Reduced Side Effects Research Articles

Abstract 6447: Development of a fully automated immunoassay system of serum pembrolizumab to forecast therapeutic effectiveness

Abstract Recently, immuno-oncology treatments utilizing immune checkpoint inhibitors including pembrolizumab have attracted attention. Such treatments demonstrate potential effectiveness against cancers resistant to existing drugs, while offering the benefit of reduced side effects. Nonetheless, there is a specific group of patients who do not experience adequate therapeutic benefits from pembrolizumab. Despite the issue, the development of a comprehensive evaluation system to forecast therapeutic effectiveness is still in development. A strategy to predict therapeutic effectiveness involves evaluating the trough level or the steady state plasma-concentration of a drug in the blood. Ohuchi, M. et al. (Lung Cancer, 2022) demonstrated that patients with lower trough levels have shorter overall survival and progression-free survival durations, according to LC-MS/MS measurement of pembrolizumab. Furthermore, Wang, N. et al (Lung Cancer, 2023) demonstrated that the optimization of the dosage interval, grounded on the steady state plasma-concentration (target concentration, 15 µg/mL) of pembrolizumab, extended the duration of progression-free survival. Despite the potential of LC-MS/MS measurements in predicting patient prognosis and the benefits of PK-guided therapy, their implementation in clinical settings is hindered by the complex and time-consuming pre-treatment process for samples. Consequently, there is a growing need for a practical and clinically feasible drug concentration measurement system. Aiming to construct a clinically applicable pembrolizumab measurement system, we have been dedicated to developing a fully automated immunoassay system using anti-pembrolizumab antibodies. We developed two measurement systems of pembrolizumab in serum using two different antibodies targeting pembrolizumab. Using less than 10 µl serum, these systems can quantify the concentration of pembrolizumab ranging from 0.125 to 500 µg/mL within an hour, thereby enabling PK-guided therapy in clinical settings. After developing the measurement systems, we performed a correlation analysis with the LC-MS/MS system, using 70 samples with varying pembrolizumab concentrations ranging from 1.80 to 85.0 µg/mL. The results demonstrated that our measurement systems have a good correlation with LC-MS/MS measurement. Furthermore, the two immunometric measurement systems demonstrated a significant correlation with each other, substantiating the accuracy of the newly developed measurement system in determining the levels of pembrolizumab in the serum. The fully automated immunoassay systems we developed can measure pembrolizumab in serum within an hour, potentially paving the way for personalized treatment based on the serum concentration of pembrolizumab in clinical practice. Citation Format: Naotaka Noda, Takuya Takahashi, Shigehiro Yagishita, Masahiro Miura, Yusuke Takahashi, Yasuhiro Irino, Masatoshi Yanagida, Akinobu Hamada. Development of a fully automated immunoassay system of serum pembrolizumab to forecast therapeutic effectiveness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6447.

A Golgi Apparatus-Targeted Photothermal Agent with Protein Anchoring for Enhanced Cancer Photothermal Therapy.

The Golgi apparatus (GA) is central in shuttling proteins from the endoplasmic reticulum to different cellular areas. Therefore, targeting the GA to precisely destroy its proteins through local heat could induce apoptosis, offering a potential avenue for effective cancer therapy. Herein, a GA-targeted photothermal agent based on protein anchoring is introduced for enhanced photothermal therapy of tumor through the modification of near-infrared molecular dye with maleimide derivative and benzene sulfonamide. The photothermal agent can actively target the GA and covalently anchor to its sulfhydryl proteins, thereby increasing its retention within the GA. Under laser irradiation, the heat generated by the photothermal agent efficiently disrupts sulfhydryl proteins in situ, leading to GA dysfunction and ultimately inducing cell apoptosis. In vivo experiments demonstrate that the photothermal agent can precisely treat tumors and significantly reduce side effects.

Optimal control in reducing side effects during and after chemotherapy of solid tumors

In this paper, we discuss the effect of optimal chemotherapy doses on solid tumor during treatment and cancer recurrence after the end of treatment. First, an optimal control problem is designed. Then, by changing the control range, the objective function, and adding a control to the previous problem, three new optimal control problems are constructed. We pursue two critical ideas for the success of the drug delivery system to the patient: minimizing the normalized global population density of cancer cells during treatment and at the end of treatment and minimizing side effects during treatment. The optimal control problem is considered with two linear and quadratic objective functions. These models are solved by a well‐established numerical method with high accuracy and a low discretization cost. The numerical results have confirmed that reducing the total dose of the cytotoxic drugs during the treatment period leads to a rapid recurrence of cancer shortly after the end of the treatment period.

