Quantitative Visualization of Lipid Nanoparticle Fusion as a Function of Formulation and Process Parameters.

Lipid nanoparticles (LNPs) have proven to be promising delivery vehicles for RNA-based vaccines and therapeutics, particularly in LNP formulations containing ionizable cationic lipids that undergo protonation/deprotonation in response to buffer pH changes. These nanoparticles are typically formulated using a rapid mixing technique at low pH, followed by a return to physiological pH that triggers LNP-LNP fusion. A detailed understanding of these dynamic processes is crucial to optimize the overall performance and efficiency of LNPs. However, knowledge gaps persist regarding how particle formation mechanisms impact drug loading and delivery functions. In this work, we employ single-molecule Convex Lens-induced Confinement (CLiC) microscopy in combination with Förster resonance energy transfer (FRET) measurements to study LNP fusion dynamics in relation to various formulation parameters, including lipid concentration, buffer conditions, drug loading ratio, PEG-lipid concentrations, and ionizable lipid selection. Our results reveal a strong correlation between the measured fusion dynamics and the formulation parameters used; these findings are consistent with DLS and Cryo-TEM-based assays. These measurements offer a cost-effective method for characterizing and screening potential drug candidates and can provide additional insights into their design, with opportunities for optimization.

Simultaneous, Single-Particle Measurements of Size and Loading Give Insights into the Structure of Drug-Delivery Nanoparticles.

Nanoparticles are a promising solution for delivery of a wide range of medicines and vaccines. Optimizing their design depends on being able to resolve, understand, and predict biophysical and therapeutic properties, as a function of design parameters. While existing tools have made great progress, gaps in understanding remain because of the inability to make detailed measurements of multiple correlated properties. Typically, an average measurement is made across a heterogeneous population, obscuring potentially important information. In this work, we develop and apply a method for characterizing nanoparticles with single-particle resolution. We use convex lens-induced confinement (CLiC) microscopy to isolate and quantify the diffusive trajectories and fluorescent intensities of individual nanoparticles trapped in microwells for long times. First, we benchmark detailed measurements of fluorescent polystyrene nanoparticles against prior data to validate our approach. Second, we apply our method to investigate the size and loading properties of lipid nanoparticle (LNP) vehicles containing silencing RNA (siRNA), as a function of lipid formulation, solution pH, and drug-loading. By taking a comprehensive look at the correlation between the intensity and size measurements, we gain insights into LNP structure and how the siRNA is distributed in the LNP. Beyond introducing an analytic for size and loading, this work allows for future studies of dynamics with single-particle resolution, such as LNP fusion and drug-release kinetics. The prime contribution of this work is to better understand the connections between microscopic and macroscopic properties of drug-delivery vehicles, enabling and accelerating their discovery and development.

PEGylated nanocapsules of perfluorooctyl bromide: Mechanism of formation, influence of polymer concentration on morphology and mechanical properties.

PEGylated nanocapsules containing a liquid core of perfluorooctyl bromide (PFOB) were formulated by an emulsion-evaporation process to be further used as ultrasound contrast agents (UCAs). In an attempt to modulate their acoustic response, related to their shell thickness-to-radius ratio, the initial concentration of polymer was varied in the formulation. Indeed, thinner shells may lead to higher echogenicity. PEGylated nanocapsules morphology was studied by electron microscopy, Small Angle Neutron Scattering and (19)F NMR spectroscopy and related to their mechanical properties to allow a better understanding of their mechanism of formation. We show that the variation of polymer concentration in the formulation impacts the formation mechanism of nanocapsules, and consequently their morphology and mechanical properties. Using low concentration of Poly(ethylene glycol)-b-poly(dl-lactide-co-glycolide) (PLGA-b-PEG), it is impossible to reduce the shell thickness of the UCA, most probably due to dewetting of the polymer layer at the PFOB/water interface. This leads to the coexistence of thick shells along with free PFOB droplets. On the other hand, for high polymer concentration, PEGylated nanocapsules with thick shells were produced with high encapsulation efficiency.

