Tumor microenvironment immunomodulation by nanoformulated TLR 7/8 agonist and PI3k delta inhibitor enhances therapeutic benefits of radiotherapy.

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.

Combination of Polymeric Micelle Formulation of TGFß Receptor Inhibitors and Paclitaxel Produce Consistent Response Across Different Mouse Models of TNBC.

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.

High-Dose Paclitaxel and its Combination with CSF1R Inhibitor in Polymeric Micelles for Chemoimmunotherapy of Triple Negative Breast Cancer.

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.

Poly(2-oxazoline)-Based Polyplexes as a PEG-Free Plasmid DNA Delivery Platform.

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.

Magnetic Control of Protein Expression via Magneto-mechanical Actuation of ND-PEGylated Iron Oxide Nanocubes for Cell Therapy.

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.

PEG-Free Polyion Complex Nanocarriers for Brain-Derived Neurotrophic Factor.

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.

Modulation of α-Chymotrypsin Conjugated to Magnetic Nanoparticles by the Non-Heating Low-Frequency Magnetic Field: Molecular Dynamics, Reaction Kinetics, and Spectroscopy Analysis.

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.

Nanoformulated Remdesivir with Extremely Low Content of Poly(2-oxazoline)-Based Stabilizer for Aerosol Treatment of COVID-19.

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.

Enhancing CDK4/6 inhibitor therapy for medulloblastoma using nanoparticle delivery and scRNA-seq-guided combination with sapanisertib.

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.

Poly(2-oxazoline)-magnetite NanoFerrogels: Magnetic field responsive theranostic platform for cancer drug delivery and imaging.

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 85-130 nm and ξ-potential of 2.7-28.3 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®) 24 h after administration. NanoFerrogels responded to super-low frequency alternating current magnetic field (50 kA m-1, 50 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.

Drug-Dependent Morphological Transitions in Spherical and Worm-Like Polymeric Micelles Define Stability and Pharmacological Performance of Micellar Drugs.

Significant advances in physicochemical properties of polymeric micelles enable optimization of therapeutic drug efficacy, supporting nanomedicine manufacturing and clinical translation. Yet, the effect of micelle morphology on pharmacological efficacy is not adequately addressed. This work addresses this gap by assessing pharmacological efficacy of polymeric micelles with spherical and worm-like morphologies. It is observed that poly(2-oxazoline)-based polymeric micelles can be elongated over time from a spherical structure to worm-like structure, with elongation influenced by several conditions, including the amount and type of drug loaded into the micelles. The role of different morphologies on pharmacological performance of drug loaded micelles against triple-negative breast cancer and pancreatic cancer tumor models is further evaluated. Spherical micelles accumulate rapidly in the tumor tissue while retaining large amounts of drug; worm-like micelles accumulate more slowly and only upon releasing significant amounts of drug. These findings suggest that the dynamic character of the drug-micelle structure and the micelle morphology play a critical role in pharmacological performance, and that spherical micelles are better suited for systemic delivery of anticancer drugs to tumors when drugs are loosely associated with the polymeric micelles.

Bioequivalence assessment of high-capacity polymeric micelle nanoformulation of paclitaxel and Abraxane® in rodent and non-human primate models using a stable isotope tracer assay.

The in vivo fate of nanoformulated drugs is governed by the physicochemical properties of the drug and the functionality of nanocarriers. Nanoformulations such as polymeric micelles, which physically encapsulate poorly soluble drugs, release their payload into the bloodstream during systemic circulation. This results in three distinct fractions of the drug-nanomedicine: encapsulated, protein-bound, and free drug. Having a thorough understanding of the pharmacokinetic (PK) profiles of each fraction is essential to elucidate mechanisms of nanomedicine-driven changes in drug exposure and PK/PD relationships pharmacodynamic activity. Here, we present a comprehensive preclinical assessment of the poly (2-oxazoline)-based polymeric micelle of paclitaxel (PTX) (POXOL hl-PM), including bioequivalence comparison to the clinically approved paclitaxel nanomedicine, Abraxane®. Physicochemical characterization and toxicity analysis of POXOL hl-PM was conducted using standardized protocols by the Nanotechnology Characterization Laboratory (NCL). The bioequivalence of POXOL hl-PM to Abraxane® was evaluated in rats and rhesus macaques using the NCL's established stable isotope tracer ultrafiltration assay (SITUA) to delineate the plasma PK of each PTX fraction. The SITUA study revealed that POXOL hl-PM and Abraxane® had comparable PK profiles not only for total PTX but also for the distinct drug fractions, suggesting bioequivalence in given animal models. The comprehensive preclinical evaluation of POXOL hl-PM in this study showcases a series of widely applicable standardized studies by NCL for assessing nanoformulations prior to clinical investigation.

