Glutathione Responsive Hyaluronic Acid Nanocapsules Obtained by Bioorthogonal Interfacial "Click" Reaction.

Azide-functionalized hyaluronic acid and disulfide dialkyne have been used for "click" reaction polymerization at the miniemulsion droplets interface leading to glutathione responsive nanocapsules (NCs). Inverse miniemulsion polymerization was chosen, due to its excellent performance properties, for example, tuning of size and size distribution, shell thickness/density, and high pay loading efficiency. The obtained size, size distribution, and encapsulation efficiency were checked via fluorescent spectroscopy, and the tripeptide glutathione was used to release an encapsulated fluorescent dye after cleavage of the nanocapsules shell. To show the glutathione-mediated intracellular cleavage of disulfide-containing NC shells, CellTracker was encapsulated into the nanocapsules. The cellular uptake in dendritic cells and the cleavage of the nanocapsules in the cells were studied using confocal laser scanning microscopy. Because of the mild reaction conditions used during the interfacial polymerization and the excellent cleavage properties, we believe that the synthesis of glutathione responsive hyaluronic acid NCs reported herein are of high interest for the encapsulation and release of sensitive compounds at high yields.

Protein corona of nanoparticles: distinct proteins regulate the cellular uptake.

Understanding nanoparticle-protein interactions is a crucial issue in the development of targeted nanomaterial delivery. Besides unraveling the composition of the nanoparticle's protein coronas, distinct proteins thereof could control nanoparticle uptake into specific cell types. Here we differentially analyzed the protein corona composition on four polymeric differently functionalized nanoparticles by label-free quantitative mass spectrometry. Next, we correlated the relative abundance of identified proteins in the corona with enhanced or decreased cellular uptake of nanoparticles into human cancer and bone marrow stem cells to identify key candidates. Finally, we verified these candidate proteins by artificially decorating nanoparticles with individual proteins showing that nanoparticles precoated with the apolipoproteins ApoA4 or ApoC3 significantly decreased the cellular uptake, whereas precoating with ApoH increased the cellular uptake.

Enhanced in vivo targeting of murine nonparenchymal liver cells with monophosphoryl lipid A functionalized microcapsules.

A broad spectrum of infectious liver diseases emphasizes the need of microparticles for targeted delivery of immunomodulatory substances to the liver. Microcapsules (MCs) are particularly attractive for innovative drug and vaccine formulations, enabling the combination of antigen, drugs, and adjuvants. The present study aimed to develop microcapsules characterized by an enhanced liver deposition and accelerated uptake by nonparenchymal liver cells (NPCs). Initially, two formulations of biodegradable microcapsules were synthesized from either hydroxyethyl starch (HES) or mannose. Notably, HES-MCs accumulated primarily in the liver, while mannose particles displayed a lung preference. Functionalization of HES-MCs with anti-CD40, anti-DEC205, and/or monophosphoryl lipid A (MPLA) enhanced uptake of MCs by nonparenchymal liver cells in vitro. In contrast, only MPLA-coated HES-MCs promoted significantly the in vivo uptake by NPCs. Finally, HES-MCs equipped with MPLA, anti-CD40, and anti-DEC205 induced the secretion of TNF-α, IL-6 by Kupffer cells (KCs), and IFN-γ and IL-12p70 by liver dendritic cells (DCs). The enhanced uptake and activation of KCs by MPLA-HES-MCs is a promising approach to prevent or treat infection, since KCs are exploited as an entry gate in various infectious diseases, such as malaria. In parallel, loading and activating liver DCs, usually prone to tolerance, bears the potential to induce antigen specific, intrahepatic immune responses necessary to prevent and treat infections affecting the liver.

Synthesis and surface immobilization of antibacterial hybrid silver-poly(l-lactide) nanoparticles.

