Heterogeneous Mixture Of Proteins Research Articles

A current perspective on snake venom composition and constituent protein families.

Snake venoms are heterogeneous mixtures of proteins and peptides used for prey subjugation. With modern proteomics there has been a rapid expansion in our knowledge of snake venom composition, resulting in the venom proteomes of 30% of vipers and 17% of elapids being characterised. From the reasonably complete proteomic coverage of front-fanged snake venom composition (179 species-68 species of elapids and 111 species of vipers), the venoms of vipers and elapids contained 42 different protein families, although 18 were only reported in < 5% of snake species. Based on the mean abundance and occurrence of the 42 protein families, they can be classified into 4 dominant, 6 secondary, 14 minor, and 18 rare protein families. The dominant, secondary and minor categories account for 96% on average of a snake's venom composition. The four dominant protein families are: phospholipase A2 (PLA2), snake venom metalloprotease (SVMP), three-finger toxins (3FTx), and snake venom serine protease (SVSP). The six secondary protein families are: L-amino acid oxidase (LAAO), cysteine-rich secretory protein (CRiSP), C-type lectins (CTL), disintegrins (DIS), kunitz peptides (KUN), and natriuretic peptides (NP). Venom variation occurs at all taxonomic levels, including within populations. The reasons for venom variation are complex, as variation is not always associated with geographical variation in diet. The four dominant protein families appear to be the most important toxin families in human envenomation, being responsible for coagulopathy, neurotoxicity, myotoxicity and cytotoxicity. Proteomic techniques can be used to investigate the toxicological profile of a snake venom and hence identify key protein families for antivenom immunorecognition.

Directed Evolution Pipeline for the Improvement of Orthogonal Translation Machinery for Genetic Code Expansion at Sense Codons.

The expansion of the genetic code beyond a single type of noncanonical amino acid (ncAA) is hindered by inefficient machinery for reassigning the meaning of sense codons. A major obstacle to using directed evolution to improve the efficiency of sense codon reassignment is that fractional sense codon reassignments lead to heterogeneous mixtures of full-length proteins with either a ncAA or a natural amino acid incorporated in response to the targeted codon. In stop codon suppression systems, missed incorporations lead to truncated proteins; improvements in activity may be inferred from increased protein yields or the production of downstream reporters. In sense codon reassignment, the heterogeneous proteins produced greatly complicate the development of screens for variants of the orthogonal machinery with improved activity. We describe the use of a previously-reported fluorescence-based screen for sense codon reassignment as the first step in a directed evolution workflow to improve the incorporation of a ncAA in response to the Arg AGG sense codon. We first screened a library with diversity introduced into both the orthogonal Methanocaldococcus jannaschii tyrosyl tRNA anticodon loop and the cognate aminoacyl tRNA synthetase (aaRS) anticodon binding domain for variants that improved incorporation of tyrosine in response to the AGG codon. The most efficient variants produced fluorescent proteins at levels indistinguishable from the E. coli translation machinery decoding tyrosine codons. Mutations to the M. jannaschii aaRS that were found to improve tyrosine incorporation were transplanted onto a M. jannaschii aaRS evolved for the incorporation of para-azidophenylalanine. Improved ncAA incorporation was evident using fluorescence- and mass-based reporters. The described workflow is generalizable and should enable the rapid tailoring of orthogonal machinery capable of activating diverse ncAAs to any sense codon target. We evaluated the selection based improvements of the orthogonal pair in a host genomically engineered for reduced target codon competition. Using this particular system for evaluation of arginine AGG codon reassignment, however, E. coli strains with genomes engineered to remove competing tRNAs did not outperform a standard laboratory E. coli strain in sense codon reassignment.

