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This study explores the potential of laser-induced nano-photon-poration as a non-invasive technique for the intracellular delivery of micro/macromolecules at the single-cell level. This research proposes the utilization of gold-coated spiky polymeric nanoparticles (Au-PNPs) and gold nanorods (GNRs) to achieve efficient intracellular micro/macromolecule delivery at the single-cell level. By shifting the operating wavelength towards the near-infrared (NIR) range, the intracellular delivery efficiency and viability of Au-PNP-mediated photon-poration are compared to those using GNR-mediated intracellular delivery. Employing Au-PNPs as mediators in conjunction with nanosecond-pulsed lasers, a highly efficient intracellular delivery, while preserving high cell viability, is demonstrated. Laser pulses directed at Au-PNPs generate over a hundred hot spots per particle through plasmon resonance, facilitating the formation of photothermal vapor nanobubbles (PVNBs). These PVNBs create transient pores, enabling the gentle transfer of cargo from the extracellular to the intracellular milieu, without inducing deleterious effects in the cells. The optimization of wavelengths in the NIR region, coupled with low laser fluence (27 mJ/cm 2 ) and nanoparticle concentrations (34 µg/mL), achieves outstanding delivery efficiencies (96%) and maintains high cell viability (up to 99%) across the various cell types, including cancer and neuronal cells. Importantly, sustained high cell viability (90-95%) is observed even 48 h post laser exposure. This innovative development holds considerable promise for diverse applications, encompassing drug delivery, gene therapy, and regenerative medicine. This study underscores the efficiency and versatility of the proposed technique, positioning it as a valuable tool for advancing intracellular delivery strategies in biomedical applications.

Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) are promising candidates for disease modeling and therapeutic purposes, however, non-viral intracellular delivery in these cells remains challenging. Gold nanoparticle (AuNP)-sensitized photoporation creates transient pores in the cell membrane by vapor nanobubble formation, allowing diffusion of extracellular biomolecules. This non-viral technique was employed to test and optimize its distinct physical mode of action in iPSC-CMs. Photoporation optimization was aimed at achieving high delivery rates while minimizing cell death. Various AuNP concentrations, in conjunction with different laser fluences, were explored to facilitate the intracellular delivery of 10 kDa and 150 kDa FITC-labelled dextran as model macromolecules. Cardiomyocyte viability was assessed using the CellTiter-Glo® viability assay, while the delivery efficiency was quantified through flow cytometry. On 30 day-old cardiomyocytes, AuNP photoporation was able to yield ∼60 % delivery efficiency while maintaining a high cell viability (∼70 %). Overall, higher AuNP concentrations resulted in greater delivery efficiencies, albeit at the expense of lower cell viability. Finally, photoporation was capable of patterning a geometric shape, demonstrating its exceptional selective resolution in delivering molecules to spatially restricted regions of the cell culture. In conclusion, AuNP-photoporation exhibits considerable potential as an effective and gentle non-viral method for intracellular delivery in iPSC-CMs.•AuNP-photoporation is a non-viral intracellular delivery method suitable for iPSC-CMs with high efficiency and cell viability•This method is capable of spatially resolved intracellular delivery with excellent resolution., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: KB declares financial interest in the company Trince which markets the LumiPore photoporation technology, (© 2024 The Authors. Published by Elsevier B.V.)

Molecular chaperones play an important part in protein folding and delivery in nature. Here, the authors report on the creation of a synthetic chaperone to control the folding of therapeutic peptides from random coil to alpha helix and demonstrate enhanced therapeutic potential in an in vivo tumour model.

Abstract Monoclonal antibodies targeting the epidermal growth factor receptor (EGFR), including cetuximab and panitumumab, have been used in clinic settings to treat cancer. They have also recently been applied to antibody–drug conjugates (ADCs); however, their clinical efficacy is limited by several issues, including lower internalization efficiency. The binding of cetuximab to the extracellular domain of EGFR suppresses ligand-induced events; therefore, we focus on ligand-independent non-canonical EGFR endocytosis for the delivery of ADCs into cells. Tumor necrosis factor-α (TNF-α) strongly induces the endocytosis of the cetuximab-EGFR complex within 15 min via the p38 phosphorylation of EGFR in a tyrosine kinase-independent manner. A secondary antibody conjugated with saporin, a ribosome-inactivating protein, also undergoes internalization with the complex and enhances its anti-proliferative activity. Anti-cancer agents, including cisplatin and temozolomide, also induce the p38-mediated internalization. The results of the present study demonstrate that synchronous non-canonical EGFR endocytosis may be a feasible strategy for promoting the therapeutic efficacy of EGFR-targeting ADCs in clinical settings.

