Light-sensitive lipid-based nanoparticles for drug delivery: design principles and future considerations for biological applications

Radiation-based therapies aided by nanoparticles have been developed for decades, and can be primarily categorized into two main platforms. First, delivery of payload of photo-reactive drugs (photosensitizers) using the conventional nanoparticles, and second, design and development of photo-triggerable nanoparticles (primarily liposomes) to attain light-assisted on-demand drug delivery. The main focus of this review is to provide an update of the history, current status and future applications of photo-triggerable lipid-based nanoparticles (light-sensitive liposomes). We will begin with a brief overview on the applications of liposomes for delivery of photosensitizers, including the choice of photosensitizers for photodynamic therapy, as well as the currently available light sources (lasers) used for these applications. The main segment of this review will encompass the details of strategies used to develop photo-triggerable liposomes for their drug delivery function. The principles underlying the assembly of photoreactive lipids into nanoparticles (liposomes) and photo-triggering mechanisms will be presented. We will also discuss factors that limit the applications of these liposomes for in vivo triggered drug delivery and emerging concepts that may lead to the biologically viable photo-activation strategies. We will conclude with our view point on the future perspectives of light-sensitive liposomes in the clinic.     

A model two-component system for studying the architecture of elastin assembly in vitro

Tropoelastin is encoded by a single human gene that spans 36 exons and is oxidized in vivo by mammalian lysyl oxidase at the epsilon amino group of available lysines to give the adipic semialdehyde, which then facilitates covalent cross-link formation in an enzyme-free process involving tropoelastin association. We demonstrate here that this process is effectively modeled by a two protein component system using purified lysyl oxidase from the yeast Pichia pastoris to facilitate the oxidation and subsequent cross-linking of recombinant human tropoelastin. The oxidized human tropoelastin forms an elastin-like polymer (EL) that is elastic, shows hydrogel behavior and contains typical elastin cross-links including lysinonorleucine, allysine aldol, and desmosine. Protease digestion and subsequent mass-spectrometry analysis of multiple ELs allowed for the identification of specific intra- and inter-molecular cross-links, leading to a model of the molecular architecture of elastin assembly in vitro. Specific intra-molecular cross-links were confined to the region of tropoelastin encoded by exons 6-15. Inter-molecular cross-links were prevalent between the regions encoded by exons 19-25. We find that assembly of tropoelastin molecules in ELs are highly enriched for a defined subset of cross-links.     

Identification of proteins that interact with exon sequences, splice sites, and the branchpoint sequence during each stage of spliceosome assembly.

We have carried out a systematic analysis of the proteins that interact with specific intron and exon sequences during each stage of mammalian spliceosome assembly. This was achieved by site-specifically labeling individual nucleotides within the 5' and 3' splice sites, the branchpoint sequence (BPS), or the exons with 32P and identifying UV-cross-linked proteins in the E, A, B, or C spliceosomal complex. Significantly, two members of the SR family of splicing factors, which are known to promote E-complex assembly, cross-link within exon sequences to a region approximately 25 nucleotides upstream from the 5' splice site. At the 5' splice site, cross-linking of the U5 small nuclear ribonucleoprotein particle protein, U5(200), was detected in both the B and C complexes. As observed in yeast cells, U5(200), also cross-links to intron/exon sequences at the 3' splice site in the C complex and may play a role in aligning the 5' and 3' exons for ligation. With label at the branch site, we detected three distinct proteins, designated BPS72,BpS70, and BPS56, which replace one another in the E, A, and C complexes. Another dynamic exchange was detected with pre-mRNA labeled at the AG dinucleotide of the 3' splice site. In this case, a protein, AG100,cross-links in the A complex and is replaced by another protein, AG75, in the C complex. The observation that these proteins are specifically associated with critical pre-mRNA sequence elements in functional complexes at different stages of spliceosome assembly implicates roles for these factors in key recognition events during the splicing pathway.     

