RESUMO
A hybrid cellulose-based programmable nanoplatform for applications in precision radiation oncology is described. Here, sugar heads work as tumor targeting moieties and steer the precise delivery of radiosensitizers, i.e. gold nanoparticles (AuNPs) into triple negative breast cancer (TNBC) cells. This "Trojan horse" approach promotes a specific and massive accumulation of radiosensitizers in TNBC cells, thus avoiding the fast turnover of small-sized AuNPs and the need for high doses of AuNPs for treatment. Application of X-rays resulted in a significant increase of the therapeutic effect while delivering the same dose, showing the possibility to use roughly half dose of X-rays to obtain the same radiotoxicity effect. These data suggest that this hybrid nanoplatform acts as a promising tool for applications in enhancing cancer radiotherapy effects with lower doses of X-rays.
Assuntos
Celulose , Ouro , Nanopartículas Metálicas , Radiossensibilizantes , Neoplasias de Mama Triplo Negativas , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Radiossensibilizantes/química , Ouro/química , Celulose/química , Humanos , Nanopartículas Metálicas/química , Nanopartículas Metálicas/uso terapêutico , Nanopartículas Metálicas/efeitos da radiação , Linhagem Celular Tumoral , Feminino , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Nanopartículas/química , Sobrevivência Celular/efeitos dos fármacosRESUMO
A deeper knowledge on the formation and biological fate of polymer based gene vectors is needed for their translation into therapy. Here, polyplexes of polyethyleneimine (PEI) and silencing RNA (siRNA) are formed with theoretical N/P ratios of 2, 4 and 12. Fluorescence correlation spectroscopy (FCS) is used to study the formation of polyplexes from fluorescently labelled PEI and siRNA. FCS proves the presence of free PEI. From the analysis of the autocorrelation functions it was possible to determine the actual stoichiometry of polyplexes. FCS and fluorescence cross correlation spectroscopy (FCCS) are used to follow the fate of the polyplexes intracellularly. Polyplexes disassemble after 1 day inside cells. Positron emission tomography (PET) studies are conducted with radiolabelled polyplexes prepared with siRNA or PEI labelled with 2,3,5,6-tetrafluorophenyl 6-[18F]-fluoronicotinate ([18F]F-PyTFP). PET studies in healthy mice show that [18F]siRNA/PEI and siRNA/[18F]PEI polyplexes show similar biodistribution patterns with limited circulation in the bloodstream and accumulation in the liver. Higher activity for [18F]PEI in the kidney and bladder suggests the presence of free PEI.
Assuntos
Polietilenoimina , RNA de Cadeia Dupla , Animais , Camundongos , Polietilenoimina/química , RNA Interferente Pequeno/química , Distribuição Tecidual , Espectrometria de Fluorescência , Tomografia por Emissão de PósitronsRESUMO
N-Acetylgalactosamine-6-sulfatase (GALNS) is an enzyme whose deficiency is related to the lysosomal storage disease Morquio A. For the development of effective therapeutic approaches against this disease, the design of suitable enzyme enhancers (i.e. pharmacological chaperones) is fundamental. The natural substrates of GALNS are the glycosaminoglycans keratan sulfate and chondroitin 6-sulfate, which mainly display repeating units of sulfated carbohydrates. With a biomimetic approach, gold nanoparticles (AuNPs) decorated with simple monosaccharides, sulfated ligands (homoligand AuNPs), or both monosaccharides and sulfated ligands (mixed-ligand AuNPs) were designed here as multivalent inhibitors of GALNS. Among the homoligand AuNPs, the most effective inhibitors of GALNS activity are the ß-D-galactoside-coated AuNPs. In the case of mixed-ligand AuNPs, ß-D-galactosides/sulfated ligands do not show better inhibition than the ß-D-galactoside-coated AuNPs. However, a synergistic effect is observed for α-D-mannosides in a mixed-ligand coating with sulfated ligands that reduced IC50 by one order of magnitude with respect to the homoligand α-D-mannoside-coated AuNPs. SAXS experiments corroborated the association of GALNS with ß-D-galactoside AuNPs. These AuNPs are able to restore the enzyme activity by almost 2-fold after thermal denaturation, indicating a potential chaperoning activity towards GALNS. This information could be exploited for future development of nanomedicines for Morquio A. The recent implications of GALNS in cancer and neuropathic pain make these kinds of multivalent bionanomaterials of great interest towards multiple therapies.
