Efficient Internalization Research Articles

Antigen targeting and anti-tumor activity of a novel anti-CD146 212Pb internalizing alpha-radioimmunoconjugate against malignant peritoneal mesothelioma

Malignant mesothelioma, a highly aggressive cancer that primarily affects the serosal membranes, has limited therapeutic options, particularly for cavitary tumors, such as peritoneal and pleural malignant mesothelioma. Intracavitary administration of a radioimmunoconjugate to locally target mesothelioma cancer cells has been proposed as a treatment. CD146, upregulated in mesothelioma but not in healthy tissues, is a promising therapeutic target. This study characterized CD146 expression and binding/internalization kinetics of the CD146-targeting antibody OI-3 coupled with 212Pb (212Pb-TCMC-OI-3) in human mesothelioma cells. Flow cytometry showed that both chimeric (chOI-3) and murine (mOI-3) antibodies rapidly bound and internalized within 1–6 h in MSTO-211H cells. 212Pb-TCMC-chOI-3 exhibited 3.1- to 13.7-fold and 3.1- to 8.5-fold increased internalized 212Pb and 212Bi atoms per cell at 2 and 24 h, respectively, compared to isotype control, underscoring enhanced internalization efficiency. Intraperitoneal administration of 212Pb-TCMC-mOI-3 to mice with intraperitoneal MSTO-211H xenografts improved median survival by a ratio of 1.3 compared to non-binding 212Pb-TCMC-mIgG1. The ability of 212Pb-TCMC-mOI-3 to target and inhibit the growth of intraperitoneal mesothelioma xenografts supports targeted radionuclide therapy’s efficacy for metastatic peritoneal mesothelioma. This study highlights the potential of localized CD146-targeted radioimmunotherapy for malignant mesothelioma, offering a new avenue for improving patient outcomes.

On the axonal transport of lipid nanoparticles in primary hippocampal neurons

Axonal transport is a crucial process in healthy neurons as it supports the intra-cellular movement of nutrients, endogenous substances, and vesicles regulating a broad set of biological functions. Notably, this physiological mechanism is efficiently exploited by a variety of viruses to infect multiple cells within the central nervous system and, thus, it has been proposed as a strategy to enhance the brain penetrance of macromolecules and nanoparticles. In this work, the retrograde and anterograde transport of lipid nanoparticles (LNP) is systematically analyzed in primary hippocampal neurons cultured in compartmentalized microfluidic chips, where neurites are left to grow within 150 μm-long channels connecting the somal and synaptic compartments. After characterizing the physico-chemical properties, toxicological profile, and cell internalization efficiency, the axonal trafficking of fluorescently labeled LNP was monitored over time via live-cell microscopy. Both naïve LNP and apolipoprotein E-coated LNP (ApoE-LNP) were considered under two different experimental configurations, with the LNP being either added to the somal or the synaptic compartment for anterograde or retrograde transport analyses, respectively. ApoE-LNP only were very efficiently uptaken by neurons and rapidly relocated in a perinuclear position. Also, ApoE-LNP incubated in the somal compartment did not translocate along the neurites (null anterograde transport), whereas ApoE-LNP added to the synaptic compartment were detected near the soma already at 30 min post incubation demonstrating retrograde transport velocities up to ∼ 160 nm/s. This preliminary study suggests that ApoE-LNP could be efficiently used to rapidly transport a variety of therapeutic and imaging cargos from the synaptic cleft to the somal compartment.

