S, N-doped carbon dots-based cisplatin delivery system in adenocarcinoma cells: Spectroscopical and computational approach.

S and N-doped carbon dots (S-CDs and N-CDs) and their cisplatin (cis-Pt) derivatives. (S-CDs@cis-Pt and N-CDs@cis-Pt) were tested on two ovarian cancer cell lines: A2780 and A2780 cells resistant to cis-Pt (A2780R). Several spectroscopic techniques were employed to check S-CDs@cis-Pt and N-CDs@cis-Pt: solid- and solution-state nuclear magnetic resonance, matrix-assisted laser desorption, ionization time-of-flight mass spectrometry, and X-ray photoelectron spectroscopy. In addition, synchrotron-based Fourier Transformed Infrared spectro-microscopy was used to evaluate the biochemical changes in cells after treatment with cis-Pt, S-CDs, N-CDs, or S-CDs@cis-Pt and N-CDs@cis-Pt, respectively. Computational chemistry was applied to establish the model for the most stable bond between S-CDs and N-CDs and cis-Pt. The results revealed the successful modification of S-CDs and N-CDs with cis-Pt and the formation of a stable composite system that can be used for drug delivery to cancer cells and likewise to overcome acquired cis-Pt resistance. Nanoparticle treatment of A2780 and A2780R cells led to the changes in their structure of lipids, proteins, and nucleic acids depending on the treatment. The results showed the S-CDs@cis-Pt and N-CDs@cis-Pt might be used in the combination with cis-Pt to treat the adenocarcinoma, thus having a potential to be further developed as drug delivery systems.

Morpholino-functionalized phosphorus dendrimers for precision regenerative medicine: osteogenic differentiation of mesenchymal stem cells.

A novel bioactive macromolecule based on morpholino-functionalized phosphorus dendrimers (generation 2, G2-Mor+) was developed for osteogenic differentiation of mesenchymal stem cells (MSCs). Interestingly, through in vitro tests, it was shown that G2-Mor+ dendrimer can strongly promote the transformation of MSCs into osteoblasts, which implies the potential application of phosphorus dendrimers in bone regeneration for precision regenerative medicine.

Zwitterion-functionalized dendrimer-entrapped gold nanoparticles for serum-enhanced gene delivery to inhibit cancer cell metastasis.

We demonstrate a novel serum-enhanced gene delivery approach using zwitterion-functionalized dendrimer-entrapped gold nanoparticles (Au DENPs) as a non-viral vector for inhibition of cancer cell metastasis in vitro. Poly(amidoamine) dendrimers of generation 5 decorated with zwitterion carboxybetaine acrylamide (CBAA) and lysosome-targeting agent morpholine (Mor) were utilized to entrap gold NPs. We show that both Mor-modified and Mor-free Au DENPs are cytocompatible and can effectively deliver plasmid DNA encoding different reporter genes to cancer cells in medium with or without serum. Strikingly, due to the antifouling property exerted by the attached zwitterion CBAA, the gene delivery efficiency of Mor-modified Au DENPs and the Mor-free Au DENPs in the serum-containing medium are 1.4 and 1.7 times higher than the corresponding vector in serum-free medium, respectively. In addition, the Mor-free vector has a better gene expression efficiency than the Mor-modified one although the Mor modification enables the polyplexes to have enhanced cancer cell uptake. Wound healing and hypermethylated in cancer 1 (HIC1) protein expression assay data reveal that the expression of HIC1 gene in cancer cells enables effective inhibition of cell migration. Our findings suggest that the created zwitterion-functionalized Au DENPs may be employed as a powerful vector for serum-enhanced gene therapy of different diseases. STATEMENT OF SIGNIFICANCE: One major challenge in the non-viral gene delivery system is that the strong interaction between serum protein and the positively charged vector/gene polyplexes neutralize the positive charge of the polyplexes and form possible protein corona, thereby significantly reducing their cellular uptake efficiency and subsequent gene transfection outcome. Here we demonstrate the conceptual advances in the serum-enhanced gene delivery using zwitterionic modification of polycationic poly(amidoamine) (PAMAM) dendrimer-entrapped gold nanoparticles (Au DENPs). We demonstrate that partial zwitterionic modification of Au DENPs is able to confer them with antifouling property to resist serum protein adsorption. Hence the vector/DNA polyplexes are able to maintain their positive potentials and small hydrodynamic size in the serum environment, where serum solely play the role as a nutrition factor for enhanced gene delivery. We demonstrate that partial modification of zwitterion carboxybetaine acrylamide (CBAA) and morpholine (Mor) onto the surface Au DENPs renders the vector with both antifouling property and lysosome targeting ability, respectively. The generated functional Au DENPs can compact pDNA to form polyplexes that enable serum-enhanced gene expression. In particular, once complexed with hypermethylated in cancer 1 (HIC1) gene, the polyplexes can significantly inhibit cancer cell migration and metastasis.

