Browsing by Subject "Liposomes"

Now showing 1 - 3 of 3
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    A platform for next-generation cancer therapies: multi-targeted nonviral vectors for site-specific gene delivery and expression
    (2013-12) Adil, Maroof Mohammad
    Advances in genetics have empowered gene therapy as a cancer treatment, however there are many challenges to delivering genes specifically to target disease sites. Presented here is the development of a new non-viral gene delivery vehicle, consisting of branched polyethyleneimine (bPEI) condensed plasmid DNA polyplexes encapsulated within a PR_b functionalized stealth liposome, for the delivery of genes specifically to &alpha5&beta1 integrin overexpressing cancer cells. This new transfection agent mediated higher gene expression than non-targeted stealth liposomes and unencapsulated polyplexes in tissue culture. In a liver-metastatic colorectal cancer mouse model, PR_b functionalized stealth liposomes outperformed non-targeted stealth liposomes and was able to specifically transfect the tumor site while avoiding healthy tissues. In addition, a comparative investigation of the transfection mechanism of PR_b functionalized nanoparticles, DOTAP/DOPE lipoplexes, bPEI polyplexes and stealth liposomes was carried out in DLD-1 cells. Results demonstrated that PR_b functionalized nanoparticles were optimally balanced for the transfection of DLD-1 cells with high colloidal stability, fast integrin mediated internalization kinetics, caveolae mediated uptake and endosomal escape. To further increase the specificity of gene expression in cancer tissue, a new therapeutic plasmid DNA (pNF-&kappaB-DTA) was developed with expression of Diphtheria toxin fragment-A (DTA) gene regulated by the transcriptional activity of NF-&kappaB, which is a transcription factor upregulated in cancer. The multi-targeted gene delivery system formed by encapsulating pNF-&kappaB-DTA/bPEI polyplexes in PR_b functionalized stealth liposomes showed more specific gene expression in cancer cells versus healthy cells compared to either individually targeted system. Transfecting cancer cells using the multi-targeted gene delivery system resulted in a dose-dependent reduction of cellular protein expression and a dose-dependent increase in cytotoxicity. Our therapeutic delivery system specifically eradicated on average 70% of a variety of cancer cells while minimally affecting healthy cells. Moving forward, the modular nature of our non-viral delivery vehicle design can facilitate targeting novel pairs of extracellular receptors and upregulated transcription factors for applications beyond cancer gene therapy.
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    Targeted drug delivery and enhanced intracellular release using functionalized liposomes.
    (2009-12) Garg, Ashish
    The ability to target cancer cells using an appropriate drug delivery system can significantly reduce the associated side effects from cancer therapies and can help in improving the overall quality of life, post cancer survival. Integrin a5b1 is expressed on several types of cancer cells, including colon cancer and plays an important role in tumor growth and metastasis. Thus, the ability to target the integrin a5b1 using an appropriate drug delivery nano-vector can significantly help in inhibiting tumor growth and reducing tumor metastasis. The work in this thesis focuses on designing and optimizing, functionalized stealth liposomes (liposomes covered with polyethylene glycol (PEG)) that specifically target the integrin a5b1. The PEG provides a steric barrier allowing the liposomes to circulate in the blood for longer duration and the functionalizing moiety, PR_b peptide specifically recognizes and binds to integrin a5b1 expressing cells. The work demonstrates that by optimizing the amount of PEG and PR_b on the liposomal interface, nano-vectors can be engineered that bind to CT26.WT colon cancer cells in a specific manner and internalize through a5b1-mediated endocytosis. To further improve the efficacy of the system, PR_b functionalized pH-sensitive stealth liposomes that exhibit triggered release under mild acidic conditions present in endocytotic vesicles were designed. The study showed that PR_b functionalized pH-sensitive stealth liposomes, undergo destabilization under mildly acidic conditions and incorporation of the PR_b peptide does not significantly affect the pH-sensitivity of the liposomes. PR_b functionalized pH-sensitive stealth liposomes bind to CT26.WT colon carcinoma cells that express integrin a5b1, undergo cellular internalization, and release their load intracellularly in a short period of time as compared to other formulations. PR_b-targeted pH-sensitive stealth liposomes encapsulating 5-fluorouracil (5-FU) show significantly higher cytotoxicity than the PR_b-targeted inert stealth liposomes and the non-targeted stealth liposomes (both pH-sensitive and inert). The studies demonstrated that optimized PR_b functionalized pH sensitive liposomes have the potential to deliver a payload, such as chemotherapeutic agents, directly to colon cancer cells in an efficient and specific manner.
