A Transdermal Prion‐Bionics Supermolecule as a RAB3A Antagonist for Enhancing Facial Youthfulness

Abstract The mechanism research of skin wrinkles, conducted on volunteers underwent high‐intensity desk work and mice subjected to partial sleep deprivation, revealed a significant reduction in dermal thickness associated with the presence of wrinkles. This can be attributed to the activation of facial nerves in a state of hysteria due to an abnormally elevated interaction between SNAP25 and RAB3A proteins involved in the synaptic vesicle cycle (SVC). Facilitated by AI‐assisted structural design, a refined peptide called RSI pep is developed to modulate this interaction and normalize SVC. Drawing inspiration from prions, which possess the ability to protect themselves against proteolysis and invade neighboring nerve cells through macropinocytosis, RSI pep is engineered to demonstrate a GSH‐responsive reversible self‐assembly into a prion‐like supermolecule (RSI prion). RSI prion showcases protease resistance, micropinocytosis‐dependent cellular internalization, and low adhesion with constituent molecules in the cuticle, thereby endowing it with the transdermic absorption and subsequent biofunction in redressing the frenzied SVC. As a facial mud mask, it effectively reduces periorbital and perinasal wrinkles in the human face. Collectively, RSI prion not only presents a clinical potential as an anti‐wrinkle prion‐like supermolecule, but also exemplifies a reproducible instance of bionic strategy‐guided drug development that bestows transdermal ability upon the pharmaceutical molecule.


Introduction
As the fundamental building blocks of living organisms, protein-protein interactions (PPIs) are intricately involved in nearly every biological process and represent a prime target for therapeutic intervention. [1]The synaptic vesicle cycle (SVC) at nerve terminals is an exemplary protein-protein interaction network that orchestrates neurotransmitter release and recovery through the processes of vesicle exocytosis, endocytosis of depleted vesicles, and regeneration of fresh ones. [2]o address the persistent and imperative therapeutic needs in diseases resulting from excessive activation of peripheral nerves, including but not limited to neurodermatitis, facial wrinkles caused by nerve tension, and involuntary muscle contractions in certain areas of the face, various efforts have emerged in the exploration and development of multiple agents such as halothane anesthetics, botulinum toxins, and nucleoid genetic tools for suppressing SVC. [3]Despite some achievements, achieving rapid and precise modulation of the SVC through transdermal drug delivery remains a formidable challenge, particularly for bio-macromolecular drugs such as peptides and proteins. [4]ue to the relentless pace of modern life and prolonged exposure to anxiety, an increasing number of individuals are experiencing facial furrows or creases caused by nerve tension derived from overactivated SVC. [5]These facial lines, particularly noticeable around the eyes and nose, can trigger excessive distress regarding one's physical appearance and result in psychosocial challenges such as diminished work performance or a significant decline in self-confidence. [6]The improvement of the psychosocial well-being of these individuals necessitates the urgent implementation of a straightforward yet effective therapy to address the concerns associated with these facial wrinkles.Therefore, the development of transdermal biomacromolecules to modulate overactivated SVC in facial peripheral nerves not only promotes the advancement of neurological drugs but also addresses real neuropsychological issues prevalent in today's society.
In the pursuit of this objective, an investigation was conducted on six volunteers and mice to delve into the potential mechanism underlying facial wrinkles caused by nerve tension.The study examined the subjects both before and after a demanding 28-day period of continuous desk work for humans, as well as a challenging 28-day period of partial sleep deprivation for mice.Astonishingly, the findings unveiled a significant reduction in dermal thickness associated with wrinkles.Subsequent investigations revealed that this dermal compression is a consequence of facial nerves being activated in a state of hysteria, which arises from an abnormally heightened interaction between SNAP25 and RAB3A proteins involved in the SVC.To alleviate these neural creases, an innovative SVC modulation was developed herein employing an AI-designed peptide (RSI pep ) with exceptional affinity for RAB3A antagonism, effectively nullifying its interaction with SNAP25.However, the remarkable biofunction of RSI pep is impeded by the formidable barriers of cuticle adhesion, proteolysis, and cytomembrane impenetrability.
Taking inspiration from prions, which possess the ability to shield themselves against proteolysis and invade neighboring nerve cells through macropinocytosis, RSI pep was ingeniously engineered to exhibit a GSH-responsive reversible self-assembly into a prion-like supermolecule (RSI prion ) that showcases remarkable resistance against proteases and relies on macropinocytosis for cellular internalization.Moreover, this prion-like nanostructure facilitates the RSI pep in overcoming adhesion with constituent molecules in the cuticle, thereby endowing it with the remarkable ability to transdermally penetrate both epidermis and dermis.As a result, RSI prion can restore the frenzied SVC to its normal state both in vitro and in vivo.Moreover, when applied as a facial mud mask with transdermal diffusion, it exhibits potent efficacy in eliminating nervous wrinkles specifically in the periorbital and perinasal region of the human face.Collectively, the report on RSI prion not only presents a clinical translational potential for the modulation of SVC as an anti-wrinkle prion-like artificial protein but also exemplifies a reproducible instance of bionic strategy-guided template-free protein design that bestows transdermal capability upon the pharmaceutical molecule.

