Berberine mediated fluorescent gold nanoclusters in biomimetic erythrocyte ghosts as a nanocarrier for enhanced photodynamic treatment.

Photodynamic therapy (PDT) is a well-established cancer treatment method that employs light to generate reactive oxygen species (ROS) causing oxidative damage to cancer cells. Nevertheless, PDT encounters challenges due to its oxygen-dependent nature, which makes it less effective in hypoxic tumor environments. To address this issue, we have developed a novel nanocomposite known as AuNC@BBR@Ghost. This nanocomposite combines the advantageous features of erythrocyte ghost membranes, the photoresponsive properties of gold nanoclusters (AuNC) and the anticancer characteristics of Berberine (BBR) for cancer treatment. Our synthesized AuNC efficiently produce ROS, with a 25% increase in efficiency when exposed to near-infrared (NIR) irradiation. By harnessing the oxygen-carrying capacity of erythrocyte ghost cells, AuNC@BBR@Ghost demonstrates a significant improvement in ROS generation, achieving an 80% efficiency. Furthermore, the AuNC exhibit tunable emission wavelengths due to their excellent fluorescent properties. In normoxic conditions, treatment of A549 lung carcinoma cells with AuNC@BBR@Ghost followed by exposure to 808 nm NIR irradiation results in a notable increase in intracellular ROS levels, accelerating cell death. In hypoxic conditions, when A549 cells were treated with AuNC@BBR@Ghost, the erythrocyte ghost acted as an oxygen supplement due to the residual hemoglobin, alleviating hypoxia and enhancing the nanocomposite's sensitivity to PDT treatment. Thus, the AuNC@BBR@Ghost nanocomposite achieves an improved effect by combining the advantageous properties of its individual components, resulting in enhanced ROS generation and adaptability to hypoxic conditions. This innovative approach successfully overcomes PDT's limitations, making AuNC@BBR@Ghost a promising nanotheranostic agent with significant potential for advanced cancer therapy.

Effect of pulsed laser parameters on photoacoustic flow cytometry efficiency in vitro and in vivo.

Photoacoustic flow cytometry is one of the most effective approaches to detect "alien" objects in the bloodstream, including circulating tumor cells, blood clots, parasites, and emboli. However, the possibility of detecting high-amplitude signals from these objects against the background of blood depends on the parameters of the laser pulse. So, the dependencies of photoacoustic signals amplitude and number on laser pulse energy (5-150 µJ), pulse length (1, 2, 5 ns), and pulse repetition rate (2, 5, 10 kHz) for the melanoma cells were investigated. First, the PA responses of a melanoma cell suspension in vitro were measured to directly assess the efficiency of converting laser light into an acoustic signal. After it, the same dependence with the developed murine model based on constant rate melanoma cell injection into the animal blood flow was tested. Both in vivo and in vitro experiments show that signal generation efficiency increases with laser pulse energy above 15 µJ. Shorter pulses, especially 1 ns, provide more efficient signal generation as well as higher pulse rates. A higher pulse rate also provides more efficient signal generation, but also leads to overheating of the skin. The results show the limits where the photoacoustic flow cytometry system can be effectively used for the detection of circulating tumor cells in undiluted blood both for in vitro experiments and for in vivo murine models.

Targeted Therapy for Glomerulonephritis Using Arterial Delivery of Encapsulated Etanercept.

Complex immunosuppressive therapy is prescribed in medical practice to patients with glomerulonephritis to help them overcome symptoms and prevent chronic renal failure. Such an approach requires long-term systemic administration of strong medications, which causes severe side effects. This work shows the efficiency of polymer capsule accumulation (2.8 ± 0.4 µm) containing labeled etanercept (100 µg per dose) in the kidneys of mice. The comparison of injection into the renal artery and tail vein shows the significant superiority of the intra-arterial administration strategy. The etanercept retention rate of 18% and 8% ID in kidneys was found 1 min and 1 h after injection, respectively. The capsules were predominantly localized in the glomeruli after injection in mice using a model of acute glomerulonephritis. Histological analysis confirmed a significant therapeutic effect only in animals with intra-arterial administration of microcapsules with etanercept. The proposed strategy combines endovascular surgery and the use of polymer microcapsules containing a high molecular weight drug that can be successfully applied to treat a wide range of kidney diseases associated with glomerular pathology.

Renal Artery Catheterization for Microcapsules' Targeted Delivery to the Mouse Kidney.

The problem of reducing the side effects associated with drug distribution throughout the body in the treatment of various kidney diseases can be solved by effective targeted drug delivery. The method described herein involves injection of a drug encapsulated in polyelectrolyte capsules to achieve prolonged local release and long-term capillary retention of several hours while these capsules are administered via the renal artery. The proposed method does not imply disruption (puncture) of the renal artery or aorta and is suitable for long-term chronic experiments on mice. In this study, we compared how capsule size and dosage affect the target kidney blood flow. It has been established that an increase in the diameter of microcapsules by 29% (from 3.1 to 4.0 µm) requires a decrease in their concentration by at least 50% with the same suspension volume. The photoacoustic method, along with laser speckle contrast imaging, was shown to be useful for monitoring blood flow and selecting a safe dose. Capsules contribute to a longer retention of a macromolecular substance in the target kidney compared to its free form due to mechanical retention in capillaries and slow impregnation into surrounding tissues during the first 1-3 h, which was shown by fluorescence tomography and microscopy. At the same time, the ability of capillaries to perform almost complete "self-cleaning" from capsular shells during the first 12 h leads to the preservation of organ tissues in a normal state. The proposed strategy, which combines endovascular surgery and the injection of polymer microcapsules containing the active substance, can be successfully used to treat a wide range of nephropathies.

Optical coherence microangiography of the mouse kidney for diagnosis of circulatory disorders.

