Exposure to zinc oxide nanoparticles affects testicular structure, reproductive development and spermatogenesis in parental and offspring male rats.

Background: This study aimed to comprehensively evaluate the toxicity exerted by zinc oxide nanoparticles (ZnO NPs) on rat testis and its effects on fertility and progeny development. Methods: Different concentrations of ZnO NPs were administered by gavage to Sprague Dawley (SD) rats to examine the adverse effects resulting from pre- and post-natal exposure. Systemic distribution of ZnO NPs, developmental performance, sperm parameters, reproductive performance, histopathological examination, and sex hormone levels were determined scheduled in the experimental rats and their male offspring. The comparative in vitro cytotoxicity of the ZnO NPs was determined among C18-4, TM3, and TM4 cells. The toxicity exerted by ZnO NPs on germ cells in vitro and the effects on the expression of cytoskeleton and blood-testis barrier (BTB)-related proteins were also determined. Results: After oral gavage, ZnO NPs mainly accumulated in the liver and testes of rats; 350 mg/kg ZnO NPs adversely affected the epididymal weight, sperm motility, and hormone levels but did not affect the fertility of rats. In addition, 350 mg/kg ZnO NPs significantly reduced the reproductive and developmental performance of offspring male rats. Testicular histopathological and electron microscopic ultrastructure examinations showed more significant abnormal structural changes than those observed in parental rats. The results of in vitro cell experiments further showed that ZnO NPs exerted cytotoxic effects on germ cells, and led to DNA damage, nucleoskeleton and cytoskeleton alterations, and could regulate actin changes through changes in LC3B. Conclusions: It is possible that ZnO NPs act directly on TM4 cells by penetrating the BTB, causing damage to the cytoskeleton and disrupting the dynamic balance of the BTB, thereby destroying the microenvironment necessary for spermatogenesis, which may lead to poor reproduction in rats.

MDM2 knockdown mediated by a triazine-modified dendrimer in the treatment of non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is the most common type of lung cancer and the five-year survival rate is lower in advanced NSCLC patients. Chemotherapy is a widely used strategy in NSCLC treatment, but is usually limited by poor therapeutic efficacy and adverse effects. Therefore, a new therapeutic regimen is needed for NSCLC treatment. Gene therapy is a new strategy in the treatment of NSCLC. However, the lack of efficient and low toxic vectors remains the major obstacle. Here, we developed a biocompatible dendrimer as a non-viral vector for the delivery of mouse double minute2 (MDM2) siRNA in vitro and in vivo to treat NSCLC. The triazine-modified dendrimer efficiently stimulates the down-regulation of MDM2 gene in NSCLC PC9 cells, which induces significant cell apoptosis through the activation of apoptosis markers such as caspase-8 and poly(ADP-ribose) polymerase (PARP) cleavage. Furthermore, the dendrimer/MDM2 siRNA polyplexes showed excellent activity in the inhibition of tumor growth in a PC9 xenograft tumor model. These results suggested that inhibition the expression of MDM2 might be a potential target in NSCLC treatment.

Screening of efficient siRNA carriers in a library of surface-engineered dendrimers.

Polymers are widely used as non-viral carriers for siRNA delivery, but concern has also arisen in their limited efficacy and inherent toxicity. Whilst many of previous efforts have been documented towards improving the performance of polymers via chemical modifications, the structure-activity relationships (SAR) of these ligand-modified polymers are not well understood. To address this issue, we systemically prepared a library of surface-engineered dendrimers (>300) as the screening pool to discover efficient siRNA carriers. The modified ligands include alkyls and fluoroalkyls, amino acids, benzene derivatives and heterocyclic compounds. Gene silencing results showed that the lead material shows excellent efficacy even in hard-to-transfect cells such as mesenchymal stem cells. The SAR studies revealed that ligands containing appropriate hydrophobicity, or ligands with both hydrophobic and functional atoms/groups are essential for polymers to achive efficient knockdown efficacy. A second-generation library designed based on the above principles further confirms the proposed design criteria. The results enable the future rational design of potent siRNA carriers.

