Phosphoprotein-based biomarkers as predictors for cancer therapy.

Disparities in cancer patient responses have prompted widespread searches to identify differences in sensitive vs. nonsensitive populations and form the basis of personalized medicine. This customized approach is dependent upon the development of pathway-specific therapeutics in conjunction with biomarkers that predict patient responses. Here, we show that Cdk5 drives growth in subgroups of patients with multiple types of neuroendocrine neoplasms. Phosphoproteomics and high throughput screening identified phosphorylation sites downstream of Cdk5. These phosphorylation events serve as biomarkers and effectively pinpoint Cdk5-driven tumors. Toward achieving targeted therapy, we demonstrate that mouse models of neuroendocrine cancer are responsive to selective Cdk5 inhibitors and biomimetic nanoparticles are effective vehicles for enhanced tumor targeting and reduction of drug toxicity. Finally, we show that biomarkers of Cdk5-dependent tumors effectively predict response to anti-Cdk5 therapy in patient-derived xenografts. Thus, a phosphoprotein-based diagnostic assay combined with Cdk5-targeted therapy is a rational treatment approach for neuroendocrine malignancies.

Rapamycin-Loaded Biomimetic Nanoparticles Reverse Vascular Inflammation.

RATIONALE: Through localized delivery of rapamycin via a biomimetic drug delivery system, it is possible to reduce vascular inflammation and thus the progression of vascular disease. OBJECTIVE: Use biomimetic nanoparticles to deliver rapamycin to the vessel wall to reduce inflammation in an in vivo model of atherosclerosis after a short dosing schedule. METHODS AND RESULTS: Biomimetic nanoparticles (leukosomes) were synthesized using membrane proteins purified from activated J774 macrophages. Rapamycin-loaded nanoparticles were characterized using dynamic light scattering and were found to have a diameter of 108±2.3 nm, a surface charge of -15.4±14.4 mV, and a polydispersity index of 0.11 +/ 0.2. For in vivo studies, ApoE-/- mice were fed a high-fat diet for 12 weeks. Mice were injected with either PBS, free rapamycin (5 mg/kg), or rapamycin-loaded leukosomes (Leuko-Rapa; 5 mg/kg) once daily for 7 days. In mice treated with Leuko-Rapa, flow cytometry of disaggregated aortic tissue revealed fewer proliferating macrophages in the aorta (15.6±9.79 %) compared with untreated mice (30.2±13.34 %) and rapamycin alone (26.8±9.87 %). Decreased macrophage proliferation correlated with decreased levels of MCP (monocyte chemoattractant protein)-1 and IL (interleukin)-b1 in mice treated with Leuko-Rapa. Furthermore, Leuko-Rapa-treated mice also displayed significantly decreased MMP (matrix metalloproteinases) activity in the aorta (mean difference 2554±363.9, P=9.95122×10-6). No significant changes in metabolic or inflammation markers observed in liver metabolic assays. Histological analysis showed improvements in lung morphology, with no alterations in heart, spleen, lung, or liver in Leuko-Rapa-treated mice. CONCLUSIONS: We showed that our biomimetic nanoparticles showed a decrease in proliferating macrophage population that was accompanied by the reduction of key proinflammatory cytokines and changes in plaque morphology. This proof-of-concept showed that our platform was capable of suppressing macrophage proliferation within the aorta after a short dosing schedule (7 days) and with a favorable toxicity profile. This treatment could be a promising intervention for the acute stabilization of late-stage plaques.

Biomimetic Concealing of PLGA Microspheres in a 3D Scaffold to Prevent Macrophage Uptake.

Scaffolds functionalized with delivery systems for the release of growth factors is a robust strategy to enhance tissue regeneration. However, after implantation, macrophages infiltrate the scaffold, eventually initiating the degradation and clearance of the delivery systems. Herein, it is hypothesized that fully embedding the poly(d,l-lactide-co-glycolide acid) microspheres (MS) in a highly structured collagen-based scaffold (concealing) can prevent their detection, preserving the integrity of the payload. Confocal laser microscopy reveals that non-embedded MS are easily internalized; when concealed, J774 and bone marrow-derived macrophages (BMDM) cannot detect them. This is further demonstrated by flow cytometry, as a tenfold decrease is found in the number of MS engulfed by the cells, suggesting that collagen can cloak the MS. This correlates with the amount of nitric oxide and tumor necrosis factor-α produced by J774 and BMDM in response to the concealed MS, comparable to that found for non-functionalized collagen scaffolds. Finally, the release kinetics of a reporter protein is preserved in the presence of macrophages, only when MS are concealed. The data provide detailed strategies for fabricating three dimensional (3D) biomimetic scaffolds able to conceal delivery systems and preserve the therapeutic molecules for release.

