On-chip fabrication of calcium carbonate nanoparticles loaded with various compounds using microfluidic approach.

Engineered calcium carbonate (CaCO3) particles are extensively used as drug delivery systems due to their availability, biological compatibility, biodegradability, and cost-effective production. The synthesis procedure of CaCO3 particles, however, suffers from poor reproducibility. Furthermore, reducing the size of CaCO3 particles to <100 nm requires the use of additives in the reaction, which increases the total reaction time. Here we propose on-chip synthesis and loading of nanoscaled CaCO3 particles using microfluidics. After the development and fabrication of a microfluidic device, we optimized the synthesis of CaCO3 NPs by varying different parameters such as flow rates in the microfluidic channels, concentration of reagents, and the reaction time. To prove the versatility of the used synthesis route, we performed single and double loading of CaCO3 NPs with various compounds (Doxorubicin, Cy5 or FITC conjugated with BSA, and DNA) using the same microfluidic device. Further, the on-chip loaded CaCO3 NPs were used as carriers to transfer compounds to model cells. We have developed a microfluidic synthesis method that opens up a new pathway for easy on-chip fabrication of functional nanoparticles for clinical use.

Size-dependent therapeutic efficiency of 223Ra-labeled calcium carbonate carriers for internal radionuclide therapy of breast cancer.

The size of drug carriers strongly affects their biodistribution, tissue penetration, and cellular uptake in vivo. As a result, when such carriers are loaded with therapeutic compounds, their size can influence the treatment outcomes. For internal α-radionuclide therapy, the carrier size is particularly important, because short-range α-emitters should be delivered to tumor volumes at a high dose rate without any side effects, i.e. off-target irradiation and toxicity. In this work, we aim to evaluate and compare the therapeutic efficiency of calcium carbonate (CaCO3) microparticles (MPs, >2 µm) and nanoparticles (NPs, <100 nm) labeled with radium-223 (223Ra) for internal α-radionuclide therapy against 4T1 breast cancer. To do this, we comprehensively study the internalization and penetration efficiency of these MPs and NPs, using 2D and 3D cell cultures. For further therapeutic tests, we develop and modify a chelator-free method for radiolabeling of CaCO3 MPs and NPs with 223Ra, improving their radiolabeling efficiency (>97%) and radiochemical stability (>97%). After intratumoral injection of 223Ra-labeled MPs and NPs, we demonstrate their different therapeutic efficiencies against a 4T1 tumor. In particular, 223Ra-labeled NPs show a tumor inhibition of approximately 85%, which is higher compared to 60% for 223Ra-labeled MPs. As a result, we can conclude that 223Ra-labeled NPs have a more suitable biodistribution within 4T1 tumors compared to 223Ra-labeled MPs. Thus, our study reveals that 223Ra-labeled CaCO3 NPs are highly promising for internal α-radionuclide therapy.

One-Pot Synthesis of Affordable Redox-Responsive Drug Delivery System Based on Trithiocyanuric Acid Nanoparticles.

Redox-responsive drug delivery systems present a promising avenue for drug delivery due to their ability to leverage the unique redox environment within tumor cells. In this work, we describe a facile and cost-effective one-pot synthesis method for a redox-responsive delivery system based on novel trithiocyanuric acid (TTCA) nanoparticles (NPs). We conduct a thorough investigation of the impact of various synthesis parameters on the morphology, stability, and loading capacity of these NPs. The great drug delivery potential of the system is further demonstrated in vitro and in vivo by using doxorubicin as a model drug. The developed TTCA-PEG NPs show great drug delivery efficiency with minimal toxicity on their own both in vivo and in vitro. The simplicity of this synthesis, along with the promising characteristics of TTCA-PEG NPs, paves the way for new opportunities in the further development of redox-responsive drug delivery systems based on TTCA.

Sm/Co-Doped Silica-Based Nanozymes Reprogram Tumor Microenvironment for ATP-Inhibited Tumor Therapy.