Ultrasound guided fluorescence molecular tomography with improved quantification by an attenuation compensated Born-normalization and in vivo preclinical study of cancer.

Ultrasound imaging, having the advantages of low-cost and non-invasiveness over MRI and X-ray CT, was reported by several studies as an adequate complement to fluorescence molecular tomography with the perspective of improving localization and quantification of fluorescent molecular targets in vivo. Based on the previous work, an improved dual-modality Fluorescence-Ultrasound imaging system was developed and then validated in imaging study with preclinical tumor model. Ultrasound imaging and a profilometer were used to obtain the anatomical prior information and 3D surface, separately, to precisely extract the tissue boundary on both sides of sample in order to achieve improved fluorescence reconstruction. Furthermore, a pattern-based fluorescence reconstruction on the detection side was incorporated to enable dimensional reduction of the dataset while keeping the useful information for reconstruction. Due to its putative role in the current imaging geometry and the chosen reconstruction technique, we developed an attenuation compensated Born-normalization method to reduce the attenuation effects and cancel off experimental factors when collecting quantitative fluorescence datasets over large area. Results of both simulation and phantom study demonstrated that fluorescent targets could be recovered accurately and quantitatively using this reconstruction mechanism. Finally, in vivo experiment confirms that the imaging system associated with the proposed image reconstruction approach was able to extract both functional and anatomical information, thereby improving quantification and localization of molecular targets.

Hybrid FMT-MRI applied to in vivo atherosclerosis imaging.

Combining Fluorescent Molecular Tomography (FMT) with anatomical imaging, e.g. MRI facilitates interpreting functional information. Furthermore, using a heterogeneous model for light propagation has been shown in simulations to be superior to homogeneous modeling to quantify fluorescence. Here, we present a combined FMT-MRI system and apply it to heart and aorta molecular imaging, a challenging area due to strong tissue heterogeneity and the presence of air-voids due to lungs. First investigating performance in a phantom and mouse corpse, the MRI-enabled heterogeneous models resulted in an improved quantification of fluorescence reconstructions. The system was then used in mice for in vivo atherosclerosis molecular imaging. Results show that, when using the heterogeneous model, reconstructions were in agreement with the ex vivo measurements. Therefore, the proposed system might serve as a powerful imaging tool for atherosclerosis in mice.

Aging-related differences in cerebral capillary blood flow in anesthetized rats.

Age-related decreases in baseline cerebral blood flow have been measured with various imaging modalities, however, the contribution of capillary flow to this phenomenon remain to elucidate. This study used 2-photon laser scanning fluorescence microscopy to measure capillary diameter, red blood cell speed, and flux in individual capillaries in the sensory-motor cortex of 12 adult (3-month-old) and 12 old (24-month-old) male Long-Evans rats under isoflurane anesthesia. The average (± standard deviation) diameter and speed over 921 capillaries were 6.4 ± 1.4 µm and 1.3 ± 1.1 mm/s, respectively. Red blood cell speed and flux were significantly higher, by 48% and 15%, respectively, in old compared with young animals (p < 5%). The diameter also showed a similar tendency (7% higher, p = 5.7%). Furthermore, capillary hematocrit and density were significantly lower in the older group (p < 5%), by 32% and 20%, respectively.

Multimodal study of the hemodynamic response to hypercapnia in anesthetized aged rats.

With aging, the brain undergoes changes in metabolism and perfusion, both of which influence the widely used blood-oxygenation-level-dependent (BOLD) MRI signal. To isolate the vascular effects associated with age, this study measured the response to a hypercapnic challenge using different imaging modalities in 19 young (3 months-old) and 13 old (24 months-old) Long-Evans rats. Intrinsic optical imaging was used to measure oxy (HbO), deoxy (HbR) and total (HbT) hemoglobin concentration changes, laser speckle for cerebral blood flow (CBF) changes, and MRI for the BOLD signal. Older rats had smaller HbO (41% smaller), HbT (50%) and CBF (34%) responses, but the temporal dynamics did not exhibit significant age differences. The ratio of CBV to CBF responses was also smaller in older adults, potentially indicating a change in the compliance of vessels.