Synthesis, Pharmacological, and Biological Evaluation of MIF-1 Picolinoyl Peptidomimetics as Positive Allosteric Modulators of D2R.

This work describes the synthesis and pharmacological evaluation of picolinoyl-based peptidomimetics of melanocyte stimulating hormone release inhibiting factor 1 (MIF-1) as dopamine modulating agents. Eight novel peptidomimetics were tested for their ability to enhance the maximal effect of tritiated N-propylapomorphine ([3H]-NPA) at dopamine D2 receptors (D2R). Methyl picolinoyl-l-valyl-l-alaninate (compound 6b) produced a statistically significant increase in the maximal [3H]-NPA response at 0.01 nM (11.9 ± 3.7%), which is close to the effect of MIF-1 in this assay at same concentration (18.3 ± 9.1%). Functional assays measuring cAMP mobilization in the presence of dopamine corroborate the activity of peptidomimetic 6b as a positive allosteric modulator (PAM) of D2R. In this assay, 6b produced a typical bell-shaped dose-response curve similar to that of the parent neuropeptide (18.3 ± 7.1% for 6b vs 15.4 ± 5.5% for MIF-1, both at 0.1 nM). Dose-response curves for dopamine in the presence of 6b show EC50 (0.33 ± 0.21 µM for 6b vs 0.17 ± 0.07 µM for MIF-1) and Emax (86.0 ± 5.4% for 6b vs 93.6 ± 4.4% for MIF-1) comparable to those of MIF-1, both at 0.01 nM. Furthermore, peptidomimetic 6b was tested for agonist activity at the human D2R and the results show that it displays no intrinsic agonism effect, endorsing its activity as a PAM of D2R. Cytotoxic and neurotoxic assays were performed for peptidomimetic 6b using HEK 293T cells and cortex neurons from 19 day old Wistar-Kyoto rat embryos, respectively, suggesting this analogue displays no toxicity effect in these assays up to 100 µM. Conformational energy minimization for 6b shows that this peptidomimetic cannot adopt the postulated type-II ß-turn bioactive conformation, endorsing the possibility of an extended bioactive conformation as claimed by other researchers as a second bioactive conformation of MIF-1. Overall, the pharmacological and toxicological profile of peptidomimetic 6b together with its favorable druglike properties and structural simplicity makes it a potential lead compound for further development and optimization.

Multifunctional Dendrimer-Entrapped Gold Nanoparticles Conjugated with Doxorubicin for pH-Responsive Drug Delivery and Targeted Computed Tomography Imaging.

Novel theranostic nanocarriers exhibit a desirable potential to treat diseases based on their ability to achieve targeted therapy while allowing for real-time imaging of the disease site. Development of such theranostic platforms is still quite challenging. Herein, we present the construction of multifunctional dendrimer-based theranostic nanosystem to achieve cancer cell chemotherapy and computed tomography (CT) imaging with targeting specificity. Doxorubicin (DOX), a model anticancer drug, was first covalently linked onto the partially acetylated poly(amidoamine) dendrimers of generation 5 (G5) prefunctionalized with folic acid (FA) through acid-sensitive cis-aconityl linkage to form G5·NHAc-FA-DOX conjugates, which were then entrapped with gold (Au) nanoparticles (NPs) to create dendrimer-entrapped Au NPs (Au DENPs). We demonstrate that the prepared DOX-Au DENPs possess an Au core size of 2.8 nm, have 9.0 DOX moieties conjugated onto each dendrimer, and are colloid stable under different conditions. The formed DOX-Au DENPs exhibit a pH-responsive release profile of DOX because of the cis-aconityl linkage, having a faster DOX release rate under a slightly acidic pH condition than under a physiological pH. Importantly, because of the coexistence of targeting ligand FA and Au core NPs as a CT imaging agent, the multifunctional DOX-loaded Au DENPs afford specific chemotherapy and CT imaging of FA receptor-overexpressing cancer cells. The constructed DOX-conjugated Au DENPs hold a promising potential to be utilized for simultaneous chemotherapy and CT imaging of various types of cancer cells.