Recent Advances and Prospects of Nucleic Acid Therapeutics for Anti-Cancer Therapy

Nucleic acid therapeutics are promising alternatives to conventional anti-cancer therapy, such as chemotherapy and radiation therapy. While conventional therapies have limitations, such as high side effects, low specificity, and drug resistance, nucleic acid therapeutics work at the gene level to eliminate the cause of the disease. Nucleic acid therapeutics treat diseases in various forms and using different mechanisms, including plasmid DNA (pDNA), small interfering RNA (siRNA), anti-microRNA (anti-miR), microRNA mimics (miRNA mimic), messenger RNA (mRNA), aptamer, catalytic nucleic acid (CNA), and CRISPR cas9 guide RNA (gRNA). In addition, nucleic acids have many advantages as nanomaterials, such as high biocompatibility, design flexibility, low immunogenicity, small size, relatively low price, and easy functionalization. Nucleic acid therapeutics can have a high therapeutic effect by being used in combination with various nucleic acid nanostructures, inorganic nanoparticles, lipid nanoparticles (LNPs), etc. to overcome low physiological stability and cell internalization efficiency. The field of nucleic acid therapeutics has advanced remarkably in recent decades, and as more and more nucleic acid therapeutics have been approved, they have already demonstrated their potential to treat diseases, including cancer. This review paper introduces the current status and recent advances in nucleic acid therapy for anti-cancer treatment and discusses the tasks and prospects ahead.

Platelet-cloaked alginate-poly (β-amino ester) a novel platform bioinspired polyelectrolyte nanoparticle for targeted delivery of carboplatin in breast cancer: An in vitro/in vivo study

Triple-negative breast cancer (TNBC) has poor prognosis. Carboplatin (Crb) is a widely used chemotherapeutic agent, in TNBC but with serious systemic toxicity and poor tumor targeting. Bioinspired drug-loaded platelets (Plt) and Plt-coated nanocarriers evade macrophage phagocytosis by membrane proteins like CD47. The goal of this study was preparation of a novel alginate-poly (β-amino ester) (PβAE) nanoparticles (NPs) for targeted delivery of Crb to TNBC cells by developing and comparison of two bioinspired carriers of Plt membrane (PltM) coated Crb-loaded alginate-poly (β-amino ester) nanoparticles (PltM@Crb-PβAE-ALG NPs) and Plt loaded Crb (Plt@Crb). The NPs were prepared by ionic gelation and subsequently were coated by platelet membrane using ultra-sonication method. The loading efficiency, release profile, and in vitro cytotoxicity of both formulations were evaluated on HUVEC and 4 T1 cells. Additionally, the in vivo tumor targeting, therapeutic efficacy, and organ toxicity of the two formulations were assessed in a murine tumor model. Results showed both Plt@Crb and (PltM@Crb-PβAE-ALG NPs) exhibited high drug loading efficiency, sustained release, enhanced cytotoxicity against 4 T1 cells, and decreased cytotoxicity in normal cells (HUVEC) in vitro. In vivo studies revealed that although both formulations considerably improved tumor inhibition compared to free Crb, but the PltM@Crb-PβAE-ALG NPs demonstrated superior cytotoxicity and therapeutic efficacy, thanks to improved Crb’s internalization efficiency, enhanced stability, and controlled release properties.

Tuning nanoparticle core composition drives orthogonal fluorescence amplification for enhanced tumour imaging

Tumour detection with high selectivity and sensitivity is crucial for delineating tumour margins and identifying metastatic foci during image-guided surgery. Optical nanoprobes with preferential tumour accumulation is often limited by inefficient amplification of biological signals. Here, we report the design of a library of hydrophobic core-tunable ultra-pH-sensitive nanoprobes (HUNPs) for orthogonally amplifying tumour microenvironmental signals on multiple tumour models. We find that tuning the hydrophobicity of nanoparticle core composition with non-ionizable monomers can enhance cellular association of HUNPs by more than ten-fold, resulting in a high cellular internalization efficiency of HUNPs with up to 50% in tumours. Combining high tumour accumulation and high cell internalization efficiency, HUNPs show orthogonally amplified fluorescence signals, permitting the precise locating and delineating margins between malignant lesions and normal tissues with high contrast-to-noise ratio and resolution. Our study provides key strategies to design nanomedicines with high intracellular bioavailability for cancer detection, drug/gene delivery, and therapy.

