Antiplatelet effect of differentially charged PEGylated lipid-polymer nanoparticles.

PEGylated nanoparticles have been extensively investigated in different platforms for drug delivery. However, the physiological effects related to platelet activation, and the potential procoagulant activity which could lead to thrombosis and further cardiovascular diseases have not been widely examined. In this work, we studied the effect of differentially charged PEGylated lipid-polymer nanoparticles in the human platelet aggregation and activation by light transmission aggregometry and flow cytometry. PEGylated nanoparticles inhibited the platelet aggregation with a dose dependency (350, 700, and 1400µg/mL) in both ADP- and collagen-induced platelet aggregation, and P-selectin expression. Charged nanoparticles (anionic and cationic) presented higher inhibitions of the platelet aggregation compared to neutral nanoparticles, and particularly the cationic particles generated a slightly higher effect. The obtained results demonstrated the safety of the differentially charged PEGylated lipid-polymer nanoparticles, and their ability to inhibit the aggregation and activation of human platelets stimulated by two classic platelet activators.

Mechanisms Involved in the Formation of Biocompatible Lipid Polymeric Hollow Patchy Particles.

Patchy polymeric particles have anisotropic surface domains that can be remarkably useful in diverse medical and industrial fields because of their ability to simultaneously present two different surface chemistries on the same construct. In this article, we report the mechanisms involved in the formation of novel lipid-polymeric hollow patchy particles during their synthesis. By cross-sectioning the patchy particles, we found that a phase segregation phenomenon occurs between the core, shell, and patch. Importantly, we found that the shear stress that the polymer blend undergoes during the particle synthesis is the most important parameter for the formation of these patchy particles. In addition, we found that the interplay of solvent-solvent, polymer-solvent, and polymer-polymer-solvent interactions generates particles with different surface morphologies. Understanding the mechanisms involved in the formation of patchy particles allows us to have a better control on their physicochemical properties. Therefore, these fundamental studies are critical to achieve batch control and scalability, which are essential aspects that must be addressed in any type of particle synthesis to be safely used in medicine.

Spontaneous formation of heterogeneous patches on polymer-lipid core-shell particle surfaces during self-assembly.

Spontaneous formation of heterogeneous patches on the surface of lipid-based nanoparticles (NPs) and microparticles (MPs) due to the segregation of two different functional groups. Patch formation is observed when tracing the functional groups with quantum dots, gold nanoparticles, and fluorescent dyes. This discovery could have important implications for the future design of self-assembled NPs and MPs for different biomedical applications.

Nanotechnology Tools Enabling Biological Discovery.

Over the years, the engineering aspect of nanotechnology has been significantly exploited. Medical intervention strategies have been developed by leveraging existing molecular biology knowledge and combining it with nanotechnology tools to improve outcomes. However, little attention has been paid to harnessing the strengths of nanotechnology as a biological discovery tool. Fundamental understanding of controlling dynamic biological processes at the subcellular level is key to developing personalized therapeutic and diagnostic interventions. Single-cell analyses using intravital microscopy, expansion microscopy, and microfluidic-based platforms have been helping to better understand cell heterogeneity in healthy and diseased cells, a major challenge in oncology. Also, single-cell analysis has revealed critical signaling pathways and biological intracellular components with key biological functions. The physical manipulation enabled by nanotools can allow real-time monitoring of biological changes at a single-cell level by sampling intracellular fluid from the same cell. The formation of intercellular highways by nanotube-like structures has important clinical implications such as metastasis development. The integration of nanomaterials into optical and molecular imaging techniques has rendered valuable morphological, structural, and biological information. Nanoscale imaging unravels mechanisms of temporality by enabling the visualization of nanoscale dynamics never observed or measured between individual cells with standard biological techniques. The exceptional sensitivity of nanozymes, artificial enzymes, make them perfect components of the next-generation mobile diagnostics devices. Here, we highlight these impactful cancer-related biological discoveries enabled by nanotechnology and producing a paradigm shift in cancer research and oncology.

Integration of Multitargeted Polymer-Based Contrast Agents with Photoacoustic Computed Tomography: An Imaging Technique to Visualize Breast Cancer Intratumor Heterogeneity.

One of the primary challenges in breast cancer diagnosis and treatment is intratumor heterogeneity (ITH), i.e., the coexistence of different genetically and epigenetically distinct malignant cells within the same tumor. Thus, the identification of ITH is critical for designing better treatments and hence to increase patient survival rates. Herein, we report a noninvasive hybrid imaging technology that integrates multitargeted and multiplexed patchy polymeric photoacoustic contrast agents (MTMPPPCAs) with single-impulse panoramic photoacoustic computed tomography (SIP-PACT). The target specificity ability of MTMPPPCAs to distinguish estrogen and progesterone receptor-positive breast tumors was demonstrated through both fluorescence and photoacoustic measurements and validated by tissue pathology analysis. This work provides the proof-of-concept of the MTMPPPCAs/SIP-PACT system to identify ITH in nonmetastatic tumors, with both high molecular specificity and real-time detection capability.

