Cellular Responses Of PEG-b-PLA Polymeric Micelles And Porous Si Nanoparticles

Cancer chemotherapy has a history of over40years and there are more than800anti-cancer agents that are currently under clinical development. However, the cancerdeaths are still overwhelming due to the multidrug resistenance （MDR） of cancer cells aswell as the severe side effects of anti-cancer agents. According to the latest releasedstatistics by the World Health Organization （WHO）, about7.6million people worldwiededied from cancer in2008and the number is expected to12million in2030. Thedevelopment of nanotechnology, especially the development of nano drug deliverysystems （NDDS） such as the non-viral gene therapy vehicles, the multifunctionalnanoparticles for simultaneous cancer diagnosis and therapy, has opened up a new field ofresearch named Nano-oncology. There are two parts in this paper; the first part puts somelight on the role of cellular uptake in the reversal of MDR by PEG-b-PLA polymericmicelles from the perspective of overcoming the MDR of conventional chemotherapeuticagents. And the second part focuses on developing a novel porous Si nanoparticles-basedphotosensitizer for photodynamic therapy （PDT）, with emphasis on diminishing the sideeffects of chemotherapeutic agents.According to the American Cancer Society, around90%of cancer patients’ deaths areassociated with MDR of cancer cells, so it becomes a matter of urgency in clinic toovercome the MDR in cancer chemotherapy. Recently, nano drug delivery systerms havebeen developed as a strategy for the reversal of MDR and the results are promising.Among the various nano drug delivery systems, polymeric micelles are extensivelystudied due to their good stability, biocompatibility, capability to solubilize hydrophobicdrugs and selective tumor deposition via the enhanced permeability and retention （EPR）effect. Currently, several Pluronic polymeric micelles-based anticancer drugs have beenunder clinical trials as a class of MDR reversal agents. In this paper, the cellular uptake pathways of PEG-b-PLA polymeric micelles were investigated and the reversal of ofMDR by paclitaxel-loaded PEG-b-PLA micelles were determined, which provides afoundation for designing more effective nano drug delivery systems.Photodynamic therapy can be an effective clinical treatment for certain types of cancerbecause of its relatively noninvasive nature. However, photosensitizers in clinic use todaydisplay several side effects that limit their use, such as high toxic, poor tumor selectivityand prolonged residence time in the normal tissues of the body. If not protected fromsunlight and other forms of bright light, the skin and eyes of the patients can becomeseverely damaged. It takes only a few minutes of exposure to induce a light sensitivityresponse, and this sensitivity can persist for4-12weeks after administration of thetherapeutic. It has been recently discovered that the quantum-confined domains in porousSi films can generate¹O₂when excited with visible light. More importantly, porous Si haslow toxicity and it is biodegradable and biocompatibile. This study represents the first invitro demonstration of photodynamic killing of cancer cells using porous Si nanoparticles,and it illustrates the potential for this nanomaterial as a nontoxic, biodegradable alternativeto molecular PDT agents used in the clinic today.In the second chapter of this dissertation, by using PEG-b-PLA polymers as the vehicle,both paclitaxel-loaded micelles （M-PTX） and nile red-loaded micelles （M-NR） wereprepared and characterized. Moreover, the in vitro release behavior and in vitrocytotoxicity against human ovarian cancer A2780cells were investigated by using M-PTX.In addition, the cellular uptake was confirmed by using M-NR. In the third chapter, thecellular uptake mechanisms were explored by using atomic force microscopy, F rsterresonance energy transfer （FRET） effect, total internal reflection fluorescence microscopy（TIRFM） and various endocytic pathway-specific inhibitors. Furthermore, the reversal ofMDR by M-PTX was investigated and its relevance with cellular uptake pathway wasfurther explored. In the fourth chapter of this dissertation, porous Si nanoparticles withdifferent average pore sizes were prepared by using various etching current densities. Moreover, the¹O₂quantum yield of porous Si nanoparticles both in ethanol and aqueoussolution were evaluated. In the chapter five, we show that porous Si nanoparticles can beused as a photosensitizer to kill both human cervical carcinoma HeLa cells and mouseembryo fibroblast NIH3T3cancer cells in vitro following