Dendrimer Nanoparticles Research Articles

Synthesis of Gold Nanoparticles with Polyamidoamine (Pamam) Generation 4 Dendrimer as Stabilizing Agent for CT Scan Contrast Agent

SummaryGold nanoparticles have great potential in biomedical applications as both a therapeutic and diagnostic agents. Pamam dendrimers are spherical, highly branched macromolecules with a narrow polydispersity to encapsulate and stabilize metal nanoparticles. In the preparation of metal nanoparticles, stabilizers play an important role in controlling particle size. The objectives of this study were to synthesize and characterize of gold nanoparticles. The gold nanoparticles were prepared by reduction of tetrachloroauric acid using sodium borohydride with and without addition of pamam generation 4 dendrimer as stabilizing agent. Gold nanoparticles stabilized pamam generation 4 dendrimer were characterized by Uv‐Vis spectrometry, transmission electron microscopy (TEM), dynamic light scattering (DLS) and zetasizer. The result showed that UV–vis absorption spectra of gold nanoparticles without pamam G4 dendrimer at 530 nm while absorption spectra of gold–pamam G4 dendrimer at 280 nm. The absorbance peak at 280 nm related to individual of gold nanoparticle encapsulated pamam dendrimer. TEM analysis exhibits the occurrence aggregation of gold nanoparticles without pamam G4 dendrimer. DLS analysis exhibit gold nanoparticles has a particles size 88‐252 nm while gold‐pamam G4 dendrimer nanoparticles 2‐15 nm. The addition of pamam generation 4 dendrimer on preparation of gold nanoparticles could prevent aggregation of nanoparticles through entrapment in cavity or core molecules. Au‐PAMAM G4 nanoparticles 0.1 mg/ml has a greater Hounsfield unit (HU) value than iodine at the same concentration, so it has better contrast properties.

Dual stimuli-sensitive dendrimers: Photothermogenic gold nanoparticle-loaded thermo-responsive elastin-mimetic dendrimers

Dendrimers are synthetic macromolecules with unique structures that can work as nanoplatforms for both photothermogenic gold nanoparticles (AuNPs) and thermosensitive elastin-like peptides (ELPs) with valine-proline-glycine-valine-glycine (VPGVG) repeats. In this study, photothermogenic AuNPs were loaded into thermo-responsive elastin-mimetic dendrimers (dendrimers conjugating ELPs at their periphery) to produce dual stimuli-sensitive nanoparticles. Polyamidoamine G4 dendrimers were modified with acetylated VPGVG and (VPGVG)2, and the resulting materials were named ELP1-den and ELP2-den, respectively. The AuNPs were prepared by the reduction of Au ions using a dendrimer-nanotemplated method. The AuNP-loaded elastin-mimetic dendrimers exhibited photothermal properties. ELP1-den and ELP2-den showed similar temperature-dependent changes in their conformations. Phase transitions were observed at around 55°C and 35°C for the AuNP-loaded ELP1-den and AuNP-loaded ELP2-den, respectively, but not for the corresponding PEGylated dendrimer. In contrast to the AuNP-loaded PEGylated dendrimer, AuNP-loaded ELP2-den readily associated with cells and induced efficient photocytotoxicity at 37°C. The cell association and the photocytotoxicity properties of AuNP-loaded ELP2-den could be controlled by temperature. These results therefore suggest that dual stimuli-sensitive dendrimer nanoparticles of this type could be used for photothermal therapy.

Abstract 13: Development of Functionalized Nanoparticle Platform for Reducing Atherosclerosis

