Biology subjects

Makela, A. V.

Publications and source records attributed to Makela, A. V..

16 recordsLinked to original sources

bioRxiv2025

VesicleVoyager: In vivo selection of surface displayed proteins that direct extracellular vesicles to tissue-specific targets

The development of technologies for screening proteins that bind to specific tissues in vivo and facilitate delivery of large cargos remains challenging, with most approaches limited to cell culture systems that often yield clinically irrelevant hits. To overcome this limitation, we developed a novel molecular screening platform using an extracellular vesicle (EV) display library. EVs are natural molecular carriers capable of delivering diverse cargos, which can be engineered to enhance specificity and targeting through surface modifications. We constructed an EV-display library presenting monobody repertoires on EV surfaces, with genetic cargo inside the EVs corresponding to the displayed proteins. These libraries were screened for tissue specific delivery through serial passage in mice via sequential intravenous administration in and recovery of tissue-selected EVs and amplification of their encapsulated monobody genes at each passage. Our results demonstrated successful selection of tissue-specific targeting proteins, as revealed by fluorescence and bioluminescence imaging followed by DNA sequencing. To understand the stochastic relationship between displayed proteins and packaged genes, we developed a Markov chain model that quantified selection dynamics and predicted enrichment patterns despite the imperfect correlation between phenotype and genotype. This EV-based monobody screening approach, combined with mathematical modeling, is a significant advancement in targeted drug delivery by leveraging the natural capabilities of EVs with the selection of targeting proteins in a physiologically relevant environment.

bioengineering↗

bioRxiv2025

Persistent endotoxin exposure in macrophages elicits an immunometabolic profile susceptible to NF-kB and p53 perturbation

The prolonged exposure of macrophages to endotoxin occurs in various chronic inflammatory conditions, including long-term inflammation triggered by particle-releasing implanted biomaterials, progressive pulmonary obstructive conditions, chronic bronchitis, colitis and hepatitis characterized by sustained exposure to gut-derived lipopolysaccharide (LPS). However, immune cellular metabolism in models of persistent inflammation remains undercharacterized, especially when compared to the well-studied immunometabolic changes following short-term LPS exposure. Here, we demonstrate that persistent LPS stimulation induces increased oxygen consumption alongside reduced levels of ATP and elevated glycolysis, with a mixed inflammatory profile indicative of endotoxin tolerance in primary bone marrow-derived macrophages. Pharmacological blockade of complex I of the mitochondrial electron transport chain lowered oxygen consumption and mitochondrial ROS by reducing NF-{kappa}B signaling. Surprisingly, NF-{kappa}B inhibition at TAK1 or I{kappa}B phosphorylation, and p53 activation, reduced both oxygen consumption and glycolysis with minimal mitochondrial ROS impact. The absence of elevated mitochondrial membrane potential in prolonged LPS exposure suggests mitochondrial ROS production is likely reverse electron transport-independent, supported by the lack of effect of a mitochondrial depolarizer on oxygen consumption. These findings reveal a distinct immunometabolic phenotype in macrophage endotoxin tolerance, extending our understanding of immunobiology in chronic inflammatory diseases.

immunology↗

bioRxiv2024

The role of mitochondrial complex I in the proinflammatory response to polylactide implants

During the foreign body response, immune cells are metabolically rewired after exposure to breakdown products of various biomaterials, including polylactide (PLA) and polyethylene. Particles of polyethylene interact with Toll-like receptor 4 on macrophages, resulting in increased oxygen consumption that forms reactive oxygen species at complex I of the mitochondrial electron transport chain (mETC). However, PLA degradation products bind to monocarboxylate transporters for downstream signaling with elevated oxygen consumption rates, whose functional implication is unclear and remains inferred from cellular responses to polyethylene biomaterials. By chemically probing the function of the mETC, we show that proinflammatory macrophages activated by exposure to amorphous PLA (aPLA) breakdown products rely on mitochondrial respiration for ATP production, independent of oxygen consumption rates. In contrast, macrophages activated by semi-crystalline PLA (cPLA) breakdown products exhibit a metabolic phenotype wherein ATP levels are unaffected by changing oxygen consumption rates. In subcutaneous implants, the incorporation of metformin in aPLA or cPLA to chemically inhibit complex I did not effectively modulate the proinflammatory response to biomaterials, suggesting that PLA degradation products elicit a distinct metabolic program, thus providing an alternative perspective on the role of mitochondrial respiration in the inflammatory response to biomaterials.

