Process Of Receptor-mediated Endocytosis Research Articles

Histopathological characteristics of PRRS and expression profiles of viral receptors in the piglet immune system

Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious viral disease that causes significant economic losses to the swine industry worldwide. PRRS virus (PRRSV) infection is a receptor-mediated endocytosis and replication process. The purpose of this study was to determine the localization and expression of four important PRRSV receptors in immunological organs of piglets. After piglets were infected with PRRSV, Hematoxylin and Eosin staining, immunofluorescence, and Western blot were used to perform histopathological examination and receptors distribution analysis. The results showed that PRRSV caused severe damage to the piglets’ immune organs, including atrophy of the thymus and swelling of lymph node. Histopathological lesions were mainly observed in the lung and lymph node and were characterized by interstitial pneumonia, collapsed follicles, exhaustion of germinal centers, and extensive hemorrhage. Immunofluorescence staining and Western blot results showed that the receptors of CD163 and NMHCII-A were mainly distributed in the thymus, hilar lymph nodes, and mesenteric lymph nodes. However, Sn and vimentin receptors were expressed at low levels in the immune organs of piglets. The distribution of the four receptors in the immune organs was more concentrated in the cortex but was more scattered in the medulla. Compared to the control group, the relative expression of the four receptors increased significantly in most immune organs after viral infection. In conclusion, our study examined the distribution and expression of four PRRSV receptors in immunological organs. We observed a significant increase in the expression of Sn, CD163, and vimentin following viral infection. These findings may provide potential targets for future antiviral reagent design or vaccine development.

Oral delivery of solid lipid nanoparticles surface decorated with hyaluronic acid and bovine serum albumin: A novel approach to treat colon cancer through active targeting

The present study aims to prepare and evaluate solid lipid nanoparticles (SLNs) loaded with irinotecan (IRN) drug and daidzein (DZN) isoflavonoid and surface coated with ligand materials such as hyaluronic acid (HA) and bovine serum albumin (BSA) with additional coating of chitosan for active targeting to receptors present on colon surface epithelium for oral targeted delivery. The optimized batch was evaluated for particle size, zeta potential exhibiting nanometric size with good entrapment efficiency. Nanoparticles were found to be spherical. FTIR and DSC revealed that all the excipients and formulation were compatabile to each other and showed better encapsulation exhibiting amorphous and crystallinity forms. In vitro drug release of SLNs confirmed that initially a burst release, followed by sustained release pattern was exhibited. Cell lines studied performed on HT-29 cells showed demonstrated that conjugated SLNs inhibited cytotoxicity at 75 μg/ml, indicating that cells were taken up through a receptor-mediated endocytosis process. Cell cycle analysis showed that cell arrest was done at 67.8 % (G0/G1 phase) and inhibited apoptosis by 56 %. Further during In vivo studies, RT-PCR study revealed downregulation of Carcinoembryonic antigen (CEA), a non-specific serum biomarker overexpressed in tumor cells and upregulation of pro-inflammatory cytokine TNF-α. Histopathological study revealed that conjugated (HA-BSA) coated with chitosan SLNs restored normal mucosa and colon architecture, depicting all mucosal layers. Hence, these conjugated SLNs may serve as a novel combination for the treatment of colon cancer.

Folic acid-decorated vitamin E Poly(ethylene glycol) monoplatinum ester with disulfide bond as theranostic nanoparticle for drug resistant tumor diagnosis and treatment

