Uptake Studies Research Articles

Precise intracellular uptake and endosomal release of diverse functional mRNA payloads via glutathione-responsive nanogels.

We present a novel, highly customizable glutathione-responsive nanogel (NG) platform for efficient mRNA delivery with precise mRNA payload release control. Optimization of various cationic monomers, including newly synthesized cationic polyarginine, polyhistidine, and acrylated guanidine monomers, allowed fine-tuning of NG properties for mRNA binding. By incorporating a poly(ethylene) glycol-based disulphide crosslinker, we achieved glutathione-triggered mRNA release, enabling targeted intracellular delivery. Our NGs demonstrated superior encapsulation (up to 89.3%) and loading (10.7%) efficiencies, with controlled mRNA release kinetics at intracellular glutathione concentrations. NGs outperformed commercial transfection reagents across multiple cell lines, including traditionally difficult-to-transfect lines. We demonstrate the platform's versatility by successfully delivering GFP mRNA, Mango II RNA aptamers, and functionally relevant β2-AMPK mRNA. Furthermore, we used TIRF microscopy to measure exact RNA copy number within the NGs. Notably, mechanistic cellular uptake studies revealed that disulphide-containing NGs exhibit enhanced cellular uptake and endosomal escape, potentially due to interactions with cell surface thiols. This work represents a highly tuneable, efficient, and biocompatible platform for mRNA delivery with relevance for gene therapy and vaccine development.

Development, Characterization, and Evaluation of Chi-Tn mAb-Functionalized DOTAP-PLGA Hybrid Nanoparticles Loaded with Docetaxel for Lung Cancer Therapy

Background/Objectives: The focus of this study was to prepare and characterize docetaxel (DCX)-loaded lipid/polymer hybrid nanoparticles (LPHNps) functionalized with the monoclonal antibody (mAb) Chi-Tn for a potential active targeting approach in lung cancer treatment. Methods: We synthesized DOTAP-PLGA hybrid nanoparticles loaded with DCX and functionalized them with Chi-Tn mAb through a biotin–avidin approach. The physicochemical characterization involved dynamic light scattering, transmission electron microscopy, Raman spectroscopy, and atomic force microscopy. The in vitro and in vivo evaluations encompassed uptake studies, cell viability tests, and the assessment of tumor growth control in a lung cancer model. Results: The nanoparticles featured a hydrophobic PLGA core with 99.9% DCX encapsulation efficiency, surrounded by a DOTAP lipid shell ensuring colloidal stability with a high positive surface charge. The incorporation of PEGylated lipids on their surface helps evade the immune system and facilitate Chi-Tn mAb attachment. The resulting nanoparticles exhibit a spherical shape with monodisperse particle sizes averaging 250 nm, and demonstrate sustained drug release. In vitro uptake studies and viability assays conducted in A549 cancer cells show that the Chi-Tn mAb enhances nanoparticle internalization and significantly reduces cell viability. In vivo studies demonstrate a notable reduction in tumor volume and an increased survival rate in the A549 tumor xenograft mice model when DCX was encapsulated in nanoparticles and targeted with Chi-Tn mAb in comparison to the free drug. Conclusions: Therefore, Chi-Tn-functionalized LPHNps hold promise as carriers for actively targeting DCX to Tn-expressing carcinomas.

Ex Vivo Biosafety and Efficacy Assessment of Advanced Chlorin e6 Nanoemulsions as a Drug Delivery System for Photodynamic Antitumoral Application

