Lipid Layer Research Articles

In Vitro Inhibition of Evaporation with Perfluorohexyloctane, an Eye Drop for Dry Eye Disease

Perfluorohexyloctane (PFHO) MIEBOTM, formerly (NOV03) is a single component, water-free eye drop approved by the Food and Drug Administration in the United States for the treatment of dry eye disease. We evaluated the in vitro inhibitory effect of PFHO on the evaporation rate (Revap) of saline. Evaporation rates were measured gravimetrically at 25°C or 35°C. The evaporation rate (Revap) of phosphate-buffered saline (PBS) was measured following the application of 11-200 µL PFHO or 100 µL artificial tears (Soothe XP [Bausch + Lomb, Bridgewater, New Jersey], Systane Balance [Alcon, Fort Worth, Texas], and Systane Ultra [Alcon]). The effect of PFHO on the Revap of PBS was further evaluated following the addition of 50 mg/mL mucin to PBS and compared with that of meibum lipid collected from a 68 year-old White volunteer. At 25°C the mean (SEM) Revap of PBS alone or PFHO alone was 4.06 (0.06) and 0.137 (0.004) µm/min, respectively. Layering 100 µL PFHO over PBS inhibited the Revap of PBS by 81% (P < 0.0001), whereas artificial tears had no effect. The presence of mucin attenuated the inhibition of the Revap of PBS by PFHO by 17% (P < 0.0001). At 35°C, the Revap of PBS was inhibited by 88% when layering 100 µL PFHO over PBS and 28% when applying a single 11µL drop of PFHO (P value < 0.0001 for both). Meibum lipid inhibited the Revap of PBS by 8% at this temperature, whereas the combination of a drop of PFHO plus meibum inhibited the Revap of PBS by 34%. PFHO significantly inhibited the Revap of saline in this in vitro model. The data support the idea that PHFO may form an antievaporative layer on the tear film surface and may be a functional substitute for the native tear-film lipid layer in patients with dry eye disease.

Diurnal Variation in Tear Film Lipid Layer Using Smartphone-based Interferometry

Diurnal Variation in Tear Film Lipid Layer Using Smartphone-based Interferometry

Therapeutic effects of orthokeratology lens combined with 0.01% atropine eye drops on juvenile myopia.

To explore the therapeutic effects of orthokeratology lens combined with 0.01% atropine eye drops on juvenile myopia. A total of 340 patients with juvenile myopia (340 eyes) treated from 2018 to December 2020 were divided into the control group (170 cases with 170 eyes, orthokeratology lens) and observation group (170 cases with 170 eyes, orthokeratology lens combined with 0.01% atropine eye drops). The best-corrected distance visual acuity, best-corrected near visual acuity, diopter, axial length, amplitude of accommodation, bright pupil diameter, dark pupil diameter, tear-film lipid layer thickness, and tear break-up time were measured before treatment and after 1 year of treatment. The incidence of adverse reactions was observed. Compared with the values before treatment, the spherical equivalent degree was significantly improved by 0.22 (0.06, 0.55) D and 0.40 (0.15, 0.72) D in the observation and control groups after the treatment, respectively (p<0.01). After the treatment, the axial length was significantly increased by (0.15 ± 0.12) mm and (0.24 ± 0.11) mm in the observation and control groups, respectively, (p<0.01). After the treatment, the amplitude of accommodation significantly declined in the observation group and was lower than that in the control group, whereas both bright and dark pupil diameters significantly increase and were larger than those in the control group (p<0.01). After the treatment, the tear-film lipid layer thickness and tear break-up time significantly declined in the two groups (p<0.01). Orthokeratology lens combined with 0.01% atropine eye drops can synergistically enhance the control effect on juvenile myopia with high safety.

Effect of water content on stratum corneum penetration mechanism of W/O type microemulsions.

The stratum corneum (SC) consists of a lipid layer that forms two types of lamellar structures: short lamellar (S-La) and long lamellar (L-La). It has been reported that S-La contains water phases in the hydrophilic region of the lipids, and that it may play an important role in regulating the water content of the SC. The amount of water in the SC can affect how a drug carrier permeates through the intercellular lipid pathway. To better understand the impact of SC water content on the skin penetration mechanism of a microemulsion (ME), we conducted a study using small-angle X-ray scattering (SAXS), wide-angle X-ray scattering (WAXS), and small-angle neutron scattering (SANS). Our results showed that MEs can enhance skin permeation under humid conditions because the lipid packing structures of the hydrated SC are more disrupted than those of the dry SC. The results also showed that the inner water of MEs was released to the SC when applying MEs to the dry SC, resulting in an increase in the repeat distance of S-La. Conversely, when MEs are applied to hydrated SC, the MEs absorb the water from the SC into their inner phases, causing a decrease in the repeat distance of S-La over time.