No therapy is currently available to enhance the removal of cholesteryl esters (CE) from existing atherosclerotic plaques to facilitate plaque regression. Such a strategy is crucial to reduce the burden of existing disease in addition to preventing the progression targeted by the current therapeutics. Earlier studies from our laboratory have established the anti-atherogenic role of CE hydrolase (CEH)-mediated CE mobilization from macrophage foam cells and final elimination of cholesterol by the liver. While transgenic expression of CEH was used in pre-clinical animal studies, increase in human CEH by activation of Liver-X-receptor (LXR) was also established. Increased lipogenesis induced by LXR ligands precludes their use. The current studies focused on the development of mannose-functionalized dendrimer nanoparticles (DNPs) for the delivery of LXR ligand (TO901317) or CEH expression vector to plaque associated macrophage foam cells. As shown in the Figure, mannose functionalization restricts the uptake of DNPs to macrophages and minimal uptake was seen with primary hepatocytes ( A ). Western diet fed LDLR-/- mice were injected (iv) with DNPs and tissues harvested 48 later to monitor gene expression by QPCR. DNP-mediated delivery of LXR ligand (DNP-LXR) increased the target gene expression (ABCA1, ABCG1) in plaque associated macrophage foam cells in the aortic arch with no effects on target gene expression in the liver ( B ) demonstrating the specific delivery of LXR ligand. Comparable increase in CEH activity was seen following exposure of macrophages to free LXR ligand and DNP-delivered LXR ligand ( C ) and DNP-mediated delivery of CEH expression vectors driven either by CMV or SR-A promoter induced dramatic increase in CEH expression ( D ). These data establish functionalized DNP as a suitable platform for specific and functional delivery of drugs or DNA to plaque associated macrophages to enhance processes involved in cholesterol removal and plaque regression.

Force spectroscopy of multivalent binding of riboflavin-conjugated dendrimers to riboflavin binding protein.

Putative riboflavin receptors are considered as biomarkers due to their overexpression in breast and prostate cancers. Hence, these receptors can be potentially exploited for use in targeted drug delivery systems where dendrimer nanoparticles with multivalent ligand attachments can lead to greater specificity in cellular interactions. In this study, the single molecule force spectroscopy technique was used to assess the physical strength of multivalent interactions by employing a riboflavin (RF)-conjugated generation 5 PAMAM dendrimer G5(RF)n nanoparticle. By varying the average RF ligand valency (n = 0, 3, 5), the rupture force was measured between G5(RF)n and the riboflavin binding protein (RFBP). The rupture force increased when the valency of RF increased. We observed at the higher valency (n = 5) three binding events that increased in rupture force with increasing loading rate. Assuming a single energy barrier, the Bell-Evans model was used to determine the kinetic off-rate and barrier width for all binding interactions. The analysis of our results appears to indicate that multivalent interactions are resulting in changes to rupture force and kinetic off-rates.

Nanoparticle Interactions With Renal Epithelial Cells in vivo

Nanotechnology approaches are actively being pursued for drug delivery, novel diagnostics, implantable devices, and consumer products. While considerable research has been performed on the effects of these materials on targeted tumor or phagocytic cells, little is known about their effects on renal cells. This becomes critical for supersmall (< 10 nm) nanoparticles (NP), designed to be renally excreted. To test NPs effect on kidney function, we injected mice with either 5 nm dextran NPs (DNP) that are similar in composition to FDA approved materials or poly(amido amine) dendrimer NPs (PNP) of comparable size. These fluorophore tagged NPs were filtered and internalized by renal cells in a dose and time dependent way. The biological effects were quantitated by immunofluorescence, measuring kidney injury markers and performing functional tests. DNP administration resulted in dose dependent increases in urinary output, while cellular albumin endocytosis was increased. The expression of megalin was also increased but AQP1 expression was unaffected. The effects after PNP administration were similar but additionally resulted in increased clathrin expression, increased endocytosis and decreased AQP1 expression. We conclude that there are no major detrimental effects of DNP on overall kidney function but changes in protein expression do occur. These studies provide a framework for the testing of additional NP preparations as they become available.

Characterizing the interactions of organic nanoparticles with renal epithelial cells in vivo.

Nanotechnology approaches are actively being pursued for drug delivery, novel diagnostics, implantable devices, and consumer products. While considerable research has been performed on the effects of these materials on targeted tumor or phagocytic cells, relatively little is known about their effects on renal cells. This becomes critical for supersmall nanoparticles (<10 nm), designed to be renally excreted. The active endocytic machinery of kidney proximal tubules avidly internalizes filtered proteins, which may also be the case for filtered nanoparticles. To test whether such interactions affect kidney function, we injected mice with either 5 nm dextran-based nanoparticles (DNP) that are similar in composition to FDA-approved materials or poly(amido amine) dendrimer nanoparticles (PNP) of comparable size. These fluorescently tagged nanoparticles were both filtered and internalized by renal tubular epithelial cells in a dose- and time-dependent fashion. The biological effects were quantitated by immunocytochemistry, measuring kidney injury markers and performing functional tests. DNP administration resulted in a dose-dependent increase in urinary output, while cellular albumin endocytosis was increased. The expression of megalin, a receptor involved in albumin uptake, was also increased, but AQP1 expression was unaffected. The effects after PNP administration were similar but additionally resulted in increased clathrin expression and increased endocytosis of dextran. We conclude that there are no major detrimental renal effects of DNP on overall kidney function, but changes in endocytosis-mediating protein expression do occur. These studies provide a framework for the testing of additional nanoparticle preparations as they become available.