Non-Heating Alternating Magnetic Field Nanomechanical Stimulation of Biomolecule Structures via Magnetic Nanoparticles as the Basis for Future Low-Toxic Biomedical Applications.

The review discusses the theoretical, experimental and toxicological aspects of the prospective biomedical application of functionalized magnetic nanoparticles (MNPs) activated by a low frequency non-heating alternating magnetic field (AMF). In this approach, known as nano-magnetomechanical activation (NMMA), the MNPs are used as mediators that localize and apply force to such target biomolecular structures as enzyme molecules, transport vesicles, cell organelles, etc., without significant heating. It is shown that NMMA can become a biophysical platform for a family of therapy methods including the addressed delivery and controlled release of therapeutic agents from transport nanomodules, as well as selective molecular nanoscale localized drugless nanomechanical impacts. It is characterized by low system biochemical and electromagnetic toxicity. A technique of 3D scanning of the NMMA region with the size of several mm to several cm over object internals has been described.

Preparation of an Orthotopic, Syngeneic Model of Lung Adenocarcinoma and the Testing of the Antitumor Efficacy of Poly(2-oxazoline) Formulation of Chemo-and Immunotherapeutic Agents.

Tumor xenograft models developed by transplanting human tissues or cells into immune-deficient mice are widely used to study human cancer response to drug candidates. However, immune-deficient mice are unfit for investigating the effect of immunotherapeutic agents on the host immune response to cancer (Morgan, 2012). Here, we describe the preparation of an orthotopic, syngeneic model of lung adenocarcinoma (LUAD), a subtype of non-small cell lung cancer (NSCLC), to study the antitumor effect of chemo and immunotherapeutic agents in an immune-competent animal. The tumor model is developed by implanting 344SQ LUAD cells derived from the metastases of KrasG12D; p53R172HΔG genetically engineered mouse model into the left lung of a syngeneic host (Sv/129). The 344SQ LUAD model offers several advantages over other models: 1) The immune-competent host allows for the assessment of the biologic effects of immune-modulating agents; 2) The pathophysiological features of the human disease are preserved due to the orthotopic approach; 3) Predisposition of the tumor to metastasize facilitates the study of therapeutic effects on primary tumor as well as the metastases ( Chen et al., 2014 ). Furthermore, we also describe a treatment strategy based on Poly(2-oxazoline) micelles that has been shown to be effective in this difficult-to-treat tumor model ( Vinod et al., 2020b ).

Preparation and Characterization of Poly(2-oxazoline) Micelles for the Solubilization and Delivery of Water Insoluble Drugs.

Many new drug development candidates are highly lipophilic compounds with low water solubility. This constitutes a formidable challenge for the use of such compounds for cancer therapy, where high doses and intravenous injections are needed ( Di et al., 2012 ). Here, we present a poly(2-oxazoline) polymer (POx)-based nanoformulation strategy to solubilize and deliver hydrophobic drugs. POx micelles are prepared by a simple thin-film hydration method. In this method, the drug and polymer are dissolved in a common solvent and allowed to mix, following which the solvent is evaporated using mild heating conditions to form a thin film. The micelles form spontaneously upon hydration with saline. POx nanoformulation of hydrophobic drugs is unique in that it has a high drug loading capacity, which is superior to micelles of conventional surfactants. Moreover, multiple active pharmaceutical ingredients (APIs) can be included within the same POx micelle, thereby enabling the codelivery of binary as well as ternary drug combinations ( Han et al., 2012 ; He et al., 2016 ).