaram características pseudoplásticas e demonstraram efeito oclusivo in vitro, o qual foi dependente da composição dos colóides. Finalmente, os estudos de permeação in vitro utilizando pele humana demonstraram que teosferas e nanocápsulas de núcleo lipídico, utilizadas neste estudo de modo comparativo, modificaram a permeção da IDB, permitindo a acumulação do ativo nas camadas superficiais da pele.
Lipid nanoparticles have been developed for administration of active substances to the skin, both for pharmaceutical and cosmetic uses. In the present work, we proposed the first use of a none-refined natural biodegradable and biocompatible lipid – Cupuaçu seed butter (Theobroma grandiflorum) – for the preparation of lipid nanoparticles, which were called theospheres. Theospheres were prepared by emulsification-solvent evaporation (ESE) and by high pressure homogenization technique (HPH), presenting size in nanometrical range and narrow particle size distribution for both methods. Taking these results into account, the next step of this work was the preparation of theospheres by ESE method using Cupuaçu seed butter with or without Brazil nut (Bertholletia excelsa) seed oil - another ingredient derived from an Amazonian fruit - intending the encapsulation of an antioxidant. Idebenone (IDB) has been selected due to its known antioxidant action and because it has been used in antiaging cosmetic formulations. IDB was incorporated in the theospheres presenting encapsulation efficiency higher than 99%. The in vitro release evaluation demonstrated that the release of IDB from theospheres was lower than that of free drug. Besides, the in vitro release study highlighted the elastic characteristics of theospheres. Additionally, IDB-loaded theospheres showed higher antioxidant activity compared to free IDB. Viewing the cutaneous administration, theosphere suspensions prepared by HPH technique were incorporated into hydrogels. The rheograms of the semi-solid formulations exhibited a non-Newtonian behavior presenting pseudoplastic characteristics. In vitro occlusion study highlighted the dependence of the occlusive effect on the lipidic composition of the theospheres. Finally, in vitro human skin permeation studies showed that theospheres and lipid-core nanocapsules, used in this study in a comparative way, changed the permeation of IDB, increasing the accumulative amount of IDB in the upper skin layer.

Extracellular vesicles (EVs), exosomes and microvesicles, transfer endogenous RNAs between neurons over short and long distances. We have explored EVs for siRNA delivery to brain. (1) We optimized siRNA chemical modifications and siRNA conjugation to lipids for EV-mediated delivery. (2) We developed a GMP-compatible, scalable method to manufacture active EVs in bulk. (3) We characterized lipid and protein content of EVs in detail. (4) We established how protein and lipid composition relates to siRNA delivering activity of EVs, and we reverse engineered natural exosomes (small EVs) into artificial exosomes based on these data. We established that cholesterol-conjugated siRNAs passively associate to EV membrane and can be productively delivered to target neurons. We extensively characterized this loading process and optimized exosome-to-siRNA ratios for loading. We found that chemical stabilization of 5'-phosphate with 5'-E-vinylphosphonate and chemical stabilization of all nucleotides with 2'-O-methyl and 2'-fluoro increases the accumulation of siRNA and the level of mRNA silencing in target cells. Therefore, we recommend using fully modified siRNAs for lipid-mediated loading to EVs. Later, we identified that α-tocopherol-succinate (vitamin E) conjugation to siRNA increases productive loading to exosomes compared to originally described cholesterol. Low EV yield has been a rate-limiting factor in preclinical development of the EV technology. We developed a scalable EV manufacturing process based on three-dimensional, xenofree culture of mesenchymal stem cells and concentration of EVs from conditioned media using tangential flow filtration. This process yields exosomes more efficient at siRNA delivery than exosomes isolated via differential ultracentrifugation from two-dimensional cultures of the same cells. In-depth characterization of EV content is required for quality control of EV preparations as well as understanding composition–activity relationship of EVs. We have generated mass-spectrometry data on more than 3000 proteins and more than 2000 lipid species detected in exosomes (small EVs) and microvesicles (large EVs) isolated from five different producer cells: two cell lines (U87 and Huh7) and three mesenchymal stem cell types (derived from bone marrow, adipose tissue and umbilical cord Wharton’s jelly). These data represent an indispensable resource for the community. Furthermore, relating composition change to activity change of EVs isolated from cells upon serum deprivation allowed us to identify essential components of siRNA-delivering exosomes. Based on these data we reverse engineered natural exosomes into artificial exosomes consisting of dioleoyl-phosphatidylcholine, cholesterol, dilysocardiolipin, Rab7, AHSG and Desmoplakin. These artificial exosomes reproduced efficient siRNA delivery of natural exosomes both in vitro and in vivo. Artificial exosomes may facilitate manufacturing, quality control and cargo loading challenge that currently impede the therapeutic EV field.