Cationic nanoparticles directly bind angiotensin-converting enzyme 2 and induce acute lung injury in mice.

BackgroundNanoparticles have become a key technology in multiple industries. However, there are growing reports of the toxicity of nanomaterials to humans. In particular, nanomaterials have been linked to lung diseases. The molecular mechanisms of nanoparticle toxicity are largely unexplored.MethodsAcute lung injury was induced in wild-type mice and angiotensin-coverting enzyme 2 (ACE2) knockout mice by the intratracheal instillation of cationic polyamidoamine dendrimer (PAMAM) nanoparticles. For rescue experiments, losartan (15 mg/kg in PBS) was injected intraperitoneally 30 min before nanoparticle administration.ResultsSome PAMAM nanoparticles, but not anionic PAMAM nanoparticles or carbon nanotubes, triggered acute lung failure in mice. Mechanistically, cationic nanoparticles can directly bind ACE2, decrease its activity and down-regulate its expression level in lung tissue, resulting in deregulation of the renin-angiotensin system. Gene inactivation of Ace2 can exacerbate lung injury. Importantly, the administration of losartan, which is an angiotensin II type I receptor antagonist, can ameliorate PAMAM nanoparticle-induced lung injury.ConclusionsOur data provide molecular insight into PAMAM nanoparticle-induced lung injury and suggest potential therapeutic and screening strategies to address the safety of nanomaterials.Electronic supplementary materialThe online version of this article (doi:10.1186/s12989-015-0080-x) contains supplementary material, which is available to authorized users.

Dendrimer‐Encapsulated Palladium Nanoparticles for Continuous‐Flow Suzuki–Miyaura Cross‐Coupling Reactions

AbstractGeneration three, four, and five (G3, G4, and G5) poly(amidoamine) dendrimers were used for the encapsulation of palladium nanoparticles (Pd NPs) and their covalent anchoring within glass microreactors. G3‐encapsulated Pd NPs showed the highest activity for a model Suzuki–Miyaura cross‐coupling (SMC) reaction of the three different encapsulated Pd NPs tested, as compared to G4 and G5. A kinetic study indicated a role of the nanoparticle as a procatalyst, from which molecular species are formed with an induction time of approximately 1 min. The dendrimer–nanoparticle catalytic platform exhibited excellent reactivity (high turnover frequencies and numbers) compared to other Pd NP flow reactors and dendrimer‐encapsulated Pd NPs at batch scale. Moreover, the Pd microreactor exhibited good stability, as witnessed by running the SMC reaction for more than 7 days with a low Pd leaching of 1.2 ppm. The covalently attached dendrimers may play a crucial role in stabilizing the Pd NPs, a critical feature in flow SMC reactions.

Antibacterial action mode of quaternized carboxymethyl chitosan/poly(amidoamine) dendrimer core–shell nanoparticles against Escherichia coli correlated with molecular chain conformation

The action mode of quaternized carboxymethyl chitosan/poly(amidoamine) dendrimer core-shell nanoparticles (CM-HTCC/PAMAM) against Escherichia coli (E. coli) was investigated via a combination of approaches including measurements of cell membrane integrity, outer membrane (OM) and inner membrane (IM) permeability, and scanning electron microscopy (SEM). CM-HTCC/PAMAM dendrimer nanoparticles likely acted in a sequent event-driven mechanism, beginning with the binding of positively charged groups from nanoparticle surface with negative cell surface, thereby causing the disorganization of cell membrane, and subsequent leakage of intracellular components which might ultimately lead to cell death. Moreover, the chain conformation of polymers was taken into account for a better understanding of the antibacterial action mode by means of viscosity and GPC measurements. High utilization ratio of positive charge and large specific surface area generated from a compacted conformation of CM-HTCC/PAMAM, significantly different from the extended conformation of HTCC, were proposed to be involved in the antibacterial action.

