Amphiphilic linear-dendritic block copolymers for drug delivery

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2007.Includes bibliographical references.Polymeric drug delivery systems have been widely used in the pharmaceutical industry. Such systems can solubilize and sequester hydrophobic drugs from degradation, thereby increasing circulation half-life and efficacy. However, there are still challenges in the design of drug delivery vehicles to achieve efficient drug delivery in a site-specific manner. In this thesis, an amphiphilic linear-dendritic block copolymer was designed, synthesized, and applied as a new polymeric drug delivery platform. First, to develop the drug delivery vehicle, an ABA dendritic-linear-dendritic block copolymer consisting of poly(amidoamine) (PAMAM) and poly(propylene oxide) (PPO) was synthesized. In order to determine the viability of the linear-dendritic block copolymer as a drug delivery vehicle, the solution-phase self-assembly behavior and the self-assembled structures were characterized experimentally and through molecular dynamics simulations. The triblock self-assembles in aqueous media to form stable micelles with low CMC values. Dynamic light scattering results and TEM indicate the formation of particles ranging from 9 to 18 nm in diameter, with smaller diameters exhibited at higher generations. Static light scattering also confirmed the trend where the aggregation number decreased with higher generations. The experimental characterization results indicated that the physical characteristics of the PPO-PAMAM micelles were desirable and within the design specifications necessary for drug delivery. The experimental results were utilized to set up simulations where further knowledge of the microstructure of the micelles formed could be gained. It was found that the block copolymers simulated formed micelles in the same size range that was seen experimentally. However, the simulations indicated that the micelles displayed greater asphericity than dendrimers.(cont) Backfolding of the terminal amine ends was encountered, which would have implications for the configuration and spacing of any additional targeting ligand attached to the dendritic ends. Further analysis revealed that with increasing generation, the porosity of the micelles increased, which could affect the diffusion rate of drugs released out of the system. Another important finding detailed the preferential localization of a model hydrophobid drug, triclosan, in an equilibrated micelle structure. Additional experiments were performed to assess the feasibility of the nanoparticles for drug delivery applications. Drug loading studies were performed with a model hydrophobic drug, triclosan, resulting in high loading efficiencies. In comparison, linear block copolymers were half as efficient in loading triclosan. It was determined that the dendritic block synergistically increased the drug loading due to either acting as an additional block capable of encapsulating drug or sterically favoring the seclusion of the drug in the core. The linear-dendritic block copolymer synthesized was found to be a promising candidate for drug delivery due to its relative stability in aqueous solution and its drug encapsulation and release properties. Overall, the linear-dendritic block copolymer displayed physical characteristics and self-assembly behavior that satisfied the design criteria for a viable drug delivery vehicle. As a further step, the potential benefits of the novel linear-dendritic architecture were explored in two different drug delivery applications. First, PPO-PAMAM was explored as a circulating nanoparticle with the capability of multivalently targeting to specific cells, due to the presence of the dense functional groups on the dendritic block forming the corona of the micelles. PPO-PAMAM was functionalized with galactose and targeted to hepatocellular carcinoma cells. It was found that the polymer was not cytotoxic and could bind to the asialoglycoprotein receptor.