Exogenous Control over Intracellular Acidification: Enhancement via Proton Caged Compounds Coupled to Gold Nanoparticles

The pH regulation has a fundamental role in several intracellular processes and its variation via exogenous compounds is a potential tool for intervening in the intracellular processes. Proton Caged Compounds (PPCs) release protons upon UV irradiation and may efficiently provoke intracellular on-command acidification. Here, we explore the intracellular pH variation, when purposely synthesized PCCs are coupled to gold nanoparticles (AuNPs) and dosed to HEK-293 cells. We detected the acidification process caused by the UV irradiation by monitoring the intensity of the asymmetric stretching mode of the CO2 molecule at 2343 cm-1. The comparison between free and AuNPs functionalized proton caged compound demonstrates a highly enhanced CO2 yield, hence pH variation, in the latter case. Finally, PCC functionalized AuNPs were marked with a purposely synthesized fluorescent marker and dosed to HEK-293 cells. The corresponding fluorescence optical images show green grains throughout the whole cytoplasm

Synthesis of proton caged disulphide compounds for gold nanoparticles functionalization

The pH plays a fundamental role in many biological systems, and it is important to be capable of monitoring and manipulating it. A method for intracellular pH control has been recently developed based on a proton caged compound (PCC), which releases protons upon irradiation with light of proper wavelength. Intracellular modulation of PCC uptake can be achieved by gold nanosized vectors. This, however, is conditioned by the possibility of conjugating PCCs and vectors. Here, we present the synthesis of purposely designed proton caged disulphide sulphonyl urethanes, which bind gold nanoparticles through the disulphide bridge and display photoreactivity through an o-nitrophenyl moiety. The new compounds have been characterized by FTIR, 1H NMR, TEM and TGA and their photoreactivity in the UV range has been probed, after functionalizing them with gold nanoparticle

Synthesis of spiropyran for gold and silver nanoparticles functionalization

The exogenous control of intracellular properties has a fundamental role in medical treatments, such as cancer photodynamic therapy, where photosensitizers are dosed or accumulated into cells and afterwards “turned on” via selective photoexcitation. Furthermore, the association of photosensitizers with gold nanoparticles (AuNPs) enhances the photodynamic effect. The pH regulation has a fundamental role in several intracellular processes and its variation via exogenous compounds is a potential tool for intervening in the intracellular mechanism. In a recent research we showed both that an exogenous control of pH is possible via purposely synthesized proton caged compounds [ , ] and that the intracellular acidification in enhanced by the association of AuNPs. Another class of pH regulating compounds of potential interest for in vitro experiments are spiropyrans and spirooxazines, because of their reversible ring-opening to the corresponding merocyanines with associated proton caption or release, depending on the direction of the following reaction . Here we report the synthesis of the photoacid 1,9-(Mercaptononyl)-3’,3’-dimethyl-6’-nitrospiro[2H-1-benzopyran-2,2’-(2H) indole], a variant of spiropyrans, functionalized with a thiol group for further functionalization of gold and silver nanoparticles

Synthesis of spiropyran for gold and silver nanoparticles functionalization

The exogenous control of intracellular properties has a fundamental role in medical treatments, such as cancer photodynamic therapy, where photosensitizers are dosed or accumulated into cells and afterwards “turned on” via selective photoexcitation. Furthermore, the association of photosensitizers with gold nanoparticles (AuNPs) enhances the photodynamic effect. The pH regulation has a fundamental role in several intracellular processes and its variation via exogenous compounds is a potential tool for intervening in the intracellular mechanism. In a recent research we showed both that an exogenous control of pH is possible via purposely synthesized proton caged compounds [ , ] and that the intracellular acidification in enhanced by the association of AuNPs. Another class of pH regulating compounds of potential interest for in vitro experiments are spiropyrans and spirooxazines, because of their reversible ring-opening to the corresponding merocyanines with associated proton caption or release, depending on the direction of the following reaction . Here we report the synthesis of the photoacid 1,9-(Mercaptononyl)-3’,3’-dimethyl-6’-nitrospiro[2H-1-benzopyran-2,2’-(2H) indole], a variant of spiropyrans, functionalized with a thiol group for further functionalization of gold and silver nanoparticles

Synthesis of caged proton disulphide compounds for gold nano particles functionalization

Synthesis of proton releasing compound, 1-(2-nitrophenyl)-carboxysulphamoyl-9-ennyldisulphide, and coupling of this Proton Caged Compounds (PCC) with gold nanoparticles (AuNPs) to allow the compound to be vehicle in cellular medium

