G4 PAMAM Dendrimers Research Articles

Poly(amidoamine) Dendrimers as Nanocarriers for 5-Fluorouracil: Effectiveness of Complex Formation and Cytotoxicity Studies.

Two generations of positively charged poly(amidoamine) dendrimers (PAMAMs) were selected for study as potential carriers for the anticancer drug 5-fluorouracil (5FU), a drug primarily used in the treatment of colorectal cancer. Analytical techniques, such as UV-Vis spectrophotometry, NMR Spectroscopy and Laser Doppler Velocimetry (LDV), have shown that the most critical factor determining the formation of a PAMAM–5FU complex is the starting components’ protonation degree. The tests confirmed the system’s ability to attach about 20 5FU molecules per one dendrimer molecule for the G4PAMAM dendrimer and about 25 molecules for the G6PAMAM dendrimer, which gives a system yield of 16% for the fourth generation and 5% for sixth generation dendrimers. Additionally, using the QCM-D method, the adsorption efficiency and the number of drug molecules immobilized in the dendrimer structure were determined. A new aspect in our study was the determination of the change in zeta potential (ζ) induced by the immobilization of 5FU molecules on the dendrimer’s outer shell and the importance of this effect in the direct contact of the carrier with cells. Cytotoxicity tests (resazurin reduction and MTS tests) showed no toxicity of dendrimers against mouse fibroblast cells (L929) and a significant decrease in cell viability in the case of four human malignant cell lines: malignant melanoma (A375), glioblastoma (SNB-19), prostate cancer (Du-145) and colon adenocarcinoma (HT-29) during incubation with PAMAM–5FU complexes. The purpose of our work was to investigate the correlation between the physicochemical properties of the carrier and active substance and the system efficiency and optimizing conditions for the formation of an efficient system based on PAMAM dendrimers as nanocarriers for 5-fluorouracil. An additional aspect was to identify potential application properties of the complexes, as demonstrated by cytotoxicity tests.

Facile Formation of PAMAM Dendrimer Nanoclusters for Enhanced Gene Delivery and Cancer Gene Therapy.

Preparation of versatile and safe nanovectors for efficient cancer gene therapy remains to be challenging in the current nanomedicine. Herein, we report the formation of dendrimer nanoclusters for enhanced gene delivery toward gene therapy of cancer. Here, poly(amidoamine) (PAMAM) dendrimers of generation 3 (G3) were cross-linked with 4,4'-dithiodibutryic acid (DA) to form nanoclusters (NCs) through 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride-induced covalent bonding. The synthesized G3-DA NCs having a hydrodynamic size of 219.3 nm possess good colloidal stability and can condense pDNA, encoding both enhanced green fluorescent protein and tumor suppressor p53 gene to form polyplexes with good cytocompatibility. Strikingly, the created NCs/pDNA polyplexes enable 2.3 and 2.1 times higher gene transfection to cancer cells than the counterpart materials of single G3 and G5 PAMAM dendrimers, respectively, under the same conditions. Furthermore, polyplex-treated cancer cells have upregulated p53 and p21 protein and mRNA expression levels and downregulated Cyclin-D1 and CDK-4 protein and mRNA expressions, thus arresting the cell cycle to the G1 phase in vitro to achieve cancer cell gene therapy. The gene delivery efficiency of the polyplexes was further validated through the in vivo tumor therapy without systemic toxicity. The synthesized highly efficient dendrimer NC-based vector system with low cytotoxicity may be extended to tackle various types of diseases related to genetic disorders.

Supramolecular assembly of rifampicin and PEGylated PAMAM dendrimer as a novel conjugate for tuberculosis

