The therapeutic efficacy of systemic drug delivery vehicles depends on their ability to evade the immune system, cross the biological barriers of the body and localize at target tissues. Leukocytes possess all of these functions and exert their targeting ability through cellular membrane interactions. Here we show that NanoPorous Silicon particles (NPS) can successfully perform all these actions when coated with cellular membranes purified from white blood cells. These hybrid particles called LeukoLike Vectors (LLV) were able to: prevent rapid clearance of phagocytic cells of the immune system; communicate with endothelial cells through receptor-ligand interaction; transport and release a payload across an inflamed reconstructed endothelium. Furthermore, LLV retained their functions when injected in vivo, showing enhanced circulation time and improved accumulation in the tumour.

A multitude of micro- and nanoparticles have been developed to improve the delivery of systemically administered pharmaceuticals, which are subject to a number of biological barriers that limit their optimal biodistribution. Bioinspired drug-delivery carriers formulated by bottom-up or top-down strategies have emerged as an alternative approach to evade the mononuclear phagocytic system and facilitate the transport across the endothelial vessel wall. Here, we describe a method that leverages the advantages of bottom-up and top-down strategies to incorporate proteins derived from the leukocyte plasma membrane into lipid nanoparticles. The resulting proteolipid vesicles - which we refer to as leukosomes - retained the versatility and physicochemical properties typical of liposomal formulations, preferentially targeted inflamed vasculature, enabled the selective and effective delivery of dexamethasone to inflamed tissues, and reduced phlogosis in a localized model of inflammation.

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The advancement of nanotechnology toward more sophisticated bioinspired approaches has highlighted the gap between the advantages of biomimetic and biohybrid platforms and the availability of manufacturing processes to scale up their production. Though the advantages of transferring biological features from cells to synthetic nanoparticles for drug delivery purposes have recently been reported, a standardizable, batch-to-batch consistent, scalable, and high-throughput assembly method is required to further develop these platforms. Microfluidics has offered a robust tool for the controlled synthesis of nanoparticles in a versatile and reproducible approach. In this study, the incorporation of membrane proteins within the bilayer of biomimetic nanovesicles (leukosomes) using a microfluidic-based platform is demonstrated. The physical, pharmaceutical, and biological properties of microfluidic-formulated leukosomes (called NA-Leuko) are characterized. NA-Leuko show extended shelf life and retention of the biological functions of donor cells (i.e., macrophage avoidance and targeting of inflamed vasculature). The NA approach represents a universal, versatile, robust, and scalable tool, which is extensively used for the assembly of lipid nanoparticles and adapted here for the manufacturing of biomimetic nanovesicles.

Activation of the vascular endothelium is characterized by increased expression of vascular adhesion molecules and chemokines. This activation occurs early in the progression of several diseases and triggers the recruitment of leukocytes. Inspired by the tropism of leukocytes, we investigated leukocyte-based biomimetic nanoparticles (i.e., leukosomes) as a novel theranostic platform for inflammatory diseases.Methods: Leukosomes were assembled by combining phospholipids and membrane proteins from leukocytes. For imaging applications, phospholipids modified with rhodamine and gadolinium were used. Leukosomes incubated with antibodies blocking lymphocyte function-associated antigen 1 (LFA-1) and CD45 were administered to explore their roles in targeting inflammation. In addition, relaxometric assessment of NPs was evaluated.Results: Liposomes and leukosomes were both spherical in shape with sizes ranging from 140-170 nm. Both NPs successfully integrated 8 and 13 µg of rhodamine and gadolinium, respectively, and demonstrated less than 4% variation in physicochemical features. Leukosomes demonstrated a 16-fold increase in breast tumor accumulation relative to liposomes. Furthermore, quantification of leukosomes in tumor vessels demonstrated a 4.5-fold increase in vessel lumens and a 14-fold increase in vessel walls. Investigating the targeting mechanism of action revealed that blockage of LFA-1 on leukosomes resulted in a 95% decrease in tumor accumulation. Whereas blockage of CD45 yielded a 60% decrease in targeting and significant increases in liver and spleen accumulation. In addition, when administered in mice with atherosclerotic plaques, leukosomes exhibited a 4-fold increase in the targeting of inflammatory vascular lesions. Lastly, relaxometric assessment of NPs demonstrated that the incorporation of membrane proteins into leukosomes did not impact the r1 and r2 relaxivities of the NPs, demonstrating 6 and 30 mM-1s-1, respectively.Conclusion: Our study demonstrates the ability of leukosomes to target activated vasculature and exhibit superior accumulation in tumors and vascular lesions. The versatility of the phospholipid backbone within leukosomes permits the incorporation of various contrast agents. Furthermore, leukosomes can potentially be loaded with therapeutics possessing diverse physical properties and thus warrant further investigation toward the development of powerful theranostic agents.