Vitamin E Poly(ethylene glycol) monoplatinum ester (TPGS) nanoparticles have attracted much attention in recent years for overcome multidrug resistance. Herein, a well-defined folic acid (FA)-conjugated and disulfide bond-linked polymer (FA-SS-TPGS) was synthesized. These polymer nanoparticles were utilized as theranostic agents for tumor-targeted magnetic resonance imaging (MRI) and chemotherapy. By loading doxorubicin (DOX) and superparamagnetic iron oxide (SPIO) particles into TPGS nanoparticles, FA-SS-TPGS@DOX/SPIO nanoparticles are obtained. In vitro drug release studies revealed that under a reducing environment in the presence of glutathione (GSH), approximately 100% of the doxorubicin (DOX) was released from the disulfide bond-linked theranostic nanoparticles within 24 h. DOX and SPIO were efficiently delivered into HepG2-ADM cells due to the folate receptor-mediated endocytosis process of the nanoparticles. Additionally, the presence of glutathione (GSH) triggered the cleaving of the disulfide bonds, further facilitating the delivery of DOX and SPIO into the cells. Furthermore, the FA-SS-TPGS @DOX-SPIO nanoparticles exhibited strong MRI contrast enhancement properties. In conclusion, FA-SS-TPGS@DOX/SPIO are potential nanoparticles for tumor-targeted MRI and chemotherapy, which can also overcome multidrug resistance.

A crustacean vitellogenin-derived peptide as an oocyte-specific delivery vehicle for gene silencing

Gene silencing by dsRNA is well documented in crustaceans, but RNA interference (RNAi) in developing oocytes is yet to be achieved. The main obstacle to RNAi in the oocytes of oviparous animals derives from their protective layers, including the cytosolic membrane, the vitelline envelope, and a layer of follicular cells. These layers form a barrier preventing the entry of large nonspecific molecules, such as double-stranded RNA (dsRNA). This article describes a sophisticated tool – designated OSDel [oocyte-specific delivery] – for the delivery of dsRNA for gene silencing in the developing oocyte. The methodology exploits the process of receptor-mediated endocytosis (RME) taking place in the crustacean female for internalizing vitellogenin (Vg) (the precursor of the major yolk protein, vitellin) into oocytes. In this process, the extracellular domain of the Vg receptor (VgR) interacts with a distinct amino acid sequence of Vg and internalizes it to form yolk droplets. Here, we tested the premise that a distinct 24 amino-acid peptide derived from Macrobrachium rosenbergii Vg (designated VgP) would interact with VgR to form particles of a size suitable for piggy-backing dsRNA into oocytes via RME. We found that fluorescently labeled VgP had a micromolar affinity for the ligand-binding domain (LBD) of the VgR and could indeed be delivered to and internalized in oocytes. As model system to illustrate the applicability of the OSDel, we injected vitellogenic females with dsRNA encoding the eye development gene PAX6 piggybacked on VgP. Proof that the dsRNA had been successfully internalized into the developing oocytes and had silenced the PAX6 gene was derived from impaired eye development in 87% of the embryos. The ability to manipulate embryos by simple injection into vitellogenic crustacean females may prove to be a powerful high throughput tool for functional genomics investigation in crustacean embryos and for silencing genes relevant to crustacean aquaculture and biotechnology.

Synthesis and Anti-Angiogenic Activity of Novel c(RGDyK) Peptide-Based JH-VII-139-1 Conjugates

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 ανβ3 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.

Effects of receptor properties on particle internalization through receptor-mediated endocytosis.

Receptor-mediated endocytosis (RME) is a highly complex process carried out by bioparticles, such as viruses and drug carriers, to enter cells. The discovery of both clathrin-dependent and clathrin-free pathways makes the RME process even more intriguing. Numerical models have been developed to facilitate the exploration of the process. However, the impacts of the receptor properties on RME have been less studied partially due to the oversimplifications of the receptor models. In this paper, we implement a stochastic model to systematically investigate the effects of mechanical (receptor flexure), geometrical (receptor length) and biochemical (ligand-receptor cutoff) properties of receptors, on RME with and without the existence of clathrin. Our simulation results show that the receptor's flexural rigidity plays an important role in RME with clathrin. There is a threshold beyond which particle internalization will not occur. Without clathrin, it is very difficult to achieve complete endocytosis with ligand-receptor interactions alone. A shorter receptor length and longer ligand-receptor reaction cutoff promote the formation of ligand-receptor bonds and facilitate particle internalization. Complete internalization can only be obtained with an extremely short receptor length and long reaction cutoff. Therefore, there are most likely some additional mechanisms to drive the membrane deformation in clathrin-free RME. Our results yield important fundamental insights into RME and provide crucial guidance when correlating the simulation results with experimental observations.