Effects of covalent functionalization on the biocompatibility characteristics of multi-walled carbon nanotubes.

We report the effect of chemical modification of multi-walled carbon nanotubes (MWNTs) on their activation of the human serum complement system, as well as the adsorption of human plasma proteins on MWNTs. Four different types of chemically-modified MWNTs were tested for complement activation via the classical and alternative pathways using haemolytic assays. Human plasma protein binding was also tested using an affinity chromatography technique based on carbon nanotube-Sepharose matrix. Covalent functionalization of MWNTs greatly altered the level of activation of the complement system via the classical pathway. For example, MWNTs functionalised with epsilon-caprolactam or L-alanine showed respectively >90% and >75% reduction in classical pathway activation compared with unmodified MWNTs. These results demonstrate for the first time that these types of chemical modification are able to alter considerably the levels of specific complement proteins bound by pristine MWNTs (used as a control experiment). The reduced levels of complement activation via the classical pathway, that are likely to increase biocompatibility, were directly correlated with the amount of C1q protein bound to chemically modified carbon nanotubes. An inverse correlation was also observed between the amount of complement factor H bound to chemically modified MWNTs and the level of complement consumption via the alternative pathway. Binding of human plasma and serum proteins to pristine and modified MWNTs was highly selective. The chemical modifications studied generally increased nanotube dispersibility in aqueous media, but diminished protein adsorption.

Engineering Atrazine Loaded Poly (lactic- co-glycolic Acid) Nanoparticles to Ameliorate Environmental Challenges.

The use of herbicides plays a vital role in controlling weeds and conserving crops; however, its usage generates both environmental and economic problems. For example, herbicides pose a financial issue as farmers must apply large quantities to protect crops due to absorption rates of less than 0.1%. Therefore, there is a great need for the development of new methods to mitigate these issues. Here, we report for the first time the synthesis of poly(lactic- co-glycolic-acid) (PLGA) nanoherbicides loaded with atrazine as an active ingredient. We used potato plants as a biological model to assess the herbicidal activity of the engineered PLGA nanoherbicides. Our method produced nanoherbicides with an average size of 110 ± 10 nm prior to lyophilization. Fifty percent of the loaded atrazine in the PLGA matrix is released in 72 h. Furthermore, we performed Monte Carlo simulations to determine the chemical interaction among atrazine, PLGA, and the solvent system. One of the most significant outcomes of these simulations was to find the formation of a hydrogen bond of 1.9 Å between PLGA and atrazine, which makes this interaction very stable. Our in vitro findings showed that as atrazine concentration is increased in PLGA nanoparticles, potato plants undergo a significant decrease in stem length, root length, fresh weight, dry weight, and the number of leaves, with root length being the most affected. These experimental results suggest the herbicidal effectiveness of atrazine-loaded PLGA nanoherbicides in inhibiting the growth of the potato plant. Hence, we present the proof-of-concept for using PLGA nanoherbicides as an alternative method for inhibiting weed growth. Future studies will involve a deep understanding of the mechanism of plant-nanoherbicide interaction as well as the role of PLGA as a growth potentiator.

Complement activation and protein adsorption by carbon nanotubes.

As a first step to validate the use of carbon nanotubes as novel vaccine or drug delivery devices, their interaction with a part of the human immune system, complement, has been explored. Haemolytic assays were conducted to investigate the activation of the human serum complement system via the classical and alternative pathways. Western blot and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) techniques were used to elucidate the mechanism of activation of complement via the classical pathway, and to analyse the interaction of complement and other plasma proteins with carbon nanotubes. We report for the first time that carbon nanotubes activate human complement via both classical and alternative pathways. We conclude that complement activation by nanotubes is consistent with reported adjuvant effects, and might also in various circumstances promote damaging effects of excessive complement activation, such as inflammation and granuloma formation. C1q binds directly to carbon nanotubes. Protein binding to carbon nanotubes is highly selective, since out of the many different proteins in plasma, very few bind to the carbon nanotubes. Fibrinogen and apolipoproteins (AI, AIV and CIII) were the proteins that bound to carbon nanotubes in greatest quantity.

Interactions of the innate immune system with carbon nanotubes.