(cont) The galactose-functionalized micelles were loaded with a chemotherapeutic, doxorubicin, and delivered to the carcinoma cells more efficiently than non-functionalized micelles and bare doxorubicin. The results from in vitro testing showed that PPO-PAMAM micelles with targeting capability are promising circulating drug delivery vehicles. In order to ensure success of subsequent testing in vivo of the targeted linear-dendritic block copolymer system, some improvements to the system were explored. First, PPO-PAMAM micelles were stabilized by physical entrapment of the hydrophobic core. An emulsion polymerization of hydrophobic methacrylate monomers created an interpenetrating polymer keeping the micelles intact at concentrations below the CMC and in a solubilizing solvent, methanol. This improvement would ensure that once injected into the bloodstream, the micelles would not destabilize and release high concentrations of drug. Another improvement that was explored was the synthesis of a new linear-dendritic block copolymer composed of a hydrophobic poly(amino acid) and a polyester dendron. Additionally, poly(ethyleneglycol) (PEG) groups were attached to the outer surface of the polyester dendron. The new system synthesized has a low CMC and is thermodynamically slow to break apart in the bloodstream. Furthermore, the micelles formed would be able to circulate for longer times with PEG aiding in evading the reticuloendothelial system. The second drug delivery application explored, which advantageously utilized the dendritic blocks on the outer surface of the block copolymer micelles was as a localized drug delivery coating created by the layer-by-layer (LbL) assembly approach. The electrostatic LbL assembly approach offers large potential in the area of drug delivery from thin films and surfaces; however, because the processing technique is aqueous-based, there have been few strategies proposed to incorporate hydrophobic molecules into these films.(cont) Here we created an LbL film that is capable of incorporating hydrophobic drug at high loadings via encapsulation with linear-dendritic block copolymer micelles and demonstrate for the first time release times of a hydrophobic antibacterial agent over a period of several weeks--a significant improvement over reports of other micelle-encapsulated thin films with release times of several minutes. The PAMAM block, which is polycationic, enabled LbL deposition with negatively charged poly(acrylic acid) (PAA). The stable PPO-PAMAM micelles incorporated into the LbL films encapsulated a hydrophobic bactericide, triclosan. Film thickness and UV-vis measurements confirm the formation of the LbL film and incorporation of triclosan into the film. Fluorescence measurements of PPO-PAMAM/PAA films with pyrene indicated the presence of hydrophobic domains in the film. GISAXS revealed regular spacing of approximately 10.5 nm in the direction parallel to the film substrate, which is approximately the same size as the PPO-PAMAM micelles in aqueous solution. Volume fraction measurements based on elemental analysis and TGA confirm the GISAXS data. An in vitro release study revealed long release times of triclosan on the order of weeks, and a Kirby Bauer test was performed on Staphylococcus Aureus demonstrating that the drug released was still active to inhibit the growth of bacteria. Linear-dendritic block copolymer micelles were successfully used in two different drug delivery applications where the dendritic block could be fully utilized. It is hoped that with the research and results presented in this thesis further development of this drug delivery platform can result in a product successfully treating a serious disease.by Phuong Nguyen.Ph.D