Laponite®: A key nanoplatform for biomedical applications?

Laponite® is a synthetic smectite clay that already has many important technological applications, which go beyond the conventional uses of clays in pharmaceutics and cosmetics. In biomedical applications, particularly in nanomedicine, this material holds great potential. Laponite® is a 2-dimensional (2D) nanomaterial composed of disk-shaped nanoscale crystals that have a high aspect ratio. These disks can strongly interact with many types of chemical entities (from small molecules or ions, to natural or synthetic polymers, to different inorganic nanoparticles) and are also easily functionalized and readily degraded in the physiological environment giving rise to non-toxic and even bioactive products. This review will highlight the potential of Laponite® as a nanomaterial in the fields of drug delivery, bioimaging, tissue engineering and regenerative medicine. New concepts, as well as novel innovative materials that stand out from the usual ones due to the unique properties of Laponite®, will also be presented and discussed.

Efficient delivery of therapeutic siRNA into glioblastoma cells using multifunctional dendrimer-entrapped gold nanoparticles.

AIM: To synthesize the arginine-glycine-aspartic (RGD) functionalized dendrimer-entrapped gold nanoparticles (Au DENPs) for siRNA delivery to induce gene silencing of cancer cells in vitro and in vivo. MATERIALS & METHODS: Au DENPs modified with RGD peptide via a polyethylene glycol spacer were used as a vector of two distinct small interfering RNAs (siRNAs) (VEGFvascular endothelial growth factor siRNA and B-cell lymphoma/leukemia-2 siRNA), and the physicochemical properties, cytocompatibility and transfection efficiency of Au DENP/siRNA polyplexes were characterized. RESULTS: The Au DENP/siRNA polyplexes with good cytocompatibility and highly efficient transfection capacity can be used for the transfection of siRNAs. CONCLUSION: The developed functional RGD-modified Au DENPs may be used for efficient gene therapy of different types of cancer.

Mechanistic Studies of Enhanced PCR Using PEGylated PEI-Entrapped Gold Nanoparticles.

The polymerase chain reaction (PCR) is considered an excellent technique and is widely used in both molecular biology research and various clinical applications. However, the presence of byproducts and low output are limitations generally associated with this technique. Recently, the use of nanoparticles (NPs) has been shown to be very effective at enhancing PCR. Although mechanisms underlying this process have been suggested, most of them are mainly based on PCR results under certain situations without abundant systematic experimental strategy. In order to overcome these challenges, we synthesized a series of polyethylene glycol (PEG)-modified polyethylenimine (PEI)-entrapped gold nanoparticles (PEG-Au PENPs), each having different gold contents. The role of the synthesized NPs in improving the PCR technique was then systematically evaluated using the error-prone two-round PCR and GC-rich PCR (74% GC content). Our results suggest a possible mechanism of PCR enhancement. In the error-prone two-round PCR system, the improvement of the specificity and efficiency of the technique using the PEG-Au PENPs mainly depends on surface-charge-mediated electrostatic interactions. In the GC-rich PCR system, thermal conduction may be the dominant factor. These important findings offer a breakthrough in understanding the mechanisms involved in improving PCR amplification, as well as in the application of nanomaterials in different fields, particularly in biology and medicine.

MMP2-Targeting and Redox-Responsive PEGylated Chlorin e6 Nanoparticles for Cancer Near-Infrared Imaging and Photodynamic Therapy.