The therapeutic application of nanomaterials requires that they are biocompatible and can reach the desired target. The innate immune system is likely to be the first defence machinery that would recognise the nanomaterials as 'non-self'. A number of studies have addressed the issue of how carbon nanotubes (CNTs) interact with phagocytic cells and their surface receptors that can impact on their intracellular processing and subsequent immune response. In addition, soluble innate immune factors also get involved in the recognition and clearance of CNTs. The interaction of CNTs with the complement system, the most potent and versatile innate immune mechanism, has shed interesting light on how complement activation on the surface of CNTs can modulate their phagocytosis and effector cytokine response. The charge or altered molecular pattern on the surface of CNTs due to functionalization and derivatization can also dictate the level of complement activation and subsequent inflammatory response. It is becoming evident that complement deposition may facilitate phagocytic uptake of CNTs through receptor routes that leads to dampening of pro-inflammatory response by complement-receptor bearing macrophages and B cells. Thus, recombinant complement regulators decorated on the CNT surface can constructively influence the therapeutic strategies involving CNTs and other nanoparticles.

Altered mitochondrial dynamics as a consequence of Venezuelan Equine encephalitis virus infection.

Mitochondria are sentinel organelles that are impacted by various forms of cellular stress, including viral infections. While signaling events associated with mitochondria, including those activated by pathogen associated molecular patterns (PAMPs), are widely studied, alterations in mitochondrial distribution and changes in mitochondrial dynamics are also beginning to be associated with cellular insult. Cells of neuronal origin have been demonstrated to display remarkable alterations in several instances, including neurodegenerative disorders. Venezuelan Equine Encephalitis Virus (VEEV) is a New World alphavirus that infects neuronal cells and contributes to an encephalitic phenotype. We demonstrate that upon infection by the vaccine strain of VEEV (TC-83), astrocytoma cells experience a robust drop in mitochondrial activity, which corresponds with an increased accumulation of reactive oxygen species (ROS) in an infection-dependent manner. Infection status also corresponds with a prominent perinuclear accumulation of mitochondria. Cellular enzymatic machinery, including PINK1 and Parkin, appears to be enriched in mitochondrial fractions as compared with uninfected cells, which is indicative of mitochondrial damage. Dynamin related protein 1 (Drp1), a protein that is associated with mitochondrial fission, demonstrated a modest enrichment in mitochondrial fractions of infected cells. Treatment with an inhibitor of mitochondrial fission, Mdivi-1, led to a decrease in caspase cleavage, suggesting that mitochondrial fission was likely to contribute to apoptosis of infected cells. Finally, our data demonstrate that mitophagy ensues in infected cells. In combination, our data suggest that VEEV infection results in significant changes in the mitochondrial landscape that may influence pathological outcomes in the infected cell.

Pulmonary surfactant protein SP-D opsonises carbon nanotubes and augments their phagocytosis and subsequent pro-inflammatory immune response.

Carbon nanotubes (CNTs) are increasingly being developed for use in biomedical applications, including drug delivery. One of the most promising applications under evaluation is in treating pulmonary diseases such as tuberculosis. Once inhaled or administered, the nanoparticles are likely to be recognised by innate immune molecules in the lungs such as hydrophilic pulmonary surfactant proteins. Here, we set out to examine the interaction between surfactant protein D (SP-D), a key lung pattern recognition molecule and CNTs, and possible downstream effects on the immune response via macrophages. We show here that a recombinant form of human SP-D (rhSP-D) bound to oxidised and carboxymethyl cellulose (CMC) coated CNTs via its C-type lectin domain and enhanced phagocytosis by U937 and THP-1 macrophages/monocytic cell lines, together with an increased pro-inflammatory response, suggesting that sequestration of SP-D by CNTs in the lungs can trigger an unwanted and damaging immune response. We also observed that functionalised CNTs, opsonised with rhSP-D, continued to activate complement via the classical pathway, suggesting that C1q, which is the recognition sub-component of the classical pathway, and SP-D have distinct pattern recognition sites on the CNTs. Consistent with our earlier reports, complement deposition on the rhSP-D opsonised CNTs led to dampening of the pro-inflammatory immune response by THP-1 macrophages, as evident from qPCR, cytokine array and NF-κB nuclear translocation analyses. This study highlights the importance of understanding the interplay between innate immune humoral factors including complement in devising nanoparticle based drug delivery strategies.

Characterization of an interaction between functionalized carbon nanotubes and an enzyme.

