Tuberculosis (TB) has been and still is a global emergency for centuries. Prevention of disease through vaccination would have a major impact on disease prevalence, but the only available current... Show moreTuberculosis (TB) has been and still is a global emergency for centuries. Prevention of disease through vaccination would have a major impact on disease prevalence, but the only available current vaccine, BCG, has insufficient impact. In this article, a novel subunit vaccine against TB was developed, using the Ag85B-ESAT6-Rv2034 fusion antigen, two adjuvants - CpG and MPLA, and a cationic pH-sensitive liposome as a delivery system, representing a new TB vaccine delivery strategy not previously reported for TB. In vitro in human dendritic cells (DCs), the adjuvanted formulation induced a significant increase in the production of (innate) cytokines and chemokines compared to the liposome without additional adjuvants. In vivo, the new vaccine administrated subcutaneously significantly reduced Mycobacterium tuberculosis (Mtb) bacterial load in the lungs and spleens of mice, significantly outperforming results from mice vaccinated with the antigen mixed with adjuvants without liposomes. In-depth analysis underpinned the vaccine's effectiveness in terms of its capacity to induce polyfunctional CD4+ and CD8+ T-cell responses, both considered essential for controlling Mtb infection. Also noteworthy was the differential abundance of various CD69+ B-cell subpopulations, which included IL17-A-producing B cells. The vaccine stimulated robust antigen-specific antibody titers, further extending its potential as a novel protective agent against TB. Show less
Nădăban, A.; Gooris, G.S.; Beddoes, C.M.; Dalgliesh, R.M.; Malfois, M.; Demé, B.; Bouwstra, J.A. 2024
The stratum corneum (SC) lipid matrix, composed primarily of ceramides (CERs), cholesterol and free fatty acids (FFA), has an important role for the skin barrier function. The presence of the long... Show moreThe stratum corneum (SC) lipid matrix, composed primarily of ceramides (CERs), cholesterol and free fatty acids (FFA), has an important role for the skin barrier function. The presence of the long periodicity phase (LPP), a unique lamellar phase, is characteristic for the SC. Insight into the lipid molecular arrangement within the LPP unit cell is imperative for understanding the relationship between the lipid subclasses and the skin barrier function. In this study, the impact of the CER head group structure on the lipid arrangement and barrier functionality was investigated using lipid models forming the LPP. The results demonstrate that the positions of CER N-(tetracosanoyl)-sphingosine (CER NS) and CER N-(tetracosanoyl)-phytosphingosine (CER NP), two essentials CER subclasses, are not influenced by the addition of another CER subclass (N-(tetracosanoyl)-dihydrosphingosine (CER NdS), N-(2R-hydroxy-tetracosanoyl)-sphingosine (CER AS) or D-(2R-hydroxy-tetracosanoyl)-phytosphingosine (CER AP)). However, differences are observed in the lipid organization and the hydrogen bonding network of the three different models. A similar localization of CER NP and CER NS is also observed in a more complex lipid model, with the CER subclass composition mimicking that of human SC. These studies show the adaptability and insensitivity of the LPP unit cell structure to changes in the lipid head group structures of the CER subclasses. Show less
Tuberculosis (TB) is still among the deadliest infectious diseases, hence there is a pressing need for more effective TB vaccines. Cationic liposome subunit vaccines are excellent vaccine... Show moreTuberculosis (TB) is still among the deadliest infectious diseases, hence there is a pressing need for more effective TB vaccines. Cationic liposome subunit vaccines are excellent vaccine candidates offering effective protection with a better safety profile than live vaccines. In this study, we aim to explore intrinsic adjuvant properties of cationic liposomes to maximize immune activation while minimizing aspecific cytotoxicity. To achieve this, we developed a rational strategy to select liposomal formulation compositions and assessed their physicochemical and immunological properties in vitro models using human monocyte-derived dendritic cells (MDDCs). A broad selection of commercially available cationic compounds was tested to prepare liposomes containing Ag85B-ESAT6-Rv2034 (AER) fusion protein antigen. 1,2-Dioleoyl-sn‑glycero-3-ethylphosphocholine (EPC)-based liposomes exhibited the most advantageous activation profile in MDDCs as assessed by cell surface activation markers, cellular uptake, antigen-specific T-cell activation, cytokine production, and cellular viability. The addition of cholesterol to 20 mol% improved the performance of the tested formulations compared to those without it; however, when its concentration was doubled there was no further benefit, resulting in reduced cell viability. This study provides new insights into the role of cationic lipids and cholesterol in liposomal subunit vaccines. Show less
