Surface antibody changes protein corona both in human and mouse serum but not final opsonization and elimination of targeted polymeric nanoparticles.

BACKGROUND: Nanoparticles represent one of the most important innovations in the medical field. Among nanocarriers, polymeric nanoparticles (PNPs) attracted much attention due to their biodegradability, biocompatibility, and capacity to increase efficacy and safety of encapsulated drugs. Another important improvement in the use of nanoparticles as delivery systems is the conjugation of a targeting agent that enables the nanoparticles to accumulate in a specific tissue. Despite these advantages, the clinical translation of therapeutic approaches based on nanoparticles is prevented by their interactions with blood proteins. In fact, the so-formed protein corona (PC) drastically alters the biological identity of the particles. Adsorbed activated proteins of the complement cascade play a pivotal role in the clearance of nanoparticles, making them more easily recognized by macrophages, leading to their rapid elimination from the bloodstream and limiting their efficacy. Since the mouse is the most used preclinical model for human disease, this work compared human and mouse PC formed on untargeted PNPs (uPNPs) and targeted PNPs (tPNPs), paying particular attention to complement activation. RESULTS: Mouse and human serum proteins adsorbed differently to PNPs. The differences in the binding of mouse complement proteins are minimal, whereas human complement components strongly distinguish the two particles. This is probably due to the human origin of the Fc portion of the antibody used as targeting agent on tPNPs. tPNPs and uPNPs mainly activate complement via the classical and alternative pathways, respectively, but this pattern did not affect their binding and internalization in macrophages and only a limited consumption of the activity of the human complement system was documented. CONCLUSIONS: The results clearly indicate the presence of complement proteins on PNPs surface but partially derived from an unspecific deposition rather than an effective complement activation. The presence of a targeting antibody favors the activation of the classical pathway, but its absence allows an increased activation of the alternative pathway. This results in similar opsonization of both PNPs and similar phagocytosis by macrophages, without an impairment of the activity of circulating complement system and, consequently, not enhancing the susceptibility to infection.

Glucosamine/L-lactide copolymers as potential carriers for the development of a sustained rifampicin release system using Mycobacterium smegmatis as a tuberculosis model.

The present study aims at developing a new, ultrafine particle-based efficient antibiotic delivery system for the treatment of tuberculosis. The carrier material to make the rifampicin (RIF)-loaded particles is a low molecular weight star-shaped polymer produced from glucosamine (core building unit) and L-lactide (GluN-LLA). Particles were made via electrohydrodynamic atomization. Prolonged release (for up to 14 days) of RIF from these particles is reported. Drug release data fits the Korsmeyer-Peppas equation, which suggests the occurrence of a modified diffusion-controlled RIF release mechanism in vitro and is also supported by differential scanning calorimetry and drug leaching tests. Cytotoxicity tests on Mycobacterium smegmatis showed that antibiotic-free GluN-LLA and polylactides (PLA) particles (reference materials) did not show any significant anti-bacterial activity. The minimum inhibitory concentration and minimum bactericidal concentration values obtained for RIF-loaded particles showed 2- to 4-fold improvements in the anti-bacterial activity relative to the free drug. Cytotoxicity tests on macrophages indicated that cell death correlates with an increase of particle concentration but is not significantly affected by material type or particle size. Confocal microscopy was used to track internalization and localization of particles in the macrophages. The uptake of GluN-LLA particles is higher than those of their PLA counterparts. In addition, after phagocytosis, the GluN-LLA particles stayed in the cytoplasm and showed favorable long-term drug release behavior, which facilitated the killing of intracellular bacteria when compared to free RIF. The present studies suggest that these drug carrier materials are potentially very attractive candidates for the development of high-payload, sustained-release antibiotic/resorbable polymer particle systems for treating bacterial lung infections.

Temozolomide Treatment Induces lncRNA MALAT1 in an NF-κB and p53 Codependent Manner in Glioblastoma.

