Chirality-Dependent Angiogenic Activity of MoS2 Quantum Dots toward Regulatable Tissue Regeneration.

Despite great advances in understanding the biological behaviors of chiral materials, the effect of chirality-configured nanoparticles on tissue regeneration-related biological processes remains poorly understood. Herein, the chirality of MoS2 quantum dots (QDs) is tailored by functionalization with l-/d-penicillamine, and the profound chiral effects of MoS2 QDs on cellular activities, angiogenesis, and tissue regeneration are thoroughly investigated. Specifically, d-MoS2 QDs show a positive effect in promoting the growth, proliferation, and migration of human umbilical vein endothelial cells. The expression of vascular endothelial growth factor (VEGF), endothelial nitric oxide synthase (eNOS), and fibroblast growth factor (FGF) in d-MoS2 QDs group is substantially up-regulated, resulting in enhanced tube formation activity. This distinct phenomenon is largely due to the higher internalization efficiency of d-MoS2 QDs than l-MoS2 QDs and chirality-dependent nano-bio interactions. In vivo angiogenic assay shows the expression level of angiogenic markers in newly-formed skin tissues of d-MoS2 QDs group is higher than that in l-MoS2 QDs group, leading to an accelerated re-epithelialization and improved skin regeneration. The findings of chirality-dependent angiogenesis activity of MoS2 QDs provide new insights into the biological activity of MoS2 nanomaterials, which also opens up a new path to the rational design of chiral nanomaterials for tissue regeneration application.

In Vivo Laser-Induced Vasoactive Microenvironmental Setting via a Stimuli-Responsive Microstructured Depot.

A stimuli-responsive polymeric three-dimensional microstructured film (PTMF) is a 3D structure with an array of sealed chambers on its external surface. In this work, we demonstrate the use of PTMF as a laser-triggered stimulus-response system for local in vivo targeted blood vessels stimulation by vasoactive substances. The native vascular networks of the mouse mesentery were used as model tissues. Epinephrine and KCl were used as vasoactive agents that were sealed into individual chambers upon precipitation in the amount of pictograms. We demonstrated the method for non-damaged one-by-one chamber activation using a focused 532 nm laser light passed through biological tissues. To avoid laser-induced photothermal damage to biological tissues, the PTMF was functionalized with Nile Red dye, which effectively absorbs laser light. Chemically stimulated blood vessel fluctuations were analyzed using digital image processing methods. Hemodynamics changes were measured and visualized using the particle image velocimetry approach.

Synthesis of Novel Antimicrobial CHX-CaCl2 Coatings on Maxillofacial Fixatures for Infection Prevention.

Maxillofacial surgery placement of fixatures (Leonard Buttons, LB) at close proximity to surgical incisions provides a potential reservoir as a secondary local factor to advanced periodontal disease, with bacterial formation around failed fixatures implicating plaque. To address infection rates, we aimed to surface coat LB and Titanium (Ti) discs using a novel form of chlorhexidine (CHX), CHX-CaCl2 and 0.2% CHX digluconate mouthwash as a comparison. CHX-CaCl2 coated, double-coated and mouthwash coated LB and Ti discs were transferred to 1 mL artificial saliva (AS) at specified time points, and UV-Visible spectroscopy (254 nm) was used to measure CHX release. The zone of inhibition (ZOI) was measured using collected aliquots against bacterial strains. Specimens were characterized using Energy Dispersive X-ray Spectroscopy (EDS), X-ray Diffraction (XRD) and Scanning Electron Microscopy (SEM). SEM displayed copious dendritic crystals on LB/ Ti disc surfaces. Drug release from double-coated CHX-CaCl2 was 14 days (Ti discs) and 6 days (LB) above MIC, compared to the comparison group (20 min). The ZOI for the CHX-CaCl2 coated groups was significantly different within groups (p < 0.05). CHX-CaCl2 surface crystallization is a new drug technology for controlled and sustained CHX release; its antibacterial effectiveness makes this drug an ideal adjunct following clinical and surgical procedures to maintain oral hygiene and prevent surgical site infections.

