fi=3111 Biolääketieteet|sv=3111 Biomedicinska vetenskaper|en=3111 Biomedicine|https://www.doria.fi:443/handle/10024/920832024-03-19T10:59:01Z2024-03-19T10:59:01ZIntracellular Delivery OF CRISPR/Cas9 Plasmid With Zif-8 Nanocarrier to Liver Cancer CellsSalo, Jessicahttps://www.doria.fi:443/handle/10024/1879172023-10-18T12:00:53Z2023-10-18T11:37:55ZIntracellular Delivery OF CRISPR/Cas9 Plasmid With Zif-8 Nanocarrier to Liver Cancer Cells
Salo, Jessica
Hepatocellular carcinomas are the fifth most common cancers with poor prognosis, inefficient drug therapy, and severe side effects. C-Myc regulates up to 20 % of all human genes. C-Myc overexpression is involved in 70 % of liver cancers. Downregulating the c-Myc expression may provide a potential therapeutic alternative for future liver cancer treatments.
CRISPR/Cas9 method enables simple, stable, and inexpensive gene editing. Zif-8 biomineralization has been utilized to encapsulate CRISPR/Cas9 edited plasmid due to its stable structure, fast and easy assembling in room temperature aqueous solution, and flexibility in payload sizes since they can directly grow a nanolayer protection shell on top of the payload. Zif-8 enables pH-mediated cargo release, which can promote endosomal escape. The aims are to deliver the CRISPR/Cas9 system to knockout c-Myc genes in HCC cells with Zif-8 nanocarrier, monitor endosomal escape, analyze the downregulated c-Myc expression, and investigate the treatment effects on cancer cells.
As a result, the CRISPR/Cas9 plasmid has been successfully and efficiently delivered into HCC cells; high ratio endosomal escape after 4 hours of transfection, whereas nuclei targeting was observed at 6 hours. The transfected HCC cells with c-Myc knockout plasmid result in lower c-Myc expression than wild-type HCC cells. Results indicate the potential of intracellular c-Myc knockout with Zif-8-mediated CRISPR/Cas9 plasmid delivery in HCC cells.
Although the c-Myc expression downregulation utilization has not yet proceeded into clinical trials, it is a potential therapy for future liver cancers. The delivery method must become more accurate and suitable for further in vivo experiments.
2023-10-18T11:37:55ZSynthesis of CuS@MSN@SpAcDex for Tacrolimus delivery to treat End Stage Renal DiseaseShrestha, Brajeshhttps://www.doria.fi:443/handle/10024/1815592021-07-06T12:00:13Z2021-07-06T11:43:43ZSynthesis of CuS@MSN@SpAcDex for Tacrolimus delivery to treat End Stage Renal Disease
Shrestha, Brajesh
The treatment of End stage renal disease (ESRD) is kidney transplantation. In order to keep the transplanted kidney safe in the patient’s body, it is necessary to kill the patient’s immune cells present in the endothelial cells. Although the use of tacrolimus and methylprednisolone drugs was found to be effective in killing patient's immune cells, the traditional oral administration makes these drugs not fully effective because of their poor water solubility and less absorption by the gastrointestinal tract. Thus, an improved therapy such as a drug delivery method is needed, where the complete dose of these drugs, with the help of nanoparticles, will reach the target site and kill the immune cells. Nanoparticles such as MSN and CuS@MSN can load tacrolimus and methylprednisolone, respectively, and deliver them to the target site.
In the present study, the MSN and CuS@MSN were synthesized in the laboratory using an easy and inexpensive method that gives a significant amount of good nanoparticles. The size of MSN and CuS@MSN were ranged from 50-80 nm and 50-120 nm, respectively. In addition, the MSN was loaded with tacrolimus, and the loading efficiency was found to be significantly high (approximately 63%) after the use of MSN and tacrolimus in a 1:5 ratio. In addition, the tacrolimus-loaded MSN was further encapsulated with SpAcDex polymer to form the MSN-tacrolimus-SpAcDex complex for further stability. Furthermore, the zeta potentials of MSN alone and MSN-tacrolimus were measured, and they were -19.1 mV and -19.5 mV.