The photosensitizer (PS) in the Photodynamic Therapy (PDT) field represents a key factor, being directly connected to the therapeutic efficacy of the process. Chlorin e6 is a second-generation photosensitizer, approved by the FDA with the most desired clinical properties for PDT applications, presenting high reactive oxygen species (ROS) generation and proven anticancer properties. However, hydrophobicity is a major limitation, leading to poor biodistribution. To overcome this condition, the present work developed an up-to-date nanoemulsion incorporating Ce6 in a new nanosystem (Ce6/NE). A comprehensive study of physicochemical properties, stability, fluorescence characteristics, the in vitro release profile, in vivo and ex vivo biocompatibility, and ex vivo efficacy was established. The nanoemulsions showed the desired particle size and stability over six months, with no spectroscopic or photophysical alterations. Uptake studies demonstrated the internalization of the Ce6/NE in monolayers, with biocompatibility at the lowest concentrations. The HET-CAM assay, however, revealed a higher biocompatibility range, also indicating Ce6/NE’s potential for cancer treatment through antiangiogenic studies. These findings highlight the use of a new promising photosensitizer for PDT modulated with nanotechnology that promotes low toxicity, higher bioavailability, and site-specific delivery.

In situ forming gels as subcutaneous delivery systems of curcumin and piperine

In this study an in situ forming gel for curcumin and piperine delivery is investigated as a long-lasting strategy in the local treatment of inflammatory and degenerative joint disease, such as osteoarthritis and rheumatoid arthritis. Particularly glyceryl monooleate, in association with phosphatidylcholine and ethanol, were employed. Different ratios between excipients were tested, with the aim to obtain a liquid form suitable for subcutaneous injection, gaining a semisolid consistency in contact with biological fluids. A formulative study was conducted to assess the composition impact on the structural properties of the formulations, particularly focusing on injectability and phase transition. Curcumin and piperine were loaded, singularly or jointly, in selected in situ forming gels. Structural characterization, performed by X-ray scattering, revealed disordered reverse micellar phases, undergoing transition to hexagonal and cubic Pn3m phase upon hydration. In vitro dialysis release study demonstrated a sustained release of both drugs over 96 h, with a faster release in the case of jointly loaded drugs. Mechanistic analysis and water uptake studies indicated a drug release governed by both diffusion and swelling/erosion of the lipid supramolecular structure. Furthermore, an ex vivo release analysis performed using human skin explants suggested the formulation suitability for subcutaneous injection, indicating that the presence of piperine in the in situ formed gel allowed to double the curcumin release with respect to the simple curcumin loaded gel.

True cancer stem cells exhibit relative degrees of dormancy and genomic stability.

Cancer stem cells in human tumors have been defined by stem cell markers, embryonal signaling pathways and characteristic biology, ie., namely the ability to repopulate the proliferating population. However, even if these properties can be demonstrated within a tumor cell subpopulation, it does not mean that they are truly hierarchical stem cells because they could have been derived from the proliferating population in a reversible manner. Using a human PDX, Mary-X, that overall expressed a strong cancer stem cell phenotype, the study conducted both GPP-labelled retroviral transfection and fluorescent microsphere uptake studies to distinguish proliferating from dormant cells and array CGH to identify regions of amplifications (gains) and deletions (losses) on the overall Mary-X population and then applied derived probes by FISH on individual cells to identify a genomically stable subpopulation. Whereas 97-99 % of the cells expressed retroviral GFP and not fluorescent particles and showed numerous gene amplifications and deletions, approximately 1-3 % of the cells showed the opposite. The subpopulation with the retained fluorescent microspheres and exhibiting genomic stability was significantly smaller in size than their GFP-expressing and genomically unstable counterparts. Sorting Mary-X spheroids on the basis of either CD133 or ALDH positivity further enriched for this subpopulation. These studies indicate that a truly biological cancer stem cell subpopulation exists that exhibits both dormancy and genomic stability. This subpopulation could not have been derived from the proliferating and resulting genomically unstable population and therefore represents a truly hierarchical stem cell subpopulation capable of only unidirectional differentiation.

Development of a phosphoaminopyrazine-loaded cellulose nanoparticle drug delivery system for targeted treatment of triple-negative breast cancer.