Flavia Ferrantelli,1,* Claudia Arenaccio,1,* Francesco Manfredi,1 Eleonora Olivetta,1 Chiara Chiozzini,1 Patrizia Leone,1 Zulema Percario,2 Alessandro Ascione,1 Michela Flego,1 Paola Di Bonito,3 Luisa Accardi,3 Maurizio Federico1 1National Center for Global Health, Istituto Superiore Di Sanità (ISS), Rome, Italy; 2Department of Science, Roma Tre University, Rome, Italy; 3Department of Infectious Diseases, Istituto Superiore Di Sanità (ISS), Rome, Italy*These authors contributed equally to this workCorrespondence: Maurizio FedericoNational Center for Global Health, Istituto Superiore Di Sanità, Viale Regina Elena, 299, Rome 00161, ItalyTel +39-06-4990-3605Fax +39-06-49903210Email maurizio.federico@iss.itPurpose: Single-chain variable fragments (scFvs) are one of the smallest antigen-binding units having the invaluable advantage to be expressed by a unique short open reading frame (ORF). Despite their reduced size, spontaneous cell entry of scFvs remains inefficient, hence precluding the possibility to target intracellular antigens. Here, we describe an original strategy to deliver scFvs inside target cells through engineered extracellular vesicles (EVs). This approach relies on the properties of a Human Immunodeficiency Virus (HIV)-1 Nef mutant protein referred to as Nefmut. It is a previously characterized Nef allele lacking basically all functions of wt Nef, yet strongly accumulating in the EV lumen also when fused at its C-terminus with a foreign protein. To gain the proof-of-principle for the efficacy of the proposed strategy, the tumor-promoting Human Papilloma Virus (HPV)16-E7 protein was considered as a scFv-specific intracellular target. The oncogenic effect of HPV16-E7 relies on its binding to the tumor suppressor pRb protein leading to a dysregulated cell duplication. Interfering with this interaction means impairing the HPV16-E7-induced cell proliferation.Methods: The Nefmut gene was fused in frame at its 3ʹ-terminus with the ORF coding for a previously characterized anti-HPV16-E7 scFv. Interaction between the Nefmut-fused anti-HPV16-E7 scFv and the HPV16-E7 protein was tested by both confocal microscope and co-immunoprecipitation analyses on co-transfected cells. The in cis anti-proliferative effect of the Nefmut/anti-HPV16-E7 scFv was assayed by transfecting HPV16-infected cells. The anti-proliferative effect of EVs engineered with Nefmut/anti-HPV16-E7 scFv on HPV16-E7-expressing cells was evaluated in two ways: i) through challenge with purified EVs by a Real-Time Cell Analysis system and ii) in transwell co-cultures by an MTS-based assay.Results: The Nefmut/anti-HPV16-E7 scFv chimeric product is efficiently uploaded in EVs, binds HPV16-E7, and inhibits the proliferation of HPV16-E7-expressing cells. Most important, challenge with cell-free EVs incorporating the Nefmut/anti-HPV16-E7 scFv led to the inhibition of proliferation of HPV16-E7-expressing cells. The proliferation of these cells was hindered also when they were co-cultured in transwells with cells producing EVs uploading Nefmut/anti-HPV16-E7 scFv.Conclusion: Our data represent the proof-of-concept for the possibility to target intracellular antigens through EV-mediated delivery of scFvs. This finding could be relevant to design novel methods of intracellular therapeutic interventions.Keywords: single-chain variable fragments, Human Papilloma Virus E7 protein, exosomes, human immunodeficiency virus Nef protein, scFv intracellular delivery