IL-7 Promotes CD95-Induced Apoptosis in B Cells via the IFN-γ/STAT1 Pathway

Interleukin-7 (IL-7) concentrations are increased in the blood of CD4+ T cell depleted individuals, including HIV-1 infected patients. High IL-7 levels might stimulate T cell activation and, as we have shown earlier, IL-7 can prime resting T cell to CD95 induced apoptosis as well. HIV-1 infection leads to B cell abnormalities including increased apoptosis via the CD95 (Fas) death receptor pathway and loss of memory B cells. Peripheral B cells are not sensitive for IL-7, due to the lack of IL-7Ra expression on their surface; however, here we demonstrate that high IL-7 concentration can prime resting B cells to CD95-mediated apoptosis via an indirect mechanism. T cells cultured with IL-7 induced high CD95 expression on resting B cells together with an increased sensitivity to CD95 mediated apoptosis. As the mediator molecule responsible for B cell priming to CD95 mediated apoptosis we identified the cytokine IFN-γ that T cells secreted in high amounts in response to IL-7. These results suggest that the lymphopenia induced cytokine IL-7 can contribute to the increased B cell apoptosis observed in HIV-1 infected individuals

Modulation of TRAIL resistance in colon carcinoma cells: Different contributions of DR4 and DR5

<p>Abstract</p> <p>Background</p> <p>rhTRAIL is a therapeutic agent, derived from the TRAIL cytokine, which induces apoptosis in cancer cells by activating the membrane death receptors 4 and 5 (DR4 and DR5). Here, we investigated each receptor's contribution to rhTRAIL sensitivity and rhTRAIL resistance. We assessed whether agonistic DR4 or DR5 antibodies could be used to circumvent rhTRAIL resistance, alone or in combination with various chemotherapies.</p> <p>Methods</p> <p>Our study was performed in an isogenic model comprised of the SW948 human colon carcinoma cell line and its rhTRAIL resistant sub-line SW948-TR. Effects of rhTRAIL and agonistic DR4/DR5 antibodies on cell viability were measured using MTT assays and identification of morphological changes characteristic of apoptosis, after acridine orange staining. Sensitivity to the different death receptor ligands was stimulated using pretreatment with the cytokine IFN-gamma and the proteasome inhibitor MG-132. To investigate the mechanisms underlying the changes in rhTRAIL sensitivity, alterations in expression levels of targets of interest were measured by Western blot analysis. Co-immunoprecipitation was used to determine the composition of the death-inducing signalling complex at the cell membrane.</p> <p>Results</p> <p>SW948 cells were sensitive to all three of the DR-targeting agents tested, although the agonistic DR5 antibody induced only weak caspase 8 cleavage and limited apoptosis. Surprisingly, agonistic DR4 and DR5 antibodies induced equivalent DISC formation and caspase 8 cleavage at the level of their individual receptors, suggesting impairment of further caspase 8 processing upon DR5 stimulation. SW948-TR cells were cross-resistant to all DR-targeting agents as a result of decreased caspase 8 expression levels. Caspase 8 protein expression was restored by MG-132 and IFN-gamma pretreatment, which also re-established sensitivity to rhTRAIL and agonistic DR4 antibody in SW948-TR. Surprisingly, MG-132 but not IFN-gamma could also increase DR5-mediated apoptosis in SW948-TR.</p> <p>Conclusions</p> <p>These results highlight a critical difference between DR4- and DR5-mediated apoptotic signaling modulation, with possible implications for future combinatorial regimens.</p

Persistent Gastric Colonization with Burkholderia pseudomallei and Dissemination from the Gastrointestinal Tract following Mucosal Inoculation of Mice

Melioidosis is a disease of humans caused by opportunistic infection with the soil and water bacterium Burkholderia pseudomallei. Melioidosis can manifest as an acute, overwhelming infection or as a chronic, recurrent infection. At present, it is not clear where B. pseudomallei resides in the mammalian host during the chronic, recurrent phase of infection. To address this question, we developed a mouse low-dose mucosal challenge model of chronic B. pseudomallei infection and investigated sites of bacterial persistence over 60 days. Sensitive culture techniques and selective media were used to quantitate bacterial burden in major organs, including the gastrointestinal (GI) tract. We found that the GI tract was the primary site of bacterial persistence during the chronic infection phase, and was the only site from which the organism could be consistently cultured during a 60-day infection period. The organism could be repeatedly recovered from all levels of the GI tract, and chronic infection was accompanied by sustained low-level fecal shedding. The stomach was identified as the primary site of GI colonization as determined by fluorescent in situ hybridization. Organisms in the stomach were associated with the gastric mucosal surface, and the propensity to colonize the gastric mucosa was observed with 4 different B. pseudomallei isolates. In contrast, B. pseudomallei organisms were present at low numbers within luminal contents in the small and large intestine and cecum relative to the stomach. Notably, inflammatory lesions were not detected in any GI tissue examined in chronically-infected mice. Only low-dose oral or intranasal inoculation led to GI colonization and development of chronic infection of the spleen and liver. Thus, we concluded that in a mouse model of melioidosis B. pseudomallei preferentially colonizes the stomach following oral inoculation, and that the chronically colonized GI tract likely serves as a reservoir for dissemination of infection to extra-intestinal sites

Infant and young child feeding practices differ by ethnicity of Vietnamese mothers