Suppression of Estrogen Receptor in Breast Cancer Cells via Transferrin Coated Gold Nanoparticles Carrying Antisens Oligonucleotides

Breast cancer is the most common cancer of women. In breast cancer treatment the estrogen receptor is an established target in receptor positive tumors. A novel approach to therapy is gene regulation by oligonucleotide drugs. Antisens oligonucleotide therapy is facing with some difficulties such as targeting a polianionic nucleotide into the tumor cell. In order to form an efficient carrier system to protect and target antisens oligonucleotides against estrogen receptor α mRNA, a gold nanoparticle based construct is formed. Uptake of amino and thiol modified gold nanoparticles was analysed by confocal microscopy. Gold nanaparticles were functionalysed by transferrin coating and estrogen receptor α antisens oligonucleotide and were applied to MCF7 breast cancer cells. It was found that funtionalized gold nanoparticles decreased the estrogen receptor expression by 51% as well as cell proliferation upto 41.00 ± 5.6 % of untreated cells. Therefore, this targeting construct using the transferrin receptor and receptor mediated endocytosis process delivers the antisens oligonucleotide efficiently. Since estrogen receptor α is an important regulator of cell proliferation, this system is able to inhibit proliferation by means of decreasing the expression level of this major regulatory molecule. It is concluded that, this functionalysed gold nanoparticle system may be designed for other targets in tumors and may also be used as a teranostic agent.

Smart Lipid-Polysaccharide Nanoparticles for Targeted Delivery of Doxorubicin to Breast Cancer Cells.

In this study, actively-targeted (CD44-receptors) and dual stimuli (pH/redox)-responsive lipid–polymer nanoparticles were proposed as a delivery vehicle of doxorubicin hydrochloride in triple negative breast cancer cell lines. A phosphatidylcholine lipid film was hydrated with a solution of oxidized hyaluronic acid and doxorubicin, chosen as model drug, followed by a crosslinking reaction with cystamine hydrochloride. The obtained spherical nanoparticles (mean diameter of 30 nm) were found to be efficiently internalized in cancer cells by a receptor-mediated endocytosis process, and to modulate the drug release depending on the pH and redox potential of the surrounding medium. In vitro cytotoxicity assays demonstrated the safety and efficacy of the nanoparticles in enhancing the cytotoxic effect of the free anticancer drug, with the IC50 values being reduced by two and three times in MDA-MB-468 and MDA-MB-231, respectively. The combination of self-assembled phospholipid molecules with a polysaccharide counterpart acting as receptor ligand, and stimuli-responsive chemical moieties, was carried out on smart multifunctional nanoparticles able to actively target breast cancer cells and improve the in vitro anticancer activity of doxorubicin.

Folic Acid-Decorated β-Cyclodextrin-Based Poly(ε-caprolactone)-dextran Star Polymer with Disulfide Bond-Linker as Theranostic Nanoparticle for Tumor-Targeted MRI and Chemotherapy.

β-cyclodextrin(βCD)-based star polymers have attracted much interest because of their unique structures and potential biomedical and biological applications. Herein, a well-defined folic acid (FA)-conjugated and disulfide bond-linked star polymer ((FA-Dex-SS)-βCD-(PCL)14) was synthesized via a couple reaction between βCD-based 14 arms poly(ε-caprolactone) (βCD-(PCL)14) and disulfide-containing α-alkyne dextran (alkyne-SS-Dex), and acted as theranostic nanoparticles for tumor-targeted MRI and chemotherapy. Theranostic nanoparticles were obtained by loading doxorubicin (DOX), and superparamagnetic iron oxide (SPIO) particles were loaded into the star polymer nanoparticles to obtain ((FA-Dex-SS)-βCD-(PCL)14@DOX-SPIO) theranostic nanoparticles. In vitro drug release studies showed that approximately 100% of the DOX was released from disulfide bond-linked theranostic nanoparticles within 24 h under a reducing environment in the presence of 10.0 mM GSH. DOX and SPIO could be delivered into HepG2 cells efficiently, owing to the folate receptor-mediated endocytosis process of the nanoparticles and glutathione (GSH), which triggered disulfide-bonds cleaving. Moreover, (FA-Dex-SS)-βCD-(PCL)14@DOX-SPIO showed strong MRI contrast enhancement properties. In conclusion, folic acid-decorated reduction-sensitive star polymeric nanoparticles are a potential theranostic nanoparticle candidate for tumor-targeted MRI and chemotherapy.

