Precision Localization of Lipid-Based Nanoparticles by Dual-Fluorescent Labeling for Accurate and High-Resolution Imaging in Living Cells

In nanomedicine, lipid-based nanoparticles (NPs) such as liposomes (LPs) have established an important position. Precise delineation of NP interaction with cells and detailed characterization of activity are becoming essential, which mainly rely on labeling with lipophilic fluorescent molecules and assuming stable association with NPs. However, because of label separation from NPs in (biological) media, or when processed by cells, fluorescence-based detection of an NP incorporating a single label may not necessarily indicate the actual presence of an NP but may be from the dissociated label, rendering results unreliable. Herein, flow cytometry and confocal microscopy are employed to demonstrate that to verify the localization of LPs in a cell with perfect accuracy, dual-labeling, and contemporaneous detection of both fluorescent signals in one pixel are required. This is combined with size exclusion chromatography (SEC) and mass spectrometry measurements to indicate factors involved in label dissociation, which helps to understand the possible conditions of dissociated label and NP. It is shown that determining label colocalization with, and label dissociation from, dual-labeled NPs are needed to provide accurate spatiotemporal insight into targeting destination (colocalized signals) and disintegration (separated signals) of NPs during intracellular processing and in studying payload delivery with precision in nanomedicine.</p

Applications of biomimetic nanoparticles in breast cancer as a blueprint for improved next-generation cervical cancer therapy

Nanomedicines are innovative and promising, but lack a convincing clinical presence. Thus, biomimetic nanoparticles (BMNPs) have been designed with functionalizations which structurally and/or functionally mimic the biological setting, endowing thereupon biological structure and functionality. These may be coated with biologically derived materials, but may also include artificial antigen-presenting cells and synthetic architectures. When applied in cancer theranostics, BMNPs show significant improvements over traditional drugs and similar non-biomimetic NPs, especially in terms of circulation time, tissue penetration, delivery, and lowered toxicity. These particles have achieved unprecedented outcomes through top-down synthesis methods (cell material to NP), which bypass complex bottom-up synthetic techniques attempting to mimic such complex and diverse biological components. Breast cancer has received much attention in this area, and as such, is studied in this paper as a template for how BMNPs could be applied in cervical cancer – an area with few BMNP applications and a dire need for efficacious and fertility-preserving therapies. This cancer remains an enormous burden globally, especially in developing countries. Being a virus-induced disease, biomimetic applications may be particularly promising, aligning with the emergence of biomimetic nanovaccines in recent years. Feasibility challenges remain within BMNPs: Extracting biological material for re-administration to patients could cause ethical debate, and the costs involved in preparing scaled up quantities of biomimetic NPs would be large. However, with a clearer understanding and tighter characterization of preparation methods and biological responses, BMNPs may add great value to the nanomedicine community.</p

HMGA2 regulation by miRNAs in cancer:Affecting cancer hallmarks and therapy response

High mobility group A 2 (HMGA2) is a protein that modulates the structure of chromatin in the nucleus. Importantly, aberrant expression of HMGA2 occurs during carcinogenesis, and this protein is an upstream mediator of cancer hallmarks including evasion of apoptosis, proliferation, invasion, metastasis, and therapy resistance. HMGA2 targets critical signaling pathways such as Wnt/β-catenin and mTOR in cancer cells. Therefore, suppression of HMGA2 function notably decreases cancer progression and improves outcome in patients. As HMGA2 is mainly oncogenic, targeting expression by non-coding RNAs (ncRNAs) is crucial to take into consideration since it affects HMGA2 function. MicroRNAs (miRNAs) belong to ncRNAs and are master regulators of vital cell processes, which affect all aspects of cancer hallmarks. Long ncRNAs (lncRNAs) and circular RNAs (circRNAs), other members of ncRNAs, are upstream mediators of miRNAs. The current review intends to discuss the importance of the miRNA/HMGA2 axis in modulation of various types of cancer, and mentions lncRNAs and circRNAs, which regulate this axis as upstream mediators. Finally, we discuss the effect of miRNAs and HMGA2 interactions on the response of cancer cells to therapy. Regarding the critical role of HMGA2 in regulation of critical signaling pathways in cancer cells, and considering the confirmed interaction between HMGA2 and one of the master regulators of cancer, miRNAs, targeting miRNA/HMGA2 axis in cancer therapy is promising and this could be the subject of future clinical trial experiments.</p

Engineering of pulmonary surfactant corona on inhaled nanoparticles to operate in the lung system