Conveyance of sofosbuvir through vesicular lipid nanocarriers as an effective strategy for management of viral meningitis.

This study aimed to deliver a potential water-soluble antiviral drug (sofosbuvir) through optimized vesicular lipid nanocarriers (LNs) to the rat brain as a novel strategy against viral meningitis. A 23 factorial design approach was established to assess the effect of formulation composition and process variables on the physicochemical properties of the LNs. Sofosbuvir-loaded LNs (SLNs) were developed by lipid layer hydration method utilizing optimized parameters and evaluated for various in vitro characterizations like FTIR, DSC, XRD, FESEM, vesicle size, zeta potential, drug carrying capacity and drug release. Plasma and brain pharmacokinetic (PK) studies were conducted in Sprague-Dawley rats. FTIR data depicted the absence of any major interaction between the drug and the excipients. DSC revealed a sharp endothermic peak for the drug. XRD showed the amorphic nature of the SLNs. Optimized SLNs were spherical as depicted from FESEM with 42.43 nm size, -49.21 mV zeta potential, 8.31% drug loading and sustained drug release in vitro. Plasma/brain PK studies depicted significant improvement in key PK parameters, viz. AUC, AUMC, MRT, and Vd, compared to those for the free drug. A more than 3.5-fold increase in MRT was observed for optimized SLNs (11.2 h) in brain tissue compared to the free drug (3.7 h). Ex vivo hemolysis data confirmed the non-toxic nature of the SLNs to human red blood cells. In silico docking study further confirmed strong interaction between the drug and selected protein 4YXP (herpes simplex) with docking score of -7.5 and 7EWQ protein (mumps virus) with docking score of -7.3. The optimized SLNs may be taken for further in vivo studies to pave the way towards clinical translation.

Синдром «сухого глаза» перед кераторефракционной хирургией: частота встречаемости и патогенетически ориентированная терапия

Aim: to assess the ocular surface in patients who were planned to undergo corneal refractive surgery (CRS), using latest diagnostic tools, and to develop a differentiated approach to the preoperative preparation of the patients with confirmed dry eye disease (DED). Patients and Methods: the study included 298 patients (298 eyes), aged 18 to 36 years, with diagnosed mild and moderate myopia, who had binocular type of vision and average keratometry readings 43.0-20.0 D. The patients were planned to undergo CRS. A standardized preoperative diagnostic examination was performed in all patients according to the protocol of refraction eye testing. The diagnostic system manufactured by MediWorks (China) was used to evaluate the ocular surface and conjunctival mucous membranes. Results: based on the results of the objective methods of examination 112 (37.6%) cases of DED were detected, including 101 (33.9%) cases of evaporative and 11 (3,7%) cases of hyposecretory forms. In addition, Schirmer-1 test was performed in patients with hyposecretory DED which showed a significantly reduced aqueous tear production. Patients with DED were advised to discontinue wearing contact lenses and to use tear substitutes containing high-molecular-weight hyaluronic acid and sodium hyaluronate in a relatively low concentration (0.15%). Also, a 0.01% solution of sulfated glycosaminoglycanes was administered to the patients. To relieve the sterile inflammatory response, the patients received a course of steroids as anti-inflammatory agents followed by cyclosporin A. An eye gel was recommended for the long- lasting overnight hydration of the corneal epithelium and its faster renewal. As a result of the positive response to the treatment, after 6–8 months of therapy it was possible to perform surgical correction in 6 (54.5%) of 11 patients of this group. In post-operative period the patients demonstrated good clinical and functional outcomes and did not report any worsening of DED clinical signs. In 89 (88.1%) of 101 patients with evaporative DED the combination therapy consisting of the eyelid self-massage and two-phase cationic emulsion (oil-water), a mimic of tear film lipid layer, which contained the positively charged nanodrops of mineral oils dispersed in water, helped to normalize the ocular surface and tear production parameters, and thus enabled to perform CRS. Conclusion: in the group of examined patients, DED was found in 37.6% of cases, and the prevalence of evaporative form was almost 10-fold higher than that of secretory form. The occurrence of two DED forms dictates a need for the differentiated approach to diagnostics and treatment. In contrast to hyposecretory form, a clear trend to the regression of symptoms was demonstrated for evaporative form of DED, when patients received pathogenesis-oriented therapy. Thus, patients with evaporative DED clinical pattern are more likely to become candidates for CRS than those with tear hyposecretion. Keywords: dry eye syndrome, corneal refractive surgery, dysfunction of the meibomian glands, tear film, nanoemulsion. For citation: Maychuk N.V., Malyshev I.S., Obraztsova M.R. Dry eye syndrome before corneal refractive surgery: the prevalence and pathogenesis- oriented therapy. Russian Journal of Clinical Ophthalmology. 2023;23(1):14–20 (in Russ.). DOI: 10.32364/2311-7729-2023-23-1-14-20.

