Carbonization of quercetin into nanogels: a leap in anticoagulant development.

Quercetin, a flavonoid abundantly found in onions, fruits, and vegetables, is recognized for its pharmacological potential, especially for its anticoagulant properties that work by inhibiting thrombin and coagulation factor Xa. However, its clinical application is limited due to poor water solubility and bioavailability. To address these limitations, we engineered carbonized nanogels derived from quercetin (CNGsQur) using controlled pyrolysis and polymerization techniques. This led to substantial improvements in its anticoagulation efficacy, water solubility, and biocompatibility. We generated a range of CNGsQur by subjecting quercetin to varying pyrolytic temperatures and then assessed their anticoagulation capacities both in vitro and in vivo. Coagulation metrics, including thrombin clotting time (TCT), activated partial thromboplastin time (aPTT), and prothrombin time (PT), along with a rat tail bleeding assay, were utilized to gauge the efficacy. CNGsQur showed a pronounced extension of coagulation time compared to uncarbonized quercetin. Specifically, CNGsQur synthesized at 270 °C (CNGsQur270) exhibited the most significant enhancement in TCT, with a binding affinity to thrombin exceeding 400 times that of quercetin. Moreover, variants synthesized at 310 °C (CNGsQur310) and 290 °C (CNGsQur290) showed the most substantial delays in PT and aPTT, respectively. Our findings indicate that the degree of carbonization significantly influences the transformation of quercetin into various CNGsQur forms, each affecting distinct coagulation pathways. Additionally, both intravenous and oral administrations of CNGsQur were found to extend rat tail bleeding times by up to fivefold. Our studies also demonstrate that CNGsQur270 effectively delays and even prevents FeCl3-induced vascular occlusion in a dose-dependent manner in mice. Thus, controlled pyrolysis offers an innovative approach for generating quercetin-derived CNGs with enhanced anticoagulation properties and water solubility, revealing the potential for synthesizing self-functional carbonized nanomaterials from other flavonoids for diverse biomedical applications.

Targeted Thrombolysis with Magnetic Nanotherapeutics: A Translational Assessment.

Plasminogen activators, such as recombinant tissue-type plasminogen activators (rtPAs), while effective in treating thromboembolic diseases, often induce hemorrhagic complications due to non-specific enzyme activities in the systemic circulation. This study evaluated the targeting efficiency, efficacy, biodistribution, and potential toxicity of a rtPA covalently attached to chitosan-coated magnetic nanoparticles (chitosan-MNP-rtPA). The thrombolytic activity of a chitosan-MNP-rtPA was preserved by protection from an endogenous plasminogen activator inhibitor-1 (PAI-1) in whole blood and after circulation in vivo, as examined by thromboelastometry. Single-photon emission computed tomography (SPECT) demonstrated real-time retention of a 99mTc-MNP-rtPA induced by magnet application in a rat embolic model; an 80% reduction in rtPA dosage for a chitosan-MNP-rtPA with magnetic guidance was shown to restore blood flow. After treatment, iron deposition was observed in the reticuloendothelial systems, with portal edema and neutrophil infiltration in the liver at a ten-fold higher dose but not the regular dose. Nevertheless, no liver or renal toxicity was observed at this higher dose. In conclusion, the liver may still be the major deposit site of rtPA nanocomposites after targeted delivery; chitosan-coated MNPs are potentially amenable to target therapeutics with parenteral administration.

The cellular responses of corneal fibroblasts to cyclic stretching loads.