Solid lipid nanoparticles (SLNPs) and lipid-drug conjugates (LDCs) are two promising lipid nanoparticle (LNP) based drug delivery systems; this thesis explores new synthetic lipids that may circumvent the limitations of currently available components for LNPs with particular focus on the stability of LNP formulations. Neutral polyethylene glycol lipids (PEG-lipids) have been designed, synthesized, and characterized with ESI-MS, for stabilizing SLNPs containing dsDNA oligomer. 1st and 2nd generation PEG-lipids investigated the effects of serinol and iminodiacetic acid backbone structures, respectively, and aliphatic chain sequences within the lipid anchors on the stability of SLNPs. Assays were developed to analyze LNP stability in both PBS buffer and PBS buffer with 10 % serum at different incubation temperatures. The results indicate that the hydrocarbon branching sequence offer additional SLNP stability over straight chain isomers. LDC monomers were designed and synthesized to allow for the formulation of LDC nanocarriers for the thiopurine drugs. These hydrophobic LDC monomers were made by linking the polar thiopurine drug to a synthetic lipid. These synthetic lipids investigated branched and straight chain derivatives – the branched isomers once again demonstrated advantages in the stability of the LDCs.
Graduate

The thesis entitled “The Role of Liposomal Hybrids and Gold Nanoparticles in the Efficacious Transport of Nucleic Acids and Small Molecular Drugs for Cancer Nanomedicine” elucidates the preparation of various liposomal formulations of cationic monomeric and gemini lipids where hydrophobic domains were consisted of tocopherol, cholesterol and pseudoglyceryl backbone for the cellular transport of nucleic acids. The thesis continues while elucidating the role of various pH sensitive molecules and gold nanoparticles in liposomes to improve the delivery efficacy levels. This thesis also elucidates the role of gold nanoparticles stabilized with natural pH sensitive molecules for efficacious drug delivery applications. Additionally, the role of such pH sensitive gold nanoparticles in association with liposomes for the co-delivery of drug and gene has been discussed. The work has been divided into six chapters. Chapter 1A: Dimeric Lipids Derived from α-Tocopherol as Efficient Gene Transfection Agents. Mechanistic Insights into Lipoplex Internalization and Therapeutic Induction of Apoptotic Activity In this chapter, we present cationic dimeric (gemini) lipids for significant plasmid DNA (pDNA) delivery to different cell lines without any marked toxicity in the presence of serum. The six gemini lipids possess α-tocopherol as their hydrophobic backbone and differ from each other in terms of their spacer chain lengths. Each of these gemini lipids mixed with a helper lipid 1, 2-dioleoyl phosphatidyl ethanolamine (DOPE), was capable of forming stable aqueous suspensions. These co-liposomal systems were examined for their potential to transfect pEGFP-C3 plasmid DNA in to nine cell lines of different origins. The transfection efficacies noticed in terms of EGFP expression levels using flow cytometry were well corroborated using independent fluorescence microscopy studies. Significant EGFP expression levels were reported using the gemini co-liposomes which counted significantly better than one well known commercial formulation lipofectamine 2000 (L2K). Transfection efficacies were also analyzed in terms of the degree of intracellular delivery of labeled plasmid DNA (pDNA) using confocal microscopy which revealed an efficient internalization in the presence of serum. The cell viability assays performed using optimized formulations demonstrated no significant toxicity towards any of the cell lines used in the study. We also had a look at the lipoplex internalization pathway to profile the uptake characteristics. A caveolae/lipid raft route was attributed to their excellent gene transfection capabilities. The study was further advanced by using a therapeutic p53-EGFP-C3 plasmid and the apoptotic activity was observed using FACS and growth inhibition assay. Figure 1. The co-liposomes of tocopheryl gemini lipids and DOPE for efficient delivery of p53-EGFP-C3 plasmid DNA that induces significant apoptotic response. Chapter 1B: Efficacious Gene Silencing in Serum and Significant Apoptotic Activity Induction by Survivin Downregulation Mediated by Cationic Gemini Tocopheryl Lipids Non-viral gene delivery offers cationic liposomes