This study explores the development of a sustainable drug delivery system using cellulose nanoparticles (CNPs) derived from potato pulp for the controlled release of phosphoaminopyrazine (PAP), a promising anticancer agent. CNPs were synthesized via nanoprecipitation, and PAP was loaded through in-situ nanoprecipitation, achieving a high loading efficiency of 79.2 %. Characterization of CNPs and PAP-loaded CNPs (PAP@CNP) using Fourier-transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDS), and thermogravimetric analysis (TGA) confirmed the structural integrity, spherical morphology (45.33 nm for CNPs, 54.6 nm for PAP@CNP), and enhanced thermal stability of PAP@CNP. In vitro, drug release studies demonstrated sustained release over 48 h, with pH-sensitive kinetics favoring the acidic tumor microenvironment. Cytotoxicity assays revealed superior efficacy of PAP@CNP against triple-negative breast cancer cells (MDA-MB-231; IC50 = 20.86 ± 0.81 μg/mL) compared to cisplatin while showing minimal toxicity to normal human umbilical vein endothelial cells (HUVECs). Cellular uptake studies using epifluorescence microscopy and flow cytometry confirmed time-dependent internalization and intracellular drug release. Potato pulp, an abundant agro-industrial waste, as a renewable source for CNPs highlights this approach's economic and environmental advantages. This study demonstrates the potential of potato pulp-derived CNPs as a sustainable and cost-effective platform for targeted cancer therapy; offering improved therapeutic outcomes and reduced environmental impact.

N-acetylcysteine and chitosan conjugate modified dexamethasone nanostructured lipid carriers: Enhanced permeability, precorneal retention and lower inflammation for the treatment of dry eye syndrome.

Dexamethasone (Dex) is a primary medication for treating dry eye syndrome, and tobramycin-dexamethasone eye drops are commercially available. However, the eye's complex physiological environment reduces its bioavailability, and repeated use can lead to significant systemic toxicity and side effects. This study introduces a novel conjugate of chitosan (CS) and N-acetylcysteine (NAC), a bioadhesive material, which was grafted onto the surface of a Dex-supported nanostructured lipid carrier (NLC) to develop an innovative nanoparticle lipid ocular drug delivery system (CS-NAC@Dex-NLC). The enhancements afforded by CS-NAC, such as adhesion, osmotic, and targeting properties, address the limitations of traditional eye drops. NMR characterization confirmed the successful synthesis of the copolymer (CS-NAC). Particle size (PS), zeta potential, and transmission electron microscopy (TEM) verified the proper formation of the CS-NAC@Dex-NLC system. Cytotoxicity tests confirmed its excellent biocompatibility and safety. Cellular uptake studies showed that CS-NAC@Dex-NLC achieved the highest efficiency. Pharmacokinetic assessments revealed a significant increase in Dex's bioavailability in tears and aqueous humor. In vitro corneal penetration and in vivo imaging experiments demonstrated effective corneal penetration and retention, enhancing the drug's duration on the ocular surface and overcoming the barrier effect. Pharmacodynamic studies in rabbits with dry eye syndrome indicated that CS-NAC@Dex-NLC effectively alleviates symptoms, repairs corneal damage, and stabilizes the tear film. ELISA results showed a reduction in inflammation caused by dry eye. These findings suggest that CS-NAC@Dex-NLC is a promising vector for dry eye treatment, offering significant clinical relevance.

Multidimensional Demographic Analyses of COVID-19 Vaccine Inequality in the United States: A Systematic Review.

COVID-19 vaccination uptake is associated with demographic characteristics such as age, sex, and ethnicity-race in the United States (U.S.). Prior research predominantly analyzed COVID-19 vaccination uptake unidimensionally, limiting insights into multidimensional demographic inequalities. Multidimensional studies provide a closer insight into vaccination inequality and assist in designing more effective vaccination strategies. Review descriptive studies of the COVID-19 vaccination uptake across combinations of at least two of the three key demographic characteristics: age, sex, and ethnicity-race in the U.S. A systematic review was performed using the Joanna Briggs Institute methodology and adhering to the PRISMA-ScR principles for reporting. Six impartial reviewers examined all of the papers. The data were obtained using a tailored data extraction template. A total of 2793 records were initially downloaded, 461 of them were dropped for duplication, and 2332 were reviewed. Based on the title and abstract reviews, 2115 records were excluded. After reviewing the full text of the remaining records, 212 more records were excluded. The remaining six records were reviewed to identify and compare their population, study period, data, the studied dose number, methodology, and results. Multidimensional COVID-19 vaccine uptake analyses are rare and mostly focused on the dose-one vaccination. Improving researchers' access to immunization registry data while preserving data security is a prerequisite for such analyses.