Light-sensitive lipid-based nanoparticles for drug delivery: design principles and future considerations for biological applications

Abstract

Radiation-based therapies aided by nanoparticles have been developed for decades, and can be primarily categorized into two main platforms. First, delivery of payload of photo-reactive drugs (photosensitizers) using the conventional nanoparticles, and second, design and development of photo-triggerable nanoparticles (primarily liposomes) to attain light-assisted on-demand drug delivery. The main focus of this review is to provide an update of the history, current status and future applications of photo-triggerable lipid-based nanoparticles (light-sensitive liposomes). We will begin with a brief overview on the applications of liposomes for delivery of photosensitizers, including the choice of photosensitizers for photodynamic therapy, as well as the currently available light sources (lasers) used for these applications. The main segment of this review will encompass the details of strategies used to develop photo-triggerable liposomes for their drug delivery function. The principles underlying the assembly of photoreactive lipids into nanoparticles (liposomes) and photo-triggering mechanisms will be presented. We will also discuss factors that limit the applications of these liposomes for in vivo triggered drug delivery and emerging concepts that may lead to the biologically viable photo-activation strategies. We will conclude with our view point on the future perspectives of light-sensitive liposomes in the clinic.     

Nanoparticle-triggered release from lipid membrane vesicles

Superparamagnetic iron oxide nanoparticles are used in a rapidly expanding number of research and practical applications in biotechnology and biomedicine. We highlight how recent developments in iron oxide nanoparticle design and understanding of nanoparticle membrane interactions have led to applications in magnetically triggered, liposome delivery vehicles with controlled structure. Nanoscale vesicles actuated by incorporated nanoparticles allow for controlling location and timing of compound release, which enables e.g. use of more potent drugs in drug delivery as the interaction with the right target is ensured. This review emphasizes recent results on the connection between nanoparticle design, vesicle assembly and the stability and release properties of the vesicles. While focused on lipid vesicles magnetically actuated through iron oxide nanoparticles, these insights are of general interest for the design of capsule and cell delivery systems for biotechnology controlled by nanoparticles.     

Development of efficient expression system for protein display on bacterial magnetic particles

Bacterial magnetic particles (BMPs) are utilized for various biomedical applications because they are easily manipulated by magnets, and functional proteins are easily displayed on BMPs. To establish highly expressed protein display on BMPs, strong promoters were identified using Magnetospirillum magneticum AMB-1 genome and proteome databases. Initially, several proteins highly expressed in AMB-1 were identified, and the upstream DNA sequences of the open-reading frames were evaluated using a luciferase-reporter gene assay to compare promoter activities. Consequently, luminescence intensity was 400 times higher due to the novel promoter identified in this study than the magA promoter previously used. Subsequently, efficient protein display on BMPs was performed using the newly identified promoter sequences. This developed display system will facilitate the assembly of various functional proteins onto BMPs to create novel magnetic nanoparticles.     

Multilayer associates based on oligonucleotides and gold nanoparticles

The development of nanodevices for the efficient transport of therapeutic molecules is one of the most urgent problems of modern molecular medicine. Noncovalent agents for the delivery of nucleic acids (NA) including those based on gold nanoparticles (GNPs) represent an attractive alternative to covalent systems, since it is easier in this case to provide the controlled release of NA. We have demonstrated the possibility to create potentially biocompatible associates of GNPs containing alternating layers of oligonucleotides and other polymers as a promising platform for the delivery of oligonucleotides into living cells. The multilayer (five layers) coated GNPs can be assembled by the sequential treatment of gold nanoparticles with nonthiolated oligonucleotide (ON), thiolated carboxyl-polyethylene glycol (SH-PEG3000-COOH), and linear polyethyleneimine (PEI). We have developed an algorithm for the analysis of multilayer coated GNPs by gel electrophoresis, photon correlation spectroscopy, and transmission electron microscopy. The assembly of associates bearing two oligonucleotide layers and having a net positive surface charge has been described. Multilayer coated GNPs were shown not to degrade in the presence of a high concentration of the major blood protein, serum albumin.     