Development of poly (I:C) modified doxorubicin loaded magnetic dendrimer nanoparticles for targeted combination therapy

The objective of this study was to develop and evaluate the anticancer activity and the safety of a combinational drug delivery system using polyamidoamine (PAMAM) dendrimer-coated iron oxide nanoparticles for doxorubicin and poly I:C delivery in vitro. Dendrimer-coated magnetic nanoparticles (DcMNPs) are suitable for drug delivery system as nanocarriers with their following properties, such as surface functional groups, symmetry perfection, internal cavities, nano-size and magnetization. These nanoparticles could be targeted to the tumor site under a magnetic field since they have a magnetic core. DcMNPs were found as a convenient vehicle for targeted doxorubicin delivery in cancer therapy. Poly (I:C) binding on doxorubicin loaded DcMNPs (DcMNPs-Dox) was reported for the first time in the literature. It was also demonstrated that loading of doxorubicin into the cavities of DcMNPs increases the binding efficiency of poly (I:C) to the surface functional groups of dendrimer up to 10 times. When we compare the in vitro cytotoxic properties of doxorubicin, poly (I:C) and poly (I:C) bound doxorubicin loaded DcMNPs (PIC-DcMNPs-Dox), it was observed that PIC-DcMNPs-Dox show the highest cytotoxic effect by passing the cell resistance mechanisms on doxorubicin resistant MCF7 (MCF7/Dox) cells. Results demonstrated that applying PIC-DcMNPs-Dox would improve the efficacy by increasing the biocompatibility of system in blood stream and the toxicity inside tumor cells. These results provide invaluable information and new insight for the design and optimization of a novel combinational drug delivery system for targeted cancer therapy.

Nanoparticle toxicity assessment using an in vitro 3-D kidney organoid culture model

Nanocarriers and nanoparticles remain an intense pharmaceutical and medical imaging technology interest. Their entry into clinical use is hampered by the lack of reliable in vitro models that accurately predict in vivo toxicity. This study evaluates a 3-D kidney organoid proximal tubule culture to assess in vitro toxicity of the hydroxylated generation-5 PAMAM dendrimer (G5-OH) compared to previously published preclinical in vivo rodent nephrotoxicity data. 3-D kidney proximal tubule cultures were created using isolated murine proximal tubule fractions suspended in a biomedical grade hyaluronic acid-based hydrogel. Toxicity in these cultures to neutral G5-OH dendrimer nanoparticles and gold nanoparticles in vitro was assessed using clinical biomarker generation. Neutral PAMAM nanoparticle dendrimers elicit in vivo-relevant kidney biomarkers and cell viability in a 3-D kidney organoid culture that closely reflect toxicity markers reported in vivo in rodent nephrotoxicity models exposed to this same nanoparticle.

Pd nanoparticles in dendrimers immobilized on silica-polyamine composites as catalysts for selective hydrogenation.

New heterogeneous hydrogenation catalysts, based on Pd nanoparticles and polypropyleneimine (PPI) dendrimers of the third generation that have been covalently grafted to a silica surface modified with polyallylamine (PAA) have been synthesized. The final products were characterized by TEM, XPS, and solid-state NMR spectroscopy. The synthesized materials are effective catalysts for selective hydrogenation of dienes to monoenes and phenyl acetylene to styrene at very high substrate/Pd ratios with turnover rates higher than related Pd nanoparticle catalysts. The synthesized catalysts can be reused without any loss of activity in the case of styrene and isoprene.

Efficient delivery of connective tissue growth factor shRNA using PAMAM nanoparticles.

