Mass spectrometryimaging (MSI) and vibrational spectroscopy imaging (VSI) complementeach other in terms of spatial resolution and molecular information.In the past decade, several combinations of such multimodal strategiesarose in research fields as diverse as microbiology, cancer, and forensics,overcoming many challenges toward the unification of these techniques.Here we focus on presenting the advantages and challenges of multimodalimaging from the point of view of studying biological samples as wellas giving a perspective on the upcoming trends regarding this topic.The latest efforts in the field are discussed, highlighting the purposeof the technique for clinical applications.Studieson complex biological phenomena often combine two or moreimaging techniques to collect high-quality comprehensive data directly  The coupling of imaging modalities startedwith optical microscopy techniques such as classical histopathology,fluorescence imaging, and immunohistochemistry being combined withmolecular imaging techniques such as infrared or Raman spectroscopyimaging and mass spectrometry imaging (MSI).24 The classical histology techniques have a major limitation whichimpedes comprehensive analysis of biological samples: they use labelsfor targeted imaging.25 This approach involves monitoringa reduced number of compounds  becausethe chemical stains, immunohistochemical tags, or other labels usedfor imaging are limited. Additionally, molecular discovery is unfeasibleas the use of labels requires rational design of targeted strategiesbased on specific binding between known molecules. Nevertheless, creativeworkflows that combine classical histology and label-free techniquescan yield valuable information that provide insight into complex biologicalsystems.A pictureis worth a thousandwords, especially when it is a molecular image that can shed lighton the composition, function, and heterogeneity of a biological tissue.However, one picture might not be enough to fully characterize complexbiosystems. That is why multimodal imaging platforms have emergedin the past decade as a coordinated combination of multiple imagingtechniques to find solutions in fields as diverse as plant-based renewableenergy, microbiology, clinical medicine, and forensics.m/z) while VSI addsinformation about the abundance and specificity of chemical familiesinstead of individual compounds . Both MSI and VSI have matured greatly in terms ofinstrumental developments in recent decades. The most commonly usedspectrometry techniques for multimodal imaging are matrix-assistedlaser desorption/ionization (MALDI), surface-assisted laser desorption/ionization(SALDI), desorption electrospray ionization (DESI), secondary ionmass spectrometry imaging (SIMS), and laser ablation-inductively coupledplasma (LA-ICP). The most commonly used vibrational spectroscopy techniques,on the other hand, are Raman spectroscopy, surface-enhanced Ramanscattering (SERS), Fourier-transform infrared spectroscopy (FTIR/IR),and attenuated total reflection-Fourier transform infrared spectroscopy(ATR-FTIR).Mass spectrometry imaging (MSI) and vibrational spectroscopyimaging(VSI) techniques are common choices for label-free imaging when studyingbiological tissues. They complement each other in terms of spatialand chemical information, and the limitations of one are complementedby the strengths of the other, so spatial resolution images are betterand unique molecular formulas can be identified more effectively.The main complementary characteristics of the two techniques are thatVSI can achieve subcellular spatial resolution while MSI is generallyused for measuring larger areas and morphologies, and that MSI collectsspecific molecular information . Analysts should keep in mind thefollowing aspects when choosing molecular imaging techniques.We have summarized in the Table S1): polarity, surfaceroughness, thickness, etc. Nevertheless, sample preparationfor multimodal imaging can be compatible.Biological samples  have to comply with the instruments’probing chamber. For all MSI techniques, samples must be vacuum compatibleexcept for DESI and for a few MALDI sources, where measurements aredone in atmospheric conditions. Similarly, all VSI techniques operatein air, however Raman and ATR-IR can also examine samples in liquid.Each technique has their own requirements regarding sample properties are generally used forlipids, peptides, and proteins; SALDI for metabolites, small moleculesand lipids; SIMS (a hard ionization technique) for biologically relevantelements, small molecules, metabolites, and sometimes lipids; andLA-ICP for inorganic compounds such as cations, metals, and otherbiologically relevant elements 1. HoweveTargeted analysespinpoint specificmolecules by looking for their molecular fingerprint or their expectedion formation . Each technique has its own strategy to achieve this:MALDI uses specific ionizing agents like 2,5-dihydroxybenzoic acid for peptides or 9-aminoacridine forlipids, SALDI uses nanomaterials to obtain a clean mass spectrum todistinguish small molecules and metabolites, while SERS employs functionalizednanomaterials to enhance the Raman signal of specific molecules. Allthese approaches demand careful design and sometimes complex samplepreparation which are rarely compatible with multiple measurementson the same sample. On the other hand, untargeted analyses need highspecificity techniques which observe many molecules simultaneouslyin order to establish molecular changes and identify key components.In this case MSI techniques are preferred; however, VSI methods canoffer complementary information.10 while smaller samples are analyzed using high-resolution methods like SIMS, LA-ICP, Raman,and SERS18 that can achieve submicrometer resolution. In multimodalimaging, one technique can be used to obtain a full scan of the sampleand another to “zoom in” on a morphologically or compositionallyinteresting area. Fortunately, both MSI and VSI can be used for high-or low-resolution imaging as depicted in Based on the sample sizeand morphology, different imaging experiments require different spatialresolution. For example, larger samples  are frequentlyimaged at lower resolution (over 50 μm per pixel) using techniquessuch as DESI, MALDI, and FTIR,Supporting Information): animal brains ,cell cultures,16 bacterial colonies,18 liver metabolism,2 cancerous tissues,17 fingermarks,12 and even plants . Label-free multimodal imaging is a new holistic approachwhich will impact analytical chemistry, especially the “omics”sciences, for two main reasons: (1) the imaging techniques providecomplementary information that identifies molecules and validatesresults and (2) the spatial and spectral information have boostedvalue.MSI and VSI can be combined to overcome the limitationsof onetechnique and complementing it by the advantages of the other. Thiscombination has been applied in various imaging studies ; the lipid signal is also specificto Raman and can inform about their level of saturation, as it isfrequently used to analyze lipid droplets, layers, and membranes.22 On the other hand, the IR spectra provide specificinformation about proteins: their composition and secondary structure.For example, Neumann et al. used two analytical methodsto obtain the chemically specific and spatially resolved informationnecessary to characterize the chemical heterogeneity of the hippocampusfrom adult rat brains: the average IR absorption spectrum revealedmorphological differences in the protein composition of the differentsegments within the hippocampus (shift between the amide I and amideII bands), while the average mass spectrum showed significant spatialdifferences among several phosphatidylcholine lipid species .4 Another complementaryfunction of multimodal imaging is to facilitate the identificationof macromolecules, such as polymers. For example, combined informationfrom ATR-IR and MALDI was used to identify condom brands on the basisof their composition: the matrix-solvent combination used for MSIfailed to show the presence of polydimethylsiloxane (present in condomlubricants) in all samples, so Raman was used to identify it usingthe characteristic 1258 cm–1 band.12 Multimodal techniques have also been used to monitor molecularbiosynthesis processes in plants. Burkhow et al.monitored the conversion of phytoenes to carotenoids directly in maizeleaves and how the rate of this conversion changed when they silencedone of the genes involved in the biosynthesis. Raman described thespatial distribution of carotenoids, but due to the lack of Ramansignal to detect phytoenes, SALDI was chosen to monitor phytoene accumulationwhen they silenced the chosen gene.6 Inanother study, combined information from SERS and LA-ICP-MS demonstratedthat hybrid nanoparticles penetrate fibroblast cells differently fromstandard silica nanoparticles.14 The SERSspectra reflected the interaction of several amino acids of the sidechains of proteins with the nanoparticles’ surface, while LA-ICPimages revealed the cellular uptake of silica nanoparticles with asilver core by monitoring the stable isotope 107Ag.14 This application promoted the use of multimodalimaging for very much needed qualitative and quantitative nanotoxicologystudies.The information about the lipid contentfrom MSI can be complemented by the protein information provided byVSI.e.g., highlighted through PCA orclustering analyses) that represent significant differences betweenphysiologically similar regions within tissues.4 Onestudy used PCA on a multimodal data set to identify the spatial distributionof different molecular classes: the antibiotic compounds quinolonesand quinolines from bacterial biofilms.18 The synergistic data processing of both VSI and MSI data highlightssmall changes that a single technique often overlooks. For example,fused MALDI and Raman images of rat hippocampus revealed differencesin protein  and lipid  compositionbetween physiologically similar regions, information otherwise inaccessibleby individual images due to lack of specificity or spatial resolutionof Raman and MALDI images, respectively.4 Small details are often crucial for correct spectral interpretationand the correct identification of molecules. The identification ofmolecules can also be validated with multimodal imaging data sets,as VSI and MSI are orthogonal methods . For example, the DESI-MSIand Raman imaging correlation map was used to assign structural featuresto individual molecular species: the multimodal analysis identifiedthe molecular weight (from MS) and the saturation levels (from Raman)of specific lipids (phosphatidylcholines and phosphatidylethanolamines)involved in the myelination process in multiple sclerosis animal modeland human brain samples.7From the viewpoint of data analysis, multimodal imagingdata sets offer a plethora of possibilities for creating both identificationand validation algorithms and strategies. The heterospectral data,colocalized mass spectra and vibrational spectra, can be used to identifysmall variations  which pinpointedthe regions of interest for higher resolution images collected withRaman  and SIMS  imaging.11 In this way high-quality spectra and high-resolution images assignedintracellular globular structures to hemicellulose-rich lignin complexesin perennial grass (Miscanthus x giganteus). Thecoregistration of the two types of image was also ingeniously usedto categorize tissue types. This time larger areas were collectedby VSI in order to guide and select the regions of interest for MSI.Specifically, FTIR microscopy was used to automatically guide high-resolutionMSI data acquisition and interpretation  through k-means segmentation algorithmsto separate tumors from healthy tissues in mouse brains.5 Another study used the histopathological analysisof Raman spectra as a guide for MALDI analysis, which differentiatedbetween healthy and altered epithelial growth from a larynx carcinomasample.17 In this study, MS revealed manyoverexpressed ions which are associated with tumor markers . So, changes occurring withinsmall areas of tissues could be monitored with both VSI and MSI, andbiomarkers were identified.Due to thelimited spatial resolution of some molecular imaging techniques (MALDIand DESI), information-rich MS images are often colocalized with higherresolution VS imagesMultimodal