bioengineering↗

bioRxiv2024

Engineered endosymbionts that modulate primary macrophage function and attenuate tumor growth by shifting the tumor microenvironment

Modulating gene expression in macrophages can be used to improve tissue regeneration and to redirect tumor microenvironments (TME) toward positive therapeutic outcomes. We have developed Bacillus subtilis as an engineered endosymbiont (EES) capable of residing inside the eukaryotic host cell cytoplasm and controlling the fate of macrophages. Secretion of mammalian transcription factors (TFs) from B. subtilis that expresses listeriolysin O (LLO; allowing the EES to escape destruction by the macrophage) modulated expression of surface markers, cytokines and chemokines, indicating functional changes in a macrophage/monocyte cell line. The engineered B. subtilis LLO TF strains were evaluated in murine bone marrow-derived macrophages (BMDMs) by flow cytometry, chemokine/cytokine profiling, metabolic assays and RNA-Seq. Delivery of TFs by the EES shifted BMDM gene expression, production of cytokine and chemokines and metabolic patterns, indicating that the TF strains could guide primary macrophage function. Thereafter, the ability of the TF strains to alter the TME was characterized in vivo, in an orthotopic murine model of triple-negative breast cancer to assess therapeutic effects. The TF strains altered the TME by shifting immune cell composition and attenuating tumor growth. Additionally, multiple doses of the TF strains were well-tolerated by the mice. The use of B. subtilis LLO TF strains as EES showed promise as a unique cancer immunotherapy by directing immune function intracellularly. The uses of EES could be expanded to modulate other mammalian cells over a range of biomedical applications.

synthetic biology↗

bioRxiv2024

Magnetic particle imaging reveals that iron-labeled extracellular vesicles accumulate in brains of mice with metastases

The incidence of breast cancer remains high worldwide and is associated with a significant risk of metastasis to the brain that can be fatal; this is due, in part, to the inability of therapeutics to cross the blood brain barrier (BBB). Extracellular vesicles (EVs) have been found to cross the BBB and further, have been used to deliver drugs to tumors. EVs from different cell types appear to have different patterns of accumulation and retention as well as efficiency of bioactive cargo delivery to recipient cells in the body. Engineering EVs as delivery tools to treat brain metastases, therefore, will require an understanding of the timing of EV accumulation, and their localization relative to metastatic sites. Magnetic particle imaging (MPI) is a sensitive and quantitative imaging method that directly detects superparamagnetic iron. Here, we demonstrate MPI as a novel tool to characterize EV biodistribution in metastatic disease after labeling EVs with superparamagnetic iron oxide (SPIO) nanoparticles. Iron-labeled EVs (FeEVs) were collected from iron-labeled parental primary 4T1 tumor cells and brain-seeking 4T1BR5 cells, followed by injection into mice with orthotopic tumors or brain metastases. MPI quantification revealed that FeEVs were retained for longer in orthotopic mammary carcinomas compared to SPIOs. MPI signal due to iron could only be detected in brains of mice bearing brain metastases after injection of FeEVs, but not SPIOs, or FeEVs when mice did not have brain metastases. These findings indicate the potential use of EVs as a therapeutic delivery tool in primary and metastatic tumors. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=99 SRC="FIGDIR/small/584146v2_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@8cf21org.highwire.dtl.DTLVardef@56b6f0org.highwire.dtl.DTLVardef@104626eorg.highwire.dtl.DTLVardef@da60cd_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2024