Stress response genes to membrane fluidizers in mycobacteria

Plasma membrane is a heterogeneous mixture of lipids and proteins that includes laterally discrete domains. Membrane domains have been extensively described in eukaryotes, but little is known in prokaryotes. Mycobacterium, a genus that includes several notorious human pathogens, organizes its plasma membrane by creating the intracellular membrane domain (IMD), which is spatially and biochemically separable from the conventional plasma membrane (PM-CW). This membrane architecture is proposed to optimize the synthesis of cell envelope precursors such as peptide glycan (PG) and arabinogalactan (AG) (Fig. 1). However, the mechanisms that govern the formation and maintenance of the IMD is completely unknown. We recently found that membrane fluidizers such as benzyl alcohol and dibucaine can reversibly disrupt the formation of the IMD. To reveal the mechanism of this stress response, in this study, we carried out transposon sequencing (Tn-seq), comparing pools of mutants that were treated or mock-treated with either benzyl alcohol or dibucaine. We found genes involved in peptidoglycan biosynthesis and outer membrane biosynthesis important for withstanding membrane fluidizer treatments, suggesting that the physical pinning by the loadbearing exoskeleton structures is critical for the IMD maintenance and recovery. We also found genes involved in modulating fatty acid structures, indicating the role of membrane lipid composition in the membrane stress response. We are currently verifying the roles of these genes by making gene deletion mutants, and testing the sensitivities of these mutants to the membrane fluidizers.

Deeper Protein Identification Using Field Asymmetric Ion Mobility Spectrometry in Top-Down Proteomics.

Field asymmetric ion mobility spectrometry (FAIMS), when used in proteomics studies, provides superior selectivity and enables more proteins to be identified by providing additional gas-phase separation. Here, we tested the performance of cylindrical FAIMS for the identification and characterization of proteoforms by top-down mass spectrometry of heterogeneous protein mixtures. Combining FAIMS with chromatographic separation resulted in a 62% increase in protein identifications, an 8% increase in proteoform identifications, and an improvement in proteoform identification compared to samples analyzed without FAIMS. In addition, utilization of FAIMS resulted in the identification of proteins encoded by lower-abundance mRNA transcripts. These improvements were attributable, in part, to improved signal-to-noise for proteoforms with similar retention times. Additionally, our results show that the optimal compensation voltage of any given proteoform was correlated with the molecular weight of the analyte. Collectively these results suggest that the addition of FAIMS can enhance top-down proteomics in both discovery and targeted applications.

Multi-epitope insert modulates solubility-based and chromatographic purification of human papilloma virus 16 L1-based vaccine without inhibiting virus-like particle assembly

The separation of heterogeneous protein mixtures has always been characterized by a trade-off between purity and yield. One way this issue has been addressed in the past is by recombinantly modifying protein to improve separations. Such modifications are mostly employed in the form of tags used specifically for affinity chromatography, though it is also possible to make changes to a protein that will have a sizeable impact on its hydrophobicity and charge/charge distribution. As such, it should also be possible to use protein tags to modulate phase separations and protein-resin binding kinetics when performing ion exchange chromatography. Here, we employed a three-step purification scheme on E. coli expressed, His-tagged, human papilloma virus 16 L1-based recombinant proteins (rHPV 16 L1) that consisted of an inclusion body (IB) wash step, a diethylaminoethyl (DEAE) anion exchange chromatography (AEX) step, and an immobilized metal affinity chromatography (IMAC) polishing step. Purification of the wild type rHPV 16 L1 protein (WT) was characterized by substantial losses during the IB wash but relatively high yield over the DEAE column. In contrast, purification of modified rHPV 16 L1, a chimeric version of the WT protein that had the last 34 amino acids replaced with an MHC class II multi-epitope insert derived from tetanus toxin and diphtheria toxin (WTΔC34-2TEp), was characterized by little to no losses in the IB wash but had a relatively low yield over the DEAE column. Since the fate of these proteins was to be used in vaccine formulations, it is important to note that the modifications made to the WTΔC34-2TEp protein had little to no effect on its ability to assemble into virus-like particles (VLPs). These results demonstrate that modifications of the WT protein via the recombinant insertion of immunofunctional polypeptides can modulate both phase-based separation and charge-based chromatographic processes. Additionally, incorporation of the specific, multi-epitope tag used in this study may prove to be beneficial in recombinant HPV vaccine development due to its potential to improve phase separation yield and vaccine immunogenicity without inhibiting VLP formation.