A tetrahedral framework nucleic acids-based gene therapeutic nanococktail alleviates cartilage damage and protects against osteoarthritis progression

Gene therapy based on therapeutic microRNAs (miRNAs) shows great potential in Osteoarthritis (OA) treatment. However, individual miRNAs application is limited by the single target, the instability, and the low efficiency of cell internalization. Here, we identified two potential candidate miRNAs, miR-140-5p and miR-455-3p, exhibited combined enhancing effects against OA progression. Furthermore, a nanococktail therapeutic platform, T-(140 + 455) was constructed by employing tetrahedral framework nucleic acids (tFNAs) to codeliver miR-140-5p/miR-455-3p and achieve enhanced gene therapy for OA. Compared with the delivery of individual miRNAs, T-(140 + 455) can effectively reverse the cell phenotype and metabolism of OA chondrocytes. Moreover, the nanococktail T-(140 + 455) reprogrammed macrophage polarization by modulating the crosstalk between chondrocytes and macrophages, combatting the damage caused by inflammation. The OA rat model treated with nanococktail T-(140 + 455) exhibited favorable performance in halting cartilage damage and regulating extracellular matrix (ECM) metabolic homeostasis. Thus, tFNA-based nanococktails enhance the delivery and efficacy of miRNAs, offering a promising strategy for OA gene therapy.

Kinetic Landscape of Single Virus-like Particles Highlights the Efficacy of SARS-CoV-2 Internalization.

The efficiency of virus internalization into target cells is a major determinant of infectivity. SARS-CoV-2 internalization occurs via S-protein-mediated cell binding followed either by direct fusion with the plasma membrane or endocytosis and subsequent fusion with the endosomal membrane. Despite the crucial role of virus internalization, the precise kinetics of the processes involved remains elusive. We developed a pipeline, which combines live-cell microscopy and advanced image analysis, for measuring the rates of multiple internalization-associated molecular events of single SARS-CoV-2-virus-like particles (VLPs), including endosome ingression and pH change. Our live-cell imaging experiments demonstrate that only a few minutes after binding to the plasma membrane, VLPs ingress into RAP5-negative endosomes via dynamin-dependent scission. Less than two minutes later, VLP speed increases in parallel with a pH drop below 5, yet these two events are not interrelated. By co-imaging fluorescently labeled nucleocapsid proteins, we show that nucleocapsid release occurs with similar kinetics to VLP acidification. Neither Omicron mutations nor abrogation of the S protein polybasic cleavage site affected the rate of VLP internalization, indicating that they do not confer any significant advantages or disadvantages during this process. Finally, we observe that VLP internalization occurs two to three times faster in VeroE6 than in A549 cells, which may contribute to the greater susceptibility of the former cell line to SARS-CoV-2 infection. Taken together, our precise measurements of the kinetics of VLP internalization-associated processes shed light on their contribution to the effectiveness of SARS-CoV-2 propagation in cells.

Ubiquitin-driven protein condensation stabilizes clathrin-mediated endocytosis.

Clathrin-mediated endocytosis is an essential cellular pathway that enables signaling and recycling of transmembrane proteins and lipids. During endocytosis, dozens of cytosolic proteins come together at the plasma membrane, assembling into a highly interconnected network that drives endocytic vesicle biogenesis. Recently, multiple groups have reported that early endocytic proteins form flexible condensates, which provide a platform for efficient assembly of endocytic vesicles. Given the importance of this network in the dynamics of endocytosis, how might cells regulate its stability? Many receptors and endocytic proteins are ubiquitylated, while early endocytic proteins such as Eps15 contain ubiquitin-interacting motifs. Therefore, we examined the influence of ubiquitin on the stability of the early endocytic protein network. In vitro, we found that recruitment of small amounts of polyubiquitin dramatically increased the stability of Eps15 condensates, suggesting that ubiquitylation could nucleate endocytic assemblies. In live-cell imaging experiments, a version of Eps15 that lacked the ubiquitin-interacting motif failed to rescue defects in endocytic initiation created by Eps15 knockout. Furthermore, fusion of Eps15 to a deubiquitylase enzyme destabilized nascent endocytic sites within minutes. In both in vitro and live-cell settings, dynamic exchange of Eps15 proteins, a measure of protein network stability, was decreased by Eps15-ubiquitin interactions and increased by loss of ubiquitin. These results collectively suggest that ubiquitylation drives assembly of the flexible protein network responsible for catalyzing endocytic events. More broadly, this work illustrates a biophysical mechanism by which ubiquitylated transmembrane proteins at the plasma membrane could regulate the efficiency of endocytic internalization.