Targeted intracellular delivery to hypoxic human breast cancer stem cells

Cancer stem cells (CSCs) drive tumorigenesis, are responsible for metastasis, and resist conventional therapies thus posing significant treatment challenges. CSCs reside in hypoxic tumor regions and therefore, effective therapies must target CSCs within this specific microenvironment. CSCs are characterized by limited distinguishable features, however, surface displayed phosphatidylserine (PS) appears to be characteristic of stem cells and offers a potential target. GlaS, a truncated coagulation protein that is internalized after binding PS, was investigated for intracellular delivery of molecular payloads to CSCs. Intracellular delivery via GlaS was enhanced in patient-derived CD44+ mammary CSCs under hypoxic conditions relative to physoxia or hyperoxia. In vivo, GlaS successfully targeted hypoxic tumor regions, and functional delivery of molecular cargo was confirmed using luciferin conjugated to GlaS via a disulfide linkage (GlaS-SS-luc), which releases luciferin upon intracellular glutathione reduction. Bioluminescence imaging demonstrated effective GlaS-mediated delivery of luciferin, a model drug, to CSCs in culture and in vivo. These findings offer the promise of directed delivery of therapeutic agents to intracellular targets in CSCs.

cancer biology↗

bioRxiv2023

Regulating the proinflammatory response to implanted composite biomaterials comprising polylactide and hydroxyapatite by targeting immunometabolism

Composite biomaterials comprising polylactide (PLA) and hydroxyapatite (HA) are applied in bone, cartilage and dental regenerative medicine, where HA confers osteoconductive properties. However, after surgical implantation, adverse immune responses to these composites can occur, which have been attributed to size and morphology of HA particles. Approaches to effectively modulate these adverse immune responses have not been described. PLA degradation products have been shown to alter immune cell metabolism, which drives the inflammatory response. Therefore, we aimed to modulate the inflammatory response to composite biomaterials by regulating glycolytic flux with small molecule inhibitors incorporated into composites comprised of amorphous PLA (aPLA) and HA (aPLA+HA). Inhibition at specific steps in glycolysis reduced proinflammatory (CD86+CD206-) and increased pro-regenerative (CD206+) immune cell populations around implanted aPLA+HA resulting in a pro-regenerative microenvironment. Notably, neutrophil and dendritic cell (DC) numbers along with proinflammatory monocyte and macrophage populations were decreased, and Arginase 1 expression among DCs was increased. Targeting immunometabolism to control the inflammatory response to biomaterial composites, and creating a pro-regenerative microenvironment, is a significant advance in tissue engineering where immunomodulation enhances osseointegration, and angiogenesis, which will lead to improved bone regeneration.

bioengineering↗

bioRxiv2023

Immunometabolic cues recompose and reprogram the microenvironment around biomaterials

Circulating monocytes infiltrate and coordinate immune responses in various inflamed tissues, such as those surrounding implanted biomaterials, affecting therapeutic, diagnostic, tissue engineering and regenerative applications. Here, we show that immunometabolic cues in the biomaterial microenvironment govern CCR2- and CX3CR1-dependent trafficking of immune cells, including neutrophils and monocytes; ultimately, this affects the composition and activation states of macrophage and dendritic cell populations. Furthermore, immunometabolic cues around implants orchestrate the relative composition of proinflammatory, transitory and anti-inflammatory CCR2+, CX3CR1+ and CCR2+CX3CR1+ immune cell populations. Consequently, modifying immunometabolism by glycolytic inhibition drives a pro-regenerative microenvironment in part by myeloid cells around amorphous polylactide implants. In addition to, Arginase 1-expressing myeloid cells, T helper 2 cells and {gamma}{delta}+ T-cells producing IL-4 significantly contribute to shaping the metabolically reprogramed, pro-regenerative microenvironment around crystalline polylactide biomaterials. Taken together, we find that local metabolic states regulate inflammatory processes in the biomaterial microenvironment, with implications for translational medicine.