Characterization of bromelain indicates a molar excess of inhibitor vs. enzyme molecules, a Jacalin-like lectin and Maillard reaction products

The phytotherapeutic bromelain is a heterogeneous protein mixture, extracted from pineapple stem, with high proteolytic activity based on cysteine proteases. Its global protein chemical composition was analyzed qualitatively and quantitatively by SDS-PAGE and RP-HPLC. A SDS-PAGE method with elaborate sample pretreatment was developed, to cope with the bromelain's self-digestion properties and the hypothetical disulfide scrambling during electrophoresis. Both can produce misleading results, if not considered. RP-HPLC was applied for its high separation power for bromelain proteinaceous compounds. A peak identification and assignment to different protein classes in bromelain was done by enzyme kinetics and MS. The method was successfully applied for the quantitative determination of the molar ratio between inhibitor and enzyme and resulted to be approximately 3:2. Bromelain contains, from a molar point of view, inhibitor molecules as major component, which thus might be considered as a natural pharmaceutical excipient in Bromelain, because it protects the enzymes against autolysis. We described two methods to separate the inhibitor fraction from the enzyme fraction, RP-HPLC and size exclusion chromatography. A pineapple derived Jacalin-like-lectin, herein called ‘Anlec’, was identified and quantified by RP-HPLC-MS in bromelain and its content was determined to be 5%, related to all proteins in bromelain. Anlec binds specifically to mannose-containing glycans and is discussed in literature to possess anti-HIV medical potential. Bromelain could therefore be a possible and economic source for the production of Anlec. An isolation strategy of Anlec from bromelain, in high purity, is shown in this work. The presented RP-HPLC results are comprehensive in chemical information, and the method is expedient to provide appropriate bromelain protein isolations but also to accomplish quality control, covering all relevant protein components. It is furthermore shown, that proteins in bromelain may react with reducing sugars in a Maillard reaction to form glycated proteins. Maillard reaction products in bromelain are detected and characterized and could be responsible for the limited stability and storage times at room temperature of bromelain. Even the active center thiol group could be potentially glycated.

A Versatile Biosynthetic Hydrogel Platform for Engineering of Tissue Analogues.

For creating functional tissue analogues in tissue engineering, stem cells require very specific 3D microenvironments to thrive and mature. Demanding (stem) cell types that are used nowadays can find such an environment in a heterogeneous protein mixture with the trade name Matrigel. Several variations of synthetic hydrogel platforms composed of poly(ethylene glycol) (PEG), which are spiked with peptides, have been recently developed and shown equivalence to Matrigel for stem cell differentiation. Here a clinically relevant hydrogel platform, based on PEG and gelatin, which even outperforms Matrigel when targeting 3D prevascularized bone and liver organoid tissue engineering models is presented. The hybrid hydrogel with natural and synthetic components stimulates efficient cell differentiation, superior to Matrigel models. Furthermore, the strength of this hydrogel lies in the option to covalently incorporate unmodified proteins. These results demonstrate how a hybrid hydrogel platform with intermediate biological complexity, when compared to existing biological materials and synthetic PEG-peptide approaches, can efficiently support tissue development from human primary cells.

Rapid two-dimensional characterisation of proteins in solution

Microfluidic platforms provide an excellent basis for working with heterogeneous samples and separating biomolecular components at high throughput, with high recovery rates and by using only very small sample volumes. To date, several micron scale platforms with preparative capabilities have been demonstrated. Here we describe and demonstrate a microfluidic device that brings preparative and analytical operations together onto a single chip and thereby allows the acquisition of multidimensional information. We achieve this objective by using a free-flow electrophoretic separation approach that directs fractions of sample into an on-chip analysis unit, where the fractions are characterised through a microfluidic diffusional sizing process. This combined approach therefore allows simultaneously quantifying the sizes and the charges of components in heterogenous mixtures. We illustrate the power of the platform by describing the size distribution of a mixture comprising components which are close in size and cannot be identified as individual components using state-of-the-art solution sizing techniques on their own. Furthermore, we show that the platform can be used for two-dimensional fingerprinting of heterogeneous protein mixtures within tens of seconds, opening up a possibility to obtain multiparameter data on biomolecular systems on a minute timescale.

Lipid Raft Phase Modulation by Membrane-Anchored Proteins with Inherent Phase Separation Properties.

