Inflammation responsive logic gate nanoparticles for the delivery of proteins

Oxidative stress and reduced pH are important stimuli targets for intracellular delivery and for delivery to diseased tissue. However, there is a dearth of materials able to deliver bioactive agents selectively under these conditions. We employed our recently developed dual response strategy to build a polymeric nanoparticle that degrades upon exposure to two stimuli in tandem. Our polythioether ketal based nanoparticles undergo two chemical transformations; the first is the oxidation of the thioether groups along the polymer backbone of the nanoparticles upon exposure to reactive oxygen species (ROS). This transformation switches the polymeric backbone from hydrophobic to hydrophilic and thus allows, in mildly acidic environments, the rapid acid-catalyzed degradation of the ketal groups also along the polymer backbone. Dynamic light scattering and payload release studies showed full particle degradation only in conditions that combined both oxidative stress and acidity, and these conditions led to higher release of encapsulated protein within 24 h. Nanoparticles in neutral pH and under oxidative conditions showed small molecule release and swelling of otherwise intact nanparticles. Notably, cellular studies show absence of toxicity and efficient uptake of nanoparticles by macrophages followed by cytoplasmic release of ovalbumin. Future work will apply this system to inflammatory diseases.     

Designing proteins for therapeutic applications

Protein design is becoming an increasingly useful tool for optimizing protein drugs and creating novel biotherapeutics. Recent progress includes the engineering of monoclonal antibodies, cytokines, enzymes and viral fusion inhibitors.     

Microparticles and nanoparticles for drug delivery

Particulate drug delivery systems have become important in experimental pharmaceutics and clinical medicine. The distinction is often made between micro- and nanoparticles, being particles with dimensions best described in micrometers and nanometers respectively. That size difference entails real differences at many levels, from formulation to in vivo usage. Here I will discuss those differences and provide examples of applications, for local and systemic drug delivery. I will outline a number of challenges of interest in particulate drug delivery.     

Magnetic nanoparticles for targeted vascular delivery

Magnetic targeting has shown promise to improve the efficacy and safety of different classes of therapeutic agents by enabling their active guidance to the site of disease and minimizing dissemination to nontarget tissues. However, its translation into clinic has proven difficult because of inherent limitations of traditional approaches inapplicable for deep tissue targeting in human subjects and a need for developing well-characterized and fully biocompatible magnetic carrier formulations. A novel magnetic targeting scheme based on the magnetizing effect of deep-penetrating uniform fields is presented as an example of a strategy providing a potentially clinically viable solution for preventing injury-triggered reobstruction of stented blood vessels (in-stent restenosis). The design of optimized magnetic carrier formulations and experimental results showing the feasibility of uniform field-controlled targeting for site-specific vascular delivery of small-molecule pharmaceuticals, biotherapeutics, and cells are discussed in the context of antirestenotic therapy. The versatility of this approach applicable to different classes of therapeutic agents exerting their antirestenotic effects through distinct mechanisms prompts exploring the utility of uniform field-mediated magnetic stent targeting for combination therapies with enhanced efficiencies and improved safety profiles. Additional improvements in terms of site specificity and protracted carrier retention at the site of injury may be expected from the development and use of magnetic carriers exhibiting affinity for arterial wall-specific antigens.     

Protein-polymer nanoparticles for nonviral gene delivery

Protein-polymer conjugates were investigated as nonviral gene delivery vectors. BSA-poly(dimethylamino) ethyl methacrylate (PDMA) nanoparticles (nBSA) were synthesized using in situ atom transfer radical polymerization (in situ ATRP) and BSA as a macroinitiator. The diameter and charge of nBSA was a function of the ATRP reaction time and ranged from 5 to 15 nm and +8.9 to +22.5, respectively. nBSA were able to condense plasmid DNA (pDNA) and form polyplexes with an average diameter of 50 nm. nBSA/pDNA polyplexes transfected cells with similar efficiencies or better as compared to linear and branched PEI. Interestingly, the nBSA particle diameter and charge did not affect pDNA complexation and transgene expression, indicating that the same gene delivery efficiency can be achieved with lower charge ratios. We believe that with the use of protein-polymer conjugates additional functionality could be introduced to polyplexes by using different protein cores and, thus, they pose an interesting alternative to the design of nonviral gene delivery vectors.     