A unique matrix metalloproteinase 2-targeted photosensitizer delivery platform was developed in this study for tumor-targeting imaging and photodynamic therapy. The model photosensitizer therapeutic agent chlorin e6 (Ce6) was first covalently conjugated with matrix metalloproteinase 2-cleavable polypeptide and then modified with polyethylene glycol via a redox-responsive cleavable disulfide linker. The resultant matrix metalloproteinase 2-cleavable polypeptide modified PEGylated Ce6 (PEG-SS-Ce6-MMP2) nanoparticles, which formed via self-assembly, were observed to be monodisperse and significantly stable in aqueous solution. In addition, owing to their cellular redox-responsiveness at the cleavable disulfide linker, the PEG-SS-Ce6-MMP2 nanoparticles were able to release Ce6 rapidly. Despite displaying enhanced intracellular internalization, the synthesized PEG-SS-Ce6-MMP2 nanoparticles did not compromise their phototoxic effects toward A549 cancer cells when compared with free Ce6 and PEGylated Ce6 nanoparticles. In vivo experiments further revealed that, in contrast with the free Ce6 or with the PEGylated Ce6 nanoparticles, the PEG-SS-Ce6-MMP2 nanoparticles showed a remarkable increase in tumor-targeting ability and a significantly improved photodynamic therapeutic efficiency in A549 tumor-bearing mice. These results suggest that the PEG-SS-Ce6-MMP2 nanoparticles hold great potential for tumor-targeting imaging and photodynamic therapy.

RGD peptide-modified dendrimer-entrapped gold nanoparticles enable highly efficient and specific gene delivery to stem cells.

We report the use of arginine-glycine-aspartic (Arg-Gly-Asp, RGD) peptide-modified dendrimer-entrapped gold nanoparticles (Au DENPs) for highly efficient and specific gene delivery to stem cells. In this study, generation 5 poly(amidoamine) dendrimers modified with RGD via a poly(ethylene glycol) (PEG) spacer and with PEG monomethyl ether were used as templates to entrap gold nanoparticles (AuNPs). The native and the RGD-modified PEGylated dendrimers and the respective well characterized Au DENPs were used as vectors to transfect human mesenchymal stem cells (hMSCs) with plasmid DNA (pDNA) carrying both the enhanced green fluorescent protein and the luciferase (pEGFPLuc) reporter genes, as well as pDNA encoding the human bone morphogenetic protein-2 (hBMP-2) gene. We show that all vectors are capable of transfecting the hMSCs with both pDNAs. Gene transfection using pEGFPLuc was demonstrated by quantitative Luc activity assay and qualitative evaluation by fluorescence microscopy. For the transfection with hBMP-2, the gene delivery efficiency was evaluated by monitoring the hBMP-2 concentration and the level of osteogenic differentiation of the hMSCs via alkaline phosphatase activity, osteocalcin secretion, calcium deposition, and von Kossa staining assays. Our results reveal that the stem cell gene delivery efficiency is largely dependent on the composition and the surface functionality of the dendrimer-based vectors. The coexistence of RGD and AuNPs rendered the designed dendrimeric vector with specific stem cell binding ability likely via binding of integrin receptor on the cell surface and improved three-dimensional conformation of dendrimers, which is beneficial for highly efficient and specific stem cell gene delivery applications.

RGD peptide-modified multifunctional dendrimer platform for drug encapsulation and targeted inhibition of cancer cells.

Development of multifunctional nanoscale drug-delivery systems for targeted cancer therapy still remains a great challenge. Here, we report the synthesis of cyclic arginine-glycine-aspartic acid (RGD) peptide-conjugated generation 5 (G5) poly(amidoamine) dendrimers for anticancer drug encapsulation and targeted therapy of cancer cells overexpressing αvß3 integrins. In this study, amine-terminated G5 dendrimers were used as a platform to be sequentially modified with fluorescein isothiocyanate (FI) via a thiourea linkage and RGD peptide via a polyethylene glycol (PEG) spacer, followed by acetylation of the remaining dendrimer terminal amines. The developed multifunctional dendrimer platform (G5.NHAc-FI-PEG-RGD) was then used to encapsulate an anticancer drug doxorubicin (DOX). We show that approximately six DOX molecules are able to be encapsulated within each dendrimer platform. The formed complexes are water-soluble, stable, and able to release DOX in a sustained manner. One- and two-dimensional NMR techniques were applied to investigate the interaction between dendrimers and DOX, and the impact of the environmental pH on the release rate of DOX from the dendrimer/DOX complexes was also explored. Furthermore, cell biological studies demonstrate that the encapsulation of DOX within the G5.NHAc-FI-PEG-RGD dendrimers does not compromise the anticancer activity of DOX and that the therapeutic efficacy of the dendrimer/DOX complexes is solely related to the encapsulated DOX drug. Importantly, thanks to the role played by RGD-mediated targeting, the developed dendrimer/drug complexes are able to specifically target αvß3 integrin-overexpressing cancer cells and display specific therapeutic efficacy to the target cells. The developed RGD peptide-targeted multifunctional dendrimers may thus be used as a versatile platform for targeted therapy of different types of αvß3 integrin-overexpressing cancer cells.