IntroductionThe study aimed to develop and explore the efficiency of a surface-modified 4.0 G PAMAM dendrimer as a novel delivery system for the anti-tuberculosis (TB) drug, rifampicin. MethodsThe 4.0 G PAMAM dendrimer having various concentrations of polyethylene glycol (PEG) 2 kDa content was synthesized using 4-nitrophenyl chloroformate as an activator, and characterized using Fourier-transform infrared (FTIR) and proton nuclear magnetic resonance (1H NMR) analysis. Thereafter, rifampicin was loaded into dendrimers via simple dissolution solvent evaporation method, and characterized using different analytical techniques. The polymer encapsulation efficiency (EE%) was determined directly using a validated HPLC method. In vitro drug release was studied at pH 7.4. The MTT technique was used to assess the cytotoxicity of the dendrimer formulations against raw 264.7 cell lines. ResultsThe percentage coverage of 4.0 G PAMAM dendrimer peripheral with PEG was achieved in a range of 38%–100%. The EE% of the native dendrimer was 7.5% (w/w), and for PEGylated dendrimers was >60% (w/w). The dynamic light scattering (DLS) verified the nanosize of dendrimer formulations, and the zeta potential was positive with values greater than 12 mV. Scanning electron microscopy (SEM) images of dendrimer confirmed the spherical or semi-spherical shape of the nanoparticles, and differential scanning calorimetry (DSC) data verified drug entrapment. The PEGylated dendrimers showed a slower release rate compared to the native/unmodified formulation and free drug. The toxicity of dendrimer was significantly improved after dendrimer PEGylation, and negligible toxicity was detected for formulations with 100% functionalization. ConclusionsThe developed PEGylated G4 PAMAM dendrimers are suggested as a suitable drug carrier with negligible cytotoxicity, high loading potential, and prolonged-release behaviour for rifampicin.

Fluorescence Spectroscopy of Porphyrins and Phthalocyanines: Some Insights into Supramolecular Self-Assembly, Microencapsulation, and Imaging Microscopy.

The molecular interactions of anionic tetrasulfonate phenyl porphyrin (TPPS) with poly(amido amine) (PAMAM) dendrimers of generation 2.0 and 4.0 (G2 and G4, respectively) forming H- or J-aggregates, as well as with human and bovine serum albumin proteins (HSA and BSA), were reviewed in the context of self-assembly molecular complementarity. The spectroscopic studies were extended to the association of aluminum phthtalocyanine (AlPCS4) detected with a PAMAM G4 dendrimer with fluorescence studies in both steady state and dynamic state, as well as due to the fluorescence quenching associated to electron-transfer with a distribution of lifetimes. The functionalization of TPPS with peripheral substituents enables the assignment of spontaneous pH-induced aggregates with different and well-defined morphologies. Other work reported in the literature, in particular with soft self-assembly materials, fall in the same area with particular interest for the environment. The microencapsulation of TPPS studies into polyelectrolyte capsules was developed quite recently and aroused much interest, which is well supported and complemented by the extensive data reported on the Imaging Microscopy section of the Luminescence of Porphyrins and Phthalocyanines included in the present review.

Core-Shell Tecto Dendrimers Enable Enhanced Tumor MR Imaging through an Amplified EPR Effect.

Development of nanoplatforms that can amplify the passive tumor targeting effect based on enhanced permeability and retention (EPR) effect is crucial for precision cancer nanomedicine applications. Herein, we present the development of core-shell tecto dendrimers (CSTDs) as a platform for enhanced tumor magnetic resonance (MR) imaging through an amplified EPR effect. In this work, poly(amidoamine) (PAMAM) dendrimers of generation 5 (G5) were decorated with β-cyclodextrin (CD) and then assembled with G3 PAMAM dendrimers premodified with adamantane (Ad) via supramolecular recognition of CD and Ad. The formed G5-CD/Ad-G3 CSTDs were conjugated with tetraazacyclododecane tetraacetic acid (DOTA)-Gd(III) chelators and further acetylated to neutralize the remaining CSTD periphery amines. We reveal that the formed CSTD.NHAc-DOTA(Gd) (CSTD-D-Gd) complexes have a narrow size distribution and satisfactory colloidal stability, and are cytocompatible within the concentration range studied. Compared to the single dendrimer counterpart of G5.NHAc-DOTA(Gd) (G5-D-Gd) complexes, the CSTD-D-Gd complexes with a higher molecular weight and volume possess a longer rotation correlation time, hence having a longitudinal relaxivity (r1) of 7.34 mM-1 s-1, which is 1.5 times larger than that of G5-D-Gd complexes (4.92 mM-1 s-1). More importantly, the CSTD-D-Gd complexes display better permeability in the three-dimensional (3D) cell spheroids in vitro through fluorescence imaging and a more significant EPR effect for improved tumor MR imaging in vivo than the G5-DOTA-Gd complexes. The generated CSTD-D-Gd complexes may be adopted for enhanced tumor MR imaging through an amplified passive EPR effect and also be further extended for different cancer theranostic applications.