Mechanical signaling plays a crucial role in maintaining extracellular matrix (ECM) homeostasis in various structures. In this study, we investigated the responses of corneal fibroblasts to cyclic stretching loads using an in vitro cell culture system. Bovine corneal fibroblasts were cultured and subjected to equibiaxial cyclic strain of 15% for 72 h at a frequency of 0.25 Hz, with bovine skin fibroblasts used as a comparison. We explored various cellular behaviors, including morphological changes, cell proliferation, and metabolism in response to mechanical stretching loads. The expression of genes, protein secretion, and enzymatic activity for several major metalloproteinases was also determined through Q-PCR, Western blot, and gel zymography. Additionally, we investigated the involvement of mitogen-activated protein kinases (MAPKs) signaling pathways in the corneal fibroblasts when subjected to mechanical stimuli. Our findings revealed that, compared to skin fibroblasts, corneal fibroblasts were reluctant to morphological changes in response to a prolonged (72 h) and high-amplitude (15% of strain) cyclic stretching load. However, cyclic stretching loads stimulated the upregulation of MMP-2 expression in corneal fibroblasts via the MAPK signaling pathways involving extracellular signal-regulated kinase and p38. Together with a lack of upregulation in type I collagen expression, our results indicate the induction of the ECM degradation process in corneal fibroblasts in response to cyclic stretching. These findings emphasize the mechanoresponsive nature of corneal fibroblasts and shed light on the potential impact of intense mechanical stress on the cornea in both normal and pathological conditions such as keratoconus, providing valuable insights for understanding corneal mechanobiology.

Laminin Receptor-Mediated Nanoparticle Uptake by Tumor Cells: Interplay of Epigallocatechin Gallate and Magnetic Force at Nano-Bio Interface.

Epigallocatechin gallate (EGCG), a major tea catechin, enhances cellular uptake of magnetic nanoparticles (MNPs), but the mechanism remains unclear. Since EGCG may interact with the 67-kDa laminin receptor (67LR) and epidermal growth factor receptor (EGFR), we investigate whether a receptor and its downstream signaling may mediate EGCG's enhancement effects on nanoparticle uptake. As measured using a colorimetric iron assay, EGCG induced a concentration-dependent enhancement effect of MNP internalization by LN-229 glioma cells, which was synergistically enhanced by the application of a magnetic field. Transmission electron microscopy demonstrated that EGCG increased the number, but not the size, of internalized vesicles, whereas EGCG and the magnet synergistically increased the size of vesicles. EGCG appears to enhance particle-particle interaction and thus aggregation following a 5-min magnet application. An antibody against 67LR, knockdown of 67LR, and a 67LR peptide (amino acid 161-170 of 67LR) attenuated EGCG-induced MNP uptake by 35%, 100%, and 45%, respectively, suggesting a crucial role of 67LR in the effects of EGCG. Heparin, the 67LR-binding glycosaminoglycan, attenuated EGCG-induced MNP uptake in the absence, but not presence, of the magnet. Such enhancement effects of EGCG were attenuated by LY294002 (a phosphoinositide 3-kinase inhibitor) and Akt inhibitor, but not by agents affecting cGMP levels, suggesting potential involvement of signaling downstream of 67LR. In contrast, the antibody against EGFR exerted no effect on EGCG-enhanced internalization. These results suggest that 67LR may be potentially amenable to tumor-targeted therapeutics.

Tannic Acid Coating Augments Glioblastoma Cellular Uptake of Magnetic Nanoparticles with Antioxidant Effects.

Coating of nanoparticles with gallates renders them antioxidant and enhances cellular internalization. In this study, (amino)silica magnetic particles modified with tannic acid (TA) and optionally with chitosan (CS) were developed, and their physicochemical properties and antioxidant activity were evaluated. The results demonstrated that the TA-modified aminosilica-coated particles, as well as the silica-coated particles with a double TA layer, exhibited high antioxidant activity, whereas the silica-coated particles with no or only a single TA layer were well-internalized by LN-229 cells. In addition, a magnet placed under the culture plates greatly increased the cellular uptake of all TA-coated magnetic nanoparticles. The coating thus had a considerable impact on nanoparticle-cell interactions and particle internalization. The TA-coated magnetic nanoparticles have great potential as intracellular carriers with preserved antioxidant activity.

Cyclic Strain Mitigates Nanoparticle Internalization by Vascular Smooth Muscle Cells.

