Investigation of interactions between biopolymer submicron particles and tumour cells carrying albumin receptors

Biopolymers are used in medicine, among other things, as a drug carrier due to their biocompatibility, biodegradability, high stability and ability to bind various drugs reversibly. At AG-Bäumler (Charité), a novel procedure for the fabrication of biopolymer particles in the submicron range was developed and published under the name CCD technology. CCD stands for co-precipitation–crosslinking-dissolution. This method delivers protein particles with a homogeneous morphology and a narrow size distribu-tion. Drugs, vitamins, nanoparticles (NP) and enzymes can be included in these parti-cles. Both the entrapment efficiency and the release of the enclosed molecules and / or NPs depend on the biopolymer used and, on the substance to be enclosed. In this work, mainly properties of human serum albumin particles (HSA-MP) loaded with dox-orubicin (DOX) are investigated. The size, zeta potential, morphology of particles as well as the entrapment efficiency and release of doxorubicin were determined. The entrapment efficiency of DOX was 25.6 ± 1.1% from the initially applied amount of the drug. The interaction of DOX-loaded HSA-MP (DOX-HSA-MPs) with human lung car-cinoma cells A549 expressing albumin receptors and non-cancerous human bronchial epithelial cells (BEAS-2B) without these receptors was also investigated. The aim of these experiments was to test the hypothesis that the DOX-HSA-MP reduce the growth of the tumor cells while the growth of the non-cancerous epithelial cells re-mains unaffected. It could be shown that the DOX-HSA-MP are endocytosed by the tumor cells and broken down in the lysosome, whereby the bound doxorubicin is re-leased and the metabolic activity of the A549 tumor cells is reduced by 60% after 72h.Biopolymere dienen in der Medizin u.a. als Drug Carrier aufgrund ihrer Biokompatibilität, Bioabbaubarkeit, hohen Stabilität und ihrer Fähigkeit verschiedene Arzneimittel reversibel zu binden. In der AG-Bäumler (Charité) wurde ein neuartiges Verfahren zur Herstellung von Biopolymerpartikeln im Submikrogrößenbereich entwickelt, welches unter der Bezeichnung CCD-Technik publiziert wurde. CCD steht für Copräzipitation-Crosslinking-Dissolution. Das Verfahren liefert Proteinpartikel mit homogener Morphologie und einer engen Größenverteilung. In diese Partikel können Arzneimittel, Vitamine, Nanopartikel (NP) und Enzyme eingeschlossen werden. Sowohl die Einschlusseffizienz als auch die Freisetzung der eingeschlossenen Moleküle und/oder NPs hängt sowohl vom Biopolymer als auch von der einzuschließenden Substanz ab. In dieser Arbeit werden hauptsächlich Eigenschaften von Humanserumalbumin Partikel (HSA-MP) beladen mit Doxorubicin (DOX) untersucht. Es wurden die Größen, Zetapotential, Morphologie, Einschlusseffizienz und Freisetzung von Doxorubicin bestimmt. Die Einschlusseffizienz von Doxorubicin betrug bezogen auf die Ausgangsmenge 25,6 ± 1,1 %. Außerdem wurde die Wechselwirkung von DOX-HSA-MPs mit Lungenkarzinomzellen A549 sowie menschlichen bronchialen Epithelzellen (BEAS-2B) untersucht, da die A549-Zellen Albuminrezeptoren exprimieren, während die BEAS-2B-Zellen keine derartigen Rezeptoren besitzen. Damit sollte die Hypothese geprüft werden, dass die DOX-HSA-MP das Wachstum der Tumorzellen verringern, während das Wachstum der Epithelzellen unbeeinflusst bleibt. Es konnte gezeigt werden, dass die DOX-HSA-MP von den Tumorzellen endozytiert und im Lysosom abgebaut werden, wodurch das gebundene Doxorubicin freigesetzt und die metabolische Aktivität der A549-Tumorzellen nach 72h Inkubation um 60% verringert wird

Doxorubicin-Loaded Human Serum Albumin Submicron Particles: Preparation, Characterization and In Vitro Cellular Uptake

Doxorubicin (DOX) is an effective anthracycline antibiotic drug which is commonly used in a broad range cancer therapy. However, due to dose depending side effects and toxicity to non-cancerous tissues, its clinical applications are restricted. To overcome these limitations, human serum albumin (HSA) has been investigated as a biocompatible drug delivery vehicle. In this study, human serum albumin submicron particles (HSA-MPs) were fabricated by using the Co-precipitation-Crosslinking-Dissolution technique (CCD technique) and DOX was loaded into the protein particles by absorption. DOX-HSA-MPs showed uniform peanut-like shape, submicron size and negative zeta-potential (-13 mV). The DOX entrapment efficiency was 25% of the initial amount. The in vitro release in phosphate buffered saline pH 7.4 was less than 1% within 5 h. In contrast, up to 40% of the entrapped DOX was released in presence of a protein digesting enzyme mixture (Pronase®) within the same time. In addition, in vitro cytotoxicity and cellular uptake of DOX-HSA-MPs were evaluated using the lung carcinoma cell line A549. The results demonstrated that DOX-HSA-MPs reduced the cell metabolic activities after 72 h. Interestingly, DOX-HSA-MPs were taken up by A549 cells up to 98% and localized in the cell lysosomal compartment. This study suggests that DOX-HSA-MPs which was fabricated by CCD technique is seen as a promising biopolymer particle as well as a viable alternative for drug delivery application to use for cancer therapy

