Konventionaalinen Raman-spektroskopia ja aika-erotteinen Raman-spektroskopia lääkeaineiden ja biomolekyylien tutkimuksessa (kirjallisuuskatsaus) : Konventionaalinen Raman-spektroskopia ja aika-erotteinen Raman-spektroskopia ei-fluoresoivien ja fluoresoivien lääkeaineiden tutkimuksessa (kokeellinen osuus)

Raman spectroscopy is based on vibrations that occur between the atoms of a compound. The overall structural energy is derived from the electronical energy as well as vibrational, rotational and translational energy. In Raman spectroscopy the vibrational and rotational energies are essential. Usually the excitation energy used in Raman spectroscopy can be either in the region of visible light or NIR. The sample absorbs the energy and energy is also scattered back to all possible directions. Elastic scattering is called the Rayleigh scattering. In that case the back-scattered photons have an equal energy as the original excitation energy. However, some of the scattering happens inelastically and it forms the basis of Raman-phenomena. If the detected photons have smaller energy than the original, it is called the Stokes scattering. If the energy is bigger, it is anti-Stokes scattering. Raman is typically very rare and weak phenomenon. The spectral features in Raman spectra consist of the intensities and energies of the back scattered photons. Raman spectroscopy provides very accurate and detailed structural information on the molecule. It is basically a label-free technique with minimal need for sample preparation and the measurements can also be carried out e.g. through container walls. Further, Raman is quite insensitive to hydrous samples and it is suitable to solutions and biological assessments. However, there are some drawbacks that are formed by the luminescence phenomena i.e. fluorescence. Strong fluorescent backgrounds can mask the relevant Raman features in spectra because Raman and fluorescence are competetive processes. For instance many drug molecules have such structures that they cause strong fluorescence. It is also one of the reasons that pharmaceutical applications and measurements have been partly limited due to this problem. There are applications to improve and enhance a Raman signal. For example resonance phenomena and SERS are favored. To solve the fluorescence-related problems there are also means; one can change the laser wavelenght, photobleach the sample or apply different kinds of data manipulation techniques to the spectral data achieved. There are drawbacks with these methods. They can be slow, complex, damage the samples and still insufficient fluorescence suppression is a problem. In this study a novel time-gated CMOS-SPAD detection technique is applied to non-fluorescent and fluorescent drug measurements. The new detection system has a programmable on-chip delay time and it is synchronized with a picosecond pulsed laser. The scattered photons can be measured in the time scale when they are simultaneously measured in traditional energy and intensity wise. Raman scattering occurs in the timescale of sub-picoseconds while the fluorescence phenomena happen typically in the order of nanoseconds. This time difference can be exploited effectively to suppress the fluorescence. In the literature review of this study the basis of vibrational spectroscopy is introduced - especially Raman spectroscopy. The techniques