Placental Nanoparticle Uptake-On-a-Chip: The Impact of Trophoblast Syncytialization and Shear Stress.

The placenta is a dynamic and complex organ that plays an essential role in the health and development of the fetus. Placental disorders can affect the health of both the mother and the fetus. There is currently an unmet clinical need to develop nanoparticle-based therapies to target and treat placental disorders. However, little is known about the interaction of nanoparticles (NPs) with the human placenta under biomimetic conditions. Specifically, the impact of shear stress exerted on the trophoblasts (placental epithelial cells) by the maternal blood flow, the gradual fusion of the trophoblasts along the gestation period (syncytialization), and the impact of microvilli formation on the cell uptake of NPs is not known. To this end, we designed dynamic placenta-on-a-chip models using BeWo cells to recapitulate the micro-physiological environment, and we induced different degrees of syncytialization via chemical induction with forskolin. We characterized the degree of syncytialization quantitatively by measuring beta human chorionic gonadotropin (ß-hCG) secretion, as well as qualitatively by immunostaining the tight junction protein, ZO-1, and counter nuclear staining. We also characterized microvilli formation under static and dynamic conditions via F-actin staining. We used these models to measure the cell uptake of chondroitin sulfate a binding protein (CSA) conjugated and control liposomes using confocal microscopy, followed by image analysis. Interestingly, exposure of the cells to a dynamic flow of media intrinsically induced syncytialization and microvilli formation compared to static controls. Under dynamic conditions, BeWo cells produced more ß-hCG in conditions that increased the cell exposure time to forskolin (p < 0.005). Our cell uptake results clearly show a combined effect of the exerted shear stress and forskolin treatment on the cell uptake of liposomes as uptake increased in forskolin exposed conditions (p < 0.05). Overall, the difference in the extent of cell uptake of liposomes among the different conditions clearly displays a need for the development of dynamic models of the placenta that consider the changes in the placental cell phenotype along the gestation period, including syncytialization, microvilli formation, and the expression of different transport and uptake receptors. Knowledge generated from this work will inform future research aiming at developing drug delivery systems targeting the placenta.

Collagen - a newly discovered major player in protein corona formation on nanoparticles.

Tracking protein corona (PC) formation on the surface of nanoparticles (NPs) is a prerequisite for successful design of next generation nanocarriers with predictable fate and behavior. However, PC formation has mostly been investigated for plasma proteins without considering potential competition with the extravascular proteins either when the NPs exit the blood circulation or when they are injected extravascularly. This study investigates the deposition of collagen, an extravascular protein that is the most abundant in the body, and albumin, the most abundant vascular protein, on the surface of gold (Au) NPs using UV-Vis and fluorescence spectroscopy with the support of mathematical modeling. Moreover, a novel spectroscopic approach to determining the protein-NP binding constants and surface occupancy is presented. We show that albumin and collagen have drastically different affinities for Au NPs. Our data demonstrates that the surface bound albumin can be exchanged with collagen confirming the dynamic nature of PC in the extravascular milieu. We propose that future PC investigations in the framework of drug delivery should rely on understanding of the NP transit in the body, and include competition experiments with relevant vascular and extravascular proteins. Furthermore, our results that reveal very strong binding of collagen to AuNPs may lay the foundation for designing long circulating collagen-coated NPs with minimal surface adsorption of plasma proteins and, thus, reduced immune recognition.

Interaction of inorganic nanoparticles with the skin barrier: current status and critical review.

