Tip-Loaded Dissolvable Microneedle Arrays Effectively Deliver Polymer-Conjugated Antibody Inhibitors of Tumor-Necrosis-Factor-Alpha Into Human Skin.

Autoinflammatory skin diseases are characterized by a disequilibrium of cytokines in the local skin microenvironment, suggesting that local delivery of therapeutics, including anticytokine antibodies, may provide benefit without the unwanted off-target effects of systemically delivered therapies. Rapid diffusion of therapeutics away from the target site has been a challenge to the development of local therapies. Conjugation of high molecular weight hydrophilic polymers to cytokine neutralizing mAbs has been shown to be an effective strategy for local control of inflammation in healing burn wounds. However, the burn application is unique because the skin barrier is already breached. For the treatment of autoinflammatory skin diseases, the major challenge for local delivery lies in penetrating the stratum corneum. Here, we investigate a new therapeutic approach combining the use of tip-loaded dissolvable microneedle arrays (TL-dMNAs) for local application of polymer-conjugated antibody inhibitors of tumor-necrosis-factor-alpha (TNF-α). Specifically, intradermal delivery and pharmacokinetics of (anti-TNF-α-Ab)-(high molecular weight hyaluronic acid [HA]) conjugates from tip-loaded, obelisk-shaped dissolvable microneedle arrays were investigated in living human skin. The results indicate (1) TL-dMNAs can be successfully fabricated to integrate (anti-TNF-α-Ab)-HA at the tip portion of the microneedles while preserving the biological activity necessary for antibody ligand binding; (2) (anti-TNF-α-Ab)-HA can be effectively delivered into human skin using obelisk-shaped TL-dMNAs; and (3) polymer conjugation effectively inhibits antibody diffusion from the delivery site. Taken together, these results support the evaluation of microneedle array-based delivery of varying polymer-antibody conjugates for the treatment of inflammatory skin diseases.

Three-dimensional printing of complex biological structures by freeform reversible embedding of suspended hydrogels.

We demonstrate the additive manufacturing of complex three-dimensional (3D) biological structures using soft protein and polysaccharide hydrogels that are challenging or impossible to create using traditional fabrication approaches. These structures are built by embedding the printed hydrogel within a secondary hydrogel that serves as a temporary, thermoreversible, and biocompatible support. This process, termed freeform reversible embedding of suspended hydrogels, enables 3D printing of hydrated materials with an elastic modulus <500 kPa including alginate, collagen, and fibrin. Computer-aided design models of 3D optical, computed tomography, and magnetic resonance imaging data were 3D printed at a resolution of ~200 µm and at low cost by leveraging open-source hardware and software tools. Proof-of-concept structures based on femurs, branched coronary arteries, trabeculated embryonic hearts, and human brains were mechanically robust and recreated complex 3D internal and external anatomical architectures.

Therapeutic intradermal delivery of tumor necrosis factor-alpha antibodies using tip-loaded dissolvable microneedle arrays.

Tumor necrosis factor-alpha (TNF-α) specific antibodies (anti-TNF-α Ab) have been shown to be potent TNF inhibitors and effective therapeutics for a range of inflammatory diseases. Typically, these drugs are administered systemically, but systemic dosing sufficient to achieve locally effective concentrations in peripheral tissues has been associated with systemic immunosuppression and related adverse events. Here, we evaluated the use of tip-loaded dissolvable microneedle arrays (MNAs) for localized intradermal delivery of anti-TNF-α Ab. MNAs with obelisk shape microneedles that incorporate the antibody cargo in the needle tips were created from carboxymethylcellulose (CMC) using a micromilling/spin-casting fabrication method. We found that anti-TNF-α Ab integrated into MNAs using this room temperature fabrication process maintained conformationally dependent TNF-α binding activity. Further, these MNAs efficiently delivered anti-TNF-α antibodies to the dermis of human skin with clinically applicable release profiles. To evaluate MNA delivered anti-TNF-α Ab function, we applied anti-TNF-α Ab containing MNAs to established psoriasiform lesions on the skin of mice. MNA anti-TNF-α Ab treatment reduced key biomarkers of psoriasiform inflammation including epidermal thickness and IL-1ß expression. Taken together, these results demonstrate efficient and biologically effective MNA delivery of anti-TNF-α Ab to the intradermal microenvironment of the skin in mice and humans, and support the development of MNA mediated antibody delivery for clinical applications. STATEMENT OF SIGNIFICANCE: Tumor necrosis factor-alpha (TNF-α) specific antibodies (anti-TNF-α Ab) have been shown to be potent TNF inhibitors and effective therapeutics for a range of inflammatory diseases. Typically, these drugs are administered systemically, but systemic dosing sufficient to achieve locally effective concentrations in peripheral tissues has been associated with systemic immunosuppression and related adverse events. Here we demonstrate efficient and biologically effective MNA delivery of anti-TNF-α Ab to the intradermal microenvironment of the skin in mice and humans. These results support the development of MNA mediated antibody delivery of therapeutic antibodies for clinical applications.