Assuntos
Condroitina Sulfatases , Nanopartículas Metálicas , Ouro , Acetilgalactosamina , Monossacarídeos , Ligantes , Sulfatos , Espalhamento a Baixo Ângulo , Difração de Raios X , LisossomosRESUMO
Polyamine-based vectors offer many advantages for gene therapy, but they are hampered by a limited knowledge on their biological fate and efficacy for nucleic acid delivery. The 18 F radiolabeled siRNA is complexed with poly(allyl amine) hydrochloride (PAH), PEGylated PAH (PAHPEG ), or oleic acid-modified PAH (PAHOleic ) to form polyplexes, and injected them intravenously into healthy rodents. The biodistribution patterns obtained by positron emission tomography (PET) imaging vary according to the polymer used for complexation. Free siRNA is quickly eliminated through the bladder. PAH and oleic acid modify PAH polyplexes accumulate in the lungs and liver. No elimination through the bladder is observed for PAH and PAHOleic within 2 h after administration. PAHPEG polyplexes accumulate in kidneys and are eliminated through the bladder. Polyplexes prepared with 18 F-labeled oleic acid-modified PAH and non-labeled siRNA show similar biodistribution to those prepared with labeled siRNA, but with more accumulation in the lungs due to the presence of non-complexed polymer. Intravenous administration of PAHOleic polyplexes in tumor models results in a limited availability of siRNA. When PAHOleic polyplexes are administered intratumorally in tumor bearing rodents, ≈40% of the radioactivity is retained in the tumor after 180 min while free siRNA is completely eliminated.
Assuntos
Neoplasias , Ácido Oleico , Humanos , RNA Interferente Pequeno , Distribuição Tecidual , Tomografia por Emissão de Pósitrons , Polímeros , PoliaminasRESUMO
Cellulose nanocrystal and gold nanoparticles are assembled, in a unique way, to yield a novel modular glyconanomaterial whose surface is then easily engineered with one or two different headgroups, by exploiting a robust click chemistry route. We demonstrate the potential of this approach by conjugating monosaccharide headgroups to the glyconanomaterial and show that the sugars retain their binding capability to C-type lectin receptors, as also directly visualized by cryo-TEM.
Assuntos
Nanopartículas Metálicas , Nanopartículas Metálicas/química , Ouro/química , Celulose/química , Química Click , Lectinas Tipo CRESUMO
Small interference RNA (siRNA) is a tool for gene modulation, which can silence any gene involved in genetic disorders. The potential of this therapeutic tool is hampered by RNA instability in the blood stream and difficulties to reach the cytosol. Polyamine-based nanoparticles play an important role in gene delivery. Polyallylamine hydrochloride (PAH) is a polycation displaying primary amines that can be easily chemically modified to match the balance between cell viability and siRNA transfection. In this work, PAH has been covalently functionalized with oleic acid at different molar ratios by carbodiimide chemistry. The substituted polymers form polyplexes that keep positive surface charge and fully encapsulate siRNA. Oleic acid substitution improves cell viability in the pulmonary cell line A549. Moreover, 6 and 14% of oleic acid substitution show an improvement in siRNA transfection efficiency. CD47 is a ubiquitous protein which acts as "don't eat me signal." SIRPα protein of macrophages recognizes CD47, leading to tumor cell phagocytosis by macrophages. By knocking down CD47 with siRNA, cancer cells become vulnerable to be eliminated by the immune system. PAH-oleic acid substitutes show high efficacy in silencing the CD47 protein, making them a potential candidate for immunotherapy.
Assuntos
Antígeno CD47 , Ácido Oleico , RNA Interferente Pequeno , Antígeno CD47/genética , Antígeno CD47/metabolismo , RNA de Cadeia Dupla , TransfecçãoRESUMO
The degradation of mesoporous silica nanoparticles (MSNs) in the biological milieu due to silica hydrolysis plays a fundamental role for the delivery of encapsulated drugs and therapeutics. However, little is known on the evolution of the pore arrangement in the MSNs in biologically relevant conditions. Small Angle X-ray scattering (SAXS) studies were performed on unmodified and PEGylated MSNs with a MCM-48 pore structure and average sizes of 140 nm, exposed to simulated body fluid solution (SBF) at pH 7.4 for different time intervals from 30 min to 24 h. Experiments were performed with silica concentrations below, at and over 0.14 mg/mL, the saturation concentration of silica in water at physiological temperature. At silica concentrations of 1 mg/mL (oversaturation), unmodified MSNs show variation in interpore distances over 6 h exposure to SBF, remaining constant thereafter. A decrease in radius of gyration is observed over the same time. Mesoporosity and radius of gyration of unmodified MSNs remain then unchanged up to 24 h. PEGylated MSNs at 1 mg/mL concentration show a broader diffraction peak but no change in the position of the peak is observed following 24 h exposure to SBF. PEGylated MSNs at 0.01 mg/mL show no diffraction peaks already after 30 min exposure to SBF, while at 0.14 mg/mL a small diffraction peak is present after 30 min exposure but disappears after 1 h.
