Cytosolic delivery of macromolecules 4. Head group-dependent membrane permeabilization by pH-sensitive detergents.

Three tertiary amine-based detergents with zero, one, or two hydroxyl groups at various positions in their head group were characterized for their ability to promote the cytosolic delivery of macromolecules. Critical micellar concentrations (CMC) and membrane-bound pKa values of the lipid constructs increased with increasing head group polarity, ranging from 1-5 microM and 5.9 to 6.3, respectively. Fluorescence resonance energy transfer (FRET) and calcein leakage experiments revealed that when the amine group is protonated introduction of -OH moieties to detergent head groups enhanced their ability to interact with and permeabilize anionic, endosome-mimicking vesicles. Different formulations of a diethanolamine-based lipid (DEL) were further evaluated for pH-dependent hemolytic activity and ability to promote cytosolic delivery of macromolecules in vitro. Intact liposomes containing DEL at its maximum limit of incorporation were less efficient than DEL-containing micelles in promoting hemoglobin leakage from human erythrocytes at acidic pH. In HeLa cells, DEL-containing detergent micelles facilitated efficient cytosolic release of endocytosed macromolecules such as fluorescein-labeled dextran of MW 10 kDa. This observation was further corroborated by a functional assay based on antisense-mediated up-regulation of enhanced green fluorescent protein (EGFP). Taken together, our findings emphasize the key role of polar head groups and micellar architecture of pH-sensitive detergents in mediating endosomal permeabilization and the efficient cytosolic delivery of macromolecules.     

Hyperthermia-triggered intracellular delivery of anticancer agent to HER2 cells by HER2-specific affibody (ZHER2-GS-Cys)-conjugated thermosensitive liposomes (HER2 affisomes)

We previously reported the formulation and physical properties of HER2 (human epidermal growth factor receptor 2)-specific affibody (ZHER2:342-Cys) conjugated thermosensitive liposomes (HER2⁺affisomes). Here we examined localized delivery potential of these affisomes by monitoring cellular interactions, intracellular uptake, and hyperthermia-induced effects on drug delivery. We modified ZHER2:342-Cys by introducing a glycine-serine spacer before the C-terminus cysteine (called ZHER2-GS-Cys) to achieve accessibility to cell surface expressed HER2. This modification did not affect HER2-specific binding and ZHER2-GS-Cys retained its ability to conjugate to the liposomes containing dipalmitoyl phosphatidyl choline: DSPE-PEG2000-Malemide, 96:04 mole ratios (HER2⁺affisomes). HER2⁺affisomes were either (i) fluorescently labeled with rhodamine-PE and calcein or (ii) loaded with an anticancer drug doxorubicin (DOX). Fluorescently labeled HER2⁺ affisomes showed at least 10-fold increase in binding to HER2⁺ cells (SK-BR-3) when compared to HER2⁻ cells (MDA-MB-468) at 37 °C. A competition experiment using free ZHER2-GS-Cys blocked HER2⁺ affisome-SK-BR-3 cell associations. Imaging with confocal microscopy showed that HER2⁺ affisomes accumulated in the cytosol of SK-BR-3 cells at 37 °C. Hyperthermia-induced intracellular release experiments showed that the treatment of HER2⁺ affisome/SK-BR-3 cell complexes with a 45 °C (± 1 °C) pre-equilibrated buffer resulted in cytosolic delivery of calcein. Substantial calcein release was observed within 20 min at 45 °C, with no effect on cell viability under these conditions. Similarly, DOX-loaded HER2⁺affisomes showed at least 2- to 3-fold higher accumulation of DOX in SK-BR-3 cells as compared to control liposomes. DOX-mediated cytotoxicity was more pronounced in SK-BR-3 cells especially at lower doses of HER2⁺affisomes. Brief exposure of liposome-cell complexes at 45 °C prior to the onset of incubations for cell killing assays resulted in enhanced cytotoxicity for affisomes and control liposomes. However, Doxil (a commercially available liposome formulation) showed significantly lower toxicity under identical conditions. Therefore, our data demonstrate that HER2⁺affisomes encompass both targeting and triggering potential and hence may prove to be viable nanodrug delivery carriers for breast cancer treatment.     

