Daniel Aili

Enzyme-responsive nanoparticles for drug release and diagnostics ☆

Enzymes are key components of the bionanotechnology toolbox that possess exceptional biorecognition capabilities and outstanding catalytic properties. When combined with the unique physical properties of nanomaterials, the resulting enzyme-responsive nanoparticles can be designed to perform functions efficiently and with high specificity for the triggering stimulus. This powerful concept has been successfully applied to the fabrication of drug delivery schemes where the tissue of interest is targeted via release of cargo triggered by the biocatalytic action of an enzyme. Moreover, the chemical transformation of the carrier by the enzyme can also generate therapeutic molecules, therefore paving the way to design multimodal nanomedicines with synergistic effects. Dysregulation of enzymatic activity has been observed in a number of severe pathological conditions, and this observation is useful not only to program drug delivery in vivo but also to fabricate ultrasensitive sensors for diagnosing these diseases. In this review, several enzyme-responsive nanomaterials such as polymer-based nanoparticles, liposomes, gold nanoparticles and quantum dots are introduced, and the modulation of their physicochemical properties by enzymatic activity emphasized. When known, toxicological issues related to the utilization nanomaterials are highlighted. Key examples of enzyme-responsive nanomaterials for drug delivery and diagnostics are presented, classified by the type of effector biomolecule, including hydrolases such as proteases, lipases and glycosidases, and oxidoreductases.

Colorimetric Protein Sensing by Controlled Assembly of Gold Nanoparticles Functionalized with Synthetic Receptors

A novel strategy is described for the colorimetric sensing of proteins, based on polypeptide-functionalized gold nanoparticles. Recognition is accomplished using a polypeptide sensor scaffold designed to specifically bind to the model analyte, human carbonic anhydrase II (HCAII). The extent of particle aggregation, induced by the Zn(2+)-triggered dimerization and folding of a second polypeptide also present on the surface of the gold nanoparticle, gives a readily detectable colorimetric shift that is dependent on the concentration of the target protein. In the absence of HCAII, particle aggregation results in a major redshift of the plasmon peak, whereas analyte binding prevented the formation of dense aggregates, significantly reducing the magnitude of the redshift. The versatility of the technique is demonstrated using a second model system based on the recognition of a peptide sequence from the tobacco mosaic virus coat protein (TMVP) by a recombinant antibody fragment (Fab57P). Concentrations down to approximately 10 nM and approximately 25 nM are detected for HCAII and Fab57P, respectively. This strategy is proposed as a generic platform for robust and specific protein analysis that can be further developed to monitor a wide range of target proteins.

Folding Induced Assembly of Polypeptide Decorated Gold Nanoparticles

Reversible assembly of gold nanoparticles controlled by the homodimerization and folding of an immobilized de novo designed synthetic polypeptide is described. In solution at neutral pH, the polypeptide folds into a helix-loop-helix four-helix bundle in the presence of zinc ions. When immobilized on gold nanoparticles, the addition of zinc ions induces dimerization and folding between peptide monomers located on separate particles, resulting in rapid particle aggregation. The particles can be completely redispersed by removal of the zinc ions from the peptide upon addition of EDTA. Calcium ions, which do not induce folding in solution, have no effect on the stability of the peptide decorated particles. The contribution from folding on particle assembly was further determined utilizing a reference peptide with the same primary sequence but containing both D and L amino acids. Particles functionalized with the reference peptide do not aggregate, as the peptides are unable to fold. The two peptides, linked to the nanoparticle surface via a cysteine residue located in the loop region, form submonolayers on planar gold with comparable properties regarding surface density, orientation, and ability to interact with zinc ions. These results demonstrate that nanoparticle assembly can be induced, controlled, and to some extent tuned, by exploiting specific molecular interactions involved in polypeptide folding.

Hybrid Nanoparticle-Liposome Detection of Phospholipase Activity

A flexible nanoparticle-based phospholipase (PL) assay is demonstrated in which the enzymatic substrate is decoupled from the nanoparticle surface. Liposomes are loaded with a polypeptide that is designed to heteroassociate with a second polypeptide immobilized on gold nanoparticles. Release of this polypeptide from the liposomes, triggered by PL, induces a folding-dependent nanoparticle bridging aggregation. The colorimetric response from this aggregation enables straightforward and continuous detection of PL in the picomolar range. The speed, specificity, and flexibility of this assay make it appropriate for a range of applications, from point of care diagnostics to high-throughput pharmaceutical screening.