Labeling and Tracking of Individual Human Mesenchymal Stromal Cells Using Photoconvertible Fluorescent Microcapsules.

The behavior and migration of human mesenchymal stromal cells (hMSCs) are focal points of research in the biomedical field. One of the major aspects is potential therapy using hMCS, but at present, the safety of their use is still controversial owing to limited data on changes that occur with hMSCs in the long term. Fluorescent photoconvertible proteins are intensively used today as "gold standard" to mark the individual cells and study single-cell interactions, migration processes, and the formation of pure lines. A crucial disadvantage of this method is the need for genetic modification of the primary culture, which casts doubt on the possibility of exploring the resulting clones in personalized medicine. Here we present a new approach for labeling and tracking hMSCs without genetic modification based on the application of cell-internalizable photoconvertible polyelectrolyte microcapsules (size: 2.6 ± 0.5 µm). These capsules were loaded with rhodamine B, and after thermal treatment, exhibited fluorescent photoconversion properties. Photoconvertible capsules demonstrated low cytotoxicity, did not affect the immunophenotype of the hMSCs, and maintained a high level of fluorescent signal for at least seven days. The developed approach was tested for cell tracking for four days and made it possible to trace the destiny of daughter cells without the need for additional labeling.

Targeted Therapy for Glomerulonephritis Using Arterial Delivery of Encapsulated Etanercept.

Complex immunosuppressive therapy is prescribed in medical practice to patients with glomerulonephritis to help them overcome symptoms and prevent chronic renal failure. Such an approach requires long-term systemic administration of strong medications, which causes severe side effects. This work shows the efficiency of polymer capsule accumulation (2.8 ± 0.4 µm) containing labeled etanercept (100 µg per dose) in the kidneys of mice. The comparison of injection into the renal artery and tail vein shows the significant superiority of the intra-arterial administration strategy. The etanercept retention rate of 18% and 8% ID in kidneys was found 1 min and 1 h after injection, respectively. The capsules were predominantly localized in the glomeruli after injection in mice using a model of acute glomerulonephritis. Histological analysis confirmed a significant therapeutic effect only in animals with intra-arterial administration of microcapsules with etanercept. The proposed strategy combines endovascular surgery and the use of polymer microcapsules containing a high molecular weight drug that can be successfully applied to treat a wide range of kidney diseases associated with glomerular pathology.

Microcapsule-Based Dose-Dependent Regulation of the Lifespan and Behavior of Adipose-Derived MSCs as a Cell-Mediated Delivery System: In Vitro Study.

The development of "biohybrid" drug delivery systems (DDS) based on mesenchymal stem/stromal cells (MSCs) is an important focus of current biotechnology research, particularly in the areas of oncotheranostics, regenerative medicine, and tissue bioengineering. However, the behavior of MSCs at sites of inflammation and tumor growth is relevant to potential tumor transformation, immunosuppression, the inhibition or stimulation of tumor growth, metastasis, and angiogenesis. Therefore, the concept was formulated to control the lifespan of MSCs for a specific time sufficient for drug delivery to the target tissue by varying the number of internalized microcontainers. The current study addressed the time-dependent in vitro assessment of the viability, migration, and division of human adipose-derived MSCs (hAMSCs) as a function of the dose of internalized polyelectrolyte microcapsules prepared using a layer-by-layer technique. Polystyrene sulfonate (PSS)­poly(allylamine hydrochloride) (PAH)-coated spherical micrometer-sized (diameter ~2−3 µm) vaterite (CaCO3) microcapsules (PAH-PSS)6 with the capping PSS layer were prepared after dissolution of the CaCO3 core template. The Cell-IQ phase contrast imaging results showed that hAMSCs internalized all (PAH-PSS)6 microcapsules saturating the intercellular medium (5−90 particles per cell). A strong (r > 0.7) linear dose-dependent and time-dependent (up to 8 days) regression was observed between the in vitro decrease in cell viability and the number of internalized microvesicles. The approximate time-to-complete-death of hAMSCs at different concentrations of microcapsules in culture was 428 h (1:5 ratio), 339 h (1:10), 252 h (1:20), 247 h (1:45), and 170 h (1:90 ratio). By varying the number of microcontainers loaded into the cells (from 1:10 to 1:90), a dose-dependent exponential decrease in both the movement rate and division rate of hAMSCs was observed. A real-time cell analysis (RTCA) of the effect of (PAH-PSS)6 microcapsules (from 1:5 to 1:20) on hAMSCs also showed a dose- and time-dependent decrease in cell longevity after a 50h study at ratios of 1:10 and 1:20. The incorporation of microcapsules (1:5, 1:20, and 1:45) resulted in a dose-dependent increase in 24−48 h secretion of GRO-α (CXCL1), MIF, and SDF-1α (CXCL12) chemokines in hAMSC culture. In turn, the normalization or inhibition of chemokine secretion occurred after 72 h, except for MIF levels below 5−20 microcapsules, which were internalized by MSCs. Thus, the proposed concept of controlling the lifespan of MSC-based DDS using a dose of internalized PAH-PSS microcapsules could be useful for biomedical applications. (PAH-PSS)6 microcapsule ratios of 1:5 and 1:10 have little effect on the lifespan of hAMSCs for a long time (up to 14−18 days), which can be recommended for regenerative therapy and tissue bioengineering associated with low oncological risk. The microcapsule ratios of 1:20 and 1:45 did not significantly restrict the migratory activity of hAMSCs-based DDS during the time interval required for tissue delivery (up to 4−5 days), followed by cell death after 10 days. Therefore, such doses of microcapsules can be used for hAMSC-based DDS in oncotheranostics.