99mTc]Technetium and Rhenium Dithiocarbazate Complexes: Chemical Synthesis and Biological Assessment.

Dithiocarbazates (DTCs) and their metal complexes have been studied regarding their property as anticancer activities. In this work, using S-benzyl-5-hydroxy-3-methyl-5-phenyl-4,5-dihydro-1H-pirazol-1-carbodithionate (H2bdtc), we prepared [ReO(bdtc)(Hbdtc)] and [[99mTc]TcO(bdtc)(Hbdtc)] complexes for tumor uptake and animal biodistribution studies. Re complex was prepared by a reaction of H2bdtc and (NBu4)[ReOCl4], the final product was characterized by IR, 1H NMR, CHN, and MS-ESI. 99mTc complex was prepared by the reaction of H2bdtc and [[99mTc]TcO4- and analyzed by planar and HPLC radiochromatography, and the stability was evaluated against amino acids and plasma. Biodistribution was performed in C57B/6 mice with B16F10 and TM1M implanted tumor. Re is asymmetric coordinated by two dithiocarbazate ligands, one with O,N,S chelation, and the other with N,S chelation; [[99mTc]TcO(bdtc)(Hbdtc)] was prepared with a radiochemical yield of around 93%. The radioactive complex is hydrophobic (LogP = 1.03), stable for 6 h in PBS and L-histidine solution; stable for 1 h in plasma, but unstable in the presence of L-cysteine. Ex vivo biodistribution demonstrated that the compound has a fast and persistent (until 2 h) uptake by the spleen (55.46%), and tumor B16F10 and TM1M uptake is lower than 1%. In vivo SPECT/CT imaging confirmed ex vivo biodistribution, except by heterogenous TM1M accumulation but not in the B16-F10 lineage. H2bdtc proved to be an interesting chelator for rhenium or [99mTc]technetium. The right spleen uptake opened the opportunity to deepen the study of the molecule in this tissue and justifies future studies to identify the reason of heterogenous uptake in TM1M tumor uptake.

Scalable Manufacturing Method for Model Protein-Loaded PLGA Nanoparticles: Biocompatibility, Trafficking and Release Properties.

Background and Objectives: Drug delivery systems (DDSs) offer efficient treatment solutions to challenging diseases such as central nervous system (CNS) diseases by bypassing biological barriers such as the blood-brain barrier (BBB). Among DDSs, polymeric nanoparticles (NPs), particularly poly(lactic-co-glycolic acid) (PLGA) NPs, hold an outstanding position due to their biocompatible and biodegradable qualities. Despite their potential, the translation of PLGA NPs from laboratory-scale production to clinical applications remains a significant challenge. This study aims to address these limitations by developing scalable PLGA NPs and evaluating their potential biological applications. Methods: We prepared blank and model-protein-loaded (albumin-FITC and wheat germ agglutinin-488 (WGA-488)) fluorescent PLGA NPs using the traditional double-emulsion method combined with the micro-spray-reactor system, a novel approach that enables fine particle production enabling scale-up applications. We tested the biocompatibility of the NPs in living RPMI 2650 and neuroblastoma cell lines, as well as their trafficking and uptake. Release kinetics of the encapsulated proteins were investigated through confocal microscopy and in vitro release studies, providing insights into the stability and functionality of the released proteins. Results: The formulation demonstrated sustained and prolonged protein release profiles. Importantly, cellular uptake studies revealed that the NPs were not internalized. Furthermore, encapsulated WGA-488 protein retained its functional activity after release, validating the integrity of the encapsulation and release processes. Conclusions: The proof-of-concept study on NP manufacturing and an innovative drug trafficking and release approach can bring new perspectives on scalable preparations of PLGA NPs and their biological applications.