Fusogenic liposome-enhanced cytosolic delivery of magnetic nanoparticles

Magnetic nanoparticles (MNPs) are widely used in cell sorting, organelle selection, drug delivery, cell delivery, and cell tracking applications. However, organelle manipulation in living cells has been limited due to the endocytic uptake and sequestration of MNPs. Here, we introduce a method for modifying MNPs with fusogenic liposomes that facilitate MNP passage directly into the cytosol. MNPs were enclosed in fusogenic liposomes that exhibit a core–shell structure under a transmission electron microscope (TEM). The lipid-to-MNP ratio was optimized for one layer of liposome coating around each MNP, so that MNPs were delivered to the cytosol without endosomal or liposomal coatings. After incubation with the retinal pigment epithelial cell line ARPE-19, single-layer liposome-coated MNPs exhibited the highest MNP delivery efficiency. Although uncoated MNPs are taken up through endocytosis, less than 15% of the fusogenic liposome-coated MNPs co-localized with early endosomes. MNPs delivered by fusogenic liposomes showed cytosolic localization early on and increased lysosomal localization at later time points. The movement of intracellular MNPs could be manipulated with an external magnet to estimate cytosolic viscosity. Bypassing endocytosis in this way allowed efficient delivery of MNPs to the cytosol, potentially allowing for the targeting of specific organelles and controlling their motion in living cells.

Fusogenic liposomes facilitate MNPs passage into the cytosol and enable direct contact between MNPs and organelles other than endosomes.     

Efficient intracellular drug and gene delivery using folate receptor-targeted pH-sensitive liposomes composed of cationic/anionic lipid combinations.

pH-sensitive liposomes are designed to promote efficient release of entrapped agents in response to low pH. In this study, novel pH-sensitive liposomes consisting of cationic/anionic lipid combinations are evaluated for intracellular drug and gene delivery. First, liposomes composed of egg phosphatidylcholine, dimethyldioctadecylammonium bromide (DDAB), cholesteryl hemisuccinate (CHEMS), and Tween-80 (25:25:49:1, mol/mol) were shown to stably entrap calcein at pH 7.4 and undergo rapid content release and irreversible aggregation under acidic pH. Compared to pH-sensitive liposomes incorporating dioleoylphosphatidylethanolamine, these liposomes showed improved retention of pH-sensitivity in the presence of serum. The folate receptor (FR), which is amplified in a wide variety of human tumors, could be targeted by incorporating 0.1 mol% folate-polyethyleneglycol-phosphatidylethanolamine (f-PEG-PE) into liposomes. f-PEG-PE has been shown to facilitate FR-mediated endocytosis of liposomes into KB human oral cancer cells, which express amplified FR. FR-targeted pH-sensitive liposomes produced increased cytosolic release of entrapped calcein, as shown by fluorescence microscopy, and enhanced cytotoxicity of entrapped cytosine-beta-D-arabinofuranoside, as shown by an 11-fold reduction in the IC(50) in KB cells, compared to FR-targeted non-pH-sensitive liposomes. Furthermore, FR-targeted pH-sensitive liposomes composed of DDAB/CHEMS/f-PEG-PE, combined with polylysine-condensed plasmid DNA, were shown to mediate FR-specific delivery of a luciferase reporter gene into KB cells in the presence of 10% serum. These findings suggest that cationic lipid-containing pH-sensitive liposomes, combined with FR targeting, are effective vehicles for intracellular drug and gene delivery.     