Infections associated with medical devices are a substantial healthcare problem. Consequently, there has been increasing research and technological efforts directed toward the development of coatings that are capable of preventing bacterial colonization of the device surface. Herein, we report on novel hybrid silver loaded poly(L-lactic acid) nanoparticles (PLLA-AgNPs) with narrowly distributed sizes (17 ± 3 nm) prepared using a combination of solvent evaporation and mini-emulsion technology. These particles were then immobilized onto solid surfaces premodified with a thin layer of allylamine plasma polymer (AApp). The antibacterial efficacy of the PLLA-AgNPs nanoparticles was studied in vitro against both gram-positive (Staphylococcus epidermidis) and gram-negative (Escherichia coli) bacteria. The minimal inhibitory concentration values against Staphylococcus epidermidis and Escherichia coli were 0.610 and 1.156 µg · mL(-1), respectively. The capacity of the prepared coatings to prevent bacterial surface colonization was assessed in the presence of Staphylococcus epidermidis, which is a strong biofilm former that causes substantial problems with medical device associated infections. The level of inhibition of bacterial growth was 98%. The substrate independent nature and the high antibacterial efficacy of coatings presented in this study may offer new alternatives for antibacterial coatings for medical devices.

Enzymatic degradation of poly(L-lactide) nanoparticles followed by the release of octenidine and their bactericidal effects.

The enzyme-triggered release of the antimicrobial agent octenidine out of poly(l-lactide)-based nanoparticles (PLLA-NPs) and their in vitro antibacterial activities in the presence of gram-positive and gram-negative bacteria are presented. The formation of the nanoparticles was achieved using a combination of the solvent evaporation and the miniemulsion approach. For the stabilization of the polymeric nanoparticles, non-ionic polymers (polyvinylalcohol [PVA], hydroxyethyl starch [HES], human serum albumin [HSA]) were successfully used for enzymatic degradation; ionic surfactants such as sodium dodecyl sulfate and cetyltrimethylammonium chloride inhibited the enzymatic degradation. The change in pH, size, size distribution and morphology during the degradation process of PLLA-NPs and the release of the antimicrobial agent was studied. The influence of the different amounts of octenidine and of the different stabilizers on the NPs' stability, size, size distribution, morphology, zeta potential and on the surface group's density is discussed. Fluorescently labeled HES-stabilized PLLA-NPs are immobilized by colloidal electrospinning. The observed data from HPLC measurements show that octenidine is released out of PLLA-NPs which are stabilized with PVA, HES or HSA. In bacteria tests the PLLA nanoparticles showed a greater ability to inhibit the growth of Staphylococcus aureus compared to Escherichia coli. FROM THE CLINICAL EDITOR: This article discusses the enzyme-triggered release and antibacterial effects of octenidine from poly(l-lactide)-based nanoparticles demonstrating the viability of this approach for potential future antibacterial therapy.

Enzyme responsive hyaluronic acid nanocapsules containing polyhexanide and their exposure to bacteria to prevent infection.

Antibacterial nanodevices could bring coatings of plastic materials and wound dressings a big step forward if the release of the antibacterial agents could be triggered by the presence of the bacteria themselves. Here, we show that novel hyaluronic acid (HA)-based nanocapsules containing the antimicrobial agent polyhexanide are specifically cleaved in the presence of hyaluronidase, a factor of pathogenicity and invasion for bacteria like Staphylococcus aureus and Escherichia coli. This resulted in an efficient killing of the pathogenic bacteria by the antimicrobial agent. The formation of different polymeric nanocapsules was achieved through a polyaddition reaction in inverse miniemulsion. After the synthesis, the nanocapsules were transferred to an aqueous medium and investigated in terms of size, size distribution, functionality, and morphology using dynamic light scattering, zeta potential measurements and scanning electron microscopy. The enzyme triggered release of a model dye and the antimicrobial polyhexanide was monitored using fluorescence and UV spectroscopy. The stability of the nanocapsules in several biological media was tested and the interaction of nanocapsules with human serum protein was studied using isothermal titration calorimetry. The antibacterial effectiveness is demonstrated by determination of the antibacterial activity and determination of the minimal bactericidal concentration (MBC).

Nanocapsules generated out of a polymeric dexamethasone shell suppress the inflammatory response of liver macrophages.

