Retinaldehyde Research Articles

Study of Phototoxic Properties of Retinal and Its Derivatives in a Photoreceptor Cell by the Method of Pulsed Photolysis

It is believed that one of the causes of photodamage to the retina is the accumulation of all-trans-retinal (ATR) in the visual cells. However, the ATR is formed during photolysis inside the disk of the photoreceptor cell and transferred to the cytoplasm of the cell, where it is converted with a high yield to retinol, a nonphototoxic compound. Inside the disk, where ATR can accumulate, it can form Schiff bases, which are also not phototoxic compounds, with the amino groups of proteins and lipids. In addition, the resulting free retinal (R) isomers bind to specific retinal-binding proteins that can shield them from oxygen. Thus, the question on the concentrations in which free ATR can accumulate inside a cell is ambiguous. In this study, it is proposed to evaluate the concentration of free ATR in a cell by the yield of the excited triplet state, since neither the Schiff bases of ATR nor retinol are transformed into an excited triplet state. It is shown that 70% of ATR form Schiff bases in the equilibrium state in the native cell. This significantly reduces the likelihood that ATR is the main inducer of photodamage. Moreover, it is shown that the quantum yield of the formation reaction of an excited triplet state of R when it is bound to interfotoreceptor proteins is significantly reduced.

The Orphan Nuclear Receptor TLX is a Receptor for Synthetic and Natural Retinoids

TLX is an orphan nuclear receptor that plays a role in neurogenesis as well in the pathogenesis of glioblastomas.TLX functions predominately as a transcriptional repressor, but no natural ligands or high affinity synthetic ligands have been identified. Here, we describe the identification retinoids as functional ligands for TLX. We identified potent synthetic retinoids that directly bind to TLX and either activate or inhibit its transcriptional repressor activity. Furthermore, we identified all-trans and 11-cis retinaldehyde, retinoids that play an essential role in the visual cycle, as the preferential natural retinoids that bind to and modulate the function of TLX. Molecular dynamics simulations followed by confirmation by mutational analysis revealed the basis of retinoid binding to TLX. Our data suggests that retinoids may play a physiological role in modulation of TLX activity and support the validity of TLX as a target for development of therapeutics to treat cognitive disorders and/or glioblastomas.

Gate-keeper of ion transport-a highly conserved helix-3 tryptophan in a channelrhodopsin chimera, C1C2/ChRWR.

Microbial rhodopsin is a large family of membrane proteins having seven transmembrane helices (TM1-7) with an all-trans retinal (ATR) chromophore that is covalently bound to Lys in the TM7. The Trp residue in the middle of TM3, which is homologous to W86 of bacteriorhodopsin (BR), is highly conserved among microbial rhodopsins with various light-driven functions. However, the significance of this Trp for the ion transport function of microbial rhodopsins has long remained unknown. Here, we replaced the W163 (BR W86 counterpart) of a channelrhodopsin (ChR), C1C2/ChRWR, which is a chimera between ChR1 and 2, with a smaller aromatic residue, Phe to verify its role in the ion transport. Under whole-cell patch clamp recordings from the ND7/23 cells that were transfected with the DNA plasmid coding human codon optimized C1C2/ChRWR (hWR) or its W163F mutant (hWR-W163F), the photocurrents were evoked by a pulsatile light at 475 nm. The ion-transporting activity of hWR was strongly altered by the W163F mutation in 3 points: (1) the H+ leak at positive membrane potential (Vm) and its light-adaptation, (2) the attenuation of cation channel activity and (3) the manifestation of outward H+ pump activity. All of these results strongly suggest that W163 has a role in stabilizing the structure involved in the gating-on and -off of the cation channel, the role of “gate keeper”. We can attribute the attenuation of cation channel activity to the incomplete gating-on and the H+ leak to the incomplete gating-off.

Diversity and Potential of Microbial Rhodopsins

One of the major topics in biophysics and physicobiology is to understand and utilize biological functions using various techniques. Rhodopsin is a photoreactive membrane-embedded protein that uses a retinal pigment (vitamin-A aldehyde) as a chromophore. By taking advantages of its photoreactivity, structure-function relationship of rhodopsins has been widely investigated with a variety of methods. Of note, recent rhodopsin research provided unexpected information regarding (i) wide distribution in various organisms, (ii) rich functional diversity and (iii) great potential for optogenetics. Here we review these three topics with future directions.

