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Nanoparticle delivery systems
Nanotechnology can be used in therapies for atherosclerosis by increasing systemic agent circulation time, lowering off-target cytotoxicity of drugs, improving drug solubility, decreasing the required dosage, combining diagnostic and therapeutic agents to form theranostics and increasing accumulation of agents at specific sites . Targeted drug delivery can be categorized as either active or passive targeting. Active targeting involves the conjugation of tissue or cell-specific ligands to either the nanocarriers or to the drugs themselves. In the passive targeting strategy, therapeutic agents or drugs are coupled with macromolecules to take advantage of the EPR effect . Recently, stimuli-responsive delivery systems have been emerged based on various triggers such as light irradiation, pH alteration, application of magnetic or electric fields, a change in temperature or response to redox potentials. These smart NPs could be potentially applied in DES for therapy of CAD. For example, Tang et al. tested pH-responsive delivery of antioxidants for treatment of cardiovascular disease such as atherosclerosis . On the other hand, localized NP delivery via stents may be a promising strategy to combat restenosis, because it could provide a sustained drug release in the target region of the artery . DES can be used to localize delivery of drugs and avoid the potential toxicity of systemic drug administration .
NPs used to inhibit restenosis are reviewed in the following paragraphs, and Figure 1 shows the schematic structure of important NPs.Figure 1. Schematic structure of important nanoparticles.
Liposomes are small vesicles, have a spherical shape and are composed of a lipid-bilayer formed from natural and nontoxic phospholipids and cholesterol . The features of liposomes such as biocompatibility (because of using natural biologically safe lipids), nanometer size, the ability to tailor the hydrophobicity and hydrophilicity can provide enhanced tissue specificity for delivery of hydrophobic drugs in the lipid environment, and for hydrophilic drugs in the aqueous core . The revascularization of occluded arteries in vivo was enhanced, along with a reduction in the risk of hemorrhagic side-effects. The effect of peptide-modified liposomes with good potential for vascular-targeted delivery of therapeutic and diagnostic agents has been studied. Ligands that recognize surface receptors on activated platelets (e.g., integrin GP IIb/IIIa and P-selectin) have been attached to liposomes to demonstrate the vital role of activated platelets in atherogenesis, atherosclerotic lesion progression and thrombosis in vascular diseases . Phase 1 results of one study show that after 28 days of follow-up in rabbit carotid artery, liposomal alendronate can reduce ISR to 40.1% in comparison to 73.5% in empty liposomal . Figures 2 & 3 demonstrate examples of liposomal delivery in CAD therapy.Figure 2. The liposomal nanoparticle with prednisolone phosphate stored in a macrophages of iliofemoral plaques.
(A) First row illustrates the plaque cells cured by LN-PLP marked for cell nuclei (DAPI), macrophages (CD68) and liposome-coating PEG. In the second row, magnified images of isolated cells are shown. (B) Third row shows CD68 cells from a plaque cured by saline, but there is no positivity for PEG.
LN-PLP: Liposomal nanoparticle with prednisolone phosphate.
Reproduced with permission from , © (2
015) Nanomedicine: Nanotechnology, Biology and Medicine.Figure 3. Delivery of tetrahydrobiopterin (BH4) by liposome nanocarrier.
(A) Improved stability of BH4 in comparison with unencapsulated BH4 after 24 h; p = 0.017; (B) BH4 concentration in ligated artery increased by liposomal delivery; p = 0.04; (C) superoxide concentration in ligated artery with targeted liposomes decreased; (D) the plaque burden was decreased by BH4 liposomal delivery in the mice ligated left carotid artery fed by 7-day high fat diet (plaque and lumen are marked by red and blue, respectively) scale bars = 100 μm; (E) the area of plaque; p = 0.0015; (F) there is no alteration in lipid metabolism via liposome deliver; p = 0.47 and 0.11, respectively).
BH4: Tetrahydrobiopterin; DHE: Dihydroethidium; FBS: Fetal bovine serum; LCA: Left common carotid artery; RCA: Right common carotid artery.
Reproduced with permission from , © (2015) ACS Nano.