Clustering Small Dendrimers into Nanoaggregates for Efficient DNA and siRNA Delivery with Minimal Toxicity.

Cationic dendrimers are widely used as nonviral gene vectors, however, current gene materials based on dendrimers are either little effective or too toxic on the transfected cells. Here, a facile strategy is presented to prepare high efficient dendrimers with low transfection toxicity. Small dendrimers with 2 nm are clustered into nanoaggregates (≈100 nm) via phenylboronic acid modification and the self-assembled materials enable efficient DNA and siRNA delivery on several cell lines. The clustered nanostructures can disassemble into small dendrimers in acidic conditions thus exerting significantly less toxicity on the transfected cells. Further structure-function relationship studies reveal that both the phenyl group and boronic acid group play essential roles in the self-assembly and gene delivery processes. The transfection efficacy of phenylboronic acid-modified dendrimers can be down-regulated by blocking the boronic acid groups on dendrimers with diols or degrading the groups with hydrogen peroxide. This study provides a facile strategy in the development of efficient and biocompatible gene vectors based on low molecular weight polymers and clearly demonstrates the structure-function relationship of phenylboronic acid-modified polymers in gene delivery.

Tumor extracellular acidity activated "off-on" release of bortezomib from a biocompatible dendrimer.

A nanoparticle with a specific response to tumor extracellular acidity provides a new option in the design of tumor-targeted delivery systems. In this study, we report such a pH-responsive polymer which realizes an "off-on" release of bortezomib in tumor acidic microenvironments. A dendrimer surface is grafted with a neutral shell to reduce its cellular uptake, and its interior is functionalized with catechol moieties. An anticancer drug, bortezomib, is loaded within the dendrimer interior via a boronate-catechol interaction. The bortezomib-loaded dendrimer is non-toxic to a number of cells under physiological conditions, but kills most of the cells in slightly acidic microenvironments. In vivo studies further prove that the bortezomib-loaded dendrimer significantly inhibits tumor growth while causing minimal systemic toxicity to the animals. Since there are a number of potent anticancer drugs containing the boronate structure, the polymeric vector in this study provides a versatile scaffold to design pH-responsive drug carriers for chemotherapy.

Hydrogen-bonding dramatically modulates the gene transfection efficacy of surface-engineered dendrimers.

Dendrimers have shown great promise in the design of efficient gene vectors. However, high transfection efficacy is usually associated with serious cytotoxicity for these cationic polymers. Here, we report a facile strategy to prepare surface-engineered dendrimers with a dramatic transfection efficacy and reduced cytotoxicity. Surface-engineered dendrimers with multiple hydrogen bonding ligands such as guanamine and nucleobase derivatives show superior efficacy and low cytotoxicity on commonly used cells as well as 3D tumor spheroids to representative transfection reagents such as Lipofectamine 2000. Complementary multiple hydrogen bonding interactions between the modified ligands and DNA nucleobases play essential roles in efficient gene transfection. The hydrogen-bond modulation strategy represents a promising tool in the design of highly efficient and less cytotoxic gene materials.

Polymers modified with double-tailed fluorous compounds for efficient DNA and siRNA delivery.

Cationic polymers are widely used as gene carriers, however, these polymers are usually associated with low transfection efficacy and non-negligible toxicity. Fluorination on polymers significantly improves their performances in gene delivery, but a high density of fluorous chains must be conjugated on a single polymer. Here we present a new strategy to construct fluorinated polymers with minimal fluorous chains for efficient DNA and siRNA delivery. A double-tailed fluorous compound 2-chloro-4,6-bis[(perfluorohexyl)propyloxy]-1,3,5-triazine (CBT) was conjugated on dendrimers of different generations and low molecular weight polyethylenimine via a facile synthesis. The yielding products with average numbers of 1-2 conjugated CBT moieties showed much improved EGFP and luciferase transfection efficacy compared to unmodified polymers. In addition, these polymers show high siRNA delivery efficacy on different cell lines. Among the synthesized polymers, generation 1 (G1) dendrimer modified with an average number of 1.9 CBT moieties (G1-CBT1.9) shows the highest efficacy when delivering both DNA and siRNA and its efficacy approaches that of Lipofectamine 2000. G1-CBT1.9 also shows efficient gene silencing in vivo. All of the CBT-modified polymers exhibit minimal toxicity on the cells at their optimal transfection conditions. This study provides a new strategy to design efficient fluorous polymers for DNA and siRNA delivery.