Caged protein nanoparticles for drug delivery

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Formulations for delivery of therapeutic proteins

Advances in biotechnology have now created a capacity to produce therapeutically active proteins on a commercial scale, opening the potential for their application in an array of disease conditions. The process of translation of the variety of different therapeutic proteins into the medicines used in clinics is now occurring. To assist in this translation, new formulations to deliver proteins could play an important role. These new formulations need to more adequately address the pharmacological and therapeutic requirement for each particular protein/peptide and, in that way, either improve present therapies or extend with new entries the current list of protein based medicines used in clinic.

Designing gene delivery vectors for cardiovascular gene therapy

Genetic therapy in the cardiovascular system has been proposed for a variety of diseases ranging from prevention of vein graft failure to hypertension. Such diversity in pathogenesis requires the delivery of therapeutic genes to diverse cell types in vivo for varying lengths of time if efficient clinical therapies are to be developed. Data from extensive preclinical studies have been compiled and a certain areas have seen translation into large-scale clinical trials, with some encouraging reports. It is clear that progress within a number of disease areas is limited by a lack of suitable gene delivery vector systems through which to deliver therapeutic genes to the target site in an efficient, non-toxic manner. In general, currently available systems, including non-viral systems and viral vectors such as adenovirus (Ad) or adeno-associated virus (AAV), have a propensity to transduce non-vascular tissue with greater ease than vascular cells thereby limiting their application in cardiovascular disease. This problem has led to the development and testing of improved vector systems for cardiovascular gene delivery. Traditional viral and non-viral systems are being engineered to increase their efficiency of vascular cell transduction and diminish their affinity for other cell types through manipulation of vector:cell binding and the use of cell-selective promoters. It is envisaged that future use of such technology will substantially increase the efficacy of cardiovascular gene therapy.     

Designing of 'intelligent' liposomes for efficient delivery of drugs

The liposome- vesicles made by a double phospholipidic layers which may encapsulate aqueous solutions- have been introduced as drug delivery vehicles due to their structural flexibility in size, composition and bilayer fluidity as well as their ability to incorporate a large variety of both hydrophilic and hydrophobic compounds. With time the liposome formulations have been perfected so as to serve certain purposes and this lead to the design of "intelligent" liposomes which can stand specifically induced modifications of the bilayers or can be surfaced with different ligands that guide them to the specific target sites. We present here a brief overview of the current strategies in the design of liposomes as drug delivery carriers and the medical applications of liposomes in humans.     

Cell-penetrating peptide-conjugated lipid nanoparticles for siRNA delivery

Lipid nanoparticles (LNP) modified with cell-penetrating peptides (CPP) were prepared for the delivery of small interfering RNA (siRNA) into cells. Lipid derivatives of CPP derived from protamine were newly synthesized and used to prepare CPP-decorated LNP (CPP-LNP). Encapsulation of siRNA into CPP-LNP improved the stability of the siRNA in serum. Fluorescence-labeled siRNA formulated in CPP-LNP was efficiently internalized into B16F10 murine melanoma cells in a time-dependent manner, although that in LNP without CPP was hardly internalized into these cells. In cells transfected with siRNA in CPP-LNP, most of the siRNA was distributed in the cytoplasm of these cells and did not localize in the lysosomes. Analysis of the endocytotic pathway indicated that CPP-LNP were mainly internalized via macropinocytosis and heparan sulfate-mediated endocytosis. CPP-LNP encapsulating siRNA effectively induced RNA interference-mediated silencing of reporter genes in B16F10 cells expressing luciferase and in HT1080 human fibrosarcoma cells expressing enhanced green fluorescent protein. These data suggest that modification of LNP with the protamine-derived CPP was effective to facilitate internalization of siRNA in the cytoplasm and thereby to enhance gene silencing.     