Intracellular Delivery System for Antibody–Peptide Drug Conjugates

Antibodies armed with biologic drugs could greatly expand the therapeutic potential of antibody–drug conjugates for cancer therapy, broadening their application to disease targets currently limited by intracellular delivery barriers. Additional selectivity and new therapeutic approaches could be realized with intracellular protein drugs that more specifically target dysregulated pathways in hematologic cancers and other malignancies. A multifunctional polymeric delivery system for enhanced cytosolic delivery of protein drugs has been developed that incorporates endosomal-releasing activity, antibody targeting, and a biocompatible long-chain ethylene glycol component for optimized safety, pharmacokinetics, and tumor biodistribution. The pH-responsive polymeric micelle carrier, with an internalizing anti-CD22 monoclonal targeting antibody, effectively delivered a proapoptotic Bcl-2 interacting mediator (BIM) peptide drug that suppressed tumor growth for the duration of treatment and prolonged survival in a xenograft mouse model of human B-cell lymphoma. Antitumor drug activity was correlated with a mechanistic induction of the Bcl-2 pathway biomarker cleaved caspase-3 and a marked decrease in the Ki-67 proliferation biomarker. Broadening the intracellular target space by more effective delivery of protein/peptide drugs could expand the repertoire of antibody–drug conjugates to currently undruggable disease-specific targets and permit tailored drug strategies to stratified subpopulations and personalized medicines.     

Lipid-drug conjugate nanoparticles of the hydrophilic drug diminazene-cytotoxicity testing and mouse serum adsorption.

Sleeping sickness is a widely distributed disease in great parts of Africa. It is caused by Trypanosoma brucei gambiense and rhodiense, transmitted by the Tse-Tse fly. After a hemolymphatic stage, the parasites enter the central nervous system where they cannot be reached by hydrophilic drugs. To potentially deliver the hydrophilic antitrypanosomal drug diminazene diaceturate to the brain of infected mice, the drug was formulated as lipid-drug conjugate (LDC) nanoparticles (NP) by combination with stearic- (SA) and oleic acid (OA). To estimate the in vivo compatibility, the particles were incubated with human granulocytes. Because as potential delivery mechanism the absorption of specific serum proteins (ApoE, Apo AI and Apo AIV) was found to be responsible for the delivery of nanoparticles to the brain, demonstrated using PBCA nanoparticles coated with polysorbate 80 (LDL uptake mechanism) the nanoparticles were incubated with mouse serum and the adsorption pattern was determined using the 2-D PAGE technique. As a result of this study, the cytotoxic potential was shown to decrease when diminazene is part of the particle matrix compared to pure fatty acid nanoparticles and the mouse serum protein adsorption pattern differs from the samples studied earlier in human serum. Especially, the fact concerning Apo-E that could be detected when the particles were incubated in human serum is absent after the mouse serum incubation, potentially, is a critical point for the delivery via the LDL-uptake mechanism but the data demonstrate that LDC nanoparticles, with 33% (wt/wt) drug loading capacity possess the potential to act as a delivery system for hydrophilic drugs like diminazene diaceturate and that further studies have to demonstrate the usability as a brain delivery system.     

Ultrasound‐Mediated Release of Hydrophilic and Lipophilic Agents From Echogenic Liposomes