A supramolecular approach to improve the gene transfection efficacy of dendrimers.

Cyanuric acid is able to form complementary hydrogen bonds with melamine. Here, the specific recognition between cyanuric acid and melamine is used to significantly improve the gene transfection efficacy of low generation dendrimers via a supramolecular approach.

Triazine-modified dendrimer for efficient TRAIL gene therapy in osteosarcoma.

Osteosarcoma is a high-grade malignant bone tumor that usually develops in the teenagers. Despite improvement in therapy, the five-year survival rate is poor for patients not responding to treatment or with metastases. Tumor necrosis factor (TNF) related apoptosis inducing ligand (TRAIL) gene therapy is a new strategy in the treatment of cancers, however, the lack of efficient and low toxic vectors remains the major obstacle in TRAIL gene therapy. In this study, a triazine-modified dendrimer G5-DAT66 was synthesized and used as a vector for TRAIL gene therapy in vitro and in vivo. The material shows much higher transfection efficacy on osteosarcoma MG-63 cell line than commercial transfection reagents such as Lipofectamine 2000 and SuperFect. It effectively induces apoptosis in MG-63 cells and three-dimensional MG-63 cell cultures when delivering a TRAIL plasmid. In vivo studies further prove that G5-DAT66 efficiently transfects TRAIL plasmid in tumors and inhibits tumor growth in osteosarcoma-bearing mice. These results suggest that triazine-modified dendrimer has promising potential for TRAIL gene therapy in osteosarcoma.

Evidence of guest encapsulation within G8 and G10 dendrimers using NMR techniques.

Encapsulation of guest molecules within the interior cavities of dendrimers is promising, but high generation dendrimers show limited encapsulation capacity due to their dense surface shell. Here, for the first time, we prove that high generation polyamidoamine dendrimers, such as generation 8 and generation 10, are able to encapsulate hydrophobic guests using NMR spectroscopy. Guest molecules such as phenylbutazone, dexamethasone sodium phosphate and 9-anthracenecarboxylic acid with molecular weights up to 516 Da are in close proximity to the interior scaffold protons of high generation dendrimers. This encapsulation behavior depends on guest hydrophobicity. Chemical defects and back-folding of terminal groups make it possible for these guest molecules to penetrate through the dense surface shell of high generation dendrimers. These results provide new insights into the host-guest chemistry of dendrimers.

Synergistic effect of amino acids modified on dendrimer surface in gene delivery.

Design of an efficient gene vector based on dendrimer remains a great challenge due to the presence of multiple barriers in gene delivery. Single-functionalization on dendrimer cannot overcome all the barriers. In this study, we synthesized a list of single-, dual- and triple-functionalized dendrimers with arginine, phenylalanine and histidine for gene delivery using a one-pot approach. The three amino acids play different roles in gene delivery: arginine is essential in formation of stable complexes, phenylalanine improves cellular uptake efficacy, and histidine increases pH-buffering capacity and minimizes cytotoxicity of the cationic dendrimer. A combination of these amino acids on dendrimer generates a synergistic effect in gene delivery. The dual- and triple-functionalized dendrimers show minimal cytotoxicity on the transfected NIH 3T3 cells. Using this combination strategy, we can obtain triple-functionalized dendrimers with comparable transfection efficacy to several commercial transfection reagents. Such a combination strategy should be applicable to the design of efficient and biocompatible gene vectors for gene delivery.

