Spontaneous persistent activity and inactivity in vivo reveals differential cortico-entorhinal functional connectivity.

Understanding the functional connectivity between brain regions and its emergent dynamics is a central challenge. Here we present a theory-experiment hybrid approach involving iteration between a minimal computational model and in vivo electrophysiological measurements. Our model not only predicted spontaneous persistent activity (SPA) during Up-Down-State oscillations, but also inactivity (SPI), which has never been reported. These were confirmed in vivo in the membrane potential of neurons, especially from layer 3 of the medial and lateral entorhinal cortices. The data was then used to constrain two free parameters, yielding a unique, experimentally determined model for each neuron. Analytic and computational analysis of the model generated a dozen quantitative predictions about network dynamics, which were all confirmed in vivo to high accuracy. Our technique predicted functional connectivity; e. g. the recurrent excitation is stronger in the medial than lateral entorhinal cortex. This too was confirmed with connectomics data. This technique uncovers how differential cortico-entorhinal dialogue generates SPA and SPI, which could form an energetically efficient working-memory substrate and influence the consolidation of memories during sleep. More broadly, our procedure can reveal the functional connectivity of large networks and a theory of their emergent dynamics.

Molecular Simulations Reveal the Free Energy Landscape and Transition State of Membrane Electroporation.

The formation of pores over lipid membranes by the application of electric fields, termed membrane electroporation, is widely used in biotechnology and medicine to deliver drugs, vaccines, or genes into living cells. Continuum models for describing the free energy landscape of membrane electroporation were proposed decades ago, but they have never been tested against spatially detailed atomistic models. Using molecular dynamics (MD) simulations with a recently proposed reaction coordinate, we computed potentials of mean force of pore nucleation and pore expansion in lipid membranes at various transmembrane potentials. Whereas the free energies of pore expansion are compatible with previous continuum models, the experimentally important free energy barrier of pore nucleation is at variance with established models. The discrepancy originates from different geometries of the transition state; previous continuum models assumed the presence of a membrane-spanning defect throughout the process, whereas, according to the MD simulations, the transition state of pore nucleation is typically passed before a transmembrane defect has formed. A modified continuum model is presented that qualitatively agrees with the MD simulations. Using kinetics of pore opening together with transition state theory, our free energies of pore nucleation are in excellent agreement with previous experimental data.

The antimicrobial fibupeptide lugdunin forms water-filled channel structures in lipid membranes.

Recently, a novel cyclo-heptapeptide composed of alternating D,L-amino acids and a unique thiazolidine heterocycle, called lugdunin, was discovered, which is produced by the nasal and skin commensal Staphylococcus lugdunensis. Lugdunin displays potent antimicrobial activity against a broad spectrum of Gram-positive bacteria, including challenging-to-treat methicillin-resistant Staphylococcus aureus (MRSA). Lugdunin specifically inhibits target bacteria by dissipating their membrane potential. However, the precise mode of action of this new class of fibupeptides remains largely elusive. Here, we disclose the mechanism by which lugdunin rapidly destabilizes the bacterial membrane potential using an in vitro approach. The peptide strongly partitions into lipid compositions resembling Gram-positive bacterial membranes but less in those harboring the eukaryotic membrane component cholesterol. Upon insertion, lugdunin forms hydrogen-bonded antiparallel ß-sheets by the formation of peptide nanotubes, as demonstrated by ATR-FTIR spectroscopy and molecular dynamics simulations. These hydrophilic nanotubes filled with a water wire facilitate not only the translocation of protons but also of monovalent cations as demonstrated by voltage-clamp experiments on black lipid membranes. Collectively, our results provide evidence that the natural fibupeptide lugdunin acts as a peptidic channel that is spontaneously formed by an intricate stacking mechanism, leading to the dissipation of a bacterial cell's membrane potential.

Acid-Sensitive Outwardly Rectifying Cl- Current in OV2944 Mouse Ovarian Cancer Cells.