Recent advances in the field of nanomedicine have demonstrated that biomimicry can further improve targeting properties of current nanotechnologies while simultaneously enable carriers with a biological identity to better interact with the biological environment. Immune cells for example employ membrane proteins to target inflamed vasculature, locally increase vascular permeability, and extravasate across inflamed endothelium. Inspired by the physiology of immune cells, we recently developed a procedure to transfer leukocyte membranes onto nanoporous silicon particles (NPS), yielding Leukolike Vectors (LLV). LLV are composed of a surface coating containing multiple receptors that are critical in the cross-talk with the endothelium, mediating cellular accumulation in the tumor microenvironment while decreasing vascular barrier function. We previously demonstrated that lymphocyte function-associated antigen (LFA-1) transferred onto LLV was able to trigger the clustering of intercellular adhesion molecule 1 (ICAM-1) on endothelial cells. Herein, we provide a more comprehensive analysis of the working mechanism of LLV in vitro in activating this pathway and in vivo in enhancing vascular permeability. Our results suggest the biological activity of the leukocyte membrane can be retained upon transplant onto NPS and is critical in providing the particles with complex biological functions towards tumor vasculature.

Nanoparticle-based drug delivery systems have been synthesized from a wide array of materials. The therapeutic success of these platforms hinges upon their ability to favorably interact with the biological environment (both systemically and locally) and recognize the diseased target tissue. The immune system, composed of a highly coordinated organization of cells trained to recognize foreign bodies, represents a key mediator of these interactions. Although components of this system may act as a barrier to nanoparticle (NP) delivery, the immune system can also be exploited to target and trigger signaling cues that facilitate the therapeutic response stemming from systemic administration of NPs. The nano-bio interface represents the key facilitator of this communication exchange, where the surface properties of NPs govern their in vivo fate. Cell membrane-based biomimetic nanoparticles have emerged as one approach to achieve targeted drug delivery by actively engaging and communicating with the biological milieu. In this review, we will highlight the relationship between these biomimetic nanoparticles and the immune system, emphasizing the role of tuning the nano-bio interface in the immunomodulation of diseases. We will also discuss the therapeutic applications of this approach with biomimetic nanoparticles, focusing on specific diseases ranging from cancer to infectious diseases. Lastly, we will provide a critical evaluation on the current state of this field of cell membrane-based biomimetic nanoparticles and its future directions in immune-based therapy.

This report describes a novel, one-pot synthesis of hybrid nanoparticles formed by a nanostructured inorganic silica core and an organic pH-responsive hydrogel shell. This easy-to-perform, oil-in-water emulsion process synthesizes fluorescently-doped silica nanoparticles wrapped within a tunable coating of cationic poly(2-diethylaminoethyl methacrylate) hydrogel in one step. Transmission electron microscopy and dynamic light scattering analysis demonstrated that the hydrogel-coated nanoparticles are uniformly dispersed in the aqueous phase. The formation of covalent chemical bonds between the silica and the polymer increases the stability of the organic phase around the inorganic core as demonstrated by thermogravimetric analysis. The cationic nature of the hydrogel is responsible for the pH buffering properties of the nanostructured system and was evaluated by titration experiments. Zeta-potential analysis demonstrated that the charge of the system was reversed when transitioned from acidic to basic pH and vice versa. Consequently, small interfering RNA (siRNA) can be loaded and released in an acidic pH environment thereby enabling the hybrid particles and their payload to avoid endosomal sequestration and enzymatic degradation. These nanoparticles, loaded with specific siRNA molecules directed towards the transcript of the membrane receptor CXCR4, significantly decreased the expression of this protein in a human breast cancer cell line (i.e., MDA-MB-231). Moreover, intravenous administration of siRNA-loaded nanoparticles demonstrated a preferential accumulation at the tumor site that resulted in a reduction of CXCR4 expression.