Antibiotic Treatment Prior to Injury Improves Post-Traumatic Osteoarthritis Outcomes in Mice.

Osteoarthritis (OA) is a painful and debilitating disease characterized by the chronic and progressive degradation of articular cartilage. Post-traumatic OA (PTOA) is a secondary form of OA that develops in ~50% of cases of severe articular injury. Inflammation and re-occurring injury have been implicated as contributing to the progression of PTOA after the initial injury. However, there is very little known about external factors prior to injury that could affect the risk of PTOA development. To examine how the gut microbiome affects PTOA development we used a chronic antibiotic treatment regimen starting at weaning for six weeks prior to ACL rupture, in mice. A six-weeks post-injury histological examination showed more robust cartilage staining on the antibiotic (AB)-treated mice than the untreated controls (VEH), suggesting slower disease progression in AB cohorts. Injured joints also showed an increase in the presence of anti-inflammatory M2 macrophages in the AB group. Molecularly, the phenotype correlated with a significantly lower expression of inflammatory genes Tlr5, Ccl8, Cxcl13, and Foxo6 in the injured joints of AB-treated animals. Our results indicate that a reduced state of inflammation at the time of injury and a lower expression of Wnt signaling modulatory protein, Rspo1, caused by AB treatment can slow down or improve PTOA outcomes.

Naphthalene DNA Adduct Formation and Tolerance in the Lung.

Naphthalene (NA) is a respiratory toxicant and possible human carcinogen. NA is a ubiquitous combustion product and significant component of jet fuel. The National Toxicology Program found that NA forms tumors in two species, in rats (nose) and mice (lung). However, it has been argued that NA does not pose a cancer risk to humans because NA is bioactivated by cytochrome P450 monooxygenase enzymes that have very high efficiency in the lung tissue of rodents but low efficiency in the lung tissue of humans. It is thought that NA carcinogenesis in rodents is related to repeated cycles of lung epithelial injury and repair, an indirect mechanism. Repeated in vivo exposure to NA leads to development of tolerance, with the emergence of cells more resistant to NA insult. We tested the hypothesis that tolerance involves reduced susceptibility to the formation of NA-DNA adducts. NA-DNA adduct formation in tolerant mice was examined in individual, metabolically-active mouse airways exposed ex vivo to 250 µΜ 14C-NA. Ex vivo dosing was used since it had been done previously and the act of creating a radioactive aerosol of a potential carcinogen posed too many safety and regulatory obstacles. Following extensive rinsing to remove unbound 14C-NA, DNA was extracted and 14C-NA-DNA adducts were quantified by AMS. The tolerant mice appeared to have slightly lower NA-DNA adduct levels than non-tolerant controls, but intra-group variations were large and the difference was statistically insignificant. It appears the tolerance may be more related to other mechanisms, such as NA-protein interactions in the airway, than DNA-adduct formation.

Disposition of the Dietary Mutagen 2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline in Healthy and Pancreatic Cancer Compromised Humans.

Pancreatic cancer is the fourth leading cause of cancer death in the U.S. Once diagnosed, prognosis is poor with a 5-year survival rate of less than 5%. Exposure to carcinogenic heterocyclic amines (HCAs) derived from cooked meat has been shown to be positively associated with pancreatic cancer risk. To evaluate the processes that determine the carcinogenic potential of HCAs for human pancreas, 14-carbon labeled 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), a putative human carcinogenic HCA found in well-done cooked meat, was administered at a dietary relevant dose to human volunteers diagnosed with pancreatic cancer undergoing partial pancreatectomy and healthy control volunteers. After (14)C-MeIQx exposure, blood and urine were collected for pharmacokinetic and metabolite analysis. MeIQx-DNA adducts levels were quantified by accelerator mass spectrometry from pancreatic tissue excised during surgery from the cancer patient group. Pharmacokinetic analysis of plasma revealed a rapid distribution of MeIQx with a plasma elimination half-life of approximately 3.5 h in 50% of the cancer patients and all of the control volunteers. In 2 of the 4 cancer patients, very low levels of MeIQx were detected in plasma and urine suggesting low absorption from the gut into the plasma. Urinary metabolite analysis revealed five MeIQx metabolites with 2-amino-3-methylimidazo[4,5-f]quinoxaline-8-carboxylic acid being the most abundant accounting for 25%-50% of the recovered 14-carbon/mL urine. There was no discernible difference in metabolite levels between the cancer patient volunteers and the control group. MeIQx-DNA adduct analysis of pancreas and duodenum tissue revealed adduct levels indistinguishable from background levels. Although other meat-derived HCA mutagens have been shown to bind DNA in pancreatic tissue, indicating that exposure to HCAs from cooked meat cannot be discounted as a risk factor for pancreatic cancer, the results from this current study show that exposure to a single dietary dose of MeIQx does not readily form measurable DNA adducts under the conditions of the experiment.

