ASCORBIC ACID MODULATES SPONTANEOUS THYMOCYTE APOPTOSIS

The aim of the paper was to analyze the effect of various concentrations of ascorbic acid on spontaneous apoptosis of lymphocytes.About 58% of all thymocytes are subjected to a spontaneous apoptosis, after 24-hour cultivation in the complete medium. The number of apoptotic thymocytes was much lower in the culture with different concentrations of ascorbic acid. The most dramatic effect was detected in the culture with the highest concentration of ascorbic acid (10000 μg/ml). In this culture, the number of apoptotic thymocytes was about 32%. These results, compared with the results of spontaneous apoptosis, show that the most significant inhibition of apoptosis was detected in the culture with 10000 μg/ml of ascorbic acid. High concentration of ascorbic acid can inhibit spontaneous apoptosis of thymocytes. Such inhibition of T cell apoptosis, as the effector cells in immune system, can represent one of the major factors by which ascorbic acid influence the immune system

Procena stabilnosti emulzionih preparata za topikalnu primenu - vrednost dinamičkomehaničkog termoanalitičkog (DMTA) testa kao brze reološke alternative konvencionalnom testu smrzavanje-odmrzavanje

The assessment of stability in emulsion-based topical preparations can be approached through real-time monitoring and/or accelerated methods, drawing predictions from pertinent stability-related physicochemical parameters. Ensuring the robustness and durability of topical products during storage, transport, and application necessitates thorough stability testing. However, due to the diversity of emulsion types and their intended use, there is no universal standard test, placing the liability on formulators/manufacturer to tailor appropriate assessments. Notably, topical emulsions, particularly cosmetic variants, often exhibit impressive stability with extended shelf lives. Nonetheless, evaluating their stability and decision-making remain challenging and time-consuming in industrial contexts. This underscores the demand for alternative testing protocols that expedite stability assessments and predict emulsion-based product stability accurately. This article comprehensively surveys literature, enriched with practical insights, exploring core mechanisms behind emulsion stability and prevention of instability. The discussion encompasses diverse approaches to stability assessment, revealing methodologies and parameters under examination during testing. Particular focus is placed on the dynamic-mechanical thermoanalysis (DMTA) method explored as a rapid, rheologically-based alternative to the conventional freeze-thaw test, emphasizing its usefulness for expediting the stability evaluation of emulsion-based topical preparations.Procena stabilnosti emulzionih preparata za topikalnu primenu može se sprovesti praćenjem promena u realnom vremenu i/ili primenom ubrzanih metoda, te predviđanjem stabilnosti i roka trajanja proizvoda na osnovu merenja relevantnih fizičkohemijskih parametara tokom ispitivanja. Kako bi se obezbedila robusnost i dugoročnost emulzionih proizvoda za kožu tokom čuvanja, transporta i primene, neophodno je sprovesti pažljivo isplanirano, opsežno ispitivanje stabilnosti. Međutim, imajući u vidu različite tipove emulzija i njihovu namenu, ne postoji univerzalni standardni protokol za ispitivanje stabilnosti, što formulatore/proizvođača čini odgovornim kada je u pitanju izbor odgovarajućeg testa i metodologije. Evidentno je da emulzije za topikalnu primenu, a posebno kozmetičke emulzije, često pokazuju visoku stabilnost sa dugim rokovima upotrebe. S druge strane, procena stabilnosti ovakvih emulzija i donošenje odgovarajućih odluka i dalje ostaje izazov u industrijskom okruženju i zahteva dosta vremena, što nameće potrebu za alternativnim protokolima koji omogućavaju ubrzano ispitivanje, ali i uspešno predviđanje stabilnosti emulzionih proizvoda. Prikazani rad daje sveobuhvatni pregled literature prožet praktičnim pogledima na ključne fenomene odgovorne za stabilnost emulzija, zatim daje uvid u različite pristupe za procenu njihove stabilnosti, uključujući metodologije koje se koriste i parametre koji se prate tokom ispitivanja. Rad u poseban fokus stavlja dinamičko-mehanički termoanalitički (DMTA) metod kao brzu reološku alternativu konvencionalnom testu smrzavanje-odmrzavanje, posebno ističući primenljivost metoda za ubrzano ispitivanje stabilnosti emulzionih preparata za topikalnu primenu