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    A Universal Delivery Platform : Near Infra-Red Activated Nanoparticles for Drug, Peptide, and Small Molecule Delivery
    (2018-10) Shin, Jeongeun
    A generic delivery platform that could deliver any biomolecule, independent of its chemical constitution, would be a significant advance not only for treatments of cancers and genetic diseases, but also for researches in cell biology and neuroscience. At present, introducing endogenous proteins, genetic materials, or other non-native species into cancer or other cells has been tested via mechanical force-driven methods (electroporation, microinjection, and membrane deformation) and nanoparticle-mediated methods (lipid, polymeric, or inorganic nanoparticles). However, mechanical force-driven delivery methods cause loss of cell viability since they brutally breach cell membrane to form transient pores and require high concentration of molecules to be delivered, compared to nanoparticle-mediated methods. Contents release from current nanoparticle-mediated delivery platforms is hard to control, because release kinetics is determined by the physicochemical properties of nanoparticles. Therefore, it is hard to trigger contents at a desired time and location. In addition, delivering functional proteins or genetic materials to cells requires bypassing the cell membrane, which is done most efficiently via endocytosis. However, endosome escape is still the most difficult to overcome bottleneck for drug, protein or nucleic acid delivery by nanoparticles. Understanding the ways in which cancer cells respond to different local concentrations of small molecules and probing the signaling pathways thus initiated often requires sub-micron and sub-millisecond resolution of physiological processes. The challenge is to perturb the cell environment with a concentration jump of a physiological ligand, cofactor or antagonist, with timing and spatial dimensions that mimic the physiological process. At present, such fast intervention is the realm of “caged” ATP, calcium and other small molecules. However, small molecule delivery via caged compounds requires each bioactive requires the chemical synthesis of its own “cage”. The use of high intensity ultraviolet (UV) light causes damage to surrounding cells and has a relatively shallow level of effectiveness in the human body. To address these issues by delivering functional proteins or small biologically active molecules to individual cells in culture, and rapidly manipulating the chemical or biological environment within and in the vicinity of a particular cell, we proposed a new platform technology based on the interactions of plasmon-resonant hollow gold nanoparticles (HGN) with physiologically friendly, highly penetrating near infrared (NIR) light. NIR light is easy to use, manipulate and target with subcellular resolution using either a conventional two-photon microscope or our customized laser set up, which opens new ways to induce events and control the level of impact in specific cell populations, even subcellular sites. HGN are thin gold shells with a hollow, water-filled core, showing the unique and highly tunable optical properties, so-called Localized Surface Plasmon Resonance (LSPR). The LSPR allows the HGN-absorbance maximum can be easily tuned to 600 – 900 nm, NIR light, by adjusting the ratio of the shell thickness to the nanoparticle diameter. The advantage of NIR is that it can penetrate several centimeters of soft tissues without showing any significance harmful effect on tissues. In previous works, the HGN synthesis, which involves the galvanic exchange of gold on a silver nanoparticle template, provides a relatively polydisperse population, which broadens the absorption maximum. We have developed new methods of creating monodispersed hollow gold nanoparticles in different sizes (25 – 40 nm) and shapes (nanospheres and nanocubes). Moreover, we optimized the reaction to synthesize highly monodispersed as small as 10 nm, with temperature control. The irradiation of picosecond NIR light pulses onto HGN shows the unique transient heat transfer dynamics. The pulse duration is significantly faster that the rate of heat dissipation into the surrounding fluid (nanosecond to microsecond). This confines the energy to the HGN, leading to a transient increase in HGN temperature above metltng point of gold-silver alloy (~ 1000 ℃) depending on the light intensity. At these temperatures, gold-thiol bonds that are used to conjugate thiol-labeled protein or siRNA to the HGN surface, are broken, releasing small molecule, protein, or siRNA cargo from the HGN. In the following nanoseconds, the hot HGN nucleates vapor nanobubbles, which rapidly grow and collapse, similar to cavitation bubbles, and induce mechanical disruption of endosome, cell or liposome membranes. Nanobubbles provide efficient endosome escape with cell-resolution