Background: Intravascular delivery of nanoparticles for theranostic application permits direct interaction of nanoparticles and vascular cells. Since vascular smooth muscle cells (VSMCs), the major components of the vascular wall, are constantly subjected to mechanical stimulation from hemodynamic influence, we asked whether cyclic strain may modulate internalization of magnetic nanoparticles (MNPs) by cultured VSMCs. Methods: Cyclic strain (1 Hz and 10%) was applied with Flexcell system in cultured VSMCs from rats, with cell-associated MNPs (MNPcell) determined by a colorimetric iron assay. Transmission and scanning electron microscopy were used for morphology studies. Confocal microscopy was used to demonstrate distribution of actin assembly in VSMCs. Results: Incubation of poly(acrylic acid) (PAA)-coated MNPs with VSMCs for 4 h induced microvilli formation and MNP internalization. Application of cyclic strain for 4-12 h significantly reduced MNPcell by up to 65% (p < 0.05), which was associated with blunted microvilli and reduced vesicle size/cell, but not vesicle numbers/cell. Confocal microscopy demonstrated that both cyclic strain and fibronectin coating of the culture plate reduced internalized MNPs, which were co-localized with vinculin. Furthermore, cytochalasin D reduced MNPcell, suggesting a role of actin polymerization in MNP uptake by VSMCs; however, a myosin II ATPase inhibitor, blebbistatin, exhibited no effect. Cyclic strain also attenuated uptake of PAA-MNPs by LN-229 cells and uptake of poly-L-lysine-coated MNPs by VSMCs. Conclusion: In such a dynamic milieu, cyclic strain may impede cellular internalization of nanocarriers, which spares the nanocarriers and augments their delivery to the target site in the lumen of vessels or outside of the circulatory system.

Preparation of Peptide and Recombinant Tissue Plasminogen Activator Conjugated Poly(Lactic-Co-Glycolic Acid) (PLGA) Magnetic Nanoparticles for Dual Targeted Thrombolytic Therapy.

Recombinant tissue plasminogen activator (rtPA) is the only thrombolytic agent that has been approved by the FDA for treatment of ischemic stroke. However, a high dose intravenous infusion is required to maintain effective drug concentration, owing to the short half-life of the thrombolytic drug, whereas a momentous limitation is the risk of bleeding. We envision a dual targeted strategy for rtPA delivery will be feasible to minimize the required dose of rtPA for treatment. For this purpose, rtPA and fibrin-avid peptide were co-immobilized to poly(lactic-co-glycolic acid) (PLGA) magnetic nanoparticles (PMNP) to prepare peptide/rtPA conjugated PMNPs (pPMNP-rtPA). During preparation, PMNP was first surface modified with avidin, which could interact with biotin. This is followed by binding PMNP-avidin with biotin-PEG-rtPA (or biotin-PEG-peptide), which was prepared beforehand by binding rtPA (or peptide) to biotin-PEG-maleimide while using click chemistry between maleimide and the single -SH group in rtPA (or peptide). The physicochemical property characterization indicated the successful preparation of the magnetic nanoparticles with full retention of rtPA fibrinolysis activity, while biological response studies underlined the high biocompatibility of all magnetic nanoparticles from cytotoxicity and hemolysis assays in vitro. The magnetic guidance and fibrin binding effects were also confirmed, which led to a higher thrombolysis rate in vitro using PMNP-rtPA or pPMNP-rtPA when compared to free rtPA after static or dynamic incubation with blood clots. Using pressure-dependent clot lysis model in a flow system, dual targeted pPMNP-rtPA could reduce the clot lysis time for reperfusion by 40% when compared to free rtPA at the same drug dosage. From in vivo targeted thrombolysis in a rat embolic model, pPMNP-rtPA was used at 20% of free rtPA dosage to restore the iliac blood flow in vascular thrombus that was created by injecting a blood clot to the hind limb area.

Targeted Delivery of Plasminogen Activators for Thrombolytic Therapy: An Integrative Evaluation.

