Intracellular Delivery Of Therapeutic Agents Research Articles

Nature-inspired peptide of MtDef4 C-terminus tail enables protein delivery in mammalian cells

Cell-penetrating peptides show promise as versatile tools for intracellular delivery of therapeutic agents. Various peptides have originated from natural proteins with antimicrobial activity. We investigated the mammalian cell-penetrating properties of a 16-residue peptide with the sequence GRCRGFRRRCFCTTHC from the C-terminus tail of the Medicago truncatula defensin MtDef4. We evaluated the peptide’s ability to penetrate multiple cell types. Our results demonstrate that the peptide efficiently penetrates mammalian cells within minutes and at a micromolar concentration. Moreover, upon N-terminal fusion to the fluorescent protein GFP, the peptide efficiently delivers GFP into the cells. Despite its remarkable cellular permeability, the peptide has only a minor effect on cellular viability, making it a promising candidate for developing a cell-penetrating peptide with potential therapeutic applications.

Cellular Uptake of Modified Mesoporous Bioactive Glass Nanoparticles for Effective Intracellular Delivery of Therapeutic Agents.

There has recently been a surge of interest in mesoporous bioactive glass nanoparticles (MBGNs) as multi-functional nanocarriers for application in bone-reconstructive and -regenerative surgery. Their excellent control over their structural and physicochemical properties renders these nanoparticles suitable for the intracellular delivery of therapeutic agents to combat degenerative bone diseases, such as bone infection, or bone cancer. Generally, the therapeutic efficacy of nanocarriers strongly depends on the efficacy of their cellular uptake, which is determined by numerous factors including cellular features and the physicochemical characteristics of nanocarriers, particularly surface charge. In this study, we have systematically investigated the effect of the surface charge of MBGNs doped with copper as a model therapeutic agent on cellular uptake by both macrophages and pre-osteoblast cells involved in bone healing and bone infections to guide the future design of MBGN-based nanocarriers. Cu-MBGNs with negative, neutral, and positive surface charges were synthesized and their cellular uptake efficiency was assessed. Additionally, the intracellular fate of internalized nanoparticles along with their ability to deliver therapeutic cargo was studied in detail. The results showed that both cell types internalized Cu-MBGNs regardless of their surface charge, indicating that cellular uptake of nanoparticles is a complex process influenced by multiple factors. This similarity in cellular uptake was attributed to the formation of a protein corona surrounding the nanoparticles when exposed to protein-rich biological media, which masks the original nanoparticle surface. Once internalized, the nanoparticles were found to mainly colocalize with lysosomes, exposing them to a more compartmentalized and acidic environment. Furthermore, we verified that Cu-MBGNs released their ionic components (Si, Ca, and Cu ions) in both acidic and neutral environments, leading to the delivery of these therapeutic cargos intracellularly. The effective internalization of Cu-MBGNs and their ability to deliver cargos intracellularly highlight their potential as intracellular delivery nanocarriers for bone-regenerative and -healing applications.

Surface conjugation of antibodies improves nanoparticle uptake in bronchial epithelial cells.

Advances in Molecular Therapy have made gene editing through systemic or topical administration of reagents a feasible strategy to treat genetic diseases in a rational manner. Encapsulation of therapeutic agents in nanoparticles can improve intracellular delivery of therapeutic agents, provided that the nanoparticles are efficiently taken up within the target cells. In prior work we had established proof-of-principle that nanoparticles carrying gene editing reagents can mediate site-specific gene editing in fetal and adult animals in vivo that results in functional disease improvement in rodent models of β-thalassemia and cystic fibrosis. Modification of the surface of nanoparticles to include targeting molecules (e.g. antibodies) holds the promise of improving cellular uptake and specific cellular binding. To improve particle uptake for diseases of the airway, like cystic fibrosis, our group tested the impact of nanoparticle surface modification with cell surface marker antibodies on uptake in human bronchial epithelial cells in vitro. Binding kinetics of antibodies (Podoplanin, Muc 1, Surfactant Protein C, and Intracellular Adhesion Molecule-1 (ICAM)) were determined to select appropriate antibodies for cellular targeting. The best target-specific antibody among those screened was ICAM antibody. Surface conjugation of nanoparticles with antibodies against ICAM improved cellular uptake in bronchial epithelial cells up to 24-fold. This is a first demonstration of improved nanoparticle uptake in epithelial cells using conjugation of target specific antibodies. Improved binding, uptake or specificity of particles delivered systemically or to the luminal surface of the airway would potentially improve efficacy, reduce the necessary dose and thus safety of administered therapeutic agents. Incremental improvement in the efficacy and safety of particle-based therapeutic strategies may allow genetic diseases such as cystic fibrosis to be cured on a fundamental genetic level before birth or shortly after birth.