selectivity, and then dissipate, leaving no toxic materials behind. Direct cargo release to the cytoplasm makes for high efficiency; reducing biomolecule concentration required more than an order of magnitude compared to commercially available chemical reagents for intracellular delivery. We detected the generation of bubble around the HGN as the evidence of fast (picoseconds) NIR absorption. We then compared the threshold energy for bubble generation in different sizes, shapes, LSPR wavelength and surface coverage of nanoparticles. The minimum fluence for nanobubble generation decreased with HGN size at a fixed LSPR wavelength, unlike solid gold nanoparticles that require an increased fluence with decreasing size. We also show that he laser fluence of NIR pulses increases as the irradiation wavelength moves off the HGN LSPR. As HGN concentration decreases, the threshold fluence necessary to induce transient vapor nanobubbles increases due to light localization through multiple scattering. However, surface treatment (citrate stabilized or thiol-linked polyethylene glycols ranging from 750 to 5000 molecular weight) made no significant difference on the threshold fluence. We then examined contents release via HGN delivery platform. We have developed HGN conjugated with thiol-labeled liposomes. As nanocarriers, liposomes can encapsulate almost any water-soluble biologically active molecule by confining high concentration. A major benefit of this technique is the universal mechanism of liposome contents release via nanobubble rupture following pulsed NIR light triggering: any molecule will be released by liposome rupture, so release rates, timing, laser fluence, etc. will be similar for all compounds of interest. By modifying the laser fluence, HGN properties, and liposome membrane composition, we can alter the energy threshold for triggering release, enabling delivery of multiple agents at different times and locations, which is impossible with current liposome or caged compound technologies. Chemically disparate calcium, ATP, and carboxyfluorescein (CF) are all released at near 100 % efficiency from liposomes within msec. For a given HGN tethered to the liposome, the threshold energy is lowest at the wavelength corresponding to the maximum adsorption wavelength of the HGN; the threshold energy increases as the wavelength of the NIR light moves away from the maximum. This allows us to create liposomes that can release at different laser fluences so that we could control release rates and windows of each biomolecule in a mixture independently, by delivering two species or even changing the order of release. In this way, we can release one compound at one place and time, then a second compound at the same place at a different time simply by modulating the laser energy. This independent release has never been demonstrated before. This independent, sequential and timely release of multiple contents shows a potential of our technology for an application in combination therapies that require multiple injections to fight against multiple drug resistance (MDR), which is still a bottle neck for cancer treatments. We presented three different applications to show the versatility of our technology. We remotely triggered calcium release from liposomes in an alginate hydrogel and induced spatially patterned alginate gelation. We also succeed to entrap mammalian cells at desired sites in alginate gel via NIR light triggered calcium release. We then showed intracellular drug delivery using our HGN tethered liposomes. Cisplatin, a prostate and ovarian cancer drug, was introduced into PC3 prostate cancer cells and showed enhanced cell killing depending on HGN size and laser fluence, compared to free cisplatin. We also demonstrated that enhanced therapeutic efficacy of cisplatin was observed when cisplatin containing liposomes were irradiated in a flow, compared to cisplatin release that happens on static cell culture condition. At last, we showed toxic KLAK peptide and Cre recombinase release from the HGN surface via nanobubble generation. With our system, we decreased IC50 of KLAK peptide by a million-fold and increased cell killing as increasing laser fluence. Upon NIR pulse irradiation, delivery of Cre recombinase into HeLa cells via nanobubble generation edited genes in cells and modified cells to express red fluorescence as an indicator of successful cell modification. Delivery of cisplatin, KLAK peptide, and Cre recombinase via our HGN platform was performed in our customized microfluidic system that shows a potential of our technology for high-throughput gene-editing, cell modification and immunotherapy for treatments of cancer and genetic diseases. This innovative delivery platform would be powerful medical and scientific tools to advance modern targeted biomolecule delivery technologies and resolve challenges for loading, delivery, and release of multiple drugs.