Exploring mitochondrial DNA copy number in circulating cell-free DNA and extracellular vesicles across cardiovascular health status: A prospective case-control pilot study.

Cardiovascular disease (CVD) is a leading global cause of mortality, difficult to predict in advance. Evidence indicates that the copy number of mitochondrial DNA (mtDNAcn) in blood is altered in individuals with CVD. MtDNA released into circulation may act as a mediator of inflammation, a recognized factor in the development of CVD, in the long distance. This pilot study aims to test if levels of mtDNAcn in buffy coat DNA (BC-mtDNA), in circulating cellfree DNA (cf-mtDNA), or in DNA extracted from plasma extracellular vesicles (EV-mtDNA) are altered in CVD patients and if they can predict heart attack in advance. A group of 144 people with different CVD statuses (50 that had CVD, 94 healthy) was selected from the LifeLines Biobank according to the incidence of new cardiovascular event monitored in 6 years (50 among controls had heart attack after the basal assessment). MtDNAcn was quantified in total cf-DNA and EV-DNA from plasma as well as in buffy coat. EVs have been characterized by their size, polydispersity index, count rate, and zeta potential, by Dynamic Light Scattering. BC-mtDNAcn and cf-mtDNAcn were not different between CVD patients and healthy subjects. EVs carried higher mtDNAcn in subject with a previous history of CVD than controls, also adjusting the analysis for the EVs derived count rate. Despite mtDNAcn was not able to predict CVD in advance, the detection of increased EV-mtDNAcn in CVD patients in this pilot study suggests the need for further investigations to determine its pathophysiological role in inflammation.

Effect of Polyplex Size on Penetration into Tumor Spheroids.

Ovarian cancer is one of the most lethal gynecological cancers in the world. In recent years, nucleic acid (NA)-based formulations have been shown to be promising treatments for ovarian cancer, including tumor nodules. However, gene therapy is not that far advanced in clinical reality due to unfavorable physicochemical properties of the NAs, such as high molecular weight, poor cellular uptake, rapid degradation by nucleases, etc. One of the strategies used to overcome these drawbacks is the complexation of anionic NAs via electrostatic interactions with cationic polymers, resulting in the formation of so-called polyplexes. In this work, the role of the size of pDNA and siRNA polyplexes on their penetration into ovarian-cancer-based tumor spheroids was investigated. For this, a methoxypoly(ethylene glycol) poly(2-(dimethylamino)ethyl methacrylate) (mPEG-pDMAEMA) diblock copolymer was synthesized as a polymeric carrier for NA binding and condensation with either plasmid DNA (pDNA) or short interfering RNA (siRNA). When prepared in HEPES buffer (10 mM, pH 7.4) at a nitrogen/phosphate (N/P) charge ratio of 5 and pDNA polyplexes were formed with a size of 162 ± 11 nm, while siRNA-based polyplexes displayed a size of 25 ± 2 nm. The polyplexes had a slightly positive zeta potential of +7-8 mV in the same buffer. SiRNA and pDNA polyplexes were tracked in vitro into tumor spheroids, resembling in vivo avascular ovarian tumor nodules. For this purpose, reproducible spheroids were obtained by coculturing ovarian carcinoma cells with primary mouse embryonic fibroblasts in different ratios (5:2, 1:1, and 2:5). Penetration studies revealed that after 24 h of incubation, siRNA polyplexes were able to penetrate deeper into the homospheroids (composed of only cancer cells) and heterospheroids (cancer cells cocultured with fibroblasts) compared to pDNA polyplexes which were mainly located in the rim. The penetration of the polyplexes was slowed when increasing the fraction of fibroblasts present in the spheroids. Furthermore, in the presence of serum siRNA polyplexes encoding for luciferase showed a high cellular uptake in 2D cells resulting in ∼50% silencing of luciferase expression. Taken together, these findings show that self-assembled small siRNA polyplexes have good potential as a platform to test ovarian tumor nodulus penetration..

Hyaluronic Acid-Based Nanosystems for CD44 Mediated Anti-Inflammatory and Antinociceptive Activity.