The Hyperactive Interaction Between SNAP25 and RAB3A may Serve as Potential Targets for Alleviating Nervous Skin Wrinkles Derived from Hysterical SVC
To investigate the impact of intensive work on facial wrinkles, the present study meticulously examined the skin wrinkle conditions of six volunteers, aged between 35 and 51 (Figure S1A, Supporting Information), who had recently returned from their winter vacation.After acquiring baseline data on canthal, infraorbital, glabella, and nasolabial wrinkles through the advanced VISIA Facial Imaging Booth (CANFIELD SCIENTIFIC, INC, USA), [7] they all embarked upon an arduous 28-day period of unwavering desk work that demanded their relentless operation for over 10 h each day.Subsequent to this period, their countenances underwent meticulous scrutiny and revealed a conspicuous augmentation in wrinkles on the canthal, infraorbital, glabella, and nasolabial regions, as evidenced by the VISIA image (Figure 1A; Figure S1B, Supporting Information) and the subsequent reduction of over 30% in facial smoothness factor (wrinkles index in Figure 1B).More notably, the reduced thickness of the dermis in the canthal, infraorbital, glabella, and nasolabial regions (Figure 1C) exhibited a significant positive correlation with changes in wrinkle indexes, which is in stark contrast to the lack of statistical association observed between collagen density changes and wrinkle indexes in these areas (Figure 1D; Figure S1C, Supporting Information).This suggests that facial wrinkles in these regions are primarily caused by dermal compression rather than collagen loss.
Moreover, this dermal compression can also be observed in the stress mice model of nervous skin wrinkles, which is established through enduring a grueling 28-day period of sleep semideprivation and anxiety stimulation with electric shock (Figure S1D, Supporting Information).The dermal thickness in the skin of stress model mice, as depicted in Figure 1E, exhibited a significant reduction of over 30% compared to that of the normalfed control mice (Ctrl), while the collagen expression remained almost constant in both groups (Figure S1E, Supporting Information).Furthermore, the outcomes of nerve silver staining unveiled that within the control group, a majority of the nerve fibers were distributed beneath or at the periphery of the muscle layer; however, in the stress model group, a significant proportion of nerve fibers deeply infiltrated into the muscular layer (Figure 1F), thereby indicating heightened excitation of nerve cells.The reaffirmation of this finding was once again demonstrated through the detection of a cluster of neurotransmitters, wherein the secretion levels of the vast majority of detectable neurotransmitters were found to be elevated within the muscular layer of the stress model group (Figure 1G), indicative of exertion of pulling force on the muscle layer to extrude muscle layer.
To further elucidate the mechanism of epidermal nerve excitation, state-of-the-art label-free quantitative high-resolution mass spectrometers were employed to discern proteomics in a contrasting manner between the stress model and control groups (Figure 1H).The performance of Gene Set Enrichment Figure 1.The hyperactive interaction between SNAP25 and RAB3A may serve as potential targets for alleviating nervous skin wrinkles derived from hysterical SVC.A) Wrinkles on the canthal, infraorbital, glabella, and nasolabial regions observed by VISIA before and after an arduous 28-day period of unwavering desk work that demanded their relentless operation for over ten hours each day.B) Facial smoothness factor (wrinkles index) before and after an arduous 28-day test.C) Thickness of the dermis in the canthal, infraorbital, glabella, and nasolabial regions.D) The relationship of wrinkles index changes to the changes of dermal thickness after 28-day test before and in the canthal, infraorbital, glabella, and nasolabial regions.E) The H&E Analysis (GSEA) revealed the presence of four neural-related signaling pathways among the Top 20 up-regulated signaling pathways, as depicted in Figure 1I.The intersection algorithm was employed to analyze the constituent proteins, unveiling four common proteins, namely SNAP25, RAB3A, GLS, and PPFIA1, which exhibited up-regulation across all four signaling pathways (Figure 1J).Among them, SNAP25 and RAB3A are directly involved in synaptic vesicle cycling (SVC) by interacting with each other to facilitate the fusion between synaptic vesicles and cytomembrane (Figure 1K). [8]Therefore, we have compelling reasons to believe that antagonizing RAB3A and subsequently nullifying its interaction with SNAP25 could potentially serve as a therapeutic strategy for alleviating cutaneous wrinkles caused by hysterical superior SVC.To validate the concept, a C14modified membrane-permeable siRNA targeting RAB3A was non-invasively introduced subcutaneously into the facial region of six volunteers with wrinkles in the infraorbital and nasolabial nerve areas using electric muscle stimulation (EMS).Three days post-treatment, all six volunteers exhibited reduced facial wrinkles, indicating the potential efficacy of RAB3A antagonism (Figure S1F, Supporting Information).

