Genetic diversity and population structure of bermudagrass (Cynodon spp.) revealed by genotyping-by-sequencing.

Bermudagrass (Cynodon spp.) breeding and cultivar development is hampered by limited information regarding its genetic and phenotypic diversity. To explore diversity in bermudagrass, a total of 206 Cynodon accessions consisting of 193 common bermudagrass (C. dactylon var. dactylon) and 13 African bermudagrass (C. transvaalensis) accessions of worldwide origin were assembled for genetic characterization. Genotyping-by-sequencing (GBS) was employed for genetic marker development. With a minor allele frequency of 0.05 and a minimum call rate of 0.5, a total of 37,496 raw single nucleotide polymorphisms (SNPs) were called de novo and were used in the genetic diversity characterization. Population structure analysis using ADMIXTURE revealed four subpopulations in this germplasm panel, which was consistent with principal component analysis (PCA) and phylogenetic analysis results. The first three principal components explained 15.6%, 10.1%, and 3.8% of the variance in the germplasm panel, respectively. The first subpopulation consisted of C. dactylon accessions from various continents; the second subpopulation was comprised mainly of C. transvaalensis accessions; the third subpopulation contained C. dactylon accessions primarily of African origin; and the fourth subpopulation represented C. dactylon accessions obtained from the Oklahoma State University bermudagrass breeding program. Genetic diversity parameters including Nei's genetic distance, inbreeding coefficient, and Fst statistic revealed substantial genetic variation in the Cynodon accessions, demonstrating the potential of this germplasm panel for further genetic studies and cultivar development in breeding programs.

Genome-wide association study reveals genomic loci influencing agronomic traits in Ethiopian sorghum (Sorghum bicolor (L.) Moench) landraces.

Uncovering the genetic basis of agronomic traits in sorghum landraces that have adapted to various agro-climatic conditions would contribute to sorghum improvement efforts around the world. To identify quantitative trait nucleotides (QTNs) associated with nine agronomic traits in a panel of 304 sorghum accessions collected from diverse environments across Ethiopia (considered to be the center of origin and diversity), multi-locus genome-wide association studies (ML-GWAS) were performed using 79,754 high quality single nucleotide polymorphism (SNP) markers. Association analyses using six ML-GWAS models identified a set of 338 significantly (LOD ≥ 3)-associated QTNs for nine agronomic traits of sorghum accessions evaluated in two environments (E1 and E2) and their combined dataset (Em). Of these, 121 reliable QTNs, including 13 for flowering time (DF), 13 for plant height (PH), 9 for tiller number (TN), 15 for panicle weight (PWT), 30 for grain yield per panicle (GYP), 12 for structural panicle mass (SPM), 13 for hundred seed weight (HSW), 6 for grain number per panicle (GNP), and 10 for panicle exertion (PE) were consistently detected by at least three ML-GWAS methods and/or in two different environments. Notably, Ethylene responsive transcription factor gene AP2/ERF, known for regulation of plant growth, and the sorghum Terminal flower1/TF1 gene, which functions in the control of floral architecture, were identified as strong candidate genes associated with PH and HSW, respectively. This study provides an entry point for further validation studies to elucidate complex mechanisms controlling important agronomic traits in sorghum. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-023-01381-5.

Comparative analysis reveals chromosome number reductions in the evolution of African bermudagrass (Cynodon transvaalensis Burtt-Davy).

African bermudagrass (Cynodon transvaalensis Burtt-Davy) (2n = 2x = 18) belongs to the genus Cynodon, tribe Cynodonteae, subfamily Chloridoideae in the grass family Poaceae. The species is frequently crossed with common bermudagrass (Cynodon dactylon Pers.) in developing high-quality hybrid turf cultivars. Molecular resources for C. transvaalensis are scarce; thus, its genomic evolution is unknown. Recently, a linkage map consisting of 1278 markers provided a powerful tool for African bermudagrass genomic research. The objective of this study was to investigate chromosome number reduction events that resulted in the nine haploid chromosomes in this species. Tag sequences of mapped single nucleotide polymorphism markers in C. transvaalensis were compared against genome sequences of Oropetium thomaeum (L.f.) Trin. (2n = 2x = 20), a genomic model in the Cynodonteae tribe. The comparative genomic analyses revealed broad collinearity between the genomes of these two species. The analyses further revealed that two major interchromosomal rearrangements of the paleochromosome ρ12 (ρ1-ρ12-ρ1 and ρ6-ρ12-ρ6) resulted in nine chromosomes in the genome of C. transvaalensis. The findings provide novel information regarding the formation of the initial diploid species in the Cynodon genus.