Оптические и трансдермальные свойства липидного барьера рогового слоя эпидермиса кожи

A study of the lipid barrier of the stratum corneum for various areas of the wrist skin was carried out using Fourier IR spectroscopy, which showed a significant difference in the optical properties depending on the dryness of the skin and the thickness of the stratum corneum. Four participants of different age took part in the experiment. Analyzing the protective function of the epidermis lipid layer was carried out on the skin subjected to various treatment methods: water, soap, Fairy detergent, and 96% ethanol. It is noted that the nature of the change in the spectrum of the barrier layer, represented by bands 1172 and 1150 cm-1, and reflecting fluctuations in the C-O groups of two alternating blocks of fatty acids – short- and longchain ones, shows a difference in its response to the chemical nature of the influencing substances. Using the example of hand skin treatment with a moisturizing cream lotion, the manifestation of the transdermal function of the lipid barrier layer and the kinetics of changes in its optical properties during the absorption of cosmetic products is shown. The well-recorded frequency of the seasonal cycle of lipid renewal for the barrier layer as well as the influence of the age factor and diet were noted.

Review Artikel: Metode Teknologi Dalam Sistem Penghantaran Obat Transdermal Sediaan Patch

&#x0D; &#x0D; &#x0D; &#x0D; Transdermal is a route of drug administration through the skin, where many lipid layers are difficult to penetrate so that transdermal preparations are needed as a place for drug entry, for example creams and gels. The mechanism of drug delivery is through diffusion of the stratum corneum layer through lipid molecules, before releasing the drug in a controlled and constant manner followed by various other benefits and aims to increase the pharmacological effect in order to reach the maximum level. However, the permeability of the drug is not strong enough to pass through the stratum corneum. So we need a transdermal preparation that can work optimally and overcome the obstacles. This study aims to demonstrate the form, evaluation, formulation, and application of transdermal preparations in new drug delivery systems. Method used literature review of the 10 most journal articles and contains related information. Based on the research results, transdermal dosage forms differ depending on the level of concentration and each technological method needs adapted to the characteristics drug and the route of drug delivery given.&#x0D; &#x0D; &#x0D; &#x0D;

Assessment of condition-induced changes on the ocular surface using novel methods to assess the tear film dynamics and the lipid layer

ObjectivesTo assess the effects of different condition-induced changes on current and novel ocular surface metrics. MethodsEighty-four healthy volunteers (22.4 ± 2.6 years) participated in this study. The ocular surface and tear film response to (1) computer use, (2) contact lens insertion and (3) tear film instillation during computer use with contact lenses were assessed. Current metrics included the ocular surface disease (OSDI) questionnaire, 5-item dry eye questionnaire (DEQ-5), bulbar redness, tear meniscus height (TMH) and non-invasive keratograph break-up time (NIKBUT). Novel metrics included the lipid layer thickness obtained from the intensity of the reflected Placido disk and the speed of tear film particles post-blink. ResultsHigher dry eye symptoms, TMH and bulbar redness, and lower values in metrics related to the intensity of the Placido disk pattern and to particle speed were found after the computer reading task (p < 0.036). When a contact lens was fitted, lower TMH, NIKBUT and particle speed metrics were obtained (p < 0.044). Mixed ANOVA revealed that artificial tears significantly ameliorated the effect of computer reading on OSDI, DEQ-5, NIKBUT, metrics related to the intensity of the Placido disk pattern and metrics related to particle speed (p < 0.033). ConclusionsComputer use and contact lens wear worsened dry eye signs and symptoms, but artificial tears ameliorated this effect. Newly developed methods can serve as a tool to detect changes in the tear film triggered by different ocular surface-disturbing conditions.