The nervous and immune systems go hand in hand in causing inflammation and pain. However, the two are not mutually exclusive. While some diseases cause inflammation, others are caused by it. Macrophages play an important role in modulating inflammation to trigger neuropathic pain. Hyaluronic acid (HA) is a naturally occurring glycosaminoglycan that has a well-known ability to bind with the cluster of differentiation 44 (CD44) receptor on classically activated M1 macrophages. Resolving inflammation by varying the molecular weight of HA is a debated concept. HA-based drug delivery nanosystems such as nanohydrogels and nanoemulsions, targeting macrophages can be used to relieve pain and inflammation by loading antinociceptive drugs and enhancing the effect of anti-inflammatory drugs. This review will discuss the ongoing research on HA-based drug delivery nanosystems regarding their antinociceptive and anti-inflammatory effects.

Interpenetrating Hydrogel Networks Enhance Mechanical Stability, Rheological Properties, Release Behavior and Adhesiveness of Platelet-Rich Plasma.

Platelet-rich plasma (PRP) has attracted much attention for the treatment of articular cartilage defects or wounds due to its intrinsic content of growth factors relevant for tissue repair. However, the short residence time of PRP in vivo, due to the action of lytic enzymes, its weak mechanical properties and the consequent short-term release of bioactive factors has restricted its application and efficacy. The present work aimed at designing new formulation strategies for PRP, based on the use of platelet concentrate (PC)-loaded hydrogels or interpenetrating polymer networks, directed at improving mechanical stability and sustaining the release of bioactive growth factors over a prolonged time-span. The interpenetrating hydrogels comprised two polymer networks interlaced on a molecular scale: (a) a first covalent network of thermosensitive and biodegradable vinyl sulfone bearing p(hydroxypropyl methacrylamide-lacate)-polyethylene glycol triblock copolymers, tandem cross-linked by thermal gelation and Michael addition when combined with thiolated hyaluronic acid, and (b) a second network composed of cross-linked fibrin. The PC-loaded hydrogels, instead, was formed only by network (a). All the designed and successfully synthesized formulations greatly increased the stability of PRP in vitro, leading to significant increase in degradation time and storage modulus of PRP gel. The resulting viscoelastic networks showed the ability to controllably release platelet derived growth factor and transforming growth factr ß1, and to improve the tissue adhesiveness of PRP. The newly developed hydrogels show great potential for application in the field of wound healing, cartilage repair and beyond.

Chitin and Chitosans: Characteristics, Eco-Friendly Processes, and Applications in Cosmetic Science.

Huge amounts of chitin and chitosans can be found in the biosphere as important constituents of the exoskeleton of many organisms and as waste by worldwide seafood companies. Presently, politicians, environmentalists, and industrialists encourage the use of these marine polysaccharides as a renewable source developed by alternative eco-friendly processes, especially in the production of regular cosmetics. The aim of this review is to outline the physicochemical and biological properties and the different bioextraction methods of chitin and chitosan sources, focusing on enzymatic deproteinization, bacteria fermentation, and enzymatic deacetylation methods. Thanks to their biodegradability, non-toxicity, biocompatibility, and bioactivity, the applications of these marine polymers are widely used in the contemporary manufacturing of biomedical and pharmaceutical products. In the end, advanced cosmetics based on chitin and chitosans are presented, analyzing different therapeutic aspects regarding skin, hair, nail, and oral care. The innovative formulations described can be considered excellent candidates for the prevention and treatment of several diseases associated with different body anatomical sectors.

Multifunctionality of cyclodextrin-based polymeric nanoparticulate delivery systems for chemotherapeutics, combination therapy, and theranostics.