Self-assembling protein hydrogels with modular integrin binding domains

Hydrogels with integrin binding activity were created from associating proteins with embedded RGD sequences. These proteins are a modified AC(10)Bcys triblock design composed of acidic A and basic B leucine zipper associating domains flanking a new soluble disordered coil block that contains nine repeats of AGAGAGPEG and three copies of the RGD integrin binding sequence. As with the original AC(10)Bcys design without the embedded RGD sequences, these proteins self-assemble into stable hydrogels at concentrations above approximately 50 mg/mL in a range of solution pH and temperature conditions. The mechanism for hydrogel assembly is the intermolecular association of A and B helical domains into bundles which act as cross-links connected by the soluble central disordered coil domains. The secondary structure of the proteins and the mechanical properties of the hydrogels they form are not adversely affected by the presence of the RGD sequences. The RGD sequences embedded in the disordered coil region support the adhesion, spreading, and polarization of human fibroblast cells on protein coated surfaces. Confocal microscopy studies demonstrated the presence of focal adhesion complexes and organized actin stress fibers in these cells. In contrast, fibroblasts seeded onto surfaces coated with the original AC(10)Bcys protein remained rounded and did not form focal adhesions, indicating that bioactivity is conferred by the presence of the embedded RGD sequences. Such hydrogel-forming bioactive proteins have potential for cell and tissue culture applications.     

Subunit structure and assembly of the globular domain of basement-membrane collagen type IV

The globular domain of collagen IV was solubilized by collagenase digestion from a mouse tumor, human placenta and bovine aorta and was purified by chromatographic methods. The materials show a unique, mainly non-collagenous amino acid composition and contain small amounts of glucosamine and galactosamine. The globular structures with Mr = 170 000 appear as a hexameric assembly originating from two collagen IV molecules. Subunits of this assembly are two different dimers Da and Db (Mr about 56 000) and monomers (Mr = 28 000). Their N-terminal amino acid sequences start with short triple-helical sequences, which overlap with the C-terminal triple helix of the alpha 1(IV) and alpha 2(IV) chain, demonstrating that the globule originates from the C terminus of collagen IV. Dimers arise from monomers by disulfide cross-linking (form Db) and/or formation of non-reducible cross-links (form Da). Reduction under non-denaturing conditions causes partial dissociation of the globule and of collagen IV dimers, indicating that reducible cross-links are formed between monomers of two different collagen IV molecules. Dissociation of the hexamer into the subunits can be achieved with 8 M urea, sodium dodecyl sulfate or in the pH range 2.5-4. The latter indicates that carboxyl groups are essential for association. Mixtures of the subunits (monomers and dimers) or purified dimers reassemble in neutral buffer into hexamers as shown by ultracentrifugation and electron microscopy. Reconstituted hexamers, however, dissociate in a much broader pH range than the native globules. Circular dichroic spectra indicate that the structure is more completely refolded from acid-treated than from urea-treated material. These data suggest that globules originating from monomers (as existing in single collagen IV molecules) are stabilized by the adjacent triple helix. Covalent cross-link formation stabilizes the globular structure and allows reconstitution in stoichiometric proportions.     