Formulation and Evaluation of Solid Lipid Nanoparticles containing Kappaphycus alvarezii Extract in A Cosmetic Gel

Objectives: The objective of the research was to prepare and evaluate the cosmetic gel containing SLNs of Kappaphycus alvarezii chloroform extract to localize the extract topically to exert its antimicrobial properties. Methodology: The antimicrobial activity evaluation for the chloroform, methanol, and aqueous K. alvarezii extracts were carried out using disc-diffusion agar plate method. The extract with maximum antimicrobial action was selected for the preparation of SLNs. Pre-formulation studies using ATR-FTIR spectrophotometer was carried out to check the compatibility of the extract with excipients in the range of 400–4000 cm−1.The SLNs were prepared by the film hydration technique with ultrasonication. The dried SLNs were evaluated for its physical characteristics, zeta potential and for the duration taken to release the encapsulated extract for the antimicrobial action. Carbopol and HPMC were chosen as the gelling agent after compatibility studies with nanoparticle dispersions.The extract loaded SLNs based gel was analysed for its pH, visual appearance, and in-vitro drug release. Results: Chloroform extract of K. alvarezii (120μg) showed maximum antimicrobial activity with cloudy and blurry zone of inhibition. The zone of inhibition was smaller for SLNs when compared to the extract. This was due to the low diffusion of encapsulated drug through the outer lipid layer of SLNs. SLNs showed a zeta potential ranging between –11.0 to –37.4mV. The formulated gel containing solid lipid nanoparticles of chloroform extract of K. alvarezii had an average pH value of 5.37, which was suitable to be used on human skin. The maximum drug release was 60.28% for over a period of 25 hours. Conclusion: In this study algal extracts were successfully encapsulated within the SLNs and when applied topically the SLNs could reside on the surface of the skin for localized action as evident from the drug release study.The zeta potential obtained for the SLNs was approximately within the limit of –30mV that yields a formulation with fairly good physical stability.

NIR-Activated Thermosensitive Liposome-Gold Nanorod Hybrids for Enhanced Drug Delivery and Stimulus Sensitivity.

Combinatorial photothermal therapy and chemotherapy is an extremely promising tumor therapeutic modality. However, such systems still remain challenges in stimulus sensitivity, avoiding drug leakage, and therapeutic safety. To solve these problems, we engineered actively loaded doxorubicin (DOX) and gold nanorod (GNR) liposomes through embedding stiff hollow mesoporous silica nanoparticles (HMSNs) in the liposomal water cavity (HMLGDB) to resist the influence of shear force of GNRs to prevent drug leakage. Under 808 nm laser irradiation, the ambient temperature was raised greatly because of the photothermal conversion of GNRs, thereby rupturing the lipid layer and then triggering the DOX release. The results of in vitro experiments showed that the low concentration of HMLGDB (15 μg/mL) could effectively overcome the MCF-7 cells (human breast cancer cell line) by the increase of DOX concentration intracellularly and the good photothermal effect of GNRs. After intravenous injection, HMLGDB exhibited intratumor aggregation and PTT capacity. Furthermore, the combined chemo-photothermal antitumor strategy demonstrated a high inhibition of tumor growth and low damage to normal tissues. The developed hybrids provide a paradigm for efficient combinatorial photothermal therapy (PTT) and chemotherapy (CT).

Clinical efficacy of 0.05% cyclosporine nano-emulsion in the treatment of dry eye syndrome associated with meibomian gland dysfunction.

To evaluate the clinical efficacy of topical 0.05% cyclosporine nano-emulsion in the treatment of dry eye syndrome (DES) with meibomian gland dysfunction (MGD). This prospective study included 64 patients with DES and MGD who were randomly assigned to three groups: Group 1 (n=24, conventional cyclosporine), Group 2 (n=21, nano-emulsion cyclosporine), and Group 3 (n=19, control). Lid margin telangiectasia (LMT), meibomian gland secretion (MGS), conjunctival injection (CI), corneal staining (CS), tear break-up time (TBUT), Schirmer test I (STI), Ocular Surface Disease Index (OSDI), and lipid layer thickness (LLT) was evaluated at 4, 8, and 12wk of treatment. In Group 3 (control), LMT, CS, and CI improved after 8wk, MGS, TBUT after 12wk of treatment. In Group 1 (conventional cyclosporine), LMT, MGS, and TBUT improved significantly after 4wk, whereas CS, CI, STI, and LLT improved significantly after 8wk, and OSDI at 12wk. In Group 2 (nano-cyclosporine), LMT, MGS, CS, CI, TBUT, and OSDI significantly improved after 4wk, and STI after 8wk. Especially, LLT was significantly higher than other groups after 4wk. Cyclosporine and nano-cyclosporine shows significant improvement in DES with MGD than the control group. In contrast, the nano-cyclosporine group shows more statistically improved CI and CS at 4wk, especially LLT at 4, 8, and 12wk compared to the conventional cyclosporine group.