Efficacy comparison in cap VLPs of PCV2 and PCV3 as swine vaccine vehicle

As one genotype of porcine circovirus (PCV) identified in 2016, PCV3 has brought huge hidden dangers to the global swine industry together with PCV2. Virus-like particles (VLPs) of capsid protein (Cap) of PCV2 serve as an alternative nano-antigen delivery strategy to efficiently induce antiviral immune response against PCV2 and/or other covalently displayed swine pathogens. However, the current understanding is limited on the capability of PCV3 as a nano-vaccine vehicle. Here we systematically compared the characteristics and the immunogenic efficacy of PCV3 Cap (Cap3) and PCV2 Cap (Cap2) in a VLP form. Cap3 VLPs presented higher internalization efficiency into cells and cytokines production compared to those of Cap2. Meanwhile, cross-reactive immunity between Cap3 VLPs and Cap2 VLPs was detected. Furthermore, to evaluate the function of Cap3 VLPs and Cap2 VLPs as vaccine vehicles carrying foreign proteins, the non-structural protein 6 of porcine reproductive and respiratory syndrome virus (PRRSV) was fused to C-terminus of Cap. Cap3-based chimeric particles induced a higher level of nsp6-specific immune response and PRRSV inhibition. Collectively, these self-assembling, Cap-based VLPs offer a compelling platform for enhancing the effectiveness of subunit vaccinations against newly emerging diseases and hold great promise for the development of Cap3-based chimeric subunit vaccines.

Coadministration of Quercetin and Indocyanine Green via PEGylated Phospholipid Micelles for Augmented Chem-Photothermal Combination Tumor Therapy.

A significant impediment persists in developing multicomponent nanomedicines designed to dismantle the heat shock protein (HSP)-based protective mechanism of malignant tumors during photothermal therapy. Herein, well-defined PEGylated phospholipid micelles were utilized to coencapsulate quercetin (QUE, a natural anticancer agent and potent HSP inhibitor) and indocyanine green (ICG, a photothermal agent) with the aim of achieving synchronized and synergistic drug effects. The subsequent investigations validated that the tailored micellar system effectively enhanced QUE's water solubility and augmented its cellular internalization efficiency. Intriguingly, the compositional PEGylated phospholipids induced extraordinary endoplasmic reticulum stress, thereby sensitizing the tumor cells to QUE. Furthermore, QUE played a crucial role in inhibiting the stress-induced overexpression of HSP70, thereby augmenting the photothermal efficacy of ICG. In systemic applications, the proposed nanotherapeutics exhibited preferential accumulation within tumors and exerted notable tumoricidal effects against 4T1 xenograft tumors under 808 nm near-infrared irradiation, facilitated by prominent near-infrared fluorescence imaging-guided chemo-photothermal therapy. Therefore, our strategy for fabricating multicomponent nanomedicines emerges as a coordinated platform for optimizing antitumor therapeutic efficacy and offers valuable insights for diverse therapeutic modalities.

Modulated Cell Internalization Behavior of Icosahedral DNA Framework with Programmable Surface Modification.