Dexamethasone (DXM) is a synthetic glucocorticoid with anti-inflammatory properties. Targeted delivery of dexamethasone to inflammatory cells, e.g. macrophages and Kupffer cells represents a promising approach to minimize side effects. The aim of the present study was to induce a targeted transport of novel DXM-based biodegradable nanocapsules to phagocytic cells. Nanocapsules (NCs) consisting of a hydroxyethylated glucose polymer (hydroxyethyl starch, HES) shell with encapsulated DXM and NCs synthesized exclusively in inverse miniemulsion out of DXM were investigated. Non-parenchymal murine liver cells served as target cells. HES-DXM NCs were predominantly incorporated by Kupffer cells (KCs). In contrast, DXM NCs were phagocytized by KCs and endothelial cells. The release of the NC-content was confirmed by incorporation of CellTracker™ into the NCs. Uptake of DXM NCs by Kupffer cells reduced significantly the release of inflammatory cytokines in response to LPS stimulation. Importantly, the DXM NCs consisting exclusively out of a dexamethasone shell offer the potential to serve as carriers for additional therapeutics. FROM THE CLINICAL EDITOR: In this paper, nanocapsule-based targeted delivery of dexamethasone to inflammatory cells is presented as a promising approach to minimize side effects and increase efficacy of this anti-inflammatory clinically used corticosteroid.

Suppressing unspecific cell uptake for targeted delivery using hydroxyethyl starch nanocapsules.

Synthesizing nanocarriers with stealth properties and delivering a "payload" to the particular organ remains a big challenge but is the prime prerequisite for any in vivo application. As a nontoxic alternative to the modification by poly(ethylene glycol) PEG, we describe the synthesis of cross-linked hydroxyethyl starch (HES, M(w) 200,000 g/mol) nanocapsules with a size range of 170-300 nm, which do not show nonspecific uptake into cells. The specific uptake was shown by coupling a folic acid conjugate as a model targeting agent onto the surface of the nanocapsules, because folic acid has a high affinity to a variety of human carcinoma cell lines which overexpress the folate receptor on the cell surface. The covalent binding of the folic acid conjugate onto HES capsules was confirmed by FTIR and NMR spectroscopy. The coupling efficiency was determined using fluorescence spectroscopy. The specific cellular uptake of the HES nanocapsules after folic acid coupling into the folate-receptor presenting cells was studied by confocal laser scanning microscopy (CLSM) and flow cytometry.

Cross-linked starch capsules containing dsDNA prepared in inverse miniemulsion as "nanoreactors" for polymerase chain reaction.

Cross-linked potato starch nanocapsules with encapsulated dsDNA (with a defined number of base pairs, i.e., 286, 476, and 790 bp) were synthesized using the miniemulsion technique. The inverse (water-in-oil) miniemulsion system was applied to create stable aqueous nanodroplets of dissolved starch in cyclohexane as a continuous phase. The amphiphilic block copolymer poly[(ethylene-co-butylene)-b-(ethylene oxide)] was used as a surfactant to stabilize the droplets. After addition of the cross-linker, 2,4-toluene diisocyanate (TDI), the polyaddition reaction took place at the droplet's interface, resulting in the formation of a polymeric cross-linked shell. The influence of starch, surfactant, and the amount of cross-linker on the average size, size distribution, and morphology of the capsules was studied by dynamic light scattering and electron microscopy. FTIR spectroscopy was used to identify the chemical composition of the capsule shell. The permeability of the shell was studied on the fluorescent dye (i.e., sulforhodamine 101) containing capsules using fluorescence spectroscopy. High thermal stability of the cross-linked capsules allows one to perform the polymerase chain reaction inside the core. The encapsulation of dsDNA and the efficiency of the PCR were confirmed by fluorescence spectroscopy after staining with the DNA-selective dye (SYBRGreen).

Enzyme-Polymer Conjugates to Enhance Enzyme Shelf Life in a Liquid Detergent Formulation.