imaging is a complex analytical tool whichinvolvesa variety of high-end instrumentation and software, so it is challengingfrom two points of view: (1) the experimental workflow and (2) thedata processing algorithms.Multimodalimaging workflowsrequire multiple optimization steps in each modality to obtain maximumoutput while maintaining sample and substrate compatibility. Therefore,sample preparation, coregistration strategies that depend on samplepreparation, and acquisition parameters have to be optimized for allinstruments.7 ITO slides,17 BVDA gelatinlifters,12 low emission glass slides,4 gold-coated slides,5 silicon wafers,18 and sterile coverslips.14 For MSI, the substrates also vary accordingto the technique and application: ITO slide,17 MALDI plate,12 custom-made SIMS plate,9 low-emission glass slide,4 gold-coatedslide,5 packing tape,6 silicon wafer,18 magnesium fluoride slide,7 and sterile coverslips.14 Choosinga substrate that worked for both imaging techniques soon became anasset, and several studies applied multimodal imaging using the samesample preparation and the same sample.17 One such substrate is the ITO-coated glassslide, on which cells,16 mouse brain,13 mammary tumor,10 and larynx17 sections are depositedright after cryosectioning. Generally, the Raman measurements (whichare nondestructive) are done first, and then the matrix is depositedon the substrate for MALDI analysis. Silicon wafers are another exampleof a commonly used substrate in SIMS, LDI, and Raman,18 while gold-coated glass slidesare used to couple SIMS and synchrotron FTIR and UV spectroscopy.2 All these substrates provide excellent spectrain both modalities with minimal preparation and interference in thedata acquisition. However, when signal enhancing agents are used (suchas organic matrixes for MALDI), the sample cannot be further usedfor other measurements without removing the agents, which may washthe analytes away. For this reason, one of the most pressing futuresteps is to develop more multimodal substrates to enhance both theRaman/IR signal and the ionization efficiency for MSI.Fortunately, MSI and VSI sample preparation iscompatible as the starting point is the same: the biological samplesare placed on a substrate. However, the substrate  is not always the same for the two techniques. Raman and IRtypically use calcium fluoride, but there are other working optionssuch as microscope slides,28 For this reason, the pretreatment of samples  has to be compatiblewith both imaging techniques. On the other hand, fresh frozen or iceembedded samples might generate artifacts in the VSI spectra due tohigh water content and autofluorescence. It is also important forthe sample to be compatible with vacuum or atmospheric conditions.Dannhorn et al. studied the sample preparation methodsused in various MSI techniques  to create auniversal embedding protocol suited for a broad range of specimens.29 The hydroxypropyl-methylcellulose and polyvinylpyrrolidonepolymer hydrogel outperformed the standard procedures , with no interferencewith MS analysis or histological stains. Although this approach stillneeds to solve issues such as cold embedding and thaw mounting samples,it also enables using the same sample section for both immunohistochemicalstaining and MSI.29Whenit comes to instrumentation, sample compatibility is as importantas choosing the right substrate. For example, biological samples mightbe formalin-fixed paraffin embedded. This affects both VSI and MSImeasurements because the signal from the paraffin overwhelms the spectrafrom the sample and conceals relevant information.16Before acquisition,however, an additional sample preparation stepmight be used for easy image registration during data analysis. Thisstep consists of marking the area to be imaged with a mask or specificpoints that are later used to coordinate the alignment of the molecularimages collected. One such strategy aimed to register images by placinga physical mask, known as a fiducial mask, on the sample to assistin locating the raster positions for both Raman and MALDI imagingtechniques.i.e.,total ablation by the laser, dehydration, chemical degradation, etc.). Therefore, when the sample is probed subsequentlyby another molecular imaging method, the quality of the collecteddata can be compromised. Neumann et al. examinedhow the IR data is altered when measuring the same sample after MALDIacquisition. The IR image quality was preserved, but a slight redshift in the spectra was associated with increased sample absorbancefrom the matrix. Other influencing factors were also noted: alteredmicroenvironment, loss of some chemical species, or exposure to atmosphere.4 Nevertheless, if the first imaging method isnondestructive, which is the case for all VSI methods, then the followingacquisition generates reliable information. As such, the latter approachis the norm (see Table S2).After ensuring compatiblesample preparation, acquisition orderis paramount. All MSI techniques are destructive so the moleculesdetected are no longer present in the remaining sample after acquisition.The sample itself may deteriorate greatly  the acquisition parameters are compatible with obtainingthe required spatial and molecular information, (2) the acquisitiontime is minimal (if sample viability is a concern), and (3) the imagesare suitable for postacquisition image coregistration to avoid resolutiondiscrepancy.Dataprocessing and analysisstrategies are needed to extract the maximum chemical information.In the case of multimodal imaging, these strategies strongly dependon the experimental workflow. Multimodal imaging data is a collectionof two datacubes which contain different spectral information butoften similar spatial information. Generally, the preprocessing stepis done separately for each datacube, in accordance with the techniques’necessities. This involves using different software for each techniqueand possibly different algorithms for similar signal processing procedures. 30 The points are usually selected on optical images, on molecularimages with single or multiple ion or band representation, and evenon score images from the principal component analysis (PCA). The simplestway to coregister multimodal images is to convert each image (of selected m/z or bands) into a single-color intensityplot and then overlap the images using the RGB color schemes.16 This approach is feasible when the images can be aligned and superimposedwithout distorting the spatial resolution of either image. Anotherstrategy for coregistration used fiducial markers. Pixel-wise coregistrationwas done with a high-resolution VSI image  and a low-resolution MSI image, wherethe sequential imaging data from the same sample was coregisteredusing a fiducial marker-based alignment7  associated with each pixel, as seen in viceversa, on the basis of the interdependence between VSI andMSI. In the scientific literature, each study tackles data fusiondifferently.10 Ryabchykov et al. fused the spectral data from MALDI and Raman imagesafter interpolating the data in the same spatial grid during coregistration.This data fusion consisted of adjusting the dimensionality and dynamicrange of each data set and then merging the two matrixes into one.8 Similarly, Neumann et al. coregisteredthe MS and IR images using anatomical features of the individual PCAscore images and then fused the data by up-sampling the MS image anddown-sampling the IR image to adjust discrepancies between the spatialresolution.4 Another data fusion strategytransformed the coordinate system of one imaging modality into theother in order to calculate the mean VSI spectra for regions whichexhibit a certain intensity of a MSI peak or MSI fingerprint.13 This concept was designed to translate any diseasemarker information from MSI into the complex Raman fingerprint, informationwhich can be further used as an in vivo applicationof the marker in a diagnostic procedure.13Data fusion consistsof merging the VSI and MSI spectra into onesingle multidimensional data set called a multimodal datacube. Themultimodal datacube describes each pixel with three components: onespatial component giving information on the pixel position and twospectral components  to classify Raman spectra for differentiating newlyformed myelin and normal myelin in remyelination;7 maximum margin criterion linear discriminant analysis (MMC-LDA)to determine the most significant mass spectrometry peaks from DESImeasurements;7 multivariate curve resolution-alternatingleast-squares (MCR-ALS) to reveal the composition of lipids and theirparticular localization;10 and clusteringalgorithms for untargeted image segmentation. The most common clusteringalgorithm is k-means clustering,7 but unsupervised hierarchical cluster analysis,which is based on interspectral distances obtained from normalizedPearson’s product-momentum correlation coefficients and Ward’salgorithm, is also used.3k-means clustering on a rat hippocampus, whereeach cluster is represented spatially into imzML, and makes it possible to use MSI-specific software forRaman imaging data. This is the first step on the way to creatinga common format for all molecular imaging data. On the other hand,multimodal imaging requires several software packages for data export,conversion, processing, and analysis. For example, WinCadence (PHI)was used to generate SIMS images, FlexImaging software (Bruker) wasused to generate LDI images, and WITec software was used for Ramandata analysis, all in the same multimodal application.11 However, some multimodal data analysis approachesuse custom-made scripts in R and Matlab,17 but data preprocessingis not always done in the same software. For this reason, multimodalimaging data analysis requires a good set of programming skills and/ormultiple licensed software.The technical challenges in terms of data processing include theformat in which the data is collected and the software used for dataanalysis. Unfortunately, for imaging experiments there is no universaldata format that encompasses both VSI and MSI data. This preventsstraightforward data fusion and analysis by a single software package.Recent progress in format compatibility between Raman and SALDI imagesproposed imzML (the standard format for MSI files) as a template forRaman imaging data as well.32 Similarly, MS techniqueshave been used for surgical guidance tools. In particular, iKnifehas identified and distinguished between primary and metastatic tumors in vivo and ex vivo.33 However, the imaging possibilities of VSI and MSI are stillbarely used in clinical medicine. We suggest some possible examplesabout the use of label-free multimodal imaging that couples VSI andMSI for the applications described below.Multimodal imaging is an analyticalapproach with benefits in theclinical environment that are as yet unexplored. Its high specificity,functional and molecular information, and the label-free aspect ofthe technique augment the histological data obtained with standardmicroscopic tools used in the clinics. This approach provides a plethoraof possibilities for biomedical applications such as diagnostics,intraoperative guidance, nanotoxicology, and even personalized medicine.For example, modern surgical tools that guide intraoperative interventionsalready use IR and Raman probing because they are affordable, offerreal-time information, and enable molecular profiles to be visualizedon the microscale.34 have arisen so that VSI can be used to screen and classify tissues using the approach knownas “spectral histopathology.”24 These applications are complementary to classic histopathology andaim to reduce the number of samples that pathologists need to examine.There are even commercial applications currently being evaluated inclinical trials that exploit IR spectroscopy in the screening of canceroustissues, such as esophageal and colon cancer, by the company DynamXMedical . The use of MSI to identify biomarkers emerged from the need notonly to distinguish between healthy and cancerous tissues but alsoto distinguish between cancer types and grades.34 As such, ambient mass spectrometry imaging analysis revealed biomarkersrepresentative of different types and subtypes of lung cancer witha sensitivity and a specificity of 93.5% and 100%, respectively.20 Therefore, multimodal imaging could be usedfor diagnostic