Topology optimization of embracing fixator considering bone remodeling to mitigate stress shielding effect.

The embracing fixator is one of the widely used internal fixation implants for bone fracture treatment. However, the stress shielding effect, a stress imbalance between the implant and bone caused by the mismatch in mechanical properties between them, is a significant and critical issue that may lead to treatment failure. Thus, it is of great importance to design the implant with an appropriate stiffness which can mitigate the stress shielding effect and provide the most favorable mechanical environment for bone healing and remodeling. To this end, a time-dependent topology optimization algorithm considering bone remodeling is proposed to optimize an embracing fixator used in the tibia fracture treatment. The change of callus density over time is simulated based on a bone remodeling model, and the callus density after a period of bone remodeling is selected to be the design objective to maximize. The design constraints include volume and the compliance of the whole fixation system. Meanwhile, the influence of the constraints on the regularity of material distribution of the optimized result is also studied. Besides, to test the effectiveness of the consideration of the bone remodeling in the embracing fixator design, a topology optimization concerning the minimization of the compliance of the entire system is also performed to make a comparison. Finally, the safety performance of optimized results considering bone remodeling is also verified by static analysis.

TLR8 activation and inhibition by guanosine analogs in RNA: Importance of functional groups and chain length.

Toll-like receptor 8 (TLR8) is an important component of the human innate immune system that recognizes single stranded RNA (ssRNA). Recent X-ray crystal structures of TLR8 bound to ssRNA revealed a previously unrecognized binding site for a 5'-UpG-3' dinucleotide. Here we use an atomic mutagenesis strategy coupled with a cellular TLR8 activation assay to probe the importance of specific functional groups present on the guanine base in RNA-mediated receptor agonism and antagonism. Results from RNA analogs containing 7-deazaguanosine, 2-aminopurine and inosine confirm the importance of guanine N7, O6 and N2, respectively, in TLR8 activation. Nevertheless, these RNAs each retained TLR8 antagonism activity. RNA containing 7-deaza-8-azainosine (7d8aI) was prepared from a novel phosphoramidite and found to be a weaker TLR8 activator than guanosine-containing RNA. However, 7d8aI-containing RNA also retained TLR8 antagonism activity indicating that removal of multiple TLR8 H-bonding sites on guanine is insufficient for blocking TLR8 antagonism by guanine-containing RNA. We also identified an oligoribonucleotide length dependence on both TLR8 activation and antagonism. These studies extend our understanding of the effects of nucleobase modification on immune stimulation and will inform the design of novel RNA-based therapeutics.

Guide Strand 3'-End Modifications Regulate siRNA Specificity.

Short interfering RNA (siRNA)-triggered gene knockdown through the RNA interference (RNAi) pathway is widely used to study gene function, and siRNA-based therapeutics are in development. However, as the guide strand of an siRNA can function like a natural microRNA (miRNA), siRNAs often repress hundreds of off-target transcripts with complementarity only to the seed region (nucleotides 2-8) of the guide strand. Here, we describe novel guide strand 3'-end modifications derived from 1-ethynylribose (1-ER) and copper-catalyzed azide-alkyne cycloaddition reactions and evaluate their impact on target versus miRNA-like off-target knockdown. Surprisingly, when positioned at the guide strand 3'-end, the parent 1-ER modification substantially reduced off-target knockdown while having no measurable effect on on-target knockdown potency. In addition, these modifications were shown to modulate siRNA affinity for the hAgo2 PAZ domain. However, the change in PAZ domain binding affinity was not sufficient to predict the modification's effect on miRNA-like off targeting.

Water soluble octa-functionalized POSS: all-click chemistry synthesis and efficient host-guest encapsulation.

A series of water soluble octa-functionalized POSSs were facilely synthesized via thiol-ene and Menschutkin click chemistry. Among them, octa-alkynyl POSS further reacted with azide-terminal alkyl long chains, resulting in a well-defined, amphiphilic octopus-like POSS. For the first time it was used for host-guest encapsulation and it exhibited an ultrahigh loading capability.

[Synthesis of a supermolecular nanoparticle γ-hy-PC/Ada-Dox and its antitumor activity].

OBJECTIVE: To synthesize a (2-Hydroxypropyl)-γ-cyclodextrin-polyethylenimine/adamantane-conjugated doxorubicin (γ-hy-PC/Ada-Dox) based supramolecular nanoparticle with host-guest interaction and to identify its physicochemical characterizations and antitumor effect. METHODS: A novel non-viral gene delivery vector γ-hy-PC/Ada-Dox was synthesized based on host-guest interaction. 1H-NMR, NOESY, UV-Vis, XRD and TGA were used to confirm the structure of the vector. The DNA condensing ability of complexes was investigated by particle size, zeta potential and gel retardation assay. Cytotoxicity of complexes was determined by MTT assay in BEL-7402 and SMMC-7721 cells. Cell wound healing assay was performed in HEK293 and BEL-7404 cells. The transfection efficiency was investigated in HEK293 cells. H/E staining and cell uptake assay was performed in BEL-7402 cells. RESULTS: The structure of γ-hy-PC/Ada-Dox was characterized by 1H-NMR, NOESY, UV-Vis, XRD, TGA. The drug loading was 0.5% and 5.5%. Gel retardation assay showed that γ-hy-PC was able to completely condense DNA at N/P ratio of 2; 0.5% and 5.5% γ-hy-PC/Ada-Dox was able to completely condense DNA at N/P ratio of 3 and 4,respectively. The cytotoxicity of polymers was lower than that of PEI25KDa. The transfection efficiency of γ-hy-PC was higher than that of γ-hy-PC/Ada-Dox at N/P ratio of 30 in HEK293 cells; and the transfection efficiency was decreasing when Ada-Dox loading was increasing. Cell uptake assay showed that γ-hy-PC/Ada-Dox was able to carry drug and FAM-siRNA into cells. CONCLUSION: The novel vector γ-hy-PC/Ada-Dox has been developed successfully, which has certain transfection efficiency and antitumor activity.