Poly(amidoamine) and poly(propyleneimine) dendrimers show distinct binding behaviors with sodium dodecyl sulfate: insights from SAXS and NMR analysis.

We investigate the interactions of generation 3 (G3) poly(amidoamine) (PAMAM) and G3 poly(propylenimine) (PPI) dendrimers with sodium dodecyl sulfate (SDS) in aqueous solution. Size and structure of the dendrimer-SDS aggregates as a function of SDS/dendrimer molar ratio were revealed by SAXS and NMR. G3 PAMAM has a relatively open and dense-core structure, while G3 PPI with the same number of surface amine groups possesses a compact and uniform structure. Upon addition of SDS, much more SDS monomers were encapsulated in the interior of PPI rather than in PAMAM. More significant size increase in PAMAM-SDS aggregate is observed at low SDS concentrations, due to the binding of SDS on PAMAM surface and further assembly into larger supramolecular structures. Both noncooperative and cooperative binding of SDS on G3 PPI surface are observed, while only noncooperative binding is proposed on G3 PAMAM, due to its open surface and large surface group distance. The size of the PPI-SDS complex is larger than that of PAMAM-SDS at higher SDS concentrations. Within the investigated SDS concentrations, SDS exhibits much stronger interactions with G3 PPI than with G3 PAMAM. These results provide new insights into dendrimer-surfactant interactions and explain why PPI is much more cytotoxic than PAMAM.

Surface-engineered dendrimers with a diaminododecane core achieve efficient gene transfection and low cytotoxicity.

Cationic dendrimers are widely used as gene vectors; however, these materials are usually associated with unsatisfied transfection efficiency and biocompatibility. In this study, we used an aliphatic hydrocarbon-cored polyamidoamine (PAMAM) dendrimer as an alternative to traditional cationic PAMAM dendrimers in the design of efficient gene vectors. Diaminododecane-cored generation 4 (C12G4) PAMAM dendrimer showed dramatically higher efficacy in luciferase and EGFP gene transfection than diaminoethane-cored generation 4 (C2G4) and diaminohexane-cored generation 4 (C6G4) PAMAM dendrimers. The viability of cells incubated with C12G4 at transfection concentrations is above 90%. The significantly improved gene transfection efficacy of C12G4 is attributed to the hydrophobic core of C12G4 which increases the cellular uptake of dendrimer/DNA polyplexes. Further modification of C12G4 with functional ligands such as arginine, 2,4-diamino-1,3,5-triazine, and fluorine compounds significantly increase its transfection efficiency on several cell lines. These results suggest that diaminododecane-cored dendrimers can be developed as a versatile scaffold in the design of efficient gene vectors.

Mitochondrial targeting dendrimer allows efficient and safe gene delivery.

Cationic dendrimers are promising tools in the introduction of nucleic acids into mammalian cells, however, present dendrimer-based gene vectors have modest transfection efficacy and significant toxicity. Here, we synthesized triphenylphosphonium (TPP) conjugated dendrimers and used them as polymeric gene vectors. TPP-conjugated dendrimers can effectively target mitochondria and showed efficient transfection efficacy on HeLa and COS-7 cells with low cytotoxicity on the transfected cells. These materials achieved much higher efficacy than the commercial transfection reagent SuperFect and comparable efficacy with Lipofectamine 2000. This study provides a new insight into the design of efficient and biocompatible gene vectors using dendrimers as scaffolds.

Glutathione-triggered "off-on" release of anticancer drugs from dendrimer-encapsulated gold nanoparticles.