Reducing protein adsorption with polymer-grafted hyaluronic acid coatings.

We report a thermoresponsive chemical modification strategy of hyaluronic acid (HA) for coating onto a broad range of biomaterials without relying on chemical functionalization of the surface. Poly(di(ethylene glycol) methyl ether methacrylate) (PMEO2MA), a polymer with a lower critical solution temperature of 26 °C in water, was grafted onto HA to allow facile formation of biopolymer coatings. While the mechanism for film formation appears to involve a complex combination of homogeneous nucleation followed by heterogeneous film growth, we demonstrate that it resulted in hydrophilic coatings that significantly reduce protein adsorption despite the high fraction of hydrophobic (PMEO2MA). Structural characterization was performed using atomic force microscopy (AFM), which showed the formation of a dense, continuous coating based on 200 nm domains that were stable in protein solutions for at least 15 days. The coatings had a water contact angle of 16°, suggesting the formation of hydrophilic but not fully wetting films. Quartz crystal microbalance with dissipation monitoring (QCM-D) as well as biolayer interferometry (BLI) techniques were used to measure adsorption of bovine serum albumin (BSA), fibrinogen (Fbg), and human immunoglobulin (IgG), with results indicating that HA-PMEO2MA-coated surfaces effectively inhibited adsorption of all three serum proteins. These results are consistent with previous studies demonstrating that this degree of hydrophilicity is sufficient to generate an effectively nonfouling surface and suggest that segregation during the solubility transition resulted in a surface that presented the hydrophilic HA component of the hybrid biopolymer. We conclude that PMEO2MA-grafted HA is a versatile platform for the passivation of hydrophobic biomaterial surfaces without need for substrate functionalization.

Polymer-conjugated inhibitors of tumor necrosis factor-α for local control of inflammation.

Burns, chronic wounds, osteoarthritis, and uveitis are examples of conditions characterized by local, intense inflammatory responses that can impede healing or even further tissue degradation. The most powerful anti-inflammatory drugs available are often administered systemically, but these carry significant side effects and are not compatible for patients that have underlying complications associated with their condition. Conjugation of monoclonal antibodies that neutralize pro-inflammatory cytokines to high molecular weight hydrophilic polymers has been shown to be an effective strategy for local control of inflammation. Lead formulations are based on antibody inhibitors of tumor necrosis factor-α conjugated to hyaluronic acid having molecular weight greater than 1 MDa. This review will discuss fundamental aspects of medical conditions that could be treated with these conjugates and design principles for preparing these cytokine-neutralizing polymer conjugates. Results demonstrating that infliximab, an approved inhibitor of tumor necrosis factor-α, can be incorporated into the conjugates using a broad range of water-soluble polymers are also presented, along with a prospectus for clinical translation.

Iron chelation by polyamidoamine dendrimers: a second-order kinetic model for metal-amine complexation.

