Increased Drug Uptake Research Articles

Mannosylated PAMAM G2 dendrimers mediated rate programmed delivery of efavirenz target HIV viral latency at reservoirs

In this current research, we conceptualized a novel nanotechnology-enabled synthesis approach of targeting HIV-harboring tissues via second-generation (G2) polyamidoamine (PAMAM) mannosylated (MPG2) dendrimers for programmed delivery of anti-HIV drugs efavirenz (EFV) and ritonavir (RTV). Briefly, here mannose served purpose of ligand in this EFV and RTV-loaded PAMAM G2 dendrimers, synthesized by divergent techniques, denoted as MPG2ER. The developed nanocarriers were characterized by different analytical tools FTIR, NMR, zeta potential, particle size, and surface morphology. The results of confocal microscopy showed substantial alterations in the morphology of H9 cells, favored by relatively higher drug uptake through the MPG2ER. Interestingly, the drug uptake study and cytotoxicity assay of MPG2ER demonstrated that it showed no significant toxicity up to 12.5 µM. A typical flow cytometry histogram also revealed that MPG2ER efficiently internalized both drugs, with an increase in drug uptake of up to 81.2 %. It also enhanced the plasma pharmacokinetics of EFV, with Cmax7.68 μg/ml, AUC of 149.19 (μg/ml) * hr, and MRT of 26.87 hrs. Subsequently, tissue pharmacokinetics further evidence that MPG2ER accumulated more in distant Human immunodeficiency virus (HIV) reservoir tissues, such as the lymph nodes and spleen, but without exhibiting significant toxicity. Abovementioned compelling evidences strongly favored translational roles of MPG2 as a potential therapeutic strategy in the clinical eradication of HIV from viral reservoir tissue.

Strategies to enhance the response of liver cancer to pharmacological treatments.

In contrast to other types of cancers, there is no available efficient pharmacological treatment to improve the outcomes of patients suffering from major primary liver cancers, i.e., hepatocellular carcinoma and cholangiocarcinoma. This dismal situation is partly due to the existence in these tumors of many different and synergistic mechanisms of resistance, accounting for the lack of response of these patients, not only to classical chemotherapy but also to more modern pharmacological agents based on the inhibition of tyrosine kinase receptors (TKIs) and the stimulation of the immune response against the tumor using immune checkpoint inhibitors (ICIs). This review summarizes the efforts to develop strategies to overcome this severe limitation, including searching for novel drugs derived from synthetic, semisynthetic, or natural products with vectorial properties against therapeutic targets to increase drug uptake or reduce drug export from cancer cells. Besides, immunotherapy is a promising line of research that is already starting to be implemented in clinical practice. Although less successful than in other cancers, the foreseen future for this strategy in treating liver cancers is considerable. Similarly, the pharmacological inhibition of epigenetic targets is highly promising. Many novel "epidrugs," able to act on "writer," "reader," and "eraser" epigenetic players, are currently being evaluated in preclinical and clinical studies. Finally, gene therapy is a broad field of research in the fight against liver cancer chemoresistance, based on the impressive advances recently achieved in gene manipulation. In sum, although the present is still dismal, there is reason for hope in the non-too-distant future.

Shikonin-loaded PLGA nanoparticles: A promising strategy for psoriasis treatment

Psoriasis is an inflammation-based skin illness marked by aggravated proliferation of epidermal cells. Shikonin is a natural naphthoquinone obtained from Arnebiae radix. It exerts anti-inflammatory and immunosuppressive effects. However, the poor water solubility and low bioavailability of shikonin limit its application. In this study, shikosin-loaded PLGA nanoparticle hydrogel was prepared and used to deliver the drug to the epidermis of psoriasis mice through local administration. The results demonstrated that shikosin-loaded PLGA nanoparticles inhibited HaCaT cell multiplication, increased drug uptake, and induced apoptosis of HaCaT cells. Results from Western blotting assays indicated that shikosin down-regulated the protein expressions of p65 and p-p65. Furthermore, shikonin mitigated psoriasis and decreased the concentrations of inflammation-inducing cytokines, i.e., IL17A, IL-17F, IL-22, IL-1β, and TNF-α. Taken together, these results suggest that shikonin-PLGA nanoparticles loaded in hydrogel system possess promising therapeutic potential for psoriasis.

