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Colloidally Stable Small Unilamellar Stearyl Amine Lipoplexes for Effective BMP-9 Gene Delivery to Stem Cells for Osteogenic Differentiation
Abstract
The biocompatibility of cationic liposomes has led to their clinical translation in gene delivery and their application apart from cancer to cardiovascular diseases, osteoporosis, metabolic diseases, and more. We have prepared PEGylated stearyl amine (pegSA) lipoplexes meticulously considering the physicochemical properties and formulation parameters to prepare single unilamellar vesicles (SUV) of < 100 nm size which retain their SUV nature upon complexation with pDNA rather than the conventional lipoplexes which show multilamellar nature. The developed PEGylated SA lipoplexes (pegSA lipoplexes) showed a lower N/P ratio (1.5) for BMP-9 gene complexation while maintaining the SUV character with a unique shape (square and triangular lipoplexes). Colloidal and pDNA complexation stability in the presence of electrolytes and serum indicates the suitability for intravenous administration for delivery of lipoplexes to bone marrow mesenchymal stem cells through sinusoidal vessels in bone marrow. Moreover, lower charge density of lipoplexes and low oxidative stress led to lower toxicity of lipoplexes to the C2C12 cells, NIH 3T3 cells, and erythrocytes. Transfection studies showed efficient gene delivery to C2C12 cells inducing osteogenic differentiation through BMP-9 expression as shown by enhanced calcium deposition in vitro, proving the potential of lipoplexes for bone regeneration. In vivo acute toxicity studies further demonstrated safety of the developed lipoplexes. Developed pegSA lipoplexes show potential for further in vivo preclinical evaluation to establish the proof of concept.
... Ligado a isso, as células tronco possuem em sua constituição a possibilidade de se regenerar com o correto estímulo biológico, bem como se diferenciar dependendo de seu potencial (PRANSKUNAS et al., 2019;LUO et al., 2021). No caso das de origem mesenquimais (CTM), é possível observar a diferenciação de diversos tecidos (VHORA et al., 2018), tais como o adiposo e osteogênico (LUO et al., 2021). Sua associação a proteínas ósseas, como a proteína morfogenética óssea 9 (BMP-9) é algo que vem sendo avaliado na ciência regenerativa (PRANSKUNAS et al., 2019;RODRÍGUEZ-MERCHÁN et al., 2021). ...
... Todavia, ainda não foram observados na literatura um estudo que evidencie de forma completa essa regeneração tecidual (REDMAN et al., 2016;PRANSKUNAS et al., 2019). Esse fator advém em decorrência comportamento biológico do corpo humano variável, podendo ser degradado de forma rápida ou possibilitar sua permanência por um longo período prejudicando o processo de regeneração do tecido ósseo (VHORA et al., 2018). ...
... Apesar dos estudos evidenciarem efeitos promissores por meios experimentais (VHORA et al., 2018), isso não reduz a necessidade de avaliação clínica, visto que é fundamental o tratamento do defeito ósseo de forma preexistente e que são mais difíceis de realização de tratamento (DENG et al., 2018). Desse modo, evidencias sugerem que essas células mesenquimais podem gerar o aumento da angiogênese quando comparado a outros meios celulares em avaliação, estimulando assim o reparo ósseo em diferentes tipos e tamanhos (REDMAN et al., 2016;DENG et al., 2018). ...
Bone tissue is composed of cells capable of tissue repair, remodeling and regeneration, but which, in large proportions, do not present satisfactory results. Thus, new therapies based on regenerative medicine using stem cells have been evaluated in order to solve the problem. The present study seeks to evaluate, through an integrative literature review, the bone formation potential of mesenchymal stem cells (MSC) over-expression of bone morphogenetic protein 9 (BMP-9). This is an exploratory, qualitative and descriptive study. For its idealization, the descriptors registered in the DeCS/MeSH “regeneration”, “mesenchymal stem cells”, “bone morphogenetic protein 9” and “growth differentiationfactor 2” were linked using the Boolean operators “and” and “or” and applied to the databases from PubMed/Medline, Scielo, Bireme and Google Scholar. Of the 142 studies located in a time frame of 10 years (2012 to 2022), 12 articles published in English and Portuguese were included in the final sample. Among the results, in vitro and in vivo studies show the therapy as promising in the process of bone tissue formation. BMP-9 and CTM increased the differentiation potency of cells in the tissue, enabling therepair of extensive bone defects. Technological tools of gene editing have suggested great possibilities to revolutionize science, since they are guides for the cleavage of DNA. Future studies are needed with a focus on cell therapy, so that the applicability of this technique is scientifically deepened and can later advance to its clinical use in the health sciences.
... The main use of genosomes is the transfer of tumor suppressor genes into cancer cells [100]. Vhora et al. developed small unilamellar lipoplexes for safe and effective delivery of pDNA expressing BMP-9 for IV administration for osteoinduction [101]. They found that lipoplexes were able to protect complexed pDNA from serum nucleases indicating the stability of the pegSA lipoplexes in blood. ...
... They found that lipoplexes were able to protect complexed pDNA from serum nucleases indicating the stability of the pegSA lipoplexes in blood. Moreover, the lipoplexes showed significantly less cytotox-icity on C2C12 stem cells and NIH 3T3 cells and lower ROS induction [101]. ...
Background
The vesicular drug delivery systems (VDDSs) are colloidal dispersions, which can be described as highly ordered assemblies composed of one or more concentric bilayers formed as a result of the self-assembling of amphiphilic building blocks in the presence of water.
Objectives
VDDSs are important to target the entrapped drugs at specific sites inside the body, control the drug release, enhance the drug bioavailability, and reduce the undesired side effects.
Methods
There are different types of VDDSs suitable for the entrapment of both hydrophilic and lipophilic drugs. According to the composition, VDDSs are classified into lipid-based and non-lipid-based VDDSs.
Results
There are different types of VDDSs which include liposomes, ethosomes, transferosomes, ufasomes, colloidosomes, cubosomes, niosomes, bilosomes, aquasomes, etc.
Conclusion
This review article aims to address the different types of VDDSs, their advantages and disadvantages, and their therapeutic applications.
... Small unilamellar liposome vesicles (SUVs) are considered as the best structures for maintaining the colloidal stability of lipoplexes. Lipoplexes having PEGylated (Polyethylene glycol) stearyl amine (supports transfection of low transfection efficacy cells) are considered optimum for SUV (< 100 nm) preparation [26]. Several studies available in support of the beneficial role of liposomes as a nonviral vector for osteoporosis treatment are discussed below. ...
... Then, the lipoplexes obtained by incubating liposomes with BMP-9 pDNA were successful in promoting osteogenic differentiation in rodents. These lipoplexes were equipped with features like significant stability, low charge density, low toxicity, and safe gene delivery by intravenous route [26]. ...
Introduction: Treatment of osteoporosis with the available drug formulations is still challenging due to multiple associated limitations such as chronic treatment, off-target side effects, poor bone targeting, and low bioavailability. Adopting advanced bone-targeted drug delivery strategies like liposomes is one of the safe and effective approaches for osteoporosis treatment.
Areas covered: This review summarizes the applications of liposomes in gene delivery, bone regeneration, bone-targeted delivery, and as a carrier for drug encapsulation in the treatment of osteoporosis. Details of all the supportive studies are discussed here and the bone-specific roles of the strategies like new generation liposomes in osteoporosis are elaborated. The future scope of performing in-depth research on the bone-targeted liposomes is discussed.
Expert opinion: Liposomes-based bone-targeted delivery of therapeutics seems to be a promising approach for the effective treatment of osteoporosis. But till date, the tremendous in vitro and in vivo research on liposomes has failed to attain significant progress in their clinical translation. From bench to bedside success of the research an interdisciplinary collaboration between the preclinical and clinical experts engaged at different stages of liposomes development is required.
... Small unilamellar liposome vesicles (SUVs) are considered as the best structures for maintaining the colloidal stability of lipoplexes. Lipoplexes having PEGylated (Polyethylene glycol) stearyl amine (supports transfection of low transfection efficacy cells) are considered optimum for SUV (< 100 nm) preparation [26]. Several studies available in support of the beneficial role of liposomes as a nonviral vector for osteoporosis treatment are discussed below. ...
... Then, the lipoplexes obtained by incubating liposomes with BMP-9 pDNA were successful in promoting osteogenic differentiation in rodents. These lipoplexes were equipped with features like significant stability, low charge density, low toxicity, and safe gene delivery by intravenous route [26]. ...
... For periodontal repair, the gene could be either injected directly into the periodontal defect via a retrovirus or incorporated into embryonic stem cells (ES) or adult stem cells, which were expanded and delivered into the defect (Figure 7) [113]. An advantage of gene therapy was that it could achieve greater bioavailability of growth factors within the periodontal wounds [114]. The delivery methods and DNA vectors in periodontal tissue engineering had the goal to maximize the duration of growth factors, optimize the delivery technique to the periodontal wounds, and minimize the patient risk [115]. ...
... An advantage of gene therapy was that it could achieve greater bioavailability of growth factors within the periodontal wounds [114]. The delivery methods and DNA vectors in periodontal tissue engineering had the goal to maximize the duration of growth factors, optimize the delivery technique to the periodontal wounds, and minimize the patient risk [115]. ...
Periodontitis is a prevalent infectious disease worldwide, causing the damage of periodontal support tissues, which can eventually lead to tooth loss. The goal of periodontal treatment is to control the infections and reconstruct the structure and function of periodontal tissues including cementum, periodontal ligament (PDL) fibers, and bone. The regeneration of these three types of tissues, including the re-formation of the oriented PDL fibers to be attached firmly to the new cementum and alveolar bone, remains a major challenge. This article represents the first systematic review on the cutting-edge researches on the regeneration of all three types of periodontal tissues and the simultaneous regeneration of the entire bone-PDL-cementum complex, via stem cells, bio-printing, gene therapy, and layered bio-mimetic technologies. This article primarily includes bone regeneration; PDL regeneration; cementum regeneration; endogenous cell-homing and host-mobilized stem cells; 3D bio-printing and generation of the oriented PDL fibers; gene therapy-based approaches for periodontal regeneration; regenerating the bone-PDL-cementum complex via layered materials and cells. These novel developments in stem cell technology and bioactive and bio-mimetic scaffolds are highly promising to substantially enhance the periodontal regeneration including both hard and soft tissues, with applicability to other therapies in the oral and maxillofacial region.
... It is a well-known fact that highly charged nanocarriers may cause higher cytotoxicity due to the disruption of the cell membrane [78]. A similar toxic effect of SA has been also reported in the literature [79,80]. Therefore, it can be said that neutral liposomes are safer than cationic ones to use as theranostic agents for infection. ...
Infectious diseases are still the major issue not only due to antibiotic resistance but also causing deaths if not diagnosed at early-stages. Different approaches including nanosized drug delivery systems and theranostics are researched to overcome antibiotic resistance, decrease the side effects of antibiotics, improve the treatment response, and early diagnose. Therefore, in the present study, nanosized, radiolabeled with 99mTc, colistin encapsulated, neutral and cationic liposome formulations were prepared as the theranostic agent for Pseudomonas aeruginosa infections. Liposomes exhibited appropriate physicochemical properties thanks to their nano-particle size (between 173 and 217 nm), neutral zeta potential value (about − 6.5 and 2.8 mV), as well as encapsulation efficiency of about 75%. All liposome formulations were radiolabeled with over 90% efficiency, and the concentration of stannous chloride was found as 1 mg.mL−1 to obtain maximum radiolabeling efficiency. In alamar blue analysis, neutral liposome formulations were found more biocompatible compared with the cationic formulations. Neutral colistin encapsulated liposomes were found to be more effective against P. aeruginosa strain according to their time-dependent antibacterial effect, in addition to their highest bacterial binding capacity. As conclusion, theranostic, nanosized, colistin encapsulated, neutral liposome formulations were found as promising agents for the imaging and treating of P. aeruginosa infections.
