PEGylated iminodiacetic acid zinc complex stabilizes cationic RNA-bearing nanoparticles

Self-assembling nanoparticles comprising cationic polymers are of interest for the delivery of oligonucleotide-based therapeutics. Unfortunately, exposure of the nanoparticle cationic surface to plasma and plasma proteins compromises particle stability and circulating half-life. Herein, we report that improved nanoparticle stability can be achieved through temporary grafting of PEG to the nanoparticle surface. Grafting is induced through zinc complexation between PEG–IDA and the exposed polyhistidylated polylysine (H-K) cationic polymer of pre-formed nanoparticles.     

Nanoparticle-triggered release from lipid membrane vesicles

Superparamagnetic iron oxide nanoparticles are used in a rapidly expanding number of research and practical applications in biotechnology and biomedicine. We highlight how recent developments in iron oxide nanoparticle design and understanding of nanoparticle membrane interactions have led to applications in magnetically triggered, liposome delivery vehicles with controlled structure. Nanoscale vesicles actuated by incorporated nanoparticles allow for controlling location and timing of compound release, which enables e.g. use of more potent drugs in drug delivery as the interaction with the right target is ensured. This review emphasizes recent results on the connection between nanoparticle design, vesicle assembly and the stability and release properties of the vesicles. While focused on lipid vesicles magnetically actuated through iron oxide nanoparticles, these insights are of general interest for the design of capsule and cell delivery systems for biotechnology controlled by nanoparticles.     

Nanoparticles containing insoluble drug for cancer therapy

Nanoparticle drug formulations have been extensively researched and developed in the field of drug delivery as a means to efficiently deliver insoluble drugs to tumor cells. By mechanisms of the enhanced permeability and retention effect, nanoparticle drug formulations are capable of greatly enhancing the safety, pharmacokinetic profiles and bioavailability of the administered treatment. Here, the progress of various nanoparticle formulations in both research and clinical applications is detailed with a focus on the development of drug/gene delivery systems. Specifically, the unique advantages and disadvantages of polymeric nanoparticles, liposomes, solid lipid nanoparticles, nanocrystals and lipid-coated nanoparticles for targeted drug delivery will be investigated in detail.     

Hemocompatible poly(NIPAm-MBA-AMPS) colloidal nanoparticles as carriers of anti-inflammatory cell penetrating peptides

Anionic copolymer systems containing sulfated monomers have great potential for delivery of cationic therapeutics, but N-isopropylacrylamide (NIPAm) 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) copolymer nanoparticles have seen limited characterization to date with regard to physical properties relevant to loading and release of therapeutics. Characterization of polymeric nanoparticles incorporating AMPS showed an increased size and decreased thermodynamic swelling ratios of AMPS containing particles as compared to NIPAm nanoparticles lacking AMPS. Particles with increasing AMPS addition showed an increased propensity for uniformity, intraparticle colloidal stability, and drug loading capacity. Peptide encapsulated in particles was shielded from peptide degradation in serum. Particles were shown not impede blood coagulation or to cause hemolysis. This study has demonstrated that AMPS incorporation into traditional NIPAm nanoparticles presents a tunable parameter for changing particle LCST, size, swelling ratio, ζ potential, and cationic peptide loading potential. This one-pot synthesis results in a thermosensitive anionic nanoparticle system that is a potentially useful platform to deliver cationic cell penetrating peptides.     

The Perforin Pore Facilitates the Delivery of Cationic Cargos

Cytotoxic lymphocytes eliminate virally infected or neoplastic cells through the action of cytotoxic proteases (granzymes). The pore-forming protein perforin is essential for delivery of granzymes into the cytoplasm of target cells; however the mechanism of this delivery is incompletely understood. Perforin contains a membrane attack complex/perforin (MACPF) domain and oligomerizes to form an aqueous pore in the plasma membrane; therefore the simplest (and best supported) model suggests that granzymes passively diffuse through the perforin pore into the cytoplasm of the target cell. Here we demonstrate that perforin preferentially delivers cationic molecules while anionic and neutral cargoes are delivered inefficiently. Furthermore, another distantly related pore-forming MACPF protein, pleurotolysin (from the oyster mushroom), also favors the delivery of cationic molecules, and efficiently delivers human granzyme B. We propose that this facilitated diffusion is due to conserved features of oligomerized MACPF proteins, which may include an anionic lumen.     