Exposure of inhaled nanoparticles (NPs) to the deep lung tissue results in the adsorption of pulmonary surfactant (PSf) on the surface of NPs and the formation of a biomolecular corona. The adsorption of the peculiar phospholipids (PLs) and surfactant proteins (SPs) provides NPs with a new bio-identity, which likely changes their corresponding interactions with cells and other bio-systems. Exploring the interaction of NPs with the PSf film at the alveolar air-fluid interface can provide valuable insights into the role of biofluids in the cellular uptake of NPs and their nanotoxic effects. Wrapping biomembranes around NPs and the formation of lipoprotein corona regulate viscoelastic changes, NP insertion into the membrane, and cellular uptake of NPs. In this review, a concise overview has been presented on the engineering of PSf on inhaled NPs to operate in lung environment. First, the physiological barriers in the pulmonary delivery of NPs and approaches to regulating their pulmonary fate are introduced and rationalized. Next, a short description is given on the different sources used for exploring the interfacial performance of inhaled NPs in vitro. A discussion is then presented on SP corona formation on the surface of inhaled NPs, coronal proteome/lipidome in respiratory tract lining fluid (RTLF), regulation of NP aggregation and surfactant flow characteristics, PSf corona and its functional role in the cellular uptake of NPs, followed by explanations on the clinical correlations of PSf corona formation/inhibition on the surface of NPs. Finally, the challenges and future perspectives of the field have been discussed. This review can be harnessed to exploit PSf for the development of safe and bio-inspired pulmonary drug delivery strategies.</p

Core–shell inorganic NP@MOF nanostructures for targeted drug delivery and multimodal imaging-guided combination tumor treatment

It is well known that metal–organic framework (MOF) nanostructures have unique characteristics such as high porosity, large surface areas and adjustable functionalities, so they are ideal candidates for developing drug delivery systems (DDSs) as well as theranostic platforms in cancer treatment. Despite the large number of MOF nanostructures that have been discovered, conventional MOF-derived nanosystems only have a single biofunctional MOF source with poor colloidal stability. Accordingly, developing core–shell MOF nanostructures with good colloidal stability is a useful method for generating efficient drug delivery, multimodal imaging and synergistic therapeutic systems. The preparation of core–shell MOF nanostructures has been done with a variety of materials, but inorganic nanoparticles (NPs) are highly effective for drug delivery and imaging-guided tumor treatment. Herein, we aimed to overview the synthesis of core–shell inorganic NP@MOF nanostructures followed by the application of core–shell MOFs derived from magnetic, quantum dots (QDs), gold (Au), and gadolinium (Gd) NPs in drug delivery and imaging-guided tumor treatment. Afterward, we surveyed different factors affecting prolonged drug delivery and cancer therapy, cellular uptake, biocompatibility, biodegradability, and enhanced permeation and retention (EPR) effect of core–shell MOFs. Last but not least, we discussed the challenges and the prospects of the field. We envision this article may hold great promise in providing valuable insights regarding the application of hybrid nanostructures as promising and potential candidates for multimodal imaging-guided combination cancer therapy.</p

C8-glycosphingolipids preferentially insert into tumor cell membranes and promote chemotherapeutic drug uptake

AbstractInsufficient drug delivery into tumor cells limits the therapeutic efficacy of chemotherapy. Co-delivery of liposome-encapsulated drug and synthetic short-chain glycosphingolipids (SC-GSLs) significantly improved drug bioavailability by enhancing intracellular drug uptake. Investigating the mechanisms underlying this SC-GSL-mediated drug uptake enhancement is the aim of this study. Fluorescence microscopy was used to visualize the cell membrane lipid transfer intracellular fate of fluorescently labeled C6-NBD-GalCer incorporated in liposomes in tumor and non-tumor cells. Additionally click chemistry was applied to image and quantify native SC-GSLs in tumor and non-tumor cell membranes. SC-GSL-mediated flip-flop was investigated in model membranes to confirm membrane-incorporation of SC-GSL and its effect on membrane remodeling. SC-GSL enriched liposomes containing doxorubicin (Dox) were incubated at 4°C and 37°C and intracellular drug uptake was studied in comparison to standard liposomes and free Dox.SC-GSL transfer to the cell membrane was independent of liposomal uptake and the majority of the transferred lipid remained in the plasma membrane. The transfer of SC-GSL was tumor cell-specific and induced membrane rearrangement as evidenced by a transbilayer flip-flop of pyrene-SM. However, pore formation was measured, as leakage of hydrophilic fluorescent probes was not observed. Moreover, drug uptake appeared to be mediated by SC-GSLs. SC-GSLs enhanced the interaction of doxorubicin (Dox) with the outer leaflet of the plasma membrane of tumor cells at 4°C. Our results demonstrate that SC-GSLs preferentially insert into tumor cell plasma membranes enhancing cell intrinsic capacity to translocate amphiphilic drugs such as Dox across the membrane via a biophysical process