GOLD NANOPARTICLES AND CHEMOTHERAPEUTIC AGENTS

Gold nanoparticles (AuNPs) receive a great deal of attention for biomedical applications due to their unique properties to monitor intracellular delivery of therapeutic agents. Evidently, drug delivery is a compelling field of research due to the need of releasing medicine at specific locations in a controlled manner with a minimum amount of side effects. The present review focuses on the combination of AuNPs with different types of chemotherapeutic agents as potential drug delivery vehicles that can be used in cancer therapy

Cyclic Cell-Penetrating Peptides as Efficient Intracellular Drug Delivery Tools.

Cyclic cell-penetrating peptides are relatively a newer class of peptides that have a huge potential for the intracellular delivery of therapeutic agents aimed at treating challenging ailments like multidrug-resistant bacterial diseases, cancer, and HIV infection. Cell-penetrating peptides (CPPs) have been extensively explored as intracellular delivery vehicles; however, they have some inherent limitations like poor stability, endosomal entrapment, toxicity, and suboptimal cell penetration. Owing to their favorable properties that avoid these limitations, cyclic CPPs can provide a good alternative to linear CPPs. Several Reviews have been published in the past decade that cover CPPs and cyclic peptides independently. To the best of our knowledge, this is one of the first Reviews that covers cyclic CPPs comprehensively in the light of studies published so far. In this Review, we have detailed examples of cyclic CPPs, their structures, and cyclization strategies followed by a detailed account of their advantages over their linear counterparts. A hot area in cyclic CPPs is the exploration of cell-penetration mechanisms; this Review highlights this topic in detail. Finally, we will review the applications of cyclic CPPs, followed by conclusions and future prospects.

Novel machine learning application for prediction of membrane insertion potential of cell-penetrating peptides.

Cell-penetrating peptides (CPPs) are often used as transporter systems to deliver various therapeutic agents into the cell. We developed a novel machine learning application which can quantitatively screen the insertion/interaction potential of various CPPs into three model phospholipid monolayers. An artificial neural network (ANN) was designed, trained, and ultimately tested on an external dataset using Langmuir experimental data for 13 CPPs (hydrophilic and amphiphilic) together with various features related to the insertion/interaction efficiency of CPPs. The trained ANN provided accurate predictions of the maximum change in surface pressure of CPPs when injected below three membrane models at pH 7.4. The accuracy of predictions was high for the dataset which was used to construct the model (r2 = 0.986) as well as for the external "prospective" dataset (r2 = 0.969). In conclusion, this study demonstrates the promising potential of ANNs for screening the insertion potential of CPPs into membrane models for efficient intracellular delivery of therapeutic agents.

Cell penetrating peptides: A concise review with emphasis on biomedical applications.

The biological membranes as natural permeable barriers are required for survival and function of living cells. However, these natural barriers could be a major obstacle for the efficient intracellular delivery of therapeutic agents. In recent two decades, the use of peptides as novel carriers for intracellular cargo delivery has been received more attention by introducing the cell penetrating peptides (CPPs). CPPs, protein transduction domains, are an attractive class of short peptide sequences which can translocate across the cell membrane. Owing to the ability of CPPs to transport across cellular membrane, they can employ as an appropriate carrier for various cargos include nucleic acid, proteins, SiRNA, therapeutic agents, nanoparticles and so on. In this review, we describe the classifications of CPPs, their uptake mechanisms as well as biomedical applications of the CPPs.

Cancer-targeted photothermal therapy using aptamer-conjugated gold nanoparticles

Targeted intracellular delivery of therapeutic agents is one of the great challenges for cancer treatment. Aptamers that bind to a variety of biological targets have emerged as new targeting moieties with high specificity for targeted cancer therapy. In this study, near-infrared (NIR) light-absorbing hollow gold nanocages (AuNCs) were synthesized and conjugated with AS1411 aptamer to achieve cancer-targeted photothermal therapy. AuNC functionalized with PEG and AS1411 (AS1411-PEG-AuNC) exhibited selective cellular uptake in breast cancer cells due to selective binding of AS1411 to nucleolin, a protein that is over-expressed in cancer cells over normal cells. As a result, AS1411-PEG-AuNC showed cancer-targeted photothermal activity. This study demonstrates that aptamer-conjugated AuNCs are effective tumor-targeting photothermal agents.