VCAM-1-targeting gold nanoshell probe for photoacoustic imaging of atherosclerotic plaque in mice.

The development of molecular probes and novel imaging modalities, allowing better resolution and specificity, is associated with an increased potential for molecular imaging of atherosclerotic plaques especially in basic and pre-clinical research applications. In that context, a photoacoustic molecular probe based on gold nanoshells targeting VCAM-1 in mice (immunonanoshells) was designed. The molecular probe was validated in vitro and in vivo, showing no noticeable acute toxic effects. We performed the conjugation of gold nanoshells displaying near-infrared absorption properties with VCAM-1 antibody molecules and PEG to increase their biocompatibility. The resulting immunonanoshells obtained under different conditions of conjugation were then assessed for specificity and sensitivity. Photoacoustic tomography was performed to determine the ability to distinguish gold nanoshells from blood both in phantoms and in vivo. Ex vivo optical projection tomography of hearts and aortas from atherosclerotic and control mice confirmed the selective accumulation of the immunonanoshells in atherosclerotic-prone regions in mice, thus validating the utility of the probe in vivo in small animals for pre-clinical research. These immunonanoshells represent an adequate mean to target atherosclerotic plaques in small animals, leading to new tools to follow the effect of therapies on the progression or regression of the disease.

Gold: a versatile tool for in vivo imaging.

Imaging for diagnostics or for evaluating the efficacy of a particular drug constitutes a key challenge, and a topical area of research in nanomedicine. There has been a tremendous effort devoted to the evaluation of a variety of contrast agents, and gold nanomaterials due to their inherent and geometrically induced optical properties, have offered significant potential for in vivo imaging. The gold based nanostructures that are most commonly employed for biological imaging include nano-spheres, -rods, -shells, -cages and -stars. This feature article provides an overview of the current state of research in utilizing these gold nano-architectures in imaging, with particular emphasis on modalities such as two-photon luminescence, computed tomography, optical coherence tomography, near infrared and photoacoustic imaging.

Imaging of an inflammatory injury in the newborn rat brain with photoacoustic tomography.

BACKGROUND: The precise assessment of cerebral saturation changes during an inflammatory injury in the developing brain, such as seen in periventricular leukomalacia, is not well defined. This study investigated the impact of inflammation on locoregional cerebral oxygen saturation in a newborn rodent model using photoacoustic imaging. METHODS: 1 mg/kg of lipopolysaccharide(LPS) diluted in saline or saline alone was injected under ultrasound guidance directly in the corpus callosum of P3 rat pups. Coronal photoacoustic images were carried out 24 h after LPS exposure. Locoregional oxygen saturation (SO2) and resting state connectivity were assessed in the cortex and the corpus callosum. Microvasculature was then evaluated on cryosection slices by lectin histochemistry. RESULTS: Significant reduction of SO2 was found in the corpus callosum; reduced SO2 was also found in the cortex ipsilateral to the injection site. Seed-based functional connectivity analysis showed that bilateral connectivity was not affected by LPS exposure. Changes in locoregional oxygen saturation were accompanied by a significant reduction in the average length of microvessels in the left cortex but no differences were observed in the corpus callosum. CONCLUSION: Inflammation in the developing brain induces marked reduction of locoregional oxygen saturation, predominantly in the white matter not explained by microvascular degeneration. The ability to examine regional saturation offers a new way to monitor injury and understand physiological disturbance non-invasively.

Two-dimensional simulation of linear wave propagation in a suspension of polymeric microcapsules used as ultrasound contrast agents.