Herein, the synthesis of enzyme-polymer conjugates is reported. Four different activated polymers (mPEG-aldehyde, mPEG-NHS, maltodextrin-aldehyde, carboxymethyl cellulose aldehyde) are conjugated to the surface of protease, α-amylase, and lipase using two different strategies (reductive amination and alkylation with NHS-activated acid). Although the chemical modification of the enzymes is accompanied by losses in enzyme activity (maximum loss 40%), the covalent attachment of polymers increases the thermal stability and the stability in a standard detergent formulation compared to the unmodified enzymes. The enzyme-polymer conjugates are characterized by asymmetrical-flow field-flow fractionation and differential scanning microcalorimetry. Furthermore, it is demonstrated that conjugated enzymes still show performance in a real washing process. Enzyme-polymer conjugates show a potential as a stabilizing system for enzymes in detergents.

Osmo-solidification of all-aqueous emulsion with enhanced preservation of protein activity.

The encapsulation of bio-active ingredients, such as proteins, in solidified particles via emulsion templating frequently induces an irreversible loss of bioactivity, because of the use of non-aqueous solvents and unfavorable conditions during the solidification process. Herein, we introduce an "osmo-solidification" approach that solidifies all-aqueous emulsion droplets by the osmotic extraction of water for encapsulating proteins and demonstrate the superior preservation of their activity. The osmo-solidification approach combines the solidification of droplets to particles and protein encapsulation in one step. Proteins encapsulated preserve their activity after osmo-solidification better than conventional solidification approaches. To the best of our knowledge, this is the first time that the osmo-solidification of all-aqueous emulsion is introduced in the fabrication of emulsion-based particles and that the stability of encapsulated proteins can be enhanced to an unprecedentedly high level.

Interleukin-2 Functionalized Nanocapsules for T Cell-Based Immunotherapy.

A major demand on immunotherapy is the direct interference with specific immune cells in vivo. In contrast to antibody-engineered nanoparticles to control dendritic cells function, targeting of T cells for biomedical applications still remains an obstacle as they disclose reduced endocytic activities. Here, by coupling the cytokine interleukin-2 (IL-2) to the surface of hydroxyethyl starch nanocapsules, we demonstrated a direct and specifc T cell targeting in vitro and in vivo by IL-2 receptor-mediated internalization. For this purpose, defined amounts of azide-functionalized IL-2 were linked to alkyne-functionalized hydroxyethyl starch nanocapsules via copper-free click reactions. In combination with validated quantification of the surface-linked IL-2 with anthracen azide, this method allowed for engineering IL-2-functionalized nanocapsules for an efficient targeting of human and murine T cell populations with various IL-2 receptor affinities. This nanocapsule-mediated technique is a promising strategy for T cell-based immunotherapies and may be translated to other cytokine-related targeting systems.

Heparin-based nanocapsules as potential drug delivery systems.

Herein, the synthesis and characterization of heparin-based nanocapsules (NCs) as potential drug delivery systems is described. For the synthesis of the heparin-based NCs, the versatile method of miniemulsion polymerization at the droplets interface was achieved resulting in narrowly distributed NCs with 180 nm in diameter. Scanning and transmission electron microscopy images showed clearly NC morphology. A highly negative charge density for the heparin-based NCs was determined by measuring the electro-kinetic potential. Measuring the activated clotting time demonstrated the biological intactness of the polymeric shell. The ability of heparin-based NCs to bind to antithrombin (AT III) was investigated using isothermal titration calorimetry and dynamic light scattering experiments. The chemical stability of the NCs was studied in physiological protein-containing solutions and also in medically interesting fluids such as sodium chloride 0.9%, Ringer's solution, and phosphate buffer saline using dynamic light scattering and measuring the fluorescence intensity. The impressive uptake of NCs in different cells was confirmed by fluorescence-activated cell sorting, confocal laser scanning microscopy, and transmission electron microscopy. The low toxicity of all types of NCs was demonstrated.

Attachment of Poly(l-lactide) Nanoparticles to Plasma-Treated Non-Woven Polymer Fabrics Using Inkjet Printing.