Magnetic Hyperthermia Nanoarchitectonics via Iron Oxide Nanoparticles Stabilised by Oleic Acid: Anti-Tumour Efficiency and Safety Evaluation in Animals with Transplanted Carcinoma.

In this study, we developed iron oxide nanoparticles stabilised with oleic acid/sodium oleate that could exert therapeutic effects for curing tumours via magnetic hyperthermia. A suspension of iron oxide nanoparticles was produced and characterised. The toxicity of the synthesised composition was examined in vivo and found to be negligible. Histological examination showed a low local irritant effect and no effect on the morphology of the internal organs. The efficiency of magnetic hyperthermia for the treatment of transplanted Walker 256 carcinoma was evaluated. The tumour was infiltrated with the synthesised particles and then treated with an alternating magnetic field. The survival rate was 85% in the studied therapy group of seven animals, while in the control group (without treatment), all animals died. The physicochemical and pharmaceutical properties of the synthesised fluid and the therapeutic results, as seen in the in vivo experiments, provide insights into therapeutic hyperthermia using injected magnetite nanoparticles.

Biodegradable Nanocarriers Resembling Extracellular Vesicles Deliver Genetic Material with the Highest Efficiency to Various Cell Types.

Efficient delivery of genetic material to primary cells remains challenging. Here, efficient transfer of genetic material is presented using synthetic biodegradable nanocarriers, resembling extracellular vesicles in their biomechanical properties. This is based on two main technological achievements: generation of soft biodegradable polyelectrolyte capsules in nanosize and efficient application of the nanocapsules for co-transfer of different RNAs to tumor cell lines and primary cells, including hematopoietic progenitor cells and primary T cells. Near to 100% efficiency is reached using only 2.5 × 10-4 pmol of siRNA, and 1 × 10-3 nmol of mRNA per cell, which is several magnitude orders below the amounts reported for any of methods published so far. The data show that biodegradable nanocapsules represent a universal and highly efficient biomimetic platform for the transfer of genetic material with the utmost potential to revolutionize gene transfer technology in vitro and in vivo.

Microchamber arrays made of biodegradable polymers for enzymatic release of small hydrophilic cargos.

