A dietary commensal microbe enhances antitumor immunity by activating tumor macrophages to sequester iron.

Innate immune cells generate a multifaceted antitumor immune response, including the conservation of essential nutrients such as iron. These cells can be modulated by commensal bacteria; however, identifying and understanding how this occurs is a challenge. Here we show that the food commensal Lactiplantibacillus plantarum IMB19 augments antitumor immunity in syngeneic and xenograft mouse tumor models. Its capsular heteropolysaccharide is the major effector molecule, functioning as a ligand for TLR2. In a two-pronged manner, it skews tumor-associated macrophages to a classically active phenotype, leading to generation of a sustained CD8+ T cell response, and triggers macrophage 'nutritional immunity' to deploy the high-affinity iron transporter lipocalin-2 for capturing and sequestering iron in the tumor microenvironment. This process induces a cycle of tumor cell death, epitope expansion and subsequent tumor clearance. Together these data indicate that food commensals might be identified and developed into 'oncobiotics' for a multi-layered approach to cancer therapy.

Ultrafast structural changes within a photosynthetic reaction centre.

Photosynthetic reaction centres harvest the energy content of sunlight by transporting electrons across an energy-transducing biological membrane. Here we use time-resolved serial femtosecond crystallography1 using an X-ray free-electron laser2 to observe light-induced structural changes in the photosynthetic reaction centre of Blastochloris viridis on a timescale of picoseconds. Structural perturbations first occur at the special pair of chlorophyll molecules of the photosynthetic reaction centre that are photo-oxidized by light. Electron transfer to the menaquinone acceptor on the opposite side of the membrane induces a movement of this cofactor together with lower amplitude protein rearrangements. These observations reveal how proteins use conformational dynamics to stabilize the charge-separation steps of electron-transfer reactions.

Role of riboflavin deficiency in malaria pathophysiology.

The emergence of resistance against antimalarials and insecticides poses a significant threat to malaria elimination strategies. It is crucial to explore potential risk factors for malaria to identify new targets and alternative therapies. Malnutrition is a well-established risk factor for malaria. Deficiencies of micronutrients such as vitamin A, zinc, iron, folic acid, and phenotypic measures of malnutrition, such as stunting and wasting, have been studied extensively in the context of malaria. Vitamin B2, also known as riboflavin, is a micronutrient involved in maintaining cellular homeostasis. Riboflavin deficiency has been shown to have an inverse correlation with malarial parasitaemia. This article reviews the role of riboflavin in maintaining redox homeostasis and probes how riboflavin deficiency could alter malaria pathogenesis by disrupting the balance between oxidants and antioxidants. Though riboflavin analogues have been explored as antimalarials, new in vivo and patient-based research is required to target riboflavin-associated pathways for antimalarial therapy.

Theranostic Fluorescent Probes.

The ability to gain spatiotemporal information, and in some cases achieve spatiotemporal control, in the context of drug delivery makes theranostic fluorescent probes an attractive and intensely investigated research topic. This interest is reflected in the steep rise in publications on the topic that have appeared over the past decade. Theranostic fluorescent probes, in their various incarnations, generally comprise a fluorophore linked to a masked drug, in which the drug is released as the result of certain stimuli, with both intrinsic and extrinsic stimuli being reported. This release is then signaled by the emergence of a fluorescent signal. Importantly, the use of appropriate fluorophores has enabled not only this emerging fluorescence as a spatiotemporal marker for drug delivery but also has provided modalities useful in photodynamic, photothermal, and sonodynamic therapeutic applications. In this review we highlight recent work on theranostic fluorescent probes with a particular focus on probes that are activated in tumor microenvironments. We also summarize efforts to develop probes for other applications, such as neurodegenerative diseases and antibacterials. This review celebrates the diversity of designs reported to date, from discrete small-molecule systems to nanomaterials. Our aim is to provide insights into the potential clinical impact of this still-emerging research direction.

scAnno: a deconvolution strategy-based automatic cell type annotation tool for single-cell RNA-sequencing data sets.