Surface modification could enhance the cell internalization efficiency of nanovehicles for targeted gene or drug delivery. However, the influence of surface modification parameters, including recognition manners, valences, and patterns, is often clouded, especially for the endocytosis of DNA nanostructures in customized shapes. Focusing on an icosahedral DNA framework, we systematically programmed three distinct types of ligands with diverse valence and spatial distribution on their outer surface to study the internalization efficiency, endocytic pathways, and postinternalization fate. The comparison in different aspects of parameters deepens our understanding of the intricate relationship between surface modification and cell entry behavior, offering insights crucial for designing and optimizing DNA framework nanostructures for potent cell-targeted purposes.

Development of Peptide Paratope Mimics Derived from the Anti-ROR1 Antibody and Long-Acting Peptide-Drug Conjugates for Targeted Cancer Therapy.

Antibody-based targeted therapy in cancer faces a challenge due to uneven antibody distribution in solid tumors, hindering effective drug delivery. We addressed this by developing peptide mimetics with nanomolar-range affinity for Receptor Tyrosine Kinase-Like Orphan Receptor 1 (ROR1) using computational methods. These peptides showed both specific targeting and deep penetration in vitro and in vivo. Additionally, we created peptide-drug conjugates (PDCs) by linking targeting peptides to toxin drugs via various linkers and enhancing their in vivo half-life with fatty side chains for albumin binding. The antitumor candidate II-3 displayed exceptional affinity (KD = 1.72 × 10-9 M), internalization efficiency, anticancer potency (IC50 = 0.015 ± 0.002 μM), and pharmacokinetics (t1/2 = 2.6 h), showcasing a rational approach for designing PDCs with favorable tissue distribution and strong tumor penetration.

Modular Fluorescent Cholesterol Naphthalimide Probes And Their Application For Cholesterol Trafficking Studies In Cells.

Development of fluorescent cholesterol analogs to better understand subcellular cholesterol trafficking is of great interest for cell biology and medicine. Our approach utilizes a bifunctional 1,8-naphthalimide scaffold with a push-pull character, modified on one side with a head group and a linker on the other side connecting it to cholesterol via an ester bond. Through structure-function studies, we've explored how different substituents-linkers and head groups-affect the ability of these fluorescent cholesterol naphthalimide analogs (CNDs) to mimic natural cholesterol behavior at both molecular and cellular levels. We categorized the resulting analogs into three groups: neutral, charged, and those featuring a hydroxyl group. Each compound was assessed for its solvatochromic behavior in organic solvents and model membranes. Extensive all-atom molecular dynamics simulations helped us examine how these analogs perform in model membranes compared to cholesterol. Additionally, we investigated the partitioning of these fluorescent probes in phase-separated giant unilamellar vesicles. We evaluated the uptake and distribution of these probes within mouse fibroblast cells and astrocytes, for their subcellular distributions in lysosomes and compared that to BODIPY-cholesterol, a well-regarded fluorescent cholesterol analog. The internalization efficiency of the fluorescent probes varies in different cell types and is affected mainly by the head groups. Our results demonstrate that the modular design significantly simplifies the creation of fluorescent cholesterol probes bearing distinct spectral, biophysical, and cellular targeting features, which makes it a valuable toolkit for the investigation of subcellular distribution and trafficking of cholesterol and its derivatives.

UV-B Radiation Disrupts Membrane Lipid Organization and Suppresses Protein Mobility of GmNARK in Arabidopsis.

While it is well known that plants interpret UV-B as an environmental cue and a potential stressor influencing their growth and development, the specific effects of UV-B-induced oxidative stress on the dynamics of membrane lipids and proteins remain underexplored. Here, we demonstrate that UV-B exposure notably increases the formation of ordered lipid domains on the plasma membrane (PM) and significantly alters the behavior of the Glycine max nodule autoregulation receptor kinase (GmNARK) protein in Arabidopsis leaves. The GmNARK protein was located on the PM and accumulated as small particles in the cytoplasm. We found that UV-B irradiation interrupted the lateral diffusion of GmNARK proteins on the PM. Furthermore, UV-B light decreases the efficiency of surface molecule internalization by clathrin-mediated endocytosis (CME). In brief, UV-B irradiation increased the proportion of the ordered lipid phase and disrupted clathrin-dependent endocytosis; thus, the endocytic trafficking and lateral mobility of GmNARK protein on the plasma membrane are crucial for nodule formation tuning. Our results revealed a novel role of low-intensity UV-B stress in altering the organization of the plasma membrane and the dynamics of membrane-associated proteins.