Integration of nanotechnology with biology leads to various advantages in applied pharmaceutical and medical sciences. In that regard, the behavior of nanoparticles (NPs) in relation to the skin, an important biological barrier, has been the target of several recent studies. Yet the potential ability of NPs to penetrate into the underlying viable tissue lies at the center of debate. This review briefly highlights the current applications of inorganic NPs, then discusses the current status of their skin penetration in view of the vast variation among the experimental setups in use. Determinants of particle penetration, adopted approaches for enhanced penetration, the underlying mechanism, as well as qualitative and quantitative analysis of NPs present in the skin are also within the scope of this review article. We emphasize analyzing the data generated from experiments on human skin, the "gold standard" for assessment of in vitro skin penetration. Based on this, we include some recommendations for future research. FROM THE CLINICAL EDITOR: Transdermal application of inorganic nanoparticle-based medications is of growing interest in nanomedicine research. This critical review discusses the knowns and the unknowns of this field, providing insightful recommendations for future research.

Microfluidics for Development of Lipid Nanoparticles: Paving the Way for Nucleic Acids to the Clinic.

Nucleic acid therapeutics hold an unprecedented promise toward treating many challenging diseases; however, their use is hampered by delivery issues. Microfluidics, dealing with fluids in the microscale dimensions, have provided a robust means to screening raw materials for development of nano delivery vectors, in addition to controlling their size and minimizing their polydispersity. In this mini-review, we are briefly highlighting the different types of nucleic acid therapies with emphasis on the delivery requirement for each type. We provide a thorough review of available methods for the development of nanoparticles, especially lipid nanoparticles (LNPs) that resulted in FDA approval of the first ever nucleic acid nanomedicine. We then focus on recent research attempts for how microfluidic synthesis of lipid nanoparticles and discuss the various parameters required for successful formulation of LPNs including chip design, flow regimes, and lipid composition. We then identify key areas of research in microfluidics and related fields that require attention for future success in clinical translation of nucleic acid nanomedicines.

Critical design parameters to develop biomimetic organ-on-a-chip models for the evaluation of the safety and efficacy of nanoparticles.

INTRODUCTION: Organ-on-a-chip (OOC) models are based on microfluidics and can recapitulate the healthy and diseased microstructure of organs1 and tissues and the dynamic microenvironment inside the human body. However, the use of OOC models to evaluate the safety and efficacy of nanoparticles (NPs) is still in the early stages. AREAS COVERED: The different design parameters of the microfluidic chip and the mechanical forces generated by fluid flow play a pivotal role in simulating the human environment. This review discusses the role of different key parameters on the performance of OOC models. These include the flow pattern, flow rate, shear stress (magnitude, rate, and distribution), viscosity of the media, and the microchannel dimensions and shape. We also discuss how the shear stress and other mechanical forces affect the transport of NPs across biological barriers, cell uptake, and their biocompatibility. EXPERT OPINION: We describe several good practices and design parameters to consider for future OOC research. We submit that following these recommendations will help realize the full potential of the OOC models in the preclinical evaluation of novel therapies, including NPs.

Omics approaches for the assessment of biological responses to nanoparticles.

Nanotechnology has enabled the development of innovative therapeutics, diagnostics, and drug delivery systems. Nanoparticles (NPs) can influence gene expression, protein synthesis, cell cycle, metabolism, and other subcellular processes. While conventional methods have limitations in characterizing responses to NPs, omics approaches can analyze complete sets of molecular entities that change upon exposure to NPs. This review discusses key omics approaches, namely transcriptomics, proteomics, metabolomics, lipidomics and multi-omics, applied to the assessment of biological responses to NPs. Fundamental concepts and analytical methods used for each approach are presented, as well as good practices for omics experiments. Bioinformatics tools are essential to analyze, interpret and visualize large omics data, and to correlate observations in different molecular layers. The authors envision that conducting interdisciplinary multi-omics analyses in future nanomedicine studies will reveal integrated cell responses to NPs at different omics levels, and the incorporation of omics into the evaluation of targeted delivery, efficacy, and safety will improve the development of nanomedicine therapies.

Characterizing Extracellular Vesicles and Particles Derived from Skeletal Muscle Myoblasts and Myotubes and the Effect of Acute Contractile Activity.