Modulation of Donor in Purely Organic Triplet Harvesting AIE-TADF Photosensitizer for Image-guided Photodynamic Therapy.

Image-guided photodynamic therapy is acknowledged as one of the most demonstrative therapeutic modalities for cancer treatment because of its high precision, non-invasiveness, and improved imaging ability. A series of purely organic photosensitizers denoted as BTMCz, BTMPTZ, and BTMPXZ, have been designed and synthesized and are found to exhibit both thermally activated delayed fluorescence and aggregation-induced emission simultaneously. Experimental and theoretical studies are combined to reveal that modulation of the donor of the photosensitizer enables distinct thermally activated delayed fluorescence via a second-order spin-orbit perturbation mechanism involving lowest singlet charge-transfer and higher-lying triplet locally excited states, respectively. Further, different donor strengths and unique aggregations (H-, J- and X-type packings) greatly influence their color-tunable up-converted luminescence and endow them with superb dispersibility in water. The confocal microscopy-based cellular uptake study confirms the successful internalization of the nano-probes, while BTMCz enables the generation of reactive oxygen species (singlet oxygen) under white-light irradiation, enabling the efficient killing of cancer cells.

SSTR2-Targeted Theranostics in Hepatocellular Carcinoma.

While the clinical use of radiolabeled somatostatin analogs is well established in neuroendocrine tumors, there is growing interest in expanding their application to other somatostatin receptor 2 (SSTR2)-expressing cancers. This study investigates the potential utility of SSTR2-targeted theranostics in hepatocellular carcinoma (HCC). SSTR2 expression in HCC cell lines and clinical samples was evaluated using qRT-PCR, Western blot analysis, and a public dataset. 67Ga-DOTATATE uptake was measured, 177Lu-DOTATATE cytotoxicity was assessed, and 68Ga-DOTATATE tumor targeting was evaluated in HCC animal models and a patient via PET/CT imaging. SSTR2 expression was confirmed in HCC cell lines and clinical samples. Radioligand uptake studies demonstrated SSTR2-mediated 67Ga-DOTATATE uptake. 177Lu-DOTATATE treatment reduced cell proliferation and enhanced the anti-tumor efficacy of the multikinase inhibitor sorafenib. 68Ga-DOTATATE PET/CT scans successfully identified tumors in HCC animal models and spinal metastases in a patient with HCC. These findings provide evidence that SSTR2-based theranostics could have significant implications for the detection and treatment of HCC.

Impact of the hydrophilic-lipophilic balance of free-base and Zn(II) tricationic pyridiniumporphyrins and irradiation wavelength in PDT against the melanoma cell lines.

The amphiphilic and asymmetric structure of porphyrins, when used as photosensitizers (PSs) for photodynamic therapy (PDT), has been shown through numerous previous studies to be a very important property that facilitates their entry into cells, which improves their efficiency in PDT. In this work, two groups of cationic AB3 pyridiniumporphyrins, free-base and chelated with Zn(II), both substituted with alkyl chains of various lengths, were studied in PDT on melanoma cell lines. The aim was to investigate the impact of hydrophilic-lipophilic balance and Zn(II) chelation, and the importance of matching the irradiation wavelength to the optical properties of the PS on in vitro PDT efficiency. Therefore, spectroscopic studies, singlet oxygen production and lipophilicity as well as cellular uptake, localization and cytotoxicity studies of the two series of porphyrins were performed. In both series of porphyrins, the longest alkyl chain (17C-atoms long) enables the greatest internalization of the PS. Chelation with Zn(II) resulted in better physicochemical properties, but slower cellular internalization. As expected, free-base porphyrins were more PDT efficient than their Zn(II) complexes after 30-min photoactivation by low-fluence (2mW/cm2) red light (643nm). However the use of orange light (606nm) with the same fluence rate was more suitable for Zn(II) porphyrins and resulted in similar overall toxicity to their free-base analogues with similar lipophilicity. Although the highest phototoxicity was achieved with the PSs carrying the longest alkyl chain, TMPyP3-C13H27 and Zn(II)-TMPyP3-C13H27 proved to be the most promising candidates for use in PDT as they exhibit high phototoxicity, but also greater selectivity towards melanoma cell lines (MeWo and A375) compared to fibroblasts (HDF).