Novel method of measurement of in vitro drug uptake in OATP1B3 overexpressing cells in the presence of dextran.

In predictions about hepatic clearance (CLH), a number of studies explored the role of albumin and transporters in drug uptake by liver cells, challenging the traditional free-drug theory. It was proposed that liver uptake can occur for transporter substrate compounds not only from the drug's unbound form but also directly from the drug-albumin complex, aphenomenon known as uptake facilitated by albumin. In contrast to albumin, dextran does not exhibit binding properties for compounds. However, as a result of its inherent capacity for stabilization, it is widely used to mimic conditions within cells. The uptake of eight known substrates of the organic anion-transporting polypeptide 1B3 (OATP1B3) was assessed using a human embryonic kidney cell line (HEK293), which stably overexpresses this transporter. An inert polymer, dextran, was used to simulate cellular conditions, and the results were compared with experiments involving human plasma and human serum albumin (HSA). This study is the first to demonstrate that dextran increases compound uptake in cells with overexpression of the OATP1B3 transporter. Contrary to the common theory that highly protein-bound ligands interact with hepatocytes to increase drug uptake, the results indicate that dextran's interaction with test compounds does not significantly increase concentrations near the cell membrane surface. We evaluated the effect of dextran on the uptake of known substrates using OATP1B3 overexpressed in the HEK293 cell line, and we suggest that its impact on drug concentrations in liver cells may differ from the traditional role of plasma proteins and albumin.

A novel octa-arginine-modified injectable self-assembling peptide hydrogel for multidrug-resistant cancer therapy

Surgery combined with systemic chemotherapy is currently the main modality of cancer treatment. However, the development of cancer multidrug resistance and inevitable surgical residual lesions lead to high rates of cancer recurrence and mortality. Meanwhile, systemic drug toxic side effects and surgical bleeding complications seriously reduce the quality of life of patients. To avoid cancer multidrug resistance and comprehensively optimize cancer treatment, this work provides a novel injectable cell penetrating peptide octa-arginine (R8)-modified RADA16 (RR) self-assembling peptide nanofiber hydrogel as an anticancer drug carrier for multidrug-resistant cancer therapy. In RR hydrogel, highly compatible RADA16 functions as the β-sheet-based self-assembling backbone which tightly adheres to cancer tissue, prevents postoperative bleeding, and sustainedly releases anticancer drugs for long-term effects. The introduced R8 motifs endow RR hydrogel with the capacity to selectively kill cancer cells and reverse cancer multidrug resistance, which minimally impact normal tissue cells. Specifically, R8 motifs on the hydrogel scaffold selectively interfere with cancer cell membranes, increase drug uptake and penetration into cancer cells and tissues by enhancing multiple internalization pathways, and reduce drug efflux by inhibiting P-gp and BCRP multidrug-resistant transporters. Therefore, this study provides a multifunctional hydrogel material with strong potential for multidrug-resistant cancer clinical translation.

Supramolecular host-guest nanosystems for overcoming cancer drug resistance.

Cancer drug resistance has become one of the main challenges for the failure of chemotherapy, greatly limiting the selection and use of anticancer drugs and dashing the hopes of cancer patients. The emergence of supramolecular host-guest nanosystems has brought the field of supramolecular chemistry into the nanoworld, providing a potential solution to this challenge. Compared with conventional chemotherapeutic platforms, supramolecular host-guest nanosystems can reverse cancer drug resistance by increasing drug uptake, reducing drug efflux, activating drugs, and inhibiting DNA repair. Herein, we summarize the research progress of supramolecular host-guest nanosystems for overcoming cancer drug resistance and discuss the future research direction in this field. It is hoped that this review will provide more positive references for overcoming cancer drug resistance and promoting the development of supramolecular host-guest nanosystems.

