[Analysis and Prevention of Gene Mutation Types of Severe Thala- ssemia in Hakka People in Gannan of Jiangxi Province].

OBJECTIVE: To investigate the types and proportion of gene mutations of thalassemia in Hakka people in Gannan Area of Jiangxi, and to provide some references for prevention and treatment of thalassemia major, genetic counseling and epidemiological studies. METHODS: 81 cases Hakka patients with severe thalassemia admitted treated in First Affiliated Hospital of Gannan Medical College from January 2009 to June 2019 were enrolled. The deletion type of α-thalassemia was detected by Gap-PCR. The point mutations of α-thalassemia and ß-thalassemia were detected by PCR-RDB. The thalassemia gene was detected and analyzed in the patients with anemia, and the frequency of gene mutation was calculated. RESULTS: Among 81 Hakka patients with thalassemia major, 4 ß-thalassemia (homozygote) genotypes were detected out, including: CD41-42（TTCT）(19 cases), ß-IVS-II-654 (C→T) (9 cases), -28M (A→G) (1 case), CD17 (A→T) (1 case); 12 ß-thalassemithalassemia (heterozygote) genotypes were detected out, including: CD41-42(-TTCT)/ß-IVS-II-654(C→T) (15 cases, 29.41%), ß-IVS-II-654(C→T)/ß-28M(A→G) (13 cases，25.49%) ； CD41-42(-TTCT)/ß-28M(A→G) (9 cases，17.65%); ß-IVS-II-654(C→T) /CD27/28(+C) (3 cases， 5.88%) ； CD41-42(-TTCT)/CD27/28(+C)(3 case，5.88%)；ß-28M(A→G)/CD17(A→T) (2 cases，3.92%)；CD41-42(-TTCT)/CD17(A→T), CD41-42(-TTCT)/Βe, ß-IVS-II-654(C→T)/ß-29、ßCD17（A→T）/CD71-72(+a), ßCD71-72/ß-28M(A→G), ß-28M(A→G) /ß-IVS-II-654(C→T)(1 cases，1.96%). There were 3 cases of ß homozygous thalassemia with α-thalassemia gene and 5 cases of ß heterozygotes thalassemia with α-thalassemia gene. CONCLUSION: The incidence rate of thalassemia in Hakka people in Gannan Area of Jiangxi is relatively high. The distribution of gene mutation types is as follows: the genotype of CD41-42 (-TTCT) is the main genotype of ß-thalassemia (homozygous); the major genotypes of ß- thalassemia (heterozygotes) are CD41-42 (-TTCT)/ß-IVS-II-654 (C→T) and ß-IVS-II-654 (C→T) /ß-28M (A→G); CD41-42 (-TTCT) gene is dominant in ß-complex α-thalassemia.

Structure-directing star-shaped block copolymers: supramolecular vesicles for the delivery of anticancer drugs.

Amphiphilic polycarbonate/PEG copolymer with a star-like architecture was designed to facilitate a unique supramolecular transformation of micelles to vesicles in aqueous solution for the efficient delivery of anticancer drugs. The star-shaped amphipilic block copolymer was synthesized by initiating the ring-opening polymerization of trimethylene carbonate (TMC) from methyl cholate through a combination of metal-free organo-catalytic living ring-opening polymerization and post-polymerization chain-end derivatization strategies. Subsequently, the self-assembly of the star-like polymer in aqueous solution into nanosized vesicles for anti-cancer drug delivery was studied. DOX was physically encapsulated into vesicles by dialysis and drug loading level was significant (22.5% in weight) for DOX. Importantly, DOX-loaded nanoparticles self-assembled from the star-like copolymer exhibited greater kinetic stability and higher DOX loading capacity than micelles prepared from cholesterol-initiated diblock analogue. The advantageous disparity is believed to be due to the transformation of micelles (diblock copolymer) to vesicles (star-like block copolymer) that possess greater core space for drug loading as well as the ability of such supramolecular structures to encapsulate DOX. DOX-loaded vesicles effectively inhibited the proliferation of 4T1, MDA-MB-231 and BT-474 cells, with IC50 values of 10, 1.5 and 1.0mg/L, respectively. DOX-loaded vesicles injected into 4T1 tumor-bearing mice exhibited enhanced accumulation in tumor tissue due to the enhanced permeation and retention (EPR) effect. Importantly, DOX-loaded vesicles demonstrated greater tumor growth inhibition than free DOX without causing significant body weight loss or cardiotoxicity. The unique ability of the star-like copolymer emanating from the methyl cholate core provided the requisite modification in the block copolymer interfacial curvature to generate vesicles of high loading capacity for DOX with significant kinetic stability that have potential for use as an anti-cancer drug delivery carrier for cancer therapy.