Abstract The transport of membrane impermeable compounds into cells is a prerequisite for the efficient cellular delivery of hydrophilic and amphiphilic compounds and drugs. Transport into the cell's cytosolic compartment should ideally be controllable and it should involve biologically compatible and degradable vehicles. Addressing these challenges, nanocontainers based on cyclodextrin amphiphiles that are stabilized by a biodegradable peptide shell are developed and their potential to deliver fluorescently labeled cargo into human cells is analyzed. Host–guest mediated self‐assembly of a thiol‐containing short peptide or a cystamine‐cross‐linked polypeptide shell on cyclodextrin vesicles produce short peptide‐shelled (SPSVss) or polypeptide‐shelled vesicles (PPSVss), respectively, with redox‐responsive and biodegradable features. Whereas SPSVss are permeable and less stable, PPSVss effectively encapsulate cargo and show a strictly regulated release of membrane impermeable cargo triggered by either reducing conditions or peptidase treatment. Live cell experiments reveal that the novel PPSVSS are readily internalized by primary human endothelial cells (human umbilical vein endothelial cells) and cervical cancer cells and that the reductive microenvironment of the cells’ endosomes trigger release of the hydrophilic cargo into the cytosol. Thus, PPSVSS represent a highly efficient, biodegradable, and tunable system for overcoming the plasma membrane as a natural barrier for membrane‐impermeable cargo.

Targeting macrophages to regulate the immune microenvironment is a new strategy for bone regeneration with nano-drugs. Nano-drugs have achieved surprising anti-inflammatory and bone-regenerative effects, however, their underlying mechanisms in macrophages remain to be clarified. Macrophage polarization, immunomodulation, and osteogenesis are governed by autophagy. Rapamycin, an autophagy inducer, has shown promising results in bone regeneration, but high dose-mediated cytotoxicity and low bioavailability hinder its clinical application. This study aimed to develop rapamycin-loaded virus-like hollow silica nanoparticles (R@HSNs) which are easily phagocytosed by macrophages and translocated to lysosomes. R@HSNs induced macrophage autophagy, promoted M2 polarization, and alleviated the degree of M1 polarization as indicated by the downregulation of inflammatory factors IL-6, IL-1β, TNF-α, and iNOS, and upregulation of anti-inflammatory factors CD163, CD206, IL-1ra, IL-10, and TGF-β. These effects were nullified by cytochalasin B-induced inhibition of R@HSNs uptake in macrophages. The conditioned medium (CM) collected from R@HSNs-treated macrophages promoted osteogenic differentiation of mouse bone marrow mesenchymal stromal cells (mBMSCs). In a mouse calvaria defect model, free rapamycin treatment was inhibited, but R@HSNs robustly promoted bone defect healing. In conclusion, silica nanocarrier-mediated intracellular rapamycin delivery to macrophages effectively triggers autophagy-mediated M2 macrophage polarization, further enhancing bone regeneration by triggering osteogenic differentiation of mBMSCs.

The therapeutic application of biofunctional proteins relies on their intracellular delivery, which is hindered by poor cellular uptake and transport from endosomes to cytoplasm. Herein, we constructed a two-dimensional (2D) ultrathin layered double hydroxide (LDH) nanosheet for the intracellular delivery of a cell-impermeable protein, gelonin, towards efficient and specific cancer treatment. The LDH nanosheet was synthesized via a facile method without using exfoliation agents and showed a high loading capacity of proteins (up to 182%). Using 2D and 3D 4T1 breast cancer cell models, LDH–gelonin demonstrated significantly higher cellular uptake efficiency, favorable endosome escape ability, and deep tumor penetration performance, leading to a higher anticancer efficiency, in comparison to free gelonin. This work provides a promising strategy and a generalized nanoplatform to efficiently deliver biofunctional proteins to unlock their therapeutic potential for cancer treatment. [ABSTRACT FROM AUTHOR]

The treatment of malignant bone tumors by chemotherapeutics often receives poor therapeutic response due to the specific physiological bone environment, and thus calls for the development of new therapeutic options. Here, we reported a bone-targeted protein nanomedicine for this purpose. Saporin, a toxin protein, was co-assembled with a boronated polymer for intracellular protein delivery, and the formed nanoparticles were further coated with an anionic polymer poly (aspartic acid) to shield the positive charges on nanoparticles and provide the bone targeting function. The prepared ternary complex nanoparticles showed high bone accumulation both in vitro and in vivo, and could reverse the surface charge property from negative to positive after locating at tumor site triggered by tumor extracellular acidity. The boronated polymer in the de-shielded nanoparticles further promote intracellular delivery of saporin into tumor cells, exerting the anticancer activity of saporin by inactivation of ribosomes. As a result, the bone-targeted and saporin-loaded nanomedicine could kill cancer cells at a low saporin dose, and efficiently prevented the progression of osteosarcoma xenograft tumors and bone metastatic breast cancer in vivo. This study provides a facile and promising strategy to develop protein-based nanomedicines for the treatment of malignant bone tumors.