Alkylating chemotherapy is a central component of the management of glioblastoma (GBM). Among the factors that regulate the response to alkylation damage, NF-κB acts to both promote and block cytotoxicity. In this study, we used genome-wide expression analysis in U87 GBM to identify NF-κB-dependent factors altered in response to temozolomide and found the long noncoding RNA (lncRNA) MALAT1 as one of the most significantly upregulated. In addition, we demonstrated that MALAT1 expression was coregulated by p50 (p105) and p53 via novel κB- and p53-binding sites in the proximal MALAT1 coding region. Temozolomide treatment inhibited p50 recruitment to its cognate element as a function of Ser329 phosphorylation while concomitantly increasing p53 recruitment. Moreover, luciferase reporter studies demonstrated that both κB and p53 cis-elements were required for efficient transactivation in response to temozolomide. Depletion of MALAT1 sensitized patient-derived GBM cells to temozolomide cytotoxicity, and in vivo delivery of nanoparticle-encapsulated anti-MALAT1 siRNA increased the efficacy of temozolomide in mice bearing intracranial GBM xenografts. Despite these observations, in situ hybridization of GBM specimens and analysis of publicly available datasets revealed that MALAT1 expression within GBM tissue was not prognostic of overall survival. Together, these findings support MALAT1 as a target for chemosensitization of GBM and identify p50 and p52 as primary regulators of this ncRNA. SIGNIFICANCE: These findings identify NF-κB and p53 as regulators of the lncRNA MALAT1 and suggest MALAT1 as a potential target for the chemosensitization of GBM.

Fluid administration rate for uncontrolled intraabdominal hemorrhage in swine.

BACKGROUND: We hypothesized that slow crystalloid resuscitation would result in less blood loss and a smaller hemoglobin decrease compared to a rapid resuscitation during uncontrolled hemorrhage. METHODS: Anesthetized, splenectomized domestic swine underwent hepatic lobar hemitransection. Lactated Ringers was given at 150 or 20 mL/min IV (rapid vs. slow, respectively, N = 12 per group; limit of 100 mL/kg). Primary endpoints were blood loss and serum hemoglobin; secondary endpoints included survival, vital signs, coagulation parameters, and blood gases. RESULTS: The slow group had a less blood loss (1.6 vs. 2.7 L, respectively) and a higher final hemoglobin concentration (6.0 vs. 3.4 g/dL). CONCLUSIONS: Using a fixed volume of crystalloid resuscitation in this porcine model of uncontrolled intraabdominal hemorrhage, a slow IV infusion rate produced less blood loss and a smaller hemoglobin decrease compared to rapid infusion.

Convection-Enhanced Delivery of Polymeric Nanoparticles Encapsulating Chemotherapy in Canines with Spontaneous Supratentorial Tumors.

BACKGROUND: Despite aggressive multimodal treatment, survival for patients with glioblastoma remains dismal. One obstacle to improving patient outcomes is the difficulty in delivering adequate therapeutic to the central nervous system due to the presence of the blood-brain barrier. Although direct drug infusion by convection-enhanced delivery (CED) can bypass the blood-brain barrier and facilitate delivery to intracranial tumors, determining the distribution of delivered therapeutic remains problematic. Image guidance is a strategy that can optimize the accuracy of therapeutic delivery. METHODS: Here we performed an open-label clinical trial in 10 pet dogs with spontaneous intracranial tumors to examine the target coverage accuracy of delivering polymeric magnetite nanoparticles (PMNPs) encapsulating temozolomide (TMZ). A modified small animal frame was applied to the head of each subject, and PMNPs were delivered stereotactically to the center of the tumor. Magnetic resonance imaging (MRI) was performed immediately postoperatively to examine PMNP distribution, and the animals were followed until death. RESULTS: Nine of the 10 dogs underwent PMNP infusion without complications. No infusate backflow was observed during any procedure. In 70% of the cases, the infusion accurately targeted the tumor mass, as determined by the presence of PMNP signal in the tumor on immediate postoperative MRI. CONCLUSIONS: These data suggest that CED of PMNPs carrying TMZ is safe in dogs with intracranial tumors and can lead to nanoparticle distribution in the region of the target. Image guidance is an important adjunct to CED, because distribution is unpredictable, with the potential for missed target delivery.