Current applications of multifunctional nanozymes for reprogramming the redox homeostasis of the tumor microenvironment (TME) have been severely confronted with low catalytic activity and the ambiguity of active sites of nanozymes, as well as the stress resistance from the rigorous physical environment of tumor cells. Herein, the Sm/Co-doped mesoporous silica with 3PO-loaded nanozymes (denoted as mSC-3PO) are rationally constructed for simultaneously inhibiting energy production by adenosine triphosphate (ATP) inhibitor 3PO and reprogramming TME by multiactivities of nanozymes with photothermal effect assist, i.e., enhanced peroxidase-like, catalase-like activity, and glutathione peroxidase-like activities, facilitating reactive oxygen species (ROS) generation, promoting oxygen content, and restraining the over-expressed glutathione. Through the optimal regulation of nanometric size and doping ratio, the fabricated superparamagnetic mSC-3PO enables the excellent exposure of active sites and avoids agglomeration owing to the large specific surface and mesoporous structure, thus providing adequate Sm/Co-doped active sites and enough spatial distribution. The constructed Sm/Co centers both participate in the simulated biological enzyme reactions and carry out the double-center catalytic process (Sm3+ and Co3+ /Co2+ ). Significantly, as the inhibitor of glycolysis, 3PO can reduce the ATP flow by cutting down the energy transform, thereby inhibiting tumor angiogenesis and assisting ROS to promote the early withering of tumor cells. In addition, the considerable near-infrared (NIR) light absorption of mSC-3PO can adapt to NIR excitable photothermal treatment therapy and photoexcitation-promoted enzymatic reactions. Taken together, this work presents a typical therapeutic paradigm of multifunctional nanozymes that simultaneously reprograms TME and promotes tumor cell apoptosis with photothermal assistance.

Calcium carbonate nanoparticles tumor delivery for combined chemo-photodynamic therapy: Comparison of local and systemic administration.

The use of nanoparticles (NPs) as delivery vehicles for multiple drugs is an intensively developing area. However, the success of NPs' accumulation in the tumor area for efficient tumor treatment has been recently questioned. Distribution of NPs in a laboratory animal is mainly related to the administration route of NPs and their physicochemical parameters, which significantly affect the delivery efficiency. In this work, we aim to compare the therapeutic efficiency and side effects of the delivery of multiple therapeutic agents with NPs by both intravenous and intratumoral injections. For this, we systematically developed universal nanosized carriers based on calcium carbonate (CaCO3) NPs (< 100 nm) that were co-loaded with a photosensitizer (Chlorin e6, Ce6) and chemotherapeutic agent (doxorubicin, Dox) for combined chemo- and photodynamic therapy (PDT) of B16-F10 melanoma tumors. By performing intratumoral or intravenous injections of NPs, we observed different biodistribution profiles and tumor accumulation efficiencies. In particular, after intratumoral administration of NPs, they mostly remained in the tumors (> 97%); while for intravenous injection, the tumor accumulation of NPs was determined to be 8.67-12.4 ID/g%. Although the delivery efficiency of NPs (presented in ID/g%) in the tumor differs, we have developed an effective strategy for tumor inhibition based on combined chemo- and PDT by both intratumoral and intravenous injections of NPs. Notably, after the combined chemo- and PDT treatment with Ce6/Dox@CaCO3 NPs, all B16-F10 melanoma tumors in mice shrank substantially, by approximately 94% for intratumoral injection and 71% for intravenous injection, which are higher values compared to mono-therapy. In addition, the CaCO3 NPs showed negligible in vivo toxicity towards major organs such as the heart, lungs, liver, kidneys, and spleen. Thus, this work demonstrates a successful approach for the enhancement of NPs' efficiency in combined anti-tumor therapy.

Ligand-free template-assisted synthesis of stable perovskite nanocrystals with near-unity photoluminescence quantum yield within the pores of vaterite spheres.

Ligand-free methods for the synthesis of halide perovskite nanocrystals are of great interest because of their excellent performance in optoelectronics and photonics. In addition, template-assisted synthesis methods have become a powerful tool for the fabrication of environmentally stable and bright nanocrystals. Here we develop a novel approach for the facile ligand-free template-assisted fabrication of perovskite nanocrystals with a near-unity absolute quantum yield, which involves CaCO3 vaterite micro- and submicrospheres as templates. We show that the optical properties of the obtained nanocrystals are affected not mainly by the template morphology, but strongly depend on the concentration of precursor solutions, anion and cation ratio, as well as on adding defect-passivating rare-earth dopants. The optimized samples are further tested as infrared radiation visualizers exhibiting promising characteristics comparable to those that are commercially available.

Synthesis of thieno[3,2-e]pyrrolo[1,2-a]pyrimidine derivatives and their precursors containing 2-aminothiophenes fragments as anticancer agents for therapy of pulmonary metastatic melanoma.

