Size-controlled granular polyphosphate (PolyP) nanoparticles were synthesized by precipitation in aqueous solutions containing physiological concentrations of calcium and magnesium. We demonstrate using dynamic light scattering (DLS) that the solubility is correlated inversely with PolyP chain length, with very long chain PolyP (PolyP1000+, more than 1000 repeating units) normally found in prokaryotes precipitating much more robustly than shorter chains like those found in human platelet dense granules (PolyP80, range 76–84 repeating units). It is believed that the precipitation of PolyP is a reversible process involving calcium coordination to phosphate monomers in the polymer chain. The particles are stable in aqueous buffer and albumin suspensions on time scales roughly equivalent to catastrophic bleeding events. Transmission electron microscopy images demonstrate that the PolyP nanoparticles are spherical and uniformly electron dense, with a particle diameter of 200–250 nm, closely resembling the content of acidocalcisomes. X-ray elemental analysis further reveals that the P/Ca ratio is 67:32. The granular nanoparticles also manifest promising procoagulant effects, as measured by in vitro clotting tests assaying contact pathway activity.

A large group of functional nanomaterials employed in biomedical applications, including targeted drug delivery, relies on amphiphilic polymers to encapsulate therapeutic payloads via self-assembly processes. Knowledge of the micelle structures will provide critical insights into design of polymeric drug delivery systems. Core-shell micelles composed of linear diblock copolymers poly(ethylene glycol)-b-poly(caprolactone) (PEG-b-PCL), poly(ethylene oxide)-b-poly(lactic acid) (PEG-b-PLA), as well as a heterografted brush consisting of a poly(glycidyl methacrylate) backbone with PEG and PLA branches (PGMA-g-PEG/PLA) were characterized by dynamic light scattering (DLS) and small angle X-ray scattering (SAXS) measurements to gain structural information regarding the particle morphology, core-shell size, and aggregation number. The structural information at this quasi-equilibrium state can also be used as a reference when studying the kinetics of polymer micellization.

Granular platelet-sized polyphosphate nanoparticles (polyP NPs) were encapsulated in sterically stabilized liposomes, forming a potential, targeted procoagulant nanotherapy resembling human platelet dense granules in both structure and functionality. Dynamic light scattering (DLS) measurements reveal that artificial dense granules (ADGs) are colloidally stable and that the granular polyP NPs are encapsulated at high efficiencies. High-resolution scanning transmission electron microscopy (HR-STEM) indicates that the ADGs are monodisperse particles with a 150 nm diameter dense core consisting of P, Ca, and O surrounded by a corrugated 25 nm thick shell containing P, C, and O. Further, the ADGs manifest promising procoagulant activity: Detergent solubilization by Tween 20 or digestion of the lipid envelope by phospholipase C (PLC) allows for ADGs to trigger autoactivation of Factor XII (FXII), the first proteolytic step in the activation of the contact pathway of clotting. Moreover, ADGs' ability to reduce the clotting time of human plasma in the presence of PLC further demonstrate the feasibility to develop ADGs into a potential procoagulant nanomedicine.

Platelet-sized polyphosphate (polyP) was functionalized on the surface of gold nanoparticles (GNPs) via a facile conjugation scheme entailing EDAC (N-(3-(dimethylamino)propyl)-N'-ethylcarbodiimide hydrochloride)-catalyzed phosphoramidation of the terminal phosphate of polyP to cystamine. Subsequent reduction of the disulfide moiety allowed for anchoring to the colloidal surface. The ability of the synthesized polyP-GNPs to initiate the contact pathway of clotting in human pooled normal plasma (PNP) was then assayed by quantifying changes in viscous, mechanical, and optical properties upon coagulation. It is revealed that the polyP-GNPs are markedly superior contact activators compared to molecularly dissolved, platelet-sized polyP (of equivalent polymer chain length). Moreover, the particles' capacity to mobilize Factor XII (FXII) and its coactivating proteins appear to be identical to very-long-chain polyP typically found in bacteria. These data imply that nanolocalization of anionic procoagulants on colloidal surfaces, achieved through covalent anchoring, may yield a robust contact surface with the ability to sufficiently cluster active clotting factors together above their threshold concentrations to cease bleeding. The polyP-GNPs therefore serve as a promising foundation in the development of a nanoparticle hemostat to treat a range of hemorrhagic scenarios.

Degradation of DPPC catalyzed by sPLA2 resulted in a mixture of highly-ordered multilayer domains and a loosely packed monolayer phase.

Utilizing synchrotron small-angle X-ray scattering (SAXS) integrated with a microfluidic device, micellization kinetics of a diblock co-polymer, poly(ethylene glycol)-b-poly(caprolactone) (PEGb-PCL) was measured in situ with millisecond temporal and micrometer spatial resolution. The evolutionary regimes of polymer micellization-nucleation, fusion, and insertion were directly observed. The five-inlet microfluidic device provided steady continuous mixing of the polymer solution and the antisolvent. Solvent replacement was mainly dominated by lateral diffusion across the hydrodynamically focused central layer, whose thickness could be precisely designed and manipulated from mass balance of the partitioning streams. Knowing the micellization kinetics of the polymers is essential for design and optimization of self-assembled polymeric nanostructures. The technique of integrating SAXS with microfluidic devices can be translatable to other systems for a breadth of applications.

To optimize the compositions of the lipid-based nanomedicine and to advance understanding of the roles of polyunsaturated phospholipids in biological membranes, this study examined the effects of polyunsaturated phospholipids on the degradation of giant unilamellar vesicles catalyzed by a secreted phospholipase A2 (sPLA 2 ) using fluorescence microscopy. Molecular interfacial packing, interaction, and degradation of the films containing various mixing ratios of saturated and polyunsaturated phospholipids were quantified using a Langmuir trough integrated with synchrotron X-ray surface scattering techniques. It was found that a high molar fraction (0.63 and above) of polyunsaturated phospholipids not only enhanced the rate of sPLA 2 -catalyzed vesicle degradation but also changed the vesicle deformation process and degradation product morphology. Hydrolysis of the saturated phospholipids generated highly ordered liquid crystal domains, which was reduced or prohibited by the presence of the polyunsaturated phospholipids in the reactant film.

Polyethylene glycol (PEG) coatings have been widely applied in pharmaceutical and biomedical systems to prevent nonspecific protein absorption, increase vesicle blood circulation time, and sustain drug release. This study systematically investigated the planar interfacial organization of phospholipid monolayers containing various amounts of PEG conjugations before and after enzyme-catalyzed degradation of the lipids using X-ray reflectivity and grazing incidence X-ray diffraction techniques. Results showed that attaching PEG to the headgroup of the lipids up to 15 mol % had limited effects on molecular packing of the lipid monolayers in the condensed phase at the gas−liquid interface and negligible effects on the enzyme adsorption to the interface. After enzyme-catalyzed degradation, equimolar fatty acids and lyso PC were generated. The fatty acids together with the subphase Ca 2+ self-assembled into highly organized multilayer domains at the interface. The X-ray measurements unambiguously revealed that the densely packed PEG markedly hindered microphase separation and formation of the palmitic acid−Ca 2+ complexes.