BACKGROUND/AIMS: Extracellular acidic conditions impair cellular activities; however, some cancer cells drive cellular signaling to adapt to the acidic environment. It remains unclear how ovarian cancer cells sense changes in extracellular pH. This study was aimed at characterizing acid-inducible currents in an ovarian cancer cell line and evaluating the involvement of these currents in cell viability. METHODS: The biophysical and pharmacological properties of membrane currents in OV2944, a mouse ovarian cancer cell line, were studied using the whole-cell configuration of the patch-clamp technique. Viability of this cell type in acidic medium was evaluated using the MTT assay. RESULTS: OV2944 had significant acid-sensitive outwardly rectifying (ASOR) Cl- currents at a pH50 of 5.3. The ASOR current was blocked by pregnenolone sulfate (PS), a steroid ion channel modulator that blocks the ASOR channel as one of its targets. The viability of the cells was reduced after exposure to an acidic medium (pH 5.3) but was slightly restored upon PS administration. CONCLUSION: These results offer first evidence for the presence of ASOR Cl- channel in ovarian cancer cells and indicate its involvement in cell viability under acidic environment.

Optical recordings of organellar membrane potentials and the components of membrane conductance in lysosomes.

The eukaryotic cell is highly compartmentalized with organelles. Owing to their function in transporting metabolites, metabolic intermediates and byproducts of metabolic activity, organelles are important players in the orchestration of cellular function. Recent advances in optical methods for interrogating the different aspects of organellar activity promise to revolutionize our ability to dissect cellular processes with unprecedented detail. The transport activity of organelles is usually coupled to the transport of charged species; therefore, it is not only associated with the metabolic landscape but also entangled with membrane potentials. In this context, the targeted expression of fluorescent probes for interrogating organellar membrane potential (Ψorg) emerges as a powerful approach, offering less-invasive conditions and technical simplicity to interrogate cellular signalling and metabolism. Different research groups have made remarkable progress in adapting a variety of optical methods for measuring and monitoring Ψorg. These approaches include using potentiometric dyes, genetically encoded voltage indicators, hybrid fluorescence resonance energy transfer sensors and photoinduced electron transfer systems. These studies have provided consistent values for the resting potential of single-membrane organelles, such as lysosomes, the Golgi and the endoplasmic reticulum. We can foresee the use of dynamic measurements of Ψorg to study fundamental problems in organellar physiology that are linked to serious cellular disorders. Here, we present an overview of the available techniques, a survey of the resting membrane potential of internal membranes and, finally, an open-source mathematical model useful to interpret and interrogate membrane-bound structures of small volume by using the lysosome as an example.

In Vivo Whole-Cell Recording from the Mouse Brain.

Measuring the membrane potential dynamics of neurons offers a comprehensive understanding of the molecular and cellular mechanisms that form their spiking activity, thus playing a crucial role in unraveling the mechanistic processes governing brain function. Techniques for intracellular recordings of membrane potentials pioneered in the 1940s have witnessed significant advancements since their inception. Among these, whole-cell patch-clamp recording has emerged as a leading method for measuring neuronal membrane potentials due to its high stability and broad applicability ranging from cultured cells to brain slices and even behaving animals. This chapter provides a detailed protocol to acquire stable whole-cell recordings from neurons in the cerebral cortex of awake, head-restrained mice. Significant enhancements to our protocol include implanting a metal head-post using adhesive resin cement and preparing a recording pipette with a long shank for targeting deeper brain regions. This protocol, once implemented, enables whole-cell recordings up to 2.5 mM beneath the cortical surface.

Enhanced Methodologies for Investigating the Electrophysiological Characteristics of Endogenous Pannexin 1 Intercellular Cell-Cell Channels.

Gap junctions, pivotal intercellular conduits, serve as communication channels between adjacent cells, playing a critical role in modulating membrane potential distribution across cellular networks. The family of Pannexin (Panx) proteins, in particular Pannexin1 (Panx1), are widely expressed in vertebrate cells and exhibit sequence homology with innexins, the invertebrate gap junction channel constituents. Despite being ubiquitously expressed, detailed functional and pharmacological properties of Panx1 intercellular cell-cell channels require further investigation. In this chapter, we introduce optimized cell culture methodologies and electrophysiology protocols to expedite the exploration of endogenous Panx1 cell-cell channels in TC620 cells, a human oligodendroglioma cell line that naturally expresses Panx1. We anticipate these refined protocols will significantly contribute to future characterizations of Panx1-based intercellular cell-cell channels across diverse cell types and offer valuable insights into both normal cellular physiology and pathophysiology.

Cloning, functional expression, and pharmacological characterization of inwardly rectifying potassium channels (Kir) from Apis mellifera.