Objective. To achieve ultrasound‐controlled drug delivery using echogenic liposomes (ELIPs), we assessed ultrasound‐triggered release of hydrophilic and lipophilic agents in vitro using color Doppler ultrasound delivered with a clinical 6‐MHz compact linear array transducer. Methods. Calcein, a hydrophilic agent, and papaverine, a lipophilic agent, were each separately loaded into ELIPs. Calcein‐loaded ELIP (C‐ELIP) and papaverine‐loaded ELIP (P‐ELIP) solutions were circulated in a flow model and treated with 6‐MHz color Doppler ultrasound or Triton X‐100. Treatment with Triton X‐100 was used to release the encapsulated calcein or papaverine content completely. The free calcein concentration in the solution was measured directly by spectrofluorimetry. The free papaverine in the solution was separated from liposome‐bound papaverine by spin column filtration, and the resulting papaverine concentration was measured directly by absorbance spectrophotometry. Dynamic changes in echogenicity were assessed with low‐output B‐mode ultrasound (mechanical index, 0.04) as mean digital intensity. Results. Color Doppler ultrasound caused calcein release from C‐ELIPs compared with flow alone (P < .05) but did not induce papaverine release from P‐ELIPs compared with flow alone (P > .05). Triton X‐100 completely released liposome‐associated calcein and papaverine. Initial echogenicity was higher for C‐ELIPs than P‐ELIPs. Color Doppler ultrasound and Triton X‐100 treatments reduced echogenicity for both C‐ELIPs and P‐ELIPs (P < .05). Conclusions. The differential efficiency of ultrasound‐mediated pharmaceutical release from ELIPs for water‐ and lipid‐soluble compounds suggests that water‐soluble drugs are better candidates for the design and development of ELIP‐based ultrasound‐controlled drug delivery systems.     

Cytosolic delivery of antisense oligonucleotides by listeriolysin O-containing liposomes

Antisense oligodeoxynucleotides (ODNs) possess great potential as sequence-specific therapeutic agents. Sufficient concentrations of intact ODN must bypass membrane barriers and access the cytosol and nucleus, for ODNs to be therapeutically effective. A cytosolic delivery strategy was designed to improve the efficiency of ODN delivery in bone-marrow-derived macrophages. This liposome-based formulation utilizes listeriolysin O (LLO), the endosomolytic hemolysin from Listeria monocytogenes, to mediate the escape of ODN from endocytic compartments into the cytosol. To monitor the cytosolic delivery of ODN, subcellular trafficking of fluorescently labeled ODNs was visualized using epifluorescence microscopy. The expression of target protein and mRNA after delivery was measured using flow cytometry and Northern blot analysis, respectively. ODN specific for murine intercellular adhesion molecule-1 (ICAM-1) encapsulated in LLO-liposomes was released to the cytosol and trafficked to the nucleus, efficiently and specifically suppressing activation-induced expression of ICAM-1 at both protein and mRNA levels. Delivery without LLO resulted in sequestration of ODN in vesicular compartments leading to little inhibition of ICAM-1 expression, which supports the requirement of LLO for efficient cytosolic delivery using this system. The data clearly demonstrate that LLO-mediated escape of ODN from intracellular vesicles is an effective approach to achieve full therapeutic antisense activity in cultured macrophages.     

Enhanced Intracellular Delivery of a Model Drug Using Microbubbles Produced by a Microfluidic Device

Focal drug delivery to a vessel wall facilitated by intravascular ultrasound and microbubbles holds promise as a potential therapy for atherosclerosis. Conventional methods of microbubble administration result in rapid clearance from the bloodstream and significant drug loss. To address these limitations, we evaluated whether drug delivery could be achieved with transiently stable microbubbles produced in real time and in close proximity to the therapeutic site. Rat aortic smooth muscle cells were placed in a flow chamber designed to simulate physiological flow conditions. A flow-focusing microfluidic device produced 8 μm diameter monodisperse microbubbles within the flow chamber, and ultrasound was applied to enhance uptake of a surrogate drug (calcein). Acoustic pressures up to 300 kPa and flow rates up to 18 mL/s were investigated. Microbubbles generated by the flow-focusing microfluidic device were stabilized with a polyethylene glycol-40 stearate shell and had either a perfluorobutane (PFB) or nitrogen gas core. The gas core composition affected stability, with PFB and nitrogen microbubbles exhibiting half-lives of 40.7 and 18.2 s, respectively. Calcein uptake was observed at lower acoustic pressures with nitrogen microbubbles (100 kPa) than with PFB microbubbles (200 kPa) (p < 0.05, n > 3). In addition, delivery was observed at all flow rates, with maximal delivery (>70% of cells) occurring at a flow rate of 9 mL/s. These results demonstrate the potential of transiently stable microbubbles produced in real time and in close proximity to the intended therapeutic site for enhancing localized drug delivery.     