bioengineering↗

bioRxiv2022

Elevated oxidative phosphorylation is critical for immune cell activation by polyethylene wear particles

Chronic inflammation is a major concern after total joint replacements (TJRs), as it is associated with bone loss, limited bone-implant integration (osseointegration), implant loosening and failure. Inflammation around implants could be directed away from adverse outcomes and toward enhanced osseointegration and improved surgical outcome. Activated macrophages exposed to polyethylene particles play a dominant inflammatory role, and exhibit elevated mitochondrial oxidative phosphorylation (OXPHOS) whose role is unclear. By probing the contribution of the electron transport chain (ETC), we show that increased oxygen consumption does not contribute to bioenergetic (ATP) levels in fibroblasts and primary bone marrow-derived macrophages activated by polyethylene particles. Rather, it generates reactive oxygen species (ROS) at complex I by increasing mitochondrial membrane potential in macrophages. Inhibition of OXPHOS in a dosedependent manner without affecting glycolysis was accomplished by targeting complex I of the ETC using either rotenone or metformin. Metformin decreased mitochondrial ROS and, subsequently, expression of proinflammatory cytokines, including IL-1{beta}, IL-6 and MCP-1 but not TNF-a in macrophages. These results highlight the contribution of mitochondrial bioenergetics to activation of immune cells by polyethylene wear particles, offering new opportunities to modulate macrophage states toward desired clinical outcomes.

bioengineering↗

bioRxiv2022

Polylactide Degradation Activates Immune Cells by Metabolic Reprogramming

Polylactide (PLA) is the most widely utilized biopolymer in medicine. However, chronic inflammation and excessive fibrosis resulting from its degradation remain significant obstacles to extended clinical use. Immune cell activation has been correlated to the acidity of breakdown products, yet methods to neutralize the pH have not significantly reduced adverse responses. Using a bioenergetic model, we observed delayed cellular changes that were not apparent in the short-term. Amorphous and semi-crystalline PLA degradation products, including monomeric L-lactic acid, mechanistically remodel metabolism in cells leading to a reactive immune microenvironment characterized by elevated proinflammatory cytokines. Selective inhibition of metabolic reprogramming and altered bioenergetics both reduce these undesirable high cytokine levels and stimulate anti-inflammatory signals. Our results present a new biocompatibility paradigm by identifying metabolism as a target for immunomodulation to increase tolerance to biomaterials, ensuring safe clinical application of PLA-based implants for soft- and hard-tissue regeneration, and advancing nanomedicine and drug delivery.

bioengineering↗

bioRxiv2022

Magnetothermal control of temperature-sensitive repressors in superparamagnetic iron nanoparticle-coated Bacillus subtilis

Superparamagnetic iron oxide nanoparticles (SPIONs) are used as contrast agents in magnetic resonance imaging (MRI) and magnetic particle imaging (MPI) and resulting images can be used to guide magnetothermal heating. Alternating magnetic fields (AMF) cause local temperature increases in regions with SPIONs, and we investigated the ability of magnetic hyperthermia to regulate temperature-sensitive repressors (TSRs) of bacterial transcription. The TSR, TlpA39, was derived from a Gram-negative bacterium, and used here for thermal control of reporter gene expression in Gram-positive, Bacillus subtilis. In vitro heating of B. subtilis with TlpA39 controlling bacterial luciferase expression, resulted in a 14.6-fold (12-hour; h) and 1.8-fold (1-h) increase in reporter transcripts with a 9.0-fold (12-h) and 11.1-fold (1-h) increase in bioluminescence. To develop magnetothermal control, B. subtilis cells were coated with three SPION variations. Electron microscopy coupled with energy dispersive X-ray spectroscopy revealed an external association with, and retention of, SPIONs on B. subtilis. Furthermore, using long duration AMF we demonstrated magnetothermal induction of the TSRs in SPION-coated B. subtilis with a maximum of 4.6-fold increases in bioluminescence. After intramuscular injections of SPION-coated B. subtilis, histology revealed that SPIONs remained in the same locations as the bacteria. For in vivo studies, 1-h of AMF is the maximum exposure due to anesthesia constraints. Both in vitro and in vivo, there was no change in bioluminescence after 1-h of AMF treatment. Pairing TSRs with magnetothermal energy using SPIONs for localized heating with AMF can lead to transcriptional control that expands options for targeted bacteriotherapies.

bioengineering↗

bioRxiv2022

MPI region of interest (ROI) analysis and quantification of iron in different volumes

MPI directly detects superparamagnetic iron oxides (SPIONs), which should enable precise, accurate, and linear quantification. However, selecting a region of interest (ROI) has strong effects on MPI quantification results. Ideally, ROI selection should be simple, user-independent, and widely applicable. In this work, we describe and compare four MPI ROI selection methods and assess their performance in vitro and in vivo. To explore the effect of ROI selection, ten ferucarbotran phantoms were imaged, each contained the same amount of iron but varied in volume. Three users tested the accuracy of the ROI methods for quantification of these samples. Lastly, the four ROI methods were applied to quantify ferucarbotran in vivo after intravenous, intramuscular, and subcutaneous injections in mice. We demonstrate that each ROI method has strengths. We conclude there is an important trade-off between ROI size and the accuracy of iron quantification, therefore the choice of ROI selection method for each study must be carefully informed.

biophysics↗

bioRxiv2021

Magnetic particle imaging of magnetotactic bacteria as living contrast agents is improved by altering magnetosome structures

Iron nanoparticles used as imaging contrast agents can help differentiate between normal and diseased tissue, or track cell movement and localize pathologies. Magnetic particle imaging (MPI) is an imaging modality that uses the magnetic properties of iron nanoparticles to provide specific, quantitative and sensitive imaging data. MPI signals depend on the size, structure and composition of the nanoparticles; MPI-tailored nanoparticles have been developed by modifying these properties. Magnetotactic bacteria produce magnetosomes which mimic synthetic nanoparticles, and thus comprise a living contrast agent in which nanoparticle formation can be modified by mutating genes. Specifically, genes that encode proteins critical to magnetosome formation and regulation, such as mamJ which helps with filament turnover. Deletion of mamJ in Magnetospirillum gryphiswaldense, MSR-1 led to clustered magnetosomes instead of the typical linear chains. Here we examined the effects of this magnetosome structure and revealed improved MPI signal and resolution from clustered magnetosomes compared to linear chains. Bioluminescent MSR-1 with the mamJ deletion were injected intravenously into tumor-bearing and healthy mice and imaged using both in vivo bioluminescence imaging (BLI) and MPI. BLI revealed the location and viability of bacteria which was used to validate localization of MPI signals. BLI identified the viability of MSR-1 for 24 hours and MPI detected iron in the liver and in multiple tumors. Development of living contrast agents offers new opportunities for imaging and therapy by using multimodality imaging to track the location and viability of the therapy and the resulting biological effects.

bioengineering↗

bioRxiv2021

Tracking the fates of iron-labeled tumor cells in vivo using Magnetic Particle Imaging

The use of imaging to detect and monitor the movement and accumulation of cells in living subjects can provide significant insights that can improve our understanding of metastasis and guide therapeutic development. For cell tracking using Magnetic Resonance Imaging (MRI), cells are labeled with iron oxides and the effects of the iron on water provides contrast. However, due to low specificity and difficulties in quantification with MRI, other modalities and approaches need to be developed. Magnetic Particle Imaging (MPI) is an emerging imaging technique which directly detects magnetic iron, allowing for a specific, quantitative and sensitive readout. Here, we use MPI to image iron-labeled tumor cells longitudinally, from implantation and growth at a primary site to movement to distant anatomic sites. In vivo bioluminescent imaging (BLI) was used to localize tumor metastases and computed tomography (CT) allowed for correlation of these signals to anatomic locations. These three imaging modalities provide information on immune escape and metastasis of iron-labeled, and unlabeled, tumor cells, and the accumulation of cell-free iron contrast over time. We identified iron signals by MPI and tumor cells via BLI, and correlated these positive contrast images with CT scans to reveal the anatomic sites with cancer cells; histologic analysis confirmed the presence of iron-labeled tumor cells in the tissues, suggesting that the metastatic cells retained enough iron for MPI detection. The use of multi-modality cell tracking reveals the movement, accumulation and fates of labeled cells that will be helpful understanding cancer progression and guiding the development of targeted therapies.

biophysics↗

bioRxiv2021

Engineered endosymbionts capable of directing mammalian cell gene expression

Modular methods for directing mammalian gene expression would enable advances in tissue regeneration, enhance cell-based therapeutics and improve modulation of immune responses. To address this challenge, engineered endosymbionts (EES) that escape endosomal destruction, reside in the cytoplasm of mammalian cells, and secrete proteins that are transported to the nucleus to control host cell gene expression were developed. Microscopy confirmed that EES escape phagosomes, replicate within the cytoplasm, and can secrete reporter proteins into the cytoplasm that were then transported to the nucleus. Synthetic operons encoding the mammalian transcription factors, Stat-1 and Klf6 or Klf4 and Gata-3 were recombined into the EES genome. Using controlled induction, these EES were shown to direct gene expression in J774A.1 macrophage/monocyte cells and modulate the host cell fates. Expressing mammalian transcription factors from engineered intracellular bacteria as endosymbionts comprises a new tool for directing host cell gene expression for therapeutic and research purposes. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=194 SRC="FIGDIR/small/463266v2_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@2b5935org.highwire.dtl.DTLVardef@1edd8edorg.highwire.dtl.DTLVardef@3cb5b9org.highwire.dtl.DTLVardef@1be9b5f_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2020

Development of Magnetic Particle Imaging (MPI) for Cell Tracking and Detection

IntroductionMagnetic particle imaging (MPI) is a new imaging modality that sensitively and specifically detects superparamagnetic iron oxide nanoparticles (SPIONs) within a sample. SPION-based MRI cell tracking has very high sensitivity, but low specificity and quantification of iron labeled cells is difficult. MPI cell tracking could overcome these challenges. MethodsMDM-AB-231BR cells labeled with MPIO, mice were intracardially injected with either 2.5 x 105 or 5.0 x 105 cells. MRI was performed in vivo the same day at 3T using a bSSFP sequence. After mice were imaged ex vivo with MPI. In a second experiment Mice received an intracardiac injection of either 2.5 x 10 5 or 5 x 10 4 MPIO-labeled 231BR cells. In a third experiment, mice were injected with 5 x 10 4 4T1BR cells, labelled with either MPIO or the SPION Vivotrax. MRI and MPI was performed in vivo. ResultsSignal from MPI and signal voids from MRI both showed more iron content in mice receiving an injection of 5.0 x 105 cells than the 2.5 x 105 injection. In the second experiment, Day 0 MRI showed signal voids and MPI signal was detected in all mouse brains. The MPI signal and iron content measured in the brains of mice that were injected with 2.5 x 10 5 cells were approximately four times greater than in brains injected with 5 x 10 4 cells. In the third experiment, in vivo MRI was able to detect signal voids in the brains of mice injected with Vivotrax and MPIO, although voids were fainter in Vivotrax labeled cells. In vivo MPI signal was only detectable in mice injected with MPIO-labeled cells. ConclusionThis is the first example of the use of MPIO for cell tracking with MPI. With an intracardiac cell injection, approximately 15% of the injected cells are expected to arrest in the brain vasculature. For our lowest cell injection of 5.0 x 104 cells this is [~]10000 cells.

biophysics↗