RESUMO
Aim: To develop a new curcumin carrier consisting of murumuru butter nanoparticles (SLN-Cs). Methods: A phase-inversion temperature method was used to produce SLN-Cs. The interaction of SLN-Cs with murine colon adenocarcinoma (CT26) cells in vitro was analyzed by confocal microscopy. Results: Stable SLN-Cs with a high curcumin-loading capacity were obtained. The SLN-Cs were more toxic to CT26 than free curcumin. Fluorescence microscopy images showed the SLN-Cs to be taken up by CT26 cells in vitro. Conclusion: These results indicate that SLN-Cs are suitable carriers of curcumin in aqueous media.
Assuntos
Curcumina , Nanopartículas , Animais , Portadores de Fármacos , Lipídeos , Lipossomos , Camundongos , Nanopartículas/toxicidade , Tamanho da PartículaRESUMO
HYPOTHESIS: Polarity in polyelectrolyte multilayers (PEMs) may vary from the inner to the top layers of the film as the charge compensation of the layers is more effective inside the PEMs than in outer layers. Doxorubicin hydrochloride (DX) is used here to sense polarity at the single polyelectrolyte level inside PEMS. EXPERIMENTAL: DX is complexed electrostatically to a polyanion, either polystyrene sulfonate (PSS) or polyacrylic acid (PAA) and assembled at selected positions in a multilayer of the polyanion and polyallylamine hydrochloride (PAH) as polycation. Local polarity in the layer domain is evaluated through changes in the intensity ratio of the first to second band of spectra of DX (I1/I2 ratio) by steady state fluorescence, and by Lifetime fluorescence. FINDINGS: PAH/PSS multilayers, show a polarity similar to water with DX/PSS as top layer, decreasing to I1/I2 ratios similar to organic solvents as the number of polyelectrolyte layers assembled on top increases. For PAH/PAA multilayers, polarity values reflect a more polar environment than water when DX/PAA is the top layer, remaining unaltered by the assembly of polyelectrolyte layers on top. Results show that different polar environments may be present in a PEM when considering polarity at the single layer level.
Assuntos
Doxorrubicina , Água , Fluorescência , Fenômenos Físicos , PolieletrólitosRESUMO
An approach for reducing toxicity and enhancing therapeutic potential of supramolecular polyamine phosphate nanoparticles (PANs) through PEGylation of polyamines before their assembly into nanoparticles is presented here. It is shown that the number of polyethylene glycol (PEG) chains for polyamine largely influence physico-chemical properties of PANs and their biological endpoints. Poly(allylamine hydrochloride) (PAH) are functionalized through carbodiimide chemistry with three ratios of PEG molecules per PAH chain: 0.1, 1, and 10. PEGylated PAH is then assembled into PANs by exposing the polymer to phosphate buffer solution. PANs decrease size and surface charge with increasing PEG ratios as evidenced by dynamic light scattering and zeta potential measurements, with the ten PEG/PAH ratio PANs having practically zero charge. Small angle X-ray scattering (SAXS) proves that PEG chains form a shell around a polyamine core, which is responsible for the screening of positive charges. MTT experiments show that the screening of amine groups decreases nanoparticle toxicity, with the lowest toxicity for the 10 PEG/PAH ratio. Fluorescence correlation spectroscopy (FCS) proves less interaction with proteins for PEGylated PANs. Positron emission tomography (PET) imaging of 18 F labelled PANs shows longer circulation time in healthy mice for PEGylated PANs than non-PEGylated ones.