related, as well as the novel time-resolved technique are covered. Further, different kinds of applications in the field of Raman spectroscopy are reviewed, mainly pharmaceutics-related and biologically relevant applications. In the experimental work the focus was to compare a continuous-wave 785 nm laser setup coupled with the CCD-detector to the pulsed picosecond 523 nm laser coupled with the CMOS-SPAD-detector. The measurements were performed on different kinds of drugs, both non-fluorescent and fluorescent. The aim was to obtain information on the effectiveness of CMOS-SPAD-technique on fluorescence suppression for solid drugs and solutions. Secondary goals were to collect knowledge on the similarities and differences between the Raman setups used for solution measurements, to optimize and discuss the key elements of setups for solids and solutions and to show preliminarily the applicability of the CMOS-SPAD-system on fluorescent drug's solutions as well as find out the requirements related to quantitative assessments using Raman spectroscopy. In drug research there is also constant need for reliable in vitro cell assays. The assessments made in this study may prove useful to the future applications e.g. measurements with living cells. An effective fluorescence suppression was achieved to strong fluorescent backgrounds using the novel time-resolved CMOS-SPAD-detection system coupled with the pulsed picosecond 532 nm laser. The setup is potentially a convenient tool to overcome many fluorescence-related limitations of Raman spectroscopy for laboratory and process analytical technology (PAT) use in the pharmaceutical setting. The results achieved encourage to consider that with careful calibration and method validation there is potential for quantitative analysis, biopharmaceutical and biological applications e.g. in vitro cell studies where most Raman techniques suffer from strong fluorescence backgrounds. Other potential fields for future applications can be also considered.Raman-spektroskopia on aineen rakenneosien värähtelyjen tutkimukseen käytettävä menetelmä. Aineen rakenteellinen kokonaisenergia muodostuu elektronisesta energiasta sekä vibraatio-, rotaatio- ja translaatioenergiasta. Näistä ennen kaikkea Raman-tekniikan kannalta keskeisiä ovat vibraatio- ja rotaatioenergiat esimerkiksi molekyyleissä. Raman-spektroskopiassa käytetään yleensä näkyvän valon ja lähi-infrapuna-alueen heräte-energiaa, joka kohdistetaan tutkittavaan näytteeseen. Näytteen rakenteeseen absorboituu heräte-energiaa, ja sitä siroaa elastisesti tai epäelastisesti takaisin kaikkiin mahdollisiin suuntiin. Elastinen sironta on Rayleigh-sirontaa, jolloin takaisin sironneiden fotonien energia on yhtä suuri kuin alkuperäinen heräte-energia. Epäelastinen sironta on Raman-sirontaa. Ilmiö on paljon harvinaisempi kuin elastinen sironta. Emittoituvat fotonit voivat olla energialtaan pienempiä (Stokes-sironta) tai suurempia (anti-Stokes) kuin alkuperäinen heräte-energia. Raman-spektrissä näkyvät spekrivyöt ja -piikit muodostuvat takaisin emittoituneiden fotonien energioiden (aaltolukujen) ja intensiteettien perusteella. Raman-spektroskopia antaa hyvin tarkkaa rakenteellista informaatiota tutkittavista molekyyleistä, se on käytännössä leima-ainevapaa tekniikka ja esimerkiksi mittaukset pakkausmateriaalien läpi onnistuvat. Näytteiden korkea vesipitoisuus ei haittaa spektrin