In thrombolytic therapy, plasminogen activators (PAs) are still the only group of drug approved to induce thrombolysis, and therefore, critical for treatment of arterial thromboembolism, such as stroke, in the acute phase. Functionalized nanocomposites have attracted great attention in achieving target thrombolysis due to favorable characteristics associated with the size, surface properties and targeting effects. Many PA-conjugated nanocomposites have been prepared and characterized, and some of them has been demonstrated with therapeutic efficacy in animal models. To facilitate future translation, this paper reviews recent progress of this area, especially focus on how to achieve reproducible thrombolysis efficacy in vivo.

Effects of PEGylation on capture of dextran-coated magnetic nanoparticles in microcirculation.

BACKGROUND: Magnetic nanoparticles (MNPs) can be localized against hemodynamic forces in blood vessels with the application of an external magnetic field. In addition, PEGylation of nanoparticles may increase the half-life of nanocomposites in circulation. In this work, we examined the effect of PEGylation on the magnetic capture of MNPs in vivo. METHODS: Laser speckle contrast imaging and capillaroscopy were used to assess the magnetic capture of dextran-coated MNPs and red blood cell (RBC) flow in cremaster microvessels of anesthetized rats. Magnetic capture of MNPs in serum flow was visualized with an in vitro circulating system. The effect of PEGylation on MNP-endothelial cell interaction was studied in cultured cells using an iron assay. RESULTS: In microcirculation through cremaster muscle, magnet-induced retention of 250 nm MNPs was associated with a variable reduction in RBC flow, suggesting a dynamic coupling of hemodynamic and magnetic forces. After magnet removal, faster restoration of flow was observed in PEG(+) than PEG(-) group, which may be attributed to a reduced interaction with vascular endothelium. However, PEGylation appears to be required for magnetic capture of 50 nm MNPs in microvessels, which was associated with increased hydrodynamic diameter to 130±6 nm in serum, but independent of the ς-potential. CONCLUSION: These results suggest that PEGylation may enhance magnetic capture of smaller MNPs and dispersion of larger MNPs after magnet removal, which may potentially affect the targeting, pharmacokinetics and therapeutic efficacy.

Scavenging of reactive oxygen species by phenolic compound-modified maghemite nanoparticles.

Maghemite (γ-Fe2O3) nanoparticles obtained through co-precipitation and oxidation were coated with heparin (Hep) to yield γ-Fe2O3@Hep, and subsequently with chitosan that was modified with different phenolic compounds, including gallic acid (CS-G), hydroquinone (CS-H), and phloroglucinol (CS-P), to yield γ-Fe2O3@Hep-CS-G, γ-Fe2O3@Hep-CS-H, and γ-Fe2O3@Hep-CS-P particles, respectively. Surface modification of the particles was analyzed by transmission electron microscopy, dynamic light scattering, attenuated total reflection Fourier transform infrared spectroscopy, and thermogravimetric analysis. Magnetic measurements indicated that the polymer coating does not affect the superparamagnetic character of the iron oxide core. However, magnetic saturation decreased with increasing thickness of the polymer coating. The antioxidant properties of the nanoparticles were analyzed using a 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. Cellular uptake and intracellular antioxidant activity of the particles were evaluated by an iron assay and flow cytometry, respectively, using L-929 and LN-229 cells. Compared to the control, the phenolic modification significantly reduced intracellular reactive oxygen species (ROS) levels to 35-56%, which was associated with a 6-8-times higher cellular uptake in L-929 cells and a 21-31-times higher cellular uptake in LN-229 cells. In contrast, γ-Fe2O3@Hep particles induced a 3.8-times and 14.9-times higher cellular uptake without inducing antioxidant activity. In conclusion, the high cellular uptake and the antioxidant properties associated with the phenolic moieties in the modified particles allow for a potential application in biomedical areas.

Gallate-induced nanoparticle uptake by tumor cells: Structure-activity relationships.

