Small-Diameter Blood Vessel Substitutes: Biomimetic Approaches to Improve Patency.

Small-dimeter blood vessels (<6 mm) are required in coronary bypass and peripheral bypass surgery to circumvent blocked arteries. However, they have poor patency rates due to thrombus formation, intimal hyperplasia at the distal anastomosis, and compliance mismatch between the native artery and the graft. This review covers the state-of-the-art technologies for improving graft patency with a focus on reducing compliance mismatch between the prosthesis and the native artery. The focus of this article is on biomimetic design strategies to match the compliance over a wide pressure range.

Cell-mimicking polyethylene glycol-diacrylate based nanolipogel for encapsulation and delivery of hydrophilic biomolecule.

Lipid based nanoparticulate formulations have been widely used for the encapsulation and sustain release of hydrophilic drugs, but they still face challenges such as high initial burst release. Nanolipogel (NLG) emerges as a potential system to encapsulate and deliver hydrophilic drug while suppressing its initial burst release. However, there is a lack of characterization of the drug release mechanism from NLGs. In this work, we present a study on the release mechanism of hydrophilic Dextran-Fluorescein Isothiocyanate (DFITC) from Poly (ethylene glycol) Diacrylate (PEGDA) NLGs by using different molecular weights of PEGDA to vary the mesh size of the nanogel core, drawing inspiration from the macromolecular crowding effect in cells, which can be viewed as a mesh network of undefined sizes. The effect is then further characterized and validated by studying the diffusion of DFITC within the nanogel core using Fluorescence Recovery after Photobleaching (FRAP), on our newly developed cell derived microlipogels (MLG). This is in contrast to conventional FRAP works on cells or bulk hydrogels, which is limited in our application. Our work showed that the mesh size of the NLGs can be controlled by using different Mw of PEGDA, such as using a smaller MW to achieve higher crosslinking density, which will lead to having smaller mesh size for the crosslinked nanogel, and the release of hydrophilic DFITC can be sustained while suppressing the initial burst release, up to 10-fold more for crosslinked PEGDA 575 NLGs. This is further validated by FRAP which showed that the diffusion of DFITC is hindered by the decreasing mesh sizes in the NLGs, as a result of lower mobile fractions. These findings will be useful for guiding the design of PEGDA NLGs to have different degree of suppression of the initial burst release as well as the cumulative release, for a wide array of applications. This can also be extended to other different types of nanogel cores and other nanogel core-based nanoparticles for encapsulation and release of hydrophilic biomolecules.

Designing siRNA/chitosan-methacrylate complex nanolipogel for prolonged gene silencing effects.

Despite immense revolutionary therapeutics potential, sustaining release of active small interfering RNA (siRNA) remains an arduous challenge. The development of nanoparticles with siRNA sustained release capabilities provides an avenue to enhance the therapeutic efficacy of gene-based therapy. Herein, we present a new system based on the encapsulation of siRNA/chitosan-methacrylate (CMA) complexes into liposomes to form UV crosslinkable Nanolipogels (NLGs) with sustained siRNA-release properties in vitro. We demonstrated that the CMA nanogel in NLGs can enhance the encapsulation efficiency of siRNA and provide sustained release of siRNA up to 28 days. To understand the particle mechanism of cellular entry, multiple endocytic inhibitors have been used to investigate its endocytosis pathways. The study saw positively charged NLGs entering cells via multiple endocytosis pathways, facilitating endosomal escape and slowly releasing siRNA into the cytoplasm. Transfection experiments confirmed that the crosslinked NLG delivery system provides effective transfection and prolonged silencing effect up to 14 days in cell cultures. We expect that this sustained-release siRNA NLG platform would be of interest in both fundamental biological studies and in clinical applications to extend the use of siRNA-based therapies.

Progress in drug-delivery systems in cardiovascular applications: stents, balloons and nanoencapsulation.