Probing exogenous-induced intracellular acidification: the effects of the incubation time

A very important characteristic in the chemistry of molecules interesting for planetary ionospheres is that they interact with the electromagnetic waves: γ, X rays and ultraviolet (UV) light. The dissociative double photoionization processes induced by VUV and EUV photons leading to the production of fragment ions with a high kinetic energy content could give in general an important contribution to the ion species escape from the atmosphere of some planets of the Solar System, like Venus, Mars and Titan. In fact, these processes occur via formation of intermediate molecular dications that can dissociate by Coulomb explosion towards the formations of two ion fragments having a kinetic energy released (KER) of several eV, and therefore much larger than the limiting thermal escape velocity. When CO2, C2H2 and N2O molecules are ionized using photons with an energy in the range of 30 to 65 eV, molecular fragmentation can be induced, with several two-body dissociation reactions leading to ion final products with high kinetic energy content [1,2]. These ionic species (CO+, O+, N2 +, N+, NO+, H+, C+, CH+, CH2 + and C2H+) are formed by Coulomb explosion of an intermediate molecular dication (CO2 2+, N2O2+ and C2H2 2+, respectively). The fragments are characterized by a translational energy ranging between 1.0 and 5.5 eV (only for H+, the measured KER reaches the maximum value of 6.0 eV), which is large enough to allow some of them to escape from the atmospheres of Mars and Titan. In the case of O+, we can conclude that the dissociative double photoionization of CO2 induced by VUV and EUV photons in the ionosphere of Mars can contribute to explain the observed behavior of the O+ ion density profile measured by the Viking 1 lander in the upper atmosphere of this planet, compared with the CO2 2+ density profile as calculated for the Viking 1 lander and Mariner 6 spacecraft geophysical conditions [1]

Probing exogenous-induced intracellular acidification: the effects of the incubation time

A very important characteristic in the chemistry of molecules interesting for planetary ionospheres is that they interact with the electromagnetic waves: γ, X rays and ultraviolet (UV) light. The dissociative double photoionization processes induced by VUV and EUV photons leading to the production of fragment ions with a high kinetic energy content could give in general an important contribution to the ion species escape from the atmosphere of some planets of the Solar System, like Venus, Mars and Titan. In fact, these processes occur via formation of intermediate molecular dications that can dissociate by Coulomb explosion towards the formations of two ion fragments having a kinetic energy released (KER) of several eV, and therefore much larger than the limiting thermal escape velocity. When CO2, C2H2 and N2O molecules are ionized using photons with an energy in the range of 30 to 65 eV, molecular fragmentation can be induced, with several two-body dissociation reactions leading to ion final products with high kinetic energy content [1,2]. These ionic species (CO+, O+, N2 +, N+, NO+, H+, C+, CH+, CH2 + and C2H+) are formed by Coulomb explosion of an intermediate molecular dication (CO2 2+, N2O2+ and C2H2 2+, respectively). The fragments are characterized by a translational energy ranging between 1.0 and 5.5 eV (only for H+, the measured KER reaches the maximum value of 6.0 eV), which is large enough to allow some of them to escape from the atmospheres of Mars and Titan. In the case of O+, we can conclude that the dissociative double photoionization of CO2 induced by VUV and EUV photons in the ionosphere of Mars can contribute to explain the observed behavior of the O+ ion density profile measured by the Viking 1 lander in the upper atmosphere of this planet, compared with the CO2 2+ density profile as calculated for the Viking 1 lander and Mariner 6 spacecraft geophysical conditions [1]

PROTON CAGED COMPOUNDS FOR INTRACELLULAR pH MONITORING

Intracellular pH plays a pivotal role in cellular processes and is highly regulated in every organelle. The structural stability and function of proteins are tightly associated with pH. Furthermore, cell cycle progression and programmed cell death have both been linked to changes in intracellular pH. Probing intracellular pH usually requires exogenous agents, such as fluorescent probes1 or Raman sensitive probes2. We have developed a new type of exogenous probe, based on Proton Caged Compounds (PCCs), which releases protons upon UV irradiation. In particular, we have purposely designed and synthesized a long alkyl-chained PCC, the 1-(2-nitrophenyl)- ethylhexadecyl sulfonate (HDNS), and probed it on 3T3-NIH cells, via infrared spectroscopy3. The light-induced pH variation may easily be monitored by infrared spectroscopy, since the release of protons acts on the equilibrium of the intracellular HCO3- . The generated H2CO3 decomposes yielding H2O and CO2, which has a characteristic signal in a clear region of the cellular infrared spectrum. Here, we report on a different mechanism of protonation for long-incubation of HDNS on 3T3-NIH cells as probe of the intracellular acidification. When the PCC is doses for more than 3 hours, the accumulation into the cells leads to a protonation of intracellular carboxylic groups upon irradiation, which is still a fingerprint of the acidification process and a probe of the intracellular pH