These MSN and MSN-tacrolimus-SpAcDex were treated with HUVECs for 2 hours, with the help of WST-1 assay, in order to determine the in vitro cell viability and cytotoxicity. The WST-1 assay was found to be precise, sensitive, reliable, inexpensive, and fast. The assay showed that both nanoparticles and nanoparticle complexes do not pose any cytotoxicity and do not inhibit the growth of HUVECs. In addition, they are biocompatible. Furthermore, the cellular uptake of these nanoparticle complexes was confirmed after the result of confocal microscopy. Thus, MSN and MSN-tacrolimus-SpAcDex complex can act as a good vehicle for drug delivery methods.
2021-07-06T11:43:43ZTargeted Sequence Delivery Platform with ROS-Response for the Treatment of OsteoarthritisBednjanec, Dorotejahttps://www.doria.fi:443/handle/10024/1815212021-06-28T13:30:57Z2021-06-28T12:07:53ZTargeted Sequence Delivery Platform with ROS-Response for the Treatment of Osteoarthritis
Bednjanec, Doroteja
Arthritis is a common problem affecting elderly people today. One specific form of arthritis is osteoarthritis, caused by lubrication deficiency at the joint surface. Due to genetics, aging, stress factors, or accidents, the cartilage layer breaks down causing inflammation of the joint. When no cartilage surrounds the bone, neighboring bones scratch each other and cause pain. To prevent and slow down those events, nanomedicine has become a popular field of study and research. The main objective of this research was to develop targeted drug delivery method for treating osteoarthritis using, small nano- and micro- compositions. Another important objective was to find a way for the composition with the lubricated surface, to target damaged tissue. The core of the built nanoparticle was Cerium oxide (CeO2), surrounded by a mesoporous silica nanoparticle (MSN) shell. Using microfluidics, the drug Agomir874 was loaded within core-shell nanoparticles, and then surrounded by the PEO-b-PFMA microparticles. The polymer contained collagen IgG2, for antibody-antigen binding to improve the elasticity of the joint. Finally, the drug 5-ASA was inserted within polymer in order to reduce inflammation in the damaged tissue. As the PEO-b-PFMA particles were diluted, the right solvent was needed to be found in order for the polymer particles to stay stable. In the future, with the help of different imaging techniques, it will be possible to determine how much drug is loaded within the particle and whether the drug loading of the particles is successful.
2021-06-28T12:07:53ZFluorescence imaging of Drosophila melanogaster tracheal system : investigating the morphology of LUBEL mutant fliesBenoit, Sabrinahttps://www.doria.fi:443/handle/10024/1777622020-08-05T12:30:17Z2020-08-03T11:42:24ZFluorescence imaging of Drosophila melanogaster tracheal system : investigating the morphology of LUBEL mutant flies
Benoit, Sabrina
Ubiquitination is a post-translational modification that is a key regulator in many pathways in the cell. Mutant LUBEL (lubelMi), linear ubiquitin E3 ligase, flies are unable to form methionine 1 (M1) linked ubiquitin chains in Drosophila and die in hypoxic conditions, whereas wild-type Cantons flies survive. The Drosophila respiratory system is examined to determine if the flies are asphyxiating due to morphological changes in the tracheal epithelial tissue. To image morphological changes in the epithelial tissue, I used chitin-specific and tubulin-specific silicone rhodamine (SiR) probes and Calcofluor White staining in combination with Zeiss LSM 880 with AiryScan microscopy techniques to evaluate the respiratory system in CantonS in basal conditions, and, thus, develop a criterion for comparison with lubelMi flies. I then used STED microscopy to image taenidia within the tracheoles and measure tracheole diameter in CantonS and lubelMi flies. A novel phenotypic abnormality was seen in LUBEL mutant flies, lubelMi and lubelRNAi flies, in comparison to the control group. lubelMi and lubelRNAi flies have discontinuous, abnormal tubulin projecting from the trachea and tracheoles and measurably thicker dorsal trunks in comparison to the control group. This abnormal tubulin phenotype and increased dorsal trunk diameter could be caused by a LUBEL-influenced developmental error during organogenesis in Drosophila melanogaster larvae.
2020-08-03T11:42:24Z