Engineering nanoparticles using chemical and biological approaches for tumor targeted delivery

Doctor of PhilosophyDepartment of ChemistrySantosh AryalNanotechnology offers exciting options for the site-selective delivery of chemotherapeutics and diagnostic agents using nanoparticles. Varieties of organic and inorganic nanomaterials have been explored extensively as a delivery system either in the form of drug carriers or imaging agents. Successful stories include the clinical translation of anticancer nanomedicines such as PEGylated liposomal doxorubicin (DOXIL®), albumin-bound paclitaxel (Abraxane®), and polymeric micelle loaded paclitaxel (Genexol®), which are currently used in the clinic as one of the first lines for cancer chemotherapies. These conventional nanomedicines rely on passive-drug targeting taking advantage of leaky tumor vasculature, called the Enhanced Permeability and Retention (EPR) effect. However, delivering biologically active components selectively to the diseased cell, for example, cancer, is highly challenging due to the biological barriers in the body including blood pool cells/proteins, heterogeneous microenvironment, and intracellular degradation. Therefore, the goal of this dissertation is to develop nanoplatforms that can deliver the agents of interest in targeted fashion to cancer while bypassing or collaborating with the biological barriers. The design consideration of these nanoplatforms centralizes on using simple chemical reactions and cell biology to engineer nanoparticles. The presented nanoparticles were extensively studied and evaluated for their biological functions using in vitro and in vivo models. These nanoconstructs described herein address current limitations of conventional nanomedicine such as (1) the lack of understanding of the interaction of nanoparticle and biological system, and (2) the lack of an effective targeting strategy to deliver drugs to the cancer cell in the tumors. The significant findings of each system will be highlighted and discussed throughout this dissertation. Results obtained highlight key findings such as NP intracellular fate, maximized tumor accumulation, and unique pharmacokinetics could open the avenues for systemic investigations for personalized medicine and lay the foundation for nanomedicine design to accelerate clinical translation

Improving indigenous Vietnamese Black Rabbit frozen sperm quality: the role of glycine and sperm selection methods

[EN] Rabbit sperm are known to undergo damage during both cryopreservation and thawing, leading to decreased viability, motility and membrane integrity. Glycine can protect sperm and reduce damage during freezing. Swim-up is a simple semen processing method for selecting good motile sperm. The study evaluated the effect of the swim-up method and glycine with different concentrations supplemented to the frozen medium. Three indigenous black rabbits were selected for semen collection by artificial vagina. Next, semen was selected by swim-up method and diluted with glycine-added frozen medium. The samples were then transferred to 0.5 mL straws, cooled to 15°C and 5°C, placed in liquid nitrogen vapour, and finally placed directly into liquid nitrogen (-196°C). The samples were thawed and evaluated for sperm quality. The results showed that the medium supplemented with 10mM glycine in combination with swim-up method for 30 min gave the best results and was significantly different from the remaining concentrations (P<0.01), with viability rate, overall mobility and membrane integrity of 68.0%, 58.7% and 49.7%, respectively. In conclusion, 10 mM glycine concentration combined with swim-up for 30 min is the optimal choice for freezing local black rabbit semen. The study highlights the importance of optimising freezing protocols to improve the quality of frozen rabbit sperm, which can have important implications for animal breeding and conservation efforts.This study is funded in part by the Can Tho University, Code: T2022-133Tran, TTT.; Duy, NLK.; Hang, NT.; Ngoc, PK.; Tuyen, DND. (2023). Improving indigenous Vietnamese Black Rabbit frozen sperm quality: the role of glycine and sperm selection methods. World Rabbit Science. 31(4):229-236. https://doi.org/10.4995/wrs.2023.1969022923631

Mutagenesis development of actinoplanes sp. KCTC 9161 by N-methyl-N'-nitro-N-nitrosoguanidine (NTG) and screening for acarbose production

Acarbose has been widely used in the therapy of type II diabetes (non-insulin dependent) because it controls blood sugar contents of patients after meals. Acarbose, a pseudo-oligosaccharide, acts as a competitive -glucosidase inhibitor. Acarbose is produced by the strains of Bacillus, Streptomyces and Actinoplanes sp. The aim of this study was to develop mutagenesis for an Actinoplanes sp. strain and screening for acarbose production. The spores of Actinoplanes sp. KCTC 9161 strain were subjected to be mutated by N-methyl-N'-nitro-N-nitrosoguanidine (NTG) for screening and finding mutant strains that were capable of production of higher acarbose (an inhibitor of α-glucosidase) higher than wild type strain. Firstly, the original NTG solution was prepared in phosphate buffer 0.05 M, pH 6.9 and the safety concentration of NTG was determined at 5 mg/ml. Then, the spores were incubated with different NTG amounts and duration. The living colonies were transferred to fermentation medium. The results obtained showed that 15 mutant strains were produced higher acarbose than wild type when used thin layer chromatography method for analysis and comparing with standard acarbose (Sigma). Three cell lines among total tested 15 mutant lines of Actinoplanes sp. KCTC 9161 produced acarbose at a higher level or indicated a higher inhibitory activity toward α-glucosidase than the original strain. Enzymatic inhibitory ativity of α-glucosidase of three mutant strains (Actinoplanes sp. KCTC- L4, L11, L14) was increased 1.3 fold higher than wild type and Actinoplanes sp. KCTC spores were very sensitive to NTG toxic, 98% spores could not survive at the treatment condition of 50 µg NTG for 30 minutes. In addition, an applicable protocol for mutating Actinoplanes sp. using NTG was suggested for further research

