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Infiltration of immunosuppressive cells into the breast tumor microenvironment (TME) is associated with suppressed effector T cell (Teff) responses, accelerated tumor growth, and poor clinical outcomes. Previous studies from our group and others identified infiltration of immunosuppressive myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs) as critical contributors to immune dysfunction in the orthotopic claudin-low tumor model, limiting the efficacy of adoptive cellular therapy. However, approaches to target these cells in the TME are currently lacking. To overcome this barrier, polymeric micellular nanoparticles (PMNPs) were used for the co-delivery of small molecule drugs activating Toll-like receptors 7 and 8 (TLR7/8) and inhibiting PI3K delta (PI3Kδ). The immunomodulation of the TME by TLR7/8 agonist and PI3K inhibitor led to type 1 macrophage polarization, decreased MDSC accumulation and selectively decreased tissue-resident Tregs in the TME, while enhancing the T and B cell adaptive immune responses. PMNPs significantly enhanced the anti-tumor activity of local radiation therapy (RT) in mice bearing orthotopic claudin-low tumors compared to RT alone. Taken together, these data demonstrate that RT combined with a nanoformulated immunostimulant diminished the immunosuppressive TME resulting in tumor regression. These findings set the stage for clinical studies of this approach.
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Nanopartículas , Receptor 7 Toll-Like , Receptor 8 Toll-Like , Microambiente Tumoral , Animais , Microambiente Tumoral/efeitos dos fármacos , Receptor 7 Toll-Like/agonistas , Feminino , Nanopartículas/química , Camundongos , Receptor 8 Toll-Like/agonistas , Imunomodulação/efeitos dos fármacos , Linhagem Celular Tumoral , Classe I de Fosfatidilinositol 3-Quinases , Células Supressoras Mieloides/efeitos dos fármacos , Células Supressoras Mieloides/imunologia , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Camundongos Endogâmicos BALB C , Micelas , HumanosRESUMO
[This corrects the article DOI: 10.1371/journal.pone.0061852.].
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Infiltration of immunosuppressive cells into the breast tumor microenvironment (TME) is associated with suppressed effector T cell (Teff) responses, accelerated tumor growth, and poor clinical outcomes. Previous studies from our group and others identified infiltration of immunosuppressive myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs) as critical contributors to immune dysfunction in the orthotopic triple-negative breast cancer (TNBC) tumor model limiting the efficacy of adoptive cellular therapy. However, approaches to target these cells specifically in the TME are currently lacking. To overcome this barrier, polymeric micelles nanoparticles (PMNPs) were used for co-delivery of small molecule drugs activating Toll-like receptors 7 and 8 (TLR7/8) and inhibiting PI3K delta. The immunomodulation of the TME by TLR7/8 agonist and PI3K inhibitor altered macrophage polarization, reduced MDSC accumulation and selectively decreased tissue-resident Tregs in the TME, while enhancing the T and B cell adaptive immune response. PMNPs significantly enhanced the anti-tumor activity of local radiation therapy (RT) in mice bearing orthotopic TNBC tumors compared to RT alone. Taken together, these data demonstrate that RT combined with a nanoformulated immunostimulant restructured the TME and has promising potential for future translation combined with RT for patients with TNBC.