purposes in clinical medicine, where VSI classifiestissues and MSI detects biomarkers of cancer grades, all in an automatedprocedure with minimum interaction from a pathologist.White light histopathologyis the gold-standard for diagnosing cancerouscells from samples of human tissues. However, it relies heavily onthe experience of the pathologist. Since experts in histopathologyare scarce and expensive to train, several applicationsin vivo but they apply MS and vibrational spectroscopy strategiesseparately: (i) iKnife for electrosurgical dissection of tissues thatinforms about the tissue lipidomic profiles and discriminates differenttypes of tumors;33 and (ii) a hand-heldcontact Raman spectroscopy probe for the in vivo,local detection of cancer cells in the human brain.35 Since both methods are hand-held, combining the iKnifeand the Raman probe into a single instrument could broaden the possibilitiesof intraoperative procedures, maximize the analysis possibilities,and give surgeons accurate guidance.Surgicalmargins, or margins of resection, are the rims of theapparently healthy tissue around a tumor that has been surgicallyremoved. Normally pathologists examine the surgical margins postoperationto check if tumorous cells are present in the margins of resection.There are devices currently used to characterize surgical margins etc., or for developing therapies and designing in vitro organoid models. For example, molecular imagescollected with SIMS MSI established the silver nanoparticle uptakeof Caco-2/TC7 and HT29-MTX cells by analyzing intracellular localizationin relation to particle size.36 Cell–cellinteraction analysis is also crucial when designing organoid modelssuch as the coculture model of the alveolar barrier, which includesseveral types of cells: alveolar type II epithelial cells, endothelialcells, macrophages, and dendritic cells.37 This model was used to discriminate chemical respiratory sensitizersfrom irritants. Multimodal imaging could be used to monitor oxidativestress, a common consequence of exposure to some toxicants, includingnanoparticles, in cells and organoid models through DNA damage andchanges in lipid peroxidation.38 For this,Raman can monitor DNA conformational changes following several bandsbetween 600 and 1700 cm–1,39 while MSI can monitor lipid peroxidation by identifying lipid speciesand their degree of saturation.40 Therefore,cell response can be characterized in situ as a livingsystem (with VSI) and as a frozen metabolic state (with MSI).Understanding cellresponse and cell–cell interactions is crucial for testingcompounds, such as drugs, cosmetics, pollutants, nanoparticles, 41 For this reason, noninvasive tests based onsweat composition analysis are becoming popular. MSI approaches wereused to monitor illicit drugs from fingerprints42 and to observe the detoxification of contaminants and medicines,41 and the biomarkers of diseases.43 For example, the sweat metabolome analysis by MS revealedthat the signal from pilocarpic acid (a metabolite of pilocarpine)and mono(2-ethylhexyl)phthalic acid (a metabolite of the plasticizerbis(2-ethylhexyl) phthalate) can be used to detect cystic fibrosisin asymptomatic infants.43 Raman spectroscopywas used to investigate the function of a single human sweat glandand the efficiency of aluminum chlorohydrate, a well-known antiperspirantingredient.44 Future applications of multimodalimaging regarding noninvasive sampling are appealing, especially becausemultimodal substrates are bioinert and compatible with aqueous samplessuch as sweat. Additionally, sweat can be collected from the skinsurface  with no need fora particular volume because both VSI and MSI have low limits of detection.Sweat is a biofluid thatcontains various excretion products: amino acids, urea, metal andnonmetal ions, metabolites, and xenobiotics (such as drug molecules).Sample preparation, coregistration, and data analysis need to bemore user-friendly if multimodal imaging is to be used more commonly.That is why standardizing multimodal imaging based on VSI and MSIprotocols and developing hybrid instrumentation and software for analysisare key to advancements in the field.in situ analysis is not always necessary. Investigating urine, saliva, sweat,blood, and even breath does not require spatial information but doesoften require multiple analytical techniques for accurate diagnosisand even biomarker identification.46 For this reason,a substrate that is compatible not only with all instruments (bothVSI and MSI techniques) but also with all types of sample  is still needed. Solid state substrates basedon gold and silicon nanostructures are promising, as they have allthe necessary requirements for LDI-MSI and SERS imaging.48 Because the fabrication processes of these substrates can be automated,and because these types of substrates are compatible with multipletechniques, they are a potential candidate for the standardized multimodalsubstrate.Here we have focused onthe imaging part of multimodal techniques, but etc. Creating a standardcoregistration protocol will make it possible to assign two qualitativelydifferent spectra to the same area of a biological sample regardlessof the imaging technique used. This will enable fast and accuratedata collection over a variety of imaging modalities including MSI,VSI, histology, and even magnetic resonance imaging and other routineclinical imaging techniques.Universal sample processing is beneficial not onlyfor multimodal imaging but also for intersectoral and multidisciplinarycollaborations. For example, samples prepared in the clinic for histologicalexamination can be stored over time and the collected consortia canlater be simultaneously analyzed by nonclinical research groups fordifferent purposes: creation of vast and unbiased spectral referencelibraries, disease biomarker identification, development of tissuetype classification algorithms, 31 In fact,the converter presented makes it possible to visualize Raman mapsin common MSI software, which provides new opportunities for dataanalysis. Although the imzML format is missing the nomenclature forRaman measurements such as “Raman Shift” units or diffractiongrating definitions (cm–1), in the future it couldadopt multiple ontology options which could be chosen during dataexport or conversion. The universal imaging file should have threecomponents: (1) a metadata section that stores all acquisition detailssuch as laser parameters ,area specifications , specific detailsof individual methods ; (2) a coordinates section containinginformation about pixel position; and (3) a data section with allthe spectral information.Once imaging datahas been collected, the acquisition software should make it possibleto export the data in a universal format for imaging. Thus, each imagingdata set, regardless of the acquisition technique, can be straightforwardlyvisualized and analyzed using a single software package. This wouldfacilitate tremendously the development of correlated data analysisalgorithms and software which can bring relevant information buriedwithin the multimodal data sets to light. Along these same lines,the use of the imzML format (common for MSI data) has been suggestedfor Raman imaging data as well.49 and optical photothermal infrared (O-PTIR) spectroscopycan reach 100 nm.50 TERS molecular imagesat 8 nm spatial resolution can be achieved through simultaneous collectionof Raman spectra and nanotopography information. TERS uses a goldor silver tip which scans the sample surface and, when illuminated,generates a locally amplified electromagnetic field resulting in anenhanced Raman spectra.49 O-PTIR has beatthe diffraction limit of conventional IR microscopes by modulatingthe changes of photothermal and photoacoustic effects from an intenseIR beam source.50 This enables collectingIR spectra in the order of hundreds of nanometers. Moreover, the instrumentcollects Raman signal simultaneously from the same location, promotingthe use of coregistered multimodal imaging. Van De Plas etal. demonstrated that fusing rich chemical information images(MALDI MSI) with high spatial information images (microscopy image)can be beneficial from three points of view: (1) the lower resolutionimage can be sharpened; (2) out-of-sample prediction can be used foracquisitions which have smaller areas; and (3) the enriched spectralinformation can be used to discover biological patterns otherwiseunnoticed.30 Therefore, fusing ultrahighresolution images (from TERS or O-PTIR) with information-rich MS images(such as SIMS) enables the exploration of uncharted territories suchas intracellular imaging.Multimodalimaging could also push the limits on spatial resolution. In fact,tip-enhanced Raman spectroscopy (TERS) can attain 8 nm,51 We could also consider the following combinations.Having a singleinstrument which combines multiple imaging methods would considerablysimplify both experimental and data analysis procedures. In fact,MS instrumentation equipped with an optical microscope is alreadyavailable on the market as iMScope QT from Shimadzu. A hybrid instrumentcombining MSI and VSI would analyze the same sample on the same substrate,it would need no coregistration strategy because the coordinate systemis identical for both imaging methods, the output data format wouldbe the same, and the data could be analyzed using the same softwarewith no need for multiple data converters. One such multimodal imaginginstrument developed in-house already combines atomic force microscopy,IR, and MS to acquire topographical and chemical images at 1.6 μmspatial resolution of PVP/PMMA polymer thin films.DESI-MSI combined with Ramanspectroscopy using a long working distance objective would enablemultimodal imaging in live samples. For example, live bacterial coloniesor frozen tissues thawed on a multimodal-compatible substrate couldfirst be sampled with Raman and then immediately sampled with DESI,so after each line acquisition the other modality would measure thesame area. The use of an LWD objective could also permit simultaneousacquisition directly from the same pixel if the laser spot size issmaller than the ESI liquid bridge (droplet) and the optical pathwayis not hindered by the presence of the capillaries.9 On the other hand, SIMS is a technique which can be usedto collect maps layer by layer or to section (or mill) the sampleusing the ion beam at the desired depth, previously determined bythe Raman profile. Therefore, the fast maps collected with Raman wouldguide the SIMS analysis, which provides exact information on elementsand small molecules.SIMS and Raman can be usedto obtain 3D profiles of tissues. The ability of confocal Raman spectroscopyto acquire in-depth information and create 3D profiles without damagingthe sample is useful for guiding sample discovery.52 can be coupled with an IR laser and detector. This canquickly scan the sample with the IR mode which can help the MALDIacquisition reduce time and increase spatial resolution to the maximum.SALDIand SERS bothneed a nanostructured agent which interacts with a laser and enhancesthe collected molecular signal. Therefore, it is reasonable to modifyan LDI instrument (such as MALDI) by coupling multiple lasers (UVfor MS and visible for Raman) and installing an additional objectiveor detector (for collecting the SERS spectra). This instrument canexplore vacuum-compatible samples such as frozen tissues sputteredwith gold or silver nanoparticles, first analyzed by SERS, becauseit is nondestructive, and then by SALDI. Similarly, a recently presentedMALDI instrument that works in transmission modehttps://www.moldiscovery.com/software/lipostarmsi/). We foresee an increase in developing such software not only formultimodal MSI and VSI data analysis but also for optical microscopyimages, fluorescence imaging, and possibly MRI.Currentmultimodal workflows collect and preprocess data sets separately andthen visualize and analyze single or coregistered images either separatelyor with data fusion methods. Unfortunately, there is no straightforwardway of doing all these data processing steps with the same software.For this reason, software in which the sophisticated computationalprocesses are automatic (such as image registration and data fusion)and users can opt for a friendly interface which requires minimalinput are in high demand, especially in clinical medicine where largedata sets are routine. For example, LipostarMSI is a software capableof raw data processing, coregistration, manually drawing or importingof regions annotated by pathologists, image visualization, univariateand multivariate image and spectral analysis, and even annotatinglipids and metabolites (Multimodal imaging based on MSI and VSI techniques blends the advantagesof each modality, specificity, sensitivity, and spatial resolution,and sheds light on details otherwise buried under thousands of pixels.The complete integration of imaging techniques from the point of viewof the experimental workflow makes it possible to adopt a multidisciplinaryapproach to sample processing, which uses not only MSI and VSI butalso MRI, histology, and other imaging techniques. As far as multimodaldata processing is concerned, universal imaging file formats and standarddata processing and analysis workflows will soon be available so thatmultimodal imaging can be used in various fields. We believe thatthe progress made in standardizing the multimodal imaging workflowand development of hybrid instrumentation will have considerable impacton research in biology, agriculture, nanotoxicology, pharmaceutics,cosmetics, and especially, clinical applications.