Natural variation further increases resilience of sorghum bred for chronically drought-prone environments.

Drought stress is one of the major constraints for crop production in the Sahel region of Africa. Here, we explore the potential to use natural genetic variation to build on the inherent drought tolerance of an elite sorghum cultivar, Teshale, that has been bred for Ethiopian conditions including chronic drought. We evaluated a backcross nested-association mapping population using 12 diverse founder lines crossed with Teshale under three drought-prone environments in Ethiopia. All 12 populations averaged higher head exsertion and lower leaf senescence than the recurrent parent in the two most stressful environments, reflecting new drought resilience mechanisms from the donors. A total of 154 quantitative trait loci (QTLs) were detected for eight drought-responsive traits, and their validity was supported by the fact that 113 (73.4%) overlapped with QTLs previously detected for the same traits, concentrated in regions previously associated with 'stay-green' traits. Allele effects showed that some favourable alleles are already present in the Ethiopian cultivar; however, the exotic donors offer rich scope for increasing drought resilience. Using model-selected SNPs associated with the eight traits identified in this study and three in a companion study, phenotypic prediction accuracies for grain yield were equivalent to genome-wide SNPs and were significantly better than random SNPs, indicating that the selected traits are predictive of sorghum grain yield.

Mapping QTLs for spring green-up, plant vigor, and plant biomass in two lowland switchgrass populations.

Switchgrass (Panicum virgatum L.) is an important perennial C4 species due to its large potential for cellulosic bioenergy feedstock production. Identification of quantitative trait loci (QTL) controlling important developmental traits is valuable to understanding the genetic basis and using marker-assisted selection (MAS) in switchgrass breeding. One F1 hybrid population derived from NL94 (♀) × SL93 (♂) and one S1 (first-generation selfed) population from NL94 were used in this study. Both the populations showed significant variations for genotype and genotype by environment interactions for three traits studied: plant vigor, spring green-up, and plant biomass. Plant vigor had strong and positive correlations with plant biomass in both populations. Broad-sense heritability estimates for plant vigor ranged from 0.46 to 0.74 and 0.45 to 0.74 in the hybrid and selfed population, respectively. Spring green-up had similar heritability estimates, 0.42-0.78 in the hybrid population, and 0.47-0.82 in the selfed population. Heritability of plant biomass was 0.54-0.64 in the hybrid population and 0.64-0.74 in the selfed population. Fifteen QTLs for spring green-up, 6 QTLs for plant vigor, and 3 QTLs for biomass yield were detected in the hybrid population, whereas 4 QTLs for spring green-up, 4 QTLs for plant vigor, and 1 QTL for biomass yield were detected in the selfed population. Markers associated with these QTLs can be used in MAS to accelerate switchgrass breeding program. This study provided new information in understanding the genetic control of biomass components and demonstrated substantial heterotic vigor that could be explored for breeding hybrid cultivars in switchgrass. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-022-01296-7.

Synthesis and evaluation of 3-(phenylethynyl)-1,1'-biphenyl-2-carboxylate derivatives as new HIF-1 inhibitors.

Selaginellins are a type of rare natural products from the genus Selaginella with unusual alkynyl phenol skeletons and extensive biological activities. Previous structural simplification of these natural compounds afforded a series of diaryl acetylene derivatives with hypoxia-inducible factor 1 (HIF-1) inhibitory activity. In this study, we synthesized thirty compounds by stepwise optimization using methyl 3-(4-methoxylphenyl ethynyl)-[4'-methoxyl-1,1'-biphenyl]-2-carboxylate (1a) as a lead compound and evaluated their HIF-1 inhibitory activity by dual luciferase reporter assay. Among them, compound 9i displayed the most potent HIF-1 inhibitory activity (IC50 = 1.5 ± 0.03 µM) with relatively low cytotoxicity. Under hypoxia, compound 9i showed no effect on the accumulation of HIF-1α protein in western blot analysis, but could down-regulate the expression of VEGF mRNA, the downstream target gene of HIF-1 pathway. Cell-based activity assay demonstrated that compound 9i could inhibit the hypoxia-induced migration, invasion and proliferation of HeLa cells at the concentrations of 1 ~ 5 µM. In mouse breast cancer xenograft model, compound 9i exhibited obvious tumor growth inhibition and very low toxicity at a dose of 15 mg/kg. The results suggested that compound 9i would be a potential antitumor agent via HIF-1 pathway inhibition.