Assuntos
Nanopartículas , Fosfatos , Animais , Camundongos , Nanopartículas/toxicidade , Poliaminas/toxicidade , Polietilenoglicóis , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
Supported Lipid Bilayers (SLBs) on Polyelectrolyte Multilayers (PEMs) have large potential as models for developing sensor devices. SLBs can be designed with receptors and channels, which benefit from the biological environment of the lipid layers, to create a sensing interface for ions and biomarkers. PEMs assembled by the Layer-by-Layer (LBL) technique and used as supports for a lipid bilayer enable an easy integration of the bilayer on almost any surface and device. For electrochemical sensors, LBL assembly enables nanoscale tunable separation of the lipid bilayer from the electrode surface, avoiding undesired effects of the electrode surface on the lipid bilayers. We study the fabrication of valinomycin-doped SLBs on PEMs as a model system for biophysical studies and for selective ion sensing. SLBs are fabricated from dioleoylphosphatidylcholine (DOPC) and dioleoylphosphatidylserine (DOPS) 50:50 vesicles doped with valinomycin, as a K+-selective carrier. SLBs were deposited on electrodes coated with poly(allyl amine hydrochloride) (PAH) and poly(styrene sodium sulfonate) (PSS) multilayers. Lipid bilayer formation was monitored by using Quartz Crystal Microbalance with Dissipation (QCMD) technique and Atomic Force Microscopy (AFM). Electrochemical impedance spectroscopy (EIS) and potentiometric measurements were performed to assess K+ selectivity over other ions and the potential of valinomycin-doped SLBs for K+-sensing.
Assuntos
Eletricidade , Bicamadas Lipídicas/química , Polieletrólitos/química , Valinomicina/química , Eletrodos , Fosfatidilcolinas/química , Propriedades de SuperfícieRESUMO
Mechanochemistry is an emerging and reliable alternative to conventional solution (batch) synthesis of complex molecules under green and solvent-free conditions. In this regard, we report here on the conjugation of a dextran polysaccharide with a fluorescent probe, a phenylboronic acid (PBA)-functionalized boron dipyrromethene (BODIPY) applying the ball milling approach. The ball milling formation of boron esters between PBA BODIPY and dextran proved to be more efficient in terms of reaction time, amount of reactants, and labelling degree compared to the corresponding solution-based synthetic route. PBA-BODIPY dextran assembles into nanoparticles of around 200 nm by hydrophobic interactions. The resulting PBA-BODIPY dextran nanoparticles retain an apolar interior as proved by pyrene fluorescence, suitable for the encapsulation of hydrophobic drugs with high biocompatibility while remaining fluorescent.
RESUMO
HYPOTHESIS: Doxorubicin hydrochloride (DX) is widely used as a chemotherapeutic agent, though its severe side-effects limit its clinical use. A way to overcome these limitations is to increase DX latency through encapsulation in suitable carriers. However, DX has a high solubility in water, hindering encapsulation. The formulation of DX with sodium cholate (NaC) will reduce aqueous solubility through charge neutralization and hydrophobic interactions thus facilitating DX encapsulation into poloxamer (F127) micelles, increasing drug latency. EXPERIMENTS: DX/NaC/PEO-PPO-PEO triblock copolymer (F127) formulations with high DX content (DX-PMs) have been prepared and characterized by scattering techniques, transmission electron microscopy and fluorescence spectroscopy. Cell proliferation has been evaluated after DX-PMs uptake in three cell lines (A549, Hela, 4T1). Cell uptake of DX has been studied by means of confocal laser scanning microscopy and flow cytometry. FINDINGS: DX-PMs formulations result in small and stable pluronic micelles, with the drug located in the apolar core of the polymeric micelles. Cell proliferation assays show a delayed cell toxicity for the encapsulated DX compared with the free drug. Data show a good correlation between cytotoxic response and slow DX delivery to nuclei. DX-PMs offer the means to restrict DX delivery to the cell interior in a highly stable and biocompatible formulation, suitable for cancer therapy.