Chitosan magnetic nanoparticles for drug delivery systems

The potential of magnetic nanoparticles (MNPs) in drug delivery systems (DDSs) is mainly related to its magnetic core and surface coating. These coatings can eliminate or minimize their aggregation under physiological conditions. Also, they can provide functional groups for bioconjugation to anticancer drugs and/or targeted ligands. Chitosan, as a derivative of chitin, is an attractive natural biopolymer from renewable resources with the presence of reactive amino and hydroxyl functional groups in its structure. Chitosan nanoparticles (NPs), due to their huge surface to volume ratio as compared to the chitosan in its bulk form, have outstanding physico-chemical, antimicrobial and biological properties. These unique properties make chitosan NPs a promising biopolymer for the application of DDSs. In this review, the current state and challenges for the application magnetic chitosan NPs in drug delivery systems were investigated. The present review also revisits the limitations and commercial impediments to provide insight for future works.     

Magnetic nanoparticles for gene and drug delivery

Investigations of magnetic micro- and nanoparticles for targeted drug delivery began over 30 years ago. Since that time, major progress has been made in particle design and synthesis techniques, however, very few clinical trials have taken place. Here we review advances in magnetic nanoparticle design, in vitro and animal experiments with magnetic nanoparticle-based drug and gene delivery, and clinical trials of drug targeting.     

Monodisperse chitosan nanoparticles for mucosal drug delivery

Chitosan nanoparticles (CS NPs) of a controlled size (below 100 nm) and narrow size distribution were obtained through the process of ionic gelation between CS and sodium tripolyphosphate (TPP). A high degree of CS deacetylation and narrow polymer molecular weight distribution were demonstrated to be critical for the controlling particle size distribution. Properties of the CS NPs were examined at different temperatures, values of pH, and ratios of CS to TPP. The model protein, bovine serum albumin, was encapsulated into the NPs, and the in vitro release profiles were examined in physiologically relevant media at 37 degrees C.     

Magnetic nanoparticles for multi-imaging and drug delivery

Various bio-medical applications of magnetic nanoparticles have been explored during the past few decades. As tools that hold great potential for advancing biological sciences, magnetic nanoparticles have been used as platform materials for enhanced magnetic resonance imaging (MRI) agents, biological separation and magnetic drug delivery systems, and magnetic hyperthermia treatment. Furthermore, approaches that integrate various imaging and bioactive moieties have been used in the design of multi-modality systems, which possess synergistically enhanced properties such as better imaging resolution and sensitivity, molecular recognition capabilities, stimulus responsive drug delivery with on-demand control, and spatio-temporally controlled cell signal activation. Below, recent studies that focus on the design and synthesis of multi-mode magnetic nanoparticles will be briefly reviewed and their potential applications in the imaging and therapy areas will be also discussed.     

Targeted albumin-based nanoparticles for delivery of amphipathic drugs

We report the preparation and physical and biological characterization of human serum albumin-based micelles of approximately 30 nm diameter for the delivery of amphipathic drugs, represented by doxorubicin. The micelles were surface conjugated with cyclic RGD peptides to guide selective delivery to cells expressing the α(v)β(3) integrin. Multiple poly(ethylene glycol)s (PEGs) with molecular weight of 3400 Da were used to form a hydrophilic outer layer, with the inner core formed by albumin conjugated with doxorubicin via disulfide bonds. Additional doxorubicin was physically adsorbed into this core to attain a high drug loading capacity, where each albumin was associated with about 50 doxorubicin molecules. The formed micelles were stable in serum but continuously released doxorubicin when incubated with free thiols at concentrations mimicking the intracellular environment. When incubated with human melanoma cells (M21+) that express the α(v)β(3) integrin, higher uptake and longer retention of doxorubicin was observed with the RGD-targeted micelles than in the case of untargeted control micelles or free doxorubicin. Consequently, the RGD-targeted micelles manifested cytotoxicity at lower doses of drug than control micelles or free drug.     

Photocontrolled nanoparticles for on-demand release of proteins

We describe here light-regulated swelling and degradation features of polymeric nanoparticles that are produced using an inverse microemulsion polymerization method. We demonstrate the phototriggered release characteristics of the nanoparticles by sequestering protein molecules and releasing them using light as a trigger. Furthermore, the intracellular translocation of the nanoparticles, along with its fluorescent protein payload, was achieved using a cell-penetrating peptide-based surface modification. We expect that the noncovalent encapsulation of proteins using nanoparticles and their photo triggered release using an external light would provide opportunities for achieving intracellular release of molecular therapeutics for on-demand requirements.     