Determining the pharmacokinetics and long-term biodistribution of SiO2 nanoparticles in vivo using accelerator mass spectrometry.

Biodistribution is an important factor in better understanding silica dioxide nanoparticle (SiNP) safety. Currently, comprehensive studies on biodistribution are lacking, most likely due to the lack of suitable analytical methods. Accelerator mass spectrometry was used to investigate the relationship between administered dose, pharmacokinetics (PK), and long-term biodistribution of (14)C-SiNPs in vivo. PK analysis showed that SiNPs were rapidly cleared from the central compartment, were distributed to tissues of the reticuloendothelial system, and persisted in the tissue over the 8 week time course, raising questions about the potential for bioaccumulation and associated long-term effects.

Development of a CNS-permeable reactivator for nerve agent exposure: an iterative, multi-disciplinary approach.

Nerve agents have experienced a resurgence in recent times with their use against civilian targets during the attacks in Syria (2012), the poisoning of Sergei and Yulia Skripal in the United Kingdom (2018) and Alexei Navalny in Russia (2020), strongly renewing the importance of antidote development against these lethal substances. The current standard treatment against their effects relies on the use of small molecule-based oximes that can efficiently restore acetylcholinesterase (AChE) activity. Despite their efficacy in reactivating AChE, the action of drugs like 2-pralidoxime (2-PAM) is primarily limited to the peripheral nervous system (PNS) and, thus, provides no significant protection to the central nervous system (CNS). This lack of action in the CNS stems from their ionic nature that, on one end makes them very powerful reactivators and on the other renders them ineffective at crossing the Blood Brain Barrier (BBB) to reach the CNS. In this report, we describe the use of an iterative approach composed of parallel chemical and in silico syntheses, computational modeling, and a battery of detailed in vitro and in vivo assays that resulted in the identification of a promising, novel CNS-permeable oxime reactivator. Additional experiments to determine acute and chronic toxicity are ongoing.

Atomic force microscopy differentiates discrete size distributions between membrane protein containing and empty nanolipoprotein particles.

To better understand the incorporation of membrane proteins into discoidal nanolipoprotein particles (NLPs) we have used atomic force microscopy (AFM) to image and analyze NLPs assembled in the presence of bacteriorhodopsin (bR), lipoprotein E4 n-terminal 22k fragment scaffold and DMPC lipid. The self-assembly process produced two distinct NLP populations: those containing inserted bR (bR-NLPs) and those that did not (empty-NLPs). The bR-NLPs were distinguishable from empty-NLPs by an average increase in height of 1.0 nm as measured by AFM. Streptavidin binding to biotinylated bR confirmed that the original 1.0 nm height increase corresponds to br-NLP incorporation. AFM and ion mobility spectrometry (IMS) measurements suggest that NLP size did not vary around a single mean but instead there were several subpopulations, which were separated by discrete diameters. Interestingly, when bR was present during assembly the diameter distribution was shifted to larger particles and the larger particles had a greater likelihood of containing bR than smaller particles, suggesting that membrane proteins alter the mechanism of NLP assembly.

Cell-free co-expression of functional membrane proteins and apolipoprotein, forming soluble nanolipoprotein particles.

Here we demonstrate rapid production of solubilized and functional membrane protein by simultaneous cell-free expression of an apolipoprotein and a membrane protein in the presence of lipids, leading to the self-assembly of membrane protein-containing nanolipoprotein particles (NLPs). NLPs have shown great promise as a biotechnology platform for solubilizing and characterizing membrane proteins. However, current approaches are limited because they require extensive efforts to express, purify, and solubilize the membrane protein prior to insertion into NLPs. By the simple addition of a few constituents to cell-free extracts, we can produce membrane proteins in NLPs with considerably less effort. For this approach an integral membrane protein and an apolipoprotein scaffold are encoded by two DNA plasmids introduced into cell-free extracts along with lipids. For this study reported here we used plasmids encoding the bacteriorhodopsin (bR) membrane apoprotein and scaffold protein Delta1-49 apolipoprotein A-I fragment (Delta49A1). Cell free co-expression of the proteins encoded by these plasmids, in the presence of the cofactor all-trans-retinal and dimyristoylphosphatidylcholine, resulted in production of functional bR as demonstrated by a 5-nm shift in the absorption spectra upon light adaptation and characteristic time-resolved FT infrared difference spectra for the bR --> M transition. Importantly the functional bR was solubilized in discoidal bR.NLPs as determined by atomic force microscopy. A survey study of other membrane proteins co-expressed with Delta49A1 scaffold protein also showed significantly increased solubility of all of the membrane proteins, indicating that this approach may provide a general method for expressing membrane proteins enabling further studies.