Lee, J.; Neustrup, M.A.; Slütter, B.A.; O'Mahony, C.; Bouwstra, J.A.; Maaden, K. van der 2024
The barrier function of the skin is primarily located in the stratum corneum (SC), the outermost layer of the skin. The SC is composed of dead cells with highly organized lipid lamellae in the... Show moreThe barrier function of the skin is primarily located in the stratum corneum (SC), the outermost layer of the skin. The SC is composed of dead cells with highly organized lipid lamellae in the intercellular space. As the lipid matrix forms the only continuous pathway, the lipids play an important role in the permeation of compounds through the SC. The main lipid classes are ceramides (CERs), cholesterol (CHOL) and free fatty acids (FFAs). Analysis of the SC lipid matrix is of crucial importance in understanding the skin barrier function, not only in healthy skin, but also in inflammatory skin diseases with an impaired skin barrier. In this review we provide (i) a historical overview of the steps undertaken to obtain information on the lipid composition and organization in SC of healthy skin and inflammatory skin diseases, ii) information on the role CERs, CHOL and FFAs play in the lipid phase behavior of very complex lipid model systems and how this knowledge can be used to understand the deviation in lipid phase behavior in inflammatory skin diseases, iii) knowledge on the role of both, CER subclasses and chain length distribution, on lipid organization and lipid membrane permeability in complex and simple model systems with synthetic CERs, CHOL and FFAs, iv) similarity in lipid phase behavior in SC of different species and complex model systems, and vi) future directions in modulating lipid composition that is expected to improve the skin barrier in inflammatory skin diseases. Show less
Rousel, J.; Saghari, M.; Pagan, L.; Nadaban, A.; Gambrah, T.; Theelen, B.; ... ; Doorn, M.B.A. van 2023
Dissolving microneedle arrays (dMNAs) can be used to deliver vaccines via the intradermal route. Fabrication of dMNAs using centrifugation is the most common preparation method of dMNAs, but it... Show moreDissolving microneedle arrays (dMNAs) can be used to deliver vaccines via the intradermal route. Fabrication of dMNAs using centrifugation is the most common preparation method of dMNAs, but it results in a substantial loss of antigens. In order to solve the issue of antigen waste, we engineered an automatic dispensing system for dMNA preparation. Here, we report on the fabrication of influenza whole inactivated virus (WIV) vaccine-loaded dMNAs (WIV dMNAs) by using the automatic dispensing system. Prior to the dispensing process, polydimethylsiloxane (PDMS) moulds were treated with oxygen plasma to increase surface hydrophilicity. WIV dMNAs were prepared with 1% (w/v) trehalose and pullulan (50 : 50 weight ratio). During the dispensing process, reduced pressure was applied to the PDMS mould via a vacuum chamber to make microneedle cavities airless. After producing dMNAs, WIV was quantified and 1.9 μg of WIV was loaded per dMNA, of which 1.3 μg was in the microneedle tips. Compared to the centrifugation method, this automatic dispensing system resulted in a 95% reduction of antigen waste. A hemagglutination assay confirmed that WIV dMNA maintained the stability of the antigen for at least four weeks of storage, even at room temperature or at 37 °C. The WIV dMNAs displayed 100% penetration efficiency in human skin, and 83% of the microneedle volume was dissolved in the skin within 10 minutes. In a vaccination study, mice immunised with WIV dMNAs showed similar IgG levels to those that received WIV intramuscularly. In conclusion, using the automatic dispensing system for dMNA production strongly reduced antigen waste and yielded dMNAs with excellent physical, mechanical, and immunological properties. Show less