Type 1 diabetes mellitus and celiac disease: endothelial dysfunction

Many reports indicate a hypercoagulative state in diabetes mellitus as result of endothelial damage. Experimental evidence suggests that a metabolic derangement triggers a cascade of biochemical events that lead to vascular dysfunction. The net effect is to convert the endothelium from thromboresistant to thrombogenic surface. In literature, a strong association between type 1 diabetes mellitus (DM1) and celiac disease (CD) has been reported. We do not have information about the hemostatic system in these associated conditions. Our study aims at evaluating whether the presence of CD in a group of DM1 patients is associated with a different expression of some hemostatic factors and with a different manifestation and/or progression of microvascular complications of DM1 in comparison with patients with only diabetes. Ninety-four adult DM1 patients were enrolled in the study and subsequently screened for CD. Anti-endomysial antibodies (EMA) were positive in 13 of 94 DM1 patients (13.8%). CD diagnosis was confirmed by histology and organ culture. The mean age and duration of DM1 of patients also affected by CD were similar to those of only diabetic patients, but the metabolic control and the hemocoagulative parameters were significantly different between the two groups: DM1 patients also affected by CD presented significantly lower concentrations of glycosylated hemoglobin (HbA1c) (P < 0.05), cholesterol (P < 0.001), triglycerides (P < 0.001), factor VII antigen (FVII:ag) (P < 0.005), factor VII coagulant activity (FVII:c) (P < 0.05), and prothrombin degradation fragments (F1+2) (P < 0.001), as well as higher values of activated C protein (APC) (<0.001). No retinal abnormalities and no signs of renal damage were observed in DM1 patients also affected by CD. Our results suggest a potential protective role of CD in the prothrombotic state of DM1

Identification of a new coeliac disease subgroup: Antiendomysial and anti-transglutaminase antibodies of IgG class in the absence of selective IgA deficiency

Objective. The aim of the present study was to increase the sensitivity of the antiendomysial antibody (EMA) test by evaluating also EMAs of IgG1 isotype. Design and subjects. Over the last 2 years, serum EMAs IgA and IgG1 were determined in 1399 patients, referred to our gastrointestinal unit due to clinical suspicion of malabsorption. Serum anti-tissue transglutaminase (tTG) antibodies IgA and IgG, as well as total IgA levels, were also investigated. Furthermore. EMAs IgA and IgG1 were evaluated in biopsy culture supernatants. Biopsy specimens were also admitted to histological and immunohistochemical evaluation. Twenty-six patients with gastroenterological disease other than coeliac disease (CD) were used as a disease control group. Ninety-nine blood donors were used as a healthy control group. Results. Diagnosis of CD was based on histological findings in the 110/1399 patients showing EMA IgA positivity, and in a further 56/1 399 patients presenting both EMA IgA and IgG1 positivity in sera as well as in culture supernatants. Of the remaining 1233 EMA IgA-negative patients. 60 showed only EMA IgG1 positivity both in sera and in culture supernatants. It is noteworthy that anti-tissue transglutaminase antibodies IgG (anti-tTG) were positive in all 60 EMA IgG1-positive patients as well. By contrast, a selective IgA deficiency was found in only 11 out of the 60 EMA IgG1-positive patients. Villous height/crypt depth ratio was < 3:1 in 38 of the 60 EMA IgG1-positive patients (63.3%), whilst overexpression of ICAM-1 and CD25 was observed in all these patients. Conclusions. In this study, we observed a group of CD patients who were EMA IgG1-positive even in the absence of EMA IgA positivity and IgA deficiency. The diagnosis was based on the finding of the gluten-dependent clinical and histological features typical of CD. Data emerging from the present investigation thus suggest that the prevalence of CD should be reassessed and that the determination of EMA IgG1 could offer a new tool in the diagnostic armamentarium of CD