muodostumista, jolloin Raman-tekniikkaa voidaan hyödyntää myös liuosnäytteille ja biologisille näytteille. Toisinaan ongelmaksi muodostuu kuitenkin luminesenssi-ilmiöt, kuten fluoresenssi. Fluoresenssi saattaa peittää alleen lähes samanaikaisesti tapahtuvan heikon Raman-signaalin. Esimerkiksi monilla lääkeaineilla on voimakkaasti fluoresoivia rakenteita ja tästä johtuen Raman-tekniikan käyttö lääkeainetutkimuksessa on osin rajoittunutta. Raman-signaalien vahvistamiseksi on kehitetty erilaisia menetelmiä. Voidaan hyödyntää esimerkiksi resonanssi-ilmiöitä tai SERS-tekniikkaa. Fluoresenssiin liittyvien ongelmien ratkaisuina on käytetty muun muassa pidemmän aallonpituuden laseria, näytteen valkaisua tai erityyppisiä signaalinkäsittelykeinoja fluoresenssitaustan poistamiseksi spektridatasta. Menetelmien haittana on kuitenkin ollut hitaus, kompleksisuus ja tehottomuus. Uudempaa tekniikkaa edustaa pikosekuntiluokan pulssilaser-herätteen käyttö sekä laserin kanssa tarkasti synkronoitu, ohjelmoitavalla viiveajalla toimiva CMOS-SPAD-detektoritekniikka. CMOS-SPAD-detektori mahdollistaa fotonien detektoinnin niiden intensiteetin ja energian mukaan sekä aikatasossa tapahtuvan detektoinnin. Raman-sironta ja fluoresenssi-ilmiöt tapahtuvat usein hieman eriaikaisesti, Raman alle pikosekunneissa ja fluoresenssi ~ nanosekunneissa. Näin ollen aika-erotteisella detektoinnilla ilmiöt voidaan erotella ja vähentää tehokkaasti fluoresenssin vaikutusta. Tämän tutkimuksen kirjallisuuskatsauksessa esitellään vibraatiospektroskopiaa, erityisesti Raman-spektroskopiaa. Katsauksessa käsitellään Raman-spektroskopian tekniikkaa, esitellään aika-erotteista Raman-tekniikkaa ja erityyppisiä Raman-spektroskopian sovelluksia lääkeaineiden sekä biomolekyylien tutkimuksessa. Tutkimuksen kokeellisessa osiossa oli tarkoituksena vertailla 785 nm jatkuvan aallonpituuden laserin ja CCD-detektorin yhdistelmää aika-erotteiseen 532 nm pulssilaseriin ja CMOS-SPAD-detektoriin erityyppisten lääkeaineiden mittauksissa. Tavoitteena oli saada kiinteiden lääkeaineiden ja niiden liuosten avulla tietoa aika-erotteisen laitteiston fluoresenssisuppression toimivuudesta. Lisäksi haluttiin selvittää alustavasti laitteistojen yhtenevyyksiä ja eroja lääkeaineliuosten mittauksissa ja optimoida kokoonpanoja kiinteiden lääkeaineiden sekä liuosten mittauksiin. Pyritiin osoittamaan uuden tekniikan soveltuvuus myös fluoresoivien lääkeaineliuosten mittauksiin ja selvittämään kvantitatiivisten mittausten asettamia vaatimuksia Raman-spektroskopialle. Lääkeainetutkimuksessa ovat usein kiinnostuksen kohteena myös in vitro suoritettavat solumääritykset. Näin ollen tutkimuksessa luotiin edellä esitettyjen määritysten avulla myös pohjaa elävillä soluilla tehtäviä mittauksia varten. Aika-erotteista Raman-spektroskopiaa käyttämällä onnistuttiin tehokkaasti vähentämään fluoresenssia niilläkin lääkeaineilla, joiden Raman-spektrin mittaus 785 nm herätteellä oli käytännössä mahdotonta. CMOS-SPAD-kokoonpanon todettiin olevan käyttökelpoinen työväline Raman-mittauksiin liittyvien fluoresenssiongelmien vähentämiseksi farmaseuttisissa sovelluksissa laboratorio-olosuhteissa sekä esimerkiksi prosessianalyysiteknologiassa (PAT). Lisäksi saatiin rohkaisevia tuloksia siitä, että tarkalla laitteiston kalibroinnilla ja menetelmien validoinnilla voidaan jatkossa suorittaa myös kvantitatiivista analyysiä ja mittauksia biologisille näytteille