Cell plasma membranes are a heterogeneous mixture of lipids and membrane proteins. The importance of heterogeneous lipid domains (also called lipid rafts) as a molecular sorting platform has been implicated in many physiological processes. Cell plasma membranes that are detached from the cytoskeletal structure spontaneously phase separate into distinct domains at equilibrium, which show their inherent demixing properties. Recently, researchers have discovered that proteins with strong interprotein interactions also spontaneously phase separate into distinct protein domains, thus enabling the maintenance of many membraneless organelles. Protein phase separation may also take place on the lipid membranes via lipid-anchored proteins, which suggests another potential molecular sorting platform for physiological processes on the cell membrane. When two-phase separation properties coexist physiologically, they may change the resulting phase behavior or serve as independent sorting platforms. In this paper, we used in vitro reconstitution and fluorescence imaging to systematically quantify the phase behavior that arises when proteins with inherent phase separation properties interact with raft mixture lipid membranes. Our observations and simulations show both that the proteins may enhance lipid phase separation and that this is a general property of phase-separating protein systems with a diverse number of components involved. This suggests that we should consider the overall effect of the properties of both membrane-anchored proteins and lipids when interpreting molecular sorting phenomena on the membranes.

Aggregation‐induced changes in the chemical exchange saturation transfer (CEST) signals of proteins

Chemical exchange saturation transfer (CEST) is an MRI technique that allows mapping of biomolecules (small metabolites, proteins) with nearly the sensitivity of conventional water proton MRI. In living organisms, several tissue-specific CEST effects have been observed and successfully applied to diagnostic imaging. In these studies, particularly the signals of proteins showed a distinct correlation with pathological changes. However, as CEST effects depend on various properties that determine and affect the chemical exchange processes, the origins of the observed signal changes remain to be understood. In this study, protein aggregation was identified as an additional process that is encoded in the CEST signals of proteins. Investigation of distinct proteins that are involved in pathological disorders, namely amyloid beta and huntingtin, revealed a significant decrease of all protein CEST signals upon controlled aggregation. This finding is of particular interest with regard to diagnostic imaging of patients with neurodegenerative diseases that involve amyloidogenesis, such as Alzheimer's or Huntington's disease. To investigate whether the observed CEST signal decrease also occurs in heterogeneous mixtures of aggregated cellular proteins, and thus prospectively in tissue, heat-shocked yeast cell lysates were employed. Additionally, investigation of different cell compartments verified the assignment of the protein CEST signals to the soluble part of the proteome. The results of in vitro experiments demonstrate that aggregation affects the CEST signals of proteins. This observation can enable hypotheses for CEST imaging as a non-invasive diagnostic tool for monitoring pathological alterations of the proteome in vivo.

Highly Sensitive and Selective Aptamer-Based Fluorescence Detection of a Malarial Biomarker Using Single-Layer MoS2 Nanosheets

We demonstrate a highly selective and sensitive aptamer-based “capture-release” fluorescence detection of a malarial biomarker, i.e., Plasmodium lactose dehydrogenase (pLDH) protein, using single-layer two-dimensional MoS2 nanosheets. The detection of the target pLDH protein utilizing the aptamer-nanosheet sensing platform is rapid and can be easily completed within 10 min. In addition, the developed biomolecular sensor is capable of detecting the target malarial biomarker in homogeneous protein solutions as well as in heterogeneous mixtures of proteins. We anticipate that the ultrathin MoS2 nanosheet-based biosensor will facilitate the further development of biomolecular nanosensors for the detection of a wide range of biomarkers for malaria and other diseases.

Investigating Lipid-Protein Interactions in a Complex Biological Membrane Model

Biological membranes are crucial as they define essential compartments for cells. Cell membranes are heterogeneous mixtures of membrane proteins and lipids. Their structure and function are fundamentally dependent on lipid-lipid and lipid-protein interactions which play a crucial role in regulating cell protein functions and are involved in many diseases when altered. However, the exact properties and the relation among the distinct components that form the biological membrane are not completely understood due the limitations of experimental methods in studying the lipid-protein interactions in living cells. Lately, computer simulations have become a promising tool to understand and clarify experimental results. The goal of our research is to identify specific lipid-protein interactions of biological relevance and work towards large-scale modeling of realistic biological membranes. In this study, we placed ten main classes of eukaryotic membrane proteins with different ratios in a prototypical plasma membrane model, containing various lipid types found in the plasma membranes of eukaryotic cells. The protein-membrane system was prepared using Martini force field. Using molecular dynamic simulations, we intend to investigate how proteins sort lipids, and to identify specific binding partners and lipids of interest for more detailed studies.

Phase separation in biology; functional organization of a higher order.

