3D-printed bioresorbable poly(lactic-co-glycolic acid) and quantum-dot nanocomposites: Scaffolds for enhanced bone mineralization and inbuilt co-monitoring.

Multifunctional 3D-printed nanocomposites based on poly(lactic-co-glycolic acid), that is, PLGA (RESOMER® LG857S) were developed for simultaneous monitoring of cells and scaffold as a function of time and spectral responses. These were achieved by impregnating carbon quantum dots (CQDs) on PLGA using melt-blending, plasticating extrusion, and 3D-printing. The nanocomposites enabled enhanced bio-affinity and cellular interactions for bone tissue engineering (TE). PLGA (control) and PLGA-CQD scaffolds were used for growing human adipose-derived-stem-cells (ADSCs) and tested for cell biocompatibility, cellular adhesion, growth, and osteogenesis. CQDs were found to enhance the hydrophilicity of nanocomposites and promote cellular nesting. MTS assays confirmed that CQDs on PLGA act as cell anchoring sites, thereby enhancing seeding efficiency and cell proliferation. Alkaline phosphate tests showed increased osteogenesis and Alizarin assays confirmed enhanced bone mineralization on PLGA-CQD. The qPCR tests based on selected mRNA expressions showed that the incorporation of CQDs significantly enhanced osteogenesis of ADSCs during all three phases of cell differentiation. The intrinsic luminescence of the composites allowed label-free monitoring of cell proliferation and bone mineralization on the scaffolds. Thus, the CQDs facilitated significant enhancements in composite processability with customized fabrication of 3D printed scaffolds, bone tissue osteoconductivity, and monitoring of cell-scaffold activities, offering multifunctional benefits for bone TE.

Delivery of pDNA to lung epithelial cells using PLGA nanoparticles formulated with a cell-penetrating peptide: understanding the intracellular fate.

The combination of nanoparticles (NPs) and cell-penetrating peptide (CPP) represents a new opportunity to develop plasmid DNA (pDNA) delivery systems with desirable properties for lung delivery. In this study, poly(lactide-co-glycolide) (PLGA) NPs containing pDNA were formulated with and without CPP using a double-emulsion technique. NPs were characterized in regards of size, surface charge, release profile, pDNA encapsulation efficiency and pDNA integrity. Cellular uptake, intracellular trafficking, uptake mechanism and pDNA expression were assessed in both A549 and Beas-2B cells. Manufactured PLGA-NPs efficiently encapsulated pDNA with approximately 50% released in the first 24 h of incubation. Addition of CPP was essential to promote NP internalization in both cell lines, with 83.85 ± 1.2% and 96.76 ± 1.7% of Beas-2B and A549 cells, respectively, with internalized NP-DNA-CPP after 3 h of incubation. Internalization appears to occur mainly via clathrin-mediated endocytosis, with other pathways also being used by the different cell lines. An endosomal-escape mechanism seems to happen in both cell lines, and eGFP expression was observed in Beas-2B after 96 h of incubation. In summary, the NP-DNA-CPP delivery system efficiently encapsulated and protected pDNA structure and is being investigated as a promising tool for gene delivery to the lungs.

Nanotoxicologic Effects of PLGA Nanoparticles Formulated with a Cell-Penetrating Peptide: Searching for a Safe pDNA Delivery System for the Lungs.