Probing Allosteric Modulation of Membrane Receptor in the Native State by Data Mining-Integrated Tracking Microscopy

Probing Allosteric Modulation of Membrane Receptor in the Native State by Data Mining-Integrated Tracking Microscopy

Unfolding of Tryptophanoctyl Ester and Elastic Deformation of Host Micelles via RR'3 N+ ⋅⋅⋅π Interaction: Conceivable Relevance to Wrapping Process of Receptor Mediated Endocytosis.

The interfacial properties of the mixed amphiphiles are modified by a stronger cation-π interaction between the quaternary ammonium head group of CTAB and the π-face of TROE, compared to the tyrosine analogue (TYOE). This eventually triggers a morphology transition through elastic deformation of the spherical micelles of CTAB to cylindrical/wormlike micelles. The unfolding of TROE and the molecular interactions in the nanoenvironment have been recognized by NMR spectroscopy and the physical characteristics of the entangled wormlike micelles are investigated by high resolution transmission electron microscopy (HRTEM), whereas the complex fluidic feature is examined by dynamic rheological measurements. Morphology tuning of the soft nanoaggregates of zwitterionic dodecylphosphocholine by the tryptophan analogue via choline-π interaction has unique biological consequences and we consider the significance of such interactions in facilitating endocytosis of a virion/nano particle(NP) in terms of a quantitative model. The implication in future research on drug development strategies is discussed.

Targeted nanoformulation of C1 inhibits the growth of KB spheroids and cancer stem cell-enriched MCF-7 mammospheres

C1, a synthetic analog of curcumin, has been reported to show potent antiproliferative effects against a variety of cancer cells. Here, we report a strong anticancer activity of the folate receptor-targeted lipid nanoparticle formulation of C1 against cancer cells and cancer stem cells both in 2D culture and 3D spheroids. The size of the C1 encapsulated folic acid functionalized nanoliposomes (Lipos-C1) was determined to be 83 ± 17 nm. Lipos-C1 nanoparticles displayed sustained C1 release kinetics at both pH 7.4 and 5.5. The folate receptor (FR) targeted nanoliposomes were internalized into FR-positive KB cells via the folate receptor-mediated endocytosis process. Lipos-C1 killed KB cells much more efficiently than C1. Lipos-C1 depolymerized microtubules, generated ROS, caused DNA damage, and induced apoptosis in KB cells. Importantly, Lipos-C1 strongly inhibited the growth of the 3D KB spheroids than C1. Interestingly, Lipos-C1 also suppressed cancer stem cells (CSCs) enriched MCF-7 mammosphere growth by impeding breast cancer stem cells (BCSCs) enrichment, growth, and proliferation. The results suggested that Lipos-C1 could be a promising nanoformulation for cancer chemotherapy.

Tagging Transferrin Receptor with a Disulfide FRET Probe To Gauge the Redox State in Endosomal Compartments.

Although the basic process of receptor-mediated endocytosis (RME) is well established, certain specific aspects, like the endosomal redox state, remain less characterized. Previous studies used chemically labeled ligands or antibodies with a FRET (fluorescence resonance energy transfer) probe to gauge the redox activity of the endocytic pathway with a limitation being their inability to track the apo receptor. New tools that allow direct labeling of a cell surface receptor with synthetic probes would aid in the study of its endocytic pathway and function. Herein, we use a peptide ligase, butelase 1, to label the human transferrin receptor 1 (TfR1) in established human cell lines with a designer disulfide FRET probe. This strategy enables us to obtain real-time live cell imaging of redox states in TfR1-mediated endocytosis, attesting a reducing environment in the endosomal compartments and the dynamics of TfR1 trafficking. A better understanding of endocytosis of different cell surface receptors has implications in designing strategies that hijack this natural process for intracellular drug delivery.

