Cellular Internalization Mechanisms Research Articles

Mesoporous silicon particles as intravascular drug delivery vectors: fabrication, in‐vitro, and in‐vivo assessments

AbstractPorous silicon is an attractive biomaterial for drug delivery thanks to its biocompatibility, biodegradability, ease of fabrication, tunable nanostructure, and porous network. Herein we briefly present the development of a multi‐stage delivery vector that leverages these advantages to enhance delivery of systemically administered therapeutic agents. We illustrate the rational design, objective‐oriented fabrication and geometric control of first stage porous silicon microparticles. We describe how geometry affects the biodistribution of first stage vectors and how their porous structure affects the loading and release of second stage theranostic payloads. We describe the mechanism of cellular internalization and intracellular trafficking of particles. Finally we present two multi‐stage vector prototypes for the delivery of magnetic resonance imaging contrast agents and small interfering RNA (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Peptide‐mediated targeted drug delivery

Targeted drug delivery to specific group of cells offers an attractive strategy to minimize the undesirable side effects and achieve the therapeutic effect with a lower dose. Both linear and cyclic peptides have been explored as trafficking moiety due to ease of synthesis, structural simplicity, and low probability of undesirable immunogenicity. Peptides derived from sequence of cell surface proteins, such as intercellular adhesion molecule-1 (ICAM-1), LHRH, Bombesin, and LFA-1, have shown potent binding affinity to the target cell surface receptors. Moreover, peptides derived from ICAM-1 receptor can be internalized by the leukemic T-cells along with the conjugated moiety offering the promise to selectively treat cancers and autoimmune diseases. Systematic analyses have revealed that physicochemical properties of the drug-peptide conjugates and their mechanism of receptor-mediated cellular internalization are important controlling factors for developing a successful targeting system. This review is focused on understanding the factors involved in the development of an effective drug-peptide conjugate with an emphasis on the chemistry and biology of the conjugates. Reported results on several promising drug-peptide conjugates have been critically evaluated. The approaches and results presented here will serve as a guide to systematically approach targeted delivery of cytotoxic drug molecules using peptides for treatment of several diseases.

Interaction of Poly(glycoamidoamine) DNA Delivery Vehicles with Cell-Surface Glycosaminoglycans Leads to Polyplex Internalization in a Manner Not Solely Dependent on Charge

Understanding the mechanisms of cellular internalization is necessary for rational design of efficient polymers for DNA delivery. In this paper, we present evidence that poly(glycoamidoamine) (PGAA)-DNA complexes (polyplexes) interact with cell-surface glycosaminoglycans (GAGs) in a manner that is not solely dependent on charge. The presence of GAGs appears to be necessary for efficient cellular uptake, as polyplex internalization was decreased in GAG-deficient CHO (pgsA-745) cells. However, uptake was nearly unaffected in cells deficient only in heparan sulfate. Internalization of PGAA polyplexes appears to be dependent on GAG sulfation in mammalian cell lines, yet the PGAA polymers are decomplexed from pDNA by high concentrations of GAGs in a charge-independent manner. This finding suggests that interactions between the carbohydrates on the polymer and GAGs may contribute to polyplex binding. Quartz crystal microbalance studies support the findings that relative PGAA polyplex-GAG binding affinities are also not completely mediated by charge. As measured by dynamic light scattering and TEM, GAGs appear to accumulate on the surface of polyplexes without disrupting them at a lower concentration, which may stimulate cellular internalization due to close interactions between the polyplexes and the GAGs. Gel electrophoresis and fluorescence measurements of an intercalating dye suggest that polyplex interaction with GAGs can induce dissociation, which could represent a potential pDNA release mechanism. These results imply that similar interactions may occur on cell surfaces, and strongly supports the hypothesis that GAGs function as cell surface receptors for polyplexes formed with PGAA vehicles.