as promising instruments for the delivery of double-stranded RNA (ds RNA) molecules for successful sequence-specific gene silencing (RNA interference). The efficient delivery of siRNA (small interfering RNA) to cells while avoiding the unexpected side effects is an important prerequisite for the exploitation of the power of this excellent tool. We discuss in this chapter about six tocopherol based cationic gemini lipids, which induce substantial gene knockdown without any obvious cytotoxicity. All the efficient co-liposomal formulations derived from each of these geminis and a helper lipid, dioleoyl phosphatidyl ethanolamine (DOPE) were well characterized using physical methods such as atomic force microscopy (AFM) and dynamic light scattering (DLS). Zeta potential measurements were conducted to estimate the surface charge of these formulations. Flow cytometric analysis showed that the optimized co-liposomal formulations could transfect anti-GFP siRNA efficiently in three different GFP expressing cell lines, viz. HEK 293T, HeLa and Caco-2 significantly better than a potent commercial standard Lipofectamine 2000 (L2K) both in the absence and presence of serum (FBS). Notably, the knockdown activity of co-liposomes of gemini lipids was not affected even in the presence of serum (10% and 50% FBS) while it dropped down for L2K significantly. Observations under a fluorescence microscope, RT-PCR and western blot analysis substantiated the flow cytometry results. The efficient cellular entry of labeled siRNA in GFP expressing cells as evidenced from confocal microscopy put forward these gemini lipids among the potent lipidic carriers for siRNA. The efficient transfection capabilities were also profiled in a more relevant fashion while performing siRNA transfections against survivin (an anti-apoptotic protein) which induced substantial apoptosis. Furthermore, the survivin downregulation improved the therapeutic efficacy levels of an anticancer drug, doxorubicin significantly. In short, the new tocopherol based gemini lipids appear to be highly promising for achieving siRNA mediated gene knockdown in various cell lines. Figure 2. The co-liposomes of tocopheryl gemini lipids and DOPE for efficient delivery of siRNA against survivin that induces significant apoptotic response. Chapter 2: Efficacious in Vitro EGFP Expression and Silencing in Serum by Cationic Pseudoglyceryl Gemini Lipids To elicit the desirable efficacy levels in cationic liposome mediated nucleic acid therapeutics has been part of extensive scientific efforts. This chapter describes three cationic gemini lipids and application of their co-liposomes with DOPE as potent pDNA (plasmid DNA) and siRNA (small interfering RNA) cytofectins for remarkably advanced efficacy levels in numerous cell lines in the presence of serum. The hydrophobic structural lineament of cationic gemini lipids is made up of pseudoglyceryl backbone linked to the hydrocarbon chains via oligo-oxyethylene units. The stable aqueous co-liposomal suspensions of gemini lipids showed an efficient binding to pDNA or siRNA and their significant intracellular delivery in various cell lines. The transfection capabilities of different co-liposomal formulations were profiled based on EGFP expression (pEGFP-C3 pDNA transfection) and EGFP knockdown (anti-GFP siRNA transfections) in EGFP expressing cell lines. The cellular EGFP expression levels and intracellular delivery of labeled nucleic acids were thoroughly studied using flow cytometry (FACS), fluorescence and confocal microscopy. The MTT based cell viability assay revealed no loss in cell viabilities for all of the transfection optimized lipoplexes of siRNA or pDNA. The transfection profile of gemini co-liposomes was noted to be significantly much better than a commercial lipofection reagent, Lipofectamine 2000 used for pDNA and siRNA applications in each of the cell lines studied. The co-liposomes and their transfection optimized lipoplexes were physiochemically characterized extensively by means of zeta potential, dynamic light scattering (DLS) and atomic force microscopy (AFM). In brief, these new gemini co-liposomal formulations seem to offer a great opportunity for successful nucleic acid (DNA and siRNA) delivery in a practical scenario. Figure 3. Efficacious EGFP expression (pDNA transfection) and EGFP silencing (anti GFP siRNA transfection) mediated by co-liposomes of pseudoglyceryl gemini lipids and DOPE. Chapter 3: Efficient Elicitation of