Spatiotemporal control of molecular delivery in the brain using photoactivable polymersomes

Spatiotemporal control of molecular delivery in the brain using photoactivable polymersomes | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Spatiotemporal control of molecular delivery in the brain using photoactivable polymersomes Clémentine bosch-bouju, Camille Ruffier, Emmanuel Ibarboure, Benjamin Chauvineau, and 14 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9232875/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Neurological diseases remain a leading cause of disability and mortality, in part because systemically administered therapies poorly access diseased brain regions and lack spatiotemporal control. While innovative nanotechnologies offer stable and versatile carriers for drug delivery, they do not inherently enable localized, on-demand release. In parallel, optical neurotechnologies provide precise control of brain activity but cannot deliver bioactive molecules. Here, we bridge these approaches by developing photoactivatable vesicles based on polymeric amphiphiles (polymersomes) that enable light-triggered, spatially confined release of encapsulated compounds in brain tissue. We assessed their safety in primary cell cultures and in vivo. Through the photorelease of CNQX, a competitive AMPA/kainate receptor antagonist, we demonstrated the stability and precise spatiotemporal control of molecular delivery. This work establishes a platform for optically-guided chemical neuromodulation. Beyond applications in basic and preclinical neuroscience, this strategy opens new avenues for targeted therapies in localized brain disorders, including glioblastoma. Biological sciences/Neuroscience Biological sciences/Chemical biology polymersomes brain photostimulation drug delivery Figures Figure 1 Figure 2 Figure 3 Figure 4 INTRODUCTION Beyond electrical and optical stimulation, the use of chemically-mediated triggering to modulate brain activity represents a fundamentally distinct and largely underexplored strategy. Neurotransmitters, neuromodulators, and pharmacological agents act through endogenous molecular pathways, offering the potential to regulate neural circuits with high biochemical specificity and sustained effects that are difficult to achieve with purely physical stimulation modalities 1,2 . However, precise spatiotemporal control over the delivery of such molecules within intact brain tissue remains a major challenge, owing to rapid diffusion, systemic side effects, and the lack of minimally invasive release technologies. Developing versatile approaches that enable on-demand, localized chemical neuromodulation could therefore open new avenues for both fundamental neuroscience and targeted therapeutic intervention. Many brain diseases remain inadequately treated because systemically administered therapies reach pathological regions only inefficiently and lack temporal control over their action 3 . As a result, access to diseased brain tissue remains a central bottleneck in the development of effective neurotherapeutics 4 , in spite of recent advances demonstrating promising outcomes 5,6 . Although drug delivery to the brain is severely constrained by the blood-brain barrier and the structural heterogeneity of neural tissue, recent advances in minimally invasive, locally targeted engineering approaches have created new opportunities for treating localized brain disorder 7 . As such, intracranial pharmacotherapy is increasingly used to target specific brain areas more precisely. In the case of focal epilepsy, anti-epileptic drugs act by reducing the global excitability of brain networks. However, it is required only in the diseased brain area, and it can be deleterious to other brain networks. For this reason, intracranial pharmacology is a therapeutic strategy already used in humans for some diseases, including focal epilepsies and glioblastoma 8,9 . Nevertheless, intracranial pharmacotherapy also has limitations, notably that drug delivery via this approach remains passive and thus is not controllable over time or in terms of dose. To improve drug delivery, encapsulation strategies based on synthetic carriers, such as polymersomes that are self-assembled vesicles from biocompatible block copolymers, have been extensively developed. Polymersomes are of particular biomedical interest owing to their high stability, chemical versatility, and ability to encapsulate and efficiently transport multiple therapeutic agents with high efficiency 10,11 . Stimuli-responsive release mechanisms for polymersomes have been developed, such as pH, temperature, or light, to better control the release of bioactive molecules 12–14 . For brain applications, stimuli-responsive polymersomes activated by light represent a particularly relevant approach for brain interventions. Indeed, neuroscience research has undergone major technological advances with the emergence of optical methods that enable in vivo recording and manipulation of neural activity, including optogenetics, fiber photometry, and two-photon photostimulation 15 . These light-based technologies are increasingly being developed to control brain circuits for clinical use 16 . However, they are primarily designed to modulate neuronal networks rather than to achieve temporally controlled delivery of therapeutic agents. Here, we leverage recent advances in optical neurotechnology to develop an innovative therapeutic strategy based on photoactivatable polymersomes 17,18 , enabling light-triggered, localized delivery of encapsulated molecules within the brain with precise control over timing, spatial distribution, and dosage. As such, we demonstrated the photorelease of CNQX, a competitive AMPA/kainate receptor antagonist, in brain tissue, in a temporally- and spatially-controlled manner. This approach establishes new experimental paradigms for preclinical research and expands the therapeutic toolkit for treating localized brain disorders. RESULTS 1- Design of photoactivable and biocompatible polymersomes Based on initial work 17 , we formulated stable and photocleavable polymersomes using an emulsion-centrifugation method ( Supplementary Fig. 1a-b ). Briefly, sucrose-containing water droplets generated by emulsion and stabilized by a copolymer in toluene were deposited onto a glucose solution-toluene interface containing the same copolymer. Upon centrifugation, the droplets crossed the interface to form polymersomes in the glucose phase, enabling quantitative encapsulation of the molecules of interest dissolved in the sucrose phase. Here, the formulation of photoactivable polymersomes was optimised to become fully compatible with brain applications: polymersomes were obtained by self-assembly of the amphiphilic block copolymer, poly(butadiene)- b -poly(ethylene oxide) (PBut n - b -PEG p ), loaded with calcein (10 mM) in a medium with an osmolarity of 300 mOsm ( Fig. 1a ). Counting polymersomes with a counting grid under a fluorescent microscope ( Supplementary Fig. 1c ) revealed a concentration of 267 ± 6.7 polymersomes / µl ( Fig. 1b ). Furthermore, polymersomes have a median diameter of 8.27 ± 0.14 µm (n = 1265 polymersomes for 6 batches) with a polydispersity of 0.36, indicating a rather homogeneous size distribution. (Fig. 1c) . Calcein is a water-soluble dye that undergoes photolysis within polymersomes upon illumination at 488 nm, a wavelength routinely used in neuroscience, thereby increasing the internal osmolarity. The resulting rapid rise in osmotic pressure generates membrane tension, leading to rapid, irreversible rupture, observed as an explosive disruption within seconds of irradiation ( Figure 1d ). As shown in Figure 1d-e , illumination of polymersomes loaded with 10 mM calcein led to the rapid rupture of the polymersome membrane and release of their contents ( Supplemental videos 1, 2, 3 ). Distribution of rupture times showed a mean rupture time of 6.39 ± 0.9 s (n = 41 polymersomes for 6 batches) under these irradiation conditions ( Fig. 1e, Supplementary Fig. 1d ). Furthermore, no morphological changes of polymersomes were observed in calcein-free polymersomes upon illumination at 488 nm, confirming the specificity of light-induced membrane rupture. Finally, when several regions of interest (ROIs) were defined, only the selected polymersomes ruptured upon illumination ( Supplementary Fig. 1e ). This approach thus enables rapid and localised release of the polymersome content, both in time and space. 2- Biosafety of photoactivable polymersomes for brain cells and brain tissue After confirming that the emulsion-centrifugation process was an appropriate method for obtaining a high yield of photoactivable polymersomes with rapid, localised release of their contents, we tested their biosafety. Biosafety of both polymersomes and photorelease process were evaluated in primary cell cultures from mouse cortices and in mouse brains. First, the effect of light stimulation alone was tested, as previous studies reported deleterious effect of 470 nm light on neuronal communication 19 . In our conditions, illumination of primary neuronal cell cultures with 470 nm light delivered through an optical fiber (Ø 400 µm, NA 0.5, 20 s) did not alter cell viability ( Fig. 2a ). Next, we evaluated the impact of calcein-loaded polymersome incubation with increasing concentrations (600 to 9000 polymersomes/well) on primary neuronal cell cultures. We found no deleterious effect on cell viability ( Fig. 2b ). Finally, we combined polymersome incubation with light illumination, at power levels that are sufficient to induce rapid photorelease (8 % of maximal power level, see Supplementary Table 2 for correspondence with light intensities) and similarly, no cytotoxic effect was observed ( Fig. 2b ). As a positive control, treatment of cells with DMSO 1% v/v and 5% v/v induces moderate and strong cell mortality, respectively ( Fig. 2b ). Finally, the biosafety of polymersomes was validated in vivo , by injecting empty polymersomes in mouse brains and assessing glial reactivity through immunostaining of IBA-1 (for microglia) and GFAP (for astrocytes) proteins at 48 h and 3 weeks after injection ( Fig. 3 ). At 48 h after injection, the injected hemisphere displayed a slight but significant increased signal for IBA1 and GFAP ( Fig. 3a,b ). However, this increase was not different between mice injected with polymersomes and those injected with vehicle solution without polymersomes (medium). This suggests that polymersomes did not induce any glial reaction beyond the stereotaxic injection itself. At 3 weeks after injection, quantification revealed no difference in IBA1 signal between the injected and non-injected hemispheres for both polymersome and medium groups ( Fig. 3a,c ). By contrast, the GFAP signal remained significantly higher in the injected hemisphere than in the non-injected one, but did not differ between the medium and polymersomes groups ( Fig. 3a,c ). This indicates that polymersomes themselves have no significant short- or long-term impact on gliosis. Altogether, these findings demonstrate that both polymersomes and calcein-mediated photorelease are well tolerated by brain cells and brain tissue. 