Exploring the Potential of Lapatinib, Fulvestrant, and Paclitaxel Conjugated with Glycidylated PAMAM G4 Dendrimers for Cancer and Parasite Treatment.

Fulvestrant (F), lapatinib (L), and paclitaxel (P) are hydrophobic, anticancer drugs used in the treatment of estrogen receptor (ER) and epidermal growth factor receptor (EGFR)-positive breast cancer. In this study, glycidylated PAMAM G4 dendrimers, substituted with F, L, and/or P and targeting tumor cells, were synthesized and characterized, and their antitumor activity against glioma U-118 MG and non-small cell lung cancer A549 cells was tested comparatively with human non-tumorogenic keratinocytes (HaCaT). All cell lines were ER+ and EGFR+. In addition, the described drugs were tested in the context of antinematode therapy on C. elegans. The results show that the water-soluble conjugates of G4P, G4F, G4L, and G4PFL actively entered the tested cells via endocytosis due to the positive zeta potential (between 13.57-40.29 mV) and the nanoparticle diameter of 99-138 nm. The conjugates of G4P and G4PFL at nanomolar concentrations were the most active, and the least active conjugate was G4F. The tested conjugates inhibited the proliferation of HaCaT and A549 cells; in glioma cells, cytotoxicity was associated mainly with cell damage (mitochondria and membrane transport). The toxicity of the conjugates was proportional to the number of drug residues attached, with the exception of G4L; its action was two- and eight-fold stronger against glioma and keratinocytes, respectively, than the equivalent of lapatinib alone. Unfortunately, non-cancer HaCaT cells were the most sensitive to the tested constructs, which forced a change in the approach to the use of ER and EGFR receptors as a goal in cancer therapy. In vivo studies on C. elegans have shown that all compounds, most notably G4PFL, may be potentially useful in anthelmintic therapy.

Development of 177Lu-Cetuximab-PAMAM dendrimeric nanosystem: a novel theranostic radioimmunoconjugate.

PURPOSE: Epidermal growth factor receptors (EGFRs) are overexpressed in a wide range of tumors and are attractive candidates to target in targeted therapies. This study aimed to introduce a novel radiolabeled compound, 177Lu-cetuximab-PAMAM G4, for the treatment of EGFR-expressing tumors. METHODS: In this study, the cetuximab mAb was bound to PAMAM G4 and labeled with 177Lu via DTPA-CHX chelator. The synthesized nanosystem was confirmed by different analyses such as DLS, FT-IR, TEM, and RT-LC. Cell viability of the radioimmunoconjugate was assessed over the EGFR-expressing cell line of SW480. The biodistribution of 177Lu-Cetuximab-PAMAMG4 was determined in different intervals after injection of the radiolabeled compound in normal and tumoral nude mice via scarification and SPECT images. RESULTS: The average size of PAMAM G4 and PAMAM-Cetuximab-DTPA-CHX nanoparticles were 2 and 70 nm, respectively. 177Lu-Cetuximab-PAMAMG4 was prepared with radiochemical purity of more than 98%. The survival rates of SW480 cells at 24, 48, and 72 h post-treatment with177Lu-Cetuximab-PAMAMG4 (500 nM) were 18%, 15%, and 14%, respectively. The biodistribution studies showed a significant accumulation of 177Lu-Cetuximab-PAMAM in the EGFR-expressing tumor. CONCLUSION: According to the results, this new agent can be considered as an efficient therapeutic complex for tumors expressing EGFR receptors.

Effect of PEGylation on drug uptake, biodistribution, and tissue toxicity of efavirenz-ritonavir loaded PAMAM G4 dendrimers.