Retinal supplementation augments optogenetic stimulation efficacy in vivo

Objective. Over the last two decades, optical control of neuronal activity in the central nervous system has seen rapid development, demonstrating the utility of optogenetics as both an experimental and therapeutic tool. Conversely, applications of optogenetics in the peripheral nervous system have been relatively constrained by the challenges of temporally variable opsin expression, light penetration and immune attack of non-native opsins. Whilst opsin expression can be increased significantly through high-concentration viral induction, subsequent attack by the immune system causes temporal decay and high variability in electrophysiological response. Approach. In this study, we present a method to circumvent the aforementioned challenges by locally supplementing all-trans-retinal (ATR) (via a slow release pellet) to increase tissue photosensitivity in transgenic mice expressing channelrhodopsin 2 (ChR2) in nerves. Main results. In mice supplemented with ATR, we demonstrate enhanced electrophysiological activation and fatigue tolerance in response to optical stimulation for six weeks. Significance. Local supplementation of ATR enables improved optogenetic stimulation efficacy in peripheral nerves. This method enables greater exploration of neurophysiology and development of clinically-viable optogenetic treatments in the peripheral nervous system.

Rhodopsin-based voltage imaging tools for use in muscles and neurons of Caenorhabditis elegans

Genetically encoded voltage indicators (GEVIs) based on microbial rhodopsins utilize the voltage-sensitive fluorescence of all-trans retinal (ATR), while in electrochromic FRET (eFRET) sensors, donor fluorescence drops when the rhodopsin acts as depolarization-sensitive acceptor. In recent years, such tools have become widely used in mammalian cells but are less commonly used in invertebrate systems, mostly due to low fluorescence yields. We systematically assessed Arch(D95N), Archon, QuasAr, and the eFRET sensors MacQ-mCitrine and QuasAr-mOrange, in the nematode Caenorhabditis elegans ATR-bearing rhodopsins reported on voltage changes in body wall muscles (BWMs), in the pharynx, the feeding organ [where Arch(D95N) showed approximately 128% ΔF/F increase per 100 mV], and in neurons, integrating circuit activity. ATR fluorescence is very dim, yet, using the retinal analog dimethylaminoretinal, it was boosted 250-fold. eFRET sensors provided sensitivities of 45 to 78% ΔF/F per 100 mV, induced by BWM action potentials, and in pharyngeal muscle, measured in simultaneous optical and sharp electrode recordings, MacQ-mCitrine showed approximately 20% ΔF/F per 100 mV. All sensors reported differences in muscle depolarization induced by a voltage-gated Ca2+-channel mutant. Optogenetically evoked de- or hyperpolarization of motor neurons increased or eliminated action potential activity and caused a rise or drop in BWM sensor fluorescence. Finally, we analyzed voltage dynamics across the entire pharynx, showing uniform depolarization but compartmentalized repolarization of anterior and posterior parts. Our work establishes all-optical, noninvasive electrophysiology in live, intact C. elegans.

Mechanisms of Transport and Delivery of Vitamin A and Carotenoids to the Retinal Pigment Epithelium.

Vision depends on the delivery of vitamin A (retinol) to the retina. Retinol in blood is bound to retinol-binding protein (RBP). Retinal pigment epithelia (RPE) cells express the RBP receptor, STRA6, that facilitates uptake of retinol. The retinol is then converted to retinyl esters by the enzyme lecithin:retinol acyltransferase. The esters are the substrate for RPE65, an enzyme that produces 11-cis retinol, which is converted to 11-cis retinaldehyde for transport to the photoreceptors to form rhodopsin. The dietary xanthophylls, lutein (LUT) and zeaxanthin (ZEA), accumulate in the macula of the eye, providing protection against age-related macular degeneration. To reach the macula, carotenoids cross the RPE. In blood, xanthophylls and β-carotene mostly associate with high-density lipoprotein (HDL) and low-density lipoprotein (LDL), respectively. Studies using a human RPE cell model evaluate the kinetics of cell uptake when carotenoids are delivered in LDL or HDL. For LUT and β-carotene, LDL delivery result in the highest rate of uptake. HDL is more effective in delivering ZEA (and meso-ZEA). This selective HDL-mediated uptake of ZEA, via a scavenger receptor and LDL-mediated uptake of LUT and β-carotene provides a mechanism for the selective accumulation of ZEA > LUT and xanthophylls over β-carotene in the macula.