Carbon nanotubes (CNTs) did not exhibit strong interactions with Biliverdin IX beta reductase enzyme (BVRB) in water. With the use of noncovalent functionalization by the surfactant Triton X-100, the surfaces of the CNTs were changed from hydrophobic to hydrophilic. The hydrophilic surface of the CNT-Triton conjugate interacts with the hydrophilic surface of BVRB, thus creating a water-soluble complex. Results from ultracentrifugation through a sucrose gradient and gel electrophoresis show the presence of the enzyme. Raman spectroscopy confirmed that the enzyme indeed interacts with CNT-Triton conjugates.

Closing the gap: accelerating the translational process in nanomedicine by proposing standardized characterization techniques.

On the cusp of widespread permeation of nanomedicine, academia, industry, and government have invested substantial financial resources in developing new ways to better treat diseases. Materials have unique physical and chemical properties at the nanoscale compared with their bulk or small-molecule analogs. These unique properties have been greatly advantageous in providing innovative solutions for medical treatments at the bench level. However, nanomedicine research has not yet fully permeated the clinical setting because of several limitations. Among these limitations are the lack of universal standards for characterizing nanomaterials and the limited knowledge that we possess regarding the interactions between nanomaterials and biological entities such as proteins. In this review, we report on recent developments in the characterization of nanomaterials as well as the newest information about the interactions between nanomaterials and proteins in the human body. We propose a standard set of techniques for universal characterization of nanomaterials. We also address relevant regulatory issues involved in the translational process for the development of drug molecules and drug delivery systems. Adherence and refinement of a universal standard in nanomaterial characterization as well as the acquisition of a deeper understanding of nanomaterials and proteins will likely accelerate the use of nanomedicine in common practice to a great extent.

Recent developments in multifunctional hybrid nanoparticles: opportunities and challenges in cancer therapy.

Multifunctional hybrid nanoparticles combine some of the unique physical and chemical characteristics of two or more classes of materials, such as polymers, liposomes, metals, quantum dots and mesoporous silica among others, to create a versatile and robust new class of nanoparticles. Here we discuss the most recent synthetic strategies to create these hybrid systems and analyze four key design aspects: stability, encapsulation of therapeutic and imaging agents, controlled release of encapsulated agents, and biocompatibility. Through the combination of multiple nanomaterials, hybrid nanoparticles aim to expand the functionality of single-component systems, using the strengths of one material to improve on weaknesses of another. We then examine how hybrid nanoparticle platforms provide unique opportunities in cancer therapy, specifically in the treatment of multidrug resistant cancer. Finally, we discuss some of the challenges hybrid nanoparticles systems might face in their large scale synthesis and commercialization in the biopharmaceutical industry.

Emerging nanotechnology approaches for HIV/AIDS treatment and prevention.

Currently, there is no cure and no preventive vaccine for HIV/AIDS. Combination antiretroviral therapy has dramatically improved treatment, but it has to be taken for a lifetime, has major side effects and is ineffective in patients in whom the virus develops resistance. Nanotechnology is an emerging multidisciplinary field that is revolutionizing medicine in the 21st century. It has a vast potential to radically advance the treatment and prevention of HIV/AIDS. In this review, we discuss the challenges with the current treatment of the disease and shed light on the remarkable potential of nanotechnology to provide more effective treatment and prevention for HIV/AIDS by advancing antiretroviral therapy, gene therapy, immunotherapy, vaccinology and microbicides.

Immunocompatibility properties of lipid-polymer hybrid nanoparticles with heterogeneous surface functional groups.

Here we report the immunological characterization of lipid-polymer hybrid nanoparticles (NPs) and propose a method to control the levels of complement activation induced by these NPs. This method consists of the highly specific modification of the NP surface with methoxyl, carboxyl, and amine groups. Hybrid NPs with methoxyl surface groups induced the lowest complement activation, whereas the NPs with amine surface groups induced the highest activation. All possible combinations among carboxyl, amine, and methoxyl groups also activated the complement system to a certain extent. All types of NPs activated the complement system primarily via the alternative pathway rather than the lectin pathway. The classical pathway was activated to a very small extent by the NPs with carboxyl and amine surface groups. Human serum and plasma protein binding studies showed that these NPs had different protein binding patterns. Studies of both complement activation and coagulation activation suggested that NPs with methoxyl surface groups might be an ideal candidate for drug delivery applications, since they are not likely to cause any immunological adverse reaction in the human body.

Multifunctional nanoparticles for prostate cancer therapy.

Multifunctional nanoparticles promise significantly better treatment for prostate cancer. This review begins with a molecular and physiological overview of prostate cancer, including current treatments in various stages of disease development. Emerging nanoparticle technology in chemotherapy, hyperthermia therapy and gene therapy will be discussed. We highlight novel advances in nanoparticle technology for prostate cancer and indicate future challenges in the rational design of multifunctional nanoparticles, such as understanding tumor characteristics and the activation of the complement system.