Micro, nano encapsulation methods for sustained release drug formulations and biomimetic applications

The Layer-by-Layer (LbL) assembly technique was used to obtain a new type of protein/polyphenol microcapsule based on naturally occurring polyphenol (-)-epigallocatechin gallate (EGCG) and gelatin, type A. The dependence of permeability on the molecular weight of permeating substances was studied and compared with commonly used polyallylamine/polystyrene sulfonate capsules. A quartz crystal microbalance was used to monitor the regularities of EGCG adsorption in alternation with type A and B Gelatins and electrophoretic mobility measurements were used that indicated that the nature of assembly was dependent on Gelatin properties. It was shown that EGCG retains its antioxidant activity in the LbL assemblies. Natural polyphenols, with previously demonstrated anti-cancer potential, EGCG, tannic acid, curcumin, and theaflavin, were encased in gelatin-based 200-nm nanoparticles consisting of a soft gel-like interior with or without a surrounding LbL-shell of polyelectrolytes (polystyrene sulfonate/polyallylamine hydrochloride, polyglutamic acid/poly-L-lysine, dextran sulfate/protamine sulfate, carboxymethyl cellulose/Gelatin, type A) assembled using the LbL technique. The characteristics of polyphenol loading and the factors affecting their release from the nanocapsules were investigated. Nanoparticle-encapsulated EGCG retained its biological activity and blocked hepatocyte growth factor (HGF) induced intracellular signaling in the breast cancer cell-line MBA-MD-231 as potently as free EGCG. Since electrostatic LbL nano-assembly is proven to be a suitable method for surface modifications on charged templates, we also used this technique for nano-coating of the phototrophic purple sulfur bacterium Allochromatium Vinosum with different synthetic and biocompatible polyelectrolyte combinations in order to investigate its biomimetic applications as related to drug delivery. The contact mechanisms between the cell surface and the insoluble elemental sulfur was investigated and studied because this step is essential for elemental sulfur uptake. Furthermore, modified uptake of sulfide by the encapsulated cells was also investigated. Growth experiments, after coating of the cells, showed that the surface charge of the bacteria neither affected the uptake of sulfide nor the contact formation between the cells and elemental sulfur. However, an increasing number of layers assembled on the cells slowed or inhibited the uptake of sulfide and elemental sulfur depending on the polymer combination used for coating. This indicated that LbL self-assembly makes it a suitable method for investigation of cell-surface related aspects in microbiology. After using LbL assembly successfully for coating microbes, we coated microbial spores in a sheath of functionalized nanofilms. Bacterial spores were encapsulated in organized ultrathin shells using LbL assembly in order to assess the biomaterial as a suitable core and determine the physiological effects of the coating. The coated spores were viable but the kinetics and extent of germination were changed from control spores in all instances. The results and insight gained from the experiments may be used to design various bioinspired systems. The spores can be made dormant for a desired amount of time using LbL encapsulation technique and can be made active when desired. In this work with LbL nanoassemly, we performed polyphenol based formulations and also modified the bacterial surface to study the effects of encapsulation on the uptake of various compounds. (Abstract shortened by UMI.

Registered Bioimaging of Nanomaterials for Diagnostic and Therapeutic Monitoring

Nanomedications can be carried by blood borne monocyte-macrophages into the reticuloendothelial system (RES; spleen, liver, lymph nodes) and to end organs. The latter include the lung, RES, and brain and are operative during human immunodeficiency virus type one (HIV-1) infection. Macrophage entry into tissues is notable in areas of active HIV-1 replication and sites of inflammation. In order to assess the potential of macrophages as nanocarriers, superparamagnetic iron-oxide and/or drug laden particles coated with surfactants were parenterally injected into HIV-1 encephalitic mice. This was done to quantitatively assess particle and drug biodistribution. Magnetic resonance imaging (MRI) test results were validated by histological coregistration and enhanced image processing. End organ disease as typified by altered brain histology were assessed by MRI. The demonstration of robust migration of nanoformulations into areas of focal encephalitis provides '"proof of concept" for the use of advanced bioimaging techniques to monitor macrophage migration. Importantly, histopathological aberrations in brain correlate with bioimaging parameters making the general utility of MRI in studies of cell distribution in disease feasible. We posit that using such methods can provide a real time index of disease burden and therapeutic efficacy with translational potential to humans

Comparative manufacture and cell-based delivery of antiretroviral nanoformulations

Nanoformulations of crystalline indinavir, ritonavir, atazanavir, and efavirenz were manufactured by wet milling, homogenization or sonication with a variety of excipients. The chemical, biological, immune, virological, and toxicological properties of these formulations were compared using an established monocyte-derived macrophage scoring indicator system. Measurements of drug uptake, retention, release, and antiretroviral activity demonstrated differences amongst preparation methods. Interestingly, for drug cell targeting and antiretroviral responses the most significant difference among the particles was the drug itself. We posit that the choice of drug and formulation composition may ultimately affect clinical utility

Mononuclear phagocyte intercellular crosstalk facilitates transmission of cell-targeted nanoformulated antiretroviral drugs to human brain endothelial cells

Despite the successes of antiretroviral therapy (ART), HIV-associated neurocognitive disorders remain prevalent in infected people. This is due, in part, to incomplete ART penetration across the blood–brain barrier (BBB) and lymph nodes and to the establishment of viral sanctuaries within the central nervous system. In efforts to improve ART delivery, our laboratories developed a macrophage-carriage system for nanoformulated crystalline ART (nanoART) (atazanavir, ritonavir, indinavir, and efavirenz). We demonstrate that nanoART transfer from mononuclear phagocytes (MP) to human brain microvascular endothelial cells (HBMEC) can be realized through cell-to-cell contacts, which can facilitate drug passage across the BBB. Coculturing of donor MP containing nanoART with recipient HBMEC facilitates intercellular particle transfer. NanoART uptake was observed in up to 52% of HBMEC with limited cytotoxicity. Folate coating of nanoART increased MP to HBMEC particle transfer by up to 77%. To translate the cell assays into relevant animal models of disease, ritonavir and atazanavir nanoformulations were injected into HIV-1-infected NOD/scid-γcnull mice reconstituted with human peripheral blood lymphocytes. Atazanavir and ritonavir levels in brains of mice treated with folate-coated nanoART were three- to four-fold higher than in mice treated with noncoated particles. This was associated with decreased viral load in the spleen and brain, and diminished brain CD11b-associated glial activation. We postulate that monocyte-macrophage transfer of nanoART to brain endothelial cells could facilitate drug entry into the brain

Comparative manufacture and cell-based delivery of antiretroviral nanoformulations

Nanoformulations of crystalline indinavir, ritonavir, atazanavir, and efavirenz were manufactured by wet milling, homogenization or sonication with a variety of excipients. The chemical, biological, immune, virological, and toxicological properties of these formulations were compared using an established monocyte-derived macrophage scoring indicator system. Measurements of drug uptake, retention, release, and antiretroviral activity demonstrated differences amongst preparation methods. Interestingly, for drug cell targeting and antiretroviral responses the most significant difference among the particles was the drug itself. We posit that the choice of drug and formulation composition may ultimately affect clinical utility