Preparation and characterization of functionalized heparin-loaded poly-Ɛ-caprolactone fibrous mats to prevent infection with human papillomaviruses.

In this study, heparin-loaded poly-ɛ-caprolactone (PCL) fibrous mats were prepared and characterized based on their physical, cytotoxic, thermal, and biological properties. The main objective of the work described here was to test the hypothesis that incorporation of heparin into a PCL carrier could serve as bio-compatible material capable of inhibiting Human Papillomavirus (HPV) infection. The idea of firmly anchoring heparin to capture soluble virus, vs. a slow heparin release to inhibit a virus in solution was tested. Thus, one material was produced via conventional heparin matrix encapsulation and electrohydrodynamic fiber processing in one step. A second type of material was obtained via heparin crosslinking. This was achieved by running a carbodiimide/N-hydroxysuccinimide (EDC/NHS) coupling reaction on preformed PCL fibers. In vitro HPV16 L1 protein binding capacity studies were performed. Infectivity assays were done using HPV16 pseudoviruses (PsVs) carrying a GFP plasmid to directly test the ability of the fibrous mats to prevent internalization of HPV PsVs. The crosslinked heparin material presented a dissociation constant (Kd) value comparable to those found in the literature for different heparin-protein L1 peptide interactions. Both materials significantly reduced internalization of HPV PsVs, with a reduction of 94% of PsVs internalization when matrix encapsulated heparin-loaded material was present. Differences in performance between the two proposed structures are discussed.

Decoy Receptor DcR1 Is Induced in a p50/Bcl3-Dependent Manner and Attenuates the Efficacy of Temozolomide.

Temozolomide is used widely to treat malignant glioma, but the overall response to this agent is generally poor. Resistance to DNA-damaging drugs such as temozolomide has been related to the induction of antiapoptotic proteins. Specifically, the transcription factor NF-κB has been suggested to participate in promoting the survival of cells exposed to chemotherapy. To identify factors that modulate cytotoxicity in the setting of DNA damage, we used an unbiased strategy to examine the NF-κB-dependent expression profile induced by temozolomide. By this route, we defined the decoy receptor DcR1 as a temozolomide response gene induced by a mechanism relying upon p50/NF-κB1. A conserved NF-κB-binding sequence (κB-site) was identified in the proximal promoter and was demonstrated to be required for DcR1 induction by temozolomide. Loss-of-function and gain-of-function studies reveal that the atypical IκB protein, Bcl3, is also required for induction of DcR1 by temozolomide. Mechanistically, DcR1 attenuates temozolomide efficacy by blunting activation of the Fas receptor pathway in p53(+/+) glioma cells. Intracranial xenograft studies show that DcR1 depletion in glioma cells enhances the efficacy of temozolomide. Taken together, our results show how DcR1 upregulation mediates temozolomide resistance and provide a rationale for DcR1 targeting as a strategy to sensitize gliomas to this widely used chemotherapy.

Development of a fatal noncompressible truncal hemorrhage model with combined hepatic and portal venous injury in normothermic normovolemic swine.

Noncompressible truncal hemorrhage and brain injury currently account for most early mortality of warfighters on the battlefield. There is no effective treatment for noncompressible truncal hemorrhage, other than rapid evacuation to a surgical facility. The availability of an effective field treatment for noncompressible truncal hemorrhage could increase the number of warfighters salvaged from this frequently-lethal scenario. Our intent was to develop a porcine model of noncompressible truncal hemorrhage with a ∼ 50% one-hour mortality so that we could develop new treatments for this difficult problem. Normovolemic normothermic domestic swine (barrows, 3 months old, 34-36 kg) underwent one of three injury types through a midline incision: 1) central stellate injury (N = 6); 2) excision of a portal vein branch distal to the main PV trunk (N = 6); or 3) hemi-transection of the left lateral lobe of the liver at its base (N = 10). The one-hour mortality of these injuries was 0, 82, and 40%, respectively; the final mean arterial pressure was 65, 24, and 30 mm Hg, respectively; and the final hemoglobin was 8.3, 2.3, and 3.6 g/dL, respectively. Hemi-transection of the left lateral lobe of the liver appeared to target our desired mortality rate better than the other injury mechanisms.