How nanoparticles interact with biological systems determines whether they can be used in theranostic applications. It has been demonstrated that tea catechins, may enhance interactions of magnetic nanoparticles (MNPs) with tumor cells and the subsequent cellular internalization of MNPs. As part of the chemical structure of the major tea catechins, gallates are found in a variety of plants and thus food components. We asked whether the structure of gallate might act as a pharmacophore in the enhancement of the effects of MNP-cell interactions. Uptake of dextran-coated MNPs by glioma cells and cell-associated MNPs (MNPcell) were respectively analyzed by confocal microscopy and a colorimetric iron assay. Co-incubation of MNPs and gallates, such as gallic acid and methyl gallate, induced a concentration-dependent increase in MNPcell, which was associated with co-localization of internalized MNPs and lysosomes. An analysis of the structure-activity relationship (SAR) revealed that the galloyl moiety exerted the most prominent enhancement effects on MNPcell which was further potentiated by the application of magnetic force; catechol coupled with a conjugated carboxylic acid side chain displayed comparable effects to gallate. Blockade or reduction in the number of hydroxyl groups rendered these compounds less effective, but without inducing cytotoxicity. The SAR results suggest that neighboring hydroxyl groups on the aromatic ring form an essential scaffold for the uptake effects; a similar SAR on antioxidant activities was also observed using a free radical-scavenging method. The results provide pivotal information for theranostic applications of gallates by facilitating nanoparticle-cell interactions and nanoparticle internalization by tumor cells.

Thrombolysis induced by intravenous administration of plasminogen activator in magnetoliposomes: dual targeting by magnetic and thermal manipulation.

In previously published studies, intra-arterial (i.a.), but not intravenous (i.v.) delivery of recombinant tissue-type plasminogen activator (rtPA) immobilized on the surface of magnetic nanoparticles induces thrombolysis by magnetic targeting. We asked whether i.v. delivery of protected rtPA in a thermosensitive magnetoliposome (TML@rtPA) may achieve target thrombolysis. PEGylated TML@rtPA was optimized and characterized; controlled release of rtPA was achieved by thermodynamic and magnetic manipulation in vitro. The lysis index of TML@rtPA incubated with blood at 43 °C vs. 37 °C was 53 ±â€¯11% vs. 81 ±â€¯3% in thromboelastograms, suggesting thermosensitive thrombolysis of TML@rtPA. In a rat embolic model with superfusion of 43 °C saline on a focal spot on the iliac artery with clot lodging, release of rtPA equivalent to 20% regular dose from TML@rtPA administered i.a. vs. i.v. significantly restored iliac blood flow 15 vs. 55 min after clot lodging, respectively. TML@rtPA with magnetic guiding and focal hyperthermia may be potentially amendable to target thrombolysis.

Interaction of poly-l-lysine coating and heparan sulfate proteoglycan on magnetic nanoparticle uptake by tumor cells.

BACKGROUND: Poly-l-lysine (PLL) enhances nanoparticle (NP) uptake, but the molecular mechanism remains unresolved. We asked whether PLL may interact with negatively charged glycoconjugates on the cell surface and facilitate uptake of magnetic NPs (MNPs) by tumor cells. METHODS: PLL-coated MNPs (PLL-MNPs) with positive and negative ζ-potential were prepared and characterized. Confocal and transmission electron microscopy was used to analyze cellular internalization of MNPs. A colorimetric iron assay was used to quantitate cell-associated MNPs (MNPcell). RESULTS: Coadministration of PLL and dextran-coated MNPs in culture enhanced cellular internalization of MNPs, with increased vesicle size and numbers/cell. MNPcell was increased by eight- to 12-fold in response to PLL in a concentration-dependent manner in human glioma and HeLa cells. However, the application of a magnetic field attenuated PLL-induced increase in MNPcell. PLL-coating increased MNPcell regardless of ζ-potential of PLL-MNPs, whereas magnetic force did not enhance MNPcell. In contrast, epigallocatechin gallate and magnetic force synergistically enhanced PLL-MNP uptake. In addition, heparin, but not sialic acid, greatly reduced the enhancement effects of PLL; however, removal of heparan sulfate from heparan sulfate proteoglycans of the cell surface by heparinase III significantly reduced MNPcell. CONCLUSION: Our results suggest that PLL-heparan sulfate proteoglycan interaction may be the first step mediating PLL-MNP internalization by tumor cells. Given these results, PLL may facilitate NP interaction with tumor cells via a molecular mechanism shared by infection machinery of certain viruses.