Extracellular vesicles (EVs), released from all cells, are essential to cellular communication and contain biomolecular cargo that can affect recipient cell function. Studies on the effects of contractile activity (exercise) on EVs usually rely on plasma/serum-based assessments, which contain EVs from many different cells. To specifically characterize skeletal muscle−derived vesicles and the effect of acute contractile activity, we used an in vitro model where C2C12 mouse myoblasts were differentiated to form myotubes. EVs were isolated from conditioned media from muscle cells at pre-differentiation (myoblasts) and post-differentiation (myotubes) and also from acutely stimulated myotubes (1 h @ 14 V, C-Pace EM, IonOptix, Westwood, MA, USA) using total exosome isolation reagent (TEI, ThermoFisher (Waltham, MA, USA), referred to as extracellular particles [EPs]) and differential ultracentrifugation (dUC; EVs). Myotube-EPs (~98 nm) were 41% smaller than myoblast-EPs (~167 nm, p < 0.001, n = 8−10). Two-way ANOVA showed a significant main effect for the size distribution of myotube vs. myoblast-EPs (p < 0.01, n = 10−13). In comparison, myoblast-EPs displayed a bimodal size distribution profile with peaks at <200 nm and 400−600, whereas myotube-Eps were largely 50−300 nm in size. Total protein yield from myotube-EPs was nearly 15-fold higher than from the myoblast-EPs, (p < 0.001 n = 6−9). Similar biophysical characteristics were observed when EVs were isolated using dUC: myotube-EVs (~195 nm) remained 41% smaller in average size than myoblast-EVs (~330 nm, p = 0.07, n = 4−6) and had comparable size distribution profiles to EPs isolated via TEI. Myotube-EVs also had 4.7-fold higher protein yield vs. myoblast EVs (p < 0.05, n = 4−6). Myotube-EPs exhibited significantly decreased expression of exosomal marker proteins TSG101, CD63, ALIX and CD81 compared with myoblast-EPs (p < 0.05, n = 7−12). Conversely, microvesicle marker ARF6 and lipoprotein marker APO-A1 were only found in the myotube-EPs (p < 0.05, n = 4−12). There was no effect of acute stimulation on myotube-EP biophysical characteristics (n = 7) or on the expression of TSG101, ARF6 or CD81 (n = 5−6). Myoblasts treated with control or acute stimulation−derived EPs (13 µg/well) for 48 h and 72 h showed no changes in mitochondrial mass (MitoTracker Red, ThermoFisher, Waltham, MA, USA), cell viability or cell count (n = 3−4). Myoblasts treated with EP-depleted media (72 h) exhibited ~90% lower cell counts (p < 0.01, n = 3). Our data show that EVs differed in size, distribution, protein yield and expression of subtype markers pre vs. post skeletal muscle−differentiation into myotubes. There was no effect of acute stimulation on biophysical profile or protein markers in EPs. Acute stimulation−derived EPs did not alter mitochondrial mass or cell count/viability. Further investigation into the effects of chronic contractile activity on the biophysical characteristics and cargo of skeletal muscle−specific EVs are warranted.

Role of drug delivery technologies in the success of COVID-19 vaccines: a perspective.

The triumphant success of mRNA vaccines is a testimony to the important role drug delivery technologies have played in protecting billions of people against SARS-CoV-2 (or the Corona Virus Disease 2019; COVID-19). Several lipid nanoparticle (LNP) mRNA vaccines were developed and have been instrumental in preventing the disease by boosting the immune system against the pathogen, SARS-CoV-2. These vaccines have been built on decades of scientific research in drug delivery of mRNA, vaccines, and other biologicals. In this manuscript, several leading and emerging scientists in the field of drug delivery share their perspective on the role of drug delivery technologies in developing safe and efficacious vaccines, in a roundtable discussion. The authors also discussed their viewpoint on the current challenges, and the key research questions that should drive this important area of research.