DDEL-04. MODULATION OF THE BLOOD-TUMOR BARRIER FOR IMPROVED DRUG DELIVERY IN GLIOBLASTOMA

Abstract Effective drug delivery remains a major challenge in glioblastoma (GBM) treatment. Specialized intercellular endothelial cell tight junctions at the blood-brain and blood-tumor barriers (BBB/BTB) represent obstacles to therapeutic efficacy by limiting intratumoral drug uptake. To understand the factors at the BTB that limit drug accessibility we performed bioinformatics analysis of TCGA data which suggested enrichment of the tight junction protein VE-cadherin (CDH5). CDH5 expression at the BTB was confirmed by immunostaining of patient GBM specimens and spatial transcriptomics using the 10X Visium platform. We found that a broadly selective protein kinase inhibitor, 6-bromoindirubin-3’-acetoxime (BIA), potently down-regulated CDH5 in cultured human cerebrovascular endothelial cells (HCMEC/D3) at the transcriptional level and this correlated with decreased trans-endothelial electrical resistance (TEER) measurement. In vivo administration of BIA (20 mg/kg IP) to mice bearing intracranial GBM xenografts showed striking downregulation of CDH5 in tumor associated endothelium. Furthermore, increased BTB permeability was shown using fluorescent tracers as well as DCE-MRI, which demonstrated elevated Ktrans values 24 hours after BIA administration. Integrated phospho-proteomic and transcriptomic analysis of BIA treated HCMEC/D3 cells support a mechanism involving p38 MAP kinase regulation of Ets-family transcription factors that drive CDH5 expression. At least a 20-fold increase in intra-tumoral accumulation of cisplatin and temozolomide was observed using G9-pCDH GBM xenografts when administered 24 h after BIA treatment. No increased drug uptake was seen in healthy brain tissue, indicating specific targeting of tumor vasculature. Efficacy studies showed extended survival when using cisplatin in combination with BIA (median 27d, p=0.0016) when compared to cisplatin (18.5d), BIA (21.5d) and control (18d). Further efficacy studies are underway. These data therefore identify the GBM-associated vasculature as a target for small molecule inhibition of tight junction formation to enhance intratumoral drug uptake. Ongoing studies are investigating the potential of this approach with a range of agents.

Macrophage targeted iron oxide nanodecoys augment innate immunological and drug killings for more effective Mycobacterium Tuberculosis clearance

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb) infection, is still one of the top killers worldwide among infectious diseases. The escape of Mtb from immunological clearance and the low targeting effects of anti-TB drugs remain the substantial challenges for TB control. Iron is particularly required for Mtb growth but also toxic for Mtb in high dosages, which makes iron an ideal toxic decoy for the ‘iron-tropic’ Mtb. Here, a macrophage-targeted iron oxide nanoparticles (IONPs)-derived IONPs-PAA-PEG-MAN nanodecoy is designed to augment innate immunological and drug killings against intracellular Mtb. IONPs-PAA-PEG-MAN nanodecoy exhibits preferential uptake in macrophages to significantly increase drug uptake with sustained high drug contents in host cells. Moreover, it can serve as a specific nanodecoy for the ‘iron-tropic’ Mtb to realize the localization of Mtb contained phagosomes surrounding the drug encapsulated nanodecoys and co-localization of Mtb with the drug encapsulated nanodecoys in lysosomes, where the incorporated rifampicin (Rif) can be readily released under acidic lysosomal condition for enhanced Mtb killing. This drug encapsulated nanodecoy can also polarize Mtb infected macrophages into anti-mycobacterial M1 phenotype and enhance M1 macrophage associated pro-inflammatory cytokine (TNF-α) production to trigger innate immunological responses against Mtb. Collectively, Rif@IONPs-PAA-PEG-MAN nanodecoy can synergistically enhance the killing efficiency of intracellular Mtb in in vitro macrophages and ex vivo monocyte-derived macrophages, and also significantly reduce the mycobacterial burdens in the lung of infected mice with alleviated pathology. These results indicate that Rif@IONPs-PAA-PEG-MAN nanodecoy may have a potential for the development of more effective therapeutic strategy against TB by manipulating augmented innate immunity and drug killings.Graphic