Circadian Rhythms of the Blood-Brain Barrier and Drug Delivery.

The blood-brain barrier (BBB) is a critical interface separating the central nervous system from the peripheral circulation, ensuring brain homeostasis and function. Recent research has unveiled a profound connection between the BBB and circadian rhythms, the endogenous oscillations synchronizing biological processes with the 24-hour light-dark cycle. This review explores the significance of circadian rhythms in the context of BBB functions, with an emphasis on substrate passage through the BBB. Our discussion includes efflux transporters and the molecular timing mechanisms that regulate their activities. A significant focus of this review is the potential implications of chronotherapy, leveraging our knowledge of circadian rhythms for improving drug delivery to the brain. Understanding the temporal changes in BBB can lead to optimized timing of drug administration, to enhance therapeutic efficacy for neurological disorders while reducing side effects. By elucidating the interplay between circadian rhythms and drug transport across the BBB, this review offers insights into innovative therapeutic interventions.

Growth differentiation factor GDF11 as a potential target for the treatment of age-related diseases

The article presents a review of literary sources dedicated to the physiological role and functions of certain proteins of the TGFβ superfamily, specifically GDF11 and GDF8, as well as their place in the pathogenesis of several diseases whose risk increases with age. Possible therapeutic applications of these proteins are described. It is shown that the role of GDF11 in the pathogenesis of the described diseases is ambiguous. GDF11 is a previously unrecognized regulator of bone remodeling, prevents myocardial hypertrophy, and improves the condition of animals with experimental diabetes or neurodegeneration. The anti-proliferative action of GDF11 is also observed in many oncological diseases. However, GDF11 may have a negative impact on the metabolism of muscle and bone tissue, which may limit its use in certain conditions. Due to differences in the expression and function of GDF11 in cardiac, nervous, muscular, and other tissues, its divergent actions, and the narrow therapeutic range of recombinant GDF11, further research is needed to determine the optimal range of indications and limitations, dosages, and methods to reduce side effects.

Chitosan in cancer therapy: a dual role as a therapeutic agent and drug delivery system.

Although chemotherapy is still the most preferred treatment for cancer, most chemotherapeutic agents target both cancer cells and healthy cells and cause serious sideeffects due to high toxicity. Improved drug delivery systems (DDSs), which enhance the efficacy of current chemotherapeutic drugs while reducing their toxicity, offer potential solutions to these challenges. Chitosan (CS) and its derivatives are biopolymers with biodegradable, biocompatible, and low-toxicity properties, and their structure allows for convenient chemical and mechanical modifications. In its role as a therapeutic agent, CS can impede the proliferation of tumor cells through the inhibition of angiogenesis and metastasis, as well as by triggering apoptosis. CS and its derivatives are also frequently preferred as DDSs due to their properties such as high drug-carrying capacity, polycationic structure, long-term circulation, and direct targeting of cancer cells. Various therapeutic agents linked to CS and its derivatives demonstrate potent anticancer effects with advantages such as reduced side effects compared to the original drugs, owing to factors like targeted distribution within cancer tissues and sustained release. This review emphasizes the utilization of CS and its derivatives, both as therapeutic agents and as carriers for established chemotherapeutic drugs.