Cyclodextrin conjugated ferritin nanocages reduce intracellular cholesterol level in foam cells

Accumulation of lipid-laden macrophages (foam cells) is characteristic of atherosclerosis development in the arterial walls. Ferritin nanocages have been found to passively accumulate in the atherosclerotic plaque. Ferritin has been actively investigated as a carrier for contrast agents in atherosclerosis diagnosis. We demonstrate the potential of ferritin as a carrier for therapeutic molecules to mediate cholesterol reduction from foam cells. Cyclodextrin molecules are chemically conjugated to the ferritin nanocages surface or encapsulated within the nanocages using metal co-loading methods. The cyclodextrin-conjugated ferritin has nanomolar affinity to cholesterol molecules. Treatment of foam cells with the conjugates shows decreased levels of intracellular accumulated cholesterol. The preferential localization of ferritin to foam cells is due to transferrin receptor-mediated endocytosis process. These findings show that ferritin nanocages as carriers localize cyclodextrin molecules to foam cells which mediate intracellular cholesterol reduction, thus highlighting its potential use as a therapeutic agent.

Mathematical modeling of the receptor-mediated endocytosis process of targeted therapeutic agents in drug delivery systems

In this paper, we propose mathematical models to describe receptor-mediated endocytosis processes. One is a stochastic differential model for the agent-target binding process. The mean extinction time and a standard variation over time profile are evaluated. The other is an age-structured model for demonstrating endocytosis and lysosome processes. A targeted drug delivery system has a complex process in how it is to deliver drug molecules in terms of administration, transportation in blood and across membranes to intracellular space, and inhibition to microtubule polymerization. In particular, receptor-mediated endocytosis of targeted therapeutic agents, such as antibody drug conjugates or ligand-targeted liposome encapsulated nanoparticles, is a key step in understanding the drug delivery mechanism. We discuss stochastic quasi steady state approximation when agent-target complex does not appreciably vary compared with the free agents. This reduces the number of the systems and the parameters; however, an initial time phase cannot be captured. In addition, we discuss the strengths and weaknesses when the age-structured model induces the reduced model compared with the full model that considers endocytosis and lysosome processes. If the total mean retention time until payload release in intracellular space is known, then the age-structured model with the Erlang distribution may fairly predict data of the released payload over time profile with far fewer parameters; however, induced compartments lose their physical meaning and describe only a delay.

Amphiphilic Glycopolypeptide Star Copolymer-Based Cross-Linked Nanocarriers for Targeted and Dual-Stimuli-Responsive Drug Delivery.

Glycopolypeptide-based nanocarriers are an attractive class of drug delivery vehicles because of the involvement of carbohydrates in the receptor-mediated endocytosis process. To enhance their efficacy toward controlled and programmable drug delivery, we have prepared stable glycopolypeptide-based bioactive dual-stimuli-responsive (redox and enzyme) micelles for delivery of anticancer drugs specifically to the cancer cells. The amphiphilic biocompatible miktoarm star copolymer, which comprises two hydrophobic poly(ε-caprolactone) blocks, a short poly(propargyl glycine) middle block, and a hydrophilic galactose glycopolypeptide block, was designed and synthesized. The star copolymer is initially self-assembled into un-cross-linked (UCL) micelles, and free alkyne groups at the core-shell interface of the UCL micelles, which were cross-linked by bis(azidoethyl) disulfide (BADS) via click chemistry to form interface cross-linked (ICL) micelles. ICL micelles were found to be stable against dilution. BADS imparted redox-responsive properties to the micelles, while PCL rendered them enzyme-degradable. Dual-stimuli-responsive release behavior with Dox as model drug was studied individually as well as synergistically by applying two stimuli in different sequences. The galactose-containing UCL and ICL micelles were shown to be nontoxic. Intracellular Dox release from UCL and ICL micelles was demonstrated in liver cancer cells (HepG2) by time-dependent cellular uptake studies, and controlled release from ICL micelles compared to UCL micelles was observed. The present report opens a new approach toward targeted and programmable drug delivery in tumor tissues via a specifically targeted (receptor-mediated), dual-responsive, and stable cross-linked nanocarrier system.