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Drug delivery systems based on amphiphilic supramolecular macrocycles have garnered increased attention over the past two decades due to their ability to successfully formulate nanoparticles. Macrocyclic (MC) materials can self-assemble at lower concentrations without the need for surfactants and polymers, but surfactants are required to form and stabilize nanoparticles at higher concentrations. Using MCs to deliver both hydrophilic and hydrophobic guest molecules is advantageous. We developed two novel types of amphiphilic macrocycle nanoparticles (MC NPs) capable of delivering either Nile Red (NR) (a hydrophobic model) or Rhodamine B (RhB) (a hydrophilic model) fluorescent dyes. We extensively characterized the materials using various techniques to determine size, morphology, stability, hemolysis, fluorescence, loading efficiency (LE), and loading capacity (LC). We then loaded the CDK4/6 inhibitor Palbociclib (Palb) into both MC NPs using a solvent diffusion method. This yielded Palb-MC NPs in the size range of 65-90 nm. They exhibited high stability over time and in fetal bovine serum with negligible toxicity against erythrocytes. Cytotoxicity was minimal when tested against RAW macrophages, human fibroblast HDFn, and adipose stromal cells (ASCs) at higher concentrations of MC NPs. Cell viability studies were conducted with different concentrations of MC NPs, Palb-MC NPs, and free Palb against RAW macrophages, human U-87 GBM, and human M14 melanoma cell lines in vitro. Flow cytometry experiments revealed that blank MC NPs and Palb-MC NPs were selectively targeted to melanoma cells, resulting in cell death compared to the other two cell lines. Future work will focus on studying the biological effect of MC NPs including their binding affinity with molecules/receptors expressed on the M14 and other melanoma cell surfaces by molecular docking simulations. Subsequently, we will evaluate the MCs as a component of combination therapy in a murine melanoma model.
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Melanoma , Nanopartículas , Piperazinas , Piridinas , Camundongos , Humanos , Animais , Melanoma/tratamento farmacológico , Simulação de Acoplamento Molecular , Sistemas de Liberação de Medicamentos , Nanopartículas/química , Tensoativos , Portadores de Fármacos/química , Quinase 4 Dependente de CiclinaRESUMO
Drug delivery systems based on amphiphilic supramolecular macrocycles have garnered increased attention over the past two decades due to their ability to successfully formulate nanoparticles. Macrocyclic (MC) materials can self-assemble at lower concentrations without the need for surfactants and polymers, but surfactants are required to form and stabilize nanoparticles at higher concentrations. Using MCs to deliver both hydrophilic and hydrophobic guest molecules is advantageous. We developed two novel types of amphiphilic macrocycle nanoparticles (MC NPs) capable of delivering either Nile Red (NR) (a hydrophobic model) or Rhodamine B (RhB) (a hydrophilic model) fluorescent dyes. We extensively characterized the materials using various techniques to determine size, morphology, stability, hemolysis, fluorescence, loading efficiency (LE), and loading capacity (LC). We then loaded the CDK4/6 inhibitor Palbociclib (Palb) into both MC NPs using a solvent diffusion method. This yielded Palb-MC NPs in the size range of 65-90 nm. They exhibited high stability over time and in fetal bovine serum with negligible toxicity against erythrocytes. Cytotoxicity was minimal when tested against RAW macrophages, human fibroblast HDFn , and adipose stromal cells (ASCs) at higher concentrations of MC NPs. Cell viability studies were conducted with different concentrations of MC NPs, Palb-MC NPs, and free Palb against RAW macrophages, human U-87 GBM, and human M14 melanoma cell lines in vitro. Flow cytometry experiments revealed that blank MC NPs and Palb-MC NPs were selectively targeted to melanoma cells, resulting in cell death compared to the other two cell lines. Future work will focus on studying the biological effect of MC NPs including their binding affinity with molecules/receptors expressed on the M14 and other melanoma cell surface by molecular docking simulations. Subsequently, we will evaluate the MCs as a component of combination therapy in a murine melanoma model.
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The present study expands the versatility of cationic poly(2-oxazoline) (POx) copolymers as a polyethylene glycol (PEG)-free platform for gene delivery to immune cells, such as monocytes and macrophages. Several block copolymers are developed by varying nonionic hydrophilic blocks (poly(2-methyl-2-oxazoline) (pMeOx) or poly(2-ethyl-2-oxazoline) (pEtOx), cationic blocks, and an optional hydrophobic block (poly(2-isopropyl-2-oxazoline) (iPrOx). The cationic blocks are produced by side chain modification of 2-methoxy-carboxyethyl-2-oxazoline (MestOx) block precursor with diethylenetriamine (DET) or tris(2-aminoethyl)amine (TREN). For the attachment of a targeting ligand, mannose, azide-alkyne cycloaddition click chemistry methods are employed. Of the two cationic side chains, polyplexes made with DET-containing copolymers transfect macrophages significantly better than those made with TREN-based copolymer. Likewise, nontargeted pEtOx-based diblock copolymer is more active in cell transfection than pMeOx-based copolymer. The triblock copolymer with hydrophobic block iPrOx performs poorly compared to the diblock copolymer which lacks this additional block. Surprisingly, attachment of a mannose ligand to either copolymer is inhibitory for transfection. Despite similarities in size and design, mannosylated polyplexes result in lower cell internalization compared to nonmannosylated polyplexes. Thus, PEG-free, nontargeted DET-, and pEtOx-based diblock copolymer outperforms other studied structures in the transfection of macrophages and displays transfection levels comparable to GeneJuice, a commercial nonlipid transfection reagent.