Poly(glycoamidoamine) Vehicles Promote pDNA Uptake through Multiple Routes and Efficient Gene Expression via Caveolae-Mediated Endocytosis

The use of synthetic polymers for the delivery of nucleic acids holds considerable promise for understanding and treating disease at the molecular level. This work aims to decipher the cellular internalization mechanisms for a series of synthetic glycopolymer DNA delivery vehicles we have termed poly(glycoamidoamine)s (PGAAs). To this end, we have performed cellular delivery experiments in the presence of pharmacological endocytosis inhibitors. Confocal microscopy analysis showed colocalization of labeled pDNA in polyplexes with immunolabeled endocytic molecules to identify the cellular internalization pathways in HeLa cells. Direct membrane penetration was also investigated through various methods, including cellular energy depletion and leakage of a cytosolic enzyme from the cell. The data suggests that the cellular internalization of PGAA polyplexes occurs through a multifaceted internalization mechanism primarily involving caveolae, yet clathrin-coated vesicles and macropinosomes were also involved to a lesser degree. The primary mechanism that leads to efficient nuclear delivery and transgene expression appears to be caveolae/raft-mediated endocytosis. The cellular internalization pathways for PGAAs were not identical to those for polyethylenimine, illustrating that differences in the chemical structure of materials directly impacts the cellular internalization mechanisms.

Gene delivery targeted to the brain using an Angiopep-conjugated polyethyleneglycol-modified polyamidoamine dendrimer

Angiopep targeting to the low-density lipoprotein receptor-related protein-1 (LRP1) was identified to exhibit high transcytosis capacity and parenchymal accumulation. In this study, it was exploited as a ligand for effective brain-targeting gene delivery. Polyamidoamine dendrimers (PAMAM) were modified with angiopep through bifunctional PEG, then complexed with DNA, yielding PAMAM–PEG–Angiopep/DNA nanoparticles (NPs). The angiopep-modified NPs were observed to be internalized by brain capillary endothelial cells (BCECs) through a clathrin- and caveolae-mediated energy-depending endocytosis, also partly through marcopinocytosis. Also, the cellular uptake of the angiopep-modified NPs were competed by angiopep-2, receptor-associated protein (RAP) and lactoferrin, indicating that LRP1-mediated endocytosis may be the main mechanism of cellular internalization of angiopep-modified NPs. And the angiopep-modified NPs showed higher efficiency in crossing blood–brain barrier (BBB) than unmodified NPs in an in vitro BBB model, and accumulated in brain more in vivo. The angiopep-modified NPs also showed higher efficiency in gene expressing in brain than the unmodified NPs. In conclusion, PAMAM–PEG–Angiopep showed great potential to be applied in designing brain-targeting drug delivery system.

The Role of Ganglioside GM1 in Cellular Internalization Mechanisms of Poly(amidoamine) Dendrimers

Generation 7 (G7) poly(amidoamine) (PAMAM) dendrimers with amine, acetamide, and carboxylate end groups were prepared to investigate polymer/cell membrane interactions in vitro. G7 PAMAM dendrimers were used in this study because higher-generation of dendrimers are more effective in permeabilization of cell plasma membranes and in the formation of nanoscale holes in supported lipid bilayers than smaller, lower-generation dendrimers. Dendrimer-based conjugates were characterized by (1)H NMR, UV/vis spectroscopy, GPC, HPLC, and CE. Positively charged amine-terminated G7 dendrimers (G7-NH(2)) were observed to internalize into KB, Rat2, and C6 cells at a 200 nM concentration. By way of contrast, neither negatively charged G7 carboxylate-terminated dendrimers (G7-COOH) nor neutral acetamide-terminated G7 dendrimers (G7-Ac) associated with the cell plasma membrane or internalized under similar conditions. A series of in vitro experiments employing endocytic markers cholera toxin subunit B (CTB), transferrin, and GM(1)-pyrene were performed to further investigate mechanisms of dendrimer internalization into cells. G7-NH(2) dendrimers colocalized with CTB; however, experiments with C6 cells indicated that internalization of G7-NH(2) was not ganglioside GM(1) dependent. The G7/CTB colocalization was thus ascribed to an artifact of direct interaction between the two species. The presence of GM(1) in the membrane also had no effect upon XTT assays of cell viability or lactate dehydrogenase (LDH) assays of membrane permeability.

