Engineered and hybrid human megakaryocytic extracellular vesicles for targeted non-viral cargo delivery to hematopoietic (blood) stem and progenitor cells.

Native and engineered extracellular vesicles generated from human megakaryocytes (huMkEVs) or from the human megakaryocytic cell line CHRF (CHEVs) interact with tropism delivering their cargo to both human and murine hematopoietic stem and progenitor cells (HSPCs). To develop non-viral delivery vectors to HSPCs based on MkEVs, we first confirmed, using NOD-scid IL2Rγnull (NSG™) mice, the targeting potential of the large EVs, enriched in microparticles (huMkMPs), chosen for their large cargo capacity. 24 h post intravenous infusion into NSG mice, huMkEVs induced a nearly 50% increase in murine platelet counts. PKH26-labeled huMkEVs or CHEVs localized to the HSPC-rich bone marrow preferentially interacting with murine HSPCs, thus confirming their receptor-mediated tropism for NSG HSPCs, and their potential to treat thromobocytopenias. We explored this tropism to functionally deliver synthetic cargo, notably plasmid DNA coding for a fluorescent reporter, to NSG HSPCs both in vitro and in vivo. We loaded huMkEVs with plasmid DNA either through electroporation or by generating hybrid particles with preloaded liposomes. Both methods facilitated successful functional targeted delivery of pDNA, as tissue weight-normalized fluorescence intensity of the expressed fluorescent reporter was significantly higher in bone marrow than other tissues. Furthermore, the fraction of fluorescent CD117+ HSPCs was nearly 19-fold higher than other cell types within the bone marrow 72-h following administration of the hybrid particles, further supporting that HSPC tropism is retained when using hybrid particles. These data demonstrate the potential of these EVs as a non-viral, HSPC-specific cargo vehicle for gene therapy applications to treat hematological diseases.

Megakaryocyte membrane-wrapped nanoparticles for targeted cargo delivery to hematopoietic stem and progenitor cells.

Hematopoietic stem and progenitor cells (HSPCs) are desirable targets for gene therapy but are notoriously difficult to target and transfect. Existing viral vector-based delivery methods are not effective in HSPCs due to their cytotoxicity, limited HSPC uptake and lack of target specificity (tropism). Poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) are attractive, nontoxic carriers that can encapsulate various cargo and enable its controlled release. To engineer PLGA NP tropism for HSPCs, megakaryocyte (Mk) membranes, which possess HSPC-targeting moieties, were extracted and wrapped around PLGA NPs, producing MkNPs. In vitro, fluorophore-labeled MkNPs are internalized by HSPCs within 24 h and were selectively taken up by HSPCs versus other physiologically related cell types. Using membranes from megakaryoblastic CHRF-288 cells containing the same HSPC-targeting moieties as Mks, CHRF-wrapped NPs (CHNPs) loaded with small interfering RNA facilitated efficient RNA interference upon delivery to HSPCs in vitro. HSPC targeting was conserved in vivo, as poly(ethylene glycol)-PLGA NPs wrapped in CHRF membranes specifically targeted and were taken up by murine bone marrow HSPCs following intravenous administration. These findings suggest that MkNPs and CHNPs are effective and promising vehicles for targeted cargo delivery to HSPCs.

Human megakaryocytic microparticles induce de novo platelet biogenesis in a wild-type murine model.

Platelet transfusions are used to treat idiopathic or drug-induced thrombocytopenia. Platelets are an expensive product in limited supply, with limited storage and distribution capabilities because they cannot be frozen. We have demonstrated that, in vitro, human megakaryocytic microparticles (huMkMPs) target human CD34+ hematopoietic stem and progenitor cells (huHSPCs) and induce their Mk differentiation and platelet biogenesis in the absence of thrombopoietin. In this study, we showed that, in vitro, huMkMPs can also target murine HSPCs (muHSPCs) to induce them to differentiate into megakaryocytes in the absence of thrombopoietin. Based on that, using wild-type BALB/c mice, we demonstrated that intravenously administering 2 × 106 huMkMPs triggered de novo murine platelet biogenesis to increase platelet levels up to 49% 16 hours after administration. huMkMPs also largely rescued low platelet levels in mice with induced thrombocytopenia 16 hours after administration by increasing platelet counts by 51%, compared with platelet counts in thrombocytopenic mice. Normalized on a tissue-mass basis, biodistribution experiments show that MkMPs localized largely to the bone marrow, lungs, and liver 24 hours after huMkMP administration. Beyond the bone marrow, CD41+ (megakaryocytes and Mk-progenitor) cells were frequent in lungs, spleen, and especially, liver. In the liver, infused huMKMPs colocalized with Mk progenitors and muHSPCs, thus suggesting that huMkMPs interact with muHSPCs in vivo to induce platelet biogenesis. Our data demonstrate the potential of huMkMPs, which can be stored frozen, to treat thrombocytopenias and serve as effective carriers for in vivo, target-specific cargo delivery to HSPCs.