Micelles are formed when amphiphilic molecules undergo self-assembly due to the energy minimization that occurs when the hydrophobic portions bunch together to form the interior. The hydrophilic shell provides the long circulation time and accounts for the relative stability in vivo. The hydrophobic core of micelles can be used for encapsulation and delivery of either bioactive therapeutic molecules, diagnostic agents or both. Attachment of moieties such as targeting ligands to the outer shell of micelles can increase active binding to disease-relevant tissues and cells . There are results that suggest phospholipid-based micelles provide better antirestenotic effects (neointimal area 0.034 in comparison to 0.046 mm2 after 14 days of implantation in rat carotid arteries) than the PEGylated liposomes; probably due to the distinctly smaller size of the phospholipid-based micelles . Micelles that had been surface-modified with anti-CD36 antibodies were loaded with Gd. These micelles could target macrophages in specimens from atherosclerotic human aortas .
Polymeric NP may be constructed in the form of solid, dense but porous structures (e.g., nanospheres and nanorods) or hollow structures (e.g., nanoshells and nanocapsules) [1,43]. Results of one study on 60-nm diameter lipid–polymeric NP functionalized by collagen IV-targeting peptides and enriched with paclitaxel demonstrate efficacious improvement. Injection of paclitaxel (0.3 mg/kg or 1 mg/kg) in a rat carotid arteries followed on days 0 and 5 resulted in lower neointima-to-media (N/M) ratio for the targeted NP group at 2 week versus the control groups. Compared with controlled groups, a 50% reduction in arterial stenosis was observed with targeted NP delivery . Another study reported that PLGA NPs containing alendronate reduced neointimal formation and restenosis up to 64% for a dose of 3 mg/kg by systemic transient depletion of monocytes in a hypercholesterolemic rabbit model . Delivery of imatinib (used as an inhibitor of PGDF receptor) by means of bioabsorbable polymeric NPs reduced the occurrence of ISR up to 50% compared with bare metal stents . Statins have been shown to prevent the proliferation of vascular SMCs (VSMC) and also to stimulate vascular healing. Researchers formulated an NP-coated DES with 20 μg pitavastatin dosage per stent and tested it in a pig coronary artery model. This coated stent inhibited ISR as effectively as a polymer-coated sirolimus-eluting stent. There was a delay in endothelial healing with the conventional sirolimus-eluting stent, whereas no delay in re-endothelialization was observed in the pitavastatin NP-eluting stent .
Dendrimers consist of a single molecule constructed from an original inner core with a series of macromolecular branches built up by successive additions of discrete units (generations). The ability to display multiple copies of functional groups on their surface makes them a unique structure for drug-delivery applications . Dendrimers are more used in cell-labeling rather than in ISR therapy. For instance, manganese G8 dendrimers  have been successfully applied in atherosclerosis detection. One study described the development of ‘tadpole’ dendrimeric materials for siRNA delivery in a rat ischemia-reperfusion model. Angiotensin II (Ang II) type 1 receptor (AT1R) has been investigated since it is the major receptor that mediates most adverse effects of Ang II. Among those tadpole dendrimers evaluated, significant effective downregulation in AT1R expression in cardiomyocytes was related to the oligo-arginine-conjugated dendrimer loaded with siRNA in vitro. Delivery of the siRNA in vivo, inhibited AT1R levels to be increased, and meaningfully cardiac function recovery was improved compared with saline injection or empty dendrimer treated groups . Additionally, polyamidoamine (PAMAM) dendrimers have been favored in recent years in CVD therapies. PAMAM zero generation dendrimers (G0) were tested as nanocarriers in drug delivery and conjugated G0 PAMAM dendrimers with a ZnPc photosensitizer were chosen to study their effects on the diseased and normal tissues extracted from human carotid arteries. Statistical analysis was carried out based on AFM images extracted through fractal analytical methodologies and Minkowski functionals. The affinity of the nanocarriers for healthy tissue and atheromatous tissue was different. Dissimilar aggregation behaviors between G0 and G0/ZnPc nanomaterials were observed. Larger G0/ZnPc aggregation on the atheromatous plaque were reported . Photodynamic therapy with PAMAM dendrimers could have a bright future in therapy of atherosclerosis.
