48 research outputs found
Liposomes
Liposomes have received increased attention in recent years. Nevertheless, liposomes, due to their various forms and applications, require further investigation. These structures can deliver both hydrophilic and hydrophobic drugs. The preparation of liposomes results in different properties for these systems. In addition, there are many factors and difficulties that affect the development of liposome drug delivery structures.The purpose of this book is to concentrate on recent developments in liposomes. The articles collected in this book are contributions by invited researchers with long-standing experience in different research areas. We hope that the material presented here is understandable to a broad audience, not only scientists but also people with a general background in many different biological sciences. This volume offers up-to-date, expert reviews of the fast-moving field of liposomes and is divided in two major sections: 1. Introduction; 2. Liposomes general propertie
KDM5B as a potential epigenetic regulator of cardiomyocyte regeneration and cell cycle activation.
The limited regenerative capacity of adult cardiomyocytes (CMs) is manifested in prevalent morbidity and mortality associated with ischemic heart disease. We previously demonstrated that a combination of four cell cycle factors (4F) promotes efficient cell division in differentiated CMs. The temporal transcriptional reprogramming in the 4F-transduced CMs indicates that epigenetic modifications mediate the cell cycle progression. Through small molecule screening, we evaluated the epigenetic impact on CM’s response in the 4F-transduced CMs. We identified the histone demethylase 5B (KDM5B), which through in vitro and ex vivo genetic knockdown and overexpression studies, exhibited a modulative effect on CM’s cell cycle plasticity along with transcriptional alteration in the cell cycle-related genes. Whereas KDM5B depletion impaired CM’s replicative capacity, its overexpression induced and augmented CM’s proliferation in a context-dependent manner. Our data indicate that KDM5B is essential for CM’s regeneration and cell cycle activation and is a potential target for myocardial repair
Determination of epithelial growth factor receptor mutations in circulatory tumour cells from non-small cell lung cancer patients isolated using a novel microfluidic device
Patients with epidermal growth factor receptor (EGFR) sensitizing mutations in non small cell lung cancer (NSCLC) receive benefit from Tyrosine Kinase inhibitors. Accurate selection of patients before treatment is highly dependent on precise molecular diagnosis of EGFR mutations. Presently in the clinic, the diagnostic samples routinely used tumour biopsy and/or cell free DNA (cfDNA), are not sufficiently effective for precise diagnosis. Circulatory tumour cells (CTC) in blood have been explored successfully as alternative and complementary diagnostic markers to the current clinical tools. However, utility in the clinics has been hampered by the relatively low concentration of CTC in blood, and the lack of robust technologies that are adaptable for routine use. The present study describes the design and optimization of an immunomagnetic based microfluidic device (Lung card version II) that isolates CTC expressing the epithelial cell adhesion molecule (EpCAM) from blood with high capture efficiency and purity. The device is a 2-part system comprising a disposable chip that is simple in design and a reusable microfluidic unit that contains a mobile magnetic arm. The simple design and work-flow process of the device ensures cost efficiency for scalability and, ultimately, use in the clinic. The device was initially validated for its capability to isolate EpCAM positive cells. Results from spiking carboxylfluorescein succinimidyl ester stained EpCAM positive cells in media/blood showed a capture efficiency of ≥ 65% and a purity ≥ 97% from a 13ml sample in 50 minutes. The isolated CTC from NSCLC patients (n=38) were analysed for mRNA markers specific to malignant cells and were characterized for EGFR mutations following PCR and next generation sequencing. The mutational status of CTC was compared to that obtained from matched, tumour biopsy, samples. Significantly more mutations (P=0.0173) were detected in CTC enriched samples than the matched biopsy. Interestingly, mutations were detected in only 4 biopsy samples and the mutations detected in the biopsy were only concordant with results from CTC enriched samples for 1 patient. Exon 19 deletion was the most frequent mutation detected (86.7%) with rare mutations such as: L792P, C797S, H509R also been detected in CTC, and the present study reports the detection of K708R mutation in NSCLC for the first time. The clinical outcomes of patients who were positive for EGFR mutation from CTC, but had been placed on therapies based on mutation results from tissue biopsy were evaluated in this study. The results showed that no significant progression free survival (PFS) benefit was attained when comparing treatment response between patients whose CTC possessed an EGFR mutation and patients whose CTC possessed no EGFR mutation (10 months vs26 months p value-0.3420 HR- 0.76 95% CI- 0.2498-2.319). In summary the results from this study showed that the microfluidic device captured CTC with efficiency equal to other immuno-affinity based devices but had better purity rates and throughput and also that the device can be utilized for CTC processing for downstream analysis. Results from this current study further demonstrated the clinical potential of CTC+NGS matrix for the detection of EGFR mutations and the prospective impact it would have for precision oncology in NSCLC are discussed
An investigation into mesenchymal stromal cells’ behaviour in 3D environment of PNIPAM-based hydrogel
Mesenchymal stromal cells (MSCs) are multipotent cells, known for the ability to differentiate into cells of bone, fat and cartilage. MSCs are commonly sourced from the bone marrow environment, where these cells reside in a 3-dimensional (3D) environment and are exposed to components of the extracellular matrix (ECM), other cells types, biochemical and mechanical stimuli. Conventional monolayer culture cannot replicate the complexity of the in vivo bone marrow environment. Therefore, a more representative MSC culture environment is required.