Inside eukaryotic cells, macromolecules are partitioned into membrane-bounded compartments and, within these, some are further organized into non-membrane-bounded structures termed membrane-less organelles. The latter structures are comprised of heterogeneous mixtures of proteins and nucleic acids and assemble through a phase separation phenomenon similar to polymer condensation. Membrane-less organelles are dynamic structures maintained through multivalent interactions that mediate diverse biological processes, many involved in RNA metabolism. They rapidly exchange components with the cellular milieu and their properties are readily altered in response to environmental cues, often implicating membrane-less organelles in responses to stress signaling. In this review, we discuss: (1) the functional roles of membrane-less organelles, (2) unifying structural and mechanistic principles that underlie their assembly and disassembly, and (3) established and emerging methods used in structural investigations of membrane-less organelles.

The impact of physiological crowding on the diffusivity of membrane bound proteins.

Diffusion of transmembrane and peripheral membrane-bound proteins within the crowded cellular membrane environment is essential to diverse biological processes including cellular signaling, endocytosis, and motility. Nonetheless we presently lack a detailed understanding of the influence of physiological levels of crowding on membrane protein diffusion. Utilizing quantitative in vitro measurements, here we demonstrate that the diffusivities of membrane bound proteins follow a single linearly decreasing trend with increasing membrane coverage by proteins. This trend holds for homogenous protein populations across a range of protein sizes and for heterogeneous mixtures of proteins of different sizes, such that protein diffusivity is controlled by the total coverage of the surrounding membrane. These results demonstrate that steric exclusion within the crowded membrane environment can fundamentally limit the diffusive rate of proteins, regardless of their size. In cells this "speed limit" could be modulated by changes in local membrane coverage, providing a mechanism for tuning the rate of molecular interaction and assembly.

Neuroinflammatory paradigms in lysosomal storage diseases.

Lysosomal storage diseases (LSDs) include approximately 70 distinct disorders that collectively account for 14% of all inherited metabolic diseases. LSDs are caused by mutations in various enzymes/proteins that disrupt lysosomal function, which impairs macromolecule degradation following endosome-lysosome and phagosome-lysosome fusion and autophagy, ultimately disrupting cellular homeostasis. LSDs are pathologically typified by lysosomal inclusions composed of a heterogeneous mixture of various proteins and lipids that can be found throughout the body. However, in many cases the CNS is dramatically affected, which may result from heightened neuronal vulnerability based on their post-mitotic state. Besides intrinsic neuronal defects, another emerging factor common to many LSDs is neuroinflammation, which may negatively impact neuronal survival and contribute to neurodegeneration. Microglial and astrocyte activation is a hallmark of many LSDs that affect the CNS, which often precedes and predicts regions where eventual neuron loss will occur. However, the timing, intensity, and duration of neuroinflammation may ultimately dictate the impact on CNS homeostasis. For example, a transient inflammatory response following CNS insult/injury can be neuroprotective, as glial cells attempt to remove the insult and provide trophic support to neurons. However, chronic inflammation, as seen in several LSDs, can promote neurodegeneration by creating a neurotoxic environment due to elevated levels of cytokines, chemokines, and pro-apoptotic molecules. Although neuroinflammation has been reported in several LSDs, the cellular basis and mechanisms responsible for eliciting neuroinflammatory pathways are just beginning to be defined. This review highlights the role of neuroinflammation in select LSDs and its potential contribution to neuron loss.

Abstract B19: Functional translation of total RNA packaged in microparticles shed from multidrug resistant cancer cells

Abstract Microparticles (MPs) are submicron (0.1 -1 µM) membrane vesicles released from various cell types including malignant cells. Long considered to be inert cellular debris, MPs have recently been identified to play a crucial role in cellular cross-talk. MPs represent a heterogeneous mixture of surface antigens, proteins, lipids and cytoplasmic components resembling its parental cell. Our previous studies have identified the packaging and transfer of the transcripts, regulatory nucleic acids and proteins which are associated with the acquirement of multidrug resistance (MDR) between cancer cells. In this study we verify the functionality of packaged transcripts via in vitro translation of extracted total RNA from MPs isolated from cancer cells. We confirm the translation of exogenous RNA in a rabbit reticulocyte translation system via Western Blot analysis, 2-D SDS and mass spectrometry. These findings have significance in understanding the impact of MPs on cancer cell biology and reiterate their potential as a target in clinical oncology. Citation Format: Jamie Fung Lu, Deep Pokharel, Frederick Luk, Mary Bebawy. Functional translation of total RNA packaged in microparticles shed from multidrug resistant cancer cells. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr B19.