Potassium channels belong to the super family of ion channels and play a fundamental role in cell excitability. Kir channels are potassium channels with an inwardly rectifying property. They play a role in setting the resting membrane potential of many excitable cells including neurons. Although putative Kir channel family genes can be found in the Apis mellifera genome, their functional expression, biophysical properties, and sensitivity to small molecules with insecticidal activity remain to be investigated. We cloned six Kir channel isoforms from Apis mellifera that derive from two Kir genes, AmKir1 and AmKir2, which are present in the Apis mellifera genome. We studied the tissue distribution, the electrophysiological and pharmacological characteristics of three isoforms that expressed functional currents (AmKir1.1, AmKir2.2, and AmKir2.3). AmKir1.1, AmKir2.2, and AmKir2.3 isoforms exhibited distinct characteristics when expressed in Xenopus oocytes. AmKir1.1 exhibited the largest potassium currents and was impermeable to cesium whereas AmKir2.2 and AmKir2.3 exhibited smaller currents but allowed cesium to permeate. AmKir1 exhibited faster opening kinetics than AmKir2. Pharmacological experiments revealed that both AmKir1.1 and AmKir2.2 are blocked by the divalent ion barium, with IC50 values of 10-5 and 10-6 M, respectively. The concentrations of VU041, a small molecule with insecticidal properties required to achieve a 50% current blockade for all three channels were higher than those needed to block Kir channels in other arthropods, such as the aphid Aphis gossypii and the mosquito Aedes aegypti. From this, we conclude that Apis mellifera AmKir channels exhibit lower sensitivity to VU041.

A potent and selective activator of large-conductance Ca2+-activated K+ channels induces preservation of mitochondrial function after hypoxia and reoxygenation by handling of calcium and transmembrane potential.

AIMS: Ischaemic heart disease remains a significant cause of mortality globally. A pharmacological agent that protects cardiac mitochondria against oxygen deprivation injuries is welcome in therapy against acute myocardial infarction. Here, we evaluate the effect of large-conductance Ca2+-activated K+ channels (BKCa) activator, Compound Z, in isolated mitochondria under hypoxia and reoxygenation. METHODS: Mitochondria from mice hearts were obtained by differential centrifugation. The isolated mitochondria were incubated with a BKCa channel activator, Compound Z, and subjected to normoxia or hypoxia/reoxygenation. Mitochondrial function was evaluated by measurement of O2 consumption in the complexes I, II, and IV in the respiratory states 1, 2, 3, and by maximal uncoupled O2 uptake, ATP production, ROS production, transmembrane potential, and calcium retention capacity. RESULTS: Incubation of isolated mitochondria with Compound Z under normoxia conditions reduced the mitochondrial functions and induced the production of a significant amount of ROS. However, under hypoxia/reoxygenation, the Compound Z prevented a profound reduction in mitochondrial functions, including reducing ROS production over the hypoxia/reoxygenation group. Furthermore, hypoxia/reoxygenation induced a large mitochondria depolarization, which Compound Z incubation prevented, but, even so, Compound Z created a small depolarization. The mitochondrial calcium uptake was prevented by the BKCa activator, extruding the mitochondrial calcium present before Compound Z incubation. CONCLUSION: The Compound Z acts as a mitochondrial BKCa channel activator and can protect mitochondria function against hypoxia/reoxygenation injury, by handling mitochondrial calcium and transmembrane potential.

Modulation of bacterial membranes and cellular macromolecules by dimethyl sulfoxide: A dose-dependent study providing novel insights.

Using Escherichia coli as a model, this manuscript delves into the intricate interactions between dimethyl sulfoxide (DMSO) and membranes, cellular macromolecules, and the effects on various aspects of bacterial physiology. Given DMSO's wide-ranging use as a solvent in microbiology, we investigate the impacts of both non-growth inhibitory (1.0 % and 2.5 % v/v) and slightly growth-inhibitory (5.0 % v/v) concentrations of DMSO. The results demonstrate that DMSO causes alterations in bacterial membrane potential, influences the electrochemical characteristics of the cell surface, and exerts substantial effects on the composition and structure of cellular biomolecules. Genome-wide gene expression data from DMSO-treated E. coli was used to further investigate and bolster the results. The findings of this study provide valuable insights into the complex relationship between DMSO and biological systems, with potential implications in drug delivery and cellular manipulation. However, it is essential to exercise caution when utilizing DMSO to enhance the solubility and delivery of bioactive compounds, as even at low concentrations, DMSO exerts non-inert effects on cellular macromolecules and processes.