Optical coherence tomography (OCT) has become widespread in clinical applications in which precise three-dimensional functional imaging of living organs is required. Nevertheless, the kidney is inaccessible for the high resolution OCT imaging due to a high light attenuation coefficient of skin and soft tissues that significantly limits the penetration depth of the probing laser beam. Here, we introduce a surgical protocol and fixation scheme that enables functional visualization of kidney's peritubular capillaries via OCT microangiography. The model of reversible/irreversible glomerulus embolization using drug microcarriers confirms the ability of OCT to detect circulatory disorders. This approach can be used for choosing optimal carriers, their dosages and diagnosis of other blood flow pathologies.

Dynamic blood flow phantom for in vivo liquid biopsy standardization.

In vivo liquid biopsy, especially using the photoacoustic (PA) method, demonstrated high clinical potential for early diagnosis of deadly diseases such as cancer, infections, and cardiovascular disorders through the detection of rare circulating tumor cells (CTCs), bacteria, and clots in the blood background. However, little progress has been made in terms of standardization of these techniques, which is crucial to validate their high sensitivity, accuracy, and reproducibility. In the present study, we addressed this important demand by introducing a dynamic blood vessel phantom with flowing mimic normal and abnormal cells. The light transparent silica microspheres were used as white blood cells and platelets phantoms, while hollow polymeric capsules, filled with hemoglobin and melanin, reproduced red blood cells and melanoma CTCs, respectively. These phantoms were successfully used for calibration of the PA flow cytometry platform with high-speed signal processing. The results suggest that these dynamic cell flow phantoms with appropriate biochemical, optical, thermal, and acoustic properties can be promising for the establishment of standardization tool for calibration of PA, fluorescent, Raman, and other detection methods of in vivo flow cytometry and liquid biopsy.

Target delivery of drug carriers in mice kidney glomeruli via renal artery. Balance between efficiency and safety.

Targeting drug delivery systems is crucial to reducing the side effects of therapy. However, many of them are lacking effectiveness for kidney targeting, due to systemic dispersion and accumulation in the lungs and liver after intravenous administration. Renal artery administration of carriers provides their effective local accumulation but may cause irreversible vessel blockage. Therefore, the combination of the correct administration procedure, suitable drug delivery system, selection of effective and safe dosage is the key to sparing local therapy. Here, we propose the 3-µm sized fluorescent capsules based on poly-L-arginine and dextran sulfate for targeting the kidney via a mice renal artery. Hemodynamic study of the target kidney in combination with the histological analysis reveals a safe dose of microcapsules (20 × 106), which has not lead to irreversible pathological changes in blood flow and kidney tissue, and provides retention of 20.5 ± 3% of the introduced capsules in the renal cortex glomeruli. Efficacy of fluorescent dye localization in the target kidney after intra-arterial administration is 9 times higher than in the opposite kidney and after intravenous injection. After 24 h microcapsules are not observed in the target kidney when the safe dose of carriers is being used but a high level of fluorescent signal persists for 48 h indicating that fluorescent cargo accumulation in tissues. Injection of non-safe microcapsule dose leads to carriers staying in glomeruli for at least 48 h which has consequences of blood flow not being restored and tissue damage being observed in histology.

Intravital molecular tagging velocimetry of cerebral blood flow using Evans Blue.

The effects of light-driven enhancement of Evans Blue dye complexes with blood plasma proteins were observed for the first time, both in vitro and in vivo. The possible background of the effect concerns the photochemical cis-trans isomerization of the azo dye molecules. The effect was induced in the solution with a red laser with a wavelength of 638 nm, which corresponds to the peak of the dye absorption. The lifetime of the enhanced fluorescence is approximately 1 second and enables its use as an optically tagged molecular flow tracer for blood flow velocity measurements. Utilizing the effect, we performed for the first time the intravital molecular tagging velocimetry of the blood velocity in blood vessels in a living animal. The results of the measurements of the blood flow velocities in the cerebral veins of a group of healthy mice are presented.

Photodynamic opening of the blood-brain barrier and pathways of brain clearing.

A new application of the photodynamic treatment (PDT) is presented for the opening of blood-brain barrier (BBB) and the brain clearing activation that is associated with it, including the use of gold nanoparticles as emerging photosensitizer carriers in PDT. The obtained results clearly demonstrate 2 pathways for the brain clearing: (1) using PDT-opening of BBB and intravenous injection of FITC-dextran we showed a clearance of this tracer via the meningeal lymphatic system in the subdural space; (2) using optical coherence tomography and intraparenchymal injection of gold nanorods, we observed their clearance through the exit gate of cerebral spinal fluid from the brain into the deep cervical lymph node, where the gold nanorods were accumulated. These data contribute to a better understanding of the cerebrovascular effects of PDT and shed light on mechanisms, underlying brain clearing after PDT-related opening of BBB, including clearance from nanoparticles as drug carriers.

Photodynamic opening of blood-brain barrier.

Photodynamic treatment (PDT) causes a significant increase in the permeability of the blood-brain barrier (BBB) in healthy mice. Using different doses of laser radiation (635 nm, 10-40 J/cm2) and photosensitizer (5-aminolevulinic acid - 5-ALA, 20 and 80 mg/kg, i.v.), we found that the optimal PDT for the reversible opening of the BBB is 15 J/cm2 and 5-ALA, 20 mg/kg, exhibiting brain tissues recovery 3 days after PDT. Further increases in the laser radiation or 5-ALA doses have no amplifying effect on the BBB permeability, but are associated with severe damage of brain tissues. These results can be an informative platform for further studies of new strategies in brain drug delivery and for better understanding of mechanisms underlying cerebrovascular effects of PDT-related fluorescence guided resection of brain tumor.