... Furthermore, in vivo, acute toxicity testing has shown that the lipoplexes produced are safe. In vivo preclinical testing of PegSA lipoplexes is promising for future proof-of-concept studies Vhora et al. (2018) gold nanoparticles (GNP-CPC) CPC containing gold nanoparticles is known as GNP-CPC. DPSCs from humans were studied to see if they could induce osteogenesis Additionally, GNP-CPC significantly enhanced osteogenic activity in hDPSCs. ...
Craniofacial deformities (CFDs) develop following oncological resection, trauma, or congenital disorders. Trauma is one of the top five causes of death globally, with rates varying from country to country. They result in a non-healing composite tissue wound as they degenerate in soft or hard tissues. Approximately one-third of oral diseases are caused by gum disease. Due to the complexity of anatomical structures in the region and the variety of tissue-specific requirements, CFD treatments present many challenges. Many treatment methods for CFDs are available today, such as drugs, regenerative medicine (RM), surgery, and tissue engineering. Functional restoration of a tissue or an organ after trauma or other chronic diseases is the focus of this emerging field of science. The materials and methodologies used in craniofacial reconstruction have significantly improved in the last few years. A facial fracture requires bone preservation as much as possible, so tiny fragments are removed initially. It is possible to replace bone marrow stem cells with oral stem cells for CFDs due to their excellent potential for bone formation. This review article discusses regenerative approaches for different types of craniofacial diseases.
... However, cytotoxicity limits the clinical use of SA as the hydrophilic nitrogen 'head' group of the molecule interacts with certain enzymes [36,42]. Other works reported apoptosis induced by SA generating reactive oxygen species, activating protein kinase C, or enhancing the release of apoptosis-dependent proteins, and hemolysis arising from the interaction between the molecule and the negatively charged erythrocyte membrane [43,44]. Human red blood cells are less sensitive to SA; thus, the addition of small amounts can be safe [45]. ...
Liposomal formulations, as versatile nanocarrier systems suitable for targeted delivery, have a highly focused role in the therapy development of unmet clinical needs and diagnostic imaging techniques. Formulating nanomedicine with suitable zeta potential is an essential but challenging task. Formulations with a minimum ±30 mV zeta potential are considered stable. The charge of the phospholipid bilayer can be adjusted with membrane additives. The present Quality by Design-derived study aimed to optimise liposomal formulations prepared via the thin-film hydration technique by applying stearylamine (SA) or dicetyl phosphate (DCP) as charge imparting agents. This 32 fractional factorial design-based study determined phosphatidylcholine, cholesterol, and SA/DCP molar ratios for liposomes with characteristics meeting the formulation requirements. The polynomials describing the effects on the zeta potential were calculated. The optimal molar ratios of the lipids were given as 12.0:5.0:5.0 for the SA-PBS pH 5.6 (optimised sample containing stearylamine) and 8.5:4.5:6.5 for the DCP-PBS pH 5.6 (optimised sample containing dicetyl phosphate) particles hydrated with phosphate-buffered saline pH 5.6. The SA-PBS pH 5.6 liposomes had a vesicle size of 108 ± 15 nm, 0.20 ± 0.04 polydispersity index, and +30.1 ± 1.2 mV zeta potential, while these values were given as 88 ± 14 nm, 0.21 ± 0.02, and −36.7 ± 3.3 mV for the DCP-PBS pH 5.6 vesicles. The prepared liposomes acquired the requirements of the zeta potential for stable formulations.
... Meanwhile, BMP9 suppresses receptor activator of nuclear factor-κB (NF-κB) ligand-(RANKL-) induced osteoclast differentiation of bone marrow macrophages (BMMs) by inhibiting the Akt-NF-κB-NFATc1 pathway [32]. Therefore, BMP9 may be explored as an effective therapeutic strategy for osteoporosis [35][36][37][38]. ...
Characteristic bone metabolism was observed in obesity and diabetes with controversial conclusions. Type 2 diabetes (T2DM) and obesity may manifest increased bone mineral density. Also, obesity is more easily to occur in T2DM. Therefore, we infer that some factors may be linked to bone and obesity as well as glucose metabolism, which regulate all of them. Bone morphogenetic proteins (BMPs), belonging to the transforming growth factor- (TGF-) beta superfamily, regulate a diverse array of cellular functions during development and in the adult. More and more studies revealed that there exists a relationship between bone metabolism and obesity as well as glucose metabolism. BMP2, BMP4, BMP6, BMP7, and BMP9 have been shown to affect the pathophysiological process of obesity and glucose metabolism beyond bone metabolism. They may exert functions in adipogenesis and differentiation as well as insulin resistance. In the review, we summarize the literature on these BMPs and their association with metabolic diseases including obesity and diabetes.
... (b) Apoptosis can be induced by the binding of Caspase 9 to cytochrome c and Apaf1. p53 may activate the expression of Apaf1 and Bax37,38 . ...
The tumor suppressor TP53 gene is one of the most frequently mutated in different types of human cancer. Particularly in colorectal cancer (CRC), it is believed that TP53 mutations play a role in the adenoma-carcinoma transition of tumors during pathological process. The TP53 mutation is the key step driving the transition from adenoma to adenocarcinoma. The functional roles of TP53 mutation in tumor development have been comprehensively investigated. In this mini review, we comprehensively summarize the p53 mutants in CRC progression and discuss the current strategies for p53 mutants in malignancies. Keywords: p53 mutants, colorectal cancer, Tp53 mutation
... Epithelial cells have a short lifetime of 5-7 days, being continuously shed and replaced. Therefore, repeated administrations of gene therapies might be necessary when these cells are targeted [35][36][37] . ...
Colorectal cancer is the third most commonly diagnosed cancer in the world characterized by neoplasia in the colon, rectum, or vermiform appendix. Current treatment approaches include chemotherapy, radiotherapy and surgery however non-specific bio-distribution of anti-cancer drug, lack of effective and safe drug delivery career, drug resistance and relapse are major limiting factors of current therapy. Gene therapy is a technique for correcting defective genes responsible for disease development. The future of gene therapy depends on achieving successful delivery of wild type gene to replace a faulty gene. Recently, there has been an increasing interest in delivery of drugs and gene via the gastrointestinal tract. Gene therapy via this route has many advantages, including non-invasive access and the versatility to treat local diseases, such as inflammatory bowel disease, colorectal cancer, as well as systemic diseases, such as haemophilia. However, the intestine presents several distinct barriers and, therefore, the design of robust non-viral delivery systems is key to future success. The review covers obstacles in the path of successful gene therapy using oral route to treat colorectal cancer as well as strategies to overcome. Keywords: Colorectal cancer, Oral gene delivery, Targeted delivery
... [20] " Targeting moieties are ligands that bind to receptors that are overexpressed on cancer cells Figure 1. Conjugating targeting moieties to the surface of particles promotes uptake and intracellular retention of particles by malignant cells, both of which enhance therapeutic efficacy, [21,22,23,24] of these ligands to drugs or particles will result in receptor-mediated active targeting and higher drug or particle concentration in malignant cells than in non-malignant cells. [25,26,27,28] Active targeting promotes internalization of ligand-conjugated drug carriers into a cell via receptor-mediated endocytosis. ...
Cancer is defined by uncontrolled growth of cells and the treatment options available include surgery, radiotherapy as well as chemotherapy. Non-specific distribution of anti-cancer drugs in the body and associated deadly adverse effects limits the use of chemotherapeutic drugs and their effectiveness. Thus, the aim of targeted site specific therapeutics is to deliver anti-cancer drug to the right site of tumour to minimize drug uptake by non-malignant cells. There are two major strategies for targeting know as passive and active targeting. The mini-review describes about both the targeting strategies, mechanisms, challenges, research outcomes and example of such delivery systems for the treatment of cancer.
... Nevertheless, these vectors have substantial drawbacks, such as immunogenic potential, oncogenic residual risk, expensive production costs, and a capsid-limited loading capacity [6][7][8][9][10]. In order to solve these problems, non-viral vectors have been developed in the last few decades, with cationic polymers or cationic lipids as the main representatives [11,12]. Theoretically, there is no limit to loading capacity. ...
One major disadvantage of nucleic acid delivery systems is the low transfection or transduction efficiency of large-sized plasmids into cells. In this communication, we demonstrate the efficient transfection of a 15.5 kb green fluorescent protein (GFP)-fused HIV-1 molecular clone with a nucleic acid delivery system prepared from the highly potent peptide-mimicking cationic lipid OH4 in a mixture with the phospholipid DOPE (co-lipid). For the transfection, liposomes were loaded using a large-sized plasmid (15.5 kb), which encodes a replication-competent HIV type 1 molecular clone that carries a Gag-internal green fluorescent protein (HIV-1 JR-FL Gag-iGFP). The particle size and charge of the generated nanocarriers with 15.5 kb were compared to those of a standardized 4.7 kb plasmid formulation. Stable, small-sized lipoplexes could be generated independently of the length of the used DNA. The transfer of fluorescently labeled pDNA-HIV1-Gag-iGFP in HEK293T cells was monitored using confocal laser scanning microscopy (cLSM). After efficient plasmid delivery, virus particles were detectable as budding structures on the plasma membrane. Moreover, we observed a randomized distribution of fluorescently labeled lipids over the plasma membrane. Obviously, a significant exchange of lipids between the drug delivery system and the cellular membranes occurs, which hints toward a fusion process. The mechanism of membrane fusion for the internalization of lipid-based drug delivery systems into cells is still a frequently discussed topic.
... Efforts were directed towards fabricating the system with an unexplored cationic agent i.e. Stearylamine, otherwise commonly employed in liposomes for targeted and gene delivery (Rajendran et al., 2015;Tahara et al., 2018;Vhora et al., 2018;Dhawan et al., 2016). ...
Aim
To investigate comparative in vitro and in vivo performance of lipid vesicular and particulate systems in escalating oral bioavailability for superior hepatoprotection.
Materials and Methods
Systems were fabricated using easy to scale up process and novel excipients to deliver Silibinin. In vitro characterization followed by pharmacokinetic and pharmacodynamic evaluation in rats was conducted to establish a correlation.
Results
Nanoformulations resulted in 20 fold increase in solubilisation and significant increase in permeation. 2.5 fold increase in bioavailability was evident in vivo. Vesicles demonstrated greatest hepatoprotective potential in efficacy study.
Conclusion
The findings establish a link between in vitro and in vivo performance to rank order lipid nanoartchitects. Concurrently, a significant potential in therapeutic intervention of hepatotoxicity is envisaged as elucidated.
... The transfection efficiency and bioavailability of DNA nanostructures can be increased by coating them with lipid membranes or proteins. 28 Linko et al. 29 coated DNA origami structures with capsid protein (CP) of cowpea chlorotic mottle virus. The coated DNA-CP structures were investigated via TEM and gel electrophoretic mobility shift assay techniques. ...
Delivery of the drug to a desired point of body and controlled release of the therapeutic agent are important features, provided by drug delivery systems (DDSs), for development of today's effective medicines. A variety of nanomaterials or nanomolecules such as lipids/liposomes, nucleic acids, peptides/proteins, composites, polymers, or carbon nanotubes can be used as DDSs. Single-molecule characterization of these small materials in terms of their size, shape, surface, encapsulation efficiency, as well as interaction with the drug-receiving cell has importance for their efficiency. The loading, distribution, or leakage of the drug as well as its interaction with DDS should also be characterized. Although diverse techniques are present for characterization of specific DDS material, methods such as electron microscopy and fluorescence microscopy are widely used. In this review, the current methodologies utilized for the single-molecule characterization of mostly preferred DDS materials were presented.
... Although this was in disagreement with that of particle size obtained by DLS, such difference can be easily realized as DLS reports the hydrodynamic diameter of the particles, whereas in TEM analysis the size obtained was of the particle fixed in grid. 26 TEM images (Fig. 3B) confirmed nanoparticles of uniform size and were in concurrence with the DLS results and were spherical with smooth surface. TEM was used to confirm the information that the nanoparticles were spherical and was not in aggregate. ...
