Loss of p53 drives neuron reprogramming in head and neck cancer.

The solid tumour microenvironment includes nerve fibres that arise from the peripheral nervous system1,2. Recent work indicates that newly formed adrenergic nerve fibres promote tumour growth, but the origin of these nerves and the mechanism of their inception are unknown1,3. Here, by comparing the transcriptomes of cancer-associated trigeminal sensory neurons with those of endogenous neurons in mouse models of oral cancer, we identified an adrenergic differentiation signature. We show that loss of TP53 leads to adrenergic transdifferentiation of tumour-associated sensory nerves through loss of the microRNA miR-34a. Tumour growth was inhibited by sensory denervation or pharmacological blockade of adrenergic receptors, but not by chemical sympathectomy of pre-existing adrenergic nerves. A retrospective analysis of samples from oral cancer revealed that p53 status was associated with nerve density, which was in turn associated with poor clinical outcomes. This crosstalk between cancer cells and neurons represents mechanism by which tumour-associated neurons are reprogrammed towards an adrenergic phenotype that can stimulate tumour progression, and is a potential target for anticancer therapy.

Rapamycin-Loaded Biomimetic Nanoparticles Reverse Vascular Inflammation.

RATIONALE: Through localized delivery of rapamycin via a biomimetic drug delivery system, it is possible to reduce vascular inflammation and thus the progression of vascular disease. OBJECTIVE: Use biomimetic nanoparticles to deliver rapamycin to the vessel wall to reduce inflammation in an in vivo model of atherosclerosis after a short dosing schedule. METHODS AND RESULTS: Biomimetic nanoparticles (leukosomes) were synthesized using membrane proteins purified from activated J774 macrophages. Rapamycin-loaded nanoparticles were characterized using dynamic light scattering and were found to have a diameter of 108±2.3 nm, a surface charge of -15.4±14.4 mV, and a polydispersity index of 0.11 +/ 0.2. For in vivo studies, ApoE-/- mice were fed a high-fat diet for 12 weeks. Mice were injected with either PBS, free rapamycin (5 mg/kg), or rapamycin-loaded leukosomes (Leuko-Rapa; 5 mg/kg) once daily for 7 days. In mice treated with Leuko-Rapa, flow cytometry of disaggregated aortic tissue revealed fewer proliferating macrophages in the aorta (15.6±9.79 %) compared with untreated mice (30.2±13.34 %) and rapamycin alone (26.8±9.87 %). Decreased macrophage proliferation correlated with decreased levels of MCP (monocyte chemoattractant protein)-1 and IL (interleukin)-b1 in mice treated with Leuko-Rapa. Furthermore, Leuko-Rapa-treated mice also displayed significantly decreased MMP (matrix metalloproteinases) activity in the aorta (mean difference 2554±363.9, P=9.95122×10-6). No significant changes in metabolic or inflammation markers observed in liver metabolic assays. Histological analysis showed improvements in lung morphology, with no alterations in heart, spleen, lung, or liver in Leuko-Rapa-treated mice. CONCLUSIONS: We showed that our biomimetic nanoparticles showed a decrease in proliferating macrophage population that was accompanied by the reduction of key proinflammatory cytokines and changes in plaque morphology. This proof-of-concept showed that our platform was capable of suppressing macrophage proliferation within the aorta after a short dosing schedule (7 days) and with a favorable toxicity profile. This treatment could be a promising intervention for the acute stabilization of late-stage plaques.

Biomimetic Nanoparticles as a Theranostic Tool for Traumatic Brain Injury.

Traumatic brain injury (TBI) triggers both central and peripheral inflammatory responses. Existing pharmacological drugs are unable to effectively and quickly target the brain inflamed regions, setting up a major roadblock towards effective brain trauma treatments. Nanoparticles (NPs) have been used in multiple diseases as drug delivery tools with remarkable success due to their rapid diffusion and specificity in the target organ. Here, leukocyte-based biomimetic NPs are fabricated as a theranostic tool to directly access inflamed regions in a TBI mouse model. This NP systemic delivery is visualized using advanced in vivo imaging techniques, including intravital microscopy and in vivo imaging system. The results demonstrate selective targeting of NPs to the injured brain and increased NPs accumulation among the peripheral organs 24 h after TBI. Interestingly, increased microglial proliferation, decreased macrophage infiltration, and reduced brain lesion following the NPs treatments compared to sham vehicle-treated mice are also found. In summary, the results suggest that NPs represent a promising future theranostic tool for TBI treatment.

Intravital imaging of vascular anomalies and extracellular matrix remodeling in orthotopic pancreatic tumors.

Pancreatic cancers, both adenocarcinomas and endocrine tumors are characterized by varying levels of aberrant angiogenesis and fibrotic microenvironment. The difficulty to deliver drugs and treat the disease has been attributed in part to the vascular architecture and tissue/ECM density. Here we present longitudinal three-dimensional intravital imaging of vascular and tumor microenvironment remodeling in spontaneous transgenic tumors (RIP1-Tag2 insulinomas) and orthotopically injected tumors (KPC adenocarcinomas). Analysis of the data acquired in insulinomas revealed major differences in tumor blood vessel branching, fraction volume, number of branch points segments, vessel straightness and length compared to the normal tissue. The aggressive adenocarcinoma presented widespread peritumoral vascular remodeling and heterogeneous vascular distribution. Longitudinal imaging was used to acquire sequential vascular remodeling data during tumor progression. This work demonstrates the potential for using a pancreatic intravital imaging window for direct visualization of the tumor heterogenic microenvironments during tumor progression.

Nanoparticles target early-stage breast cancer metastasis in vivo.

Despite advances in cancer therapy, treating cancer after it has metastasized remains an unmet clinical challenge. In this study we demonstrate that 100 nm liposomes target triple-negative murine breast-cancer metastases post intravenous administration. Metastatic breast cancer was induced in BALB/c mice either experimentally, by a tail vein injection of 4T1 cells, or spontaneously, after implanting a primary tumor xenograft. To track their biodistribution in vivo the liposomes were labeled with multi-modal diagnostic agents, including indocyanine green and rhodamine for whole-animal fluorescent imaging, gadolinium for magnetic resonance imaging (MRI), and europium for a quantitative biodistribution analysis. The accumulation of liposomes in the metastases peaked at 24 h post the intravenous administration, similar to the time they peaked in the primary tumor. The efficiency of liposomal targeting to the metastatic tissue exceeded that of a non-liposomal agent by 4.5-fold. Liposomes were detected at very early stages in the metastatic progression, including metastatic lesions smaller than 2 mm in diameter. Surprisingly, while nanoparticles target breast cancer metastasis, they may also be found in elevated levels in the pre-metastatic niche, several days before metastases are visualized by MRI or histologically in the tissue. This study highlights the promise of diagnostic and therapeutic nanoparticles for treating metastatic cancer, possibly even for preventing the onset of the metastatic dissemination by targeting the pre-metastatic niche.

Harnessing RNA Technology to Advance Therapeutic Vaccine Antigens against Chagas Disease.

Chagas disease (CD) (American trypanosomiasis caused by Trypanosoma cruzi) is a parasitic disease endemic in 21 countries in South America, with increasing global spread. When administered late in the infection, the current antiparasitic drugs do not prevent the onset of cardiac illness leading to chronic Chagasic cardiomyopathy. Therefore, new therapeutic vaccines or immunotherapies are under development using multiple platforms. In this study, we assessed the feasibility of developing an mRNA-based therapeutic CD vaccine targeting two known T. cruzi vaccine antigens (Tc24─a flagellar antigen and ASP-2─an amastigote antigen). We present the mRNA engineering steps, preparation, and stability of the lipid nanoparticles and evaluation of their uptake by dendritic cells, as well as their biodistribution in c57BL/J mice. Furthermore, we assessed the immunogenicity and efficacy of two mRNA-based candidates as monovalent and bivalent vaccine strategies using an in vivo chronic mouse model of CD. Our results show several therapeutic benefits, including reductions in parasite burdens and cardiac inflammation, with each mRNA antigen, especially with the mRNA encoding Tc24, and Tc24 in combination with ASP-2. Therefore, our findings demonstrate the potential of mRNA-based vaccines as a therapeutic option for CD and highlight the opportunities for developing multivalent vaccines using this approach.

Unleashing the potential of cell biomimetic nanoparticles: Strategies and challenges in their design and fabrication for therapeutic applications.

In this contribution to the Orations - New Horizons of the Journal of Controlled Release, I present design strategies for the two main biomimetic nanoparticles (BNP) groups: BNP composed of isolated cell membrane proteins, and BNP composed of the native cell membrane. I furthermore describe BNP fabrication methods and analyze their advantages and challenges. Finally, I suggest future therapeutic applications of each BNP group and propose a new revolutionary concept for their use.

Sex-dependent improvement in traumatic brain injury outcomes after liposomal delivery of dexamethasone in mice.

Traumatic Brain Injury (TBI) can have long-lasting physical, emotional, and cognitive consequences due to the neurodegeneration caused by its robust inflammatory response. Despite advances in rehabilitation care, effective neuroprotective treatments for TBI patients are lacking. Furthermore, current drug delivery methods for TBI treatment are inefficient in targeting inflamed brain areas. To address this issue, we have developed a liposomal nanocarrier (Lipo) encapsulating dexamethasone (Dex), an agonist for the glucocorticoid receptor utilized to alleviate inflammation and swelling in various conditions. In vitro studies show that Lipo-Dex were well tolerated in human and murine neural cells. Lipo-Dex showed significant suppression of inflammatory cytokines, IL-6 and TNF-α, release after induction of neural inflammation with lipopolysaccharide. Further, the Lipo-Dex were administered to young adult male and female C57BL/6 mice immediately after a controlled cortical impact injury. Our findings demonstrate that Lipo-Dex can selectively target the injured brain, thereby reducing lesion volume, cell death, astrogliosis, the release of proinflammatory cytokines, and microglial activation compared to Lipo-treated mice in a sex-dependent manner, showing a major impact only in male mice. This highlights the importance of considering sex as a crucial variable in developing and evaluating new nano-therapies for brain injury. These results suggest that Lipo-Dex administration may effectively treat acute TBI.

Artificially Intelligent Nanoarray Detects Various Cancers by Liquid Biopsy of Volatile Markers.

Cancer is usually not symptomatic in its early stages. However, early detection can vastly improve prognosis. Liquid biopsy holds great promise for early detection, although it still suffers from many disadvantages, mainly searching for specific cancer biomarkers. Here, a new approach for liquid biopsies is proposed, based on volatile organic compound (VOC) patterns in the blood headspace. An artificial intelligence nanoarray based on a varied set of chemi-sensitive nano-based structured films is developed and used to detect and stage cancer. As a proof-of-concept, three cancer models are tested showing high incidence and mortality rates in the population: breast cancer, ovarian cancer, and pancreatic cancer. The nanoarray has >84% accuracy, >81% sensitivity, and >80% specificity for early detection and >97% accuracy, 100% sensitivity, and >88% specificity for metastasis detection. Complementary mass spectrometry analysis validates these results. The ability to analyze such a complex biological fluid as blood, while considering data of many VOCs at a time using the artificially intelligent nanoarray, increases the sensitivity of predictive models and leads to a potential efficient early diagnosis and disease-monitoring tool for cancer.

Nanoparticles Accumulate in the Female Reproductive System during Ovulation Affecting Cancer Treatment and Fertility.

Throughout the female menstrual cycle, physiological changes occur that affect the biodistribution of nanoparticles within the reproductive system. We demonstrate a 2-fold increase in nanoparticle accumulation in murine ovaries and uterus during ovulation, compared to the nonovulatory stage, following intravenous administration. This biodistribution pattern had positive or negative effects when drug-loaded nanoparticles, sized 100 nm or smaller, were used to treat different cancers. For example, treating ovarian cancer with nanomedicines during mouse ovulation resulted in higher drug accumulation in the ovaries, improving therapeutic efficacy. Conversely, treating breast cancer during ovulation, led to reduced therapeutic efficacy, due to enhanced nanoparticle accumulation in the reproductive system rather than at the tumor site. Moreover, chemotherapeutic nanoparticles administered during ovulation increased ovarian toxicity and decreased fertility compared to the free drug. The menstrual cycle should be accounted for when designing and implementing nanomedicines for females.

Enhancing Inflammation Targeting Using Tunable Leukocyte-Based Biomimetic Nanoparticles.

Biomimetic nanoparticles aim to effectively emulate the behavior of either cells or exosomes. Leukocyte-based biomimetic nanoparticles, for instance, incorporate cell membrane proteins to transfer the natural tropism of leukocytes to the final delivery platform. However, tuning the protein integration can affect the in vivo behavior of these nanoparticles and alter their efficacy. Here we show that, while increasing the protein:lipid ratio to a maximum of 1:20 (w/w) maintained the nanoparticle's structural properties, increasing protein content resulted in improved targeting of inflamed endothelium in two different animal models. Our combined use of a microfluidic, bottom-up approach and tuning of a key synthesis parameter enabled the synthesis of reproducible, enhanced biomimetic nanoparticles that have the potential to improve the treatment of inflammatory-based conditions through targeted nanodelivery.

Humanized Biomimetic Nanovesicles for Neuron Targeting.

Nanovesicles (NVs) are emerging as innovative, theranostic tools for cargo delivery. Recently, surface engineering of NVs with membrane proteins from specific cell types has been shown to improve the biocompatibility of NVs and enable the integration of functional attributes. However, this type of biomimetic approach has not yet been explored using human neural cells for applications within the nervous system. Here, this paper optimizes and validates the scalable and reproducible production of two types of neuron-targeting NVs, each with a distinct lipid formulation backbone suited to potential therapeutic cargo, by integrating membrane proteins that are unbiasedly sourced from human pluripotent stem-cell-derived neurons. The results establish that both endogenous and genetically engineered cell-derived proteins effectively transfer to NVs without disruption of their physicochemical properties. NVs with neuron-derived membrane proteins exhibit enhanced neuronal association and uptake compared to bare NVs. Viability of 3D neural sphere cultures is not disrupted by treatment, which verifies the utility of organoid-based approaches as NV testing platforms. Finally, these results confirm cellular association and uptake of the biomimetic humanized NVs to neurons within rodent cranial nerves. In summary, the customizable NVs reported here enable next-generation functionalized theranostics aimed to promote neuroregeneration.

LDL-Based Lipid Nanoparticle Derived for Blood Plasma Accumulates Preferentially in Atherosclerotic Plaque.

Apolipoprotein-based drug delivery is a promising approach to develop safe nanoparticles capable of targeted drug delivery for various diseases. In this work, we have synthesized a lipid-based nanoparticle (NPs) that we have called "Aposomes" presenting native apolipoprotein B-100 (apoB-100), the primary protein present in Low-Density Lipoproteins (LDL) on its surface. The aposomes were synthesized from LDL isolated from blood plasma using a microfluidic approach. The synthesized aposomes had a diameter of 91 ± 4 nm and a neutral surface charge of 0.7 mV ± mV. Protein analysis using western blot and flow cytometry confirmed the presence of apoB-100 on the nanoparticle's surface. Furthermore, Aposomes retained liposomes' drug loading capabilities, demonstrating a prolonged release curve with ∼80% cargo release at 4 hours. Considering the natural tropism of LDL towards the atherosclerotic plaques, we evaluated the biological properties of aposomes in a mouse model of advanced atherosclerosis. We observed a ∼20-fold increase in targeting of plaques when comparing aposomes to control liposomes. Additionally, aposomes presented a favorable biocompatibility profile that showed no deviation from typical values in liver toxicity markers (i.e., LDH, ALT, AST, Cholesterol). The results of this study demonstrate the possibilities of using apolipoprotein-based approaches to create nanoparticles with active targeting capabilities and could be the basis for future cardiovascular therapies.

Bioinspired Extracellular Vesicles: Lessons Learned From Nature for Biomedicine and Bioengineering.

Efficient communication is essential in all layers of the biological chain. Cells exchange information using a variety of signaling moieties, such as small molecules, proteins, and nucleic acids. Cells carefully package these messages into lipid complexes, collectively named extracellular vesicles (EVs). In this work, we discuss the nature of these cell carriers, categorize them by their origin, explore their role in the homeostasis of healthy tissues, and examine how they regulate the pathophysiology of several diseases. This review will also address the limitations of using EVs for clinical applications and discuss novel methods to engineer nanoparticles to mimic the structure, function, and features of EVs. Using lessons learned from nature and understanding how cells use EVs to communicate across distant sites, we can develop a better understanding of how to tailor the fundamental features of drug delivery carriers to encapsulate various cargos and target specific sites for biomedicine and bioengineering.

Reproducible and Characterized Method for Ponatinib Encapsulation into Biomimetic Lipid Nanoparticles as a Platform for Multi-Tyrosine Kinase-Targeted Therapy.

Ponatinib (Pon) is a multi-tyrosine kinase inhibitor that demonstrated high efficiency for treating cancer. However, severe side effects caused by Pon off-targeting effects prevent its extensive use. Using our understanding into the mechanisms by which Pon is transported by bovine serum albumin in the blood, we have successfully encapsulated Pon into a biomimetic nanoparticle (NP). This lipid NP (i.e., "leukosomes") incorporates membrane proteins purified from activated leukocytes that enable immune evasion, and enhanced targeting of inflamed endothelium NPs have been characterized for their size, charge, and encapsulation efficiency. Membrane proteins enriched on the NP surface enabled modulation of Pon release. These NP formulations showed promising dose-response results on two different murine osteosarcoma cell lines, F420 and RF379. Our results indicate that our fabrication method is reproducible, nonuser-dependent, efficient in loading Pon, and applicable toward repurposing numerous therapeutic agents previously shelved due to toxicity profiles.

Leukocyte-mimicking nanovesicles for effective doxorubicin delivery to treat breast cancer and melanoma.

In the last decades, several approaches were developed to design drug delivery systems to address the multiple biological barriers encountered after administration while safely delivering a payload. In this scenario, bio-inspired and bio-mimetic approaches have emerged as promising solutions to evade the mononuclear phagocytic system while simultaneously negotiating the sequential transport across the various biological barriers. Leukocytes freely circulate in the bloodstream and selectively target the inflamed vasculature in response to injury, infection, and cancer. Recently we have shown the use of biomimetic nanovesicles, called leukosomes, which combine both the physical and biological properties of liposomes and leukocytes, respectively, to selectively deliver drugs to the inflamed vasculature. Here we report the use of leukosomes to target and deliver doxorubicin, a model chemotherapeutic, to tumors in syngeneic murine models of breast cancer and melanoma. Exploiting the inflammatory pathway responsible for recruiting immune cells to the site of injury, leukosomes exhibited increased targeting of cancer vasculature and stroma. Furthermore, delivery of doxorubicin with leukosomes enabled significant tumor growth inhibition compared with free doxorubicin in both breast and melanoma tumors. This study demonstrates the promise of using biomimetic nanovesicles for effective cancer management in solid tumors.

Tailoring the lipid composition of nanoparticles modulates their cellular uptake and affects the viability of triple negative breast cancer cells.

Lipid nanoparticles are used widely as anticancer drug and gene delivery systems. Internalizing into the target cell is a prerequisite for the proper activity of many nanoparticulate drugs. We show here, that the lipid composition of a nanoparticle affects its ability to internalize into triple-negative breast cancer cells. The lipid headgroup had the greatest effect on enhancing cellular uptake compared to other segments of the molecule. Having a receptor-targeted headgroup induced the greatest increase in cellular uptake, followed by cationic amine headgroups, both being superior to neutral (zwitterion) phosphatidylcholine or to negatively-charged headgroups. The lipid tails also affected the magnitude of cellular uptake. Longer acyl chains facilitated greater liposomal cellular uptake compared to shorter tails, 18:0 > 16:0 > 14:0. When having the same lipid tail length, unsaturated lipids were superior to saturated ones, 18:1 > 18:0. Interestingly, liposomes composed of phospholipids having 14:0 or 12:0-carbon-long-tails, such as DMPC and DLPC, decreased cell viability in a concertation dependent manner, due to a destabilizing effect these lipids had on the cancer cell membrane. Contrarily, liposomes composed of phospholipids having longer carbon tails (16:0 and 18:0), such as DPPC and HSPC, enhanced cancer cell proliferation. This effect is attributed to the integration of the exogenous liposomal lipids into the cancer-cell membrane, supporting the proliferation process. Cholesterol is a common lipid additive in nanoscale formulations, rigidifying the membrane and stabilizing its structure. Liposomes composed of DMPC (14:0) showed increased cellular uptake when enriched with cholesterol, both by endocytosis and by fusion. Contrarily, the effect of cholesterol on HSPC (18:0) liposomal uptake was minimal. Furthermore, the concentration of nanoparticles in solution affected their cellular uptake. The higher the concentration of nanoparticles the greater the absolute number of nanoparticles taken up per cell. However, the efficiency of nanoparticle uptake, i.e. the percent of nanoparticles taken up by cells, decreased as the concentration of nanoparticles increased. This study demonstrates that tuning the lipid composition and concentration of nanoscale drug delivery systems can be leveraged to modulate their cellular uptake.