Polymeric nanoparticles that can stably load anticancer drugs and release them in response to a specific trigger such as glutathione are of great interest in cancer therapy. In the present study, dendrimer-encapsulated gold nanoparticles (DEGNPs) were synthesized and used as carriers of thiolated anticancer drugs. Thiol-containing drugs such as captopril and 6-mercaptopurine loaded within DEGNPs showed an "Off-On" release behavior in the presence of thiol-reducing agents such as glutathione and dithiothreitol. Thiolated doxorubicin and cisplatin, loaded within the nanoparticle, showed much reduced cytotoxicity as compared to the free anticancer compounds. The toxicity of drug-loaded DEGNPs can be enhanced by improving the intracellular glutathione. Glutathione-triggered release of thiolated doxorubicin within cancer cells is further confirmed by flow cytometry and confocal laser scan microscopy studies. In addition, DEGNPs showed excellent biocompatibility on several cell lines. This study provides a new insight into biomedical applications of dendrimers and dendrimer-encapsulated nanoparticles.

Paramagnetic NMR investigation of dendrimer-based host-guest interactions.

In this study, the host-guest behavior of poly(amidoamine) (PAMAM) dendrimers bearing amine, hydroxyl, or carboxylate surface functionalities were investigated by paramagnetic NMR studies. 2,2,6,6-Tetramethylpiperidinyloxy (TEMPO) derivatives were used as paramagnetic guest molecules. The results showed that TEMPO-COOH significantly broaden the ¹H NMR peaks of amine- and hydroxyl-terminated PAMAM dendrimers. In comparison, no paramagnetic relaxation enhancement (PRE) was observed between TEMPO-NH2, TEMPO-OH and the three types of PAMAM dendrimers. The PRE phenomenon observed is correlated with the encapsulation of TEMPO-COOH within dendrimer pockets. Protonation of the tertiary amine groups within PAMAM dendrimers plays an important role during this process. Interestingly, the absence of TEMPO-COOH encapsulation within carboxylate-terminated PAMAM dendrimer is observed due to the repulsion of TEMPO-COO- anion and anionic dendrimer surface. The combination of paramagnetic probes and ¹H NMR linewidth analysis can be used as a powerful tool in the analysis of dendrimer-based host-guest systems.

Construction of a genetically engineered microorganism for phenanthrene biodegradation.

The bacterium Pseudomonas sp. CGMCC2953, isolated from oil-polluted soil, was used as a recipient for a biodegradative gene encoding catechol 2,3-dioxygenase (C23O), which was successfully cloned into the plasmid pK4 derived from pRK415 with a broad host range. The apparent phenanthrene biodegradation parameters of the recombinant microorganism (Pseudomonas sp. CGMCC2953-pK) were determined and compared with those of the wild type. As the key enzyme of phenanthrene degradation, C23O, could be expressed constitutively in the recombinant strain, Pseudomonas sp. CGMCC2953-pK showed an increased ability to degrade phenanthrene. The excessive production of C23O in Pseudomonas sp. CGMCC2953-pK could serve as an effective approach to construct genetically engineered microorganisms for the bioremediation of environmental contaminations.

Host-guest chemistry of dendrimer-cyclodextrin conjugates: selective encapsulations of guests within dendrimer or cyclodextrin cavities revealed by NOE NMR techniques.

In this study, G5 PAMAM dendrimer and α-, ß-, γ-cyclodextrin (CD) conjugates were synthesized. Host-guest behaviors of the conjugates toward five guest molecules including sodium methotrexate (MTX), amantadine hydrochloride (ADH), sulfamethoxazole (SMZ), sodium deoxycholate (SDC), and sodium dodecyl sulfate (SDS) were analyzed by NOE NMR techniques. Among the five guest molecules, ADH only binds with ß-CD in G5-ß-CD, SDC shows higher priority to localize within the cavity of γ-CD in G5-γ-CD, while MTX exhibits selective encapsulation within the cavities of G5 dendrimer in G5-α-CD. SDS has high binding affinity with α-CD in G5-α-CD but forms a precipitate in the complex solution. SMZ shows simultaneous encapsulation within CDs (α-, ß-, and γ-CD) or G5 in the presence of the three conjugates. The host behavior of G5-CD conjugates depends on CD cavity size, guest size, and hydrophobicity. The results obtained in this study are helpful in the optimization of dendrimer-CD conjugate-based drug delivery systems.