BACKGROUND: Limited studies have examined ethnic variation in breastfeeding and complementary feeding practices in developing countries. This study investigated ethnic variation in feeding practices in mothers with children 0–23 months old in Vietnam. METHODS: We used data on 1875 women who came from the ethnic majority, Kinh (n = 989, randomly sampled from 9875 surveyed Kinh mothers, 10 % from each province) and three ethnic minorities: E De-Mnong (n = 309), Thai-Muong (n = 229) and Tay-Nung (n = 348). Ethnic minorities were compared with the Kinh group using logistic regression model. RESULTS: Prevalence of breastfeeding initiation within an hour of birth was 69 % in Thai-Muong, but ~50 % in other ethnicities. In logistic regression, the prevalence of breastfeeding within one hour was lower in Tay-Nung (OR: 0.54; 95 % CI: 0.38, 0.77) than the majority Kinh. Prevalence of exclusive breastfeeding under 6 months was 18, 10, 17, and 33 % in Kinh, Thai-Muong, Tay-Nung, and E De-Mnong, respectively; compared to the majority Kinh, the prevalence was lower in Thai-Muong (OR: 0.42; 95 % CI: 0.25, 0.71) and higher in E De-Mnong (OR: 1.99; 95 % CI: 1.04, 3.82). Overall prevalence of bottle feeding in Thai-Muong and E De-Mnong (~20 %) was lower than in Kinh (~33 %): Thai-Muong (OR: 0.50; 95 % CI: 0.37, 0.68) and E De-Mnong (OR: 0.69; 95 % CI: 0.50, 0.95). Compared with Kinh (75 %), fewer ethnic minority children received minimum acceptable diets (33 % in Thai-Muong, 46 % in E De-Mnong, and 52 % in Tay-Nung; P < 0.05). Prevalence of minimum acceptable diet (met both dietary frequency and diversity) was lower in Thai-Muong (OR: 0.23; 95 % CI: 0.11, 0.46), Tay-Nung (OR: 0.52; 95 % CI: 0.39, 0.69), and E De-Mnong (OR: 0.55; 95 % CI: 0.33, 0.89) than the majority Kinh. CONCLUSIONS: Breastfeeding practices were suboptimal and differed by ethnicity, which suggests need for tailored interventions at multiple levels to address ethnic-specific challenges and norms. Complementary feeding practices were less optimal among ethnic minorities compared to Kinh, which suggests need for broad intervention including improved food availability, accessibility, and security. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12884-016-0995-8) contains supplementary material, which is available to authorized users

Metapopulation Dynamics Enable Persistence of Influenza A, Including A/H5N1, in Poultry

Thanks to K. Sturm-Ramirez, C. Jessup, J. Rosenthal and the staff of EcoHealth Alliance for feedback. Disclaimer: The contents are the responsibility of the authors and do not necessarily reflect the views of USAID or the United States Government.Conceived and designed the experiments: PRH TF RH DZ CSA AG MJM XX TB PD. Performed the experiments: PRH. Analyzed the data: PRH. Contributed reagents/materials/analysis tools: PRH TF RH DZ CSA AG MJM XX TB JHJ PD. Wrote the paper: PRH TF RH DZ CSA AG MJM XX TB JHJ PD.Highly pathogenic influenza A/H5N1 has persistently but sporadically caused human illness and death since 1997. Yet it is still unclear how this pathogen is able to persist globally. While wild birds seem to be a genetic reservoir for influenza A, they do not seem to be the main source of human illness. Here, we highlight the role that domestic poultry may play in maintaining A/H5N1 globally, using theoretical models of spatial population structure in poultry populations. We find that a metapopulation of moderately sized poultry flocks can sustain the pathogen in a finite poultry population for over two years. Our results suggest that it is possible that moderately intensive backyard farms could sustain the pathogen indefinitely in real systems. This fits a pattern that has been observed from many empirical systems. Rather than just employing standard culling procedures to control the disease, our model suggests ways that poultry production systems may be modified.Yeshttp://www.plosone.org/static/editorial#pee

Encouraging rational antibiotic use in childhood pneumonia: a focus on Vietnam and the Western Pacific Region

Globally, pneumonia is considered to be the biggest killer of infants and young children (aged <5 years) outside the neonatal period, with the greatest disease burden in low- and middle-income countries. Optimal management of childhood pneumonia is challenging in settings where clinicians have limited information regarding the local pathogen and drug resistance profiles. This frequently results in unnecessary and poorly targeted antibiotic use. Restricting antibiotic use is a global priority, particularly in Asia and the Western Pacific Region where excessive use is driving high rates of antimicrobial resistance. The authors conducted a comprehensive literature review to explore the antibiotic resistance profile of bacteria associated with pneumonia in the Western Pacific Region, with a focus on Vietnam. Current management practices were also considered, along with the diagnostic dilemmas faced by doctors and other factors that increase unnecessary antibiotic use. This review offers some suggestions on how these issues may be addressed