Despite all the research aiming to treat ocular diseases, age-related macular degeneration (AMD) remains one of the serious diseases worldwide, which needs to be treated. Neovascularization is a key factor in AMD and thus antiangiogenic therapy is beneficial in reducing the development of new abnormal blood vessels. Axitinib, multireceptor tyrosine kinase inhibitor, is a small molecule that works by blocking vascular endothelial growth factor receptors (VEGFR) and platelet-derived growth factor receptors (PDGFR) responsible for developing neovascularization. The goal of this study is to develop a sustained release formulation of axitinib-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles to minimize frequent administration of the drug by intravitreal injection. The nanoparticles were characterized for particle size and zeta potential, as well as using differential scanning calorimetry, transmission electrode microscope, and in vitro drug release profile. The cytotoxicity of the formulation was evaluated on human retinal pigmented epithelium ARPE19 cells by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide salt] assay. The cellular uptake, antimigration assay, and vascular endothelial growth factor (VEGF) expression levels were found out in vitro using cells. The optimized formulation was 131.33 ± 31.20 nm in size with -4.63 ± 0.76 mV zeta potential. Entrapment efficiency was found to be 87.9% ± 2.7%. The cytotoxicity of ARPE19 cells was <12% for nanoparticles suggesting the in vitro compatibility at 10 μM concentration of drug. Cellular uptake, antimigration assay, and VEGF expression levels for the nanoparticles suggested greater uptake, significant antiangiogenic potential, and inhibition of VEGF activity. The results showed successful development of axitinib-loaded PLGA nanoparticles as an alternative potential treatment for AMD.
... 2) Liposome increased stability via encapsulation. [7] 3) Liposomes are non-poisonous, bendy, biocompatible, completely biodegradable, and non-immunogenic for systemic and non-systemic administrations. [8] www.wjpr.net ...
Liposomes are spherical vesicles containing of more than one phospholipid bilayers. Liposomes are one of most efficient drug delivery systems which reduce toxicities of traditional drug and increases healing effect hence helps in reducing the dosing frequency. Today, liposomes are a very efficient and used as carrier for various drug, bacteria, virus and vaccines in various scientific disciplines amongst several different drug delivery systems, at present various liposomes formulation are in use of medical research because of their fast healing characteristic. Liposomes with changed surfaces have also been produced utilizing various atoms, alongside glycolipids or sialic acid. There are numerous liposomal formulations, which include EndoTAG1 (paclitaxel-stacked cationic liposomes), Lipoplatin (cisplatin-stacked long circling liposomes) and Stimuvax (a malignancy antibody), showing promising mending cost in logical studies. Presently, filtration and aseptic production are encouraged for the coaching of sterile liposomal merchandise. Numerous liposome primarily based drug components are authorised for medical use and many are beneath sizeable investigation. Thus, this noticeably simple technique has particular benefits for encapsulating treasured water-soluble materials. The preparation, advantages and limitations with respect to industrial application and regulatory requirement of the liposomes are defined in detail in this paper.
... Cationic lipids have been used in liposomal system to complex gene of interest and transfecting osteogenic cell lines. Delivery system for bone morphogenetic factor genes (BMP-9) have been investigated Important aspect of investigation were determination of the effect of cationic charge on complexation with plasmid; importance of PEGylation to maintain colloidal stability of lipoplexes in serum and electrolytes, effect of composition on lamellar nature of lipoplexes, and transfection potential in cell line, wherein size and zeta potential plays an important role (127). ...
Hematological cancers are a group of malignancies affecting human hematopoietic and lymphoid tissues. Although the patients respond to treatment regimen during initial phases, the hematoma tumor heterogeneity results in the presence of some minimal disease residue thereby exhibiting remission, relapses or refractoriness in disease conditions leading to poor overall survival period. The current therapeutic standard practices involve blending of conventional agents with novel targeting agents or immune-therapeutics in a cocktail to effectively reap the benefits of drugs acting through multiple signaling pathways. Considerable evaluation of the risk benefit ratio on part of clinicians is necessitated to select the best optimum therapy considering the high incidences of drug resistance. This drug resistance may be attributed to faulty upregulation or mutation of multiple drug resistance regulating genes, increased tumor cell immune system cross talk, increased expression of drug efflux pump inducers and inhibition of apoptosis among others. Conventional single drug nanotherapeutics as modulators of drug resistance have already clinically exhibited their potential by passively delivering the active cargo to desired targets in hematological neoplasms. However, with the ever-growing clinical failures of such therapies, the landscape of hematological cancer treatment has seen a plethora of changes in the last few years. The two towering changes in the treatment has been the approval of combinatorial drug nanocarrier Vyxeos™ and chimeric antigen receptor T cell (CAR-T) therapy Kymriah™ as well as Yescarta™. The approval of CAR-T therapy not only resulted in a paradigm shift in the avenues of blood cancer treatment towards personalized approaches but also saddled it with questions of economic viability and effectiveness in the entire spectrum of such neoplasms. Under such conditions, combinatorial drug nanocarriers encompassing synergistic ratios of clinically effective drug combinations affording temporal and spatial control present an exciting approach to overcome these drug resistance modalities. This platform provides increased chances of therapeutic in-vitro in-vivo correlation along with minimization of drug resistance and associated disease relapse conditions. The present review intends to present the current preclinical and clinical advances in combinatorial nanocarrier mediated management of drug resistance in hematological cancers.
... It can be considered as a most important strategy as it also protects drug from enzymatic degradation, there is reduced side effects and can also transport non-transferable molecules [2]. However, at the time of designing and developing of nanocarriers, their fate after intravenous administration including biodistribution following opsonisation should also be considered by developing stealth or PEGylated nanocarrier system to improve its circulation time [30].The olfactory pathway has also been explored wildly in recent times. It is noninvasive method of targeting a drug to the brain. ...
One of the most challenging fields for pharmaceutical and biotechnological products is the drug delivery to the central nervous system. According to the WHO records, 6.8 million people die every year with neurological disorders and the numbers are increasing every year. All the drug delivery strategies to the brain are restricted by the Blood-Brain Barrier (BBB) which limits the therapeutic drug from reaching the targeted site thus showing poor therapeutic effects. Therefore, the need for the development ofnew neurotherapeutics is important as the current treatment does not provide an effective solution to the BBB challenges. Several pharmacological, physiological and BBB temporary disruption strategies are currently under research to enhance the penetration of the drug across the BBB. The developments made using these strategies and associated challenges are discussed in detail.
Cancer is still a global health problem. Among cancer types, breast cancer is the most frequently diagnosed one, and it causes a high mortality rate if not diagnosed in the early stages. In our study, imatinib encapsulated, nanosized, neutral/cationic liposome formulations were prepared as theranostic agents for breast cancer. After the characterization studies in which all liposomes exhibited proper profile owing to their particle size between 133 and 250 nm, polydispersity index values lower than 0.4, neutral and cationic zeta potential values, and high drug encapsulation efficiency, controlled drug release behaviors with zero‐order kinetic were obtained. The higher than 90% radiolabeling efficiency values were obtained thanks to the determination of optimum radiolabeling condition (80°C temperature, 5 mCi radioactivity, and 10 min incubation period). According to the resazurin assay evaluating the cytotoxic profile of liposomes on MCF7 cells, neutral empty liposome was found as biocompatible, while both cationic liposomes (empty and drug‐loaded ones) exhibited high nonspecific cytotoxicity at even low drug concentration due to the existence of stearyl amine in the formulations. However, dose‐dependent cytotoxic effect and the highest cellular binding capacity were obtained by imatinib loaded neutral liposomes. In conclusion, ⁶⁸ Ga‐radiolabeled, imatinib‐loaded, neutral, nanosized liposome formulation is the most promising one as a theranostic agent among all formulations.
The focus of this work is on the characterization of hydrophobically-modified polyethylene glycol hydrogels, to be used as drug delivery systems, by means of the combined used of rheology and low field Nuclear Magnetic Resonance. Indeed, these two techniques allowed understanding how the transient physical bonds deriving from hydrophobic association superimpose to the pre-existing covalent bonds. We found that the improvement of physical bonds can be achieved not only by increasing the content of hydrophobic segments but also by using thermal treatments after hydrogel preparation. Moreover, we proved the reliability of an overall interpretative model linking the dependence of the shear modulus and the average magnetic relaxation time. Finally, we proposed a new mathematical approach for the determination of the magnetic relaxation spectrum. This approach reduced the computational heaviness of the procedure and allowed to easily discern the different contributes nested in the overall magnetic relaxation spectrum, an aspect that the traditional approach cannot provide directly.
Targeted gene delivery is a scientific approach with numerous advantages in the fields of bio- and personalized medicine. The delivery of genes and generally nucleic acids (i.e., DNA, RNA, etc.) into cellular and subcellular organelles for the treatment of several diseases seems to be the future in the design and development of medicines with limited adverse drug reactions and a high ratio of effectiveness and efficacy. The increased impact of the targeted gene delivery is in line with the increased usage of the nanosystems and their applications in the development of gene delivery systems. Lipoplexes and polyplexes are the most important nanosystems that are used for targeted gene delivery. They are composed of lipids (cationic and helper lipids) and polymers (cationic and/or stimuli-responsive polymers), respectively. These cationic materials can self-assemble into complexes in the presence of nucleic acids, which are negatively charged. They are also characterized as nonviral (synthetic) carriers of nucleic acids. This chapter aims to present the technology and the applications of lipoplexes and polyplexes in the field of targeted gene delivery. Special attention will be given to the mechanisms by that lipoplexes and polyplexes are utilized for the delivery and release of the complexed nucleic acids.
The ever‐evolving quest for novel approaches to treat diseases has led to the advancement of gene delivery research fueled by academic and industrial inputs. Understanding of the genetic and epigenetic control of gene expression has not only provided the mechanisms of chronic, critical, and rare diseases but has also set the groundwork for the targeted treatments. Started with viral vectors for protein expression, present gene delivery research harnesses the power of long‐known therapeutic protein‐expressing genes, RNA‐based therapeutics such as siRNA, miRNA, mRNA, piRNA, and novel CRISPER/Cas9‐based genome editing tools. This surge in research has started the evolution of nanocarriers for overcoming barriers of in vivo gene delivery and to provide effective treatment. Vast diversity of nanocarriers including liposomes, lipidic and polymeric nanoparticles, lipopolymeric systems, dendrimers, and so on have shown therapeutic potential. However, their successful clinical translation requires overcoming associated safety and toxicity concerns which range from the suitability of carrier system for the desired route of administration and acute toxicological profile to chronic safety issues related to carrier or the gene cargo. This chapter will detail the learnings from the preclinical and clinical trials on toxicity and safety concerns with different gene delivery nanocarriers and research done so far to overcome the above‐said issues.
This work reports on a novel method to synthesize hydrophobically-modified hydrogels by curing epoxy monomers with amines. The resulting networks contain hydrophilic poly(ethylene glycol) (PEG) segments, poly(propylene glycol) (PPG) segments, and C18 alkyl segments. By varying the content of C18 segments, networks with different hydrophilic-lipophilic balance (HLB) are obtained. All networks show an amphiphilic behavior, swelling considerably both in organic solvents and in aqueous media. In the latter they display a thermosensitive behavior, which is highly affected by the network HLB and the pH of the solution. A decrease in HLB results in an increment of the polymer weight content (wp) due to hydrophobic association. Furthermore, a reduction in HLB induces a remarkable increase in initial modulus, elongation at break and tensile strength, especially when wp becomes greater than about 10%. Low field nuclear magnetic resonance (LF-NMR) experiments evidence that, when HLB decreases, a sudden and considerable increase in hydrogel heterogeneity takes place due to occurrence of extensive physical crosslinking. Available data suggest that in systems with wp ≳ 10% a continuous physical network superimposes to the pre-existing chemical network and leads to a sort of double network capable of considerably improving hydrogel toughness.