Characterization of the Bacillus subtilis spore morphogenetic coat protein CotO

Bacillus spores are protected by a structurally and biochemically complex protein shell composed of over 50 polypeptide species, called the coat. Coat assembly in Bacillus subtilis serves as a relatively tractable model for the study of the formation of more complex macromolecular structures and organelles. It is also a critical model for the discovery of strategies to decontaminate B. anthracis spores. In B. subtilis, a subset of coat proteins is known to have important roles in assembly. Here we show that the recently identified B. subtilis coat protein CotO (YjbX) has an especially important morphogenetic role. We used electron and atomic force microscopy to show that CotO controls assembly of the coat layers and coat surface topography as well as biochemical and cell-biological analyses to identify coat proteins whose assembly is CotO dependent. cotO spores are defective in germination and partially sensitive to lysozyme. As a whole, these phenotypes resemble those resulting from a mutation in the coat protein gene cotH. Nonetheless, the roles of CotH and CotO and the proteins whose assembly they direct are not identical. Based on fluorescence and electron microscopy, we suggest that CotO resides in the outer coat (although not on the coat surface). We propose that CotO and CotH participate in a late phase of coat assembly. We further speculate that an important role of these proteins is ensuring that polymerization of the outer coat layers occurs in such a manner that contiguous shells, and not unproductive aggregates, are formed.     

Designing sensitive and specific spaced seeds for cross-species mRNA-to-genome alignment.

As the demand for accurately aligning gene sequences to the genome of a related species grows with the sequencing of new genomes, spaced seeds emerge as a promising vehicle for increasing alignment sensitivity. We extend the existing {0, 1} match-mismatch models for sensitivity evaluation to take into account the compositional structure of coding sequences and ultimately produce seeds better suited to this particular application. Designing seeds for alignment programs, however, needs to balance sensitivity and specificity.We assess the effects of seed variations on both sensitivity and specificity in an extended model that incorporates transitions and differentiates among the three codon positions, and show that spaced seeds with transitions offer a better sensitivity-specificity tradeoff. Furthermore, we propose a theoretical formulation for rigorously assessing seed specificity, starting from Bernoulli and Markov models of the mRNA and genomic sequences. Within this framework, we perform the first comprehensive analysis of seeds to serve as a blueprint for selecting sensitive and specific seeds for practical applications. Our analyses show that specificity is relatively constant for seeds of a given weight, while sensitivity varies widely, with the highest values attained by seeds allowing a small (2-6) number of transitions.A strategy for designing seeds, therefore, is to first select the weight of the seed by identifying the desired sensitivity-specificity tradeoff, then choose the most sensitive seed(s) within that weight group. We illustrate our methods with the alignment of chicken coding sequences against the human genome assembly version HG17.     

Influence of ancillary binding and nonspecific adsorption on bioresponsive hydrogel microlenses

We report investigations of specific and nonspecific adsorption effects on bioresponsive hydrogel microlenses to better understand their utility and potential advantages for biosensing. Bioresponsive microgels were prepared from stimuli-responsive poly(N-isopropylacrylamide-co-acrylic acid) (pNIPAM-co-AAc) microgels after functionalization with both biotin and ABP (as a photoaffinity label) via carbodiimide chemistry. Bioresponsive hydrogel microlenses were then constructed from the microgels via Coulombic assembly of the anionic microgels on a positively charged, silane-modified, glass substrate. Specific and nonspecific protein binding on the hydrogel microlenses was studied by monitoring the optical properties using brightfield and fluorescence optical microscopies. The bioresponsivity, as determined by changes in the microlensing power, is strongly coupled to the formation of cross-links via ligand-protein and/or antigen-antibody binding. However, the microlensing phenomenon and the intrinsic bioresponsivity of the hydrogels are completely insensitive to simple adsorption via nonspecific protein binding from reconstituted human serum. These results suggest that the hydrogel microlens construct may be a good candidate for a wide range of applications in which the bioresponsive material would be required to operate in complex biological media.     