Effects of manganese deficiency on chondrocyte development in tibia growth plate of Arbor Acres chicks.

The aim of this study was to investigate the effects of manganese (Mn) deficiency on chondrocyte development in tibia growth plate. Ninety 1-day-old Arbor Acres chicks were randomly divided into three groups and fed on control diet (60 mg Mn/kg diet) and manganese deficient diets (40 mg Mn/kg diet, manganese deficiency group I; 8.7 mg Mn/kg diet, manganese deficiency group II), respectively. The width of the proliferative zone of growth plate was measured by the microscope graticule. Chondrocyte apoptosis was estimated by TUNEL staining. Gene expression of p21 and Bcl-2, and expression of related proteins were analyzed by quantitative real time reverse transcription polymerase chain reaction and immunohistochemistry, respectively. Compared with the control group, manganese deficiency significantly decreased the proliferative zone width and Bcl-2 mRNA expression level, while significantly increased the apoptotic rates and the expression level of p21 gene in chondrocytes. The results indicate that manganese deficiency had a negative effect on chondrocyte development, which was mediated by the inhibition of chondrocyte proliferation and promotion of chondrocyte apoptosis.

Overcoming multidrug resistance in microbials using nanostructures self-assembled from cationic bent-core oligomers.

Novel cationic molecules based on rigid terephthalamide-bisurea cores flanked by imidazolium moieties are described. In aqueous media, these compounds self-assemble into supramolecular nanostructures with distinct morphologies. The compound with optimal hydrophilic/hydrophobic balance displays potent antimicrobial activity and high selectivity towards clinically-isolated MRSA without inducing drug-resistance. These self-assembled cationic antimicrobial nanostructures show promise for the prevention and treatment of multidrug-resistant infections.

Antimicrobial and antifouling hydrogels formed in situ from polycarbonate and poly(ethylene glycol) via Michael addition.

A novel class of antimicrobial cationic polycarbonate/PEG hydrogels are designed and synthesized by Michael addition chemistry. These hydrogels demonstrate strong broad-spectrum antimicrobial activities against various clinically isolated multidrug-resistant microbes. Moreover, they exhibit nonfouling properties and prevent the substrate from microbial adhesion. These antimicrobial and antifouling gels are promising materials as catheter coatings and wound dressings to prevent infections.

Oligomerized alpha-helical KALA peptides with pendant arms bearing cell-adhesion, DNA-binding and endosome-buffering domains as efficient gene transfection vectors.