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Manose , Polietilenoglicóis , Polietilenoglicóis/química , Ligantes , Plasmídeos/genética , Polímeros/química , DNA/química , TransfecçãoRESUMO
Triple-negative breast cancer (TNBC) is notoriously difficult to treat due to the lack of targetable receptors and sometimes poor response to chemotherapy. The transforming growth factor-beta (TGFß) family of proteins and their receptors (TGFR) are highly expressed in TNBC and implicated in chemotherapy-induced cancer stemness. Here we evaluated combination treatments using experimental TGFR inhibitors (TGFßi), SB525334 (SB), and LY2109761 (LY) with Paclitaxel (PTX) chemotherapy. These TGFßi target TGFR-I (SB) or both TGFR-I&II (LY). Due to the poor water solubility of these drugs, we incorporated each of them in poly(2-oxazoline) (POx) high-capacity polymeric micelles (SB-POx and LY-POx). We assessed their anti-cancer effect as single agents and in combination with micellar Paclitaxel (PTX-POx) using multiple immunocompetent TNBC mouse models that mimic human subtypes (4T1, T11-Apobec and T11-UV). While either TGFßi or PTX showed a differential effect in each model as single agents, the combinations were consistently effective against all three models. Genetic profiling of the tumors revealed differences in the expression levels of genes associated with TGFß, EMT, TLR-4, and Bcl2 signaling, alluding to the susceptibility to specific gene signatures to the treatment. Taken together, our study suggests that TGFßi and PTX combination therapy using high-capacity POx micelle delivery provides a robust anti-tumor response in multiple TNBC subtype mouse models.
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The presence of immunosuppressive immune cells in tumors is a significant barrier to the generation of therapeutic immune responses. Similarly, in vivo triple-negative breast cancer (TNBC) models often contain prevalent, immunosuppressive tumor-associated macrophages in the tumor microenvironment (TME), resulting in breast cancer initiation, invasion, and metastasis. Here, we test systemic chemoimmunotherapy using small-molecule agents, paclitaxel (PTX), and colony-stimulating factor 1 receptor (CSF1R) inhibitor, PLX3397, to enhance the adaptive T cell immunity against TNBCs in immunocompetent mouse TNBC models. We use high-capacity poly(2-oxazoline) (POx)-based polymeric micelles to greatly improve the solubility of insoluble PTX and PLX3397 and widen the therapeutic index of such drugs. The results demonstrate that high-dose PTX in POx, even as a single agent, exerts strong effects on TME and induces long-term immune memory. In addition, we demonstrate that the PTX and PLX3397 combination provides consistent therapeutic improvement across several TNBC models, resulting from the repolarization of the immunosuppressive TME and enhanced T cell immune response that suppress both the primary tumor growth and metastasis. Overall, the work emphasizes the benefit of drug reformulation and outlines potential translational path for both PTX and PTX with PLX3397 combination therapy using POx polymeric micelles for the treatment of TNBC.