Drug-delivery systems in cardiovascular applications regularly include the use of drug-eluting stents and drug-coated balloons to ensure sufficient drug transfer and efficacy in the treatment of cardiovascular diseases. In addition to the delivery of antiproliferative drugs, the use of growth factors, genetic materials, hormones and signaling molecules has led to the development of different nanoencapsulation techniques for targeted drug delivery. The review will cover drug delivery and coating mechanisms in current drug-eluting stents and drug-coated balloons, novel innovations in drug-eluting stent technologies and drug encapsulation in nanocarriers for delivery in vascular diseases. Newer technologies and advances in nanoencapsulation techniques, such as the use of liposomes, nanogels and layer-by-layer coating to deliver therapeutics in the cardiovascular space, will be highlighted.

Nanoparticles-reinforced poly-l-lactic acid composite materials as bioresorbable scaffold candidates for coronary stents: Insights from mechanical and finite element analysis.

Current generation of bioresorbable coronary scaffolds (BRS) posed thrombogenicity and deployment issues owing to its thick struts and overall profile. To this end, we hypothesize that the use of nanocomposite materials is able to provide improved material properties and sufficient radial strength for the intended application even at reduced strut thickness. The nanocomposite formulations of tantalum dioxide (Ta2O5), L-lactide functionalized (LA)-Ta2O5, hydroxyapatite (HA) and LA-HA with poly-l-lactic acid (PLLA) were evaluated in this study. Results showed that tensile modulus and strength were enhanced with non-functionalized nanofillers up until 15 wt% loading, whereas ductility was compromised. On the other hand, functionalized nanofillers/PLLA exhibited improved nanofiller dispersion which resulted higher tensile modulus, strength, and ductility. Selected nanocomposite formulations were evaluated using finite element analysis (FEA) of a stent with varying strut thickness (80, 100 and 150 µm). FEA data has shown that nanocomposite BRS with thinner struts (80-100 µm) made with 15 wt% LA-Ta2O5/PLLA and 10 wt% LA-HA/PLLA have increased radial strength, stiffness and reduced recoil compared to PLLA BRS at 150 µm. The reduced strut thickness can potentially mitigate issues such as scaffold thrombosis and promote re-endothelialisation of the vessel.

Spatio-Temporal In Vivo Imaging of Ocular Drug Delivery Systems using Fiberoptic Confocal Laser Microendoscopy.

Subconjunctival injection is an attractive route to administer ocular drugs due to easy trans-scleral access that bypasses anterior ocular barriers, such as the cornea and conjunctiva. While therapeutic effects and pharmacokinetics of the drugs upon subconjunctival injection have been described in some studies, very few assess the ocular distribution of drugs or drug delivery systems (DDS). The latter is critical for the optimization of intraocular DDS design and drug bioavailability to achieve the desired ocular localization and duration of action (e.g., acute versus. prolonged). This study establishes the use of fiberoptic confocal laser microendoscopy (CLM) to qualitatively study the ocular distribution of fluorescent liposomes in real-time in live mice after sub-conjunctival injection. Being designed for in vivo visual inspection of tissues at the microscopic level, this is also the first full description of the CLM imaging method to study spatio-temporal distribution of injectables in the eye after subconjunctival injection.

Liposome interaction with macrophages and foam cells for atherosclerosis treatment: effects of size, surface charge and lipid composition.

Liposomes are potential drug carriers for atherosclerosis therapy due to low immunogenicity and ease of surface modifications that allow them to have prolonged circulation half-life and specifically target atherosclerotic sites to increase uptake efficiency. However, the effects of their size, charge, and lipid compositions on macrophage and foam cell behaviour are not fully understood. In this study, liposomes of different sizes (60 nm, 100 nm and 180 nm), charges (-40 mV, -20 mV, neutral, +15 mV and +30 mV) and lipid compositions (1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, L-a-phosphatidylcholine, and egg sphingomyelin) were synthesized, characterized and exposed to macrophages and foam cells. Compared to 100 nm neutral 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) liposomes, flow cytometry and confocal imaging indicated that cationic liposomes and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DSPC) liposomes were internalized more by both macrophages and foam cells. Through endocytosis inhibition, phagocytosis and clathrin-mediated endocytosis were identified as the dominant mechanisms of uptake. Anionic and DSPC liposomes induced more cholesterol efflux capacity in foam cells. These results provide a guide for the optimal size, charge, and lipid composition of liposomes as drug carriers for atherosclerosis treatment.

Anti-inflammatory potential of simvastatin loaded nanoliposomes in 2D and 3D foam cell models.