3- Photostimulation of polymersomes in brain slices To demonstrate the efficacy of photorelease in brain tissue and quantify its ability to modulate neural processes, we developed an experimental paradigm based on measuring electrical synaptic transmission between connected neurons. Electrophysiological recordings were performed in mouse brain slices containing the synaptic connections between the CA3 and CA1 regions of the hippocampus, a key brain region involved in memory processes. Communication between these two structures relies on presynaptic glutamate release, which activates postsynaptic AMPA receptors and thereby enables the propagation of electrical signals from one neuron to the next. Therefore, blocking AMPA receptors with a competitive AMPA receptor antagonist, such as the well-established CNQX (6-cyano-7-nitroquinoxaline-2,3-dione), inhibits the electrical communication between the CA3 and CA1 regions, which can be recorded with field recordings ( Fig. 4a ). For this reason, we chose to encapsulate CNQX (100 µM) in calcein-loaded polymersomes to quantify the effect of photorelease from polymersomes on neuronal activity. We first verified that encapsulation of CNQX did not alter the photostimulation properties of polymersomes ( Supplementary Fig. 2 ). The encapsulation of CNQX slightly, but significantly, reduced the diameter of the polymersomes (7.62 ± 0.2 µm with CNQX (n = 386 polymersomes for 7 batches) vs. 8.27 ± 0.1 µm without CNQX (n = 1265 polymersomes for 6 batches), unpaired Mann-Whitney test, p < 0.0001) ( Supplementary Fig. 2a-c ). Moreover, the presence of CNQX in polymersomes did not change the rupture delay of polymersomes upon photostimulation (8.12 ± 0.9 s vs. 6.39 ± 0.9 s without CNQX, (n = 52 and polymersomes for 3 and 6 batches, respectively, unpaired Mann-Whitney test, p = 0.1192), ( Supplementary Fig. 2d ). This confirms that photostimulation was not affected by the presence of CNQX within the polymersomes and suggests that polymersomes represent a versatile platform for the encapsulation of bioactive molecules. Then, we used calcein/CNQX-loaded polymersomes to perform the photorelease of CNQX on brain slices ( Fig. 4a ). Electrical stimulation of the CA3 region induces synaptic evoked responses in CA1 region that were recorded extracellularly as field excitatory postsynaptic potentials (fEPSP) ( Fig. 4a, Supplementary Fig. 3a ). As for cell culture, the effect of light alone on fEPSPs with increasing light intensities was first quantified ( Supplementary Fig.3b ). The power levels used were similar to those used for cell culture illumination ( Supplementary Tables 1 and 2 for conversion). Under 12 % of light power, no effect was observed on fEPSP response ( Supplementary Fig. 3b-c ). However, we observed a significant inhibition of fEPSP response for light powers over 14 % with a sigmoid effect ( Supplementary Fig.3b ). This is in accordance with previous studies showing that light itself can modify neuronal excitability 19 . Given these results, we then used photostimulation at no more than 12% of maximal light power. Calcein/CNQX-loaded polymersomes were applied to the brain slice near the recording site without altering the fEPSP response ( Supplementary Fig. 3a,d ). This is a supplementary confirmation of the innocuity of polymersomes for brain tissue. Then, light at 8 % was delivered for 20 s. Shortly after, significant reduction of fEPSP was observed ( Fig. 4b, Supplementary Fig. 3e ). A similar experiment was conducted with illumination at 12 % of the maximal power level. A stronger effect was produced ( Fig. 4b ). Of note, light stimulation applied to polymersomes containing calcein alone induced only an 5.5 % reduction of fEPSP response ( Supplementary Fig. 3f ). These control experiments confirmed that the reduction in fEPSPs after polymersome photostimulation was specific to CNQX photorelease. Moreover, these results demonstrate that CNQX remains stable and bioactive following encapsulation and photorelease, further supporting the versatility of photoactivable polymersomes for drug delivery. Spatial and temporal precision of CNQX delivery To further define the spatiotemporal range of action of photoactivatable polymersomes in brain applications, the precision of CNQX photorelease was investigated in acute brain slices. First, we compared photostimulation to whole bath application of CNQX ( Supplementary Fig. 4 ). On one side, we confirmed that bath application of CNQX at 10 µM almost annihilates fEPSP response (76.5 ± 8.2% of inhibition of fEPSP baseline) while 0.5 µM of CNQX in the bath induced a minimal inhibition of fEPSP (7.61 ± 4.40 % of inhibition of fEPSP baseline) ( Supplementary Fig. 4a,b ). On the other hand, photostimulation of polymersomes containing CNQX at 100 µM induced on average a 12 % inhibition of fEPSP response as shown in Figure 4b , which equates to ~0.8 µM of CNQX bath application ( Supplementary Fig. 4b ). This indicates that only a limited number of polymersomes were locally activated, consistent with the intended spatial confinement of photorelease. Furthermore, repeated illumination after a single polymersome injection triggered successive CNQX release events ( Fig. 4c ), in accordance with the fact that not all polymersomes undergo lysis with the first illumination ( Supplementary video 3 ). Together, these results support localized photorelease and establish polymersomes as highly potent on-demand reservoirs for sustained, spatially restricted drug delivery. Finally, the spatial resolution of the photostimulation was further investigated with photostimulation spots that are distant from the recording pipette ( Fig. 4d, Supplementary Fig. 4c-e ). Reduction of fEPSP amplitude upon CNQX photorelease was evident for short distances (<200 µm) and subside over 200 µm. Remarkably, a distance-dependent gradient was observed, as shown with the non-linear fit of the data ( Fig. 4d ). These results confirmed that the photorelease of polymersomes enables local action within a diameter of less than 200 µm. DISCUSSION In this study, we introduced a unique strategy for the precise, light-controlled release of bioactive compounds within brain tissue based on photoactivatable polymersomes. Our findings establish polymersomes as robust vectors for targeted delivery to the central nervous system, combining biocompatibility, stability, and versatile encapsulation capabilities 20 . Future developments will extend this platform to deliver more complex cargos—including large or hydrophobic molecules and biologics such as enzymes — an objective supported by recent advances in polymer engineering, including a double-encapsulation strategy 21–23 . Indeed, because stimulated release from photoactive polymersomes is independent of the bioactive species itself, this approach is expected to be amenable to a range of molecules, biologics, and cocktails of active species. Equally, because light can be applied in a highly localized manner with easily controlled intensity, duration, and wavelength, precise spatio-temporal release of active molecules encapsulated in polymersomes is enabled. It is therefore a relevant tool for drug delivery in brain tissue, allowing treatment to be activated only in the targeted area, while leaving adjacent regions intact. Here, we demonstrated that our technology, which combines the precision of light stimulation with the translational advantages of nanomedicine, is adequate for brain tissue, reproducible, safe, and efficient, with high spatio-temporal resolution. While light scattering in brain tissue can be a challenge for applications requiring deeper penetration, the use of NIR- or red light-absorbing photocleavable chromophores would further enhance effective light penetration and release efficiency. Indeed, as long as photocleavage occurs, the dye itself is interchangeable in the approach described herein. Moreover, many different strategies are currently being developed to deliver light at a specific wavelength with minimal invasiveness. This includes microLED technologies and fluorescent organic nanoparticles (FONs) that can convert short-wavelength light to longer wavelengths, enabling photostimulation from outside the brain. Therefore, there is a high probability that light stimulation will become less invasive to brain tissue in the near future. Moreover, electrophysiological recordings performed in this study provided convincing evidence for the spatial and temporal precision of the photorelease process. Spatial and temporal precision of molecular photorelease in the brain could represent a breakthrough for treating localised brain diseases, such as epilepsy, stroke, and Parkinson’s disease. For example, the general mechanism of anticonvulsants in epilepsy is to reduce brain excitability, either by lowering excitatory neurotransmission or by increasing inhibitory neurotransmission 888888 . However, in the treatment of focal-onset epilepsies, only the focal area of the brain requires reduced excitability. In contrast, systemic drug administration can be pathological for the rest of the brain. In this context, local targeting of the diseased brain area may be less invasive than systemic drug administration. Furthermore, the approach developed in our study enables a temporal control over drug delivery. This also applies to pathologies such as epilepsy, where drug delivery would be ideally restricted to periods when epileptic seizures are arising 28 . In conclusion, precision light-triggered polymersome lysis to release a bio-active cargo represents a promising tool for both decrypting brain mechanisms and developing new therapeutic strategies, particularly considering recent advances in in vivo photopharmacology devices 29,30 and the growing exploration of polymer-based systems for brain disease research 31 . More broadly, this technology offers a path to closed-loop, light-guided chemical neuromodulation for the precision treatment of brain disorders. Declarations FUNDING This study received financial support from ANR (ANR-24-CE18-5491-01 and ANR-23-CE06-0008), Bordeaux Emergence, Bordeaux INP, Fondation Medisite, and the French government in the framework of the University of Bordeaux's IdEx "Investments for the Future" program / GPR BRAIN_2030. R.K. is a recipient of Clément Fayat Foundation and