The Effect of Induction Therapy on Antibody-Mediated Rejection in Kidney Transplantation: A Network Meta-Analysis Using Recent Data

Various induction regimens are available for kidney transplantation (KT); however, which is superior remains unclear. Moreover, although the induction regimens are effective and important for reducing side effects, their respective relationships with antibody-mediated rejection (AMR) after transplantation remain unclear. Therefore, this study aimed to elucidate the most effective induction regimen for AMR reduction through network analysis. We performed a comprehensive search of databases, including basiliximab, alemtuzumab, antithymocyte globulin (ATG), and daclizumab as induction regimens for KT from inception to September 1, 2022. Using a network meta-analysis, we investigated the priorities of 5 induction regimens for patient survival, graft failure, and graft rejection after ABO-incompatible KT. In total, 25 studies comprising 1768 people were included in this network meta-analysis. The primary outcome was the AMR rate of other induction regimens compared with that of basiliximab, whereas the secondary outcomes were heart failure, stroke, hospitalization, peripheral artery disease, myocardial infarction, anemia, leukopenia, herpes zoster, or adverse events. Notably, ATG reduced the AMR rate by 59% (odds ratio, 0.41; 95% credible interval, 0.20-0.90), whereas the other drugs did not show statistical significance. Furthermore, secondary outcomes did not significantly differ between the induction regimens. ATG is widely used in KT induction regimens. Our results showed that ATG reduced the risk of AMR in KT recipients when compared with other induction drugs; therefore, it appears to be an efficient choice of induction regimen to reduce AMR after KT.

Identifying microRNAs associated with tumor immunotherapy response using an interpretable machine learning model

Predicting clinical responses to tumor immunotherapy is essential to reduce side effects and the potential for sustained clinical responses. Nevertheless, preselecting patients who are likely to respond to such treatments remains highly challenging. Here, we explored the potential of microRNAs (miRNAs) as predictors of immune checkpoint blockade responses using a machine learning approach. First, we constructed random forest models to predict the response to tumor ICB therapy using miRNA expression profiles across 19 cancer types. The contribution of individual miRNAs to each prediction process was determined by employing SHapley Additive exPlanations (SHAP) for model interpretation. Remarkably, the predictive performance achieved by using a small number of miRNAs with high feature importance was similar to that achieved by using the entire miRNA set. Additionally, the genes targeted by these miRNAs were closely associated with tumor- and immune-related pathways. In conclusion, this study demonstrates the potential of miRNA expression data for assessing tumor immunotherapy responses. Furthermore, we confirmed the potential of informative miRNAs as biomarkers for the prediction of immunotherapy response, which will advance our understanding of tumor immunotherapy mechanisms.

Effectiveness of combined local therapy with antibiotics and fibrin vs. vacuum-assisted wound therapy in soft tissue infections: a retrospective study.

Soft tissue infections can be severe and life-threatening. Their treatment consists currently in radical surgical wound debridement and combined systemic antimicrobial therapy. Different side effects are possible. Local antibiotic therapy represents a new approach to reduce side effects and improve healing. The aim of this study is to assess the effectiveness of the local sprayed use of antibiotics with fibrin sealing compared with negative pressure wound therapy as an established treatment of soft-tissue infections. In this retrospective study, patients with soft tissue infections who underwent surgical treatment were analysed. One group consists of patients, who received local fibrin-antibiotic spray (FAS) (n = 62). Patients treated by vacuum-assisted wound therapy (VAWT) as the established treatment were the control group (n = 57). Main outcomes were differences in the success of healing, the duration until healing and the number of needed operations. Clinical healing could be achieved for 55 patients (98.21%) in the FAS group vs. 47 patients (92.16%) in the VAWT group (p = 0.19). Time to require this was 10.65 ± 10.38days in the FAS group and 22.85 ± 14.02days in the VAWT group (p < 0.001). In the FAS group, patients underwent an average of 1.44 ± 0.72 vs.3.46 ± 1.66 operations in the VAWT group (p < 0.001). Compared to vacuum-assisted wound therapy in soft tissue infections, local fibrin-antibiotic spray shows faster clinical healing and less needed operations. Leading to shorter hospital stays and more satisfied patients. The combination of sprayed fibrin and antibiotics can be seen as a promising and effective method.

A Deep Learning-Based Approach for Prediction of Vancomycin Treatment Monitoring: Retrospective Study Among Patients With Critical Illness.