The RAB3A-SNAP25 Inhibitor Peptide (RSI pep ) was Discovered Through AI-Assisted Structural Design
The crystal structure of PDB code 5LOW reveals that SNAP25 elegantly engages with the hydrophobic groove in RAB3A through an exquisite -helix motif composed of 17 amino acid residues, thereby forming a captivating complex with a binding area spanning 568.4 Å 2 and a remarkable Gibbs free energy of reaction measuring −10.6 kcal mol −1 (Figure 2A).The revelation of this structure has inspired us that the blockage of the SNAP25-binding domain in RAB3A through an -helix peptide could potentially suppress the interaction between SNAP25 and RAB3A, thus offering a promising avenue to rewrite the SVC.To scrutinize the minimum binding sequence, a 17-mer -helix motif derived from SNAP25 was systematically truncated residue by residue from its carboxyl terminal, resulting in the generation of a peptide library comprising 12 peptides (Figure 2B,C).The subsequent prediction of their binding structure with RAB3A, utilizing the remarkable Alpha Fold 2, unveils that a sequence comprising 15 amino acid residues known as SNAP25 pep (Seq: SEFMRNELEEMQRRA) exhibits the utmost optimal Gibbs free energy of reaction, thereby indicating an unparalleled affinity (Figure 2B,C).The identification of key binding sites in SNAP25 pep for RAB3A affinity was achieved through a meticulous alanine scanning approach, wherein each amino acid residue in SNAP25 pep was sequentially mutated to alanine (Figure 2D).The resulting mutants were subsequently subjected to binding prediction analysis employing the cutting-edge Alpha Fold 2 algorithm (Figure 2D), wherein it was discerned that E10A and Q12A exhibited a significant reduction in the Gibbs free energy of interaction (Figure 2E).The aforementioned observation strongly suggests that E10 and Q12 serve as pivotal binding sites.Subsequently, attempts were made to mutate both E10 and Q12 into other amino acid residues, revealing that E10A and Q12L exerted the most favorable influence on RAB3A binding (Figure 2F).The subsequent prediction of the binding structure between SNAP25 pep E10A/Q12L (RSI pep ) and RAB3A by Alpha Fold 2 validated the rationality and reliability of their optimized interaction (Figure 2G; Figure S2A, Supporting Information).To validate this, the binding affinity between FITClabeled RSI pep /SNAP25 pep and recombinant RAB3A were quantified using fluorescence polarization, revealing a remarkable over tenfold enhancement in RSI pep binding with RAB3A compared to SNAP25 pep (Figure 2H).The efficacy of RSI pep , as expected, was found to be approximately fourfold greater than that of SNAP25 pep in inhibiting the interaction between SNAP25 and RAB3A, as determined by competitive fluorescence polarization (Figure 2I), indicating the potent potential of RSI pep in rewriting SVC by blocking the biofunction of RAB3A.
In order to fulfill its biofunction of binding with RAB3A, RSI pep must successfully navigate through three formidable transdermal barriers before encountering RAB3A: cuticle adhesion, proteolysis, and cytomembrane impenetrability (Figure 2J). [9]Unfortunately, RSI pep can adhere to eicosapentaenoic acid (EPA), Ceratin, and Cholesterol, all of which are essential constituents in the horny layer (Figure 2K), implying the inevitable adhesion of RSI pep to the cuticle.To exacerbate the situation, even if a few RSI pep manage to break through the cuticle, the chymotrypsin, trypsin, and protease K present in the corium layer would expeditiously degrade these remnants (Figure 2L).Additionally, the impenetrability of RSI pep through the nerve cell membrane (Figure S2B, Supporting Information) was even more unfortunate, as it hindered the achievement of its biofunction in binding with RAB3A through transdermal absorption.

The RSI pep is Engineered to Undergo a GSH-Responsive Reversible Self-Assembly, Resulting in the Formation of a Prion-Like Supermolecule (RSI prion )
Drawing inspiration from prions, which possess the extraordinary ability to shield themselves against proteolysis and invade neighboring nerve cells through macropinocytosis, RSI pep has been ingeniously engineered into a prion-like supermolecule termed RSI prion through a reversible self-assembly with glutathione response (Figure 3A). [10]10b,11] The Au(I)-peptide oligomers can subsequently undergo self-assembly, forming exquisite supermolecules through staining of mice skin from the stress mice model of nervous skin wrinkles, which is established through enduring a grueling 28-day period of sleep semi-deprivation and anxiety stimulation with electric shock.F) Nerve silver staining image of mice skin of stress model and control.G) Subcutaneous neurotransmitters release.H) Proteomics results of the skin from the stress model and control groups measured by label-free quantitative high-resolution mass spectrometers.I) Top 20 up-regulated signaling pathways in GSEA analysis of the result in (H).J) Venn diagram of the four signaling pathways related to synaptic vesicle cycling in (I).The schematic diagram of SNAP25 and RAB3A function in synaptic vesicle cycling.