Convection-enhanced delivery and in vivo imaging of polymeric nanoparticles for the treatment of malignant glioma.

A major obstacle to the management of malignant glioma is the inability to effectively deliver therapeutic agent to the tumor. In this study, we describe a polymeric nanoparticle vector that not only delivers viable therapeutic, but can also be tracked in vivo using MRI. Nanoparticles, produced by a non-emulsion technique, were fabricated to carry iron oxide within the shell and the chemotherapeutic agent, temozolomide (TMZ), as the payload. Nanoparticle properties were characterized and subsequently their endocytosis-mediated uptake by glioma cells was demonstrated. Convection-enhanced delivery (CED) can disperse nanoparticles through the rodent brain and their distribution is accurately visualized by MRI. Infusion of nanoparticles does not result in observable animal toxicity relative to control. CED of TMZ-bearing nanoparticles prolongs the survival of animals with intracranial xenografts compared to control. In conclusion, the described nanoparticle vector represents a unique multifunctional platform that can be used for image-guided treatment of malignant glioma. FROM THE CLINICAL EDITOR: GBM remains one of the most notoriously treatment-unresponsive cancer types. In this study, a multifunctional nanoparticle-based temozolomide delivery system was demonstrated to possess enhanced treatment efficacy in a rodent xenograft GBM model, with the added benefit of MRI-based tracking via the incorporation of iron oxide as a T2* contrast material in the nanoparticles.

New potential therapeutic approach for the treatment of B-Cell malignancies using chlorambucil/hydroxychloroquine-loaded anti-CD20 nanoparticles.

Current B-cell disorder treatments take advantage of dose-intensive chemotherapy regimens and immunotherapy via use of monoclonal antibodies. Unfortunately, they may lead to insufficient tumor distribution of therapeutic agents, and often cause adverse effects on patients. In this contribution, we propose a novel therapeutic approach in which relatively high doses of Hydroxychloroquine and Chlorambucil were loaded into biodegradable nanoparticles coated with an anti-CD20 antibody. We demonstrate their ability to effectively target and internalize in tumor B-cells. Moreover, these nanoparticles were able to kill not only p53 mutated/deleted lymphoma cell lines expressing a low amount of CD20, but also circulating primary cells purified from chronic lymphocitic leukemia patients. Their safety was demonstrated in healthy mice, and their therapeutic effects in a new model of Burkitt's lymphoma. The latter serves as a prototype of an aggressive lympho-proliferative disease. In vitro and in vivo data showed the ability of anti-CD20 nanoparticles loaded with Hydroxychloroquine and Chlorambucil to increase tumor cell killing in comparison to free cytotoxic agents or Rituximab. These results shed light on the potential of anti-CD20 nanoparticles carrying Hydroxychloroquine and Chlorambucil for controlling a disseminated model of aggressive lymphoma, and lend credence to the idea of adopting this therapeutic approach for the treatment of B-cell disorders.

Effect of fiber diameter on the spreading, proliferation and differentiation of chondrocytes on electrospun chitosan matrices.

Tissue-engineered neocartilage with appropriate biomechanical properties holds promise not only for graft applications but also as a model system for controlled studies of chondrogenesis. Our objective in the present research study is to better understand the impact of fiber diameter on the cellular activity of chondrocytes cultured on nanofibrous matrices. By using the electrospinning process, fibrous scaffolds with fiber diameters ranging from 300 nm to 1 µm were prepared and the physicomechanical properties of the scaffolds were characterized. Bovine articular chondrocytes were then seeded and maintained on the scaffolds for 7 and 14 days in culture. An upregulation in the gene expression of collagen II was noted with decreasing fiber diameters. For cells that were cultured on scaffolds with a mean fiber diameter of 300 nm, a 2-fold higher ratio of collagen II/collagen I was noted when compared to cells cultured on sponge-like scaffolds prepared by freeze drying and lyophilization. Integrin (α(5), αv, ß(1)) gene expression was also observed to be influenced by matrix morphology. Our combined results suggest that matrix geometry can regulate and promote the retention of the chondrocyte genotype.