The aim of this study was to detect the anti-fibrosis activity of connective tissue growth factor (CTGF) small hairpin RNA (shRNA) mediated by polyamidoamine dendrimer nanoparticles in rat myocardial cell lines and myocardium. CTGF shRNAs were constructed from inverted oligonucleotides and a polyamidoamine nanoparticle vector was used to transfer shRNA into H9c2 myocardial cells and spontaneously hypertensive rats. The expression of CTGF, transforming growth factor-b1, and laminin were measured by semi-quantitative reverse transcription-polymerase chain reaction, Western blotting, and immunohistochemistry. pCTGF-shRNA significantly reduced CTGF upregulation induced by angiotensin II in H9c2 myocardial cells. The mRNA and protein expression of CTGF and laminin in pCTGF-shRNA-transferred spontaneously hypertensive rats decreased significantly compared to the control group and pHK-shRNA group (P < 0.05). The expression of transforming growth factor-b1 showed no significant difference among the 3 groups (P > 0.05). pCTGF-shRNA mediated by polyamidoamine can be used to successfully reduce myocardial CTGF and laminin expression, suggesting that this system can be used to improve myocardial fibrosis therapy.

Quantitative determination of poly(amidoamine) dendrimers in urine by liquid chromatography/electrospray ionization hybrid quadrupole linear ion trap mass spectrometry

Dendrimer nanocarriers have become of increasing interest in the field of biomedicine for their drug delivery potential. Surface modifications and optimized nanosize control are the strategies being followed to enhance drug delivery efficacy and renal clearance, especially for dendrimers of a lower generation number. The aim of this study was the development and performance evaluation of an analytical method for the quantitative determination of polyamidoamine (PAMAM) dendrimers in urine. PAMAM dendrimers (generations G0 to G3) were analyzed using liquid chromatography/electrospray ionization hybrid quadrupole linear ion trap mass spectrometry (LC/ESI-QqLIT-MS). Quantitative analysis was performed in selected reaction monitoring (SRM) mode. To confer a higher degree of confidence on the identification of PAMAM dendrimers, an SRM scan and collision-induced dissociation (CID), as a dependent scan, were performed in one single run using the information-dependent acquisition (IDA) mode. The LC/ESI-QqLIT-MS method, in SRM mode, allowed quantitative determination in urine matrix with good repeatability and reproducibility (relative standard deviation (R.S.D.) from 2 to 15%), linearity (R >0.99) over the concentration range (6∙10-4 to 5∙10-2 mmol.L-1 ), and sensitivity within the micromolar range. The detection limit values were above 1∙10-4 mmol.L-1 in both solvent and urine, for the generations studied. The developed method has demonstrated a capability for the identification and quantification of PAMAM dendrimer nanoparticles in a complex liquid matrix. The use of an LC/ESI-QqLIT-MS system, of modest m/z range and unit resolution, offers an alternative in the analysis of lower generation PAMAM dendrimers between mass analyzers of higher resolution and the conventional LC-UV method that is commonly applied for dendrimer quantification, but which lacks sufficient identification capacity. Copyright © 2013 John Wiley & Sons, Ltd.

In vitro targeting and imaging the translocator protein TSPO 18-kDa through G(4)-PAMAM–FITC labeled dendrimer

Mitochondria represent an attractive subcellular target due to its function particularly important for oxidative damage, calcium metabolism and apoptosis. However, the concept of mitochondrial targeting has been a neglected area so far. The translocator protein (TSPO) represents an interesting subcellular target not only to image disease states overexpressing this protein, but also for a selective mitochondrial drug targeting. Recently, we have delivered in vitro and in vivo small molecule imaging agents into cells overexpressing TSPO by using a family of high-affinity conjugable ligands characterized by 2-phenyl-imidazo[1,2-a]pyridine acetamide structure. As an extension, in the present work we studied the possibility to target and image TSPO with dendrimers. These nano-platforms have unique features, in fact, are prepared with a level of control not reachable with most linear polymers, leading to nearly monodisperse, globular macromolecules with a large number of peripheral groups. As a consequence, they are an ideal delivery vehicle candidate for explicit study of the effects of polymer size, charge, composition, and architecture on biologically relevant properties such as lipid bilayer interactions, cytotoxicity, cellular internalization, and subcellular compartments and organelles interactions. Here, we present the synthesis, characterization, cellular internalization, and mitochondria labeling of a TSPO targeted fourth generation [G(4)-PAMAM] dendrimer nanoparticle labeled with the organic fluorescent dye fluorescein. We comprehensively studied the cellular uptake behavior of these dendrimers, into glioma C6 cell line, under the influence of various endocytosis inhibitors. We found that TSPO targeted-G(4)-PAMAM–FITC dendrimer is quickly taken up by these cells by endocytosis pathways, and moreover specifically targets the mitochondria as evidenced from subcellular fractionation experiments and co-localization studies performed with CAT (Confocal–AFM–TIRF) microscopy.

