Albumin as a drug carrier: design of prodrugs, drug conjugates and nanoparticles.

Impact of albumin on drug delivery--new applications on the horizon.

The effectiveness of a drug is dependent on accumulation at the site of action at therapeutic levels, however, challenges such as rapid renal clearance, degradation or non-specific accumulation requires drug delivery enabling technologies. Albumin is a natural transport protein with multiple ligand binding sites, cellular receptor engagement, and a long circulatory half-life due to interaction with the recycling neonatal Fc receptor. Exploitation of these properties promotes albumin as an attractive candidate for half-life extension and targeted intracellular delivery of drugs attached by covalent conjugation, genetic fusions, association or ligand-mediated association. This review will give an overview of albumin-based products with focus on the natural biological properties and molecular interactions that can be harnessed for the design of a next-generation drug delivery platform.

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Albumin-based drug delivery: harnessing nature to cure disease

Clinical impact of serum proteins on drug delivery

Gene manipulation techniques open up the possibility of making recombinant human serum albumin (rHSA) or mutants with desirable therapeutic properties and for protein fusion products. rHSA can serve as a carrier in synthetic heme protein, thus reversibly carrying oxygen. Myristoylation of insulin results in a prolonged half-life because of self aggregation and increased albumin binding. Preferential albumin uptake by tumor cells serves as the basis for albumin-anticancer drug conjugate formulation. Furthermore, drug targeting can be achieved by incorporating drugs into albumin microspheres whereas liver targeting can be achieved by conjugating drug with galactosylated or mannosylated albumin. Microspheres and nanoparticles of different sizes can, with or without drugs and/or radioisotopes, be used for drug delivery or diagnostic purposes. In vivo implantation of albumin fusion protein expressing cells encapsulated in HSA-alginate coated beads showed promising results compared to organoids in rats. Chimeric peptide strategy with cationized albumin as the transport can deliver drugs via receptor mediated transcytosis through the blood brain barrier. Gene bearing, albumin microbubbles containing ultrasound contrast agents can non-invasively deliver gene after destruction by ultrasound. Various site-directed mutants of HSA can be tailor made depending on the application required.

Pharmaceutical Strategies Utilizing Recombinant Human Serum Albumin

Purpose The purpose of this study was to aid in the standardization of lymphoscintigraphy for detecting the sentinel node (SN) in breast cancer using Tc-99m-labeled nanocolloidal albumin. Materials and methods One hundred twenty-three women with proved breast cancer were enrolled. Four injections of 10 to 15 MBq (0.27 to 0.41 mCi) Tc-99m nanocolloid in 0.1 ml physiologic saline were administered intra- and subdermally at the margin of the skin overlying the tumor. Planar scintigraphic images in the lateral and anterior projections were obtained 2.5 to 18 hours after tracer administration. With a gamma probe used as a guide, all radioactive lymph nodes in the axilla were resected. Complete dissection then followed. Results In 116 of the 123 (94%) women, axillary nodal tracer uptake was revealed. Six of the 7 women in whom detection failure occurred had histologically proved tumor infiltration of the lymphatics and axillary involvement. In 36 (31%) of the patients with visualized lymph nodes, the SN was metastatic. The remaining 80 patients with negative SN included three cases with axillary involvement. The sensitivity of the SN with respect to the histologic status of the entire axilla thus was 92.3%, and the negative predictive value was 96.3%. The overall accuracy of the method was 97.4%. The number of hot nodes in women with and without axillary involvement was significantly different. Conclusions The described protocol represents an easily reproduced and reliable method for SN detection in breast cancer. Furthermore, the number of visualized axillary nodes reflects the histologic status of the axilla.

Lymphoscintigraphic sentinel node imaging and gamma probe detection in breast cancer with Tc-99m nanocolloidal albumin: results of an optimized protocol.