Efficient Development of Green Emulsifier/Emollient-Based Emulsion Vehicles: From RSM Optimal Experimental Design to Abridged In Vivo Assessment

Since natural-origin, sustainable ingredients are preferred by modern consumers, novel emulsifiers and emollients keep entering the market. This study hypothesizes that a combination of in silico, instrumental tools and simplified sensory studies could be used to efficiently characterize emulsions in a shorter timeframe. A total of 22 rather simple o/w emulsions were prepared by a time/energy-saving emulsification process. A natural mixed emulsifier (Lauryl Glucoside/Myristyl Glucoside/Polyglyceryl-6 Laurate) and two emollients (both with INCI name C15–19 Alkane) were used. The performed D-optimal experimental design within the response surface method (RSM) significantly narrowed down the number of samples about to enter the stage of texture, friction and sensory studies to the samples comprising 30% of a respective Emogreen emollient and 2% or 3% of the emulsifier. The sample comprising 2% emulsifier/30% Emogreen® L15 showed significantly higher firmness (42.12 mN) when compared to the one with 2% emulsifier/30% Emogreen® L19 (33.62 mN), which was somewhat unexpected considering the emollients’ inherent viscosity values (4.5 mPa·s for L15 and 9 mPa·s for L19). The sample with 2% emulsifier/30% Emogreen® L19 managed to maintain the lowest friction, while the one with 3% emulsifier/30% Emogreen® L19 released its full lubricating potential in the second part of the measurement (30–60 s). The obtained results revealed the strengths and weaknesses of each formulation, narrowing down their possible applications in the early development stage

A proposal of innovative injectability assessment method for intravenous formulations - case study on PEGylated nanoemulsions