Interaction of antimicrobial peptides, BP100 and pepR, with model membrane systems as explored by Brownian dynamics simulations on a coarse-grained model.

This work focuses on the conformational and dynamic properties of the antimicrobial peptides (AMPs), BP100 and pepR, when confined within model membrane systems. Brownian dynamics (BD) simulations of a coarse-grained model of each respective peptide in an environment reproducing the phospholipid bilayer were carried out. Simple mean-field potentials were used to reproduce three physically different model phosphatidylcholine (PC) membrane systems. Based on the simplicity of the peptide-membrane models used, 1 micros simulations were performed. With the appropriate choice of parameters, the structure and dynamics of each peptide were recovered from each of the simulated BD trajectories. BP100 was observed to adopt a alpha-helical conformation when confined in each PC membrane. For pepR under the same conditions, the formation of an N-terminal alpha-helix was detected, whereas the C-terminus appeared to be less ordered. The dynamic properties of each peptide were characterized in terms of local and global motions. BP100 tended to localize with no preferred orientation approximately halfway across each membrane leaflet, whereas pepR localized near the membrane core with no preferred orientation. Overall, the peptide dynamics were found to vary according to the size of the peptide, as well as the width of the membrane environment.

Escherichia coli cell surface perturbation and disruption induced by antimicrobial peptides BP100 and pepR.

The potential of antimicrobial peptides (AMPs) as an alternative to conventional therapies is well recognized. Insights into the biological and biophysical properties of AMPs are thus key to understanding their mode of action. In this study, the mechanisms adopted by two AMPs in disrupting the gram-negative Escherichia coli bacterial envelope were explored. BP100 is a short cecropin A-melittin hybrid peptide known to inhibit the growth of phytopathogenic gram-negative bacteria. pepR, on the other hand, is a novel AMP derived from the dengue virus capsid protein. Both BP100 and pepR were found to inhibit the growth of E. coli at micromolar concentrations. Zeta potential measurements of E. coli incubated with increasing peptide concentrations allowed for the establishment of a correlation between the minimal inhibitory concentration (MIC) of each AMP and membrane surface charge neutralization. While a neutralization-mediated killing mechanism adopted by either AMP is not necessarily implied, the hypothesis that surface neutralization occurs close to MIC values was confirmed. Atomic force microscopy (AFM) was then employed to visualize the structural effect of the interaction of each AMP with the E. coli cell envelope. At their MICs, BP100 and pepR progressively destroyed the bacterial envelope, with extensive damage already occurring 2 h after peptide addition to the bacteria. A similar effect was observed for each AMP in the concentration-dependent studies. At peptide concentrations below MIC values, only minor disruptions of the bacterial surface occurred.

The intersubunit lock-and-key motif in human glutathione transferase A1-1: role of the key residues Met51 and Phe52 in function and dimer stability.

The dimeric structure of certain cytosolic GSTs (glutathione S-transferases) is stabilized by a hydrophobic lock-and-key motif at their subunit interface. In hGSTA1-1 (human class Alpha GST with two type-1 subunits), the key consists of two residues, Met51 and Phe52, that fit into a hydrophobic cavity (lock) in the adjacent subunit. SEC (size-exclusion chromatography)-HPLC, far-UV CD and tryptophan fluorescence of the M51A and M51A/F52S mutants indicated the non-disruptive nature of these mutations on the global structure. While the M51A mutant retained 80% of wild-type activity, the activity of the M51A/F52S was markedly diminished, indicating the importance of Phe52 in maintaining the correct conformation at the active site. The M51A and M51A/F52S mutations altered the binding of ANS (8-anilinonaphthalene-l-sulphonic acid) at the H-site by destabilizing helix 9 in the C-terminal region. Data from urea unfolding studies show that the dimer is destabilized by both mutations and that the dimer dissociates to aggregation-prone monomers at low urea concentrations before global unfolding. Although not essential for the assembly of the dimeric structure of hGSTA1-1, both Met51 and Phe52 in the intersubunit lock-and-key motif play important structural roles in maintaining the catalytic and ligandin functions and stability of the GST dimer.