Insight into the function of voltage-sensing phosphatase in hindgut-derived pseudoplacenta of a viviparous teleost Xenotoca eiseni.

Nutrient absorption is essential for animal survival and development. Our previous study on zebrafish reported that nutrient absorption in lysosome-rich enterocytes (LREs) is promoted by the voltage-sensing phosphatase (VSP), which regulates phosphoinositide (PIP) homeostasis via electrical signaling in biological membranes. However, it remains unknown whether this VSP function is shared by different absorptive tissues in other species. Here, we focused on the function of VSP in a viviparous teleost Xenotoca eiseni, whose intraovarian embryos absorb nutrients from the maternal ovarian fluid through a specialized hindgut-derived pseudoplacental structure called trophotaenia. Xenotoca eiseni VSP (Xe-VSP) is expressed in trophotaenia epithelium, an absorptive tissue functionally similar to zebrafish LREs. Notably, the apical distribution of Xe-VSP in trophotaenia epithelial cells closely resembles zebrafish VSP (Dr-VSP) distribution in zebrafish LREs, suggesting a shared role for VSP in absorptive tissues between the two species. Electrophysiological analysis using a heterologous expression system revealed that Xe-VSP preserves functional voltage sensors and phosphatase activity with the leftward shifted voltage sensitivity compared with zebrafish VSP (Dr-VSP). We also identified a single amino acid variation in the S4 helix of Xe-VSP as one of the factors contributing to the leftward shifted voltage sensitivity. This study highlights the biological variation and significance of VSP in various animal species, as well as hinting at the potential role of VSP in nutrient absorption in X. eiseni trophotaenia.NEW & NOTEWORTHY We investigate the voltage-sensing phosphatase (VSP) in Xenotoca eiseni, a viviparous fish whose intraovarian embryos utilize trophotaenia for nutrient absorption. Although X. eiseni VSP (Xe-VSP) shares key features with known VSPs, its distinct voltage sensitivity arises from species-specific amino acid variation. Xe-VSP in trophotaenia epithelium suggests its involvement in nutrient absorption, similar to VSP in zebrafish enterocytes and potentially in species with similar absorptive cells. Our findings highlight the potential role of VSP across species.

Structural change of the cytoplasmic N-terminus and S1 segment of voltage-sensing phosphatase reported by Anap.

BACKGROUND: Voltage-sensing phosphatase contains a structurally conserved S1-S4-based voltage-sensor domain, which undergoes a conformational transition in response to membrane potential change. Unlike that of channels, it is functional even in isolation and is therefore advantageous for studying the transition mechanism, but its nature has not yet been fully elucidated. This study aimed to address whether the cytoplasmic N-terminus and S1 exhibit structural change. METHODS: Anap, an environment-sensitive unnatural fluorescent amino acid, was site-specifically introduced to the voltage sensor domain to probe local structural changes by using oocyte voltage clamp and photometry. Tetramethylrhodamine was also used to probe some extracellularly accessible positions. In total, 51 positions were investigated. RESULTS: We detected robust voltage-dependent signals from widely distributed positions including N-terminus and S1. In addition, response to hyperpolarization was observed at the extracellular end of S1, reflecting the local structure flexibility of the voltage-sensor domain in the down-state. We also found that the mechanical coupling between the voltage-sensor and phosphatase domains affects the depolarization-induced optical signals but not the hyperpolarization-induced signals. CONCLUSIONS: These results fill a gap between the previous interpretations from the structural and biophysical approaches and should provide important insights into the mechanisms of the voltage-sensor domain transition as well as its coupling with the effector.

Dynamic duo: Kir6 and SUR in KATP channel structure and function.

KATP channels are ligand-gated potassium channels that couple cellular energetics with membrane potential to regulate cell activity. Each channel is an eight subunit complex comprising four central pore-forming Kir6 inward rectifier potassium channel subunits surrounded by four regulatory subunits known as the sulfonylurea receptor, SUR, which confer homeostatic metabolic control of KATP gating. SUR is an ATP binding cassette (ABC) protein family homolog that lacks membrane transport activity but is essential for KATP expression and function. For more than four decades, understanding the structure-function relationship of Kir6 and SUR has remained a central objective of clinical significance. Here, we review progress in correlating the wealth of functional data in the literature with recent KATP cryoEM structures.

Orientation and Membrane Partition Free Energy of PeT-Based Voltage-Sensitive Dyes from Molecular Simulations.