Non-cryogenic structure of a chloride pump provides crucial clues to temperature-dependent channel transport efficiency

Non-cryogenic protein structures determined at ambient temperature may disclose significant information about protein activity. Chloride-pumping rhodopsin (ClR) exhibits a trend to hyperactivity induced by a change in the photoreaction rate because of a gradual decrease in temperature. Here, to track the structural changes that explain the differences in CIR activity resulting from these temperature changes, we used serial femtosecond crystallography (SFX) with an X-ray free electron laser (XFEL) to determine the non-cryogenic structure of ClR at a resolution of 1.85 Å, and compared this structure with a cryogenic ClR structure obtained with synchrotron X-ray crystallography. The XFEL-derived ClR structure revealed that the all-trans retinal (ATR) region and positions of two coordinated chloride ions slightly differed from those of the synchrotron-derived structure. Moreover, the XFEL structure enabled identification of one additional water molecule forming a hydrogen bond network with a chloride ion. Analysis of the channel cavity and a difference distance matrix plot (DDMP) clearly revealed additional structural differences. B-factor information obtained from the non-cryogenic structure supported a motility change on the residual main and side chains as well as of chloride and water molecules because of temperature effects. Our results indicate that non-cryogenic structures and time-resolved XFEL experiments could contribute to a better understanding of the chloride-pumping mechanism of ClR and other ion pumps.

Excited State Vibrational Spectra of All- trans Retinal Derivatives in Solution Revealed By Pump-DFWM Experiments.

The ultrafast structural changes during the photoinduced isomerization of the retinal-protonated Schiff base (RPSB) is still a poorly understood aspect in the retinal's photochemistry. In this work, we apply pump-degenerate four-wave mixing (pump-DFWM) to all- trans retinal (ATR) and retinal Schiff bases (RSB) to resolve coherent high- and low-frequency vibrational signatures from excited electronic states. We show that the vibrational spectra of excited singlet states in these samples exhibit pronounced differences compared to the relaxed ground state. Pump-DFWM results indicate three major features for ATR and RSB. (i) Excited state vibrational spectra of ATR and RSB consist predominately of low-frequency modes in the energetic range 100-500 cm-1. (ii) Excited state vibrational spectra show distinct differences for excitation in specific regions of electronic transitions of excited state absorption and emission. (iii) Low-frequency modes in ATR and RSB are inducible during the entire lifetime of the excited electronic states. This latter effect points to a transient molecular structure that, following initial relaxation between different excited electronic states, does not change anymore over the lifetime of the finally populated excited electronic state.

Blue light excited retinal intercepts cellular signaling

Photoreceptor chromophore, 11-cis retinal (11CR) and the photoproduct, all-trans retinal (ATR), are present in the retina at higher concentrations and interact with the visual cells. Non-visual cells in the body are also exposed to retinal that enters the circulation. Although the cornea and the lens of the eye are transparent to the blue light region where retinal can absorb and undergo excitation, the reported phototoxicity in the eye has been assigned to lipophilic non-degradable materials known as lipofuscins, which also includes retinal condensation products. The possibility of blue light excited retinal interacting with cells; intercepting signaling in the presence or absence of light has not been explored. Using live cell imaging and optogenetic signaling control, we uncovered that blue light-excited ATR and 11CR irreversibly change/distort plasma membrane (PM) bound phospholipid; phosphatidylinositol 4,5 bisphosphate (PIP2) and disrupt its function. This distortion in PIP2 was independent of visual or non-visual G-protein coupled receptor activation. The change in PIP2 was followed by an increase in the cytosolic calcium, excessive cell shape change, and cell death. Blue light alone or retinal alone did not perturb PIP2 or elicit cytosolic calcium increase. Our data also suggest that photoexcited retinal-induced PIP2 distortion and subsequent oxidative damage incur in the core of the PM. These findings suggest that retinal exerts light sensitivity to both photoreceptor and non-photoreceptor cells, and intercepts crucial signaling events, altering the cellular fate.