Genetic diversity, population structure, and selection signature in Ethiopian sorghum [Sorghum bicolor L. (Moench)] germplasm.

Ethiopia, the probable center of origin and diversity for sorghum [Sorghum bicolor L. (Moench)] and with unique ecogeographic features, possesses a large number of sorghum landraces that have not been well studied. Increased knowledge of this diverse germplasm through large-scale genomic characterization may contribute for understanding of evolutionary biology, and adequate use of these valuable resources from the center of origin. In this study, we characterized genetic diversity, population structure and selection signature in 304 sorghum accessions collected from diverse sorghum growing regions of Ethiopia using genotyping-by-sequencing. We identified a total of 108,107 high-quality single-nucleotide polymorphism (SNPs) markers that were evenly distributed across the sorghum genome. The average gene diversity among accessions was high (He = 0.29). We detected a relatively low frequency of rare alleles (26%), highlighting the potential of this germplasm for subsequent allele mining studies through genome-wide association studies. Although we found no evidence of genetic differentiation among administrative regions (FST = 0.02, P = 0.12), population structure and cluster analyses showed clear differentiation among six Ethiopian sorghum populations (FST = 0.28, P = 0.01) adapting to different environments. Analysis of SNP differentiation between the identified genetic groups revealed a total of 40 genomic regions carrying signatures of selection. These regions harbored candidate genes potentially involved in a variety of biological processes, including abiotic stress tolerance, pathogen defense and reproduction. Overall, a high level of untapped diversity for sorghum improvement remains available in Ethiopia, with patterns of diversity consistent with divergent selection on a range of adaptive characteristics.

Genetic and QTL mapping in African bermudagrass.

Cynodon transvaalensis Burtt-Davy is frequently used to cross with C. dactylon Pers. in the creation of F1 hybrid cultivars that are some of the most widely used in the worldwide turf industry. However, molecular resource development in this species is limited. Accordingly, the objectives of this study were to construct a high-density genetic map, and to identify genomic regions associated with establishment rate. In this study, we constructed the first high-density linkage map for African bermudagrass using a genotyping by sequencing approach based on 109 S1 progenies. A total of 1,246 single nucleotide polymorphisms and 32 simple sequence repeat markers were integrated in the linkage map. The total length of nine linkage groups was 882.3 cM, with an average distance of 0.69 cM per interval. Four genomic regions were identified to be associated with sod establishment rate. The results provide important genetic resources towards understanding the genome as well as marker-assisted selection for improving the establishment rate in bermudagrass breeding.

Managing flowering time in Miscanthus and sugarcane to facilitate intra- and intergeneric crosses.

Miscanthus is a close relative of Saccharum and a potentially valuable genetic resource for improving sugarcane. Differences in flowering time within and between Miscanthus and Saccharum hinders intra- and interspecific hybridizations. A series of greenhouse experiments were conducted over three years to determine how to synchronize flowering time of Saccharum and Miscanthus genotypes. We found that day length was an important factor influencing when Miscanthus and Saccharum flowered. Sugarcane could be induced to flower in a central Illinois greenhouse using supplemental lighting to reduce the rate at which days shortened during the autumn and winter to 1 min d-1, which allowed us to synchronize the flowering of some sugarcane genotypes with Miscanthus genotypes primarily from low latitudes. In a complementary growth chamber experiment, we evaluated 33 Miscanthus genotypes, including 28 M. sinensis, 2 M. floridulus, and 3 M. ×giganteus collected from 20.9° S to 44.9° N for response to three day lengths (10 h, 12.5 h, and 15 h). High latitude-adapted M. sinensis flowered mainly under 15 h days, but unexpectedly, short days resulted in short, stocky plants that did not flower; in some cases, flag leaves developed under short days but heading did not occur. In contrast, for M. sinensis and M. floridulus from low latitudes, shorter day lengths typically resulted in earlier flowering, and for some low latitude genotypes, 15 h days resulted in no flowering. However, the highest ratio of reproductive shoots to total number of culms was typically observed for 12.5 h or 15 h days. Latitude of origin was significantly associated with culm length, and the shorter the days, the stronger the relationship. Nearly all entries achieved maximal culm length under the 15 h treatment, but the nearer to the equator an accession originated, the less of a difference in culm length between the short-day treatments and the 15 h day treatment. Under short days, short culms for high-latitude accessions was achieved by different physiological mechanisms for M. sinensis genetic groups from the mainland in comparison to those from Japan; for mainland accessions, the mechanism was reduced internode length, whereas for Japanese accessions the phyllochron under short days was greater than under long days. Thus, for M. sinensis, short days typically hastened floral induction, consistent with the expectations for a facultative short-day plant. However, for high latitude accessions of M. sinensis, days less than 12.5 h also signaled that plants should prepare for winter by producing many short culms with limited elongation and development; moreover, this response was also epistatic to flowering. Thus, to flower M. sinensis that originates from high latitudes synchronously with sugarcane, the former needs day lengths >12.5 h (perhaps as high as 15 h), whereas that the latter needs day lengths <12.5 h.