Point of impact prediction in isotropic and anisotropic plates from the acoustic emission data.

It is shown in this paper that the conventional triangulation technique is not very reliable for locating the impact point even in isotropic plates when the sensors are placed close to the point of strike for two reasons: First, it is difficult to pinpoint the exact time of arrival of the signal and, second, the Lamb modes in a plate are dispersive. Dispersive signals attenuate differently at various frequencies and propagate with different speeds causing distortions in the received signals, and thus introduce error in the time of flight measurement. The triangulation technique assumes that wave speeds in all directions are the same, which is not true for anisotropic plates. Here an alternative approach based on an optimization scheme is proposed to locate the point of impact in isotropic and anisotropic plates. A formulation is presented for the general anisotropic case. Experiments are carried out with an aluminum plate by dropping balls on the plate and picking up acoustic signals at different locations. The impact points predicted by the conventional triangulation technique and the proposed modified method are compared for this isotropic plate. Then it is investigated how the prediction would change if the plate is assumed to have some anisotropy.

DPSM modeling for studying interaction between bounded ultrasonic beams and corrugated plates with experimental verification.

Periodically corrugated structures play an important role in the field of vibration control and for designing structures with desired acoustic band gaps. Analytical solutions for corrugated plates are available for well-defined, smooth corrugations, such as sinusoidal corrugations that are not very common in the real world. Often corrugated plates are fabricated by cutting grooves at regular intervals in a flat plate. No analytical solution is available to predict the wave propagation behavior in such a periodically corrugated plate in which the equation of the plate surface changes periodically between a planar fiat surface and a nonplanar parabolic groove. This problem is solved here for steady-state case by a newly developed semianalytical technique called distributed point source method (DPSM), and the theoretical predictions are compared with the experimental results generated by reflecting a bounded 2.25 MHz ultrasonic beam by a fabricated corrugated plate. The main difference that is observed in the reflected beam profile from a flat plate and a corrugated plate is that the back-scattering effect is much stronger for the corrugated plate, and the forward reflection is stronger for the flat plate. The energy distribution inside the corrugated plate also shows backward propagation of the ultrasonic energy.

Controlled Space Radiation concept for mesh-free semi-analytical technique to model wave fields in complex geometries.

Numerical modelling of the ultrasonic wave propagation is important for Structural Heath Monitoring and System Prognosis problems. In order to develop intelligent and adaptive structures with embedded damage detector and classifier mechanisms, detailed understanding of scattered wave fields due to anomaly in the structure is inevitably required. A detailed understanding of the problem demands a good modelling of the wave propagation in the problem geometry in virtual form. Therefore, efficient analytical, semi-analytical or numerical modelling techniques are required. In recent years a semi-analytical mesh-free technique called Distributed Point Source Method (DPSM) is being used for modelling various ultrasonic, electrostatic and electromagnetic wave field problems. In the conventional DPSM approach point sources are placed along the transducer faces, problem boundaries and interfaces to model incident and scattered fields. Every point source emits energy in all directions uniformly. Source strengths of these 360 degrees radiation sources are obtained by satisfying interface and boundary conditions of the problem. In conventional DPSM modelling approach it is assumed that the shadow zone does not require any special consideration. 360 degrees Radiation point sources should be capable of properly modelling shadow zones because all boundary and interface conditions are satisfied. In this paper it is investigated how good this assumption is by introducing the 'shadow zone' concept at the point source level and comparing the results generated by the conventional DPSM and by this modified approach where the conventional 360 degrees radiation point sources are replaced by the Controlled Space Radiation (CSR) sources.

Locating point of impact in anisotropic fiber reinforced composite plates.

The conventional triangulation technique cannot predict the point of impact in an anisotropic composite plate because the triangulation technique assumes that the wave speed is independent of the direction of propagation which is not the case for anisotropic plates. An alternative method based on the optimization scheme was proposed by Kundu et al. [T. Kundu, S. Das, K.V. Jata, Point of impact prediction in isotropic and anistropic plates from the acoustic emission data, J. Acoust. Soc. Am. 122, 2007, 2057-2066] to locate the point of impact in plates by analyzing the time of arrival of the ultrasonic signals received by the passive sensors attached to the plate. In this paper, that objective function is modified further to overcome the inherent difficulties associated with multiple singularities and to maximize the efficiency of the acoustic emission data for multiple receiving sensors. With this modified objective function the impact point on an anisotropic composite plate is predicted from the acoustic emission data. Experiments are carried out by dropping steel and ping pong balls on a graphite-epoxy composite plate and recording acoustic signals by passive transducers adhesively bonded to the plate at three different locations. The impact point is predicted by the proposed method and compared with the actual location of impact.