The present investigations aimed to compare the efficiency of PAMAM G4 (PG4) and PEGylated PAMAM G4 (PPG4) dendrimers as novel nanocarriers for the treatment of HIV-1. Synthesized PG4 and PPG4 dendrimers were confirmed by electrospray ionization and particle size with its morphology. The anti-human immunodeficiency virus (HIV) drug efavirenz (EFV) with a booster dose of ritonavir (RTV) was encapsulated into PG4 and PPG4 formerly noted as PG4ER and PPG4ER, respectively. Further, evaluated for dendrimers mediated solubilization, drug release, cytotoxicity, drug uptake, plasma, and tissue pharmacokinetics, and histopathology. PG4ER and PPG4ER both promoted a prolonged release of EFV in weakly acidic pH 4 up to 84 h and 132 h, respectively. The results of the cytotoxicity assay and drug uptake study showed that PPG4ER was safe and biocompatible up to 12.5 µg/ml. The plasma pharmacokinetic profile of EFV and RTV was significantly increased by PPG4ER with prolonged t1/2 up to three times as compared to free EFV-RTV and PG4ER. Histopathological analysis showed remarkably lower tissue toxicity in PPG4ER as compared to free EFV-RTV. Therefore, overall data suggested that PPG4 has a great potential for prolonged release of EFV and RTV with enhanced bioavailability and lower toxicity.

Nano-carrier Polyamidoamine Dendrimer G4 Induces Mitochondrialdependent Apoptosis in Human Multidrug-resistant Breast Cancer Cells through G0/G1 Phase Arrest.

BACKGROUND: Multidrug-resistant tumor cells have special drug detoxification/inactivation mechanisms. The terminal amino groups of the polyamidoamine (PAMAM-NH2), which is cytotoxic to tumor sensitive cells, may have no cytotoxicity in tumor resistant cells with a mechanism different from tumor sensitive cells. OBJECTIVE: This study aimed to investigate the cytotoxic effects of PAMAM-G4-NH2 on human multidrug- resistant breast cancer cells (MCF-7/ADR cells) and identify the possible molecular mechanisms. METHODS: The cytotoxicity of PAMAM-G4-NH2 (10-1000 µg/mL) against MCF-7 and MCF-7/ADR cells was detected. Then, MCF-7 and MCF-7/ADR cells were treated with PAMAM-G4-NH2 (10, 100 and 1000 µg/mL), and apoptosis, reactive oxygen species (ROS) production, mitochondrial membrane potential (MMP), activities of caspase-3, -8 and -9 and cell cycle distribution were determined. RESULTS: Within 48 h, the cell viabilities in MCF-7/ADR cells after treatment with PAMAM-G4-NH2 were significantly higher than that in MCF-7 cells in the concentration range of 200-500 µg/mL (P < 0.05). Viabilities of MCF-7/ADR cells treated with PAMAM-G4-OH and PAMAM-G4-COOH for 48 and 72 h were much higher than that of MCF-7/ADR cells treated with PAMAM-G4-NH2. Treated with high concentration (1000 µg/mL) of PAMAM-G4-NH2 for 24 h, the apoptosis ratio, ROS levels, as well as caspase-3 and -9 activities in MCF-7 and MCF-7/ADR cells increased, while MMP decreased, and the cells were arrested in the G0/G1 phase. CONCLUSION: PAMAM-G4-NH2 induced concentration-dependent cytotoxicity in MCF-7/ADR cells via G0/G1 arrest, and acted through h the mitochondria-dependent apoptotic pathway, which was similar to those in tumor sensitive cell, MCF-7 cells. The results suggest that PAMAM-G4-NH2, instead of PAMAM-G4-OH and PAMAM-G4-COOH, can be used as a carrier for drug delivery, concomitantly, it can also induce apoptosis in multidrug-resistant cancer cells in combination with the loaded drug through multiple apoptotic pathways.

Margetuximab conjugated-PEG-PAMAM G4 nano-complex: a smart nano-device for suppression of breast cancer.