Nonviral vectors which offer a safer and versatile alternative to viral vectors have been developed to overcome problems caused by viral carriers. However, their transfection efficacy or level of expression is substantially lower than viral vectors. Among various nonviral gene vectors, lipid envelops systems are an ideal platform for the incorporation of safety and efficacy into a single delivery system. Emerging strategies for gene delivery using lipid-based delivery systems mainly aim at improving the transfection efficiency and potency while reducing toxicity, achieving prolonged release, cell-specific targeting, co-delivery of drug and gene. Earlier efforts to improve the transfection efficiency while overcoming the toxicity led to the need for preparing conjugates of lipids with polyamines. In this review, we highlight current lipidic vectors that have been developed for gene therapy, challenges, and their solutions.
Osteoporosis is a worldwide disease characterized by reduction of bone mass and alteration of bone architecture resulting in increased bone fragility and increased fracture risk. Although it is seen in all age groups, gender, and races, it is more common in Caucasians (white race), older people, and women. With an aging population and
longer life span, osteoporosis is increasingly becoming a global epidemic. Currently, it has been estimated that more than 200 million people are suffering from osteoporosis. Moreover, osteoporosis results in a decreased quality of life, increased disability-adjusted life span, and big financial burden to health insurance systems of countries that are responsible for the care of such patients. Therefore, increasing awareness in medical field, which, in turn, facilitates increase awareness of the normal populace, will be effective in preventing this epidemic.
The resistance of cancer cells to chemotherapy has presented a formidable challenge. The current research aims at evaluating whether silencing of the cisplatin efflux promoter gene ABCC3 using siRNA co-loaded with the drug in a nanocarrier improves its efficacy in non-small cell lung cancer (NSCLC). Hybrid nanocarriers (HNCs) comprising lipids and poly(lactic acid-polyethylene glycol) di-block copolymer (PEG-PLA) were prepared for achieving the simultaneous delivery of cisplatin caprylate and ABCC3-siRNA to the cancer cells. PEGylation of the formulated HNCs was carried out using post-insertion technique for imparting long circulation characteristics to the carrier. The optimized formulation exhibited an entrapment efficiency of 71.9 ± 2.2% and 95.83 ± 0.39% for cisplatin caprylate and siRNA respectively. Further, the HNC was found to have hydrodynamic diameter of 153.2 ± 1.76 nm and + 25.39 ± 0.49 mV zeta potential. Morphological evaluation using cryo transmission electron microscopy confirmed the presence of lipid bilayer surrounding the polymeric core in HNCs. The in vitro cellular uptake studies showed improved uptake, while cell viability studies of the co-loaded formulation in A549 cell-line indicated significantly improved cytotoxic potential when compared with drug solution and drug-loaded HNCs; cell cycle analysis indicated increased percentage of cell arrest in G2-M phase compared with drug-loaded HNCs. Further, the gene knock-down study showed that silencing of ABCC3 mRNA might be improved in vitro efficacy of the formulation. The optimized cisplatin and ABCC3 siRNA co-loaded formulation presented significantly increased half-life and tumour regression in A549 xenograft model in BALB/c nude mice. In conclusion, siRNA co-loaded formulation presented reduced drug resistance and increased efficacy, which might be promising for the current cisplatin-based treatments in NSCLC.
Liposomes are small spherical vesicles composed mainly of phospholipids and cholesterol. Over the years, a number of liposomal formulations have shown clinical promise, but the use of liposomes in oral drug delivery is still limited. This is partly due to the vulnerability of conventional liposomes to the detrimental effect of gastrointestinal destabilizing factors and also to the poor efficiency in intestinal absorption of liposomes. Some of these issues can be ameliorated using the layer-by-layer (LbL) assembly technology, which has been widely applied in surface modification of various nanoparticulate systems. Discussions on LbL functionalization of liposomes as oral drug carriers, however, are scant in the literature. To fill this gap, this article presents an overview of the roles of LbL functionalization in the development of liposomes, followed by a discussion of major principles of molecular design and engineering of LbL-functionalized liposomes for oral drug delivery. Regarding the versatility offered by LbL assembly, it is anticipated that LbL-functionalized liposomes may emerge as one of the important carriers for oral drug administration in the future.
Cationic liposomes (CLs) have been regarded as the most promising gene delivery vector for decades with the advantages of excellent biodegradability, biocompatibility and high nucleic acids encapsulation efficiency. However, the clinical use of CLs in cancer gene therapy is limited because of many uncertain factors in vivo. Extracellular barriers such as opsonization, rapid clearance by reticuloendothelial system and poor tumor penetration, and intracellular barriers including endo/lysosomal entrapped network and restricted diffusion to nucleus, make CLs not an ideal vector for transferring extrinsic genes in the body. However, the obstacles in achieving productive therapeutic effects of nucleic acids can be addressed by tailoring the properties of CLs, which are influenced by lipid compositions and surface modification. This review has focused on the physiological barriers of CLs against cancer gene therapy and the effects of lipid compositions on governing transfection efficiency, and briefly discussed the impacts of particle size, membrane charge density and surface modification on the fate of CLs in vivo, which may provide guidance for their preclinical studies.
Exercise, low fat diet, or supplementations alone could show improvement in spermatogenesis quality afterlong period of times that could reach up to months especially in males who have high access to a high fat diet (HFD), so the current work was generated to induce this improvement in less amount of time, ~ 2 weeks, using intense exercise and cinnamaldehyde in Wistar male rats (WMR) fed an HFD. To initiate the experiment, 40 WMRs(fed an HFD for 4 weeks before the exposure to the experimental variables (EEV) and continued after that) were randomly divided into 4 groups in which a group received intense exercise and cinnamaldehyde (IECd), a group exposed to cinnamaldehyde alone (Cd), another group subjected to intense exercise alone (IE), and a group given the carrier of CD (CrCd). The EEV was lasted for 2 weeks, and the animals were subjected to measuring serum testosterone (ST), sperm counting (SpC) of motile and viable sperms, and inducible nitric oxide synthase (iNOS) gene expression (iNOSge) using real time-polymerase chain reaction (RT-PCR).The levels of the ST and SpC of motile and viable sperms were significantly (p<0.05) increased in the IECd group when compared with those in the Cd, IE, and CrCd groups. No differences (p>0.05) were noticed between Cd, IE, and CrCd groups when tested for the levels of ST and SpC. The iNOSge levels were significantly (p<0.05) decreased in the IECd group when compared with those in the Cd, IE, and CrCd groups. No differences (p>0.05) were noticed between Cd, IE, and CrCd groups when tested for the levels of iNOSge. This study indicates important effects of the intense exercise and cinnamaldehyde when applied together in improving the quality of spermatogenesis in Wistar rats that could be tested in the future on the human model.
Parenteral delivery is the second leading drug delivery approach after oral delivery. With the current surge of targeted therapeutic (proteins and peptides) and novel formulation approaches, two major sectors of parenteral controlled drug delivery, prolonged release injectables and stimuli-controlled injectables, are projected to grow extensively in the coming two decades as indicated by the current regulatory product approval and industrial pipeline. This chapter discusses these two sectors with details on the impacted therapeutic disease area, potential drug candidates, advancements in manufacturing technologies, and formulation technologies. Moreover, a comprehensive account is also given on the current and next-generation injection devices. Detailed discussions will provide a thorough guide for the development of the parenteral-prolonged and controlled drug delivery systems.
Quercetin, a natural polyphenol with strong antioxidant activity, was loaded in Eudragit-coated liposomes conceived for intestinal delivery. Eudragit was used to form a protective shell on the surface of liposomes to resist gastric environment and allow the delivery of quercetin to the intestine. The physico-chemical properties of the liposomes were assessed by light scattering and cryogenic transmission electron microscopy. Small, spherical, uni- and bilamellar liposomes were produced, with the presence of multilamellar structures in Eudragit-coated liposomes. The Eudragit coating increased the physical stability of the vesicular system in fluids mimicking the gastrointestinal environment. Further, the incorporation of quercetin in the vesicular system did not affect its intrinsic antioxidant activity, as DPPH radical was almost completely inhibited, and the vesicles were also capable of ensuring optimal protection against oxidative stress in human intestinal cells by reducing reactive oxygen species (ROS)production. The proposed approach based on quercetin vesicular formulations may be of value in the treatment of pathological conditions associated with intestinal oxidative stress.
Rational design of novel ionizable lipids for development of lipid-nucleic acid nanoparticles (LNP) is required for safe and effective systemic gene delivery for osteoporosis. LNPs require suitable characteristics for intravenous administration and effective accumulation in bone marrow for enhanced transfection. Hence, lipids with C18 tail and ionizable headgroup (Boc-His-ODA/BHODA and His-ODA/HODA) were synthesized and characterized physicochemically. LNPs were prepared with bone morphogenetic protein-9 gene (BHODA-LNP, HODA-LNP, and bone-homing peptide targeted HODA-LNP – HODA-LNPT). Thorough physicochemical (electrolyte stability, DNase I and serum stability) and biological (hemolysis, ROS induction, cytotoxicity and transfection) characterization was carried out followed by acute toxicity studies and therapeutic performance studies in ovariectomized rat model. Lipids with pH dependent ionization were successfully synthesized. LNPs thereof were ∼100 nm size with stability against electrolytes, DNase I and serum and exhibited low hemolytic potential demonstrating suitability for intravenous administration. LNPs exhibited minimal cytotoxicity, non-significant ROS induction and high transfection. In vivo studies demonstrated safety and improved bone regeneration in OVX rats with HODA-LNPT showing significantly better performance. Synthesized ionizable lipids offer safe and effective alternative for preparation of LNPs for gene delivery. Targeted BMP-9 LNP show potential for systemic osteoporosis treatment.
Gene therapy involving p11 cDNA has been thought to be a futuristic approach for the effective management of depression as the existing treatment regimen presents many issues regarding late onset of action, patient withdrawal and their side effects. For the effective transfection of p11 gene intracellularly, two cationic lipids based on phospholipid DOPE conjugated to basic amino acids histidine and arginine were synthesised, used for liposome formulation and evaluated for their ability as gene delivery vectors. They were further converted using IGF-II mAb into immunoliposomes for CNS targeting and mAb conjugation to liposomes were characterised by SDS-PAGE. They were further analysed by in vitro characterisation studies that include erythrocyte aggregation study, electrolyte-induced study, heparin compatibility study and serum stability studies. SHSY5Y cells were used for conducting cytotoxicity of synthesised lipids and live imaging of cell uptake for 25 min. Finally, the brain distribution studies and western blot were carried out in animals to evaluate them for their BBB permeation ability and effects on p11 protein which is believed to be a culprit. These formulated liposomes from synthesised lipids offer a promising approach for the treatment of depression.
Cationic liposomes have long been used as non-viral vectors for small interfering RNA (siRNA) delivery but are associated with high toxicity, less transfection efficiency, and in vivo instability. In this investigation, we have developed siRNA targeted to RRM1 that is responsible for development of resistance to gemcitabine in cancer cells. Effect of different lipid compositions has been evaluated on formation of stable and less toxic lipoplexes. Optimized cationic lipoplex (D2CH) system was comprised of dioleoyl-trimethylammoniumpropane (DOTAP), dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), hydrogenated soya phosphocholine (HSPC), cholesterol, and methoxy(polyethyleneglycol)2000-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (mPEG2000-DSPE). D2CH lipoplexes have shown particle size (147.5 ± 2.89 nm) and zeta potential (12.26 ± 0.54 mV) characteristics essential for their in vivo use. In vitro cytotoxicity study has shown low toxicity of developed lipoplexes as compared with lipofectamine-2000 up to N/P ratio as high as 7.5. Cell uptake studies and gene expression studies have confirmed intracellular availability of siRNA. In addition, developed lipoplexes also showed ~3 times less hemolytic potential as compared with DOTAP/DOPE lipoplexes at lipid concentration of 5 mg/mL. Lipoplexes also maintained particle size less than 200 nm on exposure to high electrolyte concentration and showed >70% siRNA retention in presence of serum showing siRNA protection conferred by lipoplexes. Furthermore, in vivo acute toxicity studies in mice showed that formulation was non-toxic up to a dosage of 0.75 mg of siRNA/kg as lipoplexes and 300 mg lipid/kg as blank liposomes indicating tolerability of lipoplexes at a dose much higher than required for therapeutic use. Promising results of this study warrant further investigation of developed siRNA lipoplexes for cancer treatment.