Enhanced overexpression, purification of a channelrhodopsin and a fluorescent flux assay for its functional characterization

Channelrhodopsins (ChRs) are a group of membrane proteins that allow cation flux across the cellular membrane when stimulated by light. They have been emerged as important tools in optogenetics where light is used to trigger a change in the membrane potential of live cells which induces downstream physiological cascades. There is also increased interest in their applications for generating light-responsive biomaterials. Here we have used a two-step screening protocol to develop a Pichia pastoris strain that produces superior yields of an enhance variant of CaChR2 (from Chlamydomonas reinhardtii), called ChIEF. We have also studied the effect of the co-factor, namely all-trans retinal (ATR), on the recombinant overexpression, folding, and function of the protein. We found that both ChIEF-mCitrine and CaChR2 can be overexpressed and properly trafficked to the plasma membrane in yeast regardless of the presence of the ATR. The purified protein was reconstituted into large unilamellar lipid vesicle using the detergent-assisted method. Using 9-amino-6-chloro-2-methoxyacridine (ACMA) as the fluorescent proton indicator, we have developed a flux assay to verify the light-activated proton flux in the ChIEF-mCitrine vesicles. Hence such vesicles are effectively light-responsive nano-compartments. The results presented in this work lays foundations for creating bio-mimetic materials with a light-responsive function using channelrhodopsins.

A non-retinoid antagonist of retinol-binding protein 4 rescues phenotype in a model of Stargardt disease without inhibiting the visual cycle.

A primary pathological defect in the heritable eye disorder Stargardt disease is excessive accumulation of cytotoxic lipofuscin bisretinoids in the retina. Age-dependent accumulation of lipofuscin in the retinal pigment epithelium (RPE) matches the age-dependent increase in the incidence of the atrophic (dry) form of age-related macular degeneration (AMD) and therefore may be one of several pathogenic factors contributing to AMD progression. Lipofuscin bisretinoid synthesis in the retina depends on the influx of serum retinol from the circulation into the RPE. Formation of the tertiary retinol-binding protein 4 (RBP4)-transthyretin-retinol complex in the serum is required for this influx. Herein, we report the pharmacological effects of the non-retinoid RBP4 antagonist, BPN-14136. BPN-14136 dosing in the Abca4-/- mouse model of increased lipofuscinogenesis significantly reduced serum RBP4 levels and inhibited bisretinoid synthesis, and this inhibition correlated with a partial reduction in visual cycle retinoids such as retinaldehydes serving as bisretinoid precursors. BPN-14136 administration at doses inducing maximal serum RBP4 reduction did not produce changes in the rate of the visual cycle, consistent with minimal changes in dark adaptation. Abca4-/- mice exhibited dysregulation of the complement system in the retina, and BPN-14136 administration normalized the retinal levels of proinflammatory complement cascade components such as complement factors D and H, C-reactive protein, and C3. We conclude that BPN-14136 has several beneficial characteristics, combining inhibition of bisretinoid synthesis and reduction in retinaldehydes with normalization of the retinal complement system. BPN-14136, or a similar compound, may be a promising drug candidate to manage Stargardt disease and dry AMD.

Production of a Light-Gated Proton Channel by Replacing the Retinal Chromophore with Its Synthetic Vinylene Derivative.

Rhodopsin is widely distributed in organisms as a membrane-embedded photoreceptor protein, consisting of the apoprotein opsin and vitamin-A aldehyde retinal, A1-retinal and A2-retinal being the natural chromophores. Modifications of opsin (e.g., by mutations) have provided insight into the molecular mechanism of the light-induced functions of rhodopsins as well as providing tools in chemical biology to control cellular activity by light. Instead of the apoprotein opsin, in this study, we focused on the retinal chromophore and synthesized three vinylene derivatives of A2-retinal. One of them, C(14)-vinylene A2-retinal (14V-A2), was successfully incorporated into the opsin of a light-driven proton pump archaerhodopsin-3 (AR3). Electrophysiological experiments revealed that the opsin of AR3 (archaeopsin3, AO3) with 14V-A2 functions as a light-gated proton channel. The engineered proton channel showed characteristic photochemical properties, which are significantly different from those of AR3. Thus, we successfully produced a proton channel by replacing the chromophore of AR3.

Efficacy and safety of retinaldehyde 0.1% and 0.05% creams used to treat photoaged skin: A randomized double-blind controlled trial.