Liposomal Nucleic acid delivery through the Eminence of Gold Nanoparticles Stabilized with pH Responsive Short Tripeptide Derived from Tyrosine Kinase NGF Receptors The prerequisite in the area of gene therapy today is to serve transfection efficient formulations nullifying the enduring key issues. To this end, we discuss in this chapter, the role of hybrid liposomal formulations derived from structurally distinct cationic lipids, a neutral lipid (DOPE) and pH responsive short tripeptide (KFG, Lys-Phe-Gly) capped gold nanoparticles (PAuNPs). The hybrid liposomes are presented to be efficient enough to transfect pDNA leading to remarkably high gene expression levels in various cell lines of different origins in the presence of serum (FBS). Hybrid liposomes could deliver pDNA more effectively than the native liposomes and commercial standard lipofectamine 2000 (L2K) across the entire range of N/P ratios studied under the influence of intracellular pH response and gold nanoparticles prominence. The gene transfection capabilities are profiled based on transfections performed using two different plasmids (pGL3, luciferase activity and p-EGFP-C3, green fluorescent protein expression). pDNA cellular internalization and subsequent gene expression levels are studied using flow cytometry, fluorescence microscopy and confocal microscopic studies. The extensive physiochemical characterization of hybrid liposomal formulation and their complexes with pDNA in comparison with respective native liposomes was performed using AFM, TEM, Zeta, DLS, gel retardation assay, U.V. and fluorescence emission measurements. The hybrid liposomes are shown to possess significantly higher fusion activity at lowered pH of intracellular compartments. These hybrid liposomes are fairly biocompatible across the concentration range used in transfection experiments. Precisely, introduction of these pH responsive tripeptide capped gold nanoparticles in to liposomal formulations straightforwardly must be more advantageous for a practical application in biomedical scenario to achieve therapeutic levels. Figure 4. The hybrid of liposomes and tri-peptide capped gold nanoparticles for significantly improved gene expression levels. Chapter 4: RNA Aptamer Decorated pH Sensitive Liposomes for Active Transport of Nucleic Acids in Specific Cancer Cells This chapter describes the target specific transport of pH sensitive liposomes loaded with a RNA aptamer for promising nucleic acid therapeutics. The pH sensitive liposomes are constructed from a cationic cholesteryl gemini lipid (CGL), neutral helper lipid (DOPE) and gemini analog of a pH sensitive lipid, palmitoyl homocysteine (GPHC). The liposomes are shown to be significantly fusogenic that deliver the cargoes upon lowerin the pH (6.0). The fusogenic behaviour of the liposomes was thoroughly studied by means of dynamic light scattering (DLS), zeta potential, lipid mixing, calcein dequenching and atomic force microscopy (AFM). The facile integration of cholesterol conjugated RNA aptamer in liposomes derived from cholesteryl gemini lipids was exploited for their delivery to specific cancer cells. The RNA aptamer specifically binds to epithelial cell adhesion molecule (EpCAM) with high affinity which is a cell surface marker in various solid cancers such as colorectal and breast carcinoma. These aptamer decorated pH sensitive liposomes could efficiently enter the EpCAM expressing COLO-205, Caco-2, MCF-7 and MDA-MB-231 cell lines while no such noticeable liposome transport was observed in EpCAM negative HEK 293T cells as evidenced by flow cytometry and confocal microscopy. Additionally, the liposomes are shown to be actively transported inside the cells, i.e., receptor mediated endocytosis. These liposomes could complex the nucleic acids (pDNA) in an efficient manner. The MTT based cell viability assay accounted no noticeable loss in cell viabilities for liposome treatments. Concisely, we have formulated RNA aptamer loaded pH sensitive liposomes that would certainly be promising tool in target based cancer nanomedicine. Figure 5. (A) Cellular internalization of DY-647 labeled aptamer loaded pH sensitive liposomes. (B) The liposomes were actively internalized through receptor mediated endocytosis. Each panel (A and B) represents (from left to right) bright field image, aptamer fluorescence, DAPI stained nuclei and merge of previous three impressions. Chapter 5: Natural Tri-peptide Capped Gold Nanoparticles for Efficacious