Raman Spectroscopy and Surface Plasmon Resonance as Photonic Tools for Biopharmaceutical Applications

Biophotonics is an emerging area of scientific research that uses light of photons to probe biological specimens, such as tissues, cells and molecules. The field of biophotonics is broad and considerably multidisciplinary. Therefore the prerequisite for understanding biophotonics is the capability to integrate the fundamental knowledge of the physics of light with perspectives of engineering of devices and instruments used to generate, modify, and manipulate light. Also, the fundamentals of biology and medicine are essential particularly comprehension of the biochemical and cellular phenomena that occur in living systems, and how such phenomena can be scaled up to concern the physiology of organisms, for example humans. Biological pathways and processes differ in the healthy and diseased state, and that is why it is essential to develop understanding of pathophysiology and various states of disease such as cancer, neurodegenerative disease or infectious states. Consequently, solid insights into the functions of medical treatments, including biopharmaceutics, are needed. Raman and surface plasmon resonance (SPR) are both light-based technologies enabling label-free measurements with high sensitivity. The primary aim of this Thesis was to tackle the emerging hardships encountered in the fundamental biopharmaceutical research, clinical settings, or in the pharmaceutical industry by introducing applications and data analysis methods based on the cutting edge Raman and SPR technologies. These techniques represent the biophotonic cornerstones as tools for biopharmaceutical applications throughout this Thesis. The scope of this Thesis was essentially broad. First, small drug molecules were investigated with state-of-the-art time-gated Raman technology, showing that the interfering photoluminescent backgrounds can be effectively suppressed thus improving the acquired Raman data significantly. Additionally, EVs were studied with laser tweezers Raman spectroscopy (LTRS) as larger scale analytes and representatives of a highly interesting topic in the current nanomedical field. When Raman data from several different types of single EVs was examined using sophisticated data analysis, distinct subpopulations were observed, and the differences could be related to the biochemical compositions of the vesicle membranes. For the first time, the study showed the importance of measuring single EVs instead of a pool of vesicles. Multi-parametric SPR (MP-SPR) technology was harnessed to develop applications and data analysis methods for small and larger scale analytes. Hence, a new small drug molecule, spin-labeled fluorene (SLF), was investigated in the context of Alzheimer s disease (AD) particularly its potential to interfere with the detrimental amyloid peptide aggregation processes. The developed bio-functional in vitro platform in combination with rigorous data analysis and computational simulations demonstrated the capabilities of SLF when it was employed in various biomimetic aggregation schemes. Moreover, liposomes were examined with the MP-SPR as larger scale nanomedical particles for the purposes of safe and effective nanocarrier development. The administration of a liposomal nanocarrier into the blood circulation was mimicked in the designed bionanophotonic in vitro schemes. Undiluted serum was made to interact with immobilized model liposomes in dynamic flow conditions. The findings revealed that the variation in surface chemistries of the liposomes plays a role when serum essentially immune system components are interacting with the liposomes. In particular, distinct soft and hard protein coronas were observed and characterized during the interactions. Collectively, the results and findings in this Thesis underline the broad potential of biophotonics for biopharmaceutical applications. The technical improvements in instrumentation, and creativity in the application and data analysis development make the future of biophotonics bright.Biofotoniikka on kasvava tieteenala, jossa valon fotoneja käytetään biologisten kohteiden tutkimiseen. Esimerkkeinä kudokset, solut ja molekyylit. Biofotoniikka tieteenalana on huomattavan poikkitieteellinen. Valon tuottamiseksi ja muokkaamiseksi tarvitaan perustavanlaatuista tietoa fysiikasta sekä teknisiä taitoja laitteiden suunnittelemiseksi. Lisäksi biologian, ja lääketieteen perusteet