Vancomycin pharmacokinetics are highly variable in patients with critical illnesses, and clinicians commonly use population pharmacokinetic (PPK) models based on a Bayesian approach to dose. However, these models are population-dependent, may only sometimes meet the needs of individual patients, and are only used by experienced clinicians as a reference for making treatment decisions. To assist real-world clinicians, we developed a deep learning-based decision-making system that predicts vancomycin therapeutic drug monitoring (TDM) levels in patients in intensive care unit. This study aimed to establish joint multilayer perceptron (JointMLP), a new deep-learning model for predicting vancomycin TDM levels, and compare its performance with the PPK models, extreme gradient boosting (XGBoost), and TabNet. We used a 977-case data set split into training and testing groups in a 9:1 ratio. We performed external validation of the model using 1429 cases from Kangwon National University Hospital and 2394 cases from the Medical Information Mart for Intensive Care-IV (MIMIC-IV). In addition, we performed 10-fold cross-validation on the internal training data set and calculated the 95% CIs using the metric. Finally, we evaluated the generalization ability of the JointMLP model using the MIMIC-IV data set. Our JointMLP model outperformed other models in predicting vancomycin TDM levels in internal and external data sets. Compared to PPK, the JointMLP model improved predictive power by up to 31% (mean absolute error [MAE] 6.68 vs 5.11) on the internal data set and 81% (MAE 11.87 vs 6.56) on the external data set. In addition, the JointMLP model significantly outperforms XGBoost and TabNet, with a 13% (MAE 5.75 vs 5.11) and 14% (MAE 5.85 vs 5.11) improvement in predictive accuracy on the inner data set, respectively. On both the internal and external data sets, our JointMLP model performed well compared to XGBoost and TabNet, achieving prediction accuracy improvements of 34% and 14%, respectively. Additionally, our JointMLP model showed higher robustness to outlier data than the other models, as evidenced by its higher root mean squared error performance across all data sets. The mean errors and variances of the JointMLP model were close to zero and smaller than those of the PPK model in internal and external data sets. Our JointMLP approach can help optimize treatment outcomes in patients with critical illnesses in an intensive care unit setting, reducing side effects associated with suboptimal vancomycin administration. These include increased risk of bacterial resistance, extended hospital stays, and increased health care costs. In addition, the superior performance of our model compared to existing models highlights its potential to help real-world clinicians.

Switching from immediate- to extended-release cysteamine in patients with nephropathic cystinosis: from clinical trials to clinical practice.

The purpose of this study is to evaluate the effectiveness and safety of switching from immediate-release (IR) to extended-release (ER) cysteamine in patients with nephropathic cystinosis (NC) in Spain. We conducted an observational, retrospective, multicentre study in NC patients who received IR cysteamine for at least 12months, switched to ER cysteamine, and received it for at least 6months before inclusion. Data were collected from nine patients (four children, five adults) 36months before and after the switch. Despite the highly selected population, an improvement in growth, particularly in children and a significant reduction in hospitalization days was observed. A decrease in halitosis, body odour and gastrointestinal effects was reported in most of the patients who suffered before the switch, and the use of proton pump inhibitors (PPIs) decreased in some patients. The estimated glomerular filtration rate (eGFR) remained stable in patients with preserved kidney function. No significant changes in white blood cell (WBC) cystine levels were observed after the switch. There was no significant difference in the cysteamine dose received. However, some patients were receiving <50% of the recommended dose of cysteamine before and after the switch and showed elevated levels of WBC cystine. Switching from IR to ER cysteamine in clinical practice reduces hospital stays, improves nutritional status and growth in paediatric patients and could help to enhance treatment tolerability by reducing side effects. Furthermore, the dosing of ER cysteamine could promote therapeutic compliance and positively affect the quality of life of the NC population.

Immunocytokines with target cell-restricted IL-15 activity for treatment of B cell malignancies.