Polyyne bridged AIE luminogens with red emission: design, synthesis, properties and applications.

Construction of a donor-acceptor (D-A) structure and extension of π-conjugation are the commonly used strategies to shift the emission of luminophores to the red region. However, molecules with high conjugation and a strong D-A effect tend to show weak emission due to the severe π-π interactions and twisted intramolecular charge transfer (TICT) effects. The turn-on characteristic of AIE luminogens (AIEgens) will also be lost due to the conjugation-enhanced emission in the solution state. Herein, a polyyne-bridged AIE luminogen (2TPE-4E) with long wavelength absorption and red emission has been afforded. Despite its large π-conjugation, 2TPE-4E suffers from no emission quenching resulted from strong π-π interactions and twisted intramolecular charge transfer effects. The strong red emission and the high photostability of 2TPE-4E inspired us to use it for targeted-imaging of cancer cells and monitoring the receptor mediated endocytosis process.

Apoferritin Nanocage Delivers Combination of Microtubule and Nucleus Targeting Anticancer Drugs.

An ideal nano drug delivery agent must be potent enough to carry high dose of therapeutics and competent enough in targeting specific cell of interest, having adequate optimized physiochemical properties and biocompatibility. Carrying differentially polar therapeutics simultaneously will make them superior in their class. However, it is of enormous challenge to the researchers to find such a unique nanocarrier and to engineer all of the above-mentioned features into it. In this manuscript, we have shown for the first time that apoferritin (Apf) can carry and deliver high dose of doxorubicin (Dox), docetaxel (Doc), and combination of both Dox and Doc specifically into the cancer cell and enhances killing compared to free drug without any functionalization or property modulation. In addition, we have shown that Apf alone is noncytotoxic in nature and interacts with intracellular tubulin/microtubule. Drug loaded Apf specifically bound and consequently internalized into the human breast cancer cell line (MCF7) and human cervical cancer cell line (HeLa) through receptor mediated endocytosis process and releases either single or combination of drugs in the endosome. We have also checked the binding efficacy of both drugs using molecular docking. Further, using fluorescence microscopy, we have shown that Apf can deliver combination of drugs inside cancer cells and the drugs exerts their effect thereof. Finally, we have studied the efficacy of Apf complexes with individual drugs and in combination compared to free drugs in a tumor mimicking 3D multicellular spheroid model of HeLa cell.

Abstract B21: Using computational modeling to quantify targeted agent binding and internalization in pancreatic cancers