In this study, a number of KALA-based α-helical peptides were designed and synthesized as non-viral gene carriers. The effects of lysine and histidine residues in the pendant arms and cell-binding RGD motif on DNA binding, particle size, zeta potential, cytotoxicity and gene expression efficiency were first explored. Increasing the lysine and histidine residues reduced particle size and increased zeta potential of DNA complexes, leading to greater gene expression efficiency. In addition, the introduction of RGD group further improved gene expression level. The peptide with optimal compositions, RGDN(3)K(6)H(3)CKHLAKALAKALAC (RC29), was then oligomerized to form di-, tri- and tetra-RC29 via disulfide linkage. Upon oligomerization, RC29 attained a 3-dimensional long α-helical structure with pendant arm(s) extending transversally outwards. Each arm contains a cell-adhesion motif (RGD), DNA-binding and endosome-buffering domains(.) The α-helicity of the oligomerized peptides was evaluated by circular dichroism (CD) spectroscopy, which showed that an increased oligomerization degree led to a stronger α-helical structure. These peptides form complexes with DNA efficiently. The minimum size and maximum zeta potential of tri-RC29/DNA complexes was about 200 nm and 32.5 mV, respectively. In comparison, RC29 formed DNA complexes with a similar zeta potential, but particle size was significantly larger (355 nm). DNA complexes formed at pH 7.0 yielded higher gene expressions than those formed at pH 5.5 and 6.5. Among all the oligomerized peptides, tri-RC29 provided the highest gene expression efficiency, and its peak luciferase level was 1.5 times higher than that yielded by PEI at its optimal N/P ratio (i.e. 10). Moreover, oligomerized RC29/DNA complexes were less cytotoxic than PEI/DNA complexes. These α-helical peptides can be promising carrier for delivery of therapeutic genes in the treatment of genetic disorders.

Biomimetic hydrogels for chondrogenic differentiation of human mesenchymal stem cells to neocartilage.

In this study, a collagen mimetic peptide (CMP) containing a GFOGER sequence flanked by GPO repeat units (sequence: (GPO)(4)GFOGER(GPO)(4)GCG, CMP) was synthesized and chemically incorporated into a poly(ethylene glycol) (PEG) hydrogel through Michael addition chemistry. The PEG/collagen mimetic peptide hybrid hydrogel was used as a scaffold for encapsulation, proliferation and differentiation of human mesenchymal stem cells (hMSCs) into neocartilage/chondrocytes. Biophysical studies indicated that this peptide adopts stable triple helical conformation under simulated physiological conditions. Tetra hydroxyl PEG was functionalized to generate an acrylate group and reacted with the peptide, and hydrogels were formed in situ with the addition of cells and tetra sulfhydryl PEG via Michael addition. The effect of CMP on proliferation and chondrogenesis of hMSCs was investigated. The results demonstrated that PEG-CMP hydrogels provided a natural environment, which promoted chondrogenesis of hMSCs and enhanced secretion of cartilage specific ECM as compared to PEG hydrogels without the peptide. This was attributed to enhanced cell/matrix interactions via integrin beta1/GFOGER interactions. Further, chondrogenesis was found to be affected by matrix elasticity. Soft matrix induced a greater degree of chondrogenic differentiation; however, stiff matrix had an opposite effect, inhibiting chondrogenic differentiation probably due to limited mass transport. This soft PEG/CMP hydrogel shows promise as a biomimetic scaffold that provides a desirable environment for the chondrogenic differentiation of hMSCs and is useful for the repair of cartilage defects.

Injectable Biodegradable Poly(ethylene glycol)/RGD Peptide Hybrid Hydrogels for in vitro Chondrogenesis of Human Mesenchymal Stem Cells.

In this study, an injectable and biodegradable poly(ethylene glycol) (PEG)/arginine-glycine-aspartic (RGD) peptide hybrid hydrogel has been synthesized and used as a biomimetic scaffold for encapsulation of human mesenchymal stem cells (hMSCs). Tetrahydroxyl PEG was functionalized with acrylate, and then reacted with thiol-containing peptide (RGD). Gelation occurred within 30 min with the addition of cells and PEG-dithiol via Michael addition. The hydrogels synthesized with a peptide concentration of 1.0-5.0 mM achieved significantly greater cell viability when compared to the hydrogels without the RGD peptide. However, the effect of RGD on chondrogenesis was found to be dose-dependent. Immunohistology studies demonstrated that hMSCs encapsulated in the hydrogel matrix with 1.0 mM RGD and TGF-ß3 showed enhanced positive staining for aggrecan and type II collagen as compared to that with 5.0 mM RGD and unmodified PEG hydrogels. RT-PCR results further revealed that the cells in hydrogels with 1 mM RGD expressed significantly higher levels of type II collagen than those in PEG hydogels without RGD peptide. These findings have demonstrated that the PEG-RGD hydrogels can be a promising scaffold to deliver hMSCs for cartilage repair.