It was demonstrated that hydrogel nanospheres (100 nm) made of poly (N-isopropylacrylamide) were internalized by endothelial cells and VSMC to a greater degree than microspheres (1 μm), although the cellular uptake was dependent on the incubation time, nanosphere concentration and applied shear stress levels in the medium. By contrast, microspheres were rapidly taken up by phagocytes, especially at high concentrations. These findings suggest that hydrogel nanospheres were more effective as an intravascular delivery system in terms of vascular uptake and biocompatibility . Since significant number of VSMC undergo rapid apoptosis following balloon angioplasty, Reddy and co-workers  tested the hypothesis that preventing VSMC from undergoing apoptosis could prevent intimal hyperplasia. They used rapamycin (which has antiapoptotic and antiproliferative properties) loaded into gel NPs with a mean diameter of 54 nm. When infused into a rat carotid artery model of vascular injury the authors reported significant inhibition of hyperplasia and improved re-endothelialisation of the injured artery. Furthermore, the group reported inhibition of the caspase 3/7 enzyme systems in the treated artery, thus preventing VSMC from undergoing apoptosis.
Magnetic targeting is a promising possibility for efficacious guidance of therapeutic agents to CAD diseased sites, elimination from nontargeted propagation (i.e., safety concern) and deep and long-term tissue targeting, furthermore it has suggested benefits for anti-ISR therapies, delivery of cells, gene vectors and therapeutic proteins and stented artery delivery of paclitaxel by utilization of magnetic field-guided magnetic carriers for specific-vascular delivery . Functional MNP-loaded primary endothelial cells as vectors targeting vascular stents induced gene expression related to EC growth and survival, and suppressed gene-related coagulation and suggested them for re-endothelialization by the implant and decreasing neointimal hyperplasia .
Metallic MNPs, made of iron, cobalt or nickel, are typically prepared with a core–shell structure in which gold or silica is applied as a coating material. Iron MNP composed of nanocrystalline magnetite (Fe3O4) or maghemite (γFe2O3) form a close-packed cubic lattice . Furthermore, gold shell NPs (˜120 nm) have been used for both imaging and therapy applications . Another study used paclitaxel-loaded magnetic NP with a uniform magnetic field that allowed the attachment of NP to the stent, and also drug release in order for it to be taken up by the target cells. In this case inhibition of ISR occurred with 7.5 μg paclitaxel, together with a noticeable reduction in the ratio of neointima/media that was only 63 ± 13% of that of the control group . Figure 4 reports the results of the mentioned study.Figure 4. Paclitaxel-loaded magnetic nanoparticles applied to coronary stents with a uniform magnetic field.
MNPs with PTX doses of 7.5 and 0.75 μg entered into animal bodies under magnetic versus nonmagnetic conditions. The animals sacrificed and the stented carotid segments were harvested 14 days after surgery. The control group did not receive MNP but stented. Verhoeff–van Gieson-stained section of an artery lumen treated with 7.5 μg PTX under magnetic conditions (A) demonstrated versus ‘no treatment’ control (B) (p < 0.05, Dunn’s Test Q statistic = 3.7). Original magnification 100×. Morphometric results as neointima/media ratios (C) pictured as a function of the magnetic field application and PTX dose (n ≥ 6). Data are presented as mean ± standard error.
MNP: Magnetic nanoparticle; NP: Nanoparticle; PTX: Paclitaxel.
Reprinted with permission from , © (2010) Proceedings of the National Academy of Sciences of the United States of America.
Additionally, quantum dots have been used as florescent labels to prepare traceable NP due to their tunable physicochemical features, high photostability, broad absorption spectra and narrow emission bands. Quantum dots have been proposed to monitor disease-associated events, such as macrophage cell infiltration into arterial tissues and the processes of angiogenesis and vascular remodeling . Figure 5 shows using of quantum dots to monitor monocyte-macrophages in atherosclerosis plaque. More details in plaque imaging can be found in the section ’Molecular imaging and atheroslerosis detection‘.Figure 5. Using quantum dots to image the monocyte-macrophages in atherosclerosis plaque.
The monocyte-macrophages loaded by cell penetrating quantum dots were injected to mice. Injected cells and macrophage marker CD68 are portrayed as orange and green, respectively.
Reproduced with permission from , © (2010) Current Atherosclerosis Reports.
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SOURCE https://www.ncbi.nlm.nih.gov / 2016