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Cholesterol homeostasis is essential for healthy mammalian cells and dysregulation of cholesterol metabolism contributes to the pathogenesis of various diseases including cancer. Cancer cells are dependent on cholesterol. Malignant progression is associated with high cellular demand for cholesterol, and extracellular cholesterol uptake is often elevated in cancer cell to meet its metabolic needs. Tumors take up cholesterol from the blood stream through their vasculature. Breast cancer grows in, and ovarian cancer metastasizes into fatty tissue that provides them with an additional source of cholesterol. High levels of extracellular cholesterol are beneficial for tumors whose cancer cells master the uptake of extracellular cholesterol. In this review we concentrate on cholesterol uptake mechanisms, receptor-mediated endocytosis and macropinocytosis, and how these are utilized and manipulated by cancer cells to overcome their possible intrinsic or pharmacological limitations in cholesterol synthesis. We focus especially on the involvement of lysosomes in cholesterol uptake. Identifying the vulnerabilities of cholesterol metabolism and manipulating them could provide novel efficient therapeutic strategies for treatment of cancers that manifest dependency for extracellular cholesterol.

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Background: Familial hypercholesterolemia (FH) is an autosomal dominant disorder characterized by increased LDL-cholesterol levels. About 85% of FH cases are caused by LDLR mutations encoding the low-density lipoprotein receptor (LDLR). LDLR is synthesized in the endoplasmic reticulum (ER) where it undergoes post-translational modifications and then transported through Golgi apparatus to the plasma membrane. Over 2900 LDLR variants have been reported in FH patients with limited information on the pathogenicity and functionality of many of them. This study aims to elucidate the cellular trafficking and functional implications of LDLR missense variants identified in suspected FH patients using biochemical and functional methods. Methods: We used HeLa, HEK293T, and LDLR-deficient-CHO-ldlA7 cells to evaluate the subcellular localization and LDL internalization of ten LDLR missense variants (p.C167F, p.D178N, p.C243Y, p.E277K, p.G314R, p.H327Y, p.D477N, p.D622G, p.R744Q, and p.R814Q) reported in multiethnic suspected FH patients. We also analyzed the functional impact of three variants (p.D445E, p.D482H, and p.C677F), two of which previously shown to be retained in the ER. Results: We show that p.D622G, p.D482H, and p.C667F are largely retained in the ER whereas p.R744Q is partially retained. The other variants were predominantly localized to the plasma membrane. LDL internalization assays in CHO-ldlA7 cells indicate that p.D482H, p.C243Y, p.D622G, and p.C667F have quantitatively lost their ability to internalize Dil-LDL with the others (p.C167F, p.D178N, p.G314R, p.H327Y, p.D445E, p.D477N, p.R744Q and p.R814Q) showing significant losses except for p.E277K which retained full activity. However, the LDL internalization assay is only to able evaluate the impact of the variants on LDL internalization and not the exact functional defects such as failure to bind LDL. The data represented illustrate the hypomorphism nature of variants causing FH which may explain some of the variable expressivity of FH. Conclusion: Our combinatorial approach of in silico, cellular, and functional analysis is a powerful strategy to determine pathogenicity and FH disease mechanisms which may provide opportunitites for novel therapeutic strategies.

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Mammalian cell membranes are very dynamic where they respond to several environmental stimuli by rearranging the membrane composition by basic biological processes, including endocytosis. In this context, receptor-mediated endocytosis, either clathrin-dependent or caveolae-dependent, is involved in different physiological and pathological conditions. In the last years, an important amount of evidence has been reported that kidney function involves the modulation of different types of endocytosis, including renal protein handling. In addition, the dysfunction of the endocytic machinery is involved with the development of proteinuria as well as glomerular and tubular injuries observed in kidney diseases associated with hypertension, diabetes, and others. In this present review, we will discuss the mechanisms underlying the receptor-mediated endocytosis in different glomerular cells and proximal tubule epithelial cells as well as their modulation by different factors during physiological and pathological conditions. These findings could help to expand the current understanding regarding renal protein handling as well as identify possible new therapeutic targets to halt the progression of kidney disease.