Manipulation of the Gut Microbiome Alters Acetaminophen Biodisposition in Mice.

The gut microbiota is a vast and diverse microbial community that has co-evolved with its host to perform a variety of essential functions involved in the utilization of nutrients and the processing of xenobiotics. Shifts in the composition of gut microbiota can disturb the balance of organisms which can influence the biodisposition of orally administered drugs. To determine how changes in the gut microbiome can alter drug disposition, the pharmacokinetics (PK), and biodistribution of acetaminophen were assessed in C57Bl/6 mice after treatment with the antibiotics ciprofloxacin, amoxicillin, or a cocktail of ampicillin/neomycin. Altered PK, and excretion profiles of acetaminophen were observed in antibiotic exposed animals. Plasma Cmax was significantly decreased in antibiotic treated animals suggesting decreased bioavailability. Urinary metabolite profiles revealed decreases in acetaminophen-sulfate metabolite levels in both the amoxicillin and ampicillin/neomycin treated animals. The ratio between urinary and fecal excretion was also altered in antibiotic treated animals. Analysis of gut microbe composition revealed that changes in microbe content in antibiotic treated animals was associated with changes in acetaminophen biodisposition. These results suggest that exposure to amoxicillin or ampicillin/neomycin can alter the biodisposition of acetaminophen and that these alterations could be due to changes in gut microbiome composition.

Hydrogen production by a hyperthermophilic membrane-bound hydrogenase in water-soluble nanolipoprotein particles.

Hydrogenases constitute a promising class of enzymes for ex vivo hydrogen production. Implementation of such applications is currently hindered by oxygen sensitivity and, in the case of membrane-bound hydrogenases (MBHs), poor water solubility. Nanolipoprotein particles (NLPs) formed from apolipoproteins and phospholipids offer a novel means of incorporating MBHs into a well-defined water-soluble matrix that maintains the enzymatic activity and is amenable to incorporation into more complex architectures. We report the synthesis, hydrogen-evolving activity, and physical characterization of the first MBH-NLP assembly. This may ultimately lead to the development of biomimetic hydrogen-production devices.

Immobilization of His-tagged proteins on nickel-chelating nanolipoprotein particles.

Nanolipoprotein particles (NLPs) are nanometer-sized, discoidal particles that self-assemble from purified apolipoprotein and phospholipid. Their size and facile functionalization suggest potential application of NLPs as platforms for the presentation and delivery of recombinant proteins. To this end, we investigated incorporation of nickel-chelating lipids into NLPs (NiNLPs) and subsequent sequestration of polyhistidine (His)-tagged proteins. From initial lipid screens for NLP formation, the two phospholipids DMPC and DOPC were identified as suitable bulk lipids for incorporation of the nickel-chelating lipid DOGS-NTA-Ni into NLPs, and NiNLPs were successfully formed with varying amounts of DOGS-NTA-Ni. NiNLPs consisting of 10% DOGS-NTA-Ni with 90% bulk lipid (either DMPC or DOPC) were thoroughly characterized by size exclusion chromatography (SEC), non-denaturing gradient gel electrophoresis (NDGGE), and atomic force microscopy (AFM). Three different His-tagged proteins were sequestered on NiNLPs in a nickel-dependent manner, and the amount of immobilized protein was contingent on the size and composition of the NiNLP.

Cell-free expression for nanolipoprotein particles: building a high-throughput membrane protein solubility platform.

