24 research outputs found
Hexa-arginine enhanced uptake and residualization of selective high affinity ligands by Raji lymphoma cells
<p>Abstract</p> <p>Background</p> <p>A variety of arginine-rich peptide sequences similar to those found in viral proteins have been conjugated to other molecules to facilitate their transport into the cytoplasm and nucleus of targeted cells. The selective high affinity ligand (SHAL) (DvLPBaPPP)<sub>2</sub>LLDo, which was developed to bind only to cells expressing HLA-DR10, has been conjugated to one of these peptide transduction domains, hexa-arginine, to assess the impact of the peptide on SHAL uptake and internalization by Raji cells, a B-cell lymphoma.</p> <p>Results</p> <p>An analog of the SHAL (DvLPBaPPP)<sub>2</sub>LLDo containing a hexa-arginine peptide was created by adding six D-arginine residues sequentially to a lysine inserted in the SHAL's linker. SHAL binding, internalization and residualization by Raji cells expressing HLA-DR10 were examined using whole cell binding assays and confocal microscopy. Raji cells were observed to bind two fold more <sup>111</sup>In-labeled hexa-arginine SHAL analog than Raji cells treated with the parent SHAL. Three fold more hexa-arginine SHAL remained associated with the Raji cells after washing, suggesting that the peptide also enhanced residualization of the <sup>111</sup>In transported into cells. Confocal microscopy showed both SHALs localized in the cytoplasm of Raji cells, whereas a fraction of the hexa-arginine SHAL localized in the nucleus.</p> <p>Conclusion</p> <p>The incorporation of a hexa-D-arginine peptide into the linker of the SHAL (DvLPBaPPP)<sub>2</sub>LLDo enhanced both the uptake and residualization of the SHAL analog by Raji cells. In contrast to the abundant cell surface binding observed with Lym-1 antibody, the majority of (DvLPBaPPP)<sub>2</sub>LArg6AcLLDo and the parent SHAL were internalized. Some of the internalized hexa-arginine SHAL analog was also associated with the nucleus. These results demonstrate that several important SHAL properties, including uptake, internalization, retention and possibly intracellular distribution, can be enhanced or modified by conjugating the SHALs to a short polypeptide.</p
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 resultsβindeed, 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 ΞΌm 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
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 4uC 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 utilit
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Packaging of single DNA molecules by the yeast mitochondrial protein Abf2p.
Mitochondrial and nuclear DNA are packaged by proteins in a very different manner. Although protein-DNA complexes called "nucleoids" have been identified as the genetic units of mitochondrial inheritance in yeast and man, little is known about their physical structure. The yeast mitochondrial protein Abf2p was shown to be sufficient to compact linear dsDNA, without the benefit of supercoiling, using optical and atomic force microscopy single molecule techniques. The packaging of DNA by Abf2p was observed to be very weak as evidenced by a fast Abf2p off-rate (k(off) = 0.014 +/- 0.001 s(-1)) and the extremely small forces (<0.6 pN) stabilizing the condensed protein-DNA complex. Atomic force microscopy images of individual complexes showed the 190-nm structures are loosely packaged relative to nuclear chromatin. This organization may leave mtDNA accessible for transcription and replication, while making it more vulnerable to damage
Colocalized Delivery of Adjuvant and Antigen Using Nanolipoprotein Particles Enhances the Immune Response to Recombinant Antigens
Subunit antigen-based vaccines can provide a number of
important
benefits over traditional vaccine candidates, such as overall safety.
However, because of the inherently low immunogenicity of these antigens,
methods for colocalized delivery of antigen and immunostimulatory
molecules (i.e., adjuvants) are needed. Here we report a robust nanolipoprotein
particle (NLP)-based vaccine delivery platform that facilitates the
codelivery of both subunit antigens and adjuvants. Ni-chelating NLPs
(NiNLPs) were assembled to incorporate the amphipathic adjuvants monophosphoryl
lipid A and cholesterol-modified CpG oligodeoxynucleotides, which
can bind His-tagged protein antigens. Colocalization of antigen and
adjuvant delivery using the NiNLP platform resulted in elevated antibody
production against His-tagged influenza hemagglutinin 5 and Yersinia pestis LcrV antigens. Antibody titers in
mice immunized with the adjuvanted NLPs were 5β10 times higher
than those observed with coadministration formulations and nonadjuvanted
NiNLPs. Colocalized delivery of adjuvant and antigen provides significantly
greater immune stimulation in mice than coadministered formulations
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Lipid Cross-Linking of Nanolipoprotein Particles Substantially Enhances Serum Stability and Cellular Uptake
Nanolipoprotein
particles (NLPs) consist of a discoidal phospholipid
lipid bilayer confined by an apolipoprotein belt. NLPs are a promising
platform for a variety of biomedical applications due to their biocompatibility,
size, definable composition, and amphipathic characteristics. However,
poor serum stability hampers the use of NLPs for in vivo applications
such as drug formulation. In this study, NLP stability was enhanced
upon the incorporation and subsequent UV-mediated intermolecular cross-linking
of photoactive DiynePC phospholipids in the lipid bilayer, forming
cross-linked nanoparticles (X-NLPs). Both the concentration of DiynePC
in the bilayer and UV exposure time significantly affected the resulting
X-NLP stability in 100% serum, as assessed by size exclusion chromatography
(SEC) of fluorescently labeled particles. Cross-linking did not significantly
impact the size of X-NLPs as determined by dynamic light scattering
and SEC. X-NLPs had essentially no degradation over 48 h in 100% serum,
which is a drastic improvement compared to non-cross-linked NLPs (50%
degradation by βΌ10 min). X-NLPs had greater uptake into the
human ATCC 5637 bladder cancer cell line compared to non-cross-linked
particles, indicating their potential utility for targeted drug delivery.
X-NLPs also exhibited enhanced stability following intravenous administration
in mice. These results collectively support the potential utility
of X-NLPs for a variety of in vivo applications
Evaluation of nanolipoprotein particles (NLPs) as an in vivo delivery platform.
Nanoparticles hold great promise for the delivery of therapeutics, yet limitations remain with regards to the use of these nanosystems for efficient long-lasting targeted delivery of therapeutics, including imparting functionality to the platform, in vivo stability, drug entrapment efficiency and toxicity. To begin to address these limitations, we evaluated the functionality, stability, cytotoxicity, toxicity, immunogenicity and in vivo biodistribution of nanolipoprotein particles (NLPs), which are mimetics of naturally occurring high-density lipoproteins (HDLs). We found that a wide range of molecules could be reliably conjugated to the NLP, including proteins, single-stranded DNA, and small molecules. The NLP was also found to be relatively stable in complex biological fluids and displayed no cytotoxicity in vitro at doses as high as 320 Β΅g/ml. In addition, we observed that in vivo administration of the NLP daily for 14 consecutive days did not induce significant weight loss or result in lesions on excised organs. Furthermore, the NLPs did not display overt immunogenicity with respect to antibody generation. Finally, the biodistribution of the NLP in vivo was found to be highly dependent on the route of administration, where intranasal administration resulted in prolonged retention in the lung tissue. Although only a select number of NLP compositions were evaluated, the findings of this study suggest that the NLP platform holds promise for use as both a targeted and non-targeted in vivo delivery vehicle for a range of therapeutics