Could metabolic syndrome, lipodystrophy, and aging be mesenchymal stem cell exhaustion syndromes?

One of the most important and complex diseases of modern society is metabolic syndrome. This syndrome has not been completely understood, and therefore an effective treatment is not available yet. We propose a possible stem cell mechanism involved in the development of metabolic syndrome. This way of thinking lets us consider also other significant pathologies that could have similar etiopathogenic pathways, like lipodystrophic syndromes, progeria, and aging. All these clinical situations could be the consequence of a progressive and persistent stem cell exhaustion syndrome (SCES). The main outcome of this SCES would be an irreversible loss of the effective regenerative mesenchymal stem cells (MSCs) pools. In this way, the normal repairing capacities of the organism could become inefficient. Our point of view could open the possibility for a new strategy of treatment in metabolic syndrome, lipodystrophic syndromes, progeria, and even aging: stem cell therapies.

Exploratory study on the effects of biodegradable nanoparticles with drugs on malignant B cells and on a human/mouse model of Burkitt lymphoma.

The aim of this study was to determine if Rituximab coated Biodegradable Nanoparticles (BNPs) loaded with Chlorambucil and Hydroxychloroquine could induce apoptosis of B-Chronic Lymphocytic Leukemia (B-CLL), MEC-1 and BJAB cells in vitro and evaluate their toxic and therapeutic effects on a Human/Mouse Model of Burkitt Lymphoma at an exploratory, proof of concept scale. We found that Rituximab-Chlorambucil-Hydroxychloroquine BNPs induce a decrease in cell viability of malignant B cells in a dose-dependent manner. The mediated cytotoxicity resulted from apoptosis, and was confirmed by monitoring the B-CLL cells after Annexin V/propidium iodide staining. Additional data revealed that these BNPs were non toxic for healthy animals, and had prolonged survival in this mice model of human lymphoma.

Electrospun cross-linked gelatin fibers with controlled diameter: the effect of matrix stiffness on proliferative and biosynthetic activity of chondrocytes cultured in vitro.

Nanofibrous scaffolds were prepared from gelatin solutions and were further cross-linked with glutaraldehyde (GA). The fiber diameter was varied from 100 to 1000 nm by controlling the applied voltage (4-15 kV) and the concentration of the gelatin solution (4-15%). The tensile moduli and the tensile strength of the noncross-linked scaffolds varied from 20 to 120 MPa and 0.5 to 3.5 MPa, respectively. Cross-linking with GA led to an increase in both the tensile modulus and strength and correlated with cross-linker concentration. Gelatin-based matrices were characterized by Fourier transform infrared spectroscopy and differential scanning calorimetry. High cellular viabilities and rounded morphology of chondrocytes was observed at the end of 7 days in culture with added matrix deposition and flattening of cells at 15 days. Matrix stiffness was noted to impact cell densities and the expression of chondrocytic markers, especially aggrecan. The ratios of collagen-II (C-II) to collagen-I (C-I) of 0.62 and 1.33 were noted on gelatin nanofibrous scaffolds cross-linked with 0.1% GA at the end of 7 and 15 days in culture, respectively. C-II/C-I ratios of 1.30 and 2.58 were noted on scaffolds cross-linked with 1.0% GA at the end of 7 and 15 days in culture, respectively.

Biocompatible and biodegradable ultrafine fibrillar scaffold materials for tissue engineering by facile grafting of L-lactide onto chitosan.

A chitosan derivative was prepared with good yields using a "one pot" approach by grafting L-lactide oligomers via ring opening polymerization. Side chains are primarily attached to hydroxyl groups located on carbons 3 and 6 of the glucosamine ring, while the amine group remains nonfunctionalized. By increasing the L-lactide to chitosan ratio, side chain length is controlled. This allows the manipulation of the biodegradation rate and hydrophilicity of the tissue engineering scaffold material. This general synthetic route renders functionalized chitosan soluble in a broad range of organic solvents, facilitating formation of ultrafine fibers via electrospinning. Cytotoxicity tests using fibroblasts (L929 cell line) performed on electrospun L-lactide modified chitosan fibers showed that the specimen with the highest molar ratio of L-lactide (1:24) investigated in this study is the most promising material for tissue engineering purposes, while less stable formulations might still find application in drug delivery vehicles.