illumination with either acommercial halogen light or a light-emitting diode （LED） panel.Here are the main results:（1） Paclitaxel or nile red was incorporated into PEG-b-PLA micelles by thin filmmethod. Transmission electron microscopy （TEM） showed that both M-PTX and M-NRwere monodisperse and spherical, displaying a core-shell structure. The meanhydrodynamic diameter of M-PTX and M-NR measured by dynamic light scattering were61nm and104nm, with PDI0.13and0.24, respectively. Furthermore, the entrapmentefficiency and drug-loading capacity of M-PTX were64.7%and6.08％, respectively. Thein vitro drug release result represents that a relatively less amount of paclitaxel wasreleased from M-PTX than free paclitaxel at any given time. About100%of freepaclitaxel was released from dialysis membrane and80%of paclitaxel was released fromM-PTX during24h. In addition, the paclitaxel loaded into polymer micelles still exhibitedsimilar cytotoxicity against A2780cells compared to free paclitaxel. Moreover, theconfocal microscopy results confirmed that the M-NR could be effectively taken up byhuman ovarian cancer A2780cells, C6glioma cells, as well as human lens epithelium（HLE） cells. The cell membranes and cytoplasms of the A2780, C6and HLE cells werehighly luminescent with red fluorescence. It should be noticed that cell fixation could leadto an artificial redistribution of the M-NR by increasing the cell membrane permeability.The red fluorescence in membrane disappeared after cell fixation with4%paraformaldehyde in A2780, C6and HLE cells.（2） The cellular uptake of M-NR in A2780cells at different time points were evaluatedby confocal microscope and the plasma membrane showed remarkably intensefluorescence. Large-scale images of50!50μm showed that the cells treated with M-NR or blank micelles had much rougher surface texture than those of untreated cells. To getmore detailed information about the interaction between PEG-b-PLA micelles and cellmembrane, small areas within400!400nm showed small clusters were on the surface ofthe cells treated with M-NR or blank micelles. These results indicated that the amphiphilicPEG-b-PLA micelles might insert into the amphiphilic cell membrane during cellularinternalization. Followed by clarifying the FRET between nile red （acceptor） incorporatedin PEG-b-PLA micelles and DAF （donor） inserted in cell membrane, we confirmed thecore-loaded nile red was effectively and quickly released into the cell membrane duringthe cellular uptake of M-NR, rather then the generally thought that polymeric micellescarry drug molecules until they are taken up into cells by endocytosis followed byintracellular release. In addition to this, by treating the cells with M-NR, the appearance,movement and disappearance of small vesicles with red fluorescence were observedwithin10min by using total internal reflection fluorescence microscope. The resultsindicated that endocytosis was involved in the cellular uptake of the released nile red. Tofurther elucidate the endocytic pathways of the nile red released, several specificendocytic inhibitors were used. Both the qualitative results from confocal microscopystudies and the quantitative results obtained from flow cytometry consistently indicatedthat the internalization of M-NR was an energy-dependent process and lipidraft/caveolae-mediated endocytosis may play a major role in this process. Moreimportantly, the MTT results demonstrated that M-PTX induced more cytotoxicity inA2780/T cells than free paclitaxel, indicating that PEG-b-PLA micelles reversed MDR tosome extent. The mechanism is related to the inhibition of Pgp function and Pgp ATPaseactivity by the interaction with cell membrane to induce membrane depolarization andenhance membrane microviscosity. Thus, micellar paclitaxel exhibited marked increase ofcellular accumulation of paclitaxel compared with free paclitaxel in A2780/T cells,resulted in the reversal of MDR. This study provides a new strategy for understanding themechanism of reversing MDR by nanoparticles better and designing more effective nanodrug carriers. （3） Porous Si nanoparticles were prepared by electrochemical etching of single-crystalsilicon wafers. Porous Si nanoparticles with different average hydrodynamic size （100-200nm） and different average pore sizes （7.9-17.6nm） were prepared by using various etchingcurrent densities. Scanning electron microscope （SEM） images reveal a well-orderedmesoporous nanostructure and the average pore sizes increased as the increase of theetching current densities. The as-prepared nanoparticles mainly possess ahydrogen-terminated surface while a small amount