Abstract: Breast cancer due to its high incidence and mortality is the second leading cause of death among females. On the other hand, nanoparticle-based drug delivery is one of the most promising approaches in cancer therapy, nowadays. Hence, margetuximab- and polyethylene glycol-conjugated PAMAM G4 dendrimers were efficiently synthesized for targeted delivery of quercetin (therapeutic agent) to MDA-MB-231 breast cancer cells. Synthesized nano-complexes were characterized using analytical devices such as FT-IR, TGA, DLS, Zeta potential analyzer, and TEM. The size less than 40 nm, - 18.8 mV surface charge, efficient drug loading capacity (21.48%), and controlled drug release (about 45% of drug release normal pH after 8 h) were determined for the nano-complex. In the biomedical test, the cell viability was obtained 14.67% at 24 h of post-treatment for 800 nM concentration, and IC50 was ascertained at 100 nM for the nano-complex. The expression of apoptotic Bax and Caspase9 genes was increased by more than eightfolds and more than fivefolds after treatment with an optimal concentration of nanocarrier. Also, more than threefolds of cell cycle arrest was observed at the optimal concentration synthetics, and 27.5% breast cancer cell apoptosis was detected after treatment with 100 nM nano-complex. These outputs have been indicating the potential capacity of synthesized nano-complex in inhibiting the growth of breast cancer cells.

Breast Tumor Targeting with PAMAM-PEG-5FU-99mTc As a New Therapeutic Nanocomplex: In In-vitro and In-vivo studies.

Dendrimer-based targeted drug delivery, as an innovative polymeric drug-delivery system, is promising for cancer therapy. Folate receptors (FR) are overexpressed in many types of tumor cells, such as breast cell carcinomas, which allow folate-targeted delivery. Therefor polyethylene glycol (PEG) modified-PAMAM G4 dendrimers were functionalized with folic acid (FA), as targeting agent. Then, 5-FU (5-fluorouracil) and 99mTc (technetium-99 m) as therapeutic agents were respectively loaded and conjugated to previous nano-complex (PEG-PAMAM G4-FA-5FU-99mTc). The value of drug loading was calculated by TGA analysis (16.97%). Drug release profiles of PEG-PAMAM G4-FA-5FU-99mTc and PEG-PAMAM G4-FA-5FU were evaluated. The radiochemical purity of PEG-PAMAM G4-FA-5FU-99mTc and PEG-PAMAM G4-FA-99mTc was obtained at >95% with excellent in-vitro and in-vivo stabilities. PEG-PAMAM G4-FA-5FU-99mTc was synthesized and the stability studies were carried out by the ITLC methods in serum (86.67% and 83.75%) and PBS. Combinational therapy effects of 5-FU and 99mTc containing nano-complexes were evaluated on 4 T1 (mouse breast cancer) and MDA-MB-231 (human breast adenocarcinoma) cancer cell lines. Excellent uptake values were obtained for FA-decorated nano-complexes on 4 T1 and MDA-MB-231 cell lines. Subsequently, tumor inhibition effects of PEG-PAMAM G4-FA-5FU-99mTc and PEG-PAMAM G4-FA-5FU were evaluated using the breast tumor-bearing BALB/C mice. Graphical abstract Breast Tumor Targeting with PAMAM-PEG-5FU- 99mTc As a New Therapeutic Nanocomplex: in In-vitro and In-vivo Studies was presented. This targeted drug delivery system can significantly increase the efficiency of cancer therapy, and reduce the treatment cost and time.

Interaction study of peptide-PAMAM as potential bio-nanogate for detecting anti-hepatitis B surface antigen.