Assuntos
Micelas , Poloxâmero , Disponibilidade Biológica , Doxorrubicina/farmacologia , Polietilenoglicóis , Colato de SódioRESUMO
The deposition of amyloid-ß (Aß) plaques in the brain is a significant pathological signature of Alzheimer's disease, correlating with synaptic dysfunction and neurodegeneration. Several compounds, peptides, or drugs have been designed to redirect or stop Aß aggregation. Among them, the trideca-peptide CWG-LRKLRKRLLR (mApoE), which is derived from the receptor binding sequence of apolipoprotein E, is effectively able to inhibit Aß aggregation and to promote fibril disaggregation. Taking advantage of Atomic Force Microscopy (AFM) imaging and fluorescence techniques, we investigate if the clustering of mApoE on gold nanoparticles (AuNP) surface may affect its performance in controlling Aß aggregation/disaggregation processes. The results showed that the ability of free mApoE to destroy preformed Aß fibrils or to hinder the Aß aggregation process is preserved after its clustering on AuNP. This allows the possibility to design multifunctional drug delivery systems with clustering of anti-amyloidogenic molecules on any NP surface without affecting their performance in controlling Aß aggregation processes.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Apolipoproteínas E/metabolismo , Nanopartículas Metálicas/química , Fragmentos de Peptídeos/metabolismo , Doença de Alzheimer/metabolismo , Amiloide/metabolismo , Encéfalo/metabolismo , Análise por Conglomerados , Ouro/química , Humanos , Placa Amiloide/metabolismo , Ligação Proteica/fisiologiaRESUMO
The interaction of polyamine poly(allylamine hydrochloride) with Na3PO4, Na4P2O7, Na5P3O10, Na6P6O18, and (NaPO3)26 salts and the formation of polyamine phosphate nanoparticles (PANs) are studied here. Dynamic light scattering, isothermal titration calorimetry (ITC), electrophoretical mobility measurements, atomic force microscopy, and transmission electron microscopy are used to explore the formation, stability, and pH sensitivity of PANs. An optimal concentration for PAN formation is found for each phosphate salt in terms of the most stable size and lowest polydispersity index of the nanoparticles. The minimal concentration of phosphate ions for PAN formation decreases with the increasing number of phosphate groups per phosphate salt. ITC measurements show that all polyphosphates display a characteristic endothermic peak, which is not present when monophosphates are used for PAN formation. pH stability of PANs depends on the type of phosphate salt. PANs formed with small phosphates show a small window of stability with pH from 8 to 9, while those formed with long phosphates are stable in more acidic pH environments. Our findings open multiple possibilities for fine-tuning the pH sensitivity of PANs by varying phosphate salts for potential applications in drug delivery.
RESUMO
Silencing RNA (siRNA) technologies attract significant interest as a therapeutic tool for a large number of diseases. However, the medical translation of this technology is hampered by the lack of effective delivery vehicles for siRNAs in cytosol that prevent their degradation in the bloodstream. The use of molecular complexes based on polyamines have great potential for siRNA delivery as polyamines can protect the siRNA during circulation and at the same time favor siRNA translocation in cytosol. Here, nanoparticles are prepared by complexation of poly(allylamine hydrochloride) (PAH) and siRNA varying the ratio of nitrogen groups from PAH to phosphate groups from siRNA (N/P ratio). Nanoparticles are characterized by transmission electron microscopy and dynamic light scattering. The stability of complexes of green rhodamine labelled PAH (G-PAH) and Cy5 labelled siRNA (R-siRNA) at different pHs and in cell media is studied by fluorescence cross-correlation spectroscopy (FCCS). FCCS studies show that the nanoparticles are stable at physiological pH and in cell media but they disassemble at acidic pH. An optimal N/P ratio of 2 is identified in terms of stability in media, degradation at endosomal pH and toxicity. The intracellular fate of the complexes is studied following uptake in A549 cells. The cross-correlation between G-PAH and R-siRNA decreases substantially 24â¯h after uptake, while diffusion times of siRNA decrease indicating that the complexes disassemble, liberating the siRNAs. The release of siRNAs into the cytosol is confirmed with parallel confocal laser scanning microscopy. Flow cytometry studies show that PAH/siRNA nanoparticles are effective at silencing green fluorescent protein expression at low N/P ratios at which polyethylenimine/siRNA shows no significant silencing.
Assuntos
Nanopartículas/química , Poliaminas/química , RNA Interferente Pequeno/química , RNA Interferente Pequeno/metabolismo , Células A549 , Permeabilidade da Membrana Celular , Sobrevivência Celular , Citosol/metabolismo , Corantes Fluorescentes/química , Proteínas de Fluorescência Verde/genética , Humanos , Concentração de Íons de Hidrogênio , Imagem Óptica , Polietilenoimina/química , RNA Interferente Pequeno/genética , TransfecçãoRESUMO
The surface of proteins is heterogeneous with sophisticated but precise hydrophobic and hydrophilic patches, which is essential for their diverse biological functions. To emulate such distinct surface patterns on macromolecules, we used rigid spherical synthetic dendrimers (polyphenylene dendrimers) to provide controlled amphiphilic surface patches with molecular precision. We identified an optimal spatial arrangement of these patches on certain dendrimers that enabled their interaction with human adenovirus 5 (Ad5). Patchy dendrimers bound to the surface of Ad5 formed a synthetic polymer corona that greatly altered various host interactions of Ad5 as well as in vivo distribution. The dendrimer corona (1) improved the ability of Ad5-derived gene transfer vectors to transduce cells deficient for the primary Ad5 cell membrane receptor and (2) modulated the binding of Ad5 to blood coagulation factor X, one of the most critical virus-host interactions in the bloodstream. It significantly enhanced the transduction efficiency of Ad5 while also protecting it from neutralization by natural antibodies and the complement system in human whole blood. Ad5 with a synthetic dendrimer corona revealed profoundly altered in vivo distribution, improved transduction of heart, and dampened vector sequestration by liver and spleen. We propose the design of bioactive polymers that bind protein surfaces solely based on their amphiphilic surface patches and protect against a naturally occurring protein corona, which is highly attractive to improve Ad5-based in vivo gene therapy applications.