Undoubtedly, single-cell RNA sequencing (scRNA-seq) has changed the research landscape by providing insights into heterogeneous, complex and rare cell populations. Given that more such data sets will become available in the near future, their accurate assessment with compatible and robust models for cell type annotation is a prerequisite. Considering this, herein, we developed scAnno (scRNA-seq data annotation), an automated annotation tool for scRNA-seq data sets primarily based on the single-cell cluster levels, using a joint deconvolution strategy and logistic regression. We explicitly constructed a reference profile for human (30 cell types and 50 human tissues) and a reference profile for mouse (26 cell types and 50 mouse tissues) to support this novel methodology (scAnno). scAnno offers a possibility to obtain genes with high expression and specificity in a given cell type as cell type-specific genes (marker genes) by combining co-expression genes with seed genes as a core. Of importance, scAnno can accurately identify cell type-specific genes based on cell type reference expression profiles without any prior information. Particularly, in the peripheral blood mononuclear cell data set, the marker genes identified by scAnno showed cell type-specific expression, and the majority of marker genes matched exactly with those included in the CellMarker database. Besides validating the flexibility and interpretability of scAnno in identifying marker genes, we also proved its superiority in cell type annotation over other cell type annotation tools (SingleR, scPred, CHETAH and scmap-cluster) through internal validation of data sets (average annotation accuracy: 99.05%) and cross-platform data sets (average annotation accuracy: 95.56%). Taken together, we established the first novel methodology that utilizes a deconvolution strategy for automated cell typing and is capable of being a significant application in broader scRNA-seq analysis. scAnno is available at https://github.com/liuhong-jia/scAnno.

Targeting prolyl-tRNA synthetase via a series of ATP-mimetics to accelerate drug discovery against toxoplasmosis.

The prolyl-tRNA synthetase (PRS) is a validated drug target for febrifugine and its synthetic analog halofuginone (HFG) against multiple apicomplexan parasites including Plasmodium falciparum and Toxoplasma gondii. Here, a novel ATP-mimetic centered on 1-(pyridin-4-yl) pyrrolidin-2-one (PPL) scaffold has been validated to bind to Toxoplasma gondii PRS and kill toxoplasma parasites. PPL series exhibited potent inhibition at the cellular (T. gondii parasites) and enzymatic (TgPRS) levels compared to the human counterparts. Cell-based chemical mutagenesis was employed to determine the mechanism of action via a forward genetic screen. Tg-resistant parasites were analyzed with wild-type strain by RNA-seq to identify mutations in the coding sequence conferring drug resistance by computational analysis of variants. DNA sequencing established two mutations, T477A and T592S, proximal to terminals of the PPL scaffold and not directly in the ATP, tRNA, or L-pro sites, as supported by the structural data from high-resolution crystal structures of drug-bound enzyme complexes. These data provide an avenue for structure-based activity enhancement of this chemical series as anti-infectives.

5-methylcytosine (m5C) RNA modification controls the innate immune response to virus infection by regulating type I interferons.

5-methylcytosine (m5C) is one of the most prevalent modifications of RNA, playing important roles in RNA metabolism, nuclear export, and translation. However, the potential role of RNA m5C methylation in innate immunity remains elusive. Here, we show that depletion of NSUN2, an m5C methyltransferase, significantly inhibits the replication and gene expression of a wide range of RNA and DNA viruses. Notably, we found that this antiviral effect is largely driven by an enhanced type I interferon (IFN) response. The antiviral signaling pathway is dependent on the cytosolic RNA sensor RIG-I but not MDA5. Transcriptome-wide mapping of m5C following NSUN2 depletion in human A549 cells revealed a marked reduction in the m5C methylation of several abundant noncoding RNAs (ncRNAs). However, m5C methylation of viral RNA was not noticeably altered by NSUN2 depletion. In NSUN2-depleted cells, the host RNA polymerase (Pol) III transcribed ncRNAs, in particular RPPH1 and 7SL RNAs, were substantially up-regulated, leading to an increase of unshielded 7SL RNA in cytoplasm, which served as a direct ligand for the RIG-I-mediated IFN response. In NSUN2-depleted cells, inhibition of Pol III transcription or silencing of RPPH1 and 7SL RNA dampened IFN signaling, partially rescuing viral replication and gene expression. Finally, depletion of NSUN2 in an ex vivo human lung model and a mouse model inhibits viral replication and reduces pathogenesis, which is accompanied by enhanced type I IFN responses. Collectively, our data demonstrate that RNA m5C methylation controls antiviral innate immunity through modulating the m5C methylome of ncRNAs and their expression.

Exploration of aminoacyl-tRNA synthetases from eukaryotic parasites for drug development.