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The blood-brain barrier (BBB) is a specialized semipermeable structure that highly regulates exchanges between the central nervous system parenchyma and blood vessels. Thus, the BBB also prevents the passage of various forms of therapeutic agents, nanocarriers, and their cargos. Recently, many multidisciplinary studies focus on developing cargo-loaded nanoparticles (NPs) to overcome these challenges, which are emerging as safe and effective vehicles in neurotheranostics. In this Review, first we introduce the anatomical structure and physiological functions of the BBB. Second, we present the endogenous and exogenous transport mechanisms by which NPs cross the BBB. We report various forms of nanomaterials, carriers, and their cargos, with their detailed BBB uptake and permeability characteristics. Third, we describe the effect of regulating the size, shape, charge, and surface ligands of NPs that affect their BBB permeability, which can be exploited to enhance and promote neurotheranostics. We classify typical functionalized nanomaterials developed for BBB crossing. Fourth, we provide a comprehensive review of the recent progress in developing functional polymeric nanomaterials for applications in multimodal bioimaging, therapeutics, and drug delivery. Finally, we conclude by discussing existing challenges, directions, and future perspectives in employing functionalized nanomaterials for BBB crossing.

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Dent disease type 1 is characterized by pathogenic CLCN5 gene variants and impaired receptor-mediated endocytosis in proximal tubules. However, mutation-related abnormalities in proximal tubules have not yet been described. Here, we present three patients with CLCN5 alterations and distinct morphological changes of the apical endocytic-lysosomal apparatus. The proximal tubular ultrastructure was investigated in kidney biopsy samples of three boys genotyped for non-nephrotic proteinuria. Controls: seven patients with nephrotic-range glomerular proteinuria. The genotyping findings revealed an already-known missense mutation in one patient and hitherto undescribed frameshift variants in two patients. Low-molecular-weight proteinuria, focal global glomerulosclerosis, proximal tubular changes, and tubular calcium deposits characterized each case. Three subsets of proximal tubular cells were observed: those without any abnormality, those with aplasia of apical endocytic-lysosomal apparatus and shrinkage of cells, and those with hypoplasia of apical endocytic apparatus, accumulation of proteinaceous substance in dysmorphic lysosomes, and dysmorphic mitochondria. The distribution of subsets varied from patient to patient. In one patient with a frameshift variant, an oxidative stress-like injury of proximal tubular cells and podocytes accompanied the above-mentioned alterations. Focal aplasia/hypoplasia of apical endocytic apparatus and subsequent changes in cytoplasmic organelles characterized proximal tubules in the CLCN5 pathogenic variants.

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Self-assembling nanoparticles (saNP) and nanofibers were found in the recombinant coronavirus SARS-CoV-2 S1, S2, RBD and N proteins purified by affinity chromatography using Ni Sepharose. Scanning electron (SEM), atomic force (AFM) microscopy on mica or graphite surface and in liquid as well as dynamic light scattering (DLS) revealed nanostructures of various sizes. AFM in liquid cell without drying on the surface showed mean height of S1 saNP 80.03 nm, polydispersity index (PDI) 0.006; for S2 saNP mean height 93.32 nm, PDI = 0.008; for N saNP mean height 16.71 nm, PDI = 0.99; for RBD saNP mean height 16.25 nm, PDI = 0.55. Ratios between the height and radius of each saNP in the range 0.1-0.5 suggested solid protein NP but not vesicles with internal empty spaces. The solid but not empty structures of the protein saNP were also confirmed by STEM after treatment of saNP with the standard contrasting agent uranyl acetate. The saNP remained stable after multiple freeze-thaw cycles in water and hyperosmotic solutions for 2 years at -20 °C. Receptor-mediated penetration of the SARS-CoV-2 S1 and RBD saNP in the African green mokey kidney Vero cells with the specific receptors for ß-coronavirus reproduction was more efficient compared to unspecific endocytosis into MDCK cells without the specific receptors. Amyloid-like structures were revealed in the SARS-CoV-2 S1, S2, RBD and N saNP by means of their interaction with Thioflavin T and Congo Red dyes. Taken together, spontaneous formation of the amyloid-like self-assembling nanostructures due to the internal affinity of the SARS-CoV-2 virion proteins might induce proteinopathy in patients, including conformational neurodegenerative diseases, change stability of vaccines and diagnostic systems.