Voltage measurement via small-molecule fluorescent indicators is a valuable approach in deciphering complex dynamics in electrically excitable cells. However, our understanding of various physicochemical properties governing the performance of fluorescent voltage sensors based on the photoinduced electron transfer (PeT) mechanism remains incomplete. Here, through extensive molecular dynamics and free energy calculations, we systematically examine the orientation and membrane partition of three PeT-based voltage-sensing VoltageFluor (VF) dyes in different lipid environment. We show that the symmetry of the molecular scaffold and the net charge of the hydrophilic headgroup of a given VF dye dominate its orientation and membrane partition, respectively. Our work provides a mechanistic understanding of the physical properties contributing to the voltage sensitivity, signal-to-noise ratio, as well as membrane distribution of VF dyes and sheds light onto rational design principles of PeT-based fluorescent probes in general.

Superstructured Optoionic Heterojunctions for Promoting Ion Pumping Inspired by Photoreceptor Cells.

Photoreceptor cells of vertebrates feature ultrastructural membranes interspersed with abundant photosensitive ion pumps to boost signal generation and realize high gain in dim light. In light of this, superstructured optoionic heterojunctions (SSOHs) with cation-selective nanochannels are developed for manipulating photo-driven ion pumping. A template-directed bottom-up strategy is adopted to sequentially assemble graphene oxide (GO) and PEDOT:PSS into heterogeneous membranes with sculptured superstructures, which feature programmable variation in membrane topography and contain a donor-acceptor interface capable of maintaining electron-hole separation upon photoillumination. Such elaborate design endows SSOHs with a much higher magnitude of photo-driven ion flux against a concentration gradient in contrast to conventional optoionic membranes with planar configuration. This can be ascribed to the buildup of an enhanced transmembrane potential owing to the effective separation of photogenerated carriers at the heterojunction interface and the increase of energy input from photoillumination due to a synergistic effect of reflection reduction, broad-angle absorption, and wide-waveband absorption. This work unlocks the significance of membrane topographies in photo-driven transmembrane transportation and proposes such a universal prototype that could be extended to other optoionic membranes to develop high-performance artificial ion pumps for energy conversion and sensing.

TRPM5 activation depends on a synergistic effect of calcium and PKC phosphorylation.

Transient receptor potential melastatin 5 (TRPM5) is a calcium-activated monovalent-specific ion channel involved in insulin secretion and taste transduction, making it an attractive target for drug development in various pathologies. While TRPM5 activation involves ligand binding to Gq/G-protein coupled receptors (GPCR) and subsequent elevation of intracellular calcium levels, recent reports suggest the need for additional molecular determinants. Hence, the mechanism of TRPM5 activation remains to be elucidated. Here, we show that PKC phosphorylation and the elevation of intracellular Ca2+ levels are required for TRPM5 activation, with PKC phosphorylation being crucial for channel-evoked currents, primarily at physiological membrane potentials. In contrast, physiological relevant calcium levels alone only induce TRPM5 activation at positive voltages. Our findings highlight the necessity of coordinated intracellular calcium release and PKC phosphorylation for TRPM5 activation. Thus, our results suggest that regulation of PKC activity could be a promising therapeutic target for diseases associated with TRPM5 modulation.

Dynamics of Mitochondrial DNA Copy Number and Membrane Potential in Mouse Pre-Implantation Embryos: Responses to Diverse Types of Oxidative Stress.

Mitochondria undergo a myriad of changes during pre-implantation embryo development, including shifts in activity levels and mitochondrial DNA (mtDNA) replication. However, how these distinct aspects of mitochondrial function are linked and their responsiveness to diverse stressors is not well understood. Here, we show that mtDNA content increased between 8-cell embryos and the blastocyst stage, with similar copy numbers per cell in the inner cell mass (ICM) and trophectoderm (TE). In contrast, mitochondrial membrane potential (MMP) was higher in TE than ICM. Culture in ambient oxygen (20% O2) altered both aspects of mitochondrial function: the mtDNA copy number was upregulated in ICM, while MMP was diminished in TE. Embryos cultured in 20% O2 also exhibited delayed development kinetics, impaired implantation, and reduced mtDNA levels in E18 fetal liver. A model of oocyte mitochondrial stress using rotenone showed only a modest effect on on-time development and did not alter the mtDNA copy number in ICM; however, following embryo transfer, mtDNA was higher in the fetal heart. Lastly, endogenous mitochondrial dysfunction, induced by maternal age and obesity, altered the blastocyst mtDNA copy number, but not within the ICM. These results demonstrate that mitochondrial activity and mtDNA content exhibit cell-specific changes and are differentially responsive to diverse types of oxidative stress during pre-implantation embryogenesis.