Despite the advances in cancer treatment achieved, for example, by the CD20 antibody rituximab, an urgent medical need remains to optimize the capacity of such antibodies to induce antibody-dependent cellular cytotoxicity (ADCC) that determines therapeutic efficacy. The cytokine IL-15 stimulates proliferation, activation, and cytolytic capacity of NK cells, but broad clinical use is prevented by short half-life, poor accumulation at the tumor site, and severe toxicity due to unspecific immune activation. We here report modified immunocytokines consisting of Fc-optimized CD19 and CD20 antibodies fused to an IL-15 moiety comprising an L45E-E46K double mutation (MIC+ format). The E46K mutation abrogated binding to IL-15Rα, thereby enabling substitution of physiological trans-presentation by target binding and thus conditional IL-15Rβγ stimulation, whereas the L45E mutation optimized IL-15Rβγ agonism and producibility. In vitro analysis of NK activation, anti-leukemia reactivity, and toxicity using autologous and allogeneic B cells confirmed target-dependent function of MIC+ constructs. Compared with Fc-optimized CD19 and CD20 antibodies, MIC+ constructs mediated superior target cell killing and NK cell proliferation. Mouse models using luciferase-expressing human NALM-6 lymphoma cells, patient acute lymphoblastic leukemia (ALL) cells, and murine EL-4 lymphoma cells transduced with human CD19/CD20 as targets and human and murine NK cells as effectors, respectively, confirmed superior and target-dependent anti-leukemic activity. In summary, MIC+ constructs combine the benefits of Fc-optimized antibodies and IL-15 cytokine activity and mediate superior NK cell immunity with potentially reduced side effects. They thus constitute a promising new immunotherapeutic approach shown here for B cell malignancies.

Nano-Engineering Strategies for Tumor-Specific Therapy.

Nanodelivery systems (NDSs) provide promising prospects for decreasing drug doses, reducing side effects, and improving therapeutic effects. However, the bioapplications of NDSs are still compromised by their fast clearance, indiscriminate biodistribution, and limited tumor accumulation. Hence, engineering modification of NDSs aiming at promoting tumor-specific therapy and avoiding systemic toxicity is usually needed. An NDS integrating various functionalities, including flexible camouflage, specific biorecognition, and sensitive stimuli-responsiveness, into one sequence would be "smart" and highly effective. Herein, we systematically summarize the related principles, methods, and progress. At the end of the review, we predict the obstacles to precise nanoengineering and prospects for the future application of NDSs.

The Advancement and Obstacles in Improving the Stability of Nanocarriers for Precision Drug Delivery in the Field of Nanomedicine.

Nanocarriers have emerged as a promising class of nanoscale materials in the fields of drug delivery and biomedical applications. Their unique properties, such as high surface area- tovolume ratios and enhanced permeability and retention effects, enable targeted delivery of therapeutic agents to specific tissues or cells. However, the inherent instability of nanocarriers poses significant challenges to their successful application. This review highlights the importance of nanocarrier stability in biomedical applications and its impact on biocompatibility, targeted drug delivery, long shelf life, drug delivery performance, therapeutic efficacy, reduced side effects, prolonged circulation time, and targeted delivery. Enhancing nanocarrier stability requires careful design, engineering, and optimization of physical and chemical parameters. Various strategies and cutting-edge techniques employed to improve nanocarrier stability are explored, with a focus on their applications in drug delivery. By understanding the advances and challenges in nanocarrier stability, this review aims to contribute to the development and implementation of nanocarrier- based therapies in clinical settings, advancing the field of nanomedicine.

Advanced polymeric dendrimers in the management of Alzheimer's disease

AbstractIn recent years, advanced polymeric dendrimers have emerged as a promising avenue for AD management. Dendrimers are highly branched, three‐dimensional macromolecules with precise nanoarchitectures, making them ideal candidates for the delivery of therapeutic agents and diagnostic tools. Their unique properties, such as well‐defined size, multifunctionality, and controlled surface chemistry, allow for the design of targeted and highly efficient drug delivery systems and diagnostic probes. This review aims to provide a comprehensive overview of the potential applications of advanced polymeric dendrimers in the management of Alzheimer's disease. We explored their role in drug delivery, diagnostics, and other therapeutic interventions for AD. Additionally, we will delve into the challenges and opportunities in utilizing dendrimers as a key player in the battle against this devastating disease. The review will begin by discussing the current state of Alzheimer's disease, including its pathological features, clinical manifestations, and existing treatment strategies. It will then transition to an in‐depth examination of polymeric dendrimers, highlighting their structural characteristics, synthesis methods, and biocompatibility. Subsequently, the review will delve into the various ways in which dendrimers can be tailored for AD management, including drug encapsulation and delivery, enhanced blood–brain barrier penetration, and targeted diagnostic imaging. Furthermore, we explored the potential benefits of dendrimer‐based therapies, such as improved drug efficacy, reduced side effects, and enhanced patient compliance. The review will also address the challenges associated with dendrimer‐based approaches, including toxicity concerns, regulatory hurdles, and the need for rigorous clinical evaluation.

