Optical stimulation of living cells is an innovative and successful tool, having a significant impact on neuroscience. A recently synthesized amphiphilic azobenzene, named Ziapin2, has been developed exploiting a non-covalent approach for targeting the membrane and achieving cell photostimulation. Here, we investigate, using both experimental data and mathematical modelling, the effect of repetitive light stimulation on the cell response. We discuss some possible explanations for the drop in the stimulation efficiency in reiterated stimulation processes

Lanzani G.

Magni A.

Vurro V.

English

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DOI 10.1393/ncc/i2023-23154-2Communications: SIF Congress 2022IL NUOVO CIMENTO 46 C (2023) 154The effects of reiterated cell photo-stimulationwith an azobenzeneA. Magni(1)(2), V. Vurro(1) and G. Lanzani(1)(2)(1) Center for Nano Science and Technology, Istituto Italiano di Tecnologica - Milano, Italy(2) Dipartimento di Fisica, Politecnico di Milano - Milano, Italyreceived 30 January 2023Summary. — Optical stimulation of living cells is an innovative and success-ful tool, having a significant impact on neuroscience. A recently synthesized am-phiphilic azobenzene, named Ziapin2, has been developed exploiting a non-covalentapproach for targeting the membrane and achieving cell photostimulation. Here, weinvestigate, using both experimental data and mathematical modelling, the effect ofrepetitive light stimulation on the cell response. We discuss some possible explana-tions for the drop in the stimulation efficiency in reiterated stimulation processes.1. – IntroductionSince the 18th century, scientists have been studying and controlling living organismswith external stimuli. Today, it is well known that cells can be stimulated by exploitingmany strategies. One of these is targeting the plasma membrane [1]. The plasma mem-brane is, in fact, a barrier that maintains the electrical potential of the cell and controlsthe transport of chemicals in and out of the cell itself, regulating signalling, replicationand metabolism. Therefore, an efficient way to stimulate cells is to perturb the plasmamembrane. Usually, electrical techniques are adopted to study and stimulate cells. How-ever, despite high efficiency, the insertion of electrodes and wires greatly limits thisapproach in real-life applications [2]. For this reason, researchers have focused on othercategories of stimuli, with light being one of the most studied tool [3]. Even if most livingcells are naturally not sensitive to light, a number of techniques have been developed torender cells responsive to light [4]. The employment in clinical trials of Optogenetics islimited by its invasiveness and ethical implications [5]. Interestingly, cells are responsiveto a large variety of other types of stimuli and they can, therefore, be made indirectlylight-sensitive by converting the radiation into a thermal, electrical, chemical, or mechan-ical stimulus [6]. Our approach consists in using a phototransducer which converts lightinto mechanical stimulation of the cell membrane. Ziapin2 is an amphiphilic azobenzenemolecule that positions itself in the plasma membrane, with the two ionic-terminatedCreative Commons Attribution 4.0 License (https://creativecommons.org/licenses/by/4.0) 12 A. MAGNI et al.alkyl chains aligned with the heads of the polar phospholipids. Ziapin2 is able to switchbetween two configurations. Visible light absorption (470 nm) can trigger the isomeriza-tion from the trans to the cis isomer, while the reverse transition can either be driven bylight or occur spontaneously [7, 8]. In particular, Ziapin2 forms dimer when in its transconfiguration, causing membrane thinning and consequent increase in membrane capac-itance. When exposed to visible light, trans to cis isomerisation takes place, breakingthe dimer and rapidly leading to membrane relaxation and a reduction of the membranecapacitance. The photo-mechanical modulation of the membrane capacitance generatesa fast cell hyperpolarization response followed by depolarisation. This mechanism is alsoable to trigger action potential firing in excitable cells [9,10]. In this paper, we investigatethe cell membrane potential variations in response to a series of light pulses, linking theZiapin2 molecular ensemble dynamics with the cell conduct. We performed experimen-tal measurements