Physicochemical aspects of zwitterionic core-shell tecto dendrimers characterized by a thorough NMR investigation

Core-shell tecto dendrimers (CSTDs) are a class of superstructured dendrimeric nanoconstructs with similar structural characteristics and biomedical performances to the high-generation dendrimers that can be decorated with zwitterions to be availed of good antifouling properties for different biomedical applications. Characterization of the CSTD-based dendrimeric derivatives remains to be a great challenge. Here, we attempted to utilize nuclear magnetic resonance (NMR) technologies to investigate the structure and hydrodynamic size of CSTDs functionalized with zwitterionic carboxybetaine acrylamide (CBAA) and confirmed their antifouling property through protein resistance assay. In this study, we initially prepared CSTDs by supramolecular assembly of beta-cyclodextrin (β-CD)-modified generation 5 (G5) poly(amidoamine) (PAMAM) dendrimers as cores and adamantane (Ad)-decorated G3 PAMAM dendrimers as shells, and then partially modified the CSTD terminal amines with CBAA. The dendrimeric products were thoroughly characterized. Our results show that the obtained CSTDs display strong correlation signals between the Ad and β-CD protons in the two-dimensional NMR spectrum, indicating the strong host-guest interaction existing between G3-Ad and G5-CD in the structure of CSTDs. Diffusion NMR measurements reveal that the hydrodynamic diameter of CSTDs is smaller after partial CBAA surface modification, possibly due to the tightened molecular volume after zwitterionic modification. Lastly, the CBAA-modified CSTDs were demonstrated to have good antifouling property through protein resistance assay. Our findings provide a new strategy to study the framework of the developed CSTDs and CBAA-modified CSTDs, facilitating their further design and surface modification for various biomedical applications.

Binding of Folate-G4-PAMAM dendrimer conjugate with indomethacin via ligand diffusion MD simulations

Preceding experimental findings revealed that the release of indomethacin decreased when a folate conjugate G4-PAMAM (folate-G4-PAMAM) dendrimer was used compared with its unconjugated dendrimer (G4-PAMAM). Further, better encapsulation of the conjugated dendrimer was achieved, information useful for elucidating the structural and energetic basis of indomethacin on folate-G4-PAMAM encapsulation. In this study, we employed a ligand diffusion molecular dynamic simulations (LDMDSs) strategy combined with the molecular mechanics-generalized-born surface area (MMGBSA) approach to explore the mechanism by which indomethacin conjugated to folate-G4-PAMAM dendrimer forms complexes better than G4-PAMAM dendrimer. To this, we first constructed and equilibrated the folate-G4-PAMAM dendrimer, then, this system was submitted to docking and molecular dynamics (MD) simulation to evaluate its ability to form a stable complex with the folate receptor (FR). We observed that the folate-G4-PAMAM dendrimer was able to bind FR with higher affinity than free folic acid. Based on these results, we further performed LDMDSs to assess folate-G4-PAMAM dendrimer and G4-PAMAM dendrimer contacts with indomethacin. Our results correlate with experimental data, which confirm that folate-G4-PAMAM dendrimers are capable of most rapidly binding greater numbers of indomethacin molecules than G4-PAMAM, which suggests better loading and slower release occurs when the functionalized G4-PAMAM dendrimer is used. The simulations further revealed that van der Waals interactions govern the affinity. Communicated by Ramaswamy H. Sarma

Conjugation of Gentamicin to Polyamidoamine Dendrimers Improved Anti-bacterial Properties against Pseudomonas aeruginosa.