ovat olennainen osa biofotoniikkaa. Erityisen tärkeää on ymmärrys biofysiikasta ja -kemiasta sekä molekyyli- ja solutason ilmiöistä, jotka tapahtuvat elävissä systeemeissä. Tämän väitöstyön tavoitteena oli kehittää biofotonisia tekniikoita, menetelmiä ja data-analytiikkaa nykyisen biofarmaseuttisen tutkimuksen haasteisiin. Olemassa olevien in vitro menetelmien, eli esimerkiksi koeputkessa tai maljassa tehtävien biologisten mallisysteemien ongelmana on usein se, että tulosten siirrettävyys kliinisiin tutkimuksiin ja lopullisiksi lääkevalmisteiksi ei ole suoraviivaista. Esimerkiksi leima-aineiden, kuten fluoresoivien tai radioaktiivisten molekyylien lisääminen tutkittaviin kohteisiin saattaa muuttaa niiden fysikaalista ja kemiallista käyttäytymistä elävässä ympäristössä. Raman-spektroskopia ja pintaplasmoniresonanssi ovat molemmat valoon perustuvia teknologioita, joilla mittauksia voidaan suorittaa ilman leima-aineita ja korkealla herkkyydellä. Molemmista teknologioista käytettiin kehityksen kärjessä olevia instrumentteja. Ne edustivat tässä työssä biofotonisia tekniikoita, joita täydennettiin perinteisillä tutkimusmetodeilla. Väitöstyössä tutkittiin aluksi pienlääkeaineita kiinteässä olomuodossaan Raman-spektroskopian avulla. Raman-spektroskopialla voidaan mitata erittäin tarkasti tutkittavien molekyylien kemiallinen rakenne. Tavallisesti Raman-tutkimusten ongelmana on kuitenkin voimakas fotoluminesenssi, joka tuottaa häiritsevän taustasignaalin ja haittaa Raman-spektrien tulkintaa. Uudella aika-portti Raman-tekniikalla pystyttiin erottelemaan Raman- ja fotoluminesenssisignaalit tehokkaasti. Tämä mahdollisti vaikeasti mitattavien lääkeaineiden Raman-spektrien mittaamisen ja tarkemman karakterisoinnin. Tutkimusten toisessa vaiheessa mitattiin ensimmäistä kertaa yksittäisiä ekstrasellulaarivesikkeleitä. Ekstrasellulaarivesikkelit ovat solujen tuottamia, kalvorakenteisia nanorakenteita, joiden läpimitta on noin 50 1000 nm. Niiden hyödyntäminen syöpädiagnostiikassa ja nanolääketieteellisinä lääkekantajina on kasvavan kiinnostuksen kohteena, mutta tutkimuksia rajoittaa vesikkeleiden biokemiallinen monimuotoisuus ja pieni koko. Mittaukset suoritettiin toisella uudentyyppisellä Raman-tekniikalla, laser-ansalla. Terveiden solujen ja syöpäsolujen tuottamien vesikkeleiden biokemiallista koostumusta arvioitiin kehitetyn monimuuttuja-analyysin keinoin ja havaittiin, että merkittävimmät erot vesikkeleiden välillä liittyivät niiden pintarakenteiden erilaisuuteen. Saaduilla tuloksilla on merkitystä vesikkeleiden funktioiden ymmärtämisessä ja niiden hyödyntämisessä nanolääketieteellisissä applikaatioissa. Väitöstutkimuksen kahdessa muussa osiossa kehitettiin pintaplasmoniresonanssiin pohjautuvia menetelmiä sekä data-analyysiä niin ikään pienlääkemolekyylien ja nanolääketieteellisten kohteiden tutkimukseen. Käytetty moderni pintaplasmoniresonanssitekniikka mahdollisti perinteisestä poikkeavan, perusteellisemman data-analyysin toteuttamisen. Ensin tarkasteltiin Alzheimerin taudin hoitoon kehitettyä uutta lääkemolekyyliä, jonka oli todettu hidastavan haitallista amyloid-peptidien yhteenliittymistä. Tutkimuksissa kehitetty bio-funktionaalinen in vitro alusta yhdessä tietokonesimulaatioiden kanssa osoitti, että tutkittu molekyyli vähensi selvästi amyloid-aggregaatiota. Analyysillä pystyttiin myös karakterisoimaan molekyylin interaktio-ominaisuuksia. Liposomit edustivat väitöstutkimuksen viimeisessä osuudessa nanolääketieteellisiä partikkeleita, joiden ominaisuuksia tutkittiin pintaplasmoniresonanssin avulla. Tällä hetkellä ainoat ihmiskäyttöön hyväksytyt nanolääkekantajat perustuvat liposomeihin. Kehitetyllä bionanofotonisella in vitro -alustalla matkittiin liposomikantajan annostelua verenkiertoon. Laimentamattoman seerumin annettiin vuorovaikuttaa sensoripinnalle immobilisoitujen liposomien kanssa dynaamisissa virtausolosuhteissa. Tulokset osoittivat, että liposomien pintakemialla on todennäköisesti vahva vaikutus siihen, miten seerumin komponentit, erityisesti immuunijärjestelmä, reagoivat annosteltuun liposomikantajaan. Havainnoilla on potentiaalista merkitystä turvallisten ja tehokkaiden lääkekantajien kehitystyössä. Yhteenvetona voidaan todeta, että biofotoniikkaa voidaan hyödyntää hyvin luovalla tavalla biofarmaseuttisiin applikaatioihin. Väitöstutkimuksen tulokset osoittavat, että kehitetyillä alustoilla ja data-analyysimenetelmillä on mahdollista parantaa nykyistä pienlääkekehitystä sekä nanolääketieteellistä tutkimusta