Parasitic diseases result in considerable human morbidity and mortality. The continuous emergence and spread of new drug-resistant parasite strains is an obstacle to controlling and eliminating many parasitic diseases. Aminoacyl-tRNA synthetases (aaRSs) are ubiquitous enzymes essential for protein synthesis. The design and development of diverse small molecule, drug-like inhibitors against parasite-encoded and expressed aaRSs have validated this enzyme family as druggable. In this work, we have compiled the progress to date towards establishing the druggability of aaRSs in terms of their biochemical characterization, validation as targets, inhibitor development, and structural interpretation from parasites responsible for malaria (Plasmodium), lymphatic filariasis (Brugia,Wuchereria bancrofti), giardiasis (Giardia), toxoplasmosis (Toxoplasma gondii), leishmaniasis (Leishmania), cryptosporidiosis (Cryptosporidium), and trypanosomiasis (Trypanosoma). This work thus provides a robust framework for the systematic dissection of aaRSs from these pathogens and will facilitate the cross-usage of potential inhibitors to jump-start anti-parasite drug development.

Augmenting Cancer Therapy with a Supramolecular Immunogenic Cell Death Inducer: A Lysosome-Targeted NIR-Light-Activated Ruthenium(II) Metallacycle.

Though immunogenic cell death (ICD) has garnered significant attention in the realm of anticancer therapies, effectively stimulating strong immune responses with minimal side effects in deep-seated tumors remains challenging. Herein, we introduce a novel self-assembled near-infrared-light-activated ruthenium(II) metallacycle, Ru1105 (λem = 1105 nm), as a first example of a Ru(II) supramolecular ICD inducer. Ru1105 synergistically potentiates immunomodulatory responses and reduces adverse effects in deep-seated tumors through multiple regulated approaches, including NIR-light excitation, increased reactive oxygen species (ROS) generation, selective targeting of tumor cells, precision organelle localization, and improved tumor penetration/retention capabilities. Specifically, Ru1105 demonstrates excellent depth-activated ROS production (∼1 cm), strong resistance to diffusion, and anti-ROS quenching. Moreover, Ru1105 exhibits promising results in cellular uptake and ROS generation in cancer cells and multicellular tumor spheroids. Importantly, Ru1105 induces more efficient ICD in an ultralow dose (10 µM) compared to the conventional anticancer agent, oxaliplatin (300 µM). In vivo experiments further confirm Ru1105's potency as an ICD inducer, eliciting CD8+ T cell responses and depleting Foxp3+ T cells with minimal adverse effects. Our research lays the foundation for the design of secure and exceptionally potent metal-based ICD agents in immunotherapy.

Evolving insights into the improvement of adoptive T-cell immunotherapy through PD-1/PD-L1 blockade in the clinical spectrum of lung cancer.

Undeniably, cancer immunotherapies have expanded the spectrum of cancer treatment, however, some patients do not respond to immunotherapies. This scenario is no different for lung cancer, whose two main types, non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC), still pose a serious clinical challenge. Adoptive T-cell therapies (ATC), which primarily include cytokine-induced killer (CIK) cell therapy, chimeric antigen receptor T-cell (CAR T-cell) therapy and γδ-T-cell therapy, strengthen the patient's immune system in combating cancer. Combining ATC with immune checkpoint inhibitors (ICI) further enhances the effectiveness of this approach to eradicate cancer. With a particular emphasis on CIK cell therapy, which recently completed 30 years, we highlight the role of the PD-1/PD-L1 axis in NSCLC and SCLC. Besides, we provide insights into the potential synergies of PD-1/PD-L1 inhibitors with adoptive T-cell immunotherapy in reshaping the treatment paradigm for lung cancer.

Vadadustat in Patients with Anemia and Non-Dialysis-Dependent CKD.