Characterization of the pH of folate receptor-containing endosomes and the rate of hydrolysis of internalized acid-labile folate-drug conjugates

Despite the widely accepted assumption that most endosomal compartments are acidic, evaluation of the efficiency of pH-dependent drug release from a ligand-targeted drug conjugate during receptor-mediated endocytosis is lacking. Therefore, we have characterized the kinetics of pH-dependent drug release from a model folate-drug conjugate during folate receptor (FR)-mediated endosomal trafficking. For this purpose, we synthesized an acid-labile folate-fluorescence resonance energy transfer reporter (ALFR) that emits green fluorescence (BODIPY FL, 6-((4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diazas-indacene-3-propionyl)amino)hexanoic acid) only after acid-catalyzed hydrolysis of the acyl hydrazone linker. In a cell-free system, cleavage of ALFR was found to be efficient only at acidic pH values (t1/2=1.95, 4.63, and 75 h at pH 4, 5, and 6, respectively) and essentially resistant to hydrolysis at pH 7. Curiously, when applied to folate receptor-expressing cancer cells, the acid-labile folate-linked probe exhibited little or no recovery of BODIPY FL fluorescence (green), even after 55 h of incubation, arguing very inefficient cleavage within the FR endocytic pathway. To understand this unanticipated observation, we measured the pH of FR-containing endosomes using ratiometric fluorescence microscopy and observed that most FR+ endosomes are only mildly acidic (average approximately pH 6.5). Taken together, these data argue that the FR-trafficking pathway does not involve acidic compartments and that acyl hydrazone linkers may constitute a poor option for FR-mediated drug delivery.     

Comparison of Acoustofluidic and Static Systems for Ultrasound-Mediated Molecular Delivery to T Lymphocytes

Continuous-flow acoustofluidic technologies can potentially improve processing of T lymphocytes for cell therapies by addressing the limitations with viral and non-viral delivery methods. The objective of this study was to assess the intracellular delivery efficiency with acoustofluidic treatment compared with that of static ultrasound treatment. Optimization of parameters in acoustofluidic and static configurations was performed by assessing intracellular delivery of a fluorescent compound (calcein) in viable human Jurkat T lymphocytes. Ultrasound pressure and the concentration of cationic phospholipid-coated microbubbles influenced calcein delivery in both systems. In the static system, a treatment time of 45 s increased molecular delivery compared with 0–30 s (p < 0.01). Refined parameters were used to assess molecular delivery of small and large compounds (0.6-kDa calcein and 150-kDa fluorescein isothiocyanate–dextran, respectively) after ultrasound treatment with the acoustofluidic or static systems. Molecular delivery was similar with refined parameters for acoustofluidic treatment and static treatment (p > 0.05), even though acoustofluidic treatment had lower microbubble concentration (24 μg/mL vs. 94 μg/mL) and shorter treatment time (～2–3 s vs. 45 s). This study indicates that the acoustofluidic system can significantly enhance intracellular molecular delivery, which could potentially enable acoustofluidic cell transfection during continuous flow processing for manufacture of cell therapies or other applications.     

Acid-cleavable polymeric core-shell particles for delivery of hydrophobic drugs.