Passivating Injured Endothelium with Kinexins in Thrombolytic Therapy.

Without conjunctive administration of an anticoagulant, endothelial injury-induced thrombosis is resistant to thrombolysis and prone to re-thrombosis. We hypothesized that co-delivery of recombinant tissue plasminogen activator (rtPA) with annexin V-containing anticoagulants that specifically target the injured endothelium may passivate the thrombogenic elements of the vascular injury site and enhance rtPA-induced thrombolysis. In this study, the effects of conjunctive administration of Kinexins (Kunitz inhibitor-annexin V fusion proteins) with rtPA on thrombolysis were determined in vitro and in vivo. Thromboelastometry showed that both TAP-A (tick anticoagulant peptide-annexin V fusion protein; an inhibitor of factor Xa [FXa] and prothrombinase) and A-6L15 (annexin V-6L15 fusion protein; an inhibitor of tissue factor/FVIIa) exerted concentration-dependent (10-100 nM) effects on clot formation, with TAP-A being several folds more potent than A-6L15 in whole blood. Combination of TAP-A or A-6L15 with rtPA (1 µg/mL) led to decrease in lysis index, suggesting conjunctive enhancement of thrombolysis by combined use of rtPA with TAP-A or A-6L15. In a rat cremaster muscle preparation subjected to photochemical injury, conjunctive administration of rtPA and TAP-A significantly restored tissue perfusion to 56%, which is approximately two fold of that by rtPA or TAP-A alone. Near-infrared fluorescence images demonstrated local retention of a fluorescent A-6L15-S288 at the injury site, suggesting a targeting effect of the fusion protein. Pharmacokinetic analysis showed that 123I-labelled TAP-A and A-6L15 had initial distribution half-lives (T1/2α) of approximately 6 minutes and elimination half-lives (T1/2ß) of approximately 2.3 hours. In conclusion, Kinexins were potentially useful adjunctive agents with rtPA thrombolytic therapy especially for thrombosis induced by endothelial injury.

Magnetically controlled release of recombinant tissue plasminogen activator from chitosan nanocomposites for targeted thrombolysis.

Ionic cross-linking of water-soluble chitosan with sodium tripolyphosphate in the presence of recombinant tissue plasminogen activator (rtPA) and magnetite (Fe3O4) nanoparticles could produce rtPA-encapsulated magnetic chitosan nanoparticles (MCNPs-rtPA). MCNPs do not elicit cytotoxicity and hemolysis in vitro. MCNPs-rtPA showed a negligible release of the rtPA protein when stored in phosphate buffer for 28 days. In contrast, the burst release of rtPA from MCNPs-rtPA was found in the serum with 60% of the original activity released in 30 min. The drug release into the serum is also magnet-sensitive; the release could be turned down with a magnetic field when MCNPs-rtPA was pelleted and reversibly turned on after removing the magnetic field when MCNPs-rtPA was dispersed. An in vitro thrombolytic study by thromboelastometry indicated a controlled release of rtPA from MCNPs-rtPA. In a rat embolic model where a preformed blood clot lodged in the left iliac artery upstream of the pudic epigastric branch, MCNPs-rtPA (0.2 mg kg-1 rtPA) was administered and guided magnetically to the clot, followed by mobile magnetic guidance for 60 min. Iliac blood flow increased immediately in response to the treatment, and reached a stable level ∼50 min after drug administration and the hind limb perfusion rate was restored from 53% to 75% of the basal level. Effective thrombolysis was therefore successfully demonstrated at an rtPA dose equivalent to 20% of the regular dose when the MCNPs-rtPA pellet was magnet-guided to the blood clot, followed by a triggered release of rtPA when switched to mobile magnetic guidance.

Augmented cellular uptake of nanoparticles using tea catechins: effect of surface modification on nanoparticle-cell interaction.