Doxorubicin Delivery in Vitro and in Vivo Nanotechnology has gained ever increasing interest for the successful implementation of chemotherapy based treatment of cancer. This chapter describes the role of gold nanoparticles (AuNPs) capped with a natural pH responsive short tri-peptide (Lys-Phe–Gly or KFG) for significant intracellular delivery of an anti-cancer drug, doxorubicin (DOX). A significantly increased apoptotic response was noted for DOX treatments mediated by KFG-AuNPs in comparison with drug alone treatments in various cell lines (BT-474, HeLa, HEK 293T and U251) in vitro. Furthermore, KFG-AuNPs mediated DOX treatment significantly decreased cell proliferation and tumor growth in BT-474 cell xenograft model in nude mice. In addition, KFG-AuNPs showed efficacious drug delivery in DOX-resistant HeLa cells (HeLa-DOXR) in comparison with drug alone treatments. Figure 6. Representative images of excised tumors after doxorubicin treatment mediated by pH responsive tri-peptide capped gold nanoparticles (DOX-KFG-AuNPs) (C) in comparison with doxorubicin alone treatments (B) and untreated tumors (A). Extensive cell death as observed under Hematoxylin/eosin (H&E) (D) and TUNEL (E) staining of DOX-KFG-AuNPs treated tumor sections. Chapter 6: Significant Apoptotic Activity Induction by Efficacious Co-delivery of p53 Gene and Doxorubicin Mediated by the Combination of Co-liposomes of Cationic Gemini lipid and pH Responsive Tri-peptide Combining chemotherapy with gene therapy has appeared as an efficient tool to treat complex biological disorder like cancer. Herein, we show efficient co-delivery of DNA and an anti-cancer drug, doxorubicin (DOX) by means of gemini cationic liposome (GCL) based lipoplex nanoaggregates that are coated with DOX encapsulated pH responsive tripeptide nanovesicles. The lipoplex, tripeptide vesicles and their association was thoroughly studied using dynamic light scattering (DLS), zeta potential, atomic force microscopy (AFM). Flow cytometry, fluorescence and confocal microscopic analysis revealed that the GCL-tripeptide association could significantly co-deliver the p53 expression plasmid (p53-EGFP-C3) and DOX in HeLa and HEK 293T cells in the presence of serum. A synergistic increase in gene expression level and DOX internalization was observed in co-delivery which was even substantially higher than individual lipoplex transfection and DOX treatment. The apoptosis induced due to p53 expression and DOX was profiled with the help of annexin-V positivity analysis under flow cytometry and nuclear damage analysis by DAPI nuclei counterstaining under confocal microscopy which noted to be significantly higher in cells during co-delivery. The MTT based cell viability assay revealed a significantly increased loss in cell viability counts for co-delivery treatments. Such a system delivering synergistically increased significant efficacy levels in combinatorial drug and nucleic acid therapeutics would be certainly advantageous for practical biomedical applications. Figure 7. The co-delivery of pDNA and drug (doxorubicin) mediated by GCL-tripeptide association as observed under (A) confocal microscopy (pDNA; green and doxorubicin; red) and (B) flow cytometry.

Solid lipid nanoparticles (SLN) are nanocarriers in the 10–1000 nm range of a solid core, containing both hydrophilic and hydrophobic active pharmaceutical ingredients. SLNs are composed of well-tolerated and biodegradable solid lipids such as mono-, di-, and triglycerides, fatty acids, waxes, and steroids, as well as lipophilic and hydrophilic emulsifying agents. This composition of biocompatible molecules makes SLNs one of the most successful options for the administration of drugs with different routes of administration. To determine its size, morphology, and surface charge, laser diffraction spectroscopy techniques, dynamic light scattering, coulter counter, scanning ion occlusion sensing, and advanced microscopy techniques such as scanning electron microscopy, transmission electron microscopy, and atomic force microscopy are some of the most widely used methods. Surface morphology and length can be measured by electron microscopy, while dynamic light scattering and photon correlation spectroscopy determine particle size and size distribution. In addition, colloidal stability can be determined by zeta potential analysis, indirect measurement of surface charge, and differential scanning calorimetry to characterize particles and drug interactions.