Effects of imidacloprid and fenobucarb on the dynamics of the psyllid Diaphorina citri Kuwayama and on the incidence of Candidatus Liberibacter asiaticus

Introduction. The effects of imidacloprid and fenobucarb insecticides on the dynamics of the psyllid Diaphorina citri and on the incidence of Candidatus Liberibacter asiaticus ( Ca. L. a.), the putative causal agent of Huanglongbing disease (HLB), were studied in a field experiment. Materials and methods. The experimental design consisted of three independent 0.5-ha Citrus orchards planted with disease-free HLB-susceptible orange trees, located in a Citrus producing area seriously affected by HLB. Imidacloprid was applied monthly to the trunks in one orchard at a rate of 0.15 g a.i.·tree –1 ; fenobucarb was sprayed fortnightly in a second orchard at a rate of 250 g a.i.·ha –1. The 3rd orchard was managed as a control without insecticide applications. The total number of adult D. citri specimens and the percentages of trees harbouring psyllid eggs and 5th instar nymphs were monitored at fortnightly intervals in each orchard. Ca. L. a. incidence was assessed in each orchard by PCR at 5 months, 12 months and 24 months after planting. Results. Compared with the control, both the fenobucarb and imidacloprid treatments reduced adult psyllid populations by over 90% and reduced the frequency of trees harbouring eggs and 5th instar nymphs. Only imidacloprid treatments totally prevented development of a new generation of adults from eggs. Two years after planting, the prevalence of Ca. L. a. was 0.939, 0.745 and 0.239 in the control and in the orchards treated with fenobucarb and imidacloprid, respectively. Discussion and conclusion. The results indicated that, although both the insecticides used effectively reduced D.citri populations by killing adults and nymphs and by affecting or preventing psyllid reproduction in orchards, neither of the two insecticide treatments totally prevented transmission of Ca. L. asiaticus. However, due to its long-lasting effect and systemic activity, the imidacloprid treatment provided the best protection against infections, and delayed and slowed down the spread of the pathogen. Furthermore, it reduced the number of pesticide applications needed and left the way open for biological integrated pest management programmes. (Résumé d'auteur

Cloning and expression of pigC gene in Escherichia coli

Prodigiosin (Pg), which is particularly of interest because of anticancer and antimicrobial activities, can be produced through the PigC-catalyzed condensation reaction of 4-methoxy-2, 2’-bipyrrole-5-carboxyaldehyde (MBC) and 2-methyl-3-amylpyrrole (MAP). Therefore, the PigC protein plays an important role in prodigiosin biosynthetic pathway. However, studies related to PigC protein have not been carried out in Vietnam yet. In this work, the pigC gene was cloned and expressed in Escherichia coli DH10B and BL21 (DE3), respectively. Using PCR and universal primers, we amplified a fragment of 3 kb covering entire coding region of the pigC gene from Serratia sp. strain M5. The pigC gene was inserted into pJET1.2 vector, and then transformed into E. coli DH10B. The sequence of a recombinant vector pJET1.2/pigC was evaluated by using whole colony PCR amplification. Sequence alignment results revealed that the obtained pigC gene possesses 71.5% and 75.4% of nucleotide identity in comparison with two strains, Serratia 39006 and Serratia sp. AS9 published in GenBank with their respective accession numbers of AJ833001 and CP002773. The recombinant vector pJET1.2/pigC was used to reamplify pigC, and the acquired amplicon was inserted into pET22b vector at the site of HindIII and XhoI. The clone E. coli BL21 (DE3) containing recombinant vector pET22b/pigC was expressed in the auto-induced medium. The presence of PigC protein in the lysate was identified as a 100 kDa band through Western Blot analysis using anti his-tag antibody. Afterward, the PigC protein was purified by Ni-NTA column, and its expression level was quantified through SDS-PAGE analysis. The results of our study provide a potential material for producing prodigiosin from recombinant protein in Vietnam

Child stunting is associated with child, maternal, and environmental factors in Vietnam

Child stunting in Vietnam has reduced substantially since the turn of the century but has remained relatively high for several years. We analysed data on children 6–59 months (n = 85,932) from the Vietnam Nutritional Surveillance System, a nationally representative cross‐sectional survey. Multivariable Poisson regression models were used to estimate relative risk (RR) of stunting, stratified by child age and ecological region. Covariates at the child, maternal, household, and environmental levels were included based on available data and the World Health Organization conceptual framework on child stunting. Among children 6–23 months, the strongest associations with child stunting were child age in years (RR: 2.49; 95% CI [2.26, 2.73]), maternal height < 145 cm compared with ≥150 cm (RR: 2.04; 95% CI [1.85, 2.26]), living in the Northeast compared with the Southeast (RR: 2.01; 95% CI [1.69, 2.39]), no maternal education compared with a graduate education (RR: 1.77; 95% CI, [1.44, 2.16]), and birthweight < 2,500 g (RR: 1.75; 95% CI [1.55, 1.98]). For children 24–59 months, the strongest associations with child stunting were no maternal education compared with a graduate education (RR: 2.07; 95% CI [1.79, 2.40]), living in the Northeast compared with the Southeast (RR: 1.94; 95% CI [1.74, 2.16]), and maternal height < 145 cm compared with ≥150 cm (RR: 1.81; 95% CI [1.69, 1.94]). Targeted approaches that address the strongest stunting determinants among vulnerable populations are needed and discussed. Multifaceted approaches outside the health sector are also needed to reduce inequalities in socioeconomic status.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/151838/1/mcn12826.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/151838/2/mcn12826_am.pd