Fondation pour la Recherche Médicale fellowships (France). ACKNOWLEDGEMENTS The microscopy was performed at the Bordeaux Imaging Center, a service unit of the CNRS-INSERM and Bordeaux University, and a member of the national infrastructure France BioImaging, supported by the French National Research Agency (ANR-10-INBS-04). The help of Jérémie Teillon and Sébastien Marais is acknowledged. We thank the CIRCE (Behavioural Engineering Centre) facility of Bordeaux Neurocampus. We also thank Coralie Genevois for technical assistance at the Vivoptic platform, Univ. Bordeaux, CNRS, INSERM, TBM-Core, UAR 3427, US 5, F-33000 Bordeaux. Vivoptic is a France Life Imaging (FLI) labelled platform. MATERIAL & METHODS Materials: Poly(butadiene)- block -poly(ethylene oxide) copolymers (PBD n - b -PEG p ) were obtained from Polymer Source, Inc. (Montreal, Canada). The P41745-BdEO polymers used exhibited narrow polydispersity indices (Mw/Mn = 1.01) and consisted of more than 85% 1,2-addition of butadiene units. The hydrophilic fraction accounted for approximately 33%. CNQX disodium salt hydrate, toluene, sucrose, glucose, calcein, and electrophysiology reagents were purchased from Sigma-Aldrich and used as received. Illustrative drawings were made with Biorender and Blender 5.1b. Methods: Polymersomes synthesis Emulsion-Centrifugation. Poly(butadiene)- block -poly(ethylene oxide) (PBut- b -PEG , 33% hydrophilic fraction) polymersomes were prepared using the emulsion-centrifugation method reported by Peyret et al. 17 . Briefly, 5 µL of 0.30 M sucrose was added to 500 µL of 3 mg/mL PBut- b -PEG in toluene. The solution was vigorously hand-shaken for 30 seconds to create a water-in-oil emulsion. An interface was prepared by pouring 30 µL of PBut- b -PEG(3 mg/mL) into toluene containing 30 µL of 0.30 M glucose and allowed to stabilize for 30 minutes. 100 µL of the above emulsion was slowly poured over the interface, and the sample was immediately centrifuged (3 min, 1500 g, ambient temperature). The resulting polymersomes were allowed to recover in the lower phase for several hours. For polymersomes loaded only with calcein, 5 µL of a 10 mM calcein solution in sucrose was used to prepare the emulsion. When polymersomes were loaded with both calcein (10 mM in sucrose) and CNQX (100 µM in sucrose), 2.5 µL of each solution was used to form the emulsion. The glucose concentration was adjusted to match the osmotic pressure (300 mOsm) of the dye and CNQX in the sucrose solution. Polymersome counting. The polymersome solution was homogenized, and the exact volume of the preparation was measured with a P1000 pipette. Counting was performed using an epifluorescence microscope (Eclipse E400) equipped with a pE-300 light source (CoolLED). The LED power is set to 1% to avoid photo-stimulating the polymersomes. Counting was performed using a Fast Read 102 determination slide (Biosigma, BVS100), in which each cell is filled with 7 µL of the polymersome solution. The number of polymersomes is calculated as follows: (Number of polymersomes per µL = Total number of polymersomes/Number of large squares) * dilution factor * 10. Confocal Observations. Laser scanning confocal microscopy images were acquired on an inverted Leica TCS SP5 microscope (Leica Microsystems CMS GmbH, Mannheim, Germany) equipped with an HCXPLA06x, NA 1.4 oil-immersion objective in fluorescence mode. Samples (≈20 μL) were observed on an eight-well μSlide from Ibidi (CliniSciences, 80606). The laser outputs were controlled via the Acousto-Optical Tunable filter (AOTF) and the two collection windows using the Acousto-Optical Beam Splitter (AOBS) and photomultipliers (PMT) as follows: Calcein was excited with an argon laser at 488 nm (10 %) using a 495-555 nm window. The Helium-Neon laser at 633 nm (10 %) was also used in transmission mode. Images were collected using the microscope in sequential mode to avoid emission overlap, with a line average of 4-8 and a 512*512 pixel format. The FRAP wizard, in fly mode for faster time resolution, was used to define regions of interest (ROIs) around the selected vesicles. Processing of fluorescence confocal acquisitions was performed with FIJI (ImageJ v.1.54). Biosafety evaluation in vitro Dissection. Pups (mice P0) were euthanised by decapitation, and the brain was extracted from the skull. The hemispheres were separated, and the hippocampus and meninges were removed. Cortex was cut into small pieces and incubated in papain solution (0.4 mg/mL) for 15 min at 37 °C. DNAse was added and incubated for 5 min at 37 °C. The enzymatic reaction was inactivated with an equal volume of Hibernate supplemented with 10% Fetal Bovine Serum (FBS) (Sigma-Aldrich, F9665). Final dissociation was performed by mechanically pipetting the tissue with a P1000 pipette. Cells were counted with a Luna automated Cell Counter (Logos Biosystems). Primary cell culture. Plates were incubated with coating solution containing Poly-L-Lysine (1 mg/mL) (Sigma, P2636-1G) and Laminin (3 µg/mL) (Sigma, L2020) for at least 2 h at 37 °C. All culture media were equilibrated beforehand for at least 4 h at 37 °C. Cells were seeded in seeding medium (Neurobasal-A (ThermoFischer, 12349015) supplemented with B27 plus (ThermoFisher, A3582801) and 10% FBS at 50k per well in a 96-well plate. The medium was fully replaced 1 h post-seeding to remove debris with maintenance medium (Neurobasal-A supplemented with B27 plus and 1.5% of FBS). Culture was maintained every 3-4 days by replacing half of the medium with maintenance medium, until day 14. Biosafety evaluation in primary cell culture. Cell viability was assessed using the MTT assay (Thermofisher, V13154), with P0 mice used to obtain a cell suspension from the cortex. Cells were seeded at 50k per well in 96-well plates and incubated for 14 days at 37 °C, 5% CO 2 . At maturity of the culture, two biosafety valuations were conducted, the polymersomes concentrations, respectively, 0, 600, 1500, 3000, 6000, 9000 per well and light power (0.003, 3.5, 8.7, 19.3, 30 mw), then incubated for 48 h. Half of the conditions were photo-stimulated at 3.5 mw for 20 s using a 400 mm optical fiber with a numerical aperture of 0.5 (ThorLabs, M98L01) to assess the toxicity of polymersomes post-stimulation. Post-incubation medium was fully changed with fresh medium, and MTT reagent (10 µL, final concentration 0.5 mg/mL) was added to each well and incubated at 37 °C, inducing the formation of formazan crystals. SDS-HCl (100 µL, final concentration 50 mg/mL) was added to each well to solubilize formazan, and the mixture was incubated for 10 h at 37 °C. Absorbance was measured at 570 nm in a microplate reader. All conditions were tested in triplicate. Biosafety evaluation in vivo Animals. Adult male and female C57BL/6J mice were obtained from Janvier Labs (France). All procedures were carried out in accordance with the European Communities Council Directive 2010/63/EU and approved by the French National Committee for animal experimentation (authorization APAFIS #20925 and #50835). Mice were housed under standard laboratory conditions in a temperature- and relative humidity-controlled environment and a 12-hour light/dark cycle (lights on at 07:00). Animals were group-housed, and food and water were provided ad libitum. Stereotactic Injections. Stereotaxic injections were performed in adult male and female mice under isoflurane deep anaesthesia. Animals were placed in a stereotaxic apparatus and maintained at 37 °C using a heating pad. A craniotomy was performed above the target region. The polymersomes were injected using a Hamilton syringe with a 34G blunt needle, connected to a nanoliter injector (World Precision Instruments, Nanofil) at a flow rate of 0.5 µL/min. Injections were performed into the CA1 region of the hippocampus, following stereotaxic coordinates relative to bregma: AP = –2.0 mm, ML = ±1.3 mm, DV = –1.2 mm. A volume of 2 µL of calcein-free polymersomes was injected per site. The pipette was left in place for 5 minutes after injection to minimise reflux. After surgery, the animals were monitored daily for 3 days to ensure their recovery. Tissue collection and immunofluorescence. Animals were injected with buprenorphine (0.1 mg/kg, s.c.) (Virbac) and deeply anesthetized with Euthasol (400 mg pentobarbital/kg, ip) (Dechra) 30 min later. Animals were transcardially perfused with phosphate-buffered saline (PBS), followed by 4% paraformaldehyde (PFA) (Sigma-Aldrich). Brains were extracted, post-fixed in 4% PFA at 4 °C for 48 h, and subsequently transferred to a 30% sucrose solution at 4 °C for cryoprotection. Coronal brain sections (40 µm thick) were obtained using a vibratome (Leica) and stored at −20 °C in a cryoprotective solution composed of 20% glycerol and 30% ethylene glycol (Sigma-Aldrich) in PBS. For immunofluorescence, brain slices containing the injected zone were washed 3 times in PBS (EuroMedex, ET330), then incubated in blocking solution (10% donkey serum, 0.5 % Triton X100) before incubation overnight in blocking solution containing the primary antibody (GFAP: DAKO Z033429-2 1/1000; IBA1: Fujifilm 019-19741, 1/500). The next day, slices were washed 4 times in PBS, then incubated in blocking solution containing a secondary antibody (Donkey anti-rabbit-Alexa 488, Abcam) for 2 hours. After 3 washes in PBS, slices were mounted on glass slides, dried, and finally mounted with medium DAPI Fluoromount G (EuroMedex, EM17984-24) and coverslipped. Observations and photos were made with a widefield fluorescent microscope (DM5000, Leica, Bordeaux Imaging Center). Photos were analysed with FIJI (ImageJ v.1.54). The area of interest around the injection site was manually defined, a threshold was applied, and particles were detected automatically. The mean intensity of particles, weighted by the size of each particle, was measured for each image. For each animal, 3-5 images were analysed. Ex vivo experiments Electrophysiological Recordings. Hippocampal slice preparation. Mice were anaesthetized with isoflurane, then decapitated, and the brain was quickly extracted from the skull. Coronal hippocampal slices with a 5° angle (350 µm thick) were prepared using a vibrating blade microtome (VT1000S, Leica Microsystems) in ice-cold, cutting high-sucrose artificial cerebral spinal fluid (HS-ACSF) solution containing (in mM): 125 NaCl, 25 NaHCO₃, 25 glucose, 2.5 KCl, 2 CaCl₂, 1 MgCl₂, 1.25 NaH₂PO₄, 3 pyruvate, and 75 sucrose, continuously oxygenated with 95% O 2 /5% CO 2 . Slices were incubated at 34 °C for 1 h, then maintained at room temperature. Recordings were initiated after at least 1 h of recovery. Recording ACSF (used for both storage and recording) contained (in mM): 125 NaCl, 25 NaHCO₃, 25 glucose, 2.5 KCl, 2 CaCl₂, 1 MgCl₂, and 1.25 NaH₂PO₄, and was continuously