Polyethylene glycol (PEG) conjugation is rapidly evolving strategy to solve hurdles in therapeutic delivery and is being used as an add-on tool to the traditional drug delivery methods. Chemically, PEGylation is a term used to denote modification of therapeutic molecules by conjugation with PEG. Efforts are constantly being made to develop novel strategies for conjugation of these molecules with PEG in order to increase its current applications. These strategies are specific to the therapeutic system used and also depend on the availability of activated PEGylating agents. Therefore a prior knowledge is essential in selecting appropriate method for PEGylation. Once achieved, a successful PEGylation can amend the pharmacokinetic and pharmacodynamic outcomes of therapeutics. Specifically, the primary interest is in their ability to decrease uptake by reticuloendothelial system, prolong blood residence, decrease degradation by metabolic enzymes and reduce protein immunogenicity. The extensive research in this field has resulted into many clinical studies. The knowledge of outcome of these studies gave a good feedback and lessons which helped researchers to redesign PEG conjugates with improved features which can increase the chance of hitting the market. In light of this, the current paper highlights the approaches, novel strategies and the utilization of modern concept for PEG conjugation with respect to various bioactive components of clinical relevance. Moreover, this review also discusses potential clinical outcomes of the PEG conjugation, regulatory approved PEGylated product, clinical trials for newer formulations, and also provides future prospects of this technology.
Cisplatin, first (platinum) compound to be evolved as an anticancer agent, has found its important place in cancer chemotherapy. However, the dose-dependent toxicities of cisplatin, namely nephrotoxicity, ototoxicity, peripheral neuropathy, and gastrointestinal toxicity hinder its widespread use. Liposomes can reduce the toxicity of cisplatin and provide a better therapeutic action, but the low lipid solubility of cisplatin hinders its high entrapment in such lipid carrier. In the present investigation, positively charged reactive aquated species of cisplatin were complexed with negatively charged caprylate ligands, resulting in enhanced interaction of cisplatin with lipid bilayer of liposomes and increase in its encapsulation in liposomal carrier. Prepared cisplatin liposomes were found to have a vesicular size of 107.9 ± 6.2 nm and zeta potential of -3.99 ± 3.45 mV. The optimized liposomal formulation had an encapsulation efficiency of 96.03 ± 1.24% with unprecedented drug loading (0.21 mg cisplatin / mg of lipids). The in vitro release studies exhibited a pH-dependent release of cisplatin from liposomes with highest release (67.55 ± 3.65%) at pH 5.5 indicating that a maximum release would occur inside cancer cells at endolysosomal pH. The prepared liposomes were found to be stable in the serum and showed a low hemolytic potential. In vitro cytotoxicity of cisplatin liposomes on A549 lung cancer cell line was comparable to that of cisplatin solution. The developed formulation also had a significantly higher median lethal dose (LD50) of 23.79 mg/kg than that of the cisplatin solution (12 mg/kg). A promising liposomal formulation of cisplatin has been proposed that can overcome the disadvantages associated with conventional cisplatin therapy and provide a higher safety profile.
Cationic lipids have been used in the development of non-viral gene delivery systems as lipoplexes. Stearylamine, a cationic lipid that presents a primary amine group when in solution, is able to compact genetic material by electrostatic interactions. In dispersed systems such as nanoemulsions this lipid anchors on the oil/water interface confering a positive charge to them. The aim of this work was to evaluate factors that influence DNA compaction in cationic nanoemulsions containing stearylamine. The influence of the stearylamine incorporation phase (water or oil), time of complexation, and different incubation temperatures were studied. The complexation rate was assessed by electrophoresis migration on agarose gel 0.7%, and nanoemulsion and lipoplex characterization was done by Dynamic Light Scattering (DLS). The results demonstrate that the best DNA compaction process occurs after 120 min of complexation, at low temperature (4 ± 1 °C), and after incorporation of the cationic lipid into the aqueous phase. Although the zeta potential of lipoplexes was lower than the results found for basic nanoemulsions, the granulometry did not change. Moreover, it was demonstrated that lipoplexes are suitable vehicles for gene delivery.
Bone morphogenetic proteins (BMPs) are members of the TGF-β superfamily and play a critical role in skeletal development, bone formation and stem cell differentiation. Disruptions in BMP signaling result in a variety of skeletal and extraskeletal anomalies. BMP9 is a poorly characterized member of the BMP family and is among the most osteogenic BMPs, promoting osteoblastic differentiation of mesenchymal stem cells (MSCs) both in vitro and in vivo. Recent findings from various in vivo and molecular studies strongly suggest that the mechanisms governing BMP9-mediated osteoinduction differ from other osteogenic BMPs. Many signaling pathways with diverse functions have been found to play a role in BMP9-mediated osteogenesis. Several of these pathways are also critical in the differentiation of other cell lineages, including adipocytes and chondrocytes. While BMP9 is known to be a potent osteogenic factor, it also influences several other pathways including cancer development, angiogenesis and myogenesis. Although BMP9 has been demonstrated as one of the most osteogenic BMPs, relatively little is known about the specific mechanisms responsible for these effects. BMP9 has demonstrated efficacy in promoting spinal fusion and bony non-union repair in animal models, demonstrating great translational promise. This review aims to summarize our current knowledge of BMP9-mediated osteogenesis by presenting recently completed work which may help us to further elucidate these pathways.
Liposomes have a wide array of uses that have been continuously expanded and improved upon since first being observed to self-assemble into vesicular structures. These arrangements can be found in many shapes and sizes depending on lipid composition. Liposomes are often used to deliver a molecular cargo such as DNA for therapeutic benefit. The lipids used to form such lipoplexes can be cationic, anionic, neutral, or a mixture thereof. Herein physical packing parameters and specific lipids used for gene delivery will be discussed, with lipids classified according to overall charge.
Pluripotent mesenchymal stem cells (MSCs) are bone marrow stromal progenitor cells that can differentiate into osteogenic, chondrogenic, adipogenic, and myogenic lineages. We previously demonstrated that bone morphogenetic protein (BMP) 9 is one of the most potent and yet least characterized BMPs that are able to induce osteogenic differentiation of MSCs both in vitro and in vivo. Here, we conducted gene expression-profiling analysis and identified that Hey1 of the hairy/Enhancer of split-related repressor protein basic helix-loop-helix family was among the most significantly up-regulated early targets in BMP9-stimulated MSCs. We demonstrated that Hey1 expression was up-regulated at the immediate early stage of BMP9-induced osteogenic differentiation. Chromatin immunoprecipitation analysis indicated that Hey1 may be a direct target of the BMP9-induced Smad signaling pathway. Silencing Hey1 expression diminished BMP9-induced osteogenic differentiation both in vitro and in vivo and led to chondrogenic differentiation. Likewise, constitutive Hey1 expression augmented BMP9-mediated bone matrix mineralization. Hey1 and Runx2 were shown to act synergistically in BMP9-induced osteogenic differentiation, and Runx2 expression significantly decreased in the absence of Hey1, suggesting that Runx2 may function downstream of Hey1. Accordingly, the defective osteogenic differentiation caused by Hey1 knockdown was rescued by exogenous Runx2 expression. Thus, our findings suggest that Hey1, through its interplay with Runx2, may play an important role in regulating BMP9-induced osteoblast lineage differentiation of MSCs.
Regional (intratracheal or aerosol) delivery of cationic liposome-DNA complexes for gene therapy of lung disease offers distinct advantages over systemic (intravenous) administration. However, optimal formulations for early lung cancer treatment have not been established. Therefore, we investigated >50 different liposome and micelle formulations for factors that may affect their transcription efficiency and tested the ideal formulations in an in vivo mouse model. Our data showed that cationic liposomes were generally more effective at transfecting genes than were micelles of the same lipid composition, thus suggesting a role for the bilayer structure in facilitating transfection. In addition, the transfection efficiency of liposome-delivered genes was highly dependent upon the lipid composition, lipid/DNA ratio, particle size of the liposome-DNA complex, and cell lines used. By optimizing these factors in vitro and in vivo, we developed a novel liposome formulation (DP3) suitable for intratracheal administration. Using G67 liposome as control, we found that DP3 was more effective than G67 in vitro and as effective as G67 at both preventing lung tumor growth and prolonging survival in our lung cancer mouse model. We observed a positive correlation between the in vitro p53 function and the in vivo antitumoral activities of liposome-p53 formulations, which had not been reported previously in studies of an intravenous liposome gene delivery system. This correlation may facilitate the development and optimization of a liposome-p53 formulation for aerosol use in lung cancer patients.
Mechanisms of cationic lipid-based nucleic acid delivery are receiving increasing attention, but despite this the factors
that determine high or low activity of lipoplexes are poorly understood. This study is focused on the fine structure of cationic
lipid-DNA complexes (lipoplexes) and its relevance to transfection efficiency. Monocationic (N-(1-(2,3-dioleoyloxy)propyl),N,N,N-trimethylammonium chloride,N-(1-(2,3-dimyristyloxypropyl)-N,N-dimethyl-(2-hydroxyethyl)ammonium bromide) and polycationic (2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanammonium trifluoroacetate) lipid-based assemblies, with or without neutral lipid (1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine, 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine, cholesterol) were used to prepare lipoplexes of different L+/DNA− charge ratios. Circular dichroism, cryogenic-transmission electron microscopy, and static light scattering were used for
lipoplex characterization, whereas expression of human growth hormone or green fluorescent protein was used to quantify transfection
efficiency. All monocationic lipids in the presence of inverted hexagonal phase-promoting helper lipids (1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine, cholesterol) induced appearance of Ψ− DNA, a chiral tertiary DNA structure. The formation of Ψ− DNA was also dependent on cationic lipid-DNA charge ratio. On the other hand, monocationic lipids either alone or with 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine as helper lipid, or polycationic 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanammonium trifluoroacetate-based assemblies, neither of which promotes a lipid-DNA hexagonal phase, did not
induce the formation of Ψ− DNA. Parallel transfection studies reveal that the size and phase instability of the lipoplexes, and not the formation of
Ψ− DNA structure, correlate with optimal transfection.
Interaction of cationic lipid/DNA complex with the plasma is a limiting step for the cationic lipid-mediated intravenous gene transfer and expression process. Most of the plasma components that interact with the complex and inhibit its transfection efficiency are still unknown. In the present work, human plasma proteins and lipoproteins that bind to a cationic lipid/DNA complex were isolated on a sucrose density gradient and identified by 2-D gel electrophoresis. Protein binding did not result in complex dissociation or DNA degradation. The effects of several complex-binding plasma components on the transfection efficiency were studied using lung endothelial cells cultured in vitro. Lipoprotein particles caused a drastic loss of the transfection efficiency of the complex. Surprisingly, fibrinogen was found to activate the transfection process. The roles of these complex-binding plasma components on the complex uptake efficiency were quantitatively assessed using radiolabeled plasmid DNA and qualitatively evaluated using fluorescence microscopy. A good correlation was found between the effects of the complex-binding plasma components on the transfection and on cell uptake efficiencies. In contrast to what was generally believed, our data suggest that disruption of the complex does not occur when it is in contact with the plasma and therefore could not be responsible for the loss of transfection activity. Instead, coating of complexes with plasma components seems to be responsible for reduced uptake by cells, which in turn results in reduced transfection.