Abstract Nanoparticles can acquire a plasma protein corona defining their biological identity. Corona functions were previously considered for cell‐derived extracellular vesicles (EVs). Here we demonstrate that nano‐sized EVs from therapy‐grade human placental‐expanded (PLX) stromal cells are surrounded by an imageable and functional protein corona when enriched with permissive technology. Scalable EV separation from cell‐secreted soluble factors via tangential flow‐filtration (TFF) and subtractive tandem mass‐tag (TMT) proteomics revealed significant enrichment of predominantly immunomodulatory and proangiogenic proteins. Western blot, calcein‐based flow cytometry, super‐resolution and electron microscopy verified EV identity. PLX‐EVs partly protected corona proteins from protease digestion. EVs significantly ameliorated human skin regeneration and angiogenesis in vivo, induced differential signalling in immune cells, and dose‐dependently inhibited T cell proliferation in vitro. Corona removal by size‐exclusion or ultracentrifugation abrogated angiogenesis. Re‐establishing an artificial corona by cloaking EVs with fluorescent albumin as a model protein or defined proangiogenic factors was depicted by super‐resolution microscopy, electron microscopy and zeta‐potential shift, and served as a proof‐of‐concept. Understanding EV corona formation will improve rational EV‐inspired nano‐therapy design.

https://onlinelibrary.wiley.com/doi/pdfdirect/10.1002/jev2.12207

A functional corona around extracellular vesicles enhances angiogenesis, skin regeneration and immunomodulation

Abstract Extracellular vesicle (EV) research increasingly demands for quantitative characterisation at the single vesicle level to address heterogeneity and complexity of EV subpopulations. Emerging, commercialised technologies for single EV analysis based on, for example, imaging flow cytometry or imaging after capture on chips generally require dedicated instrumentation and proprietary software not readily accessible to every lab. This limits their implementation for routine EV characterisation in the rapidly growing EV field. We and others have shown that single vesicles can be detected as light diffraction limited fluorescent spots using standard confocal and widefield fluorescence microscopes. Advancing this simple strategy into a process for routine EV quantitation, we developed ‘EVAnalyzer’, an ImageJ/Fiji (Fiji is just ImageJ) plugin for automated, quantitative single vesicle analysis from imaging data. Using EVAnalyzer, we established a robust protocol for capture, (immuno‐)labelling and fluorescent imaging of EVs. To exemplify the application scope, the process was optimised and systematically tested for (i) quantification of EV subpopulations, (ii) validation of EV labelling reagents, (iii) in situ determination of antibody specificity, sensitivity and species cross‐reactivity for EV markers and (iv) optimisation of genetic EV engineering. Additionally, we show that the process can be applied to synthetic nanoparticles, allowing to determine siRNA encapsulation efficiencies of lipid‐based nanoparticles (LNPs) and protein loading of SiO2 nanoparticles. EVAnalyzer further provides a pipeline for automated quantification of cell uptake at the single cell–single vesicle level, thereby enabling high content EV cell uptake assays and plate‐based screens. Notably, the entire procedure from sample preparation to the final data output is entirely based on standard reagents, materials, laboratory equipment and open access software. In summary, we show that EVAnalyzer enables rigorous characterisation of EVs with generally accessible tools. Since we further provide the plugin as open‐source code, we expect EVAnalyzer to not only be a resource of immediate impact, but an open innovation platform for the EV and nanoparticle research communities.

https://onlinelibrary.wiley.com/doi/pdfdirect/10.1002/jev2.12282

EVAnalyzer: High content imaging for rigorous characterisation of single extracellular vesicles using standard laboratory equipment and a new open‐source ImageJ/Fiji plugin

Thomas Heuser

Papers

Lymphoscintigraphic sentinel node imaging and gamma probe detection in breast cancer with Tc-99m nanocolloidal albumin: results of an optimized protocol.

A functional corona around extracellular vesicles enhances angiogenesis, skin regeneration and immunomodulation

EVAnalyzer: High content imaging for rigorous characterisation of single extracellular vesicles using standard laboratory equipment and a new open‐source ImageJ/Fiji plugin

Generation of Human Induced Pluripotent Stem Cell-Derived Bone Marrow Organoids