As cancer being the most difficult disease to treat, different kinds of medications and therapeutic approaches have been prominently developed by scientists. For certain families of drugs, such as immuno-therapeutics or antibody-drug conjugates, efficient delivery systems are required during administration to protect the drugs from chemical degradation or biological inactivation. Delivery systems with the ability to carry different therapeutics or diagnostic agents or both, hold promising potential to tackle the abnormalities behind cancer. In this context, this review provides updated insights on how cyclodextrin-based polymeric nanosystems have become an effective treatment approach against cancer. Cyclodextrins (CDs) are natural oligosaccharides that are famously exploited in pharmaceutical research due to their exceptional quality of entrapping water-insoluble molecules inside their hydrophobic core and providing enhanced solubility with the help of their hydrophilic exterior. Combining the properties of CDs with polymeric nanoparticles (PNPs) brings out excellent versatile and tunable profiles, thanks to the submicron-sized PNPs. By introducing the significance of CD as a delivery system, a collective discussion on different binding approaches and release mechanisms of CD-drug complexation, followed by their characterization studies has been done in this review. Further, in light of recent studies, the article majorly focuses on conveying how promoting CD to a polymeric and nanoscale elevates the multifunctional advantages against cancer that can be successfully applied in combination therapy and theranostics. Moreover, CD-based delivery systems including CALAA-01, CRLX101, and CRLX301, have demonstrated improved tumor targeting, reduced side effects, and prolonged drug release in preclinical studies and clinical trials.

Lipid nanoparticle-mediated messenger RNA delivery for ex vivo engineering of natural killer cells.

Natural killer (NK) cells participate in the immune system by eliminating cancer and virally infected cells through germline-encoded surface receptors. Their independence from prior activation as well as their significantly lower toxicity have placed them in the spotlight as an alternative to T cells for adoptive cell therapy (ACT). Engineering NK cells with mRNA has shown great potential in ACT by enhancing their tumor targeting and cytotoxicity. However, mRNA transfection of NK cells is challenging, as the most common delivery methods, such as electroporation, show limitations. Therefore, an alternative non-viral delivery system that enables high mRNA transfection efficiency with preservation of the cell viability would be beneficial for the development of NK cell therapies. In this study, we investigated both polymeric and lipid nanoparticle (LNP) formulations for eGFP-mRNA delivery to NK cells, based on a dimethylethanolamine and diethylethanolamine polymeric library and on different ionizable lipids, respectively. The mRNA nanoparticles based on cationic polymers showed limited internalization by NK cells and low transfection efficiency. On the other hand, mRNA-LNP formulations were optimized by tailoring the lipid composition and the microfluidic parameters, resulting in a high transfection efficiency (∼100%) and high protein expression in NK cells. In conclusion, compared to polyplexes and electroporation, the optimized LNPs show a greater transfection efficiency and higher overall eGFP expression, when tested in NK (KHYG-1) and T (Jurkat) cell lines, and cord blood-derived NK cells. Thus, LNP-based mRNA delivery represents a promising strategy to further develop novel NK cell therapies.

Anionic polysaccharides for stabilization and sustained release of antimicrobial peptides.

Chemical and enzymatic in vivo degradation of antimicrobial peptides represents a major challenge for their therapeutic use to treat bacterial infections. In this work, anionic polysaccharides were investigated for their ability to increase the chemical stability and achieve sustained release of such peptides. The investigated formulations comprised a combination of antimicrobial peptides (vancomycin (VAN) and daptomycin (DAP)) and anionic polysaccharides (xanthan gum (XA), hyaluronic acid (HA), propylene glycol alginate (PGA) and alginic acid (ALG)). VAN dissolved in buffer of pH 7.4 and incubated at 37 °C showed first order degradation kinetics with a reaction rate constant kobs of 5.5 × 10-2 day-1 corresponding with a half-life of 13.9 days. However, once VAN was present in a XA, HA or PGA-based hydrogel, kobs decreased to (2.1-2.3) × 10-2 day-1 while kobs was not affected in an alginate hydrogel and a dextran solution (5.4 × 10-2 and 4.4 × 10-2 day-1). Under the same conditions, XA and PGA also effectively decreased kobs for DAP (5.6 × 10-2 day-1), whereas ALG had no effect and HA even increased the degradation rate. These results demonstrate that the investigated polysaccharides (except ALG for both peptides and HA for DAP) slowed down the degradation of VAN and DAP. DSC analysis was used to investigate on polysaccharide ability to bind water molecules. Rheological analysis highlighted that the polysaccharides containing VAN displayed an increase in G' of their formulations, pointing that the peptides interaction act as crosslinker of the polymer chains. The obtained results suggest that the mechanism of stabilization of VAN and DAP against hydrolytic degradation is conferred by electrostatic interactions between the ionizable amine groups of the drugs and the anionic carboxylate groups of the polysaccharides. This, in turn, results in a close proximity of the drugs to the polysaccharide chain, where the water molecules have a lower mobility and, therefore, a lower thermodynamic activity.