Here we describe the combined use of acid-labile microgel approach and RAFT-mediated seeded dispersion polymerization technique to prepare an acid-cleavable core-shell like polymeric colloidal system for the delivery of hydrophobic drugs at slightly acidic sites. A new bisacrylate acetal crosslinker was copolymerized with n-butyl acrylate (BA) in the presence of a RAFT agent using a dispersion polymerization technique, which yielded crosslinked spherical particles with the size ranging between 150 and 500 nm. The particles were cleaved in a pH-dependent manner similar to the acid-labile hydrolysis behaviour of the crosslinker. In order to mask the hydrophobic surface of the particles, polyethylene glycol acrylate (PEG-A) was grafted onto poly(BA) seed particles via the RAFT agent groups on the particle surface. The acidic-site selective delivery potential of the poly(BA)-g-poly(PEG-A) particles was assessed in-vitro using a lipophilic fluorescent dye as a model hydrophobic drug. Ca. 73% and 34% of the total dye loaded in the particles was found to be released at pH 5.0 and 7.4 in 24 h, respectively. The growth of human neuroblastoma cells was not affected by the incubation with the core-shell particles and their cleavage by-products up to 3 mg/ml concentration. The physicochemical and the functional features support the potential value of the acid-cleavable poly(BA) core-poly(PEG-A) shell particles as carriers for the delivery of hydrophobic drugs at acidic sites.     

High cloning capacity of in vitro packaged SV40 vectors with no SV40 virus sequences

In vitro packaging of plasmid DNA using recombinant SV40 capsid proteins is a potentially useful procedure that overcomes some restrictions of the other SV40 systems such as the requirement for SV40 sequences and the limitation in size of DNA that can be packaged. The in vitro packaging system uses the four SV40 proteins (VP1, VP2, VP3, and agno) or VP1 only. The ability to confer drug resistance by three ABC transporter genes (MDR 1, MRP 1, or MXR) was determined using the surrogate fluorescent substrates rhodamine-123 or calcein AM and their specific inhibitors, or by using specific antibodies to the transporters to detect cell surface expression by fluorescence-activated cell sorter analysis (FACS). A green fluorescent protein plasmid (EGFP-C1) was also used to monitor gene transfer. The packaged plasmids ranged in size from 4.2 to 17.6 kb, and only slightly affected particle size as determined by electron microscopy. When 9.5 kb and larger plasmids were packaged using all SV40 proteins, MDR1 expression was decreased compared to VP1 alone. The size of the 15.2 kb DNA after packaging was the same as the original DNA. Packaging with SV40 capsid proteins in vitro does not require any SV40 sequences. Using either the MDR1 or the GFP gene we could demonstrate enhanced expression when cells were pretreated with phorbol 12-myristate 13-acetate (PMA) at low concentrations. Interferon-gamma did not alter expression. We conclude that in vitro packaging is more flexible then previously realized, permitting packaging of at least 17 kb plasmid DNA without the requirement for any viral sequences. This system combines efficient gene delivery of the SV40 viral vector with the presumed safety of nonviral vectors.     

Sonoporation as a Cellular Stress: Induction of Morphological Repression and Developmental Delays

For sonoporation to be established as a drug/gene delivery paradigm, it is essential to account for the biological impact of this membrane permeation strategy on living cells. Here we provide new insight into the cellular impact of sonoporation by demonstrating in vitro that this way of permeating the plasma membrane may inadvertently induce repressive cellular features even while enhancing exogenous molecule uptake. Both suspension-type (HL-60) and monolayer (ZR-75-30) cells were considered in this investigation, and they were routinely exposed to 1-MHz pulsed ultrasound (pulse length, 100 cycles; pulse repetition frequency, 1 kHz; exposure period, 60 s) with calibrated field profile (spatial-averaged peak negative pressure, 0.45 MPa) and in the presence of microbubbles (cell:bubble ratio, 10:1). The post-exposure morphology of sonoporated cells (identified as those with calcein internalization) was examined using confocal microscopy, and their cell cycle progression kinetics were analyzed using flow cytometry. Results show that for both cell types investigated, sonoporated cells exhibited membrane shrinkage and intra-cellular lipid accumulation over a 2-h period. Also, as compared with normal cells, the deoxyribonucleic acid synthesis duration of sonoporated cells was significantly lengthened, indicative of a delay in cell cycle progression. These features are known to be characteristics of a cellular stress response, suggesting that sonoporation indeed constitutes as a stress to living cells. This issue may need to be addressed in optimizing sonoporation for drug/gene delivery purposes. On the other hand, it raises opportunities for developing other therapeutic applications via sonoporation.     