1. INTRODUCTION Syringeability and injectability are recognised as fundamental performance parameters / critical quality attributes of any parenteral dosage form. Syringeability refers to the ability of an injectable preparation to transfer from a vial through a hypodermic needle prior an injection, while injectability is defined as the force, or pressure, required to inject the formulation from a syringe-needle system into the tissue [1]. When developing drug delivery systems, the priority is usually the release kinetics, biocompatibility or other factors that may come in conflict with the optimal parameters for the applicability of those systems [2]. The aim of this research was to develop a method that could be used for injectability assessment of the intravenous formulations and the application of this method on curcumin-loaded PEGylated nanoemulsions (NEs) in order to gage the impact of PEGylation on NEs injectability. 2. MATERIALS AND METHODS 2.1. Nanoemulsion preparation Nanoemulsions were prepared using high pressure homogenization method. The aqueous phase (glycerol, polysorbate 80, sodium oleate and highly purified water) was added into the oil phase (soybean oil, soybean lecithin, medium chain triglycerides, butylhydroxytoluene, benzyl alcohol, curcumin and PEGylated phospholipid – PEG2000-DSPE in 0.1 %, 0.3 % or 0.6 % concentrations) and mixed using rotor-stator homogenizer (IKA Ultra-Turrax® T25 digital, IKA®-Werke GmbH and Co. KG, Staufen, Germany), and further processed on high pressure homogenizer (EmulsiFlex-C3, Avestin Inc., Canada) at 800 bar for 10 discontinued cycles. The non PEGylated formulation was marked as CS, and the PEGylated ones were marked S1, S3 and S6, referring to the PEG2000-DSPE concentration. 2.2. Physicochemical characterization The NEs droplet size (Z-Ave) and droplet size distribution (PDI) were determined with Zetasizer Nano ZS90 (Malvern Instruments Ltd., Worcestershire). Rheological analysis was performed using MCR 302 air-bearing rheometer (Anton Paar, Graz, Austria) equipped with coaxial cylinders system (CC27 measuring bob with C-PTD 180/Air) with sheer rate range of 0.1-100 s-1 at 20°C. 2.3. Injectabilty assesment The injectability of the NEs was expressed as force (N) needed to extrude the NE in the function of the extruded volume (ml). About 10 ml of the NE was loaded into the 10 ml syringe and extruded through the 25 G scalp vein infusion set (Romed, Wilnis, Netherlands) into the blood mimicking solution, circulating through pump at 4 ml/min, in order to assess the NEs’ performance in the prospective intravenous administration. The NEs were extruded at 1 mm/s croshead speed of the loading cell of the texure analyzer (EZ-LX Compact Table-Top Testing Machine, Shimadzu, Japan) with the TrapeziumX software version 1.5 used for data collection and analysis 3. RESULTS AND DISCUSSION 3.1. Physicochemical characterization The NEs have average size of about 100 nm, with the PDI values below 0.20, indicating suitability for intravenous application. It could be observed from Fig. 1 that the addition of PEGylated phospholipids caused an increase in NE viscosity, as could be expected given that the polyethylene glycols are used in parenteral suspensions as stabilizing - rheology modifying agents [3]. 3.2. Injectability assessment The injectability assessment was performed with syringe-needle system used in our laboratory for intravenous administration in in vivo animal studies. As blood-mimicking solution, 36.6 %, v/v, glycerol solution was used [4]. It could be observed from Fig. 2 that the injectability of NEs depended on their viscosity, with the higher pressure needed to extrude the formulations with the higher PEG2000-DSPE concentration. Even though, to the best of our knowledge, there are no studies investigating the injectability of the intravenous preparations, based on some previous research on subcutaneous model [5], it is recommended the maximum force used to inject the formulations should be kept about 20 N, which would eliminate S3 and S6 from further investigation (Fig. 2). 4. CONCLUSION The injectability method used in this research proved as a useful tool in screening formulations adequate for prospective intravenous use. 5. REFERENCES 1. Cilurzo, F., et al. Injectability Evaluation: An Open Issue. AAPS PharmSciTech, 2011. 12(2): 604-609. 2. Sarmadi, M., et al. Modeling, design, and machine learning-based framework for optimal injectability of microparticle-based drug formulations. Science advances, 2020. 6: eabb6594. 3. Gullapalli, R. P., Mazzitelli, C. L. Polyethylene glycols in oral and parenteral formulations—A critical review. International Journal of Pharmaceutics, 2015. 496(2): 219-239. 4. Yousif, M. Y., et al.. Deriving a blood-mimicking fluid for particle image velocimetry in Sylgard-184 vascular models. In Annual International Conference of the IEEE Engineering in Medicine and Biology Society, 2009 (pp. 1412-1415 5. Watt, R. P., et al. (2019). Injectability as a function of viscosity and dosing materials for subcutaneous administration. International Journal of Pharmaceutics, 2019:554, 376-386. ACKNOWLEDGMENT This research was funded by the MESDT, Republic of Serbia through Grant Agreement with University of Belgrade-Faculty of Pharmacy No: 451-03-68/2022-14/200161 and supported by the Science Fund of the Republic of Serbia, GRANT No 7749108, Neuroimmune aspects of mood, anxiety and cognitive effects of leads/drug candidates acting at GABAA and/or sigma-2 receptors: In vitro/in vivo delineation by nano- and hiPSC-based platform - NanoCellEmоCog

Pun faktorski dizajn u formulaciji parenteralnih nanoemulzija sa diazepamom - fizičkohemijska karakterizacija i procena stabilnosti