A mathematical model for maximizing drug uptake in low-EPR tumors using different methods of ultrasound-mediated drug delivery

Cancer remains a significant health burden and research efforts aim to improve drug delivery options. Focused ultrasound has emerged as a potential mechanism for enhanced targeted drug delivery in tumors with a low Enhanced Permeability and Retention (EPR) effect by “sonoporation”. Traditional sonoporation has shown promise, but it can still benefit from improvements in drug uptake. The current study investigates the use of a new non-portative technique, called “ultrasonic tumor painting,” to enhance vascular retention of nanoparticles in the tumor space to achieve targeted drug delivery and increase therapeutic efficacy. An existing mathematical model was adapted to simulate the concentration of free and liposome encapsulated doxorubicin in circulation. The simulations were performed in MATLAB and specifically evaluate doxorubicin uptake in low EPR tumors. To increase drug uptake, sonoporation was employed where efficiency of intratumoral liposomal doxorubicin uptake was increased. An alternative non-permeabilizing strategy, called “tumor painting,” was utilized to improve vascular retention instead of increasing permeability. After retention, the small molecule doxorubicin is allowed to leak into the tumors space via concentration mediated Fickian Diffusion. This study demonstrates that ultrasound-based drug delivery can be improved with tumor painting. This method is a valuable tool to overcome the limitations of traditional sonoporation and has exciting potential for enhancing the therapeutic efficacy of chemotherapy.

Recent Advancements in Electroporation Technologies: From Bench to Clinic.

Over the past decade, the increased adoption of electroporation-based technologies has led to an expansion of clinical research initiatives. Electroporation has been utilized in molecular biology for mammalian and bacterial transfection; for food sanitation; and in therapeutic settings to increase drug uptake, for gene therapy, and to eliminate cancerous tissues. We begin this article by discussing the biophysics required for understanding the concepts behind the cell permeation phenomenon that is electroporation. We then review nano- and microscale single-cell electroporation technologies before scaling up to emerging in vivo applications.

The development and evaluation of hyaluronic acid coated mitochondrial targeting liposomes for celastrol delivery

In order to precisely deliver celastrol into mitochondria of tumor cells, improve antitumor efficacy of celastrol and overcome its troublesome problems in clinical application, a novel multistage-targeted celastrol delivery system (C-TL/HA) was developed via electrostatic binding of hyaluronic acid (HA) to celastrol-loaded cationic liposomes composed of natural soybean phosphatidylcholine and cholesterol modified with mitochondrial targeting molecular TPP. Study results in this article showed that C-TL/HA successfully transported celastrol into mitochondria, effectively activated apoptosis of mitochondrial pathway, exerted higher tumor inhibition efficiency and lower toxic side effects compared with free celastrol. More importantly, HA coating not only enabled this delivery system to have good stability and safety in vivo, but also increased drug uptake and facilitated tumor targeting through recognizing CD44 receptors rich on the surface of tumor cells. Conclusively, this HA-coated mitochondrial targeting liposomes may provide a prospect for the clinical application of celastrol in tumor therapy.

Doxorubicin-Loaded Polymeric Micelles Conjugated with CKR- and EVQ-FLT3 Peptides for Cytotoxicity in Leukemic Stem Cells.