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Engineered cells used as smart vehicles for delivery of secreted therapeutic proteins enable effective treatment of cancer and certain degenerative, autoimmune, and genetic disorders. However, current cell-based therapies use mostly invasive tools for tracking proteins and do not allow for controlled secretion of therapeutic proteins, which could result in unconstrained killing of surrounding healthy tissues or ineffective killing of host cancer cells. Regulating the expression of therapeutic proteins after success of therapy remains elusive. In this study, a noninvasive therapeutic approach mediated by magneto-mechanical actuation (MMA) was developed to remotely regulate the expression of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) protein, which is secreted by transduced cells. Stem cells, macrophages, and breast cancer cells were transduced with a lentiviral vector encoding the SGpL2TR protein. SGpL2TR comprises TRAIL and GpLuc domains optimized for cell-based applications. Our approach relies on the remote actuation of cubic-shape highly magnetic field responsive superparamagnetic iron oxide nanoparticles (SPIONs) coated with nitrodopamine PEG (ND-PEG), which are internalized within the cells. Cubic ND-PEG-SPIONs actuated by superlow frequency alternating current magnetic fields can translate magnetic forces into mechanical motion and in turn spur mechanosensitive cellular responses. Cubic ND-PEG-SPIONs were artificially designed to effectively operate at low magnetic field strengths (<100 mT) retaining approximately 60% of their saturation magnetization. Compared to other cells, stems cells were more sensitive to the interaction with actuated cubic ND-PEG-SPIONs, which clustered near the endoplasmic reticulum (ER). Luciferase, ELISA, and RT-qPCR analyses revealed a marked TRAIL downregulation (secretion levels were depleted down to 30%) when intracellular particles at 0.100 mg/mL Fe were actuated by magnetic fields (65 mT and 50 Hz for 30 min). Western blot studies indicated actuated, intracellular cubic ND-PEG-SPIONs can cause mild ER stress at short periods (up to 3 h) of postmagnetic field treatment thus leading to the unfolded protein response. We observed that the interaction of TRAIL polypeptides with ND-PEG can also contribute to this response. To prove the applicability of our approach, we used glioblastoma cells, which were exposed to TRAIL secreted from stem cells. We demonstrated that in the absence of MMA treatment, TRAIL essentially killed glioblastoma cells indiscriminately, but when treated with MMA, we were able to control the cell killing rate by adjusting the magnetic doses. This approach can expand the capabilities of stem cells to serve as smart vehicles for delivery of therapeutic proteins in a controlled manner without using interfering and expensive drugs, while retaining their potential to regenerate damaged tissue after treatment. This approach brings forth new alternatives to regulate protein expression noninvasively for cell therapy and other cancer therapies.
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Glioblastoma , Nanopartículas de Magnetita , Humanos , Nanopartículas de Magnetita/química , Polietilenoglicóis/química , Fenômenos MagnéticosRESUMO
Active learning (AL) has become a subject of active recent research both in industry and academia as an efficient approach for rapid design and discovery of novel chemicals, materials, and polymers. Herein, we have assessed the applicability of AL for the discovery of polymeric micelle formulations for poorly soluble drugs. We were motivated by the key advantages of this approach making it a desirable strategy for rational design of drug delivery systems due toto its ability to (i) employ relatively small datasets for model development, (ii) iterate between model development and model assessment using small external datasets that can be either generated in focused experimental studies or formed from subsets of the initial training data, and (iii) progressively evolve models towards increasingly more reliable predictions and the identification of novel chemicals with the desired properties. In this study, we compared various AL protocols for their effectiveness in finding biologically active molecules using synthetic datasets. We have investigated the dependency of AL performance on the size of the initial training set, the relative complexity of the task, and the choice of the initial training dataset. We found that AL techniques as applied to regression modeling offer no benefits over random search, while AL used for classification tasks performs better than models built for randomly selected training sets but still quite far from perfect. Using the best performing AL protocol,. Finally, the best performing AL approach was employed to discover and experimentally validate novel binding polymers for a case study of asialoglycoprotein receptor (ASGPR).