Upper urinary tract obstructions (UTOs) are blockages that inhibit the flow of urine through its normal course, leading to impaired kidney function. Imaging plays a significant role in the initial diagnosis of UTO, with anatomic imaging (primarily ultrasound (US) and non-contrast computed tomography (CT)) serving as screening tools for the detection of the dilation of the urinary collecting systems . Whether hydronephrosis represents UTO or a non-obstructive process is determined by functional imaging . If these exams reveal evidence of UTO but no discernable source, multiphase contrast enhanced CT urography and/or dynamic contrast enhanced MR urography (DCE-MRU) may be performed to delineate a cause. These are often performed in conjunction with direct ureteroscopic evaluation. While contrast-enhanced CT currently predominates, it can induce renal injury due to contrast induced nephropathy (CIN), subject patients to ionizing radiation and is limited in quantifying renal function  and establishing the extent to which hydronephrosis is due to functional obstruction. Traditional MRI is similarly limited in its ability to quantify function. DCE-MRU presents concerns regarding nephrogenic systemic fibrosis (NSF), although decreased with newer gadolinium-based contrast agents, and regarding cumulative gadolinium deposition in the basal ganglia. DCE-MR CEST urography is a promising alternative, employing new MRI contrast agents and imaging schemes and allowing for concurrent assessment of renal anatomy and functional parameters. In this review we highlight clinical challenges in the diagnosis and management of UTO, identify key advances in imaging agents and techniques for DCE-MR CEST urography and provide perspective on how this technique may evolve in clinical importance. Upper urinary tract obstructions (UTOs) are blockages in the flow of urine that result in dilation of the renal collecting system (hydronephrosis), A,B, impaThe treatment of upper UTOs depends on the cause of the obstruction and degree of renal impairment. For example, in the setting of nephrolithiasis, conservative management is often the choice for small stones, as these spontaneously pass and rarely cause permanent functional impairment. Conversely, lithotripsy and stenting are the standard treatment for larger obstructing calculi. In other settings where hydronephrosis is present without significant functional impairment, treatment centers around addressing the underlying etiology for UTO. UTO related to malignancy is typically managed surgically, irrespective of the degree of functional impairment. However, in some cases, the quantification may still play a role, particularly in determining the utility of intervening to preserve a hydronephrotic kidney. When functional impairment/acute kidney injury (AKI) is secondary to partial or complete ureteral obstruction, percutaneous nephrostomy C and retStandard blood and urine-based laboratory tests, while vital to the assessment of renal function, are somewhat limited in defining the reversibility of functional impairment related to upper UTO. Estimations of the glomerular filtration rate (eGFR) derived from serum creatinine measurements, as well as demographic and anthropomorphic information, can determine how well the kidneys remove waste and excess fluid from the blood, and are a highly cost-effective assessment of kidney function. Generally, an eGFR < 60 suggests renal impairment. Lower eGFR estimates may indicate worsening degrees of renal failure, with eGFR < 15 indicating fulminant renal failure, requiring either dialysis or transplantation. While these eGFR estimates are helpful in screening patients for renal impairment, they indicate little about the chronicity of renal injury and the reversibility of renal impairment in the setting of UTO. If prior eGFR measurements are available, values can be trended, and if a precipitous drop in eGFR accompanies the development of UTO/hydronephrosis, one can deduce a causal relationship. Generally, if the impairment is relatively new, it can be surmised that the renal impairment is largely reversible. However, in practice prior imaging and laboratory tests are often unavailable, and confounding factors ranging from medical illness, race , to hydrGFR and renal perfusion can be used for diagnosis of kidney diseases and monitoring of treatment interventions. Measuring the clearance of exogenous markers in patient’s blood is considered to be the gold standard for GFR measurement. A number of imaging options are available for patients with suspected UTOs, including ultrasound, X-ray (including intravenous urography (IVU)), computed tomography (CT), nuclear medicine renography, and magnetic resonance imaging (MRI) . We reviRenal ultrasound, which is fast, noninvasive, and easily accessible, is widely used in the initial evaluation of the urinary system A. Renal Intravenous and retrograde urography have been mainstays for renal imaging prior to the advent of cross-sectional imaging. In X-ray urography the urinary system is opacified by iodinated contrast, which is administered intravenously and excreted or retrograde via foley catheter . While rComputed Tomography (CT), which utilizes a helical array of X-rays to produce volumetric tomographic images, has largely supplanted IVU in the assessment of UTOs B. CT pro99mTc-diethylene triamine pentacetic acid, 99mTc-DTPA) or secreted by the tubules . As such, nuclear medicine scintigraphy is fundamentally based on physiology and not anatomy. In cases in which a patient has evidence of hydronephrosis from anatomic imaging, the patient should undergo nuclear medicine scintigraphy in order to determine whether the hydronephrosis is due to a UTO or a result of non-obstructive pathology. In fact, 99mTc-MAG3 is particularly useful due to its mechanism of clearance being tubular secretion-based; as a result, it is usable even with depressed eGFR due to renal dysfunction [Nuclear scintigraphy is currently the gold standard for the assessment of differential kidney function and for evaluating UTOs. The fundamental reason for the predominance of nuclear medicine scintigraphy in evaluating UTOs is the ability to differentiate a patulous renal collecting system from an obstructive uropathy . This apfunction .Scintigraphic evaluation can be enhanced by use of Lasix as a diuretic. In situations in which the clearance of radiotracer from the renal collecting systems is slow or absent, Lasix can be administered in order to increase the production of urine and overcome any intrinsic patulousness in the system. If a hydronephrotic collecting system clears rapidly after administration of Lasix, the system is dilated but not obstructed. If, on the other hand, the system clears slowly or not at all , the fin2) of urine, with heavily-fluid weighted sequences that suppress short T2 proton signals with long echo times, typically acquired using single-shot fast spin-echo (SS-FSE) and half-Fourier single-shot spin echo (HASTE) sequences to provide outstanding visualization of the urinary tract. This is of particular concern in UTO, where an obstructed kidney may generate little urine. Furthermore, dynamic cinematic acquisitions can be obtained, allowing areas of ureteral peristalsis to be distinguished from fixed stenoses. Excretory DCE-MRU urography currently relies on excreted MRI imaging agents (mainly based on gadolinium) to generate signal changes within the collecting system . Both MRU approaches avoid use of ionizing radiation and pose no risk of CIN, which are advantages over CT. They have none of the technical limitations of US. Static fluid sequences do not require administering gadolinium contrast, avoiding concerns related to NSF and cumulative gadolinium deposition. As shown in MR urography is an alternative technique for the detection of urothelial lesions and strictures. MRI utilizes magnetic fields and radiofrequency pulses without imparting ionizing radiation and displays soft tissue contrast due to differences in water density and relaxation. Two MR urography methods are currently employed: static-fluid MR urography and excretory Dynamic Contrast Enhanced MR urography (DCE-MRU). Static-fluid MR urography exploits the long transverse relaxation times  or irreversible. Additionally, in the case of urolithiasis, pH is relevant due to its relationship with stone formation, with urine acidification or alkalization employed as treatments depending on the stones’ constitution. Hence, CEST imaging is of interest in the evaluations of upper UTOs not only due to efficiency, but the ability to provide novel functional information to inform clinical therapy.CEST has emerged as an MRI contrast mechanism that can be used to detect small amounts of contrast agent through an amplified detection of low concentration protons (on the order of low mM) through their exchange with water . Based o®, +/−Mu) and high protein diet (HP +/−Mu), mild kidney disease mice kept on a regular diet (RG −/−Mu), and severe kidney disease mice on a high protein diet (HP −/−Mu). Both RD and HP +/−Mu controls displayed homogeneous pH values of 6.5 and excellent kidney perfusion of agent (~100%) while, in contrast, RD −/−Mu mice displayed a lower average pH (~6.1) and perfusion (~79%) and an order of magnitude larger range of pH values in the kidneys (±0.2). Furthermore, HP −/−Mu mice displayed a slightly lower pH (~6.0), substantially lower perfusion (~15%) and significantly larger range of pH values (±0.45). We have further tested iopamidol-based CEST MR on a complete unilateral urinary obstruction (UUO) mouse model. As shown in The triiodobenzenes iopamidol ISOVUE®, A and iopOther agents have also shown promise for kidney imaging. For example, Sherry and colleagues performed in vivo pH imaging of mouse kidneys using a frequency-dependent lanthanide-based CEST agent . Other aAt this stage, all of the major clinical scanner vendors have sequences for performing CEST imaging, allowing for the translation of pH imaging to patients ,85,86,87Early diagnosis and treatment of UTOs can salvage kidney function. CE-MRI methods have shown great potential for the evaluation of kidney function, especially CEST MRI, which can evaluate renal perfusion, quantify contrast enhancement, and effect pH mapping, which can provide more detailed information for the kidneys. In the case of upper UTOs the hope is that these new technologies might enable earlier detection of whether there is functional renal impairment and if interventions will preserve renal function. CEST MRI also shows the potential to evaluate kidney function. If CEST MRI contrast agents could provide more information that relates to renal functional impairment, DCE-MR CEST urography might become more mainstream for the assessment of upper UTOs  and be u