Complexes (lipoplexes) between cationic liposomes and single-strand oligodeoxynucleotides (ODN) are potential delivery systems for antisense therapy. The nanometer-scale morphology of these assemblies is relevant to their transfection efficiency. In this work the monocationic lipid dioleoyloxytrimethylammoniumpropane, the neutral "helper" lipid cholesterol, and an 18-mer anti-bcl2 ODN were combined at different ratios. The lipoplexes formed were characterized for the quantity of ODN bound, for the degree of lipid mixing, and for their size. The nanostructure of the system was examined by cryogenic-temperature transmission electron microscopy, augmented by small-angle x-ray scattering. Addition of ODN to cationic liposomes induced both liposome aggregation and the formation of a novel condensed lamellar phase. This phase is proposed to be stabilized by anionic single-strand ODN molecules intercalated between cationic bilayers. The proportion of cholesterol present apparently did not affect the nature of lipoplex microstructure, but changed the interlamellar spacing.
The ability of embryonic stem cells and adult stem cells to differentiate into specific cell types holds immense potential for therapeutic use in cell and gene therapy. Realization of this potential depends on efficient and optimized protocols for genetic manipulation of stem cells. In the study reported here, we demonstrate the use of nucleofection as a method to introduce plasmid DNA into embryonic and adult stem cells with significantly greater efficiency than electroporation or lipid-based transfection methods have. Using enhanced green fluorescent protein (eGFP) as a reporter gene, mouse embryonic stem cells were transfected both transiently and stably at a rate nearly 10-fold higher than conventional methods. The transfected cells retained their stem cell properties, including continued expression of the stem cell markers SSEA1, Oct4, and Rex1; formation of embryoid bodies; differentiation into cardiomyocytes in the presence of appropriate inducers; and, when injected into developing blastocysts, contribution to chimeras. Higher levels of transfection were also obtained with human embryonic carcinoma and human embryonic stem cells. Particularly hard-to-transfect adult stem cells, including bone marrow and multipotent adult progenitor cells, were also transfected efficiently by the method of nucleofection. Based on our results, we conclude that nucleofection is superior to currently available methods for introducing plasmid DNA into a variety of embryonic and adult stem cells. The high levels of transfection achieved by nucleofection will enable its use as a rapid screening tool to evaluate the effect of ectopically expressed transcription factors on tissue-specific differentiation of stem cells.
Cationic liposome-DNA complexes (lipoplexes) constitute a potentially viable alternative to viral vectors for the delivery of therapeutic genes. This review will focus on various parameters governing lipoplex biological activity, from their mode of formation to in vivo behaviour. Particular emphasis is given to the mechanism of interaction of lipoplexes with cells, in an attempt to dissect the different barriers that need to be surpassed for efficient gene expression to occur. Aspects related to new trends in the formulation of lipid-based gene delivery systems aiming at overcoming some of their limitations will be covered. Finally, examples illustrating the potential of cationic liposomes in clinical applications will be provided.
Docetaxel (DTX), one of the most effective chemotherapeutic agents for the treatment of metastatic breast cancer but finds limited clinical applications due to toxicity imparted by surfactant present in marketed formulation and non-specific bio-distribution of the drug. To address these issues, this study was undertaken by taking a novel approach of fabrication of DTX loaded scFv (Single chain variable fragment) conjugated human serum albumin (HSA) immunonanoparticles (INPs). Developed INPs were characterized for particle size, zeta potential, entrapment efficiency, in vitro drug release, stability, in vitro cell line studies and in vivo animal studies. DTX-HSA-INPs were spherical in shape with mean diameter of 160.7 ± 5.5 nm and they showed specific binding to the targeted receptor when studied in vitro in cell line culture. It showed enhanced intracellular accumulation of INPs in EGFR expressing breast carcinoma cells as compared to EGFR negative breast carcinoma cells and non-targeted nanoparticles (NPs) Improved cytotoxic potential was demonstrated by DTX-HSA-INPs compared to marketed formulation in EGFR-expressing breast carcinoma cells (MDA-MB-468 and MDA-MB-231). Improved pharmacokinetic profile and long circulation in vivo in animals also revealed that the novel scFv targeted DTX-HSA-INPs can be a better and one of the promising alternatives for breast cancer therapy.
Aim of this study was to develop anti EGFR antibody conjugated poly (lactide-co-glycolide) nanoparticles (NPs) to target epidermal growth factor receptor, highly expressed on non-small cell lung cancer cells to improve cytotoxicity and site specificity. Cetuximab was conjugated to Docetaxel loaded PLGA NPs by known EDC/NHS chemistry and characterized for size, zeta potential, conjugation efficiency and results were 128.4 ±3.6 nm, –31.0 ± 0.8 mV and 39.77 ± 3.4% respectively. In-vitro release study demonstrated sustained release of drug from NPs with 25% release at pH 5.5 after 48 hrs. In-vitro cytotoxicity studies demonstrated higher anti-proliferative activity of NPs than unconjugated NPs. Cell cycle analysis and apoptosis study were performed to evaluate extent of cell arrest at different phases and apoptotic potential for the formulations respectively. In-vivo efficacy study showed significant reduction in tumor growth and so antibody conjugated NPs present a promising active targeting carrier for tumor selective therapeutic treatment.
Combination strategy involving cyclodextrin (CD) complexation and liposomal system was investigated for Paclitaxel (PTX) to improve loading. Complexation was done using 2,6-di-O-methylbetacyclodextrin (DMβCD). Sterically stabilized double loaded PEGylated liposomes (DLPLs) containing PTX and PTX-DMβCD complex were prepared by thin film hydration. Physicochemical characterization of complex and prepared DLPLs was carried out. Cytotoxic potential, hemolytic potential and pharmacokinetics of DLPLs were tested in comparison to Taxol®. Aqueous solubility of PTX increased by almost 3x104 folds due to complexation with DMβCD as compared to pure drug solubility. Liposomal system was found to have 162.8 ± 4.1 nm size, zeta potential of -5.6 ± 0.14 mV and 2-fold increase in drug loading to 5.8 mole % for PTX due to double loading. DLPLs had low hemolytic potential and higher cytotoxicity on SKOV3 cells with improvement in IC50 value by 4.2 folds as compared to Taxol® at 48 hr. The anti-angiogenic potential of DLPLs was confirmed by 1.33 folds lesser wound recovery in SKOV3 cells compared to Taxol®. In-vivo pharmacokinetic evaluation of DLPLs in rats substantiates improvement in circulation time, higher plasma concentration and decreased clearance rate compared to Taxol®. An efficacious system with improved loading and pharmacokinetics was formulated as potential alternative for currently marketed PTX formulation.
Ordered amphiphile self-assembly materials with a tunable three-dimensional (3D) nanostructure are of fundamental interest, and crucial for progressing several biological and biomedical applications, including in meso membrane protein crystallization, as drug and medical contrast agent delivery vehicles, and as biosensors and biofuel cells. In binary systems consisting of an amphiphile and a solvent, the ability to tune the 3D cubic phase nanostructure, lipid bilayer properties and the lipid mesophase is limited. A move beyond the binary compositional space is therefore required for efficient engineering of the required material properties. In this critical review, the phase transitions upon encapsulation of more than 130 amphiphilic and soluble additives into the bicontinuous lipidic cubic phase under excess hydration are summarized. The data are interpreted using geometric considerations, interfacial curvature, electrostatic interactions, partition coefficients and miscibility of the alkyl chains. The obtained lyotropic liquid crystal engineering design rules can be used to enhance the formulation of self-assembly materials and provides a large library of these materials for use in biomedical applications (242 references).
Chitosan is among the few polymers with high biocompatibility and low toxicity. In the area of siRNA delivery, chitosan has been noted for its high buffer capacity in endosomal pH range. However, its applications are limited due to unfavorable physicochemical properties such as poor solubility, colloidal instability, unionized nature and weak binding with nucleic acids at physiologic pH which leads to premature release of nucleic acids, poor cell uptake and transfection. In order to overcome these limitations of chitosan, low molecular weight chitosan (LMWC) were prepared to improve its solubility and colloidal stability at physiologic pH. The obtained LMWC was conjugated with protamine to impart cationic charge and induce preferential binding with siRNA at physiologic pH. The polyplex were subjected to ionic cross-linking resulting in particle size of 143.7 nm and zeta potential of +12.8 mV. The polyplexes displayed enhanced resistance to displacement in heparin competition assay and to degradation in serum stability studies. Covalent conjugation also provided combined advantage of higher uptake due to cationic charge of protamine and endosomal escape capability of LMWC. The conjugate showed low cytotoxicity, high transfection efficiency and gene expression in vitro and were found safe in vivo.
Non-viral delivery using cationic solid lipid nanoparticles (SLNs) represents a useful strategy to introduce large DNA and RNA molecules to target cells. A careful selection of components and their amounts is critical to improve transfection efficiency. In this work, a selected and optimized formulation of SLNs was used to efficiently transfect circular DNA and linear RNA molecules into cells. We characterized the main physicochemical characteristics and binding capabilities of these SLNs and show that they deliver DNA and RNA molecules into cells where they display full bioactivity at nontoxic concentrations using fluorescence- and luminescence-based methodologies. Hence, we established a novel and simple SLN formulation as a powerful tool for future therapeutic use.
The ‘RNA interference’ has emerged as a potential therapeutic strategy owing to its high specificity to silent any malfunctioned gene in diseases with genetic background. Currently intravenous delivery of siRNA has been a preferred way of administration due to high access of blood to the organs where direct delivery is not possible. Among non-viral delivery systems enabling systemic delivery of siRNA, liposomes and lipid envelope systems appear to be promising due to their biocompatibility over other systems. However, these systems are still challenged by toxicity issues, instability in blood, non-specific distribution and low transfection efficiency after intravenous administration. Therefore, to increase the success of lipid based siRNA delivery, it is essential to understand the importance of various factors affecting the efficiency of siRNA delivery. The current review focuses on the formulation of lipid based siRNA formulations, the challenges posed in systemic delivery and various aspectsaffecting the transfection efficiency of such formulations. The review also focuses on emerging strategies for lipid based siRNA delivery and overviews clinical prospects for better development of siRNA delivery systems in future. Considering the current trends, it seems that liposomes and lipid based envelope systems for systemic delivery of siRNA will translate into extensive clinical application overcoming the associated challenges in near future.
Dioctadecyldimethylammonium bromide (DODAB):1-monooleoyl-rac-glycerol (MO) cationic liposomes were reported as a promising alternative to common transfection agents, showing superior effectiveness on the transfection of the 293 T mammalian cell line with pSV-β-gal plasmid DNA. The study of DODAB:MO aggregates in the absence of DNA has indicated that their morphology depends on the balance between DODAB's tendency to form bilayer structures and MO's propensity to form inverted non-lamellar structures with cubic organization. Other parameters, such as the temperature have proved to be crucial in the definition of the morphology of the developed nanocarrier. Therefore, in this work, a step forward to the current gene carrier system will be given by studying the effect of the tunable parameters (incubation temperature and MO content) on the structure of pDNA:DODAB:MO lipoplexes. More importantly, the implications that these tunable parameters could have in terms of lipoplex transfection efficiency will be investigated. Dynamic Light Scattering (DLS), Zeta (ζ) Potential, cryo-Transmission Electron Microscopy (cryo-TEM) and Ethidium Bromide (EtBr) exclusion were used to assess the formation, structure and destabilization of pDNA:DODAB:MO lipoplexes at DODAB molar fractions on (1:1) and above equimolarity (2:1, 4:1) prepared at incubation temperatures from 25 to 50 °C. Experimental results indicate that pDNA:DODAB:MO's structure is sensitive to the lipoplex incubation temperature, resulting in particles of distinct size, superficial charge and structure. These variations are also visible on the complexation dynamics of pDNA, and subsequent release upon incubation with the model proteoglycan heparin (HEP), at 25 and 50 °C. Increase in temperature leads to re-organization of DODAB and MO molecules within the liposomal formulation, causing a positive charge re-localization in the lipoplex surface, which not only alters its structure but also its transfection efficiency. Altogether, these results confirm that in the DODAB:MO carriers, an increase in the incubation temperature has a similar effect on aggregate morphology as the observed with an increase in MO content. This conclusion is extended to the pDNA:DODAB:MO lipoplexes morphology and subsequent transfection efficiency defining new strategies in lipoplexes preparation that could be used to modulate the properties of other lipid formulations for nonviral gene delivery applications.