Doxorubicin (Dox) is the standard chemotherapeutic agent for acute myeloblastic leukemia (AML) treatment. However, 40% of Dox-treated AML cases relapsed due to the presence of leukemic stem cells (LSCs). Thus, poloxamer 407 and CKR- and EVQ-FLT3 peptides were used to formulate Dox-micelles (DMs) and DM conjugated with peptides (CKR and EVQ) for improving AML-LSC treatment. Results indicated that DMs with a weight ratio of Dox to P407 of 1:200 had a particle size of 23.3 ± 1.3 nm with a high percentage of Dox entrapment. They were able to prolong drug release and maintain physicochemical stability. Following effective DM preparation, P407 was modified and conjugated with FLT3 peptides, CKR and EVQ to formulate DM-CKR, DM-EVQ, and DM-CKR+DM-EVQ. Freshly synthesized DMs displaying FLT3 peptides showed particle sizes smaller than 50 nm and a high drug entrapment level, comparable with DMs. DM-CKR+DM-EVQ was considerably more toxic to KG-1a (AML LSC-like cell model) than Dox-HCl. These FLT3-targeted DMs could increase drug uptake and induce apoptosis induction. Due to an increase in micelle-LSC binding and uptake, DMs displaying both peptides tended to improve the potency of Dox compared to a single peptide-coupled micelle.

Comprehensive Study of Antiretroviral Drug Permeability at the Cervicovaginal Mucosa via an In Vitro Model

Modulation of drug transporter activity at mucosal sites of HIV-1 transmission may be exploited to optimize retention of therapeutic antiretroviral drug concentrations at target submucosal CD4+ T cells. Previously, we showed that darunavir was a substrate for the P-glycoprotein efflux drug transporter in colorectal mucosa. Equivalent studies in the cervicovaginal epithelium have not been reported. Here, we describe the development of a physiologically relevant model to investigate the permeability of antiretroviral drugs across the vaginal epithelium. Barrier properties of the HEC-1A human endometrial epithelial cell line were determined, in a dual chamber model, by measurement of transepithelial electrical resistance, immunofluorescent staining of tight junctions and bi-directional paracellular permeability of mannitol. We then applied this model to investigate the permeability of tenofovir, darunavir and dapivirine. Efflux ratios indicated that the permeability of each drug was transporter-independent in this model. Reduction of pH to physiological levels in the apical compartment increased absorptive transfer of darunavir, an effect that was reversed by inhibition of MRP efflux transport via MK571. Thus, low pH may increase the transfer of darunavir across the epithelial barrier via increased MRP transporter activity. In a previous in vivo study in the macaque model, we demonstrated increased MRP2 expression following intravaginal stimulation with darunavir which may further increase drug uptake. Stimulation with inflammatory modulators had no effect on drug permeability across HEC-1A barrier epithelium but, in the VK2/E6E7 vaginal cell line, increased expression of both efflux and uptake drug transporters which may influence darunavir disposition.

Electrochemotherapy: An Alternative Strategy for Improving Therapy in Drug-Resistant SOLID Tumors.

Simple SummaryChemotherapy is becoming an increasingly difficult antitumor therapy to practice due to the multiple mechanisms of drug resistance. To overcome the problem, it is possible to use alternative techniques, such as electrochemotherapy, which involves the simultaneous administration of the electrical pulse (electroporation) and the treatment with the drug in order to improve the effectiveness of the drug against the tumor. Electroporation has improved the efficacy of some chemotherapeutic agents, such bleomycin, cisplatin, mitomycin C, and 5-fluorouracil. The results of in vitro, veterinary, and clinical oncology studies are promising on various cancers, such as metastatic melanoma. The purpose of this review is to give an update on the state of the art of electrochemotherapy against the main solid tumors in the preclinical, clinical, and veterinary field.Electrochemotherapy (ECT) is one of the innovative strategies to overcome the multi drug resistance (MDR) that often occurs in cancer. Resistance to anticancer drugs results from a variety of factors, such as genetic or epigenetic changes, an up-regulated outflow of drugs, and various cellular and molecular mechanisms. This technology combines the administration of chemotherapy with the application of electrical pulses, with waveforms capable of increasing drug uptake in a non-toxic and well tolerated mechanical system. ECT is used as a first-line adjuvant therapy in veterinary oncology, where it improves the efficacy of many chemotherapeutic agents by increasing their uptake into cancer cells. The chemotherapeutic agents that have been enhanced by this technique are bleomycin, cisplatin, mitomycin C, and 5-fluorouracil. After their use, a better localized control of the neoplasm has been observed. In humans, the use of ECT was initially limited to local palliative therapy for cutaneous metastases of melanoma, but phase I/II studies are currently ongoing for several histotypes of cancer, with promising results. In this review, we described the preclinical and clinical use of ECT on drug-resistant solid tumors, such as head and neck squamous cell carcinoma, breast cancer, gynecological cancer and, finally, colorectal cancer.