Lipid nanoparticle (LNP)-based drug delivery systems have become the most clinically advanced non-viral delivery technology. LNPs can encapsulate and deliver a wide variety of bioactive agents, including the small molecule drugs, proteins and peptides, and nucleic acids. However, as the physicochemical properties of small- and macromolecular cargos can vary drastically, every LNP carrier system needs to be carefully tailored in order to deliver the cargo molecules in a safe and efficient manner. Our group applied the combinatorial library synthesis approach and in vitro and in vivo screening strategy for the development of LNP delivery systems for drug delivery. In this Review, we highlight our recent progress in the design, synthesis, characterization, evaluation, and optimization of combinatorial LNPs with novel structures and properties for the delivery of small- and macromolecular therapeutics both in vitro and in vivo. These delivery systems have enormous potentials for cancer therapy, antimicrobial applications, gene silencing, genome editing, and more. We also discuss the key challenges to the mechanistic study and clinical translation of new LNP-enabled therapeutics.

Abstract Background Small extracellular vesicles (sEVs) are nanometer-sized membranous particles shed by many types of cells and can transfer a multitude of cargos between cells. Recent studies of sEVs have been focusing on their potential to be novel drug carriers due to natural composition and other promising characteristics. However, there are challenges in sEVs-based drug delivery, one of which is the inefficient loading of drugs into sEVs, especially for large biomolecules. Results In this study, we proposed a membrane-associated protein, milk fat globule–epidermal growth factor 8 protein (MFG-E8), to produce αvβ3-targeted sEVs with high delivery efficiency of interested protein. MFG-E8 is a secreted protein with NH2-terminal epidermal growth factor (EGF)–like domains, containing an Arg-Gly-Asp(RGD) sequence that binds αvβ3 and αvβ5 integrins, and COOH terminal domains C1 and C2, which can bind to lipid membrane with strong affinity. Firstly, we transiently expressed MFG-E8 in HEK293F cells and found that this protein could be secreted and adhere to the cell membrane. The recombinant MFG-E8 is also found to locate at the outer membrane of sEVs. Then we generated engineered sEVs by expressing high levels of the EGFP fused to MFG-E8 in HEK293F cells and showed that MFG-E8 could increase the delivery efficiency of EGFP into sEVs. Further delivery of Gaussia luciferase (GL) by fusion expression with MFG-E8 in donor cells demonstrated that target proteins fused with MFG-E8 still kept their activity. Finally, we identified the sEVs’ target to integrin αvβ3 by comparing the transfection efficiency with MFG-E8 loaded sEVs (MFG-E8-sEVs) in αvβ3 positive cells and αvβ3 negative cells. Analysis showed higher target protein could transfect into αvβ3 positive cells with MFG-E8-sEVs than with EGFP loaded sEVs (EGFP-sEVs), meaning the engineered sEVs with MFG-E8 not only could increase the delivery of target protein into sEVs, but also could target the αvβ3 positive cells. Conclusion This study suggests that recombinant MFG-E8 is an ideal protein to increasingly deliver the drug into sEVs and give sEVs the ability to target the αvβ3 positive cells.

Ballistic delivery with micro/nano-particles has been successfully used to transfect plant cells, however, has failed in mammalian cells due to toxic effects. Here, the authors report on a self-assembled nano-ballistic delivery system for the delivery of functional macromolecules and demonstrate efficient transfection of mammalian cells.

Dinitrosyl iron unit (DNIU), [Fe(NO) 2 ], is a natural metallocofactor for biological storage, delivery, and metabolism of nitric oxide (NO). In the attempt to gain a biomimetic insight into the natural DNIU under biological system, in this study, synthetic dinitrosyl iron complexes (DNICs) [(NO) 2 Fe(μ-SCH 2 CH 2 COOH) 2 Fe(NO) 2 ] ( DNIC-COOH ) and [(NO) 2 Fe(μ-SCH 2 CH 2 COOCH 3 ) 2 Fe(NO) 2 ] ( DNIC-COOMe ) were employed to investigate the structure-reactivity relationship of mechanism and kinetics for cellular uptake of DNICs, intracellular delivery of NO, and activation of cytoprotective heme oxygenase (HO)-1. After rapid cellular uptake of dinuclear DNIC-COOMe through a thiol-mediated pathway ( t = 0.5 h), intracellular assembly of mononuclear DNIC [(NO) max = 0.5 h), intracellular assembly of mononuclear DNIC [(NO) 2 Fe(SR)(S Cys )] n - /[(NO) 2 Fe(SR)(S Cys-protein )] n - occurred, followed by O 2 -induced release of free NO ( t max = 2-8 h) and for intracellular release of NO ( DNIC-COOH via endocytosis ( t max = 2-8 h) and for intracellular release of NO ( t max as a switch to control the mechanism, kinetics, and efficacy for cellular uptake of DNICs, intracellular delivery of NO, and activation of cytoprotective HO-1, which poses an implication on enhanced survival of postengrafted MSC for advancing the MSC-based regenerative medicine.t = 3-10 h) and on the improved survival of DNIC-COOH -primed mesenchymal stem cell (MSC)/human corneal endothelial cell (HCEC) under stressed conditions. Consequently, this study unravels the bridging thiolate ligands in dinuclear DNIC-COOH / DNIC-COOMe as a switch to control the mechanism, kinetics, and efficacy for cellular uptake of DNICs, intracellular delivery of NO, and activation of cytoprotective HO-1, which poses an implication on enhanced survival of postengrafted MSC for advancing the MSC-based regenerative medicine., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