Image-guided drug delivery in nanosystem-based cancer therapies

The past decades have shown significant advancements in the development of solid tumor treatment. For instance, implementation of nanosystems for drug delivery has led to a reduction in side effects and improved delivery to the tumor region. However, clinical translation has faced challenges, as tumor drug levels are still considered to be inadequate. Interdisciplinary research has resulted in the development of more advanced drug delivery systems. These are coined "smart" due to the ability to be followed and actively manipulated in order to have better control over local drug release. Therefore, image-guided drug delivery can be a powerful strategy to improve drug activity at the target site. Being able to visualize the inflow of the administered smart nanosystem within the tumor gives the potential to determine the right moment to apply the facilitator to initiate drug release. Here we provide an overview of available nanosystems, imaging moieties, and imaging techniques. We discuss preclinical application of these smart drug delivery systems, the strength of image-guided drug delivery, and the future of personalized treatment

An adapted dorsal skinfold model used for 4D intravital followed by whole-mount imaging to reveal endothelial cell–pericyte association

Endothelial cells and pericytes are highly dynamic vascular cells and several subtypes, based on their spatiotemporal dynamics or molecular expression, are believed to exist. The interaction between endothelial cells and pericytes is of importance in many aspects ranging from basic development to diseases like cancer. Identification of spatiotemporal dynamics is particularly interesting and methods to studies these are in demand. Here we describe the technical details of a method combining the benefits of high resolution intravital imaging and whole-mount histology. With intravital imaging using an adapted light weight dorsal skinfold chamber we identified blood flow patterns and spatiotemporal subtypes of endothelial cells and pericytes in a 4D (XYZ, spatial+T, time dimension) manner as representative examples for this model. Thereafter the tissue was extracted and stained as a whole-mount, by which the position and volumetric space of endothelial cells as well as pericytes were maintained, to identify molecular subtypes. Integration of the two imaging methods enabled 4D dissection of endothelial cell–pericyte association at the molecular level

Temperature-sensitive polymers to promote heat-triggered drug release from liposomes: Towards bypassing EPR

Heat-triggered drug release from temperature-sensitive nanocarriers upon the application of mild hyperthermia is a promising approach to achieve site-specific delivery of drugs. The combination of mild hyperthermia (41–42 °C) and temperature-sensitive liposomes (TSL) that undergo lipid phase-transition and drug release has been studied extensively and has shown promising therapeutic outcome in a variety of animal tumor models as well as initial indications of success in humans. Sensitization of liposomes to mild hyperthermia by means of exploiting the thermal behavior of temperature-sensitive polymers (TSP) provides novel opportunities. Recently, TSP-modified liposomes (TSPL) have shown potential for enhancing tumor-directed drug delivery, either by triggered drug release or by triggered cell interactions in response to heat. In this review, we describe different classes of TSPL, and analyze and discuss the mechanisms and kinetics of content release from TSPL in response to local heating. In addition, the impact of lipid composition, polymer and copolymer characteristics, serum components and PEGylation on the mechanism of content release and TSPL performance is addressed. This is done from the perspective of rationally designing TSPL, with the overall goal of conceiving efficient strategies to increase the efficacy of TSPL plus hyperthermia to improve the outcome of targeted anticancer therapy

Smart lipid-based nanosystems for therapeutic immune induction against cancers: Perspectives and outlooks

Cancer immunotherapy, a promising and widely applied mode of oncotherapy, makes use of immune stimulants and modulators to overcome the immune dysregulation present in cancer, and leverage the host’s immune capacity to eliminate tumors. Although some success has been seen in this field, toxicity and weak immune induction remain challenges. Liposomal nanosystems, previously used as targeting agents, are increasingly functioning as immunotherapeutic vehicles, with potential for delivery of contents, immune induction, and synergistic drug packaging. These systems are tailorable, multifunctional, and smart. Liposomes may deliver various immune reagents including cytokines, specific T-cell receptors, antibody fragments, and immune checkpoint inhibitors, and also present a promising platform upon which personalized medicine approaches can be built, especially with preclinical and clinical potentials of liposomes often being frustrated by inter-and intrapatient variation. In this review, we show the potential of liposomes in cancer immunotherapy, as well as the methods for synthesis and in vivo progression thereof. Both preclinical and clinical studies are included to comprehensively illuminate prospects and challenges for future research and application