Antibacterial Activities of Oral Care Products Containing Natural Plant Extracts from the Thai Highlands against <i>Staphylococcus aureus</i>: Evaluation and Satisfaction Studies

In this research, we aimed to assess antibacterial activity and develop oral care products from three natural plant extracts from the Thai highlands. The plants, including Camellia sinensis var. assamica, Zanthozylum limonella Alston, and Acorus calamus L., were extracted using two traditional extraction techniques: maceration and hydrodistillation methods. The extracts were characterized by percentage yield, total phenolic, and total flavonoid contents. Antibacterial activity against Staphylococcus aureus, which play a role in oral health and disease, was investigated. C. sinensis var. assamica extract had the highest content of phenolic acid (38.15 ± 4.12 mg GAE/g extract) and flavonoids (44.91 ± 2.76 mg QE/g extract). Interestingly, a combination of C. sinensis with Z. limonella and A. calamus provides a greater inhibitory effect against S. aureus. Furthermore, oral care products were prepared as a natural product mixture in two preparations: (i) oral ulcers gel and (ii) oral spray. Apart from antibacterial efficiency, volunteer satisfaction after the usage of oral care products containing traditional plant extracts was investigated via organoleptic evaluation. The findings of the volunteer surveys indicated positive feedback for both oral care products with high satisfaction levels. Hence, these oral care products could potentially be natural antimicrobial agents and can be further developed and applied for oral applications in the pharmaceutical and cosmetic industries

Fabrication and Characterization of Human Serum Albumin Particles Loaded with Non-Sericin Extract Obtained from Silk Cocoon as a Carrier System for Hydrophobic Substances

Non-sericin (NS) extract was produced from the ethanolic extract of Bombyx mori silk cocoons. This extract is composed of both carotenoids and flavonoids. Many of these compounds are composed of substances of poor aqueous solubility. Thus, this study focused on the development of a carrier system created from biocompatible and biodegradable materials to improve the biological activity of NS extracts. Accordingly, NS was incorporated into human serum albumin template particles with MnCO3 (NS-HSA MPs) by loading NS into the preformed HAS-MnCO3 microparticles using the coprecipitation crosslinking dissolution technique (CCD-technique). After crosslinking and template dissolution steps, the NS loaded HSA particles are negatively charged, have a size ranging from 0.8 to 0.9 &micro;m, and are peanut shaped. The degree of encapsulation efficiency ranged from 7% to 57% depending on the initial NS concentration and the steps of adsorption. In addition, NS-HSA MPs were taken up by human lung adenocarcinoma (A549 cell) for 24 h. The promotion of cellular uptake was evaluated by flow cytometry and the results produced 99% fluorescent stained cells. Moreover, the results from CLSM and 3D fluorescence imaging confirmed particle localization in the cells. Interestingly, NS-HSA MPs could not induce inflammation through nitric oxide production from macrophage RAW264.7 cells. This is the first study involving the loading of non-sericin extracts into HSA MPs by CCD technique to enhance the bioavailability and biological effects of NS. Therefore, HSA MPs could be utilized as a carrier system for hydrophobic substances targeting cells with albumin receptors

Doxorubicin–Loaded Human Serum Albumin Submicron Particles: Preparation, Characterization and In Vitro Cellular Uptake

Doxorubicin (DOX) is an effective anthracycline antibiotic drug which is commonly used in a broad range cancer therapy. However, due to dose depending side effects and toxicity to non-cancerous tissues, its clinical applications are restricted. To overcome these limitations, human serum albumin (HSA) has been investigated as a biocompatible drug delivery vehicle. In this study, human serum albumin submicron particles (HSA-MPs) were fabricated by using the Co-precipitation&ndash;Crosslinking&ndash;Dissolution technique (CCD technique) and DOX was loaded into the protein particles by absorption. DOX-HSA-MPs showed uniform peanut-like shape, submicron size and negative zeta-potential (&minus;13 mV). The DOX entrapment efficiency was 25% of the initial amount. The in vitro release in phosphate buffered saline pH 7.4 was less than 1% within 5 h. In contrast, up to 40% of the entrapped DOX was released in presence of a protein digesting enzyme mixture (Pronase&reg;) within the same time. In addition, in vitro cytotoxicity and cellular uptake of DOX-HSA-MPs were evaluated using the lung carcinoma cell line A549. The results demonstrated that DOX-HSA-MPs reduced the cell metabolic activities after 72 h. Interestingly, DOX-HSA-MPs were taken up by A549 cells up to 98% and localized in the cell lysosomal compartment. This study suggests that DOX-HSA-MPs which was fabricated by CCD technique is seen as a promising biopolymer particle as well as a viable alternative for drug delivery application to use for cancer therapy