Solid tumor antibody-drug conjugates (ADC) have experienced more clinical success in the last 5 years than the previous 18-year span since the first ADC approval in 2000. While recent advances in protein engineering, linker design, and payload variations have played a role in this success, high expression and readily internalized targets have also been crucial to solid tumor therapy. However, these factors are also paradoxically connected to poor tissue penetration and lower efficacy. Previous work shows that potent ADCs can benefit from slower internalization under subsaturating doses to improve tissue penetration and increase tumor response. In contrast, faster internalization is predicted to increase efficacy under higher, tumor saturating doses. In this work, the intracellular delivery of SN-38 conjugated to an anti-carcinoembryonic antigen (anti-CEA) antibody (Ab) is increased by coadministering a noncompeting (cross-linking) anti-CEA Ab to improve efficacy in a colorectal carcinoma animal model. The SN-38 payload enables broad tumor saturation with clinically-tolerable doses, and under these saturating conditions, using a second CEA receptor cross-linking Ab yields faster internalization, which increases tumor killing efficacy. Our spheroid results show indirect bystander killing can also occur, but the more efficient direct cell killing from targeted intracellular payload release drives a greater tumor response. These results provide a strategy to increase therapeutic effectiveness with improved intracellular delivery under tumor saturating doses with the potential to expand the ADC target repertoire., (©2023 American Association for Cancer Research.)

Introduction: We have developed a delivery approach that uses two pHLIP peptides that collaborate in the targeted intracellular delivery of a single payload, dimeric STINGa (dMSA). Methods: dMSA was conjugated with two pHLIP peptides via S-S cleavable self-immolating linkers to form 2pHLIP-dMSA. Results: Biophysical studies were carried out to confirm pH-triggered interactions of the 2pHLIP-dMSA with membrane lipid bilayers. The kinetics of linker self-immolation and dMSA release, the pharmacokinetics, the binding to plasma proteins, the stability of the agent in plasma, the targeting and resulting cytokine activation in tumors, and the biodistribution of the construct was investigated. This is the first study demonstrating that combining the energy of the membrane-associated folding of two pHLIPs can be utilized to enhance the targeted intracellular delivery of large therapeutic cargo payloads. Discussion: Linking two pHLIPs to the cargo extends blood half-life, and targeted delivery of dimeric STINGa induces tumor eradication and the development of robust anti-cancer immunity., Competing Interests: DE, OA, and YR are founders of pHLIP, Inc., and they have shares in the company. pHLIP, Inc. sponsored synthesis and purification of dMSA and modified dMSA prepared by the Iris Biotech GmbH. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Moshnikova, DuPont, Iraca, Klumpp, Visca, Allababidi, Pelzer, Engelman, Andreev and Reshetnyak.)

In vitro, drug assessment holds tremendous potential to success in novel drug development and precision medicine. Traditional techniques for drug assessment, however, face remarkable challenges to achieve high speed, as limited by incubation-based drug delivery (>several hours) and cell viability measurements (>1 d), which significantly compromise the efficacy in clinical trials. In this work, a nano-electroporation-DNA tensioner platform is reported that shortens the time of drug delivery to less than 3 s, and that of cellular mechanical force analysis to 30 min. The platform adopts a nanochannel structure to localize a safe electric field for cell perforation, while enhancing delivery speed by 10 3 times for intracellular delivery, as compared to molecular diffusion in coculture methods. The platform is further equipped with a DNA tensioner to detect cellular mechanical force for quantifying cell viability after drug treatment. Systematic head-to-head comparison, by analyzing FDA (food and drug administration)-approved drugs (paclitaxel, doxorubicin), demonstrated the platform with high speed, efficiency, and safety, showing a simple yet powerful tool for clinical drug screening and development., (© 2023 Wiley-VCH GmbH.)