The influence of pH on the vesicular traffic to the surface of the polarized epithelial cell, MDCK

The effect of pH on the secretion of the gp 80 glycoprotein complex and lysozyme from MDCK cells was examined by treatment of the cells with either NH4Cl, chloroquine or monensin. In untreated cells gp 80 is sorted with approximately 75% efficiency into the apical pathway. Lysozyme is secreted in a nonpolar fashion at both cell surfaces. Treatment of the cells with the drugs had nearly identical effects on the transport kinetics and on the ratio of the proteins released at the two plasma membrane domains. At increasing drug concentrations, the transport of both proteins to the apical and the basolateral cell surface was equally retarded. Furthermore, we observed a dose-dependent decrease in the amount of gp 80 and lysozyme released at the basolateral cell surface, which was accompanied by a nearly equivalent increase in the secretion of the two proteins at the apical plasma membrane domain. A twofold rise in the apical to basolateral ratio was already found at drug concentrations which only marginally affected the kinetics of transport. These results show that an increase in intravesicular pH not only redirects secretory proteins sorted into the basolateral pathway (Caplan et al. Nature, 329, 632 (1987] but also secretory proteins devoid of sorting information for that pathway, presumably by modulating the vesicular traffic to the basolateral cell surface.     

The calmodulin antagonist W-7 inhibits the epithelial Na+/H+ exchanger via modulating membrane surface potential

NHE3 is regulated via alterations in membrane surface charge. This is achieved through altered binding of cationic regions in the cytosolic-terminus of the exchanger with the inner leaflet of the plasma membrane. Calmodulin antagonists, including W-7, regulate surface potential and inhibit NHE3 activity. Utilizing fluorescent protein conjugated membrane probes we show that binding of cationic, but not hydrophobic peptides, to the plasma membrane is prevented by W-7. An interaction between cationic regions in the regulatory, cytosolic domain of NHE3 to anionic phospholipids in either reconstituted liposomes or the plasma membrane in cell culture is similarly prevented by W-7, at a concentration that inhibits the exchanger. We propose therefore that W-7 inhibits NHE3 activity, at least in part, by altering the association of cationic segments within the carboxy-terminus of the exchanger with anionic phospholipids in the plasma membrane.     

Toxicological effects of inorganic nanoparticles on human lung cancer A549 cells

Many researches have shown that anionic clays can be used as delivery carriers for drug or gene molecules due to their efficient cellular uptake in vitro, and enhanced permeability and retention effect in vivo. It is, therefore, highly required to establish a guideline on their potential toxicity for practical applications. The toxicity of anionic clay, layered metal hydroxide nanoparticle, was evaluated in two human lung epithelial cells, carcinoma A549 cells and normal L-132 cells, and compared with that in other human cancer cell lines such as cervical adenocarcinoma cells (HeLa) and osteosarcoma cells (HOS). The present nanoparticles showed little cytotoxic effects on the proliferation and viability of four cell lines tested at the concentrations used (<250 μg/ml) within 48 h. However, exposing cancer cells to high concentrations (250-500 μg/ml) for 72 h resulted in an inflammatory response with oxidative stress and membrane damage, which varied with the cell type (A549 > HOS > HeLa). On the other hand, the toxicity mechanism seems to be different from that of other inorganic nanoparticles frequently studied for biological and medicinal applications such as iron oxide, silica, and single walled carbon nanotubes. Iron oxide caused cell death associated with membrane damage, while single walled carbon nanotube induced oxidative stress followed by apoptosis. Silica triggered an inflammation response without causing considerable cell death for both cancer cells and normal cells, whereas layered metal hydroxide nanoparticle did not show any cytotoxic effects on normal L-132 cells in terms of inflammation response, oxidative stress, and membrane damage at the concentration of less than 250 μg/ml. It is , therefore, highly expected that the present nanoparticle can be used as a efficient vehicle for drug delivery and cancer cell targeting as well.     

The Effects of pH and Intraliposomal Buffer Strength on the Rate of Liposome Content Release and Intracellular Drug Delivery

Targeted liposomal drug formulations may enter cells by receptor-mediated endocytosis and then traffick by membrane flow into acidic intracellular compartments. In order to understand the impact of these intracellular pH changes on liposomal drug unloading, the effect of pH on the release from folate-targeted liposomes of three model compounds with distinct pH dependencies was examined. 5(6)-carboxyfluorescein, which titrates from its anionic to uncharged form following internalization by KB cells, displays strong endocytosis-dependent release, since only its uncharged (endosomal) form is membrane permeable. Endocytosis-triggered unloading of drugs of this sort is enhanced by encapsulating the drug in a weak buffer at neutral pH, so that acidification of the intraliposomal compartment following cellular uptake can occur rapidly. Sulforhodamine B, in contrast, retains both anionic and cationic charges at endosomal pH (~pH 5), and consequently, escapes the endosomes only very slowly. Doxorubicin, which is commonly loaded into liposomes in its membrane-impermeable (cationic) form using an acidic buffer, still displays endocytosis-triggered unloading, since sufficient uncharged doxorubicin remains at endosomal pHs to allow rapid re-equilibration of the drug according to the new proton gradient across the membrane. In this case, when the extraliposomal [H⁺] increases 250-fold from 4 × 10^–8 M (pH 7.4, outside the cell) to 10^–5 M (pH 5, inside the endosome), the ratio of doxorubicin inside to outside the liposome must decrease by a factor of 250. Therefore, the collapse of the transliposomal pH gradient indirectly drives an efflux of the drug molecule from the liposome. Since a change in intraliposomal pH is not required to unload drugs of this type, the intraliposomal compartment can be buffered strongly at acidic pH to prevent premature release of the drug outside the cell. In summary, pH triggered release of liposome-encapsulated drugs can be achieved both with drugs that increase as well as decrease their membrane permeabilities upon acidification, as long as the intraliposomal buffer strength and pH is rationally selected.     

Transepithelial transport of drugs by the multidrug transporter in cultured Madin-Darby canine kidney cell epithelia

We studied transepithelial transport of 3H-labeled hydrophobic cationic drugs in epithelia formed by wild-type and by drug-resistant Madin-Darby canine kidney (MDCk) cells that had been infected with a retrovirus carrying the multidrug-resistance (MDR1) cDNA which encodes the P-glycoprotein. P-glycoprotein is an ATP consuming plasma membrane multidrug transporter responsible for the efflux of cytotoxic chemotherapeutic drugs from resistant cancer cells. Wild-type MDCK cells have small amounts of P-glycoprotein detected by immunoprecipitation. Net transepithelial transport across wild-type MDCK epithelia was demonstrated. Basal to apical flux of 100 nM vinblastine was about six times higher than apical to basal flux. Addition of unlabeled vinblastine reduced basal to apical flux of tracer and increased apical to basal flux of tracer, a pattern expected if there is a saturable pump that extrudes vinblastine at the apical plasma membrane. Daunomycin, vincristine, and actinomycin D were also actively transported and at 20 microM these agents inhibited transport of vinblastine, suggesting that wild-type MDCK cells have a common transporter for all these drugs. Vinblastine transport was also inhibited by 20 microM verapamil, which inhibits the multidrug transporter and reverses multidrug-resistance in non-polarized cells. Net transepithelial transport of all these cytotoxic drugs and of verapamil was much higher in epithelia formed by MDCK cells infected with a human MDR1 virus (MDR-MDCK) which is expressed on the apical surface of MDR-MDCK monolayers. Because the transport of these cytotoxic drugs and verapamil is increased in MDR-MDCK epithelia compared to wild-type MDCK epithelia, transport in both these cell populations can be attributed to P-glycoprotein. These results are consistent with a role for P-glycoprotein in multidrug secretory transport across the epithelium of the proximal tubule since P-glycoprotein is normally expressed on the apical membrane of proximal tubule cells.     