The efficacy of ocular surface assessment approaches in evaluating dry eye treatment with artificial tears

This study evaluates the effectiveness of objective techniques in assessing dry eye disease (DED) treatment compared with the subjective assessment commonly used in clinical practice. Thirty subjects were recruited for two visits separated by 28(± 3) days of treatment with artificial tears. A buttery of common subjective assessment methods were accompanied by a set of objective techniques including measurement of noninvasive tear film break-up time (NIBUT), lipid layer thickness (LLT), and quantitative evaluation of tear film surface quality and dynamics (TFD). Additionally, meibography was performed. Two commercially available videokeratoscopes and a prototype of a lateral shearing interferometer were used for the measurements. Both subjective and objective techniques showed a positive effect of artificial tears in DED treatment. Statistically significant improvements were observed in subjective symptoms (from P < 0.001 for Ocular Surface Disease Index, OSDI to p = 0.019 for tearing), conjunctival redness (P = 0.022), ocular staining (P = 0.012), fluorescein tear film break-up time (P = 0.015), NIBUT (P = 0.037), LLT (P < 0.001), and TFD (P = 0.048). In general, weak or statistically insignificant correlations were observed between subjective and objective assessment methods. The apparent lack of correlation between these methods might indicate the complementary character of objective techniques that likely assess other characteristics of ocular surface health than those assessed subjectively.

A biophysical study of tear film lipid layer model membranes

The tear film lipid layer (TFLL), the final layer of the human tear film is responsible for surface tension reduction while blinking, water evaporation retardation and maintaining the stability of the tear film. The study of the composition-structure-function relationship of TFLL is paramount, as a compromised structure of TFLL leads to the emergence of dry eye disease (DED) which is one the most prevalent ophthalmic surface diseases of the modern world, associated with chronic pain and reduced visual capability. In this model membrane study, a systematic approach is used to study the biophysical properties of TFLL model membranes as a function of composition. Three mixed-lipid model membranes are studied along with their individual components comprising cholesteryl oleate (CO), glyceryl trioleate (GT), L-α-phosphatidylcholine (egg PC) and a free fatty acid mixture. The models become progressively more complex from binary to quaternary mixtures, allowing the role of each individual lipid to be derived. Langmuir balance, Brewster Angle Microscopy (BAM) and Profile Analysis Tensiometer (PAT) are used to study the surface activity and compression-expansion cycles, morphology, and rheological behaviour of the model membranes, respectively. Evidence of multilayering is observed with inclusion of CO and a reversible collapse is associated with the GT phase transition. An initially more coherent film is observed due to the addition of polar PC. Notably, these individual behaviours are retained in the mixed films and suggest a possible role for each physiological component of TFLL.

Tear film layers and meibomian gland assessment in patients with type 1 diabetes mellitus using a noninvasive ocular surface analyzer: a cross-sectional case–control study

PurposeTo assess the tear film layers and Meibomian glands by a noninvasive ocular surface analyzer in patients with and without type 1 diabetes mellitus (T1DM).MethodsEighty-eight participants were enrolled in this study: 44 patients with T1DM without diabetic retinopathy, and 44 patients as a control group, between 18 and 49 years old. Limbal and bulbar redness classification, lipid layer thickness (LLT), tear meniscus height (TMH), first and mean noninvasive tear break-up time (FNIBUT and MNIBUT, respectively), and Meibomian glands loss (MGL) were assessment through the ICP Ocular Surface Analyzer (OSA). Schirmer’s I test (SIT), the fluorescein tear break-up time test (TFBUT), OSDI and SPEED questionnaires, and percentage of glycosylated hemoglobin (HbA1c) were also tested.ResultsThe T1DM group showed higher limbal and bulbar redness (p = 0.010) and lower LLT (p < 0.001), TMH (p < 0.001), FNIBUT (p < 0.001), MNIBUT (p < 0.001), SIT (p = 0.001), and TFBUT (p < 0.001) than the control group. A higher percentage of MGL was found in the T1DM group in the upper (p = 0.097) and lower (p < 0.001) eyelids. No significant differences were found in dry eye symptoms across the OSDI and SPEED questionnaires between the two groups.ConclusionPatients with T1DM without signs of retinopathy showed involvement of the mucoaqueous and lipid layers of the tear film, as well as a higher percentage of MGL, using a noninvasive analyzer. Dry eye disease in people with T1DM cannot be ruled out by anamnesis and subjective symptom questionnaires alone; therefore, these patients should undergo regular anterior pole examinations.