Lozano Vigario, F.; Simo Vesperinas, I.; Depuydt, M.A.C.; Bot, I.; Veelen, P. van; Bouwstra, J.A.; ... ; Slutter, B. 2023
The lipids in the uppermost layer of the skin, the stratum corneum (SC), play an important role in the skin barrier function. The three main subclasses in the SC lipid matrix are ceramides (CER),... Show moreThe lipids in the uppermost layer of the skin, the stratum corneum (SC), play an important role in the skin barrier function. The three main subclasses in the SC lipid matrix are ceramides (CER), cholesterol and free fatty acids. In inflammatory skin diseases, such as atopic dermatitis and psoriasis, the SC lipid composition is modulated compared to the composition in healthy SC. One of the main alterations is the molar ratio between the concentration of CER N-(tetracosanoyl)-sphingosine (CER NS) and CER N-(tetracosanoyl)-phytosphingosine (CER NP), which correlated with an impaired skin barrier function. In the present study we investigated the impact of varying the CER NS:CER NP ratios on the lipid organization, lipid arrangement and barrier functionality in SC lipid model systems. The results indicate that a higher CER NS:CER NP ratio as observed in diseased skin did not alter the lipid organization or lipid arrangement in the long periodicity phase encountered in SC. The trans-epidermal water loss, an indication of the barrier functionality, was significantly higher for the CER NS:CER NP 2:1 model (mimicking the ratio in inflammatory skin diseases) compared to the CER NS:CER NP 1:2 ratio (in healthy skin). These findings provide a more detailed insight into the lipid organization in both healthy and diseased skin and suggest that in vivo the molar ratio between CER NS:CER NP contributes to barrier impairment as well, but might not be the main factor. Show less
Helder, R.W.J.; Rousel, J.; Boiten, W.A.; Gooris, G.S.; Nadaban, A.; Ghalbzouri, A. el; Bouwstra, J.A. 2022
Human skin equivalents (HSEs) are 3D-cultured human skin models that mimic many aspects of native human skin (NHS). Although HSEs resemble NHS very closely, the barrier located in the stratum... Show moreHuman skin equivalents (HSEs) are 3D-cultured human skin models that mimic many aspects of native human skin (NHS). Although HSEs resemble NHS very closely, the barrier located in the stratum corneum (SC) is impaired. This is caused by an altered lipid composition in the SC of HSEs compared with NHS. One of the most pronounced changes in this lipid composition is a high level of monounsaturation. One key enzyme in this change is stearoyl-CoA desaturase-1 (SCD1), which catalyses the monounsaturation of lipids. In order to normalize the lipid composition, we aimed to target a group of nuclear receptors that are important regulators in the lipid synthesis. This group of receptors are known as the peroxisome proliferating activating receptors (PPARs). By (de)activating each isoform (PPAR-alpha, PPAR-delta and PPAR-gamma), the PPAR isoforms may have normalizing effects on the lipid composition. In addition, another PPAR-alpha agonist Wy14643 was included as this supplement demonstrated normalizing effects in the lipid composition in a more recent study. After PPAR (ant)agonists supplementation, the mRNA of downstream targets, lipid synthesis genes and lipid composition were investigated. The PPAR downstream targets were activated, indicating that the supplements reached the keratinocytes to trigger their effect. However, minimal impact was observed on the lipid composition after PPAR isoform (de) activation. Only the highest concentration Wy14643 resulted in strong, but negative effects on CER composition. Although the novel tested modifications did not result in an improvement, more insight is gained on the nuclear receptors PPARs and their effects on the lipid barrier in full-thickness skin models. Show less