Purpose: This study aimed to design gentamicin-conjugated poly (amidoamine) (PAMAM) dendrimers to increase the therapeutic efficiency of gentamicin against Pseudomonas aeruginosa. Methods: Gentamicin-presenting dendrimers were synthesized using MAL-PEG3400-NHS as a redox-sensitive linker to attach gentamicin to the surface of G4 PAMAM dendrimers. The gentamicin molecules were thiolated by using Traut reagent. Then, the functionalized gentamicin molecules were attached to PEGylated PAMAM dendrimers through simple and high selectively maleimide (MAL)-thiol reaction. The structure of gentamicin-conjugated PAMAM dendrimers was characterized and confirmed using nuclear magnetic resonance (NMR), dynamic light scattering (DLS), zeta potential analysis, and transmission electron microscopy (TEM) imaging. The antibacterial properties of the synthesized complex were examined on P. aeruginosa and compared to gentamycin alone. Results: NMR, DLS, zeta potential analysis, and TEM imaging revealed the successful conjugation of gentamicin to PAMAM dendrimers. Data showed the appropriate physicochemical properties of the synthesized nanoparticles. We found a 3-fold increase in the antibacterial properties of gentamicin conjugated to the surface of PAMAM dendrimers compared to non-conjugated gentamicin. Based on data, the anti-biofilm effects of PAMAM-Gentamicin dendrimers increased at least 13 times more than the gentamicin in normal conditions. Conclusion: Data confirmed that PAMAM dendrimer harboring gentamicin could be touted as a novel smart drug delivery system in infectious conditions.

Nanoparticle-Delivered HIV Peptides to Dendritic Cells a Promising Approach to Generate a Therapeutic Vaccine.

Finding a functional cure for HIV-1 infection will markedly decrease the social and economic burden of this disease. In this work, we have taken advantage of the antigen presenting cell role of human dendritic cells (DCs) to try to induce an immune response to HIV-derived peptide delivered to DCs using two different polycationic nanoparticles: a G4 PAMAM dendrimer modified to a 70/30 ratio of hydroxyl groups/amines and a cyclodextrin derivative. We have studied peptide delivery using a fluorescence peptide and have studied the immune response generation by cytokine determination and flow cytometry. We have found a robust delivery of the antigenic peptide to DCs and activated dendritic cell-mediated peripheral blood mononuclear cells (PBMCs) proliferation using the mixed lymphocyte reaction. However, no expression of markers indicating activation of either B or T lymphocytes was observed. Moreover, the release of the pro-inflammatory cytokine TNF-α or IL-2 was only observed when DCs treated with either the dendrimer or the dendriplex containing the peptide. Antigenic peptide delivery to DCs is a promising approach to generate a vaccine against HIV-1 infection. However, more studies, including the simultaneous delivery of several antigenic peptides from different viral proteins, can markedly improve the immune response.

Trastuzumab Targeted Neratinib Loaded Poly-Amidoamine Dendrimer Nanocapsules for Breast Cancer Therapy.

BackgroundHuman epidermal growth factor receptor2 (Her2) positive breast cancer represents 25% of breast cancer cases. Targeted therapy with Her2 monoclonal antibody, trastuzumab (TZ), represents the first-line treatment for this type of breast cancer. In addition, neratinib, an irreversible inhibitor of the HER-2 receptor tyrosine kinase, has recently been approved as adjuvant therapy to TZ. This study aims to formulate (TZ)-grafted dendrimers loaded with neratinib, allowing a dual treatment alongside reducing the associated resistance as well as targeted therapy.MethodsTZ was conjugated on the surface of dendrimer using hetero-cross linker, MAL-PEG-NHS, and the zeta potential, and in vitro release of neratinib from dendrimers was characterized. Formulated dendrimers were also fluorescently conjugated with fluorescein isothiocyanate to visualize and quantify their SKBR-3 cellular uptake.ResultsThe G4 PAMAM dendrimer showed successful encapsulation of neratinib and a sustained release profile. Comparative in vitro studies revealed that these TZ-targeted dendrimers loaded with neratinib were more selective and have higher antiproliferation activity against SKBR-3 cells compared to neratinib alone and neratinib loaded dendrimer.ConclusionIn the current study, neratinib loaded in plain and trastuzumab-grafted dendrimer were successfully prepared. Enhanced cellular uptake of trastuzumab conjugated dendrimers was shown, together with a higher cytotoxic effect than plain neratinib dendrimers. These findings suggest the potential of TZ-conjugated dendrimers as targeting carrier for cytotoxic drugs, including neratinib.

Targeting Breast Cancer Cells with G4 PAMAM Dendrimers and Valproic Acid Derivative Complexes.