Solute Transport through Mitochondrial Porins In Vitro and In Vivo.

Mitochondria are most likely descendants of strictly aerobic prokaryotes from the class Alphaproteobacteria. The mitochondrial matrix is surrounded by two membranes according to its relationship with Gram-negative bacteria. Similar to the bacterial outer membrane, the mitochondrial outer membrane acts as a molecular sieve because it also contains diffusion pores. However, it is more actively involved in mitochondrial metabolism because it plays a functional role, whereas the bacterial outer membrane has only passive sieving properties. Mitochondrial porins, also known as eukaryotic porins or voltage-dependent anion-selective channels (VDACs) control the permeability properties of the mitochondrial outer membrane. They contrast with most bacterial porins because they are voltage-dependent. They switch at relatively small transmembrane potentials of 20 to 30 mV in closed states that exhibit different permeability properties than the open state. Whereas the open state is preferentially permeable to anionic metabolites of mitochondrial metabolism, the closed states prefer cationic solutes, in particular, calcium ions. Mitochondrial porins are encoded in the nucleus, synthesized at cytoplasmatic ribosomes, and post-translationally imported through special transport systems into mitochondria. Nineteen beta strands form the beta-barrel cylinders of mitochondrial and related porins. The pores contain in addition an α-helical structure at the N-terminal end of the protein that serves as a gate for the voltage-dependence. Similarly, they bind peripheral proteins that are involved in mitochondrial function and compartment formation. This means that mitochondrial porins are localized in a strategic position to control mitochondrial metabolism. The special features of the role of mitochondrial porins in apoptosis and cancer will also be discussed in this article.

Mean-field method for generic conductance-based integrate-and-fire neurons with finite timescales.

The construction of transfer functions in theoretical neuroscience plays an important role in determining the spiking rate behavior of neurons in networks. These functions can be obtained through various fitting methods, but the biological relevance of the parameters is not always clear. However, for stationary inputs, such functions can be obtained without the adjustment of free parameters by using mean-field methods. In this work, we expand current Fokker-Planck approaches to account for the concurrent influence of colored and multiplicative noise terms on generic conductance-based integrate-and-fire neurons. We reduce the resulting stochastic system through the application of the diffusion approximation to a one-dimensional Langevin equation. An effective Fokker-Planck is then constructed using Fox Theory, which is solved numerically using a newly developed double integration procedure to obtain the transfer function and the membrane potential distribution. The solution is capable of reproducing the transfer function and the stationary voltage distribution of simulated neurons across a wide range of parameters. The method can also be easily extended to account for different sources of noise with various multiplicative terms, and it can be used in other types of problems in principle.

In vitro and in vivo characterization of human serum albumin-based PEGylated nanoparticles for BDNF and NT3 codelivery.

The utilization of neurotrophins in medicine shows significant potential for addressing neurodegenerative conditions, such as age-related macular degeneration (AMD). However, the therapeutic use of neurotrophins has been restricted due to their short half-life. Here, we aimed to synthesize PEGylated nanoparticles based on electrostatic-driven interactions between human serum albumin (HSA), a carrier for adsorption; neurotrophin-3 (NT3); and brain-derived neurotrophic factor (BDNF). Electrophoretic (ELS) and multi-angle dynamic light scattering (MADLS) revealed that the PEGylated HSA-NT3-BDNF nanoparticles ranged from 10 to 430 nm in diameter and exhibited a low polydispersity index (<0.4) and a zeta potential of -8 mV. Based on microscale thermophoresis (MST), the estimated dissociation constant (Kd) from the HSA molecule of BDNF was 1.6 µM, and the Kd of NT3 was 732 µM. The nanoparticles were nontoxic toward ARPE-19 and L-929 cells in vitro and efficiently delivered BDNF and NT3. Based on the biodistribution of neurotrophins after intravitreal injection into BALB/c mice, both nanoparticles were gradually released in the mouse vitreous body within 28 days. PEGylated HSA-NT3-BDNF nanoparticles stabilize neurotrophins and maintain this characteristic in vivo. Thus, given the simplicity of the system, the nanoparticles may enhance the treatment of a variety of neurological disorders in the future.