Skin's effectiveness as a barrier to permeation of water and other chemicals rests almost entirely in the outermost layer of the epidermis, the stratum corneum (SC), which consists of layers of... Show moreSkin's effectiveness as a barrier to permeation of water and other chemicals rests almost entirely in the outermost layer of the epidermis, the stratum corneum (SC), which consists of layers of corneocytes surrounded by highly organized lipid lamellae. As the only continuous path through the SC, transdermal permeation necessarily involves diffusion through these lipid layers. The role of the SC as a protective barrier is supported by its exceptional lipid composition consisting of ceramides (CERs), cholesterol (CHOL), and free fatty acids (FFAs) and the complete absence of phospholipids, which are present in most biological membranes. Molecular simulation, which provides molecular level detail of lipid configurations that can be connected with barrier function, has become a popular tool for studying SC lipid systems. We review this ever-increasing body of literature with the goals of (1) enabling the experimental skin community to understand, interpret and use the information generated from the simulations, (2) providing simulation experts with a solid background in the chemistry of SC lipids including the composition, structure and organization, and barrier function, and (3) presenting a state of the art picture of the field of SC lipid simulations, highlighting the difficulties and best practices for studying these systems, to encourage the generation of robust reproducible studies in the future. This review describes molecular simulation methodology and then critically examines results derived from simulations using atomistic and then coarse-grained models. Show less
Understanding the lipid arrangement within the skin's outermost layer, the stratum corneum (SC), is important for advancing knowledge on the skin barrier function. The SC lipid matrix consists of... Show moreUnderstanding the lipid arrangement within the skin's outermost layer, the stratum corneum (SC), is important for advancing knowledge on the skin barrier function. The SC lipid matrix consists of ceramides (CERs), cholesterol, and free fatty acids, which form unique crystalline lamellar phases, referred to as the long periodicity phase (LPP) and short periodicity phases. As the SC lipid composition is complex, lipid model systems that mimic the properties of native SC are used to study the SC lipid organization and molecular arrangement. In previous studies, such lipid models were used to determine the molecular organization in the trilayer structure of the LPP unit cell. The aim of this study was to examine the location of CER N-(tetracosanoyl)-phytosphingosine (CER NP) in the unit cell of this lamellar phase and compare its position with CER N-(tetracosanoyl)-sphingosine (CER NS). We selected CER NP as it is the most prevalent CER subclass in the human SC, and its location in the LPP is not known. Our neutron diffraction results demonstrate that the acyl chain of CER NP was positioned in the central part of the trilayer structure, with a fraction also present in the outer layers, the same location as determined for the acyl chain of CER NS. In addition, our Fourier transformed infrared spectroscopy results are in agreement with this molecular arrangement, suggesting a linear arrangement for the CER NS and CER NP. These findings provide more detailed insight into the lipid organization in the SC lipid matrix. Show less
Beddoes, C.M.; Gooris, G.S.; Barlow, D.J.; Lawrence, M.J.; Dalgliesh, R.M.; Malfois, M.; ... ; Bouwstra, J.A. 2022
The stratum corneum's lipid matrix is a critical for the skin's barrier function and is primarily composed of ceramides (CERs), cholesterol (CHOL) and free fatty acids (FFAs). The lipids form a... Show moreThe stratum corneum's lipid matrix is a critical for the skin's barrier function and is primarily composed of ceramides (CERs), cholesterol (CHOL) and free fatty acids (FFAs). The lipids form a long periodicity phase (LPP), a unique trilayer unit cell structure. An enzyme driven pathway is implemented to synthesize these key lipids. If these enzymes are down- or upregulated as in inflammatory diseases, the final lipid composition is affected often altering the barrier function. In this study, we mimicked down regulation of enzymes involved in the synthesis of the sphingosine and CER amide bond. In a LPP lipid model, we substituted CER N-(tetracosanoyl)-sphingosine (CER NS) with either i) FFA C24 and free sphingosine, to simulate the loss of the CER amide bond, or ii) with FFA C24 and C18 to simulate the loss of the sphingosine headgroup. Our study shows the lipids in the LPP would not phase separate until at least 25% of the CER NS is substituted keeping the lateral packing and conformational ordering unaltered. Neutron diffraction studies showed that free sphingosine chains localized at the outer layers of the unit cell, while the remaining CER NS head group was concentrated in the inner headgroup layers. However, when FFA C18 was inserted, CER NS was dispersed throughout the LPP, resulting in an even distribution between the inner and outer water layers. The presented results highlight the importance of the CER NS headgroup structure and its interaction in combination with the carbon chain invariability for optimal lipid arrangement. Show less