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Extracellular vesicles (EVs) have recently received much attention about the application of drug carriers due to their desirable properties such as nano-size, biocompatibility, and high stability. Herein, we demonstrate orange-derived extracellular vesicles (OEV) nanodrugs (DN@OEV) by modifying cRGD-targeted doxorubicin (DOX) nanoparticles (DN) onto the surface of OEV, enabling significantly enhancing tumor accumulation and penetration, thereby efficiently inhibiting the growth of ovarian cancer. The obtained DN@OEV enabled to inducement of greater transcytosis capability in ovarian cancer cells, which presented the average above 10-fold transcytosis effect compared with individual DN. It was found that DN@OEV could trigger receptor-mediated endocytosis to promote early endosome/recycling endosomes pathway for exocytosis and simultaneously reduce degradation in the early endosomes-late endosomes-lysosome pathway, thereby inducing the enhanced transcytosis. In particular, the zombie mouse model bearing orthotopic ovarian cancer further validated DN@OEV presented high accumulation and penetration in tumor tissue by the transcytosis process. Our study indicated the strategy in enhancing transcytosis has significant implications for improving the therapeutic efficacy of the drug delivery system.

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Traumatic brain injury (TBI) induces pro-inflammatory polarization of astrocytes and causes secondary disruption of the blood-brain barrier (BBB) and brain damage. Herein, we report a successful astrocyte-targeted delivery of small interfering RNA (siRNA) by ligand functionalized lipid nanoparticles (LNPs) formulated from adenosine-conjugated lipids and a novel ionizable lipid (denoted by Ad4 LNPs). Systemic administration of Ad4 LNPs carrying siRNA against TLR4 to the mice TBI model resulted in the specific internalization of the LNPs by astrocytes in the vicinity of damaged brain tissue. A substantial knockdown of TLR4 at both mRNA and protein levels in the brain was observed, which led to a significant decrease of key pro-inflammatory cytokines and an increase of key anti-inflammatory cytokines in serum. Dye leakage measurement suggested that the Ad4-LNP-mediated knockdown of TLR4 attenuated the TBI-induced BBB disruption. Together, our data suggest that Ad4 LNP is a promising vehicle for astrocyte-specific delivery of nucleic acid therapeutics.

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The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters cells by binding to the angiotensin-converting enzyme 2 (hACE2) receptor. This process is aided by the transmembrane protease serine 2 (TMPRSS2), which enhances entry efficiency and infectiousness by cleaving the SARS-CoV-2 surface glycoprotein (Spike). The cleavage primes the Spike protein, promoting membrane fusion instead of receptor-mediated endocytosis. Despite the pivotal role played by TMPRSS2, our understanding of its non-protease distinct domains remains limited. In this report, we present evidence indicating the potential phosphorylation of a minimum of six tyrosine residues within the cytosolic tail (CT) of TMPRSS2. Via the use of TMPRSS2 CT phospho-mimetic mutants, we observed a reduction in TMPRSS2 protease activity, accompanied by a decrease in SARS-CoV-2 pseudovirus transduction, which was found to occur mainly via the endosomal pathway. We expanded our investigation beyond TMPRSS2 CT and discovered the involvement of other non-protease domains in regulating infection. Our co-immunoprecipitation experiments demonstrated a strong interaction between TMPRSS2 and Spike. We revealed a 21 amino acid long TMPRSS2-Spike-binding region (TSBR) within the TMPRSS2 scavenger receptor cysteine-rich (SRCR) domain that contributes to this interaction. Our study sheds light on novel functionalities associated with TMPRSS2's cytosolic tail and SRCR region. Both of these regions have the capability to regulate SARS-CoV-2 entry pathways. These findings contribute to a deeper understanding of the complex interplay between viral entry and host factors, opening new avenues for potential therapeutic interventions.

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The non-specificity of standard anticancer therapies has profound detrimental consequences in clinical treatment. Therapeutic specificity can be precisely achieved using cutting-edge ligands. Small synthetic oligonucleotide-ligands chosen through Systematic evolution of ligands by exponential enrichment (SELEX) would be an unceasing innovation in using nucleic acids as aptamers, frequently referred to as "chemical antibodies." Aptamers act as externally controlled switching materials that can attach to various substrates, for example, membrane proteins or nucleic acid structures. Aptamers pose excellent specificity and affinity for target molecules and can be used as medicines to suppress tumor cell growth directly. The creation of aptamer-conjugated nanoconstructs has recently opened up innovative options in cancer therapy that are more effective and target tumor cells with minor toxicity to healthy tissues. This review focuses on a comprehensive description of the most capable classes of aptamer-tethered nanocarriers for precise recognition of cancer cells with significant development in proficiency, selectivity, and targetability for cancer therapy. Existing theranostic applications with the problems and future directions are also highlighted.