Biodegradable poly(ethylene glycol)-peptide hydrogels with well-defined structure and properties for cell delivery.

In this study, biodegradable PEG-peptide hydrogels have been synthesized using Click chemistry. A series of Arg-Gly-Asp (RGD) containing peptides were prepared via a solid phase synthesis approach, which were further functionalized with azide to yield peptide azide or peptide diazide. A tetra-hydroxy terminated 4-arm PEG was functionalized with acetylene and was reacted with peptide azide/diazide and/or PEG diazide to produce hydrogels via a copper mediated 1,3-cycloaddition (Click chemistry) generating a triazole linkage as the networking forming reaction. The gelation time ranged from 2 to 30 min, depending on temperature, catalyst and precursor concentration, as well as peptide structure. The resulting hydrogels were characterized by swelling, viscoelastic properties and morphology as well as their ability for cell attachment and proliferation. Hydrogels cross-linked by peptide diazide yielded higher storage modulus (G') with shorter spacers between azide groups. As expected, the swelling degree decreased while the G' increased with increasing the concentration of the precursors as a result of increased cross-linking density. Primary human dermal fibroblasts were used as model cells to explore the possibility of using the RGD peptide hydrogels for cell-based wound healing. The attachment and proliferation of the cells on the hydrogels were evaluated. The RGD peptide hydrogels synthesized with a peptide concentration of 2.7-5.4mm achieved significantly improved cell attachment and greater cell proliferation rate when compared to the hydrogels without RGD peptides. These hydrogels may provide a platform technology to deliver cells for tissue repair.

The self-assembly of biodegradable cationic polymer micelles as vectors for gene transfection.

Cationic micelles self-assembled from a biodegradable amphiphilic copolymer, poly{(N-methyldietheneamine sebacate)-co-[(cholesteryl oxocarbonylamido ethyl) methyl bis(ethylene) ammonium bromide] sebacate} (P(MDS-co-CES)) have recently been reported for efficient gene delivery and co-delivery of drug and nucleic acid. In this study, poly(ethylene glycol) (PEG) of various molecular weights (Mn=550, 1100 and 2000) was conjugated to P(MDS-co-CES) having different cholesterol grafting degrees to improve the stability of micelle/DNA complexes in the blood for systemic in vivo gene delivery. DNA binding ability, gene transfection efficiency and cytotoxicity of P(MDS-co-CES), PMDS, PEGylated PMDS and PEGylated P(MDS-co-CES) micelles were studied and compared. As with P(MDS-co-CES), PEG-P(MDS-co-CES) polymers could also self-assemble into stable micelles of small size. However, PMDS and PEG-PMDS without cholesterol could not form stable micelles but formed large particles. PEGylation of polymers significantly decreased their gene transfection efficiency in HEK293, HepG2, HeLa, MDA-MB-231 and 4T1 cells. However, increasing N/P ratio promoted gene transfection. An increased cholesterol grafting degree led to greater gene expression level possibly because of the more stable core-shell structure of the micelles. PEG550-P(MDS-co-CES) micelles induced high gene transfection level, comparable to that provided by P(MDS-co-CES) micelles. PEGylated polymers were much less cytotoxic than P(MDS-co-CES). PEGylated P(MDS-co-CES) micelles may provide a promising non-viral vector for systemic in vivo gene delivery.

Bio-functional micelles self-assembled from a folate-conjugated block copolymer for targeted intracellular delivery of anticancer drugs.