Although topical retinoic acid effectively restores photoaged skin, the associated irritation limits the utility of the material. Retinaldehyde (RAL) is the natural precursor of retinoic acid and can also be used to treat photoaged skin; the safety profile is good. To evaluate the efficacy and safety of new anti-aging creams containing RAL at 0.1% and 0.05% used to treat photoaged skin. We enrolled 40 female Korean volunteers who applied RAL 0.1% or RAL 0.05% creams twice daily for 3 months. Wrinkles on, and the textures of, both crow's feet were quantitatively assessed using the Antera 3D® system. Transepidermal water loss (TEWL), skin hydration, the melanin index, and skin brightness were also evaluated. Overall improvement was assessed using a five-point scale by both the patients and the dermatologists. The 3-month application improved overall photoaging in both RAL 0.1% (95%) and RAL 0.05% groups (95%). Both RAL 0.1% and RAL 0.05% afforded significant textural improvements (13.7% and 12.6%, respectively), reduced the TEWL (14.5%, 17.9%), and increased hydration (10.2%, 6.0%); however, no statistical differences were observed between two groups. Only RAL 0.1% significantly improved the melanin index (by 6.5%). Both RAL 0.1% and RAL 0.05% creams were well tolerated and improved skin hydration and texture. However, only RAL 0.1% cream improved the melanin index.

Blue light‐excited retinal delocalizes plasma membrane bound PIP2 and triggers stress responses challenging the cellular integrity

The photoreceptor chromophore, retinal, an aldehyde derivative of vitamin A, harvests light to generate visual perception. Upon photon absorption, the receptor bound 11‐cis‐retinylidene (11CR) isomerizes to all trans retinal (ATR), changing the electrostatic environment in the chromophore‐binding site, subsequently transforming the inactive receptor to a momentary active state. The subsequent release of ATR allows the receptor to be relaxed to the basal state, enables a new 11CR binding and continuous photon reception. Due to the defects in retinal clearance mechanisms, higher concentrations of 11CR and ATR can be found in the retina, and a fraction of excess retinal can enter the circulation, exposing the rest of the body to retinal as well, leading to pathophysiological effects. Nevertheless, visible light induced, photoreceptor independent signaling activities of retinals and mechanisms of retinal phototoxicity are less explored. Here, we show that, retinal, in the presence of blue light, irreversibly remove the plasma membrane (PM) bound phosphatidylinositol 4,5 bisphosphate (PIP2) in photoreceptor and non‐photoreceptor cells. Interestingly, addition of retinals alone without blue light exposure as well as exposing cells to only blue light without retinal, did not show any detectable PIP2 dislodging from PM. Additionally, even in the presence of retinal, the other wavelengths of lights failed to show any effect on PM bound PIP2. Moreover, compounds that are structurally similar to retinal such as retinol and retinoic acid did not show any effect on PIP2 in the presence of blue light. Data further show that, this PIP2 removal from the PM is not due to regular enzymatic hydrolysis of PIP2, but as a result of lipid peroxidation induced by the generation of singlet oxygen and reactive oxygen species. The resultant PIP2 dislodging is followed by cellular stress responses and cell death. Clinically used photodynamic therapeutic agents (photosensitizers), also showed a unique PIP2 dislodging effect from PM in the presence of blue light as well as in other wavelengths of light, similar to blue light excited retinal. This further confirms retinal's ability to harvest light to induce photosensitization that leads to lipid peroxidation which suggests its future potential in photodynamic therapeutics in cancer as well as identification of new molecular targets for phototoxicity. Overall, here we show that, retinal exhibit light sensitivity in non‐photoreceptor cells and trigger crucial signaling pathways altering the cellular fate.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

An exploration of the algal protein channelrhodospin and the origins of optogenetics

Channelrhodopsin (ChR) is a light‐gated cation channel protein originally discovered in the algae Chlamydomonas reinhardtii. Composed of seven transmembrane helices (TM1‐7) covalently bound to all‐trans retinal (ATR), ChR permits cations to pass through cellular membranes when excited by light, functioning in algal photoreception. During excitation, ATR undergoes photoisomerization into the 13‐cis retinal form, triggering conformational changes that transfer protons between the protonated retinal Schiff base and its residue counterions. These protonation events control gating of the electronegative pore formed by TM1, 2, 3, and 7, allowing selective cation passage regulated by TM2 while excited, before thermally cycling back to ground state. The Blue Valley CAPS 2017–18 MSOE Center for BioMolecular Modeling SMART Team modeled channelrhodopsin chimaera C1C2 using JMol and 3D printing technology with the goal of exploring this protein's unique properties as utilized in the revolutionary field of optogenetics. Optogenetics uses light and genetic engineering to systematically target and manipulate cellular systems in living organisms, enabling unprecedented research and experimentation in disciplines such as neuroscience. Through Cre‐Lox site‐specific recombinase and adeno‐associated viral technology, researchers can express ChR in specific neurons, allowing light‐based experimental control of neural pathways and action potentials. Studies done with these techniques have shed new light onto neurological and psychiatric disorders, and could potentially hold the key to new effective treatment options.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Synthesis of isotopically labeled all-trans retinals for DNP-enhanced solid-state NMR studies of retinylidene proteins.