bubbled with 95 % O 2 / 5 % CO 2 . All ACSF components were purchased from Sigma-Aldrich. Electrophysiological recordings. Recordings were performed at 30 °C using a temperature-controlled system (Warner Instruments, TC-324B). Hippocampal slices were continuously superfused with oxygenated ACSF at a rate of 3–4 mL/min using a peristaltic pump (Gilson, Minipulse 3). Patch pipettes (4–6 MΩ), pulled from borosilicate glass capillaries (Sutter Instruments, GBF-150-117-10) with a horizontal puller (Sutter Instruments, P-97), were used for intracellular recordings. For extracellular recordings, borosilicate glass pipettes filled with ACSF were placed in the stratum radiatum of the CA1 region to record field excitatory postsynaptic potentials (fEPSPs). Synaptic responses were evoked by stimulation of Schaffer collateral afferents from the CA3 region using a bipolar concentric electrode (Phymep) connected to a stimulator (World Precision Instruments, A365). Polymersomes were injected directly into the slice using borosilicate glass pipettes. Photostimulation of polymersomes was performed under an upright fluorescence microscope (Nikon FN1) equipped with a 40× water-immersion objective. Fluorescence signal was detected using a pE-300 light source (CoolLED) and a CCD camera (INFINITY 3S-1UR M/C). Data acquisition and analysis . Signals were amplified using a MultiClamp 700B amplifier (Molecular Devices) and digitized via a Digidata 1440B interface (Molecular Devices), controlled by pClamp 10.7 software. Data were analysed offline using Clampfit 11.2 (Molecular Devices). Statistical analysis. For all data generated, statistical analyses were performed using GraphPad Prism 10 or 11. Normality was assessed using the Anderson–Darling, D’Agostino–Pearson, Shapiro–Wilk, and Kolmogorov–Smirnov tests. Depending on the data structure, comparisons were made using t-tests (paired or unpaired, parametric or nonparametric), one-way or two-way ANOVA, with appropriate post hoc tests. All tests were two-tailed, and p < 0.05 was considered statistically significant. REFERENCES Muir, J., Anguiano, M. & Kim, C. K. 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Hee Lee, J., Lee, S., Kim, D. & Jae Lee, K. Implantable Micro-Light-Emitting Diode (µLED)-based optogenetic interfaces toward human applications. Advanced Drug Delivery Reviews 187 , 114399 (2022). Khan, Y. et al. Synthesis of fluorescent organic nano-dots and their application as efficient color conversion layers. Nat Commun 13 , 1801 (2022). Kokkinos, V., Sisterson, N. D., Wozny, T. A. & Richardson, R. M. Association of Closed-Loop Brain Stimulation Neurophysiological Features With Seizure Control Among Patients With Focal Epilepsy. JAMA Neurology 76 , 800–808 (2019). Wu, Y. et al. Wireless multi-lateral optofluidic microsystems for real-time programmable optogenetics and photopharmacology. Nat Commun 13 , 5571 (2022). Zhang, Y. et al. Battery-free, lightweight, injectable microsystem for in vivo wireless pharmacology and optogenetics. PNAS 116 , 21427–21437 (2019). Benaicha-Fernández, A., Atkinson, S. P., Conejos-Sánchez, I., Medel, M. & Vicent, M. J. Polymer-based nanomedicines: Supporting multimodal approaches to glioblastoma Multiforme treatment. Advanced Drug Delivery Reviews 115735 (2025) doi:10.1016/j.addr.2025.115735. Additional Declarations There is NO Competing Interest. Supplementary Files Ruffieretalsupstats.pdf Supplementary statistics Ruffieretalsupinfo.docx Supplementary figures SupplementaryVideo2graychannel.avi Supplementary Video 2 SupplementaryVideo1greenchannel.avi Supplementary Video 1 SupplementaryVideo3.avi Supplementary Video 3 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9232875","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":619162082,"identity":"4fa0e7d1-9127-4fad-a06f-6e96f8c7fce1","order_by":0,"name":"Clémentine 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Bordeaux","correspondingAuthor":false,"prefix":"","firstName":"nathan","middleName":"","lastName":"McClenaghan","suffix":""},{"id":619162099,"identity":"2f338684-b8fb-4e06-9931-416fb308d1f4","order_by":17,"name":"Sébastien Lecommandoux","email":"","orcid":"","institution":"Bordeaux INP","correspondingAuthor":false,"prefix":"","firstName":"Sébastien","middleName":"","lastName":"Lecommandoux","suffix":""}],"badges":[],"createdAt":"2026-03-26 10:35:22","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-9232875/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9232875/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106387041,"identity":"0fcfda14-a8a4-42b0-8856-97ebf997be24","added_by":"auto","created_at":"2026-04-08 06:27:49","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":474973,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eConfocal imaging and quantitative characterisation of photoactivable polymersomes. a,\u003c/strong\u003e Confocal image of photoactivable polymersomes in the green channel (λ\u003csub\u003eexc\u003c/sub\u003e. = 488 nm). Scale bar, 10 µm. \u003cstrong\u003eb,\u003c/strong\u003e Relative frequency (%) of photoactivable polymersomes concentration (counts per µL). \u003cstrong\u003ec,\u003c/strong\u003e Relative frequency (%) of photoactivable polymersomes diameter (µm) displayed as a histogram with a log-normal fit (geometric mean 6.7 µm, geometric s.d. 1.8; R² = 0.68). \u003cstrong\u003ed,\u003c/strong\u003e Confocal images before (1) and after photoactivable polymersomes rupture (2) in the green channel, with corresponding 3D renderings of the polymersomes displayed above the images. Scale bar, 10 µm. \u003cstrong\u003ee,\u003c/strong\u003e Relative frequency (%) of photoactivable polymersomes release time displayed as a histogram with a log-normal fit (geometric mean 4.3 s, geometric s.d. 2.8; R² = 0.25).\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-9232875/v1/9f6749fd1cd5353c1f204b62.png"},{"id":106387018,"identity":"f9b4be63-ac70-4909-a5c7-ff1e86f68503","added_by":"auto","created_at":"2026-04-08 06:27:40","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":311374,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNo cytotoxic effect of CNQX-loaded photoactivable polymersomes (600 to 9000 polymersomes/well) before and after illumination on primary cells. a,\u003c/strong\u003e Primary cortical cells from P0 C57BL/6 mouse cortex (100 µL, Neurobasal‑A) were illuminated at 470 nm for 20 s with intensities ranging from 1% to 73%. Cell viability was measured using the Vybrant MTT assay (λ\u003csub\u003eabs\u003c/sub\u003e. = 570 nm) (Thermo Fisher). Data are mean ± SEM (n =3 wells, 3 independent experiments). No significant difference was detected versus non‑illuminated controls (one‑way ANOVA, P \u0026gt; 0.05). \u003cstrong\u003eb,\u003c/strong\u003e Cells from the same suspension were incubated for 48 h with photoactivable polymersomes loaded with CNQX (0.6-9.0 × 10³ per well) or DMSO (1% or 5%) as positive controls. Viability was assessed as in a. Data are mean ± SEM (n =3 wells, 3 independent experiments). One‑way ANOVA: **P \u0026lt; 0.01, ****P \u0026lt; 0.0001 vs. untreated.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-9232875/v1/583d204cc75424b0c2a8b680.png"},{"id":106387035,"identity":"9b41621b-0a17-4432-bb3e-0f8edba74d39","added_by":"auto","created_at":"2026-04-08 06:27:46","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1126191,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMicroglial and astrocytic reactivity after intracranial injection of polymersomes. a,\u003c/strong\u003eRepresentative IBA1 and GFAP immunofluorescence images at 48 h comparing injected vs non-injected hemispheres for polymersomes and control (medium) conditions. \u003cstrong\u003eb-c,\u003c/strong\u003e Quantification of IBA1 and GFAP signal intensity in the injected hemisphere relative to the contralateral control at 48 h (b) and 3 weeks (c); dots represent individual animals and bars show mean ± SEM, with p values indicated.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-9232875/v1/9971e46e4ac3e7f6e62fc246.png"},{"id":106387020,"identity":"cc3c8c43-0c3d-4a63-98f2-43a497ba0556","added_by":"auto","created_at":"2026-04-08 06:27:40","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":344755,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSpatial and temporal effect of CNQX photorelease on in vivo synaptic transmission at CA3-CA1 hippocampal. a,\u003c/strong\u003e Brain slices containing CA3-CA1 connections were freshly made from adult mice. The stimulation electrode was placed in the CA3 region, and the recording field electrode was placed in the CA1 region. CNQX-containing polymersomes (polymCNQX) were delivered close to the recording site with a glass pipette. Illumination (20 s) was delivered through the microscope objective. The expected effect is a reduction in field-recorded synaptic responses due to photorelease of CNQX, which blocks glutamate AMPA receptors (AMPARs). Scale bar: 10 ms, 0.5 mV. \u003cstrong\u003eb,\u003c/strong\u003e (left) Representative inhibition response of two recordings with CNQX photorelease performed with 8% (light blue) or 12% (dark blue) of maximal LED intensities. Injection of polymersomes in the slice (green line) was performed just before illumination (blue line). (right) Average inhibition response of field recordings following 8% (light blue) or 12% (dark blue) illumination with or without CNQX-containing polymersomes in brain slices. Data are represented as mean ± SEM. n = 4 to 31 trials recorded from 3 to 6 slices. Two-way ANOVA: * P \u0026lt; 0.05, **** P \u0026lt; 0.0001. \u003cstrong\u003ec,\u003c/strong\u003e Inhibition of response (%) as a function of time (min) after injection of photoactivable polymersomes loaded with CNQX (black line), followed by six illuminations at 7-min intervals of the brain slice by the microscope LED (blue line). \u003cstrong\u003ed,\u003c/strong\u003eInhibition of response (%) as a function of distance from recording (µm). Measurements were performed at four distances (d1 = 25 µm, d2 = 130 µm, d3 = 250 µm, d4 = 730 µm) and fitted with a single-phase exponential decay.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-9232875/v1/ca2fc835338a8d5f8a409a8c.png"},{"id":106960523,"identity":"22cdc132-82ff-467b-9b35-569b6a99a51d","added_by":"auto","created_at":"2026-04-15 09:21:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3316998,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9232875/v1/68b4661c-897f-43ec-9988-9460ce2a8a23.pdf"},{"id":106387033,"identity":"88771b59-8208-4292-bc9d-2e8e46f1eb40","added_by":"auto","created_at":"2026-04-08 06:27:46","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":425923,"visible":true,"origin":"","legend":"Supplementary statistics","description":"","filename":"Ruffieretalsupstats.