Assuntos
Adenovírus Humanos/genética , Dendrímeros/farmacologia , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Transdução Genética , Adenovírus Humanos/efeitos dos fármacos , Animais , Proteínas do Capsídeo/química , Dendrímeros/química , Vetores Genéticos/química , Vetores Genéticos/efeitos dos fármacos , Humanos , Interações Hidrofóbicas e Hidrofílicas/efeitos dos fármacos , Fígado/química , Fígado/efeitos dos fármacos , Polímeros/química , Polímeros/farmacologia , Receptores Virais/antagonistas & inibidores , Receptores Virais/químicaRESUMO
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease affecting motor neurons. In ALS mice, neurodegeneration is associated with the proliferative restorative attempts of ependymal stem progenitor cells (epSPCs) that normally lie in a quiescent in the spinal cord. Thus, modulation of the proliferation of epSPCs may represent a potential strategy to counteract neurodegeneration. Recent studies demonstrated that FM19G11, a hypoxia-inducible factor modulator, induces epSPC self-renewal and proliferation. The aim of the study was to investigate whether FM19G11-loaded gold nanoparticles (NPs) can affect self-renewal and proliferation processes in epSPCs isolated from G93A-SOD1 mice at disease onset. We discovered elevated levels of SOX2, OCT4, AKT1, and AKT3, key genes associated with pluripotency, self-renewal, and proliferation, in G93A-SOD1 epSPCs at the transcriptional and protein levels after treatment with FM19G11-loaded NPs. We also observed an increase in the levels of the mitochondrial uncoupling protein (UCP) gene in treated cells. FM19G11-loaded NPs treatment also affected the expression of the cell cycle-related microRNA (miR)-19a, along with its target gene PTEN, in G93A-SOD1 epSPCs. Overall our findings establish the significant impact of FM19G11-loaded NPs on the cellular pathways involved in self-renewal and proliferation in G93A-SOD1 epSPCs, thus providing an impetus to the design of novel tailored approaches to delay ALS disease progression.
Assuntos
Benzamidas/farmacologia , Autorrenovação Celular/efeitos dos fármacos , Epêndima/citologia , Ouro/química , Nanopartículas Metálicas/química , Células-Tronco/citologia , Esclerose Lateral Amiotrófica , Animais , Biomarcadores/metabolismo , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos Transgênicos , MicroRNAs/genética , MicroRNAs/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Células-Tronco Pluripotentes/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Células-Tronco/efeitos dos fármacos , Superóxido Dismutase-1/metabolismo , Proteína Desacopladora 2/metabolismoRESUMO
Polyamine Phosphate Nanoparticles (PANs) have great potential for the delivery of large therapeutics, such as plasmids and/or siRNAs. The formation of PANs by complexation of Poly(allylamine hydrochloride) (PAH) and phosphate ions from Phosphate Buffer (PB) was studied here, and how it is affected by the presence of phosphate ions from PB and ionic strength. From Transmission Electron Microscopy (TEM) and Dynamic Light Scattering (DLS) the critical PB concentration for PANs formation was determined. Below this critical point, Small Angle X-ray Scattering (SAXS) studies revealed that small PAH-phosphate aggregates coexist with not complexed or weakly complexed polymer chains in solution and that the presence of the phosphate ions increases the Kuhn length of the polymer chains until that only spherical aggregates are present in solution. TEM, DLS and SAXS showed the increase of PANs size with ionic strength up to 250â¯mM NaCl. At higher NaCl concentrations, PANs disassemble into smaller aggregates. Isothermal Titration Calorimetry (ITC) showed that PAN formation is an exothermic process and the association of phosphates below the critical PB concentration is entropically controlled.