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.

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.

Recent Advances in Chitosan-Based Carriers for Gene Delivery.

Approximately 4000 diseases are associated with malfunctioning genes in a particular cell type. Gene-based therapy provides a platform to modify the disease-causing genes expression at the cellular level to treat pathological conditions. However, gene delivery is challenging as these therapeutic genes need to overcome several physiological and intracellular barriers in order, to reach the target cells. Over the years, efforts have been dedicated to develop efficient gene delivery vectors to overcome these systemic barriers. Chitosan, a versatile polysaccharide, is an attractive non-viral vector material for gene delivery mainly due to its cationic nature, biodegradability and biocompatibility. The present review discusses the design factors that are critical for efficient gene delivery/transfection and highlights the recent progress of gene therapy using chitosan-based carriers.

Water-Responsive Shape Recovery Induced Buckling in Biodegradable Photo-Cross-Linked Poly(ethylene glycol) (PEG) Hydrogel.

The phenomenon of recovering the permanent shape from a severely deformed temporary shape, but only in the presence of the right stimulus, is known as the shape memory effect (SME). Materials with such an interesting effect are known as shape memory materials (SMMs). Typical stimuli to trigger shape recovery include temperature (heating or cooling), chemical (including water/moisture and pH value), and light. As a SMM is able not only to maintain the temporary shape but also to respond to the right stimulus when it is applied, via shape-shifting, a seamless integration of sensing and actuation functions is achieved within one single piece of material. Hydrogels are defined by their ability to absorb a large amount of water (from 10-20% up to thousands of times their dry weight), which results in significant swelling. On the other hand, dry hydrogels indeed belong to polymers, so they exhibit heat- and chemoresponsive SMEs as most polymers do. While heat-responsive SMEs have been spotted in a handful of wet hydrogels, so far, most dry hydrogels evince the heat and water (moisture)-responsive SMEs. Since water is one of the major components in living biological systems, water-responsive SMMs hold great potential for various implantable applications, including wound healing, intravascular devices, soft tissue reconstruction, and controlled drug delivery. This provides motivation to combine water-activated SMEs and swelling in hydrogels together to enhance the performance. In many applications, such as vascular occlusion via minimally invasive surgery for liver cancer treatment, the operation time (for both start and finish) is required to be well controlled. Due to the gradual and slow manner of water absorption for water-activated SMEs and swelling in hydrogels, even a combination of both effects encounters many difficulties to meet the timerequirements in real procedures of vascular occlusion. Recently, we have reported a bioabsorbable radiopaque water-responsive shape memory embolization plug for temporary vascular occlusion. The plug consists of a composite with a poly(dl-lactide-co-glycolide) (PLGA) core (loaded with radiopaque filler) and cross-linked poly(ethylene glycol) (PEG) hydrogel outer layer. The device can be activated by body fluid (or water) after about 2 min of immersion in water. The whole occlusion process is completed within a few dozens of seconds. The underlying mechanism is water-responsive shape recovery induced buckling, which occurs in an expeditious manner within a short time period and does not require complete hydration of the whole hydrogel. In this paper, we experimentally and analytically investigate the water-activated shape recovery induced buckling in this biodegradable PEG hydrogel to understand the fundamentals in precisely controlling the buckling time. The molecular mechanism responsible for the water-induced SME in PEG hydrogel is also elucidated. The original diameter and amount of prestretching are identified as two influential parameters to tailor the buckling time between 1 and 4 min as confirmed by both experiments and simulation. The phenomenon reported here, chemically induced buckling via a combination of the SME and swelling, is generic, and the study reported here should be applicable to other water- and non-water-responsive gels.

Bioprinted gelatin hydrogel platform promotes smooth muscle cell contractile phenotype maintenance.

Three dimensional (3D) bioprinting has been proposed as a method for fabricating tissue engineered small diameter vascular prostheses. This technique not only involves constructing the structural features to obtain a desired pattern but the morphology of the pattern may also be used to influence the behavior of seeded cells. Herein, we 3D bioprinted a gelatin hydrogel microchannel construct to promote and preserve the contractile phenotype of vascular smooth muscle cells (vSMCs), which is crucial for vasoresponsiveness. The microchanneled surface of a gelatin hydrogel facilitated vSMC attachment and an elongated alignment along the microchannel direction. The cells displayed distinct F-actin anisotropy in the direction of the channel. The vSMC contractile phenotype was confirmed by the positive detection of contractile marker gene proteins (α-smooth muscle actin (α-SMA) and smooth muscle-myosin heavy chain (SM-MHC)). Having demonstrated the effectiveness of the hydrogel channels bioprinted on a film, the bioprinting was applied radially to the surface of a 3D tubular construct by integrating a rotating mandrel into the 3D bioprinter. The hydrogel microchannels printed on the 3D tubular vascular construct also orientated the vSMCs and strongly promoted the contractile phenotype. Together, our study demonstrated that microchannels bioprinted using a transglutaminase crosslinked gelatin hydrogel, could successfully promote and preserve vSMC contractile phenotype. Furthermore, the hydrogel bioink could be retained on the surface of a rotating polymer tube to print radial cell guiding channels onto a vascular graft construct.