A novel paradigm examining the remote induction of nocebo effects online.

Side effect information is routinely communicated online. However, limited experimental evidence exists regarding the role of this information in generating maladaptive health outcomes (i.e., the nocebo effect). A novel paradigm was developed to remotely induce the nocebo effect via provision of online side effect information. Participants were given information regarding the positive effects of low frequency noise (LFN). A proportion were additionally warned of LFN-induced side effects. Study 1 (N = 423) investigated the source of information (listed vs. socially communicated side effects), while Study 2 (N = 560) investigated the role of positive and negative affects on attenuating and exacerbating the nocebo effect. Pooled analysis (N = 983) explored the effect of negative and positive expectations on both the nocebo effect and positive outcomes. Across studies, a significant nocebo effect in the warned side effects occurred after LFN exposure. This did not vary by source of information (Study 1) nor was it attenuated via the induction of positive affect (Study 2). Both studies demonstrated a reduction in positive outcomes among those receiving side effect information. Pooled analysis revealed that negative, but not positive, expectations mediated the nocebo effect. Positive and negative expectations interacted to predict positive outcomes. Holding negative expectations appeared to block positive health outcomes. Specifically, when negative expectations were above average, there was no effect of positive expectations on positive outcomes. Nocebo effects were remotely generated via minimal provision of side effect information. Pooled analysis revealed that future interventions should target positive and negative expectations to reduce side effects. (PsycInfo Database Record (c) 2024 APA, all rights reserved).

Development of an Intelligent Reactive Oxygen Species-Responsive Dual-Drug Delivery Nanoplatform for Enhanced Precise Therapy of Acute Lung Injury.

Acute lung injury (ALI) and its most severe form acute respiratory distress syndrome (ARDS) are commonly occurring devastating conditions that seriously threaten the respiratory system in critically ill patients. The current treatments improve oxygenation in patients with ALI/ARDS in the short term, but do not relieve the clinical mortality of patients with ARDS. To develop the novel drug delivery systems that can enhance the therapeutic efficacy of ALI/ARDS and impede adverse effects of drugs. Based on the key pathophysiological process of ARDS that is the disruption of the pulmonary endothelial barrier, bilirubin (Br) and atorvastatin (As) were encapsulated into an intelligent reactive oxygen species (ROS)-responsive nanocarrier DSPE-TK-PEG (DPTP) to form nanoparticles (BA@DPTP) in which the thioketal bonds could be triggered by high ROS levels in the ALI tissues. BA@DPTP could accumulate in inflammatory pulmonary sites through passive targeting strategy and intelligently release Br and As only in the inflammatory tissue via ROS-responsive bond, thereby enhancing the drugs effectiveness and markedly reducing side effects. BA@DPTP effectively inhibited NF-κB signaling and NLRP3/caspase-1/GSDMD-dependent pyroptosis in mouse pulmonary microvascular endothelial cells. BA@DPTP not only protected mice with lipopolysaccharide-induced ALI and retained the integrity of the pulmonary structure, but also reduced ALI-related mortality. This study combined existing drugs with nano-targeting strategies to develop a novel drug-targeting platform for the efficient treatment of ALI/ARDS.

Manganese as a Possible Anticancer Enhancer in Docetaxel Treatment of Prostate Cancer Cells.

Treatment of castration-resistant prostate cancer with docetaxel (DOC) often leads to resistance. In this study, we investigated whether manganese (Mn) has the potential to enhance treatment when combined with DOC. PC3 cells were exposed to DOC or Mn individually and in combination and cell viability was analysed in a dose- and time-dependent manner. Cell toxicity, cell cycle analysis and apoptotic protein levels were determined after 48 h of treatment. Mn in combination with different concentrations of DOC significantly enhanced the inhibitory effect on cell viability. Although the lowest dose did not cause mitotic arrest, DOC increased toxicity, which was reduced when combined with Mn. Protein analyses indicated that Mn compensates for the suppression of death receptors when combined with a low concentration of DOC and induced non-apoptotic pathways when combined with a higher concentration. Combining DOC and Mn may allow for a reduction in DOC concentration with the potential to reduce side effects.