Membrane-associated proteins and protein complexes account for approximately a third or more of the proteins in the cell (1, 2). These complexes mediate essential cellular processes; including signal transduc-tion, transport, recognition, bioenergetics and cell-cell communication. In general, membrane proteins are challenging to study because of their insolubility and tendency to aggregate when removed from their protein lipid bilayer environment. This chapter is focused on describing a novel method for producing and solubilizing membrane proteins that can be easily adapted to high-throughput expression screening. This process is based on cell-free transcription and translation technology coupled with nanolipoprotein par ticles (NLPs), which are lipid bilayers confined within a ring of amphipathic protein of defined diameter. The NLPs act as a platform for inserting, solubilizing and characterizing functional membrane proteins. NLP component proteins (apolipoproteins), as well as membrane proteins can be produced by either traditional cell-based or as discussed here, cell-free expression methodologies.

Characterization and purification of polydisperse reconstituted lipoproteins and nanolipoprotein particles.

Heterogeneity is a fact that plagues the characterization and application of many self-assembled biological constructs. The importance of obtaining particle homogeneity in biological assemblies is a critical goal, as bulk analysis tools often require identical species for reliable interpretation of the results-indeed, important tools of analysis such as x-ray diffraction typically require over 90% purity for effectiveness. This issue bears particular importance in the case of lipoproteins. Lipid-binding proteins known as apolipoproteins can self assemble with liposomes to form reconstituted high density lipoproteins (rHDLs) or nanolipoprotein particles (NLPs) when used for biotechnology applications such as the solubilization of membrane proteins. Typically, the apolipoprotein and phospholipids reactants are self assembled and even with careful assembly protocols the product often contains heterogeneous particles. In fact, size polydispersity in rHDLs and NLPs published in the literature are frequently observed, which may confound the accurate use of analytical methods. In this article, we demonstrate a procedure for producing a pure, monodisperse NLP subpopulation from a polydisperse self-assembly using size exclusion chromatography (SEC) coupled with high resolution particle imaging by atomic force microscopy (AFM). In addition, NLPs have been shown to self assemble both in the presence and absence of detergents such as cholate, yet the effects of cholate on NLP polydispersity and separation has not been systematically examined. Therefore, we examined the separation properties of NLPs assembled in both the absence and presence of cholate using SEC and native gel electrophoresis. From this analysis, NLPs prepared with and without cholate showed particles with well defined diameters spanning a similar size range. However, cholate was shown to have a dramatic affect on NLP separation by SEC and native gel electrophoresis. Furthermore, under conditions where different sized NLPs were not sufficiently separated or purified by SEC, AFM was used to deconvolute the elution pattern of different sized NLPs. From this analysis we were able to purify an NLP subpopulation to 90% size homogeneity by taking extremely fine elutions from the SEC. With this purity, we generate high quality NLP crystals that were over 100 microm in size with little precipitate, which could not be obtained utilizing the traditional size exclusion techniques. This purification procedure and the methods for validation are broadly applicable to other lipoprotein particles.

Naphthalene genotoxicity: DNA adducts in primate and mouse airway explants.

Naphthalene (NA) is a ubiquitous environmental pollutant and possible human carcinogen that forms tumors in rodents with tissue/regional and species selectivity. This study seeks to determine whether NA is able to directly adduct DNA in an ex vivo culture system. Metabolically active lung tissue was isolated and incubated in explant culture with carbon-14 labeled NA (0, 25, 250 µM) or 1,2-naphthoquinone (NQ), followed by AMS analyses of metabolite binding to DNA. Despite relatively low metabolic bioactivation in the primate airway, dose-dependent NA-DNA adduct formation was detected. More airway adducts were detected in female mice (4.7-fold) and primates (2.1-fold) than in males of the same species. Few adducts were detected in rat airway or nasal epithelium. NQ, which is a metabolic product of NA, proved to be even more potent, with levels of adduct formation 70-80-fold higher than seen when tissues were incubated with the parent compound NA. This is the first study to demonstrate NA-DNA adduct formation at a site of carcinogenesis, the mouse lung. Adducts were also detected in non-human primate lung and with a NQ metabolite of NA. Taken together, this suggests that NA may contribute to in vivo carcinogenesis through a genotoxic mechanism.

Evaluation of in vitro neuronal platforms as surrogates for in vivo whole brain systems.