Abstract This study was aimed to develop and evaluate p53 polyplex-loaded enteric-coated calcium pectinate microbeads for oral gene delivery as an effective novel alternative for colorectal cancer therapy. Mutation in p53 is the key event in colorectal cancer (CRC) and an important target for the treatment of CRC through gene therapy. Polymethacrylates-based non-viral vectors were evaluated for their ability to complex, protect and transfect p53 (wt) into colon cancer cell line. Polyplexes were formulated by complexation of cationic polymer with anionic pDNA at different N/P ratios. p53 polyplex-loaded calcium pectinate (CP) microbeads were prepared by ionotropic gelation of pectin with calcium chloride and coated with Eudragit® S100. In vitro release studies showed that enteric-coated CP microbeads protected the release of p53 polyplex in upper GIT with less than 10% release. In-vitro cell line studies and in vivo studies in rat showed that polymethacrylate carrier could transfect the pDNA effectively. Results of in vivo gene expression study further confirmed the ability of enteric-coated calcium pectinate microbeads to deliver pDNA specifically to rat colon. Conclusively, enteric-coated calcium pectinate microbeads released p53 polyplex specifically in colon and could serve as an effective alternative for CRC therapy.
Cationic lipid-nucleic acid complexes are widely used to deliver oligonucleotides, RNA and DNA into cells. Although much has been learned about the structure and forces that hold the complex together, an understanding of the mechanism of release of the nucleic acids from the complex into cells has been lacking. Recent studies have shown that anionic liposomes with compositions similar to the cytoplasmic face of the endosomal membrane are potent agents for inducing the rapid release of oligonucleotides and DNA from cationic lipid-nucleic acid complexes. Based upon these results, we propose that after the cationic lipid/nucleic complex is internalized by endocytosis it destabilizes the endosomal membrane. This destabilization induces flip-flop of anionic lipids from the cytoplasmic facing monolayer, which laterally diffuse into the complex and form a charge neutral ion-pair with the cationic lipids. This results in displacement of the nucleic acid from the cationic lipid and subsequent release of the nucleic acid into cytoplasm of the cell. We review the data that show the proposed mechanism accounts for a variety of observations on cationic lipid/nucleic acid complex-cell interactions.
In recent years the use of solid lipid nanoparticles (SLNs) as transport systems for the delivery of drugs and biomolecules has become particularly important. The use of cationic SLNs developed by the technique of microemulsion, which are complexed with DNA in order to study their application as non-viral vectors in gene therapy, is reported. The nanoparticles are characterized by scanning electron microscopy and transmission electron microscopy (SEM and TEM), atomic force microscopy (AFM) and differential scanning calorimetry (DSC). Furthermore, the process of lyophilization of the samples and their stability was studied. The nanoparticles obtained presented a particle size of 340nm with a positive surface charge of 44mV and the capability of forming lipoplexes with DNA plasmids was stated.
When ζ potentials of liposomes formed by phosphatidylcholine (PC) and stearylamine (STE) at variable concentrations were compared with those corresponding to liposomes formed by phosphatidic acid (PA), a decline and a lack of linearity in their ζ potentials compared to the logarithm of ionic strength were found at concentrations of STE above 10% (molar ratio). Despite the fact that STE is fully protonated at the interval of pH used,2-8 ζ potentials were found to be dependent not only on the ionic strength of the medium, as predicted by the classical double-layer theory, but also on the pH. Determination of the STE distribution by spectrofluorometry following the labeling of STE with fluorescamine showed that STE seemed to be preferentially located in the outer monolayer. This apparent contradiction can be explained by the migration of STE molecules from the liposomal surface to the medium, where it is organized in the form of micelles with a diameter of about 2 nm. The presence of micelles in addition to liposomes involves a large adsorption−desorption equilibrium, which, in turn, is influenced by the variation in the electrostatic free energy of the double layer on the membrane. Thus, the surface charge density varies with the change in ionic strength and pH, and consequently, the electrophoretic behavior of STE liposomes differs from that of PA liposomes.
The interaction of small cationic vesicles composed of dioctadecyldimethylammonium bromide (DODAB) with normal versus transformed mouse fibroblasts is described using cell microelectrophoresis, turbidimetry, and cell viability assays over a wide range of DODAB concentrations (10-7−10-3 M). Normal and transformed cells (104 cells/mL) attain a point of zero charge at, respectively, 18.0 and 1.6 μM DODAB. Further increasing DODAB concentration (C) generates positively charged cells. At 105 cells/mL and C ≥ 50 μM, DODAB induces cell−cell adhesion. For transformed and normal cells, peak adhesion occurs at 100 and 1000 μM DODAB, respectively. Upon 0.5 h interaction time with 100 μM DODAB, at 104 cells/mL, 20% of cell death is obtained for normal cells whereas transformed cells remain unaffected. Transformed cells have a higher affinity for DODAB vesicles than their normal counterparts but are more resistant to DODAB-induced cell death. The results indicate that DODAB vesicles interact with cells with very high affinity at low ionic strength and are not toxic below 1 mM, suggesting that they might successfully deliver oppositely charged proteins or DNA strands to cells. These results may be of importance for liposome-mediated processes currently being used for drug or gene delivery to cells.
Cationic liposomes are commonly used as a transfection reagent for DNA, RNA or proteins and as a co-adjuvant of antigens for vaccination trials. A high density of positive charges close to cell surface is likely to be recognized as a signal of danger by cells or contribute to trigger cascades that are classically activated by endogenous cationic compounds. The present review provides evidence that cationic liposomes activate several cellular pathways like pro-apoptotic and pro-inflammatory cascades. An improved knowledge of the relationship between the cationic lipid properties (nature of the lipid hydrophilic moieties, hydrocarbon tail, mode of organization) and the activation of these pathways opens the way to the use and design of cationic tailored for a specific application (e.g. for gene transport or as adjuvants).
Cationic liposome-DNA (CL-DNA) complexes are being pursued as nonviral gene delivery systems for use in applications that include clinic trials. However, to compete with viral vectors for systemic delivery in vivo, their efficiencies and pharmacokinetics need to be improved. The addition of poly (ethylene glycol)-lipids (PEGylation) prolongs circulation lifetimes of liposomes, but inhibits cellular uptake and endosomal escape of CL-DNA complexes. We show that this limits their transfection efficiency (TE) in a manner dependent on the amount of PEG-lipid, the lipid/DNA charge ratio, and the lipid membrane charge density. To improve endosomal escape of PEGylated CL-DNA complexes, we prepared an acid-labile PEG-lipid (HPEG2K-lipid, PEG MW 2000) which is designed to lose its PEG chains at the pH of late endosomes. The HPEG2K-lipid and a similar but acid-stable PEG-lipid were used to prepare PEGylated CL-DNA complexes. TLC and dynamic light scattering showed that HPEG2K-CL-DNA complexes are stable at pH 7.4 for more than 24 h, but the PEG chains are cleaved at pH 5 within 1 h, leading to complex aggregation. The acid-labile HPEG2K-CL-DNA complexes showed enhanced TE over complexes stabilized with the acid-stable PEG-lipid. Live-cell imaging showed that both types of complexes were internalized to quantitatively similar particle distributions within the first 2 h of incubation with cells. Thus, we attribute the increased TE of the HPEG2K-CL-DNA complexes to efficient endosomal escape, enabled by the acid-labile HPEG2K-lipid which sheds its PEG chains in the low pH environment of late endosomes, effectively switching on the electrostatic interactions that promote fusion of the membranes of complex and endosome.
A number of known structural properties of mixed lipid bilayer membranes and monolayers are accounted for by a model in which lipids pack into bilayers and monolayers like building blocks, each characterized by a surface head group area and characteristic solid angle. In phospholipids above the melting transition the head group area (at a given temperature and degree of hydration) is fairly invariant while the hydrocarbon region may be liquid-like so long as the molecule is not compressed beyond its characteristic solid angle.Phosphotidylcholine and phosphotidylserine are tapered lipids, i.e. their surface head group areas are greater than their non-polar end areas; cholesterol is frayed, i.e. its polar end area is less than its non-polar end area; while phosphotidylethanolamine is almost cylindrical. The “condensing” effect of cholesterol in mixed phospholipid-cholesterol films is seen as a taper-fray accomodation. The lipid distribution in erythrocyte membranes is shown to be conductive to a stable strain-free membrane.
This review discusses the gradually developing scientific understanding of emulsions and microemulsions over the past 20 years, assesses some current issues and technological challenges, and ends with some speculations on future applications of “smart” emulsion systems.
The incorporation of poly(ethylene glycol) (PEG)-conjugated lipids in lipid-based carriers substantially prolongs the circulation lifetime of liposomes. However, the mechanism(s) by which PEG-lipids achieve this have not been fully elucidated. It is believed that PEG-lipids mediate steric stabilization, ultimately reducing surface-surface interactions including the aggregation of liposomes and/or adsorption of plasma proteins. The purpose of the studies described here was to compare the effects of PEG-lipid incorporation in liposomes on protein binding, liposome-liposome aggregation and pharmacokinetics in mice. Cholesterol-free liposomes were chosen because of their increasing importance as liposomal delivery systems and their marked sensitivity to protein binding and aggregation. Specifically, liposomes containing various molecular weight PEG-lipids at a variety of molar proportions were analyzed for in vivo clearance, aggregation state (size exclusion chromatography, quasi-elastic light scattering, cryo-transmission and freeze fracture electron microscopy) as well as in vitro and in vivo protein binding. The results indicated that as little as 0.5 mol% of 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine (DSPE) modified with PEG having a mean molecular weight of 2000 (DSPE-PEG(2000)) substantially increased plasma circulation longevity of liposomes prepared of 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC). Optimal plasma circulation lifetimes could be achieved with 2 mol% DSPE-PEG(2000). At this proportion of DSPE-PEG(2000), the aggregation of DSPC-based liposomes was completely precluded. However, the total protein adsorption and the protein profile was not influenced by the level of DSPE-PEG(2000) in the membrane. These studies suggest that PEG-lipids reduce the in vivo clearance of cholesterol-free liposomal formulations primarily by inhibition of surface interactions, particularly liposome-liposome aggregation.
The polyamines spermidine, spermine, and putrescine are intimately involved in and required for cell growth and proliferation. There are also multiple effects of polyamines on other cellular processes that seem not to be a result of changes in protein expression. It is a daunting task to classify and understand cellular effects of endogenous polyamines. There has been no central hypothesis how these effects can occur or how spermine and spermidine could be targeted to various signal transduction cascades. However, now there is evidence that multiple effects of endogenous polyamines on different cellular processes may involve plasma membrane PI(4,5)P(2) and recent evidence of how polyamines could be targeted to specific cellular functions.
We present a comparative study of the physico-chemical properties, in vitro cytotoxicity and in vivo antibody production of surface-complexed DNA in EPC/DOTAP/DOPE (50/25/25% molar) liposomes and DOTAP/DOPE (50/50% molar) lipoplexes. The study aims to correlate the biological behavior and structural properties of the lipid carriers. We used DNA-hsp65, whose naked action as a gene vaccine against tuberculosis has already been demonstrated. Additionally, surface-complexed DNA-hsp65 in EPC/DOTAP/DOPE (50/25/25% molar) liposomes was effective as a single-dose tuberculosis vaccine. The results obtained showed that the EPC inclusion stabilized the DOTAP/DOPE structure, producing higher melting temperature and lower zeta potential despite a close mean hydrodynamic diameter. Resemblances in morphologies were identified in both structures, although a higher fraction of loaded DNA was not electrostatically bound in EPC/DOTAP/DOPE. EPC also induced a striking reduction in cytotoxicity, similar to naked DNA-hsp65. The proper immune response lead to a polarized antibody production of the IgG2a isotype, even for the cytotoxic DOTAP/DOPE. However, the antibody production was detected at 15 and 30 days for DOTAP/DOPE and EPC/DOTAP/DOPE, respectively. Therefore, the in vivo antibody production neither correlates with the in vitro cytotoxicity, nor with the structural stability alone. The synergistic effect of the structural stability and DNA electrostatic binding upon the surface of structures account for the immunological effects. By adjusting the composition to generate proper packing and cationic lipid/DNA interaction, we allow for the optimization of liposome formulations for required immunization or gene therapy. In a specific manner, our results contribute to studies on the tuberculosis therapy and vaccination.