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Peptide-drug conjugates are delivery systems for selective delivery of cytotoxic agents to target cancer cells. In this work, the optimized synthesis of JH-VII-139-1 and its c(RGDyK) peptide conjugates is presented. The low nanomolar SRPK1 inhibitor, JH-VII-139-1, which is an analogue of Alectinib, was linked to the ανß3 targeting oligopeptide c(RGDyK) through amide, carbamate and urea linkers. The chemostability, cytotoxic and antiangiogenic properties of the synthesized hybrids were thoroughly studied. All conjugates retained mid nanomolar-level inhibitory activity against SRPK1 kinase and two out of four conjugates, geo75 and geo77 exhibited antiproliferative effects with low micromolar IC50 values against HeLa, K562, MDA-MB231 and MCF7 cancer cells. The activities were strongly related to the stability of the linkers and the release of JH-VII-139-1. In vivo zebrafish screening assays demonstrated the ability of the synthesized conjugates to inhibit the length or width of intersegmental vessels (ISVs). Flow cytometry experiments were used to test the cellular uptake of a fluorescein tagged hybrid in MCF7 and MDA-MB231 cells that revealed a receptor-mediated endocytosis process. In conclusion, most conjugates retained the inhibitory potency against SRPK1 as JH-VII-139-1 and demonstrated antiproliferative and antiangiogenic activities. Further animal model experiments are needed to uncover the full potential of such peptide conjugates in cancer therapy and angiogenesis-related diseases.

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Epithelial cells that line the proximal tubule of the kidney rely on an intertwined ecosystem of vesicular membrane trafficking pathways to ensure the reabsorption of essential nutrients. To function effectively and to achieve homeostasis, these specialized cells require the sorting and recycling of a wide array of cell surface proteins within the endolysosomal network, including signaling receptors, nutrient transporters, ion channels, and polarity markers. The dysregulation of the endolysosomal system can lead to a generalized proximal tubule dysfunction, ultimately causing severe metabolic complications and kidney disease.In this chapter, we highlight the biological functions of the genes that code endolysosomal proteins from the perspective of understanding - and potentially reversing - the pathophysiology of endolysosomal disorders affecting the proximal tubule of the kidney. These insights might ultimately lead to potential treatments for currently intractable diseases and transform our ability to regulate kidney homeostasis and health.

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Extracellular vesicles (EVs) have recently received much attention about the application of drug carriers due to their desirable properties such as nano-size, biocompatibility, and high stability. Herein, we demonstrate orange-derived extracellular vesicles (OEV) nanodrugs (DN@OEV) by modifying cRGD-targeted doxorubicin (DOX) nanoparticles (DN) onto the surface of OEV, enabling significantly enhancing tumor accumulation and penetration, thereby efficiently inhibiting the growth of ovarian cancer. The obtained DN@OEV enabled to inducement of greater transcytosis capability in ovarian cancer cells, which presented the average above 10-fold transcytosis effect compared with individual DN. It was found that DN@OEV could trigger receptor-mediated endocytosis to promote early endosome/recycling endosomes pathway for exocytosis and simultaneously reduce degradation in the early endosomes-late endosomes-lysosome pathway, thereby inducing the enhanced transcytosis. In particular, the zombie mouse model bearing orthotopic ovarian cancer further validated DN@OEV presented high accumulation and penetration in tumor tissue by the transcytosis process. Our study indicated the strategy in enhancing transcytosis has significant implications for improving the therapeutic efficacy of the drug delivery system.