Multiscale patterning of a biomimetic scaffold integrated with composite microspheres.

The ideal scaffold for regenerative medicine should concurrently mimic the structure of the original tissue from the nano- up to the macroscale and recapitulate the biochemical composition of the extracellular matrix (ECM) in space and time. In this study, a multiscale approach is followed to selectively integrate different types of nanostructured composite microspheres loaded with reporter proteins, in a multi-compartment collagen scaffold. Through the preservation of the structural cues of the functionalized collagen scaffold at the nano- and microscale, its macroscopic features (pore size, porosity, and swelling) are not altered. Additionally, the spatial confinement of the microspheres allows the release of the reporter proteins in each of the layers of the scaffold. Finally, the staged and zero-order release kinetics enables the temporal biochemical patterning of the scaffold. The versatile manufacturing of each component of the scaffold results in the ability to customize it to better mimic the architecture and composition of the tissues and biological systems.

Evaluation of cell function upon nanovector internalization.

In vitro toxicity assays based on the evaluation and retention of advanced and specific cellular functions are proposed to investigate mesoporous silicon nanovectors. This approach provides greater insight compared to simple cellular viability and toxicity assays. Electron microscopy images demonstrate internalized nanovectors altering the curvature of the nuclear envelope with minimal effect on viability or biological function.

Short and long term, in vitro and in vivo correlations of cellular and tissue responses to mesoporous silicon nanovectors.

The characterization of nanomaterials and their influence on and interactions with the biology of cells and tissues are still partially unknown. Multistage nanovectors based on mesoporous silicon have been extensively studied for drug delivery, thermal heating, and improved diagnostic imaging. Here, the short- and long-term changes occurring in human cells upon the internalization of mesoporous silicon nanovectors (MSV) are analyzed. Using qualitative and quantitative techniques as well as in vitro and in vivo biochemical, cellular, and functional assays, it is demonstrated that MSV do not cause any significant acute or chronic effects on cells and tissues. In vitro cell toxicity and viability are analyzed, as well as the maintenance of cell phase cycling and the architecture upon the internalization of MSV. In addition, it is evaluated whether MSV produce any pro-inflammatory responses and its biocompatibility in vivo is studied. The biodistribution of MSV is followed using longitudinal in vivo imaging and organ accumulation is assessed using quantitative elemental and fluorescent techniques. Finally, a thorough pathological analysis of collected tissues demonstrates a mild transient systemic response in the liver that dissipates upon the clearance of particles. It is proposed that future endeavors aimed at understanding the toxicology of naked drug carriers should be designed to address their impact using in vitro and in vivo short- and long-term evaluations of systemic response.

Multifunctional to multistage delivery systems: The evolution of nanoparticles for biomedical applications.

Nanomaterials are advancing in several directions with significant progress being achieved with respect to their synthesis, functionalization and biomedical application. In this review, we will describe several classes of prototypical nanocarriers, such as liposomes, silicon particles, and gold nanoshells, in terms of their individual function as well as their synergistic use. Active and passive targeting, photothermal ablation, and drug controlled release constitute some of the crucial functions identified to achieve a medical purpose. Current limitations in targeting, slow clearance, and systemic as well as local toxicity are addressed in reference to the recent studies that attempted to comprehend and solve these issues. The demand for a more sophisticated understanding of the impact of nanomaterialson the body and of their potential immune response underlies this discussion. Combined components are then discussed in the setting of multifunctional nanocarriers, a class of drug delivery systems we envisioned, proposed, and evolved in the last 5 years. In particular, our third generation of nanocarriers, the multistage vectors, usher in the new field of nanomedicine by combining several components onto multifunctional nanocarriers characterized by emerging properties and able to achieve synergistic effects.

Near-infrared imaging method for the in vivo assessment of the biodistribution of nanoporous silicon particles.