In-silico research on the effects of variable voltage pulse protocols on the cell survival and permeabilization fractions in electroporated cancerous tissues

Reversible electroporation is used to increase the cell membrane permeability by the application of short duration, high voltage electric pulses with electrodes strategically positioned to induce localized drug concentration in the tumor tissue. However, inadequate selection of pulses’ characteristics can lead to relevant loss of cell viability or lack of cell permeabilization, affecting aversively the results of the therapy. Accordingly, the balance between cell membrane electro-permeabilization and cell death is crucial, and the recursive modification of electroporation parameters can favor this balance to increase the drug uptake into viable cells. Particularly, the controlled diminution of the voltage level as the number of pulses increases can reduce the cell damage without compromising the membrane electro-permeabilization. In this work, a previously developed in-silico tool, based on the Global Method of Approximate Particular Solutions (GMAPS), is calibrated, validated, and used to study the differences between constant voltage and variable voltage pulse protocols (CVP and VVP) in terms of the magnitude and distribution of cell survival and permeabilization fractions along the tissue domain, and the internalization efficiency of the therapy. In VVP protocols, the reduction of the applied voltage with the number of pulses is numerically estimated to reach a target volume-averaged survival fraction in the most sensitive zone of the tissue. Numerical results indicate that differences between the pulse protocol types (CVP and VVP) depend on the initial voltage level ∅0 and number of pulses K, with the VVP protocol bringing about superior results for the larger values of ∅0 and K considered here.

Optimizing anthocyanin Oral delivery: Effects of food biomacromolecule types on Nanocarrier performance for enhanced bioavailability

Wall material types influence the efficacy of nanocarriers in oral delivery systems. We utilized three food biomacromolecules (whey protein isolate, oxidized starch, lipids) to prepare three types of nanocarriers. Our aim was to investigate their performance in digestion, cellular absorption, mucus penetration, intestinal retention, and bioavailability of the encapsulated anthocyanins (Ant). The release rate of protein nanocarriers (Pro-NCs) was twice that of starch nanocarriers (Sta-NCs) and four times that of lipid nanocarriers (Lip-NCs) in simulated gastrointestinal fluid. Additionally, Pro-NCs demonstrated superior transmembrane transport capacity and over three times cellular internalization efficiency than Sta-NCs and Lip-NCs. Sta-NCs exhibited the highest mucus-penetrating capacity, while Pro-NCs displayed the strongest mucoadhesion, resulting in extended gastrointestinal retention time for Pro-NCs. Sta-NCs significantly enhanced the in vivo bioavailability of Ant, nearly twice that of free Ant. Our results demonstrate the critical role of wall material types in optimizing nanocarriers for the specific delivery of bioactive compounds.

Facet-engineering strategy of phosphogypsum for production of mineral slow-release fertilizers with efficient nutrient fixation and delivery

Phosphogypsum (PG) presents considerable potential for agricultural applications as a secondary primary resource. However, it currently lacks environmentally friendly, economically viable, efficient, and sustainable reuse protocols. This study firstly developed a PG-based mineral slow-release fertilizer (MSRFs) by internalization and fixation of urea within the PG lattice via facet-engineering strategy. The molecular dynamics simulations demonstrated that the binding energy of urea to the (041) facet of PG surpassed that of the (021) and (020) facets, with urea's desorption energy on the (041) facet notably higher than on the (021) and (020) facets. Guided by these calculations, we selectively exposed the (041) dominant facet of PG, and then achieving complete urea fixation within the PG lattice to form urea-PG (UPG). UPG exhibited a remarkable 48-fold extension in N release longevity in solution and a 45.77% increase in N use efficiency by plants compared to conventional urea. The facet-engineering of PG enhances the internalization and fixation efficiency of urea for slow N delivery, thereby promoting nutrient uptake for plant growth. Furthermore, we elucidated the intricate interplay between urea and PG at the molecular level, revealing the involvement of hydrogen and ionic bonding. This specific bonding structure imparts exceptional thermal stability and water resistance to the urea within UPG under environmental conditions. This study has the potential to provide insights into the high-value utilization of PG and present innovative ideas for designing efficient MSRFs.