Atherosclerosis is a multifactorial disease triggered and sustained by risk factors such as high cholesterol, high blood pressure and unhealthy lifestyle. Inflammation plays a pivotal role in atherosclerosis pathogenesis. In this study, we developed a simvastatin (STAT) loaded nanoliposomal formulation (LIPOSTAT) which can deliver the drug into atherosclerotic plaque, when administered intravenously. This formulation is easily prepared, stable, and biocompatible with minimal burst release for effective drug delivery. 2D and 3D in vitro models were examined towards anti-inflammatory effects of STAT, both free and in combination with liposomes. LIPOSTAT induced greater cholesterol efflux in the 2D foam cells and significantly reduced inflammation in both 2D and 3D models. LIPOSTAT alleviated inflammation by reducing the secretion of early and late phase pro-inflammatory cytokines, monocyte adherence marker, and lipid accumulation cytokines. Additionally, the 3D foam cell spheroid model is a convenient and practical approach in testing various anti-atherosclerotic drugs without the need for human tissue.

Layer-by-layer coated nanoliposomes for oral delivery of insulin.

Crossing the intestinal epithelial cell barrier safely and reaching the blood with therapeutic levels of bioactive insulin have been the ultimate goal of oral insulin delivery. The optimum way to overcome the barrier lies in the design of an efficient high drug loading carrier, that can protect insulin from the harsh Gastrointestinal (GI) environment and enhance its uptake and transport by epithelial cells. In the present study, we developed a multi-layered insulin loading strategy on an anionic nanoliposome surface based on electrostatic interaction with chitosan. The layer-by-layer (LbL) coated nanoliposomes achieved high insulin loading (10.7% by weight) and offered superior protection with limited release in simulated gastric fluid (SGF) (about 6% in 1 h), simulated intestinal fluid (SIF) (2% in two weeks), and phosphate buffered saline (PBS) (5% in two weeks). Intracellular imaging revealed that the LbL coated liposomes were internalized and intracellularly trafficked towards the basolateral side of the Caco-2 monolayer. Transported insulin demonstrated retention of bioactivity while crossing the epithelial barrier in the glucose uptake study in 3T3 L1-MBX adipocytes. In rat studies, oral administration of the formulation resulted in rapid absorption with a peak in plasma insulin levels 0.5 h post oral gavaging. This technology thus serves as a promising platform for potential oral insulin applications.

In vivo fate of liposomes after subconjunctival ocular delivery.

Subconjunctival administration of nanocarriers presents an alternative drug delivery strategy to overcome blood-ocular barriers to enhance drug bioavailability to specific parts of the eye. Using fiberoptic Confocal Laser Microendoscopy (CLM) and radiotracing, we describe the effects of charge, size, cholesterol content and lipid saturation on the ocular and corporal distribution of liposome nanocarriers in live mouse models. Positively charged or large (>250 nm) liposomes exhibit sustained ocular residence times in and around the injection site; cholesterol loading slows down this clearance, whereas lipid saturation accelerates clearance. Neutral, negatively charged, or smaller sized liposomes distribute to the limbus, rich in stem cells and blood capillaries. Differential lymphatic and systemic clearance from the eye to corporeal tissues was also observed across formulations. These results demonstrate the need to optimize liposome design for control over temporal and spatial nanocarrier bioavailability and clearance from the eye for improved efficacy and safety of ocular therapeutics.

Biomimetic vs. Direct Approach to Deposit Hydroxyapatite on the Surface of Low Melting Point Polymers for Tissue Engineering.

Polymers are widely used in many applications in the field of biomedical engineering. Among eclectic selections of polymers, those with low melting temperature (Tm < 200 °C), such as poly(methyl methacrylate), poly(lactic-co-glycolic acid), or polyethylene, are often used in bone, dental, maxillofacial, and corneal tissue engineering as substrates or scaffolds. These polymers, however, are bioinert, have a lack of reactive surface functional groups, and have poor wettability, affecting their ability to promote cellular functions and biointegration with the surrounding tissue. Improving the biointegration can be achieved by depositing hydroxyapatite (HAp) on the polymeric substrates. Conventional thermal spray and vapor phase coating, including the Food and Drug Administration (FDA)-approved plasma spray technique, is not suitable for application on the low Tm polymers due to the high processing temperature, reaching more than 1000 °C. Two non-thermal HAp coating approaches have been described in the literature, namely, the biomimetic deposition and direct nanoparticle immobilization techniques. In the current review, we elaborate on the unique features of each technique, followed by discussing the advantages and disadvantages of each technique to help readers decide on which method is more suitable for their intended applications. Finally, the future perspectives of the non-thermal HAp coating are given in the conclusion.