Figure 2. Discovery and pharmaceutical properties of RAB3A-SNAP25 inhibitor peptide (RSI pep ). A)
The crystal structure of PDB code 5LOW reveals the structure foundation of the interaction between SNAP25 and RAB3A.B,C) The 17-mer -helix motif derived from SNAP25 that interacts with RAB3A was systematically truncated residue by residue from its carboxyl terminal.The prediction of their binding structure with RAB3A, utilizing the remarkable Alpha Fold 2, was shown in (B) and their consequent Gibbs free energy of reaction was shown in (C).D,E) Alanine scanning of the optimal sequence from (C), characterizing by the prediction structure and consequent Gibbs free energy of reaction (E) as well as interaction area.F) The consequent the fascinating aurophilic interactions between Au(I) atoms. [10,11]y precisely adjusting the concentration of RSI pep and pH levels within the reaction system during self-assembly, a diverse range of RSI prion with hydrodynamic diameters ranging from 10 to 85 nm were successfully synthesized (Figure 3B).Subsequently, succinimide activated rhodamine (Rhb-SE) was conjugated to these RSI prion , and their uptake by HT22 nerve cells was quantified using flow cytometry (FCM) and laser scanning confocal microscopy (LSCM).The RSIprion-4, which has a hydrodynamic diameter of 38.6 nm, representing a common size of prions, [12] exhibited the highest efficiency in cellular internalization, as demonstrated by both the cellular uptake ratio measured using flow cytometry (Figure 3C) and the cellular uptake intensity measured using laser scanning confocal microscopy (Figures 3D,E).The comprehensive evaluation (product) of FCM and CLSM outcomes aligns with the extreme value distribution of the dynamic stochastic response of hydrodynamic diameter for RSI prion (Figure 3F), indicating the superiority of RSI prion -4, thus warranting its selection as the benchmark RSI prion for subsequent investigations.
Based on Fourier Transform Infrared (FT-IR) spectroscopy of RSI prion and RSI pep , it was found that there are two absorption peaks that correspond to amide bonds at 1680 cm −1 (C═O) and 3300 cm −1 (N─H) in both infrared spectra.Furthermore, Au(I)-SR showed a characteristic absorption peak at ≈2800 cm −1 , indicating infinite coordination of Auric with sulfur (Figure 3G).Additionally, UV-vis spectra of RSI prion and RSI pep confirmed these findings by revealing characteristic absorption peaks associated with peptides and Au(I)-SR bonds.Moreover, using X-ray photoelectron spectroscopy (XPS) of Au4f and S2p (Figure 3H), we observed that the sulfhydryl group is coordinating with Au(I), thus confirming the existence of a coordination between Au(I) and thiolipid that forms the basis for this aurophilic self-assembly.The successful self-assembly of RSIprion and its uniform size with monodisperse spherical structure are subsequently revealed in transmission electron microscope (TEM) images (Figure 3I), indicating a bionic prionlike architecture. [13]Moreover, high-resolution TEM elemental and diffraction analysis demonstrate the homogeneous distribution of gold (Au), nitrogen (N), oxygen (O), and sulfur (S) elements within RSI prion (Figure 3J), perfectly aligning with the findings from energy dispersive X-Ray spectroscopy (EDS) analysis results as well (Figure S3B, Supporting Information).The above-mentioned results undeniably showcase the successful transformation of RSI pep into a prion-like supermolecule boasting a spherical nanostructure.The Mark-Houwink molecular weight of this RSI prion is ≈2938.9KDa (Figure S3C, Supporting Information), suggesting an aggregation of ≈1400 RSI pep molecules.
To ascertain whether RSI prion possesses the remarkable capacity of prions to shield themselves against proteolysis, chymotrypsin, trypsin, and protease K were also employed for its treatment in a PBS buffer supplemented with essential calcium ions to activate enzymatic activity.The remarkable finding is that over 90% of RSI prion remained intact even after a 24-h exposure to the three proteases, while RSI pep exhibited a significantly shorter half-life of less than 1 h under the same conditions (Figure 3K).This can be attributed to the enhanced steric hindrance against protease recognition achieved through this engineered self-assembly. [14]Moreover, this engineered self-assembly aids in the eradication of RSI prion 's adhesion to EPA, Ceratin, and Cholesterol, as evidenced by the nearly imperceptible change in fluorescence polarization of FITC-labeled RSI prion when incubated with stratum corneum material at equi-multiplied concentration (Figure 3L).More importantly, in sharp contrast to RSI pep , RSI prion showed significant cellular internalization into HT22 nerve cells (Figure 3M).The findings demonstrated that RSIprion not only inherited the prion's characteristic of resistance to proteolysis and ability to penetrate cytomembranes, but also possessed a low affinity for cuticles, all of which contribute to the compelling potential of RSI prion in achieving transdermal absorption.

The RSI prion is Internalized into Nerve Cells via Prion-Like Macropinocytosis and Achieve Transdermal Absorption
The cunning prions skillfully exploit the intricate mechanism of macropinocytosis to infiltrate host nerve cells, while proteins or peptides aggregate into structures resembling prions, thereby cleverly stimulating macropinocytosis and facilitating their widespread dissemination into mammalian cells (Figure 4A). [15]To confirm the internalization of RSI prion into nerve cells through prion-like macropinocytosis, we initially investigated the uptake pattern of RSI prion .The cellular internalization assay during a 3-h incubation with RSI prion can be enhanced by ATP and inhibited by subzero treatment (Figure 4B).However, this process is not affected by concentration but is consistently efficient when the concentration exceeds 20 mg L −1 (Figure 4C).These findings reveal that the internalization of RSI prion into nerve cells is an active transport mechanism rather than a passive diffusion process.To investigate the active transport species of RSI prion , a repertoire of six inhibitors representing diverse mechanisms of cell internalization were employed: two inhibitors targeting clathrin-mediated endocytosis, Chlorpromazine (CPZ) and Dynasore (DYN); two inhibitors inhibiting caveolae-mediated endocytosis, Filipin and Genistein (GEN); as well as two inhibitors blocking macropinocytosis, Amiloride and Cytochalasin D (Cyto D).As expected, two macropinocytosis inhibitors, Amiloride, and Cyto D, significantly suppressed RSI prion internalization, whereas other inhibitors had no effect (Figure 4D).A GSEA analysis of cellular endocytosis pathways was performed on the proteome sequencing data of RSI priontreated and mock-treated HT22 cells to provide further evidence  for this outcome (Figure 4E,F).RSI prion treatment significantly activated pathways related to macropinocytosis (Figure 4E), with all proteins in the macropinocytosis pathway exhibiting significant upregulation (Figure 4F).Additionally, the cellular uptake of RSI prion occurred simultaneously with the internalization of 70Kd dextran, a well-established marker for macropinocytosis, [16] as demonstrated by the colocalization analysis conducted using LCSM (Figure 4G).Moreover, numerous RSI prion supramolecular nanoparticles can be observed within a single phagophore spanning hundreds of nanometers, as evidenced by the TEM image depicting the process of RSI prion internalization into HT22 cells (Figure 4H), which is indicative of characteristic subcellular morphological features associated with macropinocytosis.
The remarkable low cuticle adhesion, exceptional proteolysis resistance, and extraordinary cytomembrane penetrability derived from macropinocytosis all contribute to the convincing potential of RSI prion in achieving transdermal absorption.To validate this, a comparative investigation was conducted on the intact skin of Panama Pig using three FITC-labeled anti-wrinkle compounds.The shin longitudinal sections in Figure 4I reveal the formidable resistance of both the commercial anti-wrinkle peptide Hexa pep and RSI pep in penetrating the cuticle, while RSI prion elegantly accumulates at the depths of the corium layer.The fluorescent quantitation results further elucidated this discovery, demonstrating that RSI prion exhibited statistically significantly enhanced transdermal ability compared to Hexa pep and RSI pep (Figure 4J).

The RSI prion Disassembles into RSI pep in Response to Intracellular Glutathione and Rewrites SVC Both In Vitro and In Vivo
The chemical basis of RSI prion self-assembly involves intramolecular coordination between Au(I)-SR and intermolecular aurophilic interactions.Consequently, the assembly of RSI prion can potentially be disrupted by the cleavage of Au(I)-SR in response to a reducing environment (Figure 5A).The assembled RSI prion , as depicted in Figure 5B,C, effectively shielded the intricate helical structure of RSI pep while preserving its vital biofunction for binding with RAB3A.Remarkably, both the structural integrity and functional activity could be fully restored upon incubation with a solution containing 10 mm GSH, providing clear evidence of the disassembly process transforming RSI prion into its monomeric form -RSI pep (Figure 5B,C).The disassembly process can be further demonstrated by the release curve of RSI pep from RSI prion , as indicated by HPLC analysis: in the presence of 10 mm GSH, free RSI pep monomer is detected while RSI prion remains predominantly intact even in a GSH-free solution (Figure 5D).The disassembly of RSIp rion in response to GSH concentration was further investigated by incubating RSI prion with 2.5, 5, 10, and 20 μm GSH for 24 h while monitoring the release of RSI pep using HPLC.Figure S3D (Supporting Information) demonstrates that the time required for 50% release decreased as the GSH concentration increased, indicating a concentration-dependent mechanism for RSI prion disassembly.The aforementioned findings have demonstrated the inherent potential of RSI prion to undergo disassembly, thereby restoring its functional form as a monomeric entity known as RSI pep , within the intracellular reducing environment.
The biofunction of RSI prion in rewriting SVC was investigated through a comparative study, wherein a negative control of RSI prion called Ctrl RSI prion was synthesized using retrosynthetic RSI pep .As anticipated, the presence of 0.02 mg mL −1 Ctrl RSI prion did not exhibit any discernible impact on the inhibition of acetylcholine release from HT22 nerve cells (Figure 5F).Conversely, the isometric RSI prion demonstrated a remarkable ability to suppress acetylcholine release by 61.8%, surpassing both isotonic Hexa pep and isopycnic commercial Botox in terms of efficacy (Figure 5F).Furthermore, RSI prion exhibited a more comprehensive impact on the suppression of multiple neurotransmitter release in comparison to Hexa pep (Figure 5E), suggesting that it restrains SVC activity rather than merely inhibiting specific neurotransmitter synthesis.
3a] The mice were injected with10 μg mouse −1 RSI prion , while Hexa pep at a dosage of 10 μg mouse −1 served as the negative control and Ctrl RSI prion as the positive control.Furthermore, an additional commercial positive control was used with Botox administered at a dosage of 0.05 U mouse −1 .It is worth mentioning that the inhibitory effect on neurotransmitter release in HT22 cells by 0.05U Botox is equivalent to that of 10 μg RSI prion , which was once again confirmed by the same intramuscular median efficacy time observed for both 0.05 U mouse −1 Botox and 10 μg mouse −1 RSI prion injection (Figure 5I).Meanwhile, Ctrl RSI prion exhibited no efficacy in inducing local muscle weakening, while Hexa pep demonstrated a limited effect with a DAS score of less than two points (Figure 5G,H).The remarkable efficacy of both Botox and RSI prion , the diminished effectiveness of Hexa pep, and the complete lack of efficacy in Ctrl RSI prion were once again demonstrated by the impressive holding power exhibited by treated mice (Figure 5J), their prolonged endurance time (Figure 5K) as well as the distance covered on the rotating stick (Figure 5L).The above results collectively demonstrate that RSI prion effectively inhibits the excitation of peripheral nerves by modulating SVC both in vitro and in vivo.

The RSI prion Boasts a Highly Commendable Safety Profile
The aforementioned equivalence relation states that 10 μg of RSI prion has an equivalent effect to 0.05U of Botox.To evaluate the viability of HT22 cells, a solution containing either 10 μg of of cellular uptakes are determined by both FCM (C) and CLSM (E) outcomes.G) Fourier transform infrared (FT-IR) of RSI pep and RSI prion .H) X-ray photoelectron spectroscopy (XPS) analysis of Au 4f and S 2p of RSI prion .I) TEM images of RSI prion .J) Elemental analysis image of S, O, N, Au overlay with one representative particle of RSI prion taken by HRTEM.K) Protease hydrolysis test of RSI pep and RSI prion against the chymotrypsin, trypsin, and protease K that are present in the corium layer.L) The affinity test of RSI pep and RSI prion binding to eicosapentaenoic acid (EPA), oleic acid (OA), Ceratin, and Cholesterol measured by fluorescence polarization.M) Cellular uptakes of RSI prion and RSI pep into HT22 nerve cells measured by FCM and LSCM.RSI prion or 0.05U of Botox was meticulously prepared in a 50 μL cell culture medium, which was subsequently evenly diluted to administer treatment to the cells.As depicted in Figure 6A,B, neither RSI prion nor Botox induced necrocytosis of HT22 cells.Revealing a striking contrast to RSI prion , Botox demonstrated a dose-dependent induction of apoptosis (Figure 6A,B), aligning seamlessly with the findings of the previous report. [17]Fortunately, RSI prion does not exhibit any propensity to induce apoptosis in nerve cells, as evidenced by the FCM apoptosis analysis and GSEA analysis of the proteome from HT22 cells treated with RSI prion (Figure 6A-C).
The biosafety evaluation of RSI prion was further enhanced by subjecting protein drugs to an immunotoxicity test, which involved subcutaneous injection of varying dosages (0, 1, 5, and 25 mg kg −1 ) into immunologically sound BALB/c mice.This was followed by immune factor detection at 2 days post-injection.The administration of 1 mg Kg −1 injection, as depicted in Figure 6D, did not elicit any anaphylactic reactions in either subcutaneous tissue or blood, thereby indicating that this dosage is immunologically safe.Afterward, a meticulous assessment of acute toxicity was carried out on female and male BALB/c mice through a single subcutaneous injection of 1 mg Kg −1 RSI prion , RSI pep , or isopycnic normal saline.The mice treated with RSI prion exhibited consistent body weights and blood cell indexes, showcasing the absence of any discernible impact by RSI prion (Figure 6E).Furthermore, the biosafety of RSI prion is once again reaffirmed through the meticulous pathological section analysis of vital organs such as heart, liver, spleen, lung, and kidney, wherein no discernible pathological alterations are observed in either RSI pep -or RSI prion -treated mice (Figure 6F).The subsequent step involved a 28-day exploration of cumulative toxicity, wherein subcutaneous injections of 1 mg Kg −1 RSI prion or RSI pep were administered every alternate day.The RSI prion treatment, as anticipated, exhibited negligible impact on body weights and blood cell indexes (Figure 6G), thereby affirming its impeccable biosafety profile during prolonged usage.

The Efficacy of RSI prion in Diminishing Facial Wrinkles Induced by Excitatory Neural Activity on the Human Countenance
The remarkable performance of RSI prion in transdermal absorption, modulation of SVC, and biosafety profiles has compelled us to investigate its efficacy in reducing facial wrinkles caused by excitatory neural activity on the human countenance (Figure 7A).In pursuit of this objective, RSI prion was ingeniously transformed into a facial mud mask by seamlessly blending it with a solution of sodium hyaluronate, and delicately applying it to the visage at a dosage of 0.01 mg kg −1 , which equates to a mere one percent of the aforementioned safe dose of RSI prion .In a small-scale experiment involving only three volunteers, no allergic reactions such as redness, swelling, or itching were observed on their skin within a week of use.Revealing with great excitement, the VISIA Complexion Analysis System unveiled a remarkable 22.6% increase in dermal thickness and an astonishing 26.5% reduction in wrinkles (Figure S4A, Supporting Information).The wrinkles index and texture index measured by VISIA are indicators of facial smoothness, with a value of 100% indicating no wrinkles detected by the testing machine.For more comprehensive testing, a cohort of 20-woman volunteers aged between 32 and 60, exhibiting wrinkles induced by excitatory neural activity, were randomly assigned to partake in a 4-week double-blind trial.The trial consisted of two groups: one using the facial mud mask with RSI prion (RSI prion group) and the other without RSI prion (Ctrl group).Specifically, each subject applied the facial mud mask once a week, and their skin condition was assessed using VISIA on days 0, 7, 12, 21, and 28.The texture index and wrinkle index in RSI prion group exhibited a significant increase at day 7 compared to the baseline measured at day 0, indicating a remarkable reduction in facial wrinkles (Figure 7B,C).The significant reduction in facial wrinkles persisted throughout the entire 28-day monitoring period, as demonstrated by Figure 7B,C, where the mean texture index increased from 79.49% to 90.65%, and the mean wrinkles index increased from 58.17% to 83.07%.After the 28-day administration, the brightness index in all 13 subjects in the RSI prion group exhibited a statistically significant increase (Figure 7D), presumably attributable to the reduction of diffuse reflection resulting from the disappearance of wrinkles.Meanwhile, the remaining seven subjects in the Ctrl group exhibited no significant alterations in either texture index (Figure 7E; Figure S4B, Supporting Information) or wrinkle index (Figure 7F; Figure S4C, Supporting Information), thereby implying that the remarkable anti-wrinkle effect observed in the RSI prion group can be solely attributed to the biofunction of RSI prion rather than its excipient sodium hyaluronate.Furthermore, in order to ascertain its efficacy in the male population, a group of ten male participants were enlisted for a single-arm trial of RSI prion , mirroring the aforementioned trial conducted with female subjects.The facial mud mask derived from RSIprion demonstrated remarkable efficacy in reducing facial wrinkles caused by heightened neural activity across all ten individuals (Figure 7G-I).The mean texture index increased from 89.16% to 93.78%, and the mean wrinkles index increased from 69.68% to 92.86% (Figure 7H), thereby highlighting the gender-neutral anti-wrinkle potential of RSI prion .Furthermore, the absence of any adverse reactions such as erythema, edema, or pruritus on the skin of male and female participants in clinical trials underscores the safety and efficacy of RSI prion in reducing facial wrinkles caused by excitatory neural activity on the human countenance.

Discussion
The present study has meticulously observed the skin wrinkle conditions of six volunteers who have recently returned from mock-treated HT22 cells.F) GSEA analysis and protein levels of macropinocytosis of RSI prion -treated and mock-treated HT22 cells.G) The cellular uptake of RSI prion occurred simultaneously with the internalization of 70Kd dextran, a well-established marker for micropinocytosis, as demonstrated by the colocalization analysis conducted using LCSM.H) TEM analysis of RSI prion cellular uptakes of into HT22 cells.The red triangle symbolizes the phagosome.I) Transdermal absorption test of FITC-labeled Hexa pep, RSI pep and RSI prion using the intact skin of Panama pig.J) The fluorescent quantitation results of the fluorescent image in (I).their winter vacation, throughout an arduous 28-day period of uninterrupted desk work that has necessitated them to operate for over 10 h each day, unveiling a conspicuous reduction in dermal thickness associated with wrinkles in all participants.Interestingly, this phenomenon can also be observed in the skin of mice who endure a grueling 28-day period of partial sleep deprivation.Further bioinformatics analysis unveiled that the emergence of this skin wrinkle, derived from dermal compression, can be attributed to the activation of facial nerves in a state of hysteria due to an aberrantly elevated interaction between SNAP25 and RAB3A proteins involved in the synaptic vesicle cycle (Figure 1).Of note, the flat, discontinuous, hydrophobic, and large interfaces of PPIs, particularly in the case of SNAP25-RAB3A interaction, present a significant challenge in the discovery of conventional drugs (Mw×500).However, peptides that closely mimic the 3D conformation of proteins offer valuable insights into the development of PPI modulators. [18]The rewriting of this frenzied SVC herein involved the ingenious discovery, facilitated by AI-assisted structural design, of a refined peptide called RSI pep .This remarkable peptide exhibits a nanomolar affinity for binding with RAB3A, effectively nullifying its ability to interact with SNAP25.
Despite the development of peptide PPI modulators with some degree of success, there still exist two fundamental flaws in peptides that require addressing: their inadequate proteolytic stability and ineffective cellular internalization, [19] both of which have also contributed to the failure of RSI pep .14a,20] Another limitation arises from their inherent properties, such as hydrophobicity and electronegativity, which hinder their ability to penetrate cytomembranes and undergo cellular internalization. [21]14c,21,22] However, most of these vehicles are primarily designed to modulate PPIs in visceral or tumor cells.Unfortunately, the complex capillary network beneath the skin poses a challenge for intravenously administered peptide PPI modulators to effectively reach subcutaneous cells via blood circulation.As a result, accessible peptide PPI modulators have long been lacking for subcutaneous cells.
Drawing inspiration from prions, which elegantly orchestrate the assembly of proteins into exquisitely organized aggregates with 3D amyloid structures solely through the intricate interplay of intermolecular forces, and possess the remarkable ability to shield themselves against proteolysis and infiltrate neighboring nerve cells via macropinocytosis, RSI pep has undergone a magnificent transformation into an exquisite GSH-responsive reversible prion-like nanostructure (RSI prion ) that showcases extraordinary resilience against proteases and relies on macropinocytosis for cellular internalization, all while ensuring the absence of microbiological transmissibility within communities.Moreover, this prion-like nanostructure facilitates the RSI pep in overcoming adhesion with constituent molecules in the cuticle, thereby endowing it with the ability to penetrate both epidermis and dermis through the transappendageal pathway, which includes hair follicles, sebaceous glands, and sweat glands. [23]Thus, the remark-able efficacy of RSI prion lies in its ability to restore the frenzied SVC to a state of equilibrium, both in vitro and in vivo.
Moreover, as an exquisite modality for manipulating peripheral nerve cell protein-protein interactions (PPIs), RSI prion offers a plethora of unparalleled advantages, encompassing the painless administration, rapid cessation, sustained release, and circumvention of first-pass metabolism.Additionally, when compared to the widely used wrinkle-resistant product Botox, RSI prion demonstrates nearly identical efficacy in SVC modulation (Figure 5) and superior biosecurity (Figure 6).Furthermore, when transformed into a luxurious facial mud mask with transdermal diffusion properties, RSI prion exhibits unrivaled potency in eradicating neural wrinkles that afflict the periorbital and perinasal regions of the human face.Nevertheless, it is well-known that Botox can only be administered via subcutaneous injection.Collectively, the comprehensive report on RSI prion not only presents a clinical translational potential for the rewriting strategy of the synaptic vesicle cycle as an anti-wrinkle prion-like artificial protein but also exemplifies a reproducible instance of bionic strategy-guided drug development that confers transdermal capability upon the pharmaceutical molecule.
Gibbs free energy of reaction in response to key binding site mutations.G) The prediction binding structure of RAB3A and RSI pep .H) The affinity test of RAB3A binding to RSI pep or SNAP25 pep measured by fluorescence polarization.I) The competitive fluorescence polarization assay was conducted to authenticate the inhibitory ability of RSI pep or SNAP25 pep on the interaction between SNAP25 and RAB3A.J) The schematic diagram of three formidable transdermal barriers for RSI pep .K) The affinity test of RSI pep binding to eicosapentaenoic acid (EPA), oleic acid (OA), Ceratin, and Cholesterol measured by fluorescence polarization.L) Protease hydrolysis test of RSI pep against the chymotrypsin, trypsin, and protease K that are present in the corium layer.