PEGylated solid lipid nanoparticles for the intranasal delivery of combination antiretroviral therapy composed of Atazanavir and Elvitegravir to treat NeuroAIDS.

Intranasal drug administration offers a promising strategy for delivering combination antiretroviral therapy (cART) directly to the central nervous system to treat NeuroAIDS, leveraging the nose-to-brain route to bypass the blood-brain barrier. However, challenges such as enzymatic degradation in the nasal mucosa, low permeability, and mucociliary clearance within the nasal cavity must first be addressed to make this route feasible. To overcome these barriers, this study developed solid lipid nanoparticles (SLNs) with varying PEGylation levels (0%, 5%, 10%, and 15% w/w of PEGylated lipid), co-encapsulated with Elvitegravir (EVG) and Atazanavir (ATZ) as an integrase and protease inhibitor, respectively. Pre-formulation studies confirmed the compatibility of the drugs with the excipients. Characterization showed that PEGylation reduces SLN size by approximately up to 12% while maintaining monodispersity and a high encapsulation efficiency of over 99% for both EVG and ATZ in their amorphous forms. Incubation of the formulations in artificial nasal mucus revealed that increased PEGylation consistently reduces nanoparticle aggregation and mean aggregate size, suggesting improved SLN stability in the mucus. Importantly, higher PEGylation levels significantly enhanced model drug permeability across the nasal mucus barrier by up to 10-fold. Lastly, cellular uptake studies using the RPMI 2650 nasal epithelial cell line indicated that PEGylation does not reduce nanoparticle uptake rates. These findings highlight the potential of PEGylated SLNs as an effective vehicle for enhancing the intranasal delivery of cART to treat NeuroAIDS. However, further in vivo studies are needed to confirm the brain targeting potential of this formulation.

Enhanced luminescence and stability of TFMDSA nanoparticles via polymer-induced aggregation for bioimaging.

In recent years, fluorescence imaging has occupied a very important position in the life science and biomedical fields. However, achieving nanomaterials for bioimaging with both high fluorescence quantum efficiency and high stability remains a significant challenge. Herein, we synthesized mPEG5K-PCL10K@TFMDSA and mPEG5K-PLLA10K@TFMDSA nanoparticles using polymer-induced aggregation. This method significantly enhanced the luminescence efficiency of TFMDSA nanoparticles in solution, attributed to improved intermolecular interactions and restricted molecular vibrations. The resulting nanoparticles exhibited exceptional optical stability over a period of seven days and demonstrated low cytotoxicity towards HeLa cells, making them highly suitable for bioimaging applications. Cellular uptake studies indicated that these nanoparticles were more efficiently internalized by HeLa cells compared to their amorphous counterparts, likely due to their unique square morphology. Our findings highlight the potential of polymer-induced aggregation in enhancing the optical properties and stability of TFMDSA nanomaterials, suggesting their promise as biofluorescent probes for cancer diagnosis and other biomedical applications.

Curcumin encapsulation in self-assembled nanoparticles based on amphiphilic stearic acid-grafted inulin: Preparation, characterization, and functional evaluation.