Genome biology of the paleotetraploid perennial biomass crop Miscanthus.

Miscanthus is a perennial wild grass that is of global importance for paper production, roofing, horticultural plantings, and an emerging highly productive temperate biomass crop. We report a chromosome-scale assembly of the paleotetraploid M. sinensis genome, providing a resource for Miscanthus that links its chromosomes to the related diploid Sorghum and complex polyploid sugarcanes. The asymmetric distribution of transposons across the two homoeologous subgenomes proves Miscanthus paleo-allotetraploidy and identifies several balanced reciprocal homoeologous exchanges. Analysis of M. sinensis and M. sacchariflorus populations demonstrates extensive interspecific admixture and hybridization, and documents the origin of the highly productive triploid bioenergy crop M. × giganteus. Transcriptional profiling of leaves, stem, and rhizomes over growing seasons provides insight into rhizome development and nutrient recycling, processes critical for sustainable biomass accumulation in a perennial temperate grass. The Miscanthus genome expands the power of comparative genomics to understand traits of importance to Andropogoneae grasses.

Sequence-based genetic mapping of Cynodon dactylon Pers. reveals new insights into genome evolution in Poaceae.

Bermudagrass (Cynodon dactylon Pers.) is an important warm-season perennial used extensively for turf, forage, soil conservation and remediation worldwide. However, limited genomic information has hindered the application of molecular tools towards understanding genome evolution and in breeding new cultivars. We genotype a first-generation selfed population derived from the tetraploid (4x = 36) 'A12359' using genotyping-by-sequencing. A high-density genetic map of 18 linkage groups (LGs) is constructed with 3,544 markers. Comparative genomic analyses reveal that each of nine homeologous LG pairs of C. dactylon corresponds to one of the first nine chromosomes of Oropetium thomaeum. Two nested paleo-ancestor chromosome fusions (ρ6-ρ9-ρ6, ρ2-ρ10-ρ2) may have resulted in a 12-to-10 chromosome reduction. A segmental dissemination of the paleo-chromosome ρ12 (ρ1-ρ12-ρ1, ρ6-ρ12-ρ6) leads to the 10-to-9 chromosome reduction in C. dactylon genome. The genetic map will assist in an ongoing whole genome sequence assembly and facilitate marker-assisted selection (MAS) in developing new cultivars.

Training Population Optimization for Genomic Selection in Miscanthus.

Miscanthus is a perennial grass with potential for lignocellulosic ethanol production. To ensure its utility for this purpose, breeding efforts should focus on increasing genetic diversity of the nothospecies Miscanthus × giganteus (M×g) beyond the single clone used in many programs. Germplasm from the corresponding parental species M. sinensis (Msi) and M. sacchariflorus (Msa) could theoretically be used as training sets for genomic prediction of M×g clones with optimal genomic estimated breeding values for biofuel traits. To this end, we first showed that subpopulation structure makes a substantial contribution to the genomic selection (GS) prediction accuracies within a 538-member diversity panel of predominately Msi individuals and a 598-member diversity panels of Msa individuals. We then assessed the ability of these two diversity panels to train GS models that predict breeding values in an interspecific diploid 216-member M×g F2 panel. Low and negative prediction accuracies were observed when various subsets of the two diversity panels were used to train these GS models. To overcome the drawback of having only one interspecific M×g F2 panel available, we also evaluated prediction accuracies for traits simulated in 50 simulated interspecific M×g F2 panels derived from different sets of Msi and diploid Msa parents. The results revealed that genetic architectures with common causal mutations across Msi and Msa yielded the highest prediction accuracies. Ultimately, these results suggest that the ideal training set should contain the same causal mutations segregating within interspecific M×g populations, and thus efforts should be undertaken to ensure that individuals in the training and validation sets are as closely related as possible.