Spatial–temporal event adaptive characteristics of nanocarrier drug delivery in cancer therapy

In cancer therapy, drug delivery is a complex process that aims to transit the cargo to the destination with as little damage to the normal tissue as possible. In the last decade, tremendous development and research on nanomedicine have been exploring an ideal system with efficient drug transportation and release property. For this end, series of barriers need to be circumvented by nanomedicine, including systemic barriers, such as biosurface adsorption, phagocytic clearance, bloodstream washing, interstitial pressure, degradation, as well as intracellular barriers, such as cell membrane reorganization and internalization, endo/lysosomal escape, cytosolic or subcellular localization. Rather than being random, these barriers follow a specific spatial–temporal sequence. Therefore, the nanocarriers have to be endowed with characteristics that are adaptive to particular biological milieu on systemic and intracellular levels. To this end, we reviewed the correlations between the spatial–temporal sequences of drug delivery and nanocarrier characteristics in cancer therapy, as well as strategies to achieve efficient drug delivery upon both systemic and intracellular levels.     

Cell adherence and drug delivery from particle based mesoporous silica films

Spatially and temporally controlled drug delivery is important for implant and tissue engineering applications, as the efficacy and bioavailability of the drug can be enhanced, and can also allow for drugging stem cells at different stages of development. Long-term drug delivery over weeks to months is however difficult to achieve, and coating of 3D surfaces or creating patterned surfaces is a challenge using coating techniques like spin- and dip-coating. In this study, mesoporous films consisting of SBA-15 particles grown onto silicon wafers using wet processing were evaluated as a scaffold for drug delivery. Films with various particle sizes (100–900 nm) and hence thicknesses were grown onto trichloro(octadecyl)silane-functionalized silicon wafers using a direct growth method. Precise patterning of the areas for film growth could be obtained by local removal of the OTS functionalization through laser ablation. The films were incubated with the drug model 3,3′-dioctadecyloxacarbocyanine perchlorate (DiO), and murine myoblast cells (C2C12 cells) were seeded onto films with different particle sizes. Confocal laser scanning microscopy (CLSM) was used to study the cell growth, and a vinculin-mediated adherence of C2C12 cells on all films was verified. The successful loading of DiO into the films was confirmed by UV-vis and CLSM. It was observed that the drugs did not desorb from the particles during 24 hours in cell culture. During adherent growth on the films for 4 h, small amounts of DiO and separate particles were observed inside single cells. After 24 h, a larger number of particles and a strong DiO signal were recorded in the cells, indicating a particle mediated drug uptake. The vast majority of the DiO-loaded particles remained attached to the substrate also after 24 h of incubation, making the films attractive as longer-term reservoirs for drugs on e.g. medical implants.

Particle-based mesoporous silica films synthesized through a direct growth method were successfully used as a drug delivery system.     

Visualization of targeted transduction by engineered lentiviral vectors

We have reported a method to target lentiviral vectors to specific cell types. This method requires the incorporation of two distinct molecules on the viral vector surface: one is an antibody that renders the targeting specificity for the engineered vector, and the other is a fusogenic protein that allows the engineered vector to enter the target cell. However, the molecular mechanism that controls the targeted infection needs to be defined. In this report, we tracked the individual lentiviral particles by labeling the virus with the GFP-Vpr fusion protein. We were able to visualize the surface-displayed proteins on a single virion as well as antibody-directed targeting to a desired cell type. We also demonstrated the dynamics of virus fusion with endosomes and monitored endosome-associated transport of viruses in target cells. Our results suggest that the fusion between the engineered lentivirus and endosomes takes place at the early endosome level, and that the release of the viral core into the cytosol at the completion of the virus-endosome fusion is correlated with the endosome maturation process. This imaging study sheds some light on the infection mechanism of the engineered lentivirus and can be beneficial to the design of more efficient gene delivery vectors.     