In the development of new nanoparticle-based technologies for therapeutic and diagnostic purposes, understanding the fate of nanoparticles in the body is crucial. We recently developed a multistage vector delivery system comprising biodegradable and biocompatible nanoporous silicon particles (first-stage microparticles [S1MPs]) able to host, protect, and deliver second-stage therapeutic and diagnostic nanoparticles (S2NPs) on intravenous injection. This delivery system aims at sequentially overcoming the biologic barriers en route to the target delivery site by separating and assigning tasks to the coordinated logic-embedded vectors constituting it. In this work, by conjugating a near-infrared dye on the surface of the S1MP without compromising the porous structure and potential loading of S2NPs, we were able to monitor the in vivo distribution of S1MPs in healthy mice using an optical imaging system. It was observed that particles predominantly accumulated in the liver and spleen at the end of 24 hours. Further quantification of S1MPs in the major organs of the animals by elemental analysis of silicon using inductively coupled plasma-atomic electron spectroscopy verified the accuracy of in vivo near-infrared imaging as a tool for evaluation of nanovector biodistribution.

Lysyl oxidase engineered lipid nanovesicles for the treatment of triple negative breast cancer.

In the field of oncology research, a deeper understanding of tumor biology has shed light on the role of environmental conditions surrounding cancer cells. In this regard, targeting the tumor microenvironment has recently emerged as a new way to access this disease. In this work, a novel extracellular matrix (ECM)-targeting nanotherapeutic was engineered using a lipid-based nanoparticle chemically linked to an inhibitor of the ECM-related enzyme, lysyl oxidase 1 (LOX), that inhibits the crosslinking of elastin and collagen fibers. We demonstrated that, when the conjugated vesicles were loaded with the chemotherapeutic epirubicin, superior inhibition of triple negative breast cancer (TNBC) cell growth was observed both in vitro and in vivo. Moreover, in vivo results displayed prolonged survival, minimal cytotoxicity, and enhanced biocompatibility compared to free epirubicin and epirubicin-loaded nanoparticles. This all-in-one nano-based ECM-targeting chemotherapeutic may provide a key-enabling technology for the treatment of TNBC.

LDL-Based Lipid Nanoparticle Derived for Blood Plasma Accumulates Preferentially in Atherosclerotic Plaque.

Apolipoprotein-based drug delivery is a promising approach to develop safe nanoparticles capable of targeted drug delivery for various diseases. In this work, we have synthesized a lipid-based nanoparticle (NPs) that we have called "Aposomes" presenting native apolipoprotein B-100 (apoB-100), the primary protein present in Low-Density Lipoproteins (LDL) on its surface. The aposomes were synthesized from LDL isolated from blood plasma using a microfluidic approach. The synthesized aposomes had a diameter of 91 ± 4 nm and a neutral surface charge of 0.7 mV ± mV. Protein analysis using western blot and flow cytometry confirmed the presence of apoB-100 on the nanoparticle's surface. Furthermore, Aposomes retained liposomes' drug loading capabilities, demonstrating a prolonged release curve with ∼80% cargo release at 4 hours. Considering the natural tropism of LDL towards the atherosclerotic plaques, we evaluated the biological properties of aposomes in a mouse model of advanced atherosclerosis. We observed a ∼20-fold increase in targeting of plaques when comparing aposomes to control liposomes. Additionally, aposomes presented a favorable biocompatibility profile that showed no deviation from typical values in liver toxicity markers (i.e., LDH, ALT, AST, Cholesterol). The results of this study demonstrate the possibilities of using apolipoprotein-based approaches to create nanoparticles with active targeting capabilities and could be the basis for future cardiovascular therapies.

Enabling individualized therapy through nanotechnology.

Individualized medicine is the healthcare strategy that rebukes the idiomatic dogma of 'losing sight of the forest for the trees'. We are entering a new era of healthcare where it is no longer acceptable to develop and market a drug that is effective for only 80% of the patient population. The emergence of "-omic" technologies (e.g. genomics, transcriptomics, proteomics, metabolomics) and advances in systems biology are magnifying the deficiencies of standardized therapy, which often provide little treatment latitude for accommodating patient physiologic idiosyncrasies. A personalized approach to medicine is not a novel concept. Ever since the scientific community began unraveling the mysteries of the genome, the promise of discarding generic treatment regimens in favor of patient-specific therapies became more feasible and realistic. One of the major scientific impediments of this movement towards personalized medicine has been the need for technological enablement. Nanotechnology is projected to play a critical role in patient-specific therapy; however, this transition will depend heavily upon the evolutionary development of a systems biology approach to clinical medicine based upon "-omic" technology analysis and integration. This manuscript provides a forward looking assessment of the promise of nanomedicine as it pertains to individualized medicine and establishes a technology "snapshot" of the current state of nano-based products over a vast array of clinical indications and range of patient specificity. Other issues such as market driven hurdles and regulatory compliance reform are anticipated to "self-correct" in accordance to scientific advancement and healthcare demand. These peripheral, non-scientific concerns are not addressed at length in this manuscript; however they do exist, and their impact to the paradigm shifting healthcare transformation towards individualized medicine will be critical for its success.