Nanoparticles may serve as carriers in targeted therapeutics; interaction of the nanoparticles with a biological system may determine their targeting effects and therapeutic efficacy. Epigallocatechin-3-gallate (EGCG), a major component of tea catechins, has been conjugated with nanoparticles and tested as an anticancer agent. We investigated whether EGCG may enhance nanoparticle uptake by tumor cells. Cellular uptake of a dextran-coated magnetic nanoparticle (MNP) was determined by confocal microscopy, flow cytometry or a potassium thiocyanate colorimetric method. We demonstrated that EGCG greatly enhanced interaction and/or internalization of MNPs (with or without polyethylene glycol) by glioma cells, but not vascular endothelial cells. The enhancing effects are both time- and concentration-dependent. Such effects may be induced by a simple mix of MNPs with EGCG at a concentration as low as 1-3 µM, which increased MNP uptake 2- to 7-fold. In addition, application of magnetic force further potentiated MNP uptake, suggesting a synergetic effect of EGCG and magnetic force. Because the effects of EGCG were preserved at 4 °C, but not when EGCG was removed from the culture medium prior to addition of MNPs, a direct interaction of EGCG and MNPs was implicated. Use of an MNP-EGCG composite produced by adsorption of EGCG and magnetic separation also led to an enhanced uptake. The results reveal a novel interaction of a food component and nanocarrier system, which may be potentially amenable to magnetofection, cell labeling/tracing, and targeted therapeutics.

Retention assessment of magnetic nanoparticles in rat arteries with micro-computed tomography.

Magnetic nanoparticles (MNPs) may serve as carriers for pharmacological agents to the target in a magnetic-force guiding system. It is essential to achieve effective retention of MNPs through the external magnet placement. However, it is difficult to estimate the retention efficiency of MNPs and validate the experimental strategies. Micro-CT was used to identify the spatial distribution of MNP retention and image analysis is then extended to evaluate the MNP delivery efficiency. Male Sprague Dawley rats were anesthetized to expose abdominal arteries with an NdFeB magnet of 4.9 kG placed by the left iliac artery. After a 20 min equilibrium period, arteries were ligated, removed and fixed in a paraformaldehyde solution. Experiments were performed with intravenous injection in our platform with two independent groups. MNPs were used in the first group, while chemical compounds of recombinant tissue plaminogen activator were attached to MNPs as rtPA (recombinant tissue plaminogen activator)-MNPs in the second group. Image analysis of micro-CT shows the average retention volume of MNPs and rtPA-MNPs in the left iliac arteries is 9.3 and 6.3 fold of that in the right. Large local aggregation of MNPs and rtPA-MNPs in the left iliac arteries is the consequence of external magnet placement, suggesting feasibility of magnetic targeting through the intravenous administration. We also determined that on average 0.57% and 0.064% of MNPs and rtPA-MNPs respectively were retained in the left iliac artery. It was estimated that the average rtPA concentration of 60.16 µg mL(-1) may be achieved with rtPA-MNPs. With the micro-CT imaging approach, we accomplished visualization of the aggregation of retained particles; reconstructed 3D distribution of relative retention; estimated the average particle number of local retention; determined efficiency of targeted delivery. In particular, our quantitative image assessment suggests that intravenous administration of rtPA-MNPs may retain local concentration of rtPA high enough to induce thrombolysis.

Targeted delivery of tissue plasminogen activator by binding to silica-coated magnetic nanoparticle.