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Nature serves as a priceless source for phytomedicines to treat different types of cancer, including hepatocellular carcinoma (HCC). Apocynin (APO), an anti-cancer phytomedicine, is a particular nicotinamide adenine dinucleotide phosphate-oxidase (NADPH-oxidase) inhibitor, which has recently dawned for its multilateral pharmacological activities. As far as we are aware, no investigation has been carried out yet to develop a targeted-nanostructured delivery system of APO to HCC. Consequently, chitosan derivative with galactose groups namely; galactosylated chitosan (GC), particularly recognized by the asialoglycoprotein receptor (ASGR), was synthesized and its chemical structure was thoroughly characterized by substantial techniques. Afterwards, GC-coated nanoplatform for hepatocyte attachment "APO-loaded galactosylated chitosan-coated poly(d,l-lactide-co-glycolide) nanoparticles (APO-loaded GC-coated PLGA NPs)" was developed. The prosperous APO-loaded GC-coated PLGA NPs would be comprehensively appraised through extensive investigations. Their solid state characterization using Fourier transform-infrared spectroscopy, powder X-ray diffraction, and differential scanning calorimetry proved APO's encapsulation in the polymeric matrix. Transmission electron microscopy imaging of the investigated NPs highlighted their spherical architecture with a nanosized range and a characteristic halo-like appearance traceable to the GC coating of the NPs' surface. Saliently, the results of in vitro cytotoxicity screening revealed the spectacular anti-cancer efficacy of APO-loaded GC-coated PLGA NPs formula against the HepG2 cell line. Moreover, the fluorescence microscope disclosed the distinguished cellular uptake of such formula via ASGPR mediated endocytosis. Inclusively, a multifunctional nano-phytomedicine delivery system with a promising active hepatocyte-targeting, effective uptake into HepG2 cells, and sustained drug release pattern was successfully developed.

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Innumerable people worldwide die of cancer every year, although pharmaceutical therapy has actualized many benefits in human health. For background, anti-cancer drug development is difficult due to the multifactorial pathogenesis and complicated pathology of cancers. Cancer cells excrete hydrophobic low-molecular anti-cancer drugs by overexpressed efflux transporters such as multiple drug resistance 1 (MDR1) at the apical membrane. Mutation-driven drug resistance is also developed in cancer. Moreover, the poor distribution of drug to cancer cells is a serious problem, because patients suffer from off-target side effects. Thus, highly selective and effective drug delivery into solid cancer cells across the membrane should be established. It is known that substances (10-100 nm in diameter) such as monoclonal antibodies (mAbs) (approximately 14.2 nm in diameter) or nanoparticles spontaneously gather in solid tumor stroma or parenchyma through the capillary endothelial fenestration, ranging from 200-2000 nm, in neovasculatures due to the enhanced permeability and retention (EPR) effect. Furthermore, cancer antigens, such as HER2, Nectin-4, or TROP2, highly selectively expressed on the surface of cancer cells act as a receptor for receptor-mediated endocytosis (RME) using mAbs against such antigens. Thus, antibody-drug conjugates (ADCs) are promising anti-cancer pharmaceutical agents that fulfill accurate distribution due to the EPR effect and due to antibody-antigen binding and membrane permeability owing to RME. In this review, I introduce the implementation and possibility of highly selective anti-cancer drug delivery into solid cancer cells based on the EPR effect and RME using anti-cancer antigens ADCs with payloads through suitable linkers.

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Megalin and cubilin, endocytic proteins present in the proximal tubule of the kidney, are responsible for reabsorbing filtered proteins from urine. Our hypothesis was that potential substrates of megalin/cubilin could be identified by examining urinary protein differences between control (WT) mice and kidney-specific megalin knockdown (KD) mice. Using the IonStar proteomics approach, 877 potential megalin/cubilin substrates were discovered, with 23 of these compounds representing known megalin/cubilin substrates. Some of the proteins with the largest fold changes in the urine between KD and WT included the known megalin substrates retinol-binding protein and vitamin D-binding protein. Of the total proteins identified as novel substrates, about three-quarters of compounds had molecular weights (MWs) below 69 kDa, the MW of albumin, and the remaining had higher MWs, with about 5% of the proteins having MWs greater than 150 kDa. Sex differences in the number of identified substrates occurred, but this may be due to differences in kidney megalin expression between both male and female megalin KD and WT animals, with the ratio of megalin between WT and KD being 2.76 and 2.14 for female and male mice, respectively. The top three ingenuity canonical pathways based on the urinary proteins in both female and male KD mice were acute phase response signaling, liver X receptor/retinoid X receptor activation, and intrinsic prothrombin activation pathways. In conclusion, analysis of urine samples from kidney-specific megalin KD and WT mice was found to be useful for the identification of potential endogenous substrates for megalin and cubilin.