Polypeptide Folding-Mediated Tuning of the Optical and Structural Properties of Gold Nanoparticle Assemblies

Responsive hybrid nanomaterials with well-defined properties are of significant interest for the development of biosensors with additional applications in tissue engineering and drug delivery. Here, we present a detailed characterization using UV-vis spectroscopy and small angle X-ray scattering of a hybrid material comprised of polypeptide-decorated gold nanoparticles with highly controllable assembly properties. The assembly is triggered by a folding-dependent bridging of the particles mediated by the heteroassociation of immobilized helix-loop-helix polypeptides and a complementary nonlinear polypeptide present in solution. The polypeptides are de novo designed to associate and fold into a heterotrimeric complex comprised of two disulfide-linked four-helix bundles. The particles form structured assemblies with a highly defined interparticle gap (4.8±0.4 nm) that correlates to the size of the folded polypeptides. Transitions in particle aggregation dynamics, mass-fractal dimensions and ordering, as a function of particle size and the concentration of the bridging polypeptide, are observed; these have significant effects on the optical properties of the assemblies. The assembly and ordering of the particles are highly complex processes that are affected by a large number of variables including the number of polypeptides bridging the particles and the particle mobility within the aggregates. A fundamental understanding of these processes is of paramount interest for the development of novel hybrid nanomaterials with tunable structural and optical properties and for the optimization of nanoparticle-based colorimetric biodetection strategies.

Peptide functionalized gold nanoparticles for colorimetric detection of matrilysin (MMP-7) activity

A peptide with two cleavage sites for MMP-7 has been synthesized and immobilized on gold nanoparticles (AuNPs) through a cysteine residue. Digestion of the peptide by MMP-7 decreases its size and net charge, which leads to the aggregation of the AuNPs. The color shift caused by aggregation enables a direct and quantitative measurement of the concentration and activity of MMP-7 with an estimated limit of detection of ∼5 nM (0.1 μg mL(-1)).

Assembly of Polypeptide-Functionalized Gold Nanoparticles through a Heteroassociation-and Folding-Dependent Bridging

Gold nanoparticles were functionalized with a synthetic polypeptide, de novo-designed to associate with a charge complementary linker polypeptide in a folding-dependent manner. A heterotrimeric complex that folds into two disulphide-linked four-helix bundles is formed when the linker polypeptide associates with two of the immobilized peptides. The heterotrimer forms in between separate particles and induces a rapid and extensive aggregation with a well-defined interparticle spacing. The aggregated particles are redispersed when the disulphide bridge in the linker polypeptide is reduced.

Biofunctionalized Gold Nanoparticles for Colorimetric Sensing of Botulinum Neurotoxin A Light Chain

Botulinum neurotoxin is considered as one of the most toxic food-borne substances and is a potential bioweapon accessible to terrorists. The development of an accurate, convenient, and rapid assay for botulinum neurotoxins is therefore highly desirable for addressing biosafety concerns. Herein, novel biotinylated peptide substrates designed to mimic synaptosomal-associated protein 25 (SNAP-25) are utilized in gold nanoparticle-based assays for colorimetric detection of botulinum neurotoxin serotype A light chain (BoLcA). In these proteolytic assays, biotinylated peptides serve as triggers for the aggregation of gold nanoparticles, while the cleavage of these peptides by BoLcA prevents nanoparticle aggregation. Two different assay strategies are described, demonstrating limits of detection ranging from 5 to 0.1 nM of BoLcA with an overall assay time of 4 h. These hybrid enzyme-responsive nanomaterials provide rapid and sensitive detection for one of the most toxic substances known to man.

Self-Assembly of Fibers and Nanorings from Disulfide-Linked Helix–Loop–Helix Polypeptides