Bio-nanogate involves synthesized or natural molecules as a 'gate' towards bioreceptors and responds upon the presence of targeted analytes in nanoscale dimension. Development of bio-nanogate improves analyte selectivity and signal response across various types of biosensors. The versatility of PAMAM dendrimers to form conjugates with guest molecules, such as proteins can be utilized in forming a bio-nanogate. PAMAM interaction with peptide bioreceptor for antibody detection is of interest in this study. This study investigated the interaction of synthesized immunogenic 'a' determinant (aD) region of hepatitis B virus surface antigen (HBsAg) with PAMAM G4 and anti-HBsAg antibody, as a potential bio-nanogate for anti-HBsAg detection. The aD peptide fused with maltose binding protein (MBP), was confirmed with Western blotting. Nano-Differential Scanning Fluorimetry (nano-DSF) study revealed that the interaction of MBP-aD with anti-HBsAg indicated a higher thermal stability as compared to its interaction with PAMAM G4. Electrochemical impedance spectroscopy showed that a higher binding constant of MBP-aD interaction with anti-HBsAg (0.92 µM-1) was observed at maximum saturation, as compared with PAMAM G4 (0.07 µM-1). Thermodynamic parameters demonstrated that MBP-aD interacted with anti-HBsAg and PAMAM G4, through van der Waals and hydrogen bonding. These analyses suggest that the weak interaction of MBP-aD and PAMAM G4 may form a potential bio-nanogate. It is hypothesized that the presence of anti-HBsAg has a higher affinity towards MBP-aD which may displace PAMAM G4 in the anti-HBsAg detection system. This interaction study is crucial as an initial platform of using peptide-PAMAM as a bio-nanogate in an antibody detection system.

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.

Complexation of methotrexate via ligand diffusion molecular dynamic simulations under neutral, basic, and acidic conditions.

Methotrexate (MTX), an FDA-approved drug employed in the treatment of several types of cancer and autoimmune diseases, is characterized by its poor solubility. Therefore, new strategies have been implemented such as coupling to nanocarriers to increase its solubility. Previous experimental studies have demonstrated complexation of MTX to polyamidoamine of a generation four (PAMAM-G4) dendrimer with a complex stoichiometry of 19/22:1 under neutral conditions, providing important information that can be used to further elucidate the structural and energetic basis of the molecular binding of MTX and PAMAM-G4. In this study, we performed ligand diffusion molecular dynamic simulations (LDMDSs), using 3 µs combined with the molecular mechanics generalized surface area (MMGBSA) approach employing saturating concentrations of MTX to explore the mechanism through which MTX is complexed by PAMAM-G4 at neutral, basic, and acidic conditions. Our results reproduce the reported complex stoichiometry between MTX and PAMAM-G4 in neutral conditions. Binding free energy values suggest a much slower release in neutral and acidic conditions, consistent with the controlled rate of drug release into the bloodstream and when reaching the acidic environment of tumor tissues. Altogether, the methodology employed and the results may be useful in the evaluation of other drugs of pharmaceutical interest.

Spectroscopic and calorimetric studies on the interaction between PAMAM G4-OH and 5-fluorouracil in aqueous solutions.

The results of spectroscopic measurements (an increase in solubility, equilibrium dialysis, 1H NMR titration) and calorimetric measurements (isothermal titration ITC) indicate spontaneous (ΔG<0) binding of 5-fluorouracil molecules by PAMAM G4-OH dendrimer with terminal hydroxyl groups in an aqueous solution. PAMAM G4-OH dendrimer bonds about n=8±1 molecules of the drug with an equilibrium constant of K=70±10. The process of saturating the dendrimer active sites by the drug molecules is exothermal (ΔH<0) and is accompanied by an advantageous change in entropy (ΔS>0). The parameters of binding 5-fluorouracil by PAMAM G4-OH dendrimer were compared with those of binding this drug by the macromolecules of PAMAM G3-OH and G5-OH.

Dendrimer-Capped Gold Nanoparticles for Highly Reliable and Robust Surface Enhanced Raman Scattering.