The encapsulation of small hydrophilic molecules and response to specific biological triggers in a controlled manner have become two of the significant challenges in biomedical research, in particular in the field of localized drug delivery and biosensing. This work reports the fabrication of free-standing microchamber array films made of biodegradable polymers for the encapsulation and enzymatically triggered release of small hydrophilic molecules. Polycaprolactone (PCL) microchamber arrays were demonstrated to fully biodegrade within 5 hours of exposure to lipase from Pseudomonas cepacia (lipase PS) at a concentration of 0.5 mg ml-1, with lower concentrations producing correspondingly longer degradation times. The gradual process of deterioration was real-time monitored utilising laser Fraunhofer diffraction patterns. Additionally, a small hydrophilic molecule, 5(6)-carboxyfluorescein (CF), was loaded into the PCL microchamber arrays in a dry state; however, the substantial permeability of the PCL film led to leakage of the dye molecules. Consequently, polylactic acid (PLA) was blended with PCL to reduce its permeability, enabling blended PCL-PLA (1 : 2 ratio correspondingly) microchamber arrays to trap the small hydrophilic molecule CF. PCL-PLA (1 : 2) microchamber arrays hold potential for controlled release under the catalysis of lipase within 26 hours. Additionally, it is calculated that approximately 11 pg of CF dye crystals was loaded into individual microchambers of 10 µm size, indicating that the microchamber array films could yield a highly efficient encapsulation.

Endovascular addressing improves the effectiveness of magnetic targeting of drug carrier. Comparison with the conventional administration method.

Many nanomedicine approaches are struggling to reach high enough effectiveness in delivery if applied systemically. The perspective is sought to explore the clinical practices currently used for localized treatment. In this study, we combine in vivo targeting of carriers sensitive to the external magnetic field with clinically used endovascular delivery to specific site. Fluorescent micron-size capsules made of biodegradable polymers and containing magnetite nanoparticles incorporated in the capsule wall were explored in vivo using Near-Infrared Fluorescence Live Imaging for Real-Time. Comparison of systemic (intravenous) and directed (intra-arterial) administration of the magnetic microcapsule targeting in the hindpaw vessels demonstrated that using femoral artery injection in combination with magnetic field exposure is 4 times more efficient than tail vein injection. Thus, endovascular targeting significantly improves the capabilities of nanoengineered drug delivery systems reducing the systemic side effects of therapy.

Multilayer Capsules Inside Biological Systems: State-of-the-Art and Open Challenges.

There are many reports about the interaction of multilayer capsules with biological systems in the literature. A majority of them are devoted to the in vitro study with two-dimensional cell cultures. Multilayer capsule fabrication had been under intensive investigation from 1990s and 2000s by Prof. Helmuth Möhwald, and many of his followers further developed their own research directions, focusing on capsule implementation in various fields of biology and medicine. The aim of this future article is to consistently consider the most recent advances in cell-capsule interactions for different biomedical applications, including functionalization of clinically relevant cells, nonviral gene delivery, magnetization of cells to control their movement, and in vivo drug delivery. Finally, the description and discussion of the new trends and perspectives for improved functionalities of capsules in design and functionalization of cell-assisted drug vehicles are the major topics of this work.

Influence of Heat Treatment on Loading of Polymeric Multilayer Microcapsules with Rhodamine B.

Layer-by-layer assembled polymeric multilayer capsules (PMC) of micrometer sizes are permeable for molecules below 1 KDa; therefore, the efficacy of such capsules in the delivery of low molecular weight water soluble bioactive compounds and drugs is frequently challenged. Thermally induced contraction of hollow PMC is explored here to enhance their loading efficacy with model compound, fluorescent rhodamine B (RhB). Four bilayered capsules obtained of poly(diallyldimethylammonium chloride)/polystyrene sulfonate ([PDADMAC/PSS]4 ) or poly-l-arginine/dextran sulfate ([PARG/DS]4 ) on sacrificial CaCO3 spherical microparticles are postloaded with RhB at ambient or elevated temperatures. The influence of heat on capsule loading is determined quantitatively by varying the amounts of capsules in the batch and keeping the concentration of RhB constant. The applied heat improves the loading efficacy of [PDADMAC/PSS]4 capsules at concentrations up to 2.25 × 109 capsules mL-1 , but has a reversed effect on [PARG/DS]4 capsules at all studied concentrations ((0-3.5) × 109 capsules mL-1 ).

Polyelectrolyte-Graphene Oxide Multilayer Composites for Array of Microchambers which are Mechanically Robust and Responsive to NIR Light.