This study presents a kinetic model of the chelation of iron ions by generation 4 hydroxyl-terminated polyamidoamine (PAMAM) with ethylenediamine core (G4-OH). The coordination processes of iron ions from ferric chloride, FeCl(3), and ferrous bromide, FeBr(2), to G4-OH dendrimers were analyzed using ultraviolet-visible (UV-vis) spectroscopy, proton nuclear magnetic resonance ((1)H NMR) spectroscopy, and liquid chromatography-mass spectrometry (LC-MS). In the visible region, a charge-transfer was observed when the dendrimer was added to a ferric chloride solution. This phenomenon is a ligand-to-metal charge-transfer (LMCT) between the free electron group of the dendrimer's internal amines and the dehalogenated iron ion that takes 2 h to complete at room temperature. Analysis of potential rate laws and diffusion effects led to a second-order kinetic model for this reaction. By measuring the rate coefficients as a function of temperature (22-37 °C), an apparent activation energy of 41.5 kJ/mol was obtained using the Arrhenius method. The results of this study will fuel research of PAMAM dendrimers for environmental, pharmaceutical, and materials applications.

Assessment of the waste stabilization pond performance, New Borg El-Arab City.

New Borg Al-Arab city includes 9 residential areas and 4 industrial zones. The four industrial zones are occupied by 266 running factories belonging to different industrial sectors. All the domestic sewage as well as the industrial wastewater is treated at an oxidation pond treatment plant and the plant effluent is used in irrigation of silviculture areas. The plant consists of 2 pond complexes. Each complex comprises 9 facultative ponds, arranged in 3 parallel series. Raw wastewater is pre-treated through screens and grit removal before entering the primary ponds. The study aims at assessment of the performance of the waste stabilization ponds. The results revealed that pre-treatment units were not functioning properly. The retention time was calculated to be 6 days in each pond of the first complex and 3 days in each pond of the second complex. The mean BOD surface loading rate was calculated to be 676 kg/ha/d for the first complex and 1584 kg/ha/d for the second. The plant was hydraulically and organically overloaded. The percentage removal of BOD, COD, SS, and NH3-N had an annual mean of 57%, 56%, 44%, and 39%, respectively for the first complex and 21%, 42%, 39%, and 25%, respectively for the second. Faecal coliforms were reduced by about 1 log10 unit in both complexes. The final pond effluent was not complying with the Egyptian law for reuse in irrigation. The study proposed some recommendations which can improve the efficiency of the plant as well as the effluent quality.

Assessment of pollution levels in fish and water of main basin, Lake Mariut.

Lake Mariut has suffered over the years from the untreated sewage, agricultural and industrial wastes dumped into it. Lake Mariut fish quality and quantity were adversely affected eventually being unfit for human consumption due to its poor water quality. The present study was carried out to evaluate the levels of metals in the fish caught from Lake Mariut main basin and its fitness for human consumption, and the water quality after the enforcement of the Egyptian Law for Environmental Protection (Law 4/1994). Fish and water samples were collected form Lake Mariut main basin through out the period from May to December 2000. The results showed that mercury was totally undetected in fish and water samples. The levels of Fe obtained were very low compared to previous studies. Fe and Cr contents in fish flesh were higher compared to their levels in water samples. Also Cu has shown a decrease in both fish and water samples. Zn and Cd contents in water samples have decreased compared with former studies. However, they have not changed in fish flesh. Pb content in both fish flesh and water samples has shown a great decrease compared to previous studies. The Cd and Pb mean values in fish flesh of the present study (0.81 and 0.14 mg/kg) exceeded the Egyptian Standards No. 2360/1993 value (should not exceed 0.1 mg/kg for each). The results also showed that the water quality has changed compared with previous studies. BOD has increased in north of Main Basin, east of Main Basin, South of Main Basin, and southwest of Main Basin; and deCreased in WTP effluent; and Qalaa Drain. There has been an increase in TSS, TVSS, and NO3 levels compared to pervious studies. There has been a decrease in TS, TDS, COD,hardness, and chloride levels. This decrease, especially in COD, may be attributed to the enforcement of the Egyptian Law 4/1994. The pH and alkalinity were in agreement with the values shown by previous studies except for east of Main Basin which showed higher values. The study included some recommendations aiming at improving both water quality and fish of the Main Basin.