Active dressings that based on fabric materials are an area of interest for the treatment of wounds. Poly(l-lactide) nanoparticles containing the antimicrobial agent octenidine can be controllably lysed by toxins released by pathogenic bacteria thus releasing antimicrobial material in response to the presence of the bacterial toxins and so counteracting the infection. We developed an integrated engineering solution that allows for the stable immobilisation of nanoparticles on non-woven fabrics. The process involves coating nanoparticles on non-woven polymer surfaces by using an inkjet printing process. In order to improve the adhesion and retention of the nanoparticles on the fabric, surface pretreatment of the non-woven fabric using plasma jet treatment can be applied to increase its surface energy.

Monophosphoryl lipid A coating of hydroxyethyl starch nanocapsules drastically increases uptake and maturation by dendritic cells while minimizing the adjuvant dosage.

Enhancing delivery of antigens to dendritic cells (DCs) is essential for the induction of vigorous antigen-specific cellular immune responses. Aim of the present study was to evaluate the properties of hydroxyethyl starch nanocapsules (HES-NCs) functionalized with anti-CD40, anti-DEC205, interferon-γ (IFNγ) and/or monophosphoryl lipid A (MPLA) with respect to the overall uptake, the released cytokine profile, and the influence on phenotypic maturation of human monocyte-derived DCs using flow cytometry, confocal microscopy and enzyme-linked immunosorbent assays. NC uptake by DCs was significantly enhanced by functionalizing NCs with anti-CD40 or MPLA. With respect to the cytokine profile and the maturation status, coating with MPLA evoked a strong Th1-type cytokine response and significantly increased CD80 and CD83 expression on DCs, contrasting the moderate effects of MPLA in solution. Notably, an at least 20 fold higher amount of MPLA in solution was needed compared to the dosage of MPLA attached to HES-NCs in order to induce comparable effects, evidencing the intense dose-sparing potential of particle-bound MPLA. Reducing the amount of the vaccine adjuvant MPLA, while maintaining or even surpassing the effects on human DCs, reveals the potential of HES-NCs as a promising carrier system for the simultaneous delivery of antigen along with compounds promoting a Th1-prone cellular immune response.

Enzyme-responsive nanocomposites for wound infection prophylaxis in burn management: in vitro evaluation of their compatibility with healing processes.

Responsive, theranostic nanosystems, capable of both signaling and treating wound infections, is a sophisticated approach to reduce the most common and potentially traumatizing side effects of burn wound treatment: slowed wound healing due to prophylactic anti-infective drug exposure as well as frequent painful dressing changes. Antimicrobials as well as dye molecules have been incorporated into biodegradable nanosystems that release their content only in the presence of pathogens. Following nanocarrier degradation by bacterial enzymes, any infection will thus emit a visible signal and be effectively treated at its source. In this study, we investigated the effect of fluorescent-labeled hyaluronan nanocapsules containing polyhexanide biguanide and poly-L-lactic acid nanoparticles loaded with octenidine on primary human dermal microvascular endothelial cells, which play a major role in cutaneous wound healing. Microscopic and flow cytometric analysis indicated a time-dependent uptake of both the nanocapsules and the nanoparticles. However, enzyme immunoassays showed no significant influence on the expression of pro-inflammatory cell adhesion molecules and cytokines by the endothelial cells. Under angiogenic-stimulating conditions, the potential to form capillary-like structures in co-culture with dermal fibroblasts was not inhibited. Furthermore, cytotoxicity studies (the MTS and crystal violet assay) after short- and long-term exposure to the materials demonstrated that both systems exhibited less toxicity than solutions of the antiseptic agents alone in comparable concentrations. The results indicate that responsive antimicrobial nanocomposites could be used as an advanced drug delivery system and a promising addition to current best practice wound infection prophylaxis with few side effects.

Pharmacokinetics on a microscale: visualizing Cy5-labeled oligonucleotide release from poly(n-butylcyanoacrylate) nanocapsules in cells.