Topical Delivery of Senicapoc Nanoliposomal Formulation for Ocular Surface Treatments.

Topical ophthalmologic treatments have been facing great challenges with main limitations of low drug bioavailability, due to highly integrative defense mechanisms of the eye. This study rationally devised strategies to increase drug bioavailability by increasing ocular surface residence time of drug-loaded nanoliposomes dispersed within thermo-sensitive hydrogels (Pluronic F-127). Alternatively, we utilized sub-conjunctival injections as a depot technique to localize nanoliposomes. Senicapoc was encapsulated and sustainably released from free nanoliposomes and hydrogels formulations in vitro. Residence time increased up to 12-fold (60 min) with 24% hydrogel formulations, as compared to 5 min for free liposomes, which was observed in the eyes of Sprague-Dawley rats using fluorescence measurements. Pharmacokinetic results obtained from flushed tears, also showed that the hydrogels had greater drug retention capabilities to that of topical viscous solutions for up to 60 min. Senicapoc also remained quantifiable within sub-conjunctival tissues for up to 24 h post-injection.

Bioprinting and Differentiation of Stem Cells.

The 3D bioprinting of stem cells directly into scaffolds offers great potential for the development of regenerative therapies; in particular for the fabrication of organ and tissue substitutes. For this to be achieved; the lineage fate of bioprinted stem cell must be controllable. Bioprinting can be neutral; allowing culture conditions to trigger differentiation or alternatively; the technique can be designed to be stimulatory. Such factors as the particular bioprinting technique; bioink polymers; polymer cross-linking mechanism; bioink additives; and mechanical properties are considered. In addition; it is discussed that the stimulation of stem cell differentiation by bioprinting may lead to the remodeling and modification of the scaffold over time matching the concept of 4D bioprinting. The ability to tune bioprinting properties as an approach to fabricate stem cell bearing scaffolds and to also harness the benefits of the cells multipotency is of considerable relevance to the field of biomaterials and bioengineering.

Printing cell-laden gelatin constructs by free-form fabrication and enzymatic protein crosslinking.

Considerable interest has arisen in precision fabrication of cell bearing scaffolds and structures by free form fabrication. Gelatin is an ideal material for creating cell entrapping constructs, yet its application in free form fabrication remains challenging. We demonstrate the use of gelatin, crosslinked with microbial transglutaminase (mTgase), as a material to print cell bearing hydrogels for both 2-dimensional (2-D) precision patterns and 3-dimensional (3-D) constructs. The precision patterning was attained with 3 % gelatin and 2 % high molecular weight poly (ethylene oxide) (PEO) whereas 3-D constructs were obtained using a 5 % gelatin solution. These hydrogels, referred to as "bioinks" supported entrapped cell growth, allowing cell spreading and proliferation for both HEK293 cells and Human Umbilical Vein Endothelial Cells (HUVECs). These bioinks were shown to be dispensable by robotic precision, forming patterns and constructs that were insoluble and of suitable stiffness to endure post gelation handling. The two bioinks were further characterized for fabrication parameters and mechanical properties.

A fully biodegradable patent ductus arteriosus occlude.

The purpose of this study was to develop a fully degradable occluder for the closure of PDA, which can be deployed percutaneously. The blends of biodegradable poly(ε-caprolactone) and poly(L-lactide-co-ε-caprolactone) with various compositions were studied as the potential material. The mechanical properties, i.e. elastic modulus and strain recovery, of the blends could be largely tailored by changing the continuous phase component. Moreover, the suitable blends were selected to fabricate a prototype and its in vitro biodegradation rate and blood compatibility, was evaluated. The current results indicate that no adverse effect on the platelet and leukocyte components of the blood. Biocompatibility implantation studies of the device showed acceptable tissue response. Finally, an artificial PDA conduit was created in a pig, and the device deployment was tested from a sheath: the device recovered within 2-3 min of unsheathing and fully sealed the conduit.