Water treatment plants assessment at Talkha power plant.

Talkha power plant is the only power plant located in El-Mansoura. It generates electricity using two different methods by steam turbine and gas turbine. Both plants drew water from River Nile (208 m3 /h). The Nile raw water passes through different treatment processes to be suitable for drinking and operational uses. At Talkha power plant, there are two purification plants used for drinking water supply (100 m3/h) and for water demineralization supply (108 m3/h). This study aimed at studying the efficiency of the water purification plants. For drinking water purification plant, the annual River Nile water characterized by slightly alkaline pH (7.4-8), high annual mean values of turbidity (10.06 NTU), Standard Plate Count (SPC) (313.3 CFU/1 ml), total coliform (2717/100 ml), fecal coliform (0-2400/100 ml), and total algae (3 x 10(4) org/I). The dominant group of algae all over the study period was green algae. The blue green algae was abundant in Summer and Autumn seasons. The pH range, and the annual mean values of turbidity, TDS, total hardness, sulfates, chlorides, nitrates, nitrites, fluoride, and residual chlorine for purified water were in compliance with Egyptian drinking water standards. All the SPC recorded values with an annual mean value of 10.13 CFU/1 ml indicated that chlorine dose and contact time were not enough to kill the bacteria. However, they were in compliance with Egyptian decree (should not exceed 50 CFU/1 ml). Although the removal efficiency of the plant for total coliform and blue green algae was high (98.5% and 99.2%, respectively), the limits of the obtained results with an annual mean values of 40/100 ml and 15.6 org/l were not in compliance with the Egyptian decree (should be free from total coliform, fecal coliform and blue green algae). For water demineralization treatment plant, the raw water was characterized by slightly alkaline pH. The annual mean values of conductivity, turbidity, and TDS were 354.6 microS/cm, 10.84 NTU, and 214.6 mg/I, respectively. There was an increase in the results of conductivity, turbidity, total hardness, and TDS in carbon filter effluent which was attributed to the desorption of adsorbed ions on the carbon media. The removal efficiencies of turbidity, total hardness, and TDS indicated the high efficiency of the cationic filter. The annual removal efficiencies of conductivity, turbidity, chloride, and TDS proved the efficiency of the anionic filter for removing the dissolved and suspended ions. All of the recorded values of the pH, conductivity, turbidity, chlorides, hardness, and TDS of the mixed bed effluent indicated that the water at this stage was of high quality for boiler feed. The study recommended adjustment of coagulant and residual chlorine doses as well as contact time, and continuous monitoring and maintenance of the different units.

Quality monitoring of composting processes of wastewater sludge in Alexandria.

There are two wastewater primary treatment plants in Alexandria (west and east). The produced primary sludge is mechanically dewatered and transported to sludge disposal site 9N where composting is carried out. However, prior to 1970, composting played a very minor role in sludge or solid wastes treatment because of greatly unfavorable balance between its economics and those of the principal competing option, namely landfill disposal. This study aims at monitoring and evaluating the composting process of demitted sludge produced from Alexandria wastewater treatment plants. Ten batches of sludge were composted. During the composting process the batches were been investigated and followed up to 3 months. Representative samples (10 for each batch) were taken from these batches at the start of windrowing and after each turning (4-15 days) and were analyzed for physical; chemical; bacteriological; and parasitological characteristics, heavy metals, and plant nutrients. Results revealed that C/N ratio of the final compost product comply with the decree of the Minister of Agriculture No. 100 11967, while moisture, C%, and TKN did not. Heavy metals, faecal coliforms, and helminthes complied with the decree No. 222/2002 for the Minister of Housing, Utilities, and Urban Communities. Moisture had positive correlation with both C and VS and negative correlation with pH. Temperature had negative correlation with TKN. Both total and faecal coliforms had negative correlation with temperature and positive correlation with C, N, and VS. It is recommended to optimize the quality of the produced sludge compost by use bulking agent rich in carbon and nitrogen as Hay or Rice straw, instead of the matured sludge compost.