Gender dysphoria describes the distress associated with having a gender identity that differs from one’s birth-assigned sex. To relieve this distress, transgender, and gender diverse  individuals commonly undergo medical transition involving hormonal treatments. Current hormonal treatment guidelines cater almost exclusively for those who wish to transition from male to female or vice versa. In contrast, there is a dearth of hormonal options for those trans individuals who identify as non-binary and seek an androgynous appearance that is neither overtly male nor female. Though prolonged puberty suppression with gonadotrophin releasing hormone agonists (GnRHa) could in theory be gender-affirming by preventing the development of unwanted secondary sex characteristics, this treatment option would be limited to pre- or peri-pubertal adolescents and likely have harmful effects. Here, we discuss the theoretical use of Selective Estrogen Receptor Modulators (SERMs) for non-binary people assigned male at birth (AMAB) who are seeking an androgynous appearance through partial feminization without breast growth. Given their unique range of pharmacodynamic effects, SERMs may represent a potential gender-affirming treatment for this population, but there is a lack of knowledge regarding their use and potentially adverse effects in this context. Trans individuals have a gender identity that is different from the sex they were assigned at birth, as opposed to cisgender people, whose gender identity corresponds with their assigned sex . AlthougLike trans people with binary identities, those with a non-binary gender identity frequently face social difficulties, including social exclusion at school/work and feelings of isolation. As a result, these individuals often experience poor mental health that is worse than those with binary identities , 4. In sA common reason for poor mental health in trans (including non-binary) individuals is gender dysphoria (GD), which is the distress associated with having a gender identity that differs from one’s sex assigned at birth . Non-binMedical treatment with gender-affirming hormones, such as testosterone and estradiol may be appropriate for some non-binary individuals who wish to masculinize or feminize respectively , 11. HowCurrently, medical transition options for trans people vary depending on an individual’s pubertal stage. For trans adults, treatment options to induce secondary sex characteristics consistent with a person’s gender identity include gender-affirming hormone therapy and surgical interventions , 11. GenUnder current treatment schema, non-binary people who wish for an androgynous body have limited treatment options. For those AFAB, low-dose testosterone is being used empirically with the aim of reducing feminine features by helping to re-distribute adipose tissue and deepen the voice . Such efPubertal suppression with GnRHa blocks the release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from the pituitary gland, preventing production of testosterone and estradiol from the testes and ovaries respectively . WithoutHowever, since the pubertal increase in either testosterone or estradiol levels is important for the development and function of other body systems, use of GnRHa in trans adolescents can also cause unwanted outcomes. For example, pubertal sex hormone is essential for healthy bone development, with testosterone and estradiol acting as key promoters of new bone deposition and increased bone mass . Consistlong-term puberty suppression to maintain an androgynous body, these risks will presumably be exacerbated. Although such an approach might be ethically justified in some circumstances  and antagonistic  effects within different cell types and tissues Table 1 Given their tissue-specific antagonistic and agonistic effects, SERMs could potentially be suitable either as an independent or combined treatment modality for the gender-affirming care of non-binary individuals AMAB who desire partial feminization without breast growth. Specific SERMs Table 1 While SERMs could be used independently for non-binary adults AMAB to induce partial feminization, SERMs alone would not be expected to block unwanted pubertal masculinization in non-binary adolescents AMAB. Androgen blockade would still be required in this younger population. In the following sections, we explore the theoretical use of SERMs in this context, extrapolating from what is known about their use in other situations.Similar to estradiol hormone treatment, SERMs have been found to promote changes in body composition and skin that may be desired by non-binary people AMAB. In post-menopausal cisgender women, studies have found RLX to be an estrogen receptor agonist in adipose tissue, promoting shifts from android to gynoid fat patterns  and incrMost SERMs inhibit the effects of estradiol on breast tissue growth and consequently are used to treat estradiol-responsive breast cancers , 49. HisConsistent with their anti-estrogenic effect on breast tissue, SERMs such as tamoxifen have been reported as an effective alternative to surgery to treat persistent gynaecomastia in cisgender adolescent and adult males , 50. TheWhile promoting partial feminization without breast growth, SERMs have pro-estrogenic effects on bones and could also theoretically mitigate negative bone effects associated with GnRHa and/or anti-androgen use in non-binary adolescents and adults AMAB. A variety of SERMs have now been approved for treating osteoporosis in adults , 43 baseThough tamoxifen, RLX and lasofoxifene have all been associated with increases in BMD in post-menopausal cisgender women, there may be important differences between different generation SERMs in terms of bone health. For instance, only RLX and lasofoxifene have shown fracture benefit. Moreover, while lasofoxifene reduces the incidence of both vertebral and non-vertebral fractures , RLX wasImportantly, the positive bone health effects of SERMs in post-menopausal cisgender women cannot be directly extrapolated to gender non-binary young people AMAB. Post-menopausal cisgender women have already attained peak bone mass (PBM) in early adulthood , and it Indeed, there is evidence that questions the extent of SERMs’ pro-estrogenic effects on bones. For example, in a three-year prospective study of premenopausal cisgender women treated with tamoxifen for breast cancer, the average annual lumbar spine BMD fell 1.4%, compared to a 0.2% increase in those who received a placebo. These results prompted the authors to conclude that tamoxifen can act as a partial competitive antagonist of endogenous estradiol to cause increased bone remodelling . SimilarHence, caution is required. With no studies having assessed bone outcomes in trans people using SERMs, further research is required, including in those AMAB. Thus far, pre-clinical data has shown that lasofoxifene prevented bone loss by inhibiting bone turnover related to aging and low serum testosterone levels in male rats . In addiIn vitro studies have suggested that SERMs promote synaptic transmission, increase axonal growth, and facilitate the regeneration of damaged synapses  over a pre-specified timeline  should be determined. If treatment goals are not achieved, treatment should be ceased. Given Raloxifene is the only SERM with evidence for agonistic effects on fat and body composition , 41, it In the clinical setting, increasing numbers of non-binary individuals are seeking gender-affirming hormone therapy, and some desire androgynous physical characteristics to align with their gender identity. This can pose challenges in tailoring treatment options, and a lack of well-defined therapeutic pathways leaves non-binary people at risk of persistent GD and ongoing vulnerability to adverse mental health outcomes. SERMs may be a theoretically attractive option for non-binary people AMAB who desire partial feminization without breast growth. However, there is a dearth of evidence regarding the safety and efficacy of SERMs in this context. An individualised, shared decision-making approach to care is therefore needed when considering the use of SERMs in non-binary people, with acknowledgement of their experimental nature, significant potential risk and need for further research.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.JX performed the relevant literature review, screened studies for inclusion, extracted relevant information from the included articles, drafted the initial manuscript, and revised the manuscript. KP conceptualised the manuscript and, together with LN, AC, MO’C, and SZ reviewed and revised the manuscript. All authors contributed to the article and approved the submitted version.KP is supported by fellowships from the Royal Children’s Hospital Foundation and Hugh Williamson Trust Foundation. AC is supported by an Australian Government National Health and Medical Research Council Early Career Fellowship (#1143333) and receives support from the Viertel Charitable Foundation Clinical Investigator Award, Endocrine Society of Australia Ken-Wynne Award and Royal Australasian College of Physicians Foundation. MO’C, LN, and KP acknowledge the infrastructure funding received from the Victorian State Government through the Operational Infrastructure Support (OIS) Program.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Globally, parents and children in same-sex parent families are impacted by many laws related to the parental sexual orientation. These laws vary considerably from one country to another, ranging from full legal recognition to criminalization. The psychological consequences of living in an ambiguous or hostile legal climate likely interfere with parental health, family functioning, and child development. However, a systematic evidence synthesis of the pertinent literature and its placement within a broader psychological model are currently lacking. The aims of this review were thus (1) to systematically review qualitative and quantitative evidence on the impact of sexual orientation laws on same-sex parent families in key domains and (2) to place these findings within a broader model informed by minority stress and family theories. Our review was preregistered and conducted in line with PRISMA guidelines. We searched for qualitative, quantitative, and mixed methods studies on the impact of sexual orientation laws on target outcomes  via systematic database search  and forward-backward searches. Fifty-five studies published between 1999 and 2020 were eligible for inclusion and were synthesized using a data-based convergent synthesis design. Thirteen descriptive and three overarching analytical themes were identified through thematic synthesis. Linking our findings with minority stress and family theories, we propose a novel legal vulnerability model for same-sex parent families. The model posits that legal vulnerability constitutes an increased risk for parental and child health as well as family functioning through individual and shared pathways between family members. Additionally, the model introduces counteractions that families engage in on the personal, familial, and systemic level to mitigate the impact of legal vulnerability, as well as moderators on the contextual, familial, couple, and individual level that modulate this impact. Implications for research and clinical practice are discussed. However, much less is known about how access to equal rights (or the lack thereof) impacts both parents' and children's health in these families  systematically summarizing the pertinent evidence on the impact of laws and legal recognition on the health and family functioning of same-sex parent families, (ii) placing it within existing minority stress and family theories, and (iii) outlining implications for research and practice. We do so by introducing the concept of legal vulnerability, which – as we theorize in a novel integrative model – links the impact of laws and legal recognition of same-sex parent families with individual and family-related outcomes.Globally, the legal landscape for sexual minorities is varied and in constant flux , for example through the criminalization of the parental sexual orientation , only two  are listed as providing full equality in the category “Family” in 2020.In recent years, many countries—particularly European and North American—have seen an unprecedented shift in the legal recognition of same-sex parent families , lacking legal relationship recognition or protection from discrimination, and a country's overall (socio-)legal