Nanoplasmonic Approaches for Sensitive Detection and Molecular Characterization of Extracellular Vesicles

All cells release a multitude of nanoscale extracellular vesicles (nEVs) into circulation, offering immense potential for new diagnostic strategies. Yet, clinical translation for nEVs remains a challenge due to their vast heterogeneity, our insufficient ability to isolate subpopulations, and the low frequency of disease-associated nEVs in biofluids. The growing field of nanoplasmonics is poised to address many of these challenges. Innovative materials engineering approaches based on exploiting nanoplasmonic phenomena, i.e., the unique interaction of light with nanoscale metallic materials, can achieve unrivaled sensitivity, offering real-time analysis and new modes of medical and biological imaging. We begin with an introduction into the basic structure and function of nEVs before critically reviewing recent studies utilizing nanoplasmonic platforms to detect and characterize nEVs. For the major techniques considered, surface plasmon resonance (SPR), localized SPR, and surface enhanced Raman spectroscopy (SERS), we introduce and summarize the background theory before reviewing the studies applied to nEVs. Along the way, we consider notable aspects, limitations, and considerations needed to apply plasmonic technologies to nEV detection and analysis

Multi-parametric surface plasmon resonance platform for studying liposome-serum interactions and protein corona formation

When nanocarriers are administered into the blood circulation, a complex biomolecular layer known as the “protein corona” associates with their surface. Although the drivers of corona formation are not known, it is widely accepted that this layer mediates biological interactions of the nanocarrier with its surroundings. Label-free optical methods can be used to study protein corona formation without interfering with its dynamics. We demonstrate the proof-of-concept for a multi-parametric surface plasmon resonance (MP-SPR) technique in monitoring the formation of a protein corona on surface-immobilized liposomes subjected to flowing 100 % human serum. We observed the formation of formulation-dependent “hard” and “soft” coronas with distinct refractive indices, layer thicknesses, and surface mass densities. MP-SPR was also employed to determine the affinity (KD) of a complement system molecule (C3b) with cationic liposomes with and without polyethylene glycol. Tendency to create a thick corona correlated with a higher affinity of opsonin C3b for the surface. The label-free platform provides a fast and robust preclinical tool for tuning nanocarrier surface architecture and composition to control protein corona formation.When nanocarriers are administered into the blood circulation, a complex biomolecular layer known as the "protein corona" associates with their surface. Although the drivers of corona formation are not known, it is widely accepted that this layer mediates biological interactions of the nanocarrier with its surroundings. Label-free optical methods can be used to study protein corona formation without interfering with its dynamics. We demonstrate the proof-ofconcept for a multi-parametric surface plasmon resonance (MP-SPR) technique in monitoring the formation of a protein corona on surface-immobilized liposomes subjected to flowing 100 % human serum. We observed the formation of formulation-dependent "hard" and "soft" coronas with distinct refractive indices, layer thicknesses, and surface mass densities. MP-SPR was also employed to determine the affinity (K-D) of a complement system molecule (C3b) with cationic liposomes with and without polyethylene glycol. Tendency to create a thick corona correlated with a higher affinity of opsonin C3b for the surface. The label-free platform provides a fast and robust preclinical tool for tuning nanocarrier surface architecture and composition to control protein corona formation.Peer reviewe

Label-free characterization and real-time monitoring of cell uptake of extracellular vesicles

Extracellular vesicles (EVs) have the ability to function as molecular vehicles and could therefore be harnessed to deliver drugs to target cells in diseases such as cancer. The composition of EVs determines their function as well as their interactions with cells, which consequently affects the cell uptake efficacy of EVs. In this study, we present two novel label-free approaches for studying EVs; characterization of EV composition by time-gated surface-enhanced Raman spectroscopy (TG-SERS) and monitoring the kinetics and amount of cellular uptake of EVs by surface plasmon resonance (SPR) in real-time. Using these methods, we characterized the most abundant EVs of human blood, red blood cell (RBC)- and platelet (PLT)-derived EVs and studied their interactions with prostate cancer cells. Complementary studies were performed with nanoparticle tracking analysis for concentration and size determinations of EVs, zeta potential measurements for surface charge analysis, and fluorophore-based confocal imaging and flow cytometry to confirm EV uptake. Our results revealed distinct biochemical features between the studied EVs and demonstrated that PLT-derived EVs were more efficiently internalized by PC-3 cells than RBC-derived EVs. The two novel label-free techniques introduced in this study were found to efficiently complement conventional techniques and paves the way for further use of TG-SERS and SPR in EV studies.Peer reviewe