BACKGROUND: Vadadustat is an oral hypoxia-inducible factor (HIF) prolyl hydroxylase inhibitor, a class of drugs that stabilize HIF and stimulate erythropoietin and red-cell production. METHODS: In two phase 3, randomized, open-label, active-controlled, noninferiority trials, we compared vadadustat with the erythropoiesis-stimulating agent (ESA) darbepoetin alfa in patients with non-dialysis-dependent chronic kidney disease (NDD-CKD) not previously treated with an ESA who had a hemoglobin concentration of less than 10 g per deciliter and in patients with ESA-treated NDD-CKD and a hemoglobin concentration of 8 to 11 g per deciliter (in the United States) or 9 to 12 g per deciliter (in other countries). The primary safety end point, assessed in a time-to-event analysis, was the first major adverse cardiovascular event (MACE; a composite of death from any cause, nonfatal myocardial infarction, or nonfatal stroke), pooled across the two trials. Secondary safety end points included expanded MACE (MACE plus hospitalization for either heart failure or a thromboembolic event). The primary and key secondary efficacy end points in each trial were the mean change in hemoglobin concentration from baseline during two evaluation periods: weeks 24 through 36 and weeks 40 through 52. RESULTS: A total of 1751 patients with ESA-untreated NDD-CKD and 1725 with ESA-treated NDD-CKD underwent randomization in the two trials. In the pooled analysis, in which 1739 patients received vadadustat and 1732 received darbepoetin alfa, the hazard ratio for MACE was 1.17 (95% confidence interval [CI], 1.01 to 1.36), which did not meet the prespecified noninferiority margin of 1.25. The mean between-group differences in the change in the hemoglobin concentration at weeks 24 through 36 were 0.05 g per deciliter (95% CI, -0.04 to 0.15) in the trial involving ESA-untreated patients and -0.01 g per deciliter (95% CI, -0.09 to 0.07) in the trial involving ESA-treated patients, which met the prespecified noninferiority margin of -0.75 g per deciliter. CONCLUSIONS: Vadadustat, as compared with darbepoetin alfa, met the prespecified noninferiority criterion for hematologic efficacy but not the prespecified noninferiority criterion for cardiovascular safety in patients with NDD-CKD. (Funded by Akebia Therapeutics and Otsuka Pharmaceutical; PRO2TECT ClinicalTrials.gov numbers, NCT02648347 and NCT02680574.).

Cytokine-induced killer (CIK) cells, successes and challenges: report on the first international conference dedicated to the clinical translation of this unique adoptive cell immunotherapy.

On August 30, 2023, experts from Germany and abroad met to discuss the successes and challenges of cytokine-induced killer cell (CIK) therapy, that recently celebrated its 30th anniversary providing treatment for cancer. This first virtual conference was hosted by CIO Bonn, a certified Comprehensive Cancer Center (CCC) funded by German Cancer Aid (DKH). In addition to keynote speakers involved in CIK cell clinical trials or optimized preclinical models to improve this adoptive cell immunotherapy, more than 100 attendees from around the world also participated in this event. Initiatives to establish the International Society of CIK Cells (ISCC) and a stronger CIK cell network guiding preclinical research and future clinical trials were also announced.

Utility of scores to predict alcohol use after liver transplant (lt): take them with a grain of salt.

BACKGROUND AIMS: The Sustained Alcohol use post-Liver Transplant (SALT) and the High-Risk Alcohol Relapse (HRAR) scores were developed to predict return to alcohol use after liver transplant (LT) for alcohol associated liver disease (ALD). METHODS: A retrospective analysis of deceased donor LT 10/2018 to 4/2022 was performed. All patients (pts) underwent careful pre-LT psychosocial evaluation. Data on alcohol use, substance abuse, prior rehabilitation, and legal issues were collected. Post-LT, all were encouraged to participate in rehabilitation programs and underwent interval phosphatidylethanol (PeTH) testing. Pts with ALD were stratified by < or > 6 month sobriety prior to listing. Those with <6 month were further stratified as acute alcoholic hepatitis (AH) by NIAAA criteria and non-AH. The primary outcome was utility of the SALT (<5 vs. ≥5) and HRAR (<3 vs. ≥3) scores to predict return to alcohol use (+PeTH) within 1 year after LT. RESULTS: Of the 365 LT, 86 had > 6 month sobriety and 85 had <6 month sobriety; 41 with AH and 44 non-AH. In those with AH, the mean time of abstinence to LT was 58 days, and 71% failed prior rehabilitation. Following LT, return to drinking was similar in the AH (24%) compared to <6M non-AH (15%) and >6M ALD (22%). Only 4% had returned to heavy drinking. The accuracy of both the SALT and HRAR scores to predict return to alcohol was low (accuracy 61-63%) with poor sensitivity (46% and 37%), specificity (67-68%), positive predictive value (22-26%) with moderate negative predictive value (NPV) (81-83%), respectively with higher NPVs (95%) in predicting return to heavy drinking. CONCLUSIONS: Both SALT and HRAR scores had good NPV in identifying patients at low risk for recidivism.