The use of cell-penetrating peptides (CPPs) in combination with nanoparticles (NPs) shows great potential for intracellular delivery of DNA. Currently, its application is limited due to the potential toxicity and unknown long-term side effects. In this study NPs prepared using a biodegradable polymer, poly(lactic⁻co⁻glycolic acid (PLGA) in association with a CPP, was assessed on two lung epithelial cell lines (adenocarcinomic human alveolar basal epithelial cells (A549) and normal bronchial epithelial cells (Beas-2B cells)). Addition of CPP was essential for intracellular internalization. No effects were observed on the mitochondrial activity and membrane integrity. Cells exposed to the NPs⁻DNA⁻CPP showed low inflammatory response, low levels of apoptosis and no activation of caspase-3. Increase in necrotic cells (between 10%⁻15%) after 24 h of incubation and increase in autophagy, induced by NPs⁻DNA⁻CPP, are likely to be related to the lysosomal escape mechanism. Although oxidative stress is one of the main toxic mechanisms of NPs, NPs⁻DNA⁻CPP showed decreased reactive oxygen species (ROS) production on Beas-2B cells, with potential antioxidant effect of CPP and no effect on A549 cells. This NP system appears to be safe for intracellular delivery of plasmid DNA to the lung epithelial cells. Further investigations should be conducted in other lung-related systems to better understand its potential effects on the lungs.

Human Stimulus Factor Is a Promising Peptide for Delivery of Therapeutics.

Fluticasone propionate uptake in the presence of a proprietary cell-penetrating peptide (human stimulus factor, [HSF]) based on the N-terminal domain of lactoferrin was studied, alone and in combination with salmeterol, using an air interface Calu-3 epithelial model. The HSF enhanced uptake and transport of fluticasone propionate across the epithelial barrier when alone and in presence of salmeterol. This was attributed to transcellular-mediated uptake. This HSF is a promising peptide for delivery of therapeutics where enhanced epithelial penetrating is required.

Delivery of pDNA Polyplexes to Bronchial and Alveolar Epithelial Cells Using a Mesh Nebulizer.

PURPOSE: In this study, a cell penetrating peptide was used as an uptake enhancer for pDNA delivery to the lungs. METHODS: Polyplexes were prepared between pDNA and CPP. Intracellular delivery of pDNA was assessed in both alveolar (A549) and bronchial (Calu-3) epithelial cells. Aerosol delivery was investigated using a mesh nebulizer. RESULTS: Efficient intracellular delivery of pDNA occurs in both A549 and Calu-3 cells when delivered as polyplexes. Protection against nucleases and endosomal escape mechanism occurs when pDNA is formulated within the polyplexes. For aerosol delivery, 1% (w/v) mannitol was able to protect naked DNA structure during nebulization with a significant increase in fine particle fraction (particles <5 µm). The structure of polyplexes when delivered via a mesh nebulizer using 1% (w/v) mannitol could partially withstand the shear forces involved in aerosolization. Although some loss in functionality occurred after nebulization, membrane-associated fluorescence was observed in A549 cells. In Calu-3 cells mucus entrapment was a limiting factor for polyplex delivery. CONCLUSIONS: The presence of CPP is essential for efficient intracellular delivery of pDNA. The polyplexes can be delivered to lung epithelial cells using mesh nebulizer. The use of different excipients is essential for further optimization of these delivery systems.

Inhaled gene delivery: a formulation and delivery approach.

INTRODUCTION: Gene therapy is a potential alternative to treat a number of diseases. Different hurdles are associated with aerosol gene delivery due to the susceptibility of plasmid DNA (pDNA) structure to be degraded during the aerosolization process. Different strategies have been investigated in order to protect and efficiently deliver pDNA to the lungs using non-viral vectors. To date, no successful therapy involving non-viral vectors has been marketed, highlighting the need for further investigation in this field. Areas covered: This review is focused on the formulation and delivery of DNA to the lungs, using non-viral vectors. Aerosol gene formulations are divided according to the current delivery systems for the lung: nebulizers, dry powder inhalers and pressurized metered dose inhalers; highlighting its benefits, challenges and potential application. Expert opinion: Successful aerosol delivery is achieved when the supercoiled DNA structure is protected during aerosolization. A formulation strategy or compounds that can protect, stabilize and efficiently transfect DNA into the cells is desired in order to produce an effective, low-cost and safe formulation. Nebulizers and dry powder inhalers are the most promising approaches to be used for aerosol delivery, due to the lower shear forces involved. In this context it is also important to highlight the importance of considering the 'pDNA-formulation-device system' as an integral part of the formulation development for a successful nucleic acid delivery.