Chitosan-Modified PLGA Nanoparticles with Versatile Surface for Improved Drug Delivery

Shortage of functional groups on surface of poly(lactide-co-glycolide) (PLGA)-based drug delivery carriers always hampers its wide applications such as passive targeting and conjugation with targeting molecules. In this research, PLGA nanoparticles were modified with chitosan through physical adsorption and chemical binding methods. The surface charges were regulated by altering pH value in chitosan solutions. After the introduction of chitosan, zeta potential of the PLGA nanoparticle surface changed from negative charge to positive one, making the drug carriers more affinity to cancer cells. Functional groups were compared between PLGA nanoparticles and chitosan-modified PLGA nanoparticles. Amine groups were exhibited on PLGA nanoparticle surface after the chitosan modification as confirmed by Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy. The modified nanoparticles showed an initial burst release followed by a moderate and sustained release profile. Higher percentage of drugs from cumulative release can be achieved in the same prolonged time range. Therefore, PLGA nanoparticles modified by chitosan showed versatility of surface and a possible improvement in the efficacy of current PLGA-based drug delivery system.     

Multifunctional Nanoparticles Delivering Small Interfering RNA and Doxorubicin Overcome Drug Resistance in Cancer

Drug resistance is a major challenge to the effective treatment of cancer. We have developed two nanoparticle formulations, cationic liposome-polycation-DNA (LPD) and anionic liposome-polycation-DNA (LPD-II), for systemic co-delivery of doxorubicin (Dox) and a therapeutic small interfering RNA (siRNA) to multiple drug resistance (MDR) tumors. In this study, we have provided four strategies to overcome drug resistance. First, we formed the LPD nanoparticles with a guanidinium-containing cationic lipid, i.e. N,N-distearyl-N-methyl-N-2-(N′-arginyl) aminoethyl ammonium chloride, which can induce reactive oxygen species, down-regulate MDR transporter expression, and increase Dox uptake. Second, to block angiogenesis and increase drug penetration, we have further formulated LPD nanoparticles to co-deliver vascular endothelial growth factor siRNA and Dox. An enhanced Dox uptake and a therapeutic effect were observed when combined with vascular endothelial growth factor siRNA in the nanoparticles. Third, to avoid P-glycoprotein-mediated drug efflux, we further designed another delivery vehicle, LPD-II, which showed much higher entrapment efficiency of Dox than LPD. Finally, we delivered a therapeutic siRNA to inhibit MDR transporter. We demonstrated the first evidence of c-Myc siRNA delivered by the LPD-II nanoparticles down-regulating MDR expression and increasing Dox uptake in vivo. Three daily intravenous injections of therapeutic siRNA and Dox (1.2 mg/kg) co-formulated in either LPD or LPD-II nanoparticles showed a significant improvement in tumor growth inhibition. This study highlights a potential clinical use for the multifunctional nanoparticles with an effective delivery property and a function to overcome drug resistance in cancer. The activity and the toxicity of LPD- and LPD-II-mediated therapy are compared.     

Charged anaesthetics alter LM-fibroblast plasma-membrane enzymes by selective fluidization of inner or outer membrane leaflets.

The functional consequences of the differences in lipid composition and structure between the two leaflets of the plasma membrane were investigated. Fluorescence of 1,6-diphenylhexa-1,3,5-triene(DPH), quenching, and differential polarized phase fluorimetry demonstrated selective fluidization by local anaesthetics of individual leaflets in isolated LM-cell plasma membranes. As measured by decreased limiting anisotropy of DPH fluorescence, cationic (prilocaine) and anionic (phenobarbital and pentobarbital) amphipaths preferentially fluidized the cytofacial and exofacial leaflets respectively. Unlike prilocaine, procaine, also a cation, fluidized both leaflets of these membranes equally. Pentobarbital stimulated 5'-nucleotidase between 0.1 and 5 mM and inhibited at higher concentrations, whereas phenobarbital only inhibited, at higher concentrations. Cationic drugs were ineffective. Two maxima of (Na+ + K+)-ATPase activation were obtained with both anionic drugs. Only one activation maximum was obtained with both cationic drugs. The maximum in activity below 1 mM for all four drugs clustered about a single limiting anisotropy value in the cytofacial leaflet, whereas there was no correlation between activity and limiting anisotropy in the exofacial leaflets. Therefore, although phenobarbital and pentobarbital below 1 mM fluidized the exofacial leaflet more than the cytofacial leaflet, the smaller fluidization in the cytofacial leaflet was functionally significant for (Na+ + K+)-ATPase. Mg2+-ATPase was stimulated at 1 mM-phenobarbital, unaffected by pentobarbital and slightly stimulated by both cationic drugs at concentrations fluidizing both leaflets. Thus the activity of (Na+ + K+)-ATPase was highly sensitive to selective fluidization of the leaflet containing its active site, whereas the other enzymes examined were little affected by fluidization of either leaflet.     