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Efficient conventional chemotherapy is limited by its nonspecific nature, which causes severe systemic toxicity that can lead to patient discomfort and low therapeutic efficacy. The emergence of smart drug delivery systems (SDDSs) utilizing nanoparticles as drug nanocarriers has shown great potential in enhancing the targetability of anticancer agents and limiting their side effects. Liposomes are among the most investigated nanoplatforms due to their promising capabilities of encapsulating hydrophilic, lipophilic, and amphiphilic drugs, biocompatibility, physicochemical and biophysical properties. Liposomal nanodrug systems have demonstrated the ability to alter drugs' biodistribution by sufficiently delivering the entrapped chemotherapeutics at the targeted diseased sites, sparing normal cells from undesired cytotoxic effects. Combining liposomal treatments with ultrasound, as an external drug release triggering modality, has been proven effective in spatially and temporally controlling and stimulating drug release. Therefore, this paper reviews recent literature pertaining to the therapeutic synergy of triggering nanodrugs from liposomes using ultrasound. It also highlights the effects of multiple physical and chemical factors on liposomes' sonosensetivity, several ultrasound-induced drug release mechanisms, and the efficacy of ultrasound-responsive liposomal systems in cancer therapy. Overall, liposomal nanodrug systems triggered by ultrasound are promising cancer therapy platforms that can potentially alleviate the detriments of conventional cancer treatments.

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Effective chemotherapy for clinical treatment of brain diseases is still lacking due to the poor penetration of the blood-brain barrier (BBB). The aim of this study was to construct a folate modified pterostilbene (Pt) loaded polymeric micellar delivery system (F-Pt/M) with mPEG-PCL as carrier material to aim at penetrating the BBB for brain tissue targeting via receptor-mediated endocytosis. In this study, F-Pt/M was prepared using thin-film hydration method and then optimized by response surface methodology (RSM) with the entrapment efficiency (EE), drug loading (DL) and hydrodynamic diameter (HD) as indexes. The average hydrodynamic diameter and zeta potential of optimal F-Pt/M were 133.2 nm and 24.6 mV, respectively. DL (18.3%) and EE (98.6%) made the solubility of Pt in water about 25 times higher than that of crude Pt. Results of DSC evaluation revealed that drugs were successfully encapsulated inside the polymeric micelles. TEM images showed that homogeneous spherical micellar structures with a narrow size distribution were developed. The release result in vitro showed that F-Pt/M presented sustained release behavior compared to control free Pt solution. Compared to non-targeted Pt/M, F-Pt/M had a significantly higher cytotoxicity against FR-overexpressing A172 cells. In vitro cellular uptake tests illustrated that the micellar delivery system could significantly improve the accumulation of drugs in target cells via receptor-mediated endocytosis. BBB penetration value (P) of F-Pt/M was about 4 folds higher than that of free Pt group. In addition, drug targeting index (DTI) was calculated to determine targeting of F-Pt/M to the brain which was found to be 4.89, implying improved brain targeting was achieved. Hence, the developed F-Pt/M exhibited great potential for delivering more drug molecules across the BBB for the treatment of brain diseases.

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Nanoparticle (NP) internalization by cells is complex, highly heterogeneous, and fundamentally important for nanomedicine. We report powerful probabilistic statistics from single-cell data on quantitative NP uptake of PEG-coated transferrin receptor-targeted gold NPs for cancer-derived and fibroblast cells according to their cell size, receptor expression, and receptor density. The smaller cancer cells had a greater receptor density and more efficient uptake of targeted NPs. However, simply due to fibroblasts being larger with more receptors, they exhibited greater NP uptake. While highly heterogeneous, targeted NP uptake strongly correlated with receptor expression. When uptake was normalized to cell size, no correlation existed. Consequently, skewed population distributions in cell sizes explain the distribution in NP uptake. Furthermore, exposure to the transferrin receptor-targeted NPs alters the fibroblast size and receptor expression, suggesting that the receptor-targeted NPs may interfere with the metabolic flux and nutrient exchange, which could assist in explaining the altered regulation of cells exposed to nanoparticles.