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9232875/v1/e98bd16da6027731eb094909.pdf"},{"id":106387019,"identity":"28678b37-ea5d-46e1-aedd-606302f7f303","added_by":"auto","created_at":"2026-04-08 06:27:40","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2203040,"visible":true,"origin":"","legend":"Supplementary figures","description":"","filename":"Ruffieretalsupinfo.docx","url":"https://assets-eu.researchsquare.com/files/rs-9232875/v1/4fbf83f2228c885d4ac2e036.docx"},{"id":106387034,"identity":"142ef351-9ba2-43e6-bd93-c3edf98a3449","added_by":"auto","created_at":"2026-04-08 06:27:46","extension":"avi","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":2485968,"visible":true,"origin":"","legend":"Supplementary Video 2","description":"","filename":"SupplementaryVideo2graychannel.avi","url":"https://assets-eu.researchsquare.com/files/rs-9232875/v1/689dd849ee0a26ffcd3ee3fe.avi"},{"id":106387022,"identity":"356023b7-a328-4932-80f3-848e628c2879","added_by":"auto","created_at":"2026-04-08 06:27:40","extension":"avi","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":3009002,"visible":true,"origin":"","legend":"Supplementary Video 1","description":"","filename":"SupplementaryVideo1greenchannel.avi","url":"https://assets-eu.researchsquare.com/files/rs-9232875/v1/5dc55baa571b65218ff2f590.avi"},{"id":106387098,"identity":"4e3aab95-3a68-4fc7-9369-fb3bc635367b","added_by":"auto","created_at":"2026-04-08 06:27:56","extension":"avi","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":8468568,"visible":true,"origin":"","legend":"Supplementary Video 3","description":"","filename":"SupplementaryVideo3.avi","url":"https://assets-eu.researchsquare.com/files/rs-9232875/v1/ce0aeb421bb744908a7fca42.avi"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Spatiotemporal control of molecular delivery in the brain using photoactivable polymersomes","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eBeyond electrical and optical stimulation, the use of chemically-mediated triggering to modulate brain activity represents a fundamentally distinct and largely underexplored strategy. Neurotransmitters, neuromodulators, and pharmacological agents act through endogenous molecular pathways, offering the potential to regulate neural circuits with high biochemical specificity and sustained effects that are difficult to achieve with purely physical stimulation modalities\u003csup\u003e1,2\u003c/sup\u003e. However, precise spatiotemporal control over the delivery of such molecules within intact brain tissue remains a major challenge, owing to rapid diffusion, systemic side effects, and the lack of minimally invasive release technologies. Developing versatile approaches that enable on-demand, localized chemical neuromodulation could therefore open new avenues for both fundamental neuroscience and targeted therapeutic intervention.\u003c/p\u003e\n\u003cp\u003eMany brain diseases remain inadequately treated because systemically administered therapies reach pathological regions only inefficiently and lack temporal control over their action\u003csup\u003e3\u003c/sup\u003e. As a result, access to diseased brain tissue remains a central bottleneck in the development of effective neurotherapeutics\u003csup\u003e4\u003c/sup\u003e, in spite of recent advances demonstrating promising outcomes\u003csup\u003e5,6\u003c/sup\u003e. Although drug delivery to the brain is severely constrained by the blood-brain barrier and the structural heterogeneity of neural tissue, recent advances in minimally invasive, locally targeted engineering approaches have created new opportunities for treating localized brain disorder\u003csup\u003e7\u003c/sup\u003e. As such, intracranial pharmacotherapy is increasingly used to target specific brain areas more precisely. In the case of focal epilepsy, anti-epileptic drugs act by reducing the global excitability of brain networks. However, it is required only in the diseased brain area, and it can be deleterious to other brain networks. For this reason, intracranial pharmacology is a therapeutic strategy already used in humans for some diseases, including focal epilepsies and glioblastoma\u003csup\u003e8,9\u003c/sup\u003e. Nevertheless, intracranial pharmacotherapy also has limitations, notably that drug delivery via this approach remains passive and thus is not controllable over time or in terms of dose.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo improve drug delivery, encapsulation strategies based on synthetic carriers, such as polymersomes that are self-assembled vesicles from biocompatible block copolymers, have been extensively developed. Polymersomes are of particular biomedical interest owing to their high stability, chemical versatility, and ability to encapsulate and efficiently transport multiple therapeutic agents with high efficiency\u003csup\u003e10,11\u003c/sup\u003e. Stimuli-responsive release mechanisms for polymersomes have been developed, such as pH, temperature, or light, to better control the release of bioactive molecules\u003csup\u003e12\u0026ndash;14\u003c/sup\u003e. For brain applications, stimuli-responsive polymersomes activated by light represent a particularly relevant approach for brain interventions. Indeed, neuroscience research has undergone major technological advances with the emergence of optical methods that enable \u003cem\u003ein vivo\u003c/em\u003e recording and manipulation of neural activity, including optogenetics, fiber photometry, and two-photon photostimulation\u003csup\u003e15\u003c/sup\u003e. These light-based technologies are increasingly being developed to control brain circuits for clinical use\u003csup\u003e16\u003c/sup\u003e. However, they are primarily designed to modulate neuronal networks rather than to achieve temporally controlled delivery of therapeutic agents.\u003c/p\u003e\n\u003cp\u003eHere, we leverage recent advances in optical neurotechnology to develop an innovative therapeutic strategy based on photoactivatable polymersomes\u003csup\u003e17,18\u003c/sup\u003e, enabling light-triggered, localized delivery of encapsulated molecules within the brain with precise control over timing, spatial distribution, and dosage. As such, we demonstrated the photorelease of CNQX, a competitive AMPA/kainate receptor antagonist, in brain tissue, in a temporally- and spatially-controlled manner. This approach establishes new experimental paradigms for preclinical research and expands the therapeutic toolkit for treating localized brain disorders.\u003c/p\u003e"},{"header":"RESULTS ","content":"\u003cp\u003e\u003cstrong\u003e1-\u0026nbsp; \u0026nbsp;\u0026nbsp;Design of photoactivable and biocompatible polymersomes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on initial work\u003csup\u003e17\u003c/sup\u003e, we formulated stable and photocleavable polymersomes using an emulsion-centrifugation method (\u003cstrong\u003eSupplementary Fig. 1a-b\u003c/strong\u003e). Briefly, sucrose-containing water droplets generated by emulsion and stabilized by a copolymer in toluene were deposited onto a glucose solution-toluene interface containing the same copolymer. Upon centrifugation, the droplets crossed the interface to form polymersomes in the glucose phase, enabling quantitative encapsulation of the molecules of interest dissolved in the sucrose phase. Here, the formulation of photoactivable polymersomes was optimised to become fully compatible with brain applications: polymersomes were obtained by self-assembly of the amphiphilic block copolymer, poly(butadiene)-\u003cem\u003eb\u003c/em\u003e-poly(ethylene oxide) (PBut\u003csub\u003en\u003c/sub\u003e-\u003cem\u003eb\u003c/em\u003e-PEG\u003csub\u003ep\u003c/sub\u003e), loaded with calcein (10 mM) in a medium with an osmolarity of 300 mOsm (\u003cstrong\u003eFig. 1a\u003c/strong\u003e). Counting polymersomes with a counting grid under a fluorescent microscope (\u003cstrong\u003eSupplementary Fig. 1c\u003c/strong\u003e) revealed a concentration of 267 ± 6.7 polymersomes / µl (\u003cstrong\u003eFig. 1b\u003c/strong\u003e). Furthermore, polymersomes have a median diameter of 8.27 ± 0.14 µm (n = 1265 polymersomes for 6 batches) with a polydispersity of 0.36, indicating a rather homogeneous size distribution. \u003cstrong\u003e(Fig. 1c)\u003c/strong\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCalcein is a water-soluble dye that undergoes photolysis within polymersomes upon illumination at 488 nm, a wavelength routinely used in neuroscience, thereby increasing the internal osmolarity. The resulting rapid rise in osmotic pressure generates membrane tension, leading to rapid, irreversible rupture, observed as an explosive disruption within seconds of irradiation (\u003cstrong\u003eFigure 1d\u003c/strong\u003e). As shown in \u003cstrong\u003eFigure 1d-e\u003c/strong\u003e, illumination of polymersomes loaded with 10 mM calcein led to the rapid rupture of the polymersome membrane and release of their contents (\u003cstrong\u003eSupplemental videos 1, 2, 3\u003c/strong\u003e). Distribution of rupture times showed a mean rupture time of 6.39 ± 0.9 s (n = 41 polymersomes for 6 batches) under these irradiation conditions (\u003cstrong\u003eFig. 1e, Supplementary Fig. 1d\u003c/strong\u003e). Furthermore, no morphological changes of polymersomes were observed in calcein-free polymersomes upon illumination at 488 nm, confirming the specificity of light-induced membrane rupture. Finally, when several regions of interest (ROIs) were defined, only the selected polymersomes ruptured upon illumination (\u003cstrong\u003eSupplementary\u003c/strong\u003e \u003cstrong\u003eFig. 1e\u003c/strong\u003e). This approach thus enables rapid and localised release of the polymersome content, both in time and space.