climate have been repeatedly linked to adverse physical and mental health outcomes in sexual minority youth and adults. These include reduced life satisfaction  parental and child health, (b) family relationships and functioning , and (c) child educational and cognitive outcomes.Second, we aim at deriving an integrative model that elucidates possible pathways through which sexual orientation laws affect same-sex parent families. For this purpose, we place our findings within well-established theories and key literature pertaining to minority stress Meyer, , family https://osf.io/efgkr/). Eligibility criteria  through May 10, 2020 using multiple combinations of search terms based on free and controlled vocabulary  related to (a) sexual minorities and (b) sexual orientation laws . The systematic literature search was conducted by the first author as part of an on-going systematic review on the impact of sexual orientation laws on sexual minorities . Notably, the review by Siegel et al.  and backward searches of (i) studies eligible for inclusion retrieved via our database search, as well as (ii) unsystematic reviews on same-sex parenting  as well as the comprehensiveness of our database search.We did not systematically search gray literature databases due to the complexity of our search string and the limited possibilities of these databases to handle complex Boolean combinations and truncations  to ensure a reliable screening of the remaining records  by one rater  and subsequently via forward-backward-search (k = 31). All discrepancies in final inclusion decisions were resolved via discussion or arbitration by the third author (see OSF-Supplement S2 for references and reasons for exclusion).The first two authors independently carried out the fulltext assessments of eligible studies identified via database search  to ensure reliable extraction of the following variables: Publication source; methodology; study name and wave (if applicable); year, country, and mode of data collection; sample size and characteristics ; family type; type of law; timeframe  for qualitative studies; measurement points  for quantitative studies; outcome information on measurement and conceptual level.Md% = 97.67, range% = 87.27–100; Mdκ for categorical variables = 0.97, rangeκ = 0.79–1.00).The first and second author independently coded each study and resolved discrepancies by discussion and verification with original reports. Interrater agreement was excellent  from quantitative studies. Although not preregistered, we also extracted effect sizes on predictors of law-related health outcomes : (i) Codes that were concerned with the impact of laws, (ii) codes that were concerned with counteractions families engaged in to mitigate this impact, and (iii) codes that described possible moderators of this impact. For impact-related codes, we specified the valence . For codes relating to counteractions, we also extracted reasons not to engage in this counteraction or side-effects of this counteraction.The resulting codebook was circulated among all researchers and iteratively refined. To facilitate integration with existing minority stress and family theories, codes were labeled in line with terminology used within these frameworks where possible.Consequently, result sections of remaining studies were assigned codes using a deductive-inductive approach. That is, we chose to assign codes based on our initial codebook where possible but allowed for new codes to emerge. In quantitative studies, results from hypothesis tests of interest to our review were assigned codes. In qualitative studies, quotes from individual participants and study authors' interpretations or descriptions of themes were assigned codes. Including individual participants' quotes ensured the inclusion of studies not primarily concerned with the impact of laws or legal recognition or of studies that did not report higher-order themes relevant to the review.The first author coded all studies and refined the codebook by collapsing or further differentiating codes. Due to the heterogenous sociolegal climate described in these studies, we chose to code every study at the lowest  level. The refined codebook was again circulated among all researchers.Separating the impact of sexual orientation laws from the impact of societal prejudice, general legal challenges for stepfamilies, or lacking biological ties to a child posed a challenge during coding. Arguably, the compound impact of these related (but distinct) phenomena makes a differentiation impossible for research participants themselves. We employed a conservative coding strategy and only included quotes and study authors' statements that explicitly referenced the impact of laws or (lacking) legal recognition. While this certainly resulted in a loss of codable data, it strengthens the validity of our results.The first and second author independently coded 36% of studies according to the final codebook (OSF-Supplement S6). Remaining studies were coded by the first author and the assignment of codes was validated by the second author. Discrepancies were resolved by discussion within the research team.After completion of coding, we developed and critically discussed broader descriptive themes. While being firmly grounded in primary study results, we assigned theme labels again in line with minority stress and family theories. Lastly, three overarching analytical themes were developed that reflected our initial grouping .We conducted a negative case analysis after completion of coding Yardley,  to ensurAccording to the classification of research synthesis by Suri , we adopk = 49), book chapters (k = 3), dissertations (k = 2), and in a research report (k = 1). Forty-two studies used a qualitative design, eight a quantitative, and five a mixed-methods design. Years of data collection (if reported) ranged from 1995 to 2018 (Md = 2012).In all, the present review comprises 55 studies (49 unique samples). k = 35), 11 in one or more European countries , four in Australia and New Zealand, and one in Canada and Russia each. Three studies reported cross-country comparisons between the US and the Netherlands . Taking into account multiple assignments of codes in one study, this amounted to 458 individual codes . Thirty-five coded units stemmed from quantitative studies, from which we extracted 68 effect sizes .Our final database comprised 633 coded units of data , (Frustration with) Discrimination and Unequal Treatment (45%), and Legal Documents and Paper Trails and Reaction of Others (40% each). Six codes were only coded in one study each (2%): Acquiescence, Creation of Emotional Dependency, Creation of Financial Dependency, Emotion Regulation (Self)  incorporates themes related to the impact of sexual orientation laws, most often those that regulate the legal recognition of family relationships. These themes relate to the impact on aspects of family life and relationships , predictors and outcomes related to the health and well-being of family members , and child educational outcomes (Education). This analytical theme also incorporates the perception of no impact of legislation on any area of life or the insufficient or even detrimental consequences of (positive) legal change . It should be noted that only in two qualitative studies  describes how some families or family members are impacted more than others due to contextual , familial (Family Characteristics), couple-level (Couple Characteristics), and individual factors .The analytical theme Counteraction (135 coded units) is concerned with the numerous counteractions on the personal (Within Person), familial (Within Family), and systemic (Within System) level that same-sex parent families engage in to alleviate the impact of sexual orientation laws on their relationships, their financial and legal security, and their health and well-being.The analytical theme n = 2), growth of resilience, pride, and an improved parent-child relationship due to legal disadvantages (n = 2), negative effects of increased outness due to marriage or a positive effect of total concealment (n = 3), marginalization of same-sex couples not wanting to marry (n = 2), and exacerbated custody disputes due to a formal relationship recognition (n = 2).We identified eleven coded units from nine studies (3% of impact statements) in our negative case analysis (see OSF-Supplement S11). These include losing financial benefits due to legal change  pathways through which sexual orientation laws might impact key predictors and outcomes of parental and child health and family functioning, (ii) factors that may moderate this association, and (iii) counteractions that family members engage in to mitigate these effects. We now integrate these findings with existing empirical work and theories from the field of minority stress and family research (see below) and propose a conceptual, empirically testable model of legal vulnerability for same-sex parent families. Given the mostly qualitative or mixed methods evidence (85%) and the heterogeneous or insufficient operationalizations of legal status or sexual orientation laws in quantitative studies , we emphasize the hypothesis-generating nature of our model.legal vulnerability  in all members of the family unit (innermost circle), which impacts parental and child health and family relationships in reciprocal ways  are mutually interdependent  stress exposure  but also constitutes an independent minority stressor that impacts health-related outcomes and mediating psychological processes directly (P.2). Pathways P.3 and P.4 depict these mediational pathways  theorized to mediate the association between minority stress and  health . This hypervigilance manifested itself in diverse ways within our review, for example by always having important legal documents at hand  and general (P.6) psychological processes that parents experience jointly, as well as through mediating pathways linking minority stress to health outcomes (P.7). To conceptualize these effects, we draw on the couple minority stress model  power differentials between parents . We include this pathway for the sake of completeness  may translate into a lack of pre-defined relational roles for same-sex relationships . However, the general association between coparenting and child outcomes is a well-established finding in the family literature for both mixed-sex  can serve to validate the relationship between parents and children, as evidence suggests , but also experiences of discrimination  may have indirect ramifications for child adjustment. Specifically, a plethora of studies established that impaired parental mental health  were not uniformly found within studies  securing the family structure through obtaining wills or power of attorney  costly, time-consuming, frustrating, or shameful  should not be used as a justification for denying sexual (or other) minority groups' access to equal rights. Rather, they can be seen as evidence of the resilience that same-sex parent families show when maintaining loving and committed family relationships amidst an unfavorable legal climate and concurrent societal stigmatization.A strength of our model is its empirical evidence base gathered via systematic literature search and synthesis. Its grounding in minority stress and family theories overcomes criticism in the field . Moreover, a concomitant examination of impact-related factors, moderating characteristics, and counteractions seems warranted to advance our understanding of legal vulnerability.Quantitative investigations of the pathways outlined in the legal vulnerability model are paramount considering the current primarily qualitative evidence base.Across jurisdictions, we suggest including items related to family relationships, parenting, or child outcomes in multi-nation investigations into sexual minority health  seem warranted. Legal advances for sexual minorities are recent phenomena globally and same-sex parent families today are likely to have faced legal vulnerability at some point in their shared family biography in the past. Indeed, referenda and campaigns on sexual minority rights can exert long-lasting influences on sexual minorities legal climate for sexual minorities have been recently developed  and family members' counteractions to tackle legal