Carboxymethyl chitosan-poly(amidoamine) dendrimer core–shell nanoparticles for intracellular lysozyme delivery

Intracellular delivery of native, active proteins is challenging due to the fragility of most proteins. Herein, a novel polymer/protein polyion complex (PIC) nanoparticle with core–shell structure was prepared. Carboxymethyl chitosan-grafted-terminal carboxyl group-poly(amidoamine) (CM-chitosan-PAMAM) dendrimers were synthesized by amidation and saponification reactions. 1H NMR was used to characterize CM-chitosan-PAMAM dendrimers. The TEM images and results of lysozyme loading efficiency indicated that CM-chitosan-PAMAM dendrimers could self-assemble into core–shell nanoparticles, and lysozyme was efficiently encapsulated inside the core of CM-chitosan-PAMAM dendrimer nanoparticles. Activity of lysozyme was completely inhibited by CM-chitosan-PAMAM Dendrimers at physiological pH, whereas it was released into the medium and exhibited a significant enzymatic activity in an acidic intracellular environment. Moreover, the CM-chitosan-PAMAM dendrimer nanoparticles did not exhibit significant cytotoxicity in the range of concentrations below 3.16mg/ml. The results indicated that these CM-chitosan-PAMAM dendrimers have excellent properties as highly potent and non-toxic intracellular protein carriers, which would create opportunities for novel applications in protein delivery.

A dramatically enhanced electrochemiluminescence assay for CA125 based on dendrimer multiply labeled luminol on Fe3O4 nanoparticles

A novel electrochemiluminescence immunosensor for carbohydrate antigen 125 detection based on dendrimer and magnetic nanoparticles were developed in this study. In the assay, carbohydrate antigen 125 was used as a model analyte, and the detection was based on a sandwich structure formed by the first antibody, the carbohydrate antigen 125 and a second antibody labeled with luminol through poly(diethylenetriaminepentaacetic acid-ethylene glycol) ester dendrimer. The sandwich system was then modified on Fe3O4 magnetic nanoparticles and immobilized on a magnetic force-controlled carbon paste electrode with the help of a magnet to form the immunosensor. The result showed that the ECL immunosensor was ultrasensitive to carbohydrate antigen 125 detection at a concentration of 0.2–100μU/mL with a detection limit of 0.032μU/mL. The signal was enhanced dramatically than that produced by luminol labeled only, so was the sensitivity of the immunosensor, partly because much more probe molecules could be immobilized on the electrode surface. This also proved that dendrimers could be a useful material for the fabrication of ultrasensitive sensors. The proposed immunosensor was successfully applied to detect CA125 in real human plasma samples, which was compared with the result of conventional ELISA methods.

In vitro evaluation of the cytotoxicity and cellular uptake of CMCht/PAMAM dendrimer nanoparticles by glioblastoma cell models

Glioblastoma (GBM) is simultaneously the most common and most malignant subtype tumor of the central nervous system. These are particularly dramatic diseases ranking first among all human tumor types for tumor-related average years of life lost and for which curative therapies are not available. Recently, the use of nanoparticles as drug delivery systems (DDS) for tumor treatment has gained particular interest. In an attempt to evaluate the potential of carboxymethylchitosan/poly(amidoamine) (CMCht/PAMAM) dendrimer nanoparticles as a DDS, we aimed to evaluate its cytotoxicity and internalization efficiency in GBM cell models. CMCht/PAMAM-mediated cytotoxicity was evaluated in a GBM cell line (U87MG) and in human immortalized astrocytes (hTERT/E6/E7) by MTS and double-stranded DNA quantification. CMCht/PAMAM internalization was assessed by double fluorescence staining. Both cells lines present similar internalization kinetics when exposed to a high dose (400 μg/mL) of these nanoparticles. However, the internalization rate was higher in tumor GBM cells as compared to immortalized astrocytes when cells were exposed to lower doses (200 μg/mL) of CMCht/PAMAM for short periods (<24 h). After 48 h of exposure, both cell lines present ~100 % of internalization efficiency for the tested concentrations. Importantly, short-term exposures (1, 6, 12, 24, and 48 h) did not show cytotoxicity, and long-term exposures (7 days) to CMCht/PAMAM induced only low levels of cytotoxicity in both cell lines (~20 % of decrease in metabolic activity). The high efficiency and rate of internalization of CMCht/PAMAM we show here suggest that these nanoparticles may be an attractive DDS for brain tumor treatment in the future.