Preparation and evaluation of the electrospun chitosan/PEO fibers for potential applications in cartilage tissue engineering.

Fibrous materials have morphological similarities to natural cartilage extracellular matrix and have been considered as candidate for bone tissue engineering scaffolds. In this study, we have evaluated a novel electrospun chitosan mat composed of oriented sub-micron fibers for its tensile property and biocompatibility with chondrocytes (cell attachment, proliferation and viability). Scanning electronic microscope images showed the fibers in the electrospun chitosan mats were indeed aligned and there was a slight cross-linking between the parent fibers. The electrospun mats have significantly higher elastic modulus (2.25 MPa) than the cast films (1.19 MPa). Viability of cells on the electrospun mat was 69% of the cells on tissue-culture polystyrene (TCP control) after three days in culture, which was slightly higher than that on the cast films (63% of the TCP control). Cells on the electrospun mat grew slowly the first week but the growth rate increased after that. By day 10, cell number on the electrospun mat was almost 82% that of TCP control, which was higher than that of cast films (56% of TCP). The electrospun chitosan mats have a higher Young's modulus (P < 0.01) than cast films and provide good chondrocyte biocompatibility. The electrospun chitosan mats, thus, have the potential to be further processed into three-dimensional scaffolds for cartilage tissue repair.

Synthesis and evaluation of scaffolds prepared from chitosan fibers for potential use in cartilage tissue engineering.

Tissue engineering concepts and methodologies that employ biocompatible matrices or scaffolds have the potential to meet needs encountered in the repair of defects in articular cartilage. A desirable design parameter in the tissue engineering of cartilage in vitro is the development of seeded scaffolds with appropriate structure, composition, mechanical properties and durability that are similar to normal articular cartilage. Previous methods that have used freeze drying and lyophilization techniques to make foams and hydrogels have not met the scaffold characteristics (porosity, compressive elastic modulus, permeability and viscoelastic properties), which are required of scaffolds slated for use in cartilage tissue engineering applications. Thus there is an impetus to design and develop biomimetic scaffolds that mimic the native ECM of articular cartilage, and distribute strain in a bioresponsive manner to signal seeded chondrocytes to synthesize and organize ECM to result in material properties that are in range of natural cartilage. We have employed the method of electrospinning to prepare scaffolds with oriented and random fiber alignment.

Electrically forced coaxial nanojets for one-step hollow nanofiber design.

The outer liquid of a two-liquid coaxial electrified jet is gelled before the onset of natural instabilities to yield hollow nanofibers. By using sol-gel chemistry, innocuous solvents such as glycerol and olive oil, and electrohydrodynamics, it is possible to make such structures in a rather straightforward manner.

A method for making inorganic and hybrid (organic/inorganic) fibers and vesicles with diameters in the submicrometer and micrometer range via sol-gel chemistry and electrically forced liquid jets.

Electrically driven liquid jets are combined with sol-gel methods to design vesicles and fibers made from inorganic oxides and hybrid materials with diameters in the micrometer and submicrometer range. The proposed materials synthesis method benefits greatly from the maturity of sol-gel chemistry and the generalities of a structure-directing phenomenon that is physical in nature.

A study of cyclohexane diffusion in hybrid (organic/inorganic) gels monitored by an oscillating balance reactor.

Hybrid (organic/inorganic) gels are studied in terms of cyclohexane diffusion transients with an oscillating balance reactor (OBR) which, unlike conventional microbalances, has the well-defined gas flow profile characteristics of packed beds (PB's). The OBR-based flow-through adsorption system, when properly designed, has very good time resolution and is particularly useful in modeling adsorption processes under more realistic PB geometries. With the test sample adopted in this study, the "semirigid" nature of hybrid gels becomes apparent when simple relaxation models are chosen to fit experimental data.