Browsing by Subject "Raman-spektroskopia"
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(2018)The majority of potential new chemical entities reaching drug development phase belong to Class II the Biopharmaceutics Classification System (BCS) which complicates formulation of orally administered drugs. Therefore, there is a need to develop methods to increase the solubility and dissolution rate. Transformation of a crystalline drug into its amorphous form can be used to enhance these properties of poorly water-soluble drugs. However, amorphous drugs are thermodynamically unstable and tend to recrystallize back to the crystalline form. Coamorphous forms are a new and promising method to stabilize amorphous form. A relatively new approach is to combine the active drug compound with an amino acid to form a coamorphous system. In this study, co-amorphous systems were prepared from gamma, alpha or amorphous form of indomethacin (IND) and tryptophan (TRP) by ball milling. The solid-state changes during milling were investigated to obtain information about the co-amorphization process. The main objective was to investigate the effects of initial solid state of indomethacin on the formation pathways. In addition, different analytical methods were compared with respect to observed endpoints of the formation process. Raman spectroscopy has not been used in previous studies regarding solid state changes in co-amorphous forms. The presence of fluorescence in amorphous systems may have limited use of the method. A time-gated Raman setup together with X-ray powder diffraction and differential scanning calorimetry (DSC) was used to investigate this kind of potentially fluorescent system. Principal component analysis of spectral data revealed that the three different binary systems had individual and direct pathways towards the same end points during milling. This indicates that the co-amorphous form formed after 60 minutes of ball milling is not dependent on the initial solid-state form of IND. Straight pathways also indicated direct transformation to the coamorphous form. DSC was found to be the most sensitive method to detect changes for the longest period during co-amorphization. Conventional Raman spectroscopy was found to be suitable for investigation of the co-amorphization process. However, time-gated Raman spectroscopy did not show significant advantages compared to conventional Raman data. This study revealed that the most stable form of IND could be used for production of co-amorphous form together with TRP. Raman spectroscopy could potentially be used for investigating coamorphization also as an in-process analytical method.
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(2023)Ramansironta on sähkömagneettisen säteilyn ilmiö, jonka avulla voidaan havaita molekyylivärähdyksiä niistä sironneen valon avulla. Kun sähkömagneettinen säteily kohtaa molekyylin se voi häiritä elektronipilveä ytimen ympärillä ja prosessin lopputuloksena energiaa vapautuu sironneen säteilyn muodossa. Tätä kutsutaan epäelastiseksi sironnaksi. Mikäli sironnassa ei tapahdu fotonin energiassa muutosta kyseessä on elastinen siroaminen. Mikäli näytteeseen menevän ja sironneen fotonin välillä havaitaan energian muutos, käytetään ilmiöstä nimeä epäelastinen Raman sironta. Energian muutosta voidaan mitata, ja saatu spektri antaa tietoa kohdemolekyylistä. Tämän työn tarkoituksena oli tutkia eri Raman-spektroskopiatekniikoiden, lähinnä spontaani-Raman spektroskopian sekä koherentti anti-Stokes Ramanspektroskopian ja mikroskopian soveltuvuutta lääkemolekyylien havaitsemiseen sekä nanopartikkeleiden karakterisoinnissa ja nanopartikkeleiden ja solujen välisten interaktioiden tutkimisessa. Tekniikan vahvuuksiin kuuluu käytön suhteellinen helppous ja CARS-mikroskopian nopeus sekä korkea erotuskyky, mahdollisuus havainnoida solunäytteitä, ja epätodennäköisyys vaurioittaa tutkittavaa kohdetta. Haasteisiin lukeutuu fluoresenssin taipumus häiritä signaalia, spontaani-Raman mikroskopian heikompi erottelukyky, analyysin hitaus sekä tarve valikoida molekyyli, jonka rakenne antaisi vahvan Raman-signaalin. Tutkimuksen alatavoitteina oli sopivan lääkemolekyylin valitseminen nanopartikkeliformulaatioon, nanopartikkeleiden formulointi, niiden karakterisointi Raman-spektroskopiatekniikoilla, ja lopulta tutkia Raman spektroskopian soveltuvuutta nanopartikkeleiden ja lääkeaineen sekä solujen interaktioiden tutkimisessa ja lääkeaineen havaitsemisessa solujen sisältä. Tutkimuksessa uutta on se, että ensimmäistä kertaa polymeerinanopartikkeleiden ja niihin ladattujen lääkeaineiden soluunkulkeutumisen tutkiminen pelkällä Raman-spektroskopialla ilman esim. fluoresoivia merkkiaineita. Tutkimuksen tavoitteen pääasiallisesti täyttyivät. Valitsimme kohdemolekyyliksi klorotaloniilin, fungisidin joka on edullinen ja antaa selvästi tunnistettavan Raman-signaalin. Klorotaloniilille suoritettiin normaalit sytotoksisuuskokeet sekä vapaana lääkeaineena että formuloituina nanopartikkeleina. Nanopartikkelien formulointi onnistui ja molemmat Raman-spektroskopiatekniikat näyttivät selvästi sekä lääkemolekyylin että polymeerin kemikaalispesifiset Raman signaalit. Lääkemolekyylin havaitsemin solujen sisältä onnistui. Polymeerimolekyylit eivät missään vaiheessa kulkeutuneet solujen sisälle. Tämä oli ehkä yllättävää koska käytimme makrofageja, mutta toisaalta myös johdonmukainen muiden tutkimusten kanssa. Raman-tekniikat osoittautuivat hyviksi tutkimusmenetelmiksi, ja erityisesti CARS-mikroskoopin suuri erotuskyky osoittautui hyödylliseksi soluunkulkeutumisen kuvantamisessa.
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(2021)Extracellular vesicles (EVs) are nano-sized lipid bilayer-delimited particles, released by cells. They take part in intercellular communication by their molecular composition and are part of both physiological and pathophysiological functions. EVs can be extracted from bodily fluids, and they are particularly abundant in blood. The purpose of this thesis was to evaluate the use of Raman spectroscopy in the characterization of EVs. Raman spectroscopy is an analysis method based on the interaction of light and matter, and the inelastic scattering of light, and it is used to get information on the biochemical composition of a substance. Principal component analysis (PCA) was used to investigate if Raman spectroscopy could differentiate two different platelet-derived EV samples, a red blood cell-derived EV-sample and a red blood cell-derived reference material. Evaluation of the characterization also included a stability study of these samples, where it was examined if any temperature dependent changes occurred that could be detected by Raman spectroscopy. Additionally, the applicability of Raman spectroscopy for lipoprotein contamination detection was evaluated by examining if purification of an EV sample decreased the intensity of carotenoid peaks typical for lipoprotein spectra. Raman spectroscopy was able to differentiate all three EV samples and the red blood cell-derived reference material from each other. The most clear differences were found between red blood cell and platelet-derived samples, due to for example the characteristic haemoglobin peaks of red blood cell-derived samples. Differences were also found between the two platelet EV samples, which were thought to implicate difference in protein compositions. The characterization of red blood cell-derived samples proved to be difficult because haemoglobin contained in the samples covered most of the other signal from the samples. Stability studies implicated that during fridge storage the carotenoid peak intensity of platelet-derived EV samples decreases due to the degradation of carotenoids. In the red blood cell-derived samples, no differences assignable to changes in some specific components of the samples were observed. Contamination studies suggested the intensity of the carotenoid peaks may increase due to purification of the sample. This was counter to the assumption and may suggest the carotenoids of the EV samples are not from lipoprotein contamination, but part of the EV composition. In conclusion, Raman spectroscopy proved to be a promising method for characterization and identification of different EV samples.
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