Peptides of pHLIP family for targeted intracellular and extracellular delivery of cargo molecules to tumors

The pH (low) insertion peptides (pHLIPs) target acidity at the surfaces of cancer cells and show utility in a wide range of applications, including tumor imaging and intracellular delivery of therapeutic agents. Here we report pHLIP constructs that significantly improve the targeted delivery of agents into tumor cells. The investigated constructs include pHLIP bundles (conjugates consisting of two or four pHLIP peptides linked by polyethylene glycol) and Var3 pHLIPs containing either the nonstandard amino acid, γ-carboxyglutamic acid, or a glycine-leucine-leucine motif. The performance of the constructs in vitro and in vivo was compared with previous pHLIP variants. A wide range of experiments was performed on nine constructs including (i) biophysical measurements using steady-state and kinetic fluorescence, circular dichroism, and oriented circular dichroism to study the pH-dependent insertion of pHLIP variants across the membrane lipid bilayer; (ii) cell viability assays to gauge the pH-dependent potency of peptide-toxin constructs by assessing the intracellular delivery of the polar, cell-impermeable cargo molecule amanitin at physiological and low pH (pH 7.4 and 6.0, respectively); and (iii) tumor targeting and biodistribution measurements using fluorophore-peptide conjugates in a breast cancer mouse model. The main principles of the design of pHLIP variants for a range of medical applications are discussed.

Microparticle templating as a route to nanoscale polymer vesicles with controlled size distribution for anticancer drug delivery

Polymer vesicles are self-assembled shells of amphiphilic block copolymers (BCPs) that have attracted tremendous interest due to their encapsulation ability and intracellular delivery of therapeutic agents. However, typical processes for the formation of polymer vesicles lead to ensembles of structures with a broad size distribution (from nanometer to micrometer scale) which result in a limitation for efficient cellular uptake. In this study, we present a simple and efficient approach for the fabrication of polymer vesicles with uniform nanoscale dimensions from template formation of electrosprayed particles in a high throughput manner. First, electrospraying was applied to produce micrometer-sized templates of a block copolymer before polymer vesicles were formed from the pre-prepared microparticles via rehydration. Four different biocompatible diblock and triblock copolymers were used to successfully fabricate polymer vesicles with uniform size around 150nm using this approach. Furthermore, we encapsulate anticancer drug doxorubicin (DOX) within the polymer vesicles via this method. The kinetics of cellular uptake (HeLa cell) and intracellular distribution of DOX-loaded polymer vesicles have been quntified and monitored by flow cytometry and confocal microscopy, respectively. The results show that our new method provides a promising way to fabricate drug-loaded polymer vesicles with controllable nanoscale size for intracellular anticancer drug delivery.

Membrane-Anchoring, Comb-Like Pseudopeptides for Efficient, pH-Mediated Membrane Destabilization and Intracellular Delivery.

Endosomal release has been identified as a rate-limiting step for intracellular delivery of therapeutic agents, in particular macromolecular drugs. Herein, we report a series of synthetic pH-responsive, membrane-anchoring polymers exhibiting dramatic endosomolytic activity for efficient intracellular delivery. The comb-like pseudopeptidic polymers were synthesized by grafting different amounts of decylamine (NDA), which act as hydrophobic membrane anchors, onto the pendant carboxylic acid groups of a pseudopeptide, poly(l-lysine iso-phthalamide). The effects of the hydrophobic relatively long alkyl side chains on aqueous solution properties, cell membrane destabilization activity, and in-vitro cytotoxicity were investigated. The optimal polymer containing 18 mol % NDA exhibited limited hemolysis at pH 7.4 but induced nearly complete membrane destabilization at endosomal pH within only 20 min. The mechanistic investigation of membrane destabilization suggests the polymer-mediated pore formation. It has been demonstrated that the polymer with hydrophobic side chains displayed a considerable endosomolytic ability to release endocytosed materials into the cytoplasm of various cell lines, which is of critical importance for intracellular drug delivery applications.

Discovery and Mechanism of Highly Efficient Cyclic Cell-Penetrating Peptides.