The maternal-fetal transfer of passive immunity as a mechanism of transplacental nanoparticle drug delivery for prenatal therapies.

Nanoparticles administered into the maternal circulation and across the placenta are a potential clinical therapy to treat congenital diseases. The mechanism by which nanoparticles can safely cross the placenta for targeted drug delivery to the fetus remains poorly understood. We demonstrate that the maternal-fetal transfer of passive immunity through the neonatal Fc Receptor (FcRn) can induce the transplacental transfer of chitosan nanoparticles modifed with IgG antibodies (414 ± 27 nm). The transfer of FITC-tagged IgG-modified chitosan nanoparticles was 2.8 times higher (p = 0.0264) compared to similarly-sized unmodified chitosan nanoparticles (375 ± 17 nm). Co-administration of free IgG competitively diminished the transplacental transfer of IgG-modified nanoparticles, yet unmodified nanoparticles remained unaffected. Colocalization of the FcRn and the IgG-modified chitosan nanoparticles were observed with confocal microscopy. Barrier function before and after nanoparticle administration remained intact as determined by TEER (75-79 Ω cm2) and immmunofluorescence of ZO-1 tight junction proteins. The results provide insight into the clinical applications of nanoparticles for prenatal therapies using the mechanism of the maternal-fetal transfer of passive immunity.

Extracellular Vesicles as Predictors of Individual Response to Exercise Training in Youth Living with Obesity.

BACKGROUND: Exercise is associated with health benefits, including the prevention and management of obesity. However, heterogeneity in the adaptive response to exercise training exists. Our objective was to evaluate if changes in extracellular vesicles (EVs) after acute aerobic exercise were associated with the responder phenotype following 6-weeks of resistance training (RT). METHODS: This is a secondary analysis of plasma samples from the EXIT trial (clinical trial#02204670). Eleven sedentary youth with obesity (15.7 ± 0.5 yrs, BMI ≥95th percentile) underwent acute exercise (60% HRR, 45 min). Blood was collected at baseline [AT0 min], during [AT15-45 min], and 75 min post-recovery [AT120], and EVs purified using size exclusion chromatography from extracted plasma. Afterward, youth participated in 6-weeks RT and were categorized into responders or non-responders based on changes in insulin sensitivity. RESULTS: We assessed EV biophysical profile (size, zeta potential, protein yield, and EV subtype protein expression) in a single-blind fashion. Overall, there was a general increase in EV production in both groups. Average EV size was larger in responders (~147 nm) vs. non-responders (~124 nm; p < 0.05). EV size was positively associated with absolute change in Matsuda index (insulin sensitivity) following RT (r = 0.44, p = 0.08). EV size distribution revealed responders predominantly expressed EVs sized 150-300 nm, whereas non-responders expressed EVs sized 50-150 nm (p < 0.05). At baseline, responders had ~25% lower TSG101, ~85% higher MMP2 levels. EV protein yield was higher in responders than non-responders at AT15 (p < 0.05). CONCLUSIONS: Our data suggest that youth with obesity that respond to RT produce larger EVs that are TSG101+ and CD63+, with increased EV protein yield during acute exercise.

Gold nanoparticle penetration and reduced metabolism in human skin by toluene.