Doxorubicin-Loaded Lipid Nanoparticles Coated with Calcium Phosphate as a Potential Tool in Human and Canine Osteosarcoma Therapy.

Osteosarcoma (OSA) is the most frequently diagnosed primary malignant bone tumor in humans and dogs. In both species, standard chemotherapy can be limited by multidrug resistance of neoplastic cells, which prevents intracellular accumulation of cytotoxic drugs, resulting in chemotherapy failure. In this study, a lipophilic ester of doxorubicin (C12DOXO) was loaded into nanoparticles (NPs) using the “cold microemulsion dilution” method. The resulting NPs were then coated with calcium phosphate (CaP) in two different ways to have calcium or phosphate ions externally exposed on the surface. These systems were characterized by determining mean diameter, zeta potential, and drug entrapment efficiency; afterward, they were tested on human and canine OSA cells to study the role that the coating might play in increasing both drug uptake into tumor cells and cytotoxicity. Mean diameter of the developed NPs was in the 200–300 nm range, zeta potential depended on the coating type, and C12DOXO entrapment efficiency was in the 60–75% range. Results of studies on human and canine OSA cells were very similar and showed an increase in drug uptake and cytotoxicity for CaP-coated NPs, especially when calcium ions were externally exposed. Therefore, applications in both human and veterinary medicine can be planned in the near future.

Paclitaxel and Lapatinib dual loaded chitosan-coated PLGA nanoparticles enhance cytotoxicity by circumventing MDR1-mediated trastuzumab resistance in HER2 positive breast cancers: In-vitro and in-vivo studies

Treatment options for multidrug-resistant breast cancer are limited and ineffective. Few combinations of chemotherapeutic agents are clinically successful in treating multiple drug-resistant cancers. However, due to their varied pharmacokinetic characteristics, it is challenging to deliver dual drugs to the tumor simultaneously with the best proportion by simple co-administration. This study aimed to generate chitosan-coated PLGA nanoparticles delivering a synergistic combination of Paclitaxel (PTX) and Lapatinib (LPB) to improve efficacy in patients with multidrug-resistant breast cancer. In comparison to sensitive BT-474 cells, the 3:1 M ratio of PTX/LPB demonstrated significant synergism with a reversal index of approximately 13.4 in trastuzumab-resistant BT-474/TR cells. Particle diameters of the prepared chitosan (CS) PLGA Nps ranged from 157.4 ± 9.2 to 263.5 ± 13.5 nm. CS-coated Nps exhibited good EE and DL values of 80.8 ± 3.4% and 1.5 ± 0.12%, respectively. Moreover, CS-coating allowed successful co-delivery of PTX and LPB, resulting in the maximum in-vitro release within 72 h. Cytotoxicity against BT-474/TR cells was significantly increased when CS-coated dual loaded Nps were compared to other Nps, including free PTX, LPB, and their mixture. Quantification of the intracellular drug accumulation revealed that the encapsulation effectively increased drug uptake in BT-474/TR cells. These CS-coated dual loaded Nps exerted an excellent synergistic effect in BT-474/TR cells, leading to significantly improved cell inhibition via circumventing the MDR1 mediated trastuzumab resistance. Additionally, co-delivery of PTX and LPB demonstrated the most effective tumor growth reduction in the BT-474/TR human breast tumor xenograft compared to the saline control, single loaded PTX, and LPB Nps and a mixture of PTX and LPB combinations. The data suggest that CS-coated PLGA Nps loaded with PTX and LPB concurrently may be one of the most promising formulations for treating MDR1 mediated chemoresistance in HER2-positive breast tumors.