through the patch-clamp technique and run mathematical simulation.In the latter, we connect the amount of Ziapin2 in each of the two isomers with themodulation of the membrane capacitance and therefore to the cell membrane potential.2. – Results and discussionThe effect of Ziapin2 photostimulation on HEK cells has already been reported [7,9, 11]. Briefly, HEK-293 cell were seeded on glass substrates and treated with Ziapin2(25μM for 7 min in physiological conditions). The electrophysiological response of thecells to light stimulation was evaluated through the current-clamp measurements in wholecell configuration. The membrane potential was recorded for 800ms and during thisperiod of time a light pulse of either 20ms or 200ms with power density of 68mW mm−2was shined on the samples. The 800ms stimulation protocol was repeated 20 timeswithout any delay between subsequent repetitions.In patch-clamp measurements, a common procedure consists in acquiring severalsweeps and averaging over them in order to increase the signal to noise ratio. Thisprocedure is well accepted, as usually the cell does not change during the measurementstage. Instead, using Ziapin2 as a phototransducer and working with light pulses at1.25Hz, we noticed that the signal changes between subsequent sweeps, with the am-plitude of the hyperpolarization signal reducing after a few sweeps. We observed that,during the first pulses, there is a decrease in the hyperpolarization peak which reaches aplateau in few pulses. Moreover, the longer the light pulses (20 or 200ms), the faster theplateau is reached, at around 50% of the initial value. Using 20ms light pulses, it takesaround 10–12 sweeps to reach this plateau, while using 200ms, 3–4 sweeps are enough(fig. 1). To gain further insights in this phenomena, we adopted the following modelto reproduce the modulation of the membrane potential observed in the cells. Passiveand active ion transporting mechanisms ensure an electrical potential difference acrossthe cell membrane, with the inside of the cell negative with respect to the outside. Themembrane potential is the result of an electrochemical equilibrium of two main factors:the asymmetric distribution of ions between the intracellular and extracellular space, andthe selective permeability to ions of the membrane. This condition is modelled by a par-allel between a capacitor and a resistor. The different ionic conductances can be groupedinto an effective membrane resistance Rm. The resting membrane potential, determinedby the Goldman-Huxley-Katz equation, is represented by a voltage source Vr. The in-sulating lipid bilayer, which separates two conducting media, behaves as a capacitor.Experimental values of the membrane capacitance are in the order of 1μF cm−2.Moreover, there is an accumulation of charges on the membrane capacitance even ifTHE EFFECTS OF REITERATED CELL PHOTO-STIMULATION WITH AN AZOBENZENE 3Fig. 1. – Ziapin2-mediated membrane potential photomodulation with 20ms (A) or 200ms (B)pulses. Relative hyperpolarization values with 20ms (C) or 200ms (D) as a function of thesweep. Error bars represent the SEM, computed over n > 15 cells.the transmembrane potential is kept at zero. This is related to surface charges of thetwo leaflets of the plasma membrane (the inner side is generally more negative than theouter one) and to the different ionic composition of the extracellular and intracellularcompartments, which produces a different distribution of the ions at the two sides of themembrane. This intrinsic potential can be modelled as a voltage generator Vs in serieswith the capacitance. Translating the exogenous stimulation of the cell into a changein one of the electrical parameters of the RC circuit allows reproducing the consequentmembrane potential modulations. In our case, we assume the main effect of the changein thickness of the plasma membrane to be the change of membrane capacitance Cm. Achange in capacitance [12,13] induces variations in the membrane potential according todVmdt = −(Vm−VrRmCm(t)+ Vm−VsCm(t)dCmdt).As a first approximation, we assume proportionality between membrane capacitanceand Ziapin2 fraction molecules in cis. The overall population is ntrans + ncis = 1where ncis and ntrans are the molecules in cis and trans state, respectively. Thepopulation dynamics is described by dncisdt = kTCIntrans − (kCT I + γ)ncis = kTCI −((kCT + kTC) I + γ)ncis.Where kTC (kCT ) is the trans → cis (cis → trans) isomerization rate, normalizedby light intensity I, and γ is the thermal relaxation rate from the cis to the trans isomer.In dark conditions (I = 0), the population dynamics reduces to dncisdt = −γncis.We hypothesized that there is a fraction of Ziapin2 molecules, for which the relax-ation to the trans isomer is somehow slowed down. Thus, a certain amount of popu-lation in the cis state is long living (with a lifetime of the order of few seconds) re-ducing the amount of molecule available for photoisomerization when a second lightpulse comes. There are several phenomena that could explain this. We know thatZiapin2 isomerization photophysics is strongly affected by the different plasma mem-brane domains [8] resulting in complex dynamics. We speculate that some Ziapin2molecules end up being “trapped” in membrane domains where the higher viscosityor specific molecular interaction hinders back-isomerization. Another possible conjec-ture is that, if two molecules in cis state are sufficiently close to each other, two apolarheads of Ziapin2 weakly interact, slowing down the thermal relaxation. According to ourhypothesis, we introduce two separate Ziapin2 populations that independently evolvewith two relaxation rates according to the previous equations. Being α the fractionof molecules belonging to the slow population, the membrane capacitance is given byCm(t) = Cm,0 +ΔC (α · nSLOW,cis(t) + (1− α)nFAST,cis(t)). Exploiting a custom-madeMatlab code that solves the set of differential equations described above, we managedto nicely reproduce the experimental data. We used the following parameters, which4 A. MAGNI et al.Fig. 2. – Computed membrane potential modulation as a consequence of membrane capacitancevariations for 20ms (A) and 200ms (B) at different sweeps. Computed maximum cell hyperpo-larization ((C), (D)) as a function of the number of sweeps for 20 and 200ms, respectively.have been retrieved from previous results [7] or estimated from the electrophysiologicalmeasurements: kCT,slow = 0.004mm2 ms−1 mW−1; kTC,slow = 0.01mm2 ms−1 mW−1;γslow = 0.04ms−1; kCT,fast = 0.05mm2 s−1 mW−1; kTC,fast = 0.4mm2 s−1 mW−1;γfast = 0.1 s−1; α = 0.7; Rm = 5GΩ; Cm,0 = 25pF; ΔCm = −1.8 pF; Vr = −25mV;Vs = 100mV. The plots in fig. 2 show the results of our simulations. The model repli-cates the characteristic features of the experimental data. In particular, both using 20msand 200ms light stimulus we obtain a significant reduction of the peak hyperpolarizationamplitude due to repetitive stimulation. After 10 and 2 light pulses we reach the plateauvalue, for the 20ms and 200ms stimulations, respectively. The nice agreement betweenexperimental and simulated data supports the presence of two different Ziapin2 moleculepopulations that recover the trans state with different dynamics.3. – ConclusionIn this work, we investigated the effect of a series of light pulses over the Ziapin2-mediated membrane potential modulation. We developed a custom Matlab code thatsimulates the molecule ensemble dynamics, the induced capacitance modulation and theeffects over the membrane potential dynamics. We highlighted a correlation between theZiapin2 back-isomerization and the membrane potential modulation. Based on our previ-ous experience, we ascribed this phenomenon to different Ziapin2 populations influencedby different membrane domains.REFERENCES[1] Manfredi G. et al., APL Mater., 9 (2021) 030901.[2] Balint R. et al., Tissue Eng. Part B Rev., 19 (2013) 48.[3] Hopkins J. et al., Adv. Mater. Technol., 4 (2019) 1800744.[4] Vurro V. et al., Appl. Phys. Lett., 120 (2022) 080502.[5] Deisseroth K., Nat. Methods, 8 (2011) 26.[6] –Derek V. et al. Front. Bioeng. Biotechnol., 8 (2020) 284.[7] Paternò G. M. et al., Adv. Sci., 7 (2020) 1903241.[8] Magni A. et al., Phys. Chem. Chem. Phys., 24 (2022) 8716.[9] Vurro V. et al., Front. Mater., 7 (2021) 631567.[10] Vurro V. et al., iScience, 26 (2023) 106121.[11] Di Francesco M. L. et al., Nat. Nanotechnol., 15 (2020) 296.[12] Pinto B. I. et al., Biophys. Rev., 14 (2022) 1.[13] Martino N. et al., Sci. Rep., 5 (2015) 1.

The effects of reiterated cell photo-stimulation with an azobenzene

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The effects of reiterated cell photo-stimulation with an azobenzene

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