Linkage Mapping for Foliar Necrosis of Soybean Sudden Death Syndrome.

Sudden death syndrome (SDS) foliar symptoms consist of foliar chlorosis, foliar necrosis, leaf marginal curling, and premature defoliation, but resistance screening has been evaluated mostly based on the overall SDS foliar severity rather than on a specific foliar symptom. This study generated an F2 population derived from crossing the susceptible variety Sloan and the resistant germplasm line PI 243518, which exhibits resistance to both foliar chlorosis and necrosis. A total of 400 F2 lines were evaluated for foliar chlorosis, foliar necrosis, and overall SDS foliar symptoms, separately. Genotyping-by-sequencing was applied to obtain single nucleotide polymorphisms (SNPs) in the F2 population, and linkage mapping using 135 F2 lines with 969 high-quality SNPs identified a locus on chromosome 13 for foliar necrosis and SDS foliar symptoms. The locus partially overlaps with loci previously reported for SDS on chromosome 13, which is the third time the region from 15.98 to 21.00 Mbp has been reproduced independently and therefore qualifies this locus for a new nomenclature proposed as Rfv13-02. In summary, this study generated a new biparental population that enables not only the discovery of a locus for foliar necrosis and SDS foliar symptoms on chromosome 13 but also the potential for advanced exploration of SDS foliar resistance derived from the germplasm line PI 243518.

Genome-Wide Association Mapping Reveals Novel QTL for Seedling Leaf Rust Resistance in a Worldwide Collection of Winter Wheat.

Leaf rust of wheat ( L.) is a major disease that causes significant yield losses worldwide. The short-lived nature of leaf rust resistance () genes necessitates a continuous search for novel sources of resistance. We performed a genome-wide association study (GWAS) on a panel of 1596 wheat accessions. The panel was evaluated for leaf rust reaction by testing with a bulk of Eriks. () isolates collected from multiple fields of Oklahoma in 2013 and two predominant races in the fields of Oklahoma in 2015. The panel was genotyped with a set of 5011 single-nucleotide polymorphism (SNP) markers. A total of 14 quantitative trait loci (QTL) for leaf rust resistance were identified at a false discovery rate (FDR) of 0.01 using the mixed linear model (MLM). Of these, eight QTL reside in the vicinity of known genes or QTL, and more studies are needed to determine their relationship with known loci. is a new QTL to bread wheat but is close to a locus previously identified in durum wheat [ L. subsp. (Desf.) Husn.]. The other five QTL, including , , , , and , are likely novel loci for leaf rust resistance. The uneven distribution of the 14 QTL in the six subpopulations of the panel suggests that wheat breeders can enhance leaf rust resistance by selectively introgressing some of these QTL into their breeding materials. In addition, another 31 QTL were significantly associated with leaf rust resistance at a FDR of 0.05.

Quantitative Trait Loci Mapping for Tillering-Related Traits in Two Switchgrass Populations.

Plant tillering and related traits are morphologically important components contributing to switchgrass ( L.) biomass yield. The objectives of this study were to estimate broad-sense heritabilities for tillering-related traits, to analyze correlations between biomass yield and the traits, and to identify quantitative trait loci (QTL) for them. A first-generation selfed population of NL94 plant and a hybrid population between NL94 and SL93 plants were field established in a randomized complete block design with three replications in Stillwater and Perkins, OK. Phenotypic data were collected in 2 yr and genotypic data were obtained by genotyping simple-sequence repeat (SSR) markers in the two populations on the basis of two preexisting genetic maps. Plant base size (PBS), plant girth (PG), tillering ability (TA), tiller diameter (TD), and tiller dry weight (TDW) were positively correlated with biomass yield in both populations. Consistently, PBS had the largest correlation coefficients for biomass yield, suggesting its value as an indirect selection criterion for biomass yield. Twenty and 26 QTL for six tillering-related traits were detected in the hybrid and selfed population, respectively. Among the QTL, one on linkage group (LG) 5a between sww-2387/PVCAG-2197/2198 and PVGA-1649/1650 for PBS, PG, and TA and another on LG 2a between sww-2640/sww-2545 and PVCA-765/766 for TD and TDW were stably detected in multiple environments in the two populations. The findings add to the knowledge base regarding the genetics of tillering-related traits that could be used in accelerating the development of high-yielding cultivars through marker-assisted selection.