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2-\u0026nbsp; \u0026nbsp;\u0026nbsp;Biosafety of photoactivable polymersomes for brain cells and brain tissue\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter confirming that the emulsion-centrifugation process was an appropriate method for obtaining a high yield of photoactivable polymersomes with rapid, localised release of their contents, we tested their biosafety. Biosafety of both polymersomes and photorelease process were evaluated in primary cell cultures from mouse cortices and in mouse brains. First, the effect of light stimulation alone was tested, as previous studies reported deleterious effect of 470 nm light on neuronal communication\u003csup\u003e19\u003c/sup\u003e. In our conditions, illumination of primary neuronal cell cultures with 470 nm light delivered through an optical fiber (Ø\u0026nbsp;400 µm, NA 0.5, 20 s) did not alter cell viability (\u003cstrong\u003eFig. 2a\u003c/strong\u003e). Next, we evaluated the impact of calcein-loaded polymersome incubation with increasing concentrations (600 to 9000 polymersomes/well) on primary neuronal cell cultures. We found no deleterious effect on cell viability (\u003cstrong\u003eFig. 2b\u003c/strong\u003e). Finally, we combined polymersome incubation with light illumination, at power levels that are sufficient to induce rapid photorelease (8 % of maximal power level, see \u003cstrong\u003eSupplementary Table 2\u0026nbsp;\u003c/strong\u003efor correspondence with light intensities) and similarly, no cytotoxic effect was observed (\u003cstrong\u003eFig. 2b\u003c/strong\u003e). As a positive control, treatment of cells with DMSO 1% v/v and 5% v/v induces moderate and strong cell mortality, respectively (\u003cstrong\u003eFig. 2b\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFinally, the biosafety of polymersomes was validated\u003cem\u003e\u0026nbsp;in vivo\u003c/em\u003e, by injecting empty polymersomes in mouse brains and assessing glial reactivity through immunostaining of IBA-1 (for microglia) and GFAP (for astrocytes) proteins at 48\u0026nbsp;h and 3 weeks after injection (\u003cstrong\u003eFig. 3\u003c/strong\u003e). At 48\u0026nbsp;h after injection, the injected hemisphere displayed a slight but significant increased signal for IBA1 and GFAP (\u003cstrong\u003eFig. 3a,b\u003c/strong\u003e). However, this increase was not different between mice injected with polymersomes and those injected with vehicle solution without polymersomes (medium). This suggests that polymersomes did not induce any glial reaction beyond the stereotaxic injection itself. At 3 weeks after injection, quantification revealed no difference in IBA1 signal between the injected and non-injected hemispheres for both polymersome and medium groups (\u003cstrong\u003eFig. 3a,c\u003c/strong\u003e). By contrast, the GFAP signal remained significantly higher in the injected hemisphere than in the non-injected one, but did not differ between the medium and polymersomes groups (\u003cstrong\u003eFig. 3a,c\u003c/strong\u003e). This indicates that polymersomes themselves have no significant short- or long-term impact on gliosis.\u003c/p\u003e\n\u003cp\u003eAltogether, these findings demonstrate that both polymersomes and calcein-mediated photorelease are well tolerated by brain cells and brain tissue.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3-\u0026nbsp; \u0026nbsp;\u0026nbsp;Photostimulation of polymersomes in brain slices\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo demonstrate the efficacy of photorelease in brain tissue and quantify its ability to modulate neural processes, we developed an experimental paradigm based on measuring electrical synaptic transmission between connected neurons. Electrophysiological recordings were performed in mouse brain slices containing the synaptic connections between the CA3 and CA1 regions of the hippocampus, a key brain region involved in memory processes. Communication between these two structures relies on presynaptic glutamate release, which activates postsynaptic AMPA receptors and thereby enables the propagation of electrical signals from one neuron to the next. Therefore, blocking AMPA receptors with a competitive AMPA receptor antagonist, such as the well-established CNQX\u0026nbsp;(6-cyano-7-nitroquinoxaline-2,3-dione), inhibits the electrical communication between the CA3 and CA1 regions, which can be recorded with field recordings (\u003cstrong\u003eFig. 4a\u003c/strong\u003e). For this reason, we chose to encapsulate CNQX (100 µM) in calcein-loaded polymersomes to quantify the effect of photorelease from polymersomes on neuronal activity.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe first verified that encapsulation of CNQX did not alter the photostimulation properties of polymersomes (\u003cstrong\u003eSupplementary Fig. 2\u003c/strong\u003e). The encapsulation of CNQX slightly, but significantly, reduced the diameter of the polymersomes (7.62 ± 0.2 µm with CNQX (n = 386 polymersomes for 7 batches) vs. 8.27 ± 0.1 µm without CNQX (n = 1265 polymersomes for 6 batches), unpaired Mann-Whitney test, p \u0026lt; 0.0001) (\u003cstrong\u003eSupplementary\u003c/strong\u003e \u003cstrong\u003eFig. 2a-c\u003c/strong\u003e). Moreover, the presence of CNQX in polymersomes did not change the rupture delay of polymersomes upon photostimulation (8.12 ± 0.9 s vs. 6.39 ± 0.9 s without CNQX, (n = 52 and polymersomes for 3 and 6 batches, respectively, unpaired Mann-Whitney test, p = 0.1192), (\u003cstrong\u003eSupplementary Fig. 2d\u003c/strong\u003e). This confirms that photostimulation was not affected by the presence of CNQX within the polymersomes and suggests that polymersomes represent a versatile platform for the encapsulation of bioactive molecules.\u003c/p\u003e\n\u003cp\u003eThen, we used calcein/CNQX-loaded polymersomes to perform the photorelease of CNQX on brain slices (\u003cstrong\u003eFig. 4a\u003c/strong\u003e). Electrical stimulation of the CA3 region induces synaptic evoked responses in CA1 region that were recorded extracellularly as field excitatory postsynaptic potentials (fEPSP) (\u003cstrong\u003eFig. 4a, Supplementary Fig. 3a\u003c/strong\u003e). As for cell culture, the effect of light alone on fEPSPs with increasing light intensities was first quantified (\u003cstrong\u003eSupplementary Fig.3b\u003c/strong\u003e). The power levels used were similar to those used for cell culture illumination (\u003cstrong\u003eSupplementary Tables 1 and 2\u0026nbsp;\u003c/strong\u003efor conversion). Under 12 % of light power, no effect was observed on fEPSP response (\u003cstrong\u003eSupplementary Fig. 3b-c\u003c/strong\u003e). However, we observed a significant inhibition of fEPSP response for light powers over 14 % with a sigmoid effect (\u003cstrong\u003eSupplementary Fig.3b\u003c/strong\u003e). This is in accordance with previous studies showing that light itself can modify neuronal excitability\u003csup\u003e19\u003c/sup\u003e. Given these results, we then used photostimulation at no more than 12% of maximal light power.\u003c/p\u003e\n\u003cp\u003eCalcein/CNQX-loaded polymersomes were applied to the brain slice near the recording site without altering the fEPSP response (\u003cstrong\u003eSupplementary Fig. 3a,d\u003c/strong\u003e). This is a supplementary confirmation of the innocuity of polymersomes for brain tissue. Then, light at 8 % was delivered for 20 s. Shortly after, significant reduction of fEPSP was observed (\u003cstrong\u003eFig. 4b, Supplementary Fig. 3e\u003c/strong\u003e). A similar experiment was conducted with illumination at 12 % of the maximal power level. A stronger effect was produced (\u003cstrong\u003eFig. 4b\u003c/strong\u003e). Of note, light stimulation applied to polymersomes containing calcein alone induced only an 5.5 % reduction of fEPSP response (\u003cstrong\u003eSupplementary Fig. 3f\u003c/strong\u003e). These control experiments confirmed that the reduction in fEPSPs after polymersome photostimulation was specific to CNQX photorelease. Moreover, these results demonstrate that CNQX remains stable and bioactive following encapsulation and photorelease, further supporting the versatility of photoactivable polymersomes for drug delivery.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSpatial and temporal precision of CNQX delivery\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further define the spatiotemporal range of action of photoactivatable polymersomes in brain applications, the precision of CNQX photorelease was investigated in acute brain slices. First, we compared photostimulation to whole bath application of CNQX (\u003cstrong\u003eSupplementary Fig. 4\u003c/strong\u003e). On one side, we confirmed that bath application of CNQX at 10 µM almost annihilates fEPSP response (76.5 ± 8.2% of inhibition of fEPSP baseline) while 0.5 µM of CNQX in the bath induced a minimal inhibition of fEPSP (7.61 ± 4.40 % of inhibition of fEPSP baseline) (\u003cstrong\u003eSupplementary Fig. 4a,b\u003c/strong\u003e). On the other hand, photostimulation of polymersomes containing CNQX at 100 µM induced on average a 12 % inhibition of fEPSP response as shown in\u0026nbsp;\u003cstrong\u003eFigure 4b\u003c/strong\u003e, which equates to ~0.8 µM of CNQX bath application\u0026nbsp;(\u003cstrong\u003eSupplementary Fig. 4b\u003c/strong\u003e). This indicates that only\u0026nbsp;a limited number of polymersomes were locally activated, consistent with the intended spatial confinement of photorelease.\u0026nbsp;Furthermore,\u0026nbsp;repeated illumination after a single polymersome injection\u0026nbsp;triggered successive CNQX release events\u0026nbsp;(\u003cstrong\u003eFig. 4c\u003c/strong\u003e), in accordance with the fact that not all polymersomes undergo lysis with the first illumination (\u003cstrong\u003eSupplementary video 3\u003c/strong\u003e).\u0026nbsp;Together, these results support localized photorelease and establish polymersomes as highly potent on-demand reservoirs for sustained, spatially restricted drug delivery.\u003c/p\u003e\n\u003cp\u003eFinally, the spatial resolution of the photostimulation was further investigated with photostimulation spots that are distant from the recording pipette (\u003cstrong\u003eFig. 4d, Supplementary Fig. 4c-e\u003c/strong\u003e). Reduction of fEPSP amplitude upon CNQX photorelease was evident for short distances (\u0026lt;200 µm) and subside over 200 µm. Remarkably, a distance-dependent gradient was observed, as shown with the non-linear fit of the data (\u003cstrong\u003eFig. 4d\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThese results confirmed that the photorelease of polymersomes enables local action within a diameter of less than 200 µm.\u0026nbsp;\u003c/p\u003e"},{"header":"DISCUSSION ","content":"\u003cp\u003eIn this study, we introduced a unique strategy for the precise, light-controlled release of bioactive compounds within brain tissue based on photoactivatable polymersomes. Our findings establish polymersomes as robust vectors for targeted delivery to the central nervous system, combining biocompatibility, stability, and versatile encapsulation capabilities\u003csup\u003e20\u003c/sup\u003e. Future developments will extend this platform to deliver more complex cargos—including large or hydrophobic molecules and biologics such as enzymes — an objective supported by recent advances in polymer engineering, including a double-encapsulation strategy\u003csup\u003e21–23\u003c/sup\u003e. Indeed, because stimulated release from photoactive polymersomes is independent of the bioactive species itself, this approach is expected to be amenable to a range of molecules, biologics, and cocktails of active species.