of Si-O bond due to surface oxidationas indicated by FT-IR. It should be noted that the surface oxidiation increased thehydrophilic property of porous Si nanoparticles at some extent. The generation of¹O₂byphotoexcited porous Si nanoparticles in ethanol was detected using the chemical trappingreagent1,3-diphenylisobenzofuran （DPBF）. By taking rose bengal （RB） as a standard, the¹O₂quantum yield of the porous Si nanoparticles as a function of average pore size wasdetermined between5%to12%. In the present work, no specific dependence of¹O₂quantum yield on porosity was observed. This is attributed to the process by which theultrasonic fracture of the porous Si film into particles, the solvent deactivation as well asthe degree of oxidation during the preparation of porous Si nanoparticles. Similarly, thequantum yield for¹O₂generation by porous Si nanoparticles in water （under the currentdensity of200mA/cm2） was determined to be0.17±0.01using singlet oxygen greensensor （SOSG） as the¹O₂trapper, which is consistent with the results of0.15±0.02byusing9,10-anthracenediyl-bis（methylene） dimalonic acid （AMDA） as the¹O₂trapper.（4） The phototoxicity of porous Si nanoparticles toward HeLa and NIH3T3cancer cellsfollowing illumination with either a halogen light or a LED panel was studied. The resultsindicate that the cell viability decreased with increasing concentration of porous Sinanoparticles and with increasing light intensity for both cell types studied. Themorphology of HeLa cells exposed to porous Si nanoparticles in the dark, or with lightexposure but without nanoparticles, showed no significant change compared to anuntreated control. However, under the same light exposure conditions, HeLa cells in thepresence of porous Si nanoparticles lost their adherent nature and shrank to assume a spherical-like morphology. A second illumination protocol was tested in which thenanoparticles were not removed with a postirradiation rinse but allowed to incubate withthe cells for24h postirradiation. Both HeLa and NIH-3T3cell lines showed moresignificantly phototoxicity compared with the protocol removed the nanoparticlesimmediately postirradiation. The increased degree of cell death observed in theexperiments could be attributed to long-lived toxic species that are generated by the actionof¹O₂with amino acids or proteins present in the vicinity of the nanoparticles duringirradiation. However, in comparison with a conventional PDT agent such as methyleneblue, the porous Si nanoparticles are less effective on a mass basis. So, how to optimizethe¹O₂quantum yield of porous Si nanoparticles as well as the degradation rate, taking itsadvantage of rapid degrade property while stop being degrade too quickly to produce¹O₂,needs to be further explored by surface chemistry modification. In addition to this, porousSi nanoparticles were found to possess both photochemical and photothermal effect. Whena larger quantity of porous Si nanoparticles was present （6mg/mL,100mW/cm2halogenlamp）,23°C temperature rise was observed in10min caused by the photothermal effectof porous Si nanoparticles. Furthermore, if the irradiation power density was increased to300mW/cm2, the temperature rise was36°C greater than the control. It is worth notingthat under the highest porous Si nanoparticles concentration and strongest power intensityconditions of the cell culture experiments in the present work, the temperature rise is lessthan1°C, indicating the photothermal effect is negligible here. Thus, the cell viabilitydata presented in this work was result from photochemical （¹O₂generation） effects ratherthan from photothermal （localized heating） effects. This study affords the foundation fordeveloping the next generation of nontoxic, biodegradable nanopotosensitizers.In general, nano drug delivery systems have showed extensive potential in overcomingMDR and diminishing the toxicity of chemotherapeutic agents. With the progress made innano-oncology and the further investigation of the cellular response of nano drug deliverysystems, the next generation of nano drug delivery systems will be aimed at multi-functional, targeted delivery, as well as simultaneous diagnosis and therapy ofcancer. Thus, there would be more and more high efficiency and low toxicnanotechnology-based anti-cancer drugs emerging in clinic trials, which will benefit thepatients suffering from MDR or the severe side effects of chemotherapy. It is likely that inthe not-distant furture, personalized oncology could be achieved by designing specificnano drug delivery systems based on the phenotypical characteristics of the individualpatient.