The aim of this project was to develop a highly tunable synthetic hydrogel, on the basis of poly(N-isopropylacrylamide) (PNIPAM), to allow temperature-driven encapsulation and subsequent study of MSC behaviour in three dimensional (3D) environment. The highly branched (HB) architecture of PNIPAM polymer was obtained by means of living radical polymerisation. Further polymer functionalisation with tri-arginine peptide sequence (RRR) has stabilised hydrogel structure and reduced solvent expulsion (syneresis). Rheological studies have revealed overall resistance to deformation (G*; complex modulus) of 5wt% HB PNIPAM+RRR to be equal to 542.3 Pa at 37ËšC.
MSC single cell suspensions were successfully encapsulated in HB PNIPAM+RRR 3D droplet hydrogels, demonstrating rounded morphology, absence of proliferation and stable cell viability. Differentiation potential studies of the cell-loaded hydrogels, cultured in osteogenic or adipogenic media, demonstrated osteo-conductive, osteo-inductive and adipo-inhibitive responses.
In summary, HB PNIPAM+RRR is a novel chemical entity with a thermo-responsive nature, which forms a porous and hydrated scaffold with osteo-inductive properties for MSC encapsulation at physiologically relevant temperature. HB PNIPAM is a highly functional and amenable hydrogel platform for assessment of MSC behaviour and guidance of differentiation in 3D environment
Development of Protein Based PCBP2 siRNA Nanocomplex for Liver Fibrosis Therapy
Title from PDF of title page viewed December 19, 2019Dissertation advisor: Kun ChengVitaIncludes bibliographical references (page 147-199)Thesis (Ph.D.)--School of Pharmacy and Department of Chemistry. University of Missouri--Kansas City, 2018The objective of this dissertation is to develop a protein-based siRNA
nanocomplex for the treatment of alcoholic liver fibrosis. Our laboratory recently
discovered that silencing the poly (rC) binding protein 2 (PCBP2) gene in hepatic stellate
cells (HSCs) leads to the reversal of the accumulated extracellular matrix. We therefore
hypothesize that targeted delivery of the PCBP2 siRNA to HSCs could potentially treat
liver fibrosis. Cholesterol and IGF2R (insulin growth factor 2 receptor) specific peptide
were used as targeting ligands to deliver the siRNA to HSCs.
In Chapter 1, we briefly introduced the background about RNA interference
(RNAi), liver fibrogenesis, markers of liver fibrosis, and the role of PCBP2 in liver
fibrogenesis. We also presented the Statement of the Problems and Objectives.
In chapter 2, we reviewed the avidin-biotin technology and its potential
applications in nanotechnology, therapy and diagnosis. We also discussed the challenges
and biological barriers for siRNA delivery.
In Chapter 3, we rigorously investigated the intracellular barrier which is a rate
limiting step for the silencing activity of siRNAs. Using streptavidin as the nanocomplex
core, PCBP2 siRNA was delivered to HSC-T6 (hepatic stellate) cells. Intracellular fate of
the nanocomplex components and PCBP2 siRNA was monitored. Fluorescent probes
were tagged with the siRNA, protamine and streptavidin and analyzed under confocal
microscopy, flow cytometry and fluorescence spectroscopy. We discovered substantial
exocytosis and localization of the siRNA in recycling organelles, such as recycling
endosomes, endoplasmic reticulum and golgi apparatus. Streptavidin was found to be
colocalized with the lysosomes and in some cases along with siRNA potentially leading
to the lysosomal degradation. We found that the streptavidin, although, a very efficient
delivery carrier has reduced silencing activity at higher incubation time intervals.
In Chapter 4, we compared different variants of avidin such as avidin, neutravidin
and streptavidin for in vitro activity and cellular uptake over the extended time interval.