Self-assembly of protein superstructures by physical interactions under cytoplasm-like conditions

The structure-driven assembly of multimeric protein complexes and the formation of intracellular phase-like protein condensates have been the subject of intense research. However, the assembly of larger superstructures comprising cellular components, such as protein nanoparticles driven by general physical rather than specific biochemical interactions, remains relatively uncharacterized. Here, we use gas vesicles (GVs)—genetically encoded protein nanoparticles that form ordered intracellular clusters—as a model system to study the forces driving multiparticle assembly under cytoplasm-like conditions. Our calculations and experimental results show that the ordered assembly of GVs can be achieved by screening their mutual electrostatic repulsion with electrolytes and creating a crowding force with dissolved macromolecules. The precise balance of these forces results in different packing configurations. Biomacromolecules such as polylysine and DNA are capable of driving GV clustering. These results provide basic insights into how physically driven interactions affect the formation of protein superstructures, offer guidance for manipulating nanoparticle assembly in cellular environments through synthetic biology methods, and inform research on the biotechnology applications of GVs.     

Amplification and assembly of chip-eluted DNA (AACED): a method for high-throughput gene synthesis

A basic problem in gene synthesis is the acquisition of many short oligonucleotide sequences needed for the assembly of genes. Photolithographic methods for the massively parallel synthesis of high-density oligonucleotide arrays provides a potential source, once appropriate methods have been devised for their elution in forms suitable for enzyme-catalyzed assembly. Here, we describe a method based on the photolithographic synthesis of long (>60mers) single-stranded oligonucleotides, using a modified maskless array synthesizer. Once the covalent bond between the DNA and the glass surface is cleaved, the full-length oligonucleotides are selected and amplified using PCR. After cleavage of flanking primer sites, a population of unique, internal 40mer dsDNA sequences are released and are ready for use in biological applications. Subsequent gene assembly experiments using this DNA pool were performed and were successful in creating longer DNA fragments. This is the first report demonstrating the use of eluted chip oligonucleotides in biological applications such as PCR and assembly PCR.     

Stabilization of nanoparticles under biological assembly conditions using peptoids

Sequence-specific polymers are proving to be a powerful approach to assembly and manipulation of matter on the nanometer scale. This has been most impressive in the case of DNA, and progress has been made toward templating inorganic nanoparticles using DNA nanostructures. One obstacle to this progress is that inorganic nanomaterials are often incompatible with DNA assembly conditions, which involve aqueous solutions high in either or both monovalent and divalent salt. Synthetic oligopeptide ligands have been shown by others to improve nanoparticle stability in high concentrations of monovalent salt. Ligands that are peptoids, or sequence-specific N-functional glycine oligomers, allow precise and flexible control over the arrangement of binding groups, steric spacers, charge, and other functionality. We have synthesized short peptoids that can prevent the aggregation of gold nanoparticles in high-salt environments including divalent salt, and allow coadsorption of a single DNA molecule. This degree of precision and versatility is likely to prove essential in bottom-up assembly of nanostructures and in biomedical applications of nanomaterials.     

Use of oligonucleotides to define the site of interstrand cross-links induced by Adriamycin.

It has been known for several years that Adriamycin forms adducts and interstrand cross-links when reacted for long periods of time with bacterial and mammalian DNA in vitro, with the cross-link being restricted to 2 bp elements containing GpC sequences. The self-complementary 20mer deoxyoligonucleotide TA4T4GCA4T4A has been used in this study as a model of the apparent G-G cross-linking site at GpC sequences. The rate of formation of cross-links, as well as the dependence on both Adriamycin and Fe(III) concentration, were similar with this oligonucleotide as compared with calf thymus DNA. The cross-linking was demonstrated on both denaturing and non-denaturing sequencing gels. The half-life of the G-G cross-link was 40 h, consistent with that implied with high molecular weight, heterogeneous sequence DNA. Exonuclease III digests of adducts formed with 20mer deoxyoligonucleotides containing single, central G-G, G-I and I-I potential cross-links revealed that a guanine residue is required at both ends of the cross-link. No cross-linking was observed with a similar oligonucleotide containing only a single central (G.C) bp.     