A strategy for oral chemotherapy via dual pH-sensitive polyelectrolyte complex nanoparticles to achieve gastric survivability, intestinal permeability, hemodynamic stability and intracellular activity.

Efficient oral administration of anticancer agents requires a nanocarrier to long survive in the stomach, effectively penetrate across the small intestine, tightly retain the drug during bloodstream and quickly release drug in tumor cells. Herein a kind of dual pH-sensitive polyelectrolyte complex nanoparticles (CNPs) was developed by employing electrostatic interaction between positively charged chitosan (CS) and negative poly (L-glutamic acid) grafted polyethylene glycol-doxorubicin conjugate nanoparticles (PG-g-PEG-hyd-DOX NPs) with acid-labile hydrazone linkages. The obtained NPs and CNPs were characterized for their morphology, particle size, ζ-potential, pH-sensitivity under the simulated physiological conditions, drug release, as well as in vivo antitumor activity and biodistribution. The results indicated that CNPs can remain intact structure in pH range from 3.0 to 6.5. After detaching CS layer due to the pH-induced deprotonation with increasing pH to 7.4 in the mucus layer of the small intestine, the inner NPs would be released and effectively absorbed into blood circulation via opening the tight junctions by CS. PG-g-PEG-hyd-DOX NPs with demonstrated long-circulating properties can be accumulated in the tumor via EPR effect and dump the drug within tumor cells by acid-cleavage of hydrazone bonds between PG-g-PEG and DOX, achieving high therapeutic efficacy and low systemic toxicity. These results suggest that the design presented here, combining the functions of the gastrointestinal pH-sensitive electrostatic complex and intracellular acid-sensitive macromolecular prodrugs NPs, can sequentially overcome the biological barriers of oral anticancer drug delivery, which thus provides a promising nanomedicine platform for oral chemotherapy.

Improving the oral delivery efficiency of anticancer drugs by chitosan coated polycaprolactone-grafted hyaluronic acid nanoparticles.

Sequentially overcoming the obstacles mainly from the low water solubility of lipophilic anticancer drugs, gastrointestinal microenvironment and systemic circulation is the major concern for designing oral anticancer drug carriers. Herein, we prepared the multifunctional polyelectrolyte complex nanoparticles (CNPs), engineered by hyaluronic acid (HA) grafted polycaprolactone (PCL) nanoparticles (HA-g-PCL NPs) coated with chitosan (CS) electrostatically, as a platform to improve the oral delivery efficiency of lipophilic anticancer drugs. Paclitaxel (PTX) and doxorubicin (DOX) were used as the model medicine and fluorescence probe, respectively. The size, zeta potential, morphology and pH-sensitivity of the NPs were studied systematically. The results indicated that the core-shell structure of CS/HA-g-PCL CNPs was formed at pH 5.0, which remained intact in the pH ranging from 3.0 to 6.8, while the CS layer detached gradually with the increase of pH to 7.4 and the HA-g-PCL NPs were released. In vitro drug release studies showed that accelerated drug release was triggered by hyaluronidase-1 (Hyal-1), which was a major HA degradation enzyme abundant within tumor cells. Cell uptake studies showed that HA-g-PCL NPs were internalized into cancer cells (EC109) via receptor-mediated endocytosis, but were rarely taken up by normal fibroblasts (NIH3T3). Furthermore, intracellular drug release indicated that HA-g-PCL NPs could provide an effective approach for transport of loaded cargoes into the cytoplasm. Therefore, higher cytotoxicity for PTX loaded HA-g-PCL NPs (HA-g-PCL/PTX NPs) against cancer cells EC109 but lower cytotoxicity against normal cells NIH3T3 was observed. In vivo studies showed that CS/HA-g-PCL CNPs via oral administration were able to preferentially deliver drugs into tumor tissue with commendable antitumor efficiency and few side effects. Overall, CS/HA-g-PCL CNPs showed great potential for improving oral delivery efficiency of lipophilic anticancer drugs.