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Polímeros , Aprendizagem Baseada em Problemas , Polímeros/química , Micelas , Sistemas de Liberação de Medicamentos , PeptídeosRESUMO
In the last decade, nanomedicine has arisen as an emergent area of medicine, which studies nanometric systems, namely polymeric micelles (PMs), that increase the solubility and the stability of the encapsulated drugs. Furthermore, their application in dermal drug delivery is also relevant. PMs present unique characteristics because of their unique core-shell architecture. They are colloidal dispersions of amphiphilic compounds, which self-assemble in an aqueous medium, giving a structure-type core-shell, with a hydrophobic core (that can encapsulate hydrophobic drugs), and a hydrophilic shell, which works as a stabilizing agent. These features offer PMs adequate steric protection and determine their hydrophilicity, charge, length, and surface density properties. Furthermore, due to their small size, PMs can be absorbed by the intestinal mucosa with the drug, and they transport the drug in the bloodstream until the therapeutic target. Moreover, PMs improve the pharmacokinetic profile of the encapsulated drug, present high load capacity, and are synthesized by a reproducible, easy, and low-cost method. In silico approaches have been explored to improve the physicochemical properties of PMs. Based on this, a computer-aided strategy was developed and validated to enable the delivery of poorly soluble drugs and established critical physicochemical parameters to maximize drug loading, formulation stability, and tumor exposure. Poly(2-oxazoline) (POx)-based PMs display unprecedented high loading concerning water-insoluble drugs and over 60 drugs have been incorporated in POx PMs. Among various stimuli, pH and temperature are the most widely studied for enhanced drug release at the site of action. Researchers are focusing on dual (pH and temperature) responsive PMs for controlled and improved drug release at the site of action. These dual responsive systems are mainly evaluated for cancer therapy as certain malignancies can cause a slight increase in temperature and a decrease in the extracellular pH around the tumor site. This review is a compilation of updated therapeutic applications of PMs, such as PMs that are based on Pluronics®, micelleplexes and Pox-based PMs in several biomedical applications.
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Many therapeutic formulations incorporate poly(ethylene glycol) (PEG) as a stealth component to minimize early clearance. However, PEG is immunogenic and susceptible to accelerated clearance after multiple administrations. Here, we present two novel reformulations of a polyion complex (PIC), originally composed of poly(ethylene glycol)113-b-poly(glutamic acid)50 (PEG-PLE) and brain-derived neurotrophic factor (BDNF), termed Nano-BDNF (Nano-BDNF PEG-PLE). We replace the PEG based block copolymer with two new polymers, poly(sarcosine)127-b-poly(glutamic acid)50 (PSR-PLE) and poly(methyl-2-oxazolines)38-b-poly(oxazolepropanoic acid)27-b-poly(methyl-2-oxazoline)38 (PMeOx-PPaOx-PMeOx), which are driven to association with BDNF via electrostatic interactions and hydrogen bonding to form a PIC. Formulation using a microfluidic mixer yields small and narrowly disperse nanoparticles which associate following similar principles. Additionally, we demonstrate that encapsulation does not inhibit access by the receptor kinase, which affects BDNF's physiologic benefits. Finally, we investigate the formation of nascent nanoparticles through a series of characterization experiments and isothermal titration experiments which show the effects of pH in the context of particle self-assembly. Our findings indicate that thoughtful reformulation of PEG based, therapeutic PICs with non-PEG alternatives can be accomplished without compromising the self-assembly of the PIC.
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Enzymes conjugated to magnetic nanoparticles (MNPs) undergo changes in the catalytic activity of the non-heating low-frequency magnetic field (LFMF). We apply in silico simulations by molecular dynamics (MD) and in vitro spectroscopic analysis of the enzyme kinetics and secondary structure to study α-chymotrypsin (CT) conjugated to gold-coated iron oxide MNPs. The latter are functionalized by either carboxylic or amino group moieties to vary the points of enzyme attachment. The MD simulation suggests that application of the stretching force to the CT globule by its amino or carboxylic groups causes shrinkage of the substrate-binding site but little if any changes in the catalytic triad. Consistent with this, in CT conjugated to MNPs by either amino or carboxylic groups, LFMF alters the Michaelis-Menten constant but not the apparent catalytic constant k cat (= V max/[E]o). Irrespective of the point of conjugation to MNPs, the CT secondary structure was affected with nearly complete loss of α-helices and increase in the random structures in LFMF, as shown by attenuated total reflection Fourier transformed infrared spectroscopy. Both the catalytic activity and the protein structure of MNP-CT conjugates restored 3 h after the field exposure. We believe that such remotely actuated systems can find applications in advanced manufacturing, nanomedicine, and other areas.