Potential of subconjunctival aflibercept in treating choroidal neovascularization.

The study aimed to evaluate the intraocular pharmacokinetics and efficacy of aflibercept after subconjunctival injection in animal models for treating choroidal neovascularization (CNV) associated with Age-Related Macular Degeneration (AMD). New Zealand albino rabbits received aflibercept (2000 µg/50 µl) in one eye, and the other eye was used as control. At 7, 14, 21 and 28 days, the animals were sacrificed to dissect the ocular tissues, and serum was collected at 1hr, 3 h, 1, 7, 14, 21 and 28 days. The concentration of aflibercept in various ocular tissues and serum were measured using the immunoassay technique. The concentration maximum (Cmax) at the Retinal Pigment Epithelium (RPE)-choroid complex and retina in treated eyes was 261.55 and 33.83 ng/gm, respectively. The area under the curve (AUC0-last) for RPE-Choroid and retina were 2094.02 and 290.33 days. ng/gm respectively. The time maximum (Tmax) for the ocular tissues was reached on day 7. In the vitreous humour, a lower level of aflibercept was retrieved. The Cmax (1766.84 ng/mL) in the serum was reached on day 1, followed by a decline in the concentration till the end of the study period. In treated eyes, the levels of aflibercept in most of the ocular tissues were maintained for at least 21 days above the invitro IC50 concentration. The results of the efficacy study show that subconjunctival aflibercept could reach the therapeutic target to inhibit CNV. The subconjunctival aflibercept could be a less invasive route for treating CNV with AMD.

Interpenetrating Network of Alginate-Human Adipose Extracellular Matrix Hydrogel for Islet Cells Encapsulation.

Transplantation of microencapsulated islet cells holds great potential for the treatment of type 1 diabetes mellitus. However, its clinical translation is hampered by the peri-transplantation loss of islet viability and functionality in the microcapsules. In this work, a novel islet cells biomimetic microencapsulant material that is based on the interpenetrating networks of alginate and extracellular matrix (ECM) hydrogel composite (AEC) is presented. The ECM component is derived from human lipoaspirate. In situ encapsulation of pancreatic ß islet cells (MIN6 ß-cells) can be achieved via ionotropic gelation of the alginate matrix and thermal-induced gelation of the pepsin-solubilized ECM pre-gel. Due to the enhanced cell-matrix interaction, islets encapsulated within the AEC microcapsules (≈640 µm) display sevenfold increase in cell growth over 1 week of culture and characteristic glucose-stimulated insulin response in vitro. The results show that the AEC microcapsule is a potent platform to bioaugment the performance of islet cells.

Microfluidic-directed self-assembly of liposomes: Role of interdigitation.

Microfluidics has been used to process self-assembling liposomal systems that are commonly considered for drug delivery applications. However, it has been found that the parameters of the process are not universally suited for all lipid types. We hypothesize here that size aggregation and instability of microfluidic liposomes are a direct consequence of the presence of interdigitation in these liposomes. Interdigitation refers to the phenomenon where two opposing leaflets of a bilayer interpenetrate into one another and form a single layer. When this happens, aggregation results as the single layer is not thermodynamically stable. Such interdigitation can be induced by pressure, chemicals or by the type of lipid structure. In this study, we systematically investigate the role of lipid composition on membrane interdigitation in order to understand the dependency of lipid interdigitation on liposome formation by microfluidics. By doing so, we use nano DSC and SAXS to probe the extent of lipid interdigitation by measuring the changes in thermodynamics and membrane thickness of the lipid bilayers. Our results show that microfluidic-fabricated liposomes undergo chemical interdigitation in the presence of ethanol, in particular saturated 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). Strategies to prevent interdigitation is to either remove ethanol above the lipid's main transition temperature (Tm), preventing the formation of interdigitated structures and subsequent aggregated states or by the incorporation of the inhibiting additives, such as cholesterol.

