1,720,984 research outputs found
Biodegradable nanoparticles for restenosis following PTA: a feasibility study
No full textINTRODUCTION
Percutaneous transluminal angioplasty (PTA) is known to effectively improve the prognosis of patients with vascular diseases1. However, poor reendothelialization, and excessive migration and proliferation of vascular smooth muscular cells in the tunica media, can result in obstructive neointimal hyperplasia, and are the major mechanisms involved in restenosis following PTA2. Many animal studies indicate that local delivery of intimal hyperplasia inhibiting drugs can enhance vascular reendothelialization and prevent restenosis2, although this remains controversial. This may be due to inadequate drug concentrations or to the short period that the effective drug concentrations is available locally. The local administration of drug by biodegradable nanoparticles could enhance the drug concentrations in vessel wall and, thereby, yield the desired therapeutic effects. Recently, anti-oxidants (AA) grafted on poly(lactide-co- glycolide) (g-AA-PLGA) were proposed as novel biodegradable materials stable to gamma irradiation, characterized by different surface properties and suitable for drug delivery3,4. In this work, we investigated the feasibility to prepare g-AA-PLGA nanoparticles as carriers for the local administration of drugs in the vessel wall during PTA. To achieve this goal, the rate and extent of cellular uptake of such nanoparticles in comparison with the naïve PLGA was studied using macrophages, smooth muscular and endothelial cells as representative of the cells present in the vessel wall.
EXPERIMENTAL SET-UP
g-AA-PLGA synthesis and characterization
Caffeic acid (CA) or resveratrol (RV) were grafted to PLGA (L/G ratio 50/50, Mw=26 KDa, Tg=47.1±0.5 °C) by a free radicals-induced strategy3,4. g-AA-PLGA were characterized by the DPPH assay, GPC analysis and DSC.
Nanoparticles preparation and characterization
Surfactant-free nanoparticles (NPs) were prepared by the solvent displacement method, adding dropwise 1 mL of 1% polymeric solution in organic solvent to 10 mL of MilliQ® water. Hydrodynamic diameter (Dh) and zeta potential () were determined by a Zetasizer Nano ZS (Malvern Instrument, UK). Fluorescent NPs with 10% w/w of a FITC- PLGA conjugate were also prepared.
Cellular uptake and exocytosis
Macrophages, smooth muscular and endothelial cells were exposed to the fluorescent nanosuspension (100 μg/mL) over a 24 h period. At predetermined times, media were removed and NP uptake was determined by measuring the intracellular fluorescence. The uptake efficiency was calculated normalizing the observed fluorescence intensity in each well (IOBS) for the mean fluorescence intensity of the negative control (INC) according to equation 1:
Uptake efficiency = (IOBS - INC)/INC eq.1
The exocytosis of nanoparticles was followed for up to 7 days. The absolute number of detected photons (i.e. the derived count rate, DCR), Dh and correlogram shape were considered to qualitatively establish NPs released by the cells. The cytotoxicity of the tested formulation was also evaluated by the MTT assay5.
Statistical analysis
One-way and two-way ANOVA followed by Tukey’s test (=0.05) were performed using OriginPro 2015 (USA). Outliers were discarded according to Dixon’s T-test.
RESULTS AND DISCUSSION
Polymer synthesis and characterization
g-AA-PLGA were synthesized by performing a two-step grafting procedure at room temperature. In the former step, the oxidation of ascorbic acid by H2O2 led to the formation of hydroxyl radicals able to activate the PLGA backbone; then, the reactive sites on the preformed PLGA macroradicals react with the AA molecules resulting in CA or RV insertion. This approach avoids the degradation of the AA due to high temperature and the generation of toxic by- products. Both types of PLGA based conjugates exhibited good free radical scavenging properties since the DPPH inhibition after 1 h resulted 90.0±0.4 and 27.0±0.8 % for g- CA-PLGA and g-RV-PLGA, respectively; while the naïve PLGA was ineffective. Moreover, the grafting procedure did not affect the main physico-chemical properties of PLGA, since no significantly variations in Mw and Tg occurred in both g-AA-PLGA.
Nanoparticles characterization
Independently of the polymer used, monodispersed NPs were obtained (Table 1). The addition of 10% FITC-PLGA conjugate did not significantly modify the main features of NPs. Moreover, the MTT assay demonstrated the absence of toxicity of g-AA-PLGA (100 μg/mL) compared to the naïve PLGA.
Cellular uptake and exocytosis evaluation
Macrophages took up NPs faster than the other cell lines probably due to their physiological phagocytic activity. Indeed, in these cells no statistical differences in NP uptake were detected considering the materials and the incubation time as factors (two-way ANOVA, p=0.05).