Designing of smart nanogels based on tragacanth gum for cisplatin delivery

AbstractThe present work deals with the development of smart nanogels with a coating of a lipid layer, which synergized the triggered delivery of the anticancer drug cisplatin (CP). In the first step, the chemical initiation method was used for the grafting of poly(itaconic acid) on tragacanth gum (TG). Subsequently, this grafted TG matrix was used for the synthesis of nanoparticles. Nanoemulsion process was followed to prepare CP‐loaded grafted TG–lecithin (GTG‐LC) nanogels using dioctyl sulfosuccinate as the surfactant. These nanoparticles were characterized for size, morphology, elemental composition and crystallinity. Transmission electron microscopy images of the GTG‐LC nanogels with varying concentrations of CP demonstrate an irregular polygonal shape of the nanoparticles which is strongly influenced by the CP concentration. The smart nanogels exhibit a unique structure, nano‐within‐nano, a structure where nanosized CP is encapsulated within the GTG‐LC nanogel. Grafted nanogels have a high carboxyl functionality and exhibit pH‐sensitive behavior leading to pH‐responsive drug release behavior. These nanogels show required biocompatibility and offer interesting material for nanomedicines in chemotherapy. © 2022 Society of Industrial Chemistry.

Desiccation resistance differences in Drosophila species can be largely explained by variations in cuticular hydrocarbons.

Maintaining water balance is a universal challenge for organisms living in terrestrial environments, especially for insects, which have essential roles in our ecosystem. Although the high surface area to volume ratio in insects makes them vulnerable to water loss, insects have evolved different levels of desiccation resistance to adapt to diverse environments. To withstand desiccation, insects use a lipid layer called cuticular hydrocarbons (CHCs) to reduce water evaporation from the body surface. It has long been hypothesized that the water-proofing capability of this CHC layer, which can confer different levels of desiccation resistance, depends on its chemical composition. However, it is unknown which CHC components are important contributors to desiccation resistance and how these components can determine differences in desiccation resistance. In this study, we used machine-learning algorithms, correlation analyses, and synthetic CHCs to investigate how different CHC components affect desiccation resistance in 50 Drosophila and related species. We showed that desiccation resistance differences across these species can be largely explained by variation in CHC composition. In particular, length variation in a subset of CHCs, the methyl-branched CHCs (mbCHCs), is a key determinant of desiccation resistance. There is also a significant correlation between the evolution of longer mbCHCs and higher desiccation resistance in these species. Given that CHCs are almost ubiquitous in insects, we suggest that evolutionary changes in insect CHC components can be a general mechanism for the evolution of desiccation resistance and adaptation to diverse and changing environments.

Categorization of the Aqueous Deficient Dry Eye by a Cut-Off Criterion of TMH Measured with Tearscope.

A decrease of the Tear Meniscus Height (TMH) has been proposed as a useful indicator for Aqueous Deficient Dry Eye (ADDE) categorization. The present study aimed to calculate a TMH cut-off criterion for the categorization or severity assessment of ADDE with the Tearscope. 200 participants with a previous Dry Eye Disease (DED) diagnosis according to TFOS DEWS-II criteria were recruited. TMH by slit-lamp illumination and Lipid Layer Pattern (LLP) with Tearscope were assessed to categorise the participants into the ADDE or the Evaporative Dry Eye (EDE) group. The ADDE group was also subdivided into Mild-moderate ADDE and Moderate-severe ADDE based on TMH with slit-lamp. Additionally, the TMH was measured by Tearscope (TMH-Tc). Receiver Operating Characteristics showed that the TMH-Tc have a diagnostic capability to differentiate between ADDE and EDE participants, and between Mild-moderate or Moderate-severe ADDE, with a cut-off value of 0.159 mm (AUC = 0.843 ± 0.035, p < 0.001; sensitivity: 86.4%; specificity: 75.4%) and 0.105 mm (AUC = 0.953 ± 0.025, p < 0.001; sensitivity: 98.1%; specificity: 80.0%), respectively. The present study proposed a cut-off criterion to differentiate between ADDE and EDE participants, or between ADDE severities through TMH assessed by Tearscope.