Targeting Tumor-Associated Exosomes with Integrin-Binding Peptides

All cells expel a variety of nanosized extracellular vesicles (EVs), including exosomes, with composition reflecting the cells' biological state. Cancer pathology is dramatically mediated by EV trafficking via key proteins, lipids, metabolites, and microRNAs. Recent proteomics evidence suggests that tumor-associated exosomes exhibit distinct expression of certain membrane proteins, rendering those proteins as attractive targets for diagnostic or therapeutic application, yet it is not currently feasible to distinguish circulating EVs in complex biofluids according to their tissue of origin or state of disease. Here, peptide binding to tumor-associated EVs via overexpressed membrane protein is demonstrated. It is found that SKOV-3 ovarian tumor cells and their released EVs express alpha(3)beta(1) integrin, which can be targeted by the in-house cyclic nonapeptide, LXY30. After measuring bulk SKOV-3 EV association with LXY30 by flow cytometry, Raman spectral analysis of laser-trapped single exosomes with LXY30-dialkyne conjugate enables the differentiation of cancer-associated exosomes from noncancer exosomes. Furthermore, the foundation for a highly specific detection platform for tumor-EVs in solution with biosensor surface-immobilized LXY30 is introduced. LXY30 not only exhibits high specificity and affinity to alpha(3)beta(1) integrin-expressing EVs, but also reduces EV uptake into SKOV-3 parent cells, demonstrating the possibility for therapeutic application.Peer reviewe

Fluorescence-suppressed time-resolved Raman spectroscopy of pharmaceuticals using complementary metal-oxide semiconductor (CMOS) single-photon avalanche diode (SPAD) detector

In this work, we utilize a short-wavelength, 532-nm picosecond pulsed laser coupled with a time-gated complementary metal-oxide semiconductor (CMOS) single-photon avalanche diode (SPAD) detector to acquire Raman spectra of several drugs of interest. With this approach, we are able to reveal previously unseen Raman features and suppress the fluorescence background of these drugs. Compared to traditional Raman setups, the present time-resolved technique has two major improvements. First, it is possible to overcome the strong fluorescence background that usually interferes with the much weaker Raman spectra. Second, using the high photon energy excitation light source, we are able to generate a stronger Raman signal compared to traditional instruments. In addition, observations in the time domain can be performed, thus enabling new capabilities in the field of Raman and fluorescence spectroscopy. With this system, we demonstrate for the first time the possibility of recording fluorescence-suppressed Raman spectra of solid, amorphous and crystalline, and non-photoluminescent and photoluminescent drugs such as caffeine, ranitidine hydrochloride, and indomethacin (amorphous and crystalline forms). The raw data acquired by utilizing only the picosecond pulsed laser and a CMOS SPAD detector could be used for identifying the compounds directly without any data processing. Moreover, to validate the accuracy of this time-resolved technique, we present density functional theory (DFT) calculations for a widely used gastric acid inhibitor, ranitidine hydrochloride. The obtained time-resolved Raman peaks were identified based on the calculations and existing literature. Raman spectra using non-time-resolved setups with continuous-wave 785- and 532-nm excitation lasers were used as reference data. Overall, this demonstration of time-resolved Raman and fluorescence measurements with a CMOS SPAD detector shows promise in diverse areas, including fundamental chemical research, the pharmaceutical setting, process analytical technology (PAT), and the life sciences.Peer reviewe

Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches

© 2024 The Authors. Journal of Extracellular Vesicles, published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY), https://creativecommons.org/licenses/by/4.0/Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly.Peer reviewe

Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches

Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its ‘Minimal Information for Studies of Extracellular Vesicles’, which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly