Electrostatic adsorption of polyanions onto lipid nanoparticles controls uptake, trafficking, and transfection of RNA and DNA therapies.

Rapid advances in nucleic acid therapies highlight the immense therapeutic potential of genetic therapeutics. Lipid nanoparticles (LNPs) are highly potent nonviral transfection agents that can encapsulate and deliver various nucleic acid therapeutics, including but not limited to messenger RNA (mRNA), silencing RNA (siRNA), and plasmid DNA (pDNA). However, a major challenge of targeted LNP-mediated systemic delivery is the nanoparticles' nonspecific uptake by the liver and the mononuclear phagocytic system, due partly to the adsorption of endogenous serum proteins onto LNP surfaces. Tunable LNP surface chemistries may enable efficacious delivery across a range of organs and cell types. Here, we describe a method to electrostatically adsorb bioactive polyelectrolytes onto LNPs to create layered LNPs (LLNPs). LNP cores varying in nucleic acid cargo and component lipids were stably layered with four biologically relevant polyanions: hyaluronate (HA), poly-L-aspartate (PLD), poly-L-glutamate (PLE), and polyacrylate (PAA). We further investigated the impact of the four surface polyanions on the transfection and uptake of mRNA- and pDNA-loaded LNPs in cell cultures. PLD- and PLE-LLNPs increased mRNA transfection twofold over unlayered LNPs in immune cells. HA-LLNPs increased pDNA transfection rates by more than twofold in epithelial and immune cells. In a healthy C57BL/6 murine model, PLE- and HA-LLNPs increased transfection by 1.8-fold to 2.5-fold over unlayered LNPs in the liver and spleen. These results suggest that LbL assembly is a generalizable, highly tunable platform to modify the targeting specificity, stability, and transfection efficacy of LNPs, as well as incorporate other charged targeting and therapeutic molecules into these systems.

PTPmu-targeted nanoparticles label invasive pediatric and adult glioblastoma.

Poor prognosis for glioblastoma (GBM) is a consequence of the aggressive and infiltrative nature of gliomas where individual cells migrate away from the main tumor to distant sites, making complete surgical resection and treatment difficult. In this manuscript, we characterize an invasive pediatric glioma model and determine if nanoparticles linked to a peptide recognizing the GBM tumor biomarker PTPmu can specifically target both the main tumor and invasive cancer cells in adult and pediatric glioma models. Using both iron and lipid-based nanoparticles, we demonstrate by magnetic resonance imaging, optical imaging, histology, and iron quantification that PTPmu-targeted nanoparticles effectively label adult gliomas. Using PTPmu-targeted nanoparticles in a newly characterized orthotopic pediatric SJ-GBM2 model, we demonstrate individual tumor cell labeling both within the solid tumor margins and at invasive and dispersive sites.

Effect of Dose and Selection of Two Different Ligands on the Deposition and Antitumor Efficacy of Targeted Nanoparticles in Brain Tumors.

Deposition of nanoparticles to tumors often can be enhanced by targeting receptors overexpressed in a tumor. However, a tumor may exhibit a finite number of a biomarker that is accessible and targetable by nanoparticles, limiting the available landing spots. To explore this, we selected two different biomarkers that effectively home nanoparticles in brain tumors. Specifically, we used either an αvß3 integrin-targeting peptide or a fibronectin-targeting peptide as a ligand on nanoparticles termed RGD-NP and CREKA-NP, respectively. In mouse models of glioblastoma multiforme, we systemically injected the nanoparticles loaded with a cytotoxic drug at different doses ranging from 2 to 8 mg/kg drug. The upper dose threshold of RGD-NP is ∼2 mg/kg. CREKA-NP reached its upper dose threshold at 5 mg/kg. For both targeted nanoparticle variants, higher dose did not ensure higher intratumoral drug levels, but it contributed to elevated off-target deposition and potentially greater toxicity. A cocktail combining RGD-NP and CREKA-NP was then administered at a dose corresponding to the upper dose threshold for each formulation resulting in a 3-fold higher intratumoral deposition than the individual formulations. The combination of the two different targeting schemes at the appropriate dose for each nanoparticle variant facilitated remarkable increase in intratumoral drug levels that was not achievable by a sole targeting nanoparticle alone.

STING Protein-Based In Situ Vaccine Synergizes CD4+ T, CD8+ T, and NK Cells for Tumor Eradication.

Stimulator of interferon genes (STING) signaling is a promising target in cancer immunotherapy, with many ongoing clinical studies in combination with immune checkpoint blockade (ICB). Existing STING-based therapies largely focus on activating CD8+ T cell or NK cell-mediated cytotoxicity, while the role of CD4+ T cells in STING signaling has yet to be extensively studied in vivo. Here, a distinct CD4-mediated, protein-based combination therapy of STING and ICB as an in situ vaccine, is reported. The treatment eliminates subcutaneous MC38 and YUMM1.7 tumors in 70-100% of mice and protected all cured mice against rechallenge. Mechanistic studies reveal a robust TH 1 polarization and suppression of Treg of CD4+ T cells, followed by an effective collaboration of CD4+ T, CD8+ T, and NK cells to eliminate tumors. Finally, the potential to overcome host STING deficiency by significantly decreasing MC38 tumor burden in STING KO mice is demonstrated, addressing the translational challenge for the 19% of human population with loss-of-function STING variants.

Synergistic combination therapy delivered via layer-by-layer nanoparticles induces solid tumor regression of ovarian cancer.

The majority of patients with high grade serous ovarian cancer (HGSOC) develop recurrent disease and chemotherapy resistance. To identify drug combinations that would be effective in treatment of chemotherapy resistant disease, we examined the efficacy of drug combinations that target the three antiapoptotic proteins most commonly expressed in HGSOC-BCL2, BCL-XL, and MCL1. Co-inhibition of BCL2 and BCL-XL (ABT-263) with inhibition of MCL1 (S63845) induces potent synergistic cytotoxicity in multiple HGSOC models. Since this drug combination is predicted to be toxic to patients due to the known clinical morbidities of each drug, we developed layer-by-layer nanoparticles (LbL NPs) that co-encapsulate these inhibitors in order to target HGSOC tumor cells and reduce systemic toxicities. We show that the LbL NPs can be designed to have high association with specific ovarian tumor cell types targeted in these studies, thus enabling a more selective uptake when delivered via intraperitoneal injection. Treatment with these LbL NPs displayed better potency than free drugs in vitro and resulted in near-complete elimination of solid tumor metastases of ovarian cancer xenografts. Thus, these results support the exploration of LbL NPs as a strategy to deliver potent drug combinations to recurrent HGSOC. While these findings are described for co-encapsulation of a BCL2/XL and a MCL1 inhibitor, the modular nature of LbL assembly provides flexibility in the range of therapies that can be incorporated, making LbL NPs an adaptable vehicle for delivery of additional combinations of pathway inhibitors and other oncology drugs.

Frank's sign associated with the severity of ischemic heart disease in patients under 65 years old.

BACKGROUND AND OBJECTIVE: Frank's sign is the diagonal ear fold which has been associated with ischemic heart disease. The objective of this work was to evaluate the relationship of Frank's sign with severity of ischemic heart disease in adults ≤ 65 years old in the northeast of Mexico. PATIENTS AND METHODS: A cross-sectional study was conducted in patients ≤ 65 years old who underwent coronary angiography consecutively over a period of 5 months in 2022. Severe coronary artery disease (CAD) was associated with Frank's sign and other common cardiovascular risks. To determine the association, bivariate and multivariate analysis was performed using logistic regression that included variables with a value of p<0.05. Statistical analysis was performed with SPSS version 22. RESULTS: We included 311 patients ≤ 65 years, of whom 80% were men. The median age was 57 years (range 28-65). Frank's sign was positive in 62% of the population. The main clinical characteristics in patients with Frank's sign were type 2 diabetes mellitus (55%), p=0.003, dyslipidemia (53%), p=0.026 and smoking (68%), p=0.002. In the multivariate analysis, the independent variables associated with severe CAD were Frank's Sign OR 3.26; 95% CI (1.98-5.38), p≤0.001, male gender OR 2.28; 95% CI (1.20-4.35), p=0.012, and dyslipidemia OR 1.81; 95% CI (1.11-2.97), p=0.017. CONCLUSIONS: There is an independent association between Frank's sign with the presence of severe CAD in patients ≤ 65 years old, which may be useful for screening and prevention.

Comparison of the uptake of untargeted and targeted immunostimulatory nanoparticles by immune cells in the microenvironment of metastatic breast cancer.

To alter the immunosuppressive tumor microenvironment (TME), we developed an immunostimulatory nanoparticle (NP) to reprogram a tumor's dysfunctional and inhibitory antigen-presenting cells (APCs) into properly activated APCs that stimulate tumor-reactive cytotoxic T cells. Importantly, systemic delivery allowed NPs to efficiently utilize the entire microvasculature and gain access into the majority of the perivascular TME, which coincided with the APC-rich tumor areas leading to uptake of the NPs predominantly by APCs. In this work, a 60 nm NP was loaded with a STING agonist, which triggered robust production of interferon ß, resulting in activation of APCs. In addition to untargeted NPs, we employed 'mainstream' ligands targeting fibronectin, αvß3 integrin and P-selectin that are commonly used to direct nanoparticles to tumors. Using the 4T1 mouse model, we assessed the microdistribution of the four NP variants in the tumor immune microenvironment in three different breast cancer landscapes, including primary tumor, early metastasis, and late metastasis. The different NP variants resulted in variable uptake by immune cell subsets depending on the organ and tumor stage. Among the NP variants, therapeutic studies indicated that the untargeted NPs and the integrin-targeting NPs exhibited a remarkable short- and long-term immune response and long-lasting antitumor effect.

The effect of PEGylation on the efficacy and uptake of an immunostimulatory nanoparticle in the tumor immune microenvironment.

The efficacy of immunotherapies is often limited by the immunosuppressive tumor microenvironment, which is populated with dysfunctional innate immune cells. To reprogram the tumor-resident innate immune cells, we developed immunostimulatory silica mesoporous nanoparticles (immuno-MSN). The cargo of immuno-MSN is a Stimulator of Interferon Gene (STING) agonist, which activates innate immune cells leading to production of interferon (IFN) ß. By proficiently trafficking its cargo into immune cells, the immuno-MSN induced a 9-fold increase of IFN-ß secretion compared to free agonist. While an external PEG shield has historically been used to protect nanoparticles from immune recognition, a PEGylated immunostimulatory nanoparticle needs to strike a balance between immune evasion to avoid off-site accumulation and uptake by target immune cells in tumors. Using the 4T1 mouse model of metastatic breast cancer and flow cytometry, it was determined that the degree of PEGylation significantly influenced the uptake of 'empty' MSNs by tumor-resident innate immune cells. This was not the case for the agonist-loaded immuno-MSN variants. It should be noted the surface charge of the 'empty' MSNs was positive rather than neutral for the agonist-loaded immuno-MSNs. However, even though the cellular uptake was similar at 24 h after injection for the three immuno-MSN variants, we observed a significant beneficial effect on the activation and expansion of APCs especially in lung metastasis using the lightly PEGylated immuno-MSN variant.

Immunostimulatory silica nanoparticle boosts innate immunity in brain tumors.

The high mortality associated with glioblastoma multiforme (GBM) is attributed to its invasive nature, hypoxic core, resistant cell subpopulations and a highly immunosuppressive tumor microenvironment (TME). To support adaptive immune function and establish a more robust antitumor immune response, we boosted the local innate immune compartment of GBM using an immunostimulatory mesoporous silica nanoparticle, termed immuno-MSN. The immuno-MSN was specifically designed for systemic and proficient delivery of a potent innate immune agonist to dysfunctional antigen-presenting cells (APCs) in the brain TME. The cargo of the immuno-MSN was cyclic diguanylate monophosphate (cdGMP), a Stimulator of Interferon Gene (STING) agonist. Studies showed the immuno-MSN promoted the uptake of STING agonist by APCs in vitro and the subsequent release of the pro-inflammatory cytokine interferon ß, 6-fold greater than free agonist. In an orthotopic GBM mouse model, systemically administered immuno-MSN particles were taken up by APCs in the near-perivascular regions of the brain tumor with striking efficiency. The immuno-MSNs facilitated the recruitment of dendritic cells and macrophages to the TME while sparing healthy brain tissue and peripheral organs, resulting in elevated circulating CD8+ T cell activity (2.5-fold) and delayed GBM tumor growth. We show that an engineered immunostimulatory nanoparticle can support pro-inflammatory innate immune function in GBM and subsequently augment current immunotherapeutic interventions and improve their therapeutic outcome.

Hyperthermia-mediated changes in the tumor immune microenvironment using iron oxide nanoparticles.

Iron oxide nanoparticles (IONPs) have often been investigated for tumor hyperthermia. IONPs act as heating foci in the presence of an alternating magnetic field (AMF). It has been shown that hyperthermia can significantly alter the tumor immune microenvironment. Typically, mild hyperthermia invokes morphological changes within the tumor, which elicits a secretion of inflammatory cytokines and tumor neoantigens. Here, we focused on the direct effect of IONP-induced hyperthermia on the various tumor-resident immune cell subpopulations. We compared direct intratumoral injection to systemic administration of IONPs followed by application of an external AMF. We used the orthotopic 4T1 mouse model, which represents aggressive and metastatic breast cancer with a highly immunosuppressive microenvironment. A non-inflamed and 'cold' microenvironment inhibits peripheral effector lymphocytes from effectively trafficking into the tumor. Using intratumoral or systemic injection, IONP-induced hyperthermia achieved a significant reduction of all the immune cell subpopulations in the tumor. However, the systemic delivery approach achieved superior outcomes, resulting in substantial reductions in the populations of both innate and adaptive immune cells. Upon depletion of the existing dysfunctional tumor-resident immune cells, subsequent treatment with clinically approved immune checkpoint inhibitors encouraged the repopulation of the tumor with 'fresh' infiltrating innate and adaptive immune cells, resulting in a significant decrease of the tumor cell population.

Chronic neural activity recorded within breast tumors.

Nerve fibers are known to reside within malignant tumors and the greater the neuronal density the worse prognosis for the patient. Recent discoveries using tumor bearing animal models have eluded to the autonomic nervous system having a direct effect on tumor growth and metastasis. We report the first direct and chronic in vivo measurements of neural activity within tumors. Using a triple-negative mammary cancer mouse model and chronic neural interface techniques, we have recorded neural activity directly within the tumor mass while the tumor grows and metastasizes. The results indicate that there is a strong connection between the autonomic nervous system and the tumor and could help uncover the mechanisms of tumor growth and metastasis.

Effective treatment of cancer metastasis using a dual-ligand nanoparticle.

Metastasis is responsible for the majority of deaths of breast cancer patients. While cytotoxic drugs are available with high potency to kill breast cancer cells, they are not designed to specifically seek and navigate in the dynamic and continuously changing microenvironment of metastatic disease. To effectively delivery chemotherapeutic agents to metastasis, we designed a dual-ligand nanoparticle loaded with doxorubicin by using two different types of ligands targeting EGFR and αvß3 integrin. Metastatic cancer cells continuously change resulting in heterogeneity even across adjacent micrometastatic regions with variable expression of these targetable receptors. Using a mouse model of breast cancer metastasis, in vivo and ex vivo imaging showed that both EGFR and αvß3 integrin-targeting were required to reliably direct the nanoparticle to metastasis and capture the spread and exact topology of the disease. Survival studies compared the anticancer efficacy of the standard drug, EGFR-targeting nanoparticle, αvß3 integrin-targeting nanoparticle and the dual-ligand nanoparticle. While all the other treatments produced moderate therapeutic outcomes, treatment with the dual-ligand nanoparticle yielded significant improvement and event-free survival in a mouse model of breast cancer metastasis.

Nanoparticle Encapsulation of Synergistic Immune Agonists Enables Systemic Codelivery to Tumor Sites and IFNß-Driven Antitumor Immunity.

Effective cancer immunotherapy depends on the robust activation of tumor-specific antigen-presenting cells (APC). Immune agonists encapsulated within nanoparticles (NP) can be delivered to tumor sites to generate powerful antitumor immune responses with minimal off-target dissemination. Systemic delivery enables widespread access to the microvasculature and draining to the APC-rich perivasculature. We developed an immuno-nanoparticle (immuno-NP) coloaded with cyclic diguanylate monophosphate, an agonist of the stimulator of interferon genes pathway, and monophosphoryl lipid A, and a Toll-like receptor 4 agonist, which synergize to produce high levels of type I IFNß. Using a murine model of metastatic triple-negative breast cancer, systemic delivery of these immuno-NPs resulted in significant therapeutic outcomes due to extensive upregulation of APCs and natural killer cells in the blood and tumor compared with control treatments. These results indicate that NPs can facilitate systemic delivery of multiple immune-potentiating cargoes for effective APC-driven local and systemic antitumor immunity. SIGNIFICANCE: Systemic administration of an immuno-nanoparticle in a murine breast tumor model drives a robust tumor site-specific APC response by delivering two synergistic immune-potentiating molecules, highlighting the potential of nanoparticles for immunotherapy.

Imaging breast cancer using a dual-ligand nanochain particle.

Nanoparticles often only exploit the upregulation of a receptor on cancer cells to enhance intratumoral deposition of therapeutic and imaging agents. However, a single targeting moiety assumes that a tumor is homogenous and static. Tumoral microenvironments are both heterogenous and dynamic, often displaying variable spatial and temporal expression of targetable receptors throughout disease progression. Here, we evaluated the in vivo performance of an iron oxide nanoparticle in terms of targeting and imaging of orthotropic mouse models of aggressive breast tumors. The nanoparticle, a multi-component nanochain, was comprised of 3-5 iron oxide nanoparticles chemically linked in a linear chain. The nanoparticle's surface was decorated with two types of ligands each targeting two different upregulated biomarkers on the tumor endothelium, P-selectin and fibronectin. The nanochain exhibited improved tumor deposition not only through vascular targeting but also through its elongated structure. A single-ligand nanochain exhibited a ~2.5-fold higher intratumoral deposition than a spherical nanoparticle variant. Furthermore, the dual-ligand nanochain exhibited higher consistency in generating detectable MR signals compared to a single-ligand nanochain. Using a 7T MRI, the dual-ligand nanochains exhibited highly detectable MR signal within 3h after injection in two different animal models of breast cancer.

Vascular targeting of nanoparticles for molecular imaging of diseased endothelium.

This review seeks to highlight the enormous potential of targeted nanoparticles for molecular imaging applications. Being the closest point-of-contact, circulating nanoparticles can gain direct access to targetable molecular markers of disease that appear on the endothelium. Further, nanoparticles are ideally suitable to vascular targeting due to geometrically enhanced multivalent attachment on the vascular target. This natural synergy between nanoparticles, vascular targeting and molecular imaging can provide new avenues for diagnosis and prognosis of disease with quantitative precision. In addition to the obvious applications of targeting molecular signatures of vascular diseases (e.g., atherosclerosis), deep-tissue diseases often manifest themselves by continuously altering and remodeling their neighboring blood vessels (e.g., cancer). Thus, the remodeled endothelium provides a wide range of targets for nanoparticles and molecular imaging. To demonstrate the potential of molecular imaging, we present a variety of nanoparticles designed for molecular imaging of cancer or atherosclerosis using different imaging modalities.

Physical and Chemical Signals That Promote Vascularization of Capillary-Scale Channels.

Proper vascularization remains critical to the clinical application of engineered tissues. To engineer microvessels in vitro, we and others have delivered endothelial cells through preformed channels into patterned extracellular matrix-based gels. This approach has been limited by the size of endothelial cells in suspension, and results in plugging of channels below ~30 µm in diameter. Here, we examine physical and chemical signals that can augment direct seeding, with the aim of rapidly vascularizing capillary-scale channels. By studying tapered microchannels in type I collagen gels under various conditions, we establish that stiff scaffolds, forward pressure, and elevated cyclic AMP levels promote endothelial stability and that reverse pressure promotes endothelial migration. We applied these results to uniform 20-µm-diameter channels and optimized the magnitudes of pressure, flow, and shear stress to best support endothelial migration and vascular stability. This vascularization strategy is able to form millimeter-long perfusable capillaries within three days. Our results indicate how to manipulate the physical and chemical environment to promote rapid vascularization of capillary-scale channels within type I collagen gels.