Three all-trans retinals containing multiple 13 C labels have been synthesized to enable dynamic nuclear polarization enhanced solid-state magic angle spinning NMR studies of novel microbial retinylidene membrane proteins including proteorhodpsin and channelrhodopsin. The synthetic approaches allowed specific introduction of 13 C labels in ring substituents and at different positions in the polyene chain to probe structural features such as ring orientation and interaction of the chromophore with the protein in the ground state and in photointermediates. [10-18-13 C9 ]-All-trans-retinal (1b), [12,15-13 C2 ]-all-trans-retinal (1c), and [14,15-13 C2 ]-all-trans-retinal (1d) were synthesized in in 12, 8, and 7 linear steps from ethyl 2-oxocyclohexanecarboxylate (5) or β-ionone (4), respectively.

Rhodopsin optogenetic toolbox v2.0 for light-sensitive excitation and inhibition in Caenorhabditis elegans.

In optogenetics, rhodopsins were established as light-driven tools to manipulate neuronal activity. However, during long-term photostimulation using channelrhodopsin (ChR), desensitization can reduce effects. Furthermore, requirement for continuous presence of the chromophore all-trans retinal (ATR) in model systems lacking sufficient endogenous concentrations limits its applicability. We tested known, and engineered and characterized new variants of de- and hyperpolarizing rhodopsins in Caenorhabditis elegans. ChR2 variants combined previously described point mutations that may synergize to enable prolonged stimulation. Following brief light pulses ChR2(C128S;H134R) induced muscle activation for minutes or even for hours (‘Quint’: ChR2(C128S;L132C;H134R;D156A;T159C)), thus featuring longer open state lifetime than previously described variants. Furthermore, stability after ATR removal was increased compared to the step-function opsin ChR2(C128S). The double mutants C128S;H134R and H134R;D156C enabled increased effects during repetitive stimulation. We also tested new hyperpolarizers (ACR1, ACR2, ACR1(C102A), ZipACR). Particularly ACR1 and ACR2 showed strong effects in behavioral assays and very large currents with fast kinetics. In sum, we introduce highly light-sensitive optogenetic tools, bypassing previous shortcomings, and thus constituting new tools that feature high effectiveness and fast kinetics, allowing better repetitive stimulation or investigating prolonged neuronal activity states in C. elegans and, possibly, other systems.

Dermatoporosis: a further step to recognition.

Skin ageing has long been considered only as a cosmetic issue. However, with increasing lifespan, we experience a functional dimension of skin ageing which extends beyond cosmetics and appearance. Ten years ago, we proposed the neologism dermatoporosis to cover different characteristics of a chronic cutaneous insufficiency/fragility syndrome, to understand underlying molecular mechanisms and to develop preventative or therapeutic strategies.

Coenzyme Q10 and retinaldehyde co-loaded nanostructured lipid carriers for efficacy evaluation in wrinkles

This study depicts coenzyme Q10 (CoQ10) and retinaldehyde (RAL) co-loaded nanostructured lipid carriers (NLCs); having activity on different targets of photoageing, which can overcome deficits of conventional topical dosage forms. The developed NLCs were characterised for particle size, polydispersity index and percent entrapment efficiency (%EE), followed by their incorporation into Carbopol® 934 P-NF gel. In vitro cellular uptake and cytotoxicity assay was performed to evaluate NLCs and in vivo study on ultraviolet- (UV) induced wrinkle model to determine efficacy of NLCs. The developed stable, homogenous and spherical NLCs with size range of 200–230 nm and more than 80 %EE, showed prolonged, biphasic in vitro release pattern for CoQ10 and RAL. Ex vivo study portrayed negligible permeation through skin but appreciable penetration and distribution in skin layers. This has shown good uptake of both drugs with least cytotoxicity in cell culture studies. In vivo irritation study on Sprague Dawley (SD) rats and pharmacodynamic study on female Swiss albino mice proved it less irritant and efficacious. The developed NLCs thus hold promise in the efficient management of wrinkle and their reduction as indicated by the data obtained.