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The rise of the novel virus SARS-CoV2 which causes the disease known as COVID-19 has led to a global pandemic claiming millions of lives. With no clinically approved treatment for COVID-19, physicians initially struggled to treat the disease, and a need remains for improved antiviral therapies in this area. It is conceived early in the pandemic that an inhalable formulation of the drug remdesivir which directly targets the virus at the site of infection could improve therapeutic outcomes in COVID-19. A set of requirements are developed that would be conducive to rapid drug approval: 1) try to use GRAS reagents 2) minimize excipient concentration and 3) achieve a working concentration of 5 mg/mL remdesivir to obtain a deliverable dose which is 5-10% of the IV dose. In this work, it is discovered that Poly(2-oxazoline) block copolymers can stabilize drug nanocrystal suspensions and provide suitable formulation characteristics for aerosol delivery while maintaining antiviral efficacy. The authors believe POx block copolymers can be used as a semi-ubiquitous stabilizer for the rapid development of nanocrystal formulations for new and existing diseases.
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Tratamento Farmacológico da COVID-19 , Monofosfato de Adenosina/análogos & derivados , Alanina/análogos & derivados , Alanina/farmacologia , Antivirais/farmacologia , Antivirais/uso terapêutico , Excipientes , Humanos , Oxazóis , RNA Viral , Aerossóis e Gotículas Respiratórios , SARS-CoV-2RESUMO
The therapeutic potential of CDK4/6 inhibitors for brain tumors has been limited by recurrence. To address recurrence, we tested a nanoparticle formulation of CDK4/6 inhibitor palbociclib (POx-Palbo) in mice genetically-engineered to develop SHH-driven medulloblastoma, alone or in combination with specific agents suggested by our analysis. Nanoparticle encapsulation reduced palbociclib toxicity, enabled parenteral administration, improved CNS pharmacokinetics, and extended mouse survival, but recurrence persisted. scRNA-seq identified up-regulation of glutamate transporter Slc1a2 and down-regulation of diverse ribosomal genes in proliferating medulloblastoma cells in POx-Palbo-treated mice, suggesting mTORC1 signaling suppression, subsequently confirmed by decreased 4EBP1 phosphorylation. Combining POx-Palbo with the mTORC1 inhibitor sapanisertib produced mutually enhancing effects and prolonged mouse survival compared to either agent alone, contrasting markedly with other tested drug combinations. Our data show the potential of nanoparticle formulation and scRNA-seq analysis of resistance to improve brain tumor treatment and identify POx-Palbo + Sapanisertib as effective combinatorial therapy for SHH medulloblastoma.
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Combining diagnosis and treatment approaches in one entity is the goal of theranostics for cancer therapy. Magnetic nanoparticles have been extensively used as contrast agents for nuclear magnetic resonance imaging as well as drug carriers and remote actuation agents. Poly(2-oxazoline)-based polymeric micelles, which have been shown to efficiently solubilize hydrophobic drugs and drug combinations, have high loading capacity (above 40% w/w) for paclitaxel. In this study, we report the development of novel theranostic system, NanoFerrogels, which is designed to capitalize on the magnetic nanoparticle properties as imaging agents and the poly(2-oxazoline)-based micelles as drug loading compartment. We developed six formulations with magnetic nanoparticle content of 0.3%-12% (w/w), with the z-average sizes of 85-130â¯nm and ξ-potential of 2.7-28.3â¯mV. The release profiles of paclitaxel from NanoFerrogels were notably dependent on the degree of dopamine grafting on poly(2-oxazoline)-based micelles. Paclitaxel loaded NanoFerrogels showed efficacy against three breast cancer lines which was comparable to free paclitaxel. They also showed improved tumor and lymph node accumulation and signal reduction in vivo (2.7% in tumor; 8.5% in lymph node) compared to clinically approved imaging agent ferumoxytol (FERAHEME®) 24â¯h after administration. NanoFerrogels responded to super-low frequency alternating current magnetic field (50 kA m-1, 50â¯Hz) which accelerated drug release from paclitaxel-loaded NanoFerrogels or caused death of cells loaded with NanoFerrogels. These proof-of-concept experiments demonstrate that NanoFerrogels have potential as remotely actuated theranostic platform for cancer diagnosis and treatment.