Dissecting the mechanisms of pathogenesis in cerebral malaria.

Cerebral malaria (CM) is one of the leading causes of death due to malaria. It is characterised by coma, presence of asexual parasites in blood smear, and absence of any other reason that can cause encephalopathy. The fatality rate for CM is high, and those who survive CM often experience long-term sequelae, including cognitive and motor dysfunctions. It is unclear how parasites sequestered in the lumen of endothelial cells of the blood-brain barrier (BBB), and localised breakdown of BBB can manifest gross physiological changes across the brain. The pathological changes associated with CM are mainly due to the dysregulation of inflammatory and coagulation pathways. Other factors like host and parasite genetics, transmission intensity, and the host's immune status are likely to play a role in the development and progression of CM. This work focuses on the pathological mechanisms underlying CM. Insights from humans, mice, and in vitro studies have been summarised to present a cohesive understanding of molecular mechanisms involved in CM pathology.

Correction: Current status of therapeutic monoclonal antibodies against SARS-CoV-2.

[This corrects the article DOI: 10.1371/journal.ppat.1009885.].

Correction: India can consider integration of three eliminable disease control programmes on malaria, lymphatic filariasis, and visceral leishmaniasis.

[This corrects the article DOI: 10.1371/journal.ppat.1009492.].

Double drugging of prolyl-tRNA synthetase provides a new paradigm for anti-infective drug development.

Toxoplasmosis is caused by Toxoplasma gondii and in immunocompromised patients it may lead to seizures, encephalitis or death. The conserved enzyme prolyl-tRNA synthetase (PRS) is a validated druggable target in Toxoplasma gondii but the traditional 'single target-single drug' approach has its caveats. Here, we describe two potent inhibitors namely halofuginone (HFG) and a novel ATP mimetic (L95) that bind to Toxoplasma gondii PRS simultaneously at different neighbouring sites to cover all three of the enzyme substrate subsites. HFG and L95 act as one triple-site inhibitor in tandem and form an unusual ternary complex wherein HFG occupies the 3'-end of tRNA and the L-proline (L-pro) binding sites while L95 occupies the ATP pocket. These inhibitors exhibit nanomolar IC50 and EC50 values independently, and when given together reveal an additive mode of action in parasite inhibition assays. This work validates a novel approach and lays a structural framework for further drug development based on simultaneous targeting of multiple pockets to inhibit druggable proteins.

Nuclear Localization Signal Enhances the Targeting and Therapeutic Efficacy of a Porphyrin-Based Molecular Cargo: A Systemic In Vitro and Ex Vivo Evaluation.

The objective of the present work was to evaluate the potential of a nuclear localization signal (NLS) toward facilitating intracellular delivery and enhancement in the therapeutic efficacy of the molecular cargo. Toward this, an in-house synthesized porphyrin derivative, namely, 5-carboxymethyelene-oxyphenyl-10,15,20-tris(4-methoxyphenyl) porphyrin (UTriMA), was utilized for conjugation with the NLS sequence [PKKKRKV]. The three compounds synthesized during the course of the present work, namely DOTA-Lys-NLS, DOTA-UTriMA-Lys-NLS, and DOTA-Lys-UTriMA, were evaluated for cellular toxicity in cancer cell lines (HT1080), wherein all exhibited minimal dark toxicity. However, during photocytotoxicity studies with DOTA-Lys-UTriMA and DOTA-UTriMA-Lys-NLS conjugates in the same cell line, the latter exhibited significantly higher light-dependent toxicity compared to the former. Furthermore, the photocytotoxicity for DOTA-UTriMA-Lys-NLS in a healthy cell line (WI26VA4) was found to be significantly lower than that observed in the cancer cells. Fluorescence cell imaging studies carried out in HT1080 cancer cells revealed intracellular accumulation for the NLS-conjugated porphyrin (DOTA-UTriMA-Lys-NLS), whereas unconjugated porphyrin (DOTA-Lys-UTriMA) failed to do so. To evaluate the radiotherapeutic effects of the synthesized conjugates, all three compounds were radiolabeled with 177Lu, a well-known therapeutic radionuclide with high radiochemical purity (>95%). During in vitro studies, the [177Lu]Lu-DOTA-UTriMA-Lys-NLS complex exhibited the highest cell binding as well as internalization among the three radiolabeled complexes. Biological distribution studies for the radiolabeled compounds were performed in a fibrosarcoma-bearing small animal model, wherein significantly higher accumulation and prolonged retention of [177Lu]Lu-DOTA-UTriMA-Lys-NLS (9.32 ± 1.27% IA/g at 24 h p.i.) in the tumorous lesion compared to [177Lu]Lu-UTriMA-Lys-DOTA (2.3 ± 0.13% IA/g at 24 h p.i.) and [177Lu]Lu-DOTA-Lys-NLS complexes (0.26 ± 0.17% IA/g at 24 h p.i.) were observed. The results of the biodistribution studies were further corroborated by recording serial SPECT-CT images of fibrosarcoma-bearing Swiss mice administered with [177Lu]Lu-DOTA-UTriMA-Lys-NLS at different time points. Tumor regression studies performed with [177Lu]Lu-DOTA-UTriMA-Lys-NLS in the same animal model with two different doses [250 µCi (9.25 MBq) and 500 µCi (18.5 MBq)] resulted in a significant reduction in tumor mass in the treated group of animals. The above results revealed a definite enhancement in the targeting ability of molecular cargo upon conjugation with NLS and hence indicated that this strategy may be helpful for the preparation of drug-NLS conjugates as multimodal agents.