Previous cell-penetrating peptides (CPPs) generally have low cytosolic delivery efficiencies, because of inefficient endosomal escape. In this study, a family of small, amphipathic cyclic peptides was found to be highly efficient CPPs, with cytosolic delivery efficiencies of up to 120% (compared to 2.0% for Tat). These cyclic CPPs bind directly to the plasma membrane phospholipids and enter mammalian cells via endocytosis, followed by efficient release from the endosome. Their total cellular uptake efficiency correlates positively with the binding affinity for the plasma membrane, whereas their endosomal escape efficiency increases with the endosomal membrane-binding affinity. The cyclic CPPs induce membrane curvature on giant unilamellar vesicles and budding of small vesicles, which subsequently collapse into amorphous lipid/peptide aggregates. These data suggest that cyclic CPPs exit the endosome by binding to the endosomal membrane and inducing CPP-enriched lipid domains to bud off as small vesicles. Together with their high proteolytic stability, low cytotoxicity, and oral bioavailability, these cyclic CPPs should provide a powerful system for intracellular delivery of therapeutic agents and chemical probes.

Drug Delivery Nanocarriers from a Fully Degradable PEG-Conjugated Polyester with a Reduction-Responsive Backbone.

The remarkably high intracellular concentration of reducing agents is an excellent endogenous stimulus for designing nanocarriers programmed for intracellular delivery of therapeutic agents. However, despite their excellent biodegradability profiles, aliphatic polyesters that are fully degradable in response to the intracellular reducing environment are rare. Herein, a reduction-responsive drug delivery nanocarrier derived from a linear polyester bearing disulfide bonds is reported. The reduction-responsive polyester is synthesized via a convenient polycondensation process. After conjugation of terminal carboxylic acid groups of polyester to polyethylene glycol (PEG), the resulting polymer self-assembles into nanoparticles that are capable of encapsulating dye and anticancer drug molecules. The reduction-responsive nanoparticles display a fast payload release rate in response to the intracellular reducing environment, which translates into superior anticancer activity towards PC-3 cells.

Insights into the molecular mechanisms of action of bioportides: a strategy to target protein-protein interactions.

Cell-penetrating peptides (CPPs) are reliable vehicles for the target-selective intracellular delivery of therapeutic agents. The identification and application of numerous intrinsically bioactive CPPs, now designated as bioportides, is further endorsement of the tremendous clinical potential of CPP technologies. The refinement of proteomimetic bioportides, particularly sequences that mimic cationic α-helical domains involved in protein-protein interactions (PPIs), provides tremendous opportunities to modulate this emergent drug modality in a clinical setting. Thus, a number of CPP-based constructs are currently undergoing clinical trials as human therapeutics, with a particular focus upon anti-cancer agents. A well-characterised array of synthetic modifications, compatible with modern solid-phase synthesis, can be utilised to improve the biophysical and pharmacological properties of bioportides and so achieve cell-and tissue-selective targeting in vivo. Moreover, considering the recent successful development of stapled α-helical peptides as anti-cancer agents, we hypothesise that similar structural modifications are applicable to the design of bioportides that more effectively modulate the many interactomes known to underlie human diseases. Thus, we propose that stapled-helical bioportides could satisfy all of the clinical requirements for metabolically stable, intrinsically cell-permeable agents capable of regulating discrete PPIs by a dominant negative mode of action with minimal toxicity.

Electroporation induced delivery of siRNA-loaded copper sulfide nanoparticles

Purpose Irreversible electroporation (IRE) is a new non-thermal ablation technique. It creates both a central ablation zone and a surrounding sub-lethal region. Cancer cells within the sub-lethal region may survive and cause relapse of tumor. The electric field in sub-lethal regions may be sufficient to induce the intracellular delivery of therapeutic agents. Nanoparticles loaded with small-interfering RNA (siRNA) have been successfully used to treat various cancers. In this study, we prepared siRNA-loaded copper sulfide (CuS) nanoparticle system and tested its cellular uptake during electroporation. Materials and Methods Cytotoxicity was quantified by trypan blue staining in HepG2 cell suspensions after electroporation at different power and pulse settings. CuS nanoparticles coated with glycol chitosan (CuS-GC) were loaded with anti-luciferase siRNA via electrostatic interaction. The suspensions of HepG2 or SKOV3.ip1.luc cells were treated with electroporation at 500 V/cm for 90 pulses in the presence of CuS-siRNA. The cellular uptake of nanoparticles and siRNA were monitored by fluorescence microscope. The down-regulation of luciferase was analyzed by luminance assay. Results Electroporation at 500V/cm for 90 pulses did not cause substantial cell death (cell viability = 93.6 ± 4.5%). The CuS-siRNA nanoparticles were 50 nm in diameter. The cellular uptake of CuS nanoparticles was increased by 173% in HepG2 and 52% in SKOV3.ip1.luc cells, compared to controls without electroporation (p Conclusion We developed an in vitro cell model to simulate the peripheral sub-lethal regions of IRE. CuS-siRNA nanoparticles were delivered into carcinoma cells during electroporation and down-regulated the expression of luciferase. Future studies will evaluate the therapeutic efficacy of this combined method in in-vitro and in-vivo experiments after delivery of therapeutic nanoparticles.