Liposomal Nanotherapy for Treatment of Atherosclerosis.

Atherosclerosis is a chronic disease that can lead to life-threatening events such as myocardial infarction and stroke, is characterized by the build-up of lipids and immune cells within the arterial wall. It is understood that inflammation is a hallmark of atherosclerosis and can be a target for therapy. In support of this concept, an injectable nanoliposomal formulation encapsulating fluocinolone acetonide (FA), a corticosteroid, is developed that allows for drug delivery to atherosclerotic plaques while reducing the systemic exposure to off-target tissues. In this study, FA is successfully incorporated into liposomal nanocarriers of around 100 nm in size with loading efficiency of 90% and the formulation exhibits sustained release up to 25 d. The anti-inflammatory effect and cholesterol efflux capability of FA-liposomes are demonstrated in vitro. In vivo studies carried out with an apolipoprotein E-knockout (Apoe-/- ) mouse model of atherosclerosis show accumulation of liposomes in atherosclerotic plaques, colocalization with plaque macrophages and anti-atherogenic effect over 3 weeks of treatment. This FA-liposomal-based nanocarrier represents a novel potent nanotherapeutic option for atherosclerosis.

Targeting efficiency of nanoliposomes on atherosclerotic foam cells: polyethylene glycol-to-ligand ratio effects.

BACKGROUND: Nanoparticles that actively target tissues, with ligands attached at the extremity of polyethylene glycol (PEG) spacer, are a promising strategy to enhance target cell specificity and internalization. However, the interplay between the targeting ligands and the adjacent ligand-free PEG remains poorly understood. RESEARCH DESIGN AND METHODS: Experimentally, liposomes containing active folate ligands were firstly formulated and the optimum amount of ligand that yields the highest foam cell uptake was determined. Subsequently, ligand-free PEG was incorporated, and the effects of PEG lengths and concentrations on foam cell uptake were evaluated after the nanoparticles were incubated in human serum for 90 min. RESULTS: It was demonstrated that the targeting efficiency progressively decreased and was eventually annulled as PEG-to-ligand ratio was increased, with loss of targeting effect occurring at PEG-to-ligand ratio of >2 for PEG 750, >0.5 for PEG 2000 and <0.5 for PEG 5000. CONCLUSIONS: This work demonstrates that PEG-to-ligand ratio and serum coating on nanoparticle surface are both important features to be considered in the design of active targeting nanocarriers. This work also supports the development of novel active targeting nanotherapies for atherosclerosis.

Surface modification of corneal prosthesis with nano-hydroxyapatite to enhance in vivo biointegration.

The majority of clinical corneal prostheses (KPros) adopt a core-skirt configuration. This configuration is favored owing to the optic core (generally a cylindrical, acrylic-based material, such as PMMA), that not only provides a clear window for the patients' vision, but also confers resistance to biodegradability. The surrounding skirt (typically a biological material, such as corneal tissue) allows for host tissue integration. However, due to poor biointegration between the dissimilar core and skirt materials, it results in a weak adhesion at the interface, giving rise to clinical complications, such as bacterial infections in the tissue-PMMA interface and device extrusion. Here, we physically immobilized nano-hydroxyapatite (nHAp) on a PMMA cylinder via a dip-coating technique, to create a bioactive surface that improved biointegration in vivo. We established that the nHAp coating was safe and stable in the rabbit cornea over five weeks. More importantly, we found that apoptotic, wound healing and inflammatory responses to nHAp-coated PMMA were substantially milder than to non-coated PMMA. More mature collagen, similar to the non-operated cornea, was maintained in the corneal stroma adjacent to the nHAp-coated implant edge. However, around the non-coated cylinder, an abundant new and loose connective tissue formed, similar to bone tissue response to bioinert scaffolds. As a result of superior biointegration, tissue adhesion with nHAp-coated PMMA cylinders was also significantly enhanced compared to non-coated cylinders. This study set a precedent for the future application of the nHAp coating on clinical KPros. STATEMENT OF SIGNIFICANCE: Currently, all clinical corneal prostheses utilize as-manufactured, non-surface modified PMMA optic cylinder. The bioinert cylinder, however, has poor biointegration and adhesion with the surrounding biological tissue, which can give rise to postoperative complications, such as microbial invasion in the tissue-PMMA loose interface and PMMA optic cylinder extrusion. In the current study, we showed that surface modification of the PMMA cylinder with bioactive nano-hydroxyapatite (nHAp) significantly enhanced its biointegration with corneal stromal tissue in vivo. The superior biointegration of the nHAp-coated PMMA was signified by a more attenuated corneal wound healing, inflammatory and fibrotic response, and better tissue apposition, as well as a significantly improved corneal stromal tissue adhesion when compared to the non-coated PMMA.