Figure 3 .
Figure 3.The formation of a prion-like supermolecule (RSI prion ).A) schematic diagram of RSI pep engineering to undergo a GSH-responsive reversible self-assembly, resulting in the formation of a prion-like supermolecule (RSI prion ).B) hydrodynamic diameters of a diverse range of RSI prion ranging from 10 to 85 nm measured by dynamic light scattering.C,D) Cellular uptakes of different sizes of RSI prion into HT22 nerve cells measured by flow cytometry (FCM, C) and Laser Scanning Confocal Microscopy (LSCM, D).E) Fluorescence quantitative data of LSCM image in (D).F) The relationship between the hydrodynamic diameters of RSI prion and the scores of cellular uptakes, as fitted by the extreme value distribution.It should be noted that the scores

Figure 4 .
Figure 4.The RSI prion is internalized into nerve cells via prion-like micropinocytosis and achieves transdermal absorption.A) schematic diagram of prion and RSI prion internalize into nerve cells by micropinocytosis.B) FCM and LSCM analysis of RSI prion cellular uptakes of into HT22 cells in response to low temperature or ATP incubation.C) Cellular uptakes of RSI prion with different concentrations into HT22 nerve cells measured by FCM and LSCM.D) FCM analysis and LSCM image of RSI prion cellular uptakes of into HT22 cells in response to two clathrin-mediated endocytosis inhibitor Chlorpromazine (CPZ) and Dynasore (DYN), two caveolae-mediated endocytosis inhibitors Filipin and Genistein (GEN), as well as two macropinocytosis inhibitors Amiloride and Cytochalasin D (Cyto D).E) GSEA analysis of cellular endocytosis pathways in the proteome sequencing data of RSI prion -treated and

Figure 5 .
Figure 5.The RSI prion disassembles into RSI pep in response to intracellular glutathione (GSH) and rewrites SVC both in vitro and in vivo.A) The schematic diagram of RSI prion disassembles into RSI pep in response to intracellular glutathione.B) The circular dichroism spectrum of RSI prion measured in buffer containing 0 mm or 10 mm GSH.C) The affinity test of RSI prion binding to RAB3A measured by fluorescence polarization in buffer containing 0 mm or 10 mm GSH.D) The release curve of RSI pep from RSI prion in response to GSH.E) The neurotransmitter release from HT22 nerve cell incubating with Botox, Hexa pep, RSI pep , and RSI prion .G) The experiment to authenticate the inhibition of peripheral nerves, wherein neural sedatives were administered into the musculus gastrocnemius of C57 mice (G), followed by meticulous monitoring of Digit Abduction Score (DAS) studies to ascertain the efficacy of local muscle weakening.H) Schematic of DAS scores based on paw morphology of mice.I-K) The impressive holding power (I), endurance time (J) as well as the distance covered on the rotating stick (K), in response to the injection of Botox, Hexa pep, RSI pep , and RSI prion .

Figure 6 .
Figure 6.The RSI prion boasts a highly commendable safety profile.A,B) apoptosis and necrocytosis of HT22 nerve cell induced by Botox or RSI prion measured by FCM.C) GSEA analysis about apoptosis of the proteome from HT22 cells treated with RSI prion .D) The immunotoxicity test of RSI prion involved subcutaneous injection of varying dosages (0, 1, 5, and 25 mg kg −1 ) into immunologically sound BALB/c mice.E,F) Acute toxicity was assessed in female and male BALB/c mice through a single subcutaneous injection of 1 mg Kg −1 RSI prion , RSI pep , or isopycnic normal saline.The assessment included monitoring body weights (E), conducting blood routine examinations (E), and examining representative pathological sections of organs (F).G) A 28-day investigation of cumulative toxicity was conducted, in which subcutaneous injections of 1 mg Kg −1 RSI prion or RSI pep were administered every other day.The evaluation included monitoring body weights and conducting blood routine examinations.

Figure 7 .
Figure 7.The efficacy of RSI prion in diminishing facial wrinkles induced by excitatory neural activity on the human countenance.A) The schematic diagram of RSI prion in diminishing facial wrinkles induced by excitatory neural activity.B) Representative facial wrinkle images observed by VISIA of a female subject treated with RSI prion facial mud mask.C) Texure index and facial smoothness factor (wrinkles index) of all female subjects treated with RSI prion facial mud mask.The data were evaluated using VISIA, and a higher value indicates a lower wrinkle.D) Brightness index of all female subjects treated with RSI prion facial mud mask.E,F) Texure index(E) and wrinkles index (F) all female subjects treated with RSI prion or RSI prion -free facial mud mask.G) Representative facial wrinkle images observed by VISIA of a male subject treated with RSI prion facial mud mask.H) Texure index and facial smoothness factor (wrinkles index) of all male subjects treated with RSI prion facial mud mask.I) Brightness index of all male subjects treated with RSI prion facial mud mask.