\u003c/p\u003e\n\u003cp\u003eEqually, because light can be applied in a highly localized manner with easily controlled intensity, duration, and wavelength, precise spatio-temporal release of active molecules encapsulated in polymersomes is enabled. It is therefore a relevant tool for drug delivery in brain tissue, allowing treatment to be activated only in the targeted area, while leaving adjacent regions intact. Here, we demonstrated that our technology, which combines the precision of light stimulation with the translational advantages of nanomedicine, is adequate for brain tissue, reproducible, safe, and efficient, with high spatio-temporal resolution. While light scattering in brain tissue can be a challenge for applications requiring deeper penetration, the use of NIR- or red light-absorbing photocleavable chromophores would further enhance effective light penetration and release efficiency. Indeed, as long as photocleavage occurs, the dye itself is interchangeable in the approach described herein. Moreover, many different strategies are currently being developed to deliver light at a specific wavelength with minimal invasiveness. This includes microLED technologies and fluorescent organic nanoparticles (FONs) that can convert short-wavelength light to longer wavelengths, enabling photostimulation from outside the brain. Therefore, there is a high probability that light stimulation will become less invasive to brain tissue in the near future.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMoreover, electrophysiological recordings performed in this study provided convincing evidence for the spatial and temporal precision of the photorelease process. Spatial and temporal precision of molecular photorelease in the brain could represent a breakthrough for treating localised brain diseases, such as epilepsy, stroke, and Parkinson’s disease. For example, the general mechanism of anticonvulsants in epilepsy is to reduce brain excitability, either by lowering excitatory neurotransmission or by increasing inhibitory neurotransmission\u003csup\u003e888888\u003c/sup\u003e. However, in the treatment of focal-onset epilepsies, only the focal area of the brain requires reduced excitability. In contrast, systemic drug administration can be pathological for the rest of the brain. In this context, local targeting of the diseased brain area may be less invasive than systemic drug administration. Furthermore, the approach developed in our study enables a temporal control over drug delivery. This also applies to pathologies such as epilepsy, where drug delivery would be ideally restricted to periods when epileptic seizures are arising\u0026nbsp;\u003csup\u003e28\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn conclusion, precision light-triggered polymersome lysis to release a bio-active cargo represents a promising tool for both decrypting brain mechanisms and developing new therapeutic strategies, particularly considering recent advances in \u003cem\u003ein vivo\u003c/em\u003e photopharmacology devices\u003csup\u003e29,30\u003c/sup\u003e and the growing exploration of polymer-based systems for brain disease research\u003csup\u003e31\u003c/sup\u003e. More broadly, this technology offers a path to closed-loop, light-guided chemical neuromodulation for the precision treatment of brain disorders.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u003cu\u003eFUNDING\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study received financial support from ANR (ANR-24-CE18-5491-01 and ANR-23-CE06-0008), Bordeaux Emergence, Bordeaux INP, Fondation Medisite, and the French government in the framework of the University of Bordeaux\u0026apos;s IdEx \u0026quot;Investments for the Future\u0026quot; program / GPR BRAIN_2030. R.K. is a recipient of Cl\u0026eacute;ment Fayat Foundation and Fondation pour la Recherche M\u0026eacute;dicale fellowships (France).\u003c/p\u003e\n\u003cp\u003eACKNOWLEDGEMENTS\u003c/p\u003e\n\u003cp\u003eThe microscopy was performed at the Bordeaux Imaging Center, a service unit of the CNRS-INSERM and Bordeaux University, and a member of the national infrastructure France BioImaging, supported by the French National Research Agency (ANR-10-INBS-04). The help of J\u0026eacute;r\u0026eacute;mie Teillon and S\u0026eacute;bastien Marais is acknowledged. We thank the CIRCE (Behavioural Engineering Centre) facility of Bordeaux Neurocampus. We also thank Coralie Genevois for technical assistance at the Vivoptic platform, Univ. Bordeaux, CNRS, INSERM, TBM-Core, UAR 3427, US 5, F-33000 Bordeaux. Vivoptic is a France Life Imaging (FLI) labelled platform.\u003c/p\u003e"},{"header":"MATERIAL \u0026 METHODS","content":"\u003cp\u003e\u003cstrong\u003e\u003cu\u003eMaterials:\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePoly(butadiene)-\u003cem\u003eblock\u003c/em\u003e-poly(ethylene oxide) copolymers (PBD\u003csub\u003en\u003c/sub\u003e-\u003cem\u003eb\u003c/em\u003e-PEG\u003csub\u003ep\u003c/sub\u003e) were obtained from Polymer Source, Inc. (Montreal, Canada). The P41745-BdEO polymers used exhibited narrow polydispersity indices (Mw/Mn = 1.01) and consisted of more than 85% 1,2-addition of butadiene units. The hydrophilic fraction accounted for approximately 33%. CNQX disodium salt hydrate, toluene, sucrose, glucose, calcein, and electrophysiology reagents were purchased from Sigma-Aldrich and used as received. Illustrative drawings were made with Biorender and Blender 5.1b.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003eMethods:\u0026nbsp;\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003ePolymersomes synthesis\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEmulsion-Centrifugation.\u003c/strong\u003e Poly(butadiene)-\u003cem\u003eblock\u003c/em\u003e-poly(ethylene oxide) (PBut-\u003cem\u003eb\u003c/em\u003e-PEG\u003csub\u003e,\u003c/sub\u003e 33% hydrophilic fraction) polymersomes were prepared using the emulsion-centrifugation method reported by Peyret et al.\u003csup\u003e17\u003c/sup\u003e. Briefly, 5 µL of 0.30 M sucrose was added to 500 µL of 3 mg/mL PBut-\u003cem\u003eb\u003c/em\u003e-PEG in toluene. The solution was vigorously hand-shaken for 30 seconds to create a water-in-oil emulsion. An interface was prepared by pouring 30 µL of PBut-\u003cem\u003eb\u003c/em\u003e-PEG(3 mg/mL) into toluene containing 30 µL of 0.30 M glucose and allowed to stabilize for 30 minutes. 100 µL of the above emulsion was slowly poured over the interface, and the sample was immediately centrifuged (3 min, 1500 g, ambient temperature). The resulting polymersomes were allowed to recover in the lower phase for several hours. For polymersomes loaded only with calcein, 5 µL of a 10 mM calcein solution in sucrose was used to prepare the emulsion. When polymersomes were loaded with both calcein (10 mM in sucrose) and CNQX (100 µM in sucrose), 2.5 µL of each solution was used to form the emulsion. The glucose concentration was adjusted to match the osmotic pressure (300 mOsm) of the dye and CNQX in the sucrose solution.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePolymersome counting.\u003c/strong\u003e The polymersome solution was homogenized, and the exact volume of the preparation was measured with a P1000 pipette. Counting was performed using an epifluorescence microscope (Eclipse E400) equipped with a pE-300 light source (CoolLED). The LED power is set to 1% to avoid photo-stimulating the polymersomes. Counting was performed using a Fast Read 102 determination slide (Biosigma, BVS100), in which each cell is filled with 7 µL of the polymersome solution. The number of polymersomes is calculated as follows: (Number of polymersomes per µL = Total number of polymersomes/Number of large squares) * dilution factor * 10.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConfocal Observations.\u003c/strong\u003e Laser scanning confocal microscopy images were acquired on an inverted Leica TCS SP5 microscope (Leica Microsystems CMS GmbH, Mannheim, Germany) equipped with an HCXPLA06x, NA 1.4 oil-immersion objective in fluorescence mode. Samples (≈20 μL) were observed on an eight-well μSlide from Ibidi (CliniSciences, 80606). The laser outputs were controlled via the Acousto-Optical Tunable filter (AOTF) and the two collection windows using the Acousto-Optical Beam Splitter (AOBS) and photomultipliers (PMT) as follows: Calcein was excited with an argon laser at 488 nm (10 %) using a 495-555 nm window. The Helium-Neon laser at 633 nm (10 %) was also used in transmission mode. Images were collected using the microscope in sequential mode to avoid emission overlap, with a line average of 4-8 and a 512*512 pixel format. The FRAP wizard, in fly mode for faster time resolution, was used to define regions of interest (ROIs) around the selected vesicles. Processing of fluorescence confocal acquisitions was performed with FIJI (ImageJ v.1.54).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003eBiosafety evaluation \u003cem\u003ein vitro\u003c/em\u003e\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDissection.\u0026nbsp;\u003c/strong\u003ePups (mice P0) were euthanised by decapitation, and the brain was extracted from the skull. The hemispheres were separated, and the hippocampus and meninges were removed. Cortex was cut into small pieces and incubated in papain solution (0.4 mg/mL) for 15 min at 37 °C. DNAse was added and incubated for 5 min at 37 °C. The enzymatic reaction was inactivated with an equal volume of Hibernate supplemented with 10% Fetal Bovine Serum (FBS) (Sigma-Aldrich, F9665). Final dissociation was performed by mechanically pipetting the tissue with a P1000 pipette. Cells were counted with a Luna automated Cell Counter (Logos Biosystems).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePrimary cell culture.\u0026nbsp;\u003c/strong\u003ePlates were incubated with coating solution containing Poly-L-Lysine (1 mg/mL) (Sigma, P2636-1G) and Laminin (3 µg/mL) (Sigma, L2020) for at least 2 h at 37 °C. All culture media were equilibrated beforehand for at least 4 h at 37 °C. Cells were seeded in seeding medium (Neurobasal-A (ThermoFischer, 12349015) supplemented with B27 plus (ThermoFisher, A3582801) and 10% FBS at 50k per well in a 96-well plate. The medium was fully replaced 1 h post-seeding to remove debris with maintenance medium (Neurobasal-A supplemented with B27 plus and 1.5% of FBS). Culture was maintained every 3-4 days by replacing half of the medium with maintenance medium, until day 14.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBiosafety evaluation in primary cell culture.