Using the experimental design methodology, we have developed and characterized nanoemulsions for a parenteral delivery using diazepam as a model drug. The formulations containing 20 or 30% (w/w) of medium chain triglycerides or the mixture of medium chain triglycerides and soybean oil as the oil phase, soybean lecithin and polysorbate 80 as emulsifiers, and a phosphate buffer solution as the aqueous phase were prepared by cold high pressure homogenization. The obtained nanoemulsions were evaluated in terms of droplet size, size distribution, surface charge, drug-vehicle interactions and physical stability. To evaluate the effects of the oil phase type, oil content and drug presence, as well as their interactions on critical quality attributes of nanoemulsions, a three-factor two-level full factorial design was applied. After the preparation, all nanoemulsions revealed small spherical droplets in the range 170-210 nm, with the narrow droplet size distribution ( lt 0.15) and the surface charge about -60 mV. The experimental design results indicated that not only factors alone (oil type, oil content, presence of drug), but their interactions also had a significant effect on the nanoemulsion droplet size, polydispersity index, and zeta potential. During two months of storage at 25°C, all nanoemulsions formulated with the medium chain triglycerides-soybean oil mixture (4:1, w/w) remained physically stable, without considerable changes in monitored parameters. Physicochemical characteristics and stability of these nanoemulsions demonstrated their suitability for parenteral drug delivery.Cilj ovog rada bio je da se primenom metodologije eksperimentalnog dizajna razviju parenteralne nanoemulzije sa diazepamom kao model lekovitom supstancom i da se sprovede njihova sveobuhvatna fizičkohemijska karakterizacija. Metodom homogenizacije pod visokim pritiskom na sobnoj temperaturi izrađene su placebo i nanoemulzije sa lekom, stabilizovane smešom lecitina i polisorbata 80, variranjem udela i vrste uljane faze - 20 i 30% (m/m) triglicerida srednje dužine lanca ili smeše triglicerida srednje dužine lanca i sojinog ulja u odnosu 4:1. Dobijene nanoemulzije okarakterisane su u pogledu veličine i raspodele veličina kapi, površinskog naelektrisanja, interakcija lek-nosač i fizičke stabilnosti. U cilju procene istovremenog uticaja vrste uljane faze, udela ulja i prisustva leka, kao i njihovih interakcija, na kritične atribute kvaliteta nanoemulzija, primenjen je pun faktorski dizajn sa tri faktora na dva nivoa. Nakon izrade, sve formulacije nanoemulzija imale su malu veličinu kapi u opsegu 170-210 nm, sa veoma uskom raspodelom veličina (ispod 0,15) i površinskim naelektrisanjem oko -60 mV. Rezultati eksperimentalnog dizajna pokazali su da ne samo pojedinačni faktori (vrsta ulja, koncentracija ulja, prisustvo leka), nego i njihove interakcije, značajno utiču na veličinu kapi, indeks polidisperznosti i zeta potencijal ispitivanih nanoemulzija. Tokom 2 meseca čuvanja na 25°C, sve nanoemulzije formulisane sa smešom triglicerida srednje dužine lanca i sojinog ulja kao uljanom fazom bile su fizički stabilne, bez značajnih promena u praćenim parametrima. Fizičkohemijske karakteristike i stabilnost navedenih nanoemulzija ukazuju da one mogu biti potencijalni nosači za parenteralnu isporuku lekovitih supstanci

Parenteralne nanoemulzije diazepama - fizičkohemijska karakterizacija i in vitro ispitivanje brzine oslobađanja

The aim of the present study was to develop parenteral nanoemulsions containing increasing content of oil phase (20, 30 and 40%, w/w of medium-chain triglycerides-soybean oil mixture at 4:1 ratio), stabilized by lecithin-polysorbate 80 mixture, and to assess their feasibility as carriers for poorly water-soluble psychopharmacological drugs. To this purpose, nanoemulsions loaded with diazepam as a model drug were prepared through high pressure homogenization and characterized regarding droplet size, polydispersity, surface charge, viscosity, pH value, and electrical conductivity. Furthermore, the in vitro release of diazepam from developed nanoemulsions was examined using reverse dialysis bag technique, and drug release kinetics was evaluated through several mathematical models. After preparation, all formulations revealed small mean droplet size (206 ± 7 nm), with narrow size distribution (0.116 ± 0.012) and zeta potential around -50 mV, complying with pharmacopoeial requirements (USP 39-NF 34), wherein there were no significant changes in monitored parameters after one year of storage at 25 ± 2°C. In vitro drug release study demonstrated that 40-50% of diazepam was released from actual nanoemulsions within 1 h, while the kinetic release process could be described by Korsmeyer-Peppas model. The results obtained suggest that formulated parenteral nanoemulsions might be promising carriers for rapid delivery of lipophilic, poorly water-soluble psychopharmacological drugs.Cilj ovog istraživanja bio je da se razviju parenteralne nanoemulzije sa rastućom koncentracijom uljane faze (20, 30 i 40% smeše triglicerida srednje dužine lanca i sojinog ulja u odnosu 4:1), stabilizovane kombinacijom lecitina i polisorbata 80, i da se proceni njihova pogodnost kao nosača za slabo rastvorljive psihofarmakološke lekovite supstance. U tu svrhu, homogenizacijom pod visokim pritiskom izrađene su nanoemulzije sa diazepamom kao model lekovitom supstancom i okarakterisane u pogledu veličine kapi, indeksa polidisperznosti, površinskog naelektrisanja, viskoziteta, pH vrednosti i električne provodljivosti. Takođe, primenom reverzne tehnike sa dijaliznim vrećicama procenjena je brzina oslobađanja diazepama iz razvijenih nanoemulzija, uz karakterizaciju dobijenih profila oslobađanja primenom različitih matematičkih modela. Nakon izrade, sve formulacije imale su malu prosečnu veličinu kapi (206 ± 7 nm), sa uskom raspodelom veličina (0,116 ± 0,012) i zeta potencijalom oko -50 mV, što je u skladu sa farmakopejskim zahtevima (USP 39-NF 34) pri čemu se vrednosti navedenih parametara nisu značajno promenile nakon godinu dana čuvanja na 25 ± 2°C. In vitro ispitivanje brzine oslobađanja pokazalo je da se 40-50% diazepama oslobodi iz ispitivanih nanoemulzija tokom 1 h, pri čemu se kinetika oslobađanja može opisati Korsmeyer-Peppas modelom. Dobijeni rezultati ukazuju da formulisane parenteralne nanoemulzije predstavljaju obećavajuće nosače za brzu isporuku slabo rastvorljivih psihofarmakoloških lekovitih supstanci