Macrophage-derived nanovesicles exert intrinsic anti-inflammatory properties and prolong survival in sepsis through a direct interaction with macrophages.

Despite numerous advances in medical treatment, sepsis remains one of the leading causes of death worldwide. Sepsis is characterized by the involvement of all organs and tissues as a consequence of blood poisoning, resulting in organ failure and eventually death. Effective treatment remains an unmet need and novel approaches are urgently needed. The growing evidence of clinical and biological heterogeneity of sepsis suggests precision medicine as a possible key for achieving therapeutic breakthroughs. In this scenario, biomimetic nanomedicine represents a promising avenue for the treatment of inflammatory diseases, including sepsis. We investigated the role of macrophage-derived biomimetic nanoparticles, namely leukosomes, in a lipopolysaccharide-induced murine model of sepsis. We observed that treatment with leukosomes was associated with significantly prolonged survival. In vitro studies elucidated the potential mechanism of action of these biomimetic vesicles. The direct treatment of endothelial cells (ECs) with leukosomes did not alter the gene expression profile of EC-associated cell adhesion molecules. In contrast, the interaction of leukosomes with macrophages induced a decrease of pro-inflammatory genes (IL-6, IL-1b, and TNF-α), an increase of anti-inflammatory ones (IL-10 and TGF-ß), and indirectly an anti-inflammatory response on ECs. Taken together, these results showed the ability of leukosomes to regulate the inflammatory response in target cells, acting as a bioactive nanotherapeutic.

Sodium bicarbonate nanoparticles modulate the tumor pH and enhance the cellular uptake of doxorubicin.

Acidic pH in the tumor microenvironment is associated with cancer metabolism and creates a physiological barrier that prevents from drugs to penetrate cells. Specifically, ionizable weak-base drugs, such as doxorubicin, freely permeate membranes in their uncharged form, however, in the acidic tumor microenvironment these drugs become charged and their cellular permeability is retarded. In this study, 100-nm liposomes loaded with sodium bicarbonate were used as adjuvants to elevate the tumor pH. Combined treatment of triple-negative breast cancer cells (4T1) with doxorubicin and sodium-bicarbonate enhanced drug uptake and increased its anti-cancer activity. In vivo, mice bearing orthotropic 4T1 breast cancer tumors were administered either liposomal or free bicarbonate intravenously. 3.7 ±â€¯0.3% of the injected liposomal dose was detected in the tumor after twenty-four hours, compared to 0.17% ±â€¯0.04% in the group injected free non-liposomal bicarbonate, a 21-fold increase. Analyzing nanoparticle biodistribution within the tumor tissue revealed that 93% of the PEGylated liposomes accumulated in the extracellular matrix, while 7% were detected intracellularly. Mice administered bicarbonate-loaded liposomes reached an intra-tumor pH value of 7.38 ±â€¯0.04. Treating tumors with liposomal bicarbonate combined with a sub-therapeutic dose of doxorubicin achieved an improved therapeutic outcome, compared to mice treated with doxorubicin or bicarbonate alone. Interestingly, analysis of the tumor microenvironment demonstrated an increase in immune cell' population (T-cell, B-cell and macrophages) in tumors treated with liposomal bicarbonate. This study demonstrates that targeting metabolic adjuvants with nanoparticles to the tumor microenvironment can enhance anticancer drug activity and improve treatment.