Bioinspired Extracellular Vesicles: Lessons Learned From Nature for Biomedicine and Bioengineering.

Efficient communication is essential in all layers of the biological chain. Cells exchange information using a variety of signaling moieties, such as small molecules, proteins, and nucleic acids. Cells carefully package these messages into lipid complexes, collectively named extracellular vesicles (EVs). In this work, we discuss the nature of these cell carriers, categorize them by their origin, explore their role in the homeostasis of healthy tissues, and examine how they regulate the pathophysiology of several diseases. This review will also address the limitations of using EVs for clinical applications and discuss novel methods to engineer nanoparticles to mimic the structure, function, and features of EVs. Using lessons learned from nature and understanding how cells use EVs to communicate across distant sites, we can develop a better understanding of how to tailor the fundamental features of drug delivery carriers to encapsulate various cargos and target specific sites for biomedicine and bioengineering.

Leukocyte-mimicking nanovesicles for effective doxorubicin delivery to treat breast cancer and melanoma.

In the last decades, several approaches were developed to design drug delivery systems to address the multiple biological barriers encountered after administration while safely delivering a payload. In this scenario, bio-inspired and bio-mimetic approaches have emerged as promising solutions to evade the mononuclear phagocytic system while simultaneously negotiating the sequential transport across the various biological barriers. Leukocytes freely circulate in the bloodstream and selectively target the inflamed vasculature in response to injury, infection, and cancer. Recently we have shown the use of biomimetic nanovesicles, called leukosomes, which combine both the physical and biological properties of liposomes and leukocytes, respectively, to selectively deliver drugs to the inflamed vasculature. Here we report the use of leukosomes to target and deliver doxorubicin, a model chemotherapeutic, to tumors in syngeneic murine models of breast cancer and melanoma. Exploiting the inflammatory pathway responsible for recruiting immune cells to the site of injury, leukosomes exhibited increased targeting of cancer vasculature and stroma. Furthermore, delivery of doxorubicin with leukosomes enabled significant tumor growth inhibition compared with free doxorubicin in both breast and melanoma tumors. This study demonstrates the promise of using biomimetic nanovesicles for effective cancer management in solid tumors.

Biomimetic cellular vectors for enhancing drug delivery to the lungs.

Despite recent advances in drug delivery, the targeted treatment of unhealthy cells or tissues continues to remain a priority. In cancer (much like other pathologies), delivery vectors are designed to exploit physical and biological features of unhealthy tissues that are not always homogenous across the disease. In some cases, shifting the target from unhealthy tissues to the whole organ can represent an advantage. Specifically, the natural organ-specific retention of nanotherapeutics following intravenous administration as seen in the lung, liver, and spleen can be strategically exploited to enhance drug delivery. Herein, we outline the development of a cell-based delivery system using macrophages as a delivery vehicle. When loaded with a chemotherapeutic payload (i.e., doxorubicin), these cellular vectors (CELVEC) were shown to provide continued release within the lung. This study provides proof-of-concept evidence of an alternative class of biomimetic delivery vectors that capitalize on cell size to provide therapeutic advantages for pulmonary treatments.

Macrophage-derived nanovesicles exert intrinsic anti-inflammatory properties and prolong survival in sepsis through a direct interaction with macrophages.

Despite numerous advances in medical treatment, sepsis remains one of the leading causes of death worldwide. Sepsis is characterized by the involvement of all organs and tissues as a consequence of blood poisoning, resulting in organ failure and eventually death. Effective treatment remains an unmet need and novel approaches are urgently needed. The growing evidence of clinical and biological heterogeneity of sepsis suggests precision medicine as a possible key for achieving therapeutic breakthroughs. In this scenario, biomimetic nanomedicine represents a promising avenue for the treatment of inflammatory diseases, including sepsis. We investigated the role of macrophage-derived biomimetic nanoparticles, namely leukosomes, in a lipopolysaccharide-induced murine model of sepsis. We observed that treatment with leukosomes was associated with significantly prolonged survival. In vitro studies elucidated the potential mechanism of action of these biomimetic vesicles. The direct treatment of endothelial cells (ECs) with leukosomes did not alter the gene expression profile of EC-associated cell adhesion molecules. In contrast, the interaction of leukosomes with macrophages induced a decrease of pro-inflammatory genes (IL-6, IL-1b, and TNF-α), an increase of anti-inflammatory ones (IL-10 and TGF-ß), and indirectly an anti-inflammatory response on ECs. Taken together, these results showed the ability of leukosomes to regulate the inflammatory response in target cells, acting as a bioactive nanotherapeutic.