Triphenylphosphonium conjugation to a TRAP1 inhibitor, 2-amino-6-chloro-7,9-dihydro-8H-purin-8-one increases antiproliferative activity

Tumor-necrosis-factor-receptor associated protein 1 (TRAP1), a mitochondrial paralog of heat shock protein 90 family proteins, is overexpressed in many cancer cells and supports tumorigenesis by rewiring vital metabolic and cell death pathways. The triphenylphosphonium moiety is used to deliver therapeutic cargo to increase drug uptake into mitochondria. Various aryl- or alkyl-substituted phosphonium analogs were conjugated with TRAP1-selective inhibitors 4a-c to optimize anticancer activity. Among these various phosphonium-conjugated compounds, (6-(2-amino-9-(4-bromo-2-fluorobenzyl)-6-chloro-8-oxo-8,9-dihydro-7H-purin-7-yl)hexyl)triphenylphosphornium (6a) was identified as a potential anticancer agent. Compound 6a had IC50 values of 0.30–3.24 μM in seven different cancer cell lines and potently suppressed tumor growth without any noticeable in vivo toxicity in a nude mouse model xenografted with PC3 prostate cancer cells.

Mutually Exclusive Interactions of Rifabutin with Spatially Distinct Mycobacterial Cell Envelope Membrane Layers Offer Insights into Membrane-Centric Therapy of Infectious Diseases.

The mycobacterial cell envelope has spatially resolved inner and outer membrane layers with distinct compositions and membrane properties. However, the functional implication and relevance of this organization remain unknown. Using membrane biophysics and molecular simulations, we reveal a varied interaction profile of these layers with antibiotic Rifabutin, underlined by the structural and chemical makeup of the constituent lipids. The mycobacterial inner membrane displayed the highest partitioning of Rifabutin, which was located exclusively in the lipid head group/interfacial region. In contrast, the drug exhibited specific interaction sites in the head group/interfacial and hydrophobic acyl regions within the outer membrane. Altogether, we show that the design of membrane-active agents that selectively disrupt the mycobacterial outer membrane structure can increase drug uptake and enhance intracellular drug concentrations. Exploiting the mycobacterium-specific membrane–drug interaction profiles, chemotypes consisting of outer membrane-disruptive agents and antitubercular drugs can offer new opportunities for combinational tuberculosis (TB) therapy.

Single Cell RNA Sequencing Identifies Potential Molecular Indicators of Response to Melflufen in Multiple Myeloma