Cationic lipids have been extensively used as carriers of biologically active molecules (nucleic acids, peptides and proteins) into cells. Recent data provided evidence that cationic lipids are not just inert transporters but do activate specific cellular cascades. This review illustrates these activating properties with a few examples. Cell activation raises the question of which receptors are involved. Some cationic lipids seem to satisfy specific structural requirements of Toll-like receptors (TLR4) as they activate TLR4-dependent pathways. However, cationic lipids display a large structural diversity and it is likely that they are also recognized by receptors with a broader specificity. Alternatives are proposed and discussed to explain this broad specificity.
Recently we proposed that the antineoplastic properties observed in vivo for alkyl-lysophospholipid and alkylphosphocholine analogues are a direct consequence of the reduction of membrane stored elastic stress induced by these amphiphiles. Here we report similar behavior for a wide range of cationic surfactant analogues. Our systematic structure-activity studies show that the cytotoxic properties of cationic surfactants follow the same pattern of activity we observed previously for alkyl-lysophospholipid analogues, indicating a common mechanism of action that is consistent with the theory that these amphiphiles reduce membrane stored elastic stress. We note that several of the cationic surfactant compounds we have evaluated are also potent antibacterial and antifungal agents. The similarity of structure-activity relationships for cationic surfactants against microorganisms and those we have observed in eukaryotic cell lines leads us to suggest the possibility that the antibacterial and antifungal properties of cationic surfactants may also be due to modulation of membrane stored elastic stress.
A simple theory is developed that explains the formation of bilayers and vesicles and accounts quantitatively for many of their physical properties: Properties including vesicle size distributions and bilayer elasticity emerge from a unified theory that links thermodynamics, interaction free energy, and molecular geometry. The theory may be applied to the analysis of more complicated membrane structures and mechanisms.
The effects of non-drug-containing liposomes of different compositions and sizes on the proliferation of nine cancer-derived and one normal cultured human cell lines were determined. Stearylamine- and cardiolipin-containing liposomes were toxic (ID50) at 200 microM liposomal lipid concentrations or less, whereas phosphatidylglycerol- and phosphatidylserine-containing liposomes were toxic in the range 130-3000 microM. Phosphatidylcholine or dipalmitoylphosphatidylcholine liposomes were not toxic at 3000-4000 microM. In general, small liposomes were more toxic than large ones. The results indicate that there are wide variations in toxicity of non-drug-containing liposomes for cultured human cells. The potential for nonspecific toxicity due to the liposomes themselves should be carefully considered if human administration of drug-containing liposomes is to be done.
In order to develop the cytotoxic liposome, the cytolytic effect of polycationic liposome was examined. Upon incubation of the stearylamine-containing liposome (stearylamine-liposome) with rabbit erythrocyte, a significant extent of hemolysis was observed. Hemolytic activity of the liposome depends on the amount of stearylamine in the liposome membrane. The plots of the initial rate of hemolysis versus the concentration of stearylamine-liposome showed a sigmoidal curve, suggesting that stearylamine-liposomes act cooperatively on the erythrocyte membrane. Hemolytic activity of stearylamine-liposome was markedly influenced by the composition of hydrocarbon chains of the phospholipids in the liposome membrane, suggesting that the membrane fluidity of stearylamine-liposome is important to evoke the hemolysis. Since the liposomes containing acidic phospholipids inhibited markedly the stearylamine-liposome-caused hemolysis, it is likely that the primary target of stearylamine-liposome is the negatively charged component(s) such as acidic phospholipids on the erythrocyte membrane. Furthermore, stearylamine-liposome induced the release of the intravesicular contents from the liposome made of acidic phospholipids but not from the liposome made of phosphatidylcholine only. These results suggest that stearylamine-liposome interacted with the negative charges of the erythrocyte membrane and eventually damaged the cell. Erythrocytes from rabbit, horse and guinea pig are highly susceptible to stearylamine-liposome but those from man, sheep, cow and chicken are less so.
The inhibition of 3H-thymidine incorporation into DNA of L1210 cells in culture by liposomes was used as an index of liposome toxicity. Inhibition of 3H-thymidine incorporation appeared to be dose dependent for some lipid compositions tested. The commonly used neutral lipids, phosphatidylcholine and cholesterol did not appear to inhibit 3H-thymidine incorporation. Phosphatidic acid, an adjunct for preparing anionic liposomes, appeared to be non-toxic compared to phosphatidylserine and dicetylphosphate (alternative adjuncts for preparing anionic liposomes). Stearylamine, a synthetic lipid which continued to dominate the preparation of cationic liposome was the most toxic of the lipids tested.
The endothelium of marrow sinuses is the site of cellular and molecular exchange between the circulation and the hemopoietic compartment. Here, mature cells are delivered into circulation by passing through the cytoplasm of a single endothelial cell. Similarly, stem cells are probably trapped here. The endothelium which is structurally polarized, and shows segmental surface differentiations, can maintain a gradient for a variety of substances between blood and marrow (the marrow-blood barrier). Transendothelial cell migration involves channel formation through fusion-fission reorganization of the membrane. The endothelium is involved in other interactions particularly with perisinal macrophages. Little is known of the surface characteristics of the endothelium, an area that may help the understanding of how the cell can selectively exchange cells and molecules between the marrow and blood.
The efficiency of stearylamine (SA) liposome in transfecting DNA into eukarytic cells was evaluated and compared with that of the Lipofectin reagent and the calcium phosphate transfection method. Results with the 21 cell lines tested varied, depending on the cell type. The overall indication was that SA liposomes had certain advantages over the Lipofectin reagent and both of them were superior to the calcium phosphate method, showing the greatest effect in 8 different cell lines, while the calcium phosphate method was the best in only 4 cell lines. There were 2 cell lines which could only be transfected by SA liposomes, another 2 only by the calcium phosphate method, but none depended solely on the Lipofectin reagent. The number of cell lines failed to be transfected was 2 for SA liposomes, 3 for calcium phosphate method, and 4 for the Lipofectin reagent. The advantage of the SA liposome method is discussed.
Cationic liposomes complexed with DNA (CL-DNA) are promising synthetically based nonviral carriers of DNA vectors for gene
therapy. The solution structure of CL-DNA complexes was probed on length scales from subnanometer to micrometer by synchrotron
x-ray diffraction and optical microscopy. The addition of either linear λ-phage or plasmid DNA to CLs resulted in an unexpected
topological transition from liposomes to optically birefringent liquid-crystalline condensed globules. X-ray diffraction of
the globules revealed a novel multilamellar structure with alternating lipid bilayer and DNA monolayers. The λ-DNA chains
form a one-dimensional lattice with distinct interhelical packing regimes. Remarkably, in the isoelectric point regime, the
λ-DNA interaxial spacing expands between 24.5 and 57.1 angstroms upon lipid dilution and is indicative of a long-range electrostatic-induced
repulsion that is possibly enhanced by chain undulations.
The structure of the lipoplex formed from DNA and the sugar-based cationic gemini surfactant 1, which exhibits excellent transfection efficiency, has been investigated in the pH range 8.8-3.0 utilizing small-angle X-ray scattering (SAXS) and cryo-electron microscopy (cryo-TEM). Uniquely, three well-defined morphologies of the lipoplex were observed upon gradual acidification: a lamellar phase, a condensed lamellar phase, and an inverted hexagonal (H(II)) columnar phase. Using molecular modeling, we link the observed lipoplex morphologies and physical behavior to specific structural features in the individual surfactant, illuminating key factors in future surfactant design, viz., a spacer of six methylene groups, the presence of two nitrogens that can be protonated in the physiological pH range, two unsaturated alkyl tails, and hydrophilic sugar headgroups. Assuming that the mechanism of transfection by synthetic cationic surfactants involves endocytosis, we contend that the efficacy of gemini surfactant 1 as a gene delivery vehicle can be explained by the unprecedented observation of a pH-induced formation of the inverted hexagonal phase of the lipoplex in the endosomal pH range. This change in morphology leads to destabilization of the endosome through fusion of the lipoplex with the endosomal wall, resulting in release of DNA into the cytoplasm.
Non-viral vectors represent an important alternative in gene delivery. Among these vectors, cationic liposomes are widely studied, because of their ability to form stable complexes with DNA fragments (lipoplexes). In the present work, we report on the characterization by electron spin resonance (ESR) spectroscopy and zeta potential measurements of cationic liposomes and of their complexes with oligonucleotides. Liposomes were made with a zwitterionic lipid, DOPE, and a cationic lipid, either DOTAP or DC-Chol. Oligonucleotides were the 20-base single strand polyA, the 20-base single strand polyT, and the corresponding double strand dsAT. The zeta potential as a function of the oligonucleotide/lipid+ ratio gave an S-shaped titration curve. Well-defined surface potential changes took place upon charge compensation between the cationic lipid heads and the phosphate groups on the oligonucleotides. The inversion point depended on the specific system under study. The bilayer properties and the changes that occurred with the incorporation of DNA fragments were also monitored by ESR spectroscopy of appropriately tailored spin probes. For all the systems investigated, the ESR spectra showed that no major alteration took place after lipoplex formation and molecular packing remained substantially unchanged. Both zeta potential and ESR measurements were in favor of an external mode of packing of the lipoplexes.
Efficacious bone regeneration could revolutionize the clinical management of bone and musculoskeletal disorders. Although several bone morphogenetic proteins (BMPs) (mostly BMP-2 and BMP-7) have been shown to induce bone formation, it is unclear whether the currently used BMPs represent the most osteogenic ones. Until recently, comprehensive analysis of osteogenic activity of all BMPs has been hampered by the fact that recombinant proteins are either not biologically active or not available for all BMPs. In this study, we used recombinant adenoviruses expressing the 14 types of BMPs (AdBMPs), and demonstrated that, in addition to currently used BMP-2 and BMP-7, BMP-6 and BMP-9 effectively induced orthotopic ossification when either AdBMP-transduced osteoblast progenitors or the viral vectors were injected into the quadriceps of athymic mice. Radiographic and histological evaluation demonstrated that BMP-6 and BMP-9 induced the most robust and mature ossification at multiple time points. BMP-3, a negative regulator of bone formation, was shown to effectively inhibit orthotopic ossification induced by BMP-2, BMP-6, and BMP-7. However, BMP-3 exerted no inhibitory effect on BMP-9-induced bone formation, suggesting that BMP-9 may transduce osteogenic signaling differently. Our findings suggest that BMP-6 and BMP-9 may represent more effective osteogenic factors for bone regeneration.
Cationic liposomal lipids: from gene carriers to cell signaling
- C Lonez
- M Vandenbranden
- J-M Ruysschaert
Lonez C, Vandenbranden M, Ruysschaert J-M. Cationic liposomal lipids: from gene carriers to cell signaling. Prog Lipid Res.
2008;47(5):340-7.
Influence of poly(ethylene glycol) grafting density and polymer length on liposomes: relating plasma circulation lifetimes to protein binding
- Dos Santos
- Allen C Doppen
- A-M Anantha
- M Cox
- Kak Gallagher
Dos Santos N, Allen C, Doppen A-M, Anantha M, Cox KAK,
Gallagher RC, et al. Influence of poly(ethylene glycol) grafting
density and polymer length on liposomes: relating plasma
circulation lifetimes to protein binding. Biochim Biophys Acta
Biomembr. 2007;1768(6):1367-77.