Quantitatively benchmarking similarities and differences between the in vivo central nervous system and in vitro neuronal cultures can qualify discrepancies in functional responses and establish the utility of in vitro platforms. In this work, extracellular electrophysiology responses of cortical neurons in awake, freely-moving animals were compared to in vitro cultures of dissociated cortical neurons. After exposure to two well-characterized drugs, atropine and ketamine, a number of key points were observed: (1) significant differences in spontaneous firing activity for in vivo and in vitro systems, (2) similar response trends in single-unit spiking activity after exposure to atropine, and (3) greater sensitivity to the effects of ketamine in vitro. While in vitro cultures of dissociated cortical neurons may be appropriate for many types of pharmacological studies, we demonstrate that for some drugs, such as ketamine, this system may not fully capture the responses observed in vivo. Understanding the functionality associated with neuronal cultures will enhance the relevance of electrophysiology data sets and more accurately frame their conclusions. Comparing in vivo and in vitro rodent systems will provide the critical framework necessary for developing and interpreting in vitro systems using human cells that strive to more closely recapitulate human in vivo function and response.

The biodistribution and pharmacokinetics of the oxime acetylcholinesterase reactivator RS194B in guinea pigs.

Organophosphorus-based (OP) nerve agents represent some of the most toxic substances known to mankind. The current standard of care for exposure has changed very little in the past decades, and relies on a combination of atropine to block receptor activity and oxime-type acetylcholinesterase (AChE) reactivators to reverse the OP binding to AChE. Although these oximes can block the effects of nerve agents, their overall efficacy is reduced by their limited capacity to cross the blood-brain barrier (BBB). RS194B, a new oxime developed by Radic et al. (J. Biol. Chem., 2012) has shown promise for enhanced ability to cross the BBB. To fully assess the potential of this compound as an effective treatment for nerve agent poisoning, a comprehensive evaluation of its pharmacokinetic (PK) and biodistribution profiles was performed using both intravenous and intramuscular exposure routes. The ultra-sensitive technique of accelerator mass spectrometry was used to quantify the compound's PK profile, tissue distribution, and brain/plasma ratio at four dose concentrations in guinea pigs. PK analysis revealed a rapid distribution of the oxime with a plasma t1/2 of ∼1 h. Kidney and liver had the highest concentrations per gram of tissue followed by lung, spleen, heart and brain for all dose concentrations tested. The Cmax in the brain ranged between 0.03 and 0.18% of the administered dose, and the brain-to-plasma ratio ranged from 0.04 at the 10 mg/kg dose to 0.18 at the 200 mg/kg dose demonstrating dose dependent differences in brain and plasma concentrations. In vitro studies show that both passive diffusion and active transport contribute little to RS194B traversal of the BBB. These results indicate that biodistribution is widespread, but very low quantities accumulate in the guinea pig brain, indicating this compound may not be suitable as a centrally active reactivator.

Maternal exposure to an environmentally relevant dose of triclocarban results in perinatal exposure and potential alterations in offspring development in the mouse model.

Triclocarban (TCC) is among the top 10 most commonly detected wastewater contaminants in both concentration and frequency. Its presence in water, as well as its propensity to bioaccumulate, has raised numerous questions about potential endocrine and developmental effects. Here, we investigated whether exposure to an environmentally relevant concentration of TCC could result in transfer from mother to offspring in CD-1 mice during gestation and lactation using accelerator mass spectrometry (AMS). 14C-TCC (100 nM) was administered to dams through drinking water up to gestation day 18, or from birth to post-natal day 10. AMS was used to quantify 14C-concentrations in offspring and dams after exposure. We demonstrated that TCC does effectively transfer from mother to offspring, both trans-placentally and via lactation. TCC-related compounds were detected in the tissues of offspring with significantly higher concentrations in the brain, heart and fat. In addition to transfer from mother to offspring, exposed offspring were heavier in weight than unexposed controls demonstrating an 11% and 8.5% increase in body weight for females and males, respectively. Quantitative real-time polymerase chain reaction (qPCR) was used to examine changes in gene expression in liver and adipose tissue in exposed offspring. qPCR suggested alterations in genes involved in lipid metabolism in exposed female offspring, which was consistent with the observed increased fat pad weights and hepatic triglycerides. This study represents the first report to quantify the transfer of an environmentally relevant concentration of TCC from mother to offspring in the mouse model and evaluate bio-distribution after exposure using AMS. Our findings suggest that early-life exposure to TCC may interfere with lipid metabolism and could have implications for human health.

Infection dynamics of Sin Nombre virus after a widespread decline in host populations.