The regulation of size and form in the assembly of collagen fibrils in vivo

A possible mechanism for regulating the lateral growth of collagen fibrils in vivo is considered. A growth inhibitor associated with a particular part of the long semiflexible collagen molecule restricts that part of the molecule to the surface of the growing assembly. Lateral accretion ceases when these inhibitors form a complete circumferential layer around the fibril surface. Cell-mediated removal of the inhibitors allows lateral growth to proceed to a second limiting layer, and so on to subsequent limiting layers. In this way, cycles of inhibitor removal and limited lateral accretion permit growth to be synchronized over large populations of fibrils. Observed diameter distributions in bundles of embryonic and neonatal fibrils are those expected from a mechanism of this kind. The mechanism depends on the existence of axial order (D-periodicity) in fibrils, but not on any specific lateral packing of molecules. Rather, contacts between newly assembled molecules are presumed to be partly fluid-like in lateral directions (except where covalent cross-links have formed). Some initial fluidity in lateral packing prior to cross-linking does not preclude the subsequent emergence of quasi-crystalline packing as cross-links form. The cylindrical shape of fibrils in vivo may also be attributable in part to fluidity of intermolecular contacts at the growing surface.     

Gene-specific formation and repair of cisplatin intrastrand adducts and interstrand cross-links in Chinese hamster ovary cells

We have used three methods to study the formation and repair of intrastrand adducts and interstrand cross-links in the DNA of Chinese hamster ovary cells induced by the anticancer drug cis-diamminedichloroplatinum II (cisplatin). Using atomic absorption spectroscopy, we found that 21% of the total genomic cisplatin adducts were removed at 8 h and 42% at 24 h. We used ABC excinuclease digestion, coupled with out previously reported methodology to quantify DNA in specific genomic regions. These adducts were removed faster in the transcribed dihydrofolate reductase and c-myc genes compared to a noncoding fragment, a region containing the little or nontranscribed c-fos oncogene, and to the overall genome. Interstrand cross-links in specific sequences were quantified by Southern hybridization of denatured-renatured DNA separated on a neutral gel. We found that cross-links were removed more efficiently from the gene regions than intrastrand adducts and, at high levels of cross-linking, removal was similar from transcribed and from nontranscribed regions.     

Psoralen photo-cross-linking by triplex-forming oligonucleotides at multiple sites in the human rhodopsin gene.

Targeting DNA damage by triplex-forming oligonucleotides (TFOs) represents a way of modifying gene expression and structure and a possible approach to gene therapy. We have determined that this approach can deliver damage with great specificity to sites in the human gene for the G-protein-linked receptor rhodopsin, mutations of which can lead to the genetic disorder autosomal dominant retinitis pigmentosa. We have introduced DNA monoadducts and interstrand cross-links at multiple target sites within the gene using TFOs with a photoactivatable psoralen group at the 5'-end. The extent of formation of photoadducts (i.e., monoadducts and cross-links) was measured at target sites with a 5'-ApT sequence at the triplex-duplex junction and at a target site with 5'-ApT and 5'-TpA sequences located four and seven nucleotides away, respectively. To improve psoralen reactivity at more distant sites, psoralen moieties were attached to TFOs with nucleotide "linkers" from two to nine nucleotides in length. High-affinity binding was maintained with linkers of up to 10 nucleotides, but affinities tended to decrease somewhat with increasing linker length due to faster dissociation kinetics. DNase I footprinting indicated little, if any, interaction between linkers and the duplex. Psoralen-TFO conjugates formed DNA cross-links with high efficiency (56-65%) at 5'-ApT sequences located at triplex junctions. At a 5'-ApT site four nucleotides away, the efficiency varied with linker length; a four-nucleotide linker gave the highest efficiency. Duplexes with 5'-TpA and 5'-ApT sites two nucleotides away, in otherwise identical sequences, were cross-linked with efficiencies of 56 and 38%, respectively. These results indicate that TFO-linker-psoralen conjugates allow simultaneous, efficient targeting of multiple sites in the human rhodopsin gene.