Brain-targeting gene delivery and cellular internalization mechanisms for modified rabies virus glycoprotein RVG29 nanoparticles

A 29 amino-acid peptide derived from the rabies virus glycoprotein (RVG29) was exploited as a ligand for efficient brain-targeting gene delivery. RVG29 was modified on polyamidoamine dendrimers (PAMAM) through bifunctional PEG, then complexed with DNA, yielding PAMAM–PEG–RVG29/DNA nanoparticles (NPs). The NPs were observed to be uptaken by brain capillary endothelial cells (BCECs) through a clathrin and caveolae mediated energy-depending endocytosis. The specific cellular uptake can be inhibited by free RVG29 and GABA but not by nicotinic acetylcholine receptor (nAchR) agonists/antagonists, indicating RVG29 probably relates to the GABA B receptor besides nAchR reported previously. PAMAM–PEG–RVG29/DNA NPs showed higher blood-brain barrier (BBB)-crossing efficiency than PAMAM/DNA NPs in an in vitro BBB model. In vivo imaging showed that the NPs were preferably accumulated in brain. The report gene expression of the PAMAM–PEG–RVG29/DNA NPs was observed in brain, and significantly higher than unmodified NPs. Thus, PAMAM–PEG–RVG29 provides a safe and noninvasive approach for the gene delivery across the BBB.

Surface Modification of PAMAM Dendrimers Modulates the Mechanism of Cellular Internalization

The aim of this study was to investigate the influence of dendrimer surface properties on cellular internalization and intracellular trafficking in the human colon adenocarcinoma HT-29 cell line. Third-generation (G3) polyamidoamine (PAMAM) dendrimers were modified to contain either two lauroyl chains (G3L2), two propranolol molecules (G3P2), or two lauroyl and two propranolol molecules (G3L2P2) at the dendrimer surface. Surface-modified and unmodified dendrimers were labeled with fluorescein isothiocyanate (FITC) at an average molar ratio of 1:1. The mechanisms of cellular internalization and intracellular trafficking of dendrimers were analyzed by confocal laser scanning microscopy and flow cytometry. The internalization of G3 and G3P2 dendrimers involved both caveolae-dependent endocytosis and macropinocytosis pathways; internalization of G3L2P2 dendrimer appeared to involve caveolae-dependent, and possibly clathrin-dependent, endocytosis pathways; and internalization of G3L2 dendrimer occurred via caveolae-dependent, clathrin-dependent, and macropinocytosis pathways. Subcellular colocalization data indicated that unmodified and all surface-modified G3 PAMAM dendrimers were internalized and trafficked to endosomes and lysosomes. It is therefore apparent that the initial mode of dendrimer internalization into HT-29 cells is influenced by the surface properties of G3 PAMAM dendrimer.

Meso-tetra (carboxyphenyl) porphyrin (TCPP) nanoparticles were internalized by SW480 cells by a clathrin-mediated endocytosis pathway to induce high photocytotoxicity