Efficacy and safety of dendrimer nanoparticles with coexpression of tumor necrosis factor-&amp;alpha; and herpes simplex virus thymidine kinase in gene radiotherapy of the human uveal melanoma OCM-1 cell line

Human uveal melanoma is the most common primary intraocular tumor, and brachytherapy is one of the most common and effective treatment strategies. In order to find a safer and more effective way to increase the radio sensitivity of the tumor, we tried to use the dendrimer nanoparticle performing coexpression gene radiotherapy. In this study, we constructed recombinant DNA plasmids (early growth response-1 tumor necrosis factor-α [pEgr1-TNFα], pEgr1 thymidine kinase [TK], and pEgr1-TNFα-TK) according to the Egr1 promoter sequence. The sequences of human TNFα and herpes simplex virus (HSV) TK that were published by GenBank. Agarose gel electrophoresis and DNA sequencing had proven that we constructed the double-gene recombined plasmids pEgr1-TNF-TK correctly, as well as the plasmids pEgr1-TNFα and pEgr1-TK. The dendrimer nanoparticles combined with plasmid DNA as dendriplexes were verified with agarose gel electrophoresis and observed by transmission electron microscopy (TEM) and scanning electron microscopy to define size and shape. Zeta potential was measured using a Zetasizer analyzer. Optimal size and neutral zeta-potential characteristics of dendriplexes were achieved for the transfection studies. DNase I examination proved that the dendriplexes could protect plasmid DNA for at least 6 hours. The recombinant plasmids were transfected with dendrimer nanoparticles into the human choroidal melanoma OCM-1 cell line, followed by exposure to iodine-125 (125I) after transfection. After transfection with dendrimer nanoparticles and the irradiation of 125I, the gene expressions of TNFα and HSV1-TK were significantly increased at the protein level by enzyme-linked immunosorbent assay and Western blot analysis in OCM-1 cells. The cellular morphology of OCM-1 cells altering was observed by TEM, and a decrease in cell proliferation was revealed in cell-growth curves. Flow cytometry of annexin V/propidium iodide double-dyeing apoptosis and caspase-3 fluorescence staining showed that this treatment method could turn transfected OCM-1 cells into apoptosis and necrosis by the effects of the gene expression. This study indicated that the dendrimer nanoparticles with coexpression of TNF-α and HSV1-TK gene therapy are effective and safe and can provide us with a novel strategy to treat human uveal melanoma in the future.

Microglia Response and In Vivo Therapeutic Potential of Methylprednisolone‐Loaded Dendrimer Nanoparticles in Spinal Cord Injury

The control and manipulation of cells that trigger secondary mechanisms following spinal cord injury (SCI) is one of the first opportunities to minimize its highly detrimental outcomes. Herein, the ability of surface-engineered carboxymethylchitosan/polyamidoamine (CMCht/PAMAM) dendrimer nanoparticles to intracellularly deliver methylprednisolone (MP) to glial cells, allowing a controlled and sustained release of this corticosteroid in the injury site, is investigated. The negatively charged MP-loaded CMCht/PAMAM dendrimer nanoparticles with sizes of 109 nm enable a MP sustained release, which is detected for a period of 14 days by HPLC. In vitro studies in glial primary cultures show that incubation with 200 μg mL(-1) nanoparticles do not affect the cells' viability or proliferation, while allowing the entire population to internalize the nanoparticles. At higher concentrations, microglial cell viability is proven to be affected in response to the MP amount released. Following lateral hemisection lesions in rats, nanoparticle uptake by the spinal tissue is observed 3 h after administration. Moreover, significant differences in the locomotor output between the controls and the MP-loaded nanoparticle-treated animals one month after the lesion are observed. Therefore, MP-loaded CMCht/PAMAM dendrimer nanoparticles may prove to be useful in the reduction of the secondary injury following SCI.