The clinical application of curcumin (CUR) is restricted by its low solubility, instability, and poor bioavailability. To overcome these limitations, we developed a novel stearic acid-grafted inulin-based nano-delivery system for CUR encapsulation. The structure of stearoyl inulin (SA-IN) was characterized using Fourier-transform infrared spectroscopy, hydrogen nuclear magnetic resonance, thermogravimetric analysis, and contact angle measurements. CUR-loaded SA-IN nanoparticles (CUR@SA-IN NPs) demonstrated a high encapsulation efficiency of 91.59 % ± 3.26 %, nanoscale dispersion, and an average particle size of 190.6 ± 11.2 nm. The CUR@SA-IN NPs exhibited excellent stability and sustained-release properties. Compared with free CUR, the minimum inhibitory concentration of CUR@SA-IN NPs against Escherichia coli and Staphylococcus aureus decreased by 1.5- and 1.6-fold, respectively. The antioxidant activity increased by 2.34-fold with CUR@SA-IN NPs compared with free CUR. Also, the NPs showed superior efficacy in suppressing the expression of inflammatory cytokines and inhibiting cancer cell proliferation. The cellular uptake studies confirmed enhanced CUR absorption from the NPs compared with free CUR. The CUR@SA-IN NPs exhibited good biocompatibility. These findings highlighted the potential of amphiphilic SA-IN as an effective delivery vector for hydrophobic bioactive compounds, thereby offering a promising approach for developing efficient nanoparticle-based delivery systems.

Engineering of redox-triggered polymeric lipid hybrid nanocarriers for selective drug delivery to cancer cells.

Tunable redox-sensitive polymeric-lipid hybrid nanocarriers (RS-PLHNCs) were fabricated using homogenization and nanoprecipitation methods. These nanocarriers were composed of novel redox-cholesterol with disulfide linkages and synthesized by conjugating cholesterol with dithiodipropionic acid via esterification. Berberine (BBR) was loaded into the fabricated nanocarriers to investigate the selective uptake of BBR by cancer cells as well as its release and enhanced cytotoxicity. The optimized BBR nanocarriers BBR NP-17 and -18 exhibited a spherical shape and uniform distribution, with a particle size of 124.7 ± 1.2 nm and 185.2 ± 1.6 nm and a zeta potential of -5.9 ± 2.5 mV and -20.3 ± 1.1 mV, respectively. These NCs released >80% BBR in a simulated intracellular tumor microenvironment (TME), while only 30%-45% was released under normal physiological conditions. The accelerated drug release in the TME was due to disulfide bond cleavage and ester bond hydrolysis in the presence of GSH and acidic pH, whereas under normal conditions, the NCs remained stable/undissociated. Cellular uptake studies confirmed enhanced BBR uptake in GSH-rich cancer cells (H1975) compared with normal cells (BEAS-2B and HEK293A). Following uptake, compared with the free form of the drug, the optimized nanocarriers displayed significant selective cytotoxicity and apoptosis in cancer cells by notably downregulating anti-oxidant (NFE2L2, HO-1, NQO1, and TXRND1) and anti-apoptotic (MCL-1) genes while upregulating pro-apoptotic genes (PUMA and NOXA). This resulted in increased oxidative stress, thereby inducing selective apoptosis in the GSH-rich lung cancer cells. These results suggest that the synthesized novel NCs hold great potential for specifically delivering drugs to cancer cells (with a reduced environment) while sparing normal cells, thus ensuring safe and efficient cancer therapy.

Hyaluronic Acid-Based Hybrid Nanoparticles as Promising Carriers for the Intranasal Administration of Dimethyl Fumarate.