We studied the uptake of meso-tetra (carboxyphenyl) porphyrin (TCPP) nanoparticles by SW480 cells and carried out a systematic investigation of the cellular internalization mechanism of TCPP nanoparticles, also studied the photocytotoxicity of TCPP nanoparticles. At first, meso-tetra (carboxyphenyl) porphyrin (TCPP) nanoparticles were prepared by the method of mixing solvent techniques. SW480 cellular uptakes of photosensitizers (TCPP nanoparticles, TCPP-loaded poly (lactic-co-glycolic acid) (PLGA) nanoparticles and free TCPP) were analyzed by the method of fluorospectrophotometry. Endocytosis mechanism investigation was carried out by preincubating SW480 cells at 4 °C, and preincubating SW480 cells with sucrose, K +-free buffer solution and filipin. Clathrin HC expression after incubating SW480 cells with these three photosensitizers was analyzed by methods of Western blot and RT-PCR. At last, we analyzed the photo-cytotoxicity after incubating SW480 cells with photosensitizers and receiving irradiation. SW480 cells showed rapid uptake (0.0083 fmoles TCPP/cell) of TCPP nanoparticles after 1 h incubation. We also demonstrated that the uptake of TCPP nanoparticles by SW480 cells was a clathrin-mediated endocytosis pathway. As a result of rapid internalization of TCPP nanoparticles by SW480 cells, this special photosensitizer showed very high photocytotoxic effect on SW480 cells in vitro. The nano-sized photosensitizer with no matrix cover: TCPP nanoparticles, can produce higher photocytotoxicity than other photosensitizers (TCPP-loaded PLGA nanoparticles and free TCPP). The in vivo tumor growth inhibition experiment indicated that TCPP nanoparticles plus PDT treatment induced the most dramatic tumor-inhibiting efficacy in all TCPP treated groups. The results of this study suggest that TCPP nanoparticles represent a potential and powerful photodynamic therapy agent.

Microfabricated Particles for Engineered Drug Therapies: Elucidation into the Mechanisms of Cellular Internalization of PRINT Particles

To investigate the cellular internalization pathways of shape- and size-specific particles as a function of zeta potential in different cell types. A top-down particle fabrication technique called PRINT was utilized to fabricate monodisperse 1 microm cylindrical particles. Cellular internalization of these PRINT particles was monitored using confocal microscopy, flow cytometry, and transmission electron microscopy. The endocytic pathway used by 1 microm cationic PRINT particles was evaluated using different inhibitory strategies. Cytotoxicity assays were used to determine the toxicity of both cationic and anionic PRINT particles in multiple cell types. Particle internalization was confirmed using confocal microscopy, flow cytometry and transmission electron microscopy. The mechanism of internalization of positively charged PRINT particles was found to be predominantly clathrin-mediated endocytosis and macropinocytosis with very few particles utilizing a caveolae-mediated endocytic pathway. The exposed charge on the surface of the particles had a significant effect on the rate of endocytosis in all cell types tested, except for the macrophage cells. No significant cytotoxicity was observed for all PRINT particles used in the present study. Cylindrical 1 microm PRINT particles were readily internalized into HeLa, NIH 3T3, OVCAR-3, MCF-7, and RAW 264.7 cells. Particles with a positive zeta potential exhibited an enhanced rate of endocytosis compared to negatively charged particles with identical sizes and shapes. It was found that PRINT particles with a positive zeta potential were endocytosed into HeLa cells using predominantely clathrin-mediated and macropinocytotic pathways.

Microfabricated Particles for Engineered Drug Therapies: Elucidation into the Mechanisms of Cellular Internalization of PRINT Particles

Microfabricated Particles for Engineered Drug Therapies: Elucidation into the Mechanisms of Cellular Internalization of PRINT Particles

Imaging Gene Delivery with Fluorescence Microscopy

Gene delivery offers the promise of treatment for a range of human diseases. Although carried out initially with modified viruses, the use of synthetic molecules, including polymers, lipids and peptides, has extended the possibilities greatly for rationally designed vectors tailored to individual gene-delivery applications. Underlying the rational design of gene-delivery vectors is the need to understand the individual steps of the gene-delivery pathway. Using new methods in fluorescence microscopy, it is now possible to isolate individual steps along the gene-delivery pathway to characterize the mechanisms of cellular binding, cellular internalization and nuclear entry. This review describes the advances made in the gene-delivery field with the assistance of fluorescence microscopy. The focus of this review is the use of synthetic gene-delivery vectors, especially polyethylenimine, and the live-cell imaging and single-particle tracking techniques that reveal the intracellular dynamics of the gene-delivery process.