PURPOSE: To measure penetration and metabolic effects of ion-stabilized, polar, 15 nm gold nanoparticles in aqueous solution (AuNP-Aq) and sterically stabilized, non-polar, 6 nm gold nanoparticles in toluene (AuNP-TOL) on excised human skin. METHODS: Gold nanoparticles were characterized with dynamic light scattering and transmission electron microscopy (TEM). Skin penetration studies were done on frozen or fresh excised skin using static Franz diffusion cells. Viable treated skin was assessed by dermoscopy, reflectance confocal microscopy (RCM), multiphoton tomography (MPT) with fluorescence lifetime imaging microscopy (FLIM), and TEM. RESULTS: Dermoscopy and RCM showed large aggregates in the furrows of AuNP-Aq-treated skin. Treatment of thawed and viable skin only showed enhanced permeability to nanoparticles in the AuNP-TOL group with MPT and FLIM imaging to stratum spinosum of epidermis. TEM analysis revealed gold nanoparticles within AuNP-treated stratum corneum. FLIM analysis of NAD(P)H showed a significant decrease in total NAD(P)H in all toluene-treated groups. CONCLUSIONS: Gold nanoparticles, 15 nm, in aqueous solution aggregated on the skin surface. Toluene treatment eliminated skin metabolism; skin treated with toluene/gold nanoparticles (6 nm) for 24 h, but not at 4 h, showed increased nanoparticle permeability. These results are of value to nanotoxicology.

Localized Plasmonic Photothermal Therapy as a Life-saving Treatment Paradigm for Hospitalized COVID-19 Patients.

Lung failure is the main reason for mortality in COVID-19 patients, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To date, no drug has been clinically approved for treatment of COVID-19. Nanotechnology has a great potential in contributing significantly to the fight against COVID-19 by developing effective therapies that can selectively eradicate the respiratory virus load. We propose a novel COVID-19 management approach that is efficient in eliminating the virus load from the airways and protecting the lungs from the fatal effects of the virus. This approach relies on targeting the virus using ACE-2-functionalized gold nanorods (AuNRs) followed by irradiation with near-infrared (NIR) light for the selective eradication of SARS-CoV-2 without off-target effects, i.e., targeted plasmonic photothermal therapy. Using discrete dipole approximation (DDA), we quantitatively determined the efficiency of AuNRs (31 nm × 8 nm) in absorbing NIR when present at different orientations relative to one another on the surface of the virus. The safety and the local administration of AuNRs using a well-tolerated flexible bronchoscopy technique, commonly used for hospitalized COVID-19 patients, ensure feasibility and clinical translation. While requiring further research, we anticipate this approach to result in a first-line treatment for hospitalized COVID-19 patients that are experiencing severe respiratory conditions or belong to a high-risk population, e.g., seniors and diabetic patients.

Dynamic placenta-on-a-chip model for fetal risk assessment of nanoparticles intended to treat pregnancy-associated diseases.

Pregnant women often have to take medication either for pregnancy-related diseases or for previously existing medical conditions. Current maternal medications pose fetal risks due to off target accumulation in the fetus. Nanoparticles, engineered particles in the nanometer scale, have been used for targeted drug delivery to the site of action without off-target effects. This has opened new avenues for treatment of pregnancy-associated diseases while minimizing risks on the fetus. It is therefore instrumental to study the potential transfer of nanoparticles from the mother to the fetus. Due to limitations of in vivo and ex vivo models, an in vitro model mimicking the in vivo situation is essential. Placenta-on-a-chip provides a microphysiological recapitulation of the human placenta. Here, we reviewed the fetal risks associated with current therapeutic approaches during pregnancy, analyzed the advantages and limitations of current models used for nanoparticle assessment, and highlighted the current need for using dynamic placenta-on-a-chip models for assessing the safety of novel nanoparticle-based therapies during pregnancy.

Meta-Analysis of Nanoparticle Cytotoxicity via Data-Mining the Literature.