Development of composite polymer/graphene oxide (GO) materials attracts significant attention due to their unique properties. In this work, highly ordered arrays of hollow microchambers made of composite polyelectrolyte/GO multilayers (PEGOMs) are successfully fabricated via layer-by-layer assembly on sacrificial or sustainable templates having imprinted patterns of microwells on their surface. Mechanical and optical properties of PEGOMs are studied by nanoindentation and near-infrared (NIR) absorption spectroscopy. Incorporation of three GO layers in between the polyelectrolyte multilayer stacks increases Young's modulus and critical stress of the microchambers by a factor of 5.6 and 2.6, respectively. Optical density of this PEGOM film is found to decrease gradually from 0.14 at λ = 800 nm to 0.06 at λ = 1500 nm. Remote opening of PEGOM microchambers with NIR laser beam is also demonstrated. One of the possible applications of the developed structures includes micropackaging and delivery systems in biological tissues with remote triggering.

A fabrication method of gold coated colloidosomes and their application as targeted drug carriers.

Colloidosomes have attracted considerable attention in recent years because of their potential applications in a range of industries, such as food, bioreactors and medicine. However, traditional polymer shell colloidosomes leak low molecular weight encapsulated materials due to their intrinsic shell permeability. Here, we report aqueous core colloidosomes coated with a gold shell, which make the capsules impermeable. The shells can be ruptured using ultrasound. The gold coated colloidosomes are prepared by making an aqueous core capsule with a polymer shell and then adding HAuCl4, surfactant and l-ascorbic acid to form a second shell. We propose to use the capsules as drug carriers. The gold coated colloidosomes demonstrate a low cytotoxicity and after triggering, both encapsulated doxorubicin and broken gold fragments kill cancer cells. In addition, we set up a targeting model by modifying the gold shell colloidosomes using 4,4'-dithiodibutyric acid and crosslinking them with proteins-rabbit immunoglobulin G (IgG). Label-free surface plasmon resonance was used to test the specific targeting of the functional gold shells with rabbit antigen. The results demonstrate that a new type of functional gold coated colloidosome with non-permeability, ultrasound sensitivity and immunoassay targeting could be applied to many medical applications.

Carbon dot aggregates as an alternative to gold nanoparticles for the laser-induced opening of microchamber arrays.

Carbon dots (CDs) are usually used as an alternative to other fluorescent nanoparticles. Apart from fluorescence, CDs also have other important properties for use in composite materials, first of all their ability to absorb light energy and convert it into heat. In our work, for the first time, CDs have been proposed as an alternative to gold nanostructures for harvesting light energy, which results in the opening of polymer-based containers with biologically active compounds. In this paper, we propose a method for the synthesis of polylactic acid microchamber arrays with embedded CDs. A comparative analysis was made of the damage to microchambers functionalized with gold nanorods and with CD aggregates, depending on the wavelength and power of the laser used. The release of fluorescent cargo from the microchamber arrays with CD aggregates under laser exposure was demonstrated.

Efficient gene editing via non-viral delivery of CRISPR-Cas9 system using polymeric and hybrid microcarriers.

CRISPR-Cas9 is a revolutionary genome-editing technology that has enormous potential for the treatment of genetic diseases. However, the lack of efficient and safe, non-viral delivery systems has hindered its clinical application. Here, we report on the application of polymeric and hybrid microcarriers, made of degradable polymers such as polypeptides and polysaccharides and modified by silica shell, for delivery of all CRISPR-Cas9 components. We found that these microcarriers mediate more efficient transfection than a commercially available liposome-based transfection reagent (>70% vs. <50% for mRNA, >40% vs. 20% for plasmid DNA). For proof-of-concept, we delivered CRISPR-Cas9 components using our capsules to dTomato-expressing HEK293T cells-a model, in which loss of red fluorescence indicates successful gene editing. Notably, transfection of indicator cells translated in high-level dTomato knockout in approx. 70% of transfected cells. In conclusion, we have provided proof-of-principle that our micro-sized containers represent promising non-viral platforms for efficient and safe gene editing.