Changes in the Solid State of Nicergoline, a Poorly Soluble Drug, Under Different Grinding and Environmental Conditions: Effect on Polymorphism and Dissolution.

Nicergoline native crystals (Form I) were subjected to different grinding methods for 15, 30, 45, and 60 min: Method A, grinding at 20°C under air atmosphere; Method B, grinding in presence of liquid nitrogen under air atmosphere; Method C, grinding at 20°C under nitrogen atmosphere; and Method D, grinding in presence of liquid nitrogen under nitrogen atmosphere. Scanning electron microscopy, differential scanning calorimetry, X-ray powder diffractometry, thermogravimetry, and infrared spectroscopy were used to follow changes in the particle size and in crystalline structures. Batches from Methods A and C underwent partial amorphization immediately after grinding; Form II was obtained by heating these partially amorphous forms or after spontaneous crystallization after 1 and 5 months storage. Method B promoted the hydration of nicergoline to a monohydrate form. Batch D was stable under grinding and neither amorphization nor hydration were observed. The best intrinsic dissolution rate was that of metastable Form II, followed by Form I, while the worst was that of the Method B monohydrate form. The slowest particle dissolution was observed for hydrated particles, because of the lowest IDR, while the most rapid was exhibited by batch D, because of the very small particle size.

New Advanced Strategies for the Treatment of Lysosomal Diseases Affecting the Central Nervous System.

Lysosomal Storage Disorders (LSDs), also known as lysosomal diseases (LDs) are a group of serious genetic diseases characterized by not only the accumulation of non-catabolized compounds in the lysosomes due to the deficiency of specific enzymes which usually eliminate these compounds, but also by trafficking, calcium changes and acidification. LDs mainly affect the central nervous system (CNS), which is difficult to reach for drugs and biological molecules due to the presence of the blood-brain barrier (BBB). While some therapies have proven highly effective in treating peripheral disorders in LD patients, they fail to overcome the BBB. Researchers have developed many strategies to circumvent this problem, for example, by creating carriers for enzyme delivery, which improve the enzyme's half-life and the overexpression of receptors and transporters in the luminal or abluminal membranes of the BBB. This review aims to successfully examine the strategies developed during the last decade for the treatment of LDs, which mainly affect the CNS. Among the LD treatments, enzyme-replacement therapy (ERT) and gene therapy have proven effective, while nanoparticle, fusion protein, and small molecule-based therapies seem to offer considerable promise to treat the CNS pathology. This work also analyzed the challenges of the study to design new drug delivery systems for the effective treatment of LDs. Polymeric nanoparticles and liposomes are explored from their technological point of view and for the most relevant preclinical studies showing that they are excellent choices to protect active molecules and transport them through the BBB to target specific brain substrates for the treatment of LDs.

Thermosensitive hybrid hydrogels for the controlled release of bioactive vancomycin in the treatment of orthopaedic implant infections.

The purpose of this work was the development of antibacterial delivery systems for vancomycin, with potential application in the prevention or treatment of orthopedic implant infections. Previous studies have shown tandem thermal gelling and Michael addition cross-linking of hydrogels based on methacrylate, acrylate or vinylsulfone triblock copolymers of PEG-p(HPMAm-lac1-2) and thiolated hyaluronic acid. In this work we exploited these α-ß unsaturated derivatives of PEG-p(HPMAm-lac1-2) triblock copolymers and used them in combination with thiolated hyaluronic acid as controlled delivery systems for vancomycin. It was found that the antibiotic was sustainably released from the hydrogel networks for at least 5 days with release kinetics depending on diffusion and dissociation of the positively charged vancomycin from the negatively charged hyaluronic acid. The release of vancomycin could be tailored mainly by HA-SH solid content and degree of thiolation. The developed hydrogels were demonstrate efficacious in preserving the structural and functional integrity of the encapsulated drug by physical immobilization within the gel network and ionic interaction with hyaluronic acid, thereby preventing vancomycin deamidation processes. Furthermore, the antimicrobial activity of vancomycin loaded hydrogels was assessed, demonstrating retention of inhibitory activity towards Staphylococcus aureus during formulation and release, with slightly increased activity of vancomycin encapsulated in hydrogels of higher HA-SH content as compared to controls.