vulnerability. Researchers can utilize existing scales that measure aspects of legal vulnerability directly , particularly in their family relationships and functioning .With regard to populations, our sample is biased toward white, well-educated (presumably), cisgender, and female-headed families. Future research should strive for including diverse families in terms of parental gender and sexual orientation , race/ethnicity, and socioeconomic background, while incorporating an intersectional approach Bowleg,  that is First, legal vulnerability for same-sex parent families is caused and perpetuated by the lacking legal recognition of family relationships, just as minority-specific drivers of sexual minority health disparities are caused and perpetuated by structural and societal stigmatization  that are particularly vulnerable or, conversely, equipped with many resources . Resilience-focused therapy and counseling approaches could aim at deriving useful coping strategies for dealing with legal vulnerability as parents and outlining effective and age-appropriate strategies to address legal vulnerability in children. However, the integration of legal vulnerability into clinical practice is by no means limited to structured therapy programs. For example, informal meeting groups have been found to be an important source of social support but also legal information for same-sex parents Kazyak, . Group fIn jurisdictions with legal recognition for same-sex parent families, we argue that it is important to address possible lingering effects of legal vulnerability. For example, a study on same-sex couples (post marriage legislation) found that perceived unequal relationship recognition  predicted adverse mental health outcomes irrespective of legal relationship status  focused on the impact of sexual orientation laws on family formation, (ii) were concerned with same-sex couples with unclear parental status, (iii) investigated discrimination unrelated to the legal system, or (iv) investigated family constellations with one or more than two parents. All of these studies are beyond the scope of our review and necessitate their own synthesis. Still, some of these studies may have yielded responses by participants that would have warranted inclusion in our review .Second, we did not systematically search for gray literature excepting our forward-backward search. Publication and other dissemination biases are typically framed as a threat to the synthesis of quantitative studies  and only includes one study  on key outcomes.Fourth, synthesizing results across studies from countries with varying sociolegal climates gives rise to a possible decontextualization of findings  can be found at: MS, CA, NM, and MZ conception, design of study, analysis, interpretation of data, and approval of final version. MS, CA, and NM acquisition of data. MS drafting the manuscript. CA, NM, and MZ revised the manuscript critically for important intellectual content. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Pericytes (PCs), known as mural cells, play an important blood vessel (BV) supporting role in regulating vascular stabilization, permeability and blood flow in microcirculation as well as blood brain barrier. In carcinogenesis, defective interaction between PCs and endothelial cells (ECs) contributes to the formation of leaky, chaotic and dysfunctional vasculature in tumors. However, recent works from other laboratories and our own demonstrate that the direct interaction between PCs and other stromal cells/cancer cells can modulate tumor microenvironment (TME) to favor cancer growth and progression, independent of its BV supporting role. Furthermore, accumulating evidence suggests that PCs have an immunomodulatory role. In the current review, we focus on recent advancement in understanding PC’s regulatory role in the TME by communicating with ECs, immune cells, and tumor cells, and discuss how we can target PC’s functions to re-model TME for an improved cancer treatment strategy. Pericytes (PCs) are embedded in the basement membrane of blood microvessels , which pAnti-angiogenetic therapy is recognized as a promising treatment strategy for cancer, while many anti-angiogenic drugs have been approved for certain types of cancers, including anti-vascular endothelial growth factor (VEGF) drug  and some tyrosine kinase inhibitors  . HoweverIn this review, we will exploit the current progress of understanding the role of PC in regulating TME, and discuss its functions in regulating tumor cells and other stromal cells to dictate cancer growth and progression. For comprehensive reviews of its role in BV formation and supporting function, please see Betsholtz and Crivellato .Although the composition of TME varies in different cancer types, some features seem to be typical characteristics in most solid tumors. Indeed, TME consists of cancer cells and some non-malignant cells, including ECs, PCs, immune inflammatory cells, cancer-associated fibroblasts (CAFs), and also extracellular matrix (ECM) components  . In thisEndothelial cells are the fundamental cells lining the interior face of BV walls, which are surrounded by quiescent mural cells (including PCs). PCs are capable of interacting with newly proliferating ECs to form nascent BVs and secrete angiogenetic factors to stabilize the newly-formed vessels . In tumoAlthough PCs have been considered as a critical compartment of the TME, the underlying mechanism of tumor cell–PC interaction has yet to be elucidated. Recently, we have shown that high percentage of mural-β3-integrin negative BVs correlates with increased tumor size and progression in multiple cancers , while Ppdgfbret/ret mouse model, PCs deficiency-driven hypoxia result in IL-6 upregulation and an increased myeloid-derived suppressor cell (MDSC) transmigration in tumors, and the MDSC accumulation leads to increased tumor growth, while the amounts of circulating malignant cells can be abrogated upon the recovery of PC coverage  gene results in PC maturation, vascular normalization and consequently a marked reduction in tumor hypoxia and vessel leakiness, while these changes enhance immune cell infiltration and extend the survival of tumor bearing mice , which is an agonist of the CXC chemokine receptor 3 (CXCR3), mainly secreted by PCs and perivascular macrophages. In the perivascular TME, CXCL9 can form heterocomplex with B-cell chemoattractant CXCL12 to enhance CXCL12-induced CD8+ T cell as well as malignant B cell recruitment toward BV walls in the primary central nervous system lymphomas  have a role in modulating TME and changing the behavior of neoplastic cells in either a tumor-promoting or tumor-inhibiting manner . In the It has become a research hot topic for developing direct/indirect PC-targeted agents against angiogenesis and cancer growth in the last decades . HoweverAs an obligatory constituent of the TME, PCs modulate the TME by interacting with tumor cells, ECs, immune cells, and CAFs, beyond their BV supporting role. Recent work supports direct cross-talk between PCs and tumor cells in the TME, which can promote tumor growth independent of tumor angiogenesis. Also, the interplay between ECs and PCs in regulating vascular formation and remodeling has been demonstrated in numerous studies. Disrupting EC-PC interactions in tumor vasculature not only affects PC coverage on tumor BVs but also alter vascular and perivascular TME to influence the efficacy of anti-tumor therapies. Indeed, new studies have highlighted that PCs protect tumor cells from immune surveillance through suppressing the proliferation or response of inflammatory cells around the tumor parenchyma, which could be a new potential target for cancer immunotherapy. Besides, the observation of PC-fibroblast transition suggests the potential progenitor cell property of PC in the TME. In this review, we provide new information to support an integral role for PCs in promoting tumor progression in part through their regulatory activities of tumor cells and dominated stromal cells, suggesting that PCs can serve as a therapeutic target for anticancer treatment in addition to anti-angiogenesis. Meanwhile, the stromal cells within TME may also provide potential therapeutic targets for intending anti-angiogenesis combination therapy since their underlying relationships with PCs. Future studies should focus on exploring the underlying mechanisms of PC-stromal cell/tumor cell interaction in the TME in order to identify new therapeutic targets for an improved cancer treatment strategy.RS wrote the manuscript and made the figure as well as the table. XK, XQ, and CH reviewed the manuscript. P-PW conceptualized, wrote, and reviewed the final version. All authors approved the submission for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Drug delivery systems can be engineered to enhance the localization of therapeutics in specific tissues in response to externally applied stimuli and/or local environmental changes. In recent decades, efforts to improve drug delivery techniques at both nano- and macroscale have led to a new era of therapeutic efficacy. Such technological advancements resulted in improved drug delivery systems regularly entering the clinical setting. However, these delivery innovations are unfortunately not always readily applied to newly developed technologies. One of these new and exciting technologies that has been overlooked by drug delivery scientists is prime editing. Prime editing is a novel genome editing technology that exhibits the plug-and-play capability of CRISPR/Cas9 editors while avoiding double-strand DNA breaks throughout the entire process. This article focuses on describing the potential advantages and disadvantages of selecting nanomedicine technologies along with prime editing capabilities for the delivery of cargo. The field of drug delivery has overcome many pharmaceutical hurdles. However, the majority of drug delivery research continues to focus on only a subset of pharmaceutical agents and diseases. A chief example of this “pharmaceutical neglect” is the lack of proposed delivery strategies for the new, high-interest field of genome prime editing. Since clustered regularly interspaced short palindromic repeats (CRISPR)-inspired prime editing technology has very recently been described , its pubPrime editing technologies have the potential to essentially revolutionize current genome-editing practices. Since the CRISPR/Cas9 system has been first utilized, adoption of the technology has soared with more than $1 billion currently being spent annually by federal governments on CRISPR-based research . Howeverin vitro and theoretically also in vivo.Prime editing is a versatile genome editing method that “writes” new genetic information into precise DNA locations. This method differs from CRISPR/Cas9 systems in that it employs a catalytically impaired Cas9 endonuclease fused to a reverse transcriptase that is programmed via prime editing guide RNA (pegRNA) . pegRNA in vivo efficacy of prime editing systems is yet to be adequately demonstrated. To achieve widespread in vivo use, effective delivery of the prime editing machinery is required. Lentiviral systems have been proposed as transporters for the base editor 3 (BE3) system as its large size would not fit within generic adenoviral systems [Unfortunately, the  systems ; however systems . TherefoBefore directly identifying the requirements for a delivery vehicle for prime editing technologies, it should be acknowledged that the optimal delivery vehicle will likely change depending on the specific disease target. Unfortunately, the very advantage of broad applicability of prime editing for a plethora of diseases is a major disadvantage in selecting the proper delivery method. A single delivery approach will not enable the full-breadth adoption of prime editing for all therapies designed to treat known human pathogenic genetic variants. However, certain techniques that provide a reasonable mode of delivery to a broad subset of disease targets and their broad requirements will be discussed here.First, one of the most important considerations of prime editing delivery systems is that they must be able to deliver the entire prime editing complex. The entire complex is composed of a prime editing protein containing an RNA-guided