IntroductionMelphalan flufenamide (melflufen), is a novel peptide-drug conjugate that targets aminopeptidases and selectively delivers alkylating agents in tumors. Melflufen was recently FDA approved for the treatment of relapsed/refractory multiple myeloma (MM) patients. Considering the challenges in treating this group of patients, and the availability of several new drugs for MM, information that can support treatment selection is urgently needed. To identify potential indicators of response and mechanism of resistance to melflufen, we applied a multiparametric drug sensitivity assay to MM patient samples ex vivo and analyzed the samples by single cell RNA sequencing (scRNAseq). Ex vivo drug testing identified MM samples that were distinctly sensitive or resistant to melflufen, while differential gene expression analysis revealed pathways associated with response.MethodsBone marrow (BM) aspirates from 24 MM patients were obtained after written informed consent following approved protocols in compliance with the Declaration of Helsinki. BM mononuclear cells from 12 newly diagnosed (ND) and 12 relapsed/refractory (RR) patients were used for multi-parametric flow cytometry-based drug sensitivity and resistance testing (DSRT) evaluation to melflufen and melphalan, and for scRNAseq. Based on the results from the DSRT tests and drug sensitivity scores (DSS), we divided the samples into three groups - high sensitivity (HS, DSS > 40 (melflufen) or DSS > 16 (melphalan)), intermediate sensitivity (IS, 31 ≤ DSS ≤ 40 (melflufen) or 10 ≤ DSS ≤ 16 (melphalan)), and low sensitivity (LS, DSS < 31 (melflufen) or DSS < 10 (melphalan)). To identify genes, responsible for the general sensitivity to melphalan-based drugs we conducted differential gene expression (DGE) analyses separately for melphalan and melflufen focusing on the plasma cell populations, comparing gene expression between HS and LS samples for both drugs (“HS vs. LS melphalan” and “HS vs. LS for melflufen”, respectively). In addition, to explain the increased sensitivity of RR samples, we conducted the DGE analysis for ND vs. RR samples and searched for similarities between these three datasets.ResultsDSRT data indicated that samples from RRMM patients were significantly more sensitive to melflufen compared to samples from NDMM (Fig. 1A). In addition, we observed that samples with a gain of 1q (+1q) were more sensitive to melflufen while those with deletion of 13q (del13q) appeared to be less sensitive, although these results lacked significance (Fig. 1A). After separating the samples into different drug sensitivity groups (HS, IS, LS), DGE analysis showed significant downregulation of the drug efflux and multidrug resistance protein family member ABCB9 in the melflufen HS group opposed to the LS group (2.2-fold, p < 0.001). A similar pattern was detected for the melphalan HS vs. LS comparison suggesting that this alteration might be a common indicator of sensitivity to melphalan-based drugs. Furthermore, in the melflufen HS group we observed downregulation of the matrix metallopeptidase inhibitors TIMP1 and TIMP2 (3-fold and 1.6-fold, p < 0.001, respectively), and cathepsin inhibitors CST3 and CSTB (3.2-fold and 1.3-fold, p < 0.001, respectively) (Fig. 1B). This effect was observed in both “ND vs. RR” and “HS vs. LS for melflufen” comparisons, but not for melphalan, suggesting that these changes are associated with disease progression and specific indicators of sensitivity to melflufen. Moreover, gene set enrichment analysis (GSEA) showed activation of pathways related to protein synthesis, as well as amino acid starvation for malignant and normal cell populations in the HS group.ConclusionIn summary, our results indicate that melflufen is more active in RRMM compared to NDMM. In addition, samples from MM patients with +1q, which is considered an indicator of high-risk disease, tended to be more sensitive to melflufen. Based on differential GSEA and pathway enrichment, several synergizing mechanisms could potentially explain the higher sensitivity to melflufen, such as decreased drug efflux and increased drug uptake. Although these results indicate potential indicators of response and mechanisms of drug efficacy, further validation of these findings is required using data from melflufen treated patients. [Display omitted] DisclosuresSlipicevic: Oncopeptides AB: Current Employment. Nupponen: Oncopeptides AB: Consultancy. Lehmann: Oncopeptides AB: Current Employment. Heckman: Orion Pharma: Research Funding; Oncopeptides: Consultancy, Research Funding; Novartis: Research Funding; Celgene/BMS: Research Funding; Kronos Bio, Inc.: Research Funding.

Reversing Multidrug Resistance by Inducing Mitochondrial Dysfunction for Enhanced Chemo-Photodynamic Therapy in Tumor.

Efficiency of standard chemotherapy is dramatically hindered by intrinsic multidrug resistance (MDR). Recently, to amplify therapeutic efficacy, photodynamic therapy (PDT)-induced mitochondrial dysfunction by decorating targeting moieties on nanocarriers has obtained considerable attention. Nevertheless, low targeting efficiency, complex synthesis routes, and difficulty in releasing contents become the major obstacles in further clinical application. Herein, an ingenious liposomal-based nanomedicine (L@BP) was fabricated by encapsulating a mitochondria-anchored photosensitizer (Cy-Br) and paclitaxel (PTX) for realizing enhanced cooperation therapy. At the cellular level, L@BP could hurdle endosomal traps to localize and implement PDT in mitochondria. Intriguingly, the PDT-induced in situ mitochondrial dysfunction led to intracellular ATP reduction, which triggered the downregulated P-glycoprotein transportation capacity and thus resulted in diminishing the efflux of chemotherapeutic agents and increasing drug uptake by drug-resistant cells. The prepared nanomedicine eminently accumulated in the tumor site and acquired enhanced therapeutic efficiency on PTX-resistant lung cancer cells, which possessed great potential in circumventing MDR tumors.