In this study, a block copolymer, poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide-co-2-aminoethyl methacrylate)-b-poly(10-undecenoic acid) (P(NIPAAm-co-DMAAm-co-AMA)-b-PUA) was synthesized, and folic acid was conjugated to the hydrophilic block through the amine group in AMA. This polymer was self-assembled into micelles, which exhibited pH-induced temperature sensitivity. They were smaller in size, and possessed a better-defined core-shell structure as well as more stable hydrophobic core than the random copolymer P(NIPAAm-co-DMAAm-co-UA), and provided a shell with folate molecules. An anti-cancer drug, doxorubicin (DOX) was encapsulated into the micelles. The mean diameter of the blank and DOX-loaded micelles was less than 100 nm. DOX release was pH-dependent, being faster at low pH (endosomes/lysosomes). Therefore, DOX was readily released from the micelles into the nucleus after being taken up. More importantly, IC50 of DOX-loaded micelles with folate against folate receptor-expressing 4T1 and KB cells was much lower than that of the DOX-loaded micelles without folate (3.8 vs. 7.6 mg/L for 4T1 cells and 1.2 vs. 3.0mg/L for KB cells). In vivo experiments conducted in a 4T1 mouse breast cancer model demonstrated that DOX-loaded micelles had a longer blood circulation time than free DOX (t(1/2): 30 min and 140 min, respectively). In addition, the micelles delivered an increased amount of DOX to the tumor when compared to free DOX. These bio-functional micelles may make a promising carrier to transport anticancer drugs specifically to tumor cells and release the drug molecules inside the cells to the cytosols for improved chemotherapy.

[Synthesis and antitumor activity of selenophosphocholine analogues containing tegafur].

AIM: To synthesize the selenophosphocholine analogues containing tegafur and test their antitumor activities. METHODS: The cyclic glyceroselenophospholopid conjugate of tegafur was synthesized by the reaction of hexaethylphosphorous triamide with N1-(2-furanidyl)-N3-(hydroxyalkyl)-5-fluyorouracil and 1-O-hexadecyl glycerol as well as selenium in one-pot. Cyclic glyceroselenophospholopid conjugate of tegafur reacted with triethylamine to give title compounds. RESULTS: Six new compounds have been synthesized. Their structures were confirmed by 1H NMR, 13P NMR and elemental analysis. Antitumor activity of the title compounds against PGA1 was tested. CONCLUSION: The reaction of triethylamine with cyclic glyceroselenophospholopid conjugate of tegafur very readily occurred, which was finished within 2 h at room temperature. The opening-ring products of trans isomers showed antimutor activity against human uriaryl bladder cancer cell more effective than that of the tegafur.

Temperature- and pH-sensitive core-shell nanoparticles self-assembled from poly(n-isopropylacrylamide-co-acrylic acid-co-cholesteryl acrylate) for intracellular delivery of anticancer drugs.

Temperature- and pH-sensitive amphiphilic polymer poly(N-isopropylacrylamide-co-acrylic acid-co-cholesteryl acrylate) (P(NIPAAm-co-AA-co-CHA)) has been synthesized and employed to encapsulate paclitaxel, a highly hydrophobic anticancer drug, in core-shell nanoparticles fabricated by a membrane dialysis method. The nanoparticles are spherical in shape, and their size can be made below 200 nm by varying fabrication parameters. The lower critical solution temperature (LCST) of the nanoparticles is pH-dependent. Under the normal physiological condition (pH 7.4), the LCST is well above the normal body temperature (37 degrees C) but it is below 37 degrees C in an acidic environment (e.g. inside the endosome or lysosome). The critical association concentration of the polymer is determined to be 7 mg/L. Paclitaxel can be easily encapsulated into the nanoparticles. Its encapsulation efficiency is affected by fabrication temperature, initial drug loading and polymer concentration. In vitro release of paclitaxel from the nanoparticles is responsive to external pH changes, which is faster in a lower pH environment. Cytotoxicity of paclitaxel-loaded nanoparticles against MDA-MB-435S human breast carcinoma cells is slightly higher than that of free paclitaxel. In addition, doxorubicin is used as a probe to study cellular uptake using a confocal laser scanning microscope (CLSM). Doxorubicin molecules are able to enter the cytoplasm after escaping from the endosome and/or the lysosome. The temperature- and pH-sensitive nanoparticles would make a promising carrier for intracellular delivery of anticancer drugs.