Cellular uptake mechanism and intracellular fate of hydrophobically modified pullulan nanoparticles

The cellular uptake mechanism and intracellular fate of self-assembled nanoparticles (NPs) of cholesterol-modified pullulan (CHSP) by human hepatocellular carcinoma (HepG2) cells were investigated. Covalent conjugation with fluorescein isothiocyanate (FITC) yielded stably labeled CHSP (FITC-CHSP), which was successfully formulated into NPs (mean particle size 63.0 ± 1.9 nm) by dialysis. A cytotoxicity assay clearly indicated that the CHSP NPs did not show significant toxicity in HepG2 cells. The effects of NP concentration, incubation time, and temperature on the cellular uptake of the NPs were systematically evaluated by fluorometry, and the results suggested that cellular uptake of the NPs was concentration-,time-, and temperature-dependent. In vitro experiments with endocytic inhibitors revealed that clathrin-mediated endocytosis and macropinocytosis were involved in the internalization of CHSP NPs. The intracellular trafficking study demonstrated that CHSP NPs were entrapped in the lysosomes at 1 hour after incubation; colocalization of NPs with either the Golgi apparatus or the endoplasmic reticula was not observed during the entire course of the study. These results suggested that the CHSP NPs may serve as a versatile carrier for intracellular delivery of therapeutic agents.

Retraction Note: Rapamycin-loaded nanoparticles for inhibition of neointimal hyperplasia in experimental vein grafts

Background Nanoparticles (NPs) possess several advantages as a carrier system for intracellular delivery of therapeutic agents. Rapamycin is an immunosuppressive agent which also exhibits marked antiproliferative properties. We investigated whether rapamycin-loaded NPs can reduce neointima formation of vein graft disease in a rat model. Methods Poly(lactic-co-glycolic acid) (PLGA) NPs-containing rapamycin was prepared using an oil/water solvent evaporation technique. The size and morphology of the NP were determined by dynamic light scattering methodology and electron microscopy. In vitro cytotoxicity of blank, rapamycin-loaded PLGA NPs was studied using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Excised rat jugular vein was treated ex vivo with blank NPs, or rapamycin-loaded NPs, and then interposed back into the carotid artery position using a cuff technique. Grafts were harvested for 21 days and subjected to morphometric analysis as well as immunohistochemical analysis and Western blotting. Results Rapamycin was efficiently loaded in PLGA NPs with an encapsulation efficiency of 87.6%. The average diameter of NPs was 180.3 nm. The NPs-containing rapamycin at 1 ng/mL significantly inhibited vascular smooth muscular cells proliferation. Measurement of rapamycin levels in vein grafts showed that the concentration of rapamycin in vein grafts at 3 weeks after grafting was 0.9 ± 0.1 μg/g. In grafted veins without treatment, intima-media thickness was 300.4 ± 181.5 μm at 21 days after grafting, whereas veins treated with rapamycin-loaded NPs showed a reduction of intimal-media thickness of 150.2 ± 62.5 μm (p = 0.001). Cell proliferation was measured by proliferating cell nuclear antigen immunohistochemistry staining. As expected, proliferating cell nuclear antigen index declined from 83.4% ± 7.4% to 66.2% ± 4.5% in vein grafts after 3 weeks (p = 0.002). Platelet endothelial cell adhesion molecule (PECAM-1/CD31) staining was used to measure luminal endothelial coverage in grafts and indicated a high level of endothelialization at 21 days after grafting, with no significant effect of blank or rapamycin-loaded NPs group. Western blot analysis showed that treatment with rapamycin-loaded PLGA NPs markedly attenuated phosphorylation and activation of S6 kinase 1 phosphorylation and inactivation of 4E (eIF4E)-binding protein 1, both in vascular smooth muscular cells and vein grafts at 7 and 21 days after grafting. Conclusions We conclude that sustained-release rapamycin from rapamycin-loaded NPs inhibits vein graft thickening without affecting the endothelial cells in rat carotid vein-to-artery interposition grafts; thus, this may be a promising therapy for the treatment of vein graft disease.