[Anticancer effect of triptolide-polyethylenimine-cyclodextrin in vitro].

OBJECTIVE: To develop a drug delivery system triptolide-polyethylenimine-cyclodextrin and to evaluate its anticancer activity in vitro. METHODS: Triptolide was conjugated to polyethylenimine-cyclodextrin by N, N'-carbonyldiimidazole to form triptolide-polyethylenimine-cyclodextrin. (1)H-NMR, FT-IR and XRD were used to confirm its structure. The anticancer effect of the polymer was assessed by MTT assay, erasion trace test and hematoxylin-eosin staining. The potential to condense siRNA and to delivery siRNA into cytoplasm was demonstrated by gel retardation assay, zeta-potential determination and fluorescence staining. RESULTS: Triptolide was successfully conjugated to polyethylenimine-cyclodextrin and the conjugation rate of triptolide was 10% (w/w). siRNA was effectively condensed by the polymer at the N/P ratio of 5, and its particle size was 300 ±15 nm and zeta potential was 8 ±2.5 mV. MTT assay, erasion trace test and hematoxylin-eosin staining revealed that triptolide-polyethylenimine-cyclodextrin had anticancer effect and low cytotoxicity to normal cells. The polymer was able to deliver siRNA to the cytoplasm effectively as demonstrated by fluorescence staining. CONCLUSION: Triptolide-polyethylenimine-cyclodextrin is able to inhibit the growth and migration of cancer cells in vitro and to carry siRNA into cells effectively. It is potential to be used as a novel prodrug for co-delivery of gene and drug in cancer treatment.

[Characteristics of cationic polymers PEI-CyD, PEI-PHPA, PEE-PHPA and PEI25kD in vitro and in vivo].

OBJECTIVE: To study the characteristics of cationic polymers polyethylenimine-ß-cyclodextrin (PEI-CyD), polyethylenimine-poly-(3-hydroxypropyl)-aspartamide (PEI-PHPA), N,N-Dimethyldipropylenetriamine-Bis(3-aminopropyl)amine-aspartamide (PEE-PHPA) in vitro and in vivo. METHODS: PEI-PHPA, PEI-CyD and PEE-PHPA were synthesized and the chemistry structure of PEI-PHPA, PEI-CyD and PEE-PHPA was confirmed by (1)H-NMR. The particle size and zeta potential of these polymers were measured, and capacity of plasmid DNA condensation was tested. The inhibition of COS-7, A549, HEK293 and C6 cells was measured by MTT assay. The transfection efficiency was determined in HEK293 cell lines. The toxicity, tissue distribution and transfection efficiency of cationic polymers were tested in vivo. RESULTS: When the N/P of polymers/DNA at 30, the particle sizes were close 250 nm and the zeta-potential were near 35 mv. They were able to condense DNA at N/P ratio < 5. The MTT assay showed that the IC(50) of PEE-PHPA was 21.5, 20.2, 7.30 and 37.1 µg/ml, and that of PEI25kD was 15.8, 18.3, 11.4 and 36.7 µg/ml in C6, COS-7, A549 and HEK293cell lines, respectively. The cell viability of PEI-CyD and PEI-PHPA in above cell lines was over 60%. They had high transfection efficiency in HEK293 cell lines. The LD(50) of PEI25Kd, PEI-CyD, PEI-PHPA and PEE-PHPA in vivo was 19.50, 100.4, 521.2 and 630.0, respectively by intraperitoneal (ip) injection. The contractions of these polymers were higher in kidney than in other organs and tissues.PEE-PHPA had slight effect on kidney and liver function. CONCLUSION: PEE and PEI25kD have higher transfection efficiency and higher toxicity; while PC and PHPA-PEI have lower toxicity and higher transfection efficiency to be used as non-viral gene vector.

[Modified montmorillonite as multifunction gene and drug delivery system].

OBJECTIVE: To develop polyethylenimine-Doxorubicin-montmorillonite (PEI-Dox-MTT) as a novel multifunction delivery system. METHODS: Dox was intercalated into montmorillonite, PEI covered to the surface of Dox/MMT to make the nano-particle. XRD, FT-IR and TGA were used to confirm chemical property of the nano-particle. SEM was used to observe the morphology. The capability of drug release was investigated by PBS buffer solution (pH 7.4). The DNA binding ability of nano-particle was detected by gel electrophoresis retardation assay. The cell viability in COS-7 and SKOV3 cell lines was tested using MTT assay. The gastric mucosa protection was evaluated in vitro. RESULTS: XRD image showed that Dox was intercalated into montmorillonite, inter space of which increased to 31.3Å; the FT-IR spectra showed the vibration bands of PEI at 1 560 cm(-1) and 2 850 cm(-1), the vibration band of Dox at 1 350 cm(-1). Size analysis and SEM revealed that the size of nano-particle was 600 nm, and the zeta-potential was 30 mV. Drug release experiment explored that the nano-particle stably released drug in range of 6 X10(-4) ≊ 8 X10(-4) mg/ml within 72 h. MTT assay showed that the cell viability was over 80% in experiment condition in COS-7 and SKOV3 cell lines. 0.3 mg PEI-MMT nano-particle was able to protect gastric mucosa from alcohol. CONCLUSION: Multifunction system of PEI/Dox/MMT has been prepared successfully.