Dimethyl fumarate (DMF), the first-line oral therapy for relapsing-remitting multiple sclerosis, is rapidly metabolized into monomethyl fumarate. The DMF oral administration provokes gastrointestinal discomfort causing treatment withdrawal. The present study aimed to develop an innovative formulation for DMF nasal administration. Lipid-polymer hybrid nanoparticles (LPNs) were developed to improve DMF stability, limiting gastrointestinal side effects and increasing brain bioavailability by nose-to-brain targeting application. DMF-loaded and unloaded LPNs with or without hyaluronic acid (HA) were prepared using the nanoprecipitation via magnetic/mechanical stirring technique. Particle morphology and surface properties were evaluated; drug content, viscosity, and mucoadhesion were determined. Physico-chemical stability of LPNs and DMF in the LPNs was also explored. In vitro DMF permeation experiments were performed utilizing the PermeaPad®. The cytotoxicity and cellular uptake studies were performed using RPMI 2650 and SK-N-BE2 cell lines. DMF nose-to-brain delivery was evaluated by intranasally administering DMF-loaded LPNs to rats. LPNs with average sizes of 120-250 nm and a negative zeta potential -17.3 to -43 mV were obtained, primarily influenced by the presence of HA. HA assured drug stability up to 60 days and promoting the in vitro permeation of DMF compared to the free-DMF. HA greatly improved the viscosity and mucoadhesive properties. LPNs with and without HA did not exhibit any cytotoxicity and showed a rapid cell uptake starting from 15min to 2h with a best internalization after 1h of treatment in both epithelial and neuronal cell lines. Nasal administration of DMF-loaded LPNs allowed to quantify up to about 12μg/mL of DMF in the rat cerebrospinal fluid. The results highlight the role of HA in improving LPNs properties and performance as carrier of DMF for nasal administration. In particular, LPNs appear able to enter neurons and monolayers of epithelial cells, allowing to promote the nose-to-brain DMF delivery.

Hazard assessment of nanomaterials: how to meet the requirements for (next generation) risk assessment

BackgroundHazard and risk assessment of nanomaterials (NMs) face challenges due to, among others, the numerous existing nanoforms, discordant data and conflicting results found in the literature, and specific challenges in the application of strategies such as grouping and read-across, emphasizing the need for New Approach Methodologies (NAMs) to support Next Generation Risk Assessment (NGRA). Here these challenges are addressed in a study that couples physico-chemical characterization with in vitro investigations and in silico similarity analyses for nine nanoforms, having different chemical composition, sizes, aggregation states and shapes. For cytotoxicity assessment, three methods (Alamar Blue, Colony Forming Efficiency, and Electric Cell-Substrate Impedance Sensing) are applied in a cross-validation approach to support NAMs implementation into NGRA.ResultsThe results highlight the role of physico-chemical properties in eliciting biological responses. Uptake studies reveal distinct cellular morphological changes. The cytotoxicity assessment shows varying responses among NMs, consistent among the three methods used, while only one nanoform gave a positive response in the genotoxicity assessment performed by comet assay.ConclusionsThe study highlights the potential of in silico models to effectively identify biologically active nanoforms based on their physico-chemical properties, reinforcing previous knowledge on the relevance of certain properties, such as aspect ratio. The potential of implementing in vitro methods into NGRA is underlined, cross-validating three cytotoxicity assessment methods, and showcasing their strength in terms of sensitivity and suitability for the testing of NMs.Graphical abstractCreated with BioRender.com (publication license obtained)

Synthesis of New Promising BNCT Agents Based on Conjugates of closo-Dodecaborate Anion and Aliphatic Diamino Acids

In this work, a series of boronated amidines based on the closo-dodecaborate anion and amino acids containing an amino group in the side chain of the general formula [B12H11NHC(NH(CH2)nCH(NH3)COOH)CH3], where n = 2, 3, 4, were synthesized. These derivatives contain conserved α-amino and α-carboxyl groups recognized by the binding centers of the large neutral amino acid transporter (LAT) system, which serves as a target for the clinically applied BNCT agent para-boronophenylalanine (BPA). The paper describes several approaches to synthesizing the target compounds, their acute toxicity studies, and tumor uptake studies in vivo in two tumor models. The promising compound [B12H11NHC(NH(CH2)2CH(NH3)COOH)CH3]*3H2O demonstrates low toxicity (LD50 in a range from 150 to 300 mg/kg) and excellent solubility and also shows selective uptake in experimental melanoma in laboratory mice (T/N ratio remained >3 up to 60 min post-injection, with a maximum T/N of 6.2 ± 2.8 at 45 min).