Cell line-dependent internalization pathways and intracellular trafficking determine transfection efficiency of nanoparticle vectors

It has been suggested that cell physiology may affect the internalization pathways of non-viral vectors, leading to cell line-dependent transfection efficiency. To verify this hypothesis, fluorescently labeled alginate–chitosan nanoparticle complexes were used as non-viral vectors to transfect 293T, COS7, and CHO cells and to observe the cellular interactions and internalization mechanisms of the complexes in each cell line. 293T cells, which demonstrate the highest transfection efficiency, internalize complexes primarily through clathrin-mediated processes. COS7 cells also demonstrate some internalization of complexes through the clathrin-dependent pathway, explaining the moderate transfection exhibited. In contrast, CHO cells internalize complexes predominantly through caveolin-mediated pathways and are not transfected. Results suggest that following clathrin-mediated endocytosis, complexes are trafficked to the endo-lysosomal pathway, where the proton-sponge effect leads to their release into the cytosol. Contrarily, the absence of trafficking to this pathway following caveolin-mediated endocytosis results in vesicle-entrapped complexes that become transfection-incompetent. These results demonstrate that cell physiology is a critical factor in efficient transfection, and that trafficking to the endo-lysosomal pathway through specific internalization mechanisms is essential for transfection with alginate–chitosan nanoparticle complexes.

Cell-penetrating-peptide-based delivery of oligonucleotides: an overview

Cationic CPPs (cell-penetrating peptides) have been used largely for intracellular delivery of low-molecular-mass drugs, biomolecules and particles. Most cationic CPPs bind to cell-associated glycosaminoglycans and are internalized by endocytosis, although the detailed mechanisms involved remain controversial. Sequestration and degradation in endocytic vesicles severely limits the efficiency of cytoplasmic and/or nuclear delivery of CPP-conjugated material. Re-routing the splicing machinery by using steric-block ON (oligonucleotide) analogues, such as PNAs (peptide nucleic acids) or PMOs (phosphorodiamidate morpholino oligomers), has consequently been inefficient when ONs are conjugated with standard CPPs such as Tat (transactivator of transcription), R(9) (nona-arginine), K(8) (octalysine) or penetratin in the absence of endosomolytic agents. New arginine-rich CPPs such as (R-Ahx-R)(4) (6-aminohexanoic acid-spaced oligo-arginine) or R(6) (hexa-arginine)-penetratin conjugated to PMO or PNA resulted in efficient splicing correction at non-cytotoxic doses in the absence of chloroquine. SAR (structure-activity relationship) analyses are underway to optimize these peptide delivery vectors and to understand their mechanisms of cellular internalization.

Intracrine signaling in the mammary gland

The concept of paracrine/autocrine regulation of cellular mechanisms by peptide regulators acting through cell surface receptors and generating a cascade of intracellular second messengers is highly established. Recent evidence is accumulating that peptide regulatory factors can act within the cell (without plasma membrane signaling receptors) and this action has been coined “intracrine”. The locus of many intracrine actions appears to be the nucleus. Evidence also indicates that insulin-like growth factor binding protein-3 (IGFBP-3) directly interacts with nuclear receptors. We and others have indicated that lactoferrin (Lf) and IGFBP-3 are also intracrine factors and these proteins appear on the putative list of potential intracrine factors. These proteins have a signal sequence and are secreted from cells. Additionally, they have nuclear localization sequences (NLS) and have been shown to sequester in the nuclear compartment of some cells. While IGFBP-3 has been linked to retinoid receptors and signaling, the mechanism of cellular internalization and the resulting phenotypic effect has remained unclear. However, a recent study has indicated that nucleolin may be responsible for Lf binding as well as translocation to the nuclear compartment. We hypothesize that the high regulation, high synthesis and secretion rates of Lf and IGFBP-3 by mammary cells provide the opportunity to be critical regulators of mammary gland development and differentiation via nucleolin and retinoid receptors.

Modulation of epidermal growth factor receptors on 3T3 cells by platelet-derived growth factor.

Platelet-derived growth factor does not compete with epidermal growth factor (EGF) for binding to EGF receptors on the murine 3T3 cell surface, but it modulates EGF receptors in two ways: (i) it induces a transient down regulation of EGF receptors and (ii) it inhibits EGF-induced down regulation of EGF receptors. These data suggest a common cellular internalization mechanism for the receptors for both hormones.