Dendrimer-stabilized gold nanoparticles (Au-Den) were prepared by a facile solution based method for a highly reliable and robust surface enhanced Raman scattering (SERS) substrate. Au-Den was selectively attached on the surface of reduced graphene oxide (rGO) by noncovalent interactions between the Au capping dendrimer and the graphene surface. Au-Den/rGO exhibits the outstandingly stable and highly magnified Raman signal with an enhancement factor (EF) of 3.9 × 10(7) that enables detection of R6G dyes with concentration as low as 10 nM, retaining 95% of the Raman signal intensity after 1 year. The remarkable stability and enhancement originated not only from a simple combination of the electromagnetic and chemical mechanism of SERS but also from intensified packing density of stable Au-Den on the graphene substrate due to the firm binding between the dendrimer capped metal nanoparticles and the graphene substrate. This method is not limited to the gold nanoparticles and G4 dendrimer used herein, but also can be applied to other dendrimers and metal nanoparticles, which makes the material platform suggested here superior to other SERS substrates.

In vitro and in vivo uptake studies of PAMAM G4.5 dendrimers in breast cancer.

BACKGROUND: Breast cancer is the second leading cause of cancer death worldwide. Nanotechnology approaches can overcome the side effects of chemotherapy as well as improve the efficacy of drugs. Dendrimers are nanometric size polymers which are suitable as drug delivery systems. To the best of our knowledge, studies on the application of PAMAM G4.5 (polyamidoamine half generation 4) dendrimers as potential drug delivery systems in breast cancer have not been reported. In this work we developed a PAMAM G4.5 dendrimer containing FITC (fluorescein isothiocyanate) dye to study their uptake by murine breast cancer cells and BALB/c mice breast tumors. RESULTS: We performed a reaction between FITC and PAMAM G4.5 dendrimers which were previously derivatized with piperazine (linker molecule), characterized them by (1)H NMR (proton nuclear magnetic resonance) spectroscopy and MALDI-TOF (matrix-assisted laser desorption/ionization- time-of-flight) mass spectrometry. The experimental data indicated that 2 FITC molecules could be bound covalently at the PAMAM G4.5 dendrimer surface, with 17 FITC molecules probably occluded in PAMAM dendrimers cavity. PAMAM-FITC dendrimer (PAMAM G4.5-piperazinyl-FITC dendrimer) size distribution was evaluated by DLS (dynamic light scattering) and TEM (transmission electron microscopy). The nanoparticle hydrodynamic size was 96.3 ± 1.4 nm with a PdI (polydispersion index) of 0.0296 ± 0.0171, and the size distribution measured by TEM was 44.2 ± 9.2 nm. PAMAM-FITC dendrimers were neither cytotoxic in 4T1 cells nor hemolytic up to 24 h of incubation. In addition, they were uptaken in vitro by 4T1 cells and in vivo by BALB/c mice breast tumors. PAMAM G4.5-piperazinyl-FITC dendrimer intracellular distribution was observed through histologic analysis of the tumor by laser confocal microscopy. CONCLUSION: These results indicate that PAMAM G4.5 dendrimers enter tumor tissue cells, being good candidates to be used as antitumor drug delivery systems for breast cancer treatment and diagnosis.

Development of (177)Lu-DOTA-Dendrimer and Determination of Its Effect on Metal and Ion Levels in Tumor Tissue.

Dendrimers are synthetic nanomolecules with well-defined chemical structures. Different strategies have been used for radiolabeling dendrimers with different radioisotopes. In this study, the aim was to conjugate dendrimers with (177)Lu, to observe the in vivo behavior of the labeled compound and to measure the elementary changes in tumor tissue that could be caused by ionizing radiation. PAMAM G4 dendrimers conjugated with DOTA were labeled with (177)Lu. The radiolabeled compound was characterized and its stability was evaluated by reverse phase high performance liquid chromatography. Radiolabeling yield was >98% and stable for 24 hours. Biodistribution studies of (177)Lu-DOTA-dendrimers in C57BL/6 melanoma-bearing mice showed blood clearance with hepatic and renal depuration and tumor uptake. The concentrations of Br, Ca, Cl, Fe, K, Mg, Na, Rb, S, and Zn were determined in tumor tissues of C57BL/6 mice treated with (177)Lu-DOTA-dendrimers and in untreated mice. The results showed decreased concentrations of Br (62%), Ca (24%), Cl (51%), K (12%) and Na (60%) and increased concentrations of Fe (8%), Mg (28%), Rb (100%), S (6%) and Zn (4%) in tumor tissues of mice treated with (177)Lu-DOTA-dendrimers. These data may be useful to evaluate changes in tumor tissues as indicators of damage that could be caused by ionizing radiation.