Designing lipid nanostructures for local delivery of biologically active macromolecules

This study focuses on the possible therapeutic utility of liposomes in the local treatment of inflammatory disorders, specifically rheumatoid arthritis (RA). Our purpose was to design a depot delivery system of an anti-inflammatory glycoprotein, lactoferrin (Lf), using positive multivesicular liposomes and to investigate its in vivo efficiency. Lactoferrin (Lf) has previously been shown to have therapeutic potential in mice with collagen-induced arthritis (CIA) after intra-articular (i.a.) injection. In order to protect Lf from enzymatic degradation and to maintain an adequate concentration in the joint, liposomes have been used as carriers for controlled drug delivery. Based on our previous findings we compared the ability of free Lf and Lf encapsulated in liposomes to suppress established joint inflammation and to modulate the cytokine response of lymph node (LN) T lymphocytes in DBA/1 mice with CIA. The anti-inflammatory effect of Lf formulated in positive liposomes was more pronounced compared with the free protein. After a single i.a. injection of liposomal Lf the arthritic score significantly decreased continuously for 2 weeks while in the case of free Lf for only 3-4 days. The cytokine levels produced by LN T cells showed decreased pro-inflammatory cytokines (TNF-alpha and IFN-gamma) accompanied by increased anti-inflammatory cytokines (IL-5 and especcialy IL-10) in encapsulated compared with free Lf. When compared with free Lf, liposomal Lf decreased the expression of costimulatory molecules on DCs, reduced pro-inflammatory (TNF) and increased anti-inflammatory (IL-10) cytokine production. Using CIA model we have studied the liposome trafficking following i.a. administration and we have identified DCs as a target for liposomes in the draining LN. Our results suggest that the entrapment of Lf in liposomes may modify its pharmacodynamic profile and could have great potential as controlled delivery system in the treatment of RA and other local inflammatory conditions.     

Magnetic core-shell nanoparticles for drug delivery by nebulization

Background

Aerosolized therapeutics hold great potential for effective treatment of various diseases including lung cancer. In this context, there is an urgent need to develop novel nanocarriers suitable for drug delivery by nebulization. To address this need, we synthesized and characterized a biocompatible drug delivery vehicle following surface coating of Fe₃O₄ magnetic nanoparticles (MNPs) with a polymer poly(lactic-co-glycolic acid) (PLGA). The polymeric shell of these engineered nanoparticles was loaded with a potential anti-cancer drug quercetin and their suitability for targeting lung cancer cells via nebulization was evaluated.

Results

Average particle size of the developed MNPs and PLGA-MNPs as measured by electron microscopy was 9.6 and 53.2 nm, whereas their hydrodynamic swelling as determined using dynamic light scattering was 54.3 nm and 293.4 nm respectively. Utilizing a series of standardized biological tests incorporating a cell-based automated image acquisition and analysis procedure in combination with real-time impedance sensing, we confirmed that the developed MNP-based nanocarrier system was biocompatible, as no cytotoxicity was observed when up to 100 μg/ml PLGA-MNP was applied to the cultured human lung epithelial cells. Moreover, the PLGA-MNP preparation was well-tolerated in vivo in mice when applied intranasally as measured by glutathione and IL-6 secretion assays after 1, 4, or 7 days post-treatment. To imitate aerosol formation for drug delivery to the lungs, we applied quercitin loaded PLGA-MNPs to the human lung carcinoma cell line A549 following a single round of nebulization. The drug-loaded PLGA-MNPs significantly reduced the number of viable A549 cells, which was comparable when applied either by nebulization or by direct pipetting.

Conclusion

We have developed a magnetic core-shell nanoparticle-based nanocarrier system and evaluated the feasibility of its drug delivery capability via aerosol administration. This study has implications for targeted delivery of therapeutics and poorly soluble medicinal compounds via inhalation route.