Krem-gel sa ceramidima (II deo) - procena efekta na hidratisanost kože i transepidermalni gubitak vode iz kože

Taking into account the latest discoveries in the structure and functions of ceramides (sphingolipids, sphingoids), manufacturers of cosmetic raw materials propagate to use ceramides (nature-identical ceramides, ceramide analogues, pseudoceramides) for the purpose of restoration of the natural intercellular lipid barrier. Despite putative importance, there is no consistent data on the benefits of ceramides when used in conventional, consumer products on skin that has suffered minor barrier damage. Transepidermal water loss (TEWL) and skin moisture content are two parameters frequently used for assessment of the influence of cosmetic/pharmaceutical products on barrier function. The ceramides-containing cream-gel has been formulated, and its influence on barrier function during 14 days, compared to “placebo” containing no sphingolipids, assessed on six volunteers with no apparent barrier damage. It was shown that the cream-gel improved skin moisture content, as well as the overall barrier function quality. The lack of significant differences in the influence of the cream-gel and “placebo” on the measured parameters indicated that, for such a kind of product, there was no evidence for the beneficial effects of ceramides on the skin that has suffered minor barrier damage.Novija otkrića u vezi strukture i funkcije ceramida (sfingolipida, sfingoida) kože, poslužila su proizvođačima kozmetičkih sirovina da često nekontrolisano i neargumentovano propagiraju upotrebu ceramida u kozmetičkim proizvodima u svrhe reparacije prirodne intercelularne lipidne barijere. Rezultati ispitivanja efekata ceramida na koži često ne daju čvrste dokaze da primena konvencionalnih kozmetičkih proizvoda sa ceramidima može da popravi neznatno oštećenu kožu. Pouzdana metoda za procenu uticaja kozmetičkih/farmaceutskih preparata na funkciju kožne barijere, odnosno efikasnosti i/ili prihvatljivosti formulacije predstavlja ispitivanje transepidermalnog gubitka vode (TEGV) iz kože. Savremena formulacija krem-gela sa ceramidima je aplikovana na kožu i ispitan je njen uticaj na funkciju kožne barijere na panelu od 6 ispitanika sa zdravom kožom, u vremenskom periodu od 14 dana, prema unapred zadatom programu u odnosu na “placebo” formulaciju. Pokazano je da ispitivani krem-gel pozitivno utiče na hidratisanost (vlažnost) stratum corneuma (SC) i transepidermalni gubitak vode, odnosno kvalitet kožne barijere. Sa druge strane, odsustvo statistički značajnih razlika TEGV i vlažnosti kože pod uticajem ispitivanih formulacija ukazuje da, u ovom slučaju, nema dovoljno dokaza o prednostima prisustva ceramida u "aktivnom" krem-gelu u odnosu na “placebo” formulaciju, kada su primenjeni na neznatno oštećenu ili neoštećenu kožu ispitanika