Synthesis of Polyamidoamine Dendrimer for Encapsulating Tetramethylscutellarein for Potential Bioactivity Enhancement.

The biomedical potential of flavonoids is normally restricted by their low water solubility. However, little has been reported on their encapsulation into polyamidoamine (PAMAM) dendrimers to improve their biomedical applications. Generation four (G4) PAMAM dendrimer containing ethylenediaminetetraacetic acid core with acrylic acid and ethylenediamine as repeating units was synthesized by divergent approach and used to encapsulate a flavonoid tetramethylscutellarein (TMScu, 1) to study its solubility and in vitro release for potential bioactivity enhancement. The as-synthesized dendrimer and the dendrimer-TMScu complex were characterized by spectroscopic and spectrometric techniques. The encapsulation of 1 into dendrimer was achieved by a co-precipitation method with the encapsulation efficiency of 77.8% ± 0.69% and a loading capacity of 6.2% ± 0.06%. A phase solubility diagram indicated an increased water solubility of 1 as a function of dendrimer concentration at pH 4.0 and 7.2. In vitro release of 1 from its dendrimer complex indicated high percentage release at pH 4.0. The stability study of the TMScu-dendrimer at 0, 27 and 40 °C showed the formulations to be stable when stored in cool and dark conditions compared to those stored in light and warmer temperatures. Overall, PAMAM dendrimer-G4 is capable of encapsulating 1, increasing its solubility and thus could enhance its bioactivity.

Formation of complexes between PAMAM-NH2 G4 dendrimer and L-α-tryptophan and L-α-tyrosine in water.

Interactions between electromagnetic radiation and the side substituents of aromatic amino acids are widely used in the biochemical studies on proteins and their interactions with ligand molecules. That is why the aim of our study was to characterize the formation of complexes between PAMAM-NH2 G4 dendrimer and L-α-tryptophan and L-α-tyrosine in water. The number of L-α-tryptophan and L-α-tyrosine molecules attached to the macromolecule of PAMAM-NH2 G4 dendrimer and the formation constants of the supramolecular complexes formed have been determined. The macromolecule of PAMAM-NH2 G4 can reversibly attach about 25 L-α-tryptophan molecules with equilibrium constant K equal to 130±30 and 24±6 L-α-tyrosine molecules. This characterization was deduced on the basis of the solubility measurements of the amino acids in aqueous dendrimer solutions, the (1)H NMR and 2D-NOESY measurements of the dendrimer solutions with the amino acids, the equilibrium dialysis and the circular dichroism measurements of the dendrimer aqueous solutions with L-α-tryptophan. Our date confirmed the interactions of L-α-tryptophan and L-α-tyrosine with the dendrimer in aqueous solution and indicated a reversible character of the formed complexes.

The evaluation of the biomedical effectiveness of poly(amido)amine dendrimers generation 4.0 as a drug and as drug carriers: a systematic review and meta-analysis.

The purpose of this study was to investigate the evaluation of the biomedical effectiveness of poly(amido)amine dendrimers generation 4.0 (PAMAM G4) as a drug and as drug carriers by a systematic review of literature and meta-analysis. The results obtained from meta-analysis concluded that drug therapy reduces the change of parameters in relation to the control. The impact of the drug administered to change the test parameters are dependent on the type of tissue. PAMAM G4 may be effective in vitro and in vivo as a drug and drug carriers and may have appropriate applications in various fields of medicine. PAMAM G4 dendrimers hold promises for nanomedicine.