The design and synthesis of new promising compounds based on thienopyrimidine scaffold containing 2-aminothiophene fragments with good safety and favorable drug-like properties are highly relevant for chemotherapy. In this study, a series of 14 variants of thieno[3,2-e]pyrrolo[1,2-a]pyrimidine derivatives (11aa-oa) and their precursors (31 compounds) containing 2-aminothiophenes fragments (9aa-mb, 10aa-oa) were synthesized and screened for their cytotoxicity against B16-F10 melanoma cells. The selectivity of the developed compounds was assessed by determining the cytotoxicity using normal mouse embryonic fibroblasts (MEF NF2 cells). The lead compounds 9cb, 10ic and 11jc with the most significant antitumor activity and minimum cytotoxicity on normal non-cancerous cells were chosen for further in vivo experiments. Additional in vitro experiments with compounds 9cb, 10ic and 11jc showed that apoptosis was the predominant mechanism of death in B16-F10 melanoma cells. With support from in vivo studies, compounds 9cb, 10ic and 11jc demonstrated the biosafety to healthy mice and significant inhibition of the metastatic nodules in pulmonary metastatic melanoma mouse model. Histological analysis detected no abnormal changes in the main organs (the liver, spleen, kidneys, and heart) after the therapy. Thus, the developed compounds 9cb, 10ic and 11jc demonstrate high efficiency in the treatment of pulmonary metastatic melanoma and can be recommended for further preclinical investigation of the melanoma treatment.

Theoretical simulation and experimental design of selenium and gold incorporated polymer-based microcarriers for ROS-mediated combined photothermal therapy.

Recently, multi-modal combined photothermal therapy (PTT) with the use of photo-active materials has attracted significant attention for cancer treatment. However, drug carriers enabling efficient heating at the tumor site are yet to be designed: this is a fundamental requirement for broad implementation of PTT in clinics. In this work, we design and develop hybrid carriers based on multilayer capsules integrated with selenium nanoparticles (Se NPs) and gold nanorods (Au NRs) to realize reactive oxygen species (ROS)-mediated combined PTT. We show theoretically and experimentally that cooperative interaction of Se NPs with Au NRs improves the heat release efficiency of the developed capsules. In addition, after uptake by tumor cells, intracellular ROS level amplified by Se NPs inhibits the tumor growth. As a consequence, the synergy between Se NPs and Au NRs exhibits the advantages of hybrid carriers such as (i) improved photothermal conversion efficiency and (ii) dual-therapeutic effect. The results of in vitro and in vivo experiments demonstrate that the combination of ROS-mediated therapy and PTT has a higher tumor inhibition efficiency compared to the single-agent treatment (using only Se-loaded or Au-loaded capsules). Furthermore, the developed hybrid carriers show negligible in vivo toxicity towards major organs such as the heart, lungs, liver, kidneys and spleen. This study not only provides a potential strategy for the design of multifunctional "all-in-one" carriers, but also contributes to the development of combined PTT in clinical practice.

Development of Nanocarrier-Based Radionuclide and Photothermal Therapy in Combination with Chemotherapy in Melanoma Cancer Treatment.

Conventional cancer therapy methods have serious drawbacks that are related to the nonspecific action of anticancer drugs that leads to high toxicity on normal cells and increases the risk of cancer recurrence. The therapeutic effect can be significantly enhanced when various treatment modalities are implemented. Here, we demonstrate that the radio- and photothermal therapy (PTT) delivered through nanocarriers (gold nanorods, Au NRs) in combination with chemotherapy in a melanoma cancer results in complete tumor inhibition compared to the single therapy. The synthesized nanocarriers can be effectively labeled with 188Re therapeutic radionuclide with a high radiolabeling efficiency (94-98%) and radiochemical stability (>95%) that are appropriate for radionuclide therapy. Further, 188Re-Au NRs, mediating the conversion of laser radiation into heat, were intratumorally injected and PTT was applied. Upon the irradiation of a near-infrared laser, dual photothermal and radionuclide therapy was achieved. Additionally, the combination of 188Re-labeled Au NRs with paclitaxel (PTX) has significantly improved the treatment efficiency (188Re-labeled Au NRs, laser irradiation, and PTX) compared to therapy in monoregime. Thus, this local triple-combination therapy can be a step toward the clinical translation of Au NRs for use in cancer treatment.