Developing predictive modeling frameworks of potential cytotoxicity of engineered nanoparticles is critical for environmental and health risk analysis. The complexity and the heterogeneity of available data on potential risks of nanoparticles, in addition to interdependency of relevant influential attributes, makes it challenging to develop a generalization of nanoparticle toxicity behavior. Lack of systematic approaches to investigate these risks further adds uncertainties and variability to the body of literature and limits generalizability of existing studies. Here, we developed a rigorous approach for assembling published evidence on cytotoxicity of several organic and inorganic nanoparticles and unraveled hidden relationships that were not targeted in the original publications. We used a machine learning approach that employs decision trees together with feature selection algorithms ( e.g., Gain ratio) to analyze a set of published nanoparticle cytotoxicity sample data (2896 samples). The specific studies were selected because they specified nanoparticle-, cell-, and screening method-related attributes. The resultant decision-tree classifiers are sufficiently simple, accurate, and with high prediction power and should be widely applicable to a spectrum of nanoparticle cytotoxicity settings. Among several influential attributes, we show that the cytotoxicity of nanoparticles is primarily predicted from the nanoparticle material chemistry, followed by nanoparticle concentration and size, cell type, and cytotoxicity screening indicator. Overall, our study indicates that following rigorous and transparent methodological experimental approaches, in parallel to continuous addition to this data set developed using our approach, will offer higher predictive power and accuracy and uncover hidden relationships. Results obtained in this study help focus future studies to develop nanoparticles that are safe by design.

Surface-grafted polyethylene glycol conformation impacts the transport of PEG-functionalized liposomes through a tumour extracellular matrix model.

The effect of surface PEGylation on nanoparticle transport through an extracellular matrix (ECM) is an important determinant for tumor targeting success. Fluorescent stealth liposomes (base lipid DOPC) were prepared incorporating different proportions of PEG-grafted lipids (2.5, 5 and 10% of the total lipid content) for a series of PEG molecular weights (1000, 2000 and 5000 Da). The ECM was modelled using a collagen matrix. The kinetics of PEGylated liposome adhesion to and transport in collagen matrices were tracked using fluorescence correlation spectroscopy (FCS) and confocal microscopy, respectively. Generalized least square regressions were used to determine the temporal correlations between PEG molecular weight, surface density and conformation, and the liposome transport in a collagen hydrogel over 15 hours. PEG conformation determined the interaction of liposomes with the collagen hydrogel and their transport behaviour. Interestingly, liposomes with mushroom PEG conformation accumulated on the interface of the collagen hydrogel, creating a dense liposomal front with short diffusion distances into the hydrogels. On the other hand, liposomes with dense brush PEG conformation interacted to a lesser extent with the collagen hydrogel and diffused to longer distances. In conclusion, a better understanding of PEG surface coating as a modifier of transport in a model ECM matrix has resulted. This knowledge will improve design of future liposomal drug carrier systems.

Understanding and improving assays for cytotoxicity of nanoparticles: what really matters?

Despite years of excellent individual studies, the impact of nanoparticle (NP) cytotoxicity studies remains limited by inconsistent data collection and analysis. It is often unclear how exposure conditions can be used to determine cytotoxicity quantitatively. Discrepancies due to using different measurement conditions, readouts and controls to characterize NP interactions with cells lead to further challenges. To examine which parameters are critical in NP cytotoxicity studies, we have chosen to examine two NP types (liposomes and quantum dots) at different concentrations incubated with two primary vascular endothelial cells, HUVEC and HMVEC-C for a standard time of 24 h. We paid close attention to the effects of positive controls and cell association on interpretation of cytotoxicity data. Various cellular responses (ATP content, oxidative stress, mitochondrial toxicity, and phospholipidosis) were measured in parallel. Interestingly, cell association data varied significantly with the different image analyses. However, cytotoxicity responses could all be correlated with exposure concentration. Cell type did have an effect on cytotoxicity reports. Most significantly, NP cytotoxicity results varied with the inclusion or exclusion of positive controls. In the absence of positive controls, one tends to emphasize small changes in cell responses to NPs.

Nanoparticle localization in blood vessels: dependence on fluid shear stress, flow disturbances, and flow-induced changes in endothelial physiology.