Adipocyte-targeted delivery of rosiglitazone with localized photothermal therapy for the treatment of diet-induced obesity in mice

Obesity represents a growing public health concern and is closely associated with metabolic complications such as diabetes and fatty liver disease. Anti-obesity medications currently available have limited efficacy in weight loss and are often accompanied by adverse effects. This study proposes a localized photothermal therapy (PTT) combined with adipocyte-targeted delivery of rosiglitazone (RSG) to address obesity. Specifically, cationic albumin nanoparticles (cNPs) were synthesized to deliver RSG precisely to white adipocytes, stimulating the browning process. An IR780-loaded thermosensitive hydrogel was injected and allowed to gel in situ to afford a subcutaneous reservoir that enables localized PTT and controlled release of RSG cNPs. Notably, cNPs significantly enhanced the internalization efficiency in adipocytes in vitro and prolonged the therapeutic retention in the adipose tissue in vivo. Co-administration of RSG cNPs and PTT substantially reduced fat content, induced browning in white adipose tissue in diet-induced obese mice, and mitigated complications such as insulin resistance, fatty liver, and hyperlipidemia. The increased expression of uncoupling protein 1 contributes to enhancing energy expenditure and facilitating adipose metabolism, thereby effectively combating obesity. This therapeutic approach integrates localized PTT with adipocyte-targeted delivery to combat the global obesity epidemic thus offering a promising solution with reduced systemic toxicity and enhanced efficacy. Statement of significanceCationic albumin nanoparticles are capable of efficient internalization in adipocytes, which may enhance drug targeting to adipose tissue.The combination of rosiglitazone-loaded cationic albumin nanoparticles and local hyperthermia effectively reduces lipid accumulation in adipocytes and induces an upregulated expression of uncoupling protein 1.The combination therapy effectively inhibits fat accumulation, induces adipocyte browning, and regulates systemic metabolism in diet-induced obese mice.

Abstract LB058: N-glycan deglycosylation ameliorates anti-HER2 antibody drug conjugate-induced interstitial pneumonia in preclinical models