Gold nanorods (GNRs) are versatile asymmetric nanoparticles with unique optical properties. These properties make GNRs ideal agents for applications such as photothermal cancer therapy, biosensing,... Show moreGold nanorods (GNRs) are versatile asymmetric nanoparticles with unique optical properties. These properties make GNRs ideal agents for applications such as photothermal cancer therapy, biosensing, and in vivo imaging. However, as-synthesised GNRs need to be modified with a biocompatible stabilising coating in order to be employed in these fields as the ligands used to stabilise GNRs during synthesis are toxic. An issue is that GNR performance in the aforementioned techniques can be affected by these modified coatings. For example if coatings are too thick then GNR entry into cells, or their sensitivity in sensing applications, can be compromised. Here we show that thiolated peptide amphiphiles (PAs) can act as GNR stabilisers and provide a thin and highly-stable coating under physiologically relevant conditions. Additionally, all tested PAs formed highly ordered (51.8-58.8% β-content), and dense (2.62-3.87 peptides per nm2) monolayers on the GNR surface. Moreover, the PA-coated GNRs demonstrated no cytotoxicity in vitro and, via injection in zebrafish embryos, the behavior and cellular interactions of such PA-coated GNRs were visualised in vivo, in real time, with two-photon (2P) microscopy. Show less
Tian, Y.; Lee, J.; Maaden, K. van der; Bhide, Y.; Vries-Idema, J.J. de; Akkerman, R.; ... ; Beukema, M. 2022
Most influenza vaccines are administered via intramuscular injection which has several disadvantages that might jeopardize the compliance of vaccinees. Intradermal administration of dissolving... Show moreMost influenza vaccines are administered via intramuscular injection which has several disadvantages that might jeopardize the compliance of vaccinees. Intradermal administration of dissolving-microneedle-arrays (dMNAs) could serve as minimal invasive alternative to needle injections. However, during the production process of dMNAs antigens are subjected to several stresses, which may reduce their potency. Moreover, the needles need to have sufficient mechanical strength to penetrate the skin and subsequently dissolve effectively to release the incorporated antigen. Here, we investigated whether blends of trehalose and pullulan are suitable for the production of stable dMNA fulfilling these criteria. Our results demonstrate that production of trehalose/pullulan-based dMNAs rendered microneedles that were sharp and stiff enough to pierce into ex vivo human skin and subsequently dissolve within 15 min. The mechanical properties of the dMNAs were maintained well even after four weeks of storage at temperatures up to 37°C. In addition, immunization of mice with influenza antigens via both freshly prepared dMNAs and dMNAs after storage (four weeks at 4°C or 37°C) resulted in antibody titers of similar magnitude as found in intramuscularly injected mice and partially protected mice from influenza virus infection. Altogether, our results demonstrate the potential of trehalose/pullulan-based dMNAs as alternative dosage form for influenza vaccination. Show less
Anionic liposomal formulations have previously shown to have intrinsic tolerogenic capacity and these properties have been related to the rigidity of the particles. The combination of highly rigid... Show moreAnionic liposomal formulations have previously shown to have intrinsic tolerogenic capacity and these properties have been related to the rigidity of the particles. The combination of highly rigid anionic liposomes to deliver tolerogenic adjuvants and antigen peptides has potential applications for the treatment of autoimmune and inflammatory diseases. However, the preparation of these highly rigid anionic liposomes using traditional methods such as lipid film hydration