Nanoparticles in the bloodstream are subjected to complex fluid forces as they move through the curves and branches of healthy or tumor vasculature. While nanoparticles are known to preferentially accumulate in angiogenic vessels, little is known about the flow conditions in these vessels and how these conditions may influence localization. Here, we report a methodology which combines confocal imaging of nanoparticle-injected transgenic zebrafish embryos, 3D modeling of the vasculature, particle mapping, and computational fluid dynamics, to quantitatively assess the effects of fluid forces on nanoparticle distribution in vivo. Six-fold lower accumulation was found in zebrafish arteries compared to the lower velocity veins. Nanoparticle localization varied inversely with shear stress. Highest accumulation was present in regions of disturbed flow found at branch points and curvatures in the vasculature. To further investigate cell-particle association under flow, human endothelial cells were exposed to nanoparticles under hemodynamic conditions typically found in human vessels. Physiological adaptations of endothelial cells to 20 hours of flow enhanced nanoparticle accumulation in regions of disturbed flow. Overall our results suggest that fluid shear stress magnitude, flow disturbances, and flow-induced changes in endothelial physiology modulate nanoparticle localization in angiogenic vessels.

Setup for investigating gold nanoparticle penetration through reconstructed skin and comparison to published human skin data.

Owing to the limited source of human skin (HS) and the ethical restrictions of using animals in experiments, in vitro skin equivalents are a possible alternative for conducting particle penetration experiments. The conditions for conducting penetration experiments with model particles, 15-nm gold nanoparticles (AuNP), through nonsealed skin equivalents are described for the first time. These conditions include experimental setup, sterility conditions, effective applied dose determination, skin sectioning, and skin integrity check. Penetration at different exposure times (two and 24 h) and after tissue fixation (fixed versus unfixed skin) are examined to establish a benchmark in comparison to HS in an attempt to get similar results to HS experiments presented earlier. Multiphoton microscopy is used to detect gold luminescence in skin sections. λ(ex)=800 nm is used for excitation of AuNP and skin samples, allowing us to determine a relative index for particle penetration. Despite the observed overpredictability of penetration into skin equivalents, they could serve as a first fast screen for testing the behavior of nanoparticles and extrapolate their penetration behavior into HS. Further investigations are required to test a wide range of particles of different physicochemical properties to validate the skin equivalent-human skin particle penetration relationship.

Laser scanning microscopy approach for semiquantitation of in vitro dermal particle penetration.

Skin penetration of nanoparticles is a recent research area in focus for the aim of development of topical nanoparticulate delivery systems as well as for health risk analysis. So far, monitoring skin penetration of nanoparticles is mostly based on qualitative microscopical examination. Here, we describe an experimental approach for extracting semiquantitative data from multiphoton images of skin specimens treated with gold nanoparticles. This will aid in depicting the factors responsible for enhancing or limiting nanoparticle penetration through the skin barrier.

Depth profiling of gold nanoparticles and characterization of point spread functions in reconstructed and human skin using multiphoton microscopy.

Multiphoton microscopy has become popular in studying dermal nanoparticle penetration. This necessitates studying the imaging parameters of multiphoton microscopy in skin as an imaging medium, in terms of achievable detection depths and the resolution limit. This would simulate real-case scenarios rather than depending on theoretical values determined under ideal conditions. This study has focused on depth profiling of sub-resolution gold nanoparticles (AuNP) in reconstructed (fixed and unfixed) and human skin using multiphoton microscopy. Point spread functions (PSF) were determined for the used water-immersion objective of 63×/NA = 1.2. Factors such as skin-tissue compactness and the presence of wrinkles were found to deteriorate the accuracy of depth profiling. A broad range of AuNP detectable depths (20-100 µm) in reconstructed skin was observed. AuNP could only be detected up to ∼14 µm depth in human skin. Lateral (0.5 ± 0.1 µm) and axial (1.0 ± 0.3 µm) PSF in reconstructed and human specimens were determined. Skin cells and intercellular components didn't degrade the PSF with depth. In summary, the imaging parameters of multiphoton microscopy in skin and practical limitations encountered in tracking nanoparticle penetration using this approach were investigated.