A generation of tissue-specific stable ultrasound contrast agent (UCA) composed of a polymeric capsule with a perfluorocarbone liquid core has become available. Despite promising uses in clinical practice, the acoustical behavior of such UCA suspensions remains unclear. A simulation code (2-D finite-difference time domain, FDTD) already validated for homogeneous particles [Galaz Haiat, Berti, Taulier, Amman and Urbach, (2010). J. Acoust. Soc. Am. 127, 148-154] is used to model the ultrasound propagation in such UCA suspensions at 50 MHz to investigate the sensitivity of the ultrasonic parameters to physical parameters of UCA. The FDTD simulation code is validated by comparison with results obtained using a shell scatterer model. The attenuation coefficient (respectively, the sound velocity) increases (respectively, decreases) from 4.1 to 58.4 dB/cm (respectively, 1495 to 1428 m/s) when the concentration varies between 1.37 and 79.4 mg/ml, while the backscattered intensity increases non-linearly, showing that a concentration of around 30 mg/ml is sufficient to obtain optimal backscattering intensity. The acoustical parameters vary significantly as a function of the membrane thickness, longitudinal and transverse velocity, indicating that mode conversions in the membrane play an important role in the ultrasonic propagation. The results may be used to help manufacturers to conceive optimal liquid-filled UCA suspensions.

Experimental validation of a time domain simulation of high frequency ultrasonic propagation in a suspension of rigid particles.

Ultrasonic propagation in suspensions of particles is a difficult problem due to the random spatial distribution of the particles. Two-dimensional finite-difference time domain simulations of ultrasonic propagation in suspensions of polystyrene 5.3 mum diameter microdisks are performed at about 50 MHz. The numerical results are compared with the Faran model, considering an isolated microdisk, leading to a maximum difference of 15% between the scattering cross-section values obtained analytically and numerically. Experiments are performed with suspensions in through transmission and backscattering modes. The attenuation coefficient at 50 MHz (alpha), the ultrasonic velocity (V), and the relative backscattered intensity (I(B)) are measured for concentrations from 2 to 25 mg/ml, obtained by modifying the number of particles. Each experimental ultrasonic parameter is compared to numerical results obtained by averaging the results derived from 15 spatial distributions of microdisks. alpha increases with the concentration from 1 to 17 dB/cm. I(B) increases with concentration from 2 to 16 dB. The variation of V versus concentration is compared with the numerical results, as well as with an effective medium model. A good agreement is found between experimental and numerical results (the larger discrepancy is found for alpha with a difference lower than 2.1 dB/cm).

Phospholipid decoration of microcapsules containing perfluorooctyl bromide used as ultrasound contrast agents.

We present here an easy method to modify the surface chemistry of polymeric microcapsules of perfluorooctyl bromide used as ultrasound contrast agents (UCAs). Capsules were obtained by a solvent emulsification-evaporation process with phospholipids incorporated in the organic phase before emulsification. Several phospholipids were reviewed: fluorescent, pegylated and biotinylated phospholipids. The influence of phospholipid concentration on microcapsule size and morphology was evaluated. Only a fraction of the phospholipids is associated to microcapsules, the rest being dissolved with the surfactant in the aqueous phase. Microscopy shows that phospholipids are present within the shell and that the core/shell structure is preserved up to 0.5 mg fluorescent phospholipids, up to about 0.25 mg pegylated phospholipids or biotinylated phospholipids (for 100 mg of polymer, poly(lactide-co-glycolide) (PLGA)). HPLC allows quantifying phospholipids associated to capsules: they correspond to 10% of pegylated phospholipids introduced in the organic phase. The presence of pegylated lipids at the surface of capsules was confirmed by X-ray photon electron spectroscopy (XPS). The pegylation did not modify the echographic signal arising from capsules. Finally biotinylated microcapsules incubated with neutravidin tend to aggregate, which confirms the presence of biotin at the surface. These results are encouraging and future work will consist of nanocapsule surface modification for molecular imaging.