Cellular uptake [correction of utake] of the Tat peptide: an endocytosis mechanism following ionic interactions

The cellular delivery of various biological compounds has recently been improved by conjugating them to short peptides known as protein transduction domains or cell penetrating peptides. These peptides include Tat, Antennapedia and arginine-rich peptides. The common feature of these peptides is their highly cationic nature. Up to now, the cellular uptake of about 50 different peptides and proteins coupled to Tat or Antennapedia peptides has been reported. The ability to deliver molecules into cells is not limited to peptide moieties, since oligonucleotides, peptide nucleic acids or other low molecular weight entities have been successfully internalized. Moreover, most of these examples have been accompanied by the expected biological response. More surprisingly, the uptake of large structures such as liposomes, phages, nanoparticles or adenoviruses has also been documented. Indeed the mechanism by which these very different entities could enter cells following a putative common pathway appeared more and more intriguing after each new reported example of cellular uptake mediated by these peptides. After a long period of uncertainty regarding the mechanism of entry, data from several groups now argue for an energy-dependent process of entry. The entry of most of these molecules is likely to be inhibited by low temperature incubation or in the presence of various drugs applied to inhibit the energy-dependent pathway of cell entry. Moreover, the binding of the highly cationic Tat peptide to various anionic membrane components probably initiates the first step of the cell internalization process.     

Microtubule perturbation retards both the direct and the indirect apical pathway but does not affect sorting of plasma membrane proteins in intestinal epithelial cells (Caco-2).

Endogenous plasma membrane proteins are sorted from two sites in the human intestinal epithelial cell line Caco-2. Apical proteins are transported from the Golgi apparatus to the apical domain along a direct pathway and an indirect pathway via the basolateral membrane. In contrast, basolateral proteins never appear in the apical plasma membrane. Here we report on the effect of the microtubule-active drug nocodazole on the post-synthetic transport and sorting of plasma membrane proteins. Pulse-chase radiolabeling was combined with domain-specific cell surface assays to monitor the appearance of three apical and one basolateral protein in plasma membrane domains. Nocodazole was found to drastically retard both the direct transport of apical proteins from the Golgi apparatus and the indirect transport (transcytosis) from the basolateral membrane to the apical cell surface. In contrast, neither the transport rates of the basolateral membrane nor the sorting itself were significantly affected by the nocodazole treatment. We conclude that an intact microtubular network facilitates, but is not necessarily required for, the transport of apical membrane proteins along the two post-Golgi pathways to the brush border.     

基于核酸自组装纳米结构的siRNA药物递送研究进展

RNA干扰(RNA interference,RNAi)作为转录后调节机制,可靶向mRNA进行剪切降解从而发挥基因沉默效应.siRNA (small interference RNA)因其高效性和特异性而被广泛应用于药物研究中.目前,研究者们已开发了多种阳离子载体用于siRNA递送.但由于siRNA双链结构具有相对较强的刚性结构,且阴离子电荷密度较低,无法与阳离子载体形成稳定、致密的复合物,使得siRNA的应用仍面临诸多挑战,如细胞摄取率低、靶向特异性差、递送过程不稳定、潜在的细胞毒性以及易诱发免疫反应等.近年来,核酸自组装纳米结构由于其结构灵活且负电荷密度较高而受到广泛关注,有望实现siRNA药物的高效递送和基因沉默.本文综述了近年来基于核酸自组装纳米结构的siRNA递送的研究进展及其应用.     