Abstract The pancreatic adenocarcinoma is one of the most deadly cancers with only 6% overall 5-year survival rate. Since the current therapies fail to provide successful results, the improvement of techniques for early detection, predicting of treatment efficacy and monitoring of tumor spread during and after surgical procedures remain under focused research. The purpose of this study is to develop a novel method to assess behavior (diffusion and binding within the tissues) of imaging agents targeted to pancreatic cancer cells. Our long-term goal is to design a prospective tool for the prediction of targeted imaging agent peak concentration and cellular uptake based on individual patient data. The toll-like receptor 2 (TLR2) is known to play an important role in the immune system response. In addition, our team reported that TLR2 is a bona fide cell-surface marker for targeting pancreatic cancer. TLR2 recognizes a vast number of biomolecules, including lipoproteins, such as designed by our team, novel TLR2 ligand (TLR2L). Recent development of an intravital fluorescence microscopy method allowed for the real time in vivo imaging of the TLR2L conjugated to near-infrared fluorescent dye, Cyanine 5 (TLR2L-Cy5) and its penetration through the tissue of pancreatic adenocarcinoma tumor xenografts in mice with endogenous expression of TLR2. In order to quantify the space- and time-dependent dynamics of TLR2L we combined intravital dorsal window chamber experiments with computational modeling of TLR2L-Cy5 diffusion and internalization following intravenous administration. Our computational model accounts for an explicitly defined tissue morphology composed of individual tumor cells, extracellular matrix interpenetrated by the interstitial fluid, and tumor vasculature. We also model individual molecules of a fluorescent imaging agent that extravasate via transmural influx from blood capillaries, spread through the tumor and become internalized by the cells. The process of agonist binding to the receptor is modeled by trapping the particle by the near-by cell boundary receptor, which mimics the distance dependent electrostatic and hydrophobic interactions initiating the recognition and binding in vivo. Our studies led to quantification of the TLR2L-Cy5 intratumoral transport including agent extravasation, diffusion and intracellular accumulation in relation to tumor tissue structure and vascular architecture. The performed computational simulations allowed for detailed continuous in time assessment of the targeted imaging agent penetration on the microscopic (cell-to-tissue) level. The calibrated in silico model revealed the time-dependent dynamics of TLR2L-Cy5 agent binding, internalization and intracellular distribution. Our quantitative approach also showed a non-uniform spatial saturation of the TLR2L-Cy5 on the plasma membrane and inside the cell. Moreover, continuous simulations allowed for prediction of the maximum concentration peak in the extracellular matrix and for the uptake peak in individual cells. In addition to quantitative analysis, we provide novel (two- and three-dimensional) visualization tools for imaging agent dynamical penetration, membrane binding and receptor-mediated endocytosis process in pancreatic cells. In conclusion, we present an interdisciplinary approach to quantify diffusion and cellular uptake of an imaging agent targeted to pancreatic cancer cell lines expressing the TLR2 receptor. This integrated approach can be used in the future for the development of other targeted imaging and therapeutic agents, for other solid tumors, and for optimizing the administration schedules and time points for data collection from individual human tumor xenografts in order to improve treatment efficacy. Citation Format: Aleksandra Karolak, Veronica Estrella, Tingan Chen, Amanda Huynh, David Morse, Katarzyna Rejniak. Using computational modeling to quantify targeted agent binding and internalization in pancreatic cancers. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr B21.

Nanoparticles Interacting with Membranes: From Engulfment Patterns to Endocytosis

Nanoparticles are used to deliver drugs, imaging agents, and toxins to biological cells. The delivery requires the engulfment of the nanoparticles by cell membranes, a process that is governed by the interplay of particle adhesion and membrane bending and can be mimicked in model systems consisting of lipid or polymer vesicles. Previous theoretical studies of these systems have focussed on the engulfment of nanoparticles by planar membranes and symmetric bilayers. Here, we consider the general case of nanoparticles interacting with asymmetric bilayer membranes that form complex shapes with non-uniform curvature. Even small bilayer asymmetries lead to two critical particle sizes that determine four distinct engulfment regimes. [1] These regimes depend on two material parameters, the contact mean curvature and the spontaneous curvature, as well as on the local curvature of the membrane segments in contact with the nanoparticles. As a consequence, different segments of the same membrane can belong to different engulfment regimes and a single vesicle in contact with many nanoparticles can display ten distinct engulfment patterns as well as morphological transitions between these patterns. [2] In order to address the more complex process of receptor-mediated endocytosis, we extend our results to account for adhesion-induced segregation of the membrane components and increased spontaneous curvatures arising from protein coats. [1] We derive explicit expressions for the engulfment rate and total uptake of nanoparticles by cells, which are both predicted to depend nonmonotonically on the particle size. These expressions provide a quantitative fit to experimental data for the size-dependence of clathrin-dependent endocytosis of gold nanoparticles by HeLa cells. [1] J. Agudo-Canalejo and R. Lipowsky. ACS Nano 9, 3704 (2015) [2] J. Agudo-Canalejo and R. Lipowsky. Nano Letters (in press)