Addition of polyethylene glycol (PEG) spacer between the biotin and cholesterol ligand
was done to improve the biodistribution of the nanocomplex. We tested the live imaging
and post euthanized biodistribution of nanocomplexes and found it to be most distributed
in liver in comparison to other variants of avidin. In vitro silencing activity and cellular
uptake was also significantly higher in case of the neutravidin nanocomplex with
negligible lysosomal colocalization and exocytosis.
In Chapter 5, the neutravidin nanocomplex were further improved by using the
IGF2R-specific peptide as a targeting ligand for hepatic stellate cells. The siRNA was
also annealed to the peptide nucleic acid for attaching the biotin. PNA enhanced the
serum stability of the siRNA and helped avoid the endonuclease and chemical reagent
mediated degradation during biotin conjugation process. We developed the liver fibrosis
model by injecting CCL4/olive oil intra-peritonealy for 4-5 weeks. We started the neutravidin-PCBP2 siRNA nanocomplex treatment at the beginning of 3rd week of the
fibrosis induction to reverse the fibrosis. After the end of dosage regimen, the rats were
euthanized and the analysis was performed for the liver fibrosis molecular markers. We
found that the neutravidin PCBP2 siRNA nanocomplex successfully reversed the liver
fibrosis by significantly reducing the molecular markers of fibrosis and reduction in the
type 1 collagen.Introduction -- Literature review -- Inracellular fate and exocytosis of PCBP2 siRNA nanocomplex in hepatic stellate cells -- Conparison of avidin, neutravidin and streptavidin as nanocarriers for efficient siRNA delivery -- In vivo delivery of siRNA by protein based nanocomplex to treat aggressive liver fibrosis -- Summary and conclusion
Bacterial ghosts modulation of innate immunity: immune responses during chlamydia infection, 2015
Chlamydia trachomatis (CT) is a pestilent infection affecting upwards of 90 million people worldwide. An efficacious vaccine is needed to control the morbidities and rising healthcare cost associated with genital CT infection. We have established that protection against chlamydia infection parallels with a high frequency of T helper Type 1 cells and the associated antibodies. The current study focuses on the induction of innate immune responses involved during Chlamydia infection by a Vibrio cholera ghost-based (VCG) vaccine vector. THP-1 cells were used for dose and kinetic experiments. HeLa cells were used for infectivity assays. Based on preliminary studies, we hypothesized that the induction of immune responses by a VCG-based vaccine involves multiple innate immune signaling. Multiplex assay was used to measure T helper Type I and Type II cytokine secretion by THP-1 monocytes (Mn) or macrophages (M?). Immunostimulatory cytokine secretion was significant when both cell morphologies were pulsed with VCG or VCG/murine splenocytes. We concluded that this secretion was significant enough to compliment that which would be secreted when THP-1 cells are pulsed with Chlamydia elementary bodies alone, enhancing the innate immune response during infection. Cellular supernatants (conditioned media) containing Th1-type and Th2-type cytokines were used to culture Chlamydia-infected HeLa cell monolayers. Infected HeLa monolayers cultured in the conditioned media were significantly less infected (968 IFUs) versus HeLa monolayers cultured in Earles minimum essential media (16,486 IFUs; p<0.001). We concluded that factors contained in conditioned media prevent and/or significantly reduce infection by Chlamydia and the development of inclusion forming units. KEY TERMS: Vaccine, Innate Immunity, Chlamydia trachomatis, Bacterial Ghosts, Infectious Diseases, Immunology of Infectious Diseas
Progenitor cells in auricular cartilage demonstrate promising cartilage regenerative potential in 3D hydrogel culture
The reconstruction of auricular deformities is a very challenging surgical procedure that could benefit from a tissue engineering approach. Nevertheless, a major obstacle is presented by the acquisition of sufficient amounts of autologous cells to create a cartilage construct the size of the human ear. Extensively expanded chondrocytes are unable to retain their phenotype, while bone marrow-derived mesenchymal stromal cells (MSC) show endochondral terminal differentiation by formation of a calcified matrix. The identification of tissue-specific progenitor cells in auricular cartilage, which can be expanded to high numbers without loss of cartilage phenotype, has great prospects for cartilage regeneration of larger constructs. This study investigates the largely unexplored potential of auricular progenitor cells for cartilage tissue engineering in 3D hydrogels
Cancer Nanomedicine
This special issue brings together cutting edge research and insightful commentary on the currentl state of the Cancer Nanomedicine field