Physical characterization and cellular uptake of propylene glycol liposomes in vitro

In order to facilitate the intracellular delivery of therapeutic agents, a new type of liposomes–propylene glycol liposomes (PGL) were prepared, and their cell translocation capability in vitro was examined. PGL was composed of hydrogenated egg yolk lecithin, cholesterol, Tween 80 and propylene glycol. With curcumin as a model drug, characterization of loaded PGL were measured including surface morphology, particle size, elasticity, encapsulation efficiency of curcumin and physical stability. Using curcumin-loaded conventional liposomes as the control, the cell uptake capacity of loaded PGL was evaluated by detection the concentration of curcumin in cytoplasm. Compared with conventional liposomes, PGL exhibited such advantages as high encapsulation efficiency (92.74% ± 3.44%), small particle size (182.4 ± 89.2 nm), high deformability (Elasticity index = 48.6) and high stability both at normal temperature (about 25°C) and low temperature at 4°C. From cell experiment in vitro, PGL exhibited the highest uptake of curcumin compared with that of conventional liposomes and free curcumin solution. Little toxic effect on cellular viability was observed by methyl tetrazolium assay. In conclusion, PGL might be developed as a promising intracellular delivery carrier for therapeutic agents.

Antibodies to cell surface proteins redirect intracellular trafficking pathways

Antibody-mediated intracellular delivery of therapeutic agents has been considered for treatment of a variety of diseases. These approaches involve targeting cell-surface receptor proteins expressed by tumors or viral proteins expressed on infected cells. We examined the intracellular trafficking of a viral cell-surface-expressed protein, rabies G, with or without binding a specific antibody, ARG1. We found that antibody binding shifts the native intracellular trafficking pathway of rabies G in an Fc-independent manner. Kinetic studies indicate that the ARG1/rabies G complex progressively co-localized with clathrin, early endosomes, late endosomes, and lysosomes after addition to cells. This pathway was different from that taken by rabies G without addition of antibody, which localized with recycling endosomes. Findings were recapitulated using a cellular receptor with a well-defined endogenous recycling pathway. We conclude that antibody binding to cell-surface proteins induces redirection of intracellular trafficking of unbound or ligand bound receptors to a specific degradation pathway. These findings have broad implications for future developments of antibody-based therapeutics.

Rapamycin-loaded nanoparticles for inhibition of neointimal hyperplasia in experimental vein grafts

BackgroundNanoparticles possess several advantages as a carrier system for intracellular delivery of therapeutic agents. Rapamycin is an immunosuppressive agent which also exhibits marked antiproliferative properties. We investigated whether rapamycin-loaded nanoparticles(NPs) can reduce neointima formation in a rat model of vein graft disease.MethodsPoly(lactic-co-glycolic acid) (PLGA) NPs containing rapamycin was prepared using an oil/water solvent evaporation technique. Nanoparticle size and morphology were determined by dynamic light scattering methodology and electron microscopy. In vitro cytotoxicity of blank, rapamycin-loaded PLGA (RPLGA) NPs was studied using MTT Assay. Excised rat jugular vein was treated ex vivo with blank-NPs, or rapamycin-loaded NPs, then interposed back into the carotid artery position using a cuff technique. Grafts were harvested at 21 days and underwent morphometric analysis as well as immunohistochemical analysis.ResultsRapamycin was efficiently loaded in PLGA nanoparticles with an encapsulation efficiency was 87.6%. The average diameter of NPs was 180.3 nm. The NPs-containing rapamycin at 1 ng/ml significantly inhibited vascular smooth muscular cells proliferation. Measurement of rapamycin levels in vein grafts shown that the concentration of rapamycin in vein grafts at 3 weeks after grafting were 0.9 ± 0.1 μg/g. In grafted veins without treatment intima-media thickness was 300.4 ±181.5 μm after grafting 21 days. Whereas, Veins treated with rapamycin-loaded NPs showed a reduction of intimal-media thickness of 150.2 ± 62.5 μm (p = 0.001). CD-31 staining was used to measure luminal endothelial coverage in grafts and indicated a high level of endothelialization in 21 days vein grafts with no significant effect of blank or rapamycin-loaded NPs group.ConclusionsWe conclude that sustained-release rapamycin from rapymycin loaded NPs inhibits vein graft thickening without affecting the reendothelialization in rat carotid vein-to-artery interposition grafts and this may be a promising therapy for the treatment of vein graft disease.