In the case of endothelial cells, the uptake of g-CA-PLGA NPs was significantly higher than that of g-RV-PLGA and PLGA (two-way ANOVA: g-CA-PLGA vs PLGA p=0.028 and g-CA-PLGA vs g-RV-PLGA p=0.032).
Moreover, g-RV-PLGA and PLGA NPs were similarly taken up (p>0.05). Lastly, regarding smooth muscular cells, the uptake of g-CA-PLGA NPs occurred faster compared to the other NPs (one-way ANOVA: g-CA-PLGA vs PLGA p=0.002; g-CA-PLGA vs g-RV-PLGA p=0.004 and g-RV- PLGA vs PLGA p=0.984). As an example, Figure 1 represents the uptake efficiency of the cells after 4 h of exposition to NPs.
Furthermore, we observed a time-dependent reduction of the amount of intracellular fluorescent g-CA-PLGA NPs, indicating that this type of NPs may be also released by cells. The DLS analysis suggested that the release of NPs occurred within 8 h and 48 h in macrophages and smooth muscular cells, respectively. At both time periods, the DLS correlation functions maintained the sigmoidal shape and the Dh of the NPs was around 200 nm. These data were in agreement with the images obtained by fluorescence microscopy.
CONCLUSION
All together, these results on cellular uptake and exocytosis demonstrate that g-CA-PLGA nanoparticles may be a suitable carrier to locally administer drugs used in the prevention of restenosis following PTA.
REFERENCES
1. Singh, M.; Rihal, C.S.; Berger, P.B.; Bel, M.R.; Grill, D.E.; Garratt, K.N.; Barsenes, G.W. and Holmes, D.R. Improving outcome over time of percutaneous coronary interventions in unstable angina. J. Am. Coll. Cardiol. 36, 674-678 (2000).
2. Jukema, J.W.; Verschuren, J.J.; Ahmed, T.A. and Quax P.H. Restenosis after PCI. Part 1: pathophysiology and risk factors. Nat. Rev. Cardiol. 9, 53–62 (2001).
3. Cilurzo, F.; Puoci, F.; Selmin, F.; Iemma, F. and Minghetti, P. Pyrogallic acid-PLGA conjugate as new biodegradable material suitable for final sterilization by irradiation. Polym. Adv. Technol. 22, 2201–2205 (2011).
4. Selmin, F.; Puoci, F.; Parisi, O.I.; Franzé, S.; Musazzi, U.M. and Cilurzo, F. Caffeic acid-PLGA conjugate to design protein drug delivery systems stable to irradiation. J. Funct. Biomater. 6, 1-13 (2015).
5. Denizot,F.andLang,R.Rapidcolorimetricassayforcell growth and survival. Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability. J. Immunol. Methods 89(2), 271-277 (1986)
Pyrogallic acid-PLGA conjugate as new biodegradable material suitable for final sterilization by irradiation
No full textThis paper reports the feasibility to prepare a biodegradable material stable to γ-irradiation by grafting of poly(lactide-co-glycolide) with pyrogallic acid (PLGA-g-PA) and ferulic acid (PLGA-g-FA) in mild condition. Only the grafting procedure with PA did not modify molecular weight (Mw) of the starting polymer and PLGA-g-PA showed antioxidant properties. The polymer degradation in pH 7.4 phosphate buffer saline (PBS) was mainly governed by a random chain scission mechanism according to a first-order reaction. The irradiation at the dose of 25kGy caused only a very slight decrease of Mw and the degradation patterns of the non-irradiated and irradiated material were superimposable. PLGA-g-PA resulted a promising material to develop biodegradable drug delivery systems which would be sterilizable in the final container
Selective extraction and purification of gallic acid from actual site olive mill wastewaters by means of molecularly imprinted microparticles
No full textAn innovative biocompatible approach for the selective recovery of gallic acid (GA) from actual site olive mill wastewaters (OMWs) by means of Molecularly Imprinted Polymers (MIPs) was developed. Bulky MIPs with a high selectivity for GA were synthesized by a non-covalent approach using methacrylic acid as the functional monomer and ethylene glycol dimethacrylate as the crosslinker. Imprinting efficiency was evaluated in different water/ethanol solutions by determining a and e parameter, by using pyrogallic acid as the template analogue: the highest GA recognition, both in terms of percentage and of imprinting effect (a = 32.7; e = 4.2), was observed in pure ethanol. The efficiency of the developed extraction protocol was evaluated with an actual site OMW: a selective extraction of 80% of pure GA was achieved with high accuracy and precision, as shown by RSD% (4.3%) and recovery values (between 85% and 97%)
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Sintesi di monomeri vinilici contenenti residui 4-piridincarbonitrilici e loro trasformazione in sistemi polimerici idrofili quali modelli macromolecolari di inibitori delle amminossidasi contenenti rame
No full text- …