Layer-by-layer nanoparticles as an efficient siRNA delivery vehicle for SPARC silencing.

Efficient and safe delivery systems for siRNA therapeutics remain a challenge. Elevated secreted protein, acidic, and rich in cysteine (SPARC) protein expression is associated with tissue scarring and fibrosis. Here we investigate the feasibility of encapsulating SPARC-siRNA in the bilayers of layer-by-layer (LbL) nanoparticles (NPs) with poly(L-arginine) (ARG) and dextran (DXS) as polyelectrolytes. Cellular binding and uptake of LbL NPs as well as siRNA delivery were studied in FibroGRO cells. siGLO-siRNA and SPARC-siRNA were efficiently coated onto hydroxyapatite nanoparticles. The multilayered NPs were characterized with regard to particle size, zeta potential and surface morphology using dynamic light scattering and transmission electron microscopy. The SPARC-gene silencing and mRNA levels were analyzed using ChemiDOC western blot technique and RT-PCR. The multilayer SPARC-siRNA incorporated nanoparticles are about 200 nm in diameter and are efficiently internalized into FibroGRO cells. Their intracellular fate was also followed by tagging with suitable reporter siRNA as well as with lysotracker dye; confocal microscopy clearly indicates endosomal escape of the particles. Significant (60%) SPARC-gene knock down was achieved by using 0.4 pmole siRNA/µg of LbL NPs in FibroGRO cells and the relative expression of SPARC mRNA reduced significantly (60%) against untreated cells. The cytotoxicity as evaluated by xCelligence real-time cell proliferation and MTT cell assay, indicated that the SPARC-siRNA-loaded LbL NPs are non-toxic. In conclusion, the LbL NP system described provides a promising, safe and efficient delivery platform as a non-viral vector for siRNA delivery that uses biopolymers to enhance the gene knock down efficiency for the development of siRNA therapeutics.

Has nanomedicine lived up to its promise?

Nanomedicine for diagnosis and treatment of disease, as a discipline has been around for several years, with the first nanotherapeutic product being approved in 1995. Worldwide its importance was recognized with the setting up of several nanomedicine centres in 2004-2006. Many of these centres were set up to accelerate the speed of translation of the research. In this article we review, with a broad brush, the progress made in the last 15 years, and examine whether the translation efforts have been successful, and also evaluate whether such successes have changed the medical landscape. Possible reasons for the relatively long time to commercialization for nanomedicine products are also explored.

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.

Layer-by-layer polyelectrolyte-polyester hybrid microcapsules for encapsulation and delivery of hydrophobic drugs.

A two-step process is developed to form layer-by-layer (LbL) polyelectrolyte microcapsules, which are able to encapsulate and deliver hydrophobic drugs. Spherical porous calcium carbonate (CaCO3) microparticles were used as templates and coated with a poly(lactic acid-co-glycolic acid) (PLGA) layer containing hydrophobic compounds via an in situ precipitation gelling process. PLGA layers that precipitated from N-methyl-2-pyrrolidone (NMP) had a lower loading and smoother surface than those precipitated from acetone. The difference may be due to different viscosities and solvent exchange dynamics. In the second step, the successful coating of multilayer polyelectrolytes poly(allylamine hydrochloride) (PAH) and poly(styrene sulfonate) (PSS) onto the PLGA coated CaCO3 microparticles was confirmed with AFM and ζ-potential studies. The release of a model hydrophobic drug, ibuprofen, from these hybrid microcapsules with different numbers of PAH/PSS layers was investigated. It was found that the release of ibuprofen decreases with increasing layer numbers demonstrating the possibility to control the release of ibuprofen with these novel hybrid microcapsules. Besides loading of hydrophobic drugs, the interior of these microcapsules can also be loaded with hydrophilic compounds and functional nanoparticles as demonstrated by loading with Fe3O4 nanoparticles, forming magnetically responsive dual drug releasing carriers.

Formation of a nano-pattering NiTi surface with Ni-depleted superficial layer to promote corrosion resistance and endothelial cell-material interaction.