Preparation and characterisation of phenytoin-loaded alginate and alginate-chitosan microparticles

We aimed to prepare and investigate microparticles with the varying contents of calcium gelling ion, loaded with phenytoin, a standard antiepileptic agent, in its acidic form. Two different methods of alginate-based microparticles preparation were used: with and without treatment with chitosan. Furthermore, two standard procedures, the one-stage and the two-stage, were applied. Microparticle size of 12 one-stage formulations ranged from 466 to 636 m. Both types of formulations, chitosan-treated and nontreated, appeared to be highly loaded with the model drug ( 91-96%). The chitosan-coated alginate-based microparticles prepared by the one-stage procedure exhibited kinetics of phenytoin liberation comparable to a similar sustained release system that had been tested at pH 6.8, as published earlier. As the gel erosion of alginate-based microparticles should be potentiated by the higher pH ( used in the present study at pH 7.4), the most favorable of 12 formulations, with the liberation half-time of about 2 hr, seemed to be eligible for further modifications. Counterintuitively, the applied two-stage procedure did not appear to beneficially affect the dissolution behavior of phenytoin when tested in two formulations, which makes further modifications necessary

The importance of autochthonous breeds in sustainable production

Autochthonous breeds are the pillar of sustainable animal production worldwide. From a total of 8719 livestock breeds, 26 percent are classified as at risk of extinction, 13 percent as not at risk, 6 percent as extinct and 55 percent as being of unknown risk status. Therefore, conservation is of high priority. The Podolian cattle is one of the most endangered breed from the list of native breeds of livestock in Serbia. In 2021 only 338 breeding animals were registered. The breed is known as a precious resource of the local landscape enrichment, national heritage and history. According to the population size of the Podolian cattle, a slight increasing tendency can be evidented. The population size is variable (154 breeding animals in 2009, 252 in 2010, 270 in 2011, 260 in 2012, 264 in 2013, 306 in 2014, 258 in 2015, 263 in 2016, 338 in 2017, 317 in 2018, 357 in 2019, 416 in 2020 and 338 in 2021). The Podolian cattle population in Serbia belongs to the I group (critically endangered). Over the time the governmental subsidies influenced the overall slight increase of the number of breeding animals, but the population size is not stabile. Improved conservation program should be applied to save the autochthonous endangered breed suitable for heritage oriented sustainable production in an authentic environment

Hyperfine interactions at 181Ta solute in ferromagnetic Hf-Ni alloys with low concentration of Hf atoms

We have measured hyperfine interactions of 181Ta probe in the polycrystalline 0.2at.%Hf-Ni, 2at.%Hf-Ni and 5at.%Hf-Ni alloys, by the time differential perturbed angular correlation (TDPAC) method at room temperature (RT). The hyperfine magnetic field (Hhf) in 0.2at.%Hf-Ni alloy at the 181Ta probe is 8.66(1)T, where the small atomic concentrations of Hf atoms mainly substitute on Ni host lattice sites. Three hyperfine interactions were detected in two other alloys. In the 2at.%Hf-Ni alloy, we found existence of Larmor precession frequency ωL (1)=536(2)Mrad/s at the 181Ta probe surrounded by Ni atoms and the second electric quadrupole interaction (EQI) ωQ (2)=2.20(2)Mrad/s at the 181Ta probe in new formed HfNi5 intermetallic phase. The third EQI ωQ (3)=137.4(1)Mrad/s corresponds to the small amount of monoclinic HfO2 phase contamination at T<900K. In the 5at%Hf-Ni alloy, magnetic dipole interaction (MDI) at the 181Ta probe is ωL (1)=536(6)Mrad/s, while the second EQI at the site of the same probe in HfNi5 is ωQ (2)=2.24(2)Mrad/s. The presence of ωQ (3)=128(1)Mrad/s at the 181Ta probe originating from HfO2 contamination is in good accordance with earlier published results for this phase.52. konferencija ETRAN-a : Jun 8-12, Palić, 2008