\u0026nbsp;\u003c/strong\u003eCell viability was assessed using the MTT assay (Thermofisher, V13154), with P0 mice used to obtain a cell suspension from the cortex. Cells were seeded at 50k per well in 96-well plates and incubated for 14 days at 37 °C, 5% CO\u003csub\u003e2\u003c/sub\u003e. At maturity of the culture, two biosafety valuations were conducted, the polymersomes concentrations, respectively, 0, 600, 1500, 3000, 6000, 9000 per well and light power (0.003, 3.5, 8.7, 19.3, 30 mw), then incubated for 48 h. Half of the conditions were photo-stimulated at 3.5 mw for 20 s using a 400\u0026nbsp;mm optical fiber with a numerical aperture of 0.5 (ThorLabs, M98L01) to assess the toxicity of polymersomes post-stimulation. Post-incubation medium was fully changed with fresh medium, and MTT reagent (10 µL, final concentration 0.5\u0026nbsp;mg/mL) was added to each well and incubated at 37\u0026nbsp;°C, inducing the formation of formazan crystals. SDS-HCl (100 µL, final concentration 50\u0026nbsp;mg/mL) was added to each well to solubilize formazan, and the mixture was incubated for 10 h at 37\u0026nbsp;°C. Absorbance was measured at 570 nm in a microplate reader. All conditions were tested in triplicate.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003eBiosafety evaluation \u003cem\u003ein vivo\u003c/em\u003e\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimals.\u003c/strong\u003e Adult male and female C57BL/6J mice were obtained from Janvier Labs (France). All procedures were carried out in accordance with the European Communities Council Directive 2010/63/EU and approved by the French National Committee for animal experimentation (authorization APAFIS #20925 and #50835). Mice were housed under standard laboratory conditions in a temperature- and relative humidity-controlled environment and a 12-hour light/dark cycle (lights on at 07:00). Animals were group-housed, and food and water were provided ad libitum.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStereotactic Injections.\u003c/strong\u003e Stereotaxic injections were performed in adult male and female mice under isoflurane deep anaesthesia. Animals were placed in a stereotaxic apparatus and maintained at 37 °C using a heating pad. A craniotomy was performed above the target region. The polymersomes were injected using a Hamilton syringe with a 34G blunt needle, connected to a nanoliter injector (World Precision Instruments, Nanofil) at a flow rate of 0.5 µL/min. Injections were performed into the CA1 region of the hippocampus, following stereotaxic coordinates relative to bregma: AP = –2.0 mm, ML = ±1.3 mm, DV = –1.2 mm. A volume of 2 µL of calcein-free polymersomes was injected per site. The pipette was left in place for 5 minutes after injection to minimise reflux. After surgery, the animals were monitored daily for 3 days to ensure their recovery.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTissue collection and immunofluorescence.\u003c/strong\u003e Animals were injected with buprenorphine (0.1 mg/kg, s.c.) (Virbac) and deeply anesthetized with Euthasol (400 mg pentobarbital/kg, ip) (Dechra) 30 min later. Animals were transcardially perfused with phosphate-buffered saline (PBS), followed by 4% paraformaldehyde (PFA) (Sigma-Aldrich). Brains were extracted, post-fixed in 4% PFA at 4 °C for 48 h, and subsequently transferred to a 30% sucrose solution at 4 °C for cryoprotection. Coronal brain sections (40 µm thick) were obtained using a vibratome (Leica) and stored at −20 °C in a cryoprotective solution composed of 20% glycerol and 30% ethylene glycol (Sigma-Aldrich) in PBS.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor immunofluorescence, brain slices containing the injected zone were washed 3 times in PBS (EuroMedex, ET330), then incubated in blocking solution (10% donkey serum, 0.5 % Triton X100) before incubation overnight in blocking solution containing the primary antibody (GFAP: DAKO Z033429-2 1/1000; IBA1: Fujifilm 019-19741, 1/500). The next day, slices were washed 4 times in PBS, then incubated in blocking solution containing a secondary antibody (Donkey anti-rabbit-Alexa 488, Abcam) for 2 hours. After 3 washes in PBS, slices were mounted on glass slides, dried, and finally mounted with medium DAPI Fluoromount G (EuroMedex, EM17984-24) and coverslipped. Observations and photos were made with a widefield fluorescent microscope (DM5000, Leica, Bordeaux Imaging Center). Photos were analysed with FIJI (ImageJ v.1.54). The area of interest around the injection site was manually defined, a threshold was applied, and particles were detected automatically. The mean intensity of particles, weighted by the size of each particle, was measured for each image. For each animal, 3-5 images were analysed.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e\u003cu\u003eEx vivo\u003c/u\u003e\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cu\u003e\u0026nbsp;experiments\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eElectrophysiological Recordings. \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHippocampal slice preparation.\u003c/strong\u003e Mice were anaesthetized with isoflurane, then decapitated, and the brain was quickly extracted from the skull. Coronal hippocampal slices with a 5° angle (350 µm thick) were prepared using a vibrating blade microtome (VT1000S, Leica Microsystems) in ice-cold, cutting high-sucrose artificial cerebral spinal fluid (HS-ACSF) solution containing (in mM): 125 NaCl, 25 NaHCO₃, 25 glucose, 2.5 KCl, 2 CaCl₂, 1 MgCl₂, 1.25 NaH₂PO₄, 3 pyruvate, and 75 sucrose, continuously oxygenated with 95% O\u003csub\u003e2\u003c/sub\u003e/5%\u0026nbsp;CO\u003csub\u003e2\u003c/sub\u003e. Slices were incubated at 34 °C for 1 h, then maintained at room temperature. Recordings were initiated after at least 1 h of recovery. Recording ACSF (used for both storage and recording) contained (in mM): 125 NaCl, 25 NaHCO₃, 25 glucose, 2.5 KCl, 2 CaCl₂, 1 MgCl₂, and 1.25 NaH₂PO₄, and was continuously bubbled with 95 % O\u003csub\u003e2\u0026nbsp;\u003c/sub\u003e/ 5 % CO\u003csub\u003e2\u003c/sub\u003e. All ACSF components were purchased from Sigma-Aldrich.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eElectrophysiological recordings.\u003c/strong\u003e\u0026nbsp; \u0026nbsp;Recordings were performed at \u003cstrong\u003e30 °C\u003c/strong\u003e using a temperature-controlled system (Warner Instruments, TC-324B). Hippocampal slices were continuously superfused with oxygenated ACSF at a rate of 3–4 mL/min using a peristaltic pump (Gilson, Minipulse 3). Patch pipettes (4–6 MΩ), pulled from borosilicate glass capillaries (Sutter Instruments, GBF-150-117-10) with a horizontal puller (Sutter Instruments, P-97), were used for intracellular recordings.\u003c/p\u003e\n\u003cp\u003eFor extracellular recordings, borosilicate glass pipettes filled with ACSF were placed in the stratum radiatum of the CA1 region to record field excitatory postsynaptic potentials (fEPSPs). Synaptic responses were evoked by stimulation of Schaffer collateral afferents from the CA3 region using a bipolar concentric electrode (Phymep) connected to a stimulator (World Precision Instruments, A365).\u003c/p\u003e\n\u003cp\u003ePolymersomes were injected directly into the slice using borosilicate glass pipettes. Photostimulation of polymersomes was performed under an upright fluorescence microscope (Nikon FN1) equipped with a 40× water-immersion objective. Fluorescence signal was detected using a pE-300 light source (CoolLED) and a CCD camera (INFINITY 3S-1UR M/C).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData acquisition and analysis\u003c/strong\u003e. Signals were amplified using a MultiClamp 700B amplifier (Molecular Devices) and digitized via a Digidata 1440B interface (Molecular Devices), controlled by pClamp 10.7 software. Data were analysed offline using Clampfit 11.2 (Molecular Devices).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis.\u003c/strong\u003e For all data generated, statistical analyses were performed using GraphPad Prism 10 or 11. Normality was assessed using the Anderson–Darling, D’Agostino–Pearson, Shapiro–Wilk, and Kolmogorov–Smirnov tests. Depending on the data structure, comparisons were made using t-tests (paired or unpaired, parametric or nonparametric), one-way or two-way ANOVA, with appropriate post hoc tests. All tests were two-tailed, and p \u0026lt; 0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"REFERENCES ","content":"\u003col\u003e\n\u003cli\u003eMuir, J., Anguiano, M. \u0026amp; Kim, C. K. 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Polymer-based nanomedicines: Supporting multimodal approaches to glioblastoma Multiforme treatment. \u003cem\u003eAdvanced Drug Delivery Reviews\u003c/em\u003e 115735 (2025) doi:10.1016/j.addr.2025.115735.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"polymersomes, brain, photostimulation, drug delivery","lastPublishedDoi":"10.21203/rs.3.rs-9232875/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9232875/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Neurological diseases remain a leading cause of disability and mortality, in part because systemically administered therapies poorly access diseased brain regions and lack spatiotemporal control. While innovative nanotechnologies offer stable and versatile carriers for drug delivery, they do not inherently enable localized, on-demand release. In parallel, optical neurotechnologies provide precise control of brain activity but cannot deliver bioactive molecules. Here, we bridge these approaches by developing photoactivatable vesicles based on polymeric amphiphiles (polymersomes) that enable light-triggered, spatially confined release of encapsulated compounds in brain tissue. We assessed their safety in primary cell cultures and in vivo. Through the photorelease of CNQX, a competitive AMPA/kainate receptor antagonist, we demonstrated the stability and precise spatiotemporal control of molecular delivery. This work establishes a platform for optically-guided chemical neuromodulation. Beyond applications in basic and preclinical neuroscience, this strategy opens new avenues for targeted therapies in localized brain disorders, including glioblastoma.","manuscriptTitle":"Spatiotemporal control of molecular delivery in the brain using photoactivable polymersomes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-08 06:26:16","doi":"10.21203/rs.3.rs-9232875/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9a8de46a-9078-410d-9d08-67bec9e613ec","owner":[],"postedDate":"April 8th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":65886652,"name":"Biological sciences/Neuroscience"},{"id":65886653,"name":"Biological sciences/Chemical biology"}],"tags":[],"updatedAt":"2026-04-13T15:52:17+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-08 06:26:16","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9232875","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9232875","identity":"rs-9232875","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}