Zirconium ion implantation was performed on NiTi alloy to suppress Ni ion release as well as to improve corrosion resistance and cell-material interaction. A thicker Ni-depleted nano-scale composite layer formed after Zr implantation and the corrosion resistance was evidently increased in aspects of increased E(br) - E(corr) (difference between corrosion potential and breakdown potential) and decreased corrosion current density. 2.5/2 NiTi sample possessed the highest E(br) - E(corr), more than 500 mV higher than that of untreated NiTi, suggesting a significant improvement on pitting corrosion resistance. Ni ion release rate of Zr-NiTi was decreased due to the depletion of Ni in the superficial surface layer and the diffusion resistance effect of the ZrO(2)/TiO(2) nano-film. Increased surface wettability induced by increased surface roughness was obtained after Zr implantation. Zr-NiTi samples were found to be favorable to endothelial cells (ECs) proliferation, especially after 5 and 7 days culture.

Collagen-cellulose composite thin films that mimic soft-tissue and allow stem-cell orientation.

Mechanical properties of collagen films are less than ideal for biomaterial development towards musculoskeletal repair or cardiovascular applications. Herein, we present a collagen-cellulose composite film (CCCF) compared against swine small intestine submucosa in regards to mechanical properties, cell growth, and histological analysis. CCCF was additionally characterized by FE-SEM, NMR, mass spectrometry, and Raman Microscopy to elucidate its physical structure, collagen-cellulose composition, and structure activity relationships. Mechanical properties of the CCCF were tested in both wet and dry environments, with anisotropic stress-strain curves that mimicked soft-tissue. Mesenchymal stem cells, human umbilical vein endothelial cells, and human coronary artery smooth muscle cells were able to proliferate on the collagen films with specific cell orientation. Mesenchymal stem cells had a higher proliferation index and were able to infiltrate CCCF to a higher degree than small intestine submucosa. With the underlying biological properties, we present a collagen-cellulose composite film towards forthcoming biomaterial-related applications.

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.

Surface functionalization of nanoparticles to control cell interactions and drug release.

Nanoparticles made from poly(dl-lactide-co-glycolide) (PLGA) are used to deliver a wide range of bioactive molecules, due to their biocompatibility and biodegradability. This study investigates the surface modification of PLGA nanoparticles via the layer-by-layer (LbL) deposition of polyelectrolytes, and the effects of these coatings on the release behavior, cytotoxicity, hemolytic activity, and cellular uptake efficiency. PLGA nanoparticles are modified via LbL adsorption of two polyelectrolyte pairs: 1) poly(allylamine hydrochloride) (PAH) and poly(styrene sulfonate) (PSS) and 2) poly(L-lysine hydrobromide) (PLL) and dextran sulfate (DES). It is demonstrated that both PAH/PSS and PLL/DES coatings suppress the burst release usually observed for unmodified PLGA nanoparticles and that the release behavior can be adjusted by changing the layer numbers, layer materials, or by crosslinking the layer constituents. Neither bare nor polyelectrolyte-modified PLGA nanoparticles show any signs of cytotoxicity. However, nanoparticles with a positively charged polyelectrolyte as the outermost layer induce hemolysis, whereas uncoated particles or particles with a negatively charged polyelectrolyte as the outermost layer show no hemolytic activity. Furthermore, particles with either PAH or PLL as the outermost layer also demonstrate a higher uptake efficiency by L929 fibroblast cells, due to a higher cell-particle affinity. This study suggests that LbL coating of PLGA nanoparticles can control the release behavior of bioactive molecules as well as the surface activity, therefore providing a promising strategy to enhance the efficiency of nanoparticulate drug-delivery systems.

Engineering of erythrocyte-based drug carriers: control of protein release and bioactivity.

This work reports the fabrication of layer-by-layer (LbL) polyelectrolyte coated erythrocyte carriers that provide a simple means for controlling the burst and subsequent release of lysozyme. Erythrocytes were loaded with RITC-lysozyme as model compound via the hypotonic dialysis method. An encapsulation efficiency of 41.6% and a loading amount of 12.7 pg/cell was achieved. It is demonstrated that these carriers maintain their shape and integrity similar to natural erythrocytes after the encapsulation procedures, and achieve a uniform distribution of the encapsulated lysozyme. The erythrocyte carriers were fixed with glutaraldehyde and then successfully coated with biocompatible polyelectrolytes, poly-L: -lysine hydrobromide and dextran sulfate, using the LbL method. It is demonstrated that the release profile of the encapsulated macromolecule can be regulated by adjusting the number of polyelectrolyte layers. Furthermore by adjusting the concentrations of the cross linking agent the activity of the encapsulated lysozyme can be well preserved. These core-shell microcapsules, consisting of erythrocytes loaded with bioactive substances and coated with a polyelectrolyte multilayer shell, hold promise for a new type of biocompatible and biodegradable drug delivery system.