Many researchers have speculated that infection dynamics of Sin Nombre virus are driven by density patterns of its major host, Peromyscus maniculatus. Few, if any, studies have examined this question systematically at a realistically large spatial scale, however. We collected data from 159 independent field sites within a 1 million-hectare study area in Nevada and California, from 1995-1998. In 1997, there was a widespread and substantial reduction in host density. This reduction in host density did not reduce seroprevalence of antibody to Sin Nombre virus within host populations. During this period, however, there was a significant reduction in the likelihood that antibody-positive mice had detectable virus in their blood, as determined by reverse-transcriptase polymerase chain reaction. Our findings suggest 2 possible causal mechanisms for this reduction: an apparent change in the age structure of host populations and landscape-scale patterns of host density. This study indicates that a relationship does exist between host density and infection dynamics and that this relationship concurrently operates at different spatial scales. It also highlights the limitations of antibody seroprevalence as a metric of infections, especially during transient host-density fluctuations.

Isolation, characterization, and stability of discretely-sized nanolipoprotein particles assembled with apolipophorin-III.

BACKGROUND: Nanolipoprotein particles (NLPs) are discoidal, nanometer-sized particles comprised of self-assembled phospholipid membranes and apolipoproteins. NLPs assembled with human apolipoproteins have been used for myriad biotechnology applications, including membrane protein solubilization, drug delivery, and diagnostic imaging. To expand the repertoire of lipoproteins for these applications, insect apolipophorin-III (apoLp-III) was evaluated for the ability to form discretely-sized, homogeneous, and stable NLPs. METHODOLOGY: Four NLP populations distinct with regards to particle diameters (ranging in size from 10 nm to >25 nm) and lipid-to-apoLp-III ratios were readily isolated to high purity by size exclusion chromatography. Remodeling of the purified NLP species over time at 4 degrees C was monitored by native gel electrophoresis, size exclusion chromatography, and atomic force microscopy. Purified 20 nm NLPs displayed no remodeling and remained stable for over 1 year. Purified NLPs with 10 nm and 15 nm diameters ultimately remodeled into 20 nm NLPs over a period of months. Intra-particle chemical cross-linking of apoLp-III stabilized NLPs of all sizes. CONCLUSIONS: ApoLp-III-based NLPs can be readily prepared, purified, characterized, and stabilized, suggesting their utility for biotechnological applications.

Quantifying size distributions of nanolipoprotein particles with single-particle analysis and molecular dynamic simulations.

Self-assembly of purified apolipoproteins and phospholipids results in the formation of nanometer-sized lipoprotein complexes, referred to as nanolipoprotein particles (NLPs). These bilayer constructs are fully soluble in aqueous environments and hold great promise as a model system to aid in solubilizing membrane proteins. Size variability in the self-assembly process has been recognized for some time, yet limited studies have been conducted to examine this phenomenon. Understanding the source of this heterogeneity may lead to methods to mitigate heterogeneity or to control NLP size, which may be important for tailoring NLPs for specific membrane proteins. Here, we have used atomic force microscopy, ion mobility spectrometry, and transmission electron microscopy to quantify NLP size distributions on the single-particle scale, specifically focusing on assemblies with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and a recombinant apolipoprotein E variant containing the N-terminal 22 kDa fragment (E422k). Four discrete sizes of E422k/DMPC NLPs were identified by all three techniques, with diameters centered at approximately 14.5, 19, 23.5, and 28 nm. Computer simulations suggest that these sizes are related to the structure and number of E422k lipoproteins surrounding the NLPs and particles with an odd number of lipoproteins are consistent with the double-belt model, in which at least one lipoprotein adopts a hairpin structure.

Different apolipoproteins impact nanolipoprotein particle formation.

Spontaneous interaction of purified apolipoproteins and phospholipids results in formation of lipoprotein particles with nanometer-sized dimensions; we refer to these assemblies as nanolipoprotein particles or NLPs. These bilayer constructs can serve as suitable mimetics of biological membranes and are fully soluble in aqueous environments. We made NLPs from dimyristoylphospatidylcholine (DMPC) in combination with each of four different apolipoproteins: apoA-I, Delta-apoA-I fragment, apoE4 fragment, and apolipophorin III (apoLp-III) from the silk moth B. mori. Predominately discoidal in shape, these particles have diameters between 10 and 20 nm, share uniform heights between 4.5 and 5 nm, and can be produced in yields ranging between 40 and 60%. The particular lipoprotein, the lipid to lipoprotein ratio, and the assembly parameters determine the size and homogeneity of nanolipoprotein particles and indicate that apoA-I NLP preparations are smaller than the larger apoE422K and apoLp-III NLP preparations.