DNA-nicking domain  fused to a reverse transcriptase (RT) domain and complexed with prime editing guide RNA (pegRNA). Essentially, both protein and large RNA strand must be delivered. In theory, these components can be either codelivered in the same carrier or transported in separate carriers. However, in practice, codelivery of active agents in the same nanocarrier has generally resulted in greater therapeutic efficacy , likely Second, the delivery of the PE-pegRNA complex tends to be more difficult than that of small-molecule drugs. In general, proteins for drug delivery exhibit notoriously short circulatory half-lives, poor absorption and permeability profiles, and high rates of denaturation during transport . RNA molThird, genome editing technologies require delivery vehicles that enable intracellular and intranuclear  uptake in the target cell. While many gene delivery systems utilize viral vectors, nonviral delivery systems are often preferred because of engineered control over toxicity profiles. Nonviral vectors are more advantageous over viral vectors due to their biosafety associated with less immunotoxicity. Plasmid DNA, liposome-DNA complexes (lipoplexes), and polymer-DNA complexes are examples of commonly used nonviral vectors. However, transfection efficiency of nonviral delivery systems regularly plummets. Alterations to the formulation of nonviral delivery systems to improve solubility  often work counterproductively when intracellular entry is necessary . FurtherFourth, the delivery system should enable a path for regulatory approval and therefore cannot be designed with extreme complexities. Drug delivery scientists discreetly shy away from admitting that very few nanomedicine systems are currently available in the commercial market, despite the large growth of interest in the scientific field. Many strong nanomedicine candidates fail to achieve set regulation standards as a result of the inability to account for all the degradation products, lack of demonstrated enhanced efficacy, or the triggering of system-mediated toxicities . In the In general, a goal of nearly all drug delivery systems is to reduce off-target effects from the wide biodistribution of active pharmaceutical ingredients (APIs). Many factors need to be considered when designing an effective drug delivery system. Although it is beyond the scope of this article to fully list them here, some obvious considerations when engineering a system are to be cognizant of the nature of the drug , the particular tissues or cells being targeted, the drug modes of action, and the route of drug administration, as well as several other pharmacological factors. Despite the need for such an effort, the payoff can be monumental as the advantages of tailored drug delivery systems far outweigh nontargeted therapeutics. Even though local drug administration may aid prime editing delivery, local administration routes are not discussed here as systemic transporters likely have the broadest applicability for diseases susceptible to prime editing treatment. Accordingly, several nanotechnology-based delivery systems are addressed , and theLiposomes are vesicles composed of at least one lipid bilayer . They arWith regard to the delivery of prime editing machinery, liposomes have several distinct advantages. First, the size of the liposomes can be optimized for cargo delivery, and the interior compartment space can house both prime editing protein and pegRNA. In fact, delivery of large protein-RNA complexes has been previously demonstrated using liposomes . Second,In vivo fate of liposomes can be significantly affected by the interaction between liposomes and cells where they can be absorbed or undergo endocytosis. Stability during storage also remains an issue with most liposomal formulations requiring frozen storage conditions. If a specific prime editing therapy could initiate severe side effects without directed localization, liposomes may not provide the stability needed to adequately reduce off-target effects. Nonetheless, liposomes are likely to be strong candidate carriers for low-toxicity prime editing therapies.However, liposomal systems often have several drawbacks. While liposomes mimic natural membranes, they are still foreign materials in the body and are known to be cleared by the mononuclear phagocytic system . Effortsin vivo genome editing in the mouse brain [Micelles are the fundamental building blocks of emulsion-based formulations. A micelle is a three-dimensional assembly involving multiple amphiphilic surfactants. The hydrophobic ends (tails) of the multiple surfactant molecules arrange themselves near one another in order to minimize contact with water molecules, leading to a structure in which the hydrophilic ends encounter the water molecules at the periphery. Like liposomes, micelles are generally spherical, yet rod and planar structures can be obtained using surfactants with proper head-to-tail volume ratios and distinct solution conditions. By exchanging the solvent, inverse micelles can also be formed where the hydrophilic ends cluster and the hydrophobic ends interact with the solvent. Pharmaceutical formulations take advantage of both micelle types in the form of water-in-oil (W/O) emulsions and oil-in-water (O/W) emulsions. Advanced water-in-oil-in-water (W/O/W) and oil-in-water-in-oil (O/W/O) emulsions have also been developed to inhibit globular coalescence. Micelles carry molecular cargo by thermodynamically stabilizing the molecules in the core. Generally, fat-soluble agents are poorly soluble in surrounding solvents, leading to micelles being major applications in the transport of fat-soluble nutrients and drugs. Because of the simplicity of the system and the long history of using detergents, micelle systems are not always recognized as nanoparticle delivery systems, yet they have been shown to be effective in the delivery of genes and specific biologics . For exase brain .However, with regard to their potential for prime editing delivery, micelles face many challenges. First, micelles generally exhibit a size limitation, usually ranging from 2 to 20 nm, which can hinder the delivery applicability of larger macromolecules. In fact, amphiphilic block copolymers often self-assemble into micellar structures themselves , but rarExosomes are similar in structure to liposomes, being membrane-bound vesicles; however, one intriguing difference sets them apart. Exosomes are derived from the endosomal compartment of cells and carry unique cell biomarkers that are characteristic of the cell of origin. Therefore, exosomes often display inherent targeting molecules on the outer surface of the membrane, enabling improvement in target cell uptake . Exosomein vitro [Exosomes can be engineered in a variety of sizes, and various exosome-loading procedures have been developed . Recentlin vitro . Nanomedin vitro .While it may initially seem that exosomes are an obvious choice for prime editing delivery over liposomes, certain drawbacks limit their utility, although not entirely. First, exosome engineering remains in its infancy, and the ability to design an exosome with firm-targeting capabilities is still somewhat only theoretical. Furthermore, alterations to exosomes after synthesis, for purposes such as bloodstream cloaking, tend to result in loss of desired exosomal properties . Finallyin vivo tracking moieties, targeting molecules, and environmentally responsive entities and are thus often considered the most versatile platform for drug delivery. Upon arrival to the target cells, the most of polymer-based delivery vehicle undergo biodegradation or breakdown to initiate the delivery cargo into the cells.Polymers are long-chain molecules composed of many repeating subunits (monomers). Theoretically, polymers assume a three-dimensional architecture in solution owing to interactions with the solvent environment. Hydrophilic polymers are commonly employed to improve the solubility of drugs with low solubility, while hydrophobic polymer components are often used for drug stabilization. Many different polymer types exist and can enable a wide range of properties relevant to drug delivery, namely, polymers can be covalently attached to drugs or utilize noncovalent interactions for drug transport. Furthermore, polymer nanosystems can be designed with various architectures, sizes, and compositions. As such, many different systems can be engineered to fully encapsulate both prime editing protein and pegRNA. Additionally, polymers enable facile attachment of in vivo polymer degradation and excretion profiles is essential. Most regulatory agencies tend to look more favorably on liposomal systems than polymer-based systems, yet a few polymer-based systems have achieved US FDA approval [While a polymer-based system may be a strong candidate for prime editing delivery, several factors should be considered. First, covalent attachment of a polymer to a protein may lead to incapacitation of the protein , and neaapproval . Howeverin vivo barriers, such as the blood-brain barrier [One of the most unique nanoparticle systems is the use of dendrimers, which are macromolecules composed of repeatedly branched chains. Their divergent synthesis begins with a single molecule at the core that then branches in multiple directions. Four branches become eight in the next stage of synthesis , then sixteen, thirty-two, and so on. The dendrimer structure is unique in that the entire structure exhibits polymeric flexibility, yet the dendrimer surface exhibits an extremely high charge density. Dendrimers are known to chelate small molecules and ions within their branched structures, and their high charge density permits penetration across difficult  barrier .Dendrimers face similar issues as those of micelles when being evaluated for prime editing delivery. In theory, no dendrimer size limit should exist; however, in practice, it is very difficult to produce dendrimers beyond six generations. Quite simply, the charge density becomes too large, and steric repulsion forces restrict further branching. Therefore, the probability of developing a dendrimer system with sufficient cargo space to deliver both prime editing protein and pegRNA molecule is quite small.In this article, rigid nanoparticles are defined as nanoparticles composed of any material  that elicits a rigid morphology, including silica, metal oxide, and palladium nanoparticles. Essentially, rigid nanoparticle systems utilize conventional nanoparticles as delivery vessels. These systems can be designed using either top-down or bottom-up approaches. Top-down techniques tend to result in samples with high polydispersity in size and shape, whereas bottom-up methods can produce monodisperse samples with unique architectures. While surfaces can be chemically modified for the attachment of both proteins and nucleic acids, the attached molecules are on the surface of the particles and therefore presented to bloodstream components and rapidly disabled or cleared. Many of these systems rely solely on the adsorption of the APIs without chemical modification. However, these systems are likely to fail as prime editing delivery systems for the same reason.On the contrary, certain rigid nanoparticle systems, such as silica, can be produced with a hollow core using bottom-up approaches, allowing for the encapsulation of various molecules . As a rePrime editing technologies have the potential to alter the genome editing space and achieve biomedical treatment breakthroughs. To realize this potential, proper delivery systems must be engineered for prime editing transport and localization. Prime editing delivery systems must be able to costabilize both prime editing protein and pegRNA during transport, possess a method for cellular internalization, and maintain a straightforward path to regulatory approval. Based on these criteria, liposomes are likely to be the most promising nanomedicine candidates for prime editing delivery if their potential toxicity and instability in the circulation system are well addressed. Polymer-based carriers with lower toxicity and higher stability may represent the second best option. However, the efficiency and safety of each delivery system must be carefully considered with the inevitable variance in between systems and cell types.