Biologics, including proteins and antisense oligonucleotides (ASOs), face significant challenges when it comes to achieving intracellular delivery within specific organs or cells through systemic administrations. In this study, we present a novel approach for delivering proteins and ASOs to liver cells, both in vitro and in vivo , using conjugates that tether N -acetylated galactosamine (GalNAc)-functionalized, cell-penetrating polydisulfides (PDSs). The method involves the thiol-bearing cargo-mediated ring-opening polymerization of GalNAc-functionalized lipoamide monomers through the so-called aggregation-induced polymerization, leading to the formation of site-specific protein/ASO-PDS conjugates with narrow dispersity. The hepatocyte-selective intracellular delivery of the conjugates arises from a combination of factors, including first GalNAc binding with ASGPR receptors on liver cells, leading to cell immobilization, and the subsequent thiol-disulfide exchange occurring on the cell surface, promoting internalization. Our findings emphasize the critical role of the close proximity of the PDS backbone to the cell surface, as it governs the success of thiol-disulfide exchange and, consequently, cell penetration. These conjugates hold tremendous potential in overcoming the various biological barriers encountered during systemic and cell-specific delivery of biomacromolecular cargos, opening up new avenues for the diagnosis and treatment of a range of liver-targeting diseases.

Cell-penetrating peptides (CPPs), for example, arginine (Arg) rich peptides, are used for the intracellular delivery of nucleic acids. In this study, oligosarcosine-conjugated Arg-rich peptides were designed as plasmid DNA (pDNA) carriers, and the physicochemical parameters and transfection efficiency of the peptide/pDNA complexes were evaluated. Oligosarcosine with different lengths were conjugated to a base sequence composed of arginine and α-aminoisobutyric acid (Aib) [(Aib-Arg-Arg) 3 ]. Oligosarcosine conjugation inhibited the aggregation of the complexes after mixing with pDNA, shielded the positive charge of the complexes, and provided efficient pDNA transfection in cultured cells. The efficiency of the pDNA transfection was improved by varying the length of the oligosarcosine moiety (10-15 units were optimal). The cellular uptake efficiency and intracellular distribution of pDNA were the same regardless of oligosarcosine conjugation. These results implied that intracellular processes, including the decondensation of pDNA, contributed to the efficiency of the protein expression from pDNA. This study demonstrated the advantages of oligosarcosine conjugation to Arg-rich CPPs and provided valuable insight into the future design of CPPs.

Background: Endostatin is an antiangiogenic compound with anticancer activity. The poor stability and low half-life of endostatin are the main barriers to the clinical use of this protein. Cell-penetrating peptides (CPPs) are extensively applied as carrier in the delivery of drugs and different therapeutic agents. Therefore, they can be proper candidates to improve endostatin delivery to the target cells., Objective: In this study, we aim to computationally predict appropriate CPPs for the delivery of endostatin., Methods: Potential appropriate CPPs for protein delivery were selected based on the literature. The main parameters for detection of best CPP-endostatin fusions, including stability, hydrophobicity, antigenicity, and subcellular localization, were predicted using ProtParam, VaxiJen, and DeepLoc-1.0 servers, respectively. The 3D structures of the best CPP-Endostatin fusions were modeled by the I-TASSER server. The predicted models were validated using PROCHECK, ERRAT, Verify3D and ProSA-Web servers. The best models were visualized by the PyMol molecular graphics system., Results: Considering the principal parameters in the selection of best CPPs for endostatin delivery, endostatin fusions with four CPPs, including Cyt c-ss-MAP, TP-biot1, MPGα, and DPV1047, high stability and hydrophobicity, no antigenicity and extracellular localization were predicted as the best potential fusions for endostatin delivery. Four CPPs, including Cyt c-ss-MAP, TP-biot1, MPGα, and DPV1047, were predicted as the best potential candidates to improve endostatin delivery., Conclusion: Application of these CPPs may overcome the limitation of endostatin therapeutic applications, including poor stability and low half-life. Subsequent experimental studies will contribute to verifying these computational results., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)