BACKGROUND AND METHODS: Silica-coated magnetic nanoparticle (SiO(2)-MNP) prepared by the sol-gel method was studied as a nanocarrier for targeted delivery of tissue plasminogen activator (tPA). The nanocarrier consists of a superparamagnetic iron oxide core and an SiO(2) shell and is characterized by transmission electron microscopy, Fourier transform infrared spectroscopy, X-ray diffraction, superconducting quantum interference device, and thermogravimetric analysis. An amine-terminated surface silanizing agent (3-aminopropyltrimethoxysilane) was used to functionalize the SiO(2) surface, which provides abundant -NH(2) functional groups for conjugating with tPA. RESULTS: The optimum drug loading is reached when 0.5 mg/mL tPA is conjugated with 5 mg SiO(2)-MNP where 94% tPA is attached to the carrier with 86% retention of amidolytic activity and full retention of fibrinolytic activity. In vitro biocompatibility determined by lactate dehydrogenase release and cell proliferation indicated that SiO(2)-MNP does not elicit cytotoxicity. Hematological analysis of blood samples withdrawn from mice after venous administration indicates that tPA-conjugated SiO(2)-MNP (SiO(2)-MNP-tPA) did not alter blood component concentrations. After conjugating to SiO(2)-MNP, tPA showed enhanced storage stability in buffer and operation stability in whole blood up to 9.5 and 2.8-fold, respectively. Effective thrombolysis with SiO(2)-MNP-tPA under magnetic guidance is demonstrated in an ex vivo thrombolysis model where 34% and 40% reductions in blood clot lysis time were observed compared with runs without magnetic targeting and with free tPA, respectively, using the same drug dosage. Enhanced penetration of SiO(2)-MNP-tPA into blood clots under magnetic guidance was confirmed from microcomputed tomography analysis. CONCLUSION: Biocompatible SiO(2)-MNP developed in this study will be useful as a magnetic targeting drug carrier to improve clinical thrombolytic therapy.

Bioconjugation of recombinant tissue plasminogen activator to magnetic nanocarriers for targeted thrombolysis.

Low-toxicity magnetic nanocarriers (MNCs) composed of a shell of poly [aniline-co-N-(1-one-butyric acid) aniline] over a Fe(3)O(4) magnetic nanoparticle core were developed to carry recombinant tissue plasminogen activator (rtPA) in MNC-rtPA for targeted thrombolysis. With an average diameter of 14.8 nm, the MNCs exerted superparamagnetic properties. Up to 276 µg of active rtPA was immobilized per mg of MNCs, and the stability of the immobilized rtPA was greatly improved during storage at 4°C and 25°C. In vitro thrombolysis testing with a tubing system demonstrated that magnet-guided MNC-rtPA showed significantly improved thrombolysis compared with free rtPA and reduced the clot lysis time from 39.2 ± 3.2 minutes to 10.8 ± 4.2 minutes. In addition, magnet-guided MNC-rtPA at 20% of the regular rtPA dose restored blood flow within 15-25 minutes of treatment in a rat embolism model without triggering hematological toxicity. In conclusion, this improved system is based on magnetic targeting accelerated thrombolysis and is potentially amenable to therapeutic applications in thromboembolic diseases.

Manipulation of magnetic nanoparticle retention and hemodynamic consequences in microcirculation: assessment by laser speckle imaging.

Magnetic nanoparticles (MNPs) have been proposed for targeted or embolization therapeutics. How MNP retention occurs in circulation may critically determine local hemodynamics, tissue distribution of MNPs, and the therapeutic effects. We attempted to establish a microcirculation model to study the magnetic capture of MNPs in small vessels and to determine the factors affecting MNP retention. Two-dimensional hemodynamic changes in response to magnet-induced MNP retention in the microvessels of the cremaster muscle in vivo were observed in a real-time manner using a laser speckle imaging technique. Changes in tissue perfusion of the cremaster muscle appeared to be closely correlated with the location of the magnet placement underneath the muscle in response to intra-arterial administration of dextran-coated MNPs. Magnet-related retention was observed along the edge of the magnet, as corroborated by the results of histology analysis and microcomputed tomography. In these preparations, tissue iron content almost doubled, as revealed by inductively coupled plasma optical emission spectroscopy. In addition, MNP retention was associated with reduced downstream flow in a dose-dependent manner. Dissipation of MNPs (5 mg/kg) occurred shortly after removal of the magnet, which was associated with significant recovery of tissue flow. However, MNP dissipation did not easily occur after administration of a higher MNP dose (10 mg/kg) or prolonged exposure to the magnetic field. An ultrasound after removal of the magnet may induce the partial dispersion of MNPs and thus partially improve hemodynamics. In conclusion, our results revealed the important correlation of local MNP retention and hemodynamic changes in microcirculation, which can be crucial in the application of MNPs for effective targeted therapeutics.