Mapping malaria vectors and insecticide resistance in a high-endemic district of Haryana, India: implications for vector control strategies.

BACKGROUND: Achieving effective control and elimination of malaria in endemic regions necessitates a comprehensive understanding of local mosquito species responsible for malaria transmission and their susceptibility to insecticides. METHODS: The study was conducted in the highly malaria prone Ujina Primary Health Center of Nuh (Mewat) district of Haryana state of India. Monthly entomological surveys were carried out for adult mosquito collections via indoor resting collections, light trap collections, and pyrethrum spray collections. Larvae were also collected from different breeding sites prevalent in the region. Insecticide resistance bioassay, vector incrimination, blood meal analysis was done with the collected vector mosquitoes. RESULTS: A total of 34,974 adult Anopheles mosquitoes were caught during the survey period, out of which Anopheles subpictus was predominant (54.7%). Among vectors, Anopheles stephensi was predominant (15.5%) followed by Anopheles culicifacies (10.1%). The Human Blood Index (HBI) in the case of An. culicifacies and An. stephensi was 6.66 and 9.09, respectively. Vector incrimination results revealed Plasmodium vivax positivity rate of 1.6% for An. culicifacies. Both the vector species were found resistant to DDT, malathion and deltamethrin. CONCLUSION: The emergence of insecticide resistance in both vector species, compromises the effectiveness of commonly used public health insecticides. Consequently, the implementation of robust insecticide resistance management strategies becomes imperative. To effectively tackle the malaria transmission, a significant shift in vector control strategies is warranted, with careful consideration and adaptation to address specific challenges encountered in malaria elimination efforts.

Peptide-drug conjugate designated for targeted delivery to HER2-expressing cancer cells.

Targeted therapy of the highest globally incident breast cancer shall resolve the issue of off-target toxicity concurring with augmented killing of specific diseased cells. Thus, the goal of this study was to prepare a peptide-drug conjugate targeting elevated expression of HER2 receptors in breast cancer. Towards this, the rL-A9 peptide was conjugated with the chemotherapeutic drug doxorubicin (DOX) through a N-succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) linker. The synthesized peptide-drug conjugate, rL-A9-DOX, was characterized by mass spectrometry. Molecular docking studies, based on binding energy data, suggested a stronger interaction of rL-A9-DOX with the HER2 receptor in comparison to the unconjugated peptide, rL-A9. The cytotoxic effect of the rL-A9-DOX conjugate was observed to be higher in HER2-positive SKOV3 cells compared to HER2-negative MDA-MB-231 cells, indicating selective cell killing. Cellular internalization of the rL-A9-DOX conjugate was evident from the flow cytometry analysis, where a noticeable shift in mean fluorescent intensity (MFI) was observed for the conjugate compared to the control group. This data was further validated by confocal microscopy, where the fluorescent signal ascertained nuclear accumulation of rL-A9-DOX. The present studies highlight the promising potential of rL-A9-DOX for targeted delivery of the drug into a defined group of cancer cells.