presents problems in terms of scalability and loading efficiency of some costly tolerogenic adjuvants like 1-α,25-dihydroxyvitaminD3. Here we propose the use of an off-the-shelf staggered herringbone micromixer for the preparation of these formulations and performed a systematic study on the effect of temperature and flow conditions on the size and polydispersity index of the formulations. Furthermore, we show that the system allows for the encapsulation of a wide variety of peptides and significantly higher loading efficiency of 1-α,25-dihydroxyvitaminD3 compared to the traditional lipid film hydration method, without compromising their non-inflammatory interaction with dendritic cells. Therefore, the microfluidics method presented here is a valuable tool for the preparation of highly rigid tolerogenic liposomes in a fast, size-tuneable and scalable manner. Show less
The skin barrier function is attributed to the stratum corneum (SC) intercellular lipid matrix, which is composed primarily of ceramides (CERs), free fatty acids, and cholesterol. These lipids are... Show moreThe skin barrier function is attributed to the stratum corneum (SC) intercellular lipid matrix, which is composed primarily of ceramides (CERs), free fatty acids, and cholesterol. These lipids are organized in two lamellar phases: the short and long periodicity phases (SPP and LPP), respectively. The LPP is considered important for the skin barrier function. High levels of short-chain CERs are observed in various inflammatory skin diseases and have been correlated with barrier dysfunction. In this research, we investigated how the increase in the fraction of the short-chain CER with a nonhydroxy C16 acyl chain linked to a C18 sphingosine base CER NS(C16) at the expense of the physiological chain length CER NS with a C24 acyl chain (CER NS(C24)) impacts the microstructure and barrier function of a lipid model that mimicked certain characteristics of the SC lipid organization. The permeability and lipid organization of the model membranes were compared with that of a control model without CER NS(C16). The permeability increased significantly when ≥50% of CER NS(C24) was substituted with CER NS(C16). Employing biophysical techniques, we showed that the lipid packing density reduced with an increasing proportion of CER NS(C16). Substitution of 75% of CER NS(C24) by CER NS(C16) resulted in the formation of phase-separated lipid domains and alteration of the LPP structure. Using deuterium-labeled lipids enabled simultaneous characterization of the C24 and C16 acyl chains in the lipid models, providing insight into the mechanisms underlying the reduced skin barrier function in diseased skin. Show less
Petracca, B.; Nădăban, A.; Eeman, M.; Gooris, G.S.; Bouwstra, J.A. 2021
The stratum corneum (SC) acts as the main barrier of the skin against exogenous substances (e.g. air pollutants) and against the loss of endogenous substances such as water. The SC consists of... Show moreThe stratum corneum (SC) acts as the main barrier of the skin against exogenous substances (e.g. air pollutants) and against the loss of endogenous substances such as water. The SC consists of keratin-rich dead cells surrounded by crystalline lamellar lipid regions. The main lipid classes are ceramides (CERs), free fatty acids (FFAs), and cholesterol (CHOL). Tropospheric ozone (O3) is a potent oxidant compound that reacts instantly with biological molecules such as lipids and proteins. Although it has been reported that O3 induces biological responses at the cellular level, to the best of our knowledge, there is no information related to the damages O3 can cause at the level of the SC extracellular lipid matrix. The aim of our work was to investigate which SC lipid subclasses are prone to oxidation when exposed to O3 and how the changes in chemical structures affect the lipid organization in a stratum corneum substitute (SCS) membrane. Ultimately, the barrier properties of the SCS were examined. Our studies revealed that O3 induces chemical modifications of the unsaturated bonds in CERs and CHOL. The appearance of carbonyl groups at the headgroup level and the removal of the linoleate moiety of omega‑O‑acylceramides (CER EOS) impact the lamellar organization of the lipid assembly and to a lesser extent the lateral packing of the lipids. Unexpectedly, these changes improved the barrier function of the SCS. Show less