Matching Static and Dynamic Compliance of Small-Diameter Arteries, with Poly(lactide-co-caprolactone) Copolymers: In Vitro and In Vivo Studies.

Mechanical mismatch between vascular grafts and blood vessels is a major cause of smaller diameter vascular graft failure. To minimize this mismatch, several poly-l-lactide-co-ε-caprolactone (PLC) copolymers are evaluated as candidate materials to fabricate a small diameter graft. Using these materials, tubular prostheses of 4 mm inner diameter are fabricated by dip-coating. In vitro static and dynamic compliance tests are conducted, using custom-built apparatus featuring a closed flow system with water at 37 °C. Grafts of PLC monomer ratio of 50:50 are the most compliant (1.56% ± 0.31∙mm Hg-2 ), close to that of porcine aortic branch arteries (1.56% ± 0.43∙mm Hg-2 ), but underwent high continuous dilatation (87 µm min-1 ). Better matching is achieved by optimizing the thickness of a tubular conduit made from 70:30 PLC grafts. In vivo implantation and function of a PLC 70:30 conduit of 150 µm wall-thickness (WT) are tested as a rabbit aorta bypass. An implanted 150 µm WT PLC 70:30 prosthesis is observed over 3 h. The recorded angiogram shows continuous blood flow, no aneurysmal dilatation, leaks, or acute thrombosis during the in vivo test, indicating the potential for clinical applications.

Nanolipogels as a cell-mimicking platform for controlled release of biomacromolecules.

We present studies of protein (insulin) efflux rates from nano-sized core-shell systems with a gelled core and a lipid bilayer (nanolipogels). The efflux control mechanism is the manipulation of mesh size, and we show that diffusion control via crosslinking is the dominant mechanism for efflux control. The concept is inspired by the macromolecular crowding effect in human cells, which may be considered as a physical network of undefined mesh size. Our bio-inspired system is made of chemically crosslinked water-swellable poly(ethylene glycol) diacrylate cores, whose mesh size can be manipulated to yield a quantifiable crowding effect that then leads to predictable release rates for biomacromolecules.

Adventitial injection delivery of nano-encapsulated sirolimus (Nanolimus) to injury-induced porcine femoral vessels to reduce luminal restenosis.

Endovascular therapy in peripheral intervention has grown exponentially in the past decade, but the issue of high restenosis rates in lower extremity arteries still persist. While drug-coated balloons (DCB) have been the device of choice, recent controversary regarding the long-term safety of paclitaxel have raised concern over current DCBs. In our study, we proposed that the direct injection of a sirolimus nanoliposomal formulation (Nanolimus) using a infusion catheter can attenuate inflammation response in injured vessels. In vitro characterization showed retention of the nanoliposomes size and detectable drug amount up to 336 days in storage. For in vivo study, four female, mixed breed swines were subjected to balloon injury of the femoral arteries before treatment with either injection of saline (n = 4) or Nanolimus (n = 12) using the Bullfrog catheter. Pharmacokinetic analysis demonstrated sustained sirolimus release in the arteries and undetectable systemic drug level at 28 days. Arteries treated with Nanolimus showed significant reduction in neointima area (0.2 ± 0.3 mm2 vs 2.0 ± 1.2 mm2, p < 0.01) and luminal stenosis (14.2 ± 7.2% vs. 67.7 ± 24.8%, p < 0.01) compared to controls. In summary, adventitial delivery of sirolimus using an infusion catheter is a feasible and safe method to reduce vascular restenosis.