Lipid-Polymer Hybrid Nanoparticles for mRNA Delivery to Dendritic Cells: Impact of Lipid Composition on Performance in Different Media.

To combine the excellent transfection properties of lipids with the high stability of polymeric nanoparticles, we designed a hybrid system with a polymeric core surrounded by a shell of different lipids. The aim is to use this technology for skin vaccination purposes where the transfection of dendritic cells is crucial. Based on a carrier made of PLGA and the positively charged lipid DOTMA, we prepared a panel of nanocarriers with increasing amounts of the zwitterionic phospholipid DOPE in the lipid layer to improve their cell tolerability. We selected a nomenclature accordingly with numbers in brackets to represent the used mol% of DOPE and DOTMA in the lipid layer, respectively. We loaded mRNA onto the surface and assessed the mRNA binding efficacy and the degree of protection against RNases. We investigated the influence of the lipid composition on the toxicity, uptake and transfection in the dendritic cell line DC 2.4 challenging the formulations with different medium supplements like fetal calf serum (FCS) and salts. After selecting the most promising candidate, we performed an immune stimulation assay with primary mouse derived dendritic cells. The experiments showed that all tested lipid-polymer nanoparticles (LPNs) have comparable hydrodynamic parameters with sizes between 200 and 250 nm and are able to bind mRNA electrostatically due to their positive zetapotential (20-40 mV for most formulations). The more of DOPE we add, the more free mRNA we find and the better the cellular uptake reaching approx. 100% for LPN(60/40)-LPN(90/10). This applies for all tested formulations leading to LPN(70/30) with the best performance, in terms of 67% of live cells with protein expression. In that case, the supplements of the medium did not influence the transfection efficacy (56% vs. 67% (suppl. medium) for live cells and 63% vs. 71% in total population). We finally confirmed this finding using mouse derived primary immune cells. We can conclude that a certain amount of DOTMA in the lipid coating of the polymer core is essential for complexation of the mRNA, but the zwitterionic phospholipid DOPE is also important for the particles' performance in supplemented media.

Spatio-temporal delivery of both intra- and extracellular toll-like receptor agonists for enhancing antigen-specific immune responses.

For cancer immunotherapy, triggering toll-like receptors (TLRs) in dendritic cells (DCs) can potentiate antigen-based immune responses. Nevertheless, to generate robust and long-lived immune responses, a well-designed nanovaccine should consider different locations of TLRs on DCs and co-deliver both antigens and TLR agonist combinations to synergistically induce optimal antitumor immunity. Herein, we fabricated lipid-polymer hybrid nanoparticles (LPNPs) to spatio-temporally deliver model antigen ovalbumin (OVA) on the surface of the lipid layer, TLR4 agonist monophosphoryl lipid A (MPLA) within the lipid layer, and TLR7 agonist imiquimod (IMQ) in the polymer core to synergistically activate DCs by both extra- and intra-cellular TLRs for enhancing adaptive immune responses. LPNPs-based nanovaccines exhibited a narrow size distribution at the mean diameter of 133.23nm and zeta potential of -2.36mV, showed a high OVA loading (around 70.83μg/mg) and IMQ encapsulation efficiency (88.04%). Our data revealed that LPNPs-based nanovaccines showed great biocompatibility to immune cells and an excellent ability to enhance antigen internalization, thereby promoting DCs maturation and cytokines production. Compared to Free OVA, OVA-LPNPs promoted antigen uptake, lysosome escape, depot effect and migration to secondary lymphatic organs. Invivo immunization showed that IMQ-MPLA-OVA-LPNPs with dual agonists induced more powerful cellular and humoral immune responses. Moreover, prophylactic vaccination by IMQ-MPLA-OVA-LPNPs effectively suppressed tumor growth and increased survival efficacy. Hence, the nanovaccines we fabricated can effectively co-deliver antigens and different TLR agonists and realize coordinated stimulation of DCs in a spatio-temporal manner for enhanced immune responses, which provides a promising strategy for cancer immunotherapy.