Evaluating proteins release from, and their interactions with, thermosensitive poly (N-isopropylacrylamide) hydrogels.

Poly (N-isopropylacrylamide) (PNIPAAm) hydrogels possess a lower critical solution temperature (LCST) at around 32 degrees C. When the external temperature is raised above the LCST, the hydrogels experience abrupt and drastic shrinkage. This unique property makes them very useful for biomedical applications such as on-off switches for modulated drug delivery and tissue engineering. The aim of this work was to study the potential of using PNIPAAm hydrogels for protein delivery, and to obtain basic understandings of the protein-gel interactions as well as their effect on protein loading and release. PNIPAAm gels were synthesized with different crosslinker contents. The effects of crosslinker content, in vitro release temperature, protein loading level and molecular size as well as temperature cycling on protein release from PNIPAAm gels were examined. Greater amount of BSA was loaded using gels fabricated with lower crosslinker contents and loading solution with higher concentrations of BSA. An incomplete release of encapsulated BSA from the gels was observed in all cases. Enhanced mass transfer created by oscillating swelling-deswelling in response to temperature cycling across the LCST and lowering in vitro release temperature did not promote BSA release because of strong BSA-gel interactions. Evidence for the residual BSA in the gels after in vitro release was provided by dyeing the gels with protein determination reagent and shift in the LCST of the gels. In contrast, insulin release was much faster and more complete when compared to BSA because of its smaller size. The protein-gel interactions were analysed by investigating the LCST of, and state of water in, the blank and protein-loaded hydrogels.

Preparation and characterization of temperature-sensitive poly(N-isopropylacrylamide)-b-poly(D,L-lactide) microspheres for protein delivery.

Temperature-sensitive diblock copolymers, poly(N-isopropylacrylamide)-b-poly(D,L-lactide) (PNIPAAm-b-PLA) with different PNIPAAm contents were synthesized and utilized to fabricate microspheres containing bovine serum albumin (BSA, as a model protein) by a water-in-oil-in-water double emulsion solvent evaporation process. XPS analysis showed that PNIPAAm was a dominant component of the microspheres surface. BSA was well entrapped within the microspheres, and more than 90% encapsulation efficiency was achieved. The in vitro degradation behavior of microspheres was investigated using SEM, NMR, FTIR, and GPC. It was found that the microspheres were erodible, and polymer degradation occurred in the PLA block. Degradation of PLA was completed after 5 months incubation in PBS (pH 7.4) at 37 degrees C. A PVA concentration of 0.2% (w/v) in the internal aqueous phase yielded the microspheres with an interconnected porous structure, resulting in fast matrix erosion and sustained BSA release. However, 0.05% PVA produced the microspheres with a multivesicular internal structure wrapped with a dense skin layer, resulting in lower erosion rate and a biphasic release pattern of BSA that was characterized with an initial burst followed by a nonrelease phase. The microspheres made from PNIPAAm-b-PLA with a higher portion of PNIPAAm provided faster BSA release. In addition, BSA release from the microspheres responded to the external temperature changes. BSA release was slower at 37 degrees C (above the LCST) than at a temperature below the LCST. The microspheres fabricated with PNIPAAm-b-PLA having a 1:5 molar ratio of PNIPAAm to PLA and 0.2% (w/v) PVA in the internal aqueous phase provided a sustained release of BSA over 3 weeks in PBS (pH 7.4) at 37 degrees C.