Dual Nanozyme-Driven PtSn Bimetallic Nanoclusters for Metal-Enhanced Tumor Photothermal and Catalytic Therapy.

Specific generation of reactive oxygen species (ROS) within tumors in situ catalyzed by nanozymes is a promising strategy for cancer therapeutics. However, it remains a significant challenge to fabricate highly efficient nanozymes acting in the tumor microenvironment. Herein, we develop a bimetallic nanozyme (Pt50Sn50) with the photothermal enhancement of dual enzymatic activities for tumor catalytic therapy. The structures and activities of PtSn bimetallic nanoclusters (BNCs) with different Sn content are explored and evaluated systematically. Experimental comparisons show that the Pt50Sn50 BNCs exhibit the highest activities among all those investigated, including enzymatic activity and photothermal property, due to the generation of SnO2-x with oxygen vacancy (Ovac) sites on the surface of Pt50Sn50 BNCs. Specifically, the Pt50Sn50 BNCs exhibit photothermal-enhanced peroxidase-like and catalase-like activities, as well as a significantly enhanced anticancer efficacy in both multicellular tumor spheroids and in vivo experiments. Due to the high X-ray attenuation coefficient and excellent light absorption property, the Pt50Sn50 BNCs also show dual-mode imaging capacity of computed tomography and photoacoustic imaging, which could achieve in vivo real-time monitoring of the therapeutic process. Therefore, this work will advance the development of noble-metal nanozymes with optimal composition for efficient tumor catalytic therapy.

Overcoming the blood-brain barrier for the therapy of malignant brain tumor: current status and prospects of drug delivery approaches.

Besides the broad development of nanotechnological approaches for cancer diagnosis and therapy, currently, there is no significant progress in the treatment of different types of brain tumors. Therapeutic molecules crossing the blood-brain barrier (BBB) and reaching an appropriate targeting ability remain the key challenges. Many invasive and non-invasive methods, and various types of nanocarriers and their hybrids have been widely explored for brain tumor treatment. However, unfortunately, no crucial clinical translations were observed to date. In particular, chemotherapy and surgery remain the main methods for the therapy of brain tumors. Exploring the mechanisms of the BBB penetration in detail and investigating advanced drug delivery platforms are the key factors that could bring us closer to understanding the development of effective therapy against brain tumors. In this review, we discuss the most relevant aspects of the BBB penetration mechanisms, observing both invasive and non-invasive methods of drug delivery. We also review the recent progress in the development of functional drug delivery platforms, from viruses to cell-based vehicles, for brain tumor therapy. The destructive potential of chemotherapeutic drugs delivered to the brain tumor is also considered. This review then summarizes the existing challenges and future prospects in the use of drug delivery platforms for the treatment of brain tumors.

Biodegradable particles for protein delivery: Estimation of the release kinetics inside cells.

A methodology to quantify the efficiency of the protein loading and in-vitro delivery for biodegradable capsules with different architectures based on polyelectrolytes (dextran sulfate, poly-L-arginine and polyethylenimine) and SiO2 was developed. The capsules were loaded with model proteins such as ovalbumin and green fluorescent protein (GFP), and the protein release profile inside cells (either macrophages or HeLa cells) after endocytosis was analysed. Both, protein loading and release kinetics were evaluated by analysing confocal laser scanning microscopy images using MatLab and CellProfiler software. Our results indicate that silica capsules showed the most efficient release of proteins as cargo molecules within 48 h, as compared to their polymeric counterparts. This developed method for the analysis of the intracellular cargo release kinetics from carrier structures could be used in the future for a better control of drug release profiles.

Incorporation of Perovskite Nanocrystals into Polymer Matrix for Enhanced Stability in Biological Media: In Vitro and In Vivo Studies.

The outstanding optical properties and multiphoton absorption of lead halide perovskites make them promising for use as fluorescence tags in bioimaging applications. However, their poor stability in aqueous media and biological fluids significantly limits their further use for in vitro and in vivo applications. In this work, we have developed a universal approach for the encapsulation of lead halide perovskite nanocrystals (PNCs) (CsPbBr3 and CsPbI3) as water-resistant fluorescent markers, which are suitable for fluorescence bioimaging. The obtained encapsulated PNCs demonstrate bright green emission at 510 nm (CsPbBr3) and red emission at 688 nm (CsPbI3) under one- and two-photon excitation, and they possess an enhanced stability in water and biological fluids (PBS, human serum) for a prolonged period of time (1 week). Further in vitro and in vivo experiments revealed enhanced stability of PNCs even after their introduction directly into the biological microenvironment (CT26 cells and DBA mice). The developed approach allows making a step toward stable, low-cost, and highly efficient bioimaging platforms that are spectrally tunable and have narrow emission.