Functional Silver-Coated Colloidosomes as Targeted Carriers for Small Molecules.

Colloidosomes have attracted great interest in recent years because of their capability for storage and delivery of small molecules for medical and pharmaceutical applications. However, traditional polymer shell colloidosomes leak low molecular weight drugs due to their intrinsic shell permeability. Here, we report aqueous core colloidosomes with a silver shell, which seals the core and makes the shell impermeable. The silver-coated colloidosomes were prepared by reacting l-ascorbic acid in the microcapsule core with silver nitrate in the wash solution. The silver shell colloidosomes were then modified by using 4,4'-dithiodibutyric acid and cross-linked with rabbit Immunoglobulin G (IgG). Label-free surface plasmon resonance was used to test the specific targeting of the functional silver shell with rabbit antigen. To break the shells, ultrasound treatment was used. The results demonstrate that a new type of functional silver-coated colloidosome with immunoassay targeting, nonpermeability, and ultrasound sensitivity could be applied to many medical applications.

Gold Nanorod Mediated Chlorhexidine Microparticle Formation and Near-Infrared Light Induced Release.

Gold nanorods (GNR) are good light harvesting species for elaboration of near-infrared (NIR) responsive drug delivery systems. Herein, chlorhexidine microparticles are grown directly on the surface of gold nanorods and then stabilized with polyelectrolyte multilayer encapsulation, producing novel composite drug-GNR particles with high drug loading and NIR light sensitivity. Crystallization of chlorhexidine is caused by the ionic strength of the chloride solution that has been demonstrated via formation of a homogeneous porous spherical structure at 0.33 M CaCl2. By introducing GNRs into the CaCl2 solution, the nucleation of chlorhexidine molecules and size of produced spheres are affected, since GNRs act as sites for chlorhexidine nucleation. Similarly, when GNRs are replaced by chlorhexidine seeds (5.2 ± 1.7 µm), a core-shell crystal structure is observed. The encapsulated GNR/chlorhexidine composites are responsive to NIR light (840 nm) that increases the temperature at the chlorhexidine crystals, followed by microparticle dissolution and rupture of capsules which is illustrated with confocal microscopy and SEM. Furthermore, a stepwise burst release of chlorhexidine can be induced by multiple cycles of NIR light exposure. The GNR/chlorhexidine composites show good biocompatibility and antimicrobial activity. The proposed method of antibacterial drug release may therefore indicate that this NIR responsive chlorhexidine composite may be useful for future clinical applications.

Local and Sustained Activity of Doxycycline Delivered with Layer-by-Layer Microcapsules.

Achieving localized delivery of small molecule drugs has the potential to increase efficacy and reduce off target and side effects associated with systemic distribution. Herein, we explore the potential use of layer-by-layer (LbL) assembled microcapsules for the delivery of doxycycline. Absorbance of doxycycline onto core dextran sulfate of preassembled microcapsules provides an efficient method to load both synthetic and biodegradable microcapsules with the drug. Application of an outer layer lipid coat enhances the sustained in vitro release of doxycycline from both microcapsule types. To monitor doxycycline delivery in a biological system, C2C12 mouse myoblasts are engineered to express EGFP under the control of the optimized components of the tetracycline regulated gene expression system. Microcapsules are not toxic to these cells, and upon delivery to the cells, EGFP is more efficiently induced in those cells that contain engulfed microcapsules and monitored EGFP expression clearly demonstrates that synthetic microcapsules with a DPPC coat are the most efficient for sustain intracellular delivery. Doxycycline released from microcapsules also displayed sustained activity in an antimicrobial growth inhibition assay compared with doxycycline solution. This study reveals the potential for LbL microcapsules in small molecule drug delivery and their feasible use for achieving prolonged doxycycline activity.

Self-propelled two dimensional polymer multilayer plate micromotors.

This communication sheds light on the production method and motion patterns of autonomous moving bubble propelled two dimensional micro-plate motors. The plate motors are produced by the well-known layer-by-layer self-assembly process in combination with micro-contact printing. The motion analysis covers instances of oscillating bubble development on one or more nucleation sites, which influence the motion speed and direction.