Contribution of hydrophobic/hydrophilic modification on cationic chains of poly(ε-caprolactone)-graft-poly(dimethylamino ethylmethacrylate) amphiphilic co-polymer in gene delivery.

Nanoparticles (NPs) assembled from amphiphilic polycations have been certified as potential carriers for gene delivery. Structural modification of polycation moieties may be an efficient route to further enhance gene delivery efficiency. In this study two electroneutral monomers with different hydrophobicities, 2-hydroxyethyl methacrylate (HEMA) and 2-hydroxyethyl acrylate (HEA), were incorporated into the cationic poly(dimethylamino ethyl methacrylate) (PDMAEMA) side-chains of amphiphilic poly(ε-caprolactone)-graft-poly(dimethylamino ethylmethacrylate) (PCD) by random co-polymerization, to obtain poly(ε-caprolactone)-graft-poly(dimethylamino ethyl methacrylate-co-2-hydroxyethyl methacrylate) (PCD-HEMA) and poly(ε-caprolactone)-graft-poly(dimethylamino ethyl methacrylate-co-2-hydroxyethyl acrylate) (PCD-HEA). Minimal HEA or HEMA moieties in PDMAEMA do not lead to statistically significant changes in particle size, zeta potential, DNA condensation properties and buffering capacity of the naked NPs. However, the incorporation of HEMA and HEA lead to reductions and increases, respectively, in the surface hydrophilicity of the naked NPs and NPs/DNA complexes, which was confirmed by water contact angle assay. These simple modifications of PDMAEMA with HEA and HEMA moieties significantly affect the gene transfection efficiency on HeLa cells in vitro: PCD-HEMA NP/DNA complexes show a much higher transfection efficiency than PCD NPs/DNA complexes, while PCD-HEA NPs/DNA complexes show a lower transfection efficiency than PCD NP/DNA complexes. Fluorescence activated cell sorter and confocal laser scanning microscope results indicate that the incorporation of hydrophobic HEMA moieties facilitates an enhancement in both cellular uptake and endosomal/lysosomal escape, leading to a higher transfection efficiency. Moreover, the process of endosomal/lysosomal escape confirmed in our research that PCD and its derivatives do not just rely on the proton sponge mechanism, but also on membrane damage due to the polycation chains, especially hydrophobic modified ones. Hence, it is proved that hydrophobic modification of cationic side-chains is a crucial route to improve gene transfection mediated by polycation NPs.

Sequential thermo-induced self-gelation and acid-triggered self-release process of drug-conjugated nanoparticles: a strategy for the sustained and controlled drug delivery to tumors.

A novel strategy was purposed to sustainedly deliver drug-loaded nanoparticles (NPs) to tumor sites and further enhance intracellular drug release. NPs with the ability of sequential self-gelation and self-release in response to a tumor-specific microenvironment were developed, which involved the conjugation of doxorubicin (DOX) on a thermosensitive amphiphilic copolymer (poly(ε-caprolactone)-b-poly(ethylene glycol)-b-poly(ε-caprolactone), PCEC) via acid-cleavable hydrazone linkages. The conjugate (PCEC-co-DOX) can self-assemble into micelle-like NPs in water. Moreover, the freeze-dried PCEC-co-DOX NP powder with good dispersibility in water can easily be constructed into an injectable NP aqueous dispersion at ambient temperature, making it convenient for storage and clinical applications. After injection, the dispersion can in situ thermosensitively self-gelate, anchoring large amounts of NPs at the tumor site. Subsequently, the formed NP self-supported gel can sustainedly release NPs themselves in an acidic tumor microenvironment. The released DOX-co-PCEC NPs were taken up by tumor cells and finally realized in intracellular drug release by the acid-triggered cleavage of the hydrazone bond. Compared with the repeated injection of free DOX, a single peritumoral injection of DOX-co-PCEC NP aqueous dispersion achieved a similar tumor inhibition effect but exhibited lower systemic toxicity. In vivo biodistribution studies indicated that DOX delivered by the DOX-co-PCEC NP hydrogel accumulated mainly in tumor tissue rather than in healthy tissue in mice treated with a single peritumoral injection. These results suggest that the design presented here provides a promising nanomedicine platform for cancer therapy.