For successful design of a nanoparticulate drug delivery system, the fate of the carrier and cargo need to be followed. In this work, we fluorescently labeled poly(n-butylcyanoacrylate) (PBCA) nanocapsules as a shell and separately an oligonucleotide (20 mer) as a payload. The nanocapsules were formed by interfacial anionic polymerization on aqueous droplets generated by an inverse miniemulsion process. After uptake, the PBCA capsules were shown to be round-shaped, endosomal structures and the payload was successfully released. Cy5-labeled oligonucleotides accumulated at the mitochondrial membrane due to a combination of the high mitochondrial membrane potential and the specific molecular structure of Cy5. The specificity of this accumulation at the mitochondria was shown as the uncoupler dinitrophenol rapidly diminished the accumulation of the Cy5-labeled oligonucleotide. Importantly, a fluorescence resonance energy transfer investigation showed that the dye-labeled cargo (Cy3/Cy5-labeled oligonucleotides) reached its target site without degradation during escape from an endosomal compartment to the cytoplasm. The time course of accumulation of fluorescent signals at the mitochondria was determined by evaluating the colocalization of Cy5-labeled oligonucleotides and mitochondrial markers for up to 48 hours. As oligonucleotides are an ideal model system for small interfering RNA PBCA nanocapsules demonstrate to be a versatile delivery platform for small interfering RNA to treat a variety of diseases.

Paclitaxel-loaded polyphosphate nanoparticles: a potential strategy for bone cancer treatment.

While it has been shown that phosphates can target molecules and nanocarriers to bone we herein demonstrate the preparation of polyphosphate nanoparticles loaded with paclitaxel using a simple miniemulsion/solvent-evaporation technique as a model for chemotherapeutic delivery. Polyphosphates exhibit much higher structural versatility, relying on the pentavalence of the phosphorus center compared to conventional polyesters. This versatility allows for the development of new degradable polymeric carriers with inherent bone adhesion ability by the interaction of the nanoparticles with a calcium phosphate material used for bone regeneration. The novel polyphosphate nanoparticles were investigated in detail with respect to their size distribution, zeta-potential, thermal and morphological properties and were further proven to be efficiently loaded with a hydrophobic drug (up to 15 wt%). The in vitro cytotoxicity was assessed against human cancer cell lines (HeLa and Saos-2), and the paclitaxel-loaded nanoparticles showed a similar cytotoxicity profile similar to the commercially available formulation Taxomedac® and the pure paclitaxel for loading ratios of 10 wt% but additionally proved efficient adhesion on calcium phosphate granules allowing drug delivery to bone. This first report demonstrates that polyphosphate nanoparticles are promising materials for the development of systemic or local bone cancer treatment, even by direct application or by formation of composites with calcium phosphate cements.

Synthesis and antibacterial properties of a hybrid of silver-potato starch nanocapsules by miniemulsion/polyaddition polymerization.

Controlled synthesis of hollow polymeric nanocapsules has attracted significant attention in a wide range of applications. This paper reports a facile method for the synthesis of hybrid starch nanocapsules decorated with silver nanoparticles using the inverse miniemulsion polyaddition technique. Silver nanoparticles are formed and embedded in the shell of the nanocapsules during the polyaddition process without using any additional reducing agents. We found that silver also acts as a lipophobe that builds up osmotic pressure in the droplets facilitating the formation of stable round shaped nanocapsules. The nanocapsules' shell thickness could be tuned from 13 to 29 nm by varying the amount of cross-linker. We investigated the minimum inhibitory concentration (MIC) of the nanocapsules against Staphylococcus epidermidis ATCC 35984 and Escherichia coli ATCC 25922 which are two bacteria of medical relevance. The silver nanoparticle decorated nanocapsules showed antibacterial properties against both bacteria at the MIC of 2.315 µg mL-1 while control nanocapsules without silver had no antibacterial activity.

Towards copper-free nanocapsules obtained by orthogonal interfacial "click" polymerization in miniemulsion.

A facile method to produce nanocapsules by copper-free interfacial "click"-polymerization as orthogonal reaction for the encapsulation of functional molecules is successfully performed using stable miniemulsion droplets. Difunctional azides and alkynes have been used for polymerization around the miniemulsion droplets, leading to the formation of nanocapsules. The results were compared with copper-catalyzed systems.