Trends towards Biomimicry in Theranostics.

Over the years, imaging and therapeutic modalities have seen considerable progress as a result of advances in nanotechnology. Theranostics, or the marrying of diagnostics and therapy, has increasingly been employing nano-based approaches to treat cancer. While first-generation nanoparticles offered considerable promise in the imaging and treatment of cancer, toxicity and non-specific distribution hindered their true potential. More recently, multistage nanovectors have been strategically designed to shield and carry a payload to its intended site. However, detection by the immune system and sequestration by filtration organs (i.e., liver and spleen) remains a major obstacle. In an effort to circumvent these biological barriers, recent trends have taken inspiration from biology. These bioinspired approaches often involve the use of biologically-derived cellular components in the design and fabrication of biomimetic nanoparticles. In this review, we provide insight into early nanoparticles and how they have steadily evolved to include bioinspired approaches to increase their theranostic potential.

Design and Development of Biomimetic Nanovesicles Using a Microfluidic Approach.

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.

Biomimetic nanoparticles with enhanced affinity towards activated endothelium as versatile tools for theranostic drug delivery.

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.

Chlorin e6 Functionalized Theranostic Multistage Nanovectors Transported by Stem Cells for Effective Photodynamic Therapy.

Approaches to achieve site-specific and targeted delivery that provide an effective solution to reduce adverse, off target side effects are urgently needed for cancer therapy. Here, we utilized a Trojan-horse-like strategy to carry photosensitizer Chlorin e6 conjugated porous silicon multistage nanovectors with tumor homing mesenchymal stem cells for targeted photodynamic therapy and diagnosis. The inherent versatility of multistage nanovectors permitted the conjugation of photosensitizers to enable precise cell death induction (60%) upon photodynamic therapy, while simultaneously retaining the loading capacity to load various payloads, such as antitumor drugs and diagnostic nanoparticles. Furthermore, the mesenchymal stem cells that internalized the multistage nanovectors conserved their proliferation patterns and in vitro affinity to migrate and infiltrate breast cancer cells. In vivo administration of the mesenchymal stem cells carrying photosensitizer-conjugated multistage nanovectors in mice bearing a primary breast tumor confirmed their tropism toward cancer sites exhibiting similar targeting kinetics to control cells. In addition, this approach yielded in a > 70% decrease in local tumor cell viability after in vivo photodynamic therapy. In summary, these results show the proof-of-concept of how photosensitizer conjugated multistage nanovectors transported by stem cells can target tumors and be used for effective site-specific cancer therapy while potentially minimizing potential negative side effects.

Bio-inspired engineering of cell- and virus-like nanoparticles for drug delivery.

The engineering of future generations of nanodelivery systems aims at the creation of multifunctional vectors endowed with improved circulation, enhanced targeting and responsiveness to the biological environment. Moving past purely bio-inert systems, researchers have begun to create nanoparticles capable of proactively interacting with the biology of the body. Nature offers a wide-range of sources of inspiration for the synthesis of more effective drug delivery platforms. Because the nano-bio-interface is the key driver of nanoparticle behavior and function, the modification of nanoparticles' surfaces allows the transfer of biological properties to synthetic carriers by imparting them with a biological identity. Modulation of these surface characteristics governs nanoparticle interactions with the biological barriers they encounter. Building off these observations, we provide here an overview of virus- and cell-derived biomimetic delivery systems that combine the intrinsic hallmarks of biological membranes with the delivery capabilities of synthetic carriers. We describe the features and properties of biomimetic delivery systems, recapitulating the distinctive traits and functions of viruses, exosomes, platelets, red and white blood cells. By mimicking these biological entities, we will learn how to more efficiently interact with the human body and refine our ability to negotiate with the biological barriers that impair the therapeutic efficacy of nanoparticles.