Calcium carbonate carriers for combined chemo- and radionuclide therapy of metastatic lung cancer.

Considering the clinical limitations of individual approaches against metastatic lung cancer, the use of combined therapy can potentially improve the therapeutic effect of treatment. However, determination of the appropriate strategy of combined treatment can be challenging. In this study, combined chemo- and radionuclide therapy has been realized using radionuclide carriers (177Lu-labeled core-shell particles, 177Lu-MPs) and chemotherapeutic drug (cisplatin, CDDP) for treatment of lung metastatic cancer. The developed core-shell particles can be effectively loaded with 177Lu therapeutic radionuclide and exhibit good radiochemical stability for a prolonged period of time. In vivo biodistribution experiments have demonstrated the accumulation of the developed carriers predominantly in lungs. Direct radiometry analysis did not reveal an increased absorbance of radiation by healthy organs. It has been shown that the radionuclide therapy with 177Lu-MPs in mono-regime is able to inhibit the number of metastatic nodules (untreated mice = 120 ± 12 versus177Lu-MPs = 50 ± 7). The combination of chemo- and radionuclide therapy when using 177Lu-MPs and CDDP further enhanced the therapeutic efficiency of tumor treatment compared to the single therapy (177Lu-MPs = 50 ± 7 and CDDP = 65 ± 10 versus177Lu-MPs + CDDP = 37 ± 5). Thus, this work is a systematic research on the applicability of the combination of chemo- and radionuclide therapy to treat metastatic lung cancer.

Impact of metallic coating on the retention of 225Ac and its daugthers within core-shell nanocarriers.

Currently, alpha-emitting radionuclide 225Ac is one of the most promising isotopes in alpha therapy due to its high linear energy transfer during four sequential alpha decays. However, the main obstacle preventing the full introduction of 225Ac into clinical practice is the lack of stable retention of radionuclides, leading to free circulation of toxic isotopes in the body. In this work, the surface of silica nanoparticles (SiO2 NPs) has been modified with metallic shells composed of titanium dioxide (TiO2) and gold (Au) nanostructures to improve the retention of 225Ac and its decay products within the developed nanocarriers. In vitro and in vivo studies in healthy mice show that the metallic surface coating of SiO2 NPs promotes an enhanced sequestering of radionuclides (225Ac and its daughter isotopes) compared to non-modified SiO2 NPs for a prolonged period of time. Histological analysis reveals that for the period of 3-10 d after the injections, the developed nanocarriers have no significant toxic effects in mice. At the same time, almost no accumulation of leaked radionuclides can be detected in non-target organs (e.g., in the kidneys). In contrast, non-modified carriers (SiO2 NPs) demonstrate the release of free radionuclides, which are distributed over the whole animal body with the consequent morphological changes in the lung, liver and kidney tissues. These results highlight the potential of the developed nanocarriers to be utilized as radionuclide delivery systems and offer an insight into design rules for the fabrication of new nanotherapeutic agents.

Microfluidic synthesis of optically responsive materials for nano- and biophotonics.

Recently, nanomaterials demonstrating optical response under illumination, the so-called optically responsive nanoparticles (NPs), have found their broad application as optical switchers, gas adsorbents, data storage devices, and optical and biological sensors. Unique optical properties of such nanomaterials are strongly related to their chemical composition, geometrical parameters and morphology. Microfluidic approaches for NPs' synthesis allow overcoming the known critical stages in conventional synthesis of NPs due to a high rate of heat/mass transfer and precise regulation of synthesis conditions, which results in reproducible synthesis outcomes with the desired physico-chemical properties. Here, we review the recent advances in microfluidic approach for synthesis of optically responsive nanomaterials (plasmonic, photoluminescent, shape-changeable NPs), highlighting the general background of microfluidics, common considerations in the design of microfluidic chips (MFCs), and theoretical models of the NPs' formation mechanisms. Comparative analysis of microfluidic synthesis with conventional synthesis methods is provided further, along with the recent applications of optically responsive NPs in nano- and biophotonics.