Safety and regulatory review of dyes commonly used as excipients in pharmaceutical and nutraceutical applications.

Color selection is one of the key elements of building a strong brand development and product identity in the pharmaceutical industry, besides to prevent counterfeiting. Moreover, colored pharmaceutical dosage forms may increase patient compliance and therapy enhancement. Although most synthetic dyes are classified as safe, their regulations are stricter than other classes of excipients. Safety concerns have increased during the last years but the efforts to change to natural dyes seem to be not promising. Their instability problems and the development of "non-toxic" dyes is still a challenge. This review focuses specifically on the issues related to dye selection and summarizes the current regulatory status. A deep awareness of toxicological data based on the public domain, making sure the compliance of standards for regulation and safety for successful product development is provided. In addition, synthetic strategies are provided to covalently bind dyes on polymers to possibly overcome toxicity issues.

Toward pH-responsive coating materials--high-throughput study of (meth)acrylic copolymers.

The release behavior of a model compound (ß-naphthol orange) encapsulated in (meth)acrylate-based statistical copolymers under different environmental conditions was investigated. From monomers of varying polarity (methyl acrylate, ethyl acrylate, tert-butyl acrylate, 2-ethylhexyl methacrylate, and benzyl methacrylate) in combination with methacrylic acid, five polymer series were synthesized by free radical polymerization. The pH-dependent release kinetics were investigated via UV-vis spectroscopy at pH 1.2 and 6.8, simulating physiological conditions in the stomach and intestines. Furthermore, the influence of different ethanol contents (0 and 40 vol %) in the acidic medium was investigated. The whole approach was designed to meet the requirements of a high-throughput experimentation workflow.

Labeled Nanoparticles Based on Pharmaceutical EUDRAGIT® S 100 Polymers.

The pharmaceutically important polymer P(MAA-r-MMA)(1:2) (EUDRAGIT(®) S100) was investigated concerning its behavior to form nanoparticles via nanoprecipitation. The particles obtained were characterized regarding their size, shape, and characteristics using DLS, SEM, and AUC. Furthermore, the P(MAA-r-MMA)(1:2) copolymer was modified with different markers in order to achieve polymer-based nanocarrier systems, which are detectable and may be useful for controlled drug delivery devices to monitor the drug pathways. The particles were labeled by physical entrapment as well as by covalent attachment of various markers, e.g., radicals, fluorescent-, and near-infrared dyes, to the polymer. Physical entrapment of radicals into the polymeric units was performed by co-nanoprecipitation of P(MAA-r-MMA)(1:2) and a radical marker. By means of covalent binding of the markers to the polymer, a stable and more defined labeling of the particles was also performed, leading only to a low degree of modification of the pharmaceutical polymer. After nanoprecipitation, the resulting labeled particles were characterized by SEM and DLS, whereas their biocompatibility was proven by in vitro studies. In order to ensure the possibility of detection of the particles inside the body for drug delivery-, sensor-, and imaging applications, the polymeric carriers were also investigated by electron spin resonance, fluorescence, as well as near-infrared spectroscopy.

Rapid parallel mutation scanning of gene fragments using a microelectronic protein-DNA chip format.

We have developed a method for the de novo discovery of genetic variations, including single nucleotide polymorphisms and mutations, on microelectronic chip devices. The method combines the features of electronically controlled DNA hybridisation on open-format microarrays, with mutation detection by a fluorescence-labelled mismatch- binding protein. Electronic addressing of DNA strands to distinct test sites of the chip allows parallel analysis of several individuals, as demonstrated for mutations in different exons of the p53 gene. This microelectronic chip-based mutation discovery assay may substitute for time-consuming sequencing studies and will complement existing technologies in genomic research.