Fabrication and Characterization of Human Serum Albumin Particles Loaded with Non-Sericin Extract Obtained from Silk Cocoon as a Carrier System for Hydrophobic Substances

Non-sericin (NS) extract was produced from the ethanolic extract of Bombyx mori silk cocoons. This extract is composed of both carotenoids and flavonoids. Many of these compounds are composed of substances of poor aqueous solubility. Thus, this study focused on the development of a carrier system created from biocompatible and biodegradable materials to improve the biological activity of NS extracts. Accordingly, NS was incorporated into human serum albumin template particles with MnCO3 (NS-HSA MPs) by loading NS into the preformed HAS-MnCO3 microparticles using the coprecipitation crosslinking dissolution technique (CCD-technique). After crosslinking and template dissolution steps, the NS loaded HSA particles are negatively charged, have a size ranging from 0.8 to 0.9 mu m, and are peanut shaped. The degree of encapsulation efficiency ranged from 7% to 57% depending on the initial NS concentration and the steps of adsorption. In addition, NS-HSA MPs were taken up by human lung adenocarcinoma (A549 cell) for 24 h. The promotion of cellular uptake was evaluated by flow cytometry and the results produced 99% fluorescent stained cells. Moreover, the results from CLSM and 3D fluorescence imaging confirmed particle localization in the cells. Interestingly, NS-HSA MPs could not induce inflammation through nitric oxide production from macrophage RAW264.7 cells. This is the first study involving the loading of non-sericin extracts into HSA MPs by CCD technique to enhance the bioavailability and biological effects of NS. Therefore, HSA MPs could be utilized as a carrier system for hydrophobic substances targeting cells with albumin receptors

Albumin Submicron Particles with Entrapped Riboflavin—Fabrication and Characterization

Although riboflavin (RF) belongs to the water-soluble vitamins of group B, its solubility is low. Therefore, the application of micro-formulations may help to overcome this limiting factor for the delivery of RF. In this study we immobilized RF in newly developed albumin submicron particles prepared using the Co-precipitation Crosslinking Dissolution technique (CCD-technique) of manganese chloride and sodium carbonate in the presence of human serum albumin (HSA) and RF. The resulting RF containing HSA particles (RF-HSA-MPs) showed a narrow size distribution in the range of 0.9 to 1 μm, uniform peanut-like morphology, and a zeta-potential of −15 mV. In vitro release studies represented biphasic release profiles of RF in a phosphate buffered saline (PBS) pH 7.4 and a cell culture medium (RPMI) 1640 medium over a prolonged period. Hemolysis, platelet activation, and phagocytosis assays revealed a good hemocompatibility of RF-HSA-MPs

Surface Modification of Hemoglobin-Based Oxygen Carriers Reduces Recognition by Haptoglobin, Immunoglobulin, and Hemoglobin Antibodies

Hemoglobin-based oxygen carriers (HBOCs) represent a propitious type of blood substitute to transport oxygen throughout the body while acting as a carrier in biomedical applications. However, HBOCs in blood are recognized and rapidly scavenged by the body&rsquo;s innate immune systems. To overcome this problem, HBOCs require a surface modification that provides protection against detection and elimination in order to prolong their circulation time after administration. In this study, we investigated different surface modifications of hemoglobin submicron particles (HbMPs) by double/triple precipitation, as well as by adsorption of human serum albumin (HSA), hyaluronic acid (HA), and pluronic (Plu) to discover how diverse surface modifications influence the oxygen binding capacity and the binding of anti-hemoglobin (Hb) antibodies, immunoglobulin G (IgG), and haptoglobin (HP) to HbMPs. The particle size and zeta potential of the six types of HbMP modifications were analyzed by zeta sizer, confocal laser scanning microscopy, and transmission electron microscopy (TEM), and were compared to the unmodified HbMPs. The results revealed that all surface-modified HbMPs had a submicron size with a negative charge. A slight decrease in the oxygen binding capacity was noticed. The specific binding of anti-Hb antibodies, IgG, and HP to all surface-modified HbMPs was reduced. This indicates a coating design able to protect the particles from detection and elimination processes by the immune system, and should lead to a delayed clearance and the required and essential increase in half-life in circulation of these particles in order to fulfill their purpose. Our surface modification method reflects a promising strategy for submicron particle design, and can lead the way toward novel biomedical applications