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Alopecia areata (AA) is a prevalent autoimmune condition that causes sudden hair loss and poses significant psychological challenges to affected individuals. Current treatments, including corticosteroids and Janus kinase inhibitors, fail to provide long-term efficacy due to adverse effects and relapse after cessation. This study introduces a nanoparticle (NP) system that codeliver diphenylcyclopropenone (DPCP) and rapamycin (RAPA) prodrugs to induce immune tolerance and promote hair regeneration. The results demonstrated that the coassembled NPs exhibited uniformity and stability, were efficiently taken up by antigen-presenting cells (APCs), and successfully induced dendritic cells (DCs) to differentiate into tolerogenic phenotypes in vitro . In vivo studies on a mouse model of alopecia showed that these NPs significantly accelerated the transition of hair follicles from the telogen phase to the anagen phase, promoting hair regrowth. This research presents a promising therapeutic strategy for AA and offers insights into treating autoimmune diseases where autoantigens are unclear.

With the progress of atherosclerosis (AS), the arterial lumen stenosis and compact plaque structure, the thickening intima and the narrow gaps between endothelial cells significantly limit the penetration efficiency of nanoprobe to plaque, weakening the imaging sensitivity and therapy efficiency. Thus, in this study, a H 2 O 2 -NIR dual-mode nanomotor, Gd-doped mesoporous carbon nanoparticles/Pt with rapamycin (RAPA) loading and AntiCD36 modification (Gd-MCNs/Pt-RAPA-AC) was constructed. The asymmetric deposition of Pt on Gd-MCNs catalyzed H 2 O 2 at the inflammatory site to produce O 2 , which could promote the self-motion of the nanomotor and ease inflammation microenvironment of AS plaque. Near-infrared (NIR) laser irradiation promoted the photothermal conversion of Gd-MCNs to generate the thermal propulsion of nanomotor and photothermal ablation of inflammatory macrophages. Meanwhile, the modification of AntiCD36 to bind with inflammatory macrophages further promotes the targeting effect. The released RAPA could inhibit the inflammatory side effects caused by photothermal effects, and promote macrophage autophagy to hinder the development of AS. The dual-mode propulsion nanomotors combining with the synergistic therapy of photothermal treatment, anti-inflammatory and pro-autophagy provided improved theranositc effect of AS., Competing Interests: Declarations. Ethics approval and consent to participate: All animal experiments were conducted according to the Principles of Laboratory Animal Care of the People’s Republic of China and the Guidelines for the Care and Use of Laboratory Animals of Xuzhou Medical University, China with a permission by the Ethics Committee of Xuzhou Medical University (202307T032). Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Multiple sclerosis (MS) is a severe autoimmune disorder that wreaks havoc on the central nervous system, leading to a spectrum of motor and cognitive impairments. There is no cure, and current treatment strategies rely on broad immunosuppression, leaving patients vulnerable to infections. To address this problem, our approach aims to induce antigen-specific tolerance, a much-needed shift in MS therapy. We have engineered a tolerogenic therapy consisting of spray-dried particles made of a degradable biopolymer, acetalated dextran, and loaded with an antigenic peptide and tolerizing drug, rapamycin (Rapa). After initial characterization and optimization, particles were tested in a myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis model of MS. Representing the earliest possible time of diagnosis, mice were treated at symptom onset in an early therapeutic model, where particles containing MOG and particles containing Rapa+MOG evoked significant reductions in clinical score. Particles were then applied to a highly clinically relevant late therapeutic model during peak disease, where MOG particles and Rapa+MOG particles each elicited a dramatic therapeutic effect, reversing hind limb paralysis and restoring fully functional limbs. To confirm the antigen specificity of our therapy, we immunized mice against the influenza antigen hemagglutinin (HA) and treated them with MOG particles or Rapa+MOG particles. The particles did not suppress antibody responses against HA. Our findings underscore the potential of this particle-based therapy to reverse autoimmunity in disease-relevant models without compromising immune competence, setting it apart from existing treatments.

Iron-based chemodynamic therapy (CDT) exhibits commendable biocompatibility and selectivity, but its efficacy is constrained by the intracellular pH of tumors. To overcome this obstacle, we constructed a silica delivery platform loaded with autophagy-inducing reagents (rapamycin, RAPA) and iron-based Fenton reagents (Fe 3 O 4 ). This platform was utilized to explore a novel strategy that leverages autophagy to decrease tumor acidity, consequently boosting the effectiveness of CDT. Both in vitro and in vivo experiments revealed that RAPA prompted the generation of acidic organelles (e.g., autophagic vacuoles and autophagosomes), effectively changing the intracellular pH in the tumor microenvironment. Furthermore, RAPA-induced tumor acidification significantly amplified the efficacy of Fe 3 O 4 -based Fenton reactions, consequently increasing the effectiveness of Fe 3 O 4 -based CDT. This innovative approach, which leverages the interplay between autophagy induction and iron-based CDT, shows promise in overcoming the limitations posed by tumor pH, thus offering a more efficient approach to tumor treatments., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Scanning Transmission X-ray microscopy (STXM) is a sensitive and selective probe for the penetration of rapamycin which is topically applied to human skin ex vivo and is facilitated by skin treatment with microneedles puncturing the skin. Inner-shell excitation serves as a selective probe for detecting rapamycin by changes in optical density as well as linear combination modeling using reference spectra of the most abundant species. The results indicate that mechanical damage induced by microneedles allows this drug to accumulate in the stratum corneum without reaching the viable skin layers. This is unlike intact skin which shows no drug penetration at all and underscores the mechanical impact of microneedle skin treatment. These results are compared to drug penetration profiles of other drugs highlighting the importance of skin barriers. High spatial resolution studies also indicate that the lipophilic drug rapamycin is observed in corneocytes. Attempts in data evaluation are reported to probe rapamycin also in the lipid layers between the corneocytes, which was not accomplished before. These results are compared to recent results on rapamycin uptake in skin where barrier impairment was induced by pre-treatment with the enzyme trypsin and drug formulations leading to occlusion., (© 2024 The Authors. ChemPhysChem published by Wiley-VCH GmbH.)

Sensory capabilities are crucial for cells to interact with their environment. To mimic these functions in synthetic cells, we developed sensory globular protein vesicles (GPVs) made entirely of recombinant fusion proteins through self-assembly under aqueous conditions. GPVs demonstrate sensory functions via the formation of the FKBP-FRB ternary complex with the signaling molecule, rapamycin. The sensory domain of FKBP or FRB was genetically fused to a fluorescent protein and leucine zipper, which self-assemble into vesicles by forming amphiphilic building blocks through high-affinity binding to a counter leucine zipper fused to an elastin-like polypeptide (ELP) above its lower critical solution temperature. We observed intervesicle aggregation in a time- and concentration-dependent manner upon rapamycin binding, confirmed by colocalization studies and statistical analysis. This system enhances our understanding of protein vesicle functionality for sensing and offers a basis for exploring GPVs as models to replicate key cellular processes in synthetic cells.

Breast cancer stands as the most common form of malignancy among women globally, and it showcases commendable rates of cure when detected in early-stage and non-metastatic conditions. To overcome drug resistance and side effects observed in conventional chemotherapy, the present study aims to deliver rapamycin (RAP), a mTOR protein inhibitor, into a nanostructured lipid carrier (NLC) functionalized with folic acid for promoting active targeting to breast cancer cells. In the first step, the synthesis of 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine-N-[amino(polyethylene glycol)-2000] (ammonium salt) with folic acid (DSPE-PEG 2000 -FA) was successfully performed and characterized by UV spectroscopy, nuclear magnetic resonance, and infrared spectroscopy. Then, the folic acid-modified NLC loaded with RAP (FA-NLC-RAP) and the unmodified formulation (NLC-RAP) was developed and displayed a size of about 100 nm, negative surface charge, and high RAP encapsulation efficiency (94.92 % and 85.72 %, respectively). In vitro studies suggested that FA-NLC-RAP exhibited a higher degree of internalization in cancer cells (MCF-7) than in normal cells (MCF-10A), demonstrating the potential of folic acid as a ligand for promoting active targeting of RAP for breast cancer cells through folate receptors overexpressed in tumor cells FA-NLC-RAP significantly reduced tumor cell viability, similarly to that observed with the RAP solution. The release profile of the formulation was prolonged. Finally, studies in Caenorhabditis elegans evidenced the safety of FA-NLC-RAP characterized by a complete absence of toxicity in this animal model. Therefore, the findings imply that FA-NLC-RAP holds considerable promise for the treatment of breast cancer., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Rapamycin is a potent immunosuppressive drug that has been recently proposed for a wide range of applications beyond its current clinical use. For some of these proposed applications, encapsulation in nanoparticles is key to ensure therapeutic efficacy and safety. In this work, we evaluate the effect of pore size on mesoporous silica nanoparticles (MSN) as rapamycin nanocarriers. The successful preparation of MSN with 4 different pore sizes was confirmed by dynamic light scattering, zeta potential, transmission electron microscopy and N 2 adsorption. In these materials, rapamycin loading was pore size-dependent, with smaller pore MSN exhibiting greater loading capacity. Release studies showed sustained drug release from all MSN types, with larger pore MSN presenting faster release kinetics. In vitro experiments using the murine dendritic cell (DC) line model DC2.4 showed that pore size influenced the biological performance of MSN. MSN with smaller pore sizes presented larger nanoparticle uptake by DC2.4 cells, but were also associated with slightly larger cytotoxicity. Further evaluation of DC2.4 cells incubated with rapamycin-loaded MSN also demonstrated a significant effect of MSN pore size on their immunological response. Notably, the combination of rapamycin-loaded MSN with an inflammatory stimulus (lipopolysaccharide, LPS) led to changes in the expression of DC activation markers (CD40 and CD83) and in the production of the proinflammatory cytokine TNF-α compared to LPS-treated DC without nanoparticles. Smaller-pored MSN induced more substantial reductions in CD40 expression while eliciting increased CD83 expression, indicating potential immunomodulatory effects. These findings highlight the critical role of MSN pore size in modulating rapamycin loading, release kinetics, cellular uptake, and subsequent immunomodulatory responses., (© 2024. The Author(s).)

In the inflammatory microenvironment of atherosclerotic plaques, metabolic dysregulation of superoxide anion (O 2 - ) and hydrogen peroxide (H 2 O 2 ) leads to the activation of feedback mechanisms involving IL-1β, TNF-α, and MCP-1, which triggers inflammatory cascades between macrophages and vascular smooth muscle cells (VSMCs) in atherosclerosis (AS). To address this, a chondroitin sulfate (CS)-functionalized dual-targeted engineered nanozyme, CS-Lip/PB@Rap, was developed by encapsulating mesoporous Prussian blue nanoparticles (PBs) loaded with rapamycin (Rap) within CS-modified liposomes. CS functionalization endowed CS-Lip/PB@Rap with a specific targeting ability for CD44 receptors, thus enabling targeted delivery to inflammatory macrophages and VSMCs. Moreover, its enhanced multiple enzyme-like activities effectively modulated the imbalance of oxidative stress. The underlying mechanism of crosstalk regulation by these engineered nanozymes may inhibit the NF-κB pathway by restoring normal metabolism of O 2 - and H 2 O 2 , thereby blocking the TNF-α, IL-1β, and MCP-1 feedback loops between macrophages and VSMCs. This process reduced the production of inflammatory macrophages and inhibited the VSMC transformation from a contractile phenotype to a synthetic phenotype, preventing the formation of fibrous caps. Furthermore, the elimination of oxidative stress could decrease the production of oxygenized low-density lipoprotein (ox-LDL), which inhibited the formation of foam cells and alleviated the atherogenic progression., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Cardiovascular disease (CVD) is a leading cause of death globally, and vascular calcification (VC) is an important independent risk factor for predicting CVD. Currently, there are no established therapeutic strategies for the treatment of VC. Although recognized combination therapies of nanomedicines can provide effective strategies for disease treatment, the clinical application of nanomedicines is limited because of their complex preparation processes, low drug loading rates, and unpredictable safety risks. Thus, developing a simple, efficient, and safe nanodrug to simultaneously regulate inflammation and autophagy may be a promising strategy for treating VC. Herein, an anti-inflammatory peptide (lysine-proline-valine peptides, KPV) and the autophagy activator rapamycin (RAPA) are self-assembled to form new carrier-free spherical nanoparticles (NPs), which shows good stability and biosafety. In vivo and in vitro, KPV-RAPA NPs significantly inhibit VC in mice compared to the other treatment groups. Mechanistically, KPV-RAPA NPs inhibit inflammatory responses and activated autophagy. Therefore, this study indicates that the new carrier-free KPV-RAPA NPs have great potential as therapeutic agents for VC combination therapy, which can promote the development of nanodrugs for VC., (© 2024 Wiley‐VCH GmbH.)

Modulating protein-protein interactions (PPIs) is an attractive strategy in drug discovery. Molecular glues, bifunctional small-molecule drugs that promote PPIs, offer an approach to targeting traditionally undruggable targets. However, the efficient discovery of molecular glues has been hampered by the current limitations of conventional ensemble-averaging-based methods. In this study, we present a YaxAB nanopore for probing the efficacy of molecular glues in inducing PPIs. Using YaxAB nanopores, we demonstrate single-molecule-based, label-free monitoring of protein complex formation between mammalian target of rapamycin (mTOR) and FK506-binding proteins (FKBPs) triggered by the molecular glue, rapamycin. Owing to its wide entrance and adjustable pore size, in combination with potent electro-osmotic flow (EOF), a single funnel-shaped YaxAB nanopore enables the simultaneous detection and single-molecule-level quantification of multiprotein states, including single proteins, binary complexes, and ternary complexes induced by rapamycin. Notably, YaxAB nanopores could sensitively discriminate between the binary complexes or ternary complexes induced by rapamycin and its analogues, despite the subtle size differences of ∼122 or ∼116 Da, respectively. Taken together, our results provide proof-of-concept for single-molecule-based, label-free, and ultrasensitive screening and structure-activity relationship (SAR) analysis of molecular glues, which will contribute to low-cost, highly efficient discovery, and rational design of bifunctional modality of drugs, such as molecular glues.

In the realm of arterial disease interventions, drug-eluting stents (DES) have become a vital therapeutic choice in preventing atherosclerotic plaque formation and restenosis and facilitating vessel healing. Sirolimus-encapsulated poly Lactic-co-Glycolic acid (PLGA) Microparticles (MPs) were developed using solvent evaporation. MPs were freeze-dried with a cryoprotectant and coated on the stent surface using an efficient and reproducible nitrogen-assisted spray coating technique. The MPs displayed a uniform distribution particle size of 4.38 ± 1.1 μm, span value of 0.88 ± 0.02, coating mass transfer efficiency of 13.45 ± 1.1 % on the stent, and a coating time of ≤ 2 min per stent. Post sterilization, the particle size and morphology of the coated stents remained unchanged. Accelerated in vitro drug release profiles were evaluated under different conditions, indicating significant influences based on dissolution methods ranging from 28.2 %±4.3 %, 42.5 %±5.3 %, 76.6 %±4.7 %, and 84.25 %±3.1 % for dialysis bag (DB), vessel simulating flow-through cell (vFTC), flow-through cell (FTC), and sample and separate (SS) technique respectively for 48 h. The drug release mechanism from the coated stents is governed by the combination of the Korsmeyer Peppas and Higuchi models. The developed dissolution method exhibited discriminative effectiveness when evaluated with critical formulation attributes and process parameter variations. The 48 h accelerated drug release studies correlated well with the 6-month real-time release rate with an R 2 value of 0.9142 and Pearson's R2 of 0.9561. Ex-vivo studies demonstrated the permeation of MPs into artery tissues. Stability studies confirmed that MPs coated stents maintained desired properties at 4 °C and 30 °C/65 % RH for 6 months. Overall, these findings contribute to advancing stent technology, suggesting the potential for improvement of arterial interventions and enhanced patient outcomes., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: [Sarika Jadhav reports financial support was provided by Sahajanand Medical Technologies Pvt Ltd. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper]., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Abdominal aortic aneurysm (AAA) represents a critical cardiovascular condition characterized by localized dilation of the abdominal aorta, carrying a significant risk of rupture and mortality. Current treatment options are limited, necessitating novel therapeutic approaches. This study investigates the potential of a pioneering nanodrug delivery system, RAP@PFB, in mitigating AAA progression. RAP@PFB integrates pentagalloyl glucose (PGG) and rapamycin (RAP) within a metal-organic-framework (MOF) structure through a facile assembly process, ensuring remarkable drug loading capacity and colloidal stability. The synergistic effects of PGG, a polyphenolic antioxidant, and RAP, an mTOR inhibitor, collectively regulate key players in AAA pathogenesis, such as macrophages and smooth muscle cells (SMCs). In macrophages, RAP@PFB efficiently scavenges various free radicals, suppresses inflammation, and promotes M1-to-M2 phenotype repolarization. In SMCs, it inhibits apoptosis and calcification, thereby stabilizing the extracellular matrix and reducing the risk of AAA rupture. Administered intravenously, RAP@PFB exhibits effective accumulation at the AAA site, demonstrating robust efficacy in reducing AAA progression through multiple mechanisms. Moreover, RAP@PFB demonstrates favorable biosafety profiles, supporting its potential translation into clinical applications for AAA therapy., (© 2024 The Author(s). Small published by Wiley‐VCH GmbH.)

Purpose: Renal cell carcinoma (RCC) is the most common and lethal type of urogenital cancer, with one-third of new cases presenting as metastatic RCC (mRCC), which, being the seventh most common cancer in men and the ninth in women, poses a significant challenge. For patients with poor prognosis, temsirolimus (TEM) has been approved for first-line therapy, possessing pharmacodynamic activities that block cancer cell growth and inhibit proliferation-associated proteins. However, TEM suffers from poor water solubility, low bioavailability, and systemic side effects. This study aims to develop a novel drug formulation for the treatment of RCC., Methods: In this study, amphiphilic block copolymer (poly(ethylene glycol) monomethyl ether-poly(beta-amino ester)) (mPEG-PBAE) was utilized as a drug delivery vehicle and TEM-loaded micelles were prepared by thin-film hydration method by loading TEM inside the nanoparticles. Then, the molecular weight of mPEG-PBAE was controlled to make it realize hydrophobic-hydrophilic transition in the corresponding pH range thereby constructing pH-responsive TEM-loaded micelles. Characterization of pH-responsive TEM-loaded nanomicelles particle size, potential and micromorphology while its determination of drug-loading properties, in vitro release properties. Finally, pharmacodynamics and hepatorenal toxicity were further evaluated., Results: TEM loading in mPEG-PBAE increased the solubility of TEM in water from 2.6 μg/mL to more than 5 mg/mL. The pH-responsive TEM-loaded nanomicelles were in the form of spheres or spheroidal shapes with an average particle size of 43.83 nm and a Zeta potential of 1.79 mV. The entrapment efficiency (EE) of pH-responsive TEM nanomicelles with 12.5% drug loading reached 95.27%. Under the environment of pH 6.7, the TEM was released rapidly within 12 h, and the release rate could reach 73.12% with significant pH-dependent characteristics. In vitro experiments showed that mPEG-PBAE preparation of TEM-loaded micelles had non-hemolytic properties and had significant inhibitory effects on cancer cells. In vivo experiments demonstrated that pH-responsive TEM-loaded micelles had excellent antitumor effects with significantly reduced liver and kidney toxicity., Conclusion: In conclusion, we successfully prepared pH-responsive TEM-loaded micelles. The results showed that pH-responsive TEM-loaded micelles can achieve passive tumor targeting of TEM, and take advantage of the acidic conditions in tumor tissues to achieve rapid drug release., Competing Interests: The authors report no conflicts of interest in this work., (© 2024 Wang et al.)

Pulmonary fibrosis is a lethal interstitial lung disease, for which current treatments are inadequate in halting its progression. A significant factor contributing to the development of fibrosis is insufficient autophagy, which leads to increased fibroblast proliferation and collagen deposition. However, treatments aimed at upregulating autophagy often cause further lung pathology due to the disruption of epithelial cell balance. In response, we have developed a novel macrophage delivery system loaded with an epithelial-to-mesenchymal transition inhibitor, hyperoside (HYP), and an autophagy inducer, rapamycin (RAP). This system targets the fibrotic areas of the lung through chemotaxis, releases liposomes via macrophage extracellular traps, and effectively inhibits fibroblast proliferation while restoring the alveolar structure through the combined effects of RAP and HYP, ultimately reducing lung pathology without causing systemic toxicity. Our findings not only highlight a promising method to enhance autophagy-based treatments for pulmonary fibrosis but also demonstrate the potential of macrophages as effective nanocarriers for drug delivery.

Inducible dimerization systems, such as rapamycin-induced dimerization of FK506 binding protein (FKBP) and FKBP-rapamycin binding (FRB) domain, are widely employed chemical biology tools to manipulate cellular functions. We previously advanced an inducible dimerization system into an inducible oligomerization system by developing a bivalent fusion protein, FRB-FKBP, which forms large oligomers upon rapamycin addition and can be used to manipulate cells. However, the oligomeric structure of FRB-FKBP remains unclear. Here, we report that FRB-FKBP forms a rotationally symmetric trimer in crystals, but a larger oligomer in solution, primarily tetramers and pentamers, which maintain similar inter-subunit contacts as in the crystal trimer. These findings expand the applications of the FRB-FKBP oligomerization system in diverse biological events., (© 2024 Federation of European Biochemical Societies.)

Drug delivery systems based on biomimetic peptide nanoparticles are steadily gaining prominence in the treatment of diverse medical conditions. This study focused on the development of peptides that depend on ligand-receptor interactions to load rapamycin (RAPA). Furthermore, a multifunctional peptide was engineered to target oxidized low-density lipoprotein (oxLDL) within atherosclerotic plaques, facilitating the localized delivery of RAPA. The interactions between peptides and RAPA/oxLDL were analyzed by simulations and experimental approaches. Results show that the main amino acid residues on the mammalian target of rapamycin that bind to RAPA are constructed as peptides (P1 and P2), which have specific interactions with RAPA and can effectively improve the loading efficiency of RAPA. The encapsulation and drug loading efficiencies of P1/P2 were 68.0/47.9% and 48.3/36.5%, respectively. In addition, the interaction force of the multifunctional peptide (P3) and oxLDL surpassed that of their interaction with human umbilical vein endothelial cells by a factor of 3.6, conclusively establishing the specific targeting of oxLDL by these nanoparticles. The encapsulation and drug loading efficiencies of P3 for RAPA were determined to be 60.2% and 41.5%. P3 can effectively load RAPA and target oxLDL within the plaque, suggesting that P3 has potential as a therapeutic agent for atherosclerotic disease.

Purpose: Ovarian cancer has the highest mortality rate and lowest survival rate among female reproductive system malignancies. There are treatment options of surgery and chemotherapy, but both are limited. In this study, we developed and evaluated micelles composed of D-α-tocopheryl polyethylene-glycol (PEG) 1000 succinate (TPGS) and Soluplus ® (SOL) loaded with olaparib (OLA), a poly(ADP-ribose)polymerase (PARP) inhibitor, and rapamycin (RAPA), a mammalian target of rapamycin (mTOR) inhibitor in ovarian cancer., Methods: We prepared micelles containing different molar ratios of OLA and RAPA embedded in different weight ratios of TPGS and SOL (OLA/RAPA-TPGS/SOL) were prepared and physicochemical characterized. Furthermore, we performed in vitro cytotoxicity experiments of OLA, RAPA, and OLA/RAPA-TPGS/SOL. In vivo toxicity and antitumor efficacy assays were also performed to assess the efficacy of the mixed micellar system., Results: OLA/RAPA-TPGS/SOL containing a 4:1 TPGS:SOL weight ratio and a 2:3 OLA:RAPA molar ratio showed synergistic effects and were optimized. The drug encapsulation efficiency of this formulation was >65%, and the physicochemical properties were sustained for 180 days. Moreover, the formulation had a high cell uptake rate and significantly inhibited cell migration (** p < 0.01). In the in vivo toxicity test, no toxicity was observed, with the exception of the high dose group. Furthermore, OLA/RAPA-TPGS/SOL markedly inhibited tumor spheroid and tumor growth in vivo., Conclusion: Compared to the control, OLA/RAPA-TPGS/SOL showed significant tumor inhibition. These findings lay a foundation for the use of TPGS/SOL mixed micelles loaded with OLA and RAPA in the treatment of ovarian cancer., Competing Interests: Professor Dae Hwan Shin reports a pending patent “TPGS/Soluplus Micelles containing olaparib and rapamycin, and uses thereof”. The authors report no other conflicts of interest in this work., (© 2024 Shin et al.)

Immune rejection remains the major cause of corneal graft failure. Immunosuppressants (such as rapamycin; RAPA) adjunctive to antibiotics (such as levofloxacin hydrochloride; Lev) are a clinical mainstay after corneal grafts but suffer from poor ocular bioavailability associated with severe side effects. In this study, we fabricated a Lev@RAPA micelle loaded cationic peptide-based hydrogel (NapFFKK) as a dual-drug delivery system by integrating RAPA micelles with Lev into a cationic NapFFKK hydrogel to potentially reduced the risk of corneal graft rejection. The properties of the resulting hydrogels were characterized using transmission electronmicroscopy and rheometer. Lev@RAPA micelles loaded NapFFKK hydrogel provided sustained in vitro drug release without compromising their inherent pharmacological activities. Topical instillation of Lev@RAPA micelles loaded NapFFKK hydrogel resulted in the great ocular tolerance and extended precorneal retention over 60 min, thus significantly enhancing the ocular bioavailability of both Lev and RAPA. Overall, such dual-drug delivery system might be a promising formulation for the suppression of corneal graft failure., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Heart transplantation offers life-saving treatment for patients with end-stage heart failure; however, ischemia-reperfusion injury (IRI) and subsequent immune responses remain significant challenges. Current therapies primarily target adaptive immunity, with limited options available for addressing IRI and innate immune activation. Although plant-derived vesicle-like nanoparticles show promise in managing diseases, their application in organ transplantation complications is unexplored. Here, this work develops a novel reactive oxygen species (ROS)-responsive multifunctional fusion extracellular nanovesicles carrying rapamycin (FNVs@RAPA) to address early IRI and Ly6C + Ly6G - inflammatory macrophage-mediated rejection in heart transplantation. The FNVs comprise Exocarpium Citri grandis-derived extracellular nanovesicles with anti-inflammatory and antioxidant properties, and mesenchymal stem cell membrane-derived nanovesicles expressing calreticulin with macrophage-targeting ability. A novel ROS-responsive bio-orthogonal chemistry approach facilitates the active targeting delivery of FNVs@RAPA to the heart graft site, effectively alleviating IRI and promoting the polarization of Ly6C + Ly6G - inflammatory macrophages toward an anti-inflammatory phenotype. Hence, FNVs@RAPA represents a promising therapeutic approach for mitigating early transplantation complications and immune rejection. The fusion-targeted delivery strategy offers superior heart graft site enrichment and macrophage-specific targeting, promising improved transplant outcomes., (© 2024 Wiley‐VCH GmbH.)

Sirolimus (SR) is a macrolide with antifungal and antitumor immunosuppressant properties, classified as a selective inhibitor of mammalian target of rapamycin (mTOR). In this study, an ionic in situ gel of SR (SR-SUS-ISG) was formulated using gellan gum, exhibiting stability regardless of temperature and pH variations, causing minimal irritation. Harnessing the physiological conditions of the eye, SR-SUS-ISG underwent gelation upon contact with ions, increasing drug viscosity and prolonging retention on the ocular surface. Concurrently, SR-SUS-ISG displayed favorable shear dilution properties, reducing viscosity at ambient temperature, enhancing fluidity, and facilitating convenient packaging and transport. Biocompatibility assessments on both human corneal epithelial cells and rabbit eyes demonstrated that SR-SUS-ISG could well be tolerated. Pharmacokinetic investigations in rabbit ocular aqueous humor revealed sustained release, improved corneal penetration, and enhanced bioavailability. Additionally, in a rat corneal alkali burn model, SR-SUS-ISG exhibited inhibitory effects on corneal neovascularization, associated with decreased levels of the inflammatory factors VEGF and MMPs. These findings suggested that SR-SUS-ISG held promise as an effective ocular drug delivery system., (© 2024. The Author(s).)

In the context of arteriovenous fistula (AVF) failure, local delivery enables the release of higher concentrations of drugs that can suppress neointimal hyperplasia (NIH) while reducing systemic adverse effects. However, the radiolucency of polymeric delivery systems hinders long-term in vivo surveillance of safety and efficacy. We hypothesize that using a radiopaque perivascular wrap to deliver anti-NIH drugs could enhance AVF maturation. Through electrospinning, we fabricated multifunctional perivascular polycaprolactone (PCL) wraps loaded with bismuth nanoparticles (BiNPs) for enhanced radiologic visibility and drugs that can attenuate NIH─rosuvastatin (Rosu) and rapamycin (Rapa). The following groups were tested on the AVFs of a total of 24 Sprague-Dawley rats with induced chronic kidney disease: control (i.e., without wrap), PCL-Bi (i.e., wrap with BiNPs), PCL-Bi-Rosu, and PCL-Bi-Rapa. We found that BiNPs significantly improved the wraps' radiopacity without affecting biocompatibility. The drug release profiles of Rosu (hydrophilic drug) and Rapa (hydrophobic drug) differed significantly. Rosu demonstrated a burst release followed by gradual tapering over 8 weeks, while Rapa demonstrated a gradual release similar to that of the hydrophobic BiNPs. In vivo investigations revealed that both drug-loaded wraps can reduce vascular stenosis on ultrasonography and histomorphometry, as well as reduce [ 18 F]Fluorodeoxyglucose uptake on positron emission tomography. Immunohistochemical studies revealed that PCL-Bi-Rosu primarily attenuated endothelial dysfunction and hypoxia in the neointimal layer, while PCL-Bi-Rapa modulated hypoxia, inflammation, and cellular proliferation across the whole outflow vein. In summary, the controlled delivery of drugs with different properties and mechanisms of action against NIH through a multifunctional, radiopaque perivascular wrap can improve imaging and histologic parameters of AVF maturation.

A complete and detailed characterization of Rapamycin (1) and Prolylrapamycin (2) has been conducted by homo- and hetero-nuclear NMR experiments in DMSO-d 6 along with HRMS and FT-IR spectra and DSCs analyses. The NMR experiments allowed the assignment of every single proton and carbon atom belonging to the two structures and the definitive confirm of the presence of a pyrrolidine ring in Prolylrapamycin (2) in place of the piperidine ring that characterizes the structure of Sirolimus., (© 2024. The Author(s).)

Rapamycin (RAP) is currently being developed as potential antibreast cancer drug. However, its poor solubility completely limits its use. The aim of this study was to develop polyethylene glycol-poly(lactide-co-glycolide) (PEG-PLGA)-based nanoparticles (NPs) to load RAP via microfluidics with an appropriate polyethylene glycol (PEG) content to enhance the bioavailability of RAP. Polydimethylsiloxane (PDMS) chips with a Y-shaped channel were designed to obtain RAP-loaded PEG-PLGA NPs (RAP-PEG-PLGA). The entrapment efficiency (EE) and drug loading (DL) as well as release profile of RAP-PEG-PLGA were evaluated, and their resistance to plasma albumin adsorption of NPs with different PEG contents was evaluated and compared. RAW264.7 and 4T1 cells were used to assess the antiphagocytic and anticancer cells effect of NPs, respectively. RAP-PEG-PLGA of around 124 nm in size were successfully prepared with the EE of 82.0% and DL of 12.3%, and sustained release for around 40 d. A PEG relative content of 10% within the PEG-PLGA molecule was shown superior in resisting protein adsorption. RAP-PEG-PLGA inhibited the growth of breast cancer cells when the concentration was over 10 μg/mL, and the inhibition efficiency was significantly higher than free RAP. Hence, the current RAP-PEG-PLGA could be a potential therapeutic system for breast cancer treatment.

Stroke is a leading cause of global mortality and severe disability. However, current strategies used for treating ischemic stroke lack specific targeting capabilities, exhibit poor immune escape ability, and have limited drug release control. Herein, we developed an ROS-responsive nanocarrier for targeted delivery of the neuroprotective agent rapamycin (RAPA) to mitigate ischemic brain damage. The nanocarrier consisted of a sulfated chitosan (SCS) polymer core modified with a ROS-responsive boronic ester enveloped by a red blood cell membrane shell incorporating a stroke homing peptide. When encountering high levels of intracellular ROS in ischemic brain tissues, the release of SCS combined with RAPA from nanoparticle disintegration facilitates effective microglia polarization and, in turn, maintains blood-brain barrier integrity, reduces cerebral infarction, and promotes cerebral neurovascular remodeling in a mouse stroke model involving transient middle cerebral artery occlusion (tMCAO). This work offers a promising strategy to treat ischemic stroke therapy.

Biodegradable drug-eluting stents (DESs) are gaining importance owing to their attractive features, such as complete drug release to the target site. Magnesium (Mg) alloys are promising materials for future biodegradable DESs. However, there are few explorations using biodegradable Mg for cardiovascular stent application. In this present study, sirolimus-loaded poly D, L-lactic-co-glycolic acid (PLGA)-coated/ sirolimus-fixed/AZ91 Mg alloy-based substrate was developed via a layer-by-layer approach for cardiovascular stent application. The AZ91 Mg alloy was prepared through the squeeze casting technique. The casted AZ91 Mg alloy (Mg) was alkali-treated to provide macroporous networks to hold the sirolimus and PLGA layers. The systematic characterization was investigated via electrochemical, optical, physicochemical, and in-vitro biological characteristics. The presence of the Mg 17 Al 12 phase in the Mg sample was found in the x-ray diffraction system (XRD) spectrum which influences the corrosion behavior of the developed substrate. The alkali treatment increases the substrate's hydrophilicity which was confirmed through static contact angle measurement. The anti-corrosion characteristic of casted-AZ91 Mg alloy (Mg) was slightly less than the sirolimus-loaded PLGA-coated alkali-treated AZ91 Mg alloy (Mg/Na/S/P) substrate. However, dissolution rates for both substrates were found to be controlled at cell culture conditions. Radiographic densities of AZ91 Mg alloy substrates (Mg, Mg/Na, and Mg/Na/S/P) were measured to be 0.795 ± 0.015, 0.742 ± 0.01, and 0.712 ± 0.017, respectively. The star-shaped structure of 12% sirolimus/PLGA ensures the bioavailability of the drugs. Sirolimus release kinetic was fitted up to 80% with the "Higuchi model" for Mg samples, whereas Mg/Na/S/P showed 45% fitting with a zero-order mechanism. The Mg/Na/S/P substrate showed a 70% antithrombotic effect compared to control. Further, alkali treatment enhances the antibacterial characteristic of AZ91 Mg alloy. Also, the alkali-treated sirolimus-loaded substrates (Mg/Na/S and Mg/Na/S/P) inhibit the valvular interstitial cell's growth significantly in in-vitro. Hence, the results imply that sirolimus-loaded PLGA-coated AZ91 Mg alloy-based substrate can be a potential candidate for cardiovascular stent application., (© 2023 Wiley Periodicals LLC.)

Restenosis remains the main reason for treatment failure of arterial disease. Sirolimus (SIR) as a potent anti-proliferative agent is believed to prevent the phenomenon. The application of exosomes provides an extended-release delivery platform for SIR intramural administration. Herein, SIR was loaded into fibroblast-derived exosomes isolated by ultracentrifugation. Different parameters affecting drug loading were optimized, and exosome samples were characterized regarding physicochemical, pharmaceutical, and biological properties. Cytotoxicity, scratch wound assays, and quantitative real-time PCR for inflammation- and migration-associated genes were performed. Restenosis was induced by carotid injury in a rat carotid model and then exosomes were locally administered. After 14 days, animals were investigated by computed tomography (CT) angiography, morphometric, and immunohistochemical analyses. Western blotting confirmed the presence of specific protein markers in exosomes. Characterization of empty and SIR-loaded exosomes verified round and nanoscale structure of vesicles. Among prepared formulations, desired entrapment efficiency (EE) of 76% was achieved by protein:drug proportion of 2:1 and simple incubation for 30 min at 37 °C. Also, the optimal formulation released about 30% of the drug content during the first 24 h, followed by a prolonged release for several days. In vitro studies revealed the uptake and functional efficacy of the optimized formulation. In vivo studies revealed that %restenosis was in the following order: saline > empty exosomes > SIR-loaded exosomes. Furthermore, Ki67, alpha smooth muscle actin (α-SMA), and matrix metalloproteinase (MMP) markers were less expressed in the SIR-exosomes-treated arteries. These findings confirmed that exosomal SIR could be a hopeful strategy for the prevention of restenosis., (© 2023. Controlled Release Society.)

Single-molecule data are of great significance in biology, chemistry, and medicine. However, new experimental tools to characterize, in a multiplexed manner, protein bond rupture under force are still needed. Acoustic force spectroscopy is an emerging manipulation technique which generates acoustic waves to apply force in parallel on multiple microbeads tethered to a surface. We here exploit this configuration in combination with the recently developed modular junctured-DNA scaffold that has been designed to study protein-protein interactions at the single-molecule level. By applying repetitive constant force steps on the FKBP12-rapamycin-FRB complex, we measure its unbinding kinetics under force at the single-bond level. Special efforts are made in analyzing the data to identify potential pitfalls. We propose a calibration method allowing in situ force determination during the course of the unbinding measurement. We compare our results with well-established techniques, such as magnetic tweezers, to ensure their accuracy. We also apply our strategy to study the force-dependent rupture of a single-domain antibody with its antigen. Overall, we get a good agreement with the published parameters that have been obtained at zero force and population level. Thus, our technique offers single-molecule precision for multiplexed measurements of interactions of biotechnological and medical interest., Competing Interests: Declaration of interests PSL valorisation has submitted a patent related to the J-DNA (PCT FR2018/053533) with D.K., T.S., and C.G. among the inventors., (Copyright © 2023 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Imaging protein-protein interactions (PPIs) is a hot topic in molecular medicine in the postgenomic sequencing era. In the present study, we report bright and highly sensitive single-chain molecular strain probe templates which embed full-length Renilla luciferase 8.6-535SG (RLuc86SG) or Artificial luciferase 49 (ALuc49) as reporters. These reporters were deployed between FKBP-rapamycin binding domain (FRB) and FK506-binding protein (FKBP) as a PPI model. This unique molecular design was conceptualized to exploit molecular strains of the sandwiched reporters appended by rapamycin-triggered intramolecular PPIs. The ligand-sensing properties of the templates were maximized by interface truncations and substrate modulation. The highest fold intensities, 9.4 and 16.6, of the templates were accomplished with RLuc86SG and ALuc49, respectively. The spectra of the templates, according to substrates, revealed that the colors are tunable to blue, green, and yellow. The putative substrate-binding chemistry and the working mechanisms of the probes were computationally modeled in the presence or absence of rapamycin. Considering that the molecular strain probe templates are applicable to other PPI models, the present approach would broaden the scope of the bioassay toolbox, which harnesses the privilege of luciferase reporters and the unique concept of the molecular strain probes into bioassays and molecular imaging.

Rapalogues are powerful therapeutic modalities for breast cancer; however, they suffer from low solubility and dose-limiting side effects. To overcome these challenges, we developed a long-circulating multiheaded drug carrier called 5FA, which contains rapamycin-binding domains linked with elastin-like polypeptides (ELPs). To target these "Hydra-ELPs" toward breast cancer, we here linked 5FA with four distinct peptides which are reported to engage the cell surface form of the 78 kDa glucose-regulated protein (csGRP78). To determine if these peptides affected the carrier solubility, this library was characterized by light scattering and mass spectrometry. To guide in vitro selection of the most potent functional carrier for rapamycin, its uptake and inhibition of mTORC1 were monitored in a ductal breast cancer model (BT474). Using flow cytometry to track cellular association, it was found that only the targeted carriers enhanced cellular uptake and were susceptible to proteolysis by SubA, which specifically targets csGRP78. The functional inhibition of mTOR was monitored by Western blot for pS6K, whereby the best carrier L-5FA reduced mTOR activity by 3-fold compared to 5FA or free rapamycin. L-5FA was further visualized using super-resolution confocal laser scanning microscopy, which revealed that targeting increased exposure to the carrier by ∼8-fold. This study demonstrates how peptide ligands for GRP78, such as the L peptide (RLLDTNRPLLPY), may be incorporated into protein-based drug carriers to enhance targeting.

Loading and eluting drugs on self-expandable metallic stents (SEMSs) can be challenging in terms of fabrication, mechanical stability, and therapeutic effects. In this study, a flexible 3D nanonetworked silica film (NSF) capable of withstanding mechanical stress during dynamic expansion is constructed to function as a drug delivery platform on an entire SEMS surface. Despite covering a broad curved area, the synthesized NSF is defect-free and thin enough to increase the stent strut diameter (110 µm) by only 0.4 percent (110.45 µm). The hydrophobic modification of the surface enables loading of 4.7 times the sirolimus (SRL) concentration in NSF than Cypher, polymer-coated commercial stent, which is based on the same thickness of coating layer. Furthermore, SRL-loaded NSF exhibits a twofold delay in release compared to the control group without NSF. The SRL-loaded NSF SEMS significantly suppresses stent-induced tissue hyperplasia than the control SEMS in the rat esophagus (all variables, p < 0.05). Thus, the developed NSF is a promising polymer-free drug delivery platform to efficiently treat esophageal stricture., (© 2022 Wiley-VCH GmbH.)

We have previously developed several kinds of rapamycin-encapsulated nanoparticles to achieve sustained release of rapamycin to treat hemangioma. However, lack of intrinsic targeting and easy clearance by the immune system are major hurdles that artificial fabricated nanoparticles must overcome. We constructed rapamycin-encapsulated macrophage-derived exosomes mimic nanoparticles-in-microspheres (RNM), to achieve the goal of continuous targeted therapy of hemangiomas. The rapamycin-encapsulated exosome mimic nanoparticles (RN) were firstly prepared by the extrusion-based method from the U937 cells (the human macrophage cell line). After then, RN was encapsulated with PLGA (poly(lactic-co-glycolic acid)) microspheres to obtain RNM. The release profile, targeting activity, and biological activity of RN and RNM were investigated on hemangioma stem cells (HemSCs). RN has a size of 100 nm in diameter, with a rapamycin encapsulation efficacy (EE) of 83%. The prepared microspheres RNM have a particle size of ~30 µm), and the drug EE of RNM is 34%. The sustained release of RNM can remarkably be achieved for 40 days. As expected, RN and RNM showed effective inhibition of cellular proliferation, significant cellular apoptosis, and remarkable repressed expression of angiogenesis factors in HemSCs. Our results showed that RNM is an effective approach for prolonged and effective delivery of rapamycin to hemangiomas., (Copyright © 2022. Published by Elsevier Masson SAS.)