Immune Complex Proteomes: Tools for Vaccine Discovery

World-wide in tropical and subtropical areas, approximately 200 million cases of malaria are reported annually with about 1 million deaths occurring in children under the age of 5 years. Data from proteomic studies are providing new strategies for identifying Plasmodium falciparum proteins with malaria vaccine and therapeutic potential. Purified native and recombinant proteins, and heterogeneous mixtures of proteins have been used traditionally for proteome analysis using 2-D gel electrophoresis (2DE) followed by the identification and excision of differential protein spots stained on sodium dodecyl sulfatepolyacrylamide (SDS-PAGE) gels and analysis by mass spectrometry. The use of antibodies to add specificity to the identification of proteins analyzed by proteomics provides a practical benefit in small scale studies, in small laboratory settings, to identify changes in protein expression observed in normal and pathogenic states in humans. Antibodies can also be used to identify stage specific and developmental changes occurring in parasitic infections and in identifying expressed proteins of parasite origin present in human serum. The need for additional levels of specificity in protein identification was recognized in the initial reports of protein identification by sequencing followed by database searches for unambiguous identification of correct proteins [1]. The use of antibodies; either unlabeled or tagged to capture antigens followed by mass spectrometry

Therapeutic applications of an expanded genetic code.

To date, over 100 noncanonical amino acids (ncAAs) have been genetically encoded in living cells in order to expand the functional repertoire of the canonical 20 amino acids. More recently, this technology has been expanded to the field of protein therapeutics, where traditional chemical methods typically result in heterogeneous mixtures of proteins. The site-specific incorporation of ncAAs with orthogonal chemical groups allows unprecedented control over the site of conjugation and the stoichiometry, thus facilitating the rational optimization of the biological functions and/or pharmacokinetics of biologics. Herein, we discuss the recent contribution of ncAA technology in enhancing the pharmacological properties of current protein therapeutics as well as developing novel therapeutic modalities.

Hybrid Photopatterned Enzymatic Reaction (HyPER) for in Situ Cell Manipulation

The ability to design artificial extracellular matrices as cell-instructive scaffolds has opened the door to technologies capable of studying the fate of cells in vitro and to guiding tissue repair in vivo. One main component of the design of artificial extracellular matrices is the incorporation of biochemical cues to guide cell phenotype and multicellular organization. The extracellular matrix (ECM) is composed of a heterogeneous mixture of proteins that present a variety of spatially discrete signals to residing cell populations. In contrast, most engineered ECMs do not mimic this heterogeneity. In recent years, photo-deprotection has been used to spatially immobilize signals. However, this approach has been limited mostly to small peptides. Here we combine photo-deprotection with enzymatic reaction to achieve spatially controlled immobilization of active bioactive signals that range from small molecules to large proteins. A peptide substrate for transglutaminase factor XIII (FXIIIa) was caged with a photo-deprotectable group, which was then immobilized to the bulk of a cell-compatible hydrogel. With focused light, the substrate can be deprotected and used to immobilize patterned bioactive signals. This approach offers an innovative strategy to immobilize delicate bioactive signals, such as growth factors, without loss of activity and enables in situ cell manipulation of encapsulated cells.

Biological barriers and current strategies for modifying nanoparticle bioavailability.

Nanoparticles are diligently crafted with exacting control over size, shape, and composition. The pristine nanoparticles are rigorously characterized in vitro by numerous physical and materials science techniques. Immediately after being exposed to body fluids, nanoparticles interact with a heterogeneous mixture of proteins and numerous different cell types that modify the nanoparticle surface and affect their bioavailability. Understanding the mechanisms behind the recognition and elimination of these modified nanoparticles is the key to the successful translation of nanomaterials from preclinical to clinical applications. This paper reviews the anatomy of the primary organs (kidney, liver, and spleen) responsible for nanoparticle bioelimination and the components of the innate immune system (complement system and scavenger receptors) that indirectly and directly recognize nanoparticles as foreign. Recent results using PEG as a steric barrier, generating biomimetic nanoparticles, and the effect of nanoparticle material properties to increase the bioavailability of nanoparticles are presented.