N-acetyl histidine-conjugated glycol chitosan self-assembled nanoparticles for intracytoplasmic delivery of drugs: endocytosis, exocytosis and drug release.

Nano-sized vesicular systems (nanoparticles), ranging from 10 nm to 1000 nm in size, have potential applications as drug delivery systems. Successful clinical applications require the efficient intracellular delivery of drug-loaded nanoparticles. Here we describe N-acetyl histidine-conjugated glycol chitosan (NAcHis-GC) self-assembled nanoparticles as a promising system for intracytoplasmic delivery of drugs. Because N-acetyl histidine (NAcHis) is hydrophobic at neutral pH, the conjugates formed self-assembled nanoparticles with mean diameters of 150-250 nm. In slightly acidic environments, such as those in endosomes, the nanoparticles were disassembled due to breakdown of the hydrophilic/hydrophobic balance by the protonation of the imidazole group of NAcHis. Cellular internalization and drug release of the pH-sensitive self-assembled nanoparticles were investigated by flow cytometry and confocal microscopy. NAcHis-GC nanoparticles internalized by adsorptive endocytosis were exocytosed or localized in endosomes. The amount of exocytosed nanoparticles was dependent on the pre-incubation time prior to removal of free nanoparticles from the culture media. Flow cytometry and confocal microscopy showed that NAcHis-GC nanoparticles released drugs into the cytosol and cell cycle analysis demonstrated that paclitaxel-incorporated NAcHis-GC nanoparticles were effective in inducing arrest of cell growth.     

Development of an antisense RNA delivery system using conjugates of the MS2 bacteriophage capsids and HIV-1 TAT cell penetrating peptide

RNA-based therapeutic strategies are used widely due to their highly specific mode of action. However, the major obstacle in any RNA-based therapy is cellular delivery and stability in the cells. The self-assembly of the MS2 bacteriophage capsids has been used to develop virus-like particles (VLPs) for drug delivery. In this study, we utilized the heterobifunctional crosslinker, sulfosuccinimidyl-4-(p-maleimidophenyl)-butyrate (sulfo-SMPB), to conjugate the human immunodeficiency virus-1 (HIV-1) Tat peptide and MS2 VLPs; the antisense RNA against the 5′-untranslated region (UTR) and the internal ribosome entry site (IRES) of the hepatitis C virus (HCV) was packaged into these particles by using a two-plasmid coexpression system. The MS2 VLPs conjugated with the Tat peptide were then transferred into Huh-7 cells containing an HCV reporter system. The packaged antisense RNA showed an inhibitory effect on the translation of HCV. This paper describes our initial results with this system using the Tat peptide.     

Surface Characteristics of Nanoparticles Determine Their Intracellular Fate in and Processing by Human Blood–Brain Barrier Endothelial Cells In Vitro

A polarized layer of endothelial cells that comprises the blood–brain barrier (BBB) precludes access of systemically administered medicines to brain tissue. Consequently, there is a need for drug delivery vehicles that mediate transendothelial transport of such medicines. Endothelial cells use a variety of endocytotic pathways for the internalization of exogenous materials, including clathrin-mediated endocytosis, caveolar endocytosis, and macropinocytosis. The different modes of endocytosis result in the delivery of endocytosed material to distinctive intracellular compartments and therewith correlated differential processing. To obtain insight into the properties of drug delivery vehicles that direct their intracellular processing in brain endothelial cells, we investigated the intracellular processing of fixed-size nanoparticles in an in vitro BBB model as a function of distinct nanoparticle surface modifications. Caveolar endocytosis, adsorptive-mediated endocytosis, and receptor-mediated endocytosis were promoted by the use of uncoated 500-nm particles, attachment of the cationic polymer polyethyleneimine (PEI), and attachment of prion proteins, respectively. We demonstrate that surface modifications of nanoparticles, including charge and protein ligands, affect their mode of internalization by brain endothelial cells and thereby their subcellular fate and transcytotic potential.