脑部靶向给药技术

介于脑部毛细血管与脑组织之间的血脑屏障是一层难以通过的生理屏障 ,能够阻挡大多数外源物质进入脑内。临床上采用的中枢神经系统药物大多是能够扩散通过血脑屏障的小分子脂溶性物质 ,而这类药物已经远远不能满足临床需要 ,很多疾病的诊断、治疗需要大分子、水溶性物质。传统的将这类大分子药物导入脑部的方法效果差、危险性大 ,因此近年来针对能够通过血脑屏障脑部靶向给药技术的研究逐渐成为热点。综述了近年来国际上使用嵌合肽、免疫脂质体及纳米粒子解决脑部靶向性给药的研究进展。     

Cream Formulation Impact on Topical Administration of Engineered Colloidal Nanoparticles

In order to minimize the impact of systemic toxicity of drugs in the treatment of local acute and chronic inflammatory reactions, the achievement of reliable and efficient delivery of therapeutics in/through the skin is highly recommended. While the use of nanoparticles is now an established practice for drug intravenous targeted delivery, their transdermal penetration is still poorly understood and this important administration route remains almost unexplored. In the present study, we have synthesized magnetic (iron oxide) nanoparticles (MNP) coated with an amphiphilic polymer, developed a water-in-oil emulsion formulation for their topical administration and compared the skin penetration routes with the same nanoparticles deposited as a colloidal suspension. Transmission and scanning electron microscopies provided ultrastructural evidence that the amphiphilic nanoparticles (PMNP) cream formulation allowed the efficient penetration through all the skin layers with a controllable kinetics compared to suspension formulation. In addition to the preferential follicular pathway, also the intracellular and intercellular routes were involved. PMNP that crossed all skin layers were quantified by inductively coupled plasma mass spectrometry. The obtained data suggests that combining PMNP amphiphilic character with cream formulation improves the intradermal penetration of nanoparticles. While PMNP administration in living mice via aqueous suspension resulted in preferential nanoparticle capture by phagocytes and migration to draining lymph nodes, cream formulation favored uptake by all the analyzed dermis cell types, including hematopoietic and non-hematopoietic. Unlike aqueous suspension, cream formulation also favored the maintenance of nanoparticles in the dermal architecture avoiding their dispersion and migration to draining lymph nodes via afferent lymphatics.     

A model predicting delivery of saquinavir in nanoparticles to human monocyte/macrophage (Mo/Mac) cells

Modeling the influence of a technology such as nanoparticle systems on drug delivery is beneficial in rational formulation design. While there are many studies showing drug delivery enhancement by nanoparticles, the literature provides little guidance regarding when nanoparticles are useful for delivery of a given drug. A model was developed predicting intracellular drug concentration in cultured cells dosed with nanoparticles. The model considered drug release from nanoparticles as well as drug and nanoparticle uptake by the cells as the key system processes. Mathematical expressions for these key processes were determined using experiments in which each process occurred in isolation. In these experiments, intracellular delivery of saquinavir, a low solubility drug dosed as a formulation of poly(ethylene oxide)-modified poly(epsilon- caprolactone) (PEO-PCL) nanoparticles, was studied in THP-1 human monocyte/macrophage (Mo/Mac) cells. The model accurately predicted the enhancement in intracellular concentration when drug was administered in nanoparticles compared to aqueous solution. This simple model highlights the importance of relative kinetics of nanoparticle uptake and drug release in determining overall enhancement of intracellular drug concentration when dosing with nanoparticles.     

Delivery of raft-associated, GPI-anchored proteins to the apical surface of polarized MDCK cells by a transcytotic pathway

Epithelial cell polarity depends on mechanisms for targeting proteins to different plasma membrane domains. Here, we dissect the pathway for apical delivery of several raft-associated, glycosyl phosphatidylinositol (GPI)-anchored proteins in polarized MDCK cells using live-cell imaging and selective inhibition of apical or basolateral exocytosis. Rather than trafficking directly from the trans-Golgi network (TGN) to the apical plasma membrane as previously thought, the GPI-anchored proteins followed an indirect, transcytotic route. They first exited the TGN in membrane-bound carriers that also contained basolateral cargo, although the two cargoes were laterally segregated. The carriers were then targeted to and fused with a zone of lateral plasma membrane adjacent to tight junctions that is known to contain the exocyst. Thereafter, the GPI-anchored proteins, but not basolateral cargo, were rapidly internalized, together with endocytic tracer, into clathrin-free transport intermediates that transcytosed to the apical plasma membrane. Thus, apical sorting of these GPI-anchored proteins occurs at the plasma membrane, rather than at the TGN.