Abstract Background: Current clinical application of trastuzumab deruxtecan (T-DXd), an anti-HER2 antibody-drug conjugate (ADC) has been severely limited by interstitial lung disease (ILD). Recent studies suggest a correlation between ILD and the uptake of T-DXd by alveolar macrophages (AMs), which are major innate immune cells in lung tissues. This underlying mechanism of AMs phagocytosing T-DXd was predicted to be the binding interaction between the crystallizable fragment (Fc) of trastuzumab and the Fcγ receptors on the cell surface of AMs, exerting effector functions. Since monoclonal antibodies typically undergo N-glycosylation in the Fc fragment, here we investigate the impacts of antibody N-glycan deglycosylation on T-DXd-induced ILD in preclinical models. Methods: N-glycan removal was performed on trastuzumab and T-DXd using PNGase F. Using immunofluorescence imaging combined with flow cytometry, we conducted a comparative analysis of the binding affinity and internalization efficiency of wild type and fully N-glycan deglycosylated HER2 antibodies. Next, we determined the in vitro cytotoxicity of WT and N-glycan deglycosylated T-DXd against both macrophages and HER2-positive cancer cells using CCK-8 assay. Furthermore, the deposition of N-glycan deglycosylated T-DXd in healthy lungs and their effects in ILD incidence and severity was determined in immunocompetent BALB/c mice carrying HER2-positive tumors. Results: First, we obtained complete N-glycan deglycosylated HER2 antibodies confirmed by SDS-PAGE and mass spectrometry. Next, we showed that N-glycan removal by PNGase F did not affect the Fab fragments of HER2 antibodies binding to the HER2 antigens on HER2-expressing cancer cells (OE19, SK-OV-3 and CT26-hHER2). Importantly, we did observe an approximately 30-50% reduction in the N-glycan deglycosylated HER2 antibodies binding with a panel of three macrophages (MH-S, RAW264.7, and PMA-induced THP-1) in comparison with WT antibodies, indicating a significant impact of N-glycan removal on innate immune cell-mediated effector functions. Moreover, in vitro cytotoxicity studies further support these observations that N-glycan deglycosylated T-DXd has significantly lower cytotoxicities against MH-S (p=0.046) and RAW264.7(p=0.020) in comparison with WT T-DXd. Correlating with our in vitro findings, in vivo biodistribution data showed a reduced lung deposition of HER2 antibodies and T-DXd after N-glycan removal. Immunofluorescence staining confirms the co-localization of HER2 antibodies with CD68-positive macrophages, indicating that AMs are the major contributor to the lung deposition of HER2 antibodies and T-DXd and N-glycan removal could effectively avoid ADC being phagocytosed by AMs. We also demonstrated chronic administration of N-glycan deglycosylated T-DXd could reduce ADC-associated ILD incidence and ameliorate ILD severity. Conclusions: Collectively, our study suggests that antibody N-glycan deglycosylation has important roles in regulating interactions between ADC and innate immune cells. This finding provides valuable clues for further optimizing ADC design and treatment strategies. Citation Format: Yu-Xuan Yang, Le-Tao Ma, Peng Guo. N-glycan deglycosylation ameliorates anti-HER2 antibody drug conjugate-induced interstitial pneumonia in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB058.

Folate-modified liposomes mediate the co-delivery of cisplatin with miR-219a-5p for the targeted treatment of cisplatin-resistant lung cancer

Cisplatin (DDP) resistance, often leading to first-line chemotherapy failure in non-small cell lung cancer (NSCLC), poses a significant challenge. MiR-219a-5p has been reported to enhance the sensitivity of human NSCLC to DDP. However, free miR-219a-5p is prone to degradation by nucleases in the bloodstream, rendering it unstable. In light of this, our study developed an efficient nanodrug delivery system that achieved targeted delivery of DDP and miR-219a-5p by modifying liposomes with folate (FA). Based on the results of material characterization, we successfully constructed a well-dispersed and uniformly sized (approximately 135.8 nm) Lipo@DDP@miR-219a-5p@FA nanodrug. Agarose gel electrophoresis experiments demonstrated that Lipo@DDP@miR-219a-5p@FA exhibited good stability in serum, effectively protecting miR-219a-5p from degradation. Immunofluorescence and flow cytometry experiments revealed that, due to FA modification, Lipo@DDP@miR-219a-5p@FA could specifically bind to FA receptors on the surface of tumor cells (A549), thus enhancing drug internalization efficiency. Safety evaluations conducted in vitro demonstrated that Lipo@DDP@miR-219a-5p@FA exhibited no significant toxicity to non-cancer cells (BEAS-2B) and displayed excellent blood compatibility. Cellular functional experiments, apoptosis assays, and western blot demonstrated that Lipo@DDP@miR-219a-5p@FA effectively reversed DDP resistance in A549 cells, inhibited cell proliferation and migration, and further promoted apoptosis. In summary, the Lipo@DDP@miR-219a-5p@FA nanodrug, through specific targeting of cancer cells and reducing their resistance to DDP, significantly enhanced the anti-NSCLC effects of DDP in vitro, providing a promising therapeutic option for the clinical treatment of NSCLC.