Angiopep-2-Functionalized Lipid Cubosomes for Blood-Brain Barrier Crossing and Glioblastoma Treatment.

Glioblastoma multiforme (GBM) is an aggressive brain cancer with high malignancy and resistance to conventional treatments, resulting in a bleak prognosis. Nanoparticles offer a way to cross the blood-brain barrier (BBB) and deliver precise therapies to tumor sites with reduced side effects. In this study, we developed angiopep-2 (Ang2)-functionalized lipid cubosomes loaded with cisplatin (CDDP) and temozolomide (TMZ) for crossing the BBB and providing targeted glioblastoma therapy. Developed lipid cubosomes showed a particle size of around 300 nm and possessed an internal ordered inverse primitive cubic phase, a high conjugation efficiency of Ang2 to the particle surface, and an encapsulation efficiency of more than 70% of CDDP and TMZ. In vitro models, including BBB hCMEC/D3 cell tight monolayer, 3D BBB cell spheroid, and microfluidic BBB/GBM-on-a-chip models with cocultured BBB and glioblastoma cells, were employed to study the efficiency of the developed cubosomes to cross the BBB and showed that Ang2-functionalized cubosomes can penetrate the BBB more effectively. Furthermore, Ang2-functionalized cubosomes showed significantly higher uptake by U87 glioblastoma cells, with a 3-fold increase observed in the BBB/GBM-on-a-chip model as compared to that of the bare cubosomes. Additionally, the in vivo biodistribution showed that Ang2 modification could significantly enhance the brain accumulation of cubosomes in comparison to that of non-functionalized particles. Moreover, CDDP-loaded Ang2-functionalized cubosomes presented an enhanced toxic effect on U87 spheroids. These findings suggest that the developed Ang2-cubosomes are prospective for improved BBB crossing and enhanced delivery of therapeutics to glioblastoma and are worth pursuing further as a potential application of nanomedicine for GBM treatment.

Metabolic engineering of Streptomyces peucetius for biosynthesis of N,N-dimethylated anthracyclines.

Introduction: Daunorubicin and doxorubicin, two anthracycline polyketides produced by S. peucetius, are potent anticancer agents that are widely used in chemotherapy, despite severe side effects. Recent advances have highlighted the potential of producing improved derivatives with reduced side effects by incorporating l-rhodosamine, the N,N-dimethyl analogue of the native amino sugar moiety. Method: In this study, we aimed to produce N,N-dimethylated anthracyclines by engineering the doxorubicin biosynthetic pathway in the industrial Streptomyces peucetius strain G001. To achieve this, we introduced genes from the aclarubicin biosynthetic pathway encoding the sugar N-methyltransferases AclP and AknX2. Furthermore, the native gene for glycosyltransferase DnrS was replaced with genes encoding the aclarubicin glycosyltransferases AknS and AknT. Additionally, the gene for methylesterase RdmC from the rhodomycin biosynthetic pathway was introduced. Results: A new host was engineered successfully, whereby genes from the aclarubicin pathway were introduced and expressed. LC-MS/MS analysis of the engineered strains showed that dimethylated sugars were efficiently produced, and that these were incorporated ino the anthracycline biosynthetic pathway to produce the novel dimethylated anthracycline N,N-dimethyldaunorubicin. Further downstream tailoring steps catalysed by the cytochrome P450 monooxygenase DoxA exhibited limited efficacy with N,N-dimethylated substrates. This resulted in only low production levels of N,N-dimethyldaunorubicin and no N,N-dimethyldoxorubicin, most likely due to the low affinity of DoxA for dimethylated substrates. Discussion: S. peucetius G001 was engineered such as to produce N,N-dimethylated sugars, which were incorporated into the biosynthetic pathway. This allowed the successful production of N,N-dimethyldaunorubicin, an anticancer drug with reduced cytotoxicity. DoxA is the key enzyme that determines the efficiency of the biosynthesis of N,N-dimethylated anthracyclines, and engineering of this enzyme will be a major step forwards towards the efficient production of more N,N-dimethylated anthracyclines, including N,N-dimethyldoxorubicin. This study provides valuable insights into the biosynthesis of clinically relevant daunorubicin derivatives, highlighting the importance of combinatorial biosynthesis.