Our research group has developed some Valproic Acid (VPA) derivatives employed as anti-proliferative compounds targeting the HDAC8 enzyme. However, some of these compounds are poorly soluble in water. Employed the four generations of Polyamidoamine (G4 PAMAM) dendrimers as drug carriers of these compounds to increase their water solubility for further in vitro evaluation. VPA derivatives were subjected to Docking and Molecular Dynamics (MD) simulations to evaluate their affinity on G4 PAMAM. Then, HPLC-UV/VIS, 1H NMR, MALDI-TOF and atomic force microscopy were employed to establish the formation of the drug-G4 PAMAM complexes. The docking results showed that the amide groups of VPA derivatives make polar interactions with G4 PAMAM, whereas MD simulations corroborated the stability of the complexes. HPLC UV/VIS experiments showed an increase in the drug water solubility which was found to be directly proportional to the amount of G4 PAMAM. 1H NMR showed a disappearance of the proton amine group signals, correlating with docking results. MALDI-TOF and atomic force microscopy suggested the drug-G4 PAMAM dendrimer complexes formation. In vitro studies showed that G4 PAMAM has toxicity in the micromolar concentration in MDAMB- 231, MCF7, and 3T3-L1 cell lines. VPA CF-G4 PAMAM dendrimer complex showed anti-proliferative properties in the micromolar concentration in MCF-7 and 3T3-L1, and in the milimolar concentration in MDAMB- 231, whereas VPA MF-G4 PAMAM dendrimer complex didn't show effects on the three cell lines employed. These results demonstrate that G4 PAMAM dendrimers are capableof transporting poorly watersoluble aryl-VPA derivate compounds to increase its cytotoxic activity against neoplastic cell lines.

In silico and in vivo studies of gp120-HIV-derived peptides in complex with G4-PAMAM dendrimers.

Novel synthetic vaccines as immunotherapy approaches for HIV are interesting strategies that imply big challenges as they increase the poor immunogenic properties of peptide epitopes and their structural damage from the physiological environment. In this work, we used fourth-generation polyamidoamine dendrimers (G4-PAMAM) to increase the immunoglobulin responses (in vivo) induced by two peptide epitopes (pPGT122: DIIGDIRQAH and pVRC03: DGGANNTSNETFR), both recognized by broadly neutralizing antibodies (bNAb) on gp120-HIV type 1. pPGT122 and pVRC03 were identified on the gp120 surface via recognition by bNAb by using X-ray diffraction-derived structures obtained from the Protein Data Bank. pPGT122 and pVRC03 were coupled to the G4-PAMAM molecule by ligand diffusion using molecular dynamics (LDMDS) simulations and their energetic values were calculated by using the MMGBSA approach. Additionally, docking and MD simulations showed the affinity of pPGT122 and pVRC03 for MHC-I/II. G4-PAMAM–peptide complexes were chemically characterized through MALDI-TOF-MS, LC-ESI-QTOF-MS, atomic force microscopy (AFM) and 1H NMR spectroscopy. Then, the G4-PAMAM–peptide complexes were assayed in vivo by intranasal administration in female BALB/cJ mouse groups, showing that both peptides were immunogenic systemically and in the mucous membrane (in nasal and vaginal washes) via increase in IgG and IgA, respectively. This demonstrated that G4-PAMAM can be used as a nanocarrier for immunogenic peptides.

Complexation of peptide epitopes with G4-PAMAM dendrimer through ligand diffusion molecular dynamic simulations

Peptide epitopes from HIV-1 gp120 have been used to block the gp120-CD4 complex, whereas their poor absorbable or immunogenic properties prevent them from coupling to generation four polyamidoamine (PAMAM-G4) dendrimers. PAMAM-G4 are synthetic nanoparticles that are relatively nontoxic and nonimmunogenic have been employed as nanocarriers. In a previous study, two peptide epitopes (ABC and PGV04) from gp120 located at the protein-protein interface of the gp120-CD4 complex were identified through protein-protein dissociation. Then, their complexation with G4-PAMAM was evaluated through experimental and theoretical approaches, revealing a stoichiometry of 1:8/9 for G4-PAMAM and ABC or PGV04, respectively, providing important information that can be used to gain insight into the structural and energetic basis of the molecular binding of these G4-PAMAM-peptide systems. In this contribution, we performed ligand diffusion molecular dynamic simulations (LDMDSs) using 1.5 μs combined with the molecular mechanics generalized Born surface area (MMGBSA) approach, a strategy that successfully reproduced experimentally encapsulation on PAMAM-G4-ligand complexes, to explore the mechanism through which ABC and PGV04 are encapsulated by PAMAM-G4 under neutral and acid conditions. Our results reproduce the reported PAMAM-G4-peptide complex stoichiometry, revealing a slower peptide delivery at neutral conditions and a spontaneous release under acidic conditions. LDMDSs show that several peptides can reach stable G4-PAMAM complexes at neutral pH, and only a few are able to encapsulate on dendrimers without impacting dendrimer sphericity. Energetic analysis exploring different generalized Born models revealed that the ABC peptide has better binding properties than PGV04.