Hot Melt Extrusion: Highlighting Physicochemical Factors to Be Investigated While Designing and Optimizing a Hot Melt Extrusion Process.

Hot-melt extrusion (HME) is a well-accepted and extensively studied method for preparing numerous types of drug delivery systems and dosage forms. It offers several advantages: no solvents are required, it is easy to scale up and employ on the industrial level, and, in particular, it offers the possibility of improving drug bioavailability. HME involves the mixing of a drug with one or more excipients, in general polymers and even plasticizers, which can melt, often forming a solid dispersion of the drug in the polymer. The molten mass is extruded and cooled, giving rise to a solid material with designed properties. This process, which can be realized using different kinds of special equipment, may involve modifications in the drug physicochemical properties, such as chemical, thermal and mechanical characteristics thus affecting the drug physicochemical stability and bioavailability. During process optimization, the evaluation of the drug solid state and stability is thus of paramount importance to guarantee stable drug properties for the duration of the drug product shelf life. This manuscript reviews the most important physicochemical factors that should be investigated while designing and optimizing a hot melt extrusion process, and by extension, during the different pre-formulation, formulation and process, and post-formulation phases. It offers a comprehensive evaluation of the chemical and thermal stability of extrudates, the solid physical state of extrudates, possible drug-polymer interactions, the miscibility/solubility of the drug-polymer system, the rheological properties of extrudates, the physicomechanical properties of films produced by hot melt extrusion, and drug particle dissolution from extrudates. It draws upon the last ten years of research, extending inquiry as broadly as possible.

Nanocrystals of Poorly Soluble Drugs: Drug Bioavailability and Physicochemical Stability.

Many approaches have been developed over time to overcome the bioavailability limitations of poorly soluble drugs. With the advances in nanotechnology in recent decades, science and industry have been approaching this issue through the formulation of drugs as nanocrystals, which consist of "pure drugs and a minimum of surface active agents required for stabilization". They are defined as "carrier-free submicron colloidal drug delivery systems with a mean particle size in the nanometer range, typically between 10⁻800 nm". The primary importance of these nanoparticles was the reduction of particle size to nanoscale dimensions, with an increase in the particle surface area in contact with the dissolution medium, and thus in bioavailability. This approach has been proven successful, as demonstrated by the number of such drug products on the market. Nonetheless, despite the definition that indicates nanocrystals as a "carrier-free" system, surface active agents are necessary to prevent colloidal particles aggregation and thus improve stability. In addition, in more recent years, nanocrystal properties and technologies have attracted the interest of researchers as a means to obtain colloidal particles with modified biological properties, and thus their interest is now also addressed to modify the drug delivery and targeting. The present work provides an overview of the achievements in improving the bioavailability of poorly soluble drugs according to their administration route, describes the methods developed to overcome physicochemical and stability-related problems, and in particular reviews different stabilizers and surface agents that are able to modify the drug delivery and targeting.

Daptomycin-loaded biodegradable thermosensitive hydrogels enhance drug stability and foster bactericidal activity against Staphylococcus aureus.

A drug delivery system based on fully biodegradable thermosensitive hydrogels enabling controlled antibiotic release may support the management of implant-associated infections. In this work, the lipopeptide antibiotic daptomycin was encapsulated in hydrogel networks consisting of vinyl sulfonated triblock copolymers of PEG-p(HPMAm-lac1,2) and thiolated hyaluronic acid. High concentrations of active daptomycin exceeding the minimum biofilm eradicating concentration were sustainably eluted from the biodegradable carrier. The drug release profiles were tailored by varying the degree of substitution (DS) of thiol groups of hyaluronic acid, reaching a plateau level after 200 and 330 h for DS values of 53% and 31%, respectively. The hydrogel polymeric network preserved the structural stability of the loaded antibiotic and retained the calcium-dependent daptomycin activity, showing a noticeable biofilm bactericidal effect against a 24 h-old Staphylococcus aureus biofilm in vitro. The two-component thermosensitive hydrogels demonstrated to be an excellent antibiotic releasing scaffold with potential clinical applications in the management of implant-associated infections.