Phospholipids, which provide valuable model systems for lipid membranes, display a variety of polymorphic phases, depending on their molecular structure and on environmental conditions. High hydrostatic pressure has been used as a physical parameter to study the thermodynamic properties and phase behavior of these systems. High pressure is also a characteristic feature of certain natural membrane environments. In the first part of this article, we review our recent work on the temperature- and pressure-dependent phase behavior of phospholipid systems differing in lipid conformation and headgroup structure. In the second part, we report on the determination of the (T, x, p) phase diagrams of binary phospholipid mixtures. An additional section deals with effects of incorporating ions, small amphiphilic molecules, and steroids into the bilayer on the experimental temperature- and pressure-dependent phase behavior of lipid systems. Finally, we discuss lamellar to nonlamellar thermotropic and barotropic phase transformations, which occur for a number of lipids, such as phosphatidylethanolamines, monoacylglycerides, and lipid mixtures. It has been suggested that nonlamellar lipid structures might play an important role as transient and local intermediates in a number of biochemical processes. High-pressure smallangle x-ray (SAXS) and neutron (SANS) scattering, differential scanning calorimetry (DSC), high-pressure differential thermal analysis (DTA), and p, V, T measurements have been used as experimental methods for the investigation of these systems. Lipid bilayer dispersions, in particular the phosphatidylcholines and phosphatidylethanolamines, are the workhorses for the investigation of biophysical properties of membrane lipids because they constitute the basic structural component of biological membranes. They exhibit a rich lyotropic and thermotropic phase behavior (Cevc & Marsh, 1987; Marsh, 1991; Yeagle, 1992). Most fully hydrated saturated phospholipid bilayers exhibit two principal thermotropic lamellar phase transitions, corresponding to a gel to gel (Lβ′–Pβ′) transition and a gel to liquid-crystalline (Pβ′–Lα) main transition at a temperature Tm. In the fluid-like La phase, the hydrocarbon chains of the lipid bilayers are conformationally disordered, whereas in the gel phases the hydrocarbon chains are more extended and relatively ordered.

The delivery system plays a vital role in managing the pharmacokinetics and pharmacodynamics of a drug. The size of the carrier system contributes to its pharmacological action. Lipid-based carriers refer to the formulations containing a dissolved or suspended drug in lipidic excipients. Lipoidal systems as carriers are achieving heights due to their significant lipid nature and the size of particles in the delivery system. The micro/nano-sized lipid-based carriers possess versatility in improving the physic-chemical properties of drugs. Also, they are biocompatible and can be administered through all possible routes. Lipid-based drug delivery carrier systems of new and existing formulations can be commercialized to achieve the desired range of product specifications. Solubility of the drug in various lipids is a key factor in the development of the delivery system. Lipids as functional excipients are compatible with solid, liquid, and semi-solid dosage forms. Besides improving/enhancing the solubility and bioavailability, lipids provide multiple broad-based applications in the pharmaceutical delivery system.

Nanoparticles formed from lipids are currently applied successfully to deliver drugs. The particle size of the nanoparticle system is an essential characteristic to enhance the entrance of the drugs inside tissues and cells. Using design of experiment is appealing to find the specific conditions to optimize particle size of drug-loaded nanoparticles. Authors of this chapter applied a fractional factorial design of half fraction 24-1 with levels between continue factors, finding statistically significant differences for two factors such as concentrations of drugs and type of solvent where the organic phase is dissolved. This design shows the optimization of a formulation of capsaicin in solid lipid nanoparticles. The chapter also includes information on methods to prepare solid lipid nanoparticles (SLN), the variables involved, and a selection of studies about optimization of SLN formulations.

Nanoparticles formed from lipids are currently applied successfully to deliver drugs. The particle size of the nanoparticle system is an essential characteristic to enhance the entrance of the drugs inside tissues and cells. Using design of experiment is appealing to find the specific conditions to optimize particle size of drug-loaded nanoparticles. Authors of this chapter applied a fractional factorial design of half fraction 24-1 with levels between continue factors, finding statistically significant differences for two factors such as concentrations of drugs and type of solvent where the organic phase is dissolved. This design shows the optimization of a formulation of capsaicin in solid lipid nanoparticles. The chapter also includes information on methods to prepare solid lipid nanoparticles (SLN), the variables involved, and a selection of studies about optimization of SLN formulations.

Elevated levels of low-density lipoprotein (LDL) are associated with increased risk of coronary heart disease (CHD). Although smaller LDL particles are more atherogenic, it is not clear how LDL particle size influences atherogenesis. Smaller particles may be more prone to macrophage uptake and plaque formation. Alternatively, increased rates of lipid oxidation may explain the atherogenic effects of smaller LDL. We have developed a mimic of LDL that allows independent examination of the effect of LDL size and oxidation. We have engineered LDL mimics using liposome-encapsulated gold nanoparticles, in which the size and surface charge are independently controlled during synthesis. Here we examine the effects of lipid composition on zeta potential and electrophoretic mobility of LDL mimics. Using these mimics, we explored the effect of the lipid coating on the nanoparticles including anionic lipids and oxidized lipids. Dynamic light scattering was used to determine the size of the mimics and gel electrophoresis was used to measure the mobility and calculate zeta potential. The charge of the lipid coating influenced the mobility and we anticipate this will influence how the mimics interacts with proteins.