Generation 2 (G2) – Generation 4 (G4) PAMAM dendrimers disrupt key plasma coagulation parameters

The aim of the research was to evaluate the effects of G2 – G4 PAMAM dendrimers on basic plasma haemostasis parameters (Partially Activated Thrombin Time (APTT), Prothrombin Time (PT), Thrombin Time (TT)) as well as the activity of factor X, antithrombin III (AT), protein C and plasmin. Furthermore, tissue factor (TF) synthesis in endothelial cells and viability of smooth muscle cells in the presence of PAMAM dendrimers was investigated.APTT, PT and TT were performed according to the available commercial methods. The activity of factor X was conducted based on deficient plasma factor X. Protein C, AT and plasmin activity were measured spectrophotometrically using chromogenic substrates. Intracellular TF production in human umbilical vein endothelial cells (HUVECs) was measured using immunohistochemical method. Viability of Human Aortal Smooth Muscle cells (hAoSMCs) was established using WST-1 assay.PAMAM dendrimers decreased activity of factor X, and concomitantly prolonged PT and APTT. We also demonstrated shortened TT and increased fibrinogen concentrations in plasma treated with G4 PAMAM dendrimers, suggesting formation of fibrinogen aggregates. G2 – G4 PAMAM dendrimers decreased the activity of both naturally occurring anticoagulants AT and protein C. G2 and G3 PAMAM dendrimers did not affect the proteolytic reaction with plasmin. PAMAM dendrimers were found not to trigger TF production in undisturbed endothelial cells.PAMAM dendrimers, depending on the concentration and generation decreased viability of AoSMCs.The results presented within the current study suggest complex but mostly undesirable effect of G2 – G4 PAMAM dendrimers on plasma haemostasis and underscore the need for further in-depth research.

Molecular Beacon‐based Fluorescence Magnetic Nanoprobes for Tumor‐related HSP90 mRNA In‐suit Detection and Imaging

Heat shock proteins (HSPs) are a group of molecular chaperones that help proteins folding to their native conformation, protect cells from stressful environments. However, certain HSPs are overexpressed by malignant cells to survive under various therapies, inhibited cancer cells from senescence, apoptosis, stabilized lysosome membrane and promoted autophagy. Therefore, real‐time detection of the expression level of HSPs in living cells is required for both clinical cancer diagnosis application and oncology research to have a better understanding in anti‐apoptosis mechanism of malignant cells. Recently, delivery of molecular beacons that detect tumor‐related genes into cancer cells via nanocarriers shows promising results in the field of nanomedical diagnostics. In this study, uniformly sized and shaped superparamagnetic iron oxide nanocubes (SPIONCs) were synthesized by thermal decomposition and transferred into the aqueous phase via surface exchange with DMSA. G4 PAMAM dendrimer and Pluronic‐P123 were successively linked on SPIONCs‐DMSA in order to increase positive charge meanwhile reduce cytotoxicity. High specificity and sensitivity molecular beacon that targeting HSP90α mRNA was designed and synthesized and was condensed on PAMAM‐functionalized SPIONCs to form nanoprobes. TEM, XRD, TGA, ξ‐potential, DLS, VSM, fluorescence spectroscopy, agarose gel electrophoresis were carried out to characterize the morphology, composition, and properties of the nanoprobes. The mean diameter of as‐synthesized SPIONCs is 11 nm and increased to 100 nm after forming nanoprobes, which is stable in both water and culture media. Proliferation and hemolysis assays of the nanoprobes show low cytotoxicity and good hemocompatibility. LSCM confirmed that after 24h incubation with cells, the nanoprobes were uptake efficiently and located inside lysosome, and successfully escaped to the cytoplasm after 48h incubation. More importantly, the fluorescence of nanoprobes was observed significantly only in the HSP90 overexpressed MDA‐MB‐231 cells, instead of HSP90 low‐expressed MCF‐10A cells, which literally “highlights” a difference between normal and malignant cells, therefore provides a promising nanoplatform for real‐time gene expression level analysis and imaging in living cells.Support or Funding InformationWe would like to thank the financial supports, in whole or in part, by the National Natural Science Foundation of China (31470959, 81671821, 81471785), the Basic Research Program of Sichuan Science and Technology (2017JY0019, 2017JY0217), and the Fundamental Research Funds for the Central Universities (ZYGX2016Z001) for financial supports.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