Ingenjorer och vetenskapsman har ofta inspirerats av naturen i sokandet efter losningar pa tekniska problem. Allt ifran byggnadskonstruktioner, flygplansvingar, kompositmaterial till kardborrebandet har skapats med utgangspunkt fran forebilder i naturen. Manga av de material och konstruktioner som aterfinns i naturen har atravarda egenskaper som ar svara att erhalla i syntetiska matrial med traditionell teknik. Aven om vi i flera fall kan harma sammansattningen och formen blir resultatet inte nodvandigtvis det samma. Den storsta skillnaden mellan syntetiska material och material producerade av levande organismer ar hur deras komponenter sinsemellan ar organiserade och sammansatta. I syntetiska material ar komponenterna ofta inbordes mer eller mindre slumpvis ordnade medan de i biologiska material ar organiserade med en oerhord precision som stracker sig anda ned pa molekyl- och atomniva. Naturens byggstenar har genom evolutionens gang forfinats for att spontant kunna organisera sig och bilda komplexa material och strukturer. Denna process, som styrs genom att manga svaga krafter inom och mellan byggstenarna samverkar, kallas ofta for sjalvorganisering och ar en forutsattning for allt liv. Sjalvorganisering har ocksa blivit en allt viktigare metod inom nanotekniken for att konstruera material och strukturer med nanometerprecision. I den har avhandlingen beskrivs en typ av sjalvorganiserande material dar byggstenarna utgors av nanometerstora guldpartiklar och syntetiska proteiner. De syntetiska proteinerna ar designade for att efterlikna naturliga biomolekyler och antar en valbestamd tredimensionell struktur nar tva av dem interagerar med varandra. Denna interaktion ar mycket specifik men kan styras genom att variera kemiska parametrar som surhet och jonstyrka vilket ger en mojlighet att paverka och kontrollera proteinernas struktur. Proteinerna har vidare modifierats for att spontant organisera sig till fibrer som ar flera mikrometer langa men endast nagra nanometer tjocka. Proteinfibrer utgor en mycket viktig typ av strukturer i biologiska system och finns i alltifran spindelvav till muskler. Syntetiska proteinfibrer ar darfor bade ett intressant modellsystem och ett material med manga potentiellt intressanta anvandningsomraden. Genom att fasta de syntetiska proteinerna pa ytan av guldnanopartiklar gar interaktionerna mellan partiklarna att kontrollera pa samma satt som interaktionerna mellan proteinerna. Krafterna mellan proteinerna och interaktionerna involverade i proteinernas veckning har anvants for att reversibelt aggregera och organisera nanopartiklarna. Ett antal olika byggstenar har studerats och utvecklats till nagot som liknar ett mycket enkelt nano-Lego, som pa en given signal spontant bygger ihop sig eller trillar isar. Guldnanopartiklar ar intressanta eftersom de ar stabila och latta att modifiera kemiskt men ocksa pa grund av deras optiska egenskaper som ger dem en ovanligt vacker vinrod farg. Fargen uppstar pa grund av partiklarnas ringa storlek och varierar naturligt med egenskaperna hos den omgivande miljon. Detta gor det enkelt att studera hur partiklarna interagerar eftersom de byter farg nar de narmar sig varandra, men gor dem ocksa intressanta for sensortillampningar. En enkel och robust sensor beskrivs i avhandlingen dar syntetiska proteiner, speciellt utformade for att upptacka och binda andra molekyler, har fasts pa nanopartiklarna. Med partiklarnas hjalp gar det att med blotta ogat detektera ett manskligt protein i koncentrationer under ett tusendels gram per liter. En tidig diagnos av sjukdomstillstand kan i de flesta fall avsevart underlatta behandlingen och behovet av enkla sensorer for att bestamma narvaro och koncentration av medicinskt intressanta molekyler ar darfor mycket stort.

Critical biophysical properties in the Pseudomonas aeruginosa efflux gene regulator MexR are targeted by mutations conferring multidrug resistance

The self‐assembling MexA‐MexB‐OprM efflux pump system, encoded by the mexO operon, contributes to facile resistance of Pseudomonas aeruginosa by actively extruding multiple antimicrobials. MexR negatively regulates the mexO operon, comprising two adjacent MexR binding sites, and is as such highly targeted by mutations that confer multidrug resistance (MDR). To understand how MDR mutations impair MexR function, we studied MexR‐wt as well as a selected set of MDR single mutants distant from the proposed DNA‐binding helix. Although DNA affinity and MexA‐MexB‐OprM repression were both drastically impaired in the selected MexR‐MDR mutants, MexR‐wt bound its two binding sites in the mexO with high affinity as a dimer. In the MexR‐MDR mutants, secondary structure content and oligomerization properties were very similar to MexR‐wt despite their lack of DNA binding. Despite this, the MexR‐MDR mutants showed highly varying stabilities compared with MexR‐wt, suggesting disturbed critical interdomain contacts, because mutations in the DNA‐binding domains affected the stability of the dimer region and vice versa. Furthermore, significant ANS binding to MexR‐wt in both free and DNA‐bound states, together with increased ANS binding in all studied mutants, suggest that a hydrophobic cavity in the dimer region already shown to be involved in regulatory binding is enlarged by MDR mutations. Taken together, we propose that the biophysical MexR properties that are targeted by MDR mutations—stability, domain interactions, and internal hydrophobic surfaces—are also critical for the regulation of MexR DNA binding.