In silico search, chemical characterization and immunogenic evaluation of amino-terminated G4-PAMAM-HIV peptide complexes using three-dimensional models of the HIV-1 gp120 protein.

Peptide epitopes have been widely used to develop synthetic vaccines and immunotherapies. However, peptide epitopes may exhibit poor absorption or immunogenicity due to their low molecular weights. Conversely, fourth-generation polyamidoamine (G4-PAMAM) dendrimers are nonimmunogenic and relatively nontoxic synthetic nanoparticles that have been used as adjuvants and nanocarriers of small peptides and to improve nasal absorption. Based on this information, we hypothesized that the combination of intranasal immunization and G4-PAMAM dendrimers would be useful for enhancing the antibody responses of HIV-1 gp120 peptide epitopes. Therefore, we first used structural data, peptide epitope predictors and docking and MD simulations on MHC-II to identify two peptide epitopes on the CD4 binding site of HIV-1 gp120. The formation of G4-PAMAM-peptide complexes was evaluated in silico (molecular docking studies using different G4-PAMAM conformations retrieved from MD simulations as well as the MMGBSA approach) and validated experimentally (electrophoresis, 1H NMR and cryo-TEM). Next, the G4-PAMAM dendrimer-peptide complexes were administered intranasally to groups of female BALB/cJ mice. The results showed that both peptides were immunogenic at the systemic and mucosal levels (nasal and vaginal), and G4-PAMAM dendrimer-peptide complexes improved IgG and IgA responses in serum and nasal washes. Thus, G4-PAMAM dendrimers have potential for use as adjuvants and nanocarriers of peptides.

Characterization of G4 PAMAM dendrimer complexes with 5-fluorouracil and their interactions with bovine serum albumin

The success of novel therapeutic strategies relies strongly on the development of a reliable active agent delivery mechanism. In this work, we address the question of how G4 PAMAM dendrimers form complexes with a therapeutic agent, 5-fluorouracil (5-FU) and interact with bovine serum albumin (BSA). The analytical techniques, such as: UV–vis spectrophotometry, dynamic light scattering, electrophoretic mobility, Fourier-transform infrared spectroscopy (FTIR), multi-parametric surface plasmon resonance (MP-SPR) and quartz crystal microbalance with dissipation monitoring (QCM-D) were used to analyze the properties of the created complexes. Through running these experiments, the optimum conditions for drug-dendrimer complexation could be determined. The results show that ∼70% of the 5-FU was loaded onto the G4 PAMAM dendrimers with higher capacity at pH 7.4 in comparison to acidic conditions. Biomolecule interactions with dendrimer carriers are driven both by electrostatic and hydrophobic forces. Dendrimer surface charge is reduced upon contact with protein and changes from 61 mV to 16.9 mV. This new understanding of dendrimers as nanocarriers and their interactions with plasma proteins delivers new insight into the interaction mechanism when the nanoparticles enter physiological fluids.

Pentafluoropropionic Anhydride Functionalized PAMAM Dendrimer as miRNA Delivery Reagent

Poly(amidoamine) (PAMAM) dendrimers are good candidates for nucleic acid delivery with their well-defined characteristics. MicroRNA (miRNA) mediated regulation of biological process are also active area of investigation. Fibroblast cells, such as MRC-5, are one of the cell lines used in biological researches due to their hard to transfect nature. In this two-staged study, cystamine core G5 PAMAM dendrimers were synthesized and modified with pentafluoropropionic anhydride (PA) and subsequently tested as miRNA delivery reagent on MRC-5 cells. Effect of fluorination against to naked G5 dendrimer over transfection efficiency was also investigated by molecular docking and quantitative structure-activity relationship (QSAR) calculations. Structural characterization of the synthesized dendrimers was verified by spectroscopic techniques. Gel retardation assay, particle size and transmission electron microscopy results demonstrated polyplex formation ability of fluorinated dendrimers with miRNA at nanoscale level. Zeta potential values indicated non-aggregation and increased stability of the polyplexes. Prepared polyplexes with fluorinated dendrimer showed over 90% cell viability and transfection efficiency. In silico calculations confirmed the stable complexation with miRNA and smooth penetration capability into the cell.

Biotinylated PAMAM G3 dendrimer conjugated with celecoxib and/or Fmoc-l-Leucine and its cytotoxicity for normal and cancer human cell lines

Tumors still remain one of the main causes of mortality due to the lack of effective anti-cancer therapy. Recently it has been shown, that overexpression of inducible cyclooxygenase-2 (COX-2) and decrease of peroxisome proliferator-activated receptor γ (PPARγ) expression accompany many malignances, therefore, it has been proposed, that COX-2 inhibitors and PPARγ agonists are potential candidates for anticancer therapy and their synergistic, antineoplastic action has been described. In the present study a COX-2 inhibitor (celecoxib) and/or PPARγ agonist (Fmoc-l-Leucine) were conjugated with the biotinylated G3 PAMAM dendrimer to form a three different constructs targeted to cells with increased biotin uptake. All conjugates were characterized by the NMR spectroscopy. Investigation of three types of human cells: normal skin fibroblasts (BJ), immortalized keratinocytes (HaCaT) and cancer lines: glioblastoma (U-118 MG) and squamous cell carcinoma (SCC-15) revealed similar biotin labeled ATTO590 accumulation (after 24 h), except for SCC-15 with significantly lower loading. Constitutive expression of COX-2 protein was confirmed in all tested cells with significantly higher levels (2–2.5 times) in both cancer lines.Comparison of cytotoxicity of the new synthetized dendrimers clearly documented the highest cytotoxicity of the G31B16C15L dendrimer conjugated with both drugs (1: 1) as compared with drugs alone and single conjugates. Additive effects of construct with both compounds were shown for fibroblasts and both cancer cell lines in the order BJ > U-118 MG > SCC-15 with IC50 in the range: 0.69, 1.44 and 2.22 μM, respectively and lowest cytotoxicity in HaCaT cells (IC50 = 2.88). Our results showed, that biotinylated G3 PAMAM dendrimers substituted with COX-2 inhibitor, celecoxib, and PPARγ agonist, Fmoc-l-Leucine (1:1) may be a good candidate for local therapy of glioblastoma but not a skin cancer. Since the effect of PPARγ agonists on COX-2 expression vary depending upon the cell type, specificity of used agonist and the presence of other environmental factors, it is necessary to carefully evaluate the response of chosen drugs on the target cells.

Cytotoxicity of Silica Nanoparticles with Transcaucasian Nose-Horned Viper, Vipera ammodytes transcaucasiana, Venom on U87MG and SHSY5Y Neuronal Cancer Cells.

Highly bioactive compounds of the snake venom make them particular sources for anticancer agent development. They contain very rich peptide-protein structures. Therefore, they are very susceptible to environmental conditions such as temperature, pH, and light. In this study, Vipera ammodytes transcaucasiana venom was encapsulated in PAMAM-G4 dendrimer by sol-gel method in order to prevent degradation of venom contents from the environmental conditions. For this purpose, nanoparticles were prepared by sol-gel methodology and SEM analyses were performed. U87MG and SHSY5Y neuronal cancer cell lines were treated with different concentrations of venom-containing nanoparticles and cytotoxicity was determined by MTT assay. IC50 values of nanoparticles with snake venom were calculated as 37.24 and 44.64μg/ml for U87MG and SHSY5Y cells, respectively. The IC50 values of nanoparticles with snake venom were calculated as 10.07 and 7.9μg/ml for U87MG and SHSY5Y cells, respectively. As a result, nanoparticles with V. a. transcaucasiana venom showed remarkably high cytotoxicity. Encapsulation efficiency